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https://openalex.org/W4387299436	https://www.nature.com/articles/s41598-023-43888-z.pdf	English	null	Effect of oxygen vacancy and Si doping on the electrical properties of Ta2O5 in memristor characteristics	Scientific reports	2,023	cc-by	10,367	OPEN Md. Sherajul Islam 1,2, Jonghoon Lee 1,3, Sabyasachi Ganguli 1 & Ajit K. Roy 1 , Jonghoon Lee 1,3, Sabyasachi Ganguli 1 & Ajit K. Roy 1 The resistive switching behavior in Ta2O5 based memristors is largely controlled by the formation and annihilation of conductive filaments (CFs) that are generated by the migration of oxygen vacancies (OVs). To gain a fundamental insight on the switching characteristics, we have systematically investigated the electrical transport properties of two different Ta2O5 polymorphs ( ǫ-Ta2O5 and λ-Ta2O5), using density functional theory calculations, and associated vacancy induced electrical conductivity using Boltzmann transport theory. The projected band structure and DOS in a few types of OVs, (two-fold (O2fV), three-fold (O3fV), interlayer (OILV), and distorted octahedral coordinated vacancies (OεV)) reveal that the presence of OILV would cause Ta2O5 to transition from a semiconductor to a metal, leading to improved electrical conductivity, whereas the other OV types only create localized mid-gap defect states within the bandgap. On studying the combined effect of OVs and Si-doping, a reduction of the formation energy and creation of defect states near the conduction band edge, is observed in Si-doped Ta2O5, and lower energy is found for the OVs near Si atoms, which would be advantageous to the uniformity of CFs produced by OVs. These findings can serve as guidance for further experimental work aimed at enhancing the uniformity and switching properties of resistance switching for Ta2O5-based memristors. Of late, tantalum pentoxide (Ta2O5) has been extensively studied for its potential applications in memristors due to its high dielectric constant, high breakdown voltage, and good thermal stability1–13. One of the key advantages of Ta2O5 as a memristor material is its ability to undergo resistive switching (RS), a phenomenon in which the resistance of the material can be switched between high and low states by applying an electric field. Ta2O5 mem- ristors, like other comparable metal oxides, are important due to their potential to revolutionize memory and computing technologies, facilitate new forms of artificial intelligence, curtail energy consumption, and empower the development of intricate and complex systems8,9,14–17. Usually, the RS phenomenon in Ta2O5 is attributed to the creation and disruption of conductive filaments (CFs) within the material, which can be controlled by the applied voltage and the presence of oxygen vacancies (OVs)18–20. 1Materials and Manufacturing Directorate, Air Force Research Laboratory, Wright-Patterson Air Force Base, Dayton, OH, USA. 2Spectral Energies, LLC, Dayton, OH, USA. 3ARCTOS Technology Solutions, Dayton, OH, USA. email: sheraj_kuet@eee.kuet.ac.bd; ajit.roy@us.af.mil www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports/ of Ta2O5 based memristor are correlated with the homogeneity of the material’s nanoscale CFs. The ability of Ta2O5-based memristor to generate CFs may be attributed to the electric field-driven production and spread of OV. However, the existence of many disorders in the deposited resistive layer, the interaction of the OV with the dopant, and the difficulties in accurately defining the RS processes have made progress in this area difficult. To limit material trials and clarify the impact on system operation, it is helpful to have a theoretical knowledge of these defects. Therefore, it is critical to investigate how different dopants affect OV and how Ta2O5 -based memristor performance be improved.f of Ta2O5 based memristor are correlated with the homogeneity of the material’s nanoscale CFs. The ability of Ta2O5-based memristor to generate CFs may be attributed to the electric field-driven production and spread of OV. However, the existence of many disorders in the deposited resistive layer, the interaction of the OV with the dopant, and the difficulties in accurately defining the RS processes have made progress in this area difficult. To limit material trials and clarify the impact on system operation, it is helpful to have a theoretical knowledge of these defects. Therefore, it is critical to investigate how different dopants affect OV and how Ta2O5 -based memristor performance be improved.f p p On the other hand, depending on the fabrication method and conditions, Ta2O5 can exist in different crystal structures such as α-Ta2O5, β-Ta2O5, and ε-Ta2O5 phases27–39 that can affect the performance of the memristor. The most commonly used structure of Ta2O5 is the amorphous phase40. This is because amorphous Ta2O5 can undergo a reversible RS phenomenon that is essential for the operation of memristors. Other structures of Ta2O5, such as the orthorhombic, tetragonal, monoclinic, and cubic phases, may also be used for memristor applications32,34,37. However, these structures may require additional processing steps, such as annealing or doping, to achieve the desired properties for memristor operation. Moreover, a hidden polymorph namely called as lambda phase (λ-Ta2O5) can also exist30. Different crystal structures of Ta2O5 may exhibit different resistive switching behaviors because the breakdown voltage should be different for each structure (usually proportional to the square of the bandgap energy), which can make it challenging to optimize the performance of the device. Calculation methods All the investigations have been conducted using the ab initio first-principles plane wave pseudopotential approach executed in the Vienna Ab initio Simulation Package (VASP)44,45. The electron exchange correlation is obtained using the generalized gradient approximation of the Perdew-Burke-Ernzerhof solid (GGA-PBEsol) functional46. The structure is optimized using the RMM-DIIS algorithm with the force convergence of 0.02 eV/Å (1 × 10–5 eV). Following relaxation, the electronic structures are characterized using a kinetic energy cutoff of 400 eV. The Brillouin-zone is sampled with a k-point mesh of 17 × 13 × 9 for the structural relaxations and all the electronic properties calculations. The electrical conductivity has been calculated using the Boltzmann transport theory as implemented in MedeA VASP. Using the Boltzmann transport theory47, one can represent the electrical conductivity tensor of a solid in terms of energy dependence as (1) σαβ(E) = e2 C k n −∂f (E) ∂E vα knvβ knτkn (1) where f(E) denotes the Fermi function f (E) = 1 eβ(E−µ)+1 , with μ being the chemical potential, β = 1 kBT. τkn stands for the relaxation time, which relies on the band index (n), spin, and k-point and replicates phenomena such as electron–phonon scattering on the electronic states. vα kn represents the group velocity. The first derivative of the band energy ( ǫkn ) with respect to the appropriate Cartesian component ( α ) of the k-vector yields the vα kn for each band (n) and k-point as where f(E) denotes the Fermi function f (E) = 1 eβ(E−µ)+1 , with μ being the chemical potential, β = 1 kBT. τkn stands for the relaxation time, which relies on the band index (n), spin, and k-point and replicates phenomena such as electron–phonon scattering on the electronic states. vα kn represents the group velocity. www.nature.com/scientificreports/ To fully understand the switching mechanism of Ta2O5 in memristors, it is important to identify the mechanisms involved in the switching behavior, such as materials crystalline phase influencing oxygen migration and defect formation, and to understand how these mechanisms are affected by different experimental conditions stated above. f yf In this work, instead of studying all Ta2O5 crystalline phases we discussed above, we limit our work inves- tigating the vacancy induced electrical transport properties of orthorhombic and lambda phase Ta2O5, due to their good agreement with the experimentally observed electrical band gap of amorphous Ta2O5, using density functional theory investigations. The effect of OV on the electrical conductivity of Ta2O5 has been calculated using Boltzmann transport theory. Chemical doping, combined with OVs, is known in many cases to impact the performance of Ta2O5 memristors and help optimize key parameters such as the RS characteristics, endurance, retention, and variability. Although several molecules as dopant to Ta2O5 are feasible41–43, however, here only the combined effect of Si-doping and OV on electrical conductivity are investigated. To explore the local structural changes due to OV and doping effect, the formation energy, projected electronic band structure, and projected density of states have been calculated. This study can guide further experimental efforts aimed at enhancing the uniformity of RS and switching properties of Ta2O5-based memristor devices. OPEN However, the formation and characteristics of CFs in Ta2O5-based memristors can be complicated and greatly contingent on a number of variables, leading to several challenges in their development and optimization. Attaining consistent and replicable filament formation stands out as a primary obstacle. The formation and stability of CFs may exhibit significant susceptibility to the device configuration, the conditions of deposition, and the materials of the electrode, thereby posing challenges in attaining uniform and foreseeable performance. Hence, optimizing the device fabrication process and materials is crucial for achieving reliable and reproducible filament formation. g pi Earlier studies demonstrated that OVs are known to act as nucleation sites for the formation of CFs in Ta2O5 memristors18–21. Besides, OVs affect the stability of the CFs, which in turn can affect the endurance and retention of its resistive states, crucial to memristor’s performance reliability. OVs can also affect the switching dynamics of the memristor, including the voltage threshold, switching speed, and noise level. In this context, several stud- ies have investigated the relationship between OVs and the electrical conductivity of Ta2O5 22–26. For example, research has shown that OVs can increase electrical conductivity by creating additional charge carriers and defect states. The concentration and distribution of OVs can also affect the electrical conductivity by altering the material’s band structure and carrier mobility. To the best of our understanding, the switching properties 1Materials and Manufacturing Directorate, Air Force Research Laboratory, Wright-Patterson Air Force Base, Dayton, OH, USA. 2Spectral Energies, LLC, Dayton, OH, USA. 3ARCTOS Technology Solutions, Dayton, OH, USA. email: sheraj_kuet@eee.kuet.ac.bd; ajit.roy@us.af.mil Scientific Reports \| (2023) 13:16656 \| https://doi.org/10.1038/s41598-023-43888-z www.nature.com/scientificreports/ Results and discussionf Among many different polymorphs of Ta2O5 that have been discovered, the β-Ta2O5 (orthorhombic space group Pmmm)35,39,50–53 is the most studied polymorph because it is thought to be stable at low temperatures and experi- ence a phase transition to α-Ta2O5 at relatively high temperatures (1630 K)39. Another common phase is ǫ-Ta2O5 (space group C2/c)36, which can be produced using either a chemical method36 or a high-pressure synthesis37. The amorphous phase, the hexagonal or δ phase, the high-pressure-prepared phase, and the polymorph with a high number of OVs can also be detected by the X-ray particle diffraction observations31–33,54. In addition, λ-Ta2O5 has Among many different polymorphs of Ta2O5 that have been discovered, the β-Ta2O5 (orthorhombic space group Pmmm)35,39,50–53 is the most studied polymorph because it is thought to be stable at low temperatures and experi- ence a phase transition to α-Ta2O5 at relatively high temperatures (1630 K)39. Another common phase is ǫ-Ta2O5 (space group C2/c)36, which can be produced using either a chemical method36 or a high-pressure synthesis37. The amorphous phase, the hexagonal or δ phase, the high-pressure-prepared phase, and the polymorph with a high number of OVs can also be detected by the X-ray particle diffraction observations31–33,54. In addition, λ-Ta2O5 has drawn a lot of attention recently due to its high structural stability and good agreement with the experimentally observed electrical band gap of amorphous Ta2O5 30. We primarily concentrate on the last two demonstrative phases, specifically ǫ-Ta2O5 37,55–58 and λ-Ta2O5 19,59,60, as they can be produced under comparatively favorable circumstances and are generally thought to be sufficiently stable for use in real-world applications, as compared to the other phases. It is noted that our examination of various Ta2O5 polymorphic structures is by no means comprehensive; instead, the primary focus of this article is on analyzing the impacts of varying OVs on certain electronic transport characteristics. ǫ-Ta2O5 phase belongs to the monoclinic structure with the C2/c symmetry and λ-Ta2O5 phase belongs to the orthorhombic structure with the Pbam symmetry. In ǫ-Ta2O5 crystal structure, each Ta atom has a distorted octahedral coordination environment, with six neighboring O atoms. On the other hand, three different O sites can be found in the λ-Ta2O5 structure: the doubly and threefold coordinated sites in the Ta2O3 plane, as well as the twofold coordinated site of the Ta-O-Ta interlayer chain, as shown in Fig. 1. Calculation methods The first derivative of the band energy ( ǫkn ) with respect to the appropriate Cartesian component ( α ) of the k-vector yields the vα kn for each band (n) and k-point as (2) vα kn = 1 ℏ ∂ǫkn ∂kα (2) The electrical conductivity can further be reduced by relating the so-called transport distribution47,48 as (3) αβ(E) = 1 C k n vα knvβ knτknδ(E −ǫkn) (3) Thus, the electrical conductivity coefficient, σαβ can be represented as Thus, the electrical conductivity coefficient, σαβ can be represented as (4) σαβ = e2 ∞ −∞ dE(−∂f (E) ∂E )αβ(E) (4) To calculate electrical conductivity, the self-consistent field (SCF) charge density of the complete system is determined in the first stage with a k-point mesh of 9 × 5 × 5. Based on the SCF charge density obtained in the preceding phase, the Fermi surface and a band structure are then generated with a relatively fine and regular https://doi.org/10.1038/s41598-023-43888-z https://doi.org/10.1038/s41598-023-43888-z Scientific Reports \| (2023) 13:16656 \| https://doi.org/10.1038/s41598-023-43888-z www.nature.com/scientificreports/ k-point grids of 17 × 13 × 9. The band structure acquired in the second phase is then used to compute the elec- tronic transport characteristics using the BoltzTraP code49. k-point grids of 17 × 13 × 9. The band structure acquired in the second phase is then used to compute the elec- tronic transport characteristics using the BoltzTraP code49. Results and discussionf electron traps, leading to changes in the electronic structure of the material and altering its electrical conductivity. In the case of Ta2O5, OVs are expected to enhance the electrical conductivity of the material because the presence of OVs introduces electronic states within the band gap of Ta2O5, which can act as charge carriers and increase the conductivity. These electronic states are usually located closer to the conduction band, which means that electrons can easily move into these states and contribute positively to the conductivity. Experimental studies have confirmed that oxygen vacancies do indeed affect the electrical conductivity of Ta2O5. For example, Seki et al. found that annealing Ta2O5 films in reducing atmospheres (i.e., environments with a low oxygen partial pressure) resulted in a significant increase in the films’ electrical conductivity61. This was attributed to the crea- tion of OVs in the material. To comprehend the vacancy induced electrical conductivity in both structures, we have calculated the elec- tronic band structure for all these types of vacancies. The electronic band structure from PBEsol for pristine ǫ -Ta2O5 and λ-Ta2O5 structures are presented in supplementary information (Figure S1). The ǫ-Ta2O5 structure shows a larger band gap by pushing the conduction band minimum (CBM) to higher energy compared to the λ-Ta2O5 structure. An electronic gap of 3.18 eV and 2.16 eV is observed for ǫ-Ta2O5 and λ-Ta2O5 structures, respectively. The obtained bandgap is well matched with the previous studies19,30,37. It is worth to mention, earlier investigations demonstrated that HSE functional may estimate a higher band gap compared to PBE functional19,30. However, our focus here is to understand how conductivity is changed due to OVs, and hence the PBEsol is justifiably an acceptable choice for this study. It is evident from the band structures that the O atoms form the valence bands, while the conduction bands are formed by the delocalized electrons of the Ta atoms, as shown in Fig. 3. As evident from the data in Fig. 3, the OVs in Ta2O5 can reduce the band gap of the material through the formation of defect states within the band gap. When an O atom is missing from the crystal lattice, there is a local change in the charge balance within the crystal, leading to the formation of electronic defect states. Results and discussionf Chemical formula ǫ-Ta2O5 37 This work λ-Ta2O5 30 This work Cell setting Monoclinic Monoclinic Orthorhombic Orthorhombic Space group C2/c C2/c Pbam Pbam a (Å) 12.7853 12.74156 6.25 6.216 b (Å) 4.8537 4.8200 7.40 7.338 c (Å) 5.5276 5.49353 3.83 3.798 α (º) 90 90 90 90.00 β (º) 90 90 90 90.00 γ (º) 104.264 104.22 90 90.00 Table 1. Calculated structural parameters of ǫ-Ta2O5 and λ-Ta2O5 phase Chemical formula ǫ-Ta2O5 37 This work λ-Ta2O5 30 This work Cell setting Monoclinic Monoclinic Orthorhombic Orthorhombic Space group C2/c C2/c Pbam Pbam a (Å) 12.7853 12.74156 6.25 6.216 b (Å) 4.8537 4.8200 7.40 7.338 c (Å) 5.5276 5.49353 3.83 3.798 α (º) 90 90 90 90.00 β (º) 90 90 90 90.00 γ (º) 104.264 104.22 90 90.00 Table 1. Calculated structural parameters of ǫ-Ta2O5 and λ-Ta2O5 phase. Chemical formula ǫ-Ta2O5 37 This work λ-Ta2O5 30 This work Cell setting Monoclinic Monoclinic Orthorhombic Orthorhombic Space group C2/c C2/c Pbam Pbam a (Å) 12.7853 12.74156 6.25 6.216 b (Å) 4.8537 4.8200 7.40 7.338 c (Å) 5.5276 5.49353 3.83 3.798 α (º) 90 90 90 90.00 β (º) 90 90 90 90.00 γ (º) 104.264 104.22 90 90.00 Figure 2. Electrical conductivity of different types of oxygen vacancies induced Ta2O5 at room temperature (300 K). Table 1. Calculated structural parameters of ǫ-Ta2O5 and λ-Ta2O5 phase. Figure 2. Electrical conductivity of different types of oxygen vacancies induced Ta2O5 at room temperature (300 K). Figure 2. Electrical conductivity of different types of oxygen vacancies induced Ta2O5 at room temperature (300 K). electron traps, leading to changes in the electronic structure of the material and altering its electrical conductivity. In the case of Ta2O5, OVs are expected to enhance the electrical conductivity of the material because the presence of OVs introduces electronic states within the band gap of Ta2O5, which can act as charge carriers and increase the conductivity. These electronic states are usually located closer to the conduction band, which means that electrons can easily move into these states and contribute positively to the conductivity. Experimental studies have confirmed that oxygen vacancies do indeed affect the electrical conductivity of Ta2O5. For example, Seki et al. found that annealing Ta2O5 films in reducing atmospheres (i.e., environments with a low oxygen partial pressure) resulted in a significant increase in the films’ electrical conductivity61. This was attributed to the crea- tion of OVs in the material. Results and discussionf The calculated lattice parameters of the optimized structures of both phases along with the experimentally obtained values are presented in Table 1. The optimized lattice parameters derived from PBEsol computations for both λ-Ta2O5 and ǫ-Ta2O5 structures show excellent agreement with experiment and theoretical predictions (left column in Table 1)30,37, which also indicating that the PBEsol GGA functional performs well in forecasting structural characteristics of solid. In light of this, the PBEsol optimized structure could be used as a suitable beginning point for the ensuing electronic structure computations. The analyses start with the calculations of different types of OVs-induced electrical conductivity for two different phases of Ta2O5. In λ-Ta2O5 structure, three types of OVs are defined according to their coordination sites such as O2fV, O3fV, and OILV. The OV in ǫ -Ta2O5 structure is represented by the O ǫ V. We have taken 2 × 2 × 1 and 2 × 1 × 1 supercells for λ-Ta2O5 and ǫ -Ta2O5 structures, respectively, which contains 16 Ta atoms and 40 O atoms for both structures. The estimated electrical conductivity for different types of OVs is shown in Fig. 2.h yf yp g The calculated results revealed that with the introduction of a single OV, the electrical conductivity tends to increase for all types of vacancy defects, although the increase of conductivity for all the vacancy types is not the same. O ǫ V, O2fV, and O3fV type vacancy defects show a slight increase in conductivity compared to the pristine Ta2O5. In contrast, the OILV defect shows a considerable increase in conductivity. In general, OVs can act as Figure 1. Structural model for (a) ǫ-Ta2O5 and (b) λ-Ta2O5 phases. Upper and lower figures indicate the ‘ab’ and ‘ac’ plane views of ǫ-Ta2O5 and λ-Ta2O5 structures, respectively. Green, blue, and black circles in (b) indicate twofold, threefold, and interlayer coordinated O atoms, which are denoted as O2f, O3f, and OIL, respectively. Figure 1. Structural model for (a) ǫ-Ta2O5 and (b) λ-Ta2O5 phases. Upper and lower figures indicate the ‘ab’ and ‘ac’ plane views of ǫ-Ta2O5 and λ-Ta2O5 structures, respectively. Green, blue, and black circles in (b) indicate twofold, threefold, and interlayer coordinated O atoms, which are denoted as O2f, O3f, and OIL, respectively. https://doi.org/10.1038/s41598-023-43888-z Scientific Reports \| (2023) 13:16656 \| www.nature.com/scientificreports/ Table 1. Calculated structural parameters of ǫ-Ta2O5 and λ-Ta2O5 phase. Table 1. Calculated structural parameters of ǫ-Ta2O5 and λ-Ta2O5 phase. Results and discussionf The semiconductor to metal transition due to an OILV type defect is char- acterized by the filling of the conduction band with electrons, resulting in a partially filled conduction band that is characteristic of metals, as shown in Fig. 3d. This leads to higher electrical conductivity for this type of vacancies. It is worth noting that the effects of OVs on the bandgap of Ta2O5 are complex and can be influenced by a variety of factors, including the crystal structure of the material, the nature of the OVs themselves, and the presence of other impurities or defects in the material. Therefore, a thorough understanding of the properties of Ta2O5 and its behavior in different environments is necessary to accurately predict and control its bandgap energy. We have thus further assessed the projected density of states (PDOS) to get more information on the nature of the electronic transitions that occur in the material and the role of OV defects and impurities in the elec- tronic structure. Figure 4 shows the PDOS for various types of OV defects for both λ-Ta2O5 and ǫ-Ta2O5 phases, respectively. The presence of OVs in Ta2O5 significantly affect its PDOS due to the changes in the electronic structure caused by the introduction of defect states. It is observed from PDOS that the valence band is mainly composed of oxygen p-orbitals and the conduction band is mainly composed of Ta d-orbitals. The PDOS of Ta2O5 without any OVs typically shows a band gap between the valence band and the conduction band, as shown in supplementary information (Figure S2). When OVs are introduced into the Ta2O5 lattice, the electronic structure Fig. 3a–c. The defect states just lead to the appearance of additional electronic states within the bandgap, without necessarily changing the overall band structure of the material. The system is semiconducting in all instances because there is a gap between the highest occupied and lowest unoccupied states. The estimated energy dif- ferences between the conduction band and the defect states are 0.877 eV, 0.606 eV and 0.614 eV for O ǫ V, O2fV, and O3fV type vacancy defects, respectively. On the other hand, the introduction of the OILV defect results in a semiconductor-to-metal transition. Results and discussionf This defect state can act as an intermediate energy level between the valence and conduction bands, allowing electrons to be promoted across the bandgap more easily. This may cause an increase in the conductivity of the materials. However, the energy gap between the CBM and the defect band energy (DBE) is ~ 0.8 eV, and between the valence band maximum (VBM) and the DBE is ~ 2 eV (as shown in Fig. 3a–c). Both gaps are too large for the electron transition at room temperature (25 meV). The degree to which the bandgap is affected by OVs in Ta2O5 depends on the types of vacancies within the material. p yp Removing one O atom from O ǫ , O2f., and O3f. coordinated sites do not occupy vacant conduction band states to render the system metallic; rather they confine in a mid-gap defect state, confined at the vacancy site as shown https://doi.org/10.1038/s41598-023-43888-z Scientific Reports \| (2023) 13:16656 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. Projected electronic band structures of Ta2O5 with single (a) O ǫ V in ǫ-phase, and (b) O2fV, (c) O3fV, and (d) OILV type defects in λ-phase. The reference energy level (dotted line) is touched with the valence band maximum for all types of OVs. Yellow and blue colors represent the contribution form O and Ta atoms, respectively. Figure 3. Projected electronic band structures of Ta2O5 with single (a) O ǫ V in ǫ-phase, and (b) O2fV, (c) O3fV, and (d) OILV type defects in λ-phase. The reference energy level (dotted line) is touched with the valence band maximum for all types of OVs. Yellow and blue colors represent the contribution form O and Ta atoms, respectively. Fig. 3a–c. The defect states just lead to the appearance of additional electronic states within the bandgap, without necessarily changing the overall band structure of the material. The system is semiconducting in all instances because there is a gap between the highest occupied and lowest unoccupied states. The estimated energy dif- ferences between the conduction band and the defect states are 0.877 eV, 0.606 eV and 0.614 eV for O ǫ V, O2fV, and O3fV type vacancy defects, respectively. On the other hand, the introduction of the OILV defect results in a semiconductor-to-metal transition. Results and discussionf In terms of specific PDOS contributions, the OVs in Ta2O5 can lead to an increase in the density of states near the Fermi level associated with the Ta 5d orbitals and the O 2p orbitals. This indicates that the electronic states associated with these orbitals are more strongly affected by the OVs.h changes due to the formation of defect states within the band gap. OVs lead to the appearance of new peaks and features in the PDOS of both λ-Ta2O5 and ǫ-Ta2O5 structures. In terms of specific PDOS contributions, the OVs in Ta2O5 can lead to an increase in the density of states near the Fermi level associated with the Ta 5d orbitals and the O 2p orbitals. This indicates that the electronic states associated with these orbitals are more strongly affected by the OVs.h f y The number and position of the defect states depend on the location of the oxygen vacancies in the lattice. Although some sharp peaks are observed in the middle of the gap for O ǫ V, O2fV and O3fV defects, a broadened peak covers the entire gap region for OILV type defect, indicating the semiconductor to metal transition in this defect type. Moreover, it is observed that the Fermi level is shifted towards the conduction band with the intro- duction of OV. The Fermi level for semiconductor (insulator) at 0 K is simply at (VBM + CBM)/2. In Fig. 3a–c, the Fermi level should be at (CBM + DBE)/2 at 0 K because the highest occupied state is at the top of DBE, indi- cating that the Fermi level moves towards the conduction band for all types of OVs. The OV causes the Fermi level to move; therefore, the distribution f(E) in Eq. 4 changes with the OV. The integral sampling over E in Eq. 4 has the heavier weight of f(E) when E is near the Fermi level. Now, with OV, the Fermi level moved closer to the conduction band, which increased the weight f(E) for the conduction band. Furthermore, the Fermi level lies in the middle between DBE and CBM at 0 K, which further moves closer to CBM as temperature increases. Results and discussionf The semiconductor to metal transition due to an OILV type defect is char- acterized by the filling of the conduction band with electrons, resulting in a partially filled conduction band that is characteristic of metals, as shown in Fig. 3d. This leads to higher electrical conductivity for this type of vacancies. It is worth noting that the effects of OVs on the bandgap of Ta2O5 are complex and can be influenced by a variety of factors, including the crystal structure of the material, the nature of the OVs themselves, and the presence of other impurities or defects in the material. Therefore, a thorough understanding of the properties of Ta2O5 and its behavior in different environments is necessary to accurately predict and control its bandgap energy. f y y g gy We have thus further assessed the projected density of states (PDOS) to get more information on the nature of the electronic transitions that occur in the material and the role of OV defects and impurities in the elec- tronic structure. Figure 4 shows the PDOS for various types of OV defects for both λ-Ta2O5 and ǫ-Ta2O5 phases, respectively. The presence of OVs in Ta2O5 significantly affect its PDOS due to the changes in the electronic structure caused by the introduction of defect states. It is observed from PDOS that the valence band is mainly composed of oxygen p-orbitals and the conduction band is mainly composed of Ta d-orbitals. The PDOS of Ta2O5 without any OVs typically shows a band gap between the valence band and the conduction band, as shown in supplementary information (Figure S2). When OVs are introduced into the Ta2O5 lattice, the electronic structure https://doi.org/10.1038/s41598-023-43888-z Scientific Reports \| (2023) 13:16656 \| www.nature.com/scientificreports/ Figure 4. PDOS of Ta2O5 with single (a) O ǫ V, (b) O2fV, (c) O3fV, and (d) OILV type defects. Inset shows the zoom-in view of a broadened peak covering the entire gap region. Figure 4. PDOS of Ta2O5 with single (a) O ǫ V, (b) O2fV, (c) O3fV, and (d) OILV type defects. Inset shows the zoom-in view of a broadened peak covering the entire gap region. changes due to the formation of defect states within the band gap. OVs lead to the appearance of new peaks and features in the PDOS of both λ-Ta2O5 and ǫ-Ta2O5 structures. Results and discussionf The DFT calculated values of Fermi energy for λ and ǫ-phase pristine structures are 2.17 eV and 2.008 eV, respectively, whereas these values are 4.351 eV, 3.805 eV, 3.856 eV, and 4.5009 eV for O ǫ V, O2fV, O3fV and OILV type defects, respectively, as shown in Fig. 5. The shifting of Fermi energy towards the conduction band leads to an increase in the number of free electrons available for conduction, which can increase the electrical conductivity of the material. When OVs are present, the oxygen 2p states near the CBM become empty, and some of the electrons from the Ta 5d states may transfer to these vacant oxygen states. This transfer of electrons leads to an increase in the Fermi energy level and a shift towards the CBM. The increase in electrical conductivity due to a Fermi level shift towards the conduction band is observed in many types of materials, including semiconductors, metals, and insulators62–66. Scientific Reports \| (2023) 13:16656 \| https://doi.org/10.1038/s41598-023-43888-z www.nature.com/scientificreports/ www.nature.com/scientificreports/ 1 2 3 4 5 Pristine O3fV O2fV O V OILV ) V e( y g re n E i m re F SiD SiOV Figure 5. Changes of Fermi energy for different types of oxygen vacancies. Figure 5. Changes of Fermi energy for different types of oxygen vacancies. We have also estimated the formation energy ( Efe ) for each of the aforementioned defect types. The Efe of OVs in Ta2O5 is closely associated to the SET/RESET voltage and power utilization of the memristor during RS19, as it represents the defect-forming capability and the stability of defects. The voltage needed to move the device from a high-resistance state (HRS) to a low-resistance state (LRS) is known as the SET voltage, whereas the voltage needed to transfer the device from a LRS to a HRS is known as the RESET voltage. If the Efe of OVs is too low, it can result in a high concentration of vacancies, which can lead to poor endurance and retention characteristics. Therefore, it is important to optimize the Efe of OVs in Ta2O5-based memristors to achieve the desired SET/RESET voltage and power consumption while maintaining good endurance and retention charac- teristics. This can be achieved by tuning the material composition, crystal structure, and processing conditions to control the concentration and distribution of defects in the material. Results and discussionf The Efe of OV can be evaluated using the expression67–70 as, Efe = EOV −Epristine − nς , where EOV and Epristine represent the total energy of the OV- defected and pristine structures, respectively. The number of atoms necessary to create OV is n. If n is greater than 0 then it is necessary to add atoms to the defect-free model, and if n is less than 0 then it is necessary to delete atoms from the pristine model. ς stands for the chemical potential of the associated atom. The value of ς for the oxygen atom is used in the calculation as 4.93 eV19. The calculated Efe for the O ǫ V, O2fV, O3fV, and OILV are 6.50 eV, 6.21 eV, 5.25 eV, and 4.40 eV, respectively, which is relatively high. However, this finding is in line with the earlier investigations71. Among the Efe calculated for all OV types, the OILV shows the comparatively lowest value, indicating that this type of OV is more likely to be present in the system. While the generation and manipulation of specific types of OVs pose challenges, various processes can be employed to achieve this, including oxygen annealing or sputtering in controlled atmospheres during device fabrication, electroforming through the application of high-voltage pulses or voltage sweeps across the memristor, and the utilization of voltage or temperature variations. g p Finally, in search for lowering the high SET/REST voltage predicted above, we have investigated the doping effect and explored the combined doping and OV effect on the electrical properties in Ta2O5. To investigate the doping effect, we have considered ǫ-Ta2O5 structure as a representational unit as its electrical properties are closer to the realistic applications. It is revealed that the doping of N atom in Ta2O5 structure decreases the band gap and follows the sequence of oxide > oxynitride > nitride, which is in good agreement with experimental results as well58. It is worthy to note that several studies have been performed in improving the electronic properties of Ta2O5-based resistive memristor devices using ionic doping technology. Some successful doping strategies include Ti doping41, which has been shown to produce soft collapse with high ON/OFF ratio in Ta2O5-RRAM devices. Al doping has also been found to reduce the forming voltage required for the device to switch states42. More recently, Zr doping has been shown to produce better switching performance in Ta2O5-RRAM43. Results and discussionf However, it is important to note that metal dopants can also have negative effects on device performance. For example, metal dopants can cause field enhancement effects that would reduce the stability and uniformity of CFs pro- duced by OVs as the number of cycle increases72,73. This can lead to deterioration in the high resistive state retention of the device, which can limit its overall performance and reliability. In contrast, the use of nonmetallic Si dopants in Ta2O5-RRAM devices may offer a promising approach for improving the performance and reli- ability of these devices, particularly in terms of maintaining uniform CFs and avoiding the negative effects of field enhancement19. We have thus replaced one O atom with one Si atom in ǫ-Ta2O5 structure and then OVs are created by removing O atoms. The OV-induced projected band structure and projected density of states of Si-doped ǫ-Ta2O5 are represented in Fig. 6. An earlier study revealed that the OV in Si-doped Ta2O5 may have negligible effect on conductivity as there is no defect state in the band gap and the defect energy level is close to the VBM19. However, our calculated projected band structures and PDOS clearly state that the introduction of OVs in Si-doped Ta2O5 leads to the formation of defect states near the conduction band edge, which can par- ticipate in electronic transport. There are possibly two factors contributing to the different simulation outcomes seen in the earlier study conducted by Cai et al.19 It should firstly be noted that the positioning of the Si element https://doi.org/10.1038/s41598-023-43888-z Scientific Reports \| (2023) 13:16656 \| www.nature.com/scientificreports/ Figure 6. Projected electronic band structure of ǫ-Ta2O5 structure with (a) Si-doping and (b) combined Si-doping and one OV. PDOS of ǫ-Ta2O5 structure with (c) Si-doping and (d) combined Si-doping and one OV. Figure 6. Projected electronic band structure of ǫ-Ta2O5 structure with (a) Si-doping and (b) combined Si-doping and one OV. PDOS of ǫ-Ta2O5 structure with (c) Si-doping and (d) combined Si-doping and one is not the same. In the aforementioned work, the Ta atom was replaced with a Si dopant, but in the current inves- tigation, the O atom is replaced by the Si dopant. Furthermore, the study conducted by the Cai et al. focused on the λ-Ta2O5 phase, while our current research investigates the Si-doped features inside the ǫ-Ta2O5 structure. Results and discussionf The blue, tan, and red spheres denote the Si, Ta, and O atoms, respectively. (b) Distances of O atom from Si atom for different sites. different distances of 2.630 Å, 2.650 Å, and 3.988 Å, respectively. We observed that the O atoms located near to the Si impurity have a comparatively low Efe of OVs. These findings fit the previous research well19. This is likely due to the influence of the Si dopant on the electronic structure and bonding environment of the neighboring oxygen atoms. We have calculated the interaction energy, Einteraction between the Si dopant and OV using the relation as Einteraction = ESiOV−Eseparate . Here, ESiOV denotes the energy of the combined Si-doping and OV and Eseparate denotes the energy of the separate defect generated by the Si atom and OV. Our calculation showed that the interaction energy between the Si atom and OV is ~ 2.20 eV, demonstrating a significant attraction between OV and Si atom. Hence, OV develops in the vicinity of the Si atom, which is advantageous to the uniformity of CFs produced by OVs. This phenomenon is in line with the capacity of the Si dopant to impede the stochastic generation of CFs triggered by OVs19. The DFT simulations present here provide insight into performance trends, which is valuable in assisting in material selection and optimization for improved switching, guiding experimental efforts.h pf The determination of the doping characteristics of a defect relies heavily on the computation of the charge state of the point defect. Nevertheless, we believe that the issue of the OV charge state in memristors is more complex than the typical formation energy and transition level calculations for the single-point defect, which are often carried out for dopant analysis. The most stable charge state of OV should vary with the local environment because the formation energy of charged defects is a function of Fermi level, whereas that of neutral defects is not. We may determine the charge state of OV assuming an isolated single defect. Then, the initial charge state of OV calculated shall dictate the transport of single OV under external electrical bias. Nevertheless, the charge state of OV may change further during the course as the local concentration of OV varies along with Fermi level to form a filament where the OV charge state should return to neutral. Results and discussionf Several experimental55,74 and theoretical investigations58,75–77 have shown that the substitution of the O atom in ǫ-Ta2O5 with a N dopant may lead to significant alterations in its electrical and optical characteristics. Based on the findings of prior experimental and theoretical investigations, we have chosen to examine the impact of doping by replacing O atoms with Si in the ǫ-Ta2O5 structure. When investigating the RS of Ta2O5-based memristor, it is important to consider the Efe of OVs, the conductivity of the Ta2O5 layer, and the interaction of dopants with OV, as these factors can affect the stability and regularity of CFs in the device78,79. The calculated energy gaps between defect state and conduction band of Si-doped and combined Si-doped with OV are 0.73 eV, 0.584 eV, respectively. As can be seen in Fig. 6, with the creation of OV in Si-doped Ta2O5, the PDOS shows broadened and increased peaks in the vicinity of the Fermi level, indicating the presence of defect states that can increase the conductivity of the material.t y Additionally, the Fermi energy increases and shifts towards the conduction band, which further enhances the conductivity. Furthermore, it is observed that the calculated formation energy for single O ǫ V in Si-doped ǫ-Ta2O5 reduces to a lower value of 4.12 eV, which may cause the reduction in SET/RESET voltage in Si-doped Ta2O5-based memristor80. The presence of the silicon dopant should also have a stabilizing effect providing consistency on the OVs in the vicinity of those O atoms. To check this, we have analyzed the Efe of OVs at different positions of Si doped Ta2O5. The O atoms at three locations are removed and corresponding Efe are calculated, as shown in Fig. 7. The obtained formation energies are 4.12 eV, 5.05 eV, and 5.78 eV for the three https://doi.org/10.1038/s41598-023-43888-z Scientific Reports \| (2023) 13:16656 \| www.nature.com/scientificreports/ Figure 7. (a) Si-doped ǫ-Ta2O5 structure combined with oxygen vacancies. Different colors in the dotted circles represent different positions of the O atom with respect to the Si atom. The blue, tan, and red spheres denote the Si, Ta, and O atoms, respectively. (b) Distances of O atom from Si atom for different sites. Figure 7. (a) Si-doped ǫ-Ta2O5 structure combined with oxygen vacancies. Different colors in the dotted circles represent different positions of the O atom with respect to the Si atom. Results and discussionf Due to the greater complexity of the function of OV in memristors compared to dopants in semiconductors, we posit that a distinct examination of the charge state of OV in memristor applications is merited. Conclusions In conclusions, the electrical transport characteristics of ǫ-Ta2O5 and λ-Ta2O5 polymorphs have been thoroughly examined using density functional theory calculations. Boltzmann transport theory is used to determine the vacancy-induced electrical conductivity of both structures. Creation of vacancy defects in O ǫ , O2f, and O3f coor- dinated sites generate localized mid-gap defect states within the energy bandgaps. In contrast, the introduction of the OILV defect results in a semiconductor-to-metal transition in Ta2O5, leading to an improved electrical conductivity. The OILV also shows the lowest formation energy, indicating the lower voltage required for SET/ RESET process for this type of vacancy. The introduction of OVs leads to a shift in the Fermi level towards the conduction band for all types of OVs, which can aid in electronic transport and boost conductivity. Further, the formation energy is found to be lowered in Si-doped Ta2O5, which depends on the OV location, with the lowest energy found for the OV located closest to the Si atom- this appears to be advantageous to the uniformity of CFs produced by OVs. These results can act as guidelines for additional experimental work aimed at improving the regularity and switching characteristics of RS for Ta2O5-based resistive random-access memory. Data availabilityh y The datasets used and/or analyzed during the current study available from the corresponding author on reason- able request. Received: 12 July 2023; Accepted: 29 September 2023 Received: 12 July 2023; Accepted: 29 September 2023 https://doi.org/10.1038/s41598-023-43888-z Scientific Reports \| (2023) 13:16656 \| www.nature.com/scientificreports/ References References 1. Kao, C.-H., Chen, H., Chiu, J. S., Chen, K. S. & Pan, Y. T. Physical and electrical characteristics of the high-k Ta2O5 (tantalum pentoxide) dielectric deposited on the polycrystalline silicon. Appl. Phys. Lett. 96, 112901 (2010). p ) p p y y pp y ( ) 2. Breuer, T. et al. Realization of minimum and maximum gate function in Ta2O5-based memristive devices. Sci. Re p p p y y pp y 2. Breuer, T. et al. Realization of minimum and maximum gate function in Ta2O5-based memristive devices. Sci. Rep. 6, 23967 (2 3. Shyam, B. et al. 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https://openalex.org/W1923170586	https://biblio.ugent.be/publication/6952309/file/6960242	English	null	HtpG contributes to Salmonella Typhimurium intestinal persistence in pigs	Veterinary research	2,015	cc-by	3,904	© 2015 Verbrugghe et al. 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. HtpG contributes to Salmonella Typhimurium intestinal persistence in pigs Elin Verbrugghe1†, Alexander Van Parys1,2†, Bregje Leyman1,3, Filip Boyen1, Freddy Haesebrouck1† and Frank Pasmans1† Verbrugghe et al. Vet Res (2015) 46:118 DOI 10.1186/s13567-015-0261-5 Open Access Abstract Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella Typhimurium) contamination of pork, is one of the major sources of human salmonellosis. The bacterium is able to persist and hide in asymptomatic carrier animals, generating a reservoir for Salmonella transmission to other animals and humans. Mechanisms involved in Salmonella persistence in pigs remain poorly understood. In the present study, we demonstrate that the Salmonella htpG gene, encoding a homologue of the eukaryotic heat shock protein 90, contributes to Salmonella Typhimurium persistence in intestine-associated tissues of pigs, but not in the tonsils. HtpG does not seem to play an important role during the acute phase of infection. The contribution to persistence was shown to be associated with htpG-depend- ent Salmonella invasion and survival in porcine enterocytes and macrophages. These results reveal the role of HtpG as a virulence factor contributing to Salmonella persistence in pigs. are difficult to distinguish from uninfected pigs and they constitute a continuous reservoir of Salmonella bacteria. During periods of stress, like transport to the slaughter house, a flare up of this asymptomatic colonization may occur [6]. Until now, the mechanism of prolonged coloni- zation in carrier pigs remains poorly known, which seri- ously hampers the development of efficient mitigation measures. A thorough understanding of how Salmonella is able to persist requires the identification of bacterial genes involved. Correspondence: Elin.verbrugghe@ugent.be †Elin Verbrugghe and Alexander Van Parys shared first author Freddy Haesebrouck and Frank Pasmans shared senior authorship 1 Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium Full list of author information is available at the end of the article ole of htpG during persistence of Salmonella in pigs Role of htpG during persistence of Salmonella in pigs The animal experiment was carried out in strict accordance with the recommendation in the Euro- pean Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes. The experimental protocols and care of the animals were approved by the Ethics Committee of the Faculty of Veterinary Medicine, Ghent University (EC2010/005). Thirteen four-week-old piglets (com- mercially closed line based on Landrace) from a sero- logically Salmonella negative breeding herd were used. The Salmonella-free status of the piglets was verified serologically (IDEXX, Hoofddorp, The Netherlands) and bacteriologically via repeated faecal sampling. The animals arrived at the facility 5 days before inoculation and they were housed in isolation units at 25 °C under natural day-night rhythm in HEPA-filtered stables, with ad libitum feed and water. The piglets were ran- domly divided in a negative control group of 3 animals and 2 groups of 5 animals that were orally inoculated with a mixture of approximately 2 × 107 colony-form- ing units (CFU) Salmonella WTnal and 2 × 107 CFU Salmonella ΔhtpG:kanR. The negative control pigs were administered 2 mL HBSS. It was our hypothesis that htpG contributes to persistence but that delet- ing this gene does not result in a lack of successful initial colonization of the pig. Based on the results of Boyen et al., we determined the Salmonella numbers at day 4 (acute phase) and day 21 (persistent phase) in this experiment [5]. Therefore, one group of Salmo- nella-inoculated animals was euthanized 4 days post inoculation (pi). The negative control and the other Salmonella-inoculated group were euthanized 3 weeks pi. Samples of the palatine tonsils, ileum and contents, ileocecal lymph nodes, cecum and contents and faeces were collected and bacteriologically analyzed as pre- viously explained [6]. Briefly, 10% suspensions of all samples were prepared in buffered peptone water and the number of Salmonella bacteria was determined by plating 10-fold dilutions on BGA plates supplemented with 20 µg/mL nalidixic acid or 100 µg/mL kanamy- cin. Samples that were negative after direct plating but positive after enrichment in tetrathionate broth were presumed to contain 83 CFU per gram sample (detec- tion limit for direct plating). Salmonella Typhimurium strain 112910a phage type 120/ad, isolated from a pig stool sample, was used as the wild type strain (WT). Introduction Salmonellosis is regarded as one of the most impor- tant bacterial zoonotic diseases [1, 2]. The main route of human infection is through the consumption of con- taminated food, such as pork, with Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella Typhimurium) being the most frequently isolated sero- var from slaughter pigs [3]. Salmonella Typhimurium seldom produces systemic infections in healthy adult ani- mals. The bacterium is however able to colonize the ali- mentary tract and may cause acute enteritis followed by persistence. The ability to cause a persistent colonization in the host is a major characteristic of Salmonella viru- lence [4]. Persistence is here defined as a chronic pres- ence of Salmonella Typhimurium in the porcine host. In our infection model [5] as well as in practice, this is characterized by relatively high level infection loads in tonsils but low intestinal loads resulting in intermittent faecal shedding. Persistently colonized carrier animals g Recently, using in vivo expression technology (IVET), Van Parys et al. identified 37 Salmonella Typhimurium genes that are specifically expressed during persistence in pigs [7]. Although these are potential virulence genes, their individual contribution to Salmonella persistence remains to be further explored. In the present study, we focused on one of these 37 genes, the htpG gene that encodes a homologue of the eukaryotic heat shock pro- tein 90 (hsp90), a chaperone being important for the maintenance, activation, stabilization or maturation of proteins [8]. The aim of this study was to define the role of this gene during colonization and persistence of Sal- monella Typhimurium in pigs. Correspondence: Elin.verbrugghe@ugent.be †Elin Verbrugghe and Alexander Van Parys shared first author Freddy Haesebrouck and Frank Pasmans shared senior authorship 1 Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium Full list of author information is available at the end of the article Page 2 of 5 Page 2 of 5 Verbrugghe et al. Vet Res (2015) 46:118 ole of htpG during persistence of Salmonella in pigs A spontaneous nalidixic acid resistant derivative of the wild type strain was used in the in vivo experiment (Salmonella WTnal). Salmonella Typhimurium deletion (Salmonella ΔhtpG) and kana- mycin resistant substitution (Salmonella ΔhtpG:kanR) mutants (Table 1) were constructed according to the one- step inactivation method as described previously [9] and slightly modified for use in Salmonella Typhimurium [10]. HtpG plays a role during intestinal persistence of Salmonella Typhimurium in pigsif Four days pi, no significant differences were observed in the number of Salmonella WTnal and Salmonella ΔhtpG:kanR bacteria colonizing the tonsils, ileocecal lymph nodes, ileum and cecum and their contents and faeces (Figure 1A). In contrast, 21 days pi, significant dif- ferences in colonization were noticed between the two strains (Figure 1B). The Salmonella ΔhtpG:kanR strain was attenuated in the intestine-associated tissues and contents: ileocecal lymph nodes (decrease of 1.2 log10 (CFU/g)), ileum (decrease of 1.1 log10 (CFU/g)), cecum (decrease of 0.5 log10 (CFU/g)), ileum contents (decrease of 1.3 log10 (CFU/g)), cecal contents (decrease of 1.8 log10 (CFU/g)) and faeces (decrease of 0.3 log10 (CFU/g)). These differences were significant in the ileocecal lymph nodes (p = 0.042) and cecal contents (p = 0.018). Persistence of Salmonella is an intriguing characteristic of the bacterium that allows maintenance of the pathogen in a host population. After bacterial invasion in hosts, such as pigs, the immune system will respond to clear the infection and bacterial survival strategies for persis- tence will become important. We now showed that the Results HtpG plays a role during intestinal persistence of Salmonella Typhimurium in pigsif Cell cultures and experiments Invasion and intracellular survival of Salmonella was quantified in porcine intestinal epithelial cells (IPEC-J2) and in porcine macrophages. IPEC-J2 cells [11, 12] and primary porcine alveolar macrophages (PAM) [13] were cultured as previously described. To examine whether the ability of Salmonella Typhimurium to invade and proliferate in PAM and IPEC-J2 cells was altered after deletion of htpG, gentamicin protection assays were per- formed using Salmonella WT and Salmonella ΔhtpG. Therefore, PAM and IPEC-J2 cells were seeded in 24-well plates at a density of approximately 106 and 5 × 105 cells per well, respectively. PAM were allowed to attach for 2 h and IPEC-J2 cells were allowed to grow for 24 h. Sub- sequently, Salmonella was inoculated into the wells at a multiplicity of infection (MOI) of 10:1. To synchronize the infection, the inoculated multiwell plates were centri- fuged at 1500 rpm for 10 min and incubated for 30 min at 37 °C under 5% CO2. After washing the cells three times with Hank’s balanced salt solution (HBSS), they were supplemented for 1 h with fresh medium containing 100 µg/mL gentamicin to kill extracellular bacteria. After washing 3 times, the cells were lysed for 10 min with 1% Triton X-100 or 0.2% sodium deoxycholate, respectively. The number of invaded Salmonella bacteria was deter- mined by plating 10-fold dilutions on Brilliant Green Agar (BGA) plates. To assess intracellular proliferation, the medium containing 100 µg/mL gentamycin was replaced after 1 h with fresh medium containing 20 µg/ mL. The plates were incubated for 6 or 24 h and the num- ber of intracellular bacteria was determined as described for the invasion. Table 1 Primers used in this study to create the Salmonella ΔhtpG mutants Application Primers Sequences (5′ to 3′) Mutagenesis htpG forward CCCTCAACGTATTTTTACCATTAAAAATGGCATTGTTGAGGTCTATCCACTGTGTAGGCTGGAGCTGCTTC Mutagenesis htpG reverse CGGATAAGACGCTTCGCGTCGCCATCCGGCAGTCAGATGAGCGTTACATATGAATATCCTCCTTAG Table 1 Primers used in this study to create the Salmonella ΔhtpG mutants Sequences (5′ to 3′) Verbrugghe et al. Vet Res (2015) 46:118 Page 3 of 5 Discussionh The pathogenicity of Salmonella has been extensively studied, both in vitro and in vivo, using different animal models like the mouse, pig and chicken. Especially the mouse model is a widely used paradigm for studying the pathogenesis of systemic disease caused by Salmonella. However, investigations concerning food safety should employ natural hosts to examine gastrointestinal coloni- zation by Salmonella. A major source of Salmonella con- tamination of pork meat is cross-contamination in the slaughterhouse. A thorough understanding of the molec- ular mechanisms that Salmonella Typhimurium exploits to persist in the tonsils, gut and gut-associated lymphoid tissues, might contribute to the development of appropri- ate measures to minimize Salmonella contamination of porcine carcasses. Therefore, in the past decade, research has focused on the identification of virulence mecha- nisms contributing to its persistence in pigs. However, up to date, few data are available concerning Salmonella persistence in pigs [7, 14–17]. Statistical analysis mL)) and intracellular survival for 24 h (decrease of 0.4 log10 (CFU/mL)) of Salmonella ΔhtpG compared to the WT strain (Figure 2A). Moreover, the htpG gene was also shown to be important (p < 0.05) for Salmonella invasion (decrease of 0.1 log10 (CFU/mL)) and survival capacities at 6 h pi (decrease of 0.2 log10 (CFU/mL)) and 24 h pi (decrease of 0.3 log10 (CFU/mL)) in primary mac- rophages, in vitro (Figure 2B). All statistical analyses were performed using SPSS ver- sion 22 (SPSS Inc., Chicago, IL, USA). Normality of the data was assessed using a Kolmogorov–Smirnov and Sha- piro–Wilk test. Normally distributed data were analyzed using an unpaired Student’s t test to address the signifi- cance of difference between mean values with signifi- cance set at P ≤ 0.05. If equal variances were not assessed or if the data were not normally distributed, they were analyzed using the non-parametric Kruskal–Wallis anal- ysis, followed by a Mann–Whitney U test. HtpG is involved in invasion and intracellular replication of Salmonella in macrophages In IPEC-J2 cells, we observed a tendency (p < 0.1) towards reduced invasion (decrease of 0.1 log10 (CFU/ Figure 1 Salmonella Typhimurium WTnal and ΔhtpG:kanR colonization of pigs. Recovery of bacteria from various organs of 5 piglets orally inoculated with an equal mixture of Salmonella WTnal and ΔhtpG:kanR, A 4 days pi and B 21 days pi. The log 10 value of the number of CFU per gram sample is given as the mean + standard deviation. A non-parametric Mann–Whitney U test was performed and superscript () refers to a significant difference compared to the wild type Salmonella group (p < 0.05). Figure 1 Salmonella Typhimurium WTnal and ΔhtpG:kanR colonization of pigs. Recovery of bacteria from various organs of 5 piglets orally inoculated with an equal mixture of Salmonella WTnal and ΔhtpG:kanR, A 4 days pi and B 21 days pi. The log 10 value of the number of CFU per gram sample is given as the mean + standard deviation. A non-parametric Mann–Whitney U test was performed and superscript () refers to a significant difference compared to the wild type Salmonella group (p < 0.05). Verbrugghe et al. Vet Res (2015) 46:118 Page 4 of 5 Figure 2 Role of htpG during invasion and intracellular replication of Salmonella in host cells. Number of intracellular Salmonella WT or ΔhtpG bacteria after invasion or intracellular replication (6 and/or 24 h) in A IPEC-J2 cells or B primary macrophages. The log10 values of the number of gentamicin protected bacteria + standard deviation are shown. Results are presented as a representative experiment conducted in sixfold. An independent t-test was performed and superscript () refers to a significant difference (p < 0.05). Figure 2 Role of htpG during invasion and intracellular replication of Salmonella in host cells. Number of intracellular Salmonella WT or ΔhtpG bacteria after invasion or intracellular replication (6 and/or 24 h) in A IPEC-J2 cells or B primary macrophages. The log10 values of the number of gentamicin protected bacteria + standard deviation are shown. Results are presented as a representative experiment conducted in sixfold. An independent t-test was performed and superscript (*) refers to a significant difference (p < 0.05). Salmonella colonization of and persistence in the tonsils on one hand, and in the gut and gut-associated lymphoid tissue on the other hand [17, 19, 20]. HtpG is involved in invasion and intracellular replication of Salmonella in macrophages Although Salmo- nella persistence in tonsils represents a major factor in persistence of the bacterium in pigs, the bacterial genes involved remain largely unknown. htpG gene encodes such a survival mechanism. Deletion of the htpG gene does not affect the acute phase of infec- tion, but reduces the survival capacity of the bacterium in intestine-associated tissues of pigs. The specific role of HtpG in bacterial pathogenesis is largely unknown, but it is a homolog of the eukaryotic chaperone hsp90, being important in the adaptation of Salmonella to stress con- ditions [18]. During persistence in a certain host, Salmo- nella has to deal with numerous stresses, like the host’s immune system, decreased oxygen tension, nutrient limitation and starvation and shift in temperature and pH. Possibly, the HtpG chaperone helps Salmonella deal- ing with stressful events during persistence, in vitro and in vivo, leading to an increased survival of the bacterium. Based on the results obtained by Boyen et al., we sacri- ficed pigs 3 weeks post inoculation and used this model to examine Salmonella persistence in pigs [5]. Although its contribution to long-term (several months) persis- tence is not clear, we can conclude that htpG contributes to Salmonella Typhimurium persistence in the porcine intestine until 3 weeks after inoculation. Author details 1 Department of Pathology, Bacteriology and Avian Diseases, Faculty of Vet- erinary Medicine, Ghent University, Merelbeke, Belgium. 2 Present Address: Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, Ghent, Belgium. 3 Present Address: Department of Nutrition, Genet- ics and Ethology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium. Acknowledgements The technical assistance of Nathalie Van Rysselberghe and Anja Van den Bussche is greatly appreciated. This work was supported by the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT Vlaanderen), Brussels, Belgium (Grant IWT Landbouw 040791). Authors’ contributions , g In the past, using IVET, Van Parys et al. screened the tonsils, ileum and ileocecal lymph nodes for genes being induced during the persistent phase of colonization [7]. They showed that the htpG gene is expressed in all three organs [7]. However, we now demonstrated that the htpG gene plays a role during persistence of the bacterium in intestine-associated tissues of pigs but not in the tonsils. In contrast to the gut and gut-associated lymphoid tissue, Salmonella resides largely in an extracellular niche in the tonsils [19]. Therefore, virulence mechanisms necessary for cell invasion and intracellular survival do not contrib- ute to tonsillar colonization and persistence [17, 20]. We also showed that Salmonella Typhimurium lacking htpG was impaired in invasion and intracellular replication in epithelial cells and macrophages. 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Am J Physiol 266:828–838 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
https://openalex.org/W4386799376	https://e-journal.lp3kamandanu.com/index.php/panthera/article/download/133/183	Indonesian	null	Ecolodge sebagai Implementasi Pendidikan Sains (IPA) yang Multidimensi	Panthera	2,022	cc-by-sa	3,934	Panthera : Jurnal Ilmiah Pendidikan Sains dan Terapan E-ISSN 2808-246X; P-ISSN 2808-3636 Volume 2, Issue 4, October 2022; Page, 272-280 Email: pantherajurnal@gmail.com Panthera : Jurnal Ilmiah Pendidikan Sains da E-ISSN 2808-246X; P-ISSN 2808-3636 Volume 2, Issue 4, October 2022; Page, 272-280 Email: pantherajurnal@gmail.com Uniform Resource Locator: https://e-journal.lp3kamandanu.com/index.php/panthera ECOLODGE SEBAGAI IMPLEMENTASI PENDIDIKAN SAINS (IPA) YANG MULTIDIMENSI Iswari Pauzi1, Muhammad Sarjan2, Agus Muliadi3, Asrorul Azizi4, Hamidi5, Muhammad Yamin6, Muh. Zaini Hasanul Muttaqin7, Bakhtiar Ardiansyah8, Rindu Rahmatiah9, Sudirman10, Mulia Rasyidi11, & Yusran Khery12 1Program Studi Teknologi Laboratorium Medis, Politeknik Kesehatan Mataram, Jalan Prabu Rangkasari, Mataram, Nusa Tenggara Barat 83237, Indonesia 1,2,3,4,5,6,7,8,9,10,11,&12Program Studi Doktor Pendidikan IPA, Pascasarjana, Universitas Mataram, Jalan Pendidikan Nomor 37, Mataram, Nusa Tenggara Barat 83125, 2&5Pascasarjana, Universitas Mataram, Jalan Pendidikan Nomor 37, Mataram, Nusa Tenggara Barat 83125, Indonesia 3Program Studi Pendidikan Biologi, Fakultas Sains, Teknik, dan Terapan, Universitas Pendidikan Mandalika, Jalan Pemuda Nomor 59A, Mataram, Nusa Tenggara Barat 83125, Indonesia 83125, Indonesia 272 Panthera : Jurnal Ilmiah Pendidikan Sains dan Terapan E-ISSN 2808-246X; P-ISSN 2808-3636 Volume 2, Issue 4, October 2022; Page, 272-280 Email: pantherajurnal@gmail.com Panthera : Jurnal Ilmiah Pendidikan Sains dan Terapan E-ISSN 2808-246X; P-ISSN 2808-3636 Volume 2, Issue 4, October 2022; Page, 272-280 Email: pantherajurnal@gmail.com ABSTRACT: Science Education (IPA) is a science related to systematic natural and material symptoms that are arranged regularly, generally in the form of a collection of observations and experiments. Science learning is expected to be a vehicle for students to learn about themselves and the surrounding nature, as well as the prospect of further development in applying it in everyday life. In the world of education, environmentally sound education has begun to be integrated, for example education with the vision of SETS (Science Environment Technology and Society) is defined as science, environment, technology, and society, is a unit that in the concept of education has implementation so that students have the ability to think higher (higher order thinking) Science, Technology and Society (STS) approach or the Science, Technology and Society (STM) approach. is a combination of concept approaches, process skills, CBSA, Inquiry and disambiguation as well as environmental approaches. One example of the implementation of the learning model with the environment is lodging in an open environment with an environmentally sound concept called ecolodge. Ecolodge is a small hotel or guesthouse that combines local architecture, culture and natural characteristics, supports environmental conservation by reducing waste and energy use and provides social and economic benefits to local communities. Keywords: Ecolodge, Science Education, SETS. How to Cite: Pauzi, I., Sarjan, M., Muliadi, A., Azizi, A., Hamidi., Yamin, M., Muttaqin, M. Z. H., Ardiansyah, B., Rahmatiah, R., Sudirman., Rasyidi, M., & Khery, Y. (2022). Ecolodge sebagai Implementasi Pendidikan Sains (IPA) yang Multidimensi. Panthera : Jurnal Ilmiah Pendidikan Sains dan Terapan, 2(4), 272-280. https://doi.org/10.36312/pjipst.v2i4.133 How to Cite: Pauzi, I., Sarjan, M., Muliadi, A., Azizi, A., Hamidi., Yamin, M., Muttaqin, M. Z. H., Ardiansyah, B., Rahmatiah, R., Sudirman., Rasyidi, M., & Khery, Y. (2022). Ecolodge sebagai Implementasi Pendidikan Sains (IPA) yang Multidimensi. Panthera : Jurnal Ilmiah Pendidikan Sains dan Terapan, 2(4), 272-280. https://doi.org/10.36312/pjipst.v2i4.133 Panthera : Jurnal Ilmiah Pendidikan Sains dan Terapan is Licensed Under a CC BY-SA Creative Commons Attribution-ShareAlike 4.0 International License. 83125, Indonesia 4,7,&11Program Studi Pendidikan IPA, Institut Pendidikan Nusantara Global, Jalan Raya Praya - Mantang Nomor 07, Lombok Tengah, Nusa Tenggara Barat 85311, Indonesia 6 Praya Mantang Nomor 07, Lombok Tengah, Nusa Tenggara Barat 85311, Indonesia 6Program Studi Pendidikan Biologi, Fakultas Keguruan dan Ilmu Pendidikan, Universitas Mataram Jalan Majapahit Nomor 62 Mataram Nusa Tenggara Barat 83115 Indonesia y g g gg 6Program Studi Pendidikan Biologi, Fakultas Keguruan dan Ilmu Pendidikan, Universitas Mataram, Jalan Majapahit Nomor 62, Mataram, Nusa Tenggara Barat 83115, Indonesia 8Balai Penjaminan Mutu Pendidikan Nusa Tenggara Barat, Jalan Panji Tilar Negara 8 83114 d i gg 10Program Studi Pendidikan Matematika, Fakultas Keguruan dan Ilmu Pendidikan, Universitas Qamarul Huda Badaruddin Bagu, Jalan H. Badaruddin, Lombok Tengah, Nusa Tenggara Barat 83371, Indonesia 12Program Studi Pendidikan Kimia, Fakultas Sains, Teknik, dan Terapan, Universitas Pendidikan Mandalika, Jalan Pemuda Nomor 59A, Mataram, Nusa Tenggara Barat 83125, Indonesia 12Program Studi Pendidikan Kimia, Fakultas Sains, Teknik, dan Terapan, Universitas Pendidikan Mandalika, Jalan Pemuda Nomor 59A, Mataram, Nusa Tenggara Barat 83125, Indonesia Email: iswari.pauzi69@gmail.com Submit: 22-09-2022; Revised: 14-10-2022; Accepted: 18-10-2022; Published: 30-10-2022 ABSTRAK: Pendidikan Sains (IPA) merupakan ilmu yang berhubungan dengan gejala-gejala alam dan kebendaan yang sistematis yang tersusun secara teratur, berlaku umum yang berupa kumpulan dari hasil observasi dan eksperimen. Pembelajaran sains diharapkan dapat menjadi wahana bagi peserta didik untuk mempelajari diri sendiri dan alam sekitar, serta prospek pengembangan lebih lanjut dalam menerapkannya di dalam kehidupan sehari-hari. Dalam dunia pendidikan sudah mulai diintegrasikan pendidikan berwawasan lingkungan, misalnya Pendidikan ber-visi SETS (Science Environment Technology and Society) dimaknakan sebagai sains, lingkungan, teknologi, dan masyarakat, merupakan satu kesatuan yang dalam konsep pendidikan mempunyai implementasi agar anak didik mempunyai kemampuan berpikir tingkat tinggi (higher order thinking) Pendekatan Science, Technology and Society (STS) atau pendekatan Sains, Teknologi dan Masyarakat (STM) merupakan gabungan antara pendekatan konsep, keterampilan proses, CBSA, Inkuiri, dan diskoveri serta pendekatan lingkungan. Salah satu contoh implementasi dari model pembelajaran dengan lingkungan adalah penginapan di lingkungan terbuka dengan konsep berwawasan lingkungan yang disebut ecolodge. Ecolodge adalah sebuah hotel kecil atau guesthouse yang menggabungkan arsitektur lokal, budaya, dan karakteristik alam, mendukung konservasi lingkungan dengan mengurangi limbah dan penggunaan energi serta memberikan manfaat sosial dan ekonomi bagi komunitas lokal. Kata Kunci: Ecolodge, Pendidikan Sains, SETS. PENDAHULUAN Pendidikan Sains (IPA) didefinisikan sebagai pengetahuan yang sistematis dan tersusun secara teratur, berlaku umum (universal), dan berupa kumpulan data hasil observasi dan eksperimen. Sains juga didefinisikan sebagai pengetahuan yang diperoleh melalui pengumpulan data dengan eksperimen, pengamatan, dan deduksi untuk menghasilkan suatu penjelasan tentang sebuah gejala yang dapat dipercaya. Sains berkaitan dengan cara mencari tahu tentang alam secara sistematis, sehingga sains bukan hanya penguasaan kumpulan pengetahuan yang berupa fakta-fakta, konsep-konsep, atau prinsip-prinsip saja tetapi juga merupakan suatu proses penemuan. p p Sains memiliki catatan panjang dan penuh keberhasilan dalam menciptakan pengetahuan baru yang diaplikasikan pada berbagai pengalaman manusia dalam skala luas dan mendorong pengembangan teknologi. Sains merupakan jantung informasi baru dan teknologi komunikasi yang telah mengubah secara drastis kehidupan kita dalam dekade terakhir. Sains memiliki hubungan dengan berbagai konsep dalam berbagai disiplin ilmu. Penguasaan sains diupayakan dapat utuh, menyeluruh sehingga tidak ada ketimpangan di dalamnya. Pembelajaran sains diharapkan dapat menjadi wahana bagi peserta didik untuk mempelajari diri sendiri dan alam sekitar, serta prospek pengembangan lebih lanjut dalam menerapkannya di dalam kehidupan sehari-hari (Karaarslan & Teksöz, 2016). 273 Uniform Resource Locator: https://e-journal.lp3kamandanu.com/index.php/panthera Panthera : Jurnal Ilmiah Pendidikan Sains da E-ISSN 2808-246X; P-ISSN 2808-3636 Volume 2, Issue 4, October 2022; Page, 272-280 Email: pantherajurnal@gmail.com Proses pembelajarannya menekankan pada pemberian pengalaman langsung untuk mengembangkan kompetensi agar menjelajahi dan memahami alam sekitar secara ilmiah. Pembelajaran sains memiliki peran strategis dalam meningkatkan kualitas sumber daya manusia Indonesia. Sebagai bagian dari masyarakat dunia, kita tidak dapat lepas dari pengaruh perkembangan dan produk sains berupa teknologi yang semakin luar biasa (Indahri et al., 2021). Pendekatan Science, Technology and Society (STS) atau pendekatan Sains, Teknologi, dan Masyarakat (STM) merupakan gabungan antara pendekatan konsep, keterampilan proses, CBSA, Inkuiri, dan diskoveri serta pendekatan lingkungan. Istilah Sains Teknologi Masyarakat (STM) dalam bahasa Inggris disebut Science Technology Society (STS), Science Environtment Technology and Society (SETS) atau Sains Teknologi Lingkungan dan Masyarakat. Meskipun istilahnya banyak namun sebenarnya intinya sama yaitu Environtment, yang dalam berbagai kegiatan perlu ditonjolkan. Sains Teknologi Masyarakat (STM) merupakan pendekatan terpadu antara sains, teknologi, dan isu yang ada di masyarakat. Adapun tujuan dari pendekatan STM ini adalah menghasilkan peserta didik yang cukup memiliki bekal pengetahuan, sehingga mampu mengambil keputusan penting tentang masalah dalam masyarakat serta mengambil tindakan sehubungan dengan keputusan yang telah diambil (Usmeldi et al., 2017). Pendidikan adalah salah satu medium utama yang akan menciptakan ekowisata yang sukses, dan hal ini sangat tergantung dari pemilik atau manajer ekowisata untuk menyediakannya. PENDAHULUAN Salah satunya adalah Ecolodge yang merupakan fasilitas pariwisata yang berbasis ekologi atau lingkungan. Ecolodge adalah sebuah hotel kecil atau guesthouse yang menggabungkan arsitektur lokal, budaya dan karakteristik alam, mendukung konservasi lingkungan dengan mengurangi limbah dan penggunaan energi serta memberikan manfaat sosial dan ekonomi bagi komunitas lokal (Budiyanto et al., 2020). Manajemen dan operasi dari ecolodge berbeda dengan hotel pada umumnya karena berbagai alasan. Berdasarkan International Ecolodge Guidelines, ecolodge seringkali ditemukan di area hutan belantara yang kurang berkembang, di daerah paling terpencil di negara manapun, oleh sebab itu, area tersebut merupakan tempat terakhir yang menerima investasi pemerintah dalam bidang kesehatan, pendidikan, listrik, air minum, jalan, dan lain-lain. Hal ini menjadi tantangan khusus bagi pemilik atau manajer ecolodge yang harus mencapai pengembangan berkelanjutan dengan mendukung komunitas lokal pada program pengembangan jangka panjang dan menempatkan program konservasi di tempat yang mendapat bantuan minim dari luar (Walter et al., 2018). Uniform Resource Locator: https://e-journal.lp3kamandanu.com/index.php/panthera Uniform Resource Locator: https://e-journal.lp3kamandanu.com/index.php/panthera METODE Artikel ini disusun berdasarkan kajian dari berbagai sumber antara lain, artikel ilmiah tentang Pendidikan Sain (IPA), artikel SETS (Science Environtment Technology and Society) dan artikel Ecolodge. Studi literatur dirancang untuk memberikan gambaran tentang sumber-sumber yang telah dieksplorasi ketika meneliti/mempelajari topik tertentu dan untuk menunjukkan kepada pembaca kesesuaiannya dengan aspek topik yang lebih besar. 274 Panthera : Jurnal Ilmiah Pendidikan Sains dan Terapan E-ISSN 2808-246X; P-ISSN 2808-3636 Volume 2, Issue 4, October 2022; Page, 272-280 Email: pantherajurnal@gmail.com HASIL DAN PEMBAHASAN Pendidikan Sain (IPA) Bersifat Multidimensi Panthera : Jurnal Ilmiah Pendidikan Sains dan Terapan E-ISSN 2808-246X; P-ISSN 2808-3636 Volume 2, Issue 4, October 2022; Page, 272-280 Email: pantherajurnal@gmail.com DAN PEMBAHASAN Panthera : Jurnal Ilmiah Pendidikan Sains dan Terapan E-ISSN 2808-246X; P-ISSN 2808-3636 Volume 2, Issue 4, October 2022; Page, 272-280 Email: pantherajurnal@gmail.com Pendidikan Sain (IPA) Bersifat Multidimensi Pendidikan Sain (IPA) Bersifat Multidimensi Ilmu Pengetahuan Alam berasal dari kata “science” yaitu pengetahuan rasional mengenai alam semesta dengan segala isinya yang diperoleh melalui proses ilmiah. Berdasarkan definisi tersebut, dapat dinyatakan bahwa sains mempelajari alam dalam usahanya untuk memahami dan membentuk suatu batang tubuh pengetahuan yang dapat digunakan untuk memprediksi dan diterapkan dalam masyarakat (Khery et al., 2019). Sains memiliki catatan panjang dan penuh keberhasilan dalam menciptakan pengetahuan baru yang diaplikasikan pada berbagai pengalaman manusia dalam skala luas dan mendorong pengembangan teknologi. Sains merupakan jantung informasi baru dan teknologi komunikasi yang telah mengubah secara drastis kehidupan kita dalam dekade terakhir. Dari pandangan global dan historis, sains, sebagai suatu mata pelajaran, telah sukses dalam sejumlah bidang, mampu menyediakan secara instan metode lebih generik dalam analisis untuk menyelesaikan masalah kompleks dalam kehidupan manusia (Dewi et al., 2019). Sains bersifat dinamis, artinya selalu mengalami perkembangan dan bertambah setiap saat, sehingga dengan mempelajari sains berarti secara tidak langsung kita mengikuti perkembangan zaman. Sains memiliki hubungan dengan berbagai konsep dalam berbagai disiplin ilmu. Penguasaan sains diupayakan dapat utuh, menyeluruh sehingga tidak ada ketimpangan di dalamnya. Pembelajaran sains diharapkan dapat menjadi wahana bagi peserta didik untuk mempelajari diri sendiri dan alam sekitar, serta prospek pengembangan lebih lanjut dalam menerapkannya di dalam kehidupan sehari-hari. Proses pembelajarannya menekankan pada pemberian pengalaman langsung untuk mengembangkan kompetensi agar menjelajahi dan memahami alam sekitar secara ilmiah (Karaarslan & Teksöz, 2016). Pendidikan Sains (IPA) Berbasis SETS Pendidikan Sains (IPA) Berbasis SETS Pendekatan Science, Technology, and Society (STS) atau pendekatan Sains, Teknologi, dan Masyarakat (STM) merupakan gabungan antara pendekatan konsep, keterampilan proses, CBSA, Inkuiri, dan diskoveri serta pendekatan lingkungan. Istilah Sains Teknologi Masyarakat (STM) dalam bahasa Inggris disebut Science Technology Society (STS), Science Environtment Technology and Society (SETS) atau Sains Teknologi Lingkungan dan Masyarakat. Meskipun istilahnya banyak namun sebenarnya intinya sama yaitu Environtment, yang dalam berbagai kegiatan perlu ditonjolkan. Sains Teknologi Masyarakat (STM) merupakan pendekatan terpadu antara sains, teknologi, dan isu yang ada di masyarakat. Adapun tujuan dari pendekatan STM ini adalah menghasilkan peserta didik yang cukup memiliki bekal pengetahuan, sehingga mampu mengambil keputusan penting tentang masalah-masalah dalam masyarakat serta mengambil tindakan sehubungan dengan keputusan yang telah diambil (Usmeldi et al., 2017). Filosofi yang mendasari pendekatan STM adalah pendekatan konstruktivisme, yaitu peserta didik menyusun sendiri konsep-konsep di dalam struktur kognitifnya berdasarkan apa yang telah diketahui. Definisi SETS menurut 275 niform Resource Locator: https://e-journal.lp3kamandanu.com/index.php/panthera Panthera : Jurnal Ilmiah Pendidikan Sains dan Terapan E-ISSN 2808-246X; P-ISSN 2808-3636 Volume 2, Issue 4, October 2022; Page, 272-280 Email: pantherajurnal@gmail.com the NSTA Position Statement 1990 adalah memusatkan permasalahan dari dunia nyata yang memiliki komponen Sains dan Teknologi dari perspektif siswa, di dalamnya terdapat konsep-konsep dan proses, selanjutnya siswa diajak untuk menginvestigasi, menganalisis, dan menerapkan konsep dan proses itu pada situasi yang nyata (Karaarslan & Teksöz, 2016). Panthera : Jurnal Ilmiah Pendidikan Sains dan Terapan E-ISSN 2808-246X; P-ISSN 2808-3636 Volume 2, Issue 4, October 2022; Page, 272-280 Email: pantherajurnal@gmail.com the NSTA Position Statement 1990 adalah memusatkan permasalahan dari dunia nyata yang memiliki komponen Sains dan Teknologi dari perspektif siswa, di dalamnya terdapat konsep-konsep dan proses, selanjutnya siswa diajak untuk menginvestigasi, menganalisis, dan menerapkan konsep dan proses itu pada situasi yang nyata (Karaarslan & Teksöz, 2016). Panthera : Jurnal Ilmiah Pendidikan Sains dan Te E-ISSN 2808-246X; P-ISSN 2808-3636 Volume 2, Issue 4, October 2022; Page, 272-280 Email: pantherajurnal@gmail.com the NSTA Position Statement 1990 adalah memusatkan permasalahan dari dunia nyata yang memiliki komponen Sains dan Teknologi dari perspektif siswa, di dalamnya terdapat konsep-konsep dan proses, selanjutnya siswa diajak untuk menginvestigasi, menganalisis, dan menerapkan konsep dan proses itu pada situasi yang nyata (Karaarslan & Teksöz, 2016). y g y ( , ) Pendekatan SETS/ Salingtemas diambil dari konsep pendidikan STM (Sains, Teknologi, dan Masyarakat), pendidikan lingkungan (Environmental Education/EE), dan STL (Science, Technology, Literacy). Ecolodge adalah Implementasi dari Pendidikan Sains (IPA) yang Bersifat SETS Ecolodge adalah Implementasi dari Pendidikan Sains (IPA) yang Bersifat SETS Pendidikan Sains (IPA) Berbasis SETS Dalam pendekatan Salingtemas atau SETS (Science, Environmental, Technology, and Society) konsep pendidikan STM atau STL dan EE dipandang sebagai satu kesatuan yang tidak bisa dipisahkan (Sumarni & Kadarwati, 2020). Gambar 1. Skema Hubungan Sains dengan Sosial, Teknologi, dan Lingkungan. Gambar 1. Skema Hubungan Sains dengan Sosial, Teknologi, dan Lingkungan. Ecolodge adalah Implementasi dari Pendidikan Sains (IPA) yang Bersifat SETS Uniform Resource Locator: https://e-journal.lp3kamandanu.com/index.php/panthera 276 Pariwisata berkelanjutan saat ini tengah dipromosikan pengembangannya untuk meminimalisir dampak lingkungan serta memaksimalkan manfaat sosial- ekonomi dalam sebuah destinasi wisata. World Tourism Organization mengembangkan konsep pariwisata berkelanjutan dan mendefinisikan aktivitas wisata dengan mengarahkan manajemen seluruh sumber daya dengan cara tertentu agar kebutuhan ekonomi, sosial, dan estetika dapat terpenuhi sambil menjaga integritas budaya, proses ekologi, keberagaman Biologi, dan sistem pendukung Panthera : Jurnal Ilmiah Pendidikan Sains dan Terapan E-ISSN 2808-246X; P-ISSN 2808-3636 Volume 2, Issue 4, October 2022; Page, 272-280 Email: pantherajurnal@gmail.com kehidupan. Salah satu langkah dalam mewujudkan hal tersebut adalah dengan pengembangan ekowisata. Ekowisata sama halnya dengan pariwisata pada umumnya, harus memiliki fasilitas-fasilitas untuk mendukung produk wisatanya seperti atraksi, transportasi, dan akomodasi. Sarana akomodasi memiliki beberapa jenis dan karakteristik yang berbeda, sesuai dengan tujuan dan fasilitas yang diberikan. Penyesuaian tersebut bergantung pula pada tren kepariwisataan yang ada, saat ini pilihan wisatawan terhadap konsep berkelanjutan menjadi salah satu faktor dalam menentukan destinasi wisata, sarana akomodasi yang akan digunakan dan perencanaan perjalanan (Budiyanto et al., 2020). kehidupan. Salah satu langkah dalam mewujudkan hal tersebut adalah dengan pengembangan ekowisata. Ekowisata sama halnya dengan pariwisata pada umumnya, harus memiliki fasilitas-fasilitas untuk mendukung produk wisatanya seperti atraksi, transportasi, dan akomodasi. Sarana akomodasi memiliki beberapa jenis dan karakteristik yang berbeda, sesuai dengan tujuan dan fasilitas yang diberikan. Penyesuaian tersebut bergantung pula pada tren kepariwisataan yang ada, saat ini pilihan wisatawan terhadap konsep berkelanjutan menjadi salah satu faktor dalam menentukan destinasi wisata, sarana akomodasi yang akan digunakan dan perencanaan perjalanan (Budiyanto et al., 2020). g p p j ( y ) Pergeseran tren pariwisata mendorong terjadinya pergeseran pada pemilihan fasilitas akomodasi. Akomodasi khusus kini bukan hanya mengedepankan aspek keuntungan ekonomi dan kepuasan wisatawan, namun sesuai dengan konsep pengembangan pariwisata berkelanjutan yang mempertimbangkan aspek kelestarian lingkungan fisik maupun sosial budaya, salah satu bentuknya adalah Ecolodge (Effendi et al., 2018). Uniform Resource Locator: https://e-journal.lp3kamandanu.com/index.php/panthera 277 Gambar 2. Contoh Bentuk Ecolodge. Gambar 2. Contoh Bentuk Ecolodge. Gambar 2. Contoh Bentuk Ecolodge. 277 Uniform Resource Locator: https://e-journal.lp3kamandanu.com/index.php/panthera Panthera : Jurnal Ilmiah Pendidikan Sains dan Terapan E-ISSN 2808-246X; P-ISSN 2808-3636 Volume 2, Issue 4, October 2022; Page, 272-280 Email: pantherajurnal@gmail.com Terminologi “ecolodge” diluncurkan secara formal pada First International Ecolodge Forum and Field Seminar yang diadakan pada 1994 di Maho Bay Camps di Virgin Islands, Amerika Serikat. Uniform Resource Locator: https://e-journal.lp3kamandanu.com/index.php/panthera Ecolodge adalah Implementasi dari Pendidikan Sains (IPA) yang Bersifat SETS Dari 3 persyaratan tersebut dapat kita rincikan lagi menjadi 10 persyaratan kelayakan Ecolodge sebagai penginapan yang berwawasan ekologi, antara lain (Owusu & Boafo, 2018): 1) membantu dalam hal konservasi (perlindungan dan pelestarian) terhadap flora dan Fauna; 2) berusaha untuk dapat bekerjasama dengan masyrakat lolal; 3) memberikan penerjemahan yang sangat relevan tentang betapa pentingnya alam dan lingkungan serta budaya local kepada pengelola sendiri (karyawan) dan para pendatang (pengunjung); 4) mengerti dan memahami manfaat dari air dan sumbernya serta mengatur dalam penggunaannya; 5) memberikan penanganan yang sangat hati-hati terhadap pembuangan (pengelolaan) limbah padat, limbah cair, dan sampah; 6) menjadikan energi konvensional sebagai energi yang pasif dan menyediakan perencanaan terhadap energi alternative yang ramah lingkungan; 7) mengggunkan konsep disain Arsitektur Tradisional (vernacular), baik dalam segi bentuk, penggunaan bahan, yang bisa saja di kombinasikan 278 Panthera : Jurnal Ilmiah Pendidikan Sains dan Terapan E-ISSN 2808-246X; P-ISSN 2808-3636 Volume 2, Issue 4, October 2022; Page, 272-280 Email: pantherajurnal@gmail.com dengan segi modern asal tidak mengurangi nilai-nilai yang ramah lingkungan; 8 meminimalkan pengaruh-pengaruh negatif terhadap lingkungan selama dalam proses pembangunan; 9) mewujudkan bentuk fisik yang jelas dan mengandung arti yang spesifik dan dapat juga menerapkan nilai-nilai budaya lokal; dan 10 memberikan kontribusi dalam pelestarian budaya lokal, pengembangan, promosi pendikan, dan penelitian terhadap budaya lokal itu sendiri Panthera : Jurnal Ilmiah Pendidikan Sains dan Terapan E-ISSN 2808-246X; P-ISSN 2808-3636 Volume 2, Issue 4, October 2022; Page, 272-280 Email: pantherajurnal@gmail.com dengan segi modern asal tidak mengurangi nilai-nilai yang ramah lingkungan; 8) meminimalkan pengaruh-pengaruh negatif terhadap lingkungan selama dalam proses pembangunan; 9) mewujudkan bentuk fisik yang jelas dan mengandung arti yang spesifik dan dapat juga menerapkan nilai-nilai budaya lokal; dan 10) memberikan kontribusi dalam pelestarian budaya lokal, pengembangan, promosi, pendikan, dan penelitian terhadap budaya lokal itu sendiri dengan segi modern asal tidak mengurangi nilai-nilai yang ramah lingkungan; 8) meminimalkan pengaruh-pengaruh negatif terhadap lingkungan selama dalam proses pembangunan; 9) mewujudkan bentuk fisik yang jelas dan mengandung arti yang spesifik dan dapat juga menerapkan nilai-nilai budaya lokal; dan 10) memberikan kontribusi dalam pelestarian budaya lokal, pengembangan, promosi, pendikan, dan penelitian terhadap budaya lokal itu sendiri SARAN Penulis menyarankan kepada semua pihak untuk dapat berkontribusi dalam pelestarian budaya lokal yang mulai tergerus oleh zaman. SIMPULAN Ilmu Pengetahuan Alam berasal dari kata “science” yaitu pengetahuan rasional mengenai alam semesta dengan segala isinya yang diperoleh melalui proses ilmiah. Sains bersifat dinamis, artinya selalu mengalami perkembangan dan bertambah setiap saat, sehingga dengan mempelajari sains berarti secara tidak langsung kita mengikuti perkembangan zaman. Sains memiliki hubungan dengan berbagai konsep dalam berbagai disiplin ilmu. Proses pembelajarannya menekankan pada pemberian pengalaman langsung untuk mengembangkan kompetensi agar menjelajahi dan memahami alam sekitar secara ilmiah. Proses pembelajarannya menekankan pada pemberian pengalaman langsung untuk mengembangkan kompetensi agar menjelajahi dan memahami alam sekitar secara ilmiah. Pendekatan Science, Technology, and Society (STS) atau pendekatan Sains, Teknologi, dan Masyarakat (STM) merupakan gabungan antara pendekatan konsep, keterampilan proses, CBSA, inkuiri, dan diskoveri serta pendekatan lingkungan. UCAPAN TERIMA KASIH Penulis mengucapkan terima kasih kepada semua pihak yang telah memberikan bantuan, sehingga penelitian ini dapat terselesaikan. Uniform Resource Locator: https://e-journal.lp3kamandanu.com/index.php/panthera Ecolodge adalah Implementasi dari Pendidikan Sains (IPA) yang Bersifat SETS Ecolodge adalah fasilitas akomodasi, dengan 5-75 kamar, yang memiliki dampak kecil pada alam, finansialnya berkelanjutan, dan membantu melindungi kawasan sekitar yang sensitif, melibatkan dan menguntungkan bagi komunitas lokal, menawarkan pengalaman interpretatif dan interaktif, memberikan rasa kebersamaan dengan alam dan budaya, direncanakan, didesain, dibangun, dan bertindak dengan cara yang dapat diterima secara ekologi dan sosial (Effendi et al., 2018). Istilah ecolodge mulai banyak digunakan seiring tingginya kepedulian dan aksi masyarakat yang peduli dengan kelestarian lingkungan. Istilah ini membedakan ecolodge dengan sarana akomodasi lain, baik dari sisi proses pembangunannya hingga kegiatan operasionalnya (Sumanapala et al., 2015). Seiring maraknya pengembangan pariwisata berkelanjutan, penelitian-penelitian yang mengangkat topik ecolodge juga semakin berkembang. p g j g g Aspek fundamental untuk ecolodge selain perlindungan kawasan alam adalah pelatihan dan pendidikan. Terkait perlindungan lingkungan, mendirikan ecolodge selain harus berdampak mendekati nol pada alam, namun juga harus memiliki tujuan melindungi biodiversitas. Pelatihan dan pendidikan yang dimaksud adalah untuk warga lokal dan wisatawan. Pelatihan dilakukan untuk menambah pengetahuan dan pengalaman seputar ecolodge. Wisatawan juga mendapat kesempatan untuk belajar dari masyarakat lokal tentang lingkungan dan budaya sekitar. Apabila hal-hal tersebut dipahami dan diimplementasikan dengan benar, maka ecolodge dapat dikatakan sukses dan berkelanjutan, kemudian destinasi wisata di sekitar ecolodge dapat mengikuti prinsip yang sama dengan menciptakan jaringan kooperatif yang sehat dan adil, suatu daerah dapat mencapai pengembangan berkelanjutan (Aswita, 2018). Ecolodge harus memenuhi setidaknya 3 persyaratan global yang harus kita pahami yaitu: 1) perlindungan/pelestarian terhadap budaya dan lingkungan sekitar; 2) manfaat positif yang dapat di berikan kepada komunitas lokal yang ada di sekitarnya (Ekonomi, sosial, dan budaya); dan 3) sumber informasi bagi masyarakat lokal dan pendatang (tourits). DAFTAR RUJUKAN DAFTAR RUJUKAN Aswita, D. (2018). Environmental Education and Ecotourism for Sustainable Life: Literature Study. Jurnal Ilmiah Peuradeun, 6(1), 17-30. https://doi.org/10.26811/peuradeun.v6i1.157 Budiyanto, M. A. K., Aminah, T., & Husamah. (2020). School Strategies to Utilize the Ecotourism Potency of Songgoriti and Cangar of Batu City Tourisms as Learning Sources in High School. Jurnal Penelitian dan Pengkajian Ilmu Pendidikan: E-Saintika, 4(3), 272-283. https://doi.org/10.36312/e-saintika.v4i3.276 Dewi, C. A., Khery, Y., & Erna, M. (2019). An Ethnoscience Study in Chemistry Learning to Develop Scientific Literacy. Jurnal Pendidikan IPA Indonesia, 8(2), 279-287. https://doi.org/10.15294/jpii.v8i2.19261 p g jp Effendi, I., Elizal., Rizal, Y., Wiyati, R., & Maryanti, S. (2018). Preliminary Study on Ecotourism Potency of Tropical Forest and Coastal Area on PT p g jp Effendi, I., Elizal., Rizal, Y., Wiyati, R., & Maryanti, S. (2018). Preliminary Study on Ecotourism Potency of Tropical Forest and Coastal Area on PT Uniform Resource Locator: https://e-journal.lp3kamandanu.com/index.php/panthera 279 279 Diamond Raya Timber Concession Area, Riau Province. IOP Conference Series: Earth and Environmental Science, 216(1), 1-6. https://doi.org/10.1088/1755-1315/216/1/012046 Diamond Raya Timber Concession Area, Riau Province. IOP Conference Diamond Raya Timber Concession Area, Riau Province. IOP Conference Series: Earth and Environmental Science, 216(1), 1-6. https://doi.org/10.1088/1755-1315/216/1/012046 Diamond Raya Timber Concession Area, Riau Province. IOP Conference Series: Earth and Environmental Science, 216(1), 1-6. https://doi.org/10.1088/1755-1315/216/1/012046 Indahri, Y. (2021). Asesmen Nasional sebagai Pilihan Evaluasi Sistem Pendidikan Nasional. Aspirasi: Jurnal Masalah-masalah Sosial, 12(2), 195-215. https://doi.org/10.46807/aspirasi.v12i2.2364 Karaarslan, G., & Teksöz, G. (2016). Integrating Sustainable Development Concept in to Science Education Program is Not Enough: We Need Competent Science Teachers for Education for Sustainable Development - Turkish Experience. International Journal of Environmental & Science Education, 11(15), 8403-8424. ( ) Khery, Y., Nufida, B. A., Suryati., Rahayu, S., & Aini, M. (2019). Pemahaman Mahasiswa tentang Hakikat Sains dalam Pembelajaran Menggunakan Model Pembelajaran Mobile-NOS. Prisma Sains: Jurnal Pengkajian Ilmu dan Pembelajaran Matematika dan IPA IKIP Mataram, 7(2), 169-179. https://doi.org/10.33394/j-ps.v7i2.1771 Owusu, V., & Boafo, Y. A. (2018). Opportunities and Challenges for Multi- Level-Stakeholder Participation in Community-Based Ecotourism Development : The Case of the Boabeng-Fiema Monkey Sanctuary, Ghana. Journal of the Economic Geographical Society of Korea, 21(1), 53-68. https://doi.org/10.23841/egsk.2018.21.1.53 p g g Sumanapala, H. D. P., Perera, P. K., Kotagama, S., & Silva, D. A. C. (2015). Eco- Lodge Patrons’ Characteristics : The Sri Lankan Perspective. International Journal of Research in Social Science, 5(2), 509-525. Sumarni, W., & Kadarwati, S. (2020). 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https://openalex.org/W2617535932	http://dspace.stir.ac.uk/bitstream/1893/26660/1/8715605.pdf	English	null	Advancing Shannon Entropy for Measuring Diversity in Systems	Complexity	2,017	cc-by	7,857	Research Article Advancing Shannon Entropy for Measuring Diversity in Systems R. Rajaram,1 B. Castellani,2 and A. N. Wilson3 1Department of Mathematical Sciences, Kent State University, Kent, OH, USA 2Department of Sociology, Kent State University, 3300 Lake Rd. West, Ashtabula, OH, USA 3School of Social and Health Sciences, Abertay University, Dundee DD1 1HG, UK Correspondence should be addressed to R. Rajaram; rrajaram@kent.edu Received 31 January 2017; Revised 5 April 2017; Accepted 23 April 2017; Published 24 May 2017 Academic Editor: Enzo Pasquale Scilingo Copyright © 2017 R. Rajaram et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. From economic inequality and species diversity to power laws and the analysis of multiple trends and trajectories, diversity within systems is a major issue for science. Part of the challenge is measuring it. Shannon entropy 𝐻has been used to rethink diversity within probability distributions, based on the notion of information. However, there are two major limitations to Shannon’s approach. First, it cannot be used to compare diversity distributions that have different levels of scale. Second, it cannot be used to compare parts of diversity distributions to the whole. To address these limitations, we introduce a renormalization of probability distributions based on the notion of case-based entropy 𝐶𝑐as a function of the cumulative probability 𝑐. Given a probability density 𝑝(𝑥), 𝐶𝑐measures the diversity of the distribution up to a cumulative probability of 𝑐, by computing the length or support of an equivalent uniform distribution that has the same Shannon information as the conditional distribution of ̂𝑝𝑐(𝑥) up to cumulative probability 𝑐. We illustrate the utility of our approach by renormalizing and comparing three well-known energy distributions in physics, namely, the Maxwell-Boltzmann, Bose-Einstein, and Fermi-Dirac distributions for energy of subatomic particles. The comparison shows that 𝐶𝑐is a vast improvement over 𝐻as it provides a scale-free comparison of these diversity distributions and also allows for a comparison between parts of these diversity distributions. Hindawi Complexity Volume 2017, Article ID 8715605, 10 pages https://doi.org/10.1155/2017/8715605 Hindawi Complexity Volume 2017, Article ID 8715605, 10 pages https://doi.org/10.1155/2017/8715605 1. Diversity in Systems First, we extend the formalism in order to compute case-based entropy for continuous as well as discrete distributions. Second, we broaden our focus from complexity/complex systems to diversity in any type of statistically distributed system. That is, we start to explore distributions of diversity for systems where richness is not a function of the degree of complexity types. More specifically, as we will see later in the paper, we define the diversity of a probability distribution as the number of equivalent equiprobable types required to maintain the same amount of Shannon entropy 𝐻(i.e., the number of Shannon-equivalent equiprobable states). Given such a def- inition, a system with a high degree of richness and evenness would have a higher degree of 𝐻, whereas a system with a low degree of richness and evenness would have a low degree of 𝐻. In turn, a system with high richness but low evenness (as in the case of a skewed-right system with long tail) would have a lower degree of 𝐻than a system with high richness and high evenness. Third, the discrete indices we used had a degree of subjectivity to them, for example, how should household income be binned and what influence does that have on the distribution of diversity? As such, we wanted to see how well 𝐶𝑐worked for distributions where the unit of measurement was universally agreed upon. Fourth, we had not emphasized how 𝐶𝑐was a major advance on Shannon entropy 𝐻. As known, while 𝐻has proven useful, it compresses its measurement of diversity into a single number; it is also nonintuitive; and, as we stated above, it is not scale-free and therefore cannot be used to compare the diversity of different systems; neither can it be used to compare parts of the diversity within a system to the entire system. 1.1. Purpose of the Current Study. Recently, we have intro- duced a novel approach to representing diversity within sta- tistical distributions [5, 6], which overcomes such difficulties and allows the distribution of diversity in any given system (or cumulative portions thereof) to be directly compared to the distribution of diversity within any other system. In effect, it is a renormalization that can be applied to any probability distribution to produce a direct representation of the distribution of diversity within that distribution. 1. Diversity in Systems Examples include (a) the different levels of household income in a city, (b) the number of different species in an ecosystem, (c) the diversity of a country’s exports, (d) the distribution of 2 Complexity Complexity different nodes in a complex network, (e) the various health trends for a particular disease across time/space, or (f) the cultural or ethnic diversity of an organization or company. In all such instances, the greater the number of diversity types (be these types discrete or continuous), the greater the degree of richness in a system. In the case of the current study, for example, richness was defined as the number of different energy states. examples that are often suggested to exhibit behaviors indicative of complexity such as emergent collectivity, phase changes, or tipping points. What we found was that such systems obeyed an apparent “limiting law of restricted diver- sity” [6], which constrains the majority of cases in these complex systems to simpler types. In fact, for these types of distribution, the distributions of diversity were found to follow a scale-free 60/40 rule, with 60% or more of cases belonging to the simplest 40% or less of equiprobable diversity types. This was found to be the case regardless of whether the original distribution fit a power law or was long- tailed, making it fundamentally distinct from the well-known (but often misunderstood) Pareto Principle [7]. In turn, evenness refers to the uniformity or “equiprob- ability” of occurrence of such states. In terms of the above examples, evenness would be defined as (a) a city where household income was evenly distributed, (b) an ecosystem where the diversity of its species was equal in number, (c) a country with an even distribution of exports, (d) a complex network where all nodes had the same probability of occurrence, (e) a disease where all possible health trends were equiprobable, or (f) a company or organization where people of different cultural or ethnic backgrounds were evenly distributed. In the case of the current study, for example, evenness was defined as the uniformity or “equiprobability” of the occurrence of all possible energy states.i In the following, we continue to explore the use of case- based entropy in comparing systems described by statistical distributions. However, we now go beyond our prior work in the following ways. 1. Diversity in Systems Although statistical distributions that directly reflect the spread of key parameters (such as mass, age, wealth, or energy) provide descriptions of this diversity, it can be difficult to compare the diversity of different distributions or even the same distribution under different conditions, mostly because of differences in scales and parameters. Also, many of the measures currently available compress diversity into a single score or are not intuitive [1–4]. Although statistical distributions that directly reflect the spread of key parameters (such as mass, age, wealth, or energy) provide descriptions of this diversity, it can be difficult to compare the diversity of different distributions or even the same distribution under different conditions, mostly because of differences in scales and parameters. Also, many of the measures currently available compress diversity into a single score or are not intuitive [1–4]. Statistical distributions play an important role in any branch of science that studies systems comprised of many similar or identical particles, objects, or actors, whether material or immaterial, human or nonhuman. One of the key features that determines the characteristics and range of potential behaviors of such systems is the degree and distribution of diversity, that is, the extent to which the components of the system occupy states with similar or different features. At the outset, motivated by examples of measuring diversity in ecology and evolutionary biology from [3, 4], we sought to address these challenges. We begin with some definitions and a review of our previous research.i As Page outlined in a series of inquiries [1, 2], includ- ing The Difference and Diversity and Complexity, diversity within systems is an important concern for science, be it making sense of economic inequality, expanding the trade portfolio of countries, measuring the collapse of species diversity in various ecosystems, or determining the optimal utility/robustness of a network. However, an important major challenge in the literature on diversity and complexity, which Page also points out [1, 2], remains: the issue of measurement. i First, in terms of definitions, we follow the ecological literature, defining diversity as the interplay of “richness” and “evenness” in a probability distribution. Richness refers to the number of different diversity types in a system. 1. Diversity in Systems Arising from our work in the area of complex systems, the approach is based on the notion of case-based entropy, 𝐶𝑐[5]. This approach has two major advantages over the Shannon Entropy 𝐻, which, as we alluded to above, is one of the most commonly used measures of diversity within probability distributions and which calculates the average amount of uncertainty (or information, depending on one’s perspective) present in a given probability distribution. First, 𝐶𝑐can be used to compare distributions that have different levels of scale; and, second, 𝐶𝑐can be used to compare parts of distributions to their whole.t Hence, the purpose of the current study, as a demon- stration of the utility of 𝐶𝑐, is to renormalize and compare three physically significant energy distributions in statistical physics: the energy probability density functions for systems governed by Boltzmann, Bose-Einstein, and Fermi-Dirac statistics. 3. Case-Based Entropy of a Continuous Random Variable Our impetus for making an advance over the Shannon entropy 𝐻comes from the study of diversity in evolutionary biology and ecology, where it is employed to measure the true diversity of species (types) in a given ecological system of study [3, 4, 9, 10]. As we show here, it can also be used to measure the diversity of an arbitrary probability distribution of a continuous random variable. Hence, the uniform distribution renormalizes the effect of varying relative frequencies (or probabilities) of occurrence of the values of 𝑥without losing information (or entropy). In other words, if all choices of the random variable are equally likely, the number of values (or the length, if it is a continuous random variable) needed for the random variable to keep the same amount of information as the given distribution is a measure of diversity. In a sense, each new value (or type) is counted as adding to the diversity, only if the new value has the same probability of occurrence as the existing values. Diversity necessarily requires the values of the random variable to be equiprobable since lower probability, for example, means that such values occur rarely in the random variable and hence cannot be treated as equally diverse as other values with higher probabilities. Hence, by choosing an equiprobable (or uniform) distribution for normalization, we are counting the true diversity, that is, the number of equiprobable types that are required to match the same amount of Shannon information 𝐻as the given distribution.h Given the probability density function 𝑝(𝑥) of a random variable 𝑥in a measure space 𝑋, the Shannon-Weiner entropy index 𝐻is given by 𝐻= −∫ 𝑋 𝑝(𝑥) ln (𝑝(𝑥)) 𝑑𝑥. (1) (1) The problem, however, with the Shannon entropy index 𝐻, as we identified in our abstract and Introduction, is that while being useful for studying the diversity of a single system, it cannot be used to compare the diversity across probability distributions. In other words, 𝐻is not multiplica- tive: a doubling of value for 𝐻does not mean that the actual diversity has doubled. To address this problem, we turned to the true diversity measure 𝐷[3, 11, 12], which gives the range of equiprobable values of 𝑥that gives the same value of 𝐻: This calculation (as we have shown elsewhere [5]) can be done for parts of the distribution up to a cumulative probability of 𝑐. 3. Case-Based Entropy of a Continuous Random Variable This means that a comparison of 𝐶𝑐for a variety of distributions is actually a comparison of the variation of the fraction of diversity 𝐶𝑐contributed by values of the random variable up to 𝑐. 𝐷= 𝑒𝐻. (2) (2) The utility of 𝐷for comparing the diversity across probability distributions is that, in 𝐷, a doubling of the value means that the number of equiprobable ranges of values of 𝑥has doubled as well. 𝐷calculates the range of such equiprobable values of 𝑥that will give the same value of Shannon entropy 𝐻as observed in the distribution of 𝑥. We say that two probability densities 𝑝1(𝑥) and 𝑝2(x) are Shannon-equivalent if they have the same value of Shannon entropy. Case-based entropy is then the range of values of 𝑥for the Shannon-equivalent uniform distribution for 𝑝(𝑥). We also note that Shannon entropy can be recomputed from 𝐷by using 𝐻= ln(𝐷). p Since, regardless of the scale and units of the original distribution, 𝑐and 𝐶𝑐both vary from 0 to 1, one can plot a curve for 𝐶𝑐versus 𝑐for multiple distributions on the same axes. 𝐶𝑐thus provides us with a scale-free measure to compare distributions without omitting any of the entropy information, but by renormalizing the variable to one that has equiprobable values. What is more, it also allows us to compare different parts of the same distribution, or parts to wholes. That is, we can generate a 𝐶𝑐versus 𝑐curve for any part of a distribution (normalizing the probabilities to add up to 1 in that part) and compare the 𝐶𝑐curve of the part to the 𝐶𝑐curve of the whole or another part to see if the functional dependence of 𝐶𝑐on 𝑐is the same or different. In essence, 𝐶𝑐 has the ability to compare distributions in a “fractal” or self- similar way. In order to measure the distribution of diversity, we next need to determine the fractional contribution to overall diversity up to a cumulative probability 𝑐. In other words, we need to be able to compute the diversity contribution 𝐷𝑐up to a certain cumulative probability 𝑐. To do so, we replace 𝐻 with 𝐻𝑐, the conditional entropy, given that only the portion of the distribution up to a cumulative probability 𝑐(denoted by 𝑋𝑐) is observed with conditional probability of occurrence In [5], we showed how to carry out the renormalization for discrete probability distributions, both mathematical and empirical. 2. Renormalizing Probability: Case-Based Entropy and the Distribution of Diversity The quantity case-based entropy [5], 𝐶𝑐, renormalizes the diversity contribution of any probability distribution 𝑃(𝑥), by computing the true diversity 𝐷of an equiprobable distri- bution (called the Shannon-equivalent uniform distribution) that has the same Shannon entropy 𝐻as 𝑃(𝑥). 𝐶𝑐is precisely the number of equiprobable types in the case of a discrete distribution, or the length, support, or extent of the variable in the case of continuous distributions, which is required to keep the value of the Shannon entropy the same across the whole or any part of the distribution up to a cumulative probability After developing the concept and formalism for case- based entropy for discrete distributions [5], we first applied it to compare complexity across a range of complex systems [6]. In that work, we investigated a series of systems described by a variety of skewed-right probability distributions, choosing 3 Complexity 𝑐. We choose the Shannon-equivalent uniform distribution for two reasons: make the case for how 𝐶𝑐constitutes an advance over 𝐻, in terms of providing a scale-free comparison of probability distributions and also comparisons between parts of distri- butions. More importantly, we demonstrate how 𝐶𝑐works for continuous distributions, by examining the Maxwell- Boltzmann, Bose-Einstein, and Fermi-Dirac distributions for energy of subatomic particles. We begin with a more detailed review of 𝐶𝑐. (i) First, it is well known that, on a finite measure space, the uniform distribution maximizes entropy: that is, the uniform distribution has the maximal entropy among all probability distributions on a set of finite Lebesgue measures [8]. (ii) Second, a Shannon-equivalent uniform distribution will, by definition, count the number of values (or range of values) of 𝑥that are required to give the same information as the original distribution 𝑃(𝑥) if we assume that all the values (or range of values) are equally probable. 3. Case-Based Entropy of a Continuous Random Variable It is worth noting in passing that 𝐶𝑐reaches 40% when 𝑐≈69%, indicating that approximately 69% of the molecules in the gas are contained within the lower 40% of diversity of energy probability states at all temperatures (here, diversity is defined as the number of equivalent equiprobable energy states required to maintain the same amount of 3. Case-Based Entropy of a Continuous Random Variable In this paper, as we stated in the Introduction, we 4 Complexity Complexity with density ̂𝑝𝑐(𝑥) up to a given cumulative probability 𝑐. That is, energies 𝐸of particles in such a gas are described by the Boltzmann distribution [8]. In one dimension, this is ̂𝑝𝑐(𝑥) = 𝑝(𝑥) ∫𝑋𝑐𝑝(𝑥) 𝑑𝑥 , 𝐻𝑐= −∫ 𝑋𝑐 ̂𝑝𝑐(𝑥) ln (̂𝑝𝑐(𝑥)) , 𝑐= ∫ 𝑋𝑐 𝑝(𝑥) 𝑑𝑥, 𝐷𝑐= 𝑒𝐻𝑐. (3) 𝑝𝐵,1𝐷(𝐸) = ( 1 𝑘𝐵𝑇) 𝑒−𝐸/𝑘𝐵𝑇= 𝛽 𝑒𝛽𝐸, (6) ̂𝑝𝑐(𝑥) = 𝑝(𝑥) ∫𝑋𝑐𝑝(𝑥) 𝑑𝑥 , (6) where 𝑘𝐵is the Boltzmann constant and 𝛽= (1/𝑘𝐵𝑇).h The entropy of 𝑝𝐵,1𝐷(𝐸) can be shown to be 𝐻𝐵= 1 − ln(𝛽), and hence the true diversity of energy in the range [0, ∞) is given by (3) 𝐷𝐵,1𝐷= 𝑒𝐻= 𝑒1−ln(𝛽) = 𝑒 𝛽. (7) (7) 𝐷𝑐= 𝑒𝐻𝑐. The cumulative probability 𝑐from 𝐸= 0 to 𝐸= 𝑘is then given by The value of 𝐷𝑐for a given value of cumulative probability 𝑐is the number of Shannon-equivalent equiprobable energy states (or of values of the variable in the 𝑥-axis in general) that are required to explain the information up to a cumulative probability of 𝑐within the distribution. If 𝑐= 1, then 𝐷𝑐= 𝐷is the number of such Shannon-equivalent equiprobable energy states for the entire distribution itself. 𝑐= ∫ [0,𝑘] 𝑝𝐵,1𝐷(𝐸) 𝑑𝐸= 1 −𝑒−𝛽𝑘. (8) (8) Hence, 𝑘can be computed in terms of 𝑐as Hence, 𝑘can be computed in terms of 𝑐as 𝑘= −ln (1 −𝑐) 𝛽 . (9) gy We can then simply calculate the fractional diversity contribution or case-based entropy as (9) 𝐶𝑐= 𝐷𝑐 𝐷. (4) Equation (9) is useful for the one-dimensional Boltzmann case to eliminate the parameter 𝑘altogether in (11) to obtain an explicit relationship between 𝐶𝑐and 𝑐. It is to be noted that, in most cases, both 𝐶𝑐and 𝑐can only be parametrically related through 𝑘. The other quantities introduced in Section 3 can then be calculated as follows: (4) It is at this point that the renormalization (𝐶𝑐as a function of 𝑐) becomes scale independent as both axes range between values of 0 and 1 with the graph of 𝐶𝑐versus 𝑐passing through (0, 0) and (1, 1). Hence, irrespective of the range and scale of the original distributions, all distributions can be plotted on the same graph and their diversity contributions can be compared in a scale-free manner. 3. Case-Based Entropy of a Continuous Random Variable ̂𝑝𝑐(𝐸) = 𝑝𝐵,1𝐷(𝐸) 𝑐 = 𝛽𝑒−𝛽𝐸 1 −𝑒−𝛽𝑘, (10) ̂𝑝𝑐(𝐸) = 𝑝𝐵,1𝐷(𝐸) 𝑐 = 𝛽𝑒−𝛽𝐸 1 −𝑒−𝛽𝑘, (10) 𝐻𝑐= −∫ [0,𝑘] 𝛽𝑒−𝛽𝐸 1 −𝑒−𝛽𝑘ln ( 𝛽𝑒−𝛽𝐸 1 −𝑒−𝛽𝑘) 𝑑𝐸 = 1 + ln ( 𝑐 𝛽(1 −𝑐)(1−𝑐)/𝑐) , (11) 𝐷𝑐= 𝑒𝐻𝑐= 𝑒1+ln((𝑐/𝛽)(1−𝑐)(1−𝑐)/𝑐) = 𝑒 𝛽⋅(𝑐(1 −𝑐)(1−𝑐)/𝑐) , (12) 𝐶𝑐= 𝐷𝑐 𝐷𝐵,1𝐷 = (𝑒/𝛽) ⋅(𝑐(1 −𝑐)(1−𝑐)/𝑐) 𝑒/𝛽 = 𝑐(1 −𝑐)(1−𝑐)/𝑐. (13) (10) 𝐻𝑐= −∫ [0,𝑘] 𝛽𝑒−𝛽𝐸 1 −𝑒−𝛽𝑘ln ( 𝛽𝑒−𝛽𝐸 1 −𝑒−𝛽𝑘) 𝑑𝐸 = 1 + ln ( 𝑐 𝛽(1 −𝑐)(1−𝑐)/𝑐) , (11) 𝐷𝑐= 𝑒𝐻𝑐= 𝑒1+ln((𝑐/𝛽)(1−𝑐)(1−𝑐)/𝑐) To check the validity of our formalism, we calculate 𝐷𝑐 for the simple case of a uniform distribution given by 𝑝(𝑥) = 𝜒[0,𝐿](𝑥) on the interval 𝑋= [0, 𝐿]. Intuitively, if we choose 𝑋𝑐= [0, 𝑐], then, owing to the uniformity of the distribution, we expect 𝐷𝑐= 𝑐itself. In other words, the diversity of the part [0, 𝑐] is simply equal to 𝑐, that is, the length of the interval [0, 𝑐], and hence the 𝐶𝑐versus 𝑐curve will simply be the straight line with slope equal to 1. This can be shown as follows: (11) 𝐷𝑐= 𝑒𝐻𝑐= 𝑒1+ln((𝑐/𝛽)(1 𝑐) ) = 𝑒 𝛽⋅(𝑐(1 −𝑐)(1−𝑐)/𝑐) , (12) (12) ̂𝑝𝑐(𝑥) = 1 𝑐𝜒[0,𝐿] (𝑥) , 𝐻𝑐= −∫ [0,𝑐] 1 𝑐ln (1 𝑐) 𝑑𝑥= ln (𝑐) , 𝐷𝑐= 𝑒𝐻𝑐= 𝑒ln(𝑐) = 𝑐. (5) (5) (13) = 𝑐(1 −𝑐)(1−𝑐)/𝑐. We note that, in (13), the temperature factor 𝛽cancels out, indicating that the distribution of diversity for an ideal gas in one dimension is independent of temperature. The resulting graph of 𝐶𝑐as a function of 𝑐is shown in Figure 1. It is worth noting in passing that 𝐶𝑐reaches 40% when 𝑐≈69%, indicating that approximately 69% of the molecules in the gas are contained within the lower 40% of diversity of energy probability states at all temperatures (here, diversity is defined as the number of equivalent equiprobable energy states required to maintain the same amount of We note that, in (13), the temperature factor 𝛽cancels out, indicating that the distribution of diversity for an ideal gas in one dimension is independent of temperature.h With our formulation of 𝐶𝑐complete, we turn to the energy distributions for particles governed by Boltzmann, Bose-Einstein, and Fermi-Dirac statistics. The resulting graph of 𝐶𝑐as a function of 𝑐is shown in Figure 1. 4. Results 4.1. 𝐶𝑐for the Boltzmann Distribution in One Dimension. We first illustrate our renormalization by applying it to a relatively simple case: that of an ideal gas at temperature 𝑇. The kinetic Complexity 5 Percentage diversity contribution versus percentage of cases 0.2 0.4 0.6 0.8 0 1 Cc Case-based entropy 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Percentage of cases c Figure 1: 𝐶𝑐as a function of 𝑐for the Boltzmann distribution in one dimension. Figure 1: 𝐶𝑐as a function of 𝑐for the Boltzmann distribution in one dimension. being the parameter. Also, analytical forms are not possible, so Matlab was used to compute 𝐻𝑐, 𝐷𝑐, and 𝐶𝑐, respectively: Shannon entropy 𝐻). Thus, the one-dimensional Boltzmann distribution obeys an interesting phenomenon that we have identified in a wide range of skewed-right complex systems, which (as we briefly discussed in the Introduction) we call restricted diversity and, more technically, the 60/40 rule [6]. The independence of temperature in the 𝐶𝑐versus 𝑐curve, for the Boltzmann distribution, shows that the effect of increasing 𝑇is to shift the mean of the distribution to higher energies and to increase its standard deviation, but not to change its characteristic shape. Still, what is key to our results is that the temperature independence of the 𝐶𝑐curve for the Boltzmann distribution in one dimension validates that our renormalization preserves the fundamental features of the original distribution. 𝐷𝑐(𝑘) = 𝑒𝐻𝑐(𝑘), 𝐷𝐵,3𝐷= lim 𝑘→∞𝐷𝑐(𝑘) , 𝐶𝑐= 𝐷𝑐 𝐷𝐵,3𝐷 . (16) 𝐷𝑐(𝑘) = 𝑒𝐻𝑐(𝑘), 𝐷𝐵,3𝐷= lim 𝑘→∞𝐷𝑐(𝑘) , 𝐶𝑐= 𝐷𝑐 𝐷𝐵,3𝐷 . (16) (16) Thus, 𝐶𝑐can also only be computed in parametric form with parameter 𝑘that varies from 0 to ∞. Figure 2 shows the 𝐶𝑐curve thus calculated for the Boltzmann distribution in three dimensions. Although the temperature independence of this distribu- tion is not immediately evident from Figure 2, one would, following the same logic as for the one-dimensional case, expect the distribution of diversity to be the same for all 𝑇. That is, as in the one-dimensional case, because changes in 𝑇do not affect the original distributions characteristic shape, we expect the renormalized distribution to be independent of temperature. This does, indeed, turn out to be the case. This is illustrated in Figure 2, which overlays the results of the calculations for 𝑇= 50 K, 500 K, and 5000 K. 4. Results are indistinguishable when superimposed Boltzmann 3D curves at T = 50 K, 500 K K, and 5000 0.2 0.4 0.6 0.8 0 1 Case-based entropy Cc 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Percentage of cases c 0.2 0.4 0.6 0.8 0 1 Case-based entropy C 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Percentage of cases c are indistinguishable when superimposed Boltzmann 3D curves at T = 50 K, 500 K K, and 5000 ure 2: 𝐶𝑐versus 𝑐for Boltzmann 3D superimposed at three different temperatures: 𝑇= 50 K, 500 K, and 5000 K. BE Helium BE Photon 0.2 0.4 0.6 0.8 0 1 Cc Case-based entropy 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Percentage of cases c Figure 3: 𝐶𝑐versus 𝑐for Helium-4 and for photons. Note: the results of calculations carried out at 𝑇= 50 K, 500 K, and 5000 K are overlaid. 0.2 0.4 0.6 0.8 0 1 Cc Case-based entropy 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Percentage of cases c elium-4 and for photons. Note: the results of calculations carried out at 𝑇= 50 K, 500 K, and 5000 K are overlaid. Figure 3: 𝐶𝑐versus 𝑐for Helium-4 and for photons. Note: the results of calculations carried out at 𝑇= 50 K, 500 K, and 5000 K are overlaid. For massless bosons such as photons, the energy proba- bility density function is [13] 4.3. The Bose-Einstein Distributions for Massive and Massless Bosons. We now move on to consider the second of our example distributions. The Bose-Einstein distribution gives the energy probability density function for massive bosons above the Bose temperature 𝑇𝐵as 4.3. The Bose-Einstein Distributions for Massive and Massless Bosons. We now move on to consider the second of our example distributions. The Bose-Einstein distribution gives the energy probability density function for massive bosons above the Bose temperature 𝑇𝐵as 𝑝BE = 𝐶⋅ 𝐸2 𝑒𝛽𝐸−1. (19) (19) It is important to note that the “density of states” factors shown in (17) and (19) result in different energy distributions, despite the two types of boson obeying the same statistics.h 𝑝HB (𝐸) = 𝐶⋅ 𝐸1/2 𝐵𝑒𝛽𝐸−1, (17) (17) where 𝐶is a normalization constant and The conditional probabilities, conditional entropies, true diversities, and case-based entropies for these distributions cannot be calculated analytically but can be calculated numerically. 4. Results It is also worth noting that, just like our one-dimensional case, the curve obeys the 60/40 rule of restricted diversity [6]: regardless of temperature, over 60 percent of molecules are in the lower 40 percent of diversity of energy probability states (here again, diversity is defined as the number of equivalent equiprobable energy states required to maintain the same amount of Shannon entropy 𝐻). 4.2. 𝐶𝑐for the Boltzmann Distribution in Three Dimensions. We now turn to the calculation of 𝐶𝑐for the physically more important case of the Boltzmann distribution in three dimensions [8]: 𝑝𝐵,3𝐷(𝐸) = 2𝛽3/2𝐸1/2 √𝜋𝑒𝛽𝐸, (14) (14) where the additional factor of √4𝛽𝐸/𝜋accounts for the density of states.h y The cumulative probability 𝑐from 𝐸= 0 to 𝐸= 𝑘can be computed as follows: 𝑐= ∫ [0,𝑘] 𝑝𝐵,3𝐷(𝐸) 𝑑𝐸= √𝜋erf (√𝑘𝛽) −2𝑒−𝑘𝛽√𝑘𝛽 √𝜋 . (15) In addition, it is worth noting that as we might expect, adding more degrees of freedom increases the average energy by a factor of (1/2)𝑘𝐵𝑇per degree while maintaining the same shape for the distribution of energy. Hence, the current result will still hold true for gas molecules with higher degrees of freedom; that is, the distribution of diversity is always exactly the same for an ideal gas, whether monoatomic or polyatomic. (15) As we would hope, (15) has the property that as 𝑘→∞, the cumulative probability 𝑐→1.fi p y However, it is difficult to solve (15) for 𝑘directly in terms of 𝑐. We therefore compute 𝐶𝑐in parametric form with 𝑘 Complexity 6 Com are indistinguishable when superimposed Boltzmann 3D curves at T = 50 K, 500 K K, and 5000 0.2 0.4 0.6 0.8 0 1 Case-based entropy Cc 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Percentage of cases c Figure 2: 𝐶𝑐versus 𝑐for Boltzmann 3D superimposed at three different temperatures: 𝑇= 50 K, 500 K, and 5000 K. 4. Results The results of such calculations, using the software Matlab, are shown in Figure 3.i 𝐵= 1 𝜁(3/2) ( 𝑇 𝑇𝐵 ) 3/2 , (18) (18) where 𝜁is the Riemann zeta function. In the following calculations, we use the Bose temperature for helium, 𝑇𝐵= 3.14 K. t As with the Boltzmann distributions, we find that the distributions of diversity for the two boson systems are Complexity 7 FD Na 15000000K FD Na 6000 K FD Na 300 K FD Na 2.7K 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Percentage of cases c 1 0 0.3 0.4 0.5 0.8 0.9 0.2 0.6 0.1 0.7 Case-based entropy Cc Figure 4: Diversity curves for sodium electrons at a range of temperatures with 𝐶𝑐on the 𝑥-axis and 𝑐on the 𝑦-axis. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Percentage of cases c 1 0 0.3 0.4 0.5 0.8 0.9 0.2 0.6 0.1 0.7 Case-based entropy Cc 4: Diversity curves for sodium electrons at a range of temperatures with 𝐶𝑐on the 𝑥-axis and 𝑐on the 𝑦-axis. independent of temperature. Although the curves for the two types of boson are very similar, it is evident that the distributions of diversity do differ to some extent. For helium- 4 bosons, a slightly larger fraction of particles are contained in lower diversity energy states than is the case for photons, with 60% of atoms contained in the approximately 37% of the lowest diversity states, as compared to approximately 42% for photons. In other words, using 𝐶𝑐, we are able to identify, even in such instances where intuition might suggest it to be true, common patterns within and across these different energy systems, as well as their variations. With this point made, we move to our final energy distribution. 300 K (representing temperatures on earth), 6000 K (the temperature of the surface of the sun), and 15 × 106 K (the temperature of the core of the sun).hi This figure shows that the degree of diversity is the highest for fermions at low temperatures; for example, at 2.7 K, fully 70% of the lowest equiprobable diversity states are need to contain 60% of the particles, compared with only approximately 38% at 15×106 K. It also shows that, for sodium electrons, the diversity curve at normal temperatures on earth (300 K) is almost identical to that at very low temperatures. 4. Results That is, a room temperature Fermi gas of sodium electrons has a distribution of diversity very similar to that of a “Fermi condensate.” 4.4. The Fermi-Dirac Distribution. The final distribution we use to illustrate our approach is the Fermi-Dirac distribution: 5. Using 𝐶𝑐to Compare and Contrast Systems 𝑝FD (𝐸) = 𝐶⋅ 𝐸1/2 𝑒𝛽(𝐸−𝜇) + 1, (20) (20) With our renormalization complete for all three distributions, we sought next to demonstrate, albeit somewhat superficially, the utility of 𝐶𝑐for comparing and contrasting systems, given how widely known the results are for these three classic energy distributions. To begin with, it is usual to assume that, in the limit of high 𝑇, both Bose-Einstein and Fermi-Dirac distributions reduce to Boltzmann distributions, and so the physical properties of both bosons and fermions in this limit should be those of an ideal gas. where 𝐶is again a normalization constant and 𝜇is the Fermi energy [13]. In the following, we calculate distributions for sodium electrons, for which 𝜇= 3.4 eV. Once again, ̂𝑝, 𝐻𝑐, 𝐷𝑐, and 𝐶𝑐cannot be calculated analytically and so we rely on numerical calculations using Matlab.hf The Fermi-Dirac distribution differs from the previous examples in that it is not simply scaled by changes in energy. Instead, its shape changes, transforming from a skewed-left distribution, with a sharp cut-off at the Fermi energy at low temperatures, to a smooth, skewed-right distribution at high temperatures. Thus, unlike the situation for Boltz- mann and Bose-Einstein distributions, one would expect the distributions of diversity for fermions such as electrons to be dependent on temperature. Figure 4 compares the results of calculating 𝐶𝑐as a function of 𝑐for electrons in sodium at temperatures of 2.7 K (the temperature of space), In Figures 5 and 6, we show a comparison of all three energy distributions for temperatures of 6000 K and 15 × 106 K (the Bose-Einstein distribution for massless bosons is included for comparison). In these figures, it appears that, by 6000 K, the Bose-Einstein distribution for helium-4 is indistinguishable from the 3D Boltzmann distribution. Also, while the Fermi-Dirac distribution has clearly not reduced to the Boltzmann distribution even at 15 × 106 K, it appears to be trending towards it. 5. Using 𝐶𝑐to Compare and Contrast Systems Complexit 0 0.1 0.2 0.3 0.4 0.5 f(x) 0.6 0.7 0.8 0.9 1 5 10 15 20 0 x FD Na 6000 K BE Photon 6000 K MB 3D and BE Helium 6000 K (a) Energy density curves for Maxwell-Boltzmann 3D, Bose-Einstein Helium, Bose-Einstein Photon, and Fermi-Dirac Na 6000 K ×10−4 ×104 0 1 2 f(x) 3 0.5 1 1.5 2 2.5 3 0 x FD Na 15000000K BE Photon 15000000K MB 3D and BE Helium 15000000K (b) Energy density curves for Maxwell-Boltzmann 3D, Bose-Einstein Helium, Bose-Einstein Photon, and Fermi-Dirac Na 15000000 K Figure 5: Energy density curves. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Cumulative probability c 0.6 0.8 1 0.2 0.4 0 Case-based entropy Cc FD Na 6000 K MB 3D, BE Photon, and BE Helium 6000 K (a) 𝐶𝑐versus 𝑐curves for Maxwell-Boltzmann 3D, Bose-Einstein Helium, Bose-Einstein Photon, and Fermi-Dirac Na 6000 K 0.2 0.4 0.6 0.8 0 1 Case-based entropy Cc 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Cumulative probability c FD Na 15000000K MB 3D, BE Photon, and BE Helium at 15000000K (b) 𝐶𝑐versus 𝑐curves for Maxwell-Boltzmann 3D, Bose-Einstein Helium, Bose-Einstein Photon, and Fermi-Dirac Na 15000000 K Figure 6: 𝐶𝑐versus 𝑐curves. 8 Complexity ×10−4 ×104 0 1 2 f(x) 3 0.5 1 1.5 2 2.5 3 0 x 0 0.1 0.2 0.3 0.4 0.5 f(x) 0.6 0.7 0.8 0.9 1 5 10 15 20 0 x f(x) FD Na 15000000K BE Photon 15000000K MB 3D and BE Helium 15000000K (a) Energy density curves for Maxwell-Boltzmann 3D, Bose-Einstein Helium, Bose-Einstein Photon, and Fermi-Dirac Na 6000 K (a) Energy density curves for Maxwell-Boltzmann 3D, Bose-Einstein Helium, Bose-Einstein Photon, and Fermi-Dirac Na 6000 K (b) Energy density curves for Maxwell-Boltzmann 3D, Bose-Einstein Helium, Bose-Einstein Photon, and Fermi-Dirac Na 15000000 K (b) Energy density curves for Maxwell-Boltzmann 3D, Bose-Einstein Helium, Bose-Einstein Photon, and Fermi-Dirac Na 15000000 K Figure 5: Energy density curves. Figure 5: Energy density curves. Figure 5: Energy density curves. 5. Using 𝐶𝑐to Compare and Contrast Systems Because these renormalized distributions are independent of temperature, Complexity 9 MB 1D BE Helium and MB 3D BE Photon FD Na 15000000K FD Na 2.7K FD Na 300 K FD Na 6000 K 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Percentage of cases c 1 0 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0.1 0.2 Case-based entropy Cc Percentage diversity contribution versus percentage of cases Figure 7: Superposition of all diversity curves for Boltzmann 1D, Boltzmann 3D, Bose-Einstein Helium, Bose-Einstein Photon, and Fermi- Dirac Na at 2.7 K, 300 K, 6000 K, and 15000000 K. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Percentage of cases c 1 0 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0.1 0.2 Case-based entropy Cc Percentage diversity contribution versus percentage of cases Figure 7: Superposition of all diversity curves for Boltzmann 1D, Boltzmann 3D, Bose-Einstein Helium, Bose-Einstein Photon, and Fermi- Dirac Na at 2.7 K, 300 K, 6000 K, and 15000000 K. fermions are yet to surpass the barrier created by the Fermi energy and hence are all restricted to the lower end of the energy.h on a Shannon-equivalent uniform distribution, which comes from the work of Jost and others on the notion of true diversity in ecology and evolutionary biology [4, 9, 10]. With this approach established, we then reviewed our construction of case-based entropy 𝐶𝑐. Given a probability density 𝑝(𝑥), 𝐶𝑐 measures the diversity of the distribution up to a cumulative probability of 𝑐, by computing the length or support of an equivalent uniform distribution that has the same Shannon information as the conditional distribution of ̂𝑝𝑐(𝑥) up to a cumulative probability 𝑐. Thus, the transformation from the usual probability distribution to a distribution of case-based entropy (𝐶𝑐versus 𝑐) has allowed us to make direct scale-free comparisons, of the ways in which the Maxwell-Boltzmann, Bose-Einstein, and Fermi-Dirac energy distributions are similar or differ both internally (as a function of temperature 𝑇) and across distributions. It appears that, except for extremely high temperatures, the Fermi-Dirac distribution has a larger value of 𝐶𝑐than the others. This means that there are a larger number of Shannon-equivalent equiprobable states of energy for the Fermi-Dirac distribution as compared to the others. 5. Using 𝐶𝑐to Compare and Contrast Systems 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Cumulative probability c 0.6 0.8 1 0.2 0.4 0 Case-based entropy Cc FD Na 6000 K MB 3D, BE Photon, and BE Helium 6000 K (a) 𝐶𝑐versus 𝑐curves for Maxwell-Boltzmann 3D, Bose-Einstein Helium, Bose-Einstein Photon, and Fermi-Dirac Na 6000 K 0.2 0.4 0.6 0.8 0 1 Case-based entropy Cc 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Cumulative probability c FD Na 15000000K MB 3D, BE Photon, and BE Helium at 15000000K (b) 𝐶𝑐versus 𝑐curves for Maxwell-Boltzmann 3D, Bose-Einstein Helium, Bose-Einstein Photon, and Fermi-Dirac Na 15000000 K Figure 6: 𝐶𝑐versus 𝑐curves. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Cumulative probability c 0.6 0.8 1 0.2 0.4 0 Case-based entropy Cc 0.2 0.4 0.6 0.8 0 1 Case-based entropy Cc 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Cumulative probability c Cumulative probability c FD Na 6000 K MB 3D, BE Photon, and BE Helium 6000 K MB 3D, BE Photon, and BE Helium 6000 K FD Na 15000000K MB 3D, BE Photon, and BE Helium at 15000000K (a) 𝐶𝑐versus 𝑐curves for Maxwell-Boltzmann 3D, Bose-Einstein Helium, Bose-Einstein Photon, and Fermi-Dirac Na 6000 K (b) 𝐶𝑐versus 𝑐curves for Maxwell-Boltzmann 3D, Bose-Einstein Helium, Bose-Einstein Photon, and Fermi-Dirac Na 15000000 K (a) 𝐶𝑐versus 𝑐curves for Maxwell-Boltzmann 3D, Bose-Einstein Helium, Bose-Einstein Photon, and Fermi-Dirac Na 6000 K Figure 6: 𝐶𝑐versus 𝑐curves. Figure 6: 𝐶𝑐versus 𝑐curves. Figure 6: 𝐶𝑐versus 𝑐curves. However, comparison of the diversity distributions sug- gests that even when the energy probability density functions appear to coincide, significant physical differences remain between the systems. Figure 7 compares all the diversity curves calculated in the present work. this suggests that there is no limit in which the Bose- Einstein distribution for the photon becomes completely indistinguishable from the Boltzmann distribution. Even more strikingly, the distribution of diversity in a system obey- ing Fermi-Dirac statistics only approaches that of bosonic systems at extremely high temperatures, similar to those at the core of the sun. At lower temperatures, the Fermi gas has substantially higher degrees of diversity than all the other systems. This is because, at lower temperatures, most of the It is clear from Figure 7 that the distributions of diversity for a classical ideal gas and for both Bose-Einstein and Fermi- Dirac distributions are significantly different. 5. Using 𝐶𝑐to Compare and Contrast Systems A speculative explanation could be that Pauli’s exclusion principle does not allow for more than one fermion to occupy the same quantum state, thereby restricting the accumulation of fermions in the same state (i.e., more diversity). p y With our conceptualization of 𝐶𝑐complete, we used it to renormalize and compare three physically significant energy distributions in physics, namely, the Maxwell-Boltzmann, Bose-Einstein, and Fermi-Dirac distributions for energy of subatomic particles. We chose these three distributions for three key reasons: (1) we wanted to see if 𝐶𝑐works for continuous distribution; (2) where the focus was on diversity of types and not on their rank order in terms of complexity; and (3) where the unit order of measure was both objective and widely accepted. Based on our results, we concluded that 𝐶𝑐is a vast improvement over 𝐻as it provides an intuitively useful, scale-free comparison of probability distributions and also allows for a comparison between parts of distributions as well. [13] C. H. Tien and J. H. Lienhard, Statistical Thermodynamics, Hemisphere, 1979. Conflicts of Interest The authors declare that there are no conflicts of interest regarding the publication of this paper. 6. Conclusion As we have hopefully shown in this paper, while Shannon entropy 𝐻has been used to rethink probability distributions in terms of diversity, it suffers from two major limitations. First, it cannot be used to compare distributions that have different levels of scale. Second, it cannot be used to compare parts of distributions to the whole. The renormalization obtained will have a different shape for different distributions. In fact, a bimodal, right skewed, or other kinds of distributions will lead to a different 𝐶𝑐 versus 𝑐curve. There are two interesting points of inquiry in future papers, namely, (a) how the shape of the original distribution influences the 𝐶𝑐versus 𝑐curve and (b) whether we can reconstruct the original shape of the distribution given the 𝐶𝑐versus 𝑐curve. Because of the scale-free nature of 𝐶𝑐, To address these limitations, we introduced a renor- malization of probability distributions based on the notion of case-based entropy 𝐶𝑐(as a function of the cumulative probability 𝑐). We began with an explanation of why we rethink probability distributions in terms of diversity, based 10 Complexity Complexity all distributions can be compared in the same plot without reference to their original scales. In our future work, we will endeavor to connect the shape of the 𝐶𝑐versus 𝑐curve to the shape of the original distribution. This will allow us to locate portions of the original distribution (irrespective of their scale), where diversity is concentrated, and portions where it is sparse, even though the original distributions cannot be plotted on the same graph due to huge variation in their scales. Acknowledgments The authors would like to thank the following colleagues at Kent State University: (1) Dean Susan Stocker, (2) Kevin Acierno and Michael Ball (Computer Services), and (3) the Complexity in Health and Infrastructure Group for their support. They also wish to thank Emma Uprichard and David Byrne and the ESRC Seminar Series on Complexity and Method in the Social Sciences (Centre for Interdisciplinary Methodologies, University of Warwick, UK) for the chance to work through the initial framing of these ideas. References [1] S. E. Page, The Difference: How the Power of Diversity Creates Better Groups, Firms, Schools, and Societies, Princeton Univer- sity Press, 2008. [2] S. E. Page, Diversity and Complexity, Princeton University Press, 2008. [3] M. O. Hill, “Diversity and evenness: a unifying notation and its consequences,” Ecology, vol. 54, no. 2, pp. 427–432, 1973. [4] L. Jost, “Entropy and diversity,” Oikos, vol. 113, no. 2, pp. 363– 375, 2006. [5] R. Rajaram and B. Castellani, “An entropy based measure for comparing distributions of complexity,” Physica A. Statistical Mechanics and Its Applications, vol. 453, pp. 35–43, 2016. [6] B. Castellani and R. Rajaram, “Past the power law: complex systems and the limiting law of restricted diversity,” Complexity, vol. 21, no. 2, pp. 99–112, 2016. [7] M. E. J. Newman, “Power laws, Pareto distributions and Zipf’s law,” Contemporary Physics, vol. 46, no. 5, pp. 323–351, 2005. [8] M. C. Mackey, Time’s Arrow: The Origins of Thermodynamic Behavior, Springer Verlag, Germany, 1992. [9] T. Leinster and C. A. Cobbold, “Measuring diversity: the importance of species similarity,” Ecology, vol. 93, no. 3, pp. 477– 489, 2012. [10] J. Beck and W. Schwanghart, “Comparing measures of species diversity from incomplete inventories: an update,” Methods in Ecology and Evolution, vol. 1, no. 1, pp. 38–44, 2010. [11] R. H. Macarthur, “Patterns of species diversity,” Biological Reviews, vol. 40, pp. 510–533, 1965. [12] R. Peet, “The measurement of species diversity,” Annual Review of Ecological Systems, vol. 5, pp. 285–307, 1974. References Submit your manuscripts at https://www.hindawi.com Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Mathematics Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Mathematical Problems in Engineering Hindawi Publishing Corporation http://www.hindawi.com Diﬀerential Equations International Journal of Volume 2014 Applied Mathematics Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Probability and Statistics Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Mathematical Physics Advances in Complex Analysis Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Optimization Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Combinatorics Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 International Journal of Hindawi Publishing Corporation 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Corporation http://www.hindawi.com Volume 2014 Algebra Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Decision Sciences Advances in Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Operations Research Advances in Probability and Statistics Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Hindawi Publishing Corporation http://www hindawi com Volume 2014 Decision Sciences Advances in Probability and Statistics Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Applied Mathematics Journal of Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Operations Research Advances in The Scientific World Journal Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Hindawi Publishing Corporation http://www.hindawi.com Diﬀerential Equations International Journal of Volume 2014 The Scientific World Journal Hindawi Publishing Corporation h // hi d i V l 2014 Submit your manuscripts at https://www.hindawi.com Submit your manuscripts at https://www.hindawi.com Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Mathematics Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Mathematical Problems in Engineering Complex Analysis Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Optimization Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 Function Spaces Abstract and Applied Analysis Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 International Journal of Mathematics and Mathematical Sciences Hindawi Publishing Corporation http://www.hindawi.com Volume 201 Discrete Dynamics in Nature and Society Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 #HRBQDSDĮ,@SGDL@SHBR Journal of Hindawi Publishing Corporation http://www.hindawi.com Volume 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https://openalex.org/W3104814572	https://www.researchsquare.com/article/rs-50689/v2.pdf?c=1602824417000	English	null	Transcriptome analysis of genes and pathways associated with metabolism in Scylla paramamosain under different light intensities during indoor overwintering	BMC genomics	2,020	cc-by	10,553	Research article Posted Date: October 16th, 2020 Abstract Background: Scylla paramamosain is one of the commercially crucial marine crustaceans belonging to the genus Scylla , which is commonly distributed along the coasts of China, Vietnam, and Japan. Genomic and transcriptomic data are scarce for the mud crab. Light intensity is one of the ecological factors that affect S. paramamosain during indoor overwintering. To understand the energy metabolism mechanism adapted to light intensity, we analyzed the transcriptome of S. paramamosain hepatopancreas in response to different light intensities (0, 1.43, 40.31 μmol·m -2 ·s -1 ). Results: A total of 5052 differentially expressed genes were identified in low light group (LL group, 3104 genes were up- regulated and 1948 genes were down-regulated). A total of 7403 differentially expressed genes were identified in high light group (HL group, 5262 genes were up-regulated and 2141 genes were down- regulated). S. paramamosain adapts to different light intensity environments through the regulation of amino acids, fatty acids, carbon and energy metabolism. Different light intensities had a strong impact on the energy generation of S. paramamosain by influencing oxygen consumption rate, aerobic respiration, glycolysis/gluconeogenesis pathway, the citrate cycle (TCA cycle) and fatty acid degradation. Conclusion: Low light is more conducive to the survival of S. paramamosain , which needs to produce and consume relatively less energy to sustain physiological activities. In contrast, S. paramamosain produced more energy to adapt to the pressure of high light intensities. The findings of the study add to the knowledge of regulatory mechanisms related to S. paramamosain metabolism under different light intensities. DOI: https://doi.org/10.21203/rs.3.rs-50689/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on November 10th, 2020. See the published version at https://doi.org/10.1186/s12864-020-07190-w. Page 1/28 1. Background Therefore, understanding light intensity conditions required during the overwintering period are the focus of this study. S. paramamosain is an important marine crab species in China. This species has fast growth characteristics, large sized individuals, highly adaptable, delicious meat and high nutritional value [16]. S. paramamosain has played an important role in wild fisheries and aquaculture over the past few decades in China [17]. In 2018, the yield of S. paramamosain increased to 157,712 tons, accounting for 53.68% of the total marine crab aquaculture industry in China [18]. Although the culture of S. paramamosain has grown continuously for years, there are still very low yields, only 15–60 kg/667 m2 a year, which is unable to meet ever increasing market demand [19–21]. It is of urgent importance to improve the artificial breeding and breeding technology of S. paramamosain by improving the overwintering environment [22]. Therefore, understanding light intensity conditions required during the overwintering period are the focus of this study. In recent years, with the development of molecular sequencing technology, non-parametric transcriptome sequencing technology has gradually become the main means of studying molecular mechanisms in biological research [23]. Transcriptome sequencing is the study of all mRNAs that a particular cell can transcribe under a certain functional state. For species without reference genomes, splicing small fragments out of Unigene and constructing reference sequences for subsequent analysis is an effective means of studying the molecular mechanisms and regulatory networks of non-parametric species. Analysis by Ding et al. identified key metabolic changes in the liver of the large yellow croaker (Larimichthys crocea) in response to acute hypoxia by transcriptome [24]. Wang et al. revealed the potential influencing mechanism of dietary astaxanthin on growth and metabolism in Litopenaeus vannamei by transcriptome [25]. Research carried out by Zou et al. described the change of aroma volatile metabolism in tomato (Solanum lycopersicum) fruit under different storage temperatures and 1- MCP treatment by transcriptome [26]. While there has been a lot of research on S. paramamosain, no studies have reported the applications of the transcriptome technique for investigating molecular mechanisms associated with metabolism under different light intensities. There has been no research to date on the molecular mechanism of light intensity on S. paramamosain metabolism. In this study, the effects of different light intensities on S. paramamosain were investigated using transcriptome analysis, in particular the metabolic changes of gene expression under different light intensities. 1. Background This study will assist in explaining the potential molecular mechanisms of S. paramamosain under different light intensities. These data may help to provide the necessary theoretical basis and technical support for breeding S. paramamosain. In this study, the effects of different light intensities on S. paramamosain were investigated using transcriptome analysis, in particular the metabolic changes of gene expression under different light intensities. This study will assist in explaining the potential molecular mechanisms of S. paramamosain under different light intensities. These data may help to provide the necessary theoretical basis and technical support for breeding S. paramamosain. 1. Background There are many different stages in the life history of aquatic animals. Overwintering is an important life stage for aquatic animals living in some areas to adapt to cold winter conditions [1]. The process of overwintering determines the quality of parents and is a key factor in seedling development in the coming year [2]. There are many factors affecting the environment of aquatic animals during overwintering, such as salinity, temperature, food availability, and light. In the aquatic environment, absorption and reflection of incident light is easily changed due to the presence of plankton, suspended particulate matter and soluble organic substances [3], which influences the light intensity of water. Therefore, light is one of the most variable water quality factors that directly or indirectly affects behavior [4, 5], survival and growth [6, 7], skin color [8, 9], digestion and immunity [10], fatty acid composition [11], and oxidative stress [12] in aquatic animals. Metabolism is an extremely important activity and has been studied extensively. Nalbach et al. demonstrated that the activity of the neuron-secreting cells in the eye stalk and the thoracic brain of crabs are closely related to light intensity [13]. Increased activity of neurosecretory cells under high light intensity promoted the activity of hormone- promoting hormones and metabolism-related hormones. Most shrimp and larvae react positively to light in the larval stage and a negative reaction to light in the adult stage [14]. Appropriate light conditions can improve the feeding rate of shrimp and crab larvae and promote growth and metamorphosis [15]. This phenomenon is particularly evident in the early developmental stages of shrimp and crab larvae. Page 2/28 Page 2/28 S. paramamosain is an important marine crab species in China. This species has fast growth characteristics, large sized individuals, highly adaptable, delicious meat and high nutritional value [16]. S. paramamosain has played an important role in wild fisheries and aquaculture over the past few decades in China [17]. In 2018, the yield of S. paramamosain increased to 157,712 tons, accounting for 53.68% of the total marine crab aquaculture industry in China [18]. Although the culture of S. paramamosain has grown continuously for years, there are still very low yields, only 15–60 kg/667 m2 a year, which is unable to meet ever increasing market demand [19–21]. It is of urgent importance to improve the artificial breeding and breeding technology of S. paramamosain by improving the overwintering environment [22]. 2.1. Source, breeding and grouping of Scylla paramamosain S. paramamosain individuals were collected from the sea near Xiangshan City (121 57 14 E, 29 28 29 N) and reared in crab apartment systems. The crabs were acclimatized in the carb apartments for 10 days prior to experimentation. And the crabs (290 ± 40 g) were randomly placed into nine groups, which were placed in nine crab apartments. Three groups of crab apartments comprised an experimental group: HL Page 3/28 Page 3/28 group (high light illumination with a photon flux density of about 40.31 μmol·m-2·s-1), LL group (low light illumination with a photon flux density of about 1.43 μmol·m-2·s-1), and the control group (full darkness). The light sources of each experimental group were white LED lights, simulating natural light with full spectrum. Peak light intensity was measured using a spectroradiometer. The light cycle was controlled by a timer (10L: 14D), which turned the lights on at 07:00 every day and turned them off at 17:00. Apart from light intensities, all other daily feeding and management operations were the same for all experimental groups during the trial. The experiment was conducted in December 2018 - March 2019 for a period of four months. After the experiment, the hepatopancreas of three crabs in each group was collected for further analyses. All samples were stored at -80 °C. 2.2. RNA extraction, library construction and sequencing Total RNA was extracted with Trizol Reagent (Invitrogen, Shanghai, China) from 100 mg of hepatopancreas tissue following the manufacturer’s instructions. The sample was detected in 1.5% agarose gel electrophoresis. All RNA samples were of high quality (OD260/280 = 2.10-2.15, OD260/230 ≥ 2.0). After digesting the DNA with DNase, eukaryotic mRNA was enriched using magnetic beads with Oligo (dT). A disruption reagent was added to break the mRNA into short fragments. Using the interrupted mRNA as a template, single-strand cDNA was synthesized using a six-base random primer, and then a two-stranded reaction system was used to synthesize double-stranded cDNA, which was purified using the kit. The purified double-stranded cDNA was subjected to end repair, a tail was added and the sequencing linker was ligated. The fragment size was then selected, and finally PCR amplification was performed. The constructed library was qualified with an Agilent 2100 Bioanalyzer and sequenced using an Illumina HiSeq X Ten sequencer to generate 150 bp double-ended data. 2.3. Gene annotation and classification After removing adaptor sequences, ambiguous N nucleotides (with a ratio of N > 10%) and low-quality sequences (with a quality score < 5), the remaining clean reads were assembled using Trinity software as described for de novo transcriptome assembly without a reference genome, and the longest copies of redundant transcripts were regarded as unigenes. Using diamond software, Unigenes were compared to the NR, KOG (http://www.ncbi.nlm.nih.gov/COG/), GO (http://www.geneontology.org/), Swiss-Prot (http://www.ebi.ac.uk/uniprot/), and KEGG (http://www.genome.jp/kegg/) databases, and PFMER software was used to compare the Pfam database for unigene functional analysis. 2.4. Differential gene identification, enrichment, and pathway analysis Gene expression levels were positively correlated with their abundance. In transcriptome sequencing analysis, Unigene expression levels could be estimated by counting the sequencing reads of Unigenes. Page 4/28 Page 4/28 The reads count was directly proportional to the true expression level of the gene and also positively correlated with the length of the gene and the depth of sequencing. The uniplexed Unigene database was used to identify the abundance of each Unigene by sequence similarity alignment. Bowtie2 software was used to get the number of Unigene reads in each sample, and Express software was applied to calculate the Unigene expression FPKM value. The number of Unigene counts for each sample was normalized using DESeq software to calculate the difference multiple. The negative binomial distribution test was used to test the significance of the difference of read numbers. Finally, the differentially expressed Unigenes were screened based on the difference multiple and the difference significance test results. The threshold for screening differences was p value < 0.05 and a fold change  2. The differential expression of Unigenes was analyzed by GO enrichment and its function was described. The number of differential mRNAs included in each GO term was counted and the significance of the differential Unigene enrichment in each GO entry was calculated using the hypergeometric distribution test. The pathway analysis of the differential Unigenes was performed based on the KEGG database, and the significance of the differential Unigene enrichment in each pathway entry was calculated by the hypergeometric distribution test. 2.5. Quantitative real-time PCR (qRT-PCR) analysis Trizol reagent (Takara Bio, Otsu, Japan) was used to extract the total RNA from the hepatopancreas samples following the manufacturer’s instructions. Electrophoresis using 1.0% agarose gel helped to assess the quality of RNA. After the purification of total RNA, PrimeScript®RT Reagent Kit With gDNA Eraser (Takara Bio Inc., Shiga, Japan) was used to synthesize first-strand cDNA following the manufacturer’s instructions. ddH2O was used to dilute the cDNA obtained to 1:10, which was used as a template for RT-PCR. Table 1 lists the primer information. SYBR®Premix Ex Taq™ (QIGAN) was used for conducting the qRT-PCR ampliﬁcations with a 20-mL reaction mixture on the ABI7500 Real-Time PCR Detection System (Applied Biosystems). All of these experiments were conducted in triplicate. The comparative threshold cycle method (2-△△CT) was used to analyze the expression levels of target genes, taking β-actin as the reference gene. Each experiment involved three biological replicates. All the data underwent tests to meet the requirements for ANOVA, and a one-way ANOVA together with Tukey's HSD test were carried out to analyze the data. All data were represented by the mean ± standard deviation. SPSS statistical software 22.0 was used for statistical analyses. P values less than 0.05 were considered to be statistically significant. 3.1. Differentially expressed genes (DEGs) in the hepatopancreas of S. paramamosain A total of nine samples were used in this analysis. The Q30 of raw data for each sample was distributed from 93.00 to 94.09%, the effective data was distributed from 6.97 to 8.06 G, and the average GC content was 45.90% (Additional file: Table S3). Unigene 54537 pieces were spliced with a total length of Page 5/28 Page 5/28 60890086 bp and an average length of 1116 bp (Additional file: Table S2). As the genome sequencing of S. paramamosain has not yet been elucidated, after reads filtering, Trinity was used to perform de novo assembly with clean reads. Tgicl was used on cluster transcripts to remove abundance and obtain Unigenes. 60890086 bp and an average length of 1116 bp (Additional file: Table S2). As the genome sequencing of S. paramamosain has not yet been elucidated, after reads filtering, Trinity was used to perform de novo assembly with clean reads. Tgicl was used on cluster transcripts to remove abundance and obtain Unigenes. The reads were compared to Unigene and the alignment was 90.63-93.10% (Additional file: Table S3). Stochasticity evaluation of sequencing data showed that the distribution of reads in the various parts of the gene was relatively uniform, indicating that the randomness of the break was good. The RPKM values of the individual genes determined under each 5% increment were compared to the expression levels of the final corresponding genes. The results differed by less than 15%, and therefore, sequencing data met quantitative requirements. The distribution of FPKM mean values for Unigenes ranged from 11.50 to 13.33 (Additional file: Fig. S1). Inter-sample correlation tests confirmed that the study was reliable and the sample selection was reasonable. Principal component analysis showed that each group of samples was distributed in different regions, and the same group of samples was concentrated spatially. Cluster analysis also showed that similar samples grouped together (Fig. 3). Finally, high-quality transcripts were obtained and used as reference sequences. For database annotations of Unigenes, 16049 (29.43%) genes were annotated into the NR library, 11876 (21.78%) genes were annotated into the Swissprot library, 7881 (14.45%) genes were annotated into the KEGG library, 10586 (19.41%) were annotated into the KOG library, 13351 (24.48%) genes were annotated into the eggNOG library, 11204 (20.54%) genes were annotated into the GO library, and 284 (0.52%) genes were annotated into the Pfam library (Additional file: Table S4). 3.1. Differentially expressed genes (DEGs) in the hepatopancreas of S. paramamosain In the HL group, the three most highly down-regulated genes were triosephosphate isomerase B (ID: Q90XG0 0.33-fold), aconitate hydratase (ID: Q99KI0; 0.37-fold), and glyceraldehyde-3-phosphate dehydrogenase (ID: P56649; 0.38-fold) (Table 1). Glycine, serine and threonine metabolism (ko00260) and beta-Alanine metabolism (ko00410) are important pathways in amino acid metabolism. 2-amino-3-ketobutyrate coenzyme A ligase (ID: O75600; LL: 2.13-fold, HL: 2.49- fold) and cystathionine gamma-lyase (ID: Q8VCN5; LL: 4.08-fold, HL: 16.10-fold) were up-regulated in both pathways. Betaine--homocysteine S-methyltransferase 1 (ID: Q5XGM3) was down-regulated in the LL group (0.53-fold) and up-regulated in the HL group (7.34-fold). In the LL group, the three most highly down-regulated genes were cytosolic non-specific dipeptidase (ID: Q96KP4; 0.13-fold), peroxisomal sarcosine oxidase (ID: Q29RU9; 0.16-fold), and aldehyde dehydrogenase (ID: P81178; 0.25-fold). In the HL group, the three most highly down-regulated genes were serine--pyruvate aminotransferase (ID: P41689; 0.19-fold), cytosolic non-specific dipeptidase (ID: Q96KP4; 0.21-fold) and D-3-phosphoglycerate dehydrogenase (ID: A5GFY8; 0.30-fold) (Table 1). Two important pathways in lipid metabolism are fatty acid elongation (ko00062) and fatty acid degradation (ko00071). Lysosomal thioesterase PPT2 (ID: O70489; LL: 3.47-fold, HL: 3.12-fold) and lysosomal thioesterase PPT2-B (ID: Q6GNY7; LL: 1.97-fold, HL: 3.00-fold) were up-regulated in both the LL and HL groups. In the LL group, fatty aldehyde dehydrogenase (ID: P47740) was the most down-regulated gene (0.31-fold), followed by alcohol dehydrogenase class-3 (ID: P79896, 0.38-fold) and hydroxyacyl-coenzyme A dehydrogenase (ID: Q16836; 0.44-fold) (Table 1). In the HL group, the three most down-regulated genes were hydroxyacyl-coenzyme A dehydrogenase (ID: Q16836; 0.35-fold), alcohol dehydrogenase class-3 (ID: P79896; 0.40-fold) and enoyl-CoA delta isomerase 2 (ID: Q9WUR2; 0.41-fold) (Table 1). oxidoreductase chain 3 (ID: P18930; 0.21-fold), NADH-ubiquinone oxidoreductase chain 2 (ID: P34848; 0.24-fold), and V-type proton ATPase subunit F (ID: Q1HQK8; 0.31-fold) (Table 1). In the HL group, the three most highly down-regulated genes were NADH-ubiquinone oxidoreductase chain 2 (ID: P34848; 0.10-fold), cytochrome c oxidase subunit 1 (ID: P00399; 0.17-fold), and NADH-ubiquinone oxidoreductase chain 4 (ID: Q34048; 0.17-fold) (Table 1). Two important pathways in energy metabolism are glycolysis / gluconeogenesis (ko00010) and the citrate cycle (TCA cycle, ko00020). In this process, only pyruvate dehydrogenase E1 component (ID: P49432) was significantly up-regulated. The fold change was 3.26 in the LL group and 2.64 in the HL group. In the LL group, the three most highly down-regulated genes were glyceraldehyde-3-phosphate dehydrogenase (ID: P56649; 0.25-fold), hexokinase type 2 (ID: Q9NFT7; 0.28- fold) and enolase (ID: P56252; 0.34-fold). 3.1. Differentially expressed genes (DEGs) in the hepatopancreas of S. paramamosain In the HL group, the three most highly down-regulated genes were triosephosphate isomerase B (ID: Q90XG0 0.33-fold), aconitate hydratase (ID: Q99KI0; 0.37-fold), and glyceraldehyde-3-phosphate dehydrogenase (ID: P56649; 0.38-fold) (Table 1). Glycine, serine and threonine metabolism (ko00260) and beta-Alanine metabolism (ko00410) are important pathways in amino acid metabolism. 2-amino-3-ketobutyrate coenzyme A ligase (ID: O75600; LL: 2.13-fold, HL: 2.49- fold) and cystathionine gamma-lyase (ID: Q8VCN5; LL: 4.08-fold, HL: 16.10-fold) were up-regulated in both pathways. Betaine--homocysteine S-methyltransferase 1 (ID: Q5XGM3) was down-regulated in the LL group (0.53-fold) and up-regulated in the HL group (7.34-fold). In the LL group, the three most highly down-regulated genes were cytosolic non-specific dipeptidase (ID: Q96KP4; 0.13-fold), peroxisomal sarcosine oxidase (ID: Q29RU9; 0.16-fold), and aldehyde dehydrogenase (ID: P81178; 0.25-fold). In the HL group, the three most highly down-regulated genes were serine--pyruvate aminotransferase (ID: P41689; 0.19-fold), cytosolic non-specific dipeptidase (ID: Q96KP4; 0.21-fold) and D-3-phosphoglycerate dehydrogenase (ID: A5GFY8; 0.30-fold) (Table 1). Two important pathways in lipid metabolism are fatty acid elongation (ko00062) and fatty acid degradation (ko00071). Lysosomal thioesterase PPT2 (ID: O70489; LL: 3.47-fold, HL: 3.12-fold) and lysosomal thioesterase PPT2-B (ID: Q6GNY7; LL: 1.97-fold, HL: 3.00-fold) were up-regulated in both the LL and HL groups. In the LL group, fatty aldehyde dehydrogenase (ID: P47740) was the most down-regulated gene (0.31-fold), followed by alcohol dehydrogenase class-3 (ID: P79896, 0.38-fold) and hydroxyacyl-coenzyme A dehydrogenase (ID: Q16836; 0.44-fold) (Table 1). In the HL group, the three most down-regulated genes were hydroxyacyl-coenzyme A dehydrogenase (ID: Q16836; 0.35-fold), alcohol dehydrogenase class-3 (ID: P79896; 0.40-fold) and enoyl-CoA delta isomerase 2 (ID: Q9WUR2; 0 41 fold) (Table 1) 3.1. Differentially expressed genes (DEGs) in the hepatopancreas of S. paramamosain For functional annotation results, there were 54287 SSRs predicted, including 25,440 Unigenes containing SSRs, 13,056 Unigenes containing more than one SSR and 11,842 composite SSRs (Additional file: Table S5). A total of 29,221 CDS sequences were predicted, of which 16111 were predicted by the database comparison method and 13110 were predicted by ESTScan. A total of 1672 Unigenes were annotated to the transcription factor database and distributed across 63 families. To compare the difference between the experimental group and control group in terms of the change in global proteomes in the hepatopancreas, volcano plots were created of P-value (-log10 P-value) versus the log2fold change for each gene analyzed. Accordingly, relative to the control group, the LL and HL group exhibited a change of a certain proportion of proteins (Fig. 2). A total of 5052 differentially expressed Unigenes were obtained between the LL group and the control, including 3104 up-regulated Unigenes and 1948 down-regulated Unigenes, while 7403 differentially expressed Unigenes were obtained between the HL group and the control, including 5262 up-regulated Unigenes and 2141 down-regulated Unigenes (p value<0.05 & \|log2FC\|>1) (Fig. 1A). Interestingly, 2480 differentially expressed Unigenes were intersected (Fig. 1B). It is well known that carbon metabolism plays a key role in metabolic regulation. An important pathway in carbon metabolism is oxidative phosphorylation (ko00190), in which all significantly different genes are down-regulated. In the LL group, the three most highly down-regulated genes were NADH-ubiquinone Page 6/28 Page 6/28 oxidoreductase chain 3 (ID: P18930; 0.21-fold), NADH-ubiquinone oxidoreductase chain 2 (ID: P34848; 0.24-fold), and V-type proton ATPase subunit F (ID: Q1HQK8; 0.31-fold) (Table 1). In the HL group, the three most highly down-regulated genes were NADH-ubiquinone oxidoreductase chain 2 (ID: P34848; 0.10-fold), cytochrome c oxidase subunit 1 (ID: P00399; 0.17-fold), and NADH-ubiquinone oxidoreductase chain 4 (ID: Q34048; 0.17-fold) (Table 1). Two important pathways in energy metabolism are glycolysis / gluconeogenesis (ko00010) and the citrate cycle (TCA cycle, ko00020). In this process, only pyruvate dehydrogenase E1 component (ID: P49432) was significantly up-regulated. The fold change was 3.26 in the LL group and 2.64 in the HL group. In the LL group, the three most highly down-regulated genes were glyceraldehyde-3-phosphate dehydrogenase (ID: P56649; 0.25-fold), hexokinase type 2 (ID: Q9NFT7; 0.28- fold) and enolase (ID: P56252; 0.34-fold). 3.2. Functional annotation Gene Ontology (GO) terms could be divided into three ontologies: cellular components, molecular function, and biological processes. In the LL group, 49 GO terms were assigned to the up-regulated group and 54 to the down-regulated group (Fig. 4A). In the HL group, 48 GO terms were assigned to the up- regulated group and 52 to the down-regulated group (Fig. 4B). A total of 23 processes were identified in the biological process category, comprising three biological processes (cellular process, metabolic process and biological regulation) as the most strongly affected in the hepatopancreas of S. paramamosain under different light intensities (Fig. 4). In the cellular component category, cells, cell Page 7/28 Page 7/28 Page 7/28 parts, and membranes were most involved (Fig. 4). In the molecular function category, two items comprising binding and catalytic activities were most involved (Fig. 4). 3.3. Metabolic relatedDEG pathway analysis KEGG pathway classification (Fig. 5) was performed according to the KEGG database website. All DEGs were graded into five categories according to their biological function, including cellular processes (LL, 150; HL, 175), environmental information processing (LL, 119; HL, 121), genetic information processing (LL, 198; HL, 309), metabolism (LL, 426; HL, 519) and organismal systems (LL, 227; HL, 247) (Fig. 5). The pathways related to metabolism were subdivided into twelve subsets (level 2): xenobiotic biodegradation and metabolism (LL, 23; HL, 25), nucleotide metabolism (LL, 18; HL, 26), metabolism of terpenoids and polyketides (LL, 5; HL, 7), metabolism of other amino acids (LL, 24; HL, 26), metabolism of cofactors and vitamins (LL, 37; HL, 34), lipid metabolism (LL, 63; HL, 66), glycan biosynthesis and metabolism (LL, 27; HL, 36), global and overview maps (LL, 42; HL, 57), energy metabolism (LL, 55; HL, 99), carbohydrate metabolism (LL, 72; HL, 75), biosynthesis of secondary metabolism (LL, 4; HL, 3) and amino acid metabolism (LL, 56; HL, 65) (Fig. 5A&B and Additional file 1: Table S6). Functional enrichment was also performed on DEGs according to the above KEGG pathway classification. The top 20 pathways showed that there were 15 and 12 pathways directly related to metabolism in the LL and HL groups, respectively. The most common metabolic pathways were biosynthesis of amino acids (ko01230), degradation of aromatic compounds (ko01220), carbon metabolism (ko01200), methane metabolism (ko00680), oxidative phosphorylation (ko00190) and glycolysis/Gluconeogenesis (ko00010) (Fig. 5C&D). The pathways related to metabolism in the LL group were pentose and glucuronate interconversions (ko00040), drug metabolism-cytochrome P450 (ko00982), starch and sucrose metabolism (ko00500), metabolism of xenobiotics by cytochrome P450 (ko00980), porphyrin and chlorophyll metabolism (ko00860), retinol metabolism (ko00830), glycine, serine and threonine metabolism (ko00260), steroid hormone biosynthesis (ko00140) and ascorbate and aldarate metabolism (ko00053) (Fig. 5C). The pathways related to metabolism in the HL group were caprolactam degradation (ko00930), carbon fixation in photosynthetic organisms (ko00710), aminobenzoate degradation (ko00627), geraniol degradation (ko00281), fatty acid elongation (ko00062) and the TCA cycle (ko00020) (Fig. 5D). Seven important metabolic pathways were selected for further analysis: Glycine, serine and threonine metabolism (ko00260), beta-Alanine metabolism (ko00410), fatty acid elongation (ko00062), fatty acid degradation (ko00071), oxidative phosphorylation (ko00190), glycolysis / gluconeogenesis (ko00010) and the TCA cycle (ko00020) (Table 1). We focused on these pathways and the differential genes they contain. 3.4. 3.3. Metabolic relatedDEG pathway analysis Validity of DEGs in transcriptomic data Page 8/28 To validate the transcriptome results, qRT-PCR was used to check the transcript levels in six identiﬁed DEGs (PPT2, ODPB, KBL, ATP5H, G3P, and SPYA). As indicated by qRT-PCR, in the six genes selected in a random manner, the majority of the genes exhibited a similar transcription level expression to the transcriptome results in different light intensities, with the exception of G3P in the HL group and SPYA in the LL group (Fig. 6). The qRT-PCR results showed a high level of consistency with the transcriptome data obtained from transcriptome, indicating that the transcriptome results were reliable. 4. Discussion Light is an important and variable factor in both natural and farmed ponds [27]. S. paramamosain living in water is easily impacted by light intensity and the impacts of different light intensities on aquatic crustaceans have received some attention in recent years. One of the main functions of the hepatopancreas is metabolism [28, 29]. This study focuses on using transcriptome technology to investigate metabolism gene expression changes in the hepatopancreas of S. paramamosain in different light intensities. Four functional categories were selected for evaluating the dataset, mainly referring to carbohydrate metabolism, energy metabolism, amino acid metabolism, as well as lipid metabolism. The following sections will focus on discussing the biological relevance exhibited by the above-mentioned seven metabolic pathways. 4.1. Effect of light intensity on energy metabolism of S. paramamosain Among the energy metabolism pathways that were enriched, oxidative phosphorylation (ko00190) was the most affected. Five types of genes (NADH dehydrogenase, NADH-ubiquinone oxidoreductase, ATP synthase, cytochrome c oxidase, inorganic pyrophosphatase) were significantly down-regulated, and the fold change of the HL group was significantly higher than that of the LL group. Mitochondria are the driving force for all living things to carry out life activities, and are the main sites for intracellular oxidative phosphorylation and formation of ATP which drives cellular functions [30]. O id i h l i i li i b h l d b h b d ATP d Mitochondria are the driving force for all living things to carry out life activities, and are the main sites for intracellular oxidative phosphorylation and formation of ATP which drives cellular functions [30]. Oxidative paosphorylation is a coupling reaction between the energy released by the body, ATP, and inorganic phosphate synthesis through the respiratory chain [31]. NADH-ubiquinone oxidoreductase and cytochrome c oxidase (COX) participate in the transfer of electrons to oxygen, and thus, are considered the key components of the respiratory chain [32]. Previous studies have shown that light affects oxygen consumption in the respiratory chain of crustaceans. Crayfish (Procambarus clarkia and Procambarus digueti) have a reduced oxygen consumption rate during long light periods [33]. The oxygen consumption rate of Chinese prawn and Orconectes nais was not affected by the light cycle [34]. The larvae of Macrobrachium rosenbergii have a high oxygen consumption rate in the dark, while lobster larvae and Chinese prawn exhibit high oxygen consumption under light conditions [35]. The oxygen consumption rate of P. vannamei was not affected by light intensity [36]. The average oxygen consumption rate of Penaeus chinensis in different shades of light was different under dark and light conditions. In this study, the change of NADH-ubiquinone oxidoreductase and COX genes showed that different light intensities affected the respiratory process of S. paramamosain. Significant down-regulation of NADH-ubiquinone Page 9/28 Page 9/28 oxidoreductase and COX genes indicated the reduced transfer of electrons to oxygen and a reduced oxygen consumption rate under light conditions. In addition, the oxygen consumption rate under high light intensity was lower than that under low light intensity The reaction catalyzed by the inorganic pyrophosphatase produces inorganic pyrophosphate, which relies on several important nucleotide triphosphates and is necessary for the synthesis of RNA, DNA, proteins, and lipids. 4.1. Effect of light intensity on energy metabolism of S. paramamosain While providing Pi for biomolecules, the inorganic pyrophosphatase can synthesize ATP, a terminal product of cellular energy metabolism [37]. NADH dehydrogenase which is produced by reduced coenzyme NADH is involved in many biochemical metabolic processes, such as glycolysis, the Krebs cycle, and beta oxidation [38]. This coenzyme contains electrons with a high electrode potential, which release a lot of energy when oxidized [39]. The results of the current study showed that inorganic pyrophosphatase genes in both experimental groups were significantly down-regulated, indicating that the amount of ATP synthesis was significantly reduced under light conditions. This conclusion can also be illustrated from the negative regulation of NADH dehydrogenase complex and ATP synthase subunit enrichment under light conditions in the current study. 4.2. Effect of light intensity on carbohydrate metabolism of S. paramamosain The hepatopancreas is the main organ of carbohydrate metabolism in crustaceans, and facilitates the maintenance of blood glucose levels through four metabolic compounds and the metabolic pathways they participate in; glycosylation, glycogenolysis, glycolysis and gluconeogenesis [40, 41]. According to enrichment analysis of the KEGG pathway, two important pathways related to carbohydrate metabolism were found: glycolysis/gluconeogenesis (ko00010) and the TCA cycle (ko00020). Many of the genes in these pathways changed significantly, indicating that carbohydrate metabolism is of great significance for S. paramamosain to adapt to different light conditions. Because the hepatopancreas stores a limited amount of glucose or glycogen, once this has been used up the body needs to synthesize glucose through gluconeogenesis to maintain normal energy requirements. During the process of gluconeogenesis, glucone-6-phosphatase catalyzes the conversion of non-sugar substances (such as lactic acid, pyruvate, propionic acid, glycerol, etc.) into glucose or glycogen [42, 43]. Triosephosphate isomerase, an important enzyme of the glycolysis/gluconeogenesis pathway, interconverts dihydroxyacetone phosphate and glyceraldehyde-3-phosphate dehydrogenase (G3P) to prevent the accumulation of cell-toxic dihydroxyacetone phosphate [44]. Under light conditions, the glycolysis /gluconeogenesis-related enzymes, such as triosephosphate isomerase, G3P, L-lactate dehydrogenase, acetyl-coenzyme A (acetyl-coA) and glucone-6-phosphatase were significantly down- regulated, indicating that the synthesis pathway of glycolysis /gluconeogenesis was inhibited to some extent. It can be seen from the oxidative phosphorylation pathway of energy metabolism that aerobic respiration is more vigorous under light conditions. This process produces carbon dioxide and water, releases energy and synthesizes large amounts of ATP by completely oxidizing and breaking down organic matter (usually decomposing glucose). This may be the main reason for inhibiting the glycolysis/gluconeogenesis pathway. In this study, pyruvate kinase gene expression was up-regulated by Because the hepatopancreas stores a limited amount of glucose or glycogen, once this has been used up the body needs to synthesize glucose through gluconeogenesis to maintain normal energy requirements. During the process of gluconeogenesis, glucone-6-phosphatase catalyzes the conversion of non-sugar substances (such as lactic acid, pyruvate, propionic acid, glycerol, etc.) into glucose or glycogen [42, 43]. Triosephosphate isomerase, an important enzyme of the glycolysis/gluconeogenesis pathway, Page 10/28 Page 10/28 9.7-fold in the HL group, which promoted the production of pyruvate which can be converted into acetyl- coA and enter the fatty acid biosynthesis pathway [45]. 9.7-fold in the HL group, which promoted the production of pyruvate which can be converted into acetyl- coA and enter the fatty acid biosynthesis pathway [45]. 4.2. Effect of light intensity on carbohydrate metabolism of S. paramamosain The tricarboxylic acid (TCA) cycle is the third stage of catabolism, in which acetyl-coA is completely oxidized and decomposed into carbon dioxide [46]. The related genes, except for the pyruvate dehydrogenase (PDH) gene, were significantly down-regulated. PDH is one of the key enzymes in the central metabolic system. When pyruvate enters the TCA cycle, it first forms acetyl-coA under the action of PDH in the mitochondrial inner membrane, and then enters the TCA cycle [47]. The PDH gene was significantly up-regulated by 2.6-fold in the HL group and 3.2-fold in the LL group. However, there was no significant difference in acetyl-coA gene between the HL and LL groups, indicating that not enough pyruvate entered the TCA cycle to produce acetyl-coA. From the experimental results, the main genes that were down-regulated in this cycle were catalytic enzymes genes, such as malate dehydrogenase, isocitrate dehydrogenase, ATP-citrate synthase, aconitate hydratase, and fumarate hydratase. This indicates that light conditions limit the TCA cycle of the organism and the specific mechanism behind this requires further study. 4.3. Effect of light intensity on amino acid metabolism in S. paramamosain In terms of amino acid metabolism, two genes were significantly up-regulated in the two groups: 2-amino- 3-ketobutyrate coenzyme A ligase and cystathionine gamma-lyase. Betaine--homocysteine S- methyltransferase 1 and sarcosine dehydrogenase were significantly up-regulated in the HL group, but significantly down-regulated in the LL group. From the results of this study, it can be seen that the regulation patterns of amino acids under different light intensities have similarities and differences. In terms of amino acid metabolism, two genes were significantly up-regulated in the two groups: 2-amino- 3-ketobutyrate coenzyme A ligase and cystathionine gamma-lyase. Betaine--homocysteine S- methyltransferase 1 and sarcosine dehydrogenase were significantly up-regulated in the HL group, but significantly down-regulated in the LL group. From the results of this study, it can be seen that the regulation patterns of amino acids under different light intensities have similarities and differences. Cystathionine is a metabolic intermediate of sulfur-containing amino acids, which is involved in the conversion between cysteine and methionine. Cystathionine gamma-lyase acts on cystathionine so that cystathionine eliminates pyruvate and NH3 and produces homocysteine [48]. The fold change of GCL in the HL group (16.10-fold change) was significantly higher than that in the LL group (4.08-fold change), indicating that the HL group produced more homocysteine. Homocysteine has been widely studied in human diseases, but it has not been studied in crustaceans. Sarcosine usually exists in the form of creatine phosphate, and organisms mainly rely on ATP to provide energy under stress [49]. However, ATP reserves in the body are very small, so continuous synthesis is needed, and creatine phosphate can promote the synthesis of ATP. Creatine dehydrogenase is an enzyme that catalyzes chemical reactions, and its elevation significantly reduces the level of creatine. The sarcosine content in high light conditions was significantly higher than that in low-light conditions, which may be related to the fact that S. paramamosain requires more energy under high light conditions. Some studies have shown that some glycosylated amino acids (such as alanine, glycine and serine) are decomposed in the hepatopancreas, and the carbon skeleton can be converted into metabolites such as pyruvate, ketoglutaric acid, succinyl coA, fumaric acid and oxaloacetic acid through catabolism under stress conditions, thus, affecting amino acid metabolism [24]. However, under opposite environmental conditions, these metabolites are restricted from entering the TCA cycle to produce ATP for energy or Page 11/28 Page 11/28 glucose through the gluconeogenic pathway. 4.4. Effect of light intensity on lipid metabolism in S. paramamosain Lipids are the most important substrates for maintaining tissue function and are also important energy stores, providing energy for organisms [50]. In this study, KEGG analysis screened two key metabolic pathways related to the synthesis of fatty acids including fatty acid elongation and fatty acid degradation. A total of 14 DEGs were found to be involved in these two metabolic pathways, indicating that light intensity significantly affects lipids metabolism. Elongation of fatty acids is an important biological process in the biosynthesis of polyunsaturated fatty acids [51, 52]. Polyunsaturated fatty acids play an important role in maintaining the relative fluidity of cell membranes and ensuring the normal physiological function of cells. In this study, four genes were found to be significantly correlated with the content of polyunsaturated fatty acids: lysosomal thioesterase PPT2, lysosomal thioesterase PPT2-B, 3-ketoacyl-CoA thiolase and hydroxyacyl-coA. The lysosomal thiesterase gene plays an important role in fatty acid elongation, but the specific mechanism has not been reported and needs further study. Thiesterase plays an important catalytic role in the synthesis and degradation of fatty acids. Thiesterases can be divided into two groups according to their different functions, in which 3-ketoacyl-CoA thiolase plays an important role in fatty acid decomposition [53]. The significant down-regulation of the 3-ketoacyl-CoA thiolase gene indicates that the solution rate of fatty acids decreases and more fatty acids might be accumulated under light conditions. In addition, different genes in fatty acid degradation pathways differed between high-light and low-light conditions, but were significantly down-regulated in both. This result indicates that fatty acid degradation was somewhat inhibited in the presence of light, but the specific mechanisms of these actions requires further study. 4.3. Effect of light intensity on amino acid metabolism in S. paramamosain This conclusion was confirmed by a significant decrease of the gluconeogenic pathway. The combination of amino acid catabolism and gluconeogenic pathways may be a mechanism for adaptation to light intensity. However, the specific role of some amino acids needs further study to fully understand amino acid dynamics in different light intensities. Funding This work was supported by the Major Agricultural Technology Collaborative Extension Program of Zhejiang province (2020XTTGSC03), Basic Public Welfare Research Program of Zhejiang Province (LY20D060001), Major Sci & Tech Special Project of Zhejiang Province (2016C02055-8), Ministry of Agriculture of China & China Agriculture Research System (CARS-48), the K. C. Wong Magna Fund in Ningbo University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Ethics approval The animal subjects used in the present study are crabs, which are invertebrates and are exempt from this requirement. Availability of data and materials Raw Illumina sequences were deposited in the National Center for Biotechnology Information (NCBI) and our SRA records will be accessible with the following link after the indicated release date: https://www.ncbi.nlm.nih.gov/sra/PRJNA640880, SRA accession: PRJNA640880; Temporary Submission ID: SUB7645760. 5. Conclusions In this study, we used RNA-sequencing to construct gene expression profiles related to metabolism mechanisms in S. paramamosain. To our knowledge, this is the first transcriptome study of these mechanisms in S. paramamosain. The results of this study revealed that glycine, serine and threonine metabolism, beta-Alanine metabolism, fatty acid elongation, fatty acid degradation, oxidative phosphorylation, glycolysis/gluconeogenesis, and the TCA cycle are involved in metabolism mechanism in S. paramamosain. In conclusion, energy metabolism, amino acid metabolism, carbohydrate metabolism, and lipid metabolism are important means of regulation to cope with different light intensities. These results contribute to the existing knowledge of energy metabolism mechanisms in aquatic animals when adapting to different light intensities. These data may help to understand the molecular mechanisms of metabolism under different light intensities in S. paramamosain from a new perspective. Page 12/28 Page 12/28 Authors’ contributions C Wang and H Wang conceived and designed the study. N Li performed the cultivation of experimental animals. All other experiments were performed and analyzed by N Li. N Li, J Zhou, H Wang, C Shi, L Liu, C Mu and C Wang were also involved in the experimental work. N Li wrote the manuscript with support from all authors. All authors read and approved the final manuscript. References 1. Fasola M, Biddau CL. 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Tables Page 17/28 The metabolic related differentially expressed genes (DEGs) identified in hepatopancreas of Scylla paramamosain under different light intensity by transcriptome. Tables Swissprot annotation Swissprot ID LL/Control HL/Control FoldChange P val FoldChange P val Oxidative phosphorylation (ko00190) NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 11 Q9D8B4 0.4244 0.0069 0.3038 4.64E- 05 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 12 O97725 0.5027 0.0320 0.4272 0.0143 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 7 Q9Z1P6 0.4448 0.0117 0.4501 0.0075 NADH dehydrogenase [ubiquinone] 1 subunit C2 Q02827 0.5304 0.0372 0.3998 0.0007 NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 4 P0CB71 0.4975 0.0300 0.4757 0.0405 NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 7 Q02368 0.4824 0.0272 0.4284 0.0164 NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 3 Q02365 0.4691 0.0283 0.3656 0.0098 NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8 Q02372 0.4436 0.0078 0.3871 0.0028 NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 2 Q0MQC7 0.4078 0.0034 0.3620 0.0001 Page 18/28 Swissprot annotation Swissprot ID LL/Control HL/Control FoldChange P val FoldChange P val NADH dehydrogenase [ubiquinone] iron-sulfur protein 4, mitochondrial Q9CXZ1 0.4943 0.0210 0.3934 0.0004 NADH dehydrogenase [ubiquinone] iron-sulfur protein 7, mitochondrial O75251 0.4492 0.0101 0.3609 0.0006 NADH-ubiquinone oxidoreductase chain 4L P18934 0.3750 0.0089 0.3079 0.0023 NADH-ubiquinone oxidoreductase chain 6 P18933 0.3685 0.0005 0.2222 3.38E- 09 NADH-ubiquinone oxidoreductase chain 4 Q34048 0.3959 0.0099 0.1789 0.0487 NADH-ubiquinone oxidoreductase chain 2 P34848 0.2425 0.0004 0.1031 0.0055 NADH-ubiquinone oxidoreductase chain 3 P18930 0.2116 4.61E- 05 0.2649 0.0001 ATP synthase subunit beta Q5ZLC5 0.5238 0.0250 0.4125 0.0004 ATP synthase subunit g Q5RFH0 0.3822 0.0011 0.3295 0.0001 ATP synthase subunit gamma O01666 0.5652 0.0488 0.4692 0.0028 ATP synthase subunit d Q24251 0.4474 0.0082 0.3143 0.0004 ATP synthase subunit b Q94516 0.5377 0.0353 0.4163 0.0007 ATP synthase subunit delta P35434 0.4663 0.0110 0.3982 0.0040 ATP synthase-coupling factor 6 Q24407 0.5513 0.0446 0.4851 0.0045 ATP synthase lipid-binding protein Q9U505 0.5480 0.0355 0.1381 0.0260 Cytochrome c oxidase subunit 6B1 P14854 0.4849 0.0155 0.4162 0.0036 Cytochrome c oxidase subunit 1 P00399 0.4428 0.0028 0.1772 1.59E- Page 19/28 Page 19/28 Swissprot annotation Swissprot ID LL/Control HL/Control FoldChange P val FoldChange P val Cytochrome c oxidase subunit 3 P00417 0.3186 0.0002 0.2095 0.0002 V-type proton ATPase subunit F Q1HQK8 0.3166 9.16E- 05 0.3091 0.0001 V-type proton ATPase subunit e 2 Q8NHE4 0.4510 0.0203 0.2667 0.0004 Inorganic pyrophosphatase O77460 0.3413 0.0011 0.3730 0.0006 Glycolysis / Gluconeogenesis (ko00010) Aldose 1-epimerase Q9GKX6 0.4300 0.0136 0.5034 0.0424 Phosphoglycerate kinase 1 Q5NVB5 0.4353 0.0035 0.4346 0.0010 Enolase P56252 0.3451 0.0003 0.4208 0.0014 Glyceraldehyde-3-phosphate dehydrogenase P56649 0.2565 0.0006 0.3811 0.0061 Glucose-6-phosphatase O42153 0.4234 0.0031 0.4551 0.0040 Triosephosphate isomerase B Q90XG0 0.3375 0.0002 0.3313 1.10E- 05 Acetyl-coenzyme A synthetase Q9NR19 - - 0.5993 0.0439 L-lactate dehydrogenase Q95028 - - 0.5656 0.0288 Pyruvate dehydrogenase protein X component Q8BKZ9 - - 0.4991 0.0309 Hexokinase type 2 Q9NFT7 0.2888 0.0282 - - Citrate cycle (TCA cycle, ko00020) Pyruvate dehydrogenase E1 component subunit beta P49432 3.2622 0.0022 2.6429 0.0005 Malate dehydrogenase Q5NVR2 0.5229 0.0204 0.4403 0.0009 Succinate–CoA ligase [ADP/GDP-forming] subunit alpha, mitochondrial P13086 0.5567 0.0452 0.4452 0.0016 Malate dehydrogenase Q3T145 0.3996 0.0010 0.4326 0.0006 Swissprot annotation Swissprot annotation Swissprot ID LL/Control HL/Control FoldChange P val FoldChange P val Cytoplasmic aconitate hydratase Q0VCU1 0.4902 0.0109 0.5738 0.0262 Isocitrate dehydrogenase [NADP] Q04467 0.4560 0.0079 0.5210 0.0216 Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex Q9D2G2 - - 0.4379 0.0020 Succinate dehydrogenase cytochrome b560 subunit P70097 - - 0.4841 0.0074 Aconitate hydratase Q99KI0 - - 0.3799 0.0001 Fumarate hydratase Q60HF9 - - 0.5111 0.0082 ATP-citrate synthase Q91V92 0.4900 0.0239 - - Pyruvate carboxylase Q29RK2 0.4938 0.0171 - - Glycine, serine and threonine metabolism (ko00260) Serine dehydratase-like Q96GA7 0.4796 0.0457 0.3862 0.0043 Glycine cleavage system H protein P23434 0.3558 0.0003 0.3919 0.0002 Phosphoglycerate mutase 2 Q32KV0 0.3546 0.0055 0.4024 0.0159 Serine–pyruvate aminotransferase P41689 0.4186 0.0181 0.1968 2.98E- 05 D-3-phosphoglycerate dehydrogenase A5GFY8 0.3899 0.0042 0.3054 2.12E- 05 2-amino-3-ketobutyrate coenzyme A ligase O75600 2.1360 0.0470 2.4998 0.0070 Cystathionine gamma-lyase Q8VCN5 4.0899 0.0094 16.1023 7.93E- 05 Peroxisomal sarcosine oxidase Q29RU9 0.1632 2.73E- 09 0.4559 0.0016 Betaine–homocysteine S- methyltransferase 1 Q5XGM3 0.5321 0.0447 7.3483 0.0184 Swissprot annotation Page 21/28 Page 21/28 Swissprot annotation Swissprot ID LL/Control HL/Control FoldChange P val FoldChange P val Aldehyde dehydrogenase P81178 0.2589 3.45E- 06 0.4912 0.0037 Cytosolic non-specific dipeptidase Q96KP4 0.1312 0.0006 0.2145 0.0087 Spermidine synthase Q64674 0.5022 0.0310 0.5562 0.0409 Enoyl-CoA hydratase P14604 - - 0.4812 0.0056 Alpha-aminoadipic semialdehyde dehydrogenase Q2KJC9 0.4374 0.0041 - - Fatty acid elongation (ko00062) Lysosomal thioesterase PPT2 O70489 3.4756 0.0236 3.1206 0.0462 Lysosomal thioesterase PPT2-B Q6GNY7 1.9706 0.0252 3.0013 1.27E- 05 3-ketoacyl-CoA thiolase Q3T0R7 0.4633 0.0070 0.4285 0.0006 Hydroxyacyl-coenzyme A dehydrogenase Q16836 0.4400 0.0349 0.3574 0.0132 Fatty acid degradation (ko00071) Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial P45954 - - 0.5031 0.0261 Aldehyde dehydrogenase family 9 member A1-A Q7ZVB2 - - 0.4922 0.0255 Enoyl-CoA delta isomerase 2 Q9WUR2 - - 0.4154 0.0024 Glutaryl-CoA dehydrogenase Q2KHZ9 - - 0.5238 0.0112 Long-chain specific acyl-CoA dehydrogenase P51174 - - 0.5771 0.0348 Trifunctional enzyme subunit beta Q99JY0 - - 0.4686 0.0035 Alcohol dehydrogenase class-3 P79896 0.3836 0.0010 0.4089 0.0005 Fatty aldehyde dehydrogenase P47740 0.3161 0.0193 - - Swissprot annotation Figures Figure 1 Distribution of diﬀerentially expressed genes (DEGs) with different light intensity eﬀects in mud crab hepatopancreas. Tables (A) The number of DEGs (fold changes > 2 and Q-values < 0.05) under different light intensities. (B) Venn diagram of the up-regulated and down-regulated genes in the HL and LL groups. Figure 1 Distribution of diﬀerentially expressed genes (DEGs) with different light intensity eﬀects in mud crab hepatopancreas. (A) The number of DEGs (fold changes > 2 and Q-values < 0.05) under different light intensities. (B) Venn diagram of the up-regulated and down-regulated genes in the HL and LL groups. Distribution of diﬀerentially expressed genes (DEGs) with different light intensity eﬀects in mud crab hepatopancreas. (A) The number of DEGs (fold changes > 2 and Q-values < 0.05) under different light intensities. (B) Venn diagram of the up-regulated and down-regulated genes in the HL and LL groups. Page 23/28 Figure 2 Volcano plots showing DEPs in mud crab hepatopancreas in the LL group vs the control (A) and the HL group vs the control (B). Dots highlighted in red (FC > 1.2) and green (FC < 0.83) indicate genes significantly altered in abundance (P ≤ 0.05). Figure 2 Volcano plots showing DEPs in mud crab hepatopancreas in the LL group vs the control (A) and the HL group vs the control (B). Dots highlighted in red (FC > 1.2) and green (FC < 0.83) indicate genes significantly altered in abundance (P ≤ 0.05). Page 24/28 Page 24/28 Figure 3 Hierarchical cluster analysis of DEGs in mud crab hepatopancreas in the LL group vs the control (A) and the HL group vs the control (B). Diﬀerent colors represent different relative abundance of genes, where red represents higher intensity and green represents lower intensity. Figure 3 Figure 3 Hierarchical cluster analysis of DEGs in mud crab hepatopancreas in the LL group vs the control (A) and the HL group vs the control (B). Diﬀerent colors represent different relative abundance of genes, where red represents higher intensity and green represents lower intensity. Hierarchical cluster analysis of DEGs in mud crab hepatopancreas in the LL group vs the control (A) and the HL group vs the control (B). Diﬀerent colors represent different relative abundance of genes, where red represents higher intensity and green represents lower intensity. Page 25/28 4 ntology (GO) classiﬁcation showing DEGs in mud crab hepatopancreas in the LL group vs the (A) and the HL group vs the control (B). Figure 4 Gene Ontology (GO) classiﬁcation showing DEGs in mud crab hepatopancreas in the LL group vs Figure 6 Quantitative real-time PCR (qRT-PCR) analysis of six differentially expressed genes in mud crab hepatopancreas under different light intensities using the 2 −ΔΔCt method. Data are presented as means (± SD). Means denoted by * showed significant differences (P < 0.05). Definition of abbreviations: PPT2, lysosomal thioesterase PPT2; ODPB, pyruvate dehydrogenase E1 component subunit beta; KBL, 2-amino- 3-ketobutyrate coenzyme A ligase; ATP5H, ATP synthase subunit d; G3P, Glyceraldehyde-3-phosphate dehydrogenase; SPYA, Serine--pyruvate aminotransferase. Figure 4 Gene Ontology (GO) classiﬁcation showing DEGs in mud crab hepatopancreas in the LL group vs the control (A) and the HL group vs the control (B). Page 26/28 Page 26/28 Figure 5 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of DEPs in mud crab hepatopancreas hepatopancreas in the LL group vs the control (A&C) and the HL group vs the control (B&D). Figure 5 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of DEPs in mud crab hepatopancreas hepatopancreas in the LL group vs the control (A&C) and the HL group vs the control (B&D). Page 27/28 Page 27/28 Page 27/28 Figure 6 Quantitative real-time PCR (qRT-PCR) analysis of six differentially expressed genes in mud crab hepatopancreas under different light intensities using the 2 −ΔΔCt method. Data are presented as means (± SD). Means denoted by * showed significant differences (P < 0.05). Definition of abbreviations: PPT2, lysosomal thioesterase PPT2; ODPB, pyruvate dehydrogenase E1 component subunit beta; KBL, 2-amino- 3-ketobutyrate coenzyme A ligase; ATP5H, ATP synthase subunit d; G3P, Glyceraldehyde-3-phosphate dehydrogenase; SPYA, Serine--pyruvate aminotransferase. Supplementary Files Additionalfile.docx Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Page 28/28
https://openalex.org/W4362462207	https://figshare.com/articles/journal_contribution/Supplementary_Figure_3_from_Neoadjuvant_Vaccination_Provides_Superior_Protection_against_Tumor_Relapse_following_Surgery_Compared_with_Adjuvant_Vaccination/22381227/1/files/39826626.pdf	English	null	Supplementary Figure 2 from Neoadjuvant Vaccination Provides Superior Protection against Tumor Relapse following Surgery Compared with Adjuvant Vaccination	null	2,023	cc-by	144	Supplementary Figure 3. Quantification of vitiligo in vaccinated animals post surgery. WT mice were inoculated with 105 B16F10 cells i.d. on day -14 and vaccinated with 108 pfu of AdhDCT or AdLCMV on different days before or after the surgery. Appearance of vitiligo was recorded 3 weeks post surgery and representative picture for each group of mice is shown. Incidence of vitiligo is indicated in the upper right corner on each photograph. Supplementary Figure 3. Quantification of vitiligo in vaccinated animals post surgery. WT mice were inoculated with 105 B16F10 cells i.d. on day -14 and vaccinated with 108 pfu of AdhDCT or AdLCMV on different days before or after the surgery. Appearance of vitiligo was recorded 3 weeks post surgery and representative picture for each group of mice is shown. Incidence of vitiligo is indicated in the upper right corner on each photograph.
https://openalex.org/W3008983702	https://europepmc.org/articles/pmc7076446?pdf=render	English	null	First Phylogeny of Bitterbush Family, Picramniaceae (Picramniales)	Plants	2,020	cc-by	6,346	Received: 17 December 2019; Accepted: 19 February 2020; Published: 21 February 2020 Received: 17 December 2019; Accepted: 19 February 2020; Published: 21 February 2020 Abstract: Picramniaceae is the only member of Picramniales which is sister to the clade (Sapindales (Huerteales (Malvales, Brassicales))) in the rosidsmalvids. Not much is known about most aspects of their ecology, geography, and morphology. The family is restricted to American tropics. Picramniaceae representatives are rich in secondary metabolites; some species are known to be important for pharmaceutical purposes. Traditionally, Picramniaceae was classiﬁed as a subfamily of Simaroubaceae, but from 1995 on, it has been segregated containing two genera, Picramnia and Alvaradoa, with the recent addition of a third genus, Nothotalisia, described in 2011. Only a few species of the family have been the subject of DNA-related research, and fewer than half of the species have been included in morphological phylogenetic analyses. It is clear that Picramniaceae remains a largely under-researched plant group. Here we present the ﬁrst molecular phylogenetic tree of the group, based on both chloroplast and nuclear markers, widely adopted in the plant DNA barcoding. The main ﬁndings are: The family and its genera are monophyletic and Picramnia is sister to two other genera; some clades corroborate previous assumptions of relationships made on a morphological or geographical basis, while most parts of the molecular topology suggest high levels of homoplasy in the morphological evolution of Picramnia. Keywords: Alvaradoa; Nothotalisia; Picramnia; Sapindales; rosids; rbcL; trnL-F; IT Keywords: Alvaradoa; Nothotalisia; Picramnia; Sapindales; rosids; rbcL; trnL-F; ITS plants plants First Phylogeny of Bitterbush Family, Picramniaceae (Picramniales) Alexey Shipunov 1,, Shyla Carr 1, Spencer Furniss 1, Kyle Pay 1 and José Rubens Pirani 2 1 Minot State University, Minot, ND 58707, USA; shyla.simmons@ndus.edu (S.C.); spencer.furniss@minotstateu.edu (S.F.); kyle.pay@minotstateu.edu (K.P.) 2 University of São Paulo, São Paulo 01000-000, Brazil; pirani@usp.br Correspondence: dactylorhiza@gmail.com Plants 2020, 9, 284; doi:10.3390/plants9020284 plants plants 1. Introduction Most ﬂowering plants families described by the end of the 18th century have proven to be robust, stable groups. However, molecular tools have also been used to ﬁnd less stable assemblages. One example of this is Simaroubaceae, a family in the Sapindales which was long suspected of being “non-natural” [1], and ﬁnally, in 1995 the two genera then forming subfamilies Picramnioideae and Alvaradoideae, Picramnia Sw. and Alvaradoa Liebm., were separated as Picramniaceae family [2]. Picramniaceae Fernando and Quinn is a rosid family ofca. 50 known species. They are dioecious, neotropical plants, with alternate, pinnately compound leaves, minute ﬂowers with stamens opposite the petals, and a syncarpous gynoeceum with an inconspicuous style [3]. It is the only member of Picramniales which is robustly supported [4] as a sister group to the clade (Sapindales (Huerteales (Malvales, Brassicales))) [5]. Both family and order are restricted to American tropics, with centers of diversity in Mexico, the Amazonian region, and Southern Brazil (Figure 1). Whereas not much is known about many aspects of their life, Picramniaceae representatives are rich in secondary metabolites, and some species are known to be important for pharmaceutical purposes (as anti-malaria and anti-cancer drugs), even though their chemistry is not thoroughly studied [6]. Plants 2020, 9, 284; doi:10.3390/plants9020284 www.mdpi.com/journal/plants www.mdpi.com/journal/plants Plants 2020, 9, 284 2 of 13 2 of 13 Figure 1. Geographic distribution of Picramnia (green), Alvaradoa (black), and Nothotalisia (red). Figure 1. Geographic distribution of Picramnia (green), Alvaradoa (black), and Nothotalisia (red). Due to their small, actinomorphic ﬂowers, bitter bark, and pinnately compound leaves, they were initially placed in Simaroubaceae [7]. However, the structure of ﬂowers within Picramniaceae is seriously diﬀerent [1,2]; the most striking is their syncarpous ovary with 2–3 ovules per locule while gynoecium of Simaroubaceae is typically almost apocarpous, with 1–2 ovules per carpel. Picramniaceae was formerly circumscribed with two genera, Picramnia [8] and Alvaradoa [9]. Picramnia is small or mid-size trees that grow either in the lower stories of moist tropical forests (e.g., in Amazonia and Choco) or in more dry habitats, from Mexico and Caribbeans to Southern Brazil and Northern Argentina. Some of them (like the northernmost Picramnia pentandra Sw. from South Florida) frequently grow as shrubs. Gross morphology of Picramnia is rather uniform and non-distinct, this was probably a reason of some notorious mistakes. 1. Introduction One of examples is that Hispaniola generic endemic Casabitoa perfae described as a plant with putative aﬃnities to Phyllanthaceae, is actually identical to Picramnia dictyoneura (Urb.) Urb. and Ekman [10]. Alvaradoa is signiﬁcantly more xerophilous and shrubby, with wind-dispersed fruits (contrary to the animal-dispersed fruits of Picramnia and Nothotalisia). The range of distribution of the genus is conspicuously disjunct: Mexico and Caribbeans (Cuba, Hispaniola, and Jamaica), and Bolivia, Argentina, and South Brazil, as depicted by Thomas [11]. Plants 2020, 9, 284 3 of 13 Recently, the third genus, Nothotalisia W.W.Thomas [12], was discovered among herbarium samples of TalisiaAubl. (Sapindaceae). Nothotalisia, like some species of Picramnia, includes mid-size forest trees that exhibits a broad distribution, from Panama to Peru, and likely associated with wet tropical forests of Choco and Amazonia. Interestingly, Nothotalisia appears not to be rare in collections: For example, it is abundant in herbarium samples collected in the Darien province of Panama (42 samples only in MO herbarium, dated from 1959 to 2011). In all, we believe that diversity of Picramniaceae is far from being exhausted, and we can always expect new discoveries. g y p We should mention here also the monotypic Gumillea Ruiz and Pav. [13] which might also belong to Picramniaceae [14]. This genus was described from some unknown place in Peru and unfortunately not recollected so far. In general, Gumillea auriculata Ruiz and Pav. reminds Picramnia but the plant bears stipules and bisexual ﬂowers. Even though there is no recent specimen of that plant nor any molecular data, the morphology of inﬂorescence, ﬂowers, and leaves suggests a relationship to Picramniales. The structures described and illustrated as “stipules” look like pseudostipules found in some species of Picramnia (e.g., P. campestris Rizzini and Occhioni, P. guianensis (Aubl.) Jans.-Jac.) where petiole is reduced and modiﬁed basal leaﬂets deﬂected to protect axillary buds [15,16]. Only a few species of Picramniaceae have been the subject of DNA-related research, and there are no molecular phylogenetic analyses so far. No generic subdivision was established; even the total number of species is not absolutely clear. Apparently, Picramniaceae remains an under-researched group. To ﬁll this gap, we performed a broad sampling of the group, aiming at collecting as many tissue samples as possible to amplify several widely used DNA barcoding markers in order to cover the species diversity in the group. 1. Introduction We believe that our reconstruction of phylogenetic trees may become a basis for future studies in the group. 2. Material and Methods We collected 276 tissue samples (Table S1) from representatives of Picramniaceae and some other species (used as outgroups in the following analyses) and produce the ﬁrst molecular phylogenetic trees of the group. Our strategy was developed with an idea of the broadest sampling. We typically obtained multiple samples per species and attempted to extract DNA and sequence our markers multiple times until we reach satisfactory results. As we work with under-studied group, it was also especially important to provide photographic vouchers for each sample, and to be sure of the correct identiﬁcations, so either determination from known experts or our own determinations were preferred. At the end of this process, only a few species were missed from our samples, and most of them are either rare or local. As of today, our sequencing list contains 41 species, which is about 80% of the supposed group diversity. Most of them are newly sequenced material, only eight species represented with information in the public databases (GenBank and BOLD). In all, we were able to increase the amount of available information ﬁve-fold. Due to the apparent problems with identiﬁcation, we always trusted our samples ﬁrst. To improve the quality of databases, we constructed the working classiﬁcation of Picraminiaceae (Table S2), which now also contains the results of this phylogenetic study. DNA extraction of the Picramniaceae herbarium samples was not always straightforward. Less than 20% of herbarium samples, fresh or old, yielded the DNA of the appropriate quality. We believe that the main reasons are the conditions of their collection and preparation. We were able also to extract DNA from living samples (collected in silica gel or just air-dried) taken from 8 species, and in all these cases the DNA extraction, ampliﬁcation and sequencing were successful. The DNA extraction was made using NUCLEOSPIN Plant II Kit (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany), which we believe is a good trade-oﬀbetween eﬃciency and simplicity. We improved the protocol to increase the lysis time to 30 min and employ thermomixer on the slow rotation speed (350 rpm) instead of a water bath. We used Nanodrop 1000 Spectrophotometer (Thermo Scientiﬁc, Wilmington, DE, USA), to estimate concentration and purity (the 260/280 nm ratio of absorbance) of DNA. 4 of 13 Plants 2020, 9, 284 We employed three of the most widely used barcoding DNA markers, chloroplast trnL-F [17] and rbcL [18], and nuclear ITS. 2. Material and Methods These short barcoding DNA markers are thought to be the best to amplify from herbarium samples.Typically, our reaction mixture had a total volume of 20 µL which contained 5.2 µL of PCR Master Mix (components mostly from Thermo Fisher Scientiﬁc, Waltham, Massachusetts supplied with Platinum DNA Taq Polymerase; this was chosen after the series of experiments with other polymerases), 1 µL of 10 µM forward and reverse primers, 2 µL of DNA solution from the extraction and 10.8 µL of MQ puriﬁed water (obtained from a Barnstead GenPure Pro system, Thermo Scientiﬁc, Langenselbold, Germany) in the TBT-PAR water mix [19]. The latter was speciﬁcally developed to improve ampliﬁcation from the “recalcitrant” herbarium samples. Thermal cycler programs were mostly 94◦for 5 min, then 35 cycles of 94◦for 1 min; 51◦(or similar, depends on the primer) for 1 min, 72◦for 2 min, and ﬁnally 72◦for 10 min. PCR products were sent for puriﬁcation and sequencing to Functional Biosciences, Inc. (Madison, WY, USA) and sequenced there following standard Sanger-based protocol. Sequences were obtained, assembled, and edited using Sequencher™ 4.5 (Genes Codes Corporation, Ann Arbor, MI, USA). This is how we ended with almost 780 sequencing chromatograms, of which we selected the 140 with the highest signal intensity for the next steps. These sequences represented 33 plant species. To make this research portable and expandable, we automated most of the steps with the“Ripeline” workﬂow (Figure 2). This is the collection of UNIX shell and R [20] scripts that automate many processes related with sequence selection, quality checking, alignments, gap coding, concatenation, and phylogenetic tree production. Ripeline involves multiple pieces of software, for example, AliView [21], MUSCLE [22], APE [23], MrBayes [24], shipunov [25], and phangorn [26]. The Ripeline example, which uses its essential features and includes associated R code and documentation, is freely available from the Github [27]. Figure 2. Ripeline: workﬂow and basic features. Figure 2. Ripeline: workﬂow and basic features. With the help of Ripeline, we were able to create the super-matrix, which includes all three DNA markers and obtain maximal parsimony (MP) and Bayesian (MB) phylogeny trees. Maximum parsimony analyses were run with the help of R phangorn package [26] using parsimony ratchet [28] with 2000 iterations and then 1000 bootstrap replicates. Bayesian analyses were run through the combination of MrBayes 3.2.6 [24] and R shipunov [25] packages. 2. Material and Methods MCMC chains were run for 1,000,000 generations, sampling every 10th generation resulting in 100,000 trees. The ﬁrst 25% of trees were 5 of 13 Plants 2020, 9, 284 discarded as burn-in, and the remaining trees were summed to calculate the posterior probabilities. Trees were rooted with representatives of Sapindales (one out of the four orders forming the sister group of Picramniales) as outgroups: Leitneria ﬂoridana Chapm. (currently included in Simaroubaceae), Kirkia acuminata Oliv. (Kirkiaceae) and Talisia nervosa Radlk. (Sapindaceae), or with L. ﬂoridana alone. To stabilize the tree and to increase branch support, we used the hyper-matrix approach [29] and concatenated several alternative alignments of the ITS data part. Plants 2019, 8, x 5 of 13 1,000,000 generations, sampling every 10th generation resulting in 100,000 trees. The first 25% of trees were discarded as burn-in, and the remaining trees were summed to calculate the posterior probabilities. Trees were rooted with representatives of Sapindales (one out of the four orders forming the sister group of Picramniales) as outgroups: Leitneria floridana Chapm. (currently included in Simaroubaceae), Kirkia acuminata Oliv. (Kirkiaceae) and Talisia nervosa Radlk. No morphological matrix is available for the whole family, so one of the most straightforward ways to assess its morphological diversity was to use the hierarchical components of identiﬁcation keys [30]. To see how well our results correspond with the accumulated knowledge about the family, we employed these keys (along with data from morphological descriptions when available) from the three publications covering the diversity of Picramniaceae in Central America [31], Western South America [32], and Brazil [15]. We also used geographical data to see if any patterns of that type might be observed on our phylogeny trees. Ancestral character estimation was done with the help of APE package [23]; we encoded polymorphisms as most ancestral states. i u e i i a ou a eae), i ia a u i a a O i ( i ia eae) a a i ia e o a a (Sapindaceae), or with L. floridana alone. To stabilize the tree and to increase branch support, we used the hyper-matrix approach [29] and concatenated several alternative alignments of the ITS data part. No morphological matrix is available for the whole family, so one of the most straightforward ways to assess its morphological diversity was to use the hierarchical components of identification keys [30]. 2. Material and Methods To see how well our results correspond with the accumulated knowledge about the family, we employed these keys (along with data from morphological descriptions when available) from the three publications covering the diversity of Picramniaceae in Central America [31], Western South America [32], and Brazil [15]. We also used geographical data to see if any patterns of that i h b b d h l A l h i i d i h h Datasets, scripts, trees for individual markers and other information used in preparation of this publication are available as one zip archive from the ﬁrst author’s Open Repository [33]. We encourage readers to reproduce our results and develop our methods further. All sequences were deposited into the GenBank (Table S3). type might be observed on our phylogeny trees. Ancestral character estimation was done with the help of APE package [23]; we encoded polymorphisms as most ancestral states. Datasets, scripts, trees for individual markers and other information used in preparation of this publication are available as one zip archive from the first author’s Open Repository [33]. We encourage readers to reproduce our results and develop our methods further. All sequences were 3.1. Picramniaceae in General 3 1 Picramniaceae in Gene With the outgroups mentioned above, Picramniaceae formed a stable clade. First branches (Figure 3) are Picramnia and Nothotalisia + Alvaradoa, with suﬃcient (close to 100%) support. 3.1. Picramniaceae in General With the outgroups mentioned above, Picramniaceae formed a stable clade. First branches (Figure 3) are Picramnia and Nothotalisia + Alvaradoa, with sufficient (close to 100%) support. Figure 3. The overview of our phylogeny trees depicting the relationships and relative size of the three strongly supported monophyletic genera of Picramniaceae. Each triangle is the result of concatenation applied to the branches of the phylogenetic tree. Figure 3. The overview of our phylogeny trees depicting the relationships and relative size of the three strongly supported monophyletic genera of Picramniaceae. Each triangle is the result of concatenation applied to the branches of the phylogenetic tree. 6 of 13 Plants 2020, 9, 284 3.1.1. Alvaradoa This part of the tree is strongly supported (Figure 4). South American Alvaradoa subovata Cronquist and A. puberulenta (Monach.) Sleumer form the ﬁrst clade whereas West Indian species—the second clade. Alvaradoa amorphoides Liebm., which grows in Mexico and Florida, is basal to West Indian species. Figure 4. MrBayes tree of concatenated (“semi-strict”) loci.Numbers are Bayesian support (when ≥60%). Figure 4. MrBayes tree of concatenated (“semi-strict”) loci.Numbers are Bayesian support (when ≥60%). 3.1.2. Nothotalisia Nothotalisia peruviana (Standl.) W.W. Thomas and N. cancellata W.W. Thomas form the stable clade to (Figure 4) which N. piranii W.W. Thomas is the sister group. 3.2. Morphological and Geographical Patterns Even after extensive sampling, some species of Picramnia are still missing in our molecular dataset. To test the possible placement of these species, we used the hierarchical component of identiﬁcation keys together with phylogeny trees and employed k-nearest neighbor machine learning with bootstrap to point on possible sister species. Since super-species level classiﬁcation is not developed, we decided to return trios of putative neighbor species which the highest bootstrap conﬁdence (Table 1). Table 1. Placements of Picramnia species based on k-nearest neighbor machine learning. Species to Place Most Likely Neigbor Probability, % Most Likely Neigbor Probability, % Most Likely Neigbor Probability, % P. coccinea P. latifolia 31 P. caracasana 18 P. juniniana 16 P. ferrea P. juniniana 18 P. oreadica 15 P. campestris 14 P. gardneri P. latifolia 32 P. caracasana 19 P. juniniana 14 P. gardneri P. caracasana 34 P. latifolia 27 P. sellowii 27 P. grandiﬂora P. latifolia 26 P. juniniana 15 P. oreadica 15 P. matudai P. gracilis 17 P. hirsuta 15 P. teapensis 14 P. spruceana P. latifolia 36 P. caracasana 27 P. sellowii 20 P. spruceana P. caracasana 18 P. sellowii 17 P. latifolia 15 Table 1. Placements of Picramnia species based on k-nearest neighbor machine learning. There are multiple morphological and geographical patterns observed on the ancestral character estimations (Figure 5a–f). For example, 3–4 carpels recognized as the most ancestral type (Figure 5a), whereas monocarpellate (Alvaradoa) and bicarpellate (part of Picramnia) are likely advanced character states. There is no visible pattern for the position of inﬂorescence (Figure 5b). Reversely, the phylogenetic distribution of the inﬂorescence type is rich in patterns (Figure 5c), but this ancestral type is not well deﬁned. Ancestral merosity was estimated as 5–6 merous, whereas tetramerous or trimerous ﬂowers evolved independently in several Picramnia clades (Figure 5d). A type of trichome (Figure 5e) signiﬁcantly deviates from the ancestral “attenuate” type only twice within the genus. Finally, regional geography estimation (Figure 5f) suggests domination of the Central American and West Indies regions, with more recent origin of two other areas (West South American and Amazonian, and Bolivian plus extra-Amazonian Brazil) centers. 3.1.3. Picramnia Generally, there is much less stability in that part of the tree. Many clades are not reliably supported. The most supported are the following groupings: (1) Picramnia antidesma Sw. + P. deﬂexa W.W. Thomas+ P. latifolia Tul. + P. thomasii Gonzalez-Martinez and J. Jimenez Ram.; (2) P. andrade-limae 7 of 13 Plants 2020, 9, 284 Pirani + P. elliptica Pirani and W.W. Thomas + P. bahiensis Turcz. + P. grandifolia Engl. (latter two with less conﬁdence); (3) P. dictyoneura Planch. + P. sphaerocarpa Planch. and (4) P. glazioviana Engl. + P. parvifolia Engl.; (5) P. oreadica Pirani + P. ramiﬂora Planch. + P. campestris + P. juniniana J.F.Macbr. However, some lower supported groups also make sense, especially (6) P. excelsa Kuhlm. ex Pirani + P. gracilis Tul. + P. nuriensis Steyerm. +P. tumbesina Cornejo. 3.2. Morphological and Geographical Patterns Remarkably, parts of the topology seem to be more or less geographically structured: two clades are mostly composed by Bolivian to extra-Amazonian Brazilian species; one clade is mostly composed by species ranging from Darien (Panama) to Peru extending to the Amazonian region; and two other clades composed of a mixture of species either showing the latter distribution pattern or species ranging from Central American to the West Indies (Figure 5f). Plants 2020, 9, 284 8 of 13 Plants 2020, 9, 284 8 of 13 (a) (b) (c) (d) Figure 5. Cont. 8 of 13 (a) (b) (c) (d) Figure 5. Cont. (b) (a) (d) Figure 5. Cont. Plants 2020, 9, 284 9 of 13 lants 2020, 9, 284 9 of 13 (e) (f) Figure 5. Ancestral character estimation on six different types of characters: (a) number of carpels, (b) position of the inflorescence, (c) type of the inflorescence, (d) merosity of flower, (e) type of trichomes, (f) geography on the regional level. Figure 5. Ancestral character estimation on six diﬀerent types of characters: (a) number of carpels, (b) position of the inﬂorescence, (c) type of the inﬂorescence, (d) merosity of ﬂower, (e) type of trichomes, (f) geography on the regional level. 9 of 13 (f) (e) (e) (f) (e) Figure 5. Ancestral character estimation on six different types of characters: (a) number of carpels, (b) position of the inflorescence, (c) type of the inflorescence, (d) merosity of flower, (e) type of trichomes, (f) geography on the regional level. Figure 5. Ancestral character estimation on six diﬀerent types of characters: (a) number of carpels, (b) position of the inﬂorescence, (c) type of the inﬂorescence, (d) merosity of ﬂower, (e) type of trichomes, (f) geography on the regional level. 4.1. Picramniaceae in General 4.1. Picramniaceae in General Picramniaceae is here supported with three monophyletic genera. The monophyly of each genus was expected since they are clearly distinct taxa on morphological grounds. However, the molecular evidence of a closer relationship between Nothotalisia and Alvaradoa seems unnatural as we compare the general morphology of the threegenera. As Thomas [12] already pointed out, “Nothotalisia is strikingly similar to many species of Picramnia” since both genera bear alternate leaflets with oblique bases and attenuate tips, racemose inflorescences with congested lateral cymes, and berry-like fruits. Alvaradoa is easily distinguished from the former two genera by its much smaller, oblong leaflets with symmetric bases, and flattened, winged, dry fruits. On the other hand, Nothotalisia is unique in having an androgynophore on male flowers; this peculiar structure bears the five stamens united by their filaments and a small pistillode on top. Picramniaceae is here supported with three monophyletic genera. The monophyly of each genus was expected since they are clearly distinct taxa on morphological grounds. However, the molecular evidence of a closer relationship between Nothotalisia and Alvaradoa seems unnatural as we compare the general morphology of the threegenera. As Thomas [12] already pointed out, “Nothotalisia is strikingly similar to many species of Picramnia” since both genera bear alternate leaﬂets with oblique bases and attenuate tips, racemose inﬂorescences with congested lateral cymes, and berry-like fruits. Alvaradoa is easily distinguished from the former two genera by its much smaller, oblong leaﬂets with symmetric bases, and ﬂattened, winged, dry fruits. On the other hand, Nothotalisia is unique in having an androgynophore on male ﬂowers; this peculiar structure bears the ﬁve stamens united by their ﬁlaments and a small pistillode on top. 4.2. Alvaradoa and Nothotalisia Within Alvaradoa the Ca 4.2. Alvaradoa and Nothotalisia Within Alvaradoa, the Caribbean clade is well supported, whereas A. amorphoides + South American species are less robust. Nevertheless, this latter clade might point to the concurrent hypotheses on the causes of the disjunct pattern between Central America and subtropical South American Cone, observed in several groups of angiosperms (e.g., [34]): ancient connection (vicariance) vs. long-distance dispersal. A reappraisal of such an American Amphitropical disjunct pattern was recently carried out by Simpson et al. [35], suggesting dispersal as the leading cause in most of the lineages. Within Nothotalisia, Amazonian N. piranii is distinct from the more widespread N. cancellata and N. peruviana. Within Alvaradoa, the Caribbean clade is well supported, whereas A. amorphoides + South American species are less robust. Nevertheless, this latter clade might point to the concurrent hypotheses on the causes of the disjunct pattern between Central America and subtropical South American Cone, observed in several groups of angiosperms (e.g., [34]): ancient connection (vicariance) vs. long-distance dispersal. A reappraisal of such an American Amphitropical disjunct pattern was recently carried out by Simpson et al. [35], suggesting dispersal as the leading cause in most of the lineages. Within Nothotalisia, Amazonian N. piranii is distinct from the more widespread N. cancellata and N. peruviana. 4.3. Picramnia 4.3. Picramnia Several of the smaller clades within Picramnia seem to be strongly correlated with morphology and congruent with geography. Other clades are composed by a mixture of species that had not so far been considered as closely related on a morphological basis. 10 of 13 Plants 2020, 9, 284 Pirani [15,16] proposed that P. ramiﬂora and P. latifolia could be related since they share several morphological features, and some herbarium specimens may be hard to ascribe to one or the other species. However, they emerge far away in the molecular topology. P. latifolia presents as main diagnostic characters distinguishing it from the remaining species the mostly lateral inﬂorescences bearing (sub)sessile ﬂowers and fruits, although pedicellate ﬂowers occur in some specimens. It is a widespread, mostly Amazonian species that reach Central America; therefore, its closer relationship to Central American to Andean species (P. antidesma, P. deﬂexa, and P. thomasii) makes sense on a geographical basis. P. ramiﬂora, however, is a quite disjunct species from the Brazilian Atlantic Forest. The clade formed by (P. thomasii (P.deﬂexa (P.latifolia, and P.antidesma))) is morphologically heterogeneous, with a mixture of species bearing pentamerous ﬂowers (P. latifolia and P. thomasii) and species with trimerous or tetramerous ﬂowers (the other two). This fact implies that even though the numbers of sepals and petals have a consistent relevance for species recognition within the genus, often used in keys (e.g., [15,31,36]), it seems to hold little phylogenetic signal. Other examples of clades containing either trimerous, tetrameous or pentamerous species are: (a) P. guerrerensis W.W. Thomas (5-merous) nested with P. hirsuta W.W. Thomas and P. teapensis Tul. (3–4-merous); (b) P. elliptica and P. grandifolia (5-merous) closely related to P. bahiensis and P. andrade-limae (3-merous); (c) P. excelsa (3-merous) nested within a clade with P. sellowii and P. guianensis (both 5-merous), along with P. sphaerocarpa which is particularly polymorphic producing 3–4–5-merous ﬂowers. Other morphological features that have long been used in the taxonomy of Picramnia, as well as to generate assumptions of relationships, concern to the inﬂorescence type and position (e.g., [15,31]); these characters may be critically considered, as indicated by the molecular data. For example, P. hirsuta, with long simple racemes, maybe sister to P. polyantha (Benth.) Planch., which bears profusely branched thyrses. In the same way, racemose P. elliptica and P. grandifolia here emerged within a clade with P. andrade-limae and P. bahiensis; both provided with thyrses. 4.3. Picramnia 4.3. Picramnia Nonetheless, some assumptions of relationships previously made on morphological grounds are here corroborated by the molecular data. For example, the close relationship between P. glazioviana and P. parvifolia proposed by Pirani [15], based on several features, was here recovered; and both are partially sympatric species of the Atlantic Forest in Eastern to Southern Brazil. However, it seems awkward the fact that a species so similar to P. parvifolia as is P. excelsa [15], both sympatric inside the Araucaria forests of Southern Brazil, did not emerge in that same clade. Such reasoning may also be pointed to P. glazioviana, which is quite similar and sympatric to P. ciliata in the Brazilian coastal moist forests, but this latter emerged near the base of the molecular tree. P. andrade-limae and P. bahiensis also have trimerous ﬂowers with peculiar obcordate petals and assumed as related to the aforementioned Atlantic Forest group with P. glazioviana [15], but emerged on other branches of the phylogenetic tree. g p g p y g Astonishingly, the two samples of P. oreadica did not emerge together, but the fact that each of them is currently ascribed to distinct allopatric subspecies may be considered here [15]. These are heliophilous shrubs inhabiting the open cerrados of Central Brazilian Plateau [16], known so far from few herbarium specimens, and our results show that further studies on taxonomic circumscription are here necessary. Also, it should be expected that P. guianensis would be nested in the clade including P. oreadica and P. campestris, because they share a special type of indumentum on the ovary (peculiar clavate trichomes), and are all mostly related to sandy or rocky substrates. These species live mostly as heliophytic shrubs, contrasting with the typical understory habit prevailing in the genus. However, the fact the P. guianensis emerged in another clade, that feature may have appeared independently. P. ferrea Pirani and W.W.Thomas, unsuccessfully sampled in this analysis, could probably belong to the P. guianensis clade on a biogeographical basis, or the P. campestris clade on morphological grounds (e.g., clavate trichomes on ovary surface). Hence, there is some evidence which corroborates previous assumptions of relationships made on a morphological basis, while most parts of the molecular topology suggest high levels of homoplasy in the structural evolution of Picramnia. 4.3. Picramnia 4.3. Picramnia On the other hand, as we pointed out previously, it is remarkable that parts of the topology seem to be more or less geographically structured since two clades are mostly 11 of 13 Plants 2020, 9, 284 composed of Bolivian to extra-Amazonian Brazilian species. Another clade is mostly composed of species ranging from Darien (Panama) to Peru extending to the Amazonian region. In contrast, the next two clades are composed of a mixture of species, either showing the latter distribution pattern or species ranging from Central American to the West Indies. Even though we are aware that the deﬁnition of these areas of distribution is somewhat artiﬁcial, they are mostly in agreement with biogeographical regions and provinces previously proposed for the Americas [27]. The spatial component revealed in our phylogenetic results suggests that the diversiﬁcation of some of the lineages within Picramnia may reﬂect some of the geomorphological, climatic, and ecological grounds that helped to shape or drive the history of the neotropical biota. Final Remarks Even with extensive sampling and continuous sequencing eﬀorts, our data is still incomplete. Only 20% of our sequencing eﬀorts ended with reliable sequences. GenBank data generally lack sequences of Picramniaceae. Some rare species are still not sampled. Nevertheless, we think that our phylogeny results might serve as a backbone for future research. Our best hope is that this work represents the beginning of the second, molecular stage in Picramniaceae research. One of the most apparent next steps is to check how morphology and DNA go together, the molecular “weight” of traditional morphological characters. Supplementary Materials: The following are available online at http://www.mdpi.com/2223-7747/9/2/284/s1, Table S1: Vouchers of Picramniaceae samples, Table S2: Working classiﬁcation of Picramniaceae, Table S3: Genbank IDs. Author Contributions: Conceptualization, A.S. and J.R.P.; methodology, A.S. and J.R.P.; software, A.S.; validation, J.R.P.; formal analysis, A.S., S.C., S.F., K.P.; investigation, A.S., S.C., S.F., K.P.; resources, A.S., K.P.; data curation, A.S., K.P.; writing—original draft preparation, A.S.; writing—review and editing, A.S., J.R.P.; visualization, A.S.; supervision, A.S., J.R.P.; project administration, A.S.; funding acquisition, A.S., J.R.P. All authors have read and agreed to the published version of the manuscript. Funding: From May 2014, this research is supported by North Dakota INBRE. JRP is supported by a producti grant from CNPq—Conselho Nacional de Desenvolvimento Cientíﬁco e Tecnológico. Acknowledgments: We are grateful to the curators of NY, MO, COL, HUH, US, UC/JEPS, CAS, F, BRIT, SPF, SP, USM herbarium collections and living collections of the Fairchild Botanical Garden, Missouri Botanical Garden and Jardín Botánico de Bogotá José CelestinoMutis (Bogota, Colombia) for the permission to work with their material. Our special thanks to Wayt Thomas (NYBG), to César Adrián González Martínez (UNAM) for the great help with material, and to Mark Chase (Kew Gardens) for the help with ﬁrst rbcL sequences. Conﬂicts of Interest: The authors declare no conﬂict of interest. s of Interest: The authors declare no conﬂict of interest. References 1. Fernando, E.S.; Gadek, P.A.; Quinn, C.J. Simaroubaceae, an artiﬁcial construct: Evidence from rbcL sequence variation. Am. J. Bot. 1995, 82, 92–103. [CrossRef] 1. Fernando, E.S.; Gadek, P.A.; Quinn, C.J. 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https://openalex.org/W3037930425	https://europepmc.org/articles/pmc7353653?pdf=render	English	null	Utilization of Bioelectrical Impedance to Predict Intramuscular Fat and Physicochemical Traits of the Beef Longissimus Thoracis et Lumborum Muscle	Foods	2,020	cc-by	7,956	Received: 3 June 2020; Accepted: 24 June 2020; Published: 25 June 2020 Abstract: The bioelectrical impedance analysis (BIA) is a non-destructive technique that has been successfully used to assess the body and carcass composition of farm species. This study aimed to predict intramuscular fat (IMF) and physicochemical traits in the longissimus thoracis et lumborum muscle (LM) of beef, using BIA. These traits were evaluated in LM samples of 52 crossbred heifer carcasses. The BIA was performed in LM, using a 50 Hz frequency high precision impedance converter system. A correlation analysis of the studied variables was performed. Then a stepwise with a k-folds cross validation procedure was used to modelling the prediction of IMF and physicochemical traits from BIA parameters (24.5% ≤CV ≤47.3%). Wide variation was found for IMF and BIA parameters. In general, correlations of BIA parameters with IMF and physicochemical traits were moderate to high and were similar for all BIA parameters (−0.50 ≤r ≤0.50 only for total pigments, a* and pH48). It was possible to predict IMF and physicochemical traits from BIA. The best ﬁt explained 79.3% of the variation in IMF, while for physicochemical traits the best ﬁts were for sarcomere length and shear force (64.4% and 60.5%, respectively). The results conﬁrmed the potential of BIA for objective measurement of meat quality. Keywords: bioelectrical impedance analysis; meat quality; beef João Afonso 1,* , Cristina Guedes 2, Virgínia Santos 2, Raul Morais 3 , José Silva 2 , Alfredo Teixeira 4 and Severiano Silva 2 João Afonso 1,* , Cristina Guedes 2, Virgínia Santos 2, Raul Morais 3 , José Silva 2 , Alfredo Teixeira 4 and Severiano Silva 2 1 Faculdade de Medicina Veterinária, ULisboa, Avenida da Universidade Técnica, 1300-477 Lisboa, Portu 2 culdade de Medicina Veterinária, ULisboa, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal ntro de Ciência Animal e Veterinária, Universidade de Trás-os-Montes e Alto Douro, 01-801 Vila Real, Portugal; cguedes@utad.pt (C.G.); vsantos@utad.pt (V.S.); jasilva@utad.pt (J.S.); lva@utad.pt (S.S.) , , , , g 2 Centro de Ciência Animal e Veterinária, Universidade de Trás-os-Montes e Alto Douro, 5001-801 Vila Real, Portugal; cguedes@utad.pt (C.G.); vsantos@utad.pt (V.S.); jasilva@utad.pt (J.S.); ssilva@utad.pt (S.S.) , , , , g 2 Centro de Ciência Animal e Veterinária, Universidade de Trás-os-Montes e Alto Douro, 5001-801 Vila Real, Portugal; cguedes@utad.pt (C.G.); vsantos@utad.pt (V.S.); jasilva@utad.pt (J.S.); ssilva@utad.pt (S.S.) 3 p 3 INESC TEC-INESC Technology and Science and Universidade de Trás-os-Montes e Alto Douro, 5001-801 Vila Real, Portugal; rmorais@utad.pt 4 CIMO, Instituto Politécnico de Bragança, 5300-253 Bragança, Portugal; teixeira@ipb.pt * Correspondence: jafonso@fmv.ulisboa.pt foods foods foods foods www.mdpi.com/journal/foods 2.1. Muscle Sample Preparation After slaughter, the carcasses were dressed and centrally-split into two sides. Then entered the chill room and were refrigerated for two days at 2 ◦C. After the chill period, carcasses were deboned in accordance with the scheme used by the abattoir, whereas no primal cuts were removed prior to deboning. During this process, using a metallic ruler, a cut of 10 cm length of LM at the level of 3rd and 5th lumbar vertebrae was removed from the carcass. This cut was divided into two slices, which were identiﬁed, weighed, individually vacuum-packed, placed in insulated cooled containers and transported to the laboratory, for an aging period of ten days, at 2 ◦C. After this period the BIA and physicochemical analyses were carried out, and the corresponding results recorded. 2. Materials and Methods Fifty-two Limousine x Holstein crossbred heifers with 355.5±35.0 kg of live weight and with age ranging from 9–12 months were slaughtered at the PEC Nordeste (Penaﬁel, Portugal) abattoir. All animals were cared for and killed in compliance with the welfare regulations and respecting EU Council Regulation (EC) No. 1099/2009. The animals were stunned using captive-bolt pistol prior to exsanguination and dressed according to standard commercial practices. The transport time of the animals from the farm to the slaughterhouse was 1–2 h and the pre-slaughter resting duration in lairage was approximately 3 h. 2.2. Bioelectrical Impedance Analysis After removal of subcutaneous and intermuscular fat, one 3 cm thick slice sample of LM from each carcass was placed over a ﬂat surface for BIA measurements. The bioelectrical impedance device used in the present study was a single frequency unit built speciﬁcally for this purpose, around a high precision impedance converter AD5933 system (Analog Devices, Norwood, MA, USA). After calibration, and at a frequency of 50 kHz, the magnitude of the impedance and relative phase of the impedance was calculated, using a Discrete Fourier Transform algorithm to obtain the resistance (Rs) and reactance (Xc) values. The impedance (Z) was calculated as Z = (Rs2 + Xc2)0.5. As detector terminals, two hypodermic needles were inserted 2 cm into each 3 cm thick LM slice, 10 cm apart, providing an attachment for the connecting clips of the bioelectrical impedance terminal leads. For each sample, four measurements were made and the values of Rs and Xc correspond to their average. All bioelectrical impedance measurements were made in LM slices with temperature ranging between 2 and 6 ◦C. 1. Introduction Meat is a nutritious and popular food, which provides high biological value proteins and important micronutrients, and in recent years increasing attention has been put on its quality and safety [1]. Meat quality is aﬀected by several factors throughout the production line and identifying and ensuring meat quality remains an ongoing challenge for meat processors [2]. The traditional evaluation of meat quality such as intramuscular fat (IMF) and physicochemical traits involves destructive, expensive and time-consuming methods [3]. To overcome these limitations, several non-destructive, precise, and fast techniques have been developed and successfully applied in meat quality assessment. Over the past two decades, there have been several emerging techniques, particularly those based on imaging and spectroscopic principles [4,5]. Among the imaging techniques, computed tomography [6], computer vision [7], and ultrasound [5] stand out. Concerning spectroscopic techniques, the emphasis has been put on near-infrared spectroscopy (NIRS) techniques [8,9]; hyperspectral imaging systems [10,11], Foods 2020, 9, 836; doi:10.3390/foods9060836 www.mdpi.com/journal/foods 2 of 11 Foods 2020, 9, 836 and Raman spectroscopy [2]. For all these techniques, attributes of accuracy, precision, cost, and ease of use have been considered. However, some attributes, particularly those related to cost and ease of use, are not achieved in most of the previously mentioned techniques. This encourages further research to investigate the application of other techniques in the assessment of meat quality traits. The bioelectrical impedance analysis (BIA) is an example of an unexpansive and easy to use technique that has been used successfully to assess the body and carcass composition of several farm species [12–14]. However, not much information has been reported on the ability of such technique to assess IMF and/or physicochemical meat quality traits. Therefore, our objective was to examine the usefulness of BIA to predict IMF and physicochemical traits of the beef Longissimus thoracis et lumborum muscle. 2.4. Total Collagen Two sub-samples of approximately 4 g were obtained from each meat sample. The sub-samples were ﬁnely chopped, while frozen, and each one put into a ﬂask with a lid. To each ﬂask were added 30 mL of 7 N sulfuric acid. The ﬂasks were closed and placed in an oven at 105 ◦C for 16 h, and the sample was hydrolyzed. Then, the hot hydrolyzate was transferred to 200 mL volumetric ﬂasks, the ﬂasks being washed with distilled water and ﬁlled to a volume of 200 mL. After homogenization, the contents of the ﬂasks were ﬁltered on Whatman ﬁlter paper No. 4, up to a volume of 100 mL, and stored at a temperature of approximately 4 ◦C, for some time that did not exceed two weeks. The previous ﬁltrate was pipetted and diluted in distilled water, in 100 mL ﬂasks, so that the concentration of L-hydroxyproline in the ﬁnal dilution was between 0.6 and 2.4 µg/mL. To 2 mL of the ﬁnal dilution, 1 mL of an oxidizing solution of chloramine T was added, homogenized and left to stand for 20 min at room temperature. Then, 1 mL of colorimetric reagent was added and homogenized, 25 mL of which was prepared by diluting 2.5 g of 4-dimethylaminobenzaldehyde in 8.75 mL of 60% perchloric acid to which it was added, slowly with stirring, 16.25 mL of 2-propanol. The covered test tubes were quickly placed in a Digiterm 100 water bath (J.P. Selecta, Barcelona, Spain) at 60 ◦C for exactly 15 min. Then they were quickly cooled under running water for at least 3 min. Absorbance was measured at 558 nm against a blank 1 cm cell optical path assay on a Jasco V-530 UV/VIS spectrophotometer (JASCO Corporation, Tokyo, Japan). The absorbance of 0.6, 1.2, 1.4, and 2.4 µg/mL L-hydroxyproline solutions were also measured in order to construct the standard curve for each assay. For the preparation of L-hydroxyproline solutions, an intermediate solution of L-hydroxyproline with 6 µg/mL was initially prepared, pipetting 5 mL of the L-hydroxyproline stock solution (600 µg/mL) into a 500 mL ﬂask and making up the remaining volume with distilled water. From this solution, solutions with four diﬀerent concentrations of L-hydroxyproline (0.6, 1.2, 1.4, and 2.4 µg/mL) were then prepared, having been pipetted 10, 20, 30, and 40 mL. To, respectively, of the intermediate solution, for 100 mL. 2.3. Intramuscular Fat For IMF chemical determinations, vacuum-packed samples were unpacked and intermuscular and the subcutaneous fats were carefully trimmed from the LM samples. The samples were minced and homogenized, using an Ultra Turrax T25 apparatus (IKA, Germany), prior to the chemical analysis. Chemical IMF content of LM samples was obtained in triplicates after ether-extraction in a Tecator Soxtec HT 1043 (Höganäs, Sweden), using petroleum ether as the solvent, and was determined gravimetrically, after evaporating the solvent according to AOAC (2000) [15] procedure. The content of the chemical IMF was expressed as a percentage of LM weight. 3 of 11 Foods 2020, 9, 836 2.4. Total Collagen To ﬂasks, making up the remaining volume of the ﬂasks with distilled water. After obtaining the standard curve, the concentration of L-hydroxyproline (µg/mL. To) of each sample was calculated using the corrected absorbance. The concentration of total collagen in the sub-samples was obtained by multiplying the concentration of L-hydroxyproline by factor 7.25 [16]. 2.5. Total Pigments Total pigments were determined using the method of Wierbicki et al. (1955) [17] modiﬁed by Boccard et al. (1981) [18]. In this method, total pigments are dosed as cyanmetamyoglobin. Approximately 5.0 g was obtained in duplicate of each meat sample of frozen chopped muscle, to which 40 mL of cold distilled water were added and homogenized at a rapid speed for 30 s with Ultra-Turrax T25B (Kika Labortechnik, Staufen, Germany). The pH of each sample was checked and samples with a pH higher than 6.0 were acidiﬁed with 0.1 M HCl to pH 5.5. The homogenate was ﬁltered on medium porosity paper with a high retention time (Whatman No. 3), with the additional use of 5 mL of distilled water to remove all residues of the homogenate from the ﬂask for ﬁltration. Being the ﬁltrate perfectly translucent and red, 25 mL of the ﬁltrate was transferred to the test tube. Otherwise, the ﬁltrate obtained was re-ﬁltered and, in case the situation remained, the procedure was repeated. A test tube with an equal amount of distilled water was prepared for the blank test. Then, 0.1 mL of 2.5% potassium ferricyanide solution (K3Fe(CN)6) was added to each of the tubes and homogenized. Afterwards, 0.1 mL of 5% potassium cyanide (KCN) was added to each of the tubes and homogenized, and the solution should turn red due to the formation of cyanmethamyoglobin. The absorbance was measured at 540 nm, in a 1 cm optical cell, in a Jasco V-530 UV/VIS spectrophotometer (JASCO Corporation, Tokyo, Japan). The concentration of myoglobin in the fresh product (mg/g) was obtained by multiplying the corrected absorbance by 14.56; this factor is an empirical value for the conditions in which the analysis was carried out and for a given standard curve. 4 of 11 Foods 2020, 9, 836 2.6. Cooking Losses From each sample, 70–100 g of meat was weighed, and a Digi-Sense Thermocouple Scanning Thermometer (Cole Parmer, Niles, IL, USA) was placed in the thermal center of each meat block. Each meat block was then put in a plastic bag which was closed in order to remove the air, to facilitate immersion, preventing water from entering and moving the probe, and was placed in a Digiterm 100 water bath (JP Selecta, Barcelona, Spain) at 75 ◦C, until the internal temperature of 70 ◦C was reached. Subsequently, it was placed in running water until the internal temperature reached 15 ◦C. The meat was removed from the bag, dried carefully and weighed, thus obtaining the ﬁnal weight. Cooking losses were determined by the diﬀerence between the initial weight (Wi) and the ﬁnal weight (Wf) after cooking, and were expressed as a percentage of the initial weight [19]: Cooking losses (%) = [(Wi −Wf)/Wi] × 100. (1) (1) Cooking losses (%) = [(Wi −Wf)/Wi] × 100. These samples were subsequently packaged and stored at 4 ◦C to be used in determining the shear force. These samples were subsequently packaged and stored at 4 ◦C to be used in determining the shear force. 2.8. Warner–Bratzler Shear Force Meat samples used to determine the cooking losses were removed from the refrigerator and placed at room temperature for temperature equilibrium. Approximately ten sub-samples of meat were cut in cuboid shape with 1 cm2 of side and about 3–5 cm in length. The cut was made so that the muscle ﬁbers were parallel to the length of the cuboid. The sub-samples were placed in the Stevens QTS25 Texturometer tray with the Warner–Bratzler rectangular hole probe (Stevens Advanced Weighing Systems Ltd., Great Dunmow, UK), with the ﬁbers arranged perpendicularly to the blade direction, and the probe was used at a speed of 100 mm/min [21]. The mean values of the maximum shear force (kg/cm2) were then obtained. 2.7. Sarcomere Length To determine the length of the sarcomeres was used the method described by Cross et al. (1981) [20]. Brieﬂy, approximately 2 g of LM was cut with a scalpel in small portions, to which 30 mL of a cold 0.25 M sucrose solution was added and subsequently homogenized at slow speed for 60 seconds with the Ultra-Turrax T25B (Kika Labortechnik, Staufen, Germany). When the ﬁbers were not suﬃciently broken, they were further homogenized for 20 to 30 s. This procedure was repeated with the care that the obtained myoﬁbrils had no less than 10 sarcomeres. A drop of the homogenate was placed on a slide, using a Pasteur pipette, and it was covered with a coverslip, having been observed under the Nikon Labophot-2 optical microscope in phase 1, with the 40× objective with attached camera. The length of 10 consecutive sarcomeres was measured, and approximately 15 groups of 10 sarcomeres per sample were measured using the image analysis software Matrox Inspector 4.1 (Matrox Electronic Systems Ltd., Dorval, QC, Canada). The average length of the sample sarcomeres was subsequently determined. 2.10. Statistical Analysis A descriptive analysis of the data was performed, using Microsoft Excel, to obtain the mean, standard deviation, maximum value, minimum value and coeﬃcient of variation of the variables under study. Correlations were established between the BIA parameters and IMF and the physicochemical characteristics of the LM. A stepwise regression with k-fold cross-validation procedure was used for modelling of the predictions of IMF and physicochemical characteristics of the LM with BIA parameters. The accuracy of the estimates was based on the k-fold coeﬃcient of determination (k-fold-R2), while the root mean square error of the cross-validation (RMSE) was used to determine the precision of the prediction model. Additionally, as an indicator of the overall prediction ability of k-fold cross-validation models, the ratio of prediction to deviation (RPD) was evaluated. The RPD is calculated as the ratio of standard deviation (SD) of the reference values to the RMSE (RPD = SD/RMSE). An RPD > 2.5 indicates excellent prediction models, 2.0 < RPD < 2.5 indicates very good prediction models, 1.8 < RPD < 2.0 indicates good prediction models still allowing quantitative predictions, 1.4 < RPD < 1.8 indicates fair prediction models, still useful for assessment, and 1.0 < RPD < 1.4 indicates poor prediction models [24]. All statistical procedures were carried out using the John’s Macintosh Project (JMP) software version 14 (SAS Institute, Cary, NC, USA). 2.9. Color and pH Measurements Color and pH measurements were made 48 h after slaughter. The LM surface color measurements were obtained with the handheld CR-10 colorimeter (Konica Minolta Sensing Inc., Osaka, Japan) and assessed using the L, a, and b* three-dimensional color space, deﬁned by the Commission Internationale de l‘Éclairage [22]. In this system, L, a, and b* represent the measurements of luminosity, red-green, and yellow-blue, respectively. The LM slices were held in open plastic bags at 2 ◦C for two hours (blooming time) to ensure optimal myoglobin oxygenation [23]. Then, the determinations of L* a* and b* parameters were carried out randomly, in three locations at the cutting surface of the LM slices, using measurement geometry 0◦/45◦, D65 illuminant and 10◦observer. The colorimeter was calibrated before the usage with a standard white ceramic disk and a trap opening for black. The average pH values resulted from three readings performed directly on each LM slice, using a drilling electrode 5 of 11 Foods 2020, 9, 836 coupled to a Hanna HI 9025 pH meter (Hanna Instruments, Woonsocket, RI, USA) with automatic temperature compensation. coupled to a Hanna HI 9025 pH meter (Hanna Instruments, Woonsocket, RI, USA) with automatic temperature compensation. SD = standard deviation; IMF = intramuscular fat; WBSF = Warner–Bratzler shear force; L, a and b* = trichromatic coordinates (brightness, red and yellow, respectively); pH48 = pH 48 h after slaughter; Rs = resistance; Xc = reactance; Z = impedance, calculated as (Rs2 + Xc2)0.5. 3. Results 6 of 11 Foods 2020, 9, 836 As shown in Table 2, with the exception of the moderate correlations of WBSF with total collagen (r = 0.697; p < 0.01) and sarcomere length (r = −0.569; p < 0.01), the other correlations between IMF, total collagen, sarcomere length and WBSF were all high – positive between IMF and sarcomere length (r = 0.830; p < 0.01) and negative in the remaining cases (−0.869 ≤r ≤−0.740; p < 0.01). All the other correlations between the diﬀerent LM traits, although signiﬁcant, were negligible to moderate (−0.707 ≤r ≤0.708; p < 0.01). Table 2. Coeﬃcients of correlation between the diﬀerent longissimus thoracis et lumborum muscle (LM) physicochemical traits (n = 52) 1. Total Collagen (mg/g) Total Pigments (mg/g) Cooking Losses (%) Sarcomere Length (µm) WBSF (kg/cm2) L* a* b* pH48 IMF (%) −0.869 0.386 −0.707 0.830 −0.798 0.642 0.424 0.708 −0.540 Total collagen (mg/g) −0.252 0.647 −0.740 0.697 −0.565 −0.367 −0.654 0.476 Total pigments (mg/g) −0.212 0.193 −0.417 0.223 −0.047 0.315 −0.334 Cooking losses (%) −0.556 0.569 −0.432 −0.468 −0.604 0.414 Sarcomere length (µm) −0.569 0.487 0.338 0.603 −0.362 WBSF(kg/cm2) −0.457 −0.439 −0.700 0.640 L* 0.230 0.460 −0.319 a* 0.282 −0.280 b* −0.471 IMF = intramuscular fat; WBSF = Warner–Bratzler shear force; L, a and b* = trichromatic coordinates (brightness, red and yellow, respectively); pH48 = pH 48 h after slaughter. 1 p < 0.01. Table 2. Coeﬃcients of correlation between the diﬀerent longissimus thoracis et lumborum muscle (LM) physicochemical traits (n = 52) 1. IMF = intramuscular fat; WBSF = Warner–Bratzler shear force; L, a and b* = trichromatic coordinates (brightness, red and yellow, respectively); pH48 = pH 48 h after slaughter. 1 p < 0.01. IMF = intramuscular fat; WBSF = Warner–Bratzler shear force; L, a and b* = trichromatic coordinates (brightness, red and yellow, respectively); pH48 = pH 48 h after slaughter. 1 p < 0.01. Overall, the correlations with LM physicochemical traits were quite similar for the three BIA parameters (Table 3). 3. Results As shown in Table 1, there was a low coeﬃcient of variation (CV) for LW, dressing and HCW (respectively 9.8%, 2.2%, and 8.7%) but, with the exception of pH 48 h after slaughter (pH48; CV = 4.0%), all the other LM physicochemical traits and all BIA parameters showed higher CV (12.5% ≤CV ≤47.3%). Most physicochemical traits showed a CV between 4.0% and 27.2%, lower or quite similar to the CV for the BIA parameters (24.5% ≤CV ≤31.4%). The exception was the CV of 47.3% for IMF, with the IMF content of the samples ranging between 1.65% and 16.53%. Table 1. Descriptive statistics for live weight (LW), dressing, hot carcass weight (HCW), longissimus thoracis et lumborum muscle (LM) physicochemical traits, and bioelectrical impedance (BIA) parameters (n = 52). Table 1. Descriptive statistics for live weight (LW), dressing, hot carcass weight (HCW), longissimus thoracis et lumborum muscle (LM) physicochemical traits, and bioelectrical impedance (BIA) parameters (n = 52). Table 1. Descriptive statistics for live weight (LW), dressing, hot carcass weight (HCW), longissimus thoracis et lumborum muscle (LM) physicochemical traits, and bioelectrical impedance (BIA) parameters (n = 52). Trait Mean SD Minimum Maximum CV (%) LW (kg) 355.5 35.0 291.0 433.0 9.8 Dressing (%) 60 1.32 56 62 2.2 HCW (kg) 210.3 18.2 176.7 249.9 8.7 LM physicochemical traits IMF (%) 7.48 3.54 1.65 16.53 47.3 Total collagen (mg/g) 7.52 1.19 4.74 9.43 15.8 Cooking losses (%) 18.0 2.59 13.2 23.4 14.4 Sarcomere length (µm) 1.83 0.21 1.48 2.23 11.4 WBSF (kg/cm2) 5.51 0.99 3.72 7.62 18.0. Total pigments (mg/g) 3.01 0.82 1.57 4.40 27.2 L* 31.0 3.89 25.1 41.8 12.5 a* 13.27 2.63 8.23 16.87 19.8 b* 8.99 1.47 3.49 12.1 16.3 pH48 5.95 0.24 5.55 6.61 4.0 BIA parameters Rs (Ω) 86.8 21.2 58.6 138.1 24.5 Xc (Ω) 108.9 34.1 65.1 161.5 31.4 Z (Ω) 139.4 39.5 89.7 212.1 28.4 SD = standard deviation; IMF = intramuscular fat; WBSF = Warner–Bratzler shear force; L, a and b* = trichromatic coordinates (brightness, red and yellow, respectively); pH48 = pH 48 h after slaughter; Rs = resistance; Xc = reactance; Z = impedance, calculated as (Rs2 + Xc2)0.5. 3. Results Even so, Rs was the BIA parameter showing the highest correlation with IMF (r = 0.854; p < 0.01), WBSF (r = −0.778; p < 0.01), b* (r = 0.610; p < 0.01) and total pigments (r = 0.388; p < 0.01), Xc was the BIA parameter showing the highest correlation with sarcomere length (r = 0.803; p < 0.01), L* (r = 0.567; p < 0.01) and pH48 (r = −0.489; p < 0.01), and Z was the BIA parameter showing the highest correlation with total collagen (r = −0.716; p < 0.01), cooking losses (r = −0.607; p < 0.01) and a* (r = 0.431; p < 0.01). All BIA parameters were signiﬁcantly correlated with LM physicochemical traits, although the correlations of BIA parameters with cooking losses and pH48 (−0.607 ≤r ≤−0.460; p < 0.01), and with total pigments, L, a, and b* (0.361 ≤r ≤0.610; p < 0.01) were poor to moderate. The correlations with BIA parameters were high and negative for total collagen and WBSF (−0.778 ≤r ≤−0.705; p < 0.01), and high and positive for IMF and sarcomere length (0.748 ≤r ≤0.854; p < 0.01). Table 3. Coeﬃcients of correlation of bioelectrical impedance (BIA) parameters with longissimus thoracis et lumborum muscle (LM) physicochemical traits (n = 52) 1. Table 3. Coeﬃcients of correlation of bioelectrical impedance (BIA) parameters with longissimus thoracis et lumborum muscle (LM) physicochemical traits (n = 52) 1. Rs (Ω) Xc (Ω) Z (Ω) IMF (%) 0.854 0.835 0.851 Total collagen (mg/g) −0.709 −0.705 −0.716 Cooking losses (%) −0.605 −0.598 −0.607 Sarcomere length (µm) 0.748 0.803 0.795 WBSF (kg/cm2) −0.778 −0.747 −0.763 Total pigments (mg/g) 0.388 0.361 0.374 L* 0.541 0.567 0.565 a* 0.421 0.426 0.431 b* 0.610 0.569 0.590 pH48 −0.460 −0.489 −0.486 Rs = resistance; Xc = reactance; Z = impedance, calculated as (Rs2 + Xc2)0.5; IMF = intramuscular fat; WBSF = Warner–Bratzler shear force; L, a and b* = trichromatic coordinates (brightness, red and yellow, respectively); pH48 = pH 48 h after slaughter.1 p < 0.01. Rs = resistance; Xc = reactance; Z = impedance, calculated as (Rs2 + Xc2)0.5; IMF = intramuscular fat; WBSF = Warner–Bratzler shear force; L, a and b* = trichromatic coordinates (brightness, red and yellow, respectively); pH48 = pH 48 h after slaughter.1 p < 0.01. 3. Results 7 of 11 Foods 2020, 9, 836 The best multiple regression models for predicting physicochemical traits of LM, obtained by the stepwise regression procedure, are presented in Table 4. The multiple regression analysis of the data showed that a model with Rs and Xc explained 79.3% of the variation observed in IMF, with an RMSE of 1.631 and an RPD of 2.2. The main predictor was Rs, but the inclusion of Xc in the model increased the accuracy of the prediction by about six percentage points. The models for estimation of the other physicochemical traits showed much lower accuracy. Nevertheless, while the models for estimation of cooking losses, total pigments, L, a, and b, and pH48 only explained 13.4–36.0% (p < 0.01) of the variation observed in these traits, the models for estimation of total collagen, sarcomere length and WBSF still explained a considerable amount of the variation observed, respectively 51.3%, 64.4%, and 60.5% (p < 0.01). Table 4. Best multiple regression models for predicting longissimus thoracis et lumborum muscle (LM) physicochemical traits (n = 52) 1. Table 4. Best multiple regression models for predicting longissimus thoracis et lumborum muscle (LM) physicochemical traits (n = 52) 1. TRAIT Intercept Rs Xc Z k-Fold-R2 RMSE RPD IMF (%) −5.117 0.129 0.014 0.793 1.631 2.2 Total collagen (mg/g) 10.529 −0.0215 0.513 0.839 1.4 Cooking losses (%) 24.041 0.041 −0.075 0.346 2.096 1.2 Sarcomere length (µm) 1.291 0.0049 0.644 0.127 1.7 WBSF (kg/cm2) 8.612 −0.0358 0.605 0.631 1.6 Total pigments (mg/g) 1.709 0.015 0.134 0.762 1.1 L 23.958 0.064 0.308 3.232 1.2 a* 8.528 −3.149 −4.015 5.129 0.223 2.318 1.1 b* 5.338 0.042 0.360 1.174 1.3 pH48 6.382 0.300 0.377 −0.484 0.287 0.202 1.2 Rs = resistance; Xc = reactance; Z = impedance, calculated as (Rs2 + Xc2)0.5; IMF = intramuscular fat; WBSF = Warner–Bratzler shear force; L, a and b* = trichromatic coordinates (brightness, red and yellow, respectively); pH48 = pH 48 hours after slaughter. 1 All models are signiﬁcant at p < 0.01. 4. Discussion The value now obtained for the correlation between sarcomere length and WBSF is in line with the correlations obtained by Muchenje et al. (2008) [25] for the same traits (r = −0.47 and −0.58, respectively for meat aged for 2 and 21 days; p < 0.05). However, with this exception, the results obtained by Muchenje et al. (2008) [25] are quite diﬀerent from the present ones, with those authors reporting that most meat quality traits were not correlated (p > 0.05), while the present study showed signiﬁcant correlations between all meat quality traits analyzed. The negative correlation now obtained between IMF and total collagen is in agreement with Ponnampalam et al. (2015) [26]. Unlike the present study, Silva et al. (1999) [27] reported the absence of a signiﬁcant correlation between WBSF and IMF, but this may be related to the low level of IMF (1.9%) in the samples tested by those authors. Silva et al. (1999) [27] also reported the absence of a signiﬁcant correlation between WBSF and total collagen, as well as Maher et al. (2005) [28], but the positive correlation now shown between these two traits is close to the one observed by Torrescano et al. (2003) [29]. Torrescano et al. (2003) [29] showed no signiﬁcant correlation between sarcomere length and WBSF, but other authors have already shown a negative correlation between sarcomere length and WBSF (e.g., Ahnström et al., 2006 [30], and Silva et al., 1999 [27]) and, in fact, muscles with short sarcomere length are generally tough [31]. The signiﬁcant correlations now obtained, between BIA parameters and all the LM physicochemical traits analyzed, conﬁrmed the relationship, already shown by previous studies, between impedance and meat quality traits. In the case of IMF, total collagen, WBSF and sarcomere length, the high correlations now veriﬁed between these traits were reﬂected in moderate to high correlations of BIA parameters with the same four traits. Moreover, the sign of these correlations with BIA parameters reﬂected the sign of the correlations between the diﬀerent meat quality traits correctly, except in the case of a*, that just showed a negligible correlation with BIA parameters. 8 of 11 Foods 2020, 9, 836 Being fat an electrical insulator, the fat content of meat samples inﬂuences their bioelectrical impedance and Slanger and Marchello (1994) [32] developed a prediction equation that explained 83% of the variation observed in percentage fat of LM. Madsen et al. 4. Discussion (1997) [33], using their patented apparatus for measuring the IMF in carcasses or parts of carcasses, or the total content of fat in minced meat, explained 70.6% and 86.5% of the variation observed in IMF, respectively with one and two insertions. Damez and Clerjon (2013) [34] refer the lack of consistency in the measurement of fat content after rigor, due to the inﬂuence of membrane state on impedance, relating the good results of Slanger and Marchello (1994) [32] to the absence of membrane or extracellular compartment modiﬁcations immediately after slaughter and to the fact that the measurements were made at a stable temperature. The present results were obtained with samples kept at 2 ◦C for 12 days after slaughter and they are in the range of the results presented by Slanger and Marchello (1994) [32] and Madsen et al. (1997) [33]. Another question was raised by Ponnampalam et al. (2015) [26]—having tested meat samples with a fat content of 4.9% ± 2.5% wet weight; they concluded that bioimpedance spectroscopy was not suitable to predict fat content of meat samples low in fat. Altmann and Pliquett (2006) [35], comparing their percentage of 62.5% for correct classiﬁcations in beef above a threshold value of 2% with that of 80.2% obtained by Madsen et al. (1999) [36], for correct classiﬁcations above a threshold value of 3.5%, had already concluded that, depending on the level of IMF within a breed, low IMF was often predicted as too high or vice versa. Working with meat samples with a much wider range of fat content, Marchello et al. (1999) [37] also showed a remarkable diﬀerence in the accuracy of their impedance models to predict IMF. In fact, depending on the level of IMF, their impedance models explained 80% and 84–95% of the variation observed, respectively in beef trim and ground beef with 4.2 to 48.5% fat, but only explained 36% and 60–86% of the variation observed, respectively in beef trim and ground beef with less than 35% fat. In the present study, the IMF content ranged between 1.65% and 16.53%, and the best model still explained 79.3% of the variation observed, showing an RPD value of 2.2, which qualiﬁes as a very good prediction model. Considering only samples above threshold values of 2%, as Altmann and Pliquett (2006) [35], and 3.5%, as Madsen et al. 4. Discussion (1999) [36], there was no signiﬁcant change in the predictive value of the best model for the present data set (data not shown). The high correlation now obtained between BIA parameters and total collagen contrasts with the results of Lepetit et al. (2002) [38], that showed no trends between post-rigor impedance and the rank of muscles on their collagen content, ﬁnding support in the observation of Swatland (1980) [39] that a sheet of connective tissue has a similar impedance to meat. The fact is that, in the present study, the multiple regression analysis resulted in the development of a signiﬁcant prediction model that, with an RPD value of 1.4 qualiﬁes as a model of low to moderate predictive value. While Hopkins and Wang (2012) [40], showed no signiﬁcant correlation between impedance and 5-day shear force, Byrne et al. (2000) [41] had already reported a moderate to high correlation between impedance measurements and WBSF (r = 0.68, p < 0.001), and the present study showed a correlation signiﬁcantly higher. The best predictive impedance model only explained 60.5% of the variation observed in WBSF, but its RPD value of 1.6 means that it can still be used for assessment of WBSF. Concerning sarcomere length, Byrne et al. (2000) [41] showed no signiﬁcant correlations with impedance measurements, but the present study showed a high positive correlation between sarcomere length and BIA parameters. This resulted in a prediction model still useful for assessment of sarcomere length (RPD = 1.7). The moderate positive correlation now observed between WBSF and cooking losses compare to the moderate to high correlations (0.67 ≤r ≤0, 79; p < 0.001) observed by Silva et al. (1999) [27] and, similarly to WBSF, cooking losses also showed a moderate correlation with BIA parameters. This contrasts with the results of Byrne et al. (2000) [41], showing just a few signiﬁcant and poor correlations of cooking losses with impedance measured at diﬀerent times. The fact is that the best prediction model now developed, with RPD = 1.2, showed to have poor predictive value. Foods 2020, 9, 836 9 of 11 The low to moderate correlations of total pigments and trichromatic coordinates with BIA parameters are in line with the conclusion of Byrne et al. (2000) [41] that the few signiﬁcant correlations found with impedance measurements at speciﬁc times post mortem were of little value. 4. Discussion This was conﬁrmed by multiple regression analysis, showing RPD values between 1.1 and 1.4 for the best prediction models. p Callow (1936) [42] noticed that electrical impedance was related to ultimate pH and Bendall and Swatland (1988) [43] stated that electrical impedance was directly proportional to pH, with a curvilinear relationship, but Swatland (1995) [44] proposed that the relationship between impedance and pH was just indirect, due to a common relationship with ATP. While most studies in this area have focused on early detection of dark, ﬁrm and dry (DFD) meat and pale, soft and exudative (PSE) meat, Damez and Clerjon (2008) [45], besides pointing out that Guerrero et al. (2004) [46], for instance, showed no ability of electrical measurements in early detection of DFD, relate diﬃculties in the detection of PSE meats, during the period when rigor sets in, due to the fact that pH and temperature change rapidly in that period, while the associated structural changes that aﬀect the electric properties of meat evolve more slowly. Concurring to the results of the present study, Byrne et al. (2000) [41], studying the correlations of pH measurements taken at 2, 5, and 7 h, with impedance measurements made between 7 h and 14 days post mortem, also obtained signiﬁcant but low correlations between pH and impedance measurements. The fact is that the best impedance model now obtained must be qualiﬁed as a poor predictor of pH48 (RPD = 1.2). 5. Conclusions The present results conﬁrmed the potential interest of using BIA parameters for objective measurement of meat characteristics, since all traits of meat quality analyzed were correlated with BIA parameters, concurring to the type of correlation observed between each pair of traits. Moreover, the correlations with BIA parameters were validated with the development of signiﬁcant BIA prediction models for all traits. From the present data set, it can be concluded that BIA was able to predict the IMF in LM samples. Concerning the other physicochemical traits of LM samples, it was also possible to develop models that provided useful information for the assessment of total collagen, sarcomere length and WBSF, but further research is needed to develop BIA equipment for use in meat processing plants. Author Contributions: Conceptualization, S.S., J.A. and V.S.; methodology, S.S., V.S., J.S. and R.M.; validation, R.M. and A.T.; formal analysis, S.S., C.G. and A.T.; investigation, J.A.; resources, C.G.; writing—original draft preparation, J.A., S.S. and C.G.; writing—review and editing, C.G., V.S., J.S. and A.T.; visualization, J.A.; supervision, S.S.; project administration, C.G. and V.S.; funding acquisition, C.G. All listed authors meet the ICMJE criteria and all who meet the four criteria are identiﬁed as authors. We attest that all authors contributed signiﬁcantly to the creation of this manuscript, each having fulﬁlled criteria as established by the ICMJE. We conﬁrm that the manuscript has been read and approved by all named authors. We conﬁrm that the order of authors listed in the manuscript has been approved by all named authors. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the project UIDB/CVT/00772/2020 funded by the Fundação para a Ciência e Tecnologia (FCT). Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References Cheng, J.H.; Nicolai, B.; Sun, D.W. Hyperspectral imaging with multivariate analysis for technological applications in detecting quality attributes of red meats: A review. J. Food Eng. 2014, 132, 1–13. [CrossRef] 11. Cheng, J.H.; Nicolai, B.; Sun, D.W. Hyperspectral imaging with multivariate analysis for technological parameters prediction and classiﬁcation of muscle foods: A review. Meat Sci. 2017, 123, 182–191. [CrossRef] 12 Sil S R Af J M i A M i R C b A B i A C G d C M T i i A A li i 11. Cheng, J.H.; Nicolai, B.; Sun, D.W. Hyperspectral imaging with multivariate analysis for technological parameters prediction and classiﬁcation of muscle foods: A review. Meat Sci. 2017, 123, 182–191. [CrossRef] 12. 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https://openalex.org/W4377820239	https://www.researchsquare.com/article/rs-2913209/latest.pdf	English	null	Health Seeking Behaviour and Knowledge on Neonatal Danger Signs Among Neonatal Caregivers in Upper Denkyira East Municipality, Ghana	Research Square (Research Square)	2,023	cc-by	10,788	Health Seeking Behaviour and Knowledge on Neonatal Danger Signs Among Neonatal Caregivers in Upper Denkyira East Municipality, Ghana Philip Gyaase (  philipgyaase2@gmail.com ) Nursing and Midwifery Training College, Dunkwa-On-Offin Edward Aduse-Poku Nursing and Midwifery Training College, Dunkwa-On-Offin Mavis Opoku Lanquaye Nursing and Midwifery Training College, Dunkwa-On-Offin Emmanuel Boateng Acheampong Nursing and Midwifery Training College, Dunkwa-On-Offin David Ben Sampson Nursing and Midwifery Training College, Dunkwa-On-Offin Research Article Keywords: Health seeking behaviour, Knowledge and neonatal danger signs, lactating mothers Posted Date: May 23rd, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2913209/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at BMC Pediatrics on January 8th, 2024. See the published version at https://doi.org/10.1186/s12887-023-04430-2. Page 1/24 Page 1/24 Abstract Background: The purpose of the project was to assess the health seeking behaviour and knowledge on neonatal danger signs among lactating mothers in the Upper Denkyira East Municipality. Background: The purpose of the project was to assess the health seeking behaviour and knowledge on neonatal danger signs among lactating mothers in the Upper Denkyira East Municipality. Methods: The study used quantitative and employed a descriptive cross sectional survey to recruit mothers and neonatal care takers visiting postnatal clinic. The target population was mothers and neonatal care takers above 18 years visiting the facility and were willing to be part of the study. Total population for the study was 387 however, 381 responded to the questionnaire. Structured questionnaire was the main data collection tool for the study. Data were analysed with SPSS version 23.0. Logistic regression with Pearson Chi square, p-value and odd ratio were the main statistical methods for the data analysis. Results: The results showed that almost half of the respondents 138 (36.2%) stated that diarrhoea and vomiting constituted the major danger signs that sent their neonates to the hospital. Also the health seeking behaviour of the mothers showed positive results as most of the mothers (77.2%) attended hospital on their own volition. Finally, the association between mothers’ socio-demographic characteristics and recognition of neonatal danger signs showed that mothers educational level and occupation were statistically significant (p-value=0.000). Conclusion: The study concluded that mothers’ knowledge on neonatal danger signs was high and good health seeking behaviour. It is recommended that community health nurses and midwives should embark on home visits to encourage mothers to practice the knowledge and skills acquired during counselling at the hospital. Background Due to the high prevalence of newborn danger signs in this region, which accounts for 39% of all neonatal deaths worldwide, there are 31 deaths per 1000 live births in sub-Saharan Africa [9]. If prompt and appropriate healthcare measures are implemented, the majority of neonatal danger signs' avoidable causes, which account for around two thirds of neonatal danger signs, might be avoided [10]. The MDG- was created with a focus on lowering newborn morbidities and mortalities since Ghana has acknowledged the catastrophic effects of neonatal danger signals [11]. Neonatal mortality has maintained at 59 deaths per 1000 live births despite the nation's target for infant mortality being 26 deaths per 1000 live births [12]. Improving newborn health and survival requires timely and appropriate health seeking behavior and timely treatments [13]. This is only possible if moms have good awareness of and behavior about infant risk indications [14]; [15]. One of the emerging nations in Africa with a high newborn death rate is Ghana [16]. In Ghana, four out of ten (40%) deaths among children under five are attributable to neonatal mortality [17]. Neonatal mortality increased from 30 to 32 per 1000 live births as of 2019 [4] during the previous five years. Almost half (43%) of all births in Ghana today take place at home [18]. Many of these infants could be subjected to unwholesome care methods, especially if their moms are inexperienced [19]. International organizations have created a number of treatments to lower newborn morbidity and death [20]. The Every Newborn Action Plan (ENAP) of the United Nations International Children's Emergency Fund (UNICEF) and the World Health Organization (WHO) are two examples of such programs [20]. The fundamental goal of this action plan is to address the prevention and management of the major causes of neonatal death [21]. Promoting postnatal care throughout the newborn period is another area of cooperation between these two organizations [22]. The advice of visits by trained health workers to postnatal mothers' homes, especially during a baby's first week of life, is one important aspect of the promotion of postnatal care during the neonatal period [23]. However, there is no proof in writing that Ghanaian healthcare professionals are following this advice [24]. Background One of the most serious public health issues in the world today is neonatal mortality. Seventy-five percent of neonatal deaths occur in the first week of life, according to estimates that four million deaths occur worldwide each year during the first four weeks of life [1]. Ninety-nine percent of all newborn deaths take place in low- and middle-income nations, primarily in sub-Saharan Africa [2]. Most of these neonatal fatalities take place at home, which suggests that either few families are aware of the symptoms of neonatal sickness or that most newborns are not transferred to medical facilities when they are ill [3]. Neonatal danger signs are signs of serious illness that affects neonates during their neonatal period and that neonatal periods mark the first 28 days of neonatal life. Globally, neonatal danger signs are known to be associated health complications among neonates and among these complications are decreased neonatal growth and mental development, cerebral palsy and others among the notifiable cause of neonatal danger signs are neonatal jaundice, vomiting, cord sepsis, inability to suck breast milk, convulsions, hyperthermia, hypothermia, etc [4]. Findings have also indicated that, poor infrastructure in terms of poor resourced antenatal and post-natal services, poor home care for neonates by mothers due to information gap and decreased nutritional status of mothers contributes to increased occurrence of neonatal danger signs among neonates [5]. It is also found that, some socio demographic and sociocultural practices which defines health seeking behavior of neonatal mothers has a significant contribution to the occurrence of health related danger signs among neonates [6]. Worldwide, it is known that, about 7000 neonatal mortalities are recorded every day of which most of these deaths occurs in the first 24 hours of delivery or within the first of neonatal birth life. Scientific data have also estimated that, majority of under-five mortalities are attributed neonatal danger signs. The World Health Organization reported that about four million neonates lose their life during their neonatal period and 98% of these deaths occur in developing countries [7]. The report also estimated that, the risk of developing neonatal anger sign in a developing is six times higher than in the developed countries [8]. Page 2/24 Page 2/24 An estimated 1.16 million newborn deaths occur in Africa each year, with roughly one million of those deaths occurring in the first week of the neonatal period [8]. Background Another pertinent advice made by the WHO is that mothers and their newborns should be released from the hospital after 24 hours following an uncomplicated vaginal delivery, however this recommendation also doesn't seem to be followed [25]. Due to inadequate hospital facilities, many hospitals discharge mothers who delivered earlier than is advised [26]. Because there aren't enough beds in the maternity hospitals to monitor both laboring women and those in the fourth stage of labor, moms in the Upper Denkyira East, for instance, who have a normal delivery and their infants appear to be in good health, are released after twelve (12) hours [27]. Numerous of these beneficial suggestions that are intended to address the medical requirements of newborns do not appear to be receiving the required compliance [28]. The Ghana National Newborn Health Strategy and Action Plan, 2018– 2022, set a target of lowering newborn death to 21 per 1000 live births [29]. This is what they hoped to accomplish by endorsing postnatal care. The postpartum period is the perfect time to teach new moms about newborn care [30]. Global, regional, and local public health concerns surround neonatal morbidity and death [31]. About 38% of neonatal deaths worldwide, primarily in underdeveloped nations, are caused by neonatal danger signals, which are on the rise [32]. The majority of these deaths occur in the first 24 hours and the first week of newborn life, according to the World Health Organization, which estimates that 45% of under-five mortalities occur during the neonatal period [32]. According to estimates, around 10 million children die before turning one each year, and 98% of those deaths have place in developing nations [33]. According to reports, there are 130 million newborns born each year, and over 4 million infants die during the first 28 days, with the majority of these deaths occurring in less developed nations [34]. Additionally, it has been discovered that a newborn neonate's chance of having neonatal danger signs is around six times higher in developing countries than in industrialized ones [35]. The risk of perinatal death is increased by five times during pregnancy and during labor, which has a detrimental effect on the health of the newborn [36]. According to research, the first seven days following delivery account for around 75% of infant fatalities [37]. The study Area The study took place in the Upper Denkyira East Municipality (UDEM) of Ghana. This municipality is one of the twenty-two (22) Administrative Metropolitan, Municipalities and Districts of the Central Region of Ghana. The Municipality was established in 2007 by Legislative Instrument (LI 1877) from the then Upper Denkyira District in pursuance of deepening Decentralization process in Ghana. It was inaugurated in February, 2008. The Administrative Capital is Dunkwa-On-Offin. It lies within latitudes 50.30’ and 60.02’ north of the equator and longitudes 10 W and 20 W of the Greenwich Meridian. It shares common boundaries with Adansi South in the north, Assin central Municipal in the east and Twifo Atti-Morkwa District in the west and Upper Denkyira West District in the north-west. The upper Denkyira East Municipality covers a total land area of 1,020 square kilometres, which is about 10% of the total land area of the Central Region [44]. The population of Upper Denkyira East Municipality according to the 2021 Population and Housing Census, is 110,141 representing 3.3 percent of the region’s total population. Fifty percent of the Municipality’s population is rural with poor health care. The study Area has twenty three health institutions made up of hospitals, health Centers, rural Clinics, and private clinics. Others are private maternity homes and Community Health Planning and Services (CHPS) Compounds. The study site was Presbyterian hospital. The Hospitals have an Out-patient Department (OPD), an Antenatal Clinic (ANC), Obstetric and Gynaecological unit, post-natal unit, Neonatal Intensive Care unit, wards and functional Medical Laboratory Unit. , post-natal unit, Neonatal Intensive Care unit, wards and functional Medical Laboratory Background Page 3/24 Neonatal mortality is on the rise, and this rise is perceived as a threat rather than a burden, according to a global agenda on the subject [38]. According to studies, Nigeria has the highest rate of neonatal deaths, and if prompt recognition, diagnosis, and treatment are not provided, a neonate will die within a minute [38]. Another study estimated that about 28% of deaths in children under the age of five are caused by neonatal danger signs. The majority of neonatal deaths in Ghana are due to neonatal danger signs like infections, birth asphyxia, preterm, and low birth weight [39]. Ghana has a high rate of neonatal danger signs. Due to neonatal danger indications, there has been an increase in hospitalizations and fatalities. Data from the Upper Denkyira East Municipal Health Directorate are already available, and they show that there were 656 admissions overall in 2020, with 41 (6.3%) of those resulting in newborn fatalities [40]. Between January 2020 and May 2020, there were 264 neonatal admissions to hospitals, out of which 18 (6.8%) fatalities were noted [40]. Although studies have demonstrated that neonates can display dangerous neonatal symptoms, other researchers believe that this issue is more prevalent in the study site [41]. Postpartum mothers' awareness of neonatal care procedures is improved by health education [42]. If moms are knowledgeable on how to provide good infant care, these neonatal deaths may be decreased [41]. Some women lack knowledge and expertise about newborn routines and care. Some adolescent moms exhibit poor newborn care, as evidenced by their delayed breastfeeding beginning and incorrect feeding habits [43]. After birth, the health of the neonate largely depends upon the care and practices adopted by their mothers; hence, the need to assess health seeking behaviour, knowledge and association between the socio-demographic characteristics of mothers and the knowledge on neonatal danger signs on neonatal danger signs among mothers in the Upper Denkyira East Municipality to provide information to educate and empower mothers to reduce morbidity and mortality during neonatal period. Sample size and sampling technique A simple random sampling technique was employed in the sampling of study participants. A random sampling of mothers/caregivers of neonatal babies who delivered within the 28 days and were 18 years and above were chosen from neonatal unit, clinic or ward in Upper Denkyira East Municipality. The researcher reviewed the daily charts to determine the list of eligible participants. Using a flip of the coin, a list of potential participants was generated for the study. Data were collected over a period of three weeks. The study employed Cochran Formula to determining the study minimum sample size; . Where n was the minimum sample population thus proportion of population that has the variable of interest. (p=0.40) q was the proportion of the population without the actual variable of interest. (q=1-p) Z was the confidence level set at 95% e was the sampling error or level of precision set at 5% using the above formulae with 5% unresponsive rate the required sample size for the study was 387. This was calculated from the total caregivers/mothers of 2650 from January to December, 2022. n = ZΛ2 (pq) ÷ (eΛ22) Study population The study used mothers with neonates and other neonatal caregivers above eighteen years who were visiting the various postnatal clinics in the Upper Denkyira East Municipality and were willing to take part in the study. Study design Page 4/24 Page 4/24 Page 4/24 Page 4/24 The study employed a descriptive cross sectional study designs. Descriptive survey deals with situations that occur, performance that prevail and beliefs that are on-going and styles that are emerging [45]. The descriptive survey design method deals with questions concerning the pertaining issues or situation. The design was chosen due to its ability to take data about people for a specific period of time without embarking on any follow-up. A descriptive cross sectional design gave the researchers the opportunity to examine the health seeking behaviours and knowledge of lactating mothers in their natural environment. The features of the population were also described as well as their responses. The quantitative method approach was adopted for the study. This approach allowed the researchers to use a broader sample size which was most likely ensured a more representative view of the study population. With this approach, observable data were gathered to answer the research questions using statistical, computational or mathematical techniques. Study Variables The study variables were divided into two, the dependent and the independent variables. The dependent variables were variables that are tested and measured in a research work whilst the independent variables were the variables that were controlled in a research work to test the effects on the dependent variables. Page 5/24 Page 5/24 Table 1 Table 1 Logical Framework of the study variables Variable Operational Definition Level of Measurement Type of Variable Age Above eighteen years Ratio Continuous Marital status Married, Single, Co habiting Nominal Categorical Education level No Formal Education, Formal Education. Nominal Categorical Employment Employed, Unemployed Nominal Categorical Umbilical Discharge Presence of wet and smelly umbilical cord. Nominal Categorical Yellowing of neonatal body Yellowish of neonatal skin and face Nominal Categorical Temperature Neonatal body too cold and hot Ratio Continuous Poor Feeding Neonatal inability to adequately suck breast milk Nominal Categorical Convulsion Continuous involuntary movement of the body Nominal Categorical Vomiting Excess fluid from neonatal mouth Nominal Categorical Cultural practices, beliefs Practices that prevent mothers to seek healthcare for sick neonates. Nominal Categorical Accessibility to healthcare Closeness of health facility for medical care Nominal Categorical Altitude of health staff Health workers behavior that prevent mothers to seek health for their neonates when sick. Nominal Categorical Decision making with regards to neonatal health Power to make decision when baby is sick and when to take to hospital Nominal Categorical Logical Framework of the study variables Operational Definition Level of Measurement Type of Variable Data Collection Data collection was done by the use of a structured questionnaire. The questionnaire comprised four sections which are: personal and general information on respondents, knowledge of mothers on neonatal danger signs, health seeking behaviours of mothers on neonatal danger signs and Socio-demographic characteristics of mothers that were associated with neonatal danger signs. Bias in the data collection was as much as possible avoided. This was done as the principal investigator visited the facilities during the data collection to review secondary data of the health facilities. This helped compliment the main data to avoid or reduce bias to the minimum level. Pre-testing In the Diaso Hospital in the Upper Denkyira West District, pre-testing of the data collection instruments took place. The pre- test facility was situated outside the study area, but in terms of personnel, facilities given to clients and the configuration of the wards, it had similar characteristics. The pre-testing helped classify certain difficulties that were linked to the understanding of the respondents. The researcher pre-tested the questionnaire on 30 neonatal caregiver or mothers to check for reliability and validity of the instruments. Corrections in the questionnaire were made by experts and the research team members. The Cronbach Alpha co-efficient was calculated for the questionnaire and yielded 0.803 hence reliable and valid study instrument. Data Analysis Data Analysis Page 6/24 Page 6/24 The data for the study were analyzed using SPSS version 23.0. The categorical variables analysed using descriptive statistics and data presented in percentages, mean, standard deviation. The analysis of association between normally distributed variable was done using Pearson Chi-square and p-value were used to assess the association between the respondents’ sociodemographic characteristics and knowledge on neonatal danger signs. A probability value of less than 0.05 was considered as having a significant statistical association. Missing data were addressed by referring to the primary data to enter any oversight data. Any data genuinely missing from the primary data were excluded. To reduce missing data, the investigator made sure respondents completed all items on the questionnaire before retrieval. Ethical approval of the study was obtained from the Ghana Health Service Ethics Review Committee with identification number: GHS-ERC 037/10/22. Data Collection commenced after ethical approval was granted. Permission was also sought from the Municipal Director of Health Services (UGEM) and the Management of the various Health Facilities used for the study. The purpose of the study was discussed with the respondents for their informed consent. Written informed consent was signed by the respondents prior to participating in the study. Since the study used some illiterates an informed consent was obtained from their legal guardians. Each participant was taking through the study purpose and eligible persons were made to sign a consent form to indicate their acceptance to be part of the study. Respondents who could not read or write were given interpreters to translate the English to their local language (Twi). Respondents were assured of confidentiality and anonymity for the information provided. The respondents were given the free will to take part in the study and stopped whenever they desired to stop. Socio-demographic Characteristics of the Respondents Most of the respondents 207(54.3%) were within the age range of 21–30 while 63(16.5%) were below 21 years. Majority of the respondents 247(64.8%) were Christians as against 134(35.2%) who were Moslems. An overwhelming majority of the respondents 260(68.2%) were Akans with 33 (8.7%) been Ga-Adangbe. Again, 138(36.2%) of the respondents had Tertiary educational certificate followed by Senior High while the minority had no formal education. About half of the respondents 145(38.1%) were public workers while 46(12.1%) were farmers. Lastly, an overwhelming majority of the respondents 347(91.1%) delivered at the hospital whist 34(8.9%) delivered at home as shown in Table 2. Page 7/24 Page 7/24 Table 2 Socio-demographic Characteristics of the Respondents Variables Frequency n = 381 Percent Age range Below 21 years 21–30 years 31–40 years Mean = 27; SD 15.5 63 207 111 16.5 54.3 29.1 Religion Christianity Moslem 247 134 64.8 35.2 Tribe Akan Ewe Ga-Adangbe Northerner 260 48 33 40 68.2 12.6 8.7 10.5 Educational level No formal education Primary JHS SHS Tertiary 30 31 86 96 138 7.9 8.1 22.6 25.2 36.2 Occupation Trader Artisan Farmer Public worker Unemployed 58 75 46 145 57 15.2 19.7 12.1 38.1 15.0 Place of delivery Hospital Home 347 34 91.1 8.9 rs on Neonatal Danger Signs mothers on neonatal danger signs; majority of the respond ( ) Table 2 Moslem Tribe Akan Ewe Ga-Adangbe Northerner 260 48 33 40 68.2 12.6 8.7 10.5 Educational level No formal education Primary JHS SHS Tertiary 30 31 86 96 138 7.9 8.1 22.6 25.2 36.2 Occupation Trader Artisan Farmer Public worker Unemployed 58 75 46 145 57 15.2 19.7 12.1 38.1 15.0 Place of delivery Hospital Home 347 34 91.1 8.9 Knowledge of Mothers on Neonatal Danger Signs Knowledge of Mothers on Neonatal Danger Signs Knowledge of Mothers on Neonatal Danger Signs Table 3 shows the knowledge of mothers on neonatal danger signs; majority of the respondents 244(64.0%) practiced exclusive breastfeeding whereas 137(36.0%) practiced mixed feeding with the reason been insufficient breastmilk production 67(48.9%) as against 16(11.6%) who said nipple problem. Also, an overwhelming majority of the respondents Page 8/24 375(98.4%) stated that their babies were able to feed on breastmilk whilst 6(1.6%) said no. Furthermore, most of the respondents 347(91.1%) gave the yellowish breastmilk to their babies whilst 34(8.9%) expressed and discarded the yellowish breastmilk. Again, almost half of the respondents 187(49.1%) used the healthy looks of their babies to determine whether the breastfeeding was going on well whilst 15(3.9%) used the weight of their babies. Moreover, majority of the respondents 254(66.7%) strongly agreed that the cord of their babies must be dressed and covered whilst 3(0.8%) strongly disagreed. Also, most of them 249(65.4%) disagreed that bleeding, discharging, redness and swelling of the baby’s cord was normal as against 6(1.6%) who saw it to be normal. An overwhelming majority of the respondents 324(85.0%) used methylated spirit as the main medication used for dressing their babies’ cords with most of them 240(63.0%) washing their hands sometimes before caring for their babies’ cords. 375(98.4%) stated that their babies were able to feed on breastmilk whilst 6(1.6%) said no. Furthermore, most of the respondents 347(91.1%) gave the yellowish breastmilk to their babies whilst 34(8.9%) expressed and discarded the yellowish breastmilk. Again, almost half of the respondents 187(49.1%) used the healthy looks of their babies to determine whether the breastfeeding was going on well whilst 15(3.9%) used the weight of their babies. Moreover, majority of the respondents 254(66.7%) strongly agreed that the cord of their babies must be dressed and covered whilst 3(0.8%) strongly disagreed. Also, most of them 249(65.4%) disagreed that bleeding, discharging, redness and swelling of the baby’s cord was normal as against 6(1.6%) who saw it to be normal. An overwhelming majority of the respondents 324(85.0%) used methylated spirit as the main medication used for dressing their babies’ cords with most of them 240(63.0%) washing their hands sometimes before caring for their babies’ cords. 375(98.4%) stated that their babies were able to feed on breastmilk whilst 6(1.6%) said no. Furthermore, most of the respondents 347(91.1%) gave the yellowish breastmilk to their babies whilst 34(8.9%) expressed and discarded the yellowish breastmilk. Conditions that would make mother send her neonate immediately to a health facility From Fig. 1, majority of the respondents 138(36.2%) stated that diarrhoea/vomiting constituted the major danger sign that sent their neonates in to health care facilities followed by crying 87(22.8%) whilst the few of them 3(0.8%) said when the neonates were looking weak in appearance. Knowledge of Mothers on Neonatal Danger Signs Again, almost half of the respondents 187(49.1%) used the healthy looks of their babies to determine whether the breastfeeding was going on well whilst 15(3.9%) used the weight of their babies. Moreover, majority of the respondents 254(66.7%) strongly agreed that the cord of their babies must be dressed and covered whilst 3(0.8%) strongly disagreed. Also, most of them 249(65.4%) disagreed that bleeding, discharging, redness and swelling of the baby’s cord was normal as against 6(1.6%) who saw it to be normal. An overwhelming majority of the respondents 324(85.0%) used methylated spirit as the main medication used for dressing their babies’ cords with most of them 240(63.0%) washing their hands sometimes before caring for their babies’ cords. Page 9/24 Table 3 Knowledge of Mothers on Neonatal Danger Signs Variables F = 381 Percent (%) What type of breastfeeding are you practicing? Exclusive breastfeeding Mixed feeding 244 137 64.0 36.0 Reasons for practicing mixed feeding Insufficient breastmilk Working situation Nipple problem 67 54 16 48.9 39.4 11.6 Did you cover your baby after birth? Yes No 375 6 98.4 1.6 What did you do with the yellowish breastmilk? Given to the baby Expressed and discarded 347 34 91.1 8.9 How will you know if your breastfeeding is doing well? Baby looks healthy Baby is calmed and relaxed Baby reaches or roots for breast if hungry Baby gains weight 187 161 18 15 49.1 42.3 4.7 3.9 The cord of your baby must be dressed and covered Strongly agree Agree Disagree Strongly disagree 254 97 27 3 66.7 25.5 7.1 0.8 Babies should be bathed immediately after birth Strongly agree Agree Disagree Strongly disagree 18 6 249 108 4.7 1.6 65.4 28.3 Knowledge of Mothers on Neonatal Danger Signs Page 10/24 Page 10/24 What main medication/substance do you use in dressing your baby’s cord? Methylated spirit Chlorhexidine Coconut oil Brine (salt water) 324 45 9 3 85.0 11.8 2.4 0.8 How often do you wash your hands before caring for your baby’s cord? Always Sometimes Scarcely Not at all 110 240 25 6 28.9 63.0 6.6 1.6 Conditions that would make mother send her neonate immediately to a health facility Who normally advised caregivers/mothers to go to the hospital Figure 2 below indicates those who advised respondents to go to the hospital; an overwhelming majority of the respondents 294(77.2%) indicated that they went to the hospital on their own volition whilst 18(4.7%) went to the hospital upon the advice of their parents. Health Seeking Behaviours of caregivers/mothers with neo Table 4 depicts the health seeking behaviours of the respondents; almost all the respondents 360(94.5%) attended postnatal clinic at the Mission hospital 178(49.4%) followed by Public hospital 110(30.5%) whilst few of them 72(20.0%) attended Private hospitals. Again, almost half of the respondents 120(33.3%) attended postnatal clinic thrice as against 33(9.1%) who attended five times. Majority of the respondents 274(76.1%) started their antenatal visits during the first trimester whilst 7(1.9%0 started during 3rd trimester. Also, an overwhelming majority of the respondents 327(85.8%) delivered at term whilst 54(14.2%) delivered preterm. Page 11/24 Page 11/24 Table 4 Table 4 Health seeking behaviours of caregivers/mothers with neonatal danger signs Variables Frequency Percent (%) Postnatal care Attendant Non-attendant Total 360 21 381 94.5 5.5 100.0 If attendant where did you receive care? Mission hospital Public hospital Private hospital Total 178 110 72 360 49.4 30.5 20.0 100.0 Number of postnatal visits Once Twice Thrice Four Five Total 45 50 120 112 33 360 12.5 13.8 33.3 31.1 9.1 100.0 Which trimester did you start antenatal clinic? 1st trimester 2nd trimester 3rd trimester Total 274 79 7 360 76.1 21.9 1.9 100.0 Gestational age at delivery Term Preterm Total 327 54 381 85.8 14.2 100.0 advised caregivers/mothers to go to the hospital tes those who advised respondents to go to the hospital; an overwhelming majorit 2%) indicated that they went to the hospital on their own volition whilst 18(4.7%) w eir parents. The Association between the Socio-demographic characteristics of mothers and the knowledge on neonatal danger signs Page 12/24 Page 12/24 Table 5 shows the bivariate analysis of the association between the socio-demographic characteristics of mothers and their knowledge on neonatal danger signs, the following variables proved statistically significant; age (p-value = 0.005), marital status (p-value = 0.000), religion (p-value = 0.004), tribe (p-value = 0.000), educational level (p-value = 0.000) and occupation (p-value = 0.000) whereas place of birth was not statistically significant with p-value = 0.165. Table 5 shows the bivariate analysis of the association between the socio-demographic characteristics of mothers and their knowledge on neonatal danger signs, the following variables proved statistically significant; age (p-value = 0.005), marital status (p-value = 0.000), religion (p-value = 0.004), tribe (p-value = 0.000), educational level (p-value = 0.000) and occupation (p-value = 0.000) whereas place of birth was not statistically significant with p-value = 0.165. Table 5 shows the bivariate analysis of the association between the socio-demographic characteristics of mothers and their knowledge on neonatal danger signs, the following variables proved statistically significant; age (p-value = 0.005), marital status (p-value = 0.000), religion (p-value = 0.004), tribe (p-value = 0.000), educational level (p-value = 0.000) and occupation (p-value = 0.000) whereas place of birth was not statistically significant with p-value = 0.165. Table 5 shows the bivariate analysis of the association between the socio-demographic characteristics of mothers and their knowledge on neonatal danger signs, the following variables proved statistically significant; age (p-value = 0.005), marital status (p-value = 0.000), religion (p-value = 0.004), tribe (p-value = 0.000), educational level (p-value = 0.000) and occupation (p-value = 0.000) whereas place of birth was not statistically significant with p-value = 0.165. The Association between the Socio-demographic characteristics of mothers and the knowledge on neonatal danger signs Page 13/24 Table 5 d The bivariate analysis of the association between the socio-demographic characteristics of mothers and the knowledge on neonatal danger signs neonatal danger signs Variables Knowledge on conditions that neonates would be sent to the hospital χ2(p-value) Seizures f(%) Diarrhoea/ Vomiting f(%) Crying f(%) Not feeding well f(%) Looking weak f(%) Fever f(%) Discharging cord f(%) Age range Below 21years 21-30years 31-40yreas Total 6(1.6) 25(6.6) 18(4.7) 49(12.9) 15(3.9) 76(19.9) 47(12.3) 138(36.2) 21(5.5) 45(11.8) 21(5.5) 87(22.8) 3(0.8) 9(2.4) 10(2.6) 22(5.8) 0(0.0) 3(0.8) 0(0.0) 3(0.8) 18(4.7) 39(10.2) 12(3.1) 69(18.1) 0(0.00) 10(2.6) 3(0.8) 13(3.4) 28.278(0.005) Marital status Single Married Co-habiting Divorced Total 6(1.6) 37(9.7) 6(1.6) 0(0.00) 49(12.9) 30(7.9) 69(18.1) 39(10.2) 0(0.00) 138(36.2) 36(9.4) 36(9.4) 15(3.9) 0(0.00) 87(22.8) 3(0.8) 16(4.2) 3(0.8) 0(0.0) 22(5.8) 3(0.8) 0(0.0) 0(0.0) 0(0.0) 3(0.8) 21(5.5) 36(9.4) 9(2.4) 3(0.8) 69(18.1) 7(1.8) 6(1.6) 0(0.0) 0(0.0) 13(3.4) 56.906(0.000) Religion Christianity Moslem Total 33(8.7) 16(4.2) 49(12.9) 93(24.4) 45(11.8) 138(36.2) 48(12.6) 39(10.2) 87(22.8) 16(4.2) 6(1.6) 22(5.8) 3(0.8) 0(0.0) 3(0.8) 51(13.4) 18(4.7) 69(18.1) 3(0.8) 10(2.6) 13(3.4) 19.280(0.004) Tribe Akan Ewe Ga-Adangbe Northerner Total 34(8.9) 12(3.1) 3(0.8) 0(0.0) 49(12.9) 95(24.9) 15(3.9) 18(4.7) 10(2.6) 138(36.2) 57(15.0) 18(4.7) 3(0.8) 9(2.4) 87(22.8) 10(2.6) 0(0.0) 6(1.6) 6(1.6) 22(5.8) 3(0.8) 0(0.0) 0(0.0) 0(0.0) 3(0.8) 54(14.2) 0(0.0) 3(0.8) 12(3.1) 69(18.1) 7(1.8) 3(0.8) 0(0.0) 3(0.8) 13(3.4) 74.182(0.000) Page 14/24 Page 14/24 Educational level No formal edu. Primary JHS SHS Tertiary Total 3(0.8) 3(0.8) 3(0.8) 9(2.4) 31(8.1) 49(12.9) 15(3.9) 6(1.6) 31(8.1) 27(7.1) 59(15.5) 138(36.2) 0(0.0) 9(2.4) 21(5.5) 24(6.3) 33(8.7) 87(22.8) 0(0.0) 0(0.0) 4(1.0) 12(3.1) 6(1.6) 22(5.8) 0(0.0) 3(0.8) 0(0.0) 0(0.0) 0(0.0) 3(0.8) 9(2.4) 6(1.6) 24(6.3) 21(5.5) 9(2.4) 69(18.1) 3(0.8) 4(1.0) 3(0.8) 3(0.8) 0(0.0) 13(3.4) 105.475(0.000) Occupation Trader Artisan Farmer Public worker Unemployed Total 0(0.0) 6(1.6) 6(1.6) 28(7.3) 9(2.4) 49(12.9) 21(5.5) 24(6.3) 21(5.5) 63(16.5) 9(2.4) 138(36.2) 9(2.4) 15(3.9) 6(1.6) 36(9.4) 21(5.5) 87(22.8) 0(0.0) 9(2.4) 4(1.0) 9(2.4) 0(0.0) 22(5.8) 3(0.8) 0(0.0) 0(0.0) 0(0.0) 0(0.0) 3(0.8) 18(4.7) 21(5.5) 3(0.8) 9(2.4) 18(4.7) 69(18.1) 7(1.8) 0(0.0) 6(1.6) 0(0.0) 0(0.0) 13(3.4) 136.249(0.000) Place of delivery Hospital Home Total 46(12.1) 3(0.8) 49(12.9) 125(32.8) 13(3.4) 138(36.2) 78(20.5) 9(2.4) 87(22.8) 22(5.8) 0(0.0) 22(5.8) 3(0.8) 0(0.0) 3(0.8) 60(15.7) 9(2.4) 69(18.1) 13(3.4) 0(0.0) 13(3.4) 9.156(0.165) Knowledge of mothers on neonatal danger signs Regarding heat control, it is advised that the newborn be wrapped right away and placed on the mother's chest or belly, and that the first bath be postponed ideally for 24 hours. The vast majority of moms in this survey said that the infant should be wrapped and placed on the mother's chest, which is in line with the WHO's 2018 recommendation. The time of the baby's first bath may not be right because just 6% of the moms thought that babies should be bathed right away after delivery. This was discovered to be different from research results by [9], which showed that just 7% of moms knew the right moment for the baby's first bath. Early bathing puts the newborn at risk for hypothermia, which could have detrimental effects on their health. More over half of the moms (64.0%) were aware of the ideal feeding techniques for newborns (exclusive breastfeeding). It was discovered that this matched up with studies done in Ghana [4]. There are several benefits to starting nursing early. Early breastfeeding benefits mothers because it increases milk production and encourages the release of oxytocin, which aids in uterine contraction and lessens postpartum blood loss. Colostrum, which is particularly nutrient-rich and contains antibodies that shield the infant from illnesses, is found in the first breast milk. This result is consistent with the current study, in which the majority of women (91.1%) gave their infants the yellowish colostrum. In relation to how mothers got to know whether their babies were breastfeeding well, most of the mothers stated when the baby looked healthy was the indication that he/she was breastfeeding well. This finding is in line with a number of empirical studies that have been conducted on the breastfeeding practices of postnatal mothers in general. Using secondary data analysis from a randomized control trial, a study on how inadequate postpartum breastfeeding among Nigerian moms contributes to early neonatal danger signs [9] was conducted. Primips made comprised the population being researched, with 97 women in the experimental group and 115 in the control group. Analysis revealed that neonates with bad eating habits were at higher risk than those with good feeding habits, and mothers' awareness of inadequate feeding aided in the management of newborn danger indicators. Mothers were able to identify the following neonatal warning signs: diarrhoea/vomiting, crying, fever, convulsions, not feeding properly, discharging cord, and appearing weak. Knowledge of mothers on neonatal danger signs Typically, a condition causes vomiting. Spitting up and vomiting may typically be distinguished by seasoned mothers, but first-time parents may need to consult a doctor or nurse. Dehydration can result from vomiting because fluids are lost. Children occasionally struggle to drink enough to replace lost fluids, either because they are still throwing up or because they do not want to. Typically, children who are vomiting do not want to eat, although this is rarely an issue. In order to get rid of poisonous drugs that have been ingested, vomiting can be helpful [18]. However, the most frequent cause of vomiting is diseases. Vomiting can occasionally indicate a serious problem, such as a blockage in the stomach or intestines or increased pressure inside the brain (intracranial hypertension), even though the illness is normally very innocuous [19]. Knowledge of mothers on neonatal danger signs The findings in this study revealed that mothers had good knowledge on essential newborn care and neonatal danger signs that call for immediate care at health facility. This suggests that mothers can recognize the warning signals of newborn illnesses and take their children to the hospital. Contrarily, a comparable study conducted by [5] in rural Ghana's Northern region indicated that 20% of mothers could name at least four newborn danger indicators, which was based on the mothers' knowledge of neonatal danger signs as a proxy. The reason for the difference may be because women today are more conscious than they were seven years ago of the need of providing for their newborns. Additionally, the knowledge findings in this study are comparable to those of a study [8] conducted in Ethiopia, where it was discovered that 84% of mothers had enough knowledge of crucial newborn care. Specifically, on cord care, more than half of the mothers (63.0%) in this study indicated that they sometimes washed their hands before caring for their baby’s cord while few (6%) of the mothers also indicated that they never washed their hands at all. On the medication to apply on the cord, majority of the mothers used methylated spirit as compared to chlorhexidine (11.8%). This is inconsistent with Word Health Organization’s 2018 recommendation on cord care especially with the used of the methylated spirit [3]. However, this could be due to the fact that the chlorhexidine slows the hearing of the cord. Page 15/24 Page 15/24 Page 15/24 Regarding heat control, it is advised that the newborn be wrapped right away and placed on the mother's chest or belly, and that the first bath be postponed ideally for 24 hours. The vast majority of moms in this survey said that the infant should be wrapped and placed on the mother's chest, which is in line with the WHO's 2018 recommendation. The time of the baby's first bath may not be right because just 6% of the moms thought that babies should be bathed right away after delivery. This was discovered to be different from research results by [9], which showed that just 7% of moms knew the right moment for the baby's first bath. Early bathing puts the newborn at risk for hypothermia, which could have detrimental effects on their health. The health seeking behaviour of neonatal caregivers Similar findings were made by [12] in their research in Ethiopia, which indicated that postnatal counselling received & the location of delivery were independent predictors of early breastfeeding. first trimester were more likely to provide their babies with high-quality newborn feedings than mothers who started ANC in the second and third trimesters. Similar findings were made by [12] in their research in Ethiopia, which indicated that postnatal counselling received & the location of delivery were independent predictors of early breastfeeding. The findings again showed that majority of the respondents attended the postnatal clinic three times. Regular attendance made the mothers more knowledgeable on the danger signs of the neonates. This result is consistent with a study done in India, where it was discovered that a mother's postpartum attendance had a substantial relationship to safe cord care and the identification of other neonatal risk indicators. The same study discovered that women with one attendance had lower rates of recommended newborn care practices than mothers with many attendances [33]. Again, it was discovered that few mothers gave birth prematurely whereas most did so at term. Preterm infants appeared to be less healthy than full-term infants. In order to improve newborn health in poor nations, gestational age is a crucial element to take into account. This is because there aren't enough or any basic obstetric health services, which leads to the majority of preterm births. However, healthy habits and practices when they return home have an impact on babies who are born at term [5]. Numerous nations have included various methods into their health programs to make it easier to identify these health issues and lower newborn mortality [6]. The assessment of general danger signals in the examination of neonates presenting with illness at healthcare facilities is a key component of the Integrated Management of Newborn and Childhood Illness (IMNCI) developed by the WHO [2]. The incidence of under-five mortality has steadily declined worldwide during the past few decades. Nearly two-thirds of all deaths in the first year of life and 40% of deaths before the age of five occur during the neonatal era. Due to a shift in certain healthcare professionals' attitudes about unwell babies and their mothers, this achievement has been curtailed. There is evidence that reducing the percentage of home deliveries could cut prenatal deaths in half [4]. The health seeking behaviour of neonatal caregivers This objective sought to assess mothers’ health seeking behaviour with regards to the danger signs associated with their neonates. In this study 94.5% of the mothers attended postnatal clinic. Most of them attended the mission hospital and this could be due to the fact that the major hospital in the municipality is a mission hospital. Similarly a study in India, Uttar Pradesh showed that postnatal attendance by mothers and skilled attendance at delivery were significantly associated with newborn care practice when compared to mothers who did not attend postnatal and antenatal clinics and mothers who were attended at delivery by either relatives or traditional birth attendants [30]. This implies that both antenatal and postnatal attendance is crucial in the care of the neonates. This objective sought to assess mothers’ health seeking behaviour with regards to the danger signs associated with their neonates. In this study 94.5% of the mothers attended postnatal clinic. Most of them attended the mission hospital and this could be due to the fact that the major hospital in the municipality is a mission hospital. Similarly a study in India, Uttar Pradesh showed that postnatal attendance by mothers and skilled attendance at delivery were significantly Uttar Pradesh showed that postnatal attendance by mothers and skilled attendance at delivery were significantly associated with newborn care practice when compared to mothers who did not attend postnatal and antenatal clinics and mothers who were attended at delivery by either relatives or traditional birth attendants [30]. This implies that both antenatal and postnatal attendance is crucial in the care of the neonates. In a related study, it was discovered that mothers who visited a postnatal clinic (PNC) after giving birth and who received advice on how to care for the newborn were more likely to practice the advised newborn care than mothers who did not visit a PNC and who did not receive newborn care advice [15]. The majority of the women attended an antennal clinic during the first trimester, according to the findings. This backs up a study by [24] that found that moms who started ANC early in the Page 16/24 Page 16/24 Page 16/24 first trimester were more likely to provide their babies with high-quality newborn feedings than mothers who started ANC in the second and third trimesters. The association between mothers’ socio-demographic characteristics and neonatal danger signs In this study the only socio-demographic variable that showed significant association were educational level and occupation of the mothers. Mothers with JHS education were four times likely to have knowledge about neonatal danger signs as compare to mothers who had primary or no formal education. This was found to be similar to finding by [4] where they found that women whose least educational level was JHS were more likely to know about danger signs of their babies and good feeding practices than mothers who had no formal education. Similarly studies conducted in Uganda and India showed that mothers’ education status had significant association with newborn care practices such as delay bathing, umbilical care, vaccination and proper eye care [12]. Studies conducted in Ethiopia on newborn care practice also corroborated finding from other studies that found mother’s level of education to be positively associated with good newborn practice [23]. In this study the only socio-demographic variable that showed significant association were educational level and occupation of the mothers. Mothers with JHS education were four times likely to have knowledge about neonatal danger signs as compare to mothers who had primary or no formal education. This was found to be similar to finding by [4] where they found that women whose least educational level was JHS were more likely to know about danger signs of their babies and good feeding practices than mothers who had no formal education. Similarly studies conducted in Uganda and India showed that mothers’ education status had significant association with newborn care practices such as delay bathing, In contrast to this study other studies found a relationship between other socio-demographic characteristics of mother. Similar study in their study found that maternal age was associated with early initiation of breast feeding, health seeking behaviour and cord care [8]. Studies also found significant association between marital statuses of women with neonatal care and exclusive breastfeeding [11]. Again mothers’ occupation had significant association with knowledge on neonatal danger signs. This supports [19], stating that salary mothers are about seven times likely to practice essential newborn care compared to unemployed and other workers. Also employed mothers were ten times likely to practice essential neonatal care compared to unemployed mothers [13]. The association between mothers’ socio-demographic characteristics and neonatal danger signs The observed differences in the findings of this study and others in literature regarding relationship between socio- demographic characteristics of mothers and neonatal danger signs may be due to differences in socio-demographic characteristics. There could be other powerful factors that influence mother knowledge on neonatal danger signs that were not explored. It was expected that mother’s place of birth would have a strong effect on their knowledge on neonatal danger signs. However, this study found no significant association between mothers’ place of birth and knowledge on neonatal danger signs. This contradicts other findings reported in literature. According to [20], mothers who gave birth at the hospital had good knowledge about neonatal danger signs and were about six times more likely to practice recommended newborn care practices compared to mothers with mothers who delivered at home. Similarly, [28] found that place of birth had significant association with mother knowledge on neonatal care. Mothers with good knowledge from the midwives had good practices and recognition of danger signs. A study in northern Ghana also found place of birth of mothers to be one of the main predictors of good neonatal care [17]. The observed differences in finding may be due to differences in power relations regarding decision on how the neonates should be cared for. In the Northern region of Ghana, mother in-laws and father in-lows exert more influence on decision regarding the care of newborns although the mother may have good knowledge on essential newborn care. Seeking timely medical attention for the sick newborn depends heavily on the mother’s ability to recognize danger sings in the newborn. In this study it was found that, all the mothers were able to mention some of the danger signs associated with the neonates. delayed healthcare seeking. Understanding care-seeking behavior reduces possible delays and significantly enhances the health of neonates [2]. delayed healthcare seeking. Understanding care-seeking behavior reduces possible delays and significantly enhances the health of neonates [2]. The health seeking behaviour of neonatal caregivers In terms of being persuaded to visit the hospital, the vast majority of respondents (77.2%) did so of their own free will. Understanding the patterns and factors that influence how mothers, families, and other newborn caregivers seek care for their babies is essential to designing effective measures to increase infant survival. One of the key methods to lower infant mortality in underdeveloped countries is to improve families' care-seeking behaviors [18]. According to the WHO, obtaining quick and proper care might minimize the mortality rate of children with acute respiratory infections by 20%. Early detection of neonatal danger signs and the provision of high-quality curative healthcare treatments for unwell newborns are significant strategies to lower newborn mortality [2]. The likelihood that an infant would survive could be considerably increased by early diagnosis of newborn morbidity. However, the mother, father, grandparents, and other close relatives may have an impact on a child's early interest in health. In Africa, grandparents play a significant role as gatekeepers for child care. Their activities can occasionally prevent children from obtaining medical attention [19]. This finding is contrary to the current study's findings, where most women visited the hospital of their own free will. WHO reports that due to poor health seeking behavior, children had the highest risk of dying in the first 28 days of life, with an average global rate of 18 fatalities per 1000 live births in 2018. One of the most frequent causes of newborn mortality in underdeveloped countries is health seeking behavior. Some people believe that the current efforts to lower newborn mortality are hampered by an inadequate understanding of the social and cultural factors that influence health, as well as the danger signs of the neonate and the implementation of effective ways to lessen their effects. At home, where few women seek medical attention for symptoms of neonatal sickness and almost no newborns are admitted to hospitals when they are ill, the highest rate of neonatal mortality occurs. Neonatal mortality can be impacted by Page 17/24 Page 17/24 Implications of the study Child birth and neonatal period have great emotional effect on families, especially first- time mothers and it could be considered as a new experience in life. The need to pursue studies in the area of neonatal care is important as poor Page 18/24 Page 18/24 Page 18/24 knowledge by parents, especially the mothers regarding neonatal care could pose a threat to neonatal health [8]. The Sustainable Development Goal 3 seeks to ensuring wellbeing for all of all ages and encourages health for all. This study would go a long way to improve neonatal health and help achieve the goal by 2030. knowledge by parents, especially the mothers regarding neonatal care could pose a threat to neonatal health [8]. The Sustainable Development Goal 3 seeks to ensuring wellbeing for all of all ages and encourages health for all. This study would go a long way to improve neonatal health and help achieve the goal by 2030. It is imperative to provide comprehensive training in the field of neonatal care for mothers. For such trainings to be effective, it is important to determine the current neonatal care practices and identify deficiencies among mothers through research like this in the study area. This will enable health workers to clearly know which areas to emphasize in educating and empowering mothers to enable them improve their health care and ultimately reduce neonatal morbidity and mortality. Findings from the study could assist policy makers to develop appropriate policies and interventions pertaining to neonatal health and survival. Also, stakeholders will be empowered to implement the policies to bring to the fore neonatal survival issues. The goal of the Ghana National Newborn Health Strategy and Action Plan, is to reduce neonatal mortality to 21 per 1000 live births [6]. It is hoped that the findings would inform the Regional Health Directorate and facilities to re-strategize to increase mothers’ knowledge on neonatal danger signs. Also,the findings of the study would be useful for nurse managers and health administrators to formulate policies to enhance quality health care delivery in relation to maternal and child health services. Limitations of the study The study could not be used to analyze study participants over a period of time and this was a significant obstacle in finding a trend and a meaningful association that exists between two variables. Some of the weaknesses of the design and type of the study methodology included lack of in-depth or thick description of analysis and difficulty in generalizing the findings were some of the limitations faced however, findings can be used in areas with similar health characteristics. Conclusion In general, knowledge on neonatal danger signs was good among the study participants. However, there are knowledge gaps in some components of essential newborn care. The study found good health seeking behaviour of mothers with mothers attending health facilities on their own volition. With the association between socio-demographic characteristics of the mothers and the knowledge on neonatal danger signs, educational level and occupation of mothers were statistically significant. However, there may be other factors that influence mothers’ knowledge on neonatal danger signs that could be explored. The study therefore recommends the following: Community Health Nurses and Midwives should follow up by home visits and encourage mothers to practice the knowledge and skills they have acquired during counseling to detect danger signs in their children. There is also the need for health managers in the municipality to develop strategies to encourage mothers to deliver at the health facility where appropriate newborn care and counseling are provided. Mothers should be empowered to make decisions concerning their children’s health care. Mothers should be empowered to make decisions concerning their children’s health care. Further studies should be conducted to explore other household and community factors that could influence mother’s knowledge on neonatal danger signs. Abbreviations Page 19/24 CHPS Community Based Health Planning and services HIV Human Imuno-deficiency Virus ICU Intensive Care Unit SPSS Statistical Package of Social Sciences TBA Traditional Birth Attendant WHO World Health Organisation CHPS Community Based Health Planning and services HIV Human Imuno-deficiency Virus ICU Intensive Care Unit SPSS Statistical Package of Social Sciences TBA Traditional Birth Attendant WHO World Health Organisation Acknowledgements The authors wish to express their gratitude to the caregivers for their assistance and also authorities of all the facilities used for this study, we say thank you. Funding The authors had no financial support for the research, authorship or publication of this article. Data analyzed during this study may be available from the corresponding author upon reasonable request. Data analyzed during this study may be available from the corresponding author upon reasonable request. Ethics approval and consent to participate All methods were carried out in accordance with relevant guidelines and regulations. Ethics approval was obtained from the Ghana Health Service Ethics Review Committee with reference number: GHS-ERC 037/10/22. Permission was also sought from the Municipal Director of Health Services and the Management of the various Health Facilities used for the study. Respondents also signed an informed consent form before participating in the study. Since the study used some illiterates an informed consent was obtained from their legal guardians. service of an interpreter was sought so that the respondents who could not read or write were assisted by way of interpretation of questions to their local language (Twi). Researchers had custody of identifiable information. Furthermore, no relationship was established between researchers and participants during the study. Consent for publication Not applicable Authors’ contributions This work was carried out in collaboration between all authors. Author PG designed the study, performed the statistical analysis, wrote the protocol, and wrote the first draft of the manuscript. Authors EAP and MOL managed the analyses and editing of the study. Author EBA and DBS managed the literature searches and selection of journal. All authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. 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A rapid assessment of the quality of neonatal healthcare in Kilimanjaro region, Northeast Tanzania BMC Pediatr.2013,12(1):182. 41. Misgna HG, Gebru HB, Birhanu MM. Knowledge, practice and associated factors of essential newborn care at home among mothers in Gulomekada District, Eastern. BMC Pregnancy Childbirth, 2017. 1–8. 42. Nepal HG, Joshi HB, Sharma MM, Teijingen P. Knowledge, practice and associated factors of essential newborn care at home among mothers in Gulomekada District, Eastern. BMC Pregnancy Childbirth, 2018; 1–8. Page 22/24 Page 22/24 43. Nigatu SG, Worku AG, Dadi AF. Level of mother’s knowledge about neonatal danger signs and associated factors in North West of Ethiopia: a community based study. BMC Res Notes, 2015; 4–9. 44. Padiyath DA, Bhat PR, Ekambaram BP. Trends and Determinants of Neonatal Mortality in Nepal, -2019; 45–79. 45. UNICEF. The State of World Children. 2017; https://doi.org/10.4172/2090-4.1000172. 43. Nigatu SG, Worku AG, Dadi AF. Level of mother’s knowledge about neonatal danger signs and associated factors in North West of Ethiopia: a community based study. BMC Res Notes, 2015; 4–9. 45. UNICEF. The State of World Children. 2017; https://doi.org/10.4172/2090-4.1000172. Figures Figures Figure 2 Who Normally Advised Respondents to go to the Hospital Figure 1 Figure 1 Conditions that would make Mother send her Neonate Immediately to a Health Facility Conditions that would make Mother send her Neonate Immediately to a Health Facility Page 23/24 Page 23/24 Page 23/24 Page 23/24 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. PhilipGyaaserawdata.pdf StudyTools.pdf Page 24/24
https://openalex.org/W2126856190	https://publications.aston.ac.uk/id/eprint/20096/1/Role_of_ECL2_in_CGRP_receptor_activation.pdf	English	null	The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach	Journal of the Royal Society interface	2,013	cc-by	10,619	Research 1Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK 2School of Biological Sciences, University of Auckland, 3 Symonds Street, Private Bag 92 019, New Zealand 3School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK 4School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, UK 1Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK Cite this article: Woolley MJ, Watkins HA, Taddese B, Karakullukcu ZG, Barwell J, Smith KJ, Hay DL, Poyner DR, Reynolds CA, Conner AC. 2013 The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach. J R Soc Interface 10: 20130589. The calcitonin gene-related peptide (CGRP) receptor is a complex of a cal- citonin receptor-like receptor (CLR), which is a family B G-protein-coupled receptor (GPCR) and receptor activity modifying protein 1. The role of the second extracellular loop (ECL2) of CLR in binding CGRP and coupling to Gs was investigated using a combination of mutagenesis and modelling. An alanine scan of residues 271–294 of CLR showed that the ability of CGRP to produce cAMP was impaired by point mutations at 13 residues; most of these also impaired the response to adrenomedullin (AM). These data were used to select probable ECL2-modelled conformations that are involved in agonist binding, allowing the identification of the likely contacts between the peptide and receptor. The implications of the most likely structures for receptor activation are discussed. http://dx.doi.org/10.1098/rsif.2013.0589 Received: 3 July 2013 Accepted: 29 August 2013 Received: 3 July 2013 Accepted: 29 August 2013 Subject Areas: computational biology, biochemistry The extracellular loops of G protein-coupled receptors (GPCRs) are important for receptor function. They contribute to protein folding, provide structure to the extra- cellular region and mediate movement of the transmembrane (TM) helices on activation. The second extracellular loop (ECL2) is of significance for ligand binding andreceptoractivation [1–3]. Infamily B (orsecretin-like) GPCRs, it isthemost con- served and often the longest of all the ECLs, and so is in a good position to interact with the endogenous peptide agonists for these receptors and is in a prominent cen- tral position to mediate conformational changes [1]. The peptide ligands typically contain 30–50 amino acids. The family B receptors are characterized by a large N-terminal domain (approx. 100 amino acids), which interacts with the C-termini of their cognate peptide ligands [4]. The N-termini of these peptides interact with the ECLs and the TM region of the receptors resulting in activation [4,5]. ECL2 has been implicated in agonist binding at the GLP-1, secretin and CRF1 receptors [6–10]. For the GLP-1 receptor, ECL2 plays an important role in directing coupling towards stimulation of ERK1/2 activation versus Gs and activation of adenylate cyclase [7]. However, the molecular basis for this observation remains obscure. rsif.royalsocietypublishing.org Michael. J. Woolley1, Harriet A. Watkins2, Bruck Taddese3, Z. Gamze Karakullukcu3, James Barwell4, Kevin J. Smith3, Debbie L. Hay2, David R. Poyner4, Christopher A. Reynolds3 and Alex C. Conner1 Michael. J. Woolley1, Harriet A. Watkins2, Bruck Taddese3, Z. Gamze Karakullukcu3, James Barwell4, Kevin J. Smith3, Debbie L. Hay2, David R. Poyner4, Christopher A. Reynolds3 and Alex C. Conner1 Michael. J. Woolley1, Harriet A. Watkins2, Bruck Taddese3, Z. Gamze Karakullukcu3, James Barwell4, Kevin J. Smith3, Debbie L. Hay2, David R. Poyner4, Christopher A. Reynolds3 and Alex C. Conner1 Research Cite this article: Woolley MJ, Watkins HA, Taddese B, Karakullukcu ZG, Barwell J, Smith KJ, Hay DL, Poyner DR, Reynolds CA, Conner AC. 2013 The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach. J R Soc Interface 10: 20130589. 1Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK 2School of Biological Sciences, University of Auckland, 3 Symonds Street, Private Bag 92 019, New Zealand 3School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK 4School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, UK 2.4. CLR models In previous work, we have examined the role of the first and third ECLs (ECL1/3) of the CGRP receptor. However, only a small number of residues within ECLs 1 and 3 were implicated in CGRP binding [18]. Consequently, we have now addressed the role of ECL2 in this receptor. In an extension of the strategy usedtoexaminetheTMresiduesofCLR,wehaveexperimentally identified CLR residues that reside within the last turn of TM4, ECL2 and the first turn of the turn of TM5 and that are key for CGRP and AM interactions with the CGRP receptor. We have then used all of our data for each ECL and the TM domain, in combination with heuristic loop modelling, molecular dynamics, docking and sequence analysis to model the interaction between the N-terminus of the CGRP peptide and CLR, based on a model of the Gs-coupled state of the CGRP receptor. The starting point for the models indicating the interaction between CGRP and ECL2 was a recent model of the active state [19]. The most appropriate X-ray crystal structure model for a fully active GPCR is that of the b2-adrenergic receptor (b2-AR) coupled to Gs [27], which is similar to the b2-AR nanobody stabil- ized [28] and the rhodopsin active structures stabilized by a C-terminal peptide from transducin [29,30]; these active structures are characterized by the outward tilt of the intracellularend of TM6 that is necessary for G protein binding. In the absence of G protein or G protein-derived peptides, X-ray crystal structures of agonists bound to GPCRs stabilize substates that are not too different to the inactive form [31], i.e. they lack the outward tilt of TM6; in such agonist-bound structures, the conformation of Y5.58 is taken to be indicative of the state because in the inactive form, it usually inter- acts with L1.63 and L8.50, whereas in the active substate, it stabilizes the active conformation of R3.50. Our active structures are stabilized by the Gs C-terminal peptide (R373-L394), and hence included the outward tilt of TM6 [27]. Nevertheless, the conformation of Y5.58 (and the tilt of TM6) was monitored to check that active state char- acter was maintained as fully as possible and that the simulations could be terminated should the active structure begin to acquire inactive character. expression and radioligand binding p g g The preparation of mutants and molecular biology was as descri- bed earlier [23]. An HA-tagged CLR construct was used to allow measurement of cell surface receptor expression by ELISA [20,23]. Mutants were transfected into Cos 7 cells using PEI [20,23]. Radio- ligand binding was carried out on cell membranes using [125I] iodohistidyl-human alpha CGRP (Perkin Elmer) [20,23]. 2.4. CLR models The underlying alignment was based on a novel approach to the class A–class B alignment, aided by a GCR1/class E alignment that was used as a bridge between the class A and class B sequences. This has been repeated using improved methodology but the alignment remains unaltered [32]. The status of GCR1, GCR2 and other putative plant GPCRs has been questioned [33–35], but GCR1 has all the features expected for a GPCR fold [32], while GCR2 was predicted [36] and later shown to be a lanbiotic cyclase [37]. The simulations that underlie the CLR modelling [19] were extended to 100 ns (see the electronic supplementary material). rsif.royalsocietypublishing.org J R Soc Interface 10: 20130589 2 rsif.royalsocietypublishing.org J R Soc Interface 10: 20130589 We have previously used a combination of mutagenesis and computation to produce a model indicating how the TM domain of the calcitonin receptor-like receptor (CLR) can inter- act with Gs [19]. CLR is the GPCR component of the calcitonin gene-related peptide (CGRP) receptor.CGRPispart ofa peptide family that also includes adrenomedullin (AM), calcitonin and amylin and is involved in heart disease and migraine [20]. CLR interacts with a single TM protein, receptor activity- modifying protein 1 (RAMP1), in order to bind CGRP and also AM [21], though AM interacts with CLR more strongly in the presence of RAMP2. Using an independent modelling approach, Wootten et al. [22] have produced a broadly similar model for the GLP-1 receptor, which indicates how agonists can activate the receptor by interacting with different TM resi- dues. Using both mutagenesis and computation is a powerful strategy for studying the activation of GPCRs [12], especially given that structural techniques for instance crystallography give static pictures of what is fundamentally a dynamic process. org J R Soc Interface 10: 20130589 2.3. Data analysis Curve fitting was done with GRAPHPAD PRISM 5 or 6 (GraphPad Software Inc., San Diego, CA, USA). Both this and statistical analysis were as described previously [20]. 2.3. Data analysis Curve fitting was done with GRAPHPAD PRISM 5 or 6 (GraphPad Software Inc., San Diego, CA, USA). Both this and statistical analysis were as described previously [20]. Keywords: calcitonin gene-related peptide, class B G-protein-coupled receptor, loop modelling, site-directed mutagenesis, calcitonin receptor-like receptor Authors for correspondence: David R. Poyner e-mail: d.r.poyner@aston.ac.uk Christopher A. Reynolds e-mail: reync@essex.ac.uk Alex C. Conner e-mail: a.c.conner@bham.ac.uk Several distinct conformations have been recognized for ECL2 in family A GPCRs, ranging from the ‘lid’ seen in rhodopsin, which encloses the bound reti- nal ligand, to the extended sheet seen for most of the peptide GPCRs [11–13]. Movement of ECL2 seems to be important in the activation of family A GPCRs; this is linked to agonist-induced changes in TM5 and helps propagate these changes to other parts of the GPCR [11,14]. Electronic supplementary material is available at http://dx.doi.org/10.1098/rsif.2013.0589 or via http://rsif.royalsocietypublishing.org. Thereiscurrentlynocrystalstructureshowinghowapeptideagonistbindstothe ECLs or TM domain of a family B GPCR. A number of models have been proposed based on cross-linking and mutagenesis data [8,15,16] but it has proved extremely & 2013 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0/, which permits unrestricted use, provided the original author and source are credited. 1026 to 10212 M with SB as a basal point. The plate was incubated in the absence of light for 30 min at room temperature before 20 ml/well of detection mix was added. The plate was incubated in the absence of light for a further 60 min. FRET was recorded by excitation at 320 nm and emission at 665 nm. The experimental pEC50 values for wild-type (WT) receptor in the electronic sup- plementary materials, table S1 show some variation, reflecting differences in coupling efficiencies between cells as the data were collectedin excess ofayear.Such drift has beenobservedpreviously with CGRPreceptors [24,25]. To control for this, a paired design-test was used so that each mutant was compared in the same exper- iment against a corresponding WT control. For the investigation of AM-mediated stimulation of the CGRP receptor, cAMP was measured with alphascreen, as described previously [26]. Data were normalized against the maximum fitted response for CGRP or AM; basal cAMP was taken as the fitted minimum. difficult to accurately predict the ECL conformations by this approach or bysimulation [17]. Given the variety in familyB pep- tide sequences [5,17], it seems likely that there is no single mode of peptide binding to the ECLs of family B GPCRs [18]. 2 2.2. Measurement of cAMP y yp g g 3 rsif.royalsocietypublishing.org J R Soc Interface 10: 20130589 3 rg J R Soc Interface 10: 20130589 Figure 1. Effect of mutations on the pEC50 for AM (white bars) and CGRP (grey bars) at cAMP production. The difference in mean pEC50 between the mutant and WT receptor is shown, hence a negative value shows a decrease in potency. Where there was no detectable stimulation of the mutant by peptide (#), an arbitrary value of –3 has been shown in the figure. Data are shown in the electronic supplementary material, table S1. p , 0.05, p , 0.01, pEC50 of mutant significantly different from that of WT by paired Student’s t-test or repeated measures ANOVA followed by Dunnett’s test as appropriate. supplementary material, figure S1). This is well before it would be possible to fully sample the loop conformations, despite high principle components for ECL2 residues; see the electronic sup- plementary material, figure S1D. Thus, given that the MODELLER scoring functions are approximate, we have taken a heuristic approach to determining the ECL2 conformation. We have therefore generated and refined100 independentloop conformations; suitable conformations were selected if key loop residues identified by the mutagenesis interacted with CGRP or AM to within a cut-off of 5.5 A˚ . There are many assumptions inherent in this approach, as discussed below in §3.2.5; this is apparent because it was impossible to generate conformations where each significant ECL2 residue interacted with CGRP. Thus, multiple conformations of ECL1 (resi- dues 202–212), ECL2 (residues 274–293) and ECL3 (residues 353–363) were simultaneously generated in the presence of CGRP1–32 using MODELLER [41–43]. Each conformation is character- ized by its DOPE score; a lower score corresponds to a more likely conformation. AM binding to CLR was similarly modelled by sim- ultaneously mutating CGRP to AM and generating the loop conformations within MODELLER. 2.6. Residue numbering For amino acids within the proposed ECLs of CLR, only the resi- due numbers are shown. For residues that are within the TM helices of CLR, a superscript denotes their position using an adap- tion of the Ballasteros–Weinstein numbering proposed elsewhere [19]. The peptide residue numbers are given as superscripts. 3.1.1. Effects of alanine substitution on CGRP-mediated cAMP production Twenty-four individual residues of CLR ECL2 from A271 to I294 were mutated to alanine (alanine residues were mutated to leucine) and their ability to respond to CGRP and stimulate cAMP production was investigated when co- expressed with human RAMP1. These residues were selected as they are most likely to incorporate the whole of ECL2 and approximately one turn of helices 4 and 5 immediately adja- cent to the loop, based on our previous analysis [19]. Figures 1 and 2 and the electronic supplementary materials, table S1 and figure S2 show that the pEC50 values of 14 of the 24 mutants were significantly different to WT, indica- ting that ECL2 is particularly important for CGRP receptor function. Seven mutants resulted in significant cAMP reduct- ion of more than 10-fold. These are R274A, Y278A, D280A, C282A, W283A, I284A and T288A. Of the remaining seven mutations, N281A showed a small increase in potency and six mutants had small but significant reductions in pEC50. Y292A and I294A showed significant but small increases in maximal cAMP response (Emax) (see the electronic supplementary material, table S2). 2.5. Sequence analysis q y The sequences of CGRP and amylin (which binds to the calcito- nin receptor (CTR)) from several species were aligned [5,44]. The sequences of amylin and CGRP and the sequences of CTR and CLR were analysed in parallel to identify mutations that are cor- related between the CTR–amylin and CLR–CGRP systems, with a view to identifying contact points. 3.1.2. Cell surface expression of receptors and CGRP binding Owing to the likelihood of attenuated binding of CGRP, radio- ligand binding could not be reliably used to provide an estimate of receptor expression for these constructs. Accord- ingly, an ELISA to measure receptors at the cell surface was used (see the electronic supplementary materials, table S3). All of the mutants were expressed at the cell surface to within approximately 60% of WT levels. Reductions to this level of expression has been previously shown to have little effect on the potency of CGRP-mediated cAMP signalling for this or CTR expressed in Cos 7 cells [45,46] and in this study, there was only a very low correlation between expression and pEC50 (r2 ¼ 0.23). Mutated receptors that had large impairments of cAMP production were examined for their ability to bind CGRP using a radioligand-binding assay (see the electronic supplementary materials, table S4). All of the mutants examined, except Y277A had reduced affinity for the radioligand. 2.2. Measurement of cAMP For the investigation of CGRP-mediated activation of the recep- tor, cAMP was measured using a FRET-based PerkinElmer LANCE cAMP 384 kit according to manufacturer’s instructions. Briefly, transfected cells were removed from the plate with trypsin EDTA, washed with phosphate-buffered saline and resuspended in assay stimulation buffer (SB; phosphate-buffered saline þ 0.1% (w/v) bovine serum albumin þ 0.5 mM isobutylmethylxanthine). Cells were counted with a haemocytometer and the appropriate cell number resuspended in SB þ 1/100 AlexaFluor 647-anti cAMP antibody at an assay concentration of 2000 cells/10 ml. A total of 2000 cells/well were loaded onto a 96-well white Optiplate (PerkinElmer) and were concentration-dependently stimulated in triplicate with a logarithmic increase of CGRP diluted in SB from To model the peptide binding, disulfide-bonded cyclic CGRP1–7 was constructed in Maestro and docked to the active CLR model (after 80 ns, i.e. just before the first signs of the onset of inactive character; [19]) using Glide SP [38,39], as described in the electronic supplementary material. The best-scoring pose was verified by sequence analysis, as described below. The CGRP extension (up to residue 32) was modelled onto CGRP1–7 using the NMR solution structure of salmon calcitonin (PDB code 2glh) [40], using the alignment of Watkins et al. [5]. In the molecular dynamics simulations, the active structure begins to acquire some inactive character after 80 ns (i.e. F7.53 switched towards the inactive conformation; see the electronic 1 0 –1 –2 –3 A271L I272A A273L R274A S275A S285A S286A D287A T288A H289A L290A L291A Y292A I293A I294A L276A Y277A Y278A N279A D280A N281A C282A W283A I284A change in pEC50 CGRP * # # # # * ** * * * ** * * * * * * * * mutant AM Figure 1. Effect of mutations on the pEC50 for AM (white bars) and CGRP (grey bars) at cAMP production. The difference in mean pEC50 between the mutant and WT receptor is shown, hence a negative value shows a decrease in potency. Where there was no detectable stimulation of the mutant by peptide (#), an arbitrary value of –3 has been shown in the figure. Data are shown in the electronic supplementary material, table S1. p , 0.05, *p , 0.01, pEC50 of mutant significantly different from that of WT by paired Student’s t-test or repeated measures ANOVA followed by Dunnett’s test as appropriate. cAMP production The CGRP receptor also acts as a functional AM receptor with an affinity of approximately 10-folds less than that for CGRP [47]. To explore the effects of the above mutants further, their ability to respond to AM through cAMP production was sub- sequently investigated. The majority of the effects were in line with those seen with CGRP (figure 1 and the electronic sup- plementary material, table S2), the most notable differences being the lack of effects of AM at Y277 and Y278 and for some mutants, it was impossible to measure any activation of the receptor (figure 3). In the case of R274A, W283A and D280A, the response was so low that an Emax could not be reliably determined. There was especially good agreement between the two agonists for the central area of functional importance ranging from D280 to T288. As with CGRP, many of these alanine substitutions reduced AM potency. 3. Results and discussion 3.1. Experimental analysis of the CGRP receptor In this section, the results of an alanine scan of the CGRP receptor will be presented. The implications of these data will be discussed alongside the modelling in §3.2. 150 100 50 –50 0 150 100 50 –50 0 150 100 50 –50 0 150 100 50 –50 0 WT WT WT WT WT R274A WT Y277A % WT cAMP % WT cAMP % WT cAMP % WT cAMP 0 –12 log (haCGRP) M log (haCGRP) M –11 –10 –9 –8 –7 –6 0 –12 –11 –10 –9 –8 –7 –6 R274A Y278A Y278A C282A C282A WT I284A I284A WT Y277A D280A D280A W283A W283A T288A T288A (b) (a) (c) (g) (h) (d) (e) ( f ) 2. (a–h) Concentration–response curves of mutants showing changes in pEC50 in response to CGRP. Representative curves are shown from experiments at least three times. The curves are normalized to the fitted Emax for CGRP on the WT receptor, which is defined as 100%. 150 100 50 –50 0 WT R274A % WT cAMP R274A (b (a) rsif.royalsocietypublishing.org J R Soc Interface 10: 20130589 4 rsif.royalsocietypublishing.org J R Soc Interface 10: 20130589 4 WT D280A D280A –50 150 100 50 –50 0 WT % WT cAMP Y278A Y278A (c) (d) (d) WT W283A W283A 50 150 100 50 –50 0 WT % WT cAMP C282A C282A (e) ( f ( f ) log (haCGRP) M 0 –12 –11 –10 –9 –8 –7 –6 WT T288A T288A ) 150 100 50 –50 0 WT % WT cAMP 0 –12 log (haCGRP) M –11 –10 –9 –8 –7 –6 I284A I284A (g) ( log (haCGRP) M log (haCGRP) M Figure 2. (a–h) Concentration–response curves of mutants showing changes in pEC50 in response to CGRP. Representative curves are shown from experiments repeated at least three times. The curves are normalized to the fitted Emax for CGRP on the WT receptor, which is defined as 100%. 3.1.3. Effects of alanine substitution on AM-mediated The effect of mutations at the N- and C-termini of ECL2 was more variable between the two peptides. At the N-terminus, the CGRP-affecting mutants Y277A or Y278A did not show AM-mediated effects on pEC50. However, there were approxi- mately 25% reductions in the maximal cAMP response. At the C-terminus of ECL2, the two leucine mutants (L290A and L291A), which showed a reduction in potency with CGRP, were not significantly affected when stimulated with AM. 3.1.4. Basal activity 9.62+0.76, C212A 8.10+0.43, n ¼ 3, p , 0.05, Student’s t-test); however, the double mutant C212AC282A showed a WT response (pEC50 values: WT 9.49+0.11 n ¼ 3, C212AC282A 9.41+0.09, n ¼ 3), thus implying that the disul- fide bond itself is not crucial for CGRP binding or signalling. for AM responsiveness (using Cos 7 cells from a New Zea- land source and Alphascreen), small but statistically significant elevations were noted for A273L, Y277A and I284A but not N281A and I294A. Thus, the increase in basal cAMP depended on experimental conditions and was always small. In the course of analysing over 200 mutants of CLR [19,20,48], we have observed very few that showed elevated activity, possibly indicating that there are multiple locks in place to keep the receptor in an inactive form; it is possible that the RAMPs might contribute to this. for AM responsiveness (using Cos 7 cells from a New Zea- land source and Alphascreen), small but statistically significant elevations were noted for A273L, Y277A and I284A but not N281A and I294A. Thus, the increase in basal cAMP depended on experimental conditions and was always small. In the course of analysing over 200 mutants of CLR [19,20,48], we have observed very few that showed elevated activity, possibly indicating that there are multiple locks in place to keep the receptor in an inactive form; it is possible that the RAMPs might contribute to this. 3.1.4. Basal activity In the investigation of CGRP (with Cos 7 cells from a UK source and measuring cAMP with a LANCE assay), N281A and I294A showed a significant increase in basal cAMP signalling (i.e. constitutive ligand-independent signalling acti- vity; see the electronic supplementary material, table S2). These values increased by 9.9+ 0.9% and 21.8+2.9% above WT, respectively. When the cells were investigated 150 100 50 –50 0 150 100 50 –50 0 150 100 50 –50 0 150 100 50 –50 0 WT WT % WT cAMP % WT cAMP % WT cAMP % WT cAMP 0 –12 log (hAM) M log (hAM) M –11 –10 –9 –8 –7 –6 0 –12 –11 –10 –9 –8 –7 –6 R274A R274A WT C282A C282A I284A I284A Y277A Y277A D280A D280A WT WT WT WT WT W283A W283A S285A S285A T288A T288A (b) (a) (c) (g) (h) (d) (e) ( f ) ure 3. (a–h) Concentration–response curves of mutants showing changes in cAMP in response to AM. Data are mean + s.e.m. of combined normalized data m at least three independent experiments. The curves are normalized to the fitted Emax for AM on the WT receptor, which is defined as 100%. rsif.royalsocietypublishing.org J R Soc Interface 10: 20130589 5 (b) I284A I284A WT 150 100 50 –50 0 % WT cAMP WT W283A W283A (e) ( f ) ( f ) log (hAM) M 0 –12 –11 –10 –9 –8 –7 –6 WT T288A T288A ) 150 100 50 –50 0 % WT cAMP 0 –12 log (hAM) M –11 –10 –9 –8 –7 –6 WT S285A S285A (g) (h) log (hAM) M Figure 3. (a–h) Concentration–response curves of mutants showing changes in cAMP in response to AM. Data are mean + s.e.m. of combined normalized data from at least three independent experiments. The curves are normalized to the fitted Emax for AM on the WT receptor, which is defined as 100%. 9.62+0.76, C212A 8.10+0.43, n ¼ 3, p , 0.05, Student’s t-test); however, the double mutant C212AC282A showed a WT response (pEC50 values: WT 9.49+0.11 n ¼ 3, C212AC282A 9.41+0.09, n ¼ 3), thus implying that the disul- fide bond itself is not crucial for CGRP binding or signalling. 3.1.6. The importance of the ECL2/TM5 junction As noted above, L290A and L291A both showed small but significant reductions in CGRP potency. As movements at the top of TM5 have been implicated in the early stages of activation of the b2-AR and rhodopsin [11], the role of residues in this region was probed by further mutagenesis. The individual reduction in EC50 values for L290A (approx. eightfold) and L291A (approx. fourfold) was highly exacer- bated by an L290AL291A double mutant that reduced the EC50 by approximately 500-fold compared with WT (WT 3.1.5. The importance of the ECL2–TM3 disulfide linkage As C282 is predicted to form a disulfide bond with C2123.25 [19], further mutagenesis was used to explore this, using CGRP as the agonist. The mutant C212A impaired cAMP pro- duction in much the same way as C282A (pEC50 values: WT 9 (a) (c) (b) 8 7 6 5 number of interactions 4 3 2 1 –4400 –4000 –3600 modeller DOPE score –3200 –2800 A1 R274 S275 L276 Y277 Y278 N279 D280 A281 C282 W283 I284 S285 S286 D287 T288 H289 L290 C2 D3 T4 A5 T6 C7 V8 T9 H10 R11 L12 A13 G14 L15 R18 Figure 4. (a) A sample (20/100) of ECL1, ECL2 and ECL3 conformations generated by MODELLER for the CLR : CGRP complex; each of the 20 loops is shown in a different colour. CLR is shown in blue, helices are shown as cylinders, CGCR1–12 is shown in red and CGRP12–18, which has relatively few interactions to ECL2, is shown in mauve. The C2–C7 disulfide bond is shown in yellow. (b) A plot of the number of residue–residue interactions between ECL2 and CGRP against the MODELLER DOPE score for the CLR conformation; the lower the score, the more probable the loop conformation. Conformations that interact with T6 via T288, D287, D280 or both D287 and T288 are shown as red, blue green and purple crosses (þ), respectively. Conformation were these residues do not interact with T6 are shown as grey crosses (). (c) The interactions between ECL2 residues and CGRP residues as observed over the full set of 100 ECL2 conformations; the thickness of the line is broadly in line with the frequency of the interactions. For clarity interactions observed in fewer than 10 structures are omitted (but see the electronic supplemen- tary materials, figure S3 for the full set of interactions). 3.1.6. The importance of the ECL2/TM5 junction (b) A plot of the number of residue–residue interactions between ECL2 and CGRP against the MODELLER DOPE score for the CLR conformation; the lower the score, the more probable the loop conformation. Conformations that interact with T6 via T288, D287, D280 or both D287 and T288 are shown as red, blue green and purple crosses (þ), respectively. Conformation were these residues do not interact with T6 are shown as grey crosses (). (c) The interactions between ECL2 residues and CGRP residues as observed over the full set of 100 ECL2 conformations; the thickness of the line is broadly in line with the frequency of the interactions. For clarity interactions observed in fewer than 10 structures are omitted (but see the electronic supplemen- tary materials, figure S3 for the full set of interactions). The coloured lines indicate interactions of specific CGRP residues: D3 (brown), A5 (purple), T6 (black), T9 (orange), H10 (grey), R11, R18 (red) and L12, L15 (green). pEC50 10.6 +0.11, L290AL291A pEC50 7.93+0.13, n ¼ 3, p , 0.01, Student’s t-test). peptide–receptor contacts [12]. Here, we re-interpret several previously reported experimental observations that were made in the absence of a modelled structure [44] and that are consistent with our models. D3 of CGRP in our model is not in a constrained pocket, hence it makes few interactions with ECL2; indeed this position can accommodate a photo- affinity probe [44]. The fact that it can be readily replaced by arginine in the AM model provides further justification for our model. By contrast, A5 of CGRP is sterically constrained and indeed makes a relatively large number of interactions (figure 4c), showing that it has many close neighbours. T6 is discussed in more detail below; here, the preferred interactions are to T288. In several loop conformations, T9 of CGRP inter- acts with polar residues such as D280, D287 and T288. There is some indirect evidence that H10 may also be part of this net- work that responds to negative charges [44]. L15 of CGRP can be replaced by a large benzoyl-phenylalanine moiety with 3.1.6. The importance of the ECL2/TM5 junction The coloured lines indicate interactions of specific CGRP residues: D3 (brown), A5 (purple), T6 (black), T9 (orange), H10 (grey), R11, R18 (red) and L12, L15 (green). rsif.royalsocietypublishing.org J R Soc Interface 10: 20130589 6 9 (b) 8 7 6 5 number of interactions 4 3 2 1 –4400 –4000 –3600 modeller DOPE score –3200 –2800 (a) (c) (a) (c) A1 R274 S275 L276 Y277 Y278 N279 D280 A281 C282 W283 I284 S285 S286 D287 T288 H289 L290 C2 D3 T4 A5 T6 C7 V8 T9 H10 R11 L12 A13 G14 L15 R18 Figure 4. (a) A sample (20/100) of ECL1, ECL2 and ECL3 conformations generated by MODELLER for the CLR : CGRP complex; each of the 20 loops is shown in a different colour. CLR is shown in blue, helices are shown as cylinders, CGCR1–12 is shown in red and CGRP12–18, which has relatively few interactions to ECL2, is shown in mauve. The C2–C7 disulfide bond is shown in yellow. (b) A plot of the number of residue–residue interactions between ECL2 and CGRP against the MODELLER DOPE score for the CLR conformation; the lower the score, the more probable the loop conformation. Conformations that interact with T6 via T288, D287, D280 or both D287 and T288 are shown as red, blue green and purple crosses (þ), respectively. Conformation were these residues do not interact with T6 are shown as grey crosses (). (c) The interactions between ECL2 residues and CGRP residues as observed over the full set of 100 ECL2 conformations; the thickness of the line is broadly in line with the frequency of the interactions. For clarity interactions observed in fewer than 10 structures are omitted (but see the electronic supplemen- tary materials, figure S3 for the full set of interactions). The coloured lines indicate interactions of specific CGRP residues: D3 (brown), A5 (purple), T6 (black), T9 (orange), H10 (grey), R11, R18 (red) and L12, L15 (green). Figure 4. (a) A sample (20/100) of ECL1, ECL2 and ECL3 conformations generated by MODELLER for the CLR : CGRP complex; each of the 20 loops is shown in a different colour. CLR is shown in blue, helices are shown as cylinders, CGCR1–12 is shown in red and CGRP12–18, which has relatively few interactions to ECL2, is shown in mauve. The C2–C7 disulfide bond is shown in yellow. rsif.royalsocietypublishing.org J R Soc Interface 10: 20130589 7 rsif.royalsocietypublishing.org J R Soc Interface 10: 20130589 7 rsif.royalsocietypublishing.org CLR residues CGRP effect AM effect A273 p p R274 p p Y277 p Y278 p D280 p p C282 p W283 p p I284 p p S285 p p D287 p T288 p p L290 p L291 p CLR residues CGRP effect AM effect A273 p p R274 p p Y277 p Y278 p D280 p p C282 p W283 p p I284 p p S285 p p D287 p T288 p p L290 p L291 p Figure 5. The highest scoring docked pose of CGRP1–7 (mauve). Resides A1 and T6 reside close to Ala203 and Thr288, respectively (blue). The CGRP dis- ulfide bond is shown in yellow. The loop conformation shown here for ECL2 was a high scoring (i.e. favoured) conformation. TM7 is shown as transparent. J R Soc Interface 10: 20130589 Figure 5 shows that the best-scoring docked conformation of CGRP1–7 satisfies the T6 criteria (by interacting with D280/ D287/T288) in the presence of a sample ECL2 conformation (figure 4), and that D3 is not in a sterically crowded region (see also figure 4c). This indicates that the docked CGRP1–7 has provided a suitable template for modelling the full CGRP peptide and hence modelling the conformations of ECL2. In the docked conformation, A1 of CGRP is not only close to A203 of ECL1 of CGRP, but also able to accommo- date N-terminal extensions. As discussed in the electronic supplementary material, this is consistent with mutagenesis and bioinformatic analysis of CGRP and AM binding. only small changes in affinity (it makes few interactions to ECL2); however, replacement of L12 of CGRP causes around a 10-fold decrease (it makes more interactions with ECL2) [44]. Replacement of R18 of CGRP by alanine has virtually no effect, and indeed it makes few interactions, but the double alanine mutant R11AR18A shows 100-fold reduction in affinity [44]. Replacing either of these arginine residues with glutamate caused over a 10-fold reduction in affinity; but replacement with glutamine led to retention of high affi- nity binding [24,25,49], presumably because glutamine can still donate hydrogen bonds. Indeed, we see that both D280 and D287 are able to interact with R11, but as D287 is less sig- nificant for AM (where K substitutes for R11), it is more likely that D280 is the preferred partner to R11, even though D287 makes more interactions. 3.2.2. Filtering the ECL2 conformations 3.2.2. Filtering the ECL2 conformations The number of interactions made by key ECL2 residues (table 1) to CGRP for each of the 100 loop conformations are displayed in figure 4b (y-axis). The ideal loop confor- mation should make an interaction between T6 of CGRP and D280, D287 or T288, have a large negative DOPE score, make a high number of key interactions and ideally have W283 in a vertical orientation (see below). The majority of conformations, denoted by a grey cross in figure 4b, do not make an appropriate interaction with T6 and are discarded. Several conformations make 6–9 interactions, including those to T6 via D280, D287 or T288. Because interactions to D280, D287 or T288 are observed in the top 15% of the most energetically preferred conformations and because the DOPE score is an empirical rather than a rigorously accurate score, it is not advisable to use energy (i.e. the DOPE score) as the sole criteria to identify the preferred binding mode, hence the importance of filtering the loop conformations using the mutagenesis data. Only one conformation (conformation 34, top left of figure 4b) records a direct interaction with R2744.64, for either CGRP or AM, but this interaction is to R11 of CGRP and the distance is long; closer inspection shows that D280 bridges between R2744.64 and R11 of CGRP. Several other conformations of R2744.64 act in this way, and could help to orientate a D280-R11 interaction. Thus, a direct interaction between CGRP and R2744.64 is probably an unrealistic selection criterion. R2744.64 is highly conserved as arginine or lysine across the class B GPCRs; mutation in the GLP-1 and secretin receptors also impairs function [6,7,51]. It is possible that the positively charged head group may also interact with the phospholipid bilayer [52]. W283 and I284, which reside in the centre of ECL2, make the most interactions; these are either to the region around R11 or to the N-terminal region (see below). Having analysed the pattern of interactions over all loop conformations, we see that this pattern is consistent with known experimental data on the CGRP peptide [44] and so we can now seek to identify preferred conformations and interactions from the mutagenesis data given in table 1, which indicates the most important residues for CGRP or AM binding. rsif.royalsocietypublishing.org J R Soc Interface 10: 20130589 7 CGRP residues 1, 4, 8, 13 and beyond are predicted to make few, if any contacts with ECL2; this is consistent with the known structure activity relationships for CGRP where they seem to be of only minor importance [44]; this observation justifies the orientation of the helical extension to CGRP1–7. 3.2. Modelling of ECL2 and interactions with CGRP and AM 3.2.1. Analysis of the MODELLER-generated ECL2 conformations The loop conformations spanned a large proportion of the space available to ECL2 (figure 4a). The analysis of the full set of interactions for all loop conformations is given in figure 4b,c. Although this analysis includes high-energy con- formations, it is interesting as it highlights general interaction preferences of several amino acids in CGRP and ECL2. ECL2 forms an extended loop; this conformation is often seen as two antiparallel beta-strands in all peptide GPCRs for which structures are available, creating a large interface for Figure 5. The highest scoring docked pose of CGRP1–7 (mauve). Resides A1 and T6 reside close to Ala203 and Thr288, respectively (blue). The CGRP dis- ulfide bond is shown in yellow. The loop conformation shown here for ECL2 was a high scoring (i.e. favoured) conformation. TM7 is shown as transparent. Table 1. Data used to select loop conformations. 3.2.2. Filtering the ECL2 conformations The residue that is the equivalent to position 6 in CGRP is conserved as threonine in all members of the CGRP/CT/ AM/amylin family of peptides and is essential for CGRP agonist activity [44,50]. Sequence analysis shows that the most likely candidates that are (i) conserved in CLR and CTR and (ii) able to donate or accept an H-bond are in ECL2 where D280, D287 and T288 are the best candidates. As D280 makes few interactions to T6 (see above) and D287 is not significant in AM binding (figure 1) and is of moderate importance in CGRP activation, it would seem that T288 is the most promising candidate. Based on the data in figure 4b (and similarly for AM inter- actions with the CGRP receptor which altered pEC50), we selected six conformations for CGRP and seven for AM (see the electronic supplementary material, figure S4). Apart from one CLR/CGRP structure (conformation 34), these all have a similar conformation for ECL2. However, despite a similar (a) (b) (c) Figure 6. The interactions in the high scoring models for CGRP model. The interactions include D280 with H10 and R11, T288 with T6 (and A5), S285 with A5 and D287 with A5 (shown in (b); the ECL2 hydrophobic residue interactions include W283 with D3 and T4 as well as residues on TM2 and TM3, and I284 with A5. (b) Hydrogen bonds between significant ECL2 residues include R274 interacting with Y277 and D287 (close to A5) interacting with Y278, which seem to stabilize the ECL2 fold. (c) The remaining significant ECL2 residues, namely A273, L290 and L291; A273 and L291 do not interact with CGRP, L290 interacts with L12 in some ECL2 conformations while L291 points towards TM7, but could also affect the RAMP interaction; it is notable that most of these non-interacting residues plus Y277 and Y278 are not sig- nificant in the binding of AM and so it is likely that in CLR they are important in RAMP1-directed indirect effects on the binding. These structures have not been refined by molecular dynamics simulations for reasons discussed above and so the molecular information should not be overinterpreted. model, CGRP can interact with L1952.61, V1982.64, A1992.65 but not T1912.57 and H2193.32 because they lie too deeply within the helical bundle. If CGRP were positioned to interact with these residues, it would then most likely not satisfy the interactions of A1 and T6. rsif.royalsocietypublishing.org J R Soc Interface 10: 20130589 8 (a) (b) 8 (a) (b) (b) J R Soc Interface 10: 20130589 (c) (c) (c) 3.2.5. Implications for receptor activation p p The binding of an agonist has to stabilize an active confor- mation of the receptor, increasing the activity of effector proteins. For the CGRP receptor, the best characterized inter- action is with Gs, leading to the production of cAMP and, based on homology with the active crystal structures of rho- dopsin and the b2-AR and extensive mutagenesis, we produced a model of how this can take place [19]. Based on the current data, it is possible to make some broad points in regard to possible agonist activation mechanisms in CLR. A change in the C-terminal region of ECL2 could easily be propagated to TM5 and changes in this helix that are linked to movement of TM6 have been identified as one of the ear- liest steps leading to receptor activation in family A GPCRs [11,53]. It may be significant that T6 of CGRP, important for agonist activity in both CGRP and AM [44,50], is in close proximity to the C-terminal region of ECL2 where D287 or preferably T288 are its most likely interaction partners. Further support for an important role of TM5 in receptor acti- vation comes from the disrupting effect of the double mutant of L220/L221A. A shift in TM5 in CLR could subsequently allow movement of TM6. In family B GPCRs, there is a con- served proline residue in TM6, which is likely to produce a kink [23] analogous to the situation found in family A GPCRs, so displacement of TM6 will lead to the opening of a G protein-binding pocket between it and TM3 [11]. ECL2 conformation, there are differences in the orientations of the W283 side chain, and only one high scoring CGRP and one high scoring AM conformation have W283 in a vertical orien- tation (a range of W283 interactions is shown in the electronic supplementary material, figures S4 and S5). The preferred CGRP structure (figure 6) satisfies 6/13 of the mutagenesis results given in table 1, whereasthe AM model (seethe electronic supplementary material, figure S6) satisfies 4/7. 3.2.4. AM binding (b) Hydrogen bonds between significant ECL2 residues include R274 interacting with Y277 and D287 (close to A5) interacting with Y278, which seem to stabilize the ECL2 fold. (c) The remaining significant ECL2 residues, namely A273, L290 and L291; A273 and L291 do not interact with CGRP, L290 interacts with L12 in some ECL2 conformations while L291 points towards TM7, but could also affect the RAMP interaction; it is notable that most of these non-interacting residues plus Y277 and Y278 are not sig- nificant in the binding of AM and so it is likely that in CLR they are important in RAMP1-directed indirect effects on the binding. These structures have not been refined by molecular dynamics simulations for reasons discussed above and so the molecular information should not be overinterpreted. 3.2.2. Filtering the ECL2 conformations The alanine-substitution effect at T1912.57 and H2193.32 may instead result from interactions with W283 of ECL2 as suggested by analysis of inactive CLR simulations and selected high scoring loop conforma- tions (figure 6; see the electronic supplementary material, figures S5–S7). The hydrophobic patch of L1952.61, V1982.64 and A1992.65 on TM2 is important in CLR for CGRP- mediated activation of cAMP production [20] but while this region is more polar in some GPCRs, alternative hydrophobic regions reside nearby in other family B GPCRs where W283 could bind. Consequently, we prefer the vertical confor- mation of W283 as no other conformation satisfies the mutation data on T1912.57 and H2193.32. 3.2.4. AM binding Our models can also explain the mutagenesis data for AM- mediated activation of the same receptor. It is proposed that AM sits in a very similar orientation to CGRP in the pre- sumed binding pocket (see the supplementary material) but accesses a slightly different subset of ECL conformations. There are no direct interactions with R2744.64, Y277 and Y278, but in the case of CGRP, the model suggests that there are also few, if any, direct interactions with these residues and instead they work primarily to stabilize pro- ductive conformations of ECL2. In the case of CGRP, we suggest that the consequence of this is to strengthen the inter- actions between the contact points between the peptide and ECL2 and so enhance potency. AM probably makes fewer contacts with ECL2; RAMP2 may be required to generate a full complement of interactions with this loop. Figure 6. The interactions in the high scoring models for CGRP model. The interactions include D280 with H10 and R11, T288 with T6 (and A5), S285 with A5 and D287 with A5 (shown in (b); the ECL2 hydrophobic residue interactions include W283 with D3 and T4 as well as residues on TM2 and TM3, and I284 with A5. (b) Hydrogen bonds between significant ECL2 residues include R274 interacting with Y277 and D287 (close to A5) interacting with Y278, which seem to stabilize the ECL2 fold. (c) The remaining significant ECL2 residues, namely A273, L290 and L291; A273 and L291 do not interact with CGRP, L290 interacts with L12 in some ECL2 conformations while L291 points towards TM7, but could also affect the RAMP interaction; it is notable that most of these non-interacting residues plus Y277 and Y278 are not sig- nificant in the binding of AM and so it is likely that in CLR they are important in RAMP1-directed indirect effects on the binding. These structures have not been refined by molecular dynamics simulations for reasons discussed above and so the molecular information should not be overinterpreted. Figure 6. The interactions in the high scoring models for CGRP model. The interactions include D280 with H10 and R11, T288 with T6 (and A5), S285 with A5 and D287 with A5 (shown in (b); the ECL2 hydrophobic residue interactions include W283 with D3 and T4 as well as residues on TM2 and TM3, and I284 with A5. rsif.royalsocietypublishing.org J R Soc Interface 10: 20130589 9 CGRP with its receptor involving 13 ECL2 residues in the loop out of 24 residues studied. By contrast, only two resi- dues in ECL1 and one in ECL3 influence the interaction of CGRP with its receptor [19,20]. Within ECL2, R274, W283, D280, D287 and T288 are of particular importance. We have suggested mechanisms where binding to this loop causes changes to the extracellular ends of TMs 2, 3 and 5, which in turn can be linked to movements of the cytoplasmic ends of TMs 3, 6 and 7, to allow G protein binding and activation. 9 While ECL2 appears to be involved in the binding of many peptide agonists to family B GPCRs, its precise role is probably receptor and ligand dependent. Models have been produced based on either mutagenesis or cross-linking data for the bind- ing of secretin, GLP-1, VIP and PTH to their receptors [8,15,16,57,58]. While there are of course limitations to any modelling study, it is striking that there is no agreement as to the mode of binding. In the case of some models developed for GLP-1, secretin and VIP, their N-termini penetrate more deeply to interact with TM2, providing a more direct method of altering the conformation of both this helix and probably the adjacent TM3 during the process of receptor activation. While GLP-1 may not bind in the same way as CGRP (where the disulfide bond makes the N-terminus more bulky), a study of this receptor has provided the clearest evidence yet that ECL2 is an important determinant of signalling specificity [6,7]. Even in possession of crystal structures, the flexible nature of ECLs requires studies such as this to shed more light on how GPCRs respond to peptide agonists. Our approach used to model the interaction between either CGRP or AM and the receptor may be applicable to other GPCRs. The full set of structures is available from ftp.essex.ac.uk/pub/oyster/ CLR_ECL2_2013/CLR_ECL2_structures.tar.gz (see also the electronic supplementary material); these structures compare favourably to the recent class B X-ray structures of glucagon and the corticotropin-releasing factor-1 receptors as described in the electronic supplementary material. J R Soc Interface 10: 20130589 The model developed for CGRP binding is therefore con- sistent with what is known about how CGRP activates its receptor, although it is speculative. It is also important to note that we have interpreted the effects of the mutations directly on the conformation of ECL2 itself. References 1. Wheatley M, Wootten D, Conner MT, Simms J, Kendrick R, Logan RT, Poyner DR, Barwell J. 2012 Lifting the lid on GPCRs: the role of extracellular loops. Br. J. Pharmacol. 165, 1688–1703. (doi:10.1111/j.1476-5381.2011.01629.x) 1. Wheatley M, Wootten D, Conner MT, Simms J, Kendrick R, Logan RT, Poyner DR, Barwell J. 2012 Lifting the lid on GPCRs: the role of extracellular loops. Br. J. Pharmacol. 165, 1688–1703. (doi:10.1111/j.1476-5381.2011.01629.x) 4. Hoare SR. 2005 Mechanisms of peptide and nonpeptide ligand binding to Class B G-protein- coupled receptors. Drug Discov. Today 10, 417–427. 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Conner M, Hawtin SR, Simms J, Wootten D, Lawson Z, Conner AC, Parslow RA, Wheatley M. 2007 Systematic analysis of the entire second extracellular loop of the V1a vasopressin receptor: key residues, conserved throughout a G-protein-coupled receptor family, identified. J. Biol. Chem. 282, 17 405–17 412. (doi:10.1074/jbc.M702151200) 10. Gkountelias K, Tselios T, Venihaki M, Deraos G, Lazaridis I, Rassouli O, Gravanis A, Liapakis G. 4. Conclusion We have evaluated the role of ECL2 in the binding of both CGRP and AM to the CGRP receptor and have interpreted the results by means of molecular modelling. The study indi- cates that ECL2 is particularly important for the interaction of Funding statement. This work was supported by grants from the Well- come Trust to D.R.P. (091496), the New Zealand Heart Foundation to H.A.W., the MRC to C.A.R. (G1001812) and a grant of computer time at Montpellier under the HPC Europa2 programme (B.T.). rsif.royalsocietypublishing.org J R Soc Interface 10: 20130589 9 We cannot rule out that some of the effects may be on the TM bundle or extra- cellular domains of CLR or even of the RAMP. The data can be used to address these issues more fully once a crystal structure of the active CLR/RAMP1 complex becomes available. 3.2.3. The orientation of W283 The loop generation alone does not help to address the orientation of W283 as it interacts with CGRP in most confor- mations of ECL2. However, the docking raises an interesting question with regard to the site-directed mutagenesis data on TM2 and TM3, as shown in the electronic supplementary material, figure S7. A number of residues on TM2 and TM3 (namely T1912.57, L1952.61, V1982.64, A1992.65 and H2193.32) show reduced cAMP production on mutation [19]. In our The conserved N-terminal part of ECL2 [1] links ECL2 not only with TM4 but is also likely to influence ECL1 and hence TM2 and 3. While ECL1 is of only minor importance in the binding of CGRP, residues just below it in TM2 and H2193.32 of TM3 are of considerable significance for receptor activation [20]. Similar clusters are not obvious in the upper regions of the other TMs [19], although systematic mutagen- esis is needed to test this. These residues at the tops of TM2 and 3 may be in a position to make contacts with residues at the base of ECL2, such as W283 and I284 as proposed in this study (see the electronic supplementary material, figure S7). Thus, CGRP has the potential to influence TM2 and 3 both directly and indirectly via ECL2. In family A GPCRs, TM3 is of particular importance for receptor acti- vation partly owing to its angle of tilt across the TM bundle linking different parts of the bundle together. 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https://openalex.org/W2800731880	https://europepmc.org/articles/pmc5916944?pdf=render	English	null	Integrative network analyses of wilt transcriptome in chickpea reveal genotype dependent regulatory hubs in immunity and susceptibility	Scientific reports	2,018	cc-by	12,497	Integrative network analyses of wilt transcriptome in chickpea reveal genotype dependent regulatory hubs in immunity and susceptibility Received: 23 May 2017 Accepted: 5 January 2018 Published: xx xx xxxx Received: 23 May 2017 Accepted: 5 January 2018 Published: xx xx xxxx Received: 23 May 2017 Accepted: 5 January 2018 Published: xx xx xxxx Host specific resistance and non-host resistance are two plant immune responses to counter pathogen invasion. Gene network organizing principles leading to quantitative differences in resistant and susceptible host during host specific resistance are poorly understood. Vascular wilt caused by root pathogen Fusarium species is complex and governed by host specific resistance in crop plants, including chickpea. Here, we temporally profiled two contrasting chickpea genotypes in disease and immune state to better understand gene expression switches in host specific resistance. Integrative gene- regulatory network elucidated tangible insight into interaction coordinators leading to pathway determination governing distinct (disease or immune) phenotypes. Global network analysis identified five major hubs with 389 co-regulated genes. Functional enrichment revealed immunome containing three subnetworks involving CTI, PTI and ETI and wilt diseasome encompassing four subnetworks highlighting pathogen perception, penetration, colonization and disease establishment. These subnetworks likely represent key components that coordinate various biological processes favouring defence or disease. Furthermore, we identified core 76 disease/immunity related genes through subcellular analysis. Our regularized network with robust statistical assessment captured known and unexpected gene interaction, candidate novel regulators as future biomarkers and first time showed system-wide quantitative architecture corresponding to genotypic characteristics in wilt landscape. Plants frequently encounters to wide range of patho-stresses that modulate growth and development thereby affecting the overall productivity. Counter action strategies are pre-requisite for assault and defense against viru- lence factors of pathogen and innate immune system of plants. Several of these stresses are united by the fact that at least part of their detrimental effect on plant performance is caused by the deregulation of the immune status. It is a monolayer paradigm in which mounting resistance in host is counter-balanced by deregulated pathogen virulence. Although sequence of recognition, signal transduction and response is common theme but selection in plant is unique to type of pathogen attack contributing to disease or resistance1. Plant innate immunity can be activated by microbe, chitin or pathogen-associated molecular patterns (M/C/PAMPs) in accurate manner and determined by transcription factors and extensive transcriptional reprogramming. Fungal invasion is orches- trated by set of genes that exhibit induction and/or repression during infection dictating host’s ability to mitigate and pathogen to propagate in plant-microbe interactions2. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 23 May 2017 Accepted: 5 January 2018 Published: xx xx xxxx National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, 110067, India. Correspondence and requests for materials should be addressed to S.C. (email: subhrac@hotmail.com) Results F i Fusarium subverts physiological processes in chickpea. Fusarium infects through roots, enters xylem vessels, produce pathotoxins affecting plant metabolism. Furthermore, pathogen invasion also induce membrane injury and affect water conductance and potential that regulate stomatal opening and obstruct water and min- eral transport leading to wilting and subsequent death of host plant32,33. To investigate genotype specific cellu- lar responses during host specific resistance, we screened different chickpea varieties challenged with Fusarium oxysporum. Sharply contrasting phenotype and considerable symptomatic differences were observed amongst the studied genotypes. WR-315 and CPS1 appeared to be the most resistant variety while JG-62 was found to be most susceptible and early wilting genotype. C-104 and K850 showed late wilting phenotype. No symptoms were observed on mock-inoculated seedlings. No visible changes were observed in WR-315 seedlings till one month post inoculation, but in JG-62 wilting started after 48 hpi and the symptoms were further intensified at 96–120 hpi exhibiting severe wilting. To understand pathogen progression quantitatively, fungal biomass was determined in Fusarium challenged resistant and susceptible chickpea genotypes. We found significant increase in fungal biomass over time in susceptible genotype (JG-62) in contrast to the resistant genotype (WR-315) (Fig. 1i). Furthermore, we examined ROS production in both the genotypes till 120 hpi as qualitative measure. A large ROS burst was observed in patho-stressed resistant WR-315 roots at 12–48 hpi, while ROS production was less pronounced in the susceptible genotype, JG-62. Fusarium induced ROS production was not detected in control roots by DAB staining (Supplementary Fig. S1). Plants adapt to water stress condition by increased concentration of intracellular solutes, such as proline, which facilitate the maintenance of cell pressure potential34. Moreover, role of proline as osmoprotectant in plant defense against invading pathogen has earlier been documented35,36. We observed that WR-315 was able to main- tain Relative Water Content (RWC) considerably at higher level while JG-62 showed the maximum decline, sug- gesting inability of susceptible genotype to take up water due to maximum colonization of the pathogen in the xylem (Fig. 1a). Concomitantly, WR-315 showed significant increase in endogenous free proline followed by CPS1 during patho-stress. This may be related to ~90% recovery in RWC (Fig. 1b). Plasma membrane integrity and lipid peroxidation due to fungal attack often leads to pathogen invasion. Marked difference in electrolyte leakage was observed between genotypes with maximum in JG-62 and least in WR-315 and CPS1 (Fig. 1c). www.nature.com/scientificreports/ categorized into >120 formae speciales (f. spp.)11. Host genotype specificity and hemibiotrophic pathogen interac- tion depends on gene-for-gene model12–14. Despite earlier study showing that resistance to Fusarium in chickpea is host specific, polygenic and under complex genetic control15, the mechanism controlling effective resistance and genotype-pathotype interaction remains largely unknown.f g yp p yp g y Advances in high-throughput omics technologies offer unparalleled opportunities to evaluate patho-stress response at system level. These responses are characterized by dynamic and variable gene expression changes leading to reprogramming of many cellular functions16. It is known that differences in the transcriptome between PTI and ETI are largely quantitative17,18. Further, interconnectivity between subcellular compartments dictate organelle specific gene interaction spread along the gene regulatory network modules19. Various schemes have been proposed from differential transcriptome to identify features/genes that are dramatically different in dis- ease and immune state20. Consequently, network-based protein-protein interaction (PPI) characterize intricate and interwoven relationships that govern cellular functions. Thus, integrating PPI and gene-expression profile provides novel insights into functional interactions amongst deregulated genes21–25. This combined approach can provide insights into regulation of cellular processes and identify the interaction architecture and the under- lying gene regulatory networks26. To elucidate molecular mechanism of diseasome or immunome conventional statistical methods and computational approaches have been employed27,28. Regulatory relationships among 22 immune-related genes in Arabidopsis were elucidated based on sector switching model derived from network analysis29. Thus, transcript profiling assist in elucidating logic of regulatory circuits to provide insights into cel- lular processes and identification of interaction architecture to manage plant immune response30. Earlier, we reported expressed sequence tags (EST) and comprehensive insight into identity and function of immune respon- sive root transcriptome in chickpea31. p p Here, we present integrative functional network analyses based on cDNA microarray temporal datasets con- sisting of 6072 spots representing 1749 unigenes to examine the common and discrete features of chickpea gene network during host specific resistance in response to Fusarium wilt. The aim was to create a signal transduction catalogue for chickpea defense and/or disease signaling and develop a snapshot of transcriptional regulatory programs underlying the immune response. We assembled gene network by integrating temporal gene expres- sion data from two contrasting chickpea genotypes differing in patho-stress response. Data analysis revealed that molecules belonging to several biological processes were preferentially and differentially expressed during patho-stress. A diseasome and immunome was constructed to reveal invasion or resistance mechanism. www.nature.com/scientificreports/ Corollary of this hypothesis was elucidated in unbiased manner to determine significant difference in gene expression and interdependencies among cellular components to determine relationships among variables. Further, the host specific response seemed to be tightly regulated by transcriptional regulators to execute condition-specific and complex biological functions in eukaryotes. Finally, the study underpins genotype dependent transcriptional reg- ulation during patho-stress and highlights the importance of module coordination in host specific plant defense. Integrative network analyses of wilt transcriptome in chickpea reveal genotype dependent regulatory hubs in immunity and susceptibility Thus, host immunome and diseasome should be pre- cisely regulated by gene networks to maintain balance between immune response and pathogen colonization. Identification of genotype specific molecular events may provide unique insight into the effect of genotypic vari- ation on the plant-pathogen interaction. However, the difference and the overlap in the downstream components of CTI, PTI and ETI are largely unknown1,3–10. g y Chickpea (Cicer arietinum) is the second most important legume worldwide, but its production is highly threatened due to vascular wilt. Fusarium oxysporum, causal agent of vascular wilt is a soil borne fungus and adapt hemibiotrophic mode of invasion. According to host specificity, wilt-inducing F. oxysporum isolates are National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, 110067, India. Correspondence and requests for materials should be addressed to S.C. (email: subhrac@hotmail.com) Scientific REPOrTS \| (2018) 8:6528 \| DOI:10.1038/s41598-018-19919-5 www.nature.com/scientificreports/ Results F i Lipid peroxidation also showed a similar trend with increased malondialdehyde (MDA) levels (~4.5-fold) in JG-62 (Fig. 1d). Photosynthetic ability is one of the most significant parameter in stress response37. WR-315 and CPS1 maintained significantly higher chlorophyll a, chlorophyll b and chlorophyll c in addition to protein content than other genotypes (Fig. 1e–h). Taken together, these results confirm WR-315 as most resistant and JG-62 as most susceptible to Fusarium wilt which has earlier been well documented38–44. Scientific REPOrTS \| (2018) 8:6528 \| DOI:10.1038/s41598-018-19919-5 2 www.nature.com/scientificreports/ Figure 1. Physiological and biochemical analysis of chickpea varieties in response to percent RWC, (b) endogenous free proline content, (c) estimation of electrolyte leaka (e,f,g) measurement of photosynthetic pigments chlorophyll A, chlorophyll B and ch (h) total protein, (i) relative quantification of fungal biomass by real-time PCR on DN oxysporum-infected roots of JG-62 and WR-315 at 6, 12, 24, 48 and 120 hpi. Amplific were normalized to the abundance of chickpea 18S sequence. Each replicate is a pool independent experiments with three biological replicates. Lines and vertical bars den Expression changes were analyzed by ANOVA and Tukey post-hoc test (p < 0.05) and “” indicates statistical significance of relative amount of fungal DNA. Figure 1. Physiological and biochemical analysis of chickpea varieties in response to Fusarium attack. (a) percent RWC, (b) endogenous free proline content, (c) estimation of electrolyte leakage, (d) MDA levels, (e,f,g) measurement of photosynthetic pigments chlorophyll A, chlorophyll B and chlorophyll C, respectively, (h) total protein, (i) relative quantification of fungal biomass by real-time PCR on DNA extracted from F. oxysporum-infected roots of JG-62 and WR-315 at 6, 12, 24, 48 and 120 hpi. Amplification values for FoGDP were normalized to the abundance of chickpea 18S sequence. Each replicate is a pool of five plants of three independent experiments with three biological replicates. Lines and vertical bars denotes the mean values ± SE. Expression changes were analyzed by ANOVA and Tukey post-hoc test (p < 0.05) and vertical bars indicate SE. “” indicates statistical significance of relative amount of fungal DNA. Figure 1. Physiological and biochemical analysis of chickpea varieties in response to Fusarium attack. (a) percent RWC, (b) endogenous free proline content, (c) estimation of electrolyte leakage, (d) MDA levels, (e,f,g) measurement of photosynthetic pigments chlorophyll A, chlorophyll B and chlorophyll C, respectively, (h) total protein, (i) relative quantification of fungal biomass by real-time PCR on DNA extracted from F. Results F i oxysporum-infected roots of JG-62 and WR-315 at 6, 12, 24, 48 and 120 hpi. Amplification values for FoGDP were normalized to the abundance of chickpea 18S sequence. Each replicate is a pool of five plants of three independent experiments with three biological replicates. Lines and vertical bars denotes the mean values ± SE. Expression changes were analyzed by ANOVA and Tukey post-hoc test (p < 0.05) and vertical bars indicate SE. “” indicates statistical significance of relative amount of fungal DNA. Scientific REPOrTS \| (2018) 8:6528 \| DOI:10.1038/s41598-018-19919-5 3 www.nature.com/scientificreports/ Figure 2. Gene expression pattern of DEGs and qRT-PCR analysis. (a) regulation of DEGs for JG-62 (wilt susceptible) and WR-315 (wilt resistant) genotypes of chickpea over the time course after inoculation with Fusarium, (b) venn diagram depicting exclusive and overlapping DEGs. (c,d,e) venn diagram representing regulation of IDEGs, DDEGs and CDEGs. (f) relative mRNA levels of eight candidate DEGs involving PR10, pectinesterase (PE), uncharacterized protein, aquaporin, cystatin, DnaJ, PR5, ERF5 were assessed by qRT-PCR. Statistical significance of expression changes were analyzed by ANOVA and indicated by for p < 0.05 (Tukey post-hoc test). Vertical bars denote SE. Figure 2. Gene expression pattern of DEGs and qRT-PCR analysis. (a) regulation of DEGs for JG-62 (wilt susceptible) and WR-315 (wilt resistant) genotypes of chickpea over the time course after inoculation with Fusarium, (b) venn diagram depicting exclusive and overlapping DEGs. (c,d,e) venn diagram representing regulation of IDEGs, DDEGs and CDEGs. (f) relative mRNA levels of eight candidate DEGs involving PR10, pectinesterase (PE), uncharacterized protein, aquaporin, cystatin, DnaJ, PR5, ERF5 were assessed by qRT-PCR. Statistical significance of expression changes were analyzed by ANOVA and indicated by * for p < 0.05 (Tukey post-hoc test). Vertical bars denote SE. Mutual exclusivity and consistency of transcriptional reprogrammers in disease vs immune state. Temporal gene expression profiles in Fusarium infected chickpea seedlings of two contrasting genotypes, wilt-susceptible (JG-62) and wilt-resistant (WR-315) were assessed using microarray analysis. Among 6072 qual- ified probes representing 1749 genes present in the microarray, compared to the control mock inoculated, a set of 1200 immune responsive factors (IRFs) and 77 fungal genes were differed by a factor of two or more (fold change > 2.5, p < 0.05) in at least one of the six time points in either genotypes (Supplementary Dataset 1; Fig. 2a). Results F i From the identified 77 fungal genes, 17 were exclusively expressed in JG-62, including 11 downregulated and 6 upregulated genes. 11 fungal genes (4 downregulated and 7 upregulated) were specifically expressed in WR-315. However, 43 fungal genes with 12 and 31 genes exhibiting downregulation and upregulation, respectively were differentially expressed in both the genotype. Three fungal genes, namely exosome complex component, mtr3 and uncharacterized fungal genes exhibited upregulation in JG-62 but downregulated in WR-315. A fungal ATP synthase subunit showed downregulation at early time points and was upregulated in later time point in JG-62, whereas in WR-315 it showed upregulation till 120 hpi. Gene with unknown function and v-type proton ATPase catalytic subunit A were upregulated in early time point and downregulatd in later time point in JG-62, whereas in resistant genotype they showed upregulation till 120 hpi or showed no expression, respectively. Of the IRFs, 391 disease associated differentially expressed genes (DDEGs) and 216 immunity associated differ- entially expressed genes (IDEGs) were unique to JG-62 and WR-315, respectively while 593 common differen- tially expressed genes (CDEGs) were found to be shared between the genotypes (Fig. 2b). The analysis revealed that each stage of disease development in JG-62 or immune response in WR-315 was represented by distinct transcription profile. Out of 391 DDEGs in JG-62, 119 genes were upregulated and 250 were downregulated (Fig. 2c). In WR-315, among 216 IDEGs 52 exhibited upregulation and 151 showed downregulation (Fig. 2d). Of note, although in both genotypes, percentage of up- or down-regulated DEGs was different but the number was similar at each post-inoculation time. Meanwhile, the total up- and down-regulated DEGs were comparable between two genotypes. However, in JG-62, more number of genes (337 DEGs) showed altered expression at 6 hpi than WR-315 (75 DEGs) possibly due to the fact that pathogen sensing and perception promote gene expres- sion regulation in susceptible genotype at early stage of invasion. Similar number of genes were differentially Scientific REPOrTS \| (2018) 8:6528 \| DOI:10.1038/s41598-018-19919-5 4 www.nature.com/scientificreports/ regulated at 12 hpi and 24 hpi in both the genotypes reflecting the transition point for disease or immune state. At 48 hpi, 537 DEGs were identified in JG-62 and 283 DEGs in WR-315, suggesting that in later stages clear signal separation represented by distinct transcriptional reprogrammers might regulate disease or immune pathways in contrasting genotypes. Results F i According to the gene expression pattern of two contrasting genotypes, we classified the DEGs into two subsets of SOTA clusters (SC): (i) DDEGs exclusive to JG-62 representing SC1, SC3, SC5 and SC8 and (ii) IDEGs exclusive to WR-315 representing SC4 and SC7. Further analysis of the CDEGs identified 266 genes that act as core genes shared by JG-62 and WR-315 confirming that combinatorial interactions of DEGs drive reprogramming events during patho-stress. Validation of microarray data as recurrent transcript. The effect of genotype and temporal kinetics under patho-stress was further assessed by qRT-PCR (Fig. 2f, Supplementary Table S1). IDEGs like PR10, pec- tinesterase, uncharacterized protein showed elevated expression in WR-315. Aquaporin, a DDEG was mark- edly repressed in JG-62. Transcripts belonging to CDEGs, namely cystatin and DnaJ exhibited upregulation or mixed expression while PR4 showed repression in both genotypes. Notably, ERF5 involved in hormone signaling showed upregulation in susceptible genotype and downregulation in resistant genotype. Thus, relative expres- sion levels of DEGs showed strong positive correlation and similar trends as compared with microarray analysis (Supplementary Fig. S3). Cannonical and non-cannonical pathways modulated by patho-stress. To assess the biological relevance of the transcriptional reprogrammers, we performed gene ontology analysis of DEGs using Blast2GO46. Functional analysis of the DDEGs, IDEGs and CDEGs enriched GO terms belonged to biological process, cellular component and molecular function (Fig. 4). The DEGs in the cluster of biological process was primarily involved in cellular metabolism (251 IDEGs, 432 DDEGs, 2329 CDEGs) and oxidation reduction process (7 IDEGs, 15 DDEGs, 74 CDEGs). This is consistent with the fact that carbon and nitrogen metabolism were greatly affected during patho-stress irrespective of genotypes47,48. The DEGs were also found to be members of variety of cellular components with a large representation from cytoplasm. As JG-62 and WR-315 displayed distinct molecular properties in response to Fusarium, we intended to identify key genes involved in maintaining cellular function- ality. It was observed that genes related to nucleic acid binding (3 IDEGs, 12 DDEGs, 56 CDEGs), nucleoside binding (6 IDEGs, 11 DDEGs, 78 CDEGs), ribonucleotide binding (5 IDEGs, 13 DDEGs, 81 CDEGs), anion binding (7 IDEGs, 17 DDEGs, 97 CDEGs) and nucleoside phosphate binding (7 IDEGs, 17 DDEGs, 102 CDEGs) may determine disease or immune state of the respective genotypes. Identification of gene network and mapping functional transcription factor networks during vascular wilt. Results F i Interestingly, opposite trend in gene expression was observed at 24 hpi showing more upregulated DEGs and 120 hpi depicting more downregulated IDEGs in resistant genotype confirming a link between number of DEGs and immune processes. To further assess the similar or diverse effects of the two contrasting genotypes, venn diagrams were used to depict the overlap of CDEGs that were significantly up- and down-regulated. Among upregulated CDEGs, 231 genes and in the downregulated CDEGs, expression of 250 genes was commonly regulated in both genotypes (Fig. 2e). While 28 were preferentially upregulated in JG-62 and downregulated in WR-315 and 19 showed upregulation in WR-315 and downregulation in JG-62. Furthermore, in both the genotypes, cell wall remodelers like pectinesterase and xyloglucosyl transferase were upregulated while cytoskeleton associated genes like actin and profilin-like genes were downregulated. Also ROS associated genes like cytochrome P450, cytochrome P450 monooxygenase and peroxidase showed upregulation in both genotypes (Supplementary Dataset 1). This is consistent with the fact that distinct counter regulatory pathways affect the set-point in immune/disease homeostatic control that is governed by the net balance between inhibitory and stimulatory responses in two genotypes.i y p g yp Principal component analysis (PCA) signifies variation in gene expression in which each dimension repre- sents the variability45. We conducted PCA to evaluate the degree of differences and relatedness of transcription profiles among different conditions. It was observed that maximum variation was accounted for by first compo- nent PC1 (eigenvalue 13.698; 35.12%) and second component PC2 (eigenvalue 2.428; 11.47%). Although 2-D plot revealed similar variance across time points in both early and later stages of invasion, but the transcripts were of variable nature between the genotypes (Fig. 3a). Further, to identify genes that significantly regulate expression during patho-stress, we conducted two-way ANOVA and found 31 genes that had significant expression differ- ence between the genotypes (Fig. 3b). g yp ( g ) Next, to facilitate biological interpretation of the dataset, we generated an unbiased framework that group similar expression across time points and genotypes using SOTA analysis (Supplementary Fig. S2). Clustering of DDEGs, IDEGs and CDEGs resulted in ten distinct clusters distinguishing the five JG-62-treated and five WR-315-treated seedlings and the two remaining untreated seedlings from each of the control genotype. Lists of commonly and differentially expressed genes are provided in Supplementary Datasets 2, 3, 4. Scientific REPOrTS \| (2018) 8:6528 \| DOI:10.1038/s41598-018-19919-5 Results F i Characteristic of disease and immune state can be governed by gene expression dynamics as a cause and a consequence of patho-stress49, therefore, we analyzed integrated gene expression profile in two contrasting genotypes to understand functionality and modularity of gene interactions. We found variability in co-regulated gene expression across time points and between genotypes, suggesting that attributes of disease and immunity is dictated by functionality and modularity of gene interactions and often share common and diverse function. Further, we constructed a maximum continuous network consisted of 389 nodes connected via 9079 edges based on temporal gene expression data and known PPIs (Fig. 5). Visual inspection of the resulting interac- tome revealed highly connected functional and regulatory network and identified key exclusive or specific IRFs that play important role in disease and immune state. Resulting network was densely organized and consisted of four biologically significant coherent subnetworks with functional sub-specialization. These subnetworks Scientific REPOrTS \| (2018) 8:6528 \| DOI:10.1038/s41598-018-19919-5 5 www.nature.com/scientificreports/ Figure 3. Investigation of identified DEGs. (a) PCA of the data set shows that the expression profiles of all 12 conditions are different from each other. X-axis and y-axis denotes principal component 1 (PC1) and principal component 2 (PC2), respectively. Number refers to IDs depicted in Supplementary Table S1. (b) Heat map of DEGs between two genotypes. Significant differences (p < 0.05) were estimated using two-way ANOVA. Figure 3. Investigation of identified DEGs. (a) PCA of the data set shows that the expression profiles of all 12 conditions are different from each other. X-axis and y-axis denotes principal component 1 (PC1) and principal component 2 (PC2), respectively. Number refers to IDs depicted in Supplementary Table S1. (b) Heat map of DEGs between two genotypes. Significant differences (p < 0.05) were estimated using two-way ANOVA. were significantly enriched for IRFs belonging to biological pathways involving cell organization and biogenesis (43 nodes, 354 edges) (Post-translational protein modification) (SN1); transcription and translation regulation (SN2) (55 nodes, 235 edges) (Translation); signal transduction (SN3) (50 nodes, 165 edges) (Transcription); Nucleic acid processing, cell cycle replication and metabolism response (SN4) (50 nodes, 180 edges) (Cellular assembly and transport) (Fig. 6a–d; Supplementary Dataset 5).fi y p g pp y Subnetwork SN1 was associated with vesicular trafficking and might be involved in sorting of mRNAs which remain untranslated due to downregulation of many ribosomal proteins during stress. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 4. Functional enrichment of the DEGs. Distribution of transcripts based on Blast2GO analyses. Y-axis indicates significant Blast2GO functional categories (p < 0.05) and the X-axis shows number of transcripts. Figure 4. Functional enrichment of the DEGs. Distribution of transcripts based on Blast2GO analyses. Y-axis indicates significant Blast2GO functional categories (p < 0.05) and the X-axis shows number of transcripts. be placed distantly from the central cluster and included protein kinase (PK) domain (IRF870). Both APK1 and PK were induced more in susceptible genotype suggesting that distinct signaling components may be activated in diseased state. Subnetwork SN2 (transcription and translation regulation) linked to SN1 was dominated by ribo- somal proteins and translation factors. Ribosomal proteins like L35, L37a, L23, L15, S8 (IRF922, 789, 1134, 505) were downregulated while S13–2 (IRF324) was upregulated in both genotypes. This may be attributed to stress conditions inducing a radical reprogramming to inhibit the translation and promote the repair of defense-related genes. Ubiquitin encoding genes (IRF705, IRF1204) may act as a regulator for activation of many ribosomal proteins which perform distinct roles in specific cellular processes. Ribosomal protein S3a (IRF682) is a key player in cell transformation52 while ribosomal protein L27 (IRF254) is involved in mRNA degradation triggered by genotoxic stress53. Suppression of the ribosomal L2 (IRF26) was identified as a novel mechanism for stress adaptation54. This is consistent with the notion that shared and distinct set point governed by transcription and translation dictates disease or immune response. p Subnetwork SN3 was enriched in GTP binding proteins, chromatin remodelers, ribosomal proteins and ubiquitin factors. Histone variants (histone H3.2) (IRF796) and deacetylases (HDT1, HDA2) (IRF670, IRF504) were clustered along with ubiquitin (IRF1204) and were further connected to GTP binding proteins, namely ADP-ribosylation factor 1 (IRF810) and ribosomal proteins (IRF717, 922, 941, 1075). Our data showed induction of Ras encoding gene (IRF297), golgi SNAP receptor complex member 1 (IRF310) and vesicle sorting (GOS11) (IRF474) during early time points. Two of the histone deacetylase HDT1 (IRF200, 504, 670) showed interaction with uncharacterized protein (IRF1238) that act as versatile modulator of chromatin function and affect the struc- tural flexibility of DNA. Histone deacetylase HDT1 showed downregulation, which indicates that chromatin decondensation drives distinct transcription patterns. Co-regulation and interaction of these genes suggest modu- lation of signal transduction pathways and plethora of metabolism shared in both genotypes. Results F i Macromolecule biogen- esis is known to have prominent role in gene regulation during stress50. We found that 20 S proteasome A and B subunit (IRF1042) was upregulated while ubiquitin-conjugating enzyme E2 (IRF12) and 26 S protease regulatory subunit 6 (IRF1054) were downregulated in the JG-62. However, ubiquitin (IRF1204) was first induced and then repressed in both genotypes. Notably, SN1 was also markedly enriched for genes involved in phosphorylation cascade. Many of which are core stress associated signaling component in addition implicated in the regula- tion of nucleic acid processing, including serine/threonine kinases and other related kinases (IRF1, 48, 185, 410, 470, 586, 656, 792, 1021, 1150, 1251), CIPK (IRF410), leucine-rich repeat (LRR) transmembrane protein kinase (IRF1259), shaggy-related protein kinase gamma (IRF767, 869), calcium-dependent calmodulin-independent kinase (IRF847) and putative receptor-like protein kinase (IRF731). While LRR TM PK (IRF314) is known to play role in plant defense51, involvement of APK1 (IRF32) is a novel finding. A few of these kinases were seen to Scientific REPOrTS \| (2018) 8:6528 \| DOI:10.1038/s41598-018-19919-5 6 www.nature.com/scientificreports/ SN3 was functionally linked to SN4 (nucleic acid processing, cell cycle replication and metabolism response). Association of disease or immunity with cell cycle checkpoint was found for tubulin alpha-1 chain (IRF701) in SN4. It was downregulated in both the genotypes, indicating that cell cycle regulator recycling might play vital role during patho-stress. Cellular processes are governed by complex gene expression programs regulated by transcription factors (TFs). To identify TF dynamics, we segregated TFs, chromatin-related proteins, transcriptional machinery com- ponents and protein kinases and mapped their differential expression into transcriptional regulatory networks (TRNs) (Fig. 6e). Expression based regulatory links predicted by interactions comprised of 39 distinct TFs regu- lating 186 distinct targets with p < 0.05 supporting that perturbation of TF is directly related to immunity or dis- ease state. We recovered a number of well-characterized complexes, such as zinc finger family, 14–3–3, histones and TF complex. Finally, TF interactions were used to functionally interrogate the mastermind signaling net- work. Transcription factor reprograming observed in network (39 nodes) contributed centrally to the robustness. Transcription factor bHLH122 (IRF165), zinc finger A20 and AN1 SAP8 (IRF703) and nuclear transcription fac- tor Y subunit A-7 (IRF723) were among the top transcription factor while serine/threonine kinases were among Scientific REPOrTS \| (2018) 8:6528 \| DOI:10.1038/s41598-018-19919-5 7 www.nature.com/scientificreports/ the top protein kinases associated with the DEGs across the datasets. Unique observation was predominant clus- ter of genes belonged to homeodomain leucine zipper family (IRF667) and BEL1 (IRF160) while the other cluster was centered around bHLH (IRF188, 751, 817, 1048, 1264) alongwith MYB transcription factor MYB2 (IRF203), Figure 5. Gene network analysis. Network was constructed using gene expression data of 12 conditions from susceptible and resistant genotypes. Changes in expression profiles at different time points from two genotypes were captured in co-expression network. Each node represents a given protein associated with an EST (based on top BLAST hit against SwissProt) and an edge denotes a probability of two given proteins (nodes) potentially interacting based on the cytoprophet algorithm. Figure 5. Gene network analysis. Network was constructed using gene expression data of 12 conditions from susceptible and resistant genotypes. Changes in expression profiles at different time points from two genotypes were captured in co-expression network. www.nature.com/scientificreports/ Each node represents a given protein associated with an EST (based on top BLAST hit against SwissProt) and an edge denotes a probability of two given proteins (nodes) potentially interacting based on the cytoprophet algorithm. Figure 5. Gene network analysis. Network was constructed using gene expression data of 12 conditions from susceptible and resistant genotypes. Changes in expression profiles at different time points from two genotypes were captured in co-expression network. Each node represents a given protein associated with an EST (based on top BLAST hit against SwissProt) and an edge denotes a probability of two given proteins (nodes) potentially interacting based on the cytoprophet algorithm. the top protein kinases associated with the DEGs across the datasets. Unique observation was predominant clus- ter of genes belonged to homeodomain leucine zipper family (IRF667) and BEL1 (IRF160) while the other cluster was centered around bHLH (IRF188, 751, 817, 1048, 1264) alongwith MYB transcription factor MYB2 (IRF203), Hy5 transcription factor (IRF276) and BEL 1 domain (IRF160). Although these TFs shows development related function55, their involvement in disease/immunity would open up possibility for exploring their functionality. the top protein kinases associated with the DEGs across the datasets. Unique observation was predominant clus- ter of genes belonged to homeodomain leucine zipper family (IRF667) and BEL1 (IRF160) while the other cluster was centered around bHLH (IRF188, 751, 817, 1048, 1264) alongwith MYB transcription factor MYB2 (IRF203), Hy5 transcription factor (IRF276) and BEL 1 domain (IRF160). Although these TFs shows development related function55, their involvement in disease/immunity would open up possibility for exploring their functionality. 8 Scientific REPOrTS \| (2018) 8:6528 \| DOI:10.1038/s41598-018-19919-5 www.nature.com/scientificreports/ Network specializations of wilt diseaseome and immunome. Analyses of modular network and machine learning methods dissect role of disease/immune-related gene network. We designed an integrative multistep framework combining gene expression analysis and modular co-expression network to systematically characterize the organizing principles of wilt diseasome and immunome. E h di d h d fi i d di i i i d h h di k b di Figure 6. Gene sub-networks associated with pathostress and mapping transcription factor network. (a) cell organization and biogenesis, (b) transcription, and translation regulation, (c) signal transduction, (d) nucleic acid processing, cell cycle replication and metabolism response, (e) transcription factor network. Nodes and edges represent genes and coexpression between genes, respectively. Figure 6. Gene sub-networks associated with pathostress and mapping transcription factor network. www.nature.com/scientificreports/ Further, heterogeneity and complex association involving calcium-dependent protein kinase 8, nucleoside diphosphate kinase, G-protein signaling elements, phytochrome-associated serine/threonine-protein phosphatase 3 were observed in module 2 (M2) consisting of 54 DEGs for fungal penetration. The most highly active set of colonization associated genes (144 DEGs) like proteases, transferases, ribosome machinery and transporters were abundant in module 3 (M3). Finally, module 4 (M4) was enriched with genes involved in disease development like TFs, coactivators, translocases and signal elements functional in disease pathway. Some of the genes matching these criteria are involved in DNA damage responses and cell cycle checkpoint regulation57. Thus, it can be concluded that DEGs associated with signaling components were co-expressed during perception and penetration of Fusarium, whereas in colonization pro- tein fate, transport and transcription related DEGs were majorly represented. As expected, varied families of transcription factors and signaling pathways components showed interactions in disease development module. Consistent with morphological and biochemical changes diseasome showed diverse signaling and regulatory pathways in each of the four modules. Triggering innate immune response reveals homeostatic points that regulate cellular pathway6. Although, PTI and ETI are two main plant immune responses to counter pathogen invasion, however, in response to fun- gal pathogens CTI is considered separately as immune response to chitin polysaccharides of fungal cell wall9. To determine immunity-related pathways, we designed and assembled gene expression profiles in correlation network framework delineated into four hubs encompassing PTI, ETI and CTI (Fig. 7b). We adopted a scoring strategy to reduce false positives and remove unreliable gene association data. The network comprised of 207 nodes and 2482 edges was enriched with genes implicated in pattern-, effector- or R-mediated signaling and homeostasis. For PTI, CTI and ETI modules, there were 23, 3 and 35 DEGs, respectively. Also, we identified shared DEGs amongst PTI, CTI and ETI encompassing 146 genes and 916 interactions. The module referred as “common” might represents a core component involved in several biological processes that favour immune response over development. p p Immune response and metabolic changes were two major themes in the “common” module. Delineating reg- ulatory interactions between nodes showed major connected components involved in ROS generation, protein fate determination, cell rescue and defense and intruder perception. Minor components in immunome involved in cell wall reinforcement and macromolecular trafficking. www.nature.com/scientificreports/ (a) cel organization and biogenesis, (b) transcription, and translation regulation, (c) signal transduction, (d) nuclei acid processing, cell cycle replication and metabolism response, (e) transcription factor network. Nodes and edges represent genes and coexpression between genes, respectively. Figure 6. Gene sub-networks associated with pathostress and mapping transcription factor network. (a) cell organization and biogenesis, (b) transcription, and translation regulation, (c) signal transduction, (d) nucleic acid processing, cell cycle replication and metabolism response, (e) transcription factor network. Nodes and edges represent genes and coexpression between genes, respectively. Network specializations of wilt diseaseome and immunome. Analyses of modular network and machine learning methods dissect role of disease/immune-related gene network. We designed an integrative multistep framework combining gene expression analysis and modular co-expression network to systematically characterize the organizing principles of wilt diseasome and immunome.i g g p p Each disorder has a definite and discrete genetic origin, and thus the disease network seems to be disc nected into several nodes linked to well-defined events of disease cycle grouped into small hubs of clo Scientific REPOrTS \| (2018) 8:6528 \| DOI:10.1038/s41598-018-19919-5 9 www.nature.com/scientificreports/ Figure 7. Modular network of wilt diseaseome and immunome. (a) wilt diseasome segregated into perception, penetration, colonization and disease development. (b) immunome assembled from gene expression data in correlation network framework segregated into four hubs encompassing PTI, ETI and CTI. Nodes and edges denote genes and interactions between genes, respectively. Figure 7. Modular network of wilt diseaseome and immunome. (a) wilt diseasome segregated into perception, penetration, colonization and disease development. (b) immunome assembled from gene expression data in correlation network framework segregated into four hubs encompassing PTI, ETI and CTI. Nodes and edges denote genes and interactions between genes, respectively. related phases56. We constructed wilt diseasome having four biologically relevant modules, encompassing pathogen perception, penetration, colonization and disease development (Fig. 7a). Although the wilt disea- some layout was generated based on the knowledge of disease event, major goal was to identify target genes that may function selectively in susceptible genotype. Observed four modules had linear dependencies of 233 statistically significant genes. These modules uncover inconsistencies associated with disease state. Perception related genes (3 DEGs) were associated with module 1 (M1) including receptor-like protein kinase, brassinos- teroid insensitive1-associated receptor kinase 1 (BAK1) and LRR receptor-like serine/threonine protein kinase indicative of stress regulation for initiation of disease signaling. www.nature.com/scientificreports/ Of the three major hubs, hub1 mapped to PAMPs and PRR interaction, including tetratricopeptide repeat protein (IRF7) and putative lipid-transfer protein DIR1 (IRF260); processing and presentation via complex formation encompassing type I inositol 1,4,5-trisphosphate 5-phosphatase CVP2 (IRF163), coronatine-insensitive protein 1 (IRF81) and phospholipase D alpha 1 (IRF544); differentiation, activation and cell receptor signaling comprised of Ras-related protein RABG3f (IRF638), Ras-related protein RABA1b (IRF121), GTP-binding protein SAR1B (IRF319), developmentally-regulated G-protein 3 (IRF1043), calnexin homolog (IRF918) etc. Hub 2 mapped to effector and resistance protein inter- actions, including receptor kinase mediated signal transducer and transcriptional activator alongwith associ- ated signaling events that include disease resistance response protein DRRG49-C (DRRP; IRF1202), DRRP Pi49 Scientific REPOrTS \| (2018) 8:6528 \| DOI:10.1038/s41598-018-19919-5 10 www.nature.com/scientificreports/ Figure 8. Subcellular layers illustrating the PPI sub-network. Layered PPI network assembled from microarray data was separated into four cellular organelles. Degree of connectivity is mentioned in Supplementary Table S7. Figure 8. Subcellular layers illustrating the PPI sub-network. Layered PPI network assembled from microar data was separated into four cellular organelles. Degree of connectivity is mentioned in Supplementary Table Figure 8. Subcellular layers illustrating the PPI sub-network. Layered PPI network assembled from microarray data was separated into four cellular organelles. Degree of connectivity is mentioned in Supplementary Table S7. (IRF1186), probable receptor-like protein kinase (IRF1030), BAK1 (IRF495), probable LRR receptor-like serine/ threonine-protein kinase (IRF1259). Hub 3 forms molecular signature anchored around extracellular matrix genes associated with chitin mediated activation of immune response comprised of endochitinase (IRF141), beta-glucosidase 13 (IRF1057), probable polygalacturonase (IRF1205) etc. Other expressed genes include fatty acid anchor proteins that links matrix to plasma membrane and transduce the signal to cell interior. By observing PTI modules, we found that metabolism, transcription, signaling and cell rescue and defense related DEGs were the most predominant, whereas in ETI signalling protein fate related DEGs and their association were majorly represented. Interestingly, out of three DEGs in CTI module one of them was endochitinase, a key player in chitin mediated immune response. As proposed by Tsuda and Katagiri (2010), in plants immune-related transcriptional reprogramming is a shared response during PTI and ETI and separate signalling events regulate PTI and ETI. Identification of regulatory hubs and subcellular layering in network. Large number of regula- tory genes, their overlapping expression pattern and localization demonstrates complexity of cellular elements to environmental stresses. We built layered interectome to analyze organeller dynamics during fungal invasion. www.nature.com/scientificreports/ The assembled PPI network from microarray data was divided into four layers: Cell wall (1%), cell membrane (6%), cytoplasm (78%) and nucleus (15%) (Fig. 8; Supplementary Dataset 6). Correlation was estimated for every gene pair using in-house program named COREL_FIND. In order to highlight shared and distinct biological processes governing disease or immunity, we selected gene pairs with a minimum expression value difference of 1.5 for each time point over a minimum of three time points. We pruned the initial network and constructed simpler gene regulatory model with 76 nodes and 197 edges. An important observation was that some hubs were common in both genotypes but showed interaction with different proteins, wherein some of the interacting partners were common. Next, we evaluated our data to identify the genes co-expressing in at least two time points in either of the genotypes with a difference of 2 between their expression values. The resulting set of genes was analyzed with BINGO to prepare a combined process map (Supplementary Fig. S4). Results showed that translation initiation and elongation seemed to be predominant in susceptible genotype while protein modification, particularly phos- phorylation was predominant in the resistant genotype. This suggests that apart from transcriptional control, translational and post translational modifications are major players in mediating plant immune responses. Scientific REPOrTS \| (2018) 8:6528 \| DOI:10.1038/s41598-018-19919-5 www.nature.com/scientificreports/ generate a probabilistic network model. This analysis assesses the impact of deregulated genes and their interac- tions on specific biological response. It identifies immune- or disease-responsive genes and highlights advantages of this methodology over available plant systems biology approaches. Compared to other methods of differential gene expression analyses60, the major advantage of our strategy is that the gene is included in the network based on prior evidence narrowing the assumption based conclusions of interactions between candidate genes and at the same platform discover novel candidate with known interactions. We identified a large gene co-expression network that was enriched for transcripts related to shared and distinct patho-stress associated processes. In keeping with similar network-based studies of complex diseases of human and yeast, our approach leverages the combined evidences from biochemical analysis and gene expression profiling. Within the network, we identified functionally coherent and coordinated cell organization and transcriptional programmers, signaling hubs, repli- cation, metabolic regulators and modulators segregated into sub-networks. The finding of increased expression of ethylene-responsive TF, zinc finger A20 and AN1 SAP8 and selenium-binding protein 2 in susceptible gen- otype and its downregulation in resistant genotype is in accordance with earlier reports describing the effect of genotypes in fungal pathogenesis. Therefore, targeting both disease and immune signaling has been proposed as a possible avenue for identification of disease or immune regulators. pi g Functional feedback loops and hormonal rebalancing is achieved through collective activities of genes and its product61. System level analysis of biological processes provides an endeavor to assess interactions between molecular entities60. Pathways significantly enriched in CDEGs, IDEGs and DDEGs include family of 40 S ribo- somal proteins, 60 S ribosomal proteins and ubiquitin suggesting that shared functional features provide insight into the fundamental aspects of cell organization during patho-stress. Surprisingly, GO analysis performed on active network showed significant representation of certain transcripts, namely agglutinin (IRF884), allantoinase (IRF1072) and neutral ceramidase (IRF179), which showed variations in fold-change, form, or cellular compart- mentalization. These non-canonical proteins might be a new potential target for biomarker discovery. We also found many shared and distinct protein kinases in the co-regulatory network those might be involved in perceiv- ing and relaying the signals to the downstream components. In our data, protein kinase (IRF114) was connected with ribosomal protein S10 (IRF116), T-complex protein 1β (IRF757), elongation factor (IRF1229) and GTP binding protein indicating that this may regulate protein synthesis in stress. www.nature.com/scientificreports/ Also protein kinase (IRF114) showed interaction with cell division protease (IRF143) through translation initiation factor 5 A (IRF293) that might have role in modeling cell division during stress. Yet another hub protein kinase, namely shaggy-related protein kinase gamma (IRF767) interacted with polyadenylate-binding protein (IRF695) and MyB transcription factor (IRF924). RNA binding protein and Myb further interact with ubiquitin (IRF705) and HDA2 (IRF504). Cross interactions suggest that shaggy-related protein kinase gamma might regulate defense response at transcriptional and post transcriptional level. We also identified shared coregulated hubs in both the genotypes enriched in APK protein kinase (IRF32), kinetochore SKP1 (IRF290), elongation factor EF1 (IRF1229) and DnaJ (IRF615). APK1 occu- pied the central position and was connected to oligosaccharyl transferase (IRF158) and peptidyl-prolyl cis-trans isomerase (IRF99) through putative leucine repeat protein (IRF657) suggesting its role in protein modifications. Further, APK1 was connected to kinetochore SKP1 (IRF290) through ubiquitin (IRF705) and DnaJ (IRF615) through elongation factor 1 A protein (IRF1229). Association of these proteins might be required to impart resist- ance to plants. Interestingly, APK1 was found to be connected to subtilisin-like protease (IRF625) and inorganic phosphate transporter PHO84 (IRF468) of fungal origin, suggesting thereby pathogen induced phosphorus star- vation mediated by APK1 in plant during fungal stress. We also found syntaxin (IRF193) highly connected to cys- tatin (IRF208) through APK1 Kinase (IRF32). Interaction between syntaxin and cystatin suggests that syntaxin might be involved in secretion of cystatin, an antifungal protein to the cell periphery during defense response. Regulatory hubs coregulated only in the resistant genotype included serine/threonine protein kinase (IRF48), glu- tathione S transferase (IRF117), Ras GTPase (IRF718) and putative DNA binding protein (IRF863). Aquaporin (IRF2132) had connections with unknown protein (IRF505) alongwith GTP binding protein, namely obg ATPase (IRF295) and putative DNA binding protein (IRF89). This DNA binding protein was also connected to ribosomal proteins (IRF505, 1183) and a Ras GTPase (IRF718). Interconnection of putative DNA binding protein (IRF89) with ribosomal proteins (IRF505, 1183) and Ras GTPase (IRF718) reflected that translation processes were gov- erned by signal transduction and protein-DNA interactions during stress. Results showed that translation initia- tion and elongation seemed to be predominant in susceptible genotype while protein modification, particularly phosphorylation was predominant in the resistant genotype. This suggests that apart from transcriptional control, translational and post translational modifications play a significant role in plant immunity. Discussion Interplay of defensive and offensive strategies regulate immune responses in plants against pathogens. Successful invasion suppress basal defense and reprogram host cellular machinery to restructure plant biological processes9. Stimulation of chitin, PAMP and effector-triggered immunity (CTI, PTI and ETI) involve cellular reprogram- ming leading to defense circuits, self-tolerance and pathogen resistance6,58,59. Understanding these regulatory processes associated with pathophysiological pathways and host specific resistance remains a challenge but can have important mechanistic implications in crop improvement. In this study, we identify patho-stress respon- sive genes, molecular pathways and biomarkers relevant for wilt disease and immune response. We performed Go annotation and network-based analysis using Cytoprophet Maximum Likelihood Approach algorithm to Scientific REPOrTS \| (2018) 8:6528 \| DOI:10.1038/s41598-018-19919-5 11 www.nature.com/scientificreports/ Methods Pl oxysporum DNA was conducted using primers spanning internal transcribed sequences of fungal gene. Approximately, 20 ng of genomic DNA was used as the template for each sample. Sequence of the primers used in the study is given in Supplementary Table S1.i p y g pp y Microarray analysis. Amplification of cDNA clones from the susceptible and resistant subtracted cDNA librar- ies and preparation of cDNA microarrays has been done from two biological replicates as described previously by Ashraf et al.31. cDNA microarray having 6072 probes that correspond to 1749 unigenes, including 166 unigenes which showed homology to Fusarium sp. RNA from control and Fusarium infected WR-315 and JG-62 roots at different post infection time points was extracted using Trizol reagent (Invitrogen, CA) and reverse-transcribed followed by labelling of cDNA samples with Cy3 and Cy5 fluorescent dyes, respectively. Hybridization of purified cDNA onto microarray slides were carried out in hybridization chambers (Corning, USA) at 65 °C for 16 h as described in Ashraf et al.31. Scan array 5000 scanner and Scan array express software (PerkinElmer, MA) was used for scanning and analyzing microarrays. Raw data was processed by excluding spots of poor morphology, high local background, bubbles and channel intensities less than 500 for further analysis. Background correction by subtracting local background intensity of each spot from its foreground intensity value and intensity dependent Lowess normalization was performed using Avadis software (PerkinElmer, MA). Quantification of spots were performed by an adaptive method and Cy5/Cy3 signal ratio was obtained as described previously31. Significance of the data was calculated by Benjamini and Horchberg FDR for multiple corrections in cross-slide one class t-test on two biological replicates hybridized on two microarray slides each having individual clones spotted in dupli- cates with p ≤ 0.05 and fold change of 2.5. Statistical analysis. We used MEV software (TIGR) for K means clustering analysis of 1287 genes showing altered expression in at least one time point in one of the genotypes. Expression values of the individual gene measured in two genotypes represented as independent sets of values were used for analysis to identify the sim- ilarity and differences in expression pattern between two contrasting chickpea genotypes. Principal component analysis and two-way ANOVA was performed on the same set of genes using the same program. The PCA was done with the parameters SAMPLE SELECTION as Cluster genes and CENTERING MODE as Mean. Construction of regulatory gene networks. Methods Pl Plant material and in planta infection. Chickpea (Cicer arietinum. L) seeds of wilt susceptible genotype JG-62 and resistant genotype WR-315 were surface sterilized. Seedlings were grown at 25 ± 2 °C on MS basal medium (agar 0.6% w/w) under 16 h/8 h (light/dark) photoperiod with 50 ± 5% relative humidity as described by Ashraf et al.31. Fusarium oxysporum ciceri Race 1 (Foc1) was cultured at 28 °C in potato dextrose broth. After 3 weeks of germination, seedlings were treated with either Fusarium spore suspension (1 × 106/ml) or water as con- trol. Both control and infected plants from both the genotypes were grown under same conditions. Root and col- lar tissues were collected at 6, 12, 24, 48 h and 5 days post inoculation. All the samples were snap-freezed in liquid nitrogen before storing at −80 °C. Experiments were performed thrice for three independent biological replicates. Physiological and biochemical analyses. Physiological and biochemical analyses were performed for control and fungal treated chickpea root tissue collected after post-infection time points for both genotypes. RWC was measured by determining dry weight (DW), fresh weight (DW) and turgid weight (TW) of each sample as described in (20). Ions leaching into the MQ water were measured to estimate electrolyte leakage from the leaf sections. Samples placed in MQ were incubated for 4 h at room temperature and conductivity before (C1) and after (C2) autoclaving was recorded using conductivity62. Experiments were carried out in triplicates. Free proline content, photosynthetic pigments and lipid peroxidation were measured against corrected weight of the actual moisture content of tissue as described earlier62. All the experiments were repeated three times. DAB Staining. ROS was detected using 3,3′ diaminobenzidine (DAB) staining. In brief, chickpea seedlings of resistant and susceptible genotypes were treated with F. oxysproum and the roots were stained in 1 mg/mL of DAB buffer solution and kept for 10 h at RT. After staining, the tissues were boiled in acetic acid and lactophenol for 2 min and rinsed with 50% ethanol for 5 times. Finally, the roots were observed under Nikon Eclipse 80i Microscope (Nikon). Quantification of F. oxysporum DNA. Genomic DNA was extracted from infected plant roots using DNeasy Plant Mini Kit (Qiagen). Real-time PCR was performed with Fusarium specific as well as chickpea spe- cific gene primers and the normalized Ct values were used for the estimation of fungal biomass. The amplification and quantification of F. www.nature.com/scientificreports/ Dissection of shared and distinct aspects of immunity and susceptibility is valuable and can provide important insight for breeding disease-resistant crops. Further, defining the nature of causative and descriptive markers is a major challenge. To investigate causative and descriptive markers for immunity or disease response, we focused on immune adapta- tion and disease development events, including hormone and signaling response, accumulation and allocation of biomolecules and effect on cellular organization. Subtle expression changes occur from perception until coloni- zation and disease state in JG-62, whereas WR-315 exhibit a modular PTI, ETI or CTI immunity. In the immune state, more prominent genes were peroxidase, PAL, NAD(P)H-dependent 6′-deoxychalcone synthase, PR10 can be observed as descriptive markers. Transcription factor bHLH115, heat shock transcription factor B2A, prob- able methyltransferase PMT21, NOT2/NOT3/NOT5 and AT-rich interactive domain-containing protein 3 are uniquely characterized by a coordinated regulation in both genotypes and might have implications in disease development and thus can be considered as causative markers. Instead, genes exhibiting up/downregulation in both genotypes like APK1A, Zinc finger protein (GATA-type) BEL1-like homeodomain transcription factor and probable nucleolar protein 5–2 are associated with cellular reprogramming and can be causative or descriptive markers. Genes allied with disease progression (e.g. NAD(P)H-dependent 6′-deoxychalcone synthase, PMT21, NOT2/NOT3/NOT5) and immunity manifestation (e.g. PAL, peroxidase, PR10) both spatially and temporally might be associated with biomarker development. Scientific REPOrTS \| (2018) 8:6528 \| DOI:10.1038/s41598-018-19919-5 12 www.nature.com/scientificreports/ Taken together, our data provide the first evidence of transcriptional plasticity in regulating disease and immune pathways using gene expression changes and network analysis in wilt disease. We identified disease or immune pathways previously reported to be relevant, as well as novel potential players of Fusarium pathogenesis. Strikingly, we explored a balanced homeostatic innate immune and disease response and demonstrated changes in signal signature and sharing of primary metabolic components in defense and disease. More generally, the network framework described here can be employed across diverse diseases and host range. Methods Pl Construction of subcellular layered network. Apart from the network groups, the complete microarray expression data for all EST’s falling under regulation class were analyzed with a simple self-developed program called COREL_FIND. For a set of microarray expression values it gives an output of probable co-expressing pair of candidates if provided a threshold difference to be considered between the expression values for two genes at same time point and a minimum number of times such threshold is crossed across a time series. This program was used on five time point expression values of susceptible and resistant genotype with a parameter of Difference = 1.5 and Minimum Number of Hits = 3. The set of results obtained were transformed into a sin- gle column of non-redundant ID’s and superimposed on the topology of the network previously generated. For these ID’s Statistical Betweeness score was calculated by CentiScale Cytoscape plugin within the main regulation network. The sub-network for these ID’s was extracted from the main regulation network. The nodes with a high betweeness score were predicted to be playing an active role in the plant immunity system.hi y g y y This program was again used on five time point expression values of susceptible and resistant genotype with a parameter of Difference = 2 and Minimum Number of Hits = 2 to identify the genes co-expressing in at least two time points in either of the genotypes with a difference of 2 between their expression values. The resulting set of genes was analyzed with BINGO to prepare a combined process map. Network visualization. Networks were visualized in cytoscape and its plugins. However, to create shared network, output of GO enrichment obtained from BiNGO was imported into Enrichment Map plugin and was visualized in cerebral plugins (http://www.pathogenomics.ca/cerebral/) in pathway style. Real time PCR. Expression profiles of few genes selected from the microarray dataset were validated by per- forming quantitative real time PCR. For each genotype and time point, total RNA quantification was done on a NanoDrop Spectrophotometer (Nanodrop Technologies) and 5 µg of total RNA was used for cDNA preparation using Reverse transcriptase kit (Applied biosystems). For qRT-PCR, cDNA was diluted ten times and reaction mixture was prepared for individual genes in triplicates using Sybr Green Mastermix (Applied biosystems). PCR was performed on an ABI 7500 sequence detection system according to the manufacturer’s protocol (Applied Biosystems). ΔΔCt method was used for relative quantification. Methods Pl We have built network based on the microarray analysis in association with known protein-protein interaction data from (Database of Interacting Proteins) DIP data- bases in absence of chickpea interactome. We followed a following strategy: homology search of EST sequences associated with the microarray probe IDs was performed using BLASTx against the SwissProt database. The top SwissProt BLAST hit was in turn assigned to each EST sequence and consequently to the corressponding probe IDs. GO ontology mapping and annotation was also performed for the sequences using BLAST2GO software46. A table of SWISS PROT IDs and the corresponding expression data was submitted to Cytoprophet Network Scientific REPOrTS \| (2018) 8:6528 \| DOI:10.1038/s41598-018-19919-5 13 www.nature.com/scientificreports/ inference plugin of Cytoscape. The Cytoprophet Maximum Likelihood Approach algorithm63 was used to gener- ate a probabilistic model of network on 572 differential genes falling under regulatory category where the network assigned 391 proteins within it rejecting others on the basis of lack of interaction data or low probability score on which further emphasis was given. Further we submitted the assigned SwissProt IDs assigned to the cytoprophet plugin in Cytoscape (https://www.ncbi.nlm.nih.gov/pubmed/18653520) which computes the probability of two proteins to potentially interact with each other with DIP (https://www.ncbi.nlm.nih.gov/pubmed/11752321) as its reference. Further, we have built diseasome and immmunome based on the microarray analysis. Modules were computed on the PPI network using the cytoscape MCODE plugin (https://www.ncbi.nlm.nih.gov/pmc/ articles/PMC149346/), which clusters nodes in a network based on several parameters of their connectivity with all other nodes. GO ontology mapping and literature search was performed on susceptible and resistant genotype datasets. SWISS PROT IDs and the corresponding expression data was submitted to Cytoscape and analysed using the Network Analyser plug in. Further, Network Analyser plug in was used to designate network genes in the respective modules. Plant transcription factor database (Plant TFDB) was used to perform regulatory gene network analysis to identify regulators responsible for observed patterns in gene expression. A culmination of the Cytoprophet and BLAST2GO results was brought about through BINGO plugin of Cytoscape64. The complex net- work of 389 EST’s was supplied to BINGO along with BLAST2GO mapping data in form of a BINGO annotation file. From the BINGO run we got the results in form of statistically prominent biological process groups as per GO Ontology within the network predicted. These groups were divided into four major categories each including a sub category best suiting its parent. Methods Pl Normalization of the data was done using 18 S as an endogenous control. Primers sequences are listed in Supplementary Table S1. Data availability statement. All data generated or analysed during this study are included in this pub- lished article (and its Supplementary Information files). 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Acknowledgementsh g This work was supported by grants from the Department of Biotechnology (DBT) (BT/PR8472/AGR/02/389/2006 nd BT/PR8467/AGR/02/387/06). Government of India and the National Institute of Plant Genome Research g This work was supported by grants from the Department of Biotechnology (DBT) (BT/PR8472/AGR/02/389/2006 and BT/PR8467/AGR/02/387/06). Government of India and the National Institute of Plant Genome Research, and BT/PR8467/AGR/02/387/06). Government of India and the National Institute of Plant Genome Research, New Delhi, India to S.C. N.A., G.N. and P.A. are the recipient of pre-doctoral fellowship from the Council of Scientific and Industrial research (CSIR), Govt. of India. K.N. is the recipient of post-doctoral fellowship from the Department of Biotechnology (DBT), Govt. of India. Authors also thank Mr. Jasbeer Singh for illustrations and graphical representations in the manuscript. New Delhi, India to S.C. N.A., G.N. and P.A. are the recipient of pre-doctoral fellowship from the Council of Scientific and Industrial research (CSIR), Govt. of India. K.N. is the recipient of post-doctoral fellowship from the Department of Biotechnology (DBT), Govt. of India. Authors also thank Mr. Jasbeer Singh for illustrations and New Delhi, India to S.C. N.A., G.N. and P.A. are the recipient of pre doctoral fellowship from the Council of Scientific and Industrial research (CSIR), Govt. of India. K.N. is the recipient of post-doctoral fellowship from the Department of Biotechnology (DBT) Govt of India Authors also thank Mr Jasbeer Singh for illustrations and www.nature.com/scientificreports/ 2 Naora, H Involvement of ribosomal proteins in regulating cell growth and apoptosis: Translational modulation or recruitment for 51. Diévart, A. & Clark, S. E. LRR-containing receptors regulating plant development and defense. Development 131, 251–261 (2004). 52. Naora, H. 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Plant Physiol 164, 481–495 (2014). y g y 1. Kim, Y. et al. Mechanisms underlying robustness and tunability in a plant immune signaling network. Cell Host Microbe 15, 84–94 (2014).f 62. Bhushan, D. et al. Comparative proteomics analysis of differentially expressed proteins in chickpea extracellular matrix during dehydration stress. Scientific REPOrTS \| (2018) 8:6528 \| DOI:10.1038/s41598-018-19919-5 Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-19919-5. Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-19919-5. Competing Interests: The authors declare no competing interests. Competing Interests: The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps an institutional affiliations. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the 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/. 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) 8:6528 \| DOI:10.1038/s41598-018-19919-5 16
https://openalex.org/W4281482996	https://biblio.ugent.be/publication/8755545/file/8755548	English	null	Teaching Integer Programming by Scheduling the Belgian Soccer League	Transactions on education	2,023	cc-by	8,669	Teaching Integer Programming by Scheduling the Belgian Soccer League Dries Goossens, Jeroen Beliën Dries Goossens, Jeroen Beliën This article was downloaded by: [2a02:1811:9c1f:7200:f5d7:b1a3:6f8:d6b2] On: 07 June 2022, At: 06:42 Publisher: Institute for Operations Research and the Management Sciences (INFORMS) INFORMS is located in Maryland, USA This article was downloaded by: [2a02:1811:9c1f:7200:f5d7:b1a3:6f8:d6b2] On: 07 June 2022, At: 06:42 Publisher: Institute for Operations Research and the Management Sciences (INFORMS) INFORMS is located in Maryland, USA This article was downloaded by: [2a02:1811:9c1f:7200:f5d7:b1a3:6f8:d6b2] On: 07 June 2022, At: 06:42 Publisher: Institute for Operations Research and the Management Sciences (INFORMS) INFORMS is located in Maryland, USA This article was downloaded by: [2a02:1811:9c1f:7200:f5d7:b1a3:6f8:d6b2] On: 07 June 2022, At: 06:42 Publisher: Institute for Operations Research and the Management Sciences (INFORMS) INFORMS is located in Maryland, USA Full terms and conditions of use: https://pubsonline.informs.org/Publications/Libr Conditions This article may be used only for the purposes of research, teaching, and/or private study. 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Copyright © 2022 The Author(s) Please scroll down for article—it is on subsequent pages INFORMS Transactions on Education Publication details, including instructions for authors and subscription information: http://pubsonline.informs.org Teaching Integer Programming by Scheduling the Belgian Soccer League To cite this article: Dries Goossens, Jeroen Beliën (2022) Teaching Integer Programming by Scheduling the Belgian Soccer League. INFORMS Transactions on Education Dries Goossens, Jeroen Beliën (2022) Teaching Integer Programming by Scheduling the Belgian Soccer League. INFORMS Transactions on Education Published online in Articles in Advance 25 May 2022 . https://doi.org/10.1287/ited.2022.0269 Full terms and conditions of use: https://pubsonline.informs.org/Publications/Librarians-Portal/PubsOnLine-Terms-and Conditions Full terms and conditions of use: https://pubsonline.informs.org/Publications/Librarians-Portal/PubsOnLine-Terms-and- Conditions i i http://pubsonline.informs.org/journal/ited Dries Goossens,a,b,* Jeroen Beli¨enc The spreadsheet exercises ﬁle and solution ﬁles are available at https://www.informs.org/Publications/ Subscribe/Access-Restricted-Materials. that is based on the well-known assignment model. Later, a more sophisticated two-phased approach is discussed, in which ﬁrst the home advantage is settled for each team on each round. This serves as input for the second phase, where the opponents are determined. Both phases are again solved using integer programming. Dries Goossens,a,b,* Jeroen Beli¨enc aDepartment of Business Informatics and Operations Management, Ghent University, 9000 Ghent, Belgium; bFlandersMake@UGent – core lab CVAMO, 9000 Ghent, Belgium; cResearch Centre for Operations Management, KU Leuven, Campus Brussels, 1000 Brussels, Belgium *Corresponding author Contact: dries.goossens@ugent.be, https://orcid.org/0000-0003-0224-3412 (DG); jeroen.belien@kuleuven.be, https://orcid.org/0000-0003-2349-7390 (JB) Received: December 28, 2020 Revised: June 7, 2021; October 15, 2021 Accepted: January 10, 2022 Published Online in Articles in Advance: https://doi.org/10.1287/ited.2022.0269 Copyright: © 2022 The Author(s) May 25, 2022 Abstract. This paper presents a didactic approach for teaching integer programming start- ing from a real-life case on scheduling the Belgian soccer league. We share our experiences as well as didactic resources for two teaching formats. The ﬁrst format involves hands-on exercises and is more appropriate for small student groups (up to 40 students). The second format is an interactive lecture that focuses more on current research challenges and is better suited for large student groups. During the last decade, both formats have been used repeat- edly with consistent, positive feedback from students. The combination of a hands-on, inter- active approach that actively involves students with the particularities and experiences of solving a real-life case makes our approach unique and positively affects students’ attention. Received: December 28, 2020 Revised: June 7, 2021; October 15, 2021 Accepted: January 10, 2022 Published Online in Articles in Advance: https://doi.org/10.1287/ited.2022.0269 Copyright: © 2022 The Author(s) May 25, 2022 Received: December 28, 2020 History: This paper has been accepted for the INFORMS Transactions on Education Special Issue on Cases Based on Real-World Projects from the INFORMS Journal on Applied Analytics. Open Access Statement: This work is licensed under a Creative Commons Attribution 4.0 International License. You are free to copy, distribute, transmit and adapt this work, but you must attribute this work as “INFORMS Transactions on Education. Copyright © 2022 The Author(s). https://doi.org/10. 1287/ited.2022.0269, used under a Creative Commons Attribution License: https://creativecommons. org/licenses/by/4.0/.” Open Access Statement: This work is licensed under a Creative Commons Attribution 4.0 International License. You are free to copy, distribute, transmit and adapt this work, but you must attribute this work as “INFORMS Transactions on Education. Copyright © 2022 The Author(s). https://doi.org/10. 1287/ited.2022.0269, used under a Creative Commons Attribution License: https://creativecommons. org/licenses/by/4.0/.” g y Supplemental Material: The supplemental material is available at https://doi.org/10.1287/ited.2022.0269. The spreadsheet exercises ﬁle and solution ﬁles are available at https://www.informs.org/Publications/ Subscribe/Access-Restricted-Materials. Supplemental Material: The supplemental material is available at https://doi.org/10.1287/ited.2022.0269. With 12,500 members from nearly 90 countries, INFORMS is the largest international association of operations research (O.R.) and analytics professionals and students. INFORMS provides unique networking and learning opportunities for individual professionals, and organizations of all types and sizes, to better understand and use O.R. and analytics tools and methods to transform strategic visions and achieve better outcomes. g For more information on INFORMS, its publications, membership, or meetings visit http:/ INFORMS TRANSACTIONS ON EDUCATION Articles in Advance, pp. 1–9 ISSN 1532-0545 (online) Keywords: integer programming • sports scheduling • spreadsheet optimization • real-life case 2. Related Literature Several studies have shown the importance of using real-life examples for motivating students in developing quantitative skills. Popular approaches to bring real-life practice to the classroom are case-based teaching and ﬁeld-based education. We refer to Drake (2019) for a recent overview on teaching operations research (OR)/ management science with cases. The effectiveness of ﬁeld-based education is described in Gorman (2018a), while Gorman (2018b) presents a recent overview of related literature. Capstone projects, where students work on real-world industry-sponsored projects, are an important manifestation of ﬁeld-based education. Konrad et al. (2018) provide an interesting study on the impact of capstone projects on student skills develop- ment. They turn out to be particularly important for developing skills that go beyond the purely technical capabilities of mastering analytical methodologies and developing models. They train students in deﬁning the problem from a business context; distilling the deci- sion space and objective priorities; collecting and clean- ing data; and presenting solutions to stakeholders. Such skills are often neglected in theoretical OR courses. Although direct contact with industrial partners is extremely important, real-life practice can also be intro- duced in the classroom by less comprehensive and less time-consuming activities as hands-on lectures around real-life applications. Teaching quantitative methods in general can beneﬁt from the students’ inherent interest in sports. For instance, Willoughby (2004) presents a well-received one-semester undergraduate course de- voted to decision analysis and statistical methodologies in sports. Cochran (2004) also recognizes sports as a con- text that is more familiar and interesting to many stu- dents and can therefore help in improving their under- standing and retention. Bickel (2004) demonstrates the use of a baseball example to teach fundamental probabil- istic and decision analysis. Sports scheduling is a particularly fruitful didactic application for teaching integer programming, as the problem is intuitive for students to grasp immediately, but, at the same time, the domain is rich enough to illustrate various IP concepts (Trick 2004). Hence, it comes as no surprise that we are not the ﬁrst to make use of a sports optimization problem in a classroom setting. Chlond (2011) presents a hypothetical, but original, puzzle related to sports league tables that can be solved using an IP formulation. 1. Introduction In sports scheduling, the problem is to decide for each match when it is to be played and which team gets the advantage of playing at its own venue. In a schedule, matches are grouped in rounds, which typically corre- spond to weekends, such that each team plays, at most, one game per round. In this paper, we focus on a case study from the Jupiler Pro League, the highest professio- nal soccer division in Belgium, whose scheduling process is described in a paper by Goossens and Spieksma (2009) in Interfaces (now known as the INFORMS Journal on Applied Analytics). This league consists of 18 teams, which play a so-called double-round-robin tournament (i.e., a tournament where each team plays against each other team twice). Finding a good schedule can be quite a chal- lenge, as there is a large variety and number of—often conﬂicting—wishes and requirements from stakeholders (clubs, fans, broadcasters, police, etc.) to be taken into account. The paper describes the transition from a man- ual scheduling procedure to an integer-programming (IP) approach. First, the authors describe an IP formulation Shortly after the publication by Goossens and Spieksma (2009), the competition design of the Jupiler Pro League changed. The number of teams was reduced to 16, and after these teams ﬁnish a regular double- round-robin season, a postseason competition with the best six teams now follows. This postseason competition is again organized as a double-round-robin tournament and termed “play-off 1” (which is, in fact, an incorrect name because the term play-off is normally only used for elimination tournaments). Play-off 1 determines the champion as well as the teams that will represent Bel- gium in the European tournaments in the next season. A particularity of play-off 1 is that the participating teams start with half of the points they collected in the regular season. Goossens (2018) gives a follow-up on the impact 1 Goossens and Beli¨en: Teaching IP by Scheduling Belgian Soccer INFORMS Transactions on Education, Articles in Advance, pp. 1–9, © 2022 The Author(s) 2 of these changes on the scheduling process, as described by Goossens and Spieksma (2009). An important challenge in teaching IP is to train stu- dents in transforming conditional statements of logic into linear relations involving binary decision variables. 1. Introduction Stevens and Palocsay (2017) propose a two-step peda- gogical approach, in which a conditional requirement is ﬁrst decomposed into a group of elementary implica- tions, which are subsequently translated into linear con- straints. The effectiveness of this approach for instructing business students is demonstrated through an analysis of pretest and posttest results. Many educational papers on teaching IP involve spreadsheet models. Example appli- cations range from simple games or puzzles to functional area business problems and sports, either purely ﬁcti- tious or (based on) a real-life case. Rasmussen and Weiss (2007) and Weiss and Rasmussen (2007) use a spread- sheet implementation of the Sudoku puzzle to teach IP modeling. Based on a real-life application, Rao and Beli¨en (2014) present a production-planning case offered in a three-step approach, in which each step introduces a next layer of modeling complexity. Three spreadsheet imple- mentations, as well as their corresponding algebraic for- mulations, are provided. Pachamanova (2006) presents a distribution-channel selection case that illustrates model- ing ﬁxed (entry) costs. Particularly for the education of business students, she emphasizes the importance of the management side, giving priority to making assumptions, modeling, validation, and business decision making, rather than purely focusing on the (spreadsheet) optimi- zation model. Beli¨en et al. (2013) present spreadsheet optimization models for an online energy-supply game. Compared with industrial applications, these non- traditional applications lie closer to the students’ reality, in particular for undergraduate students. As a result, an increase in the students’ motivation to develop IP models has been observed. Similar to Rao and Beli¨en (2014), Beli¨en et al. (2013) add complexity in a step-by-step approach. We acknowledge the importance of a stepwise approach and adopt a four-step decomposition in this paper. y p In this paper, we focus on play-off 1. Appealing for its all-decisive nature, yet manageable from a compu- tational point of view because of its limited number of teams, this league has interesting assets for teaching integer programming. This paper describes an interac- tive lecture that is built around four modeling exer- cises, where students develop a spreadsheet IP model for scheduling play-off 1. Section 2 discusses the liter- ature on using real-life examples for teaching integer programming. A short introduction to sports schedul- ing is given in Section 3. The spreadsheet models are discussed in Section 4, followed by a description of our classroom experience in Section 5. We conclude in Section 6. 3. Sports Scheduling • Team \|T\| plays against team r, • Team \|T\| plays against team r, This section introduces a number of important con- cepts in sports timetabling. In particular, the (mir- rored) double-round-robin tournament format and the clock method are essential for the hands-on soccer league scheduling class (ﬁrst format of this case; see Section 5). The carry-over effect, home-away patterns, and the phased approach are also touched on in the ﬁrst format, but are discussed more in-depth in the interactive lecture (second format of this case; see Sec- tion 5). For more background, we refer to an extended surveys of the literature and sports-scheduling termi- nology by Kendall et al. (2010), Rasmussen and Trick (2008), and Ribeiro (2012). A classiﬁcation of sports- scheduling problems and a repository of problem instances is given by Van Bulck et al. (2020). Goossens and Spieksma (2011) present an overview of schedules used by professional soccer leagues in Europe and dis- cuss their properties. • Team [r + k]+ plays against team [r −k]−, for each k ∈{1,2,:::, \|T\|=2 −1}, where [x]+ x if x \|T\| −1 and [x]+ x −\|T\| + 1 otherwise, while [x]− x if x1 and [x]− x + \|T\| −1 otherwise. Graphically, the clock method is easier to comprehend, as illustrated in Figure 1, where nodes represent teams, and edges correspond to matches. Each round leads to a next round by shifting all edges one stroke clockwise. • Team [r + k]+ plays against team [r −k]−, for each k ∈{1,2,:::, \|T\|=2 −1}, where [x]+ x if x \|T\| −1 and [x]+ x −\|T\| + 1 otherwise, while [x]− x if x1 and [x]− x + \|T\| −1 otherwise. Graphically, the clock method is easier to comprehend, as illustrated in Figure 1, where nodes represent teams, and edges correspond to matches. Each round leads to a next round by shifting all edges one stroke clockwise. It is a common idea in sports that the strength or ﬁt- ness of an opponent can be impacted by its previous match. For instance, if team A is very strong, its oppo- nent, team B, may be exhausted after playing against team A, or its line-up in the next match may be weak- ened by suspensions incurred because of (provoked) foul play against team A. 2. Related Literature Given a ﬁnal rank- ing with summary data (including number of points gained, number of wins, losses, goals for, and goals against) as typically provided in soccer, the challenge of the puzzle is to reconstruct the actual scores for each of the matches. Beli¨en et al. (2011) present spreadsheet optimization models that can be used to win a fantasy cycling game. The paper by Trick (2004) on teaching integer programming using sports scheduling is most Goossens and Beli¨en: Teaching IP by Scheduling Belgian Soccer INFORMS Transactions on Education, Articles in Advance, pp. 1–9, © 2022 The Author(s) 3 related to our work. It uses a round-robin scheduling problem to illustrate IP modeling, symmetry-breaking constraints, cut generation, and alternative formula- tions both through a spreadsheet model and algebraic modeling approach. Birge (2004) provides a spread- sheet IP model for a simpliﬁed case of developing a schedule for Major League Football involving ﬁve teams of the Western Division, where the objective is to minimize the total travel distance. Besides the trav- eling salesman problem solution methodology, the case illustrates the use of decomposition, column gen- eration, and IP formulation. majority of the tournaments used a mirrored format: It is easier to compute (the second half of the season can simply be copied from the ﬁrst), and it creates an equal (and maximal) number of rounds between each match A versus B and its counterpart B versus A. p In 1847, Reverend Kirkman, though unconcerned by sports scheduling, published a method that can be used for constructing a schedule for single-round-robin com- petitions (Kirkman 1847). This method is known as the clock method (or circle method), and its outcome has been referred to as a canonical schedule. A precise description of the circle method for a single round-robin tourna- ment for an even set of teams T is as follows. For each round r ∈{1,:::, \|T\| −1}, we have: 3. Sports Scheduling Hence, if team C plays against team B in the next round, team C may have a beneﬁt from team A being B’s previous opponent. We say that there is a carry-over effect from team A to C (Russell 1980). A fair schedule should avoid the situation in which team C frequently plays against the team that played against a strong team (team A) in the previous match. Although there have been attempts to develop schedules that balance the carry-over effects as much as possible (e.g., Anderson 1999), an interesting result is A schedule for a double-round-robin tournament is often phased, in the sense that no team plays any other team for the second time before it has faced all other opponents once. Hence, a phased double-round-robin tournament can be split into two single-round-robin tournaments. If the order of the opponents in each tournament half is identical for each team, we say that the schedule is mirrored. Until recently, the vast Figure 1. Representation of the Canonical Schedule for a Competition with Eight Teams; Only Rounds 1, 2, and 7 are Depicted igure 1. Representation of the Canonical Schedule for a Competition with Eight Teams; Only Rounds 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 Notes. In round 2, game 8-2 is obtained after shifting the hand of the clock, which starts at 8 and points at 1 in round 1, one stroke clockwise to 2. For the games that do not involve team 8, both ends of the edge are shifted one stroke clockwise; for example, game 7-2 in round 1 becomes game 1-3 in round 2. 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 Notes. In round 2, game 8-2 is obtained after shifting the hand of the clock, which starts at 8 and points at 1 in round 1, one stroke clockwise to 2 For the games that do not involve team 8, both ends of the edge are shifted one stroke clockwise; for example, game 7-2 in round 1 becomes game 1-3 in round 2. Figure 1. Representation of the Canonical Schedule for a Competition with Eight Teams; Only Rounds 1, 2, and 7 are Depicted n of the Canonical Schedule for a Competition with Eight Teams; Only Rounds 1, 2, and 7 are Depicted 3. Sports Scheduling 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 8 4 5 4 5 Notes. In round 2, game 8-2 is obtained after shifting the hand of the clock, which starts at 8 and points at 1 in round 1, one stroke clockwise to 2. For the games that do not involve team 8, both ends of the edge are shifted one stroke clockwise; for example, game 7-2 in round 1 becomes game 1-3 in round 2. Goossens and Beli¨en: Teaching IP by Scheduling Belgian Soccer INFORMS Transactions on Education, Articles in Advance, pp. 1–9, © 2022 The Author(s) 4 scheduling the ﬁrst half of the tournament (the second half of the tournament is assumed to be mirrored from the ﬁrst), the optimization models are limited in terms of the number of decision variables and con- straints, making them easily solvable with Microsoft Excel’s standard solver. that the canonical schedule actually has the most unbal- anced carry-over effects (Lambrechts et al. 2017). In that sense, it is remarkable that this particular schedule is so popular (Goossens and Spieksma 2011). p p ( p ) The sequence of home matches (“H”) and away matches (“A”) played by a single team is called its home- away pattern (HAP). Given such a HAP, the occurrence of two consecutive home matches or two consecutive away matches is called a break. Nemhauser and Trick (1998) were the ﬁrst to take advantage of HAPs by split- ting the sports-scheduling problem into subproblems. A ﬁrst subproblem consists of ﬁnding a set of HAPs that allows one to construct a schedule. Note that not each set of HAPs is feasible, with each round having an equal number of H’s and A’s being a simple necessary condi- tion. Another condition is that each pair of HAPs differs, which immediately explains why breaks are unavoid- able (for more conditions, see Miyashiro et al. 2003). Next, the opponents are determined for each pattern, taking into account that on any given round, a pattern with an H can only play against a pattern with an A. Finally, the patterns are assigned to actual teams in a third subproblem. Several of the most successful appli- cations in sports scheduling make use of a phased approach, even though the order of the subproblems and the methods used to solve them may differ. Minimize Minimize (1) Subject to n∈N xtn 1 ∀t ∈T, (2) t∈T xtn 1 ∀n ∈N, (3) xtn ∈{0,1} ∀n ∈N, ∀t ∈T: (4) (2) (4) Constraints (2) ensure that each team is assigned to exactly one number, while Constraints (3) guarantee that each number is assigned to exactly one team. The spreadsheet exercise ﬁle and solution ﬁle are provided in the accompanying ﬁles soccer_template_1.xlsx and soccer_sol_1.xlsx, respectively, in the supplemental material. 3. Sports Scheduling The ﬁrst exercise asks students to model an assign- ment problem. The students are given a so-called basic match schedule, which is essentially a schedule in which the teams have been replaced by numbers (also referred to as “placeholders”). A basic match schedule has the advantage that it already satisﬁes the single-round-robin conditions—that is, each number plays exactly once against each other number, and every number plays exactly one game in each round. For six teams, this leads to ﬁve rounds and three matches per round. An example of a basic match schedule is given in Table 1. This is, in fact, a canoni- cal schedule (generated with the clock method). The idea is to assign a unique number n ∈N (with N the set of numbers) to each team t ∈T (with T the set of teams) to obtain a schedule. So, we have deci- sion variables xtn equal to one if team t is assigned to number n, and zero otherwise. Each possible assign- ment of a number to a team could lead to a number of wishes that are violated, leading to a certain penalty cost. All such penalty costs ctn are provided. Our objective is to minimize the total penalty cost. This leads to the following assignment problem: y Integer programming plays an important role in solv- ing sport-scheduling problems. In fact, several real-life sport-scheduling applications that have been tackled with integer programming have been reported in the academic literature. We mention soccer leagues in Italy (Della Croce and Oliveri 2006), Chile (Dur´an et al. 2007), and Ecuador (Recalde et al. 2013); the German basketball league (Westphal 2014); volleyball leagues in Argentina (Bonomo et al. 2012) and Italy (Cocchi et al. 2018); the Canadian football league (Kostuk and Willoughby 2012); and the South American FIFA World Cup quali- ﬁers (Dur´an et al. 2017) as examples. Integer program- ming has also been used to solve related problems like referee scheduling (Alarc´on et al. 2014), determining whether a team is eliminated for play-offs (Ribeiro and Urrutia 2005), and grouping teams into divisions or ser- ies (Toffolo et al. 2019). t∈T n∈N ctnxtn, (1) 4. Hands-On Spreadsheet Exercises Following the general format of Constraints (14), this could be modeled by adding ﬁve constraints, one for each of the ﬁve rounds: xGEN,2 + xSTA,5 ≤1, (5) xGEN,4 + xSTA,1 ≤1, (6) xGEN,6 + xSTA,3 ≤1: (7) XAND,1 + XAND,3 + XAND,5 + XCLU,1 + XCLU,3 + XCLU,5 −2XSTA,2 −2XSTA,4 −2XSTA,6 ≤0, (15) XAND,2 + XAND,4 + XAND,6 + XCLU,2 + XCLU,4 + XCLU,6 −2XSTA,5 −2XSTA,1 −2XSTA,3 ≤0, (16) XAND,1 + XAND,2 + XAND,5 + XCLU,1 + XCLU,2 + XCLU,5 −2XSTA,6 −2XSTA,4 −2XSTA,3 ≤0, (17) XAND,3 + XAND,5 + XAND,6 + XCLU,3 + XCLU,5 + XCLU,6 −2XSTA,2 −2XSTA,1 −2XSTA,4 ≤0, (18) XAND,2 + XAND,4 + XAND,1 + XCLU,2 + XCLU,4 + XCLU,1 −2XSTA,6 −2XSTA,5 −2XSTA,3 ≤0: (19) Constraints (5)–(7) ensure that whenever Gent is coupled to a number that plays a home game in round 2, Stand- ard cannot be assigned to the number of the opponent in the corresponding game. The spreadsheet exercise ﬁle and solution ﬁle are provided in the accompanying ﬁles soccer_template_2.xlsx and soccer_sol_2.xlsx, re- spectively, in the supplemental material. (15) In the third exercise, students are asked to add a constraint that forbids that two speciﬁc teams play a home game in the same round (e.g., because they share the same stadium or the teams are geographically so close that the police cannot guarantee safety if both teams play a home game at the same time). If this requirement holds for the teams Standard and Ant- werp, given the basic match schedule in Table 1, one possible way to address this is to add: (18) (19) A solution to the infeasibility problem is to treat the constraints as soft constraints. We introduce penalty variables yr for each round r and add them with a pen- alty cost coefﬁcient to the objective function. 4. Hands-On Spreadsheet Exercises The hands-on part entails developing a schedule for play-off 1 of the highest professional Belgian soccer league. We consider the edition of season 2018–19, which involves the teams Club Bruges FC (CLU), RSC Anderlecht (AND), AA Gent (GEN), Standard Li`ege (STA), FC Antwerp (ANT), and KRC Genk (GNK). The exercises are incremental: The second, third, and fourth exercise start from the preceding solution and each time add a new modeling challenge. As play-off 1 involves only six teams and the exercises focus on The second exercise introduces a ﬁrst extension of a constraint that involves more than one team and can Table 1. Example of a Basic Match Schedule for a Single- Round-Robin Tournament Involving Six Teams Round 1 Round 2 Round 3 Round 4 Round 5 1-2 2-5 1-6 3-2 2-6 3-4 4-1 2-4 5-1 4-5 5-6 6-3 5-3 6-4 1-3 Table 1. Example of a Basic Match Schedule for a Sing Round-Robin Tournament Involving Six Teams Goossens and Beli¨en: Teaching IP by Scheduling Belgian Soccer INFORMS Transactions on Education, Articles in Advance, pp. 1–9, © 2022 The Author(s) 5 therefore not be handled using the penalty costs ctn. More speciﬁcally, the students are asked to extend the previous model, so that a particular team (Gent) does not play a home game against a particular opponent (Standard) in a speciﬁc round (round 2). Given the basic match schedule in Table 1, this leads to the fol- lowing constraints to be added: So far, each added constraint has increased the objective value. However, it is not hard to realize that as more such constraints are added, the model may become infeasible. This happens, for instance, if, on top of the previous constraints, we need to enforce that in the ﬁrst ﬁve rounds, when either Club or Anderlecht (or both) play at home, Standard wants to play away. 4. Hands-On Spreadsheet Exercises The Con- straints (15)–(19) become: xSTA,1 xANT,6, (8) xSTA,6 xANT,1, (9) xSTA,2 xANT,3, (10) xSTA,3 xANT,2, (11) xSTA,4 xANT,5, (12) xSTA,5 xANT,4: (13) xSTA,1 xANT,6, (8) xSTA,6 xANT,1, (9) xSTA,2 xANT,3, (10) xSTA,3 xANT,2, (11) xSTA,4 xANT,5, (12) xSTA,5 xANT,4: (13) XAND,1 + XAND,3 + XAND,5 + XCLU,1 + XCLU,3 + XCLU,5 −2XSTA,2 −2XSTA,4 −2XSTA,6 ≤2y1, (20) XAND,2 + XAND,4 + XAND,6 + XCLU,2 + XCLU,4 + XCLU,6 −2XSTA,5 −2XSTA,1 −2XSTA,3 ≤2y2, (21) XAND,1 + XAND,2 + XAND,5 + XCLU,1 + XCLU,2 + XCLU,5 −2XSTA,6 −2XSTA,4 −2XSTA,3 ≤2y3, (22) XAND,3 + XAND,5 + XAND,6 + XCLU,3 + XCLU,5 + XCLU,6 −2XSTA,2 −2XSTA,1 −2XSTA,4 ≤2y4, (23) XAND,2 + XAND,4 + XAND,1 + XCLU,2 + XCLU,4 + XCLU,1 −2XSTA,6 −2XSTA,5 −2XSTA,3 ≤2y5: (24) (20) (21) This model starts from the observation that in the basic match schedule of Table 1, whenever number 1 plays a home game, the only number that in every round plays an away game is number 6. Thus, if Standard is coupled to number 1, Antwerp must be coupled to number 6 (Constraint (8)) and vice versa (Constraint (9)). The same observation holds for the numbers 2 and 3 (Constraints (10) and (11)) and the numbers 4 and 5 (Constraints (12) and (13)). The spreadsheet assignment and solution of the third exercise are provided in the accompanying ﬁles soccer_template_3.xlsx and soccer_sol_3.xlsx, re- spectively, in the supplemental material. (24) In general, we introduce additional binary variables yc equal to one if constraint c is not satisﬁed, and zero otherwise. If pc denotes the penalty for violating con- straint c, Objective (1) becomes: The additional constraints of exercise 2 and exercise 3 can be generalized as follows: Minimize t∈T n∈N ctnxtn + c∈C pcyc: (25) Minimize (25) t∈T n∈N atncxtn ≤bc ∀c ∈C, (14) ∀c ∈C, (14) And the additional Constraints (14) are replaced by: And the additional Constraints (14) are replaced by: t∈T n∈N atncxtn ≤bc + Myc ∀c ∈C, (26) ∀c ∈C, (26) (26) with c ∈C the set of additional constraints and atnc and bc the technological coefﬁcients and right-hand side corresponding to constraint c. with M a big number. Goossens and Beli¨en: Teaching IP by Scheduling Belgian Soccer INFORMS Transactions on Education, Articles in Advance, pp. 5. Classroom Experience The case has been given as a two-hour lecture in two different formats: a hands-on computer class session and an interactive lecture without hands-on exercises, but addressing issues beyond the assignment method. Whereas in both formats, the main teaching objective is to show how optimization methods can be successfully applied to real-life problems, the secondary teaching goals slightly differ between both formats. In the ﬁrst format, the secondary objective is to give students a hands-on training in developing IP models, while in the second format, the secondary objective is to discuss more advanced methodologies and highlight current research challenges. Next, we outline the approach of solving this prob- lem by means of an IP formulation. We explain that even though it sounds very reasonable, we faced a number of issues in real life to convince the league owners to adopt this modeling approach. These involve the loss of insight in the scheduling process when moving from a manual scheduling process to a “black box” and having to explicitly list the require- ments and their importance instead of sticking to an “I’ll tell you whether or not I like the schedule once you propose one” attitude. Determining the relative importance of the requirements was a difﬁcult exercise for the league owners. At ﬁrst, their reaction was that simply giving a maximal weight to each constraint would result in the best schedule. At the same time, it became clear that, in the past, some teams had been considered “more equal than others.” We believe that these issues are common to many settings where oper- ations research replaces legacy decision processes. g In both formats, the case starts with an interactive presentation on the challenges, main objectives, and constraints related to developing a new schedule for the Belgian professional soccer league. The PowerPoint ﬁle soccer_scheduling_ITE.pptx (see supplemental material) can be used to guide this lecture and is pro- vided as additional material for interested instructors. A lively discussion starts, in which the students are confronted with two questions: (1) Which stakeholders are involved; and (2) what makes up a good schedule? To direct the discussion, students are asked to come up with speciﬁc examples of issues that should be taken into account when building a schedule and which stakeholders are affected. During this discussion, stu- dents almost always succeed in bringing up all impor- tant constraints and related parties themselves. 4. Hands-On Spreadsheet Exercises 1–9, © 2022 The Author(s) 6 alternatives like the number of stars in the universe, the number of living insects, the number of people ever born on planet Earth, etc., and the number of fea- sible schedules for a double-round-robin tournament involving 14 teams. That the latter is by far the biggest number is a real eye-opener to many students. With respect to (b), a small example is given involving only six teams, for which a single-round-robin schedule is built from scratch. Already in round 4 of this schedule, the manual procedure gets stuck into an infeasibility with respect to the constraint that every team plays exactly once against each other team, which can only be ﬁxed by altering earlier rounds. At this point, the clock method (see Section 3) is introduced, and it is shown how this method succeeds in developing a fea- sible round-robin schedule quickly. The spreadsheet assignment and solution of the fourth exercise are provided in the accompanying ﬁles soccer_template_4.xlsx and soccer_sol_4.xlsx, respec- tively, in the supplemental material. Note that in case there are multiple optimal solu- tions, the Excel solver reports only one of them. These exercises could easily be extended with an assignment of identifying all alternative optima (if any), which entails an iterative approach that adds in each itera- tion a constraint that cuts away the preceding optimal solution. This constraint ensures that the sum of all x- variables equal to one in the previous solution is smaller or equal than the number of teams minus one. 5. Classroom Experience They are classiﬁed by the instructor in four categories, objec- tives and constraints related to: (1) mayors and police, (2) television, (3) clubs, and (4) the league organizer. We point out that it is typically impossible to satisfy all these wishes, as some of them are clearly conﬂicting. p g y p From this point on, the formats differ. The ﬁrst format continues with the four Excel IP modeling exercises, where the students are given time to tackle them on their own. Feedback is provided during class, and the solutions are gradually presented for each exercise (because each exercise builds on the following). In the second format, the different modeling examples of the assignment method are presented by the teacher, without the implementation in Excel. An interactive dis- cussion starts on the limitations of the assignment method. Issues like the carry-over effect, the fact that there are numerous other basic match schedules, and the consequences in terms of solution quality of mirror- ing the ﬁrst half of the season to obtain the second half schedule are raised. More advanced solution methodol- ogies as the two-phase decomposition approach start- ing from a set of home-away patterns, as well as local search approaches, are subsequently discussed. We refer to Goossens and Spieksma (2009) for a more detailed discussion of these issues and the IP models for the two- phase decomposition approach. A ﬁrst take-away is to show students how complex the problem is in terms of (a) the total number of possi- ble solutions and (b) the difﬁculty to even ﬁnd a solu- tion that satisﬁes the basic round-robin requirements. With respect to (a), a small quiz is done, in which the students need to select the highest number among Goossens and Beli¨en: Teaching IP by Scheduling Belgian Soccer INFORMS Transactions on Education, Articles in Advance, pp. 1–9, © 2022 The Author(s) 7 In both formats, the case is concluded with a brief discussion on the reception of the solution. Besides the number of violated constraints (and the total pen- alty cost), which are clearly much lower than the man- ual approach could obtain, we show the impact of the improved schedule in terms of reduced cost of polic- ing, increased stadium attendance, and revenue from the TV broadcasting contract. 5. Classroom Experience Obviously, the schedule is not the only factor in this story (the numbers are, for instance, also determined by the fanbase of the teams that promote to or are relegated from the ﬁrst division), but the difference before and after the adop- tion of the IP modeling approach is unmistakable. Nevertheless, we point out that coaches tend to look at the schedule from the point of view of their team’s performance: A good schedule is one where their team wins many games. In that respect, no schedule can be acceptable for all teams, and given that coaches typically get more media attention than other (more important) stakeholders, a negative perception of the schedule with the general public remains a possibility. this format has been brought eight times in an intro- ductory course on operations research for postgradu- ate business students with limited background in mathematics (in this case, the focus was on the ﬁrst exercise; the other exercises were left for the stronger students to explore on their own). The second format (interactive lecture) was brought for an audience of 120–300 graduate business students and was used in total six times. For both formats, we regularly received positive (informal) feedback from the students. After having given the interactive lecture (second format) in March 2021 as an online class (due to the COVID-19 sit- uation at the time), we distributed a questionnaire that has been ﬁlled out by 112 students. This survey con- sists of eight questions with answers in the format of a Likert scale. The questions and the responses are listed in Table 2. The results conﬁrm our feeling that the soc- cer league scheduling case is well appreciated by our students. Students see closing the gap between theory and practice as the strongest asset of the case. 5. Classroom Experience More than 90% of the respondents either agree or strongly agree with question 2 (on the use of optimization mod- eling for solving real-life problems), question 5 (on the use of optimization to reconcile conﬂicting interests in real-life settings), and question 6 (on the surplus value of the case, as compared with typical textbook exer- cises, to learn how to successfully apply optimization g p p y We have used the ﬁrst teaching format (hands-on computer class) nine times as part of a course that focuses on real-life applications of operations research for groups of 20–40 graduate business students who already completed a basic course on operations re- search in their undergraduate studies. Furthermore, Table 2. Questions and Results of a Survey Filled Out by 112 Students After Having Followed the Interactive Lecture (Second Format) Question Strongly agree Agree Neutral Disagree Strongly disagree 1. The soccer league scheduling case has increased my interest in optimization modeling. 22 (19.6%) 68 (60.7%) 19 (17.0%) 2 (1.8%) 1 (0.9%) 2. The soccer league scheduling case has increased my insight in the use of optimization modeling for solving real-life problems. 37 (33.0%) 60 (53.6%) 14 (12.5%) 1 (0.9%) 0 (0.0%) 3. The soccer league scheduling case has made me aware of the vast number of possible solutions an integer programming problem may have. 48 (42.9%) 52 (46.4%) 10 (8.9%) 1 (0.9%) 0 (0.0%) 4. The soccer league scheduling case has improved my optimization modeling skills. 3 (2.7%) 21 (18.8%) 65 (58.0%) 21 (18.8%) 2 (1.8%) 5. The soccer leagues scheduling case has shown me how optimization can be used to reconcile conﬂicting interests of various stakeholders in real- life problems. 36 (32.1%) 64 (57.1%) 12 (10.7%) 0 (0.0%) 0 (0.0%) 6. The soccer league scheduling case has a surplus value compared with typical textbook examples with respect to learning how to successfully apply optimization modeling in practice. 39 (34.8%) 53 (47.3%) 17 (15.2%) 3 (2.7%) 0 0.0%) 7. In the soccer league scheduling case, progressively adding complexities to the basic scheduling problem improved my understanding of the modeling process. 32 (28.6%) 57 (50.9%) 19 (17.0%) 3 (2.7%) 0 (0.0%) 8. The soccer league scheduling case has increased my interest in a job (academic, consultancy, business) within the ﬁeld of operations research. 7 (6.3%) 33 (29.5%) 58 (51.8%) 13 (11.6%) 1 (0.9%) Note. Questions 3 and 7 have only been answered by 111 students. Note. Questions 3 and 7 have only been answered by 111 students. 6. Conclusion h h This paper has presented our 10-year experience with two teaching formats of an integer-programming class around a real-life sports-scheduling problem. In both formats, students are actively involved, either through an interactive class discussion or through several hands- on modeling exercises, or a combination of both. From both the informal, positive student feedback we consis- tently receive for these classes and the formal results of a survey, we strongly believe that the combination of active involvement and the real-life application posi- tively affects students’ attention and interest in optimiza- tion modeling. Our hands-on exercises are incremental and do not require students to solve a large, complex scheduling problem from scratch. Nevertheless, by solv- ing these (small) modeling exercises, meaningful results arise that could be presented to the league owners. Although there is room for discussion on issues that complicate the adoption of these models in practice, both teaching formats are mainly instructor-driven. Developing a case for students to solve (either individu- ally or in group) with no or minimal support from the instructor would better reﬂect the real-life experience of optimization professionals. For instance, our class does not teach students how to cope with missing or incorrect data or translating fuzzy wishes into speciﬁc constraints and objectives. Developing such a comprehensive case around this real-life problem is an interesting avenue for future work. pp y • “I really found this case very interesting. The pro- fessor has made me more curious about real-life opti- mization problems. Thank you for that!” • “Even though it was an evening lecture, it was really better than an average lecture. The professor got my attention although I am not really interested in foot- ball nor optimization.” p • “Choosing another sport could have been more gender-neutral.” g Also from this open feedback, we conclude that many students value the soccer league scheduling case. The last comment, however, raises the important issue of this case’s potential to reach out to female students or minor- ity groups. Let us start with saying that in Belgium, there is also a (nonprofessional) women’s soccer league. The reason why this league was not studied in this case study is that many of the constraints that make the case chal- lenging from a modeling (or computational) point of view do not apply to the women’s league. 5. Classroom Experience Thank you very much for this incredible opportunity!” • “Constructing the session with the underlying story, step by step discoveries, as well as the results of each trial made the approach very clear and attractive! pp y The presentation has really enhanced my interest in the various applications of optimization problems and in research. Thank you very much for this incredible opportunity!” 6. Conclusion h h Indeed, matches attract far fewer spectators (none of them hooli- gans); hence, the police do not forbid certain games at particular moments. Furthermore, apart from a handful of games since season 2020–21, the league is not broad- cast on TV. Consequently, there are no wishes related to a favorable broadcasting scheme to be taken into account. In this respect, the women’s highest soccer league is comparable to nonprofessional men’s soccer leagues in Belgium, for which building a league schedule is far less complex. Also, given that there is much less money involved, fairness issues (e.g., avoiding carry- over and breaks) tend to receive much less attention. All in all, the only aspect that remains is developing a dou- ble-round-robin schedule that satisﬁes the basic con- straints (every team meets each team exactly twice), which is fairly easy (any assignment of teams to numbers in the basic match schedule will do). The same observa- tions hold for most parasports (e.g., the Belgium wheel- chair basketball league) and nonprofessional sports. To Alarc´on F, Dur´an G, Guajardo M (2014) Referee assignment in the Chilean football league using integer programming and pat- terns. Internat. Trans. Oper. Res. 21:415–438. Anderson I (1999) Balancing carry-over effects in tournaments. Holroyd F, Quinn K, Rowley C, Webb B, eds. Combinatorial Designs and Their Applications (CRC Press, Boca Raton, FL), 1–16. 5. Classroom Experience Results of a Survey Filled Out by 112 Students After Having Followed the Interactive Lecture Goossens and Beli¨en: Teaching IP by Scheduling Belgian Soccer INFORMS Transactions on Education, Articles in Advance, pp. 1–9, © 2022 The Author(s) 8 decrease the observed “distance” with this case for stu- dents from minority groups, we added an extra slide to the PowerPoint. We advise using this slide to: modeling in practice). The responses suggest that the case is less successful in actually improving the stu- dents’ optimization-modeling skills (question 4). This is probably due to the second format (interactive lec- ture) for which the survey was taken. This format focuses on the real-life aspect and interactive discus- sion at the cost of hands-on modeling exercises (ﬁrst format). g • Make students aware that the methodology of this case does not merely apply to men’s leagues, but can be extended to all sports leagues that attract many spectators, of which the games are broadcast on TV, and that are organized using a round-robin format (e.g., women’s soccer in the United States or wheelchair basketball in Germany). • Make students aware that the methodology of this case does not merely apply to men’s leagues, but can be extended to all sports leagues that attract many spectators, of which the games are broadcast on TV, and that are organized using a round-robin format (e.g., women’s soccer in the United States or wheelchair basketball in Germany). At the end of the survey, students could share some additional feedback in an open format. As an illustra- tion, we list some students’ quotes that we received: • Initiate a discussion on which stakeholders or con- straints are less important for sports leagues that attract fewer spectators and which aspects still remain in the problem. • Initiate a discussion on which stakeholders or con- straints are less important for sports leagues that attract fewer spectators and which aspects still remain in the problem. • “Constructing the session with the underlying story, step by step discoveries, as well as the results of each trial made the approach very clear and attractive! The presentation has really enhanced my interest in the various applications of optimization problems and in research. References Alarc´on F, Dur´an G, Guajardo M (2014) Referee assignment in the Chilean football league using integer programming and pat- terns. Internat. Trans. Oper. Res. 21:415–438. Anderson I (1999) Balancing carry-over effects in tournaments. Holroyd F, Quinn K, Rowley C, Webb B, eds. 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https://openalex.org/W2991875373	https://www.frontiersin.org/articles/10.3389/fpsyg.2019.02665/pdf	English	null	Gratitude Moderates the Mediating Effect of Deliberate Rumination on the Relationship Between Intrusive Rumination and Post-traumatic Growth	Frontiers in psychology	2,019	cc-by	7,204	ORIGINAL RESEARCH published: 03 December 2019 doi: 10.3389/fpsyg.2019.02665 Eunseung Kim1 and Sungman Bae2* Eunseung Kim and Sungman Bae 1 Department of Psychology, Dankook University, Cheonan, South Korea, 2 Department of Psychology and Psychotherapy, College of Health Sciences, Dankook University, Cheonan, South Korea 1 Department of Psychology, Dankook University, Cheonan, South Korea, 2 Department of Psychology and Psychotherapy, College of Health Sciences, Dankook University, Cheonan, South Korea Purpose: This study examines the moderating effect of gratitude on the mediating effect of deliberate rumination on the relationship between intrusive rumination and post-traumatic growth (PTG). Methods: We used self-report questionnaires to collect data from 450 18–68-year- old participants (Mage = 39.73, SD = 13.73) residing in major cities and regions across South Korea. Data that were collected from 411 participants were subjected to analysis. Version 25 of SPSS (Statistical Package for Social Science) and PROCESS macro were used to analyze mediation, moderation, and moderated mediation effects. Abbreviations: PTG, post-traumatic growth; SPSS, Statistical Package for Social Science. Edited by: Edited by: Jamil Ahmad Malik, Quaid-i-Azam University, Pakistan Jamil Ahmad Malik, Quaid-i-Azam University, Pakistan Reviewed by: Giulia Fioravanti, University of Florence, Italy Gianluca Seraﬁni, San Martino Hospital (IRCCS), Italy Correspondence: Sungman Bae spirit73@dankook.ac.kr Reviewed by: Giulia Fioravanti, University of Florence, Italy Gianluca Seraﬁni, San Martino Hospital (IRCCS), Italy Conclusion: Deliberate rumination and gratitude facilitate PTG for traumatized adults. In particular, gratitude reinforces the effect of deliberate rumination on PTG. Keywords: post-traumatic growth, intrusive rumination, deliberate rumination, gratitude, trauma Correspondence: Sungman Bae spirit73@dankook.ac.kr INTRODUCTION Specialty section: This article was submitted to Health Psychology, a section of the journal Frontiers in Psychology Nationwide surveys indicate that 71.9% of Korean (Seo et al., 2012) adults and 89% of American adults (Kilpatrick et al., 2013) experience at least one traumatic experience during their lifetime. Traumatic events can cause serious psychological problems (Christopher, 2004), and approximately 10–15% of those who experience trauma are diagnosed with post-traumatic stress disorder (Green and Lindy, 1994). Received: 08 August 2019 Accepted: 12 November 2019 Published: 03 December 2019 While past studies have focused on clinical symptoms (e.g., reexperiencing symptoms, hyperarousal) that result from the experience of a traumatic event, recent research has begun to focus on positive changes and growth that occur after a traumatic experience (Joseph et al., 1993; Nerken, 1993; Tedeschi and Calhoun, 1995). Indeed, researchers have found that PTG can occur after exposure to various types of traumatic events such as bereavement (Taku et al., 2008), cancer (Cormio et al., 2017), physical violence (Kleim and Ehlers, 2009), and traﬃc accidents (Nishi et al., 2010). Results: Deliberate rumination mediated the relationship between intrusive rumination and PTG. Gratitude moderated the effect of deliberate rumination on PTG. Finally, gratitude moderated the mediating effect of deliberate rumination on the relationship between intrusive rumination and PTG. Citation: Recent studies have also shown that gratitude moderates the eﬀect of post- traumatic stress on PTG (Vieselmeyer et al., 2017; Leppma et al., 2018). Highly grateful individuals tend to be more likely to appreciate everyday events (McCullough et al., 2002), which in turn can facilitate adaptive coping (Fredrickson, 2004). Such individuals experience gratitude more frequently in daily life and across a wider array of circumstances compared to those with lower levels of gratitude. Further, experiences of gratitude in the midst of trauma may be signiﬁcant because gratitude not only inspires and transforms individuals, it also oﬀers meaning in life by helping people interpret their life as a gift. Gratefulness may also have long-term survival beneﬁts by making people more open-minded and ﬂexible, ultimately allowing them to better perceive and take advantage of opportunities, which in turn can facilitate adaptive coping (Johnson and Fredrickson, 2005). Past studies have also shown that gratitude promotes deliberate rumination, which promotes PTG (Wood et al., 2010; Zhou and Wu, 2015; Kim and Lee, 2016). Rumination plays a key role in the process of PTG and is divided into two categories: intrusive and deliberate rumination (Calhoun and Tedeschi, 2006). Intrusive rumination is a process by which one automatically reexperiences images, emotions, and thoughts that are related to an event. On the other hand, deliberate rumination refers to an intentional thought process through which one attempts to understand the cause and meaning of an incident. After a traumatic event, aﬀected individuals may experience intrusive rumination and extreme emotional distress, but they may also simultaneously attempt to engage in deliberate rumination to alleviate psychological distress (Wu et al., 2015; Zhou and Wu, 2015; Zhang et al., 2018). Deliberate rumination can facilitate the expansion of existing schemas and help one better understand a traumatic experience (Triplett et al., 2012). An individual’s beliefs and values may be changed and enriched through the process of deliberate rumination, and this in turn may promote PTG. PTG can lead to various positive changes such as an increased appreciation of life and self-understanding as well as changes in life priorities (Calhoun et al., 2010). In particular, Zhou and Wu (2015) longitudinally examined the relationship between gratitude, deliberate rumination, and PTG among people who had experienced an earthquake. Speciﬁcally, the participants were assessed three and a half years (T1), four and a half years (T2), and ﬁve and a half years (T3) after the earthquake. Citation: Kim E and Bae S (2019) Gratitude Moderates the Mediating Effect of Deliberate Rumination on the Relationship Between Intrusive Rumination and Post-traumatic Growth. Front. Psychol. 10:2665. doi: 10.3389/fpsyg.2019.02665 December 2019 \| Volume 10 \| Article 2665 1 Frontiers in Psychology \| www.frontiersin.org Deliberate Rumination, Gratitude and PTG Kim and Bae 2004; Taku et al., 2008; Triplett et al., 2012; Zhou and Wu, 2015; Cárdenas et al., 2019). In particular, Triplett et al. (2012) found that deliberate rumination mediates the relationship between intrusive rumination and PTG. Post-traumatic growth refers to the positive changes and growth that occur as a result of experiencing trauma (Tedeschi and Calhoun, 2004). PTG can lead to an enhanced sense of personal strength, changes in one’s perspective toward life, and improved interpersonal relationships. Some individuals experience positive changes and growth after trauma, although others experience intrusive symptoms such as nightmares, hypo/hyperarousal, depression, and negative changes in cognition and mood. Persistent depression is linked to worse outcomes such as maladaptive coping and maladjustment (Ghio et al., 2015). Gratitude has been identiﬁed as a factor that promotes PTG. Gratitude refers to an attitude of thankfulness and joy for the beneﬁts and blessings that one has received from other people and nature (Emmons and Shelton, 2002). Adler and Fagley (2005) noted that gratitude involves “noticing and acknowledging its value and meaning of something—an event, behavior, object—and feeling a positive emotional connection to it.” Gratitude includes cognitive, emotional, and behavioral factors (McCullough et al., 2001; Watkins et al., 2003; Adler and Fagley, 2005) and is an attribution-dependent characteristic (Weiner, 1985) that allows one to perceive the beneﬁts that have been gained from others and various life experiences (Emmons, 2007). Gratitude consists of an appreciation of others and various aspects of daily life and the ability to recall positive past experiences (Watkins et al., 2003). Why do some people experience PTG? According to previous studies, factors related to PTG can be classiﬁed into three categories: the characteristics of the event, personal characteristics, and cognitive processing of the traumatic experience (Calhoun et al., 2000; Linley and Joseph, 2004; Tedeschi and Calhoun, 2004; Kim and Lee, 2016). In particular, many researchers have focused on the ways in which individuals cognitively process a traumatic event (e.g., intrusive and deliberate rumination). p p ( ) A recent meta-analytic study found that gratitude is a signiﬁcant predictor of PTG (Jang and Kim, 2017). Frontiers in Psychology \| www.frontiersin.org Citation: N = 411 Frequency percent Sex Male 166 40.4% Female 245 59.6% Age 20–29 123 29.9% 30–39 98 23.8% 40–49 83 20.2% 50–59 69 16.8% 60–69 38 9.2% Education No experience 2 0.5% Primary School and Middle school 53 12.9% Community college graduate 41 10.0% College undergraduate 56 13.6% College graduation 155 37.7% Graduate grad school 105 25.3% Marriage Single 163 39.7% Marriage 230 56.0% Divorced 14 3.4% Bereavement 4 1.0% Income Status High 5 1.2% Middle-high 83 20.2% Middle 210 51.1% Middle-low 94 22.9% Low 19 4.6% Religious No religion 159 38.7% Catholic 26 6.3% Protestant 197 47.9% Buddhism 28 6.8% Won Buddhism 1 0.2% FIGURE 1 \| Hypothetical Model. FIGURE 1 \| Hypothetical Model. FIGURE 1 \| Hypothetical Model. Hypotheses 2: Deliberate rumination will mediate the relationship between intrusive rumination and PTG. Hypothesis 3: Gratitude will be positively related to PTG. Hypothesis 3: Gratitude will be positively related to PTG. Hypothesis 4: Gratitude will moderate the relationship between deliberate rumination and PTG. Participants and Procedures Participants in this study were recruited from community centers in major cities and regions across South Korea. Trained investigators (e.g., psychology graduate students) provided participants with a detailed introduction to the study. All participants provided written informed consent in accordance with the Declaration of Helsinki and signed a consent form before completing the questionnaires. After the participants completed the questionnaires, they were compensated. This study was approved by the Institutional Review Board of Dankook University. TABLE 2 \| Correlations, mean, and standard deviation among variables (N = 411). ations, mean, and standard deviation among variables (N = 411) TABLE 2 \| Correlations, mean, and standard deviation among v TABLE 2 \| Correlations, mean, and standard deviation among variables (N = 411). Intrusive rumination Deliberate rumination Gratitude Post-traumatic growth Intrusive rumination Deliberate rumination 0.36∗∗ Gratitude −0.29∗∗ 0.02 Post-traumatic growth −0.03 0.36∗∗ 0.45∗∗ Mean 17.94 22.98 34.95 79.47 SD 6.97 7.01 5.96 22.60 ∗∗P < 0.01. y This study was conducted among 450 adults with an age range of 19–68 years; the mean age of participants was 39.73 years (SD = 13.73). The sample consisted of 245 women (59.6%) and 166 men (40.4%). The age distribution of the participants was as follows: 123 (29.9%), 98 (23.8%), 83 (20.2%), 69 (16.8%), and 38 (9.2%) participants were in their 20s, 30s, 40s, 50s, and 60s, respectively. Further, 155 (37.7%), 105 (25.3%), 56 (10.6%), 41 (10.0%), and 53 (12.9%) participants were college, graduate school, university, professional college, and high school graduates, respectively; 0.5% had no educational experience. Additionally, 230 (56%) and 163 (36.7%) participants were married and unmarried, respectively (Table 1). Finally, using Allen’s (2005) categorization of trauma into three types – interpersonal, impersonal, and interpersonal related trauma – 143 (34.8%) participants of this study experienced interpersonal trauma inﬂicted by human perpetrators (e.g., physical or sexual abuse), 146 (35.5%) experienced impersonal trauma caused by human or natural origins (e.g., accidents or natural disasters), and 122 (29.7%) experienced interpersonal related trauma (e.g., death of a partner, severe illness). Citation: The results showed that gratitude predicted PTG from T2 and T3. This result indicates that gratitude is a stable predictor of PTG. Additionally, gratitude at T1 predicted PTG at T3 through deliberate rumination at T2. Tedeschi and Calhoun (1996, 2004) noted that the process of PTG occurs across several stages. Individuals possess a schema that corresponds to a cognitive framework of the self, others, and the world. After traumatic events, aﬀected individuals experience distortions of their belief systems, and intrusive rumination is activated in response to extreme psychological stress (Zhou and Wu, 2015; Zhang et al., 2018). However, intrusive thoughts may activate deliberate rumination, which may help individuals better understand their traumatic experience and rebuild a new schema (Triplett et al., 2012; Tsai et al., 2016; Gwak and Park, 2018). In summary, the mediating eﬀect of deliberate rumination on the relationship between intrusive rumination and PTG has been established in the literature. In the present study, gratitude was expected to moderate the relationship between deliberate rumination and PTG. Accordingly, ﬁve research hypotheses were formulated (Figure 1). Deliberate rumination is an attempt to accommodate trauma into one’s cognitive schema. It is an active cognitive process that involves reconstructing an existing schema to promote PTG. The eﬀects of deliberate rumination on PTG have been demonstrated in the literature (Tedeschi and Calhoun, 2004; Calhoun and Tedeschi, 2006), and higher levels of deliberate rumination have been found to be associated with greater PTG (Linley and Joseph, Hypothesis 1: Intrusive rumination will be negatively related to PTG. December 2019 \| Volume 10 \| Article 2665 Frontiers in Psychology \| www.frontiersin.org 2 Deliberate Rumination, Gratitude and PTG Kim and Bae IGURE 1 \| Hypothetical Model. Hypotheses 2: Deliberate rumination will mediate the relationship between intrusive rumination and PTG. Hypothesis 3: Gratitude will be positively related to PTG. Hypothesis 4: Gratitude will moderate the relationship between deliberate rumination and PTG. Hypothesis 5: The mediating eﬀect of deliberate rumination on the relationship between intrusive rumination and PTG will be moderated by gratitude. ATERIALS AND METHODS articipants and Procedures rticipants in this study were recruited from community nters in major cities and regions across South Korea. Trained vestigators (e.g., psychology graduate students) provided rticipants with a detailed introduction to the study. All rticipants provided written informed consent in accordance th the Declaration of Helsinki and signed a consent form TABLE 1 \| Demographic Information. Data Analysis Version 25.0 of SPSS and PROCESS macro 2.16 for SPSS (Hayes, 2013) were used to test the research hypotheses. First, frequencies and descriptive statistics were computed to examine the demographic characteristics of the sample, and Pearson’s correlation analysis was conducted to examine the relationships between the study variables. Second, model 4 of PROCESS macro for SPSS was used to examine the mediating eﬀect of deliberate rumination on the relationship between intrusive rumination and PTG (Zhao et al., 2010; Rucker et al., 2011). Next, a bootstrap test was conducted and the resultant 95% conﬁdence intervals were inspected to examine the signiﬁcance of the indirect eﬀects that resulted from mediation analysis (Preacher et al., 2007). Measures Trauma Experience Questionnaire In the present study, the Trauma Experience Questionnaire, which was developed by Song et al. (2009) and revised by Shin and Chung (2012), was used to obtain information about traumatic events participants had experienced (e.g., type, duration, and severity of the traumatic event). The questionnaire contained seven items. Participants were asked to disclose the most painful traumatic event that they had experienced and to categorize the type of the event. They were then instructed to respond to the items of the assessment based December 2019 \| Volume 10 \| Article 2665 Frontiers in Psychology \| www.frontiersin.org 3 Deliberate Rumination, Gratitude and PTG Kim and Bae TABLE 3 \| Mediating effects of deliberate rumination in relationship between intrusive rumination and post-traumatic growth). DV IV B S.E t p 95% CI F R2 Lower Upper Deliberate rumination (constant) 16.33 0.90 18.10 0.00 14.56 18.10 62.51∗∗∗ 0.13 Intrusive rumination 0.37 0.05 7.91 0.00 0.28 0.46 Post-traumatic growth Intrusive rumination 0.60 0.16 −3.80 0.00 −0.91 −0.29 38.63∗∗∗ 0.16 Deliberate rumination 1.36 0.16 8.77 0.00 1.06 1.67 ∗∗∗P < 0.001. TABLE 4 \| Indirect effects on post-traumatic growth (Bootstrapping). Pathway Coefﬁcient SE 95% CI Lower Upper Intrusive rumination →Deliberate rumination → Post-traumatic growth 0.50 0.08 0.36 0.67 on the aforementioned event. They reported whether they experienced psychological pain and scored the severity of the subjective pain that they had experienced at the time of the event and more recently on a scale of 1–7 (1 = no pain; 7 = very painful). Korean Version of the Event-Related Rumination Inventory The Korean version of the Event-Related Rumination Inventory was developed by Cann et al. (2011) and revised by Ahn et al. (2013). The scale consists of 20 items that assess intrusive and deliberate rumination using a 4-point Likert scale (0 = not at all; 3 = always). A few examples of items that assess intrusive rumination are as follows: “Thoughts, memories, or images of the event came to mind even when I did not want them” and “I could not keep images or thoughts about the event from entering my mind.” A few examples of items that assess deliberate rumination are as follows: “I thought about whether I could ﬁnd meaning from my experience,” “I thought about the event and tried to understand what happened,” and “I deliberately thought about how the event had aﬀected me.” In the present study, the Cronbach’s alphas of the intrusive and deliberate rumination subscales were 0.93 and 0.91, respectively. extent, and density of gratitude, consisting of 6 items that require responses to be recorded on a 7-point Likert scale (0 = strongly disagree; 6 = strongly agree). The following are representative scale items: “I have so much in life to be thankful for” and “As I get older, I ﬁnd myself better able to appreciate the people, events, and situations that have been part of my life.” Two items are reverse- scored, and higher total scores are indicative of greater gratitude. The Cronbach’s alpha of this scale was 0.89 in the present study. Korean Version of the Post-traumatic Growth Inventory The Korean version of the Post-Traumatic Growth Inventory that was developed by Tedeschi and Calhoun (1996) and validated by Song et al. (2009) was used to measure individual perceptions of positive changes after a traumatic experience. The scale consists of 21 items with four subscales: relating to others, new possibilities, personal strength, and spiritual change. For each of the subscales, responses are recorded on a 6-point Likert scale (0 = no change; 5 = very high degree of change). Higher total scores are indicative of greater PTG. The following are representative scale items: “I changed my priorities about what is important in my life,” “I established a new path for my life,” and “I am more likely to try to change things that need changing.” The Cronbach’s alpha of this scale was 0.94 in the present study. Model 1 of PROCESS macro for SPSS was used to examine the moderating eﬀect of gratitude on the relationship between deliberate rumination and PTG. Additionally, simple regression was conducted to examine the eﬀect of deliberate rumination on PTG as a function of the level of gratitude (i.e., high vs. low). Finally, model 14 of PROCESS macro for SPSS was used to examine whether gratitude moderates the mediating eﬀect of deliberate rumination on the relationship between intrusive rumination and PTG. The Mediating Effect of Deliberate Rumination on the Relationship Between Intrusive Rumination and PTG The mediating eﬀect of deliberate rumination on the relationship between intrusive rumination and PTG was examined (Table 3). Intrusive rumination had a negative eﬀect on PTG (B = 0.60, t = −3.80, p < 0.001) and a positive eﬀect on deliberate rumination (B = 0.37, t = 7.91, p < 0.001). Deliberate rumination had a positive eﬀect on PTG (B = 1.36, t = 8.77, p < 0.001). In addition, the mediating eﬀect of deliberate rumination on the relationship between intrusive rumination and PTG was statistically signiﬁcant (95% conﬁdence interval = 0.36, 0.67) (Table 4). FIGURE 2 \| Gratitude moderated the relationship between deliberated rumination and post-traumatic growth. the moderating eﬀect of gratitude on the relationship between deliberate rumination and PTG. The main eﬀects of deliberate rumination (B = 1.12, t = 8.55, p < 0.001) and gratitude (B = 1.61, t = 10.27, p < 0.001) as well as their interaction eﬀect (i.e., deliberate rumination × gratitude) (B = −0.04, t = −2.10, p < 0.05) were statistically signiﬁcant. Simple regression analysis was conducted to examine the eﬀect of deliberate rumination on PTG as a function of the level of gratitude (Table 6). The results revealed that the 95% conﬁdence intervals did not include a 0. In other words, the eﬀect of deliberate rumination on PTG diﬀered between individuals who reported low (i.e., M −1 SD) and high (i.e., M + 1 SD) levels of gratitude (Figure 2). Gratitude reinforced the eﬀect of deliberate rumination on PTG. Correlation Analysis Correlation Analysis Emergent correlations between the study variables are shown in Table 2. Intrusive rumination correlated positively with deliberate rumination (r = 0.36, p < 0.01) and negatively with gratitude (r = −0.29, p < 0.01). On the other hand, deliberate rumination was unrelated to PTG. Deliberate rumination correlated positively with PTG (r = 0.36, p < 0.01) but was unrelated to gratitude. Finally, there was a positive correlation (r = 0.45, p < 0.01) between gratitude and PTG. Korean Version of the Gratitude Questionnaire Korean Version of the Gratitude Questionnaire We used the Korean version of the Gratitude Questionnaire that was developed by McCullough et al. (2002) and validated by Kwon et al. (2006). It measures the intensity, frequency, December 2019 \| Volume 10 \| Article 2665 Frontiers in Psychology \| www.frontiersin.org 4 Deliberate Rumination, Gratitude and PTG Kim and Bae TABLE 5 \| The moderating effects of gratitude on the relationship between intentional rumination and post-traumatic growth). DV IV B S.E t p 95% CI F R2 Lower Upper Post-traumatic growth (constant) 79.50 0.92 86.79 0.00 77.20 81.30 66.79∗∗∗ 0.33 Deliberate rumination 1.12 0.13 8.55 0.00 0.85 1.36 Gratitude 1.61 0.16 10.27 0.00 1.30 1.91 Deliberate rumination × Gratitude −0.04 0.02 −2.10 0.03 −0.08 −0.01 ∗∗∗p < 0.001. RESULTS FIGURE 2 \| Gratitude moderated the relationship between deliberated rumination and post-traumatic growth. FIGURE 2 \| Gratitude moderated the relationship between deliberated rumination and post-traumatic growth. The Moderating Effect of Gratitude on the Relationship Between Deliberate Rumination and PTG The moderating eﬀect of gratitude was examined by entering deliberate rumination, gratitude, and their interaction terms (i.e., deliberate rumination × gratitude) into the model. Table 5 shows the results of the analysis that was conducted to examine TABLE 6 \| Conditional indirect effect at speciﬁc levels of the gratitude when deliberate rumination as a mediator. Conditional effect of gratitude Estimate S.E t p 95% CI Lower Upper −1SD −5.95 0.17 7.99 0.00 1.02 1.68 M 1.05 0.13 8.06 0.00 0.80 1.32 +1SD 0.86 0.18 4.83 0.00 0.51 0.1.21 The Moderating Effect of Gratitude on the Mediating Effect of Deliberate Rumination on the Relationship Between Intrusive Rumination and PTG The Moderating Effect of Gratitude on the Mediating Effect of Deliberate Rumination on the Relationship Between Intrusive Rumination and PTG Table 7 shows the results of the analysis that was conducted to examine the moderating eﬀect of gratitude on the mediating December 2019 \| Volume 10 \| Article 2665 5 Deliberate Rumination, Gratitude and PTG Kim and Bae TABLE 7 \| Moderated mediation effects of gratitude on the relationship between intrusive rumination, deliberate rumination, and post-traumatic growth. Mediator variables models (DV: Deliberate rumination) B S.E t p Lower Upper (constant) −0.0005 0.33 −0.0017 0.9987 −0.6421 0.6410 Intrusive rumination 0.37 0.05 7.91 0.00 0.28 0.46 Moderator variable models (DV: Post-traumatic growth) B S. E t p Lower Upper (constant) 0.03 0.92 0.04 0.97 −1.76 1.84 Intrusive rumination −0.18 0.15 −1.19 0.23 −0.47 0.12 Deliberate rumination 1.17 0.14 8.36 0.00 0.90 1.45 Gratitude 1.54 0.16 9.29 0.00 1.21 1.87 Deliberate rumination × Gratitude −0.04 0.02 −2.25 0.03 −0.08 −0.01 Moderate mediation index S.E Lower Upper −0.016 0.008 −0.033 −0.002 eﬀect of deliberate rumination on the relationship between intrusive rumination and PTG. The direct eﬀect of intrusive rumination on PTG was not signiﬁcant (B = −0.18, t = −1.19, p = 0.23). However, deliberate rumination had a positive eﬀect on PTG (B = 1.17, t = 3.88, p < 0.001), the interaction term (deliberate rumination × gratitude) (B = −0.04, t = −2.25, p < 0.05) was signiﬁcant, and the index of the moderated mediation eﬀect was signiﬁcant (95% conﬁdence interval = −0.033, −0.002) (Figure 3). Further, Table 8 shows the results of the bootstrap test, which revealed the conditional indirect eﬀect of gratitude. The 95% conﬁdence intervals did not include a 0 In other words, the moderating eﬀect of gratitude FIGURE 3 \| Gratitude moderated the mediating effect of deliberate rumination between intrusive rumination and post-traumatic growth. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. TABLE 7 \| Moderated mediation effects of gratitude on the relationship between intrusive rumination, deliberate rumination, and post-traumatic growth. Mediator variables models (DV: Deliberate rumination) B S.E t p Lower Upper (constant) −0.0005 0.33 −0.0017 0.9987 −0.6421 0.6410 Intrusive rumination 0.37 0.05 7.91 0.00 0.28 0.46 Moderator variable models (DV: Post-traumatic growth) B S. The Moderating Effect of Gratitude on the Mediating Effect of Deliberate Rumination on the Relationship Between Intrusive Rumination and PTG E t p Lower Upper (constant) 0.03 0.92 0.04 0.97 −1.76 1.84 Intrusive rumination −0.18 0.15 −1.19 0.23 −0.47 0.12 Deliberate rumination 1.17 0.14 8.36 0.00 0.90 1.45 Gratitude 1.54 0.16 9.29 0.00 1.21 1.87 Deliberate rumination × Gratitude −0.04 0.02 −2.25 0.03 −0.08 −0.01 Moderate mediation index S.E Lower Upper −0.016 0.008 −0.033 −0.002 TABLE 7 \| Moderated mediation effects of gratitude on the relationship between intrusive rumination, deliberate rumination, and post-traumatic growth. eﬀect of deliberate rumination on the relationship between intrusive rumination and PTG. The direct eﬀect of intrusive rumination on PTG was not signiﬁcant (B = −0.18, t = −1.19, p = 0.23). However, deliberate rumination had a positive eﬀect on PTG (B = 1.17, t = 3.88, p < 0.001), the interaction term (deliberate rumination × gratitude) (B = −0.04, t = −2.25, p < 0.05) was signiﬁcant, and the index of the moderated mediation eﬀect was signiﬁcant (95% conﬁdence interval = −0.033, −0.002) (Figure 3). Further, Table 8 shows the results of the bootstrap test, which revealed the conditional indirect eﬀect of gratitude. The 95% conﬁdence intervals did not include a 0. In other words, the moderating eﬀect of gratitude through deliberate rumination on PTG diﬀered for individuals who reported low (i.e., M −1 SD), average (i.e., mean), and high (i.e., M + 1 SD) levels of gratitude. FIGURE 3 \| Gratitude moderated the mediating effect of deliberate rumination between intrusive rumination and post-traumatic growth. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. an incident and correct dysfunctional beliefs about a traumatic event (Seo and Chae, 2006). Second, in the present study, gratitude had a positive impact on PTG, and deliberate rumination and gratitude had an interaction eﬀect on PTG. These results concur with past ﬁndings that the inﬂuence of deliberate rumination on PTG is strengthened by high levels of gratitude (Zhou and Wu, 2015; Kim and Lee, 2016). This suggests that the eﬀect of deliberate rumination on PTG is reinforced by gratitude. Frontiers in Psychology \| www.frontiersin.org December 2019 \| Volume 10 \| Article 2665 DISCUSSION The major ﬁndings of the present study are discussed in this section. The previously observed mediating eﬀect of deliberate rumination on the relationship between intrusive rumination and PTG was veriﬁed. Intrusive rumination facilitated PTG by promoting deliberate rumination (Taku et al., 2008; Triplett et al., 2012; Wu et al., 2015). These results are consistent with past ﬁndings that post-traumatic intrusive rumination stimulates deliberate rumination, which in turn promotes PTG (Triplett et al., 2012; Tsai et al., 2016; Zhou and Wu, 2016; Zhang et al., 2018). Past studies have only examined the direct relationship between gratitude and PTG. In this regard, the present ﬁndings further our understanding of the speciﬁc role that gratitude plays in PTG by delineating the moderating eﬀect of gratitude on the relationship between deliberate rumination and PTG. The results of this study support the PTG model that was proposed by Tedeschi and Calhoun (2004). According to this model, deliberate rumination plays a key role in the PTG process. Many individuals experience intrusive rumination after traumatic events (Scarpa et al., 2009), and intrusive rumination can activate deliberate rumination. Persistent intrusive rumination can adversely impact PTG and is likely to cause post-traumatic stress disorder (Triplett et al., 2012). In particular, deliberate rumination can help one reconstruct TABLE 8 \| Conditional indirect effect of gratitude when deliberate rumination mediated between intrusive rumination and post-traumatic growth. Mediator Gratitude Estimates S.E 95% CI Lower Upper Deliberate rumination −1SD 0.53 0.09 0.36 0.73 M 0.42 0.07 0.29 0.56 +1SD 0.34 0.07 0.20 0.49 December 2019 \| Volume 10 \| Article 2665 TABLE 8 \| Conditional indirect effect of gratitude when deliberate rumination mediated between intrusive rumination and post-traumatic growth. 6 Deliberate Rumination, Gratitude and PTG Kim and Bae Gratitude can alter the perspectives from which traumatic experiences are interpreted (Watkins, 2014). Highly grateful individuals tend to ﬁnd positive resources in their lives after a traumatic event and perceive themselves and their environments positively (Fredrickson, 2004). In other words, gratitude can help individuals ﬁnd new meaning and value after a traumatic experience and accept painful experiences as a part of their lives. should use behavioral observations and the reports of family members and acquaintances to measure the study variables. Second, as this study used a cross-sectional research design, inferences cannot be drawn about the causality of emergent relationships. Future studies should use a longitudinal research design to test the validity of the present ﬁndings. ETHICS STATEMENT All subjects gave written informed consent in accordance with the Declaration of Helsinki. This study was approved by the Institutional Review Board of Dankook University. ACKNOWLEDGMENTS The authors wish to thank editage staﬀfor their translation of the manuscript. This study has several limitations. First, self-report measures were used to collect data in the present study; thus, future studies DISCUSSION Third, we did not distinguish between diﬀerent types of trauma and the period after trauma occurrence; we weighed all traumatic experiences equally, although research suggests that diﬀerent types of trauma and the period after occurrence might diﬀerently inﬂuence PTG. One of the key ﬁndings of this study pertains to the moderating eﬀect of gratitude on the mediating eﬀect of deliberate rumination on the relationship between intrusive rumination and PTG. This result is consistent with past ﬁndings that highly grateful individuals experience greater PTG as a result of their cognitive eﬀorts to understand a traumatic event (McCullough et al., 2006; Chun and Lee, 2013; Watkins, 2014). This suggests that the impact of deliberate rumination on PTG varies as a function of the level of gratitude. In this regard, gratitude can activate deliberate rumination and act as a buﬀer against the psychological distress that is caused by intrusive thoughts (Tsai et al., 2016; Leppma et al., 2018). Despite its limitations, this is the ﬁrst study to assess the moderating eﬀect of gratitude on the mediating eﬀect of deliberate rumination on the relationship between intrusive rumination and PTG. These ﬁndings shed light on how and when gratitude is associated with PTG via deliberate rumination. Further, this study provides grounds for gratitude interventions for adults who have experienced trauma to facilitate growth. It also implies that in the midst of distress, those who have experienced trauma may be able to consider their life more meaningful through gratitude. This study makes several contributions to the literature. First, past studies that have examined the impact of gratitude on PTG have been conducted using developmentally homogeneous samples such as adolescents and middle-aged adults. However, in this study, we used a heterogeneous sample of participants who represented all the developmental stages ranging from youth to older adulthood. This enhances the generalizability of the observed eﬀect of gratitude on PTG to people of all ages. Second, the association between the gratitude and PTG has been observed in previous studies, but the moderating eﬀect of gratitude on PTG has not been clearly delineated. In addition, there has been a lack of understanding about how deliberate rumination contributes to PTG. In this study, we have delineated the moderating eﬀect that gratitude has on the mediating eﬀect of deliberate rumination on the relationship between intrusive rumination and PTG. DATA AVAILABILITY STATEMENT The datasets generated for this study are available on request to the corresponding author. AUTHOR CONTRIBUTIONS The present ﬁndings have clinical implications. Speciﬁcally, therapists must be informed that repetitive and intrusive thoughts are natural reactions to a traumatic event and that traumatized individuals do not need to excessively suppress or avoid thoughts about their traumatic experience. In addition, it is necessary to implement training and intervention programs that can help individuals engage in deliberate rather than intrusive rumination. In particular, the present ﬁnding that gratitude enhances the eﬀect of deliberate rumination on PTG suggests that psychological interventions should aim to promote gratitude among traumatized individuals (e.g., gratitude writing). EK collected and analyzed the data and wrote the ﬁrst draft of the manuscript. SB led manuscript writing and revised it critically for important content. 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The relationship between posttraumatic cognitive change, posttraumatic stress disorder, and posttraumatic growth among Chinese adolescents after the Yancheng December 2019 \| Volume 10 \| Article 2665 Frontiers in Psychology \| www.frontiersin.org 9
https://openalex.org/W3034981634	https://europepmc.org/articles/pmc7353362?pdf=render	English	null	Black Sorghum Phenolic Extract Modulates Platelet Activation and Platelet Microparticle Release	Nutrients	2,020	cc-by	6,390	Received: 20 May 2020; Accepted: 10 June 2020; Published: 12 June 2020 Received: 20 May 2020; Accepted: 10 June 2020; Published: 12 June 2020 Abstract: Platelet hyper-activation and platelet microparticles (PMPs) play a key role in the pathogenesis of cardiovascular diseases. Dietary polyphenols are believed to mimic antiplatelet agents by blunting platelet activation receptors via its antioxidant phenomenon. However, there is limited information on the anti-platelet activity of grain-derived polyphenols. The aim of the study is to evaluate the eﬀects of sorghum extract (Shawaya short black 1 variety), an extract previously characterised for its high antioxidant activity and reduction of oxidative stress-related endothelial dysfunction, on platelet aggregation, platelet activation and PMP release. Whole blood samples collected from 18 healthy volunteers were treated with varying non-cytotoxic concentrations of polyphenol-rich black sorghum extract (BSE). Platelet aggregation study utilised 5 µg/mL collagen to target the GPVI pathway of thrombus formation whereas adenine phosphate (ADP) was used to stimulate the P2Y1/P2Y12 pathway of platelet activation assessed by ﬂow cytometry. Procaspase-activating compound 1 (PAC-1) and P-selectin/CD62P were used to evaluate platelet activation- related conformational changes and degranulation respectively. PMPs were isolated from unstimulated platelets and quantiﬁed by size distribution and binding to CD42b. BSE treatment signiﬁcantly reduced both collagen-induced platelet aggregation and circulatory PMP release at 40 µg/mL (p < 0.001) when compared to control. However, there was no signiﬁcant impact of BSE on ADP-induced activation-dependent conformational change and degranulation of platelets. Results of this study suggest that phenolic rich BSE may confer cardio-protection by modulating speciﬁc signalling pathways involved in platelet activation and PMP release. Keywords: black sorghum; polyphenols; platelets; platelet microparticles; atherosclerosis Black Sorghum Phenolic Extract Modulates Platele Activation and Platelet Microparticle Release Borkwei Ed Nignpense 1 , Kenneth A Chinkwo 1,2 , Christopher L Blanchard 1,2 and Abishek B Santhakumar 1,2,* 1 School of Biomedical Sciences, Charles Sturt University, Locked Bag 588, Wagga Wagga, NSW 2678, Australia; bednignpense@csu.edu.au (B.E.D.); kchinkwo@csu.edu.au (K.A.C.); CBlanchard@csu.edu.au (C.L.B.) 2 Australian Research Council (ARC) Industrial Transformation Training Centre (ITTC) for Functional Grains, Graham Centre for Agricultural Innovation, Charles Sturt University, Wagga Wagga, NSW 2650, Australia * Correspondence: asanthakumar@csu.edu.au; Tel.: +61-2-6933-2678 1 School of Biomedical Sciences, Charles Sturt University, Locked Bag 588, Wagga Wagga, NSW 2678, Australia; bednignpense@csu.edu.au (B.E.D.); kchinkwo@csu.edu.au (K.A.C.); CBlanchard@csu.edu.au (C.L.B.) 2 Australian Research Council (ARC) Industrial Transformation Training Centre (ITTC) for Functional Grains, Graham Centre for Agricultural Innovation, Charles Sturt University, Wagga Wagga, NSW 2650, Australia * Correspondence: asanthakumar@csu.edu.au; Tel.: +61-2-6933-2678 1 School of Biomedical Sciences, Charles Sturt University, Locked Bag 588, Wagga Wagga, NSW 2678, Australia; bednignpense@csu.edu.au (B.E.D.); kchinkwo@csu.edu.au (K.A.C.); CBlanchard@csu.edu.au (C.L.B.) 2 Australian Research Council (ARC) Industrial Transformation Training Centre (ITTC) for Functional Grains, Graham Centre for Agricultural Innovation, Charles Sturt University, Wagga Wagga, NSW 2650, Australia * Correspondence: asanthakumar@csu.edu.au; Tel.: +61-2-6933-2678 nutrients nutrients 1. Introduction According to a World Health Organisation report, cardiovascular diseases accounted for an estimated 31% of deaths globally with majority being a result of stroke or heart attack [1,2]. In clinical settings, treatment involves blunting the activity of platelets using antiplatelet drugs. These drugs interfere with the thrombotic pathophysiology—wherein a rupture of an atherosclerotic plaque triggers platelet hyper-activation resulting in unwanted clot formation and occlusion of the blood vessel. Macrovesicles referred to as platelet microparticles (PMPs) are released following platelet activation and can contribute to the thrombotic situation [3,4]. The several signalling pathways involved in platelet activation and thrombus formation include receptor-agonist pathways such as P2Y1/P2Y12-ADP, GPVI-collagen, PAR1-thrombin and the COX-1-thromboxane [5]. An agonist such as collagen when exposed by atherosclerotic plaque may Nutrients 2020, 12, 1760; doi:10.3390/nu12061760 www.mdpi.com/journal/nutrients 2 of 11 Nutrients 2020, 12, 1760 activate nearby platelets by binding to their GPVI receptor resulting in complex intracellular signalling that produce a conformational change (indicated by GPIIb/IIIa receptor expression), degranulation (indicated by P-selectin secretion) and subsequent formation of platelet aggregates [5]. In addition, PMP released upon activation possess adhesive and pro-coagulant platelet-derived receptors that further enhance thrombus formation, thereby acting as biomarkers of platelet activation [3]. The common antiplatelet agents, clopidogrel and aspirin, used in clinical treatments inhibit platelet activation and its circulating biomarkers by selectively targeting P2Y1/P2Y12-ADP and COX-1-thromboxane respectively [6]. Unfortunately, because of the unresponsiveness and side eﬀects associated with administration there have been considerable research in dietary bioactive agents known as polyphenols [7]. One such example of a polyphenol-rich functional food is sorghum whole grain. Although mainly used as animal feed, studies have demonstrated that it possesses anti-inﬂammatory, anti-cancer and antioxidant properties which add value to it as a food for human consumption [8]. Sorghum of diﬀerent types exist that are classiﬁed based on the pigmentation of the pericarp and vary in their phenolic content [9]. The polyphenols found in sorghum that contribute to its bioactivity include ﬂavonoids, hydroxybenzoic acids and hydroxycinnamic acid [8]. Furthermore Francis et al. [10] recently demonstrated that black sorghum rich in catechins and their derivatives may confer cardioprotective properties. The treatment of human umbilical vein cells with ﬂavonoid-rich extract was found to prevent oxidative stress-related endothelial dysfunction through the modulation of gene expression. Furthermore, these cardio-protective beneﬁts of polyphenols apply in the context of platelet function. 1. Introduction Several studies have demonstrated that polyphenols may inhibit platelet activation, adhesion, degranulation and aggregation by targeting speciﬁc thrombogenic pathways for example P2Y1/P2Y12-ADP, GPVI-collagen, PAR1-thrombin and the COX-1-thromboxane. As reviewed by Ed Nignpense et al. [11] many of the studies that investigate the polyphenol impact on platelet function and PMP generation utilise aggregometry and ﬂow cytometry. However there is limited research on sorghum-derived polyphenols in modulating biomarkers of platelet activation. This study aims to investigate the impact of black sorghum derived polyphenol extracts on collagen-induced platelet aggregation, ADP-induced platelet activation and PMP generation. 2.1. Research Ethics The study protocol was approved by the Charles Sturt University Human Research Ethics Committee (HR17012) and the Institutional Biosafety Committee (19HB02). The study was performed in compliance with relevant laws and institutional guidelines. 2.2. Volunteer Recruitment Eighteen healthy volunteers between 18–65 years of age (9 males and 9 females) were recruited from Charles Sturt University and the local community. Informed consent was obtained from all participants prior to commencement of the study. The criteria for recruitment involved normal health status with no history of conditions such as cardiovascular, metabolic, liver or lung disease. Other parameters that could aﬀect the integrity of the analysis such as alcohol consumption, smoking, pregnancy, allergies or venepuncture diﬃculty were considered during the recruitment process. A health screening questionnaire was used to assess the already mentioned parameters. A dietary questionnaire (adapted from WINTEC and NZ academy of sport) was used to assess the usual dietary intake of volunteers and to avoid recruitment of participants on a high antioxidant diet. The cut-oﬀ ﬁgure for each type of food listed in the questionnaire was based on nutrient reference ranges for Australia and New Zealand—recommended daily nutrient intake values. 3 of 11 Nutrients 2020, 12, 1760 2.4. Full Blood Examination Using an Abbott CELL-DYN Emerald 22 Haematology Analyser (Abbott Diagnostics, Illinois, USA), a full blood examination (FBE) was performed on all samples. The FBE results of volunteers indicated that the blood cell parameters were within normal reference ranges as determined by the Royal College of pathologists of Australia. Individuals with cell counts, especially platelet counts, outside of the reference range were excluded from the study. Quality control validation and maintenance were all performed according to the Abbott CELL-DYN Emerald 22 Haematology Analyser manual. 2.3. Blood Collection and Processing After fasting for at least 8 h, whole blood was collected from each participant by a trained phlebotomist into a tri-potassium ethylene diamine tetra-acetic acid (EDTA-1.8 mg/mL concentration) anticoagulant tube (Vacuette Greiner Bio-one, Interpath Services, Heidelberg West, VIC, Australia) and a tri-sodium citrate (28.12 g/L concentration) anticoagulant tube (Becton, Dickson and Company, North Ryde, NSW, Australia). A 20-mL syringe (Becton, Dickson and Company, North Ryde, NSW, Australia) and 21-gauge 1.5-inch needle (Terumo Medical Corporation, Macquarie Park, Australia) were used to draw blood from the median cubital vein. The purpose of choosing a larger gauge was to avoid the activation of platelets while drawing or dispensing blood. Utmost care was taken to ensure samples were not obtained through a traumatic collection and that none contained obvious clots. In addition, care was taken to ensure minimal specimen handling and agitation in order to prevent artefactual platelet activation. The ﬁrst 2 mL of blood was discarded before drawing into the tri-sodium citrate tubes in order to avoid the risk of collecting platelets activated by venepuncture. Tri-sodium tubes were used for aggregometry and ﬂow cytometry assays whereas the EDTA was used to perform full blood examinations. 2.5. Extraction of Black Sorghum Polyphenols Sorghum (Sorghum bicolor) samples of six different pericarp varieties were obtained from glasshouse trials conducted by Curtin University, Perth, Western Australia. Six pigmented varieties of sorghum were cultivated under the same conditions, grown in a glasshouse equipped with Lumisol Clear AF cover (200 µm thick, at a transparency of ca UV-A 94%, UV-B 84% and photosynthetic active radiation (PAR, 400-700 nm) 93% [8]. Extraction and analysis of phenolic composition and antioxidant activity were performed previously using methods described by Rao et al. [8]. Among the different sorghum varieties, the black pericarp variety (Shawaya short black 1) was selected for this study because of its relatively high antioxidant activity when analysed with ferric reducing antioxidant power (FRAP; 20.19 ± 2.69 mg/g TE) and 2,2-dipheny-1-picrylhydrazyl (DPPH; 18.04 ± 3.53 mg/g TE) antioxidant assays (Supplementary Figure S1, Tables S1 and S2). The highest level of polyphenols found in the BSE included catechin derivatives, catechins and pentahydroxyflavanone-(3->4)-catechin-7-O- glucoside (Supplementary Table S2). Stock concentrations of BSE (20 mg/mL in 50% DMSO) were diluted in phosphate buffered saline (PBS) to achieve desired concentrations (5 µg/mL, 20 µg/mL and 40 µg/mL) in whole blood. Desired concentrations were selected based on viability studies done by Francis et al. [10]. 2.6. Whole Blood Platelet Aggregometry Platelets in whole blood were stimulated for aggregation using 5 µg/mL collagen exogenous platelet agonists (DSKH Australia Pty. Ltd., Hallam, VIC, Australia) to investigate the effect of BSE treatment on the platelet aggregation. Five hundred microliters of citrated whole blood were added to 100 µL of 0.1% DMSO control (Sigma-Aldrich, Castle Hill, NSW, Australia) or BSE stock concentrations (5 µg/mL, 20 µg/mL and 40 µg/mL) and mixed with 400 µL of saline. The sample was then incubated at 37 ◦C for 20 min. Using a Chrono-log model 700 lumi-aggregometer (DKSH Australia Pty. Ltd., Hallam, VIC, Australia) the samples were analysed by means of electrical impedance (ohms) to determine the amount of platelet aggregation occurring in the sample over a 6-min time period (Supplementary Figure S2). 4 of 11 Nutrients 2020, 12, 1760 2.7. Flow Cytometry 2.7. Flow Cytometry 2.7.2. Measurement of Platelet Activation-Dependent Conformational Change and Degranulation The eﬀects of BSE on ADP-induced platelet activation were analysed and interpreted using a Gallios ﬂow cytometer (Beckman Coulter, Inc., Lane Cove NSW, Australia). The protocols were adopted from the method described by Santhakumar et al. [12] with some modiﬁcations. Platelet activation and thrombogenic indicators were assessed via activation-dependent platelet monoclonal antibodies (mAbs) purchased from Becton, Dickinson and Company, North Ryde, NSW, Australia. Procaspase activating compound-1 (PAC-1)-ﬂuorescein isothiocyanate-ﬂuorescein isothiocyanate was used to detect platelet activation-related conformational change and P-selectin/CD62P-allophycocyanin highlighted activation dependent degranulation. CD42b-phycoerythrin identiﬁed the GPIb-IX-V receptor, a common receptor found on the surface of all platelets, activated and resting included. A decreased expression of mAb exhibits alleviation of thrombogenesis. Within 5 min of collection tri-sodium citrated whole blood was used for assay preparation to avoid artefactual activation of platelets. A volume of 40 µL of blood was incubated with DMSO control or the various BSE concentration for 20 min at 37 ◦C in the dark. A 10-µL mixture of all three monoclonal antibodies (3.33 µL each of PAC-1, CD62P and CD42b) was added to blood samples and incubated for 20 min at room temperature in the dark. To induce platelet activation, 10 µM ADP (Helena laboratories Pty. Ltd., Mt Waverly, VIC, Australia) was added, and samples were incubated for a further 15 min in the dark at room temperature, after which erythrocytes were lysed (575 µL of 10 % lysing solution). Samples were thoroughly vortexed to ensure homogeneity and incubated in the dark at room temperature for a further 15 min and then analysed. In all, 10,000 platelet events were acquired, gated based on light scatter and CD42b mAb expression and activated platelets were articulated as mean ﬂuorescence intensity (MFI) (Supplementary Figure S3). 2.7.3. Measurement of Circulatory PMPs Using the microparticle gating established with Megamix beads, PMPs were identiﬁed and quantiﬁed by size distribution and binding to CD42b (Supplementary Figure S4). The protocol for circulatory PMP analysis was adapted from Lu et al. [13]. A volume of 1 mL whole blood was added to micro-centrifuge tubes in the presence of PGE1 (120 nmol/L; Sigma-Aldrich, Castle Hill, NSW, Australia). PGE1 was added to prevent artefactual activation during centrifugation. The blood samples were incubated with the respective 100 µL BSE concentrations and DMSO control for 20 min at 37 ◦C in the dark. Each sample was then centrifuged for 15 min at a 1000 rpm and the resultant platelet rich plasma (PRP) was discarded. The remaining blood was spun a further 15 min at 3000 rpm. The supernatant rich in PMPs (40 µL) was collected into ﬂow tubes and incubated with 4 µL of CD42b and 6 µL of stain buﬀer (Becton, Dickson and Company, North Ryde, NSW, Australia) in dark room for 15 min. Four percent formaldehyde was used to ﬁx any activation of platelets left in the supernatant. After a 10-min incubation period the samples were run for PMP analysis on the ﬂow cytometer. 2.7.1. Standardisation Flow-check ﬂuorospheres were run as quality control for optimal laser alignments. Antibody capture beads (Anti-Mouse Ig, K CompBeads, BD Biosciences, North Ryde, NSW, Australia) were used for single colour compensation controls in order to achieve optimal compensation. Megamix beads (0.1 µm, 0.3 µm, 0.5 µm, 1 µm) from Biocytex, Marseille, France were used as per manufacturer’s instructions to set up an appropriate gating to detecting microparticles. They were run before each PMP analysis. 2.8. Statistical Analysis A two-way ANOVA following Tukey’s post comparison test was performed using GraphPad Prism version 8.0 for Windows (GraphPad Software, La Jolla, California, USA). A minimum sample size of 14 participants in total was required for 80% power to detect a 5% variation in the laboratory 5 of 11 Nutrients 2020, 12, 1760 parameters measured where a 3–5% standard deviation exists in the population, assuming an alpha error of 0.05. All the data were expressed as mean ± standard deviation (SD). Diﬀerences between the groups were signiﬁcant when p < 0.05. Any signiﬁcant statistical interactions were included in the analysis where applicable. 3. Results The baseline parameters including full blood counts for all 18 participants were within normal reference ranges set by the Royal College of Pathologists of Australasia (Table 1) [14]. The baseline parameters including full blood counts for all 18 participants were within normal reference ranges set by the Royal College of Pathologists of Australasia (Table 1) [14]. Table 1. Baseline full blood counts of participants. Parameters Mean ± SD Age (years) 26 ± 8 WBC (× 109/L) 5.5 ± 1.3 Neutrophil (%) 48.6 ± 9.5 Lymphocytes (%) 37.8 ± 9.2 Monocytes (%) 10.6 ± 2.3 Eosinophils (%) 2.8 ± 1.3 Basophils (%) 0.1 ± 0.1 RBC (× 1012/L) 4.6 ± 0.5 Haemoglobin (g/L) 147.9 ± 16.1 PCV (%) 0.41 ± 0.04 MCV (fL) 90.0 ± 3.5 MCH (pg) 35.8 ± 15.2 MCHC (g/dL) 360.1 ± 6.0 RDW (%) 14.9 ± 0.8 Platelet count (× 109/L) 248.3 ± 50.0 MPV 8.41 ± 0.89 Values are represented as mean ± Standard deviation (SD). RBC, red blood cell, PCV, packed cell volume, MCV, mean cell volume, MCH, mean cell haemoglobin, MCHC, mean cell haemoglobin concentration, RDW, red cell distribution width, MPV, mean platelet volume. Table 1. Baseline full blood counts of participants. Values are represented as mean ± Standard deviation (SD). RBC, red blood cell, PCV, packed cell volume, MCV, mean cell volume, MCH, mean cell haemoglobin, MCHC, mean cell haemoglobin concentration, RDW, red cell distribution width, MPV, mean platelet volume. 3.1. Eﬀect of BSE on Whole Blood Platelet Aggregation and Platelet Activation BSE at 40 µg/mL concentration signiﬁcantly reduced platelet aggregation stimulated by collagen by 19 % (p = 0.0004) (Figure 1). BSE at lower concentrations did not exhibit any signiﬁcant reduction in aggregation. BSE at 40 µg/mL concentration signiﬁcantly reduced platelet aggregation stimulated by collagen by 19 % (p = 0.0004) (Figure 1). BSE at lower concentrations did not exhibit any signiﬁcant reduction in aggregation. It was observed that whole blood treatment with the varying concentrations of BSE did not signiﬁcantly aﬀect ADP-induced platelet conformational change and degranulation indicated by PAC-1 and P-selectin expression respectively (Supplementary Figures S5 and S6). It was observed that whole blood treatment with the varying concentrations of BSE did not signiﬁcantly aﬀect ADP-induced platelet conformational change and degranulation indicated by PAC-1 and P-selectin expression respectively (Supplementary Figures S5 and S6). 3.2. Eﬀect of BSE on Circulatory PMPs 3.2. Eﬀect of BSE on Circulatory PMPs BSE at a concentration of 40 µg/mL signiﬁcantly reduced the amount of circulatory PMPs in whole blood by 47% (p = 0.0008). Lower concentrations of BSE did not exhibit any signiﬁcant reduction to the amount circulatory PMPs (Figure 2). 6 of 11 Nutrients 2020, 12, 1760 Figure 1. The eﬀect of varying concentrations of BSE on collagen-induced aggregation. BSE at 40 µg/mL signiﬁcantly reduced platelet aggregation (5.3 ± 1.3; p value = 0.0004). BSE at 5 µg/mL and 20 µg/mL did not reduce platelet aggregation when compared to control (p value > 0.1). N = 18 and data is represented in aggregation (Ohms) versus BSE concentrations. * signiﬁes statistical signiﬁcance p < 0.001 compared to control. Error bars expressed as mean ± SD. Figure 1. The eﬀect of varying concentrations of BSE on collagen-induced aggregation. BSE at 40 µg/mL signiﬁcantly reduced platelet aggregation (5.3 ± 1.3; p value = 0.0004). BSE at 5 µg/mL and 20 µg/mL did not reduce platelet aggregation when compared to control (p value > 0.1). N = 18 and data is represented in aggregation (Ohms) versus BSE concentrations. * signiﬁes statistical signiﬁcance p < 0.001 compared to control. Error bars expressed as mean ± SD. Figure 2. The eﬀect of varying concentrations of BSE on circulatory PMP production in vitro. BSE at 40 µg/mL signiﬁcantly reduced the amount of circulatory PMPs (<24190 ± 4935, p = 0.0008). BSE at 5 µg/mL and 20 µg/mL did not reduce platelet aggregation when compared to control (p value > 0.1). N = 14 and data are represented in number of PMP events versus BSE concentrations. * signiﬁes statistical signiﬁcance p < 0.001 compared to control. Error bars expressed as mean ± SD. Figure 2. The eﬀect of varying concentrations of BSE on circulatory PMP production in vitro. BSE at 40 µg/mL signiﬁcantly reduced the amount of circulatory PMPs (<24190 ± 4935, p = 0.0008). BSE at 5 µg/mL and 20 µg/mL did not reduce platelet aggregation when compared to control (p value > 0.1). N = 14 and data are represented in number of PMP events versus BSE concentrations. * signiﬁes statistical signiﬁcance p < 0.001 compared to control. Error bars expressed as mean ± SD. 4. Discussion It has been suggested that the total plasma polyphenol concentration rarely exceeds 1 µM and that their antiplatelet eﬀects are only found at high non-physiological concentrations (greater than 50 µM) [17,18]. However, it is likely that these plasma concentrations are underestimations due to the ability of polyphenols to bind to the surface of red blood cells and thereby exert their bioactivity [19]. Furthermore polyphenols (structurally related to catechin) and their metabolites have been shown to inhibit platelet function in vitro [20]. This highlights the possibility of sorghum catechins and their metabolites having antiplatelet eﬀects in vivo despite bioavailability concern. Interestingly, an in vivo human dietary intervention trial compared consumption of red and white wholegrain sorghum-based pasta to a control pasta in order to investigate its acute eﬀect on the total phenol content and antioxidant activity in the plasma of healthy subjects [21]. Results showed that when compared to the control pasta, the red sorghum pasta showed signiﬁcantly increased net plasma phenolic content and antioxidant activity post consumption (from 216.90 ± 2.62 at baseline to 269.40 ± 2.33 at 2 h; p < 0.001), thus demonstrating a plausible correlation between antioxidant activity and sorghum polyphenol consumption—which in turn may contribute to antiplatelet eﬀects. The antiplatelet eﬀects was observed with BSE-included inhibition of collagen-induced aggregation and circulatory PMP production but no eﬀect on ADP-induced platelet activation. The absence of antiplatelet eﬀects on the P2Y1/ P2Y12-ADP activation pathway suggests that BSE polyphenols are not mimicking the action of drugs such as clopidogrel that antagonise P2Y12 receptor activation [22]. However, the inhibition of collagen-induced aggregation suggest that BSE polyphenols interfere with GPVI-collagen signalling pathways by either blunting the GPVI receptor directly or by other mechanisms [5]. Previous studies have demonstrated that ﬂavonoids, speciﬁcally quercetin and catechin, can act synergistically to inhibit collagen-induced aggregation by blunting the associated burst of H2O2 and subsequent PLC activation [23,24]. Thus, a possible mechanism of inhibition BSE ﬂavonoids may be a synergistic antagonism of the positive feedback activation of intracellular signals triggered by H2O2. Moreover, it has been shown that the phosphorylation cascade initiated by collagen can be inhibited by ﬂavonoids [25,26]. Flavones, especially apigenin and luteolin, by virtue of a double bond in the C2-C3 and the keto group in C4 can also inhibit collagen-induced activation by antagonizing the TxA2 receptor activation which is also involved in the positive feedback loop [27]. 4. Discussion There is growing interest in understanding the therapeutic beneﬁts of functional foods. Sorghum for example is one of the functional foods that is showing promise in this area. With sorghum-derived polyphenols already having demonstrated anti-inﬂammatory, anti-cancer and antioxidant properties, the current study aimed to evaluate the eﬀects of polyphenol-rich BSE on platelet function in terms of aggregation, conformational change, degranulation and circulatory PMP production [8–10]. It was observed that BSE signiﬁcantly inhibited collagen-induced platelet aggregation and decreased the release of circulatory PMPs but did not have a signiﬁcant eﬀect on ADP-induced platelet conformational change or degranulation. Although these results do not reﬂect a typical dose-dependent inhibition, they suggest a potential role of BSE polyphenols at optimum concentrations to interfere with pathways in the GPVI-collagen signalling and the release of circulatory PMPs but little or no eﬀect on P2Y1/ P2Y12-ADP pathway. 7 of 11 Nutrients 2020, 12, 1760 To the best of our knowledge only a few studies have investigated the antiplatelet eﬀects of sorghum extracts. Li, Yu and Fan et al. [15] extracted alditols and monosaccharides from sorghum vinegar to evaluate their anti-aggregation activity using the turbidimetric method. Results from their study indicated a signiﬁcant dose-dependent inhibition of aggregation via multiple agonists, arachidonic acid, collagen, ADP and thrombin. Furthermore, a diﬀerent study by Fan et al. [16] reported in vitro inhibition of ADP- and thrombin- induced rabbit platelet aggregation by methanolic extracts of aged sorghum vinegar with the half maximal inhibitory concentrations (IC50) of 1.7 ± 0.3 and 8.9 ± 1.9 mg/mL respectively. When rats were orally administered the extracts (>100 mg/kg), both collagen- and epinephrine-induced pulmonary thrombosis were inhibited. In comparison with the present study it is to be noted that these studies employed platelet-rich plasma rather than whole blood hence not accounting for the possible involvement of other blood cells and extracellular mediators involved in thrombus formation. In addition, sorghum vinegar extracts were used at higher concentrations; milligrams compared to micrograms used in this study. This raises the question of bioavailability and the importance of employing physiological concentrations of extracts. Although the BSE concentration of 40 µg/mL at which antiplatelet eﬀect were observed is relatively lesser in concentration than used in the other studies, the most bioactive compounds with respect to antioxidant activity were catechins and other ﬂavonoids which are usually considered to have low bioavailability [8]. 5. Conclusion and Future Considerations In summary, the present study contributes to the growing body of literature on bioactivity of sorghum polyphenols and highlights possible mechanisms of antiplatelet action that may result in cardiovascular health beneﬁts. Because of the ability to reduce collagen-induced platelet activation and circulatory PMP generation, BSE polyphenols demonstrate the potential to interfere with pathological processes involved in vascular disorders and thrombotic complications. However, a larger panel of agonist for the ﬂow cytometry and aggregometry studies will aid to further elucidate antiplatelet mechanisms. Because of the bioavailability concerns, well-controlled dietary intervention trials using larger sample sizes to evaluate the antiplatelet eﬀects of sorghum consumption in healthy and pro-thrombotic populations are warranted to justify our ﬁndings. Because of the varied phenolic proﬁles of the diﬀerent sorghum varieties, further research comparing the antiplatelet therapeutic potential of diﬀerent grains is also warranted. Furthermore, this study attest to the measurement of circulatory PMPs as a biomarker of platelet activation to assess the bioactivity of functional foods. Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/12/6/1760/s1, Figure S1: Characterisation of phenolic compounds in BSE. Ultra-high-performance liquid chromatography (UHPLC) was employed to quantify the diﬀerent phenolic compounds identiﬁed by the peak (on top). An online 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was coupled with UHPLC to quantify the relative antioxidant activity (peaks below) of each compound identiﬁed, Figure S2: A report derived from the collagen induced platelet aggregation study using the Chrono-log model 700 lumi-aggregometer (DKSH Australia Pty. Ltd, Hallam, VIC, Australia). The blue tracing represents the control (whole blood with no BSE) and the black tracing represents the whole blood pre-treated with 5 µg/mL BSE. The addition of BSE reduced the maximum platelet aggregation expressed in Ohms from 15 ohms to 11 ohms, Figure S3: A report from the ADP-induced platelet activation analysis using Kaluza Flow Cytometry Software (Beckman Coulter, Brea, CA, USA). Results indicate the gating of whole platelet population (CD42b positive events) and the proportion of activated platelets indicated by PAC-1 and P-selectin expression, Figure S4: A report from the PMP analysis using Kaluza Flow Cytometry Software (Beckman Coulter, Brea, CA, USA). Microparticle gating was established using Megamix beads of standard sizes. PMPs were distinguished from other microparticles by size (0.5 µm - 0.9 µm) and expression of CD42b. The number of CD42b positive events in the microparticle gate was used to quantify the PMPs, Figure S5: The eﬀect of varying concentrations of BSE on PAC-1 expression. 4. Discussion Besides inhibition of the GPVI-collagen signalling, BSE polyphenols showed inhibition of the circulatory PMP production. To the best of our knowledge, this is the ﬁrst study investigating the eﬀect of sorghum-derived polyphenols on PMP production. In contrast to this study, other PMP studies have employed the use of Annexin V as well as the platelet speciﬁc antibody CD42b, to identify pro-coagulant PMPs 8 of 11 Nutrients 2020, 12, 1760 by their phosphatidylserine expression and to limit background noise [28]. However, because of the heterogeneity of PMPs, not all PMPs express phosphatidylserine [29]. Moreover, the measurement of CD42b-positive PMPs alone is signiﬁcant as its increase has been associated with an increased risk of coronary heart disease [30]. From the current study, the signiﬁcant inhibition of CD42b-positive circulatory PMPs observed in vitro may be attributed to the antioxidant properties of BSE polyphenols. It is believed that the inhibition of PMP generation may be the result of neutralisation of H2O2, scavenging of other free radicals or interaction with intracellular signalling leading to PMP release. g g g g g The juxtaposition of both the present study and that of an earlier study by Francis et al. [10] highlights the multifaceted role of BSE polyphenols in cardio-protection. The group investigated the eﬀects of BSE polyphenols on the expression of antioxidant- and inﬂammatory-linked genes involved in endothelial dysfunction under oxidative stress. Results indicated that BSE polyphenols alleviate oxidative stress–induced damage to endothelial cells. Since vascular dysfunction is a precursor to cardiovascular diseases, the current study builds upon earlier ﬁndings by exhibiting the antiplatelet eﬀects of BSE. In the context of endothelial dysfunction, platelet activation and circulatory PMPs play central roles in the pathogenesis of atherothrombosis. The disruption of the plaque exposes collagen that binds to the GPVI receptor resulting in platelet activation and subsequent thrombus formation [31]. Circulatory PMPs may contribute to thrombosis via GPIb-IX-V receptor binding and have a pro-inﬂammatory eﬀect to promote the development of the plaque [32]. Therefore, by reducing collagen-induced platelet aggregation and circulatory PMP generation, BSE polyphenols may be displaying the potential to augment thrombosis. 6. Sambu, N.; Curzen, N. Monitoring the Eﬀectiveness of Antiplatelet Therapy: Opportunities and Limitations. Br. J. Clin. Pharmacol. 2011, 72, 683–696. [CrossRef] 5. Conclusion and Future Considerations BSE did not signiﬁcantly reduce ADP-induced platelet conformational change detected by PAC-1 expression (p values > 0.1 compared to control) N=14 and data is represented in mean ﬂuorescence intensity (MFI) versus BSE concentrations. Error bars expressed as mean ± SD, Figure S6: The eﬀect of varying concentrations of BSE on P-selectin expression. BSE did not signiﬁcantly reduce ADP-induced platelet degranulation detected by P-selectin expression (p values > 0.1 compared to control ) N=14 9 of 11 Nutrients 2020, 12, 1760 and data is represented in mean ﬂuorescence intensity (MFI) versus BSE concentrations Error bars expressed as mean ± SD, Table S1: Phenolic composition and antioxidant activity of sorghum varieties on as is basis, Table S2: List of top ten phenolic compounds identiﬁed in the black sorghum phenolic rich extracts by Q-TOF LC/MS and quantiﬁed using UHPLC-Online ABTS system (Adapted from Rao et al., 2018). Author Contributions: B.E.N. conducted the experiments outlined in this study and drafted the manuscript. K.A.C., C.L.B. and A.B.S. were involved in the designing and critical reviewing of the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This study was funded by the Australian Research Council Industrial Transformations Training Centre for Functional Grains (Project ID 100737). Acknowledgments: The authors would like to acknowledge Graham Centre for Agricultural Innovation for providing funding towards open access publication of this article. Borkwei Ed Nignpense is a recipient of a PhD Scholarship from the Australian Government Research Training Program and also a recipient of a top-up scholarship by the Graham Centre. Conﬂicts of Interest: The authors declare no conﬂict of interest. Abbreviations ADP Adenosine diphosphate ATP Adenosine triphosphate BSE Black sorghum extract COX-1 Cyclooxygenase-1 DMSO Dimethyl sulfoxide EDTA Ethylene diamine tetra-acetic acid FBE Full blood examination GAE Gallic acid equivalents MCH Mean cell haemoglobin MCHC Mean cell haemoglobin concentration MCV Mean cell haemoglobin MFI Mean ﬂuorescence intensity MPV Mean platelet volume PAR Protease-activated receptor PAC-1 Procaspase activating compound-1 PBS Phosphate buﬀered saline PCV Packed cell volume PGE1 Prostaglandin E1 PMP Platelet microparticle RBC Red blood cell SD Standard deviation WBC White blood cell References 2005, 53, 6813–6818. [CrossRef] 10. Francis, N.; Rao, S.; Blanchard, C.; Santhakumar, A. Black Sorghum Phenolic Extract Regulates Expression of Genes Associated with Oxidative Stress and Inﬂammation in Human Endothelial Cells. Molecules 2019, 24, 3321. [CrossRef] 11. Ed Nignpense, B.; Chinkwo, K.A.; Blanchard, C.L.; Santhakumar, A.B. 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https://openalex.org/W2797823821	https://zenodo.org/record/2015986/files/article.pdf	English	null	Syncopation and Emphasis. I	The musical times/Musical times	1,912	public-domain	5,309	Syncopation and Emphasis. I Author(s): Reginald Gatty Source: The Musical Times, Vol. 53, No. 832 (Jun. 1, 1912), pp. 369-372 Published by: Musical Times Publications Ltd. Stable URL: http://www.jstor.org/stable/907325 Accessed: 15-02-2016 08:28 UTC Syncopation and Emphasis. I Author(s): Reginald Gatty Source: The Musical Times, Vol. 53, No. 832 (Jun. 1, 1912), pp. 369-372 Published by: Musical Times Publications Ltd. Stable URL: http://www.jstor.org/stable/907325 Accessed: 15-02-2016 08:28 UTC Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at http://www.jstor.org/page/ info/about/policies/terms.jsp JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact support@jstor.org. Musical Times Publications Ltd. is collaborating with JSTOR to digitize, preserve and extend access to The Musical Times. http://www.jstor.org * 'Grammar of Music.' London, T. Hurst. ? 86. t 'Musik-Lexikon.' Article Syncoje. S'Grove's Dictionary' (Article Syncofation) defines the effect as 'An alteration of regular rhythm, produced by placing the strongest emphasis on part of the bar not usually accented,' but in this case, I ~ I must alsobe an example of syncopation, which is thus confused with emphasis. y In the following articles an attempt has been made to co-ordinate the various scattered explanations with regard to syncopation and emphasis, and to reconcile the discrepancies that exist between them. It has further been sought to show that although vagueness of definition is partly responsible for the confusion, there also prevails a widespread misconception as to the very nature of some of the effects in question. r y d question. First of all it must be pointed out that the one essential feature of syncopation and emphasis is that, being concerned with the so-called rhetorica (or oratorical) accents of music, they depend for their very existence on their unambiguous differentiation from the natural (grammatical or metrical) accents. It is only because they are recognised as dependent contrasts to the regular series of accents associated with the bar-lines that they are able to have their proper effect. Once the rhythm, and consequently the music, becomes obscure, by reason of the fact that they can no longer be differentiated from the natural accents, they have lost their raison d'ttre, unless we can be sure that this obscurity was the actual intention of the composer. It may seem superfluous to labour the point, but its importance will be realised later on, when it will be found that it is just here where differencesof opinionactuallyoccur. g It is time that these great men came to occupy their true place in the Kingdom ofArt. The England of the time has been aptly called a nest of singing birds, and the phrase is equally true of the musicians as of the poets. Of course there is no Shakespeare-- he comes only once in the world's history. But not only did these men lead the musical world of their time, they produced work of high perfection of a distinct character, and which the world cannot afford to lose-work which is among the richest treasures of art, and should be not only the fruit but the food, of the human spirit. , , opinion actually Coming now to syncopation, I define it with Callcott* and Riemannt as the sustaining of a sound that begins on a weak beat (or the weak portion of a divided beat) and ends on a strong one (or the strong portion of a divided beat). , t 'Musik-Lexikon.' Article Syncoje. This content downloaded from 150.131.192.151 on Mon, 15 Feb 2016 08:28:04 UTC All use subject to JSTOR Terms and Conditions This content downloaded from 150.131.192.151 on Mon, 15 Feb 2016 08:28:04 UTC All use subject to JSTOR Terms and Conditions THE MUSICAL TIMES.-JUNE I, 1912. 369 and proved interesting. More mature in style is the 'Fantasia of Foure Parts' which appears in 'Parthenia,' a collection of Virginal music published in i6ii. It consisted of works by Byrd (8), Bull ('), and Gibbons (6)-twenty-one in all. The Fantasia is not strongly individualised as instrumental music, but is an interesting work grave and serious in character, and has very markedly the characteristic just mentioned, viz., that a fresh figure appears near the end, and is worked imitationally to make a Coda. Of the other pieces by Gibbons in 'Parthenia,' distinctly the most attractive is'Lord Salisbury his Pavan.' This appears also in the 'Fitzwilliam Virginal Book,' which contains 297 pieces, and was probably copied out in prison (1608 or 1609-16) as a pastime by the younger Tregian, a Catholic who suffered for his faith. Byrd (59), Bull (36), Farnaby (55), Dowland (3), Morley (9), and others appear; but Gibbons, strangely enough, has only two pieces. The other is 'The Woods so wilde,' which shows traces of the immature instrumental technique of the time. Semiquaver passages of no particular design, above and below the melody, apparently gave pleasure by their mere rapidity. The sound of the instrument seems of itself to have satisfied the hearers. Still, in the quieter portion, there is a noticeable feeling for the sentiment of the title. and proved interesting. More mature in style is the 'Fantasia of Foure Parts' which appears in 'Parthenia,' a collection of Virginal music published in i6ii. It consisted of works by Byrd (8), Bull ('), and Gibbons (6)-twenty-one in all. The Fantasia is not strongly individualised as instrumental music, but is an interesting work, grave and serious in character, and has very markedly the characteristic just mentioned, viz., that a fresh figure appears near the end, and is worked imitationally to make a Coda. Of the other pieces by Gibbons in 'Parthenia,' distinctly the most attractive is'Lord Salisbury his Pavan.' This appears also in the 'Fitzwilliam Virginal Book,' which contains 297 pieces, and was probably copied out in prison (1608 or 1609-16) as a pastime by the younger Tregian, a Catholic who suffered for his faith. * 'Grammar of Music.' London, T. Hurst. ? 86. This content downloaded from 150.131.192.151 on Mon, 15 Feb 2016 08:28:04 UTC All use subject to JSTOR Terms and Conditions Byrd (59), Bull (36), Farnaby (55), Dowland (3), Morley (9), and others appear; but Gibbons, strangely enough, has only two pieces. The other is 'The Woods so wilde,' which shows traces of the immature instrumental technique of the time. Semiquaver passages of no particular design, above and below the melody, apparently gave pleasure by their mere rapidity. The sound of the instrument seems of itself to have satisfied the hearers. Still, in the quieter portion, there is a noticeable feeling for the sentiment of the title. f - -_ (b) On weak beats, or the weak portions of divided beats: (b) On weak beats, or the weak portions of divided beats: (b) On weak beats, or the weak portions of divided beats: (b) Through the bars: Ex. 6. HANDEL. 'Athalia,' No. 3. 'The rising world.' won - - drous, won - - - drous Ex. 7. SCHUMANN. Sonata in G minor, Op. 22. Ex. 8. SCHUMANN. Pianoforte Concerto, Op. 54. Allegro vivace. p3 -c== Ex. 9. SCHUMANN. 'Phantasiestiicke,' Op. z2, No. 4. ----4 -------r-I--- Ex. zo. BEETHOVEN. Pianoforte Sonata, Op. 28, Ist movement. + ..-, -=- 1 - -... =# ---- We have now to consider the class of kindred effects, dependent on emphasis, as indicated by phrasing slurs and sforzato-signs(sf >- A) d HAYDN. Quartet, Op. 54, No. 2 (Ist movement). Ex. i4. Ex. I5. SCHUBERT. Quartet in A minor, Op. 29. BEETHOVEN. Pianoforte Sonata, Op. 27, No. I, Finale. Ex. 16. _fC Ex ?t. BEETHOVEN. 3rd Symphony, Scherzo. f sf slf sf Ex. z8. BEETHOVEN. Pianoforte Trio, Op. z, No. 2. s/ sf sf It frequently happens, however, that the phrasing HAYDN. Quartet, Op. 54, No. 2 (Ist movement). Ex. i4. Ex. I5. SCHUBERT. Quartet in A minor, Op. 29. BEETHOVEN. Pianoforte Sonata, Op. 27, No. I, Finale. Ex. 16. _fC Ex ?t. BEETHOVEN. 3rd Symphony, Scherzo. f sf slf sf Ex. z8. BEETHOVEN. Pianoforte Trio, Op. z, No. 2. s/ sf sf It frequentlyhappens howeverthatthephrasing HAYDN. Quartet, Op. 54, No. 2 (Ist movement). Ex. i4. Ex. I5. SCHUBERT. Quartet in A minor, Op. 29. BEETHOVEN. Pianoforte Sonata, Op. 27, No. I, Finale. HAYDN. Quartet, Op. 54, No. 2 (Ist movement) SCHUBERT. Quartet in A minor, Op. 29. BEETHOVEN. Pianoforte Sonata, Op. 27, No. I, Finale. Ex. 16. _fC Ex ?t. BEETHOVEN. 3rd Symphony, Scherzo. f sf slf sf Ex. z8. BEETHOVEN. Pianoforte Trio, Op. z, No. 2. s/ sf sf BEETHOVEN. Pianoforte Sonata, Op. 27, No. I, Finale. BEETHOVEN. Pianoforte Trio, Op. z, No. 2. We have now to consider the class of kindred effects, dependent on emphasis, as indicated by phrasing slurs and sforzato-signs (sf >-, A). Moreover, the resemblance is an accidental rather than a vital one. On this principle of analysis, is really 4, ~' ,'the length of the bar being doubled.' It is perfectly proper to point out the resemblance between the two forms of notation, but it can only lead to confusion to explain all the' subtler effects of one time in terms of another, besides ignoring the differences of subjective expression suggested to the player by the particular notation employed. that (b) and (c) are much more akin, by reason of tIke accented notes ending on te strong/3eat ofeack bar, than (a) and (c), where we find the note on the strong beat sounded in (a) but silent in (c). This being the case, it seems less confusing to consider (a) merely as an example of emphasis in triple time rather than as a special kind of syncopation in triple time that still kcas to be dziferentiated from (b). The displacement of the accent on to this weak beat from the following (silent) strong beat is rather an accompanying feature of the device than an essential one, as is shown by the fact that suspensions are also syncopations, although the change of harmony preserves the strong accent on the strong beat. This may be seen in the following example quoted from Pauer ('Musical Forms,' Novello, p. I7) : A proposal of some novelty was made at a recent meeting of the Liverpool Philharmonic Society. It seems that the system of guest-conductors has not met with unmixed approval, as it involves extra expense in fees, and the conductors have, in the opinion of some, shown a tendency to be autocratic at the expense of the Society's profits. From among the floatin discontent arose the suggestion: g s e Ex. A. _ __ -_ - - - t gg (i) That it be advertised in the Musical Press that the Liverpool Philharmonic Society is open to receive application from conductors willing to offer their services to conduct one or more concerts, and to submit beforehand their programm to the committee; (2) that if the programme be approved the conductor shall be paid only a percentage of the profi after all the expenses have been met; (3) should the programme meet the disapproval of any member he shall be entitled to return his ticket to the secretary for sale, and if such ticket be sold one-half of the proceeds shall go to the credit of the member's subscription of the following season, and the remainder to the Society'sfunds. Syncopations may be divided into two main classes: those that occur within the bar, and those that go 'through the bars' for their completion. Both may occur either singly or in sequence, in It would have been interesting to watch the strenuous rivalry of our best English conductors, and perhaps many from abroad, for the rewards of this democratic competition, but unfortunately the suggestion was not taken. This content downloaded from 150.131.192.151 on Mon, 15 Feb 2016 08:28:04 UTC All use subject to JSTOR Terms and Conditions THE MUSICAL TIMES.-JUNE I, 1912. 370o which case there may be one or more in each bar. The following examples are adduced. (a) Within the bar : which case there may be one or more in each bar. The following examples are adduced. 4, ~' ,'the length of the bar being doubled.' It is perfectly proper to point out the resemblance between the two forms of notation, but it can only lead to confusion to explain all the' subtler effects of one time in terms of another, besides ignoring the differences of subjective expression suggested to the player by the particular notation employed. t This passage is quoted by Corder (Grove's Dictionary, Article Syncofjation) as an example of syncopation, but see note f on p. 369. C. F. Abdy Williams ('The rhythm of modern music '), Macmillan, 1909, p. 98), also cites (-= )asanexampleof syncopa- tion, and gives (p. 97) the following definition: 'An accented note is cut short, or is represented by a short rest, and the succeeding note, entering before its expected time, is made longer than the shortened accented note or rest.' It will be evident, however, if we compare examples (a), (b) and (c), (a) Within the bar : These are of two main kind&, according as they occur on strong or weak beats, or the weak portions of divided beats. (a) On strong beats : g , portions of divided beats. (a) On strong beats : Ex. ii. SCHUMANN. Fantasie, Op. 17. -sf sf Ex 12. SCHUMANN. Novellete, No. 8, Trio II. (Op. 21). SCHUMANN. Op. 26, No. I (' Faschingsschwank '). Ex. _3. f - -_ (b) On weak beats, or the weak portions of divided beats: HAYDN. Quartet, Op. 54, No. 2 (Ist movement). Ex. i4. Ex. I5. SCHUBERT. Quartet in A minor, Op. 29. BEETHOVEN. Pianoforte Sonata, Op. 27, No. I, Finale. Ex. 16. _fC Ex ?t. BEETHOVEN. 3rd Symphony, Scherzo. f sf slf sf Ex. z8. BEETHOVEN. Pianoforte Trio, Op. z, No. 2. s/ sf sf It frequently happens, however, that the phrasing accents, instead of being placed on strong or t This passage is quoted by Corder (Grove's Dictionary, Article Syncofjation) as an example of syncopation, but see note f on p. 369. C. F. Abdy Williams ('The rhythm of modern music '), Macmillan, 1909, p 98) also cites ( = )asanexampleof syncopa- Ex. 2. HANDEL. ' Joshua.' Air, ' Heroes, when with glory.' re . quites, . . . . . . . love the 'Judas Maccabaeus,' Act II. Ex. 3. How vain., is man, . . who 'Tristan und Isolde,' Act II. Ex. 4, TCHAIKOVSKY, Op. IX. Ex. 5. (b) Through the bars: Ex. 6. HANDEL. 'Athalia,' No. 3. 'The rising world.' won - - drous, won - - - drous Ex. 7. SCHUMANN. Sonata in G minor, Op. 22. Ex. 8. SCHUMANN. Pianoforte Concerto, Op. 54. Allegro vivace. p3 -c== Ex. 9. SCHUMANN. 'Phantasiestiicke,' Op. z2, No. 4. ----4 -------r-I--- Ex. zo. BEETHOVEN. Pianoforte Sonata, Op. 28, Ist movement. + ..-, -=- 1 - -... =# ---- We have now to consider the class of kindred effects, dependent on emphasis, as indicated by phrasing slurs and sforzato-signs (sf >-, A). g Ex. ii. SCHUMANN. Fantasie, Op. 17. -sf sf Ex 12. SCHUMANN. Novellete, No. 8, Trio II. (Op. 21). SCHUMANN. Op. 26, No. I (' Faschingsschwank '). Ex. _3. It frequently happens, however, that the phrasing accents, instead of being placed on strong or It frequently happens, however, that the phrasing accents, instead of being placed on strong or * Franklin Taylor says of this passage (' Technique and Expression in Pianoforte Playing,' Novello, p. 32): 'The length of the bar is doubled, becoming, as it were, a bar of 3-2 instead of 3-4,' and shows 'the effect of this change' as follows: Ex. 8A. .________, , -t--M- -- - -- But this conversion should rather be I d . J& &c. Moreover, the resemblance is an accidental rather than a vital one. On this principle of analysis, is really 4, ~' ,'the length of the bar being doubled.' It is perfectly proper to point out the resemblance between the two forms of notation, but it can only lead to confusion to explain all the' subtler effects of one time in terms of another, besides ignoring the differences of subjective expression suggested to the player by the particular notation employed. t This passage is quoted by Corder (Grove's Dictionary, Article Syncofjation) as an example of syncopation, but see note f on p. 369. C. F. Abdy Williams ('The rhythm of modern music '), Macmillan, 1909, p. 98), also cites (-= )asanexampleof syncopa- tion, and gives (p. 97) the following definition: 'An accented note is cut short, or is represented by a short rest, and the succeeding note, entering before its expected time, is made longer than the shortened accented note or rest.' It will be evident, however, if we compare examples (a), (b) and (c), Ex. i8a. _(c) - that (b) and (c) are much more akin, by reason of tIke accented notes ending on te strong/3eat ofeack bar, than (a) and (c), where we find the note on the strong beat sounded in (a) but silent in (c). This being the case, it seems less confusing to consider (a) merely as an example of emphasis in triple time rather than as a special kind of syncopation in triple time that still kcas to be dziferentiated from (b). Ex. i8a. _(c) - But this conversion should rather be I d . J& &c. Moreover, the resemblance is an accidental rather than a vital one. On this principle of analysis, is really But this conversion should rather be I d . J& &c. This content downloaded from 150.131.192.151 on Mon, 15 Feb 2016 08:28:04 UTC All use subject to JSTOR Terms and Conditions . h.,_ S__ , 3? (h.S 3.) Model 7. In quadruple time the scheme of this will be: r W 3. 3. W 3. h-S 3. (W 3. S 3, &c.) Model 8. S 3.11 W 3. (S 3. W 3.) 1 I1 :/l~ l ll~? ~ 11~~ BEETHOVEN. 4th Symphony, Ist movement. Ex. 22. S 3. W3. S3. W3. S3. W3. cres. cre - - sce - - do. q y ( ) p g p > > > > > S 2t. W 2. W 2. (S 2. W 2, &c.) Model I. J 21 ,IIJ J 1c See Ex. 3r. W 2, W 2. S 2. (W 2. W 2, &c.) Model 2. 1 > > > "&c See Exs. 28 and 29. W 2. S 2. W 2. (W 2. S 2, &c.) Model 3. 1 &c. See Ex. 34 In the following example we have yet another nuance applicable to this whole class of effects-the nuance of cross motive-accents already mentioned The music suggests the phrasing scheme just quoted (Model 8), and the accents are made to contradict it, in this case synchronising with the natural accents. It must also be pointed out that as no phrasing-accents are appended to Exs. 20, 21 and 22, it rests with the interpreter whether he adopts what are here termed motive-accents given above each example, or elects to contradict the implied phrasing scheme by the use of cross motive-accents, such as is expressly called for in Ex. 23. The normal motive-accents are appended in brackets: See Ex. 34. (b) Duple and quadruple time: Model 4. S 3. W 3 h-S 3. W 3. S 3. W 3, &c.) CLEMENTI. Sonata, Op. 36, No. 3. Ex. 20. W? 3 3 SCHUMANN. 'Von Schlaraffenland ' Op 79 No 5 SCHUMANN. 'Von Schlaraffenland,' Op. 79, No. 5. Ex. z3-? (S 3.) (w 3.) (s 3.) sf ( w- s.)s (W 3.) sJP II In quadruple time, S 6, h-S 6, &c. ? Prout says of this example (' Musical Form,'Augener, ? 291): 'Here the first six bars are in reality four bars of f: time, and might be written : Ex. 23a. sfP As this is the beginning of the song, there can be no feeling of cross- accent here, because the regular accent of time has never been established. This content downloaded from 150.131.192.151 on Mon, 15 Feb 2016 08:28:04 UTC All use subject to JSTOR Terms and Conditions The position of the cadences in the second and fifth bars would also be wrong were the time here really duple.' But see noteon P. 370. Of these various terms tempo d'imbroglio seems the best. Tempo rubato is already used in another sense; temjo della confusione, 'false accents' and 'cross-accents' seem less happy. The peculiar feature about tem15o d' imbroglio is that it is the melodic motives that are 'cross' with the natural accents, and it is quite possible to have, by a further nuance, the phrasing-accents 'cross' with the motives (see Ex. 23). Under these circlumstances it seems better to term the normal phrasing-accents of tempo d' imbroglio 'motive-accents,' and to use the expression 'cross motive-accents' for those phrasing accents that, as in Ex. 23, supplant the expected motive-accents, and in consequence seem generally to synchronise with the natural accents. The melodic motives of tempo d' imbroglio will be called 'cross- motives.' As this is the beginning of the song, there can be no feeling of cross- accent here, because the regular accent of * time has never been established. The position of the cadences in the second and fifth bars would also be wrong were the time here really duple.' But see noteon P. 370. This content downloaded from 150.131.192.151 on Mon, 15 Feb 2016 08:28:04 UTC All use subject to JSTOR Terms and Conditions This content downloaded from 150.131.192.151 on Mon, 15 Feb 2016 08:28:04 UTC All use subject to JSTOR Terms and Conditions THE MUSICAL TIMES.-JUNE I, 1912 37I Model 5. In quadruple time the scheme of this will be : weak beats, or on the weak portions of divided beats, as in the above examples, or on a series of strong and weak beats as in Ex. I9: weak beats, or on the weak portions of divided beats, as in the above examples, or on a series of strong and weak beats as in Ex. I9: W 3, h.S 3, W 3, S3, (W 3, h-S 3, &c.) Model 6. h-S 3. w 3. s 3. w 3. (h-S 3, &c.) BEETHOVEN. Sonata in F, Op. 5, No I. Ex. 21. W3. S3. 3 . h.S 3. . h.,_ S__ , 3? (h.S 3.) g I9: Ex. 9. SCHUMANN. 'Carnaval,' Op. 9. . ... . . . .. l []--- -- - are placed on both strong and weak beats at certain definite intervals, and when we examine the thematic construction of such passages we find that in every case the melodic outline would seem to fit more naturally into bars with a different time- signature than that which is assigned to them. This is of course the familiar device known by the names of 'cross-accents' or 'false-accents,' tempo ruzbato, tempo d' imbroglio, or tempo della confusione For the sake of greater clearness I give skeleton phrasing models of the different varieties, and begin with triple time by reason of its greater frequency. (a) Tempo d' imbroglio in triple time: are placed on both strong and weak beats at certain definite intervals, and when we examine the thematic construction of such passages we find that in every case the melodic outline would seem to fit more naturally into bars with a different time- signature than that which is assigned to them. This is of course the familiar device known by the names of 'cross-accents' or 'false-accents,' tempo ruzbato, tempo d' imbroglio, or tempo della confusione. For the sake of greater clearness I give skeleton phrasing models of the different varieties, and begin with triple time by reason of its greater frequency. (a) Tempod' imbroglioin tripletime: BEETHOVEN. Sonata in F, Op. 5, No I. o * BEETHOVEN. Sonata in F, Op. 5, No I. Ex. 21. W3. S3. 3 . h.S 3. * It will be clear on a moment's consideration that the effect of syncopation will also be obtained, if a note instead of being sustained throughout, under the conditions proper to syncopation, is shortened of its length and a corresponding rest is substituted. Model 9. h-S 6. S 6. (or W 3. S 3.) 3.) In compound duple ( ) the phrasing accents admit of a three-fold treatment, owing to the fact that the six crochets are susceptible to a dual grouping : Hence we get the further models for tempo d' imbroglioin tripletime: g p Model i8. S ?. W 4. Model 9. ~ W i. S z. Model 2o. S W 2. W 2. Model 2l. W 2~. W 2. S 2. Model 22. W 2. S 2. W 2. and In the first place (corresponding with Model 8 in simpledupletime)we get: p ( p g simple duple time) we get : Model io. S 9. h-S 9. (S 9, &c.) =z~ = _I I1 rr' iir Iit Ii ac. and Model ii. h-S 9. S 9. (h-S 9, &c.) Then, corresponding with (b) above, we get: s 4. W4. W4. (S 4. W 4, &c.) Model I2. > > Model 3. W 4. W 4- S 4, &c. and Model I4. W 4. S 4. W 4, &c. Finally, corresponding with (a) above, we get : S 2. W2. W 2. S 2. W2. W 2., &c. Model I5. i 4 =I > : Ex. 24. WEBER. 'Concertstiick.' 2. W2 W2 2 W2 W 2. 8va .................................................................. S2. W2. W2. S 2. W2. W2. 8va 8 v a ....... ........ ...... ......-... .................. .................. ................. >_~ >5 .>_ > Variants of this type will be, of course : Model z6. W 2. W 2. S 2, &c., and Model 7. W 2. S 2. WV 2, &c. f h simple duple time) we get : Model io. S 9. h-S 9. (S 9, &c.) =z~ = _I I1 rr' iir Iit Ii ac. and Model ii. h-S 9. S 9. (h-S 9, &c.) Then, corresponding with (b) above, we get: s 4. W4. W4. (S 4. W 4, &c.) Model I2. > > Model 3. W 4. W 4- S 4, &c. and Model I4. W 4. S 4. W 4, &c. Finally, corresponding with (a) above, we get : S 2. W2. W 2. S 2. W2. W 2., &c. Model I5. i 4 =I > : Ex. 24. WEBER. 'Concertstiick.' 2. W2 W2 2 W2 W 2. 8va .................................................................. S2. W2. W2. S 2. W2. W2. 8va 8 v a ....... ........ ...... ......-... .................. .................. ................. z _I I1 rr' iir Iit Ii ac. and Model ii. h-S 9. S 9. (h-S 9, &c.) Then, corresponding with (b) above, we get: e g examples triple appended Syn 2. W 2. S 2. (Syn 2. W 2, &c.) Model 23. I > : L " r re a?(is ac. SCHUMANN. Op. 21, NO. 4. Syn 2. W 2. S 2. Syn 2. Ex 25a._ BEETHOVEN. 3rd Symphony, ist movement. Syn2. W2. S2. Syn2. W2. S2. Syn2. Ex. 25. sf s s __ .f S f--, CS --_ ?-, -- -C. ,z. W 2. S2. Syn 2. (W 2. S 2. Syn 2, &c.) 1Model 24. > > > > oSCHUMANN, ' Carnaval,' Op. 9. W2. S 2. Syn 2. W 2. S 2. Syn 2. W 2. S 2. Syn 2. Ex. 26. ' ~ ~ ~ ? I-) L_ - S 2. Syn 2. W 2. (S2. Syn 2. W 2, &c.) Model 25. S . 3 g d d g gc. >- > >STEFFANI. Ex. 27.* S 2. Syn 2. W 2. Nb, nb, nb, mai nol di - ro . In the next article we will consider the mental effect intended by the employment of tempo d' imbroglio. ( b d ) Variants of this type will be, of course : A parallel grouping of the six half-beats in triple time gives the following var:eties of phrasing effects : ( )I ( )--- (a)I ! . In the next article we will consider the mental effect intended by the employment of tempo d' imbroglio. . In the next article we will consider the mental effect intended by the employment of tempo d' imbroglio. * Prout ;emarks of this effect (' Musical Form,' Augener, ? 290), that it is ' analogous to the introduction of triplets in common time, e.g., instead of ,--- 7 -- --- - - g- - __ - -we have __ - A glance at M d l ll k l h h f th * Prout ;emarks of this effect (' Musical Form,' Augener, ? 290), that it is ' analogous to the introduction of triplets in common time, e.g., instead of ,--- 7 -- --- - - g- - __ - -we have __ - A glance at (To be continued.) Model x5 will make it clear that the grouping of the crotchets actually does amount to the division of compound duple time into triplets. This content downloaded from 150.131.192.151 on Mon, 15 Feb 2016 08:28:04 UTC All use subject to JSTOR Terms and Conditions This content downloaded from 150.131.192.151 on Mon, 15 Feb 2016 08:28:04 UTC All use subject to JSTOR Terms and Conditions THE MUSICAL TIMES.--JUNE I, 1912. 372 and Hence we get the further models for tempo d' imbroglioin tripletime: Model 9. h-S 6. S 6. (or W 3. S 3.) Model 9. h-S 6. S 6. (or W 3. S 3.) >_~ >5 .>_ > Variants of this type will be, of course : Model z6. W 2. W 2. S 2, &c., and Model 7. W 2. S 2. WV 2, &c. A parallelgroupingofthesixh lf b tin triple simple duple time) we get Model io. S 9. h-S 9. (S 9, &c.) =z~ = _I I1 rr' iir Iit Ii ac. and Model ii. h-S 9. S 9. (h-S 9, &c.) Then, corresponding with (b) above, we get: s 4. W4. W4. (S 4. W 4, &c.) Model I2. > > Model 3. W 4. W 4- S 4, &c. and Model I4. W 4. S 4. W 4, &c. Finally, corresponding with (a) above, we get : S 2. W2. W 2. S 2. W2. W 2., &c. Model I5. i 4 =I > : Ex. 24*. WEBER. 'Concertstiick.' 2. W2 W2 2 W2 W 2. 8va .................................................................. S2. W2. W2. S 2. W2. W2. 8va 8 v a ....... ........ ...... ......-... .................. .................. ................. >_~ >5 .>_ > Variants of this type will be, of course : Model z6. W 2. W 2. S 2, &c., and Model 7. W 2. S 2. WV 2, &c. A parallel grouping of the six half-beats in triple th f h The above list is by no means exhaustive of the possibilities of emphasis in the service of tempo d' imnbroglizo, but the theorist is well-advised not to cite too many theoretic schemes without the corresponding musical examples. There is, The above list is by no means exhaustive of the possibilities of emphasis in the service of tempo d' imnbroglizo, but the theorist is well-advised not to cite too many theoretic schemes without the corresponding musical examples. There is, however, still one more recognised variety of tempo d' imbroglio to be mentioned-that obtained by a combination of syncopation and emphasis. Three examplesin tripletime are appended: =z~ = _I I1 rr' iir Iit Ii ac. and This content downloaded from 150.131.192.151 on Mon, 15 Feb 2016 08:28:04 UTC All use subject to JSTOR Terms and Conditions This content downloaded from 150.131.192.151 on Mon, 15 Feb 2016 08:28:04 UTC All use subject to JSTOR Terms and Conditions This content downloaded from 150.131.192.151 on Mon, 15 Feb 2016 08:28:04 UTC All use subject to JSTOR Terms and Conditions
https://openalex.org/W4396588829	https://www.frontiersin.org/articles/10.3389/fcimb.2024.1386483/pdf?isPublishedV2=False	English	null	Effect of Ducrosia anethifolia methanol extract against methicillin resistant Staphylococcus aureus and Pseudomonas aeruginosa biofilms on excision wound in diabetic mice	Frontiers in cellular and infection microbiology	2,024	cc-by	9,440	OPEN ACCESS OPEN ACCESS EDITED BY Nayeem Ahmad, Arabian Gulf University, Bahrain REVIEWED BY Azna Zuberi, Northwestern University, United States Sanjay Kumar, Sharda University, India Farah Rehan, Arabian Gulf University, Bahrain CORRESPONDENCE Yasir Almuhanna yalmuhanna@su.edu.sa RECEIVED 15 February 2024 ACCEPTED 11 April 2024 PUBLISHED 02 May 2024 CITATION Almuhanna Y (2024) Effect of Ducrosia anethifolia methanol extract against methicillin resistant Staphylococcus aureus and Pseudomonas aeruginosa bioﬁlms on excision wound in diabetic mice. Front. Cell. Infect. Microbiol. 14:1386483. doi: 10.3389/fcimb.2024.1386483 EDITED BY Nayeem Ahmad, Arabian Gulf University, Bahrain REVIEWED BY Azna Zuberi, Northwestern University, United States Sanjay Kumar, Sharda University, India Farah Rehan, Arabian Gulf University, Bahrain Yasir Almuhanna Department of Clinical Laboratory Science, College of Applied Medical Sciences, Shaqra University, Shaqra, Saudi Arabia C O Almuhanna Y (2024) Effect of Ducrosia anethifolia methanol extract against methicillin resistant Staphylococcus aureus and Pseudomonas aeruginosa bioﬁlms on excision wound in diabetic mice. Front. Cell. Infect. Microbiol. 14:1386483. doi: 10.3389/fcimb.2024.1386483 Background: Ducrosia anethifolia is an aromatic desert plant used in Saudi folk medicine to treat skin infections. It is widely found in Middle Eastern countries. Methods: A methanolic extract of the plant was prepared, and its phytoconstituents were determined using LC-MS. In-vitro and in-vivo antibacterial and antibioﬁlm activities of the methanolic extract were evaluated against multidrug-resistant bacteria. The cytotoxic effect was assessed using HaCaT cell lines in-vitro. Diabetic mice were used to study the in-vivo antibioﬁlm and wound healing activity using the excision wound method. © 2024 Almuhanna. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Results: More than 50 phytoconstituents were found in the extract after LC-MS analysis. The extract exhibited antibacterial activity against both the tested pathogens. The extract was free of irritant effects on mice skin, and no cytotoxicity was observed on HaCaT cells with an IC50 value of 1381 µg/ml. The ointment formulation of the extract increased the healing of diabetic wounds. The microbial load of both pathogens in the wounded tissue was also reduced after the treatment. The extract was more effective against methicillin- resistant Staphylococcus aureus (MRSA) than MDR-P. TYPE Original Research PUBLISHED 02 May 2024 DOI 10.3389/fcimb.2024.1386483 TYPE Original Research PUBLISHED 02 May 2024 DOI 10.3389/fcimb.2024.1386483 TYPE Original Research PUBLISHED 02 May 2024 DOI 10.3389/fcimb.2024.1386483 TYPE Original Research PUBLISHED 02 May 2024 DOI 10.3389/fcimb.2024.1386483 KEYWORDS Frontiers in Cellular and Infection Microbiology Effect of Ducrosia anethifolia methanol extract against methicillin resistant Staphylococcus aureus and Pseudomonas aeruginosa bioﬁlms on excision wound in diabetic mice Effect of Ducrosia anethifolia methanol extract against methicillin resistant Staphylococcus aureus and Pseudomonas aeruginosa bioﬁlms on excision wound in diabetic mice 1 Introduction grata, and Clematis viticella, are reported to inhibit P. aeruginosa bioﬁlms (Alam et al., 2020). Similarly, African medical plants such as Alchornea laxiﬂora, Ficus exasperata, Morinda lucida, Jatropha gossypiifolia, Ocimum gratissimum, and Acalypha wilkesiana were shown to inhibit bioﬁlm formation by various pathogens (Olawuwo et al., 2022). Medical plants from Argentina, such as Lycium chilense and Schinus fasciculatus, have also been reported for anti-bioﬁlm effects against various pathogens (Romero et al., 2016). Most of the studies on the antibioﬁlm activities of plant products have been carried out using in-vitro methods that do not provide sufﬁcient evidence that these plants will be effective antibioﬁlm agents in vivo (Lu et al., 2021; Younis et al., 2021; Priyanto et al., 2022). Furthermore, phytoconstituents present in some of the extracts are not known (Alam et al., 2020; Zammuto et al., 2022). The active chemical constituents present in the plant extracts help in the development of novel molecules (Harikrishnan et al., 2021; Oselusi et al., 2021). Medicinal plants with potent antimicrobial effects are used traditionally in Middle Eastern countries (Ullah et al., 2020). Earlier reports show that Saudi medicinal plants have good antibacterial and anti-inﬂammatory effects and are widely used in traditional medicine to treat infections and wounds (Shahat et al., 2017; El-Seedi et al., 2022). However, these plants have not been explored for their antimicrobial effects, especially against multidrug-resistant pathogenic infections and bioﬁlm formation. One of the plants commonly used in the Kingdom of Saudi Arabia for wound treatment is the leaves of Ducrosia anethifolia Bois, belonging to the family- Apiaceae (Flora of Saudi Arabia by Ahmed Mohammed Migahid \| Open Library). The plant is also used to treat skin infections in several other countries, including Afghanistan, Pakistan, Iran, Iraq, and other Arabian countries (Mottaghipisheh et al., 2020). It is locally called ‘Al-Haza’ in Arabic and is a desert plant that grows in Saudi Arabia’s volcanic cinders. This plant is a biennial herb and is drought-resistant. Earlier reports show that the plant possesses different pharmacological effects. Some of the activities reported include anti-diabetic and antiulcer effects (Unissa Syed et al., 2022), analgesic, central nervous system depressant actions such as anti- anxiety, sedative, and anti-depressant effects (Abbaszadeh et al., 2019), carminative, relief of colic pain and as a ﬂavoring agent (Mottaghipisheh et al., 2020). Further there are reports on phytoconstituents present in Ducrosia anethifolia showing antibacterial activity against MRSA (Mahboubi et al., 2014). 2.2 Animals Swiss albino mice (27 to 30 g) maintained under a controlled environment were utilized. The experimental procedure was approved by the Ethical Research Committee of Shaqra University (No. ERC SU_20220066). OPEN ACCESS aeruginosa in both in vitro and in vivo experiments. Further, skin regeneration was also observed in histological studies. Conclusions: The results showed that D. anethifolia methanol extract supports wound healing in infected wounds in diabetic mice through antibacterial, antibioﬁlm, and wound healing activities. LCMS analysis, cytotoxicity, epithelization, HaCaT (human keratinocyte), skin irritation 01 frontiersin.org Frontiers in Cellular and Infection Microbiology 10.3389/fcimb.2024.1386483 Almuhanna grata, and Clematis viticella, are reported to inhibit P. aeruginosa bioﬁlms (Alam et al., 2020). Similarly, African medical plants such as Alchornea laxiﬂora, Ficus exasperata, Morinda lucida, Jatropha gossypiifolia, Ocimum gratissimum, and Acalypha wilkesiana were shown to inhibit bioﬁlm formation by various pathogens (Olawuwo et al., 2022). Medical plants from Argentina, such as Lycium chilense and Schinus fasciculatus, have also been reported for anti-bioﬁlm effects against various pathogens (Romero et al., 2016). Most of the studies on the antibioﬁlm activities of plant products have been carried out using in-vitro methods that do not provide sufﬁcient evidence that these plants will be effective antibioﬁlm agents in vivo (Lu et al., 2021; Younis et al., 2021; Priyanto et al., 2022). Furthermore, phytoconstituents present in some of the extracts are not known (Alam et al., 2020; Zammuto et al., 2022). The active chemical constituents present in the plant extracts help in the development of novel molecules (Harikrishnan et al., 2021; Oselusi et al., 2021). Frontiers in Cellular and Infection Microbiology 2.1 Chemicals Chemicals of analytical grade purchased from local chemical suppliers were used. 1 Introduction The present study evaluated the unexplored antimicrobial, antibioﬁlm, and wound healing of Ducrosia anethifolia to conﬁrm its traditional use as an anti-infective agent on skin wounds in diabetic animals. Furthermore, an attempt was made to identify phytoconstituents present in the methanolic extract of the leaves through liquid chromatography-mass spectrometry (LC-MS) analysis that may help in the identiﬁcation of lead molecules. The skin irritant effect of the prepared extract formulation was evaluated on the mouse skin in-vivo and on human keratinocytes (HaCaT) in- vitro to determine the safety. Infections in wounds are prevalent due to exposure of wounded tissue to bacteria. The infectious organism usually forms a bioﬁlm over the wounded tissue within 24 hours to escape the attack from the patient’s immune system and attenuate the effect of antimicrobial agents. Bioﬁlms are bacteria aggregates embedded in a barrier consisting of sugars and proteins (Flemming et al., 2016). These are considered the single most common cause of delay in wound healing, and they delay the wound healing process through an inappropriate inﬂammatory response that damages the wounded tissue (Darvishi et al., 2022). Hence, agents used in the treatment of wounds should not only possess antimicrobial effects but should effectively prevent and eradicate bioﬁlm formation over the wounded tissues (Thapa et al., 2023). The two most common pathogens causing skin infections include Methicillin-resistant Staphylococcus aureus (MRSA) and multi-drug-resistant- Pseudomonas aeruginosa (MDR- P. aeruginosa). MRSA is associated with community-acquired skin and soft tissue infections as well as nosocomial infections (Odell, 2010; Pannewick et al., 2021). Furthermore, there are earlier reports on the effect of essential oils and decanal, a component of D. anethifolia against MRSA, wherein it was shown that more than one phytoconstituent of D. anethifolia is responsible for its antimicrobial effect (Mahboubi and Feizabadi, 2009). MDR-P. aeruginosa is one of the most common infective organisms for skin and soft tissue infections (Wu et al., 2011). An earlier study indicates that hydroalcoholic extract of D. anethifolia from Jordan inhibits P. aeruginosa in-vitro (Nawash et al., 2013). frontiersin.org 2.3 Extract preparation and phytochemical analysis The herb was collected in August 2022, followed by authentication in the institute by a botanist. A specimen of the herb (No. SU/CAMS/09/2022) is maintained in the institute as a reference. The plant was shade-dried, coarsely powdered, subjected to Soxhlet extraction using methanol, and dried in a rotavapor (Mukherjee, 2019). The extract yield obtained was 26.34% w/w. The extract was injected into the waters LC instrument (XEVO- TQD#QCA1232) having a C18 column (250 mm X 2.1 mm, 2.6 µm). Many plant extracts have been reported for antibioﬁlm effects. Traditional plants from Pakistan, such as Bergenia ciliata, Clematis 02 frontiersin.org Almuhanna 10.3389/fcimb.2024.1386483 The ﬂow rate was maintained at 0.2 ml/min, and detection was carried out at 280 nm. Acetonitrile and ammonium formate buffer were used as solvents with gradient conditions as reported by Al- Ghanayem et al (Al-Ghanayem et al., 2022a). The spectra were recorded at ionization modes from m/z 150 to 2000. animals. Group I was an untreated control, while group II was applied with the emulsifying base. The extract ointment at 5% w/w and 10% w/w was applied to animals of groups III and IV, and the last group received the local application of mupirocin 2% or gentamicin 0.1%. In six animals from each group, the wounded area was measured every 4th day for 20 days, and these animals were sacriﬁced to determine the bacterial count (CFU/g). Tissues from these animals were also subjected to histological examination by ﬁxing them in neutral formalin. Sections were stained using H and E stain, and skin epithelium regeneration was observed under 200X using a microscope (Leica DM 2500) with a camera (DFC 295). The epithelization period was monitored in the remaining six animals, which indicated complete healing of the wounds. 2.7 Cytotoxic assay on HaCaT cell lines The SRB assay was used to determine the cytotoxicity of the extract (Denzinger et al., 2022). The HaCaT cells were grown in 96- well plates in Dulbecco’s Modiﬁed Eagle’s Medium supplemented with fetal bovine serum (10%), and antibiotic (1%) at 37°C with 5% CO2. Next day, extract prepared in an incomplete medium at different concentrations starting from 1 µg/ml to 1000 µg/ml was added, followed by 24 h incubation. Trichloroacetic acid - 10% (100 µl) was added, followed by incubation for another 1 h. The cells were washed in distilled water and dried, followed by the addition of sulforhodamine solution (ﬁnal concentration of 0.04%) and incubation for 1 h. Following this, the cells were washed with acetic acid (1% v/v) and Tris base solution (pH=10.5) was added. This was shaken on an orbital shaker to solubilize the protein- bound dye. The optical density was read at 510 nm in an ELISA plate reader. 2.8 Statistical analysis Mean ± SEM values were used for comparison, and one-way ANOVA followed by Tukey’s test was used to determine the level of signiﬁcance. Instat software was used for statistical analysis (GraphPad Prism version 6.04 for Windows). 3.1 Phytochemical analysis This was done using a method standardized in our laboratory (Alrouji et al., 2023). Streptozocin and nicotinamide were used to induce diabetes (Yan, 2022). Mice were considered diabetic if the fasting blood sugar level exceeded 150 mg/dL. A coverslip containing bioﬁlm formed by the bacteria that was conﬁrmed by crystal violet assay (Mohamed et al., 2014) was applied to the excision wounds under anesthesia (Anesthesia (Guideline) \| Vertebrate Animal Research). The bioﬁlm formation was conﬁrmed after 72 h by carefully removing and examining the thin bioﬁlm layer that developed on the wounded tissue. The animals were then divided into two groups, one each for MRSA and MDR-P. aeruginosa, with ﬁve subgroups containing twelve The methanolic extract of D. anethifolia showed the presence of a large number of phytoconstituents in LC-MS analysis (Figures 1, 2). In the positive (Table 1) and negative (Table 2) modes, 14 and 37 suspected molecules were identiﬁed, respectively. 2.4 Antibacterial activity and antibioﬁlm activity in-vitro Antibacterial effects of the extract were carried out against MRSA and MDR-P. aeruginosa using conventional methods to detect the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) (Ekom et al., 2022). The pathogens (106 CFU/mL) were inoculated into Luria Bertani (LB) broth, and the antibioﬁlm effect was determined using the crystal violet binding assay (O’Toole, 2011). Different extract concentrations, starting from 6.25 µg/ml up to 400 µg/ml in geometrical dilution along with bacterial culture, were added to each well of the microtire plate followed by incubation at 37 °C for 24 h. The planktonic cells were discarded, and crystal violet (20 mL) was added to the wells and allowed to stain for 15 min. The excess stain was removed, rinsed with potassium phosphate buffer (10 mM), and dried. Ethanol (96% v/v) was added to the wells to solubilize the crystal violet, and the optical density was read at 570 nm. 2.5 Ointment formulation and skin irritation test The D. anethifolia extract formulation at two different concentrations was prepared (5% w/w and 10% w/w) employing liquid parafﬁn, emulsifying wax, and soft parafﬁn by fusion method (Nayeem et al., 2008). All the constituents of the ointment base were melted and mixed with the extract with constant stirring to obtain a uniform ointment. The physicochemical characteristics of the ointment formulation were evaluated (Kolhe et al., 2018). The formulation was applied on the mouse skin for irritation test and observed every 12 h until 72 h. Frontiers in Cellular and Infection Microbiology 3.2 Antibacterial and antibioﬁlm activity The minimum inhibitory concentration was 256 µg/ml for MRSA and 512 µg/ml for MDR-P. aeruginosa. The minimum 03 frontiersin.org frontiersin.org Almuhanna 10.3389/fcimb.2024.1386483 FIGURE 1 Chromatogram of D. anethifolia methanolic extract in positive mode. Retention times are shown in X axis and the base peak intensity of major peaks are marked. FIGURE 1 Chromatogram of D. anethifolia methanolic extract in positive mode. Retention times are shown in X axis and the base peak intensity of major peaks are marked. FIGURE 1 Chromatogram of D. anethifolia methanolic extract in positive mode. Retention times are shown in X axis and the base peak intensity of major peaks are marked 3.3 Physicochemical properties and skin irritation test bactericidal concentration was 512 µg/ml for MRSA and 1024 µg/ml for MDR-P. aeruginosa. A concentration of 50 µg/ml exhibited signiﬁcant antibioﬁlm activity against MRSA while MDR-P. aeruginosa bioﬁlm formation was signiﬁcantly affected at 100 µg/ ml, and these effects were concentration-dependent (Figure 3). The ointment formulation was homogenous with excellent stability and diffusion. The spreadability was 10 seconds with a FIGURE 2 Chromatogram of D. anethifolia methanolic extract in negative mode. Retention times are shown in X axis and the base peak intensity of major peaks are marked. FIGURE 2 Chromatogram of D. anethifolia methanolic extract in negative mode. Retention times are shown in X axis and the base peak intensity of major peaks are marked. 04 Frontiers in Cellular and Infection Microbiology frontiersin.org 10.3389/fcimb.2024.1386483 Almuhanna TABLE 1 List of suspected molecules identiﬁed in D. anethifolia methanolic extract in positive mode. TABLE 1 List of suspected molecules identiﬁed in D. anethifolia methanolic extract in positive mode. S.No. R.Time Score Compound Name Formula Exact Mass Observed Mass Mass Diff 1. 1.27 0.978 1,10-Phenanthroline monohydrate C12H8N2 180.068 179.1104 0.96 2. 6.29 0.957 Adenosine C10H13N5O4 267.096 263.1652 3.93 3. 10.32 0.979 D-erythro-Dihydrosphingosine C18H39NO2 301.298 305.1769 -3.88 4. 14.95 0.935 Scoulerin C19H21NO4 327.147 323.1966 3.95 5. 15.36 0.928 Methyl Jasmonate C13H20O3 224.141 224.1574 -0.02 6. 17.72 0.934 DL-Dihydrozeatin C10H15N5O 221.127 224.1236 -3 7. 18.16 0.978 Etidronic acid C2H8O7P2 205.974 203.0679 2.91 8. 18.50 0.975 L-Carnosine C9H14N4O3 226.23 229.1514 -2.92 9. 19.42 0.592 1-Isothiocyanato-8-(methylsulﬁnyl)-octane C10H19NOS2 233.09 235.1915 -2.1 10. 19.66 0.676 Melatonin C13H16N2O2 232.121 235.2590 -3.14 11. 23.10 0.902 Riboﬂavin-5′-monophosphate sodium salt hydrate C17H21N4O9P 456.104 459.2601 -3.16 12. 23.17 0.887 peonidin-3-o-beta-d-glucopyranoside C22H23O11 463.124 459.3276 3.8 13. 23.72 0.685 Hydroxypyruvic acid dimethyl ketal phosphate tri (cyclohexylammonium) salt C5H11O8P 230.019 329.2370 -99.22 14. 27.91 0.767 n-Butyryl coenzyme A lithium salt hydrate C25H42N7O17P3S 837.157 834.7177 2.44 diffusion of 0.6 cm. The prepared ointment was stable at 24°C, 37°C and 40°C. Extract formulation, when applied on intact skin, showed no obvious irritation or inﬂammation for 72 h. the wounded tissue (Table 3). Skin sections obtained from animals receiving different treatments showed various degrees of skin regeneration. The skin damage was more in the MDR-P. aeruginosa infected control animals compared to MRSA-infected control animals, indicating severe skin damage due to Gram-negative MDR-P. aeruginosa as compared to Gram-positive MRSA. Similarly, skin regeneration after treatment with antibiotic or D. anethifolia extract ointment was noticeably more in MRSA-infected animals than MDR-P. 3.4 Antibioﬁlm and wound healing effects The D. anethifolia ointment formation improved the healing of wounds in diabetic mice. The extract formulation (10% w/w) signiﬁcantly supported wound healing from the 8th day onwards in MRSA-induced bioﬁlm wounds. However, the lower concentration of the extract formulation (5% w/w) showed a signiﬁcant wound- healing effect from the 12th day. The antibiotic mupirocin signiﬁcantly affected wound contraction from the 4th day. There was no signiﬁcant difference in the infected wound in animals that did not receive any treatment and the base-treated wounds, indicating that the base is inert (Figure 4). The epithelization period was signiﬁcantly reduced in low (5% w/w) and high (10% w/w) concentration extract-treated groups compared to the control. As expected, the epithelization period was signiﬁcantly less in the antibiotic-treated group than in the base-treated control group (Figure 5). These effects were similar in MDR-P. aeruginosa induced bioﬁlm wounds, but the effect of the extract was noticeably less than that observed with MRSA-infected wounds (Figures 6, 7). The microbial load in the wounded tissue after 20 days of treatment was reduced after treatment with both concentrations of D. anethifolia extract ointment in case of MRSA-infected wounds. However, in MDR-P. aeruginosa infected wounds, there was a signiﬁcant decrease only in wounds treated with the high concentration of D. anethifolia extract ointment (10% w/w). Antibiotic treatments signiﬁcantly reduced the microbial load in 3.3 Physicochemical properties and skin irritation test aeruginosa infected animals (Figure 8). 3.5 Effect on HaCaT cells in-vitro Ducrosia anethifolia did not induce signiﬁcant toxicity to the HaCaT cell lines in-vitro, as indicated by a high IC50 value of 1381 µg/ml (Figure 9). The extract was tested up to a concentration of 1000 µg/ml, and a signiﬁcant reduction in cell viability was observed at 500 µg/ml. Frontiers in Cellular and Infection Microbiology 4 Discussion Ducrosia anethifolia is traditionally used in different regions of the world for the treatment of skin infections and pain relief (Mottaghipisheh et al., 2020). The current study was undertaken because this herb is widely used in Saudi folk medicine to treat skin infections. The results of the current study supported its traditional use as indicated by its antimicrobial, antibioﬁlm, and wound- healing effects. Though there are earlier reports on the antimicrobial effect of Ducrosia anethifolia, none of these studies determined the antibioﬁlm activity and wound healing effect 05 frontiersin.org 10.3389/fcimb.2024.1386483 Almuhanna TABLE 2 List of suspected molecules identiﬁed in D. anethifolia methanolic extract in negative mode. S.No. R.Time Score Compound Name Formula Exact Mass Observed Mass Mass Diff 1. 1.23 0.757 D(-)-Gulono-gamma-lactone C6H10O6 178.047 181.1687``` 178.05 2. 1.30 0.467 Galactinol Dihydrate C12H22O11 342.116 375.3669 -33.25 3. 1.57 0.752 Chlorogenic acid Hemihydrate C16H18O9 354.095 357.2455 -3.15 4. 1.91 0.938 R-2-hydroxy-3-butenyl glucosinolate (progoitrin) C11H19NO10S2 389.045 389.2364 -0.19 5. 6.17 0.926 Lignoceric Acid C24H48O2 368.365 367.2005 1.16 6. 6.38 0.914 Gluconasturtiin C15H21NO9S2 423.065 423.1850 -0.12 7. 7.26 0.976 Sebacic acid C10H18O4 202.12 201.0433 1.08 8. 7.37 0.799 6-(gamma,gamma-Dimethylallylamino)purine C10H13N5 203.117 201.1445 1.97 9. 10.33 0.694 S-Sulfocysteine C3H7NO5S2 200.976 201.1445 -0.17 10. 13.91 0.759 DL-4-Hydroxy-3-methoxymandelic acid C9H10O5 198.052 201.1445 -3.09 11. 15.38 0.678 Petunidin C16H13O7 317.066 315.1653 1.9 12. 15.69 0.658 zearalenone C18H22O5 318.146 321.2057 -3.06 13. 16.23 0.93 Kaempferol-3-O-alpha-L-rhamnoside C21H20O10 432.105 433.4439 -1.34 14. 16.81 0.804 Sodium Cholate Hydrate C24H40O5 408.57 409.3091 -0.74 15. 17.36 0.816 Sodium gluconate C6H12O7 196.058 199.1874 -3.13 16. 17.43 0.783 Syringic Acid C9H10O5 198.052 199.1874 -1.14 17. 17.77 0.739 Pyridoxal-5’-phosphate hydrate C8H10NO6P 247.024 249.3301 -2.31 18. 19.95 0.679 Uridine-5′-diphosphoglucuronic acid trisodium salt C15H22N2O18P2 580.034 579.5029 0.53 19. 20.60 0.988 6-Phosphogluconic acid Barium salt hydrate C6H13O10P 276.024 277.3377 -1.31 20. 20.70 0.894 Phloridzin C21H24O10 436.136 277.3714 158.76 21. 20.77 0.957 L-saccharopine C11H20N2O6 276.132 277.3714 -1.24 22. 20.97 0.882 2’-Deoxycytidine C9H13N3O4 227.09 277.2955 -50.21 23. 21.04 0.876 L-Carnosine C9H14N4O3 226.106 227.3293 -1.22 24. 21.11 0.801 Sinapic acid C11H12O5 224.068 227.2618 -3.19 25. 22.17 0.892 D-Glucosamine-6-phosphate sodium salt C6H14NO8P 259.045 253.3457 5.7 26. 22.92 0.976 6-Phosphogluconic acid Barium salt hydrate C6H13O10P 276.024 279.3624 -3.34 27. 23.02 0.957 gamma-Linolenic acid C18H30O2 278.43 279.3961 -0.97 28. 23.12 0.735 acacetin C16H12O5 284.068 279.3286 4.74 29. 23.22 0.694 Guanosine-5’-triphosphate sodium salt C10H16N5O14P3 522.99 517.3729 5.62 30. 23.29 0.673 Piperacillin sodium salt C23H27N5O7S 517.163 517.3391 -0.18 31. 4 Discussion All values are mean ± SEM, n=6, **P<0.001 as compared to untreated infected control. (Mottaghipisheh et al., 2020). The traditional use of this herb in skin infection may not be due only to antimicrobial effects, without considerable antibioﬁlm and wound healing properties, which were conﬁrmed in the current study. It is also reported for antibacterial effects (Jeyaraj et al., 2022). These effects help in wound healing. L-carnosine, a dipeptide composed of amino acids, b-alanine, and histidine have been reported for antioxidant, anti-inﬂammatory, and antibacterial actions (Kandhasamy et al., 2021). The anti-inﬂammatory effect may have increased the healing of wounded tissue with the contribution of antioxidant and antibacterial actions that inhibited oxidative stress and microbial load, respectively. Melatonin is a hormone found in both animals and plants. It has potent antioxidant, anti-inﬂammatory, and immunomodulatory properties that aid in the healing of wounds (Ganganna et al., 2021). Ducrosia anethifolia extract was prepared using methanol that extracts several secondary and primary metabolites (Jones et al., 2006). Analysis of the prepared extract using LC-MS revealed the presence of many constituents. Some of the suspected phytoconstituents identiﬁed in the current study have been reported earlier for antimicrobial and antibioﬁlm effects. These phytoconstituents include D-erythro- dihydrosphingosine, petunidin, L-carnosine, and melatonin. D- erythro-dihydrosphingosine is a sphingolipid that has been reported to inhibit the growth of several strains of bacteria by increasing the permeability of the bacterial cell membrane (Shin et al., 2022). Petunidin, an anthocyanidin ﬂavonoid, has a good antioxidant effect. Another important phytoconstituent identiﬁed in the plant was chlorogenic acid. It is a polyphenol found in several plants, including vegetables and fruits. There are several reports on the antibacterial Day 4 Day 8 Day 12 Day 16 Day 20 0 50 100 150 Wound contraction after different treatments Duration Wound contraction (%) Untreated control Pa+ Base Pa + Da (5%) Pa + Da (10%) Pa + GEN (0.1%) * * * * * * * * * * FIGURE 6 Contraction of excision wound infected with P. aeruginosa in mice treated with D. anethifolia methanolic extract and gentamicin. The extract (Da -10% w/w) showed effect from Day 8 onwards while the gentamicin (GEN) was more effective than either concentration of the extract showing signiﬁcant effect from Day 4 onwards. All values are mean ± SEM, n=6, P<0.05, *P<0.001 as compared to untreated infected control. 4 Discussion 25.68 0.975 alpha-D-glucose-1-phosphate dipotassium salt dihydate C6H13O9P 260.029 255.4040 4.63 32. 25.81 0.982 D-Glucose-6-phosphate sodium salt C6H13O9P 260.029 260.03 0.001 33. 25.88 0.966 D-Mannose-6-phosphate barium salt hydrate C6H13O9P 260.029 255.3703 4.66 34. 26.22 0.911 Luteolin C15H10O6 286.047 281.3533 4.69 35. 26.50 0.96 Xanthosine C10H12N4O6 284.075 281.3533 2.72 36. 27.83 0.566 Glycyrrhizin C42H62O16 822.403 815.6823 6.72 37. 27.89 0.485 Glycyrrhizic acid ammonium salt C42H62O16 822.403 815.6823 6.72 Frontiers in Cellular and Infection Microbiology 06 frontiersin.org Almuhanna 10.3389/fcimb.2024.1386483 FIGURE 3 Antibioﬁlm activity of D. anethifolia methanolic extract in crystal violet assay, n= 4 (biological repeats) P<0.05, P0.01, P<0.001 as compared to untreated control. A concentration-dependent anti- bioﬁlm effect was observed. Activity against Gram-positive MRSA was noticeably more compared to that observed against Gram- negative MDR-P. aeruginosa. Untreated control MRSA+base MRSA+ Da(5%) MRSA+ Da (10%) MRSA+MPN (2%) 0 10 20 30 Epithelization period Epithelization period (days) * FIGURE 5 Period of epithelisation in excision wound infected with MRSA after different treatments. The extract (Da -10% w/w) and mupirocin (MPN-2% w/w) showed signiﬁcant action on wound contraction while extract at lower concentration (Da -5% w/w) did not show signiﬁcant effect. All values are mean ± SEM, n=6, **P<0.001 as compared to untreated infected control. FIGURE 3 Antibioﬁlm activity of D. anethifolia methanolic extract in crystal violet assay, n= 4 (biological repeats) P<0.05, P0.01, P<0.001 as compared to untreated control. A concentration-dependent anti- bioﬁlm effect was observed. Activity against Gram-positive MRSA was noticeably more compared to that observed against Gram- negative MDR-P. aeruginosa. Epithelization period Epithelization period (days) FIGURE 3 Antibioﬁlm activity of D. anethifolia methanolic extract in crystal violet assay, n= 4 (biological repeats) P<0.05, P0.01, P<0.001 as compared to untreated control. A concentration-dependent anti- bioﬁlm effect was observed. Activity against Gram-positive MRSA was noticeably more compared to that observed against Gram- negative MDR-P. aeruginosa. FIGURE 3 Antibioﬁlm activity of D. anethifolia methanolic extract in crystal violet assay, n= 4 (biological repeats) P<0.05, P0.01, P<0.001 as compared to untreated control. A concentration-dependent anti- bioﬁlm effect was observed. Activity against Gram-positive MRSA was noticeably more compared to that observed against Gram- negative MDR-P. aeruginosa. FIGURE 5 FIGURE 5 FIGURE 5 Period of epithelisation in excision wound infected with MRSA after different treatments. The extract (Da -10% w/w) and mupirocin (MPN-2% w/w) showed signiﬁcant action on wound contraction while extract at lower concentration (Da -5% w/w) did not show signiﬁcant effect. 4 Discussion Day 4 Day 8 Day 12 Day 16 Day 20 0 50 100 150 Wound contraction after different treatments Duration Wound contraction (%) Untreated control Pa+ Base Pa + Da (5%) Pa + Da (10%) Pa + GEN (0.1%) * * * * * * * * * * FIGURE 6 Day 4 Day 8 Day 12 Day 16 Day 20 0 50 100 150 Wound contraction after different treatments Duration Wound contraction (%) Untreated control MRSA + Base MRSA + Da (5%) MRSA + Da (10%) MRSA + MPN (2%) * * * * * * * ** FIGURE 4 Contraction of excision wound infected with MRSA in mice treated with D. anethifolia. methanolic extract. The extract (Da -10% w/w) showed effect from Day 8 onwards while the mupirocin (MPN) was more effective than either concentration of the extract. All values are mean ± SEM for six animals, P<0.01, *P<0.001 as compared to untreated infected control. Day 4 Day 8 Day 12 Day 16 Day 20 0 50 100 150 Wound contraction after different treatments Duration Wound contraction (%) Untreated control MRSA + Base MRSA + Da (5%) MRSA + Da (10%) MRSA + MPN (2%) * * * * * * * **** FIGURE 4 Contraction of excision wound infected with MRSA in mice treated with D. anethifolia. methanolic extract. The extract (Da -10% w/w) showed effect from Day 8 onwards while the mupirocin (MPN) was more effective than either concentration of the extract. All values are mean ± SEM for six animals, P<0.01, *P<0.001 as compared to untreated infected control. Wound contraction after different treatments Day 4 Day 8 Day 12 Day 16 Day 20 0 50 100 150 Wound contraction after different treatments Duration Wound contraction (%) Untreated control MRSA + Base MRSA + Da (5%) MRSA + Da (10%) MRSA + MPN (2%) * * * * * * * ****** FIGURE 4 Wound contraction after different treatments FIGURE 6 Contraction of excision wound infected with P. aeruginosa in mice treated with D. anethifolia methanolic extract and gentamicin. The extract (Da -10% w/w) showed effect from Day 8 onwards while the gentamicin (GEN) was more effective than either concentration of the extract showing signiﬁcant effect from Day 4 onwards. All values are mean ± SEM, n=6, P<0.05, *P<0.001 as compared to untreated infected control. 4 Discussion Day 4 Day 8 Day 12 Day 16 Day 20 0 50 100 150 Wound contraction after different treatments Duration Wound contraction (%) Untreated control Pa+ Base Pa + Da (5%) Pa + Da (10%) Pa + GEN (0.1%) * * * * * * * * * * FIGURE 6 Contraction of excision wound infected with P. aeruginosa in mice treated with D. anethifolia methanolic extract and gentamicin. The extract (Da -10% w/w) showed effect from Day 8 onwards while the gentamicin (GEN) was more effective than either concentration of the extract showing signiﬁcant effect from Day 4 onwards. All values are mean ± SEM, n=6, P<0.05, *P<0.001 as compared to untreated infected control. Day 4 Day 8 Day 12 Day 16 Day 20 0 50 100 150 Wound contraction after different treatments Duration Wound contraction (%) Untreated control Pa+ Base Pa + Da (5%) Pa + Da (10%) Pa + GEN (0.1%) * * * * * * * * * * FIGURE 6 Contraction of excision wound infected with P. aeruginosa in mice treated with D. anethifolia methanolic extract and gentamicin. The extract (Da -10% w/w) showed effect from Day 8 onwards while the gentamicin (GEN) was more effective than either concentration of the extract showing signiﬁcant effect from Day 4 onwards. All values are mean ± SEM, n=6, P<0.05, P<0.001 as compared to untreated infected control. Day 4 Day 8 Day 12 Day 16 Day 20 0 50 100 150 Wound contraction after different treatments Duration Wound contraction (%) Untreated control MRSA + Base MRSA + Da (5%) MRSA + Da (10%) MRSA + MPN (2%) * * * * * * * **** FIGURE 4 Contraction of excision wound infected with MRSA in mice treated with D. anethifolia. methanolic extract. The extract (Da -10% w/w) showed effect from Day 8 onwards while the mupirocin (MPN) was more effective than either concentration of the extract. All values are mean ± SEM for six animals, P<0.01, *P<0.001 as compared to untreated infected control. 4 Discussion However, this could be due to the place and time of collection of the plant material and method of analysis, as some of these studies were carried out using gas chromatography-mass spectrometry (GC- MS). Many reports are from Iran, which has different weather conditions than Saudi Arabia. Further, the current study was carried out using methanol extract, and there are no earlier reports on the phytochemicals present in the methanolic extract of D. anethifolia. FIGURE 7 Period of epithelisation in excision wound infected with P. aeruginosa after treatment with D. anethifolia extract and gentamicin. The extract (Da -10% w/w) and gentamicin (GEN-0.1% w/w) showed signiﬁcant effect on wound contraction while extract at lower concentration (Da -5% w/w) did not show signiﬁcant effect. All values are mean ± SEM, n=6, P<0.01, **P<0.001 as compared to untreated infected control. effect of chlorogenic acid, and it is reported to inhibit several strains of bacteria, conﬁrming its broad-spectrum antibacterial action (Sun et al., 2020). The extract also showed the presence of kaempferol, which is a known antibacterial agent. It is reported to increase cell membrane permeability, inhibit bacterial enzyme activity, and have a strong antioxidant effect (Periferakis et al., 2022). Similar to kaempferol, syringic acid, and sinapic acid is found in several plant species, and these are known to inhibit bacterial growth by a mechanism similar to kaempferol (Pandi and Kalappan, 2021; Meng et al., 2022). Sodium gluconate is abundantly found in several plants. It is a chelating agent that chelates ions essential for bacterial growth, and there are few reports on the antibacterial effect of this compound (Kapanya et al., 2020). Luteolin is an important ﬂavonoid that is reported for antibacterial activity against a wide Many plants have been reported for antibacterial and wound healing effects in normal and diabetic rats (Al-Ghanayem et al., 2022b; Almuhanna et al., 2023). However, there are very few reports on the in-vivo antibioﬁlm effects of plants and phytoconstituents (Lu et al., 2019). Several plant-based formulations are reported to control infection in diabetic wounds but their efﬁcacy on bioﬁlm is unknown. To overcome antimicrobial resistance, environmental degradation, and pollution, plant-based formulations are becoming safer alternatives for antibiotics and have gained importance in recent. Apart from antibacterial activity, many of the plant components are reported for enhancing ﬁbroblast proliferation, a main step in wound healing (Thakur et al., 2011). 4 Discussion FIGURE 6 Contraction of excision wound infected with P. aeruginosa in mice treated with D. anethifolia methanolic extract and gentamicin. The extract (Da -10% w/w) showed effect from Day 8 onwards while the gentamicin (GEN) was more effective than either concentration of the extract showing signiﬁcant effect from Day 4 onwards. All values are mean ± SEM, n=6, P<0.05, *P<0.001 as compared to untreated infected control. FIGURE 4 Contraction of excision wound infected with MRSA in mice treated with D. anethifolia. methanolic extract. The extract (Da -10% w/w) showed effect from Day 8 onwards while the mupirocin (MPN) was more effective than either concentration of the extract. All values are mean ± SEM for six animals, P<0.01, *P<0.001 as compared to untreated infected control. 07 Frontiers in Cellular and Infection Microbiology frontiersin.org Almuhanna 10.3389/fcimb.2024.1386483 range of both gram-positive and gram-negative bacteria. It also has anti-inﬂammatory and antioxidant properties that help in wound healing (Guo et al., 2020). Glycyrrhizin, commonly found in licorice, was found to be present in D. anethifolia. There are many reports on the broad-spectrum antibacterial effect of glycyrrhizin (Eynde et al., 2023). It also possesses antioxidant, anti-inﬂammatory, and immunomodulatory effects (Feng et al., 2022). Furthermore, glycyrrhizin has been reported to enhance the antibacterial effects of many conventionally used antimicrobial agents (Hazlett et al., 2019). The presence of various phytoconstituents with diverse pharmacological effects that include antioxidant, anti-inﬂammatory, immunomodulatory, and antibacterial effects might have contributed to the overall observed effects. Untreated control Pa+base Pa+ Da(5%) Pa+ Da (10%) Pa+GEN (0.1%) 0 10 20 30 Epithelization period Epithelization period (days) * FIGURE 7 Period of epithelisation in excision wound infected with P. aeruginosa after treatment with D. anethifolia extract and gentamicin. The extract (Da -10% w/w) and gentamicin (GEN-0.1% w/w) showed signiﬁcant effect on wound contraction while extract at lower concentration (Da -5% w/w) did not show signiﬁcant effect. All values are mean ± SEM, n=6, P<0.01, *P<0.001 as compared to untreated infected control. Epithelization period The phytochemical analysis of D. anethifolia has been carried out by several authors in different extracts prepared using different solvents such as aqueous, ethanol, and ethyl acetate. A comparison of the phytoconstituents reported by these authors with those found in this study did not match any of the constituents (Zamyad et al., 2019; Mottaghipisheh et al., 2020; Arabsalehi et al., 2022). The reason for this cannot be explained by the present data. frontiersin.org 4 Discussion In Middle East traditional plants including Ducrosia anethifolia are used as a traditional medicine. Management of wounds in diabetic conditions is a serious concern as pathogens such as MRSA and MDR-P. aeruginosa are resistant to conventionally used antibiotics. Both these pathogens were selected based on literature and as a representative strain from Gram-positive bacteria and Gram-negative bacteria to establish the wide spectrum of activity. The extract showed a more antibacterial effect on MRSA when compared to MDR-P. aeruginosa. Usually, Gram-negative bacteria are more tolerant to phytochemicals and natural compounds compared to Gram-positive bacteria due to the different physiological structures of the cell walls. The lipopolysaccharide layer and periplasmic space of the cell wall help the Gram-negative bacteria to show resistance against natural compounds (Al-Ghanayem et al., 2022b). TABLE 3 Microbial load in the wounded tissue after different treatments for 20 days in infected mice. TABLE 3 Microbial load in the wounded tissue after different treatments for 20 days in infected mice. Group Log10 CFU/g of tissue MRSA P. aeruginosa Untreated control 5.23 ± 0.054 5.38 ± 0.063 Control (base) 5.12 ± 0.086 5.23 ± 0.082 D.anethifolia ointment (5%w/w) 3.25 ± 0.092* 5.09 ± 0.085NS D.anethifolia ointment (10%w/w) 2.04 ± 0.024* 4.78 ± 0.092 #Antibiotic 1.24 ± 0.046* 1.82 ± 0.054* #Antibiotic-mupirocin (2%) for the MRSA-infected group and gentamicin (0.1%) for P. aeruginosa-infected group. Data are mean ± SEM, n=6,P<0.01;P<0.001 in comparison to the control (base); NSNon signiﬁcant. Treatment of bioﬁlm-formed wounds requires the use of strong antimicrobials and proper care, and in a few cases, surgery may be Frontiers in Cellular and Infection Microbiology 08 frontiersin.org frontiersin.org Almuhanna 10.3389/fcimb.2024.1386483 FIGURE 8 Representative images of skin section after treatment with higher concentrations of D. anethifolia extract (H and E stained, 200 X). In the control animals, the skin epithelial width is less when compared to the treated animals (arrow indicates skin epithelium). FIGURE 8 Representative images of skin section after treatment with higher concentrations of D. anethifolia extract (H and E stained, 200 X). In the control animals, the skin epithelial width is less when compared to the treated animals (arrow indicates skin epithelium). required (Ruhal and Kataria, 2021). Herbs and phytochemicals have been reported for antibioﬁlm and wound-healing properties. This includes Aloe vera, curcumin, allicin, and many essential oils. 4 Discussion It is believed that herbs and phytochemicals may hold promising beneﬁts in the management of bioﬁlm infections and wound care (Karygianni et al., 2016). FIGURE 9 Cell viability of HaCaT cells after treatment with different concentrations of D. anethifolia extract in SRB assay, n=4, P<0.001 as compared to untreated control. There was no cytotoxic effect up to concentrations of 100 µg/ml and the IC50 value was 1381 µg/ml. In the current study, bioﬁlms were induced on excision wound in diabetic animals. Wounds in diabetic condition provide a suitable environment for the formation of bioﬁlms, and if untreated, it may lead to gangrene. There are several animal models for the development of bioﬁlm. The method adopted in this study was developed and validated in our laboratory (Alrouji et al., 2023). The selection of two different concentrations was based on pilot studies and skin irritation studies. There are several studies on different plant extracts using the same concentrations (Taddese et al., 2021; Tekleyes et al., 2021). The ointment in a suitable base was used to increase the stability, spreadability, and diffusion (Kolhe et al., 2018). The MIC of the extract was 256 µg/ml for MRSA and 512 µg/ml for MDR-P. aeruginosa, which shows that the pathogens are precisely inhibited at different concentrations. These values are higher compared to conventionally used antibiotics that are pure FIGURE 9 Cell viability of HaCaT cells after treatment with different concentrations of D. anethifolia extract in SRB assay, n=4, P<0.001 as compared to untreated control. There was no cytotoxic effect up to concentrations of 100 µg/ml and the IC50 value was 1381 µg/ml. FIGURE 9 Cell viability of HaCaT cells after treatment with different concentrations of D. anethifolia extract in SRB assay, n=4, **P<0.001 as compared to untreated control. There was no cytotoxic effect up to concentrations of 100 µg/ml and the IC50 value was 1381 µg/ml. 09 frontiersin.org Frontiers in Cellular and Infection Microbiology Almuhanna 10.3389/fcimb.2024.1386483 evaluating individual bioactive phytoconstituents may help in the discovery of novel antibacterial and antibioﬁlm agent(s). The results of the study may help in identifying novel molecules that may positively affect the different phases of the wound healing process. chemicals. The MIC values are always higher for crude extracts that contain several phytoconstituents as compared to pure chemicals and isolated phytoconstituents. Isolation of active constituents from this crude extract may lead to new lead molecules having potent antibacterial effects. Data availability statement The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. 5 Conclusion The methanolic extract of Ducrosia anethifolia showed good antibacterial, antibioﬁlm, and wound healing properties. The antibacterial effect was dose-dependent, and the effect was more against MRSA than MDR-P. aeruginosa. The extract did not produce any skin irritation and was also safe on HaCaT cell lines. The LC-MS analysis of the extract revealed the presence of several phytochemicals, some of which have been reported for antibacterial, antioxidant, and anti-inﬂammatory actions. The effects observed in the current study could be due to multiple phytoconstituents, and 4 Discussion The present study is on crude methanol extracts of Ducrosia anethifolia. Identifying potential phytochemicals possessing antibacterial and antibioﬁlm effects may further help to explore novel compounds for treating MRSA or MDR- P. aeruginosa- infected diabetic wounds. The wounds were infected with single pathogens, either MRSA or MDR- P. aeruginosa; however, in diabetic wounds, polymicrobial infections and bioﬁlms were also formed. Further studies on polymicrobial antibioﬁlm activity and infection control may provide in-depth knowledge on the efﬁcacy of the Ducrosia anethifolia extract. The study conducted was focused on the excision wound model. Extending the studies on different wound models may also provide insight into the wound-healing properties of the extract. Conﬂict of interest The author declares that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Funding The author(s) declare that no ﬁnancial support was received for the research, authorship, and/or publication of this article. Acknowledgments The author would like to thank the Deanship of Scientiﬁc Research at Shaqra University for supporting this work. Author contributions YA: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Though this study determined both in-vivo and in-vitro antibioﬁlm activity of D. anethifolia extract, it has a few limitations. The present work determined the activity of the crude extract of the plant and the contribution of each phytoconstituent present in the extract to the observed effects was not assessed. This is important to determine the synergistic and antagonistic effects of the combination of phytoconstituents, as earlier reports on D. anethifolia showed that volatile oils are effective antimicrobial agents while its main phytoconstituent-decanal was less effective suggesting synergistic effects of different molecules present in the extract (Mahboubi and Feizabadi, 2009). The present study was done using only one model of wound healing. Effect on other models of wound healing such as the incision-wound model, and burn-wound model may help to substantiate the effect of D. anethifolia on the wound healing process (Sami et al., 2019). Ethics statement The animal study was approved by Ethical Research Committee Shaqra University. The study was conducted in accordance with the local legislation and institutional requirements. This study determined antibacterial, antibioﬁlm and wound healing properties of the crude methanolic extract of Ducrosia anethifolia. 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https://openalex.org/W4387401071	https://jelectrochem.xmu.edu.cn/cgi/viewcontent.cgi?article=1643&context=journal	Chinese	null	Influence of DC Electric Field on the Oil-water Relative Permeability in Water-drive Reservoir	Deleted Journal	2,005	cc-by	1,407	Recommended Citation Recommended Citation ZHANGJi-hong ZHANGJi-hong. Influence of DC Electric Field on the Oil-water Relative Permeability in Water-drive Reservoir[J]. Journal of Electrochemistry, 2005 , 11(2): 215-218. DOI: 10.61558/2993-074X.1643 Available at: https://jelectrochem.xmu.edu.cn/journal/vol11/iss2/19 Journal of Electrochemistry Journal of Electrochemistry Journal of Electrochemistry Journal of Electrochemistry Volume 11 Issue 2 Journal of Electrochemistry Journal of Electrochemistry Journal of Electrochemistry Journal of Electrochemistry Influence of DC Electric Field on the Oil-water Relative Influence of DC Electric Field on the Oil-water Relative Permeability in Water-drive Reservoir Permeability in Water-drive Reservoir ZHANGJi-hong ZHANGJi-hong Available at: https://jelectrochem.xmu.edu.cn/journal/vol11/iss2/19 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 ELECTROCHEM ISTRY 第11卷　第2期 2005年5月第11卷　第2期 2005年5月 Vol. 11　No. 2 May 2005 ELECTROCHEM ISTRY 收稿日期: 2004210225,修订日期: 2004212214, 3 通讯联系人: Tel: (86210) 289734056, E2mail: dqzhjh@126. com 973重大基础研究前期专项(2002CCA00700)资助直流电场对水驱油藏油水相对渗透率的影响研究张继红 3 1, 2 ,岳湘安 1 ,杨　晶 2 ,刘　颖 2 ,李宏岭 1 (1. 中国石油大学提高采收率中心,北京102249, 2. 大庆石油学院石油工程学院,黑龙江大庆, 163318) 摘要: 　研究、分析了直流电场强度、方向等因素对油藏岩石油水相对渗透率的影响及其作用机理. 实验表明,在直流电场作用下,岩心呈强水湿性,束缚水饱和度增大,残余油饱和度降低;随着电场强度的增加,油相相对渗透率增大,水相相对渗透率降低;其极性相和非极性相各自呈现的不同渗流特性、壁面双电层结构变化以及原油的电粘效应等都是影响油水相对渗透率的因素. 关键词: 　直流电场;相对渗透率;渗流特性在直流电场作用下,油藏可以产生许多有利于油气开采的物理和化学效应,从而提高原油采收率 [ 1, 2 ]. 深入剖析外加电场下水驱油藏岩石中油水相对渗透率的影响因素和作用机理是此一有潜力课题的关键 [ 3 ]. 但迄今,有关此方面的研究国内外文献罕见报道. 1. 2　实验流程 1)将实验流体装入相应的容器中,岩心放在岩心夹持器中,安装好阀门及相应管线. 2)加环压至4 MPa. 将抽空后岩心饱和实验用水,同时测量岩心孔隙体积,计算岩心孔隙度. 3)按图1,在恒温箱中连接好管路,测量岩心的水测渗透率,之后将系统恒温至45℃约3 h. 4)将饱和模拟油的岩心作水驱油实验,记录压力和流出的油、水量随时间的变化. 5)同样,将另外两块渗透率及孔隙度相近的岩心重复上述步骤,之后再依次分别在外加正、反向电场作用下记录压力、电流及产出的油、水量随时间的变化. 本文应用非稳定法测定了在外加电场下的油水相对渗透率,并分析它的基本特征及其影响因素和作用机理. 5)同样,将另外两块渗透率及孔隙度相近的岩心重复上述步骤,之后再依次分别在外加正、反向电场作用下记录压力、电流及产出的油、水量随时间的变化. 1. 1　实验方案设计应用非稳定法作直流电场下的油水相对渗透率实验 [ 4 ]. 实验中考虑了地层及地层流体的组成、物性和流动特性等因素,并将水动力方向与电动力方向相同的电场指定为正向电场,反之为反向电场. 每组实验均选取渗透率及孔隙度基本相同的3 块岩心,分别作无电场作用、正向电场( + )作用和反向电场( - )作用下的相对渗透率实验. 1. 3　实验结果分析　　图2为外加电场作用下的水驱采收率实验结果. 由图可见,在正向电场作用下,水驱采收率随电场强度的增大而提高,而且矿化度越高这种效应越强. 在反向电场作用下,其效果与正向电场的相反. 如图,在10 V /cm的正向电场下,其水驱采收率比不加电场时的提高约6%. 上述这一现象主要是由于电化学效应、界面效应及电动力效应综合作用的结果 [ 5 ]. 实验用油系大庆榆树林油田原油配制的模拟油,粘度8. 0 mPa· S (45 ℃) ;实验用水为参照榆树林油田地层水平均组成配制的模拟水(矿化度 3 848 mg/L) ,稀释后其矿化度为770 mg/L (表1). 收稿日期: 2004210225,修订日期: 2004212214, 3 通讯联系人: Tel: (86210) 289734056, E2mail: dqzhjh@126. com 973重大基础研究前期专项(2002CCA00700)资助电　化　学 2005年 · 6 1 2 · · 6 1 2 · 表1　大庆榆树林油田模拟地层水矿物含量表1　大庆榆树林油田模拟地层水矿物含量 Tab. 1　M ineral content of the simulated formation water in Yu Shu L in oil field of Daqing Salinity/mg· L - 1 M ineral contents /mg· L - 1 NaCl KCl CaCl2 MgCl2 Na2CO3 NaHCO3 Na2 SO4 3848 3. 259 0. 037 0. 677 0. 975 1. 113 0. 642 3. 081 770 0. 652 0. 007 0. 133 0. 195 0. 223 0. 128 0. 616 图1　岩心中油水相对渗透率实验装置简图 Fig. 1　D iagram of the oil2water relative permeability experiment in cores 1) pulseless pump, 2, 3 ) m iddle Container, 4 ) core clamper, 5) amperemeter, 6 ) voltage gauge, 7) high voltage electric supply, 8) p ressure sensor, 9) measuring cylinder, 10) thermostat 图2　直流电场作用下的水驱采收率曲线 Fig. 2　Curves of the water drive recovery underDC electric field 外加电场下油水相对渗透率变化曲线如图3 图3　直流电场作用下水驱油藏油水相对渗透率曲线 (◆:油相▲:水相) Fig. 3　Curves of the oil2water relative permeability for water drive reservoir under DC electric field (◆: oil phase 表1　大庆榆树林油田模拟地层水矿物含量 Tab. 1　M ineral content of the simulated formation water in Yu Shu L in oil field of Daqing 田模拟地层水矿物含量 ation water in Yu Shu L in oil field of Daqing M ineral contents /mg· L - 1 MgCl2 Na2CO3 NaHCO3 Na2 SO4 7 0. 975 1. 113 0. 642 3. 081 3 0. 195 0. 223 0. 128 0. 616 图3　直流电场作用下水驱油藏油水相对渗透率曲线 (◆:油相▲:水相) Fig. 3　Curves of the oil2water relative permeability for wate 表1　大庆榆树林油田模拟地层水矿物含量 Tab. 1　M ineral content of the simulated formation water in Yu Shu L in oil field of Daqing Tab. 1　M ineral content of the simulated formation water in Yu Shu L in oil field of Daqing 滞留的水分子,使水溶液表面膜减小,从而扩大了流体的流通面积. 另外,在电场作用下油和水的附加电动力也不同,结果是促使了油把水从孔道中排出,即以油置换水. 滞留的水分子,使水溶液表面膜减小,从而扩大了流体的流通面积. 另外,在电场作用下油和水的附加电动力也不同,结果是促使了油把水从孔道中排出,即以油置换水. 可见,在直流电场作用下,其油水相对渗透率均表现为岩石强水湿,即等渗点饱和度大于60% ,况且残余油饱和度Sor处的水相相对渗透率krw < 0. 1, 这就是使用直流电场提高采收率的原因之一. 图3 实验结果同时表明,不论外加正向或反向电场,束缚水饱和度均增大,残余油饱和度均降低. 而在外加正向电场作用下,束缚水饱和度Swc处的油相相对渗透率kro增大,而Sor处的krw下降,这对采油是有利的. 滞留的水分子,使水溶液表面膜减小,从而扩大了流体的流通面积. 另外,在电场作用下油和水的附加电动力也不同,结果是促使了油把水从孔道中排出,即以油置换水. 可见,在直流电场作用下,其油水相对渗透率均表现为岩石强水湿,即等渗点饱和度大于60% ,况且残余油饱和度Sor处的水相相对渗透率krw < 0. 1, 这就是使用直流电场提高采收率的原因之一. 图3 实验结果同时表明,不论外加正向或反向电场,束缚水饱和度均增大,残余油饱和度均降低. 而在外加正向电场作用下,束缚水饱和度Swc处的油相相对渗透率kro增大,而Sor处的krw下降,这对采油是有利的. 2. 3　原油的电粘效应使用RS2150H流变仪测定榆树林油田原油在直流电场作用下的视粘度,结果如图4所见. 原油的低剪切视粘度受电场强度的影响较明显,且随着电场强度的增加有所下降. 而在高剪切速率( Û r)条件下,电场强度对原油的视粘度影响不大. 图5示出原油低剪切速率视粘度随直流电场强度的变化曲线. 由图可见,在7. 0 V的外加电场下,当剪切速率为0. 036 s - 1时, 45℃原油的视粘度由771 mPa· S降至310 mPa· S,幅度很大. 这一特性,对于油层中原油的流动十分有利,能较大幅度地提高地下原图4　直流电场作用下原油的电粘效应曲线(45 ℃) Fig. 4　Curves of the electro2viscous effect of oil under DC e2 lectric field potential/V: ◆0, ▲7, ●75. 2, □191, ◇794, △1 200, ○2 500 图5　原油低剪切视粘度随直流电场强度的变化曲线 Fig. 5　Influence of DC electric field on the viscosity of oil at low shear rate 使用RS2150H流变仪测定榆树林油田原油在直流电场作用下的视粘度,结果如图4所见. 原油的低剪切视粘度受电场强度的影响较明显,且随着电场强度的增加有所下降. 而在高剪切速率( Û r)条件下,电场强度对原油的视粘度影响不大. 图5示出原油低剪切速率视粘度随直流电场强度的变化曲线. 由图可见,在7. 0 V的外加电场下,当剪切速率为0. 036 s - 1时, 45℃原油的视粘度由771 mPa· S降至310 mPa· S,幅度很大. 这一特性,对于油层中原油的流动十分有利,能较大幅度地提高地下原水驱结束时,与Sor处对应的驱替压差有所增加,这可能是由于在外加电场下因采收率较高,剩余油较少,且在岩石孔道内呈滴状,表现出较强的液阻效应. 与常规水驱相比,在直流电场作用下,其水驱过程的水相相对渗透率显著降低,残余油饱和度明显减小,等渗点下含水饱和度有所增大(向右移动) ,岩石呈强水湿性. 图4　直流电场作用下原油的电粘效应曲线(45 ℃) 2　油水相对渗透率影响因素分析 2. 1　极性相与非极性相的水2电渗流特性对水驱油过程,地层流体主要为油、水两相混合物. 其中水相的极性一般较强,为极性相;油相的极性相对较弱(烃类中一些含氧、含氮化物是具有极性的) ,可视为非极性相. 极性相与非极性相对外加电场的响应不同,前者在极化油层中的渗流实际上是受水动力和静电力综合作用的控制,而某些非极性介质则可在电场作用下极化. 因此,若对油层施加一个方向和强度适当的直流电场,就可以改变极性相与非极性相在水力和电动力综合作用下渗流特性的差异,从而相应地调整极性介质的渗流速率 [ 2 ]. 这是利用外加电场改变油、水相渗透率的主要机理之一. 图4　直流电场作用下原油的电粘效应曲线(45 ℃) 图4　直流电场作用下原油的电粘效应曲线(45 ℃) Fig. 4　Curves of the electro2viscous effect of oil under DC e2 lectric field potential/V: ◆0, ▲7, ●75. 2, □191, ◇794, △1 200, ○2 500 图1　岩心中油水相对渗透率实验装置简图 Fig. 1　D iagram of the oil2water relative permeability experiment in cores 1) pulseless pump, 2, 3 ) m iddle Container, 4 ) core clamper, 5) amperemeter, 6 ) voltage gauge, 7) high voltage electric supply, 8) p ressure sensor, 9) measuring cylinder, 10) thermostat 图2　直流电场作用下的水驱采收率曲线图3　直流电场作用下水驱油藏油水相对渗透率曲线 (◆:油相▲:水相) Fig. 2　Curves of the water drive recovery underDC electric field Fig. 3　Curves of the oil2water relative permeability for water drive reservoir under DC electric field. (◆: oil phase, 外加电场下油水相对渗透率变化曲线如图3 所示,本实验的岩心渗透率为0. 198μ m 2 , 孔隙度为20. 74% , 驱替水矿化度为3 848 mg/L. 由图3 外加电场下油水相对渗透率变化曲线如图3 所示,本实验的岩心渗透率为0. 198μ m 2 , 孔隙度为20. 74% , 驱替水矿化度为3 848 mg/L. 由图3 ▲: water phase) ▲: water phase) 张继红等:直流电场对水驱油藏油水相对渗透率的影响研究第2期 ·7 1 2 · 3　结　论在直流电 1)在直流电场作用下,油藏岩石油水相对渗透率表现出岩心的强水湿性,束缚水饱和度增加, 残余油饱和度降低,且在正向电场作用下,随电场强度的增大,油相相对渗透率明显增大,水相相对渗透率降低. [ 3 ] 　Honapo M, Kerdolits L, Havy A H. Relative Permeabili2 ty in Reservoir (Ma Zhiyuan, Gao Yawen translated. [M ]. Beijing: Petroleum Industry Publisher, 1989. 33 ～35. [ 3 ] 　Honapo M, Kerdolits L, Havy A H. Relative Permeabili2 ty in Reservoir (Ma Zhiyuan, Gao Yawen translated. [M ]. Beijing: Petroleum Industry Publisher, 1989. 33 ～35. 2)在正向电场作用下,水驱采收率随电场强度的增加而增加;而在反向电场作用下,效果相反. 3)在低剪切速率下,原油的电粘效应较为明显,即使在较弱的直流电场作用下,其视粘度便有较大幅度的降低. 这一特性,对于油层中原油的流动十分有利,可较大幅度地提高地下原油的相渗透 [ 4 ] 　Shen Pingping (沈平平). Petrophysics Experimental Technology[M ]. Beijing: Petroleum Industry Publish2 er, 1995. 134～176. [ 4 ] 　Shen Pingping (沈平平). Petrophysics Experimental Technology[M ]. Beijing: Petroleum Industry Publish2 er, 1995. 134～176. [ 5 ]　Zhang Jihong(张继红). Charaterstics of electrodynam ic seepage flow of one phase fluid in porous media [ J ]. Journal of Daqing Petroleum Institute, 2002, 26 (1) : 24 ～26. [ 5 ]　Zhang Jihong(张继红). Charaterstics of electrodynam ic seepage flow of one phase fluid in porous media [ J ]. Journal of Daqing Petroleum Institute, 2002, 26 (1) : 24 ～26. 参考文献(Refe rence s) : [ 1 ]　Г. Г. Васитов , Э. М. Симзий. Develop O il From Res2 ervoir byU tilizing Physical Field (Cai Tiancheng transla2 ted) [M ]. Beijing: Petroleum Industry Publisher, 1993. [ 2 ]　W ang D ianSheng (王殿生) , Guan Jiteng (关继腾) , Huang L iubin (黄柳宾). Ehanced oil recovery ( EOR) method of direct current electric field[ J ]. O il D rilling & Production Technology, 1997, 19 (6) : 23～26. 4)机理分析表明,外加电场条件下油水相对渗透率的变化主要与极性相与非极性相的渗流特性、双电层结构以及原油的电粘效应等因素有关. 综上所述,外加电场对水驱油过程具有显著的影响. 岩心中极性相与非极性相的水—电渗流特性、流体与壁面间双电层结构的变化、原油的电粘效应等都是影响油水相对渗透率的主要因素. 对此,可从电化学效应、界面电现象及油水界面性质作更深入的研究,为电场采油新技术的开发奠定基础. 参考文献(Refe rence s) : 2. 2　双电层结构的变化岩石骨架与地层流体相接触时,在两相界面处必然产生双电层. 油藏孔隙中的电动力学现象与双电层密切相关. 矿物表面通常吸附负电荷,这些带负电荷的表面与盐水中阳离子之间的吸引力导致扩散层的形成. 该双电层被分成不动层和可动层, 不动层由孔隙壁面的负电荷与其紧邻的正离子层组成. 当于岩心两端施加直流电场时,随着电场强度的增大,将会导致不动层厚度减小,并同时减弱双电层的电粘效应,不动层变为可动层,渗流速率增加. 当可动层中的阳离子向阴极迁移时,它会拖动图5　原油低剪切视粘度随直流电场强度的变化曲线图5　原油低剪切视粘度随直流电场强度的变化曲线 Fig. 5　Influence of DC electric field on the viscosity of oil at low shear rate 电　化　学 2005年 · 8 1 2 · 4)机理分析表明,外加电场条件下油水相对渗透率的变化主要与极性相与非极性相的渗流特性、双电层结构以及原油的电粘效应等因素有关. Influence of DC Electric Field on the Oil2water Relative Permeability in Water2drive Reservoir ZHANG Ji2hong 3 1, 2 , YUE Xiang2an 1 , YANG J ing 2 , L IU ying 2 , L I Hong2ling 1 (1. Reserch Center of Enhanced O il Recovery, Petroleum University of China, B eijing 102249, China, 2. Departm ent of Petroleum Engineering of Daqing University, Daqing 163318, China) Abstract: The effects of DC electric field strength and direction on oil2water relative permeability and the in2 fluence mechanism in reservoir were studied and analysed. The results of experiments indicated that, with the app lication of external electric field, the core samp les becomes more hydrohilic, irreducible water saturation in2 creases and residual oil saturation decreases. Meanwhile, oil relative permeability increases and water relative permeability decreases with increasing of the strength of external electric field. It is demonstrated aswell that the different seepage flow behaviors, the structure change of double2electric2layer surface and the electric2viscous effect of polar and non2polar phase are the main factors influencing oil2water relative permeability. Key wo rds: DC electric field, Relative permeability, Seepage flow behavior
https://openalex.org/W3158771256	https://hrcak.srce.hr/file/362479	Bosnian	null	Prilog poznavanju hrvatsko-srbijanskoga graničnog spora u Hrvatskom podunavlju – historijsko-geografski i suvremeni aspekti	Geoadria	2,021	cc-by	20,830	BRANIMIR VUKOSAV1, ZVONIMIR MATIJEVIĆ2 1 Sveučilište u Zadru, Odjel za geografiju, Ulica Dr. Franje Tuđmana 24 i, 23 000 Zadar, Hrvatska / University of Zadar, Department of Geography, Zadar, Croatia, e-mail: bvukosav@unizd.hr 2 Gimnazija Nova Gradiška, Trg kralja Tomislava 9, 35 400 Nova Gradiška, Hrvatska / Nova Gradiška Gymnasium, Nova Gradiška, Croatia, e-mail: zamtij96@gmail.com autor za kontakt / corresponding author Višestoljetno postojanje hrvatskih zemalja u različitim političko-geografskim okvirima, kao i njihov granični položaj između civilizacijski i kulturno različi tih i često suprotstavljenih političko-teritorijalnih cjelina uvjetovali su proces vrlo dinamičnih političkih, teritorijalnih i demografskih mijena koje su postupno oblikovale granice suvremene Republike Hrvatske. Od takvoga historijsko-geo grafskog dinamizma nije bilo iznimka ni područje hrvatskoga Podunavlja, odno sno prostor suvremene državne granice Republike Hrvatske sa Srbijom. Geneza hrvatske državne granice u tom području najvećim je dijelom novijega datuma, no njezin nastanak 1945. kao republičke granice između Hrvatske i Srbije u sklopu novonastale socijalističke Jugoslavije posredno je uvjetovan historijsko-geograf skim procesima koji su mu prethodili. Osamostaljenjem Republike Hrvatske i drugih republika bivše Jugoslavije dolazi do niza otvorenih granično-teritorijalnih pitanja stvorenih promjenom republičkih granica u međudržavne, od čega nije izuzeta ni navedena istočna granica Hrvatske. Njezin sjeverni dio koji je vezan uz tok rijeke Dunav postaje predmetom spora Hrvatske prvo sa SR Jugoslavijom, a potom i sa Srbijom, ponajprije zbog činjenice da čin preciznog određenja granice nije proveden za vrijeme socijalističke Jugoslavije te zbog suprotstavljenih geograf skih i pravnih interpretacija o njezinu pružanju. Historijsko-geografskom anali zom nastanka i razvoja hrvatsko-srbijanske granice u hrvatskome Podunavlju u 20. stoljeću, u radu se kontekstualizira aktualni hrvatsko-srbijanski granični spor na dijelu međusobne granice vezanom uz rijeku Dunav. Pregledom historijsko-ge ografskih čimbenika, kao i vrednovanjem stajališta dviju zemalja po pitanju spora u svjetlu međunarodno-pravne prakse nastoji se donijeti relevantne zaključke o razgraničenju Hrvatske i Srbije u području hrvatskoga Podunavlja. Ključne riječi: hrvatsko Podunavlje, granice, Dunav, historijsko-geografski ra zvoj, granični spor 177 25/2 (2020) 177-208 B. Vukosav, Z. Matijević The centuries-long existence of Croatian lands in different political and geograph ical frameworks, as well as their bordering position between civilizational and cul turally diverse and often opposing political and territorial units, have conditioned the process of very dynamic political, territorial and demographic changes that gradually shaped the borders of the modern Republic of Croatia. The area of the Croatian Danube region with the country’s eastern national border to Serbia, has not been exempt from such historical and geographical dynamism. INTRODUCTION With the length of 317.6 km from the tripoint with Hungary in the north to the tripoint with Bosnia-Herzegovina in the south, the eastern border of the Republic of Croatia with Serbia in the Croatian Danube area is among the youngest Croatian borders with an exception of its central segment that stretches from the confluence of the river Drava into Danube to a point in the course of Danube near Ilok (Fig. 1). The border got its provisory shape in the first years after the end of the World War II, during the administrative-terri torial definition of the constituents of the former Federative National Republic of Yugoslavia. Dur ing most of the second half of the 20th century the border had a function of an administrative border between the republic of the former Yugoslavia un til Croatian independence in 1991. At that point of time it changed into a national border, and in 2013 became a border of the European Union, with a perspective of becoming a border of the so- called ‘Schengen area.’1 However, since the border was nominally defined during the time of former Yugoslavia, it has never actually been precisely delineated, and this omis sion of the former regime caused the contemporary border dispute between the Republic of Croatia and the Republic of Ser bia. S duljinom od 317,6 km od tromeđe s Mađar skom na sjeveru do tromeđe s Bosnom i Herce govinom na jugu, istočna državna granica Repu blike Hrvatske sa Srbijom u području hrvatskoga Podunavlja dijelom svoje crte pružanja jedna je od najmlađih hrvatskih granica, uz iznimku nje zina središnjega odsjeka od ušća Drave u Dunav do točke na Dunavu nedaleko od Iloka (Sl. 1.). Svoj načelni oblik granica je dobila u prvim go dinama nakon završetka Drugoga svjetskog rata, tijekom upravno-teritorijalnog definiranja sastav nica tadašnje FNRJ, te je većinom druge polovice 20. stoljeća imala funkciju međurepubličke gra nice u okvirima bivše države sve do 1991. i osa mostaljenja Hrvatske. Tada poprima ulogu držav ne granice, a od 2013. godine i ulogu izvanjske granice Europske unije, s perspektivom međe tzv. „schengenskog prostora“1. No, od vremena biv še države kada je granica nominalno definirana, nikad zapravo nije precizno delimitirana, a takav propust bivšega režima prouzročio je suvremeni granični spor između Republike Hrvatske i Re Slika 1. 1 ‘Schengen area’ encompasses the territories of 26 European countries that adopted an agreement signed in 1985 in Schen gen, Luxemburg. The Schengen area functions as a single coun try, with outer border controls for those that enter or exit the area, but without the inner border controls between the countries that have signed the agreement. As a member of the European union, Croatia is also obliged to join the Schengen area (URL 1). BRANIMIR VUKOSAV1*, ZVONIMIR MATIJEVIĆ2 The genesis of the Croatian national border in this area is mostly recent, but its emergence in 1945 as the republic border between Croatia and Serbia within the newly es tablished socialist Yugoslavia was indirectly conditioned by the historical and ge ographical processes that preceded it. With the independence of the Republic of Croatia and other republics of the former Yugoslavia, a number of open border and territorial disputes created by the change of the republic’s borders to national ones occurred. The above mentioned eastern national border of Croatia is among such disputed areas. Its northern segment which is related to the stream of the Danube river became an object of Croatian territorial dispute, firstly with FR Yugoslavia and afterwards with its successor Serbia, mostly due to the opposing geographic and legal interpretations concerning borderline, and the fact that the precise delineation was not conducted during the time of socialist Yugoslavia. h The actual border dispute between Croatia and Serbia in the Danube river region is contextualized in the paper through a historical and geographical analysis of the border’s genesis. A review of historical and geographical facts, as well as a valori sation of both countries’ views on the matter of the disputed border segments are used to reach relevant conclusions regarding the dispute in question. g g p q Key words: Croatian Danube region, borders, Danube, historical and geograph ical development, border dispute 178 25/2 (2020) 177-208 B. Vukosav, Z. Matijević INTRODUCTION Granice Istočne Hrvatske Figure 1 Borders of Eastern Croatia The key problem of the border between Croatia and Serbia is related to its segment from the tripoint with Hungary to the point near Ilok. In that segment, the middle of the course of the Danube was in principle defined Slika 1. Granice Istočne Hrvatske F B d f E t C ti Slika 1. Granice Istočne Hrvatske Figure 1 Borders of Eastern Croatia Figure 1 Borders of Eastern Croatia 1 Schengenski prostor obuhvaća teritorije 26 europskih zema lja koje su usvojile sporazum potpisan 1985. u Schengenu u Luk semburgu. Schengenski prostor funkcionira kao područje jedne države, s kontrolama za one koji ulaze i izlaze iz područja, ali bez unutarnjih graničnih kontrola među zemljama potpisnicama. Hrvatska se, kao članica Europske unije, također obvezala na pri stupanje Schengenskom prostoru (URL 1). 179 B. Vukosav, Z. Matijević 25/2 (2020) 177-208 as the border of the two federal units of Yugoslavia in 1945 based on the decision of a special commis sion called ‘The AVNOJ presidency commission for reaching a proposal for delimiting the territo ries of Vojvodina and Croatia’ (the so-called ‘Đilas2 commission’). Although in 1946 the Constitution of the Federative National Republic of Yugoslavia confirmed the proposal, such a ‘solution’ has left an open issue of the precise delimitation of the border line by either taking into consideration or revising the cadastral municipalities which followed the for mer course of the Danube river before it changed due to anthropogenic alterations and natural pro cesses. This discrepancy between the ‘Đilas’ border and the cadastral municipalities’ borders was not resolved until the 1990s and Croatia’s proclamation of independence. The Danube region becomes the borderland of the independent Croatia and the unresolved issue of the precise delimitation of the border become a legacy in the form of border dis pute among the two contemporary countries that have the opposing views about the border line. The additional problem in the final delimitation is the fact that the Danube is a plain river that meanders and changes its course naturally due to the Coriolis force, eroding the right (west) bank and accumu lating material on the left (east) bank. Due to that particular natural feature there has been additional natural movement of the Danube’s course towards west, at the expense of Croatian national territory. 2 U literaturi se komisiju najčešće naziva „Đilasovom komisi jom“ po Milovanu Đilasu, članu Centralnog komiteta Komuni stičke partije Jugoslavije i saveznim ministrom za Crnu Goru koji je imenovan njezinim predsjednikom. 2 In the literature, the AVNOJ commission is commonly re ferred to as the ‘ Đilas commission’, after its president Milovan Đi las, a member of the Central Committee of the Communist Party of Yugoslavia, who was also a federal minister for Montenegro. INTRODUCTION This particular natural feature makes any insisting on the course of Danube being a fixed internation al border problematic in the long run (Matijević, 2020). publike Srbije. Temeljni problem razgraničenja Hrvatske i Sr bije vezan je uz dio granice od tromeđe s Mađar skom do Iloka gdje je, na temelju rješenja u tu svrhu osnovane Komisije Predsjedništva AVNOJ-a za izradu prijedloga razgraničenja teritorije između Vojvodine i Hrvatske (tzv. „Đilasove2 komisije“) godine 1945. sredina toka rijeke Dunav načelno definirana kao granica dviju federalnih jedinica. Iako je potvrđeno Ustavom Federativne Narodne Republike Jugoslavije godine 1946., takvo „rje šenje“ ostavilo je otvorenim problem preciznog određenja granične crte i uzimanja u obzir ili re vidiranja katastarskih općina koje su, pak, pratile stari tok Dunava od prije antropogenih i prirodnih promjena njegova toka. Ova diskrepancija između „Đilasove“ granice i granica katastarskih općina nije riješena sve do 1990-ih i do osamostaljenja Hrvatske. Područje Dunava postaje tako granič nim područjem neovisne Hrvatske, a neriješeno pitanje preciznog utvrđenja granice naslijeđeno je u vidu graničnog spora između današnjih dviju dr žava koje imaju različite stavove o tome kuda bi se granična crta trebala pružati. Dodatni problem pri konačnom razgraničenju je i činjenica da je Dunav ravničarska rijeka koja meandrira i mijenja tok pod utjecajem Coriolisove sile pri čemu dolazi do ero zije na desnoj (zapadnoj) obali, te akumulacije na lijevoj obali rijeke. Zbog navedene prirodno-geo grafske značajke, došlo je i do dodatnog pomicanja toka Dunava prema zapadu, a nauštrb hrvatskoga državnog teritorija. Ova značajka svako inzistiranje na toku Dunava kao fiksnoj međudržavnoj grani ci čini dugoročno problematičnim (Matijević, 2020.). The paper consists of two parts – a historical and geographical review of the border development and an analysis of the border dispute. In the first part, the available literature and sources are used to analyse the historical and geographical process es and border changes in the area of the Croatian Danube region throughout the 20th century, with an aim of understanding the broader context of the forming of the eastern Croatian border, mainly in terms of the political and geographical circum Rad je podijeljen u dva dijela: historijsko-geo grafski pregled razvoja granice i analizu graničnog spora. U prvomu dijelu se, uz pomoć dostupne li terature i izvora, analiziraju historijsko-geografski procesi i granične mijene u prostoru hrvatskoga Podunavlja u 20. stoljeću radi razumijevanja ši rega konteksta nastanka istočne hrvatske granice, poglavito političko-geografskih okolnosti u kojima se u prošlom stoljeću razvijao ovaj dio hrvatskih 180 25/2 (2020) 177-208 B. Vukosav, Z. Matijević zemalja. publike Srbije. U drugome se dijelu opisuje i analizira problem razgraničenja dviju država u aktualnim geopolitičkim okolnostima, te se iznose dostupni argumenti jedne i druge strane u hrvatsko-srbijan skom graničnom sporu radi donošenja relevantnog zaključka o mogućem ishodu spora. stances in which this part of the Croatian lands has been developing. The second part describes and analyses the problem of border delimitation be tween Croatia and Serbia within the actual geopo litical circumstances, and includes the arguments of both sides in their border dispute with an aim of reaching a relevant conclusion about its possible outcomes. Temeljna hipoteza historijsko-geografskoga di jela analize jest da je u području hrvatskoga Po dunavlja ishod teritorijalnih mijena u 20. stoljeću za Hrvatsku bio većim dijelom nepovoljan. Na historijsko-geografsku analizu potom se nadove zuje analiza aktualnoga graničnog spora Hrvatske sa Srbijom gdje se pregledom argumentacije obiju strana nastoji utvrditi kako činjenično stanje tako i mogući ishodi spora, imajući u vidu sve relevantne historijsko-geografske i međunarodnopravne čim benike. Pritom je temeljna hipoteza da su u raz doblju nakon okvirnog definiranja granice izme đu Hrvatske i Srbije u vremenu SFRJ sva sporna područja upravno, gospodarski i infrastrukturno pripadala upravo Hrvatskoj. The main hypothesis of the historical and ge ographical part of the paper is that the outcome of the territorial changes in the 20th century was generally unfavourable for Croatia. In the analysis of the actual border dispute between Croatia and Serbia, the review of both sides’ arguments is used in order to determine facts and possible outcomes, keeping in mind all the relevant factors regarding history and international law. The main hypothe sis of the second part is that in the period after the nominal definition of the border between Croatia and Serbia during the former Yugoslavia all the disputed areas administratively, economically and in terms of infrastructure belonged to Croatia. PREVIOUS RESEARCH Problem razgraničenja Hrvatske i Srbije do sada je obrađivan u nizu radova i drugih publikacija te mu je pristupano s gledišta različitih znanstvenih disciplina. Od djela koja valja posebno spomenuti ističe se knjiga S. Sršana (2003.) u kojoj autor, uz mnoštvo vrijednih kartografskih priloga, daje deta ljan pregled razvoja hrvatskih granica na sjeveroi stoku, uz referiranje na pojedine činjenice koje su, prema njegovu sudu, ključne za njihovo konačno uređenje. Posebno treba spomenuti i djelo auto ra M. Klemenčića i C. Schofielda (2001.), gdje je problematika razvoja istočnih hrvatskih granica posebno sagledana u vidu političko-geografskog pristupa proučavanja graničnih i teritorijalnih sporova, uz poseban osvrt na kontekst relativno nedavnih historijsko-geografskih zbivanja nakon hrvatskoga osamostaljenja. So far, the issue of delimitation of the border between Croatia and Serbia has been dealt with in many papers and other publications and has been approached from the point of view of various disciplines. Among the works that should be spe cifically mentioned is a book by S. Sršan (2003) in which the author provides many valuable car tographic sources and a detailed review of the de velopment of Croatian borders in the northeast, with references to the factors that are, according to him, crucial for the borders’ final definition. Fur thermore, a book that stands out is the one by M. Klemenčić and C. Schoefield (2001) in which the problems of the development of eastern Croatian borders is focused on in terms of a political and geographical approach to border and territorial disputes, with a special view on the context of the relatively recent historical and geographical events after Croatia declared its independence. Što se tiče objavljenih znanstvenih radova koji se izravno bave problematikom razgraničenja u hrvat skomu Podunavlju, posebno su važni rad I. Jelića (1991.) koji posebno obrađuje povijesna zbivanja iz vremena pokušaja utvrđivanja hrvatsko-srbijan ske granice 1945. – 1947., rad M. Štambuk-Škalić When it comes to the published scientific pa pers that deal with the issues of defining the bor ders in the Croatian Danube region, among the 181 B. Vukosav, Z. Matijević 25/2 (2020) 177-208 most important are the one by I. Jelić (1991) that specifically deals with historical events from the 1945-1947 period when an attempt was made to define the Croatia-Serbia border. Furthermore, the work of M. Štambuk-Škalić (1995) provides re prints of the most important documents from the mentioned period, while a more recent paper by S. PREVIOUS RESEARCH Mrduljaš (2017) views the issue of border changes in the east of Croatia from the demographic point of view. E. Kraljević (2007) describes and analyses the formation and the development of the ‘Đilas commission’ in 1945, which is especially impor tant in terms of historical and geographical con textualization of the border dispute. Other authors that deal with the topic of Croatian borders in the Danube region are D. Pavličević (1993), M. Bara (2007), Z. Radelić (2006) and others. (1995.) koja donosi i obrađuje niz vrijednih doku menata iz navedenog razdoblja te rad S. Mrdulja ša (2017.) koji problematici graničnih mijena na istoku Hrvatske pristupa iz demografske perspekti ve. E. Kraljević (2007.) detaljno opisuje i analizira nastanak i djelovanje Komisije za razgraničenje iz 1945., što je od posebne važnosti za historijsko- geografsku kontekstualizaciju pograničnog spora između Hrvatske i Srbije. Od ostalih autora koji sagledavaju problematiku hrvatske granice u Po dunavlju ističu se D. Pavličević (1993.), M. Bara (2007.), Z. Radelić (2006.) i drugi. HISTORIJSKO-GEOGRAFSKI PREGLED MIJENA ISTOČNE HRVATSKE GRANICE U 20. STOLJEĆU HISTORICAL AND GEOGRAPHICAL ANALYSIS OF CHANGES OF THE EASTERN CROATIAN BORDER IN THE 20TH CENTURY Def iniranje granica između dvaju svjetskih ratova Suvremeni razvoj i nastanak istočne hrvatske granice značajno je uvjetovan zbivanjima nakon Prvoga svjetskog rata. Godine 1918., porazom i raspadom Austro-Ugarske, hrvatske zemlje s cjelo kupnim teritorijem Srijema našle su se u okvirima Države Slovenaca, Hrvata i Srba koja je bila krat kog vijeka. Nedugo nakon njezina osnutka, bez pristanka Hrvatskoga sabora, spomenuta država s kraljevinama Srbijom i Crnom Gorom ujedinjuje se u Kraljevinu Srba, Hrvata i Slovenaca (od 1929. Kraljevinu Jugoslaviju) pod srpskim kraljem i sa središtem u Beogradu. Prostori Baranje, Bačke i Banata, dotad u sastavu Mađarske, Trianonskim sporazumom iz 1920. također ulaze u sastav nove države. Unatoč idealističkim gledištima pojedinih zagovornika ujedinjenja, Kraljevina SHS bila je centralizirana monarhija u kojoj je prevladavala srpska kulturna, etnička i politička hegemonija te u kojoj drugim narodima nisu priznavana nacio nalna prava (Pavličević, 1998.). Defining the borders between the world wars Modern development and the formation of the eastern Croatian border was significantly affect ed by the events that took place after the World War I. In the year 1918, after the defeat and the disintegration of the Austro-Hungarian Empire, Croatian lands that encompassed the whole terri tory of Syrmia (Srijem) were included in the State of Slovenes, Croats and Serbs for a short period. Shortly after that state’s creation, the mentioned state was united with the kingdoms of Serbia and Montenegro into a new state called the Kingdom of Serbs, Croats and Slovenes (after 1929 – King dom of Yugoslavia) under the Serbian king, and with Belgrade as its capital. With the Treaty of Trianon in 1920, the regions of Baranja, Bačka and Banat that previously belonged to Hungary were included in the new state as well. Despite the idealistic views of the advocates of the unification, the Kingdom of Serbs, Croats and Slovenes was a centralized monarchy where a Serbian cultur al, ethnic and political hegemony prevailed, with other nations not having their national rights rec Novonastale političke prilike odrazile su se i na granice hrvatskih zemalja u hrvatskome Podunav lju koje doživljavaju značajne teritorijalne pro mjene u okvirima upravnoga ustroja nove države nekoliko puta. Prvi upravni ustroj Kraljevine SHS iz 1922. temeljio se na oblastima kao upravnim 182 25/2 (2020) 177-208 B. Vukosav, Z. Matijević ognized (Pavličević, 1998). ognized (Pavličević, 1998). jedinicama najvišeg reda, s očitim ciljem poništa vanja dotadašnje povijesne teritorijalnosti zemalja sastavnica. Uvedene su ukupno trideset tri oblasti, a hrvatske su zemlje nekadašnje trojedine kralje vine Hrvatske-Slavonije i Dalmacije rascijepljene između Zagrebačke, Primorsko-krajiške, Osječke, Srijemske, Splitske i Dubrovačke oblasti. Povijesno hrvatski prostori kao što su Boka kotorska i Međi murje našli su se izvan upravnog područja navede nih oblasti (Matijević, 2020.). Prostor hrvatskoga Podunavlja obuhvaćen je Osječkom i Srijemskom, dok su Baranja i Bačka pripale Novosadskoj oblasti (Klemenčić, Schofield, 2001.). New political circumstances also affected the borders of the Croatian lands in the Croatian Dan ube region on several occasions, in form of signif icant territorial and administrative changes within the new state. The first administrative division of the Kingdom of Serbs, Croats and Slovenes from 1922 was based on regions (oblasts) as administra tive units of the highest rank, with an obvious aim to annul previous historical territories and bor ders of its constituents. Defining the borders between the world wars The state was divided into 33 regions and the Croatian lands of the former Kingdom of Croatia-Slavonia and Dalmatia were divided between the regions of Zagreb, Primor je-Krajina, Osijek, Syrmia, Split and Dubrovnik. Historical Croatian territories of Međimurje and Boka kotorska were not even included in the men tioned regions (Matijević, 2020). The area of the Croatian Danube region was included in the regions of Osijek and Syrmia, while Baranja and Bačka were added to the region of Novi Sad (Kle menčić, Schofield, 2001). Nakon kraljeva ukidanja ustava i skupštine pro glašenjem „šestosiječanjske diktature“ 1929., usli jedio je preustroj te je novoimenovana Kraljevina Jugoslavija podijeljena na devet banovina s imeni ma vezanim uz rijeke i bez ikakva etničkoga ili hi storijsko-geografskog uporišta, pri čemu su se hr vatske zemlje našle u okvirima Savske i Primorske banovine. U novim upravnim okolnostima, grani ce u hrvatskom Podunavlju utvrđene su tako da su pojedini tradicionalno hrvatski krajevi upravno iz dvojeni iz većinski hrvatskih područja. Primjerice, povijesno hrvatsko područje Srijema našlo se ve ćim dijelom izuzeto iz spomenutih dviju banovina s hrvatskom većinom te su priključeni Dunavskoj banovini koja je obuhvaćala i Vojvodinu, Banat, Bačku, Baranju i Šumadiju te imala sjedište u No vom Sadu (Boban, 1995.). Grad Zemun također je izdvojen iz nekadašnjega povijesnog područja Srijema te je s Beogradom činio posebnu upravnu jedinicu. Zakratko su čak i dijelovi istočne Slavo nije (Vukovar, Vinkovci i Županja) bili uvršteni u sastav Drinske banovine, da bi nakon korekcija 1931. ipak pripali Savskoj, a Šid i Srijemska Mitro vica Dunavskoj banovini (Jelić, 1991.). After the king proclaimed the ‘6th of January dictatorship’ which abolished the constitution and dissolved the parliament, an administrative reor ganization followed. The country changed its name into Kingdom of Yugoslavia and was divided into nine banovinas (provinces) that were named after the rivers and lacked any ethnic or historical ba sis. Within such organization, Croatian lands were included in the Sava Banovina and Littoral Bano vina. The borders in the Croatian Danube region were defined in a way that some of the tradition ally Croatian areas were administratively detached from the areas with the Croatian ethnic majority that they were previously a part of. Defining the borders between the world wars Consequently, in the spring of 1939, it led to a series of negotiations between Dragiša Cvetković, the prime minister of Yugoslavia, and Vladko Maček, the leader of the Croatian Peasant Party, in order to find a solution to the so called ‘Croatian question’ and to preserve the country in the midst of the contemporary turbulent circumstances in Europe and the world. The matter of acquiring a larger autonomy and the self-determination of the Croatian people within the common state was in trinsically related to the issue of borders, given that the key intention of the negotiations was to form a new and unique administrative unit which would affirm the Croatian national and territorial iden tity. The Cvetković-Maček agreement was signed on 23 August 1939, administratively and territo rially establishing Banovina Croatia which was to have wide administrative authority for most of the economic, social and public matters, excluding the foreign, military and security affairs. Its borders included the existing banovina Savska and bano vina Primorska, as well as the districts Ilok, Šid, Brčko, Gradačac, Derventa, Travnik, Fojnica and Dubrovnik. With the accession of districts Ilok and Šid, the Croatian lands restored parts Syrmia that had been lost during their adjoining into the existing country and its first two administrative di visions (Boban, 1995). Hrvatske granice i teritorij tijekom Drugoga svjetskog rata U travnju 1941., tijekom Drugoga svjetskog rata, osovinske sile predvođene nacističkom Nje mačkom izvršile su invaziju na Kraljevinu Jugosla viju nakon državnog udara kojim su svrgnuti re gent Pavle Karađorđević i Vlada Cvetković-Maček. Pojedini dijelovi Jugoslavije pripojeni su Njemač kom Reichu, Italiji, Bugarskoj i Mađarskoj, u Srbi ji je uspostavljena njemačka vojna uprava u kom binaciji s kvislinškim Nedićevim režimom, a na teritoriju Banovine Hrvatske te dijelova Vrbaske, Dunavske, Drinske i Zetske banovine proglašena je Nezavisna Država Hrvatska (NDH), kvislinška i satelitska država Njemačke i Italije. Bio je to po kušaj realizacije hrvatske države u okvirima osovin skog poretka, a u njoj je jedini nositelj vlasti bio radikalni Ustaški pokret. Iako je NDH površinom bila veća u odnosu na Banovinu Hrvatsku (jer je u njezin sastav, među ostalim, ušla i cijela Bosna i Hercegovina), Rimskim ugovorima iz svibnja 1941. Italiji je prepustila velike dijelove jadranske obale i otoka, povijesno i etnički hrvatskih prosto ra. U kontekstu granica u hrvatskomu Podunavlju, NDH je pripao cijeli istočni Srijem te su se grani ce u ovom području podudarale s onima od prije Defining the borders between the world wars For instance, a historically Croatian region of Syrmia was separat ed from the two banovinas with Croatian majority and included in Danube Banovina which also en compassed the regions of Vojvodina, Banat, Bačka, Baranja and Šumadija with the city of Novi Sad as its administrative centre (Boban, 1995). The city of Zemun was also administratively excluded from the rest of Syrmia and was joined to the capital of Belgrade to form a special administrative unit. For a short period of time, even the parts of Eastern Slavonia (Vukovar, Vinkovci and Županja) were included into Drina banovina. They were, howev Godine 1934. kralj Aleksandar I. ubijen je u atentatu u Marseilleu, nakon čega je na čelu drža ve regent Pavle Karađorđević kao jedan od trojice namjesnika. Regent je težio neutralnosti Jugosla vije, a provodio je popustljivu politiku radi oču vanja Jugoslavije. Posljedično, u proljeće 1939. započinju pregovori predsjednika vlade Jugosla vije Dragiše Cvetkovića i vođe Hrvatske seljačke stranke Vladka Mačeka u pokušaju pronalaženja rješenja „hrvatskoga pitanja“, cilj kojeg je očuva nje zajedničke države u svjetlu novih zaoštrenih 183 B. Vukosav, Z. Matijević 25/2 (2020) 177-208 er, added to Sava banovina, while the towns of Šid and Srijemska Mitrovica were included in Dan ube banovina after the corrections in 1931 (Jelić, 1991). prilika u Europi i svijetu. Pitanje postizanja veće autonomije i samoodređenja hrvatskoga naroda u sastavu zajedničke države neizbježno je povlačilo sa sobom i pitanje granica s obzirom na to da je temeljna namjera pregovora bila ustrojiti novu, je dinstvenu upravnu jedinicu koja bi afirmirala hr vatski nacionalni i teritorijalni identitet. Sporazum Cvetković-Maček potpisan je 23. kolovoza 1939. te je u upravnom i teritorijalnom smislu uspostav ljena Banovina Hrvatska koja je zadobila vrlo ši roke upravne ovlasti u vidu većine gospodarskih, društvenih i javnih poslova, osim vanjskih, vojnih i sigurnosnih. Što se tiče granica, obuhvatila je dotadašnju Savsku i Primorsku banovinu te kota reve Ilok, Šid, Brčko, Gradačac, Derventu, Trav nik, Fojnicu i Dubrovnik. Pripajanjem Iločkog i Šidskog kotara Banovini Hrvatskoj, hrvatske su zemlje vratile dio Srijema prethodno izgubljenog ulaskom u zajedničku državu i njezinim prvotnim dvama upravnim ustrojima (Boban, 1995.). In 1934, king Alexander I was assassinated in Marseille. After this event, the country was gov erned by Pavle Karađorđević as one of the three re gents. Pavle Karađorđević preferred Yugoslavia to remain neutral, and his inner policies were marked by permissiveness in order to preserve the country. Croatian borders and territory during the World War II In April of 1941, during the World War II, the axis powers led by the Nazi Germany invaded the Kingdom of Yugoslavia after the coup d’etat in the country which resulted in overthrowing the regent Pavle Karađorđević and Cvetković-Maček government. Parts of Yugoslavia were annexed 184 25/2 (2020) 177-208 B. Vukosav, Z. Matijević and incorporated in the German Reich, Italy, Bul garia and Hungary. Serbia was occupied by the German armed forces and a puppet Nedić regime was established in it. In the territory of Banovina Croatia, and the territories of banovinas Danube, Drina, Zeta, a new country was proclaimed – In dependent State of Croatia (ISC), a puppet state of Germany and Italy. It was an attempt to establish a Croatian state within the frames of the axis world order, and the sole authority in it was the radical Ustaša movement. Although the ISC encompassed larger area than Banovina Croatia (given that it en compassed the whole Bosnia and Herzegovina), in May 1941 its leadership signed the Treaties of Rome and ceded large parts of the Adriatic coast and islands, historically and ethnically Croatian ar eas, to fascist Italy. In Croatian Danube region, the ISC included the eastern Syrmia, with the borders matching those prior to 1918. The inner admin istrative organization of the ISC was based on 22 great counties. In Croatian Danube region, those were the county of Baranja with the centre in Osi jek, and Vuka with the centre in Vukovar (Mati jević, 2020). In the beginning, the area of eastern Syrmia was under German military jurisdiction but was eventually given over to the ISC author ities by the end of 1941. Such borders nominally existed until May 1945 and the final downfall of the axis powers together with the ISC, when the Croatian lands became a federal part of the new Yugoslavia under the communist social and eco nomic system. 1918. Unutarnji upravni ustroj NDH temeljio se na uspostavi dvadeset dvije velike župe. U hrvat skom Podunavlju to su bile Baranja sa sjedištem u Osijeku i župa Vuka sa sjedištem u Vukovaru (Matijević, 2020.). Područje istočnoga Srijema u početku je također bilo pod njemačkom vojnom upravom, te je predano NDH tek potkraj 1941. godine. Takvo je stanje granica nominalno potra jalo do svibnja 1945., kada sile Osovine, a s njima i kvislinška NDH, doživljavaju konačni slom. Croatian borders and territory during the World War II Hr vatske zemlje tada ulaze u sastav nove Jugoslavije s komunističkim društvenim i gospodarskim ure đenjem. Valja istaknuti da su temelji za buduće utvrđenje granica federalne Hrvatske, te kasnije i neovisne Republike Hrvatske, dijelom nastali kroz postupnu teritorijalnu organizaciju partizanske borbe tije kom rata te tijekom pojedinih zasjedanja AVNOJ- a (Antifašističko vijeće narodnog oslobođenja Ju goslavije) i ZAVNOH-a (Zemaljsko antifašističko vijeće narodnog oslobođenja Hrvatske) u ratnom vremenu. Ključnu ulogu u tom procesu imao je ZAVNOH, tijelo koje je na prvom zasjedanju u Otočcu i Plitvičkim jezerima u lipnju 1943. preu zelo funkciju najvišega političkog tijela NOP-a. Za buduću teritorijalnu organizaciju Hrvatske ključ no je Drugo zasjedanje ZAVNOH-a održano 12. – 15. listopada 1943. kada je potvrđena prethodna Odluka o priključenju Istre, Rijeke, Zadra i ostalih okupiranih krajeva Hrvatskoj (URL 2). U kontekstu položaja Srijema u okvirima orga nizacije Narodnooslobodilačkog pokreta (NOP) ključno je spomenuti naredbu Titova Vrhovnog štaba iz 1942. upućenu Glavnom štabu Hrvatske o osnivanju operativnih zona s ciljem uspješni je organizacije borbe u Hrvatskoj. U toj naredbi istaknuta je potreba za formiranjem jedinstvenoga operativnog rukovodstva s četiri partizanska odre da na području Slavonije, uključujući i prostor Srijema (Treća hrvatska operativna zona). Unatoč osnivanju novoga pokrajinskog komiteta za Voj vodinu, ondje je ingerenciju imala Komunistička partija Hrvatske (KPH) koja je uvelike doprinijela obnovi oružanog otpora i organizaciji partizan skih snaga koje su prethodno bile razbijene u voj nim akcijama njemačkih i snaga NDH. Ovdje je ključno spomenuti da su ZAVNOH i slavonsko rukovodstvo KPH s jedne, te vojvođansko ruko It should be noted that the basis to define fu ture borders of the federal Croatia (and later of the Republic of Croatia) was partially formed through the gradual territorial organization of the partisan resistance during the war, as well as during the AVNOJ (Anti-Fascist Council for the Nation al Liberation of Yugoslavia) and the ZAVNOH (State Anti-Fascist Council for the National Lib eration of Croatia) wartime sessions. ZAVNOH had a key role in that process, given the fact that it assumed the role of the highest political authority of the anti-fascist movement in Croatia during the First session in Otočac and Plitvice lakes in June 1943. During its Second session that took place in October 1943, the council confirmed the previous decision ‘to reintegrate Istria, Rijeka, Zadar and 185 B. Vukosav, Z. Matijević 25/2 (2020) 177-208 other occupied territories into Croatia’ (URL 2). Croatian borders and territory during the World War II uputio pismo pokrajinskom komi tetu za Vojvodinu u ime CK KPJ (Centralni ko mitet Komunističke Partije Jugoslavije) s odlukom prema kojoj sav teritorij istočno od crte Vukovar – Vinkovci – Županja ulazi politički i vojno u in gerenciju vojvođanskoga pokrajinskog komiteta. Takva je odluka izazvala negodovanje slavonsko ga partijskog rukovodstva koje je izrazilo poseban otpor tumačenju odluke vojvođanskog rukovod stva prema kojemu bi se Vojvodini trebalo pripo jiti kotare Vukovar, Vinkovce i Županju. Nakon zahtjeva KPH za intervencijom CK KPJ, najviše rukovodstvo predložilo je razgraničenje koje bi po toj crti uzimalo u obzir etničku većinu, odnosno pripajanje većinski hrvatskih područja Slavoniji, a većinski srpskih Srijemu. S ovim se pak nije slaga lo vojvođansko rukovodstvo. Ovaj će spor ostati aktualan sve do završetka Drugoga svjetskog rata (Jelić, 1991.). vodstvo s druge strane imale suprotna stajališta o nadležnosti nad područjem Srijema, što je često rezultiralo negodovanjima nakon pojedinih odlu ka nadležnih tijela glede istoga. Primjerice, Tito je u srpnju 1943. uputio pismo pokrajinskom komi tetu za Vojvodinu u ime CK KPJ (Centralni ko mitet Komunističke Partije Jugoslavije) s odlukom prema kojoj sav teritorij istočno od crte Vukovar – Vinkovci – Županja ulazi politički i vojno u in gerenciju vojvođanskoga pokrajinskog komiteta. Takva je odluka izazvala negodovanje slavonsko ga partijskog rukovodstva koje je izrazilo poseban otpor tumačenju odluke vojvođanskog rukovod stva prema kojemu bi se Vojvodini trebalo pripo jiti kotare Vukovar, Vinkovce i Županju. Nakon zahtjeva KPH za intervencijom CK KPJ, najviše rukovodstvo predložilo je razgraničenje koje bi po toj crti uzimalo u obzir etničku većinu, odnosno pripajanje većinski hrvatskih područja Slavoniji, a većinski srpskih Srijemu. S ovim se pak nije slaga lo vojvođansko rukovodstvo. Ovaj će spor ostati aktualan sve do završetka Drugoga svjetskog rata (Jelić, 1991.). vodstvo s druge strane imale suprotna stajališta o nadležnosti nad područjem Srijema, što je često rezultiralo negodovanjima nakon pojedinih odlu ka nadležnih tijela glede istoga. Primjerice, Tito je u srpnju 1943. uputio pismo pokrajinskom komi tetu za Vojvodinu u ime CK KPJ (Centralni ko mitet Komunističke Partije Jugoslavije) s odlukom prema kojoj sav teritorij istočno od crte Vukovar Croatian borders and territory during the World War II Considering the position of Syrimia within the territorial organization of the anti-fascist move ment, it is crucial to mention the order issued by the Tito’s Supreme headquarters in 1942 to the Main HQ of Croatia about founding the opera tive zones with an aim to organize the resistance in Croatia more effectively. The order emphasized the need to form a sole operational leadership with four partisan detachments in the area of Slavonia (eastern Croatia), which would also include the area of Syrmia (Third Croatian Operational Zone). Regardless of the founding of a new province com mittee in Vojvodina, the authority in that prov ince was the Communist Party of Croatia (CPC) which largely contributed to the armed resistance and the reorganization of the partisan forces which were previously scattered during the German and the ISC armed forces’ military operations against them. It is important to mention that ZAVNOH and the Slavonian leadership of the CPC on one side, and the Vojvodina committee on the other side, had opposing views on authority over the area of Syrmia, which often resulted in resentments af ter certain decisions of the supreme authorities about the area. For example, in July 1943 Tito sent a letter to the province committee of Vojvodina on behalf of the Central Committee of the Commu nist Party of Yugoslavia (CC CPY) with a decision that all of the territory on the east of the line Vuk ovar – Vinkovci – Županja is to be politically and martially put under the Vojvodina committee’s authority. This decision caused complaints from the CPC leadership in Slavonia, which expressed a firm opposition to the interpretation of the de cision by the Vojvodina committee, according to which the counties of Vukovar, Vinkovci and Žu panja shoud be incorporated into Vojvodina. The CPC demanded the intervention on behalf of the CC CPY, which resulted in the highest authority suggesting a delimitation that would take ethnic majority into consideration and integrate the areas with the predominant Croat majority to Slavonia, and those with the predominant Serb majority to Syrmia. However, this did not resonate well with other occupied territories into Croatia’ (URL 2). vodstvo s druge strane imale suprotna stajališta o nadležnosti nad područjem Srijema, što je često rezultiralo negodovanjima nakon pojedinih odlu ka nadležnih tijela glede istoga. Primjerice, Tito je u srpnju 1943. Defining the borders of Croatia within the socialist Yugoslavia Defining the borders of Croatia within the socialist Yugoslavia Hrvatsku, izgubila dio Srijema, kao i dijelove BiH (Amanović, 2013.). Hrvatsku, izgubila dio Srijema, kao i dijelove BiH (Amanović, 2013.). Područja zapadnog Srijema, Baranje, Banata i sjeverozapadnog dijela Bačke bila su u pogledu novoga razgraničenja problematična s obzirom na miješani etnički sastav i kontekst hrvatsko-srpskih odnosa koji su pogoršani tijekom netom završeno ga rata. Stoga je u svrhu određivanja granice iz među Hrvatske i Vojvodine u budućoj državi 19. lipnja 1945. osnovana već spomenuta Komisija Predsjedništva AVNOJ-a za izradu prijedloga raz graničenja teritorije između Vojvodine i Hrvatske (tzv. „Đilasova komisija“) koja je trebala donijeti odluku o razgraničenju, utemeljenu na težnjama stanovništva te geografsko-funkcionalnim značaj kama prostora (Mrduljaš, 2017.). Uz Milovana Đilasa u komisiji su bili Milentije Popović i Vic ko Krstulović, ministri unutrašnjih poslova Srbi je i Hrvatske, zatim Jerko Zlatarić, potpredsjed nik Okružnoga narodnooslobodilačkog odbora u Somboru te sekretar Jedinstvenoga narodnooslo bodilačkog fronta (JNOF) Vojvodine Jovan Veseli nov (Matijević, 2020.). Immediately following the end of the World War II in Europe, the leadership of the newly es tablished Democratic Federative Yugoslavia (Fed erative National Republic of Yugoslavia from 1946 and Socialist Federative Republic of Yugoslavia from 1963) made specific moves in order to define the administrative borders in the new country. The definition of the borders of its six federal states (later national, then socialist republics) was to be based on historical and ethnic principles. The key principle was the ethnic one, except in cases when it contradicted the higher principles such as economic and developmental factors (Bagarić, 2017). In accordance to the mentioned decision of the ZAVNOH from 1943, the areas of Dalmatia, Kvarner and the islands that were ceded to Italy in 1943, as well as Istria and Zadar which Italy gained in 1922 by the Treaty of Rapallo, were all to be re integrated into Croatia. Also, the post-war delim itation in the east of Slavonia would result in the addition of Baranja region to Croatia. At the same time, in comparison to the territory of Banovina Croatia, the areas of Bosnia and Herzegovina, as well as Syrmia, were excluded from the new federal Croatia (Amanović, 2013). Već 1. srpnja 1945. Komisija donosi izvješće ko jim se utvrđuje privremeno razgraničenje između Hrvatske i Vojvodine. Def iniranje granica Hrvatske u okvirima socijalističke Jugoslavije Def iniranje granica Hrvatske u okvirima socijalističke Jugoslavije Neposredno nakon završetka Drugoga svjetskog rata u Europi pred ljeto 1945., čelništvo nove De mokratske Federativne Jugoslavije (od 1946. Fede rativna Narodna Republika Jugoslavija, a od 1963. Socijalistička Federativna Republika Jugoslavi ja) započelo je s konkretnim potezima uređenja upravnih granica u novoj državi. Utvrđenje novih granica šest federalnih država (kasnijih narodnih, pa socijalističkih republika) nastojalo se temeljiti na povijesnom i etničkom načelu. Etničko načelo pritom je bilo ključno, osim ako nije bilo u su protnosti s načelima višeg reda, pri čemu se mislilo na gospodarske i slične razvojne čimbenike (Baga rić, 2017.). Hrvatskoj su, u skladu s prethodnom odlukom ZAVNOH-a donesenoj 1943., ponovno vraćena područja Dalmacije, Kvarnera te otoka koji su dvije godine ranije pripale Italiji, kao i Istra i Zadar koji su Italiji pripali još 1920. Rapalskim ugovorima. Hrvatskoj će, poslijeratnim razgra ničenjem na istoku Slavonije, pripasti i Baranja. Istodobno, Hrvatska je, u odnosu na Banovinu 186 25/2 (2020) 177-208 B. Vukosav, Z. Matijević Defining the borders of Croatia within the socialist Yugoslavia On 1 July 1945, the Commission issued a report in which it determined the temporary delimitation between Croatia and Vojvodina. The Commission proposed that the northern segment of the border follows the course of the Danube up to the point between the settlements of Bačko Novo Selo and Bukin in the district of Bačka Palanka, from where further delimitation was to be generally done ac cording to the ethnic majority principle. Within that proposed course of action, the Commission defined disputable areas related to certain districts: 1. Bačka: the districts of Subotica, Sombor, Apa tin and Odžaci (in the north and the northeast of the Danube), 2. Baranja: districts Batina and Darda in the con fluence area of the Drava and the Danube riv ers, Što se tiče područja Baranje, Komisija je utvrdila da srezovi Batina i Darda, etnički gledano, ima ju relativnu hrvatsku većinu. Također je utvrđeno da gospodarski gravitiraju području zapadno od Dunava te je predloženo da oba sreza uđu u sastav Hrvatske, kako iz etničkih tako i iz gospodarskih razloga (Štambuk-Škalić, 1995.). 3. Syrmia: districts Vukovar, Ilok and Šid in the south and the southwest of the Danube (Sršan, 2003; Matijević, 2020). The first group of the disputable areas, namely the districts in Bačka, were to remain in Vojvodina, regardless of their mixed ethnic composition (at the time the situation was as follows: the district of Subotica had an absolute Croat majority; the dis trict of Sombor had a relative Hungarian majority, while the ratio of Croat and Serb populations was such that Serbs were a majority in the city, while Croats were majority in the surrounding villages; Hungarians were a majority in the district of Apat in, while Croats were the most numerous group of the Slavic population; in the district of Odžaci the majority of population was Slovak, and after them the Serbs). In the explanation, the Commission stated that, regardless of the absolute Croat major ity in Subotica, ‘it could not reach the conclusion that a territorial belt could be established north of Sombor, which would include the city of Subotica and belong to Croatia’ and that, in case of the dis trict’s inclusion into Croatia, ‘large economic and cultural centre such as Subotica would become a peripheral city’ (Jelić, 1991, 18). Defining the borders of Croatia within the socialist Yugoslavia Komisija je predložila da sjeverni dio granice ide tokom Dunava do međe između sela Bačko Novo Selo i Bukin u Bačko-pa lanačkom srezu, odakle se u većoj ili manjoj mjeri razgraničenje na prostoru Srijema trebalo izvršiti po etničkom načelu. No u tom smislu u sve tri povijesne regije (Bačka, Baranja, Srijem) Komisija je definirala sporna područja vezana uz pojedine srezove: The regions of west Syrmia, Baranja, Banat and northeastern Bačka proved to be problematic when it comes to the new delimitation, given their mixed ethnic structure and the relations between Croats and Serbs that became troubled during the period of the Kingdom of Yugoslavia and further wors ened after the events that occurred in the World War II. Thus, in order to determine the border be tween Croatia and Vojvodina in the future state, a special body called ‘The AVNOJ presidency com mission for reaching a proposal for delimiting the territories of Vojvodina and Croatia’ (the so-called ‘Đilas commission’) was founded. The Commis sion’s task was to bring the decision on the delimi tation, based on the aspirations of the population, as well as the geographic and functional features of the area (Mrduljaš, 2017). Besides Milovan Đilas, the commission consisted of ministers of internal affairs of Serbia and Croatia Milentije Popović and Vicko Krstulović, vice-president of the Sombor 1. Bačka: srezovi Subotica, Sombor, Apatin i Od žaci (sjeverno i sjeveroistočno od Dunava) 2. Baranja: srezovi Batina i Darda u području sliva Drave i Dunava 3. Srijem: srezovi Vukovar, Ilok i Šid južno i jugo zapadno od Dunava (Sršan, 2003.; Matijević, 2020.). 3. Srijem: srezovi Vukovar, Ilok i Šid južno i jugo zapadno od Dunava (Sršan, 2003.; Matijević, 2020.). Za prvu skupinu spornih područja, srezove u Bačkoj, Komisija je odredila da ostanu u Vojvo dini, unatoč iznimno „šarolikom“ etničkom sasta vu (srez Subotica je tada bio apsolutnom većinom naseljen Hrvatima; u srezu Sombor relativna ve 187 B. Vukosav, Z. Matijević 25/2 (2020) 177-208 ćina bili su Mađari, dok je međuodnos Hrvata i Srba takav da su Srbi bili više zastupljeni u gradu, a Hrvati na selima; u srezu Apatin također su bili najbrojniji Mađari, a od slavenskog stanovništva Hrvati; dok su u Odžacima većina bili Slovaci, a nakon njih Srbi). Defining the borders of Croatia within the socialist Yugoslavia U obrazloženju, Komisija navodi da, unatoč apsolutnoj hrvatskoj većini u Suboti ci, „nije mogla doći do zaključka da bi severno od grada Sombora mogao da se uspostavi pojas koji bi zajedno s gradom Suboticom pripadao Hrvatskoj“ te da bi se u slučaju priključenja toga sreza Hr vatskoj „Subotica, kao krupni privredni i kulturni centar, pretvorila u periferni grad“ (Jelić, 1991., 18). No naglašeno je da je rješenje privremeno i otvoreno odnosno da ovisi o budućem uspostavlja nju vanjske jugoslavenske granice prema Mađar skoj, te bi se, u slučaju pripajanja područja obližnje regije Baje u južnoj Mađarskoj gdje živi velik broj Hrvata, ovo pitanje trebalo ponovno razmotriti (Klemenčić, Schofield, 2001.). ćina bili su Mađari, dok je međuodnos Hrvata i Srba takav da su Srbi bili više zastupljeni u gradu, a Hrvati na selima; u srezu Apatin također su bili najbrojniji Mađari, a od slavenskog stanovništva Hrvati; dok su u Odžacima većina bili Slovaci, a nakon njih Srbi). U obrazloženju, Komisija navodi da, unatoč apsolutnoj hrvatskoj većini u Suboti ci, „nije mogla doći do zaključka da bi severno od grada Sombora mogao da se uspostavi pojas koji bi zajedno s gradom Suboticom pripadao Hrvatskoj“ te da bi se u slučaju priključenja toga sreza Hr vatskoj „Subotica, kao krupni privredni i kulturni centar, pretvorila u periferni grad“ (Jelić, 1991., 18). No naglašeno je da je rješenje privremeno i otvoreno odnosno da ovisi o budućem uspostavlja nju vanjske jugoslavenske granice prema Mađar skoj, te bi se, u slučaju pripajanja područja obližnje regije Baje u južnoj Mađarskoj gdje živi velik broj Hrvata, ovo pitanje trebalo ponovno razmotriti (Klemenčić, Schofield, 2001.). County Anti-Fascist Committee Jerko Zlatarić, and the secretary of the United Anti-Fascist front of Vojvodina Jovan Veselinov (Matijević, 2020). On 1 July 1945, the Commission issued a report in which it determined the temporary delimitation between Croatia and Vojvodina. The Commission proposed that the northern segment of the border follows the course of the Danube up to the point between the settlements of Bačko Novo Selo and Bukin in the district of Bačka Palanka, from where further delimitation was to be generally done ac cording to the ethnic majority principle. Within that proposed course of action, the Commission defined disputable areas related to certain districts: County Anti-Fascist Committee Jerko Zlatarić, and the secretary of the United Anti-Fascist front of Vojvodina Jovan Veselinov (Matijević, 2020). Defining the borders of Croatia within the socialist Yugoslavia U Ustavu, pak, NR Hrvatske iz siječnja 1947., njezin je teritorij definiran kao „područje sadašnje oblasti Dalmacije i sadašnjih okruga: Osijek, Slavonski Brod, Daru var, Bjelovar, Varaždin, Zagreb, Sisak, Karlovac, Sušak i Gospić, te područje grada Zagreba“, dok je u članku 13. istaknuto da se „granice Narodne Re publike Hrvatske ne mogu mijenjati bez pristanka Sabora Narodne Republike Hrvatske“ (URL 3). U bivšoj državi republičke granice nisu bile određene na terenu, već je državno zakonodavstvo upućivalo na republičko. Zakonodavstvo, pak, SR Hrvatske, kao i SR Srbije, ali i SR Slovenije jasno je kazalo kako teritorij republike čine općine koje pak čine katastarske općine (URL 4). Regarding the Baranja area, the Commission found that the districts of Batina and Darda have a Croat majority. Moreover, it concluded that they economically gravitate towards the areas on the west of the Danube, and proposed that both districts be added to Croatia for both ethnic and economic reasons (Štambuk-Škalić, 1995). The Commission’s most challenging task was to delineate Croatia and Vojvodina in the area of Syr mian districts. Namely, in 1945 the distribution of ethnic Croats and ethnic Serbs in the area was very complex. The Commission determined that the territory from Vukovar and Vinkovci to Šid was to be integrated with Croatia given that it was set tled mostly by Croats (with an exception of a small northern part of the district of Vukovar with the Serb majority), and concluded that the district of Ilok was ethnically complex (Croats were a major ity in the town of Ilok and the villages in the west, while Serbs were a majority in the villages in the east) but should nevertheless have been integrated with Vojvodina due to economic and functional reasons, i.e. due to the fact that, from the view point of the Commission, integrating Ilok with Croatia would mean its slower development due to ‘bad administrative connections.’ However, the possibility of the later integrating of the western part of the district to Croatia was left open. An im portant factor in the future negotiations will turn out to be the decision of AVNOJ reached during its Third session held on 10 August 1945 to accept the proposal of accession of Vojvodina to Serbia, which would make the temporarily determined border an issue between the republics of Croatia and Serbia (Boban, 1995). Defining the borders of Croatia within the socialist Yugoslavia However, it was emphasized that the proposal was only temporary g Najzahtjevniji posao komisije bio je razgraničiti Hrvatsku i Vojvodinu na području su srijemskih srezova. Ovdje je, naime, etnička situacija 1945. bila izrazito kompleksna kad je riječ o prostornom rasporedu hrvatskoga i srpskoga stanovništva. Ko misija je, pak, odredila da prostor od Vukovara i Vinkovaca do Šida, naseljen velikom većinom Hrvatima (uz iznimku jednoga manjeg sjevernog dijela sreza Vukovar gdje su većina Srbi) pripad ne Hrvatskoj, dok je za srez Ilok konstatirala da je etnički podijeljen (Hrvati su većina u gradu Iloku i pojedinim zapadnim selima, a Srbi u istočnim selima), ali je predložila održanje teritorijalnog obujma sreza i njegovo pripajanje Vojvodini, no minalno iz gospodarskih i funkcionalnih razloga, odnosno jer bi Ilok po njezinu mišljenju imao ote žan razvoj „zbog težine administrativnih veza“. No ostavljena je mogućnost otvaranja pitanja naknad nog priključenja zapadnog dijela sreza Hrvatskoj. Važan čimbenik za buduću nadležnost u pregovo rima bit će kasnija odluka AVNOJ-a na Trećem 188 25/2 (2020) 177-208 B. Vukosav, Z. Matijević and thus open to changes, depending on the de limitation of the outer Yugoslav border with Hun gary, meaning that the issue could be reconsidered if the nearby region of Baja in southern Hungary with a significant Croat population was included in the territory of Yugoslavia (Klemenčić, Schof ield, 2001). zasjedanju u Beogradu 10. kolovoza 1945., kojom je prihvaćen prijedlog o priključenju Vojvodine Sr biji te je u tom smislu predložena granica postala privremenom hrvatsko-srbijanskom međurepu bličkom granicom (Boban, 1995.). Prije konačnog utvrđenja granice, privremeno rješenje Đilasove komisije doživjelo je određene promjene. Naime, naknadno je referendumskim izjašnjavanjem stanovništva odlučeno da se dio Iločkog sreza pripoji Hrvatskoj (naselje Ilok i za padna sela), što je bilo i očekivano, imajući u vidu da je spomenuti dio ove upravne cjeline bio ve ćinski naseljen Hrvatima. Druga korekcija bilo je pripajanje sela Bapska-Novak Hrvatskoj, te sela Ja mena Srbiji (Vojvodini), također po načelu etnič ke pripadnosti (Klemenčić, Schofield, 2001.; Sršan, 2003.). Tako utvrđene granice konačno su potvrđene Ustavom FNRJ donesenim 1946., a u siječnju 1947. i Ustav NR Srbije prihvatio takvo razgraničenje s Hrvatskom (Bara, 2007.). Valja istaknuti da je u Nacrtu Ustava FNRJ članom 12. određeno da razgraničenja između narodnih republika provodi Narodna skupština FNRJ, a naknadno usvojenim amandmanima i stav da se granice narodne republike ne mogu mijenjati bez njezina pristanka (Jelić, 1991.). Defining the borders of Croatia within the socialist Yugoslavia Propust jasnoga i preciznoga usklađivanja „Đi lasovih“ i katastarskih granica godinama je stvarao niz praktičnih poteškoća u društvenom i javnom životu stanovništva s obiju strana Dunava tijekom razdoblja socijalističke Jugoslavije poput problema s utvrđivanjem nadležnosti sudova, inspekcijskim nadzorom, konflikata s nadležnošću u lovu i ribo lovu i slično (Sršan, 2003.). Imajući to u vidu, Before the final decision on the border, the tem porary decision of the Đilas commission was to be somewhat revised. Namely, following the referen dum, it was decided that the town of Ilok and the western villages join Croatia. This course of events was to be expected, given that the mentioned part 189 B. Vukosav, Z. Matijević 25/2 (2020) 177-208 smatranje toka Dunava kakav je bio 1945. konač nim razgraničenjem može se opravdano smatrati upitnim. Posljedično, postoji pravni temelj za tvrd nju da se dio granice gdje nije došlo do preciznijeg utvrđivanja ni do korekcije katastarskih granica smatra privremenim rješenjem. Nepostojanje ko načnog razgraničenja koje bi uzelo sve čimbenike u obzir stvorilo je mogućnost različitih pravnih tu mačenja zaključaka Đilasove komisije, što će dove sti do toga da pitanje utvrđivanja granice opstane sve do danas u obliku međudržavnoga graničnog spora između Hrvatske i Srbije. of this administrative unit had mainly Croat pop ulation. The second modification was the joining of the village Bapska-Novak with Croatia and the village of Jamena to Serbia (Vojvodina), also based on the principle of ethnic majority (Klemenčić, Schofield, 2001; Sršan, 2003). After the revi sions, all the borders were confirmed by the 1946 Constitution of the Federative National Republic of Yugoslavia, and afterwards by the 1947 Consti tution of the National Republic of Serbia (Bara, 2007). It is important to note that the article 12 of the draft of the Constitution of the FNRY states that the delimitation between the republics must be confirmed by the parliament, and the later amendments also clearly state that the borders of a national republic cannot be altered without its consent (Jelić, 1991). Defining the borders of Croatia within the socialist Yugoslavia The 1947 Constitution of NR Croatia defines its territory as ‘an area consist ing of the present region of Dalmatia and the pres ent counties of Osijek, Slavonski brod, Daruvar, Bjelovar, Varaždin, Zagreb, Sisak, Karlovac, Sušak, Gospić, and the city of Zagreb,’ while the article 13 emphasizes that’ ‘the borders of the National Republic of Croatia cannot be changed without the consent of the Parliament of the National Re public of Croatia’ (URL 3). Essentially, the borders between the former country’s republics were not demarcated on the terrain, but the federal legisla tion referred the issue to the legislation of the re publics. Legislations of both Croatia and Serbia, as well as Slovenia clearly stated that territory of a republic is comprised of municipalities which are comprised of cadastral municipalities (URL 4). Promjena funkcije granice u Podunavlju nastankom samostalne Republike Hrvatske Dana 19. svibnja 1991. održan je referendum o budućem političkom položaju Republike Hrvat ske na kojemu su se građani golemom većinom izjasnili za samostalnost Hrvatske (više od 94 %), te je Hrvatski sabor 25. lipnja 1991. donio odluku o raskidu državnopravnih veza s ostalim republika ma i pokrajinama SFRJ. Tada započinje razdoblje velikosrpske agresije na Hrvatsku. Idućih četiri godine JNA i pobunjeni Srbi okupirali su trećinu hrvatskoga državnog teritorija. Na okupiranom teritoriju osnovana je srpska pseudodržava (tzv. „Republika Srpska Krajina“). Unatoč međuna rodnom priznanju Republike Hrvatske od brojnih zemalja svijeta 15. siječnja 1992. granatiranje i bombardiranje hrvatskih gradova i sela, kao i ma sovna ubojstva i progone hrvatskoga stanovništva iz okupiranih područja (Magaš, 2013.). Što se tiče hrvatskoga Podunavlja, pobunjeni su Srbi ovladali Baranjom, dijelovima istočne Slavonije i zapadnog Srijema, kao i cijelom obalom Dunava. Ratno sta nje potrajat će sve do proljeća i ljeta 1995. i voj no-redarstvenih operacija „Bljesak“ i „Oluja“ kada hrvatska vojska oslobađa najveći dio okupiranih teritorija, uz iznimku upravo hrvatskoga Podunav lja koje će biti vraćeno Hrvatskoj tek 1998., po završetku procesa mirne reintegracije. The hesitation of a clear and precise revision of the ‘Đilas’ borders with the existing cadastral bor ders generated a number of practical problems in social and public life of the populations on both banks of the Danube during the era of the socialist Yugoslavia, such as unclear jurisdiction of courts, inspections, jurisdictions in hunting, fishing, etc. (Sršan, 2003). Keeping that in mind, referring to the 1945 delimitation between Croatia and Serbia as ‘final’ can be called into question. In fact, there are clear legal grounds for the assertion that those borders that were not demarcated, nor aligned with the cadastral borders, can be still referred to as ‘provisional.’ The lack of final demarcation that would take all factors into consideration generat Granice samostalne Republike Hrvatske pri marno su određene dijelom teksta Deklaracije o neovisnosti u stavku IV.: „Ovom odlukom sadaš nje granice Republike Hrvatske postaju granice prema drugim republikama i susjednim državama dosadašnje SFRJ“ (URL 5). No ova teritorijalna 190 25/2 (2020) 177-208 B. Vukosav, Z. Matijević činjenica bit će međunarodno potvrđena tek mi šljenjima arbitražne komisije za bivšu Jugoslavi ju, odnosno tzv. „Badinterove komisije“ (nazvane tako po predsjedniku francuskoga ustavnog suda Robertu Badinteru koji je predsjedao arbitražnom komisijom) i posljedičnim priznanjem Republike Hrvatske 15. siječnja 1992. 3 Uti possidetis (lat. kako posjedujete), načelo međunarodnog prava na temelju kojega se unutrašnja razgraničenja između ad ministrativnih jedinica priznaju kao međunarodne granice novih država u času stjecanja neovisnosti. Nastalo je i primijenjeno u 19. stoljeću u procesu osamostaljivanja bivših kolonija u Latin skoj Americi kako bi se spriječilo izbijanje sukoba između novo nastalih država oko spornih područja (URL 6). Change of the border’s function after the independence of the Republic of Croatia On 19 May 1991 a referendum about the future political position of the Republic of Croatia was held, with a great majority of citizens (over 94%) opting for Croatia’s independence. Accordingly, on 25 June 1991 the Croatian Sabor (parliament) verified the decision of Croatia’s severing all of the public law relations with the other republics of Yugoslavia. Afterwards, a Greater-Serbian aggres sion was launched against Croatia. In the follow ing four years, in spite of international recognition of the Republic of Croatia by many countries of the world on 15 January 1992, approximately one third of the Croatian national territory was occu pied by the Yugoslav National Army and the rebel Serb forces, and a Serb pseudo-state (the so-called ‘Republic of Serbian Krajina’) was proclaimed in the occupied territories, shelling and bombing of Croatian cities and villages occured daily, as well as mass murders and expulsions of the ethnic Croat population from the occupied territories (Magaš, 2013). In the Croatian Danube region, the rebel Serbs occupied Baranja, parts of eastern Slavonia and western Syrmia, including all of the Danube’s west bank. Such conditions lasted until spring and summer of 1995 when most of the occupied terri tories were liberated by the Croatian Army by the military operations ‘Blitz’ and ‘Storm’, with an ex ception of the Danube region which would not be returned to Croatia until 1998, after the process of peaceful reintegration. Svakako najznačajnija promjena za hrvatsko Po dunavlje u 1990-ima bila je promjena dotadašnje međurepubličke granice između SR Hrvatske i SR Srbije u državnu granicu između Republike Hrvat ske i Savezne Republike Jugoslavije (kasnije Repu blike Srbije). U području hrvatskoga Podunavlja istočna je hrvatska granica od početka velikosrpske agresije pa sve do mirne reintegracije ove regije tri godine nakon rata bila nedostupna hrvatskoj up ravi s obzirom na okupaciju Baranje, te dijelova istočne Slavonije i Srijema. Službeno je proces mir ne reintegracije hrvatskoga Podunavlja završio 15. siječnja 1998., te je tog trenutka Republika Hrvat ska ostvarila upravno-teritorijalnu cjelovitost prvi put nakon osamostaljenja. Novonastala granica postala je, međutim, predmetom spora između Hrvatske i susjedne Srbije, s obzirom na to da bivša zajednička država nije pristupila preciznoj delimi taciji granice na području rijeke Dunav. Hrvatska je identifikaciji granica pristupila nedugo nakon stjecanja neovisnosti i međunarodnoga priznanja, osnivanjem Državne komisije za granice (od 2001. Komisija za granice). U sklopu navedene komisi je je dana 8. veljače 1996. Promjena funkcije granice u Podunavlju nastankom samostalne Republike Hrvatske Vodeći se pravnim na čelom uti possidetis3, Badinterova komisija istaknu la je kako se granice bivših federalnih sastavnica Jugoslavije smatraju državnim granicama te se ne mogu mijenjati silom, već isključivo sporazumno. Ovo je dalo važan pravni temelj ne samo prizna nju Hrvatske, već i diplomatskom djelovanju, te u konačnici legitimnosti oslobađanja hrvatskoga teritorija. ed a possibility of contrasted legal interpretations, which is why the issue of delineating the border in the Danube area will be inherited by the independ ent Croatia and Serbia as their border dispute. 3 Uti possidetis (lat. ‘as you posses’), a principle in international law according to which borders of administrative units are reco gnized as international borders of the new countries in the mo ment of gaining independence. It was created and applied in the 19th century, in the process of the former colonies in Latin Ame rica acquiring independence, in order to prevent conflicts between the new countries regarding their disputed areas (URL 6). Change of the border’s function after the independence of the Republic of Croatia u Zagrebu osnovana i Ekspertna skupina za identificiranje hrvatsko-srp ske granice na području tadašnjih pet općina koje su graničile sa Srbijom – Beli Manastir, Vukovar, Borders of the Republic of Croatia are primarily determined by a part of the Declaration of inde pendence in article IV: ‘With this decision, the present borders of the Republic of Croatia become national borders with other republics and neigh bouring states of the former Yugoslavia’ (URL 5). However, this territorial fact was not interna tionally recognized until the conclusions of the Arbitration Commission of the Conference on Yugoslavia, or the so-called ‘Badinter commission’ 191 25/2 (2020) 177-208 B. Vukosav, Z. Matijević (named after Robert Badinter, the president of the French Constitutional court who was named pres ident of this commission) and the consequential recognition of the Republic of Croatia on 15 Jan uary 1992. Referring to the legal principle uti pos sidetis,3 the Badinter commission concluded that the borders of the former federal constituents of Yugoslavia are to be considered national borders and cannot be changed by force, but only through agreement. This was an important legal basis not only to the recognition to Croatia, but also to Cro atian diplomatic efforts, and finally to the legiti macy of liberating Croatian territory. Vinkovci, Ilok i Županja. Ova je ekspertna skupi na pribavila svu relevantnu dokumentaciju poput katastarskih planova, zemljišnih knjiga, dokaza o posjedovanju itd., radi utvrđivanja teritorijalnog prava Hrvatske (Sršan, 2003.). Početkom 2000. Hrvatska i tadašnja SR Jugo slavija (od 2006. neovisna Srbija) osnovale su za jedničko Međudržavno diplomatsko povjerenstvo koje je počelo s radom 2002., a kojemu je cilj bio definirati granicu, no u prvih deset godina nave deno se povjerenstvo sastalo samo dva puta, uz donošenje zaključka da u stavovima dviju država postoji razilaženje u stavovima o granici na Duna vu (URL 7). The most significant change for the Croatian Danube region during the 1990s was translation of the existing republican border between the so cialist republics of Croatia and Serbia into a na tional border between Republic of Croatia and the Federal Republic of Yugoslavia (later Republic of Serbia). Since the beginning of the Greater-Serbi an aggression until the peaceful reintegration of the Croatian Danube region, the eastern Croatian border was mostly unavailable to the Croatian au thorities due to occupation of Baranja as well as parts of eastern Slavonia and Syrmia. SUVREMENI ASPEKTI GRANIČNOG SPORA NA DUNAVU I ANALIZA GRANIČNE CRTE Republika Hrvatska je donošenjem Ustava 22. prosinca 1990. utvrdila svoje granice, među koji ma i istočnu. Ustavom je predviđeno da granice kakve je imala kao federalna jedinica u Jugoslaviji postaju državnim granicama, što znači da je sa dašnji ocrt granice utemeljen najvećim dijelom na rezultatima rada Đilasove komisije koji su naknad no pretočeni u zakone bivše države. No upravo su nepotpunost zaključaka komisije te mogućnost njihove različite pravne interpretacije, uz činjenicu da katastarska korekcija tijekom postojanja bivše države nikad nije izvršena, generirale spomenuti međudržavni granični spor nakon hrvatskoga osa mostaljenja. Dok oko 120 kilometara južnog dijela granice koja prilazi područjem zapadnoga Srijema nije sporno (iako se obje strane slažu da postoji potreba za manjim korekcijama), problematičan je sjeverni dio u duljini od oko 140 kilometara, a koji je neposredno vezan uz tok rijeke Dunav od tromeđe s Mađarskom do Iloka. At the beginning of the year 2000, Croatia and FR Yugoslavia (since 2006 independent Serbia) founded a mutual Interstate diplomatic commis sion that started with its activities in 2002, and its aim was to define the border line. However, in the first ten years, the commission held meetings only on two occasions, and the only conclusion that was reached was that there is a dissent regarding their positions about the border on the Danube (URL 7). Although in the year 2001 both countries signed the Protocol of border delimitation and have reached an agreement that they would use all the relevant documents including cadastres in the pro cess of defining the border, no concrete moves or arrangements with regards to the final delimitation have been made since. In other words, by signing this Protocol, the FR Yugoslavia (as well as its suc cessor Serbia) in a sense acknowledged cadastre as being relevant in border delimitation, but subse quently dismissed this argument and began insist ing solely on other legal arguments that ultimately support its own position and interest (URL 7). Korijen problema je u činjenici da su na po dručju Dunava potkraj 19. stoljeća izvršeni meli oracijski radovi kako bi se promijenio tok rijeke, odnosno skratio i pojednostavio plovni put. No od tada nije učinjena korekcija postojećih kata starskih granica koje su se prethodno poklapale s tokom Dunava i nisu se mijenjale od vremena austrougarske uprave4 te su katastarske općine zadržale svoj prethodni teritorijalni ocrt. 4 Osnivanje katastara u Hrvatskoj započelo je proglašenjem Carskog patenta 23. prosinca 1817., čime je određeno da se iz vrši katstarska izmjera i klasiranje zemljišta, uz izradu katastar skih operata u svim zemljama Austro-Ugarske Monarhije (Roić, Paar, 2018.). rodnom sudu pravde u Den Haagu (URL 9). – Beli Manastir, Vukovar, Vinkovci, Ilok and Žu panja. This expert group acquired all the relevant documentation such as cadastral plans, land regis tries, evidence of property, etc. with an aim to de termine Croatia’s territorial rights (Sršan, 2003). Change of the border’s function after the independence of the Republic of Croatia The process of the peaceful reintegration of the Croatian Dan ube region officially ended on 15 January 1998. At that moment, Republic of Croatia achieved its administrative and territorial integrity for the first time since declaring its independence. However, the newly formed border became the object of dis pute between Croatia and Serbia, given that their former country failed to precisely delimit the bor der in the Danube area. Croatia approached the issue of identification of its borders shortly after gaining independence and international recogni tion by founding the State commission for borders (after 2001 Commission for borders). As a part of that commission, on 8 February 1996 an Ex pert group was set up for identifying the border between Croatia and Serbia in the area of the con temporary five municipalities that bordered Serbia Iako su obje zemlje godine 2001. potpisale Pro tokol o razgraničenju i složile su se o tome da će se prilikom utvrđivanja granice koristiti katastar i ostala relevantna dokumentacija, konkretni potezi i dogovori o konačnom razgraničenju nikad nisu implementirani. Drugim riječima, SR Jugoslavija (odnosno njezin pravni slijednik Srbija) je pot pisivanjem ovoga protokola u određenom smislu uvažila katastar kao relevantan za određivanje gra nica, da bi kasnije relativizirala navedeni argument i inzistirala na drugim pravnim argumentima koji podržavaju njezin stav i interes (URL 7). Od 2010. navedeni je spor sve više u fokusu jav nosti u objema zemljama, posebice otkad je Srbija donijela odluku da izgradi riječnu luku nedaleko od Apatina, na dijelu spornog teritorija. Iste se go dine sastala i zajednička komisija, no jedini done seni zaključak bio je taj da o navedenom pitanju postoje različita mišljenja dviju strana. Godine 2011. srbijanska je diplomacija podnijela zahtjev Europskoj uniji, tražeći da Europska unija inzisti ra na razrješenju spora prije ulaska Hrvatske u tu europsku integraciju. Takav potez Srbija je tada poduzela zbog mišljenja kako će nakon ulaska Hr vatske u Europsku uniju situacija za Srbiju biti ne povoljnija. No taj je zahtjev Europska unija odbila, a daljnji pokušaji postizanja dogovora zasad su još uvijek daleko od realizacije (URL 8). Unatoč navedenoj srbijanskoj diplomatskoj ak ciji, s obzirom na trenutačne stavove o mogućem načinu razrješenja spora, srbijanska strana drži da je dogovor moguć bilateralno ili arbitražom. Sta jalište je, pak, Hrvatske da ako izostane dogovor stručnjaka, obje strane prepuste rješenje Međuna 192 25/2 (2020) 177-208 B. Vukosav, Z. Change of the border’s function after the independence of the Republic of Croatia Matijević rodnom sudu pravde u Den Haagu (URL 9). rodnom sudu pravde u Den Haagu (URL 9). SUVREMENI ASPEKTI GRANIČNOG SPORA NA DUNAVU I ANALIZA GRANIČNE CRTE To se odrazilo u vidu „džepova teritorija“ na istočnoj obali rijeke koji su i dalje pripadali katastarskim općinama na zapadu, i obrnuto. Pritom je najveći broj tih „džepova“ ostao na lijevoj, istočnoj obali Dunava (Matijević, 2020.). Prema hrvatskom tumačenju, nastali džepovi teritorija na istočnoj obali pripadaju Hrvatskoj, a na desnoj obali Sr biji. Srbijansko tumačenje jest da aktualno raz graničenje prati tok Dunava te nijedna od država Since 2010, the dispute has been increasingly in public focus in both countries, especially since Ser bia made plans to build a river port near Apatin, in one of the zones of the disputed territory. In the same year there was a meeting of the two coun tries’ mutual commission, but the only conclusion was that the sides have different opinions on the issue. In 2011, Serbian diplomacy filed a claim to the European union, asking that the EU insists on the border dispute being resolved prior to the ac cession of Croatia into this European integration. Serbia opted for this course of action because it held an opinion that its own position would be unfavourable after Croatia’s accession to the EU. However, the EU denied the claim and any further attempts to reach an agreement are still far from realization (URL 8). Regardless of the mentioned Serbian diplomatic attempt, the Serbian side still think that the pos sible ways of resolving the dispute are within the 193 B. Vukosav, Z. Matijević 25/2 (2020) 177-208 frame of a bilateral agreement or, alternatively, via an arbitration. On the other hand, the position of Croatia is that, in case the experts fail to provide a solution acceptable to both sides, the issue should be handled by the International Court of Law in The Hague (URL 9). nema teritorij na suprotnoj obali. Poseban pro blem su i Šarengradska te Vukovarska ada koje po katastarskim granicama pripadaju Hrvatskoj, dok bi srbijanskom interpretacijom one pripadale Srbiji, baš kao i spomenuti teritorijalni dijelovi na lijevoj obali (Sl. 2.). S obzirom na to da ni vlasti bivše države nakon 1945. nikad nisu pristupile preciznom definiranju katastarskih granica, ukupno je oko 90 km2 terito rija hrvatskih katastarskih općina ostalo na lijevoj strani Dunava, a na desnoj samo 9 km2 srbijan skih katastarskih općina (Klemenčić, Schofield, 2001.). SUVREMENI ASPEKTI GRANIČNOG SPORA NA DUNAVU I ANALIZA GRANIČNE CRTE Većinom je u oba slučaja riječ o posjedi ma u vidu šuma, pašnjaka i obradivog zemljišta, te u manjoj mjeri trsticima i neobradivom zemlji štu (Sršan, 2003.). Hrvatska i Srbija danas imaju pravno suprotstavljena mišljenja o tome koje je pravno tumačenje ispravno i kakav je stvarni ocrt granice na Dunavu. S obzirom na to da o preci znom i dogovorenom razgraničenju ovise i čimbe CONTEMPORARY ASPECTS OF THE DANUBE BORDER DISPUTE AND THE ANALYSIS OF THE BORDER LINE its Constitution on 22 December ublic of Croatia defined its borders eastern one. According to the Con borders it had as a federal constit rmer Yugoslavia are translated into rs, which means that the current border is mostly based on the work of the Đilas commission, that was subsequently reflected in the legislature of the former country. However, the incompleteness of the commission’s conclusions, as well as both the possibility of their different legal interpreta tion and the fact that the former country failed to carry out the necessary cadastral correction, generated the above mentioned international border dispute af ter Croatia proclaimed its inde pendence. And while there are no disputes along the 120 km long southern segment of the border that passes through the area of the western Syrmia (al though both sides agree upon the need of its smaller correc tions), the northern 140 km long segment of the border relat ed to the course of the Danube extending from the border with Hungary in the north to Ilok in the south is still unsolved. O f h f h di By adopting its Constitution on 22 December 1990, the Republic of Croatia defined its borders including the eastern one. According to the Con stitution, the borders it had as a federal constit uent of the former Yugoslavia are translated into national borders, which means that the current outline of the border is mostly based on the work of the Đilas commission, that was subsequently reflected in the legislature of the former country. However, the incompleteness of the commission’s conclusions, as well as both the possibility of their different legal interpreta tion and the fact that the former country failed to carry out the necessary cadastral correction, generated the above mentioned international border dispute af ter Croatia proclaimed its inde pendence. And while there are no disputes along the 120 km long southern segment of the border that passes through the area of the western Syrmia (al though both sides agree upon the need of its smaller correc tions), the northern 140 km long segment of the border relat ed to the course of the Danube extending from the border with Hungary in the north to Ilok in Slika 2. Hrvatski teritorij u Podunavlju prema stajalištu Republike Hrvatske Figure 2 Croatian territory in the Danube region according to the viewpoint of the Republic of Croatia Slika 2. 4 The establishment of the cadastre in Croatia began with the proclamation of the Imperial Patent on December 23, 1817, which determined that the cadastral survey and land classification would be made, followed by cadastral operations in all countries of the Austro-Hungarian Monarchy (Roić, Paar, 2018). CONTEMPORARY ASPECTS OF THE DANUBE BORDER DISPUTE AND THE ANALYSIS OF THE BORDER LINE Hrvatski teritorij u Podunavlju prema stajalištu Republike Hrvatske Slika 2. Hrvatski teritorij u Podunavlju prema stajalištu Republike Hrvatske One of the roots of the dispute is the fact that at the end of the Figure 2 Croatian territory in the Danube region according to the viewpoint of the Republic of Croatia of the Republic of Croatia 194 25/2 (2020) 177-208 B. Vukosav, Z. Matijević 19th century a series of works on land reclama tion and changes in the river course in order to shorten the waterway was undertaken. However, a correction of the existing cadastral borders that followed the old course of the Danube has never been made, and these borders have retained the same territorial outline ever since the time of the Austro-Hungarian administration.4 It resulted in a series of ‘territorial pockets’ on the east side of the river belonging to cadastral municipalities in the west, and vice versa. The largest number of territo rial pockets were created on the left, eastern side of the Danube (Matijević, 2020). According to the Croatian interpretation, the territorial pockets in the east side of the river belong to Croatia, and those in the west side to Serbia. On the other hand, Ser bia insists that the border follows the actual course of the Danube and that neither country can claim any territories on opposite banks of the river. Another issue is related to the river islands Šarengrad and Vukovar which, according to the cadastral borders, belong to Croa tia, while according to the Serbian interpretation of the border, belong to Serbia (Fig. 2). nici sigurnosti, odnosno nadzor nad teritorijem, između dviju država postoji privremeni dogovor o tome da se trenutačni tok Dunava koristi kao pri vremena granica nadzora te do rješenja spora Hr vatska nadzire teritorij na desnoj, a Srbija na lijevoj obali rijeke (URL 10). Opis granica katastarskih općina i spornih područja Prema dostupnim prostornim podacima (URL 11), granica hrvatskih katastarskih općina na sjeveru započinje tromeđom (Hrvatska-Srbija- Mađarska) 5,5 km istočno od trenutačnog toka Slika 3. Prikaz spornih područja na satelitskoj snimci od tromeđe s Mađarskom do Apatina – džepovi teritorija na lijevoj (žuto) i desnoj (crveno) obali Dunava Figure 3 View of the disputed areas on the satellite image from the border with Hungary to Apatin - pockets of territory on the left (yellow) and right (red) banks of the Danube Izvor: / Source: URL 12 Since the authorities of the for mer country never approached the precise definition of cadastral boundaries after 1945, a total of about 90 km2 of Croatian cadas tral municipalities remained on the left side of the Danube, and only 9 km2 of Serbian cadastral munic ipalities on the right (Klemenčić, Schofield, 2001). In both cases, these are mostly estates consisting of forests, pastures and arable land, and to a lesser extent of reeds and uncultivated land (Sršan, 2003). Today, Croatia and Serbia have le Slika 3. Prikaz spornih područja na satelitskoj snimci od tromeđe s Mađarskom do Apatina – džepovi teritorija na lijevoj (žuto) i desnoj (crveno) obali Dunava Figure 3 View of the disputed areas on the satellite image from the border with Hungary to Apatin - pockets of territory on the left (yellow) and right (red) banks of the Danube Izvor: / Source: URL 12 Dunava te se, prateći bivši tok zapadno od srbi janskog naselja Kolut spaja s Dunavom kod mje sta Batina zatvarajući džep teritorija Karapanđa/ Kenđija na istočnoj obali Dunava koji pripada katastarskim općinama Draž i Batina, te dalje 195 B. Vukosav, Z. Matijević 25/2 (2020) 177-208 Slika 4. Prikaz spornih područja na satelitskoj snimci od Apatina do Aljmaša. Džepovi teritorija na lijevoj (žuto) i desnoj (crveno) obali Dunava Figure 4 A satellite image of disputed areas from Apatin to Aljmaš. Pockets of territory on the left (yellow) and right (red) banks of the Danube Izvor: / Source: URL 12 gally conflicting opinions on what the actual outline of the border on the Danube is. Opis granica katastarskih općina i spornih područja Given that the pre cise demarcation of the border also determines the security factors such as surveillance the territory, there is a temporary agreement between the two countries that the current course of the Danube be used as a temporary border of surveillance, and until the dispute is resolved, Croatia controls the territory on the right and Serbia on the left bank of the river (URL 10). The description of the cadastral municipalities’ borders and the disputed areas According to available spatial data (URL 11), the eastern border of Croatian cadastral municipalities in the north starts at the tripoint (Croatia-Serbia-Hungary) 5.5 km east of the current course of the Danube and, following the former course west of the Serbian settle ment of Kolut, joins the Danube near Batina, closing the pocket of the territory of Karapanđa / Kenđi ja on the eastern bank of the Dan ube which belongs to the cadastral municipalities of Draž and Batina, and further follows the course in the length of about 3.5 km. From there, it turns east and northeast along the former course, following the former meander, and south to wards Bački Monoštor, after which southwest, east and northeast back to the current course of the Dan ube, closing the pocket of Blaževica / Kolonđoš on the left bank. At that point, about 5.5 km east-southeast of Zmajevac in Kneževi Vinogra di municipality, the border crosses the Danube and stretches along its right bank first to the southwest Slika 4. Prikaz spornih područja na satelitskoj snimci od Apatina do Aljmaša. Džepovi teritorija na lijevoj (žuto) i desnoj (crveno) obali Dunava Figure 4 A satellite image of disputed areas from Apatin to Aljmaš. Pockets of territory on the left (yellow) and right (red) banks of the Danube Izvor: / Source: URL 12 Figure 4 A satellite image of disputed areas from Apatin to Aljmaš. Pockets of territory on the left (yellow) and right (red) banks of the Danube Izvor: / Source: URL 12 Slika 5. Vukovarska ada na satelitskoj snimci Figure 5 A satellite image of the Vukovar island Izvor: / Source: URL 12 Slika 5. Vukovarska ada na satelitskoj snimci Figure 5 A satellite image of the Vukovar island Izvor: / Source: URL 12 196 25/2 (2020) 177-208 B. Vukosav, Z. Matijević Slika 6. Šarengradska ada na satelitskoj snimci, sa žuto označenim spornim područjem Figure 6 A satellite image of the Šarengrad island, with the disputed area marked yellow Izvor: / Source: URL 12 and then to the southeast, closing Siga, the largest pocket of territory on that bank. About 8.5 km on the southeast of Zmajevac, the border again intersects the course of the Danube and meanders to the east, returning twice again to the Dan ube and closing two spacious pock ets of territory on the left bank be tween Bački Monoštor and Apatin. The description of the cadastral municipalities’ borders and the disputed areas Otud ponovno zaokreće istočno i sjeveroistočno bivšim tokom prateći bivši meandar, te južno prema Bačkom Monoštoru, nakon kojega jugozapadno, istočno i sjeveroistočno natrag prema aktualnom toku Du nava, zatvarajući na lijevoj obali džep teritorija Blaževica/Kolonđoš. Na tom mjestu, oko 5,5 km istočno-jugoistočno od naselja Zmajevac u općini Kneževi Vinogradi, granica presijeca tok Dunava te se proteže njegovom desnom obalom najpri je prema jugozapadu, a zatim prema jugoistoku, zatvarajući Sigu, najprostraniji džep teritorija na spomenutoj obali. Na oko 8,5 km jugoistočno od Zmajevca, granica ponovno presijeca tok Dunava te vijuga prema istoku vraćajući se dva puta po novno prema Dunavu i zatvarajući dva prostrana džepa teritorija na lijevoj obali između Bačkog Monoštora i Apatina. Prema katastarskim gra nicama, svi teritoriji na lijevoj obali Dunava iz među Zmajevca i Apatina pripadaju katastarskim općinama Zmajevac I. i Zmajevac II. (Sl. 3.). From a place about 2 km northwest of Aljmaš, at the confluence of the Drava and the Danube, the border follows the course of the Danube in the length of 94 km to the point between Ilok and Bač ka Palanka. On this stretch, it includes the Vukovar island within the cadastral municipality of Vukovar (Fig. 5) and Šarengrad island within the cadastral municipalities of Mohovo and Šarengrad (Fig. 6). The description of the cadastral municipalities’ borders and the disputed areas According to the cadastral bounda ries, all territories on the left bank of the Danube between Zmajevac and Apatin belong to the cadastral municipalities of Zmajevac I and Zmajevac II (Fig. 3). Slika 6. Šarengradska ada na satelitskoj snimci, sa žuto označenim spornim područjem Figure 6 A satellite image of the Šarengrad island, with the disputed area marked yellow Izvor: / Source: URL 12 Slika 6. Šarengradska ada na satelitskoj snimci, sa žuto označenim spornim područjem About 4 km west-northwest of Apatin, the border again follows the course of the Danube, and next to the settlement closes the pocket territory on the left bank (Poluostrvo), then me anders again closing first a small pocket of Serbi an territory on the right bank, and then two more pockets of territory on the left bank (Zverinjak). Further south and southeast, the border crosses the Danube twice more, closing another smaller pocket on the right and a larger pocket of territory (Srebrenica) on the left bank. All these parts of the territory on the left bank of the Danube belong to the cadastral municipality of Kopačevo (Fig. 4). načenim e disputed area About 4 km west-northwest of Apatin, the border again follows the course of the Danube, and next to the settlement closes the pocket territory on the left bank (Poluostrvo), then me anders again closing first a small pocket of Serbi an territory on the right bank, and then two more pockets of territory on the left bank (Zverinjak). Further south and southeast, the border crosses the Danube twice more, closing another smaller pocket on the right and a larger pocket of territory (Srebrenica) on the left bank. All these parts of the territory on the left bank of the Danube belong to the cadastral municipality of Kopačevo (Fig. 4). From a place about 2 km northwest of Aljmaš, at the confluence of the Drava and the Danube, the border follows the course of the Danube in the length of 94 km to the point between Ilok and Bač ka Palanka. On this stretch, it includes the Vukovar island within the cadastral municipality of Vukovar (Fig. 5) and Šarengrad island within the cadastral municipalities of Mohovo and Šarengrad (Fig. 6). načenim e disputed area Izvor: / Source: URL 12 prati tok u duljini od oko 3,5 km. Position and argumentation of the Republic of Croatia Na oko 4 km zapadno-sjeverozapadno od Apa tina, granica ponovno prati tok Dunava te uz samo naselje zatvara džep teritorij na lijevoj obali (Poluostrvo), zatim ponovno vijuga zatvarajući najprije jedan omanji džep srbijanskoga terito rija na desnoj obali, a nakon toga još dva džepa teritorija na lijevoj obali (Zverinjak). Dalje pre In all territorial disputes created by the dissolu tion of the socialist Yugoslavia, the Croatian po sition on borders is the same, and it is based on the fact that the Republic’s borders in the former country at the moment of independence became national. According to the official position of the 197 B. Vukosav, Z. Matijević 25/2 (2020) 177-208 ma jugu i jugoistoku granica još dvaput presijeca Dunav zatvarajući još jedan manji džep na de snoj i veći džep teritorija (Srebrenica) na lijevoj obali. Navedeni dijelovi teritorija na lijevoj obali Dunava pripadaju katastarskoj općini Kopačevo (Sl. 4.). Republic of Croatia on the border with Serbia, the northern segment of the border generally follows the course of the Danube. However, as already stated, the border line does not overlap precisely with the current course of the river, but rather intersects the river in several places, extending to a greater extent into the land are as on the left and to a lesser extent on the right bank. It is a border line that follows the border of cadastral municipalities, defined on the basis of the old course of the Danube. The results of the work of the Đilas Commission are considered from the Croatian perspective to be provisional conclusions aimed at generally determining the border between the two federal units, after which a detailed specifying of the border line had yet to be approached, bearing in mind the aforemen tioned cadastral boundaries, which was never done. The tentativeness of the conclusions of the Đilas Commission is best reflected in a series of disputes in the area after 1945, occurring in the zones of territorial pockets, as well as the Dan ube river islands that were also occasionally dis puted. Position and argumentation of the Republic of Croatia Croatia bases its position on international law, specifically on the principle of uti possidetis according to which the real situation territorial possession is transferred to the newly emerging state at the time of gaining independence, fol lowing the opinion of the Badinter Commission of 1991, according to which the borders of the former republics became state borders and bear ing in mind all the evidence of the previous func tional and administrative connection between the river islands and the territories on the left bank of the Danube with Croatia during the period of the socialist Yugoslavia (Klemenčić, Schofield, 2001; Matijević, 2020). Od mjesta na oko 2 km sjeverozapadno od Alj maša, na mjestu utoka Drave u Dunav, granica prati tok Dunava u duljini od 94 km do točke iz među Iloka i Bačke Palanke. Na tom potezu obu hvaća Vukovarsku adu u sastavu katastarske općine Vukovar (Sl. 5.) i Šarengradsku adu u sastavu kata starskih općina Mohovo i Šarengrad (Sl. 6.). Stajalište i argumentacija Republike Hrvatske U svim teritorijalnim sporovima nastalima ras padom socijalističke Jugoslavije, hrvatski stav o granicama je isti, a utemeljen je na činjenici da su republičke granice bivše države u trenutku osamostaljenja postale državnima. Prema službe nom stajalištu Republike Hrvatske o granici sa Srbijom, sjeverni segment granice načelno slijedi pravac toka Dunava. No, kako je rečeno, granič na crta nije istovjetna sa sadašnjim tokom rijeke, već granica na više mjesta presijeca rijeku zadirući u većoj mjeri u kopnena područja na lijevoj i u manjoj mjeri na desnoj obali. Riječ je o crti gra nice koja prati granicu katastarskih općina, defi niranih na temelju starog toka Dunava. Rezultati rada Đilasove komisije iz hrvatske se perspektive pritom smatraju privremenim zaključcima čija je svrha bila načelno odrediti razgraničenje između dviju federalnih jedinica, a nakon kojega je tek trebalo pristupiti detaljnom određenju granične crte imajući u vidu spomenute katastarske gra nice, što nikada nije učinjeno. Provizornost za ključaka Đilasove komisije najbolje se odražava u nizu sporova u spomenutom području nakon 1945. u područjima teritorijalnih džepova, kao i dunavskih ada koje su se našle u sličnoj teritori jalnoj diskrepanciji. Svoj stav Hrvatska temelji na međunarodnom pravu, konkretno na načelu uti possidetis prema kojemu se realno stanje posjedo vanja teritorija prenosi na novonastalu državu u trenutku stjecanja neovisnosti, nastavno na mi šljenje Badinterove komisije iz 1991. prema ko jemu su granice bivših republika postale državne M. Klemenčić and C. Schofield (2001) mention a number of examples of disputes over jurisdic tion over the disputed zones between Croatia and Vojvodina at the time of the Yugoslav state, most often at the level of local government, as well as documents proving that the areas on the left bank of the Danube, as well as the disputed river islands in the time of the former state, were most often managed by the Croatian republic and local au thorities. In 1950, for example, local authorities of Osijek district informed the republic authorities 198 25/2 (2020) 177-208 B. Vukosav, Z. Matijević of the then NR Croatia of the damage caused by floods in the Kenđija area on the left bank of the Danube, seeking financial support. Although the republic authorities did not react on this occasion, a document from 1965, regarding a similar situa tion after the flood in the same area, testifies that money was provided for the repair of damages by the Ministry of Agriculture of the Socialist Repub lic of Croatia. Stajalište i argumentacija Republike Hrvatske The authors also cite a document from 1961 according to which the Osijek National Council gave the area of Kenđija into a concession to the Hunting Association Batina. The document clearly lists the Kenđija area as part of the territory under the jurisdiction of the National Council in Osijek. granice, a imajući u vidu sve dokaze o prethodnoj funkcionalnoj i upravnoj povezanosti teritori ja i ada na lijevoj obali Dunava s Hrvatskom u vremenu socijalističke Jugoslavije (Klemenčić, Schofield, 2001.; Matijević, 2020.). M. Klemenčić i C. Schofield (2001.) navode niz primjera sporova zbog nadležnosti nad spornim zonama između Hrvatske i Vojvodine u vrijeme jugoslavenske države, najčešće na razini lokalne uprave, kao i dokumente koji dokazuju da su po dručjima na lijevoj obali Dunava, kao i spornim adama u vremenu bivše države većinom upravlja le hrvatske republičke i lokalne vlasti. Primjerice, 1950. godine lokalne vlasti Osječkog kotara izvi jestile su republičke vlasti tadašnje NR Hrvatske o šteti nastaloj poplavama u području Kenđije na lijevoj obali Dunava, tražeći financijsku potporu. Iako tom prigodom republičke vlasti nisu reagi rale, dokument iz 1965. u sličnoj situaciji nakon poplave u istom području svjedoči o tome da je za sanaciju štete novac osiguralo Ministarstvo poljo privrede SR Hrvatske. Autori navode i dokument iz 1961. prema kojemu je Narodno vijeće Osije ka dalo područje Kenđije u koncesiju lovačkom društvu Batina. U dokumentu se jasno navodi po dručje Kenđije kao dio teritorija Narodnog vijeća u Osijeku. It is also worth mentioning the documents that clearly point to disagreements over territorial ju risdiction over the Danube river islands during the time of socialist Yugoslavia, as well as docu ments on the positions of Croatian and Serbian republican authorities and Vojvodina provincial authorities on this issue. For example, M. Kle menčić and C. Schofield (2001) cite a 1947 com plaint by the Vojvodina Agriculture Executive Council to the competent Ministry of Forestry in Serbia over the refusal of local authorities in Vukovar to leave four river islands to the manage ment of AP Vojvodina, citing the Consritutional law of AP Vojvodina on the border between Cro atia and Serbia. In the complaint, they ask the Serbian ministry to raise the problem to the level of the Federal Ministry of Forestry. Stajalište i argumentacija Republike Hrvatske Štambuk-Škalić (1995, 318) quotes the full text of a 1947 letter by the Vukovar National Committee to the Presidency of the Government of the National Republic of Croatia on the man agement and use of forest areas on the Danube islands, which corresponds to the standpoint that the Croatian authorities then took regarding the border: M. Štambuk-Škalić (1995., 318) citira cjelovit tekst dopisa Kotarskoga narodnog odbora Vuko var Predsjedništvu Vlade NR Hrvatske iz 1947. o upravljanju i korištenju šumskih predjela ada na Dunavu, a što odgovara i stavu koji su hrvatske vlasti potom zauzele o granicama: ‘Since they exist, the Vukovar (Ćifut) island, Mo hovski rit, the Šarengrad island and Hagel, regard less of whether they are located on the left or right side of the course Danube, have been in the area of cadastral municipalities on the right side of the Danube, i.e. on the territory of the NR Croatia. The Danube, changing its course, changed its po sition in relation to the Danube islands, but never crossed from its base area. In addition to the above, the last mentioned three islands have shifted to the left side of the course of Danube in recent years by artificial and unnatural means, because a canal was dug between Mohov and Šarengrad to shorten the Danube flow. This only cut the way for the boats not to go around the Danube meanders. This can not possibly be the reason for these islands to be handed over to an area that has never been related to them, nor has it managed these islands at any time and which cannot justify its request. „Vukovarska (Ćifutska) ada, Mohovski rit, Ša rengradska ada i Hagel od kako postoje, bez ob zira da li se nalaze na lijevoj ili desnoj strani živog Dunava, bile su na području katastralnih općina s desne strane Dunava, tj. na području NR Hrvat ske. Dunav mijenjajući svoj tok mijenjao je svoj položaj prema dunavskim otocima, ali nikada nije prelazio sa svog temeljnog područja. Posljednje tri ade osim navedenog prešle su posljednjih godina na lijevu stranu živog Dunava umjetnim nenarav nim putem, a to s razloga, što je između Mohova i Šarengrada prokopan kanal radi skraćenja toka Dunava. Time je samo presječen put da lađe ne obilaze dunavske zavoje. Stajalište i argumentacija Republike Hrvatske Among the documents attached to the complaint is a corre spondence between the aforementioned Execu tive Council and the Ministry of Forestry of the National Republic of Croatia, which was previ ously asked to intervene in the matter and which refused to do so. The Federal Ministry informed the Serbian ministry in a consequent commu nication that it recommended that the relevant ministries of Serbia and Croatia resolve the dis pute by mutual agreement, but that the Croatian Ministry maintained its initial position that the islands territorially belong to Vukovar district and that they cannot be handed over to the authori ties of AP Vojvodina until the relevant commis sions clearly delineate the border between the two republics (Klemenčić, Schofield, 2001). This Valja spomenuti i dokumente koji jasno upu ćuju na nesuglasice zbog teritorijalne nadležnosti nad dunavskim adama u vremenu socijalističke Ju goslavije, kao i dokumente o stavovima hrvatskih, odnosno vojvođanskih i srbijanskih republičkih i pokrajinskih vlasti o navedenom pitanju. Primje rice, M. Klemenčić i C. Schofield (2001.) navode pritužbu Izvršnog vijeća za poljoprivredu Vojvo dine iz 1947. nadležnom Ministarstvu šumarstva u Srbiji zbog odbijanja lokalne vlasti u Vukovaru da prepuste četiri ade na upravljanje AP Vojvodi ni pozivajući se na Ustavni zakon AP Vojvodine o granici između Hrvatske i Srbije. U pritužbi traže od srbijanskoga ministarstva da problem podignu na razinu Saveznog ministarstva šumarstva. Među dokumentima priloženim pritužbi je i korespon dencija između navedenoga Izvršnog vijeća i Mini starstva šumarstva NR Hrvatske od kojega su pret hodno tražili da intervenira, a koje je to odbilo. Savezno ministarstvo informiralo je u posljedičnoj komunikaciji ministarstvo NR Srbije da je prepo 199 B. Vukosav, Z. Matijević 25/2 (2020) 177-208 ručilo da relevantna ministarstva Srbije i Hrvatske riješe spor sporazumom, ali da je hrvatsko mini starstvo ostalo pri svojemu prvotnom stavu da ade teritorijalno pripadaju vukovarskom kotaru te da se ne mogu predati na korištenje vlastima AP Voj vodine sve dok relevantne komisije jasno ne odre de granicu između dviju republika (Klemenčić, Schofield, 2001.). Ova rečenica posebno je važ na jer odražava tadašnji stav vlasti NR Hrvatske o zaključcima Đilasove komisije, odnosno o viđenju pravnog stanja dunavskog dijela istočne hrvatske granice. last sentence is particularly important because it reflects the position of the Croatian authorities at the time on the conclusions of the Đilas Com mission, i.e. on the perceived legal state of the Danube part of the eastern Croatian border. M. Stajalište i argumentacija Republike Hrvatske They also cite the re port of Hrvatske šume on the islands’ management from 1970, when part of Šarengrad island eroded and endangered the safety of navigation on the Danube, after which Hrvatske šume sent workers from Vukovar to intervene (Klemenčić, Schof ield 2001 according to Reba, 1999). Considering relevant documents, it is worth to mention the ex istence of two reports of the Republic’s Office for Public Administration in Zagreb, one of which, from the 1970s, proves that the disputed areas were managed by Croatia, including the collection of taxes. Vrijedi istaknuti još neke činjenice koje spomi nju autori: 1947. godine šume na području Ša rengrada su nacionalizirane, a Šarengradska ada je dodijeljena vukovarskom ogranku Hrvatskih šuma na upravljanje. Također, navode i izvještaj Hrvatskih šuma o upravljanju adama iz 1970., kada je dio Šarengradske ade erodirao i ugrozio sigurnost plovidbe Dunavom, nakon čega su Hr vatske šume poslale radnike iz Vukovara u inter venciju (Klemenčić, Schofield, 2001., prema Reba, 1999.). Od relevantnih dokumenata treba spomenuti i postojanje dvaju izvješća Republič kog ureda za javnu upravu u Zagrebu, od kojih jedan iz 1970-ih dokazuje da je spornim područ jima upravljala Hrvatska, uključujući i prikuplja nje poreznih davanja. These cases clearly indicate not only the func tional and administrative connection of the terri tory on the left bank of the Danube with the then NR, i.e. SR Croatia, but also the position of the federal authorities in Yugoslavia on the border be tween Croatia and Serbia (Vojvodina) at the time. It was indirectly but quite clearly obvious that they considered the conclusions of the Đilas Commis sion, at least regarding their relevance to the Dan ube, tentative solutions rather than a definitive delimitation. Navedeni slučajevi jasno upućuju ne samo na funkcionalnu i upravnu povezanost teritorija na lijevoj obali Dunava s tadašnjom NR, odnosno SR Hrvatskom, nego i na tadašnji stav federalnih vlasti u Jugoslaviji o granici između Hrvatske i Sr bije (Vojvodine) pri čemu je neizravno ali sasvim jasno da je zaključke Đilasove komisije, barem što se tiče njihove relevantnosti u odnosu na Dunav, smatrala provizornim rješenjima, a ne konačnom delimitacijom. Stajalište i argumentacija Republike Hrvatske To nikako ne može biti razlog, da se ti otoci predaju području koje nije na njih nikada bilo upućeno, niti je tim otocima upravljalo u nikoje vrijeme i koje ne može svoje traženje ničim opravdati. The population of this area has always used these islands, and its existence has been related to the islands, as well as to other parts of its area. All these islands (ada) are mostly overgrown with forests, and are considered forest areas. Vukovar is land is managed by the Federal Agricultural Prop erty – Management of Vukovar, and other by the National Committee of the Vukovar district.’ Ovo područno žiteljstvo od vajkada uživa te ade, te je njegova egzistencija vezana s tim adama, kao i ostalim površinama svog hatara. Od navedenih otoka (ada) svi su uglavnom obra sli šumama, te se smatraju šumskim površinama. Vukovarskom adom upravlja Savezno poljopri vredno dobro – Direkcija Vukovar, a ostalim ko tarski NO Vukovar.“ It is also important to emphasize the position of the federal ministry, which, according to M. Kle menčić and C. Schofield (2001), emphasizes in the correspondence that followed that the Serbian ministry’s position is that the Law on the Estab lishment and Organisation of AP Vojvodina is not relevant in this regard, since it does not explicitly state that the aforementioned border necessari Važno je istaknuti i stav saveznog ministar stva koje, prema M. Klemenčiću i C. Schofieldu (2001.), u korespondenciji koja je uslijedila nagla šava kako je stav srbijanskoga ministarstva o tome 200 25/2 (2020) 177-208 B. Vukosav, Z. Matijević ly follows the middle of the main course of the Danube. The Federal Ministry also confirms that, consequently, the dispute can only be resolved by precisely delimiting the federal units of Croatia and Serbia. da Zakon o ustanovljenju i ustrojstvu AP Vojvo dine nije u tom smislu relevantan jer iz njega izri jekom ne proizlazi da spomenuta granica nužno prolazi sredinom glavnog toka Dunava. Federalno ministarstvo također potvrđuje da se, slijedom na vedenoga, spor može razriješiti jedino preciznim razgraničenjem federalnih jedinica Hrvatske i Sr bije. Some other facts mentioned by the authors should also be noted: in 1947 forests in the area of Šarengrad were nationalized, and the Šarengrad island was assigned to be managed by the Vukovar branch of Hrvatske šume. 5 Talveg (njem. Thalweg) – srednja crta glavnoga plovnog ka nala, odnosno krivudava spojna crta najdubljih točaka u koritu plovnih rijeka; najsigurniji nizvodni plovni put koji je istovreme no i granica između dviju susjednih država (Cvitanović, 2002.). Stajalište i argumenti Republike Srbije f p f The Serbian view is that the national border be tween Croatia and Serbia should be the current course of the Danube river, that is, the line that passes through the middle of the thalweg5 (since the Danube is a navigable river). Serbia bases its arguments on the practice of the international law on demarcation on rivers according to which the middle of the river flow is usually considered a bor der (in the navigable rivers it is usually the above mentioned thalweg line), as well as on the already mentioned Act on the Establishment and Organi zation of AP Vojvodina from 1945. In this regard, given the aforementioned practice of the interna tional law on demarcation, the Serbian side legally interprets the conclusions of the Djilas Commis sion as definitive. Srbijansko je stajalište da međudržavnom grani com Hrvatske i Srbije treba smatrati sadašnji tok rijeke Dunav, odnosno crtu koja prolazi sredinom talvega5 (s obzirom na to da je Dunav plovna ri jeka). Srbija svoju argumentaciju temelji na me đunarodno-pravnoj praksi o razgraničenju na ri jekama prema kojoj se za granicu obično koristi sredina riječnog toka (odnosno kod plovnih rijeka već spomenuta crta talvega), kao i na već spome nutom Zakonu o ustanovljavanju i ustrojstvu AP Vojvodine iz 1945. Prema stajalištu Srbije, rješe nje Đilasove komisije definira Dunav kao granicu riječima „Privremena granica između Vojvodine i Hrvatske treba da ide: od mađarske granice, rekom Dunavom do međe između sela Bačko Novo Selo i Bukin“ (Jelić, 1991., 17). U tom smislu, imajući u vidu i navedenu međunarodno-pravnu praksu o razgraničenju, srbijanska strana pravno tumači za ključke Đilasove komisije kao konačne. The principle of international law on demar cation on navigable rivers, according to which in international practice thalweg is most often des ignated as the demarcation line, has proved to be the best solution for river traffic during low water levels in many cases. Therefore, this is the strong est argument of the Republic of Serbia, with the most relevance in the international law. 5 Thalweg (germ.) – the middle line of the main waterway, i.e. the curving connecting line of the deepest points in the river bed; the safest downstream waterway, which is also the border betwe en the two neighbouring countries (Cvitanović, 2002). teritorija. Position and arguments of the Republic of Serbia Stajalište i argumentacija Republike Hrvatske Bearing in mind the legal ‘incompleteness’ of the conclusions of the Đilas Commission with re gard to the cadastral structure, as well as the above mentioned documents testifying to the views of individual political entities in the former State on the dispute, parts of the territory on the left bank of the Danube, the territorial pockets and islands in question can at least be considered as the area where the border has yet to be established. Tak ing into account the examples that testify to the management of Croatia in these areas, i.e. the in frastructure connection of the disputed zones with Croatia, and applying the principle of uti possidetis, there is also a clear legal basis that the disputed areas can today be considered as parts of Croatian Imajući u vidu pravnu „nedorečenost“ zaklju čaka Đilasove komisije s obzirom na katastarski ustroj, kao i navedene dokumente koji svjedoče o stavovima pojedinih političkih subjekata u biv šoj državi o navedenom sporu, dijelovi teritori ja na lijevoj obali Dunava, navedeni teritorijalni džepovi i sporne ade mogu se u najmanju ruku smatrati područjem na kojemu tek treba utvrditi granicu. Uzimajući u obzir i primjere koji svjedo če o upravljanju Hrvatske tim područjima, odno sno infrastrukturnoj povezanosti spornih zona s Hrvatskom te primjenjujući načelo uti possidetis, postoji i jasna pravna osnova da se sporna pod ručja danas mogu smatrati dijelovima hrvatskoga 201 B. Vukosav, Z. Matijević 25/2 (2020) 177-208 Vrednovanje argumenata obiju strana The arguments of Croatia and Serbia show that both invoke certain principles of the international law, as well as certain historical and geographical factors and events by which they contextualize them. But in doing so, both Croatia and Serbia in sist on the ‘weight’ of certain arguments that corre spond to their territorial interests. The arguments of both countries can be summed up as follows: Iz argumentacija Hrvatske i Srbije vidljivo je da se obje pozivaju na određena međunarodno-prav na načela, kao i na pojedine historijsko-geografske čimbenike i događaje kojima ih kontekstualiziraju. No pritom i Hrvatska i Srbija inzistiraju na „te žini“ određenih argumenata koje odgovaraju nji hovim teritorijalnim interesima. Argumenti obiju zemalja mogu se sažeti na sljedeći način: – Croatia: Badinter Commission Opinion No. 3, and the principle of uti possidetis applied in the context of the borders of cadastral municipalities, as well as in the context of Croatia’s infrastructural and administrative connection with the disputed areas between 1945 and independence. – Hrvatska: Mišljenje Badinterove komisije br. 3 te načelo uti possidetis u kontekstu granica kata starskih općina, kao i u kontekstu infrastrukturne i upravne povezanosti Hrvatske sa spornim područ jima u razdoblju od 1945. do osamostaljenja. – Serbia: the principle of uti possidetis in the con text of the conclusions of the 1945 Đilas Commis sion, the Act establishing the AP Vojvodina, and the principle of the international law on demar cation of countries on waterway rivers along the thalweg line. – Srbija: načelo uti possidetis u kontekstu zaklju čaka Đilasove komisije iz 1945., Zakon o usta novljenju AP Vojvodine te međunarodno-pravno načelo razgraničenja država na plovnim rijekama crtom talvega. Općenito, obje zemlje suglasne su o zaključa ku Badinterove komisije iz 1992., pri čemu je već spomenuto načelo uti possidetis prihvaćeno kao ključno načelo. Drugim riječima, u trenutku osamostaljenja pojedinih republika, dotadašnje republičke granice postaju državne. No ključna je razlika u interpretaciji spomenutog načela, pa se postavlja pitanje: Koja i kakva granična crta se može smatrati „naslijeđenom granicom“ s obzi rom na historijsko-geografske čimbenike i dostu pnu dokumentaciju? Overall, both countries agree on the conclusions of the Badinter Commission from 1992, with the already mentioned principle of uti possidetis ac cepted as a key principle. In other words, at the moment of independence of individual republics, the republics’ borders became national borders. Stajalište i argumenti Republike Srbije In essence, Serbia considers that the (new) Danube flow was defined as a boundary by the conclusions of the Djilas Commission in 1945, which was confirmed by the law on the organisation of Vojvodina in the same year, and that in this sense the principle of uti possidetis must be applied with regards of these two facts, while respecting the aforementioned thalweg principle as a line of demarcation on navigable riv ers. Regarding the natural and geographical fact of the Danube flow naturally shifting westwards, Serbia’s position is that gradual and ‘“evolutionary’ changes in the flow of rivers also change the state border (Dimitrijević, 2003). Načelo međunarodnoga prava o razgraničenju na plovnim rijekama prema kojemu se za crtu razgraničenja najčešće uzima talveg u međuna rodnoj praksi u mnogim se slučajevima pokazalo najboljim rješenjem s obzirom na prometovanje rijekama u vremenu niskog vodostaja. Stoga je to ujedno i najsnažniji argument Republike Srbije, uz najviše uporišta u međunarodnom pravu. U suštini, Srbija smatra da je (novi) tok Dunava kao granica definiran zaključcima Đilasove komisije 1945., potvrđen zakonom o ustrojstvu Vojvodine iste godine te da se u tom smislu načelo uti po ssidetis mora primijeniti s obzirom na te dvije či njenice, uz poštovanje spomenutog načela talvega kao crte razgraničenja na plovnim rijekama. Što se tiče prirodno-geografske činjenice prirodnog pomicanja toka Dunava prema zapadu, stajalište je Srbije da postupne i „evolutivne“ promjene toka rijeka mijenjaju i državnu granicu (Dimitri jević, 2003.). According to this position, all areas of the Cro atian cadastral municipalities on the eastern bank of the Danube (Karapanđa/Kenđija, Blaževica/ Prema navedenom stajalištu, sva su područja hr 202 25/2 (2020) 177-208 B. Vukosav, Z. Matijević Kolonđoš, Zmajevac I and II, Poluostrvo, Varošviz, Zverinjak, Srebrenica) are actually parts of the territory of the Republic of Serbia, while smaller pockets of cadastral Serbian territory, the largest of which is Siga belong to Croatia. The same would be applied in case of the river islands Vukovar and Šarengrad, which Serbia considers to be integral parts of its national territory. vatskih katastarskih općina na istočnoj obali Du nava (Karapanđa/Kenđija, Blaževica/Kolonđoš, Zmajevac I. i II., Poluostrvo, Varošviz, Zverinjak, Srebrenica) zapravo dijelovi teritorija Republike Srbije, dok manji džepovi katastarski srbijanskoga teritorija od kojih je najveća Siga pripadaju Hr vatskoj. Isto je i s Vukovarskom i Šarengradskom adom za koje Srbija smatra da su integrali dijelovi njezina državnog teritorija. Vrednovanje argumenata obiju strana In the case of the Danube region, it is the cadastral evidence that can be a criterion indicating the historical and factual affiliation of the disputed territories in Croatia. Furthermore, the above mentioned doc umentation testifies that these territories in many segments were under the jurisdiction of the NR (SR) Croatia. Važno je razmotriti i činjenicu prirodnog pomi canja toka Dunava prema zapadu zbog Coriolisove sile. Iako postoje pravni presedani koji upućuju na to da je u određivanju eventualne granične kom penzacije bitna brzina promjene (je li promjena toka nagla ili evolutivna), međunarodna praksa u sporovima između neovisnih država, posebice u domeni međunarodnih pravnih tijela koja su po sredovala u sporovima, bila je poticanje bilateral nih sporazuma i zajedničkih rješenja s naglaskom na učvršćivanje granica na rijekama (Donaldson, 2011.; Matijević, 2020.). To u praksi znači da, unatoč određenim presedanima na koje se poziva Srbija, a koji upućuju na to da se u slučaju postu pne promjene toka rijeke kojim ide granica, pri stupa korekciji granice u skladu s rijekom, postoji praksa favoriziranja međusobnog dogovora zema lja među kojima postoji takva granica radi prona laženja rješenja kojim bi se išlo prema učvršćivanju granične crte (npr. antropogenim učvršćivanjem vodotoka). It is also important to consider the fact that the Danube flow is naturally shifting westwards due to Coriolis’ force. While there are legal precedents to suggest that the pace of change (whether the change in flow is abrupt or evolutional) is essen tial in determining possible border compensa tion, international practice in disputes between independent states, in particular in the domain of international legal bodies that have mediated disputes, was to foster bilateral agreements and common solutions focusing on making the border stationary (Donaldson, 2011; Matijević, 2020). In practice, this means that, despite certain prec edents invoked by Serbia, which indicate that, in the event of a gradual change in the flow of the river which the border follows, border correction is approached in a way that the border changes with the river course, there is a practice of favouring mutual agreement between countries which share such a border, with the aim of finding a solution that would go towards fixing the border line (e.g. anthropogenic fixing of the watercourse). Vrednovanje argumenata obiju strana But the key difference is in the interpretation of said principle, so the question arises – which and what boundary line can be considered an ‘inher ited boundary’ with regard to historical and geo graphical factors and available documentation? Jasno je da stajalište Republike Srbije naglašava opću međunarodno-pravnu praksu o razgraničenju na rijekama, kao i načelno spominjanje Dunava kao granice u tekstu rješenja „Đilasove komisije“ iz 1945. S obzirom na plovnost Dunava i njegovu međunarodnu važnost, to je ujedno i najsnažniji argument srbijanske strane u sporu. S druge stra On the one hand, it is clear that the position of the Republic of Serbia underlines the general prac tice of the international law on demarcation on rivers, as well as the general mention of the Dan ube as a border in the text of the 1945 solution of the Đilas Commission. Furthermore, the Dan 203 25/2 (2020) 177-208 B. Vukosav, Z. Matijević ube’s international importance as a waterway is the Serbian side’s strongest argument in the dispute. On the other hand, cadastral evidence, as well as numerous documentation, prove Croatia’s infra structural, administrative and economic connec tions with the areas on the left bank of the current Danube flow. ne, katastarski dokazi, kao i brojna dokumentacija svjedoče, pak, o infrastrukturnoj, upravnoj i gos podarskoj povezanosti Hrvatske s područjima na lijevoj obali trenutačnog toka Dunava. M. Klemenčić i C. Schofield (2001.) navode kako je granica između Hrvatske i Bosne i Her cegovine vrlo precizno određena pri čemu su se nadležne vlasti dviju republika kod spornih od sjeka koristile upravo katastarskim dokumentima kao sredstvom preciznog razgraničenja. S tim u vezi, korištenje katastarskih dokaza može se sma trati „državnom praksom“, koja je u slučaju raz graničenja Hrvatske i Srbije izostala. Upravo ovi katastarski dokazi u slučaju Podunavlja mogu biti kriterij koji upućuje na povijesnu i činjeničnu pri padnost spornih područja Hrvatskoj. Nadalje, na vedena dokumentacija svjedoči da su ti teritoriji u mnogim segmentima bili u nadležnosti NR (SR) Hrvatske. M. Klemenčić and C. Schofield (2001) note that the border between Croatia and Bosnia and Herzegovina is very precisely determined, with the competent authorities of the two republics using cadastral documents in the disputed sections as a means of precise demarcation. In this regard, the use of cadastral evidence can be considered ‘state practice’, which in the case of the demarcation of Croatia and Serbia has been absent. Vrednovanje argumenata obiju strana U skladu s tim, jasno je da bi u eventualnom međunarodno-pravnom postupku katastarske granice i dokumentacija o upravnoj nadležnosti u vrijeme bivše države mogle biti presudni ar gumenti i kriteriji kod međusobnog razgraniče nja dviju država kojima se hrvatska strana u bilo kakvoj arbitraži može ravnopravno suprotstaviti načelu crte talvega. Stoga se može zaključiti da Accordingly, it is clear that in any international legal procedure cadastral borders and administra tive jurisdiction documentation from the time of 204 25/2 (2020) 177-208 B. Vukosav, Z. Matijević the former State could be the key arguments and criteria for the mutual delimitation of two coun tries, with which the Croatian side can challenge the principle of the thalweg line on equal grounds in any arbitration. It can therefore be concluded that there is a strong basis upon which a future final demarcation could follow the borders of ca dastral municipalities, and with the necessary legal corrections that would regulate the unhindered right to sail on the Danube for both countries. postoji snažna osnova na temelju koje bi buduće konačno razgraničenje moglo pratiti granice kata starskih općina, a uz nužne pravne korekcije koje bi regulirale nesmetano pravo plovidbe Dunavom za obje države. ZAKLJUČAK Historijsko-geografska analiza razvoja istočne granice hrvatskih zemalja u prvom dijelu rada upućuje na vrlo dinamičan historijsko-geografski i teritorijalni razvoj hrvatskoga Podunavlja koji se odrazio na promjenama granica nekoliko puta. S obzirom na povijesno stanje, ključne promjene koje su zadesile istočnu granicu hrvatskih zemalja dogodile su se nakon Prvoga svjetskog rata u vri jeme Kraljevine SHS/Jugoslavije, te nakon Drugo ga svjetskog rata kada je iz njihova sastava trajno izuzet prostor istočnoga Srijema. S druge strane, teritorijalni dobitak za Hrvatsku u prostoru Podu navlja bila je Baranja, odnosno „trokut“ teritorija omeđen Dunavom, Dravom i hrvatsko-mađar skom granicom, nekadašnji dio mađarske povije sne regije Baranya. Imajući u vidu ne samo omjer teritorijalnih dobitaka i gubitaka već i gospodarsku razvijenost i potencijal priključenih i izgubljenih područja, načelno je ishod teritorijalnih promjena bio razmjerno nepovoljan te je u interesu suvre mene Republike Hrvatske stabilizacija graničnog stanja i uređenje odnosa sa susjednim zemljama, posebno oko područje rijeke Dunav koja posjedu je veliko geostrateško značenje za čitavu Srednju i Jugoistočnu Europu. CONCLUSION The historical and geographical analysis of the development of the eastern border of the Croatian lands in the first part of the paper points to the very dynamic historical, geographical and territo rial development of the Croatian Danube region, which has been reflected in the changes of borders on several occasions. Given the historical situation, the key changes that affected the eastern border of Croatian countries occurred after the First World War at the time of the Kingdom of SHS/Yugosla via, and especially after World War II, when the area of Eastern Syrmia was permanently excluded from their territories. Moreover, the territorial gain for Croatia in the Danube region was Baranja, i.e. the ‘triangle’ of territory bordered by the Danube, the Drava and the Croatian-Hungarian border, formerly a part of Hungary’s historic Baranya re gion. Bearing in mind not only the ratio of territo rial gains and losses, but also the economic devel opment and potential of connected and lost areas, in principle the outcome of territorial changes was relatively unfavourable, and it is in the interest of the modern Republic of Croatia to stabilize the border situation and regulate relations with the neighbouring countries, especially around the Danube River area, which has a great geostrategic significance for the whole of Central and South Eastern Europe. U drugom dijelu rada provedena je analiza gra ničnog spora između Hrvatske i Srbije. Potvrđena je upravna, gospodarska i infrastrukturna pove zanost spornih područja s Hrvatskom na temelju analize historijsko-geografskih činjenica. Dijelo vi granice uz Dunav, posebice „najmlađi“ dio od Mađarske granice do ušća Drave u Dunav, a koji je postao republičkom granicom 1945. sadrži naj više spornih područja na lijevoj obali Dunava za koja se nedvojbeno može utvrditi da pripadaju katastarskim općinama u Hrvatskoj. Također, niz dostupne dokumentacije upućuje na upravljanje i gospodarenje hrvatskih republičkih tijela spornim područjima kako u teritorijalnim džepovima na li In the second part of the paper, an analysis of the border dispute between Croatia and Serbia was carried out. The administrative, economic and in frastructural connection of the disputed areas to Croatia was confirmed on the basis of an analysis of historical and geographical facts. Parts of the 205 25/2 (2020) 177-208 B. Vukosav, Z. IZVORI I LITERATURA Amanović, B. (2013): Teritorijalne promjene u Hrvatskoj od 1941. do 1991., Diplomski rad, Sveučilište Josipa Jurja Strossmayera u Osijeku, Filozofski fakultet; pp. 74. Bagarić, P. (2017): Hrvatske granice nakon Drugoga svjetskog rata 1945.-1956., Hrvatska revija Bara, M. (2007): Đilasova komisija i sudbina bačkih Hrvata, Pro Tempore, 4 (4), 47-58. Boban, Lj. (1995): Hrvatske granice od 1918. do 1993. godine, Školska knjiga, Zagreb, pp. 68. Cvitanović, A. (2002): Geografski rječnik. Hrvatsko geografsko društvo – Zadar, Filozofski fakultet u Za dru, Matica Hrvatska – Zadar, ZADIZ d.o.o. Zadar, pp. 683. Dimitrijević, D. (2003): Međunarodno-pravni tretman graničnih sporova na prostoru bivše Jugoslavije, Međunarodni problemi, 55 (3-4), 354-373. p Donaldson, J. W. (2011): Paradox of the Moving Boundary: Legal Heredity of River Accretion and Avulsion, Water Alternatives, 4 (2), 155-170. Jelić, I. (1991): O nastanku granice između Hrvatske i Srbije, Časopis za suvremenu povijest, 23 (1-3), 1-32. Klemenčić, M., Schofield, C. (2001): War and Peace on the Danube: The Evolution of Croatia-Serbia Boundary, Boundary and territory briefing, 3 (3), Durham University, pp. 61. Kraljević, E. (2007): Prilog za povijest uprave: Komisija za razgraničenje pri Predsjedništvu Vlade Narod ne Republike Hrvatske 1945.-1946., Arhivski vjesnik, 50 (1), 121-130. Magaš, D. (2013): Geografija Hrvatske, Sveučilište u Zadru (Odjel za geografiju) i Meridijani, Zadar, Sa mobor, pp. 597. Matijević, Z. (2020): Suvremene političko-geografske implikacije razvoja državne granice u hrvatskom Podu navlju, Diplomski rad, Sveučilište u Zadru, Odjel za geografiju, Zadar, pp. 72. Mrduljaš, S. (2017): Etnička struktura Istočnog Srijema (1921.) i razgraničenje između Hrvatske i Srbije (1945.-1947.), Scrinia Slavonica, 17, pp. 181-210. Pavličević, D. (1993): Hrvatske granice u podunavlju, u: Slavonija, Srijem, Baranja i Bačka,( ur. Sekulić, A.), Matica hrvatska, Zagreb, 9-31. Pavličević, D. (1998): Kratka politička i kulturna povijest Hrvatske, Hrvatski informativni centar, Zagreb, pp. 76. Radelić, Z. (2006): Hrvatska u Jugoslaviji 1945.-1991. Od zajedništva do razlaza, Školska knjiga, Zagreb, pp. 701. Reba, M. (1999): Croatia – Yugoslavia boundary on the river Danube, neobjavljeni magistarski rad, Dur ham: International Boundaries Research Unit, Department of Geography, University of Durham. Roić, M., Paar, R. (2018): 200 godina katastra u Hrvatskoj, Zbornik radova – VI. Hrvatski kongres o kata stru (ur. M. Raić), Hrvatsko geodetsko društvo, Zagreb, 37-50. Sršan, S. (2003): Sjeveroistočne granice Hrvatske, Osijek: Državni arhiv u Osijeku, pp.103. Štambuk-Škalić, M. (1995): Hrvatska istočna granica u dokumentima 1945-1947., Fontes: Izvori za hr vatsku povijest, 1 (1), 153-329. CONCLUSION Matijević Danube border, especially the ‘youngest’ segment that stretches from the Hungarian border to the mouth of the Drava river into the Danube, which became the republic border in 1945, contain the most of the disputed areas on the left bank of the Danube, which can undoubtedly be found to be long to the Croatian cadastral municipalities. In addition, a lot of available documentation indi cates the jurisdiction of the Croatian republic and local bodies in the disputed areas both in case of the territorial pockets on the Danube’s left bank and in the case of the two river islands (Vukovar and Šarengrad). Therefore, Croatia’s historical and legal arguments in the border dispute with Serbia have a valid historical, geographical and inter national legal basis, and the Republic of Croatia should continue to insist on them in the process of reaching any bilateral agreement on the border, as well as in the case of a possible legal process, es pecially bearing in mind the protection of its own national and territorial interests. jevoj strani Dunava, tako i na dvjema adama (Vu kovarska i Šarengradska). Stoga povijesni i pravni argumenti Hrvatske u graničnom sporu sa Srbijom imaju valjanu historijsko-geografsku i međunarod no-pravnu osnovu te Republika Hrvatska na njima treba nastaviti inzistirati kako u procesu postizanja bilo kakva bilateralnog sporazuma o granici tako i u eventualnom pravnom procesu, posebno imajući u vidu zaštitu vlastitih nacionalnih i teritorijalnih interesa. 206 25/2 (2020) 177-208 B. Vukosav, Z. Matijević IZVORI I LITERATURA URL 1, Što predstavlja Schengenski prostor?, Republika Hrvatska, Ministarstvo pravosuđa i uprave, https:// uprava.gov.hr/sto-predstavlja-schengenski-prostor-14047/14047, 12. 11. 2020. URL 2, Zemaljsko antifašističko vijeće narodnog oslobođenja Hrvatske, Hrvatska enciklopedija, mrež no izdanje, Leksikografski zavod Miroslav Krleža, 2020., http://www.enciklopedija.hr/Natuknica. aspx?ID=67105, 22. 10. 2020. p URL 3, Ustav Narodne Republike Hrvatske (1947.), Wikizvor https://hr.wikisource.org/wiki/Ustav_Na rodne_Republike_Hrvatske_(1947.), 3. 11. 2020. URL 4, Granični spor s Hrvatskom usporit će put Srbije u EU, Večernji list, https://www.vecernji.hr/vijesti/ granicni-spor-s-hrvatskom-usporit-ce-put-srbije-u-eu-154535, 4. 11. 2020. URL 5, Deklaracija o proglašenju suverene i samostalne Republike Hrvatske (25. lipnja 1991.), Hrvatski sabor, https://www.sabor.hr/hr/deklaracija-o-proglasenju-suverene-i-samostalne-republike-hrvatske- 207 B. Vukosav, Z. Matijević 25/2 (2020) 177-208 25-lipnja-1991, 1. 4. 2020. p j URL 6, uti possidetis, Hrvatska enciklopedija, Leksikografski zavod Miroslav Krleža, 2020. http://www. enciklopedija.hr/Natuknica.aspx?ID=63502, 7. 4. 2020. URL 7, Granica Hrvatske i Srbije nije Dunav, već katastarska općina, Večernji list, https://www.vecernji.hr/ vijesti/granica-hrvatske-i-srbije-nije-dunav-vec-katastarska-opcina-1184569, 1. 4. 2020. URL 8, Croatia–Serbia border dispute, Wikipedia, https://en.wikipedia.org/wiki/ Croatia%E2%80%93Serbia_border_dispute, 1. 4. 2020. URL 9, Granica Srbije i Hrvatske na Dunavu dogovorom ili u Den Haagu – mediji, Ius info, https://www. iusinfo.hr/aktualno/dnevne-novosti/21358, 1. 4. 2020. URL 10, Hrvatsko selo zaglavio u Srbiji, Jutarnji list, https://www.jutarnji.hr/vijesti/hrvatska/hrvatsko-selo- zaglavilo-u-srbiji-tuzna-prica-o-zaboravljenom-mjestu-u-sastavu-rh-ali-s-lijeve-strane-dunava-sve-nas- vise-uzima-drzavljanstvo-srbije-5635511, 6. 11. 2020. URL 10, Hrvatsko selo zaglavio u Srbiji, Jutarnji list, https://www.jutarnji.hr/vijesti/hrvatska/hrvatsko-selo- zaglavilo-u-srbiji-tuzna-prica-o-zaboravljenom-mjestu-u-sastavu-rh-ali-s-lijeve-strane-dunava-sve-nas- vise-uzima-drzavljanstvo-srbije-5635511, 6. 11. 2020. URL 11, Uređena zemlja, Republika Hrvatska, Ministarstvo pravosuđa i uprave, Državna geodetska upra va, https://oss.uredjenazemlja.hr/public/cadServices.jsp?action=dkpViewerPublic, 6. 11. 2020. URL 11, Uređena zemlja, Republika Hrvatska, Ministarstvo pravosuđa i uprave, Državna geodetska upra va, https://oss.uredjenazemlja.hr/public/cadServices.jsp?action=dkpViewerPublic, 6. 11. 2020. URL 12, Google Earth, Google, https://earth.google.com/web, siječanj – rujan 2020. 208
https://openalex.org/W2954889917	https://cyberleninka.ru/article/n/angular-photometry-of-biological-tissue-by-ellipsoidal-reflector-method/pdf	English	null	Angular Photometry of Biological Tissue by Ellipsoidal Reflector Method	Pribory i metody izmerenij	2,019	cc-by	5,884	Angular Photometry of Biological Tissue by Ellipsoidal Reflector Method M.A. Bezuglyi, N.V. Bezuglaya, A.V. Ventsuryk, K.P. Vonsevych National Technical University of Ukraine «Igor Sikorsky Kyiv Polytechnic Institute», Peremohy Ave., 37, Kyiv 03056, Ukraine M.A. Bezuglyi, N.V. Bezuglaya, A.V. Ventsuryk, K.P. Vonsevych National Technical University of Ukraine «Igor Sikorsky Kyiv Polytechnic Institute», Peremohy Ave., 37, Kyiv 03056, Ukraine Received 01.02.2019 Accepted for publication 29.05.2019 Приборы и методы измерений 2019. – Т. 10, № 2. – С. 160–168 M.A. Bezuglyi et al. Приборы и методы измерений 2019. – Т. 10, № 2. – С. 160–168 M.A. Bezuglyi et al. Devices and Methods of Measurements 2019, vol. 10, no. 2, pp. 160–168 M.A. Bezuglyi et al. Abstract Angular measurements in optics of biological tissues are used for different applied spectroscopic task for roughness surface control, define of refractive index and for research of optical properties. Purpose of the research is investigation of the reflectance of biologic tissues by the ellipsoidal reflector method under the variable angle of the incident radiation.l The research investigates functional features of improved photometry method by ellipsoidal reflectors. The photometric setup with mirror ellipsoid of revolution in reflected light was developed. Theoretical foundations of the design of an ellipsoidal reflector with a specific slot to ensure the input of laser radiation into the object area were presented. Analytical solution for calculating the angles range of incident radiation depending on the eccentricity and focal parameter of the ellipsoid are obtained. Also created the scheme of image processing at angular photometry by ellipsoidal reflector. The research represents results of experimental series for samples of muscle tissues at wavelengths 405 nm, 532 nm, 650 nm. During experiment there were received photometric images on the equipment with such parameters: laser beam incident angles range 12.5–62.5°, ellipsoidal reflector eccentricity 0.6, focal parameter 18 mm, slot width 8 mm. The nature of light scattering by muscle tissues at different wavelengths was represented by graphs for the collimated reflection area. The investigated method allows qualitative estimation of influence of internal or surface layers of biologic tissues optical properties on the light scattering under variable angles of incident radiation by the shape of zone of incident light. Keywords: ellipsoidal reflector, angular measurements, optical properties, muscle tissue. DOI: 10.21122/2220-9506-2019-10-2-160-168 Адрес для переписки: М.А. Безуглый Национальный технический университет Украины «Киевский политехнический институт», пр. Победы, 37, г. Киев 03056, Украина e-mail: mikhail_bezuglyy@ukr.net Address for correspondence: M.A. Bezuglyi National Technical University of Ukraine «Igor Sikorsky Kyiv Polytechnic Institute», Peremohy Ave., 37, Kiev 03056, Ukraine e-mail: mikhail_bezuglyy@ukr.net Для цитирования: M.A. Bezuglyi, N.V. Bezuglaya, A.V. Ventsuryk, K.P. Vonsevych. Angular Photometry of Biological Tissue by Ellipsoidal Reflector Method. Приборы и методы измерений. 2019. – Т. 10, № 2. – С. 160–168. DOI: 10.21122/2220-9506-2019-10-2-160-168 For citation: M.A. Bezuglyi, N.V. Bezuglaya, A.V. Ventsuryk, K.P. Vonsevych. Angular Photometry of Biological Tissue by Ellipsoidal Reflector Method. Devices and Methods of Measurements. 2019, vol. 10, no. 2, рр. 160–168. DOI: 10.21122/2220-9506-2019-10-2-160-168 Адрес для переписки: М.А. Безуглый Национальный технический университет Украины «Киевский политехнический институт», пр. Победы, 37, г. Киев 03056, Украина e-mail: mikhail_bezuglyy@ukr.net Address for correspondence: M.A. Угловая фотометрия биологических тканей методом эллипсоидальных рефлекторов M А Безуглый Н В Безуглая А В Венцурик К П Вонсевич M.А. Безуглый, Н.В. Безуглая, А.В. Венцурик, К.П. Вонсевич Национальный технический университет Украины «Киевский политехнический институт имени Игоря Сикорского», пр. Победы, 37, г. Киев 03056, Украина Национальный технический университет Украины «Киевский политехнический институт имени Игоря Сикорского», пр. Победы, 37, г. Киев 03056, Украина Поступила 01.02.2019 Принята к печати 29.05.2019 Угловые измерения в оптике биологических тканей широко применяются для решения различ- ных прикладных спектроскопических задач для контроля шероховатой поверхности, определения показателя преломления, а также для изучения оптических свойств. Целью данной работы являлось исследование отражающей способности биологических тканей методом зеркальных эллипсоидов вращения при переменных углах падения зондирующего изучения. В работе исследуются функциональные возможности усовершенствованного метода фотометрии эллипсоидальными рефлекторами. Разработана и представлена установка для фотометрии зеркаль- ным эллипсоидом вращения в отраженном свете. Для обеспечения работы метода представлены те- оретические основы конструирования эллипсоидального рефлектора с характерным пазом для обе- спечения ввода лазерного излучения в исследуемую область. Получены аналитические выражения для вычисления диапазона углов падающего излучения в зависимости от эксцентриситета и фокаль- ного параметра эллипсоида. Также представлена усовершенствованная схема обработки изображе- ний, получаемых при угловой фотометрии эллипсоидальными рефлекторами. Представлены результаты серии экспериментальных исследований для мышечной ткани на дли- нах волн 405 нм, 532 нм и 650 нм. В ходе эксперимента были получены фотометрические изображе- ния при использовании фотометра со следующими конструктивными параметрами: диапазон угла па- дения лазерного излучения 12.5–62.5°, эксцентриситет эллипсоидального рефлектора 0,6, фокальный параметр 18 мм и ширина паза 8 мм. Характер светорассеяния мышечными тканями на различных длинах волн представлен графика- ми для зоны коллимированного отражения. При этом форма зоны падающего потока позволяет оце- нить влияние оптических свойств внутреннего или приповерхностного слоя биологической тканине на светорассеяние при различных углах падения. Ключевые слова: эллипсоидальный рефлектор, угловые измерения, оптические свойства, мышечная ткань Ключевые слова: эллипсоидальный рефлектор, угловые измерения, оптические свойства, мышечная ткань. DOI: 10.21122/2220-9506-2019-10-2-160-168 Адрес для переписки: М.А. Безуглый Национальный технический университет Украины «Киевский политехнический институт», пр. Победы, 37, г. Киев 03056, Украина e-mail: mikhail_bezuglyy@ukr.net Address for correspondence: M.A. Bezuglyi National Technical University of Ukraine «Igor Sikorsky Kyiv Polytechnic Institute», Peremohy Ave., 37, Kiev 03056, Ukraine e-mail: mikhail_bezuglyy@ukr.net Для цитирования: M.A. Bezuglyi, N.V. Bezuglaya, A.V. Ventsuryk, K.P. Vonsevych. Angular Photometry of Biological Tissue by Ellipsoidal Reflector Method. Приборы и методы измерений. 2019. – Т. 10, № 2. – С. 160–168. DOI: 10.21122/2220-9506-2019-10-2-160-168 For citation: M.A. Bezuglyi, N.V. Bezuglaya, A.V. Ventsuryk, K.P. Vonsevych. Angular Photometry of Biological Tissue by Ellipsoidal Reflector Method. Devices and Methods of Measurements. 2019, vol. 10, no. 2, рр. 160–168. Abstract Bezuglyi National Technical University of Ukraine «Igor Sikorsky Kyiv Polytechnic Institute», Peremohy Ave., 37, Kiev 03056, Ukraine e-mail: mikhail_bezuglyy@ukr.net Для цитирования: M.A. Bezuglyi, N.V. Bezuglaya, A.V. Ventsuryk, K.P. Vonsevych. Angular Photometry of Biological Tissue by Ellipsoidal Reflector Method. Приборы и методы измерений. 2019. – Т. 10, № 2. – С. 160–168. DOI: 10.21122/2220-9506-2019-10-2-160-168 For citation: M.A. Bezuglyi, N.V. Bezuglaya, A.V. Ventsuryk, K.P. Vonsevych. Angular Photometry of Biological Tissue by Ellipsoidal Reflector Method. Devices and Methods of Measurements. 2019, vol. 10, no. 2, рр. 160–168. DOI: 10.21122/2220-9506-2019-10-2-160-168 160 Приборы и методы измерений 2019. – Т. 10, № 2. – С. 160–168 M.A. Bezuglyi et al. Devices and Methods of Measurements 2019, vol. 10, no. 2, pp. 160–168 M.A. Bezuglyi et al. Introduction The progress in laser and computer technologies allow implementing of optical methods in different areas of non-destructive control. Among other, such methods applies for investigation of dispersive mediums, materials, and coating [1–5], or diagnostics of tissues and organs condition in biology and medicine [6–12]. Distribution of the electromagnetic radiation along optical range in biologic tissues (BT) is complicated and stochastic processes. Such distribution defines analytically by the classic electromagnetic theory, or extrapolates by the radiative transfer theory [13, 14]. Mathematical solution of subordinated to such theories systems of equations performs by the variety of theoretic and numeric methods [15]. Despite of that, the unified system of received results comparison criterion is still not established. Typical reasons are significant limitations of features of measuring and computation instruments intended for determination of indexes and coefficients of equations.i – put the sample of BT between glass elements with polished surfaces [23–25] with the sizes of fine irregularities less than the radiation wavelength Full-wave simulation like finite different time domain [16, 17] or finite element method [18, 19] applies more often for rigorous solution of Maxwell’s equations. However, it is still perspective for practical application in optics of biological tissues. In biomedical optics the radiative transfer theory is more popular. It provides results that are more precise and corresponds to reliability criteria in clinical application during investigation of pathologies. For direct and inverse problem solution following methods are typical: Monte Carlo simulation, adding-doubling method, Kubelka–Munk function, diffusion approximation, and other. Direct problem solution is determination of transmittance, reflectance and absorption of the BT, while inverse is determination of the BT optical properties – absorption and scattering coefficients, anisotropy factor. – creation of physiologic experiment conditions by placing of the sample inside the physiologic liquids [26]. Experiment under in vivo conditions involve thick (semi-infinite) biologic sample under investigation. The light flux, registered in opposite to incident radiation direction, consist of a passed through the surface and interacted with the media part, and a reflected by the surface light. The nature of angular distribution of reflected flux defines the type of the surface and its relief [27]. The probability of light diffusion backward to the surface depends on the quantity of interactions with the media, when the photon direction changes before the moment of its absorption in scattering, and depends on the optical properties of BT [22]. Model experiment in both cases satisfies considering Fresnel conditions. Угловая фотометрия биологических тканей методом эллипсоидальных рефлекторов M А Безуглый Н В Безуглая А В Венцурик К П Вонсевич DOI: 10.21122/2220-9506-2019-10-2-160-168 Адрес для переписки: М.А. Безуглый Национальный технический университет Украины «Киевский политехнический институт», пр. Победы, 37, г. Киев 03056, Украина e-mail: mikhail_bezuglyy@ukr.net Address for correspondence: M.A. Bezuglyi National Technical University of Ukraine «Igor Sikorsky Kyiv Polytechnic Institute», Peremohy Ave., 37, Kiev 03056, Ukraine e-mail: mikhail_bezuglyy@ukr.net Для цитирования: M.A. Bezuglyi, N.V. Bezuglaya, A.V. Ventsuryk, K.P. Vonsevych. Angular Photometry of Biological Tissue by Ellipsoidal Reflector Method. Приборы и методы измерений. 2019. – Т. 10, № 2. – С. 160–168. DOI: 10.21122/2220-9506-2019-10-2-160-168 For citation: M.A. Bezuglyi, N.V. Bezuglaya, A.V. Ventsuryk, K.P. Vonsevych. Angular Photometry of Biological Tissue by Ellipsoidal Reflector Method. Devices and Methods of Measurements. 2019, vol. 10, no. 2, рр. 160–168. DOI: 10.21122/2220-9506-2019-10-2-160-168 161 Devices and Methods of Measurements 2019, vol. 10, no. 2, pp. 160–168 M.A. Bezuglyi et al. Приборы и методы измерений 2019. – Т. 10, № 2. – С. 160–168 M.A. Bezuglyi et al. Devices and Methods of Measurements 2019, vol. 10, no. 2, pp. 160–168 M.A. Bezuglyi et al. Devices and Methods of Measurements 2019, vol. 10, no. 2, pp. 160–168 M.A. Bezuglyi et al. Приборы и методы измерений 2019. – Т. 10, № 2. – С. 160–168 M.A. Bezuglyi et al. Приборы и методы измерений 2019. – Т. 10, № 2. – С. 160–168 M.A. Bezuglyi et al. The document represents investigation of biological tissues by methods of light scattering optics in experimental conditions in vitro or ex vivo. In the research there were set hypotheses and limitations for proper introduction of measured values into mathematical model. The layer of tissue under investigation contains top and bottom boundaries, and internal region. The thickness of internal region is small relatively to its perimeter. BT surface (the boundary between the media) usually have roughness of different size comparing to wavelength. In such case, the external specular and/or diffuse reflection from it is possible. There occurs scattering inside the BT during the passing of light beam through the surface. Such process occurs until the total absorption, or appearing of light beam outside of the sample in the form of internal diffuse reflection or transmission. The main goal of such experiment is minimizing of external component impact (especially diffuse) on the resulting spatial distribution of scattered light. Such conditions can be created experimentally by several ways: Introduction Continuing investigation of features and properties of optical electronic devices, which uses ellipsoidal reflectors (ER) as the optical core of informational and measuring system of biomedical photometer [20–22], the current report represents results of series of practical experiments. These results allow investigating additional information about the interaction of electromagnetic radiation with turbid biological media according to the light scattering optics. Light scattering by BT samples depends on the illumination type and have significant differences during application of diffuse or collimated flux. There apply different measuring standards for determination of reflective ability and reflection coefficient for these light methods [28]. Reflectance and the reflection coefficient depends 162 Devices and Methods of Measurements 2019, vol. 10, no. 2, pp. 160–168 M.A. Bezuglyi et al. Приборы и методы измерений 2019. – Т. 10, № 2. – С. 160–168 M.A. Bezuglyi et al. on the incidence angle, radiation polarization, and refractive index. These parameters determine the boundary between the two media. Refractive index and optical properties of media significantly depends from the wavelength. Optical properties of BT can be determined in the indirect method only. Thus, the measurement of the refractive index should be performed only experimentally. For many types of tissues, the problem is in the absorption and scattering. Thus, the reflection coefficient for skin equals to 10–55 % and depends on the radiation spectrum, pigmentation rate and rugosity, presence of adipose and moisture. Mentioned factors depend on the gender, age, type, and color of the skin [29]. Figure 1 – Scheme of unit for angular photometry by ellipsoidal reflector: 1 – base; 2 – bar; 3 – height regulation mechanism; 4 – flywheel; 5 – worm gear with dovetail; 6 – cap disc; 7 – laser module; 8 – CCD camera; 9 – ellipsoidal reflector; 10 – biological tissue holder; 11 – subject stage; 12 – adjustment screws; 13 – horizontal regulation block; 14 – worm gear with dovetail; 15 – flywheel Considering the mentioned information, the purpose of the research is investigation of the reflectance of biologic tissues by the ellipsoidal reflector method under the variable angle of the incident radiation. Methods and tools Considering axial symmetry of vertically oriented ellipsoid of revolution, the determination of operating range of incident angles performs with using of ellipse equation: x a z b 2 2 2 2 1 + = , Heights h1 and h2 are technological dimensions and are selected for ensuring of ER shape support depending on the detail production material. For current investigation it was used construction alloy ENAW-2024; and heights h1 and (f – h1) were selected in the range 2–4 mm. where a and b – semi-minor and semi-major axis of ellipse. Considering expression of ellipse through its eccentricity e, focal parameter p and focal distance f, it is possible to determine the points of its intersection with straight lines, which contains ultimate points of critical angles (Figure 3): Based on the mechanism of image processing at ellipsoidal photometry [32], the methodology of photometric image analysis during angular photometry by ellipsoidal reflector was improved. The processing scheme (Figure 4), except of region of interest and external ring A1, also contains ellipse of incident flux A2 and area of collimated reflection A3. Figure 3 – Determination of critical angles of incident light in ellipsoidal reflector with slot Figure 4 – The processing scheme of photometric images during angular photometry by ellipsoidal reflectors: ROI – region of interest; A1 – external ring; A2 – ellipse of incident flux; A3 – area of collimated reflection Th h t i ti l f di ti fl Figure 3 – Determination of critical angles of incident light in ellipsoidal reflector with slot Figure 4 – The processing scheme of photometric images during angular photometry by ellipsoidal reflectors: ROI – region of interest; A1 – external ring; A2 – ellipse of incident flux; A3 – area of collimated reflection Figure 4 – The processing scheme of photometric images during angular photometry by ellipsoidal reflectors: ROI – region of interest; A1 – external ring; A2 – ellipse of incident flux; A3 – area of collimated reflection x p e e f h 1 2 2 2 1 2 1 1 = − − − ⋅ − ( ) ( ) ( ) , x p e e h f 2 2 2 2 2 2 1 1 = − − − ⋅ − ( ) ( ) ( ) . Methods and tools Devices and Methods of Measurements 2019, vol. 10, no. 2, pp. 160–168 M.A. Bezuglyi et al. stand have subject stage 11, which is fastened to the horizontal regulation block 13 by worm gear and dovetail 14. Longitudinal displacement performs during rotation of flywheel 15. Subject stage fits horizontally, and precise regulation performs by adjustment screws 12. The measuring base of the stand is ellipsoidal reflector 9, which is mounted on the BT holder 10 during experiment. The unit adjustment performs before measurements for ensuring of horizontal mounting of subject stage, sample, and reflector. The height of laser block regulates for variable thickness of BT sample. In the holder installs reflector 9 align with CCD camera 8 for ellipsoidal CCD-reflectometry [20]. Images are registered under different incident angles of laser beam and analyzed by specialized software. stand have subject stage 11, which is fastened to the horizontal regulation block 13 by worm gear and dovetail 14. Longitudinal displacement performs during rotation of flywheel 15. Subject stage fits horizontally, and precise regulation performs by adjustment screws 12. The measuring base of the stand is ellipsoidal reflector 9, which is mounted on the BT holder 10 during experiment. The unit adjustment performs before measurements for ensuring of horizontal mounting of subject stage, sample, and reflector. The height of laser block regulates for variable thickness of BT sample. In the holder installs reflector 9 align with CCD camera 8 for ellipsoidal CCD-reflectometry [20]. Images are registered under different incident angles of laser beam and analyzed by specialized software. Considering (1), critical angles of incident radiation for ellipsoidal reflector with its set of constructive parameters can be determined using equations: q αmin , =     arcctg h x 2 2 αmax . =     arcctg h x 1 1 (2.a) (2.b) (2.a) (2.b) It is important to note that the coordinate x2 determines the value of minimum angle in case of slant height for top slot forming part (Figure 3). If slant height is parallel to the axis x (Figure 2), the minimum angle determines from the simple trigonometric dependency. Equations (2) are valid for lines, which contains optical axis of incident ray. During the calculation of critical incident angles there considers radius of real laser beam and increase minimal and decrease maximal angles correspondingly. Methods and tools The adjustment device aligns reflector, optical system, and CCD camera referring to horizontal baseline. The change of energetic characteristics of incident radiation performs by regulation of power by universal power source, which further supplies to laser diodes with the wavelength 405 nm, 532 nm, and 650 nm. The nominal power of each diode equal to 5 mW. Unit consists of base 1 with fitted perpendicular to it bar 2. To this bar attached the height regulation mechanism 3. It is worm gear with dovetail 5, and is actuated by the flywheel 4. The research focused on specific features of realization of improved method of photometry by ellipsoidal reflectors. The target is investigation of its use for measurements of reflected and backscattered light. The method allows investigating of the optical properties of scattered media under different incident angles, and energy characteristics of incident optical radiation. For research of the method the experimental unit was constructed (Figure 1). The unit use photometric system with ellipsoid of revolution with internal mirror surface. The mirror is truncated by the focal planes orthogonally to the semi-major axis and contains specific longitudinal slot (Figure 2) for receiving of optical radiation under variable angles. Ellipsoidal reflector was produced by the method of trajectory copying [30]. Additionally, authors investigated other technologies of shape formation of internal ellipsoidal mirror surface [31] and photometer production [22]. Figure 2 – Ellipsoidal reflector for angular photometry: 3D model (a); end-product (b) a b b a b a Figure 2 – Ellipsoidal reflector for angular photometry: 3D model (a); end-product (b) The unit contain mechanism for micrometric height regulation for investigation of BT with various thicknesses. Experimental unit operates by the method of photometry in reflected light [20]. The condition of semi-infinite thickness of BT sample satisfies by the application of black opaque lining with absorption coefficient close to 0.99. Mechanism 3 consists of three disks, one of which is a cap 6 for fixing of all disks, and two others acts as a fastening for laser module 7. The aperture in the bottom part of the module applies for ensuring of necessary laser beam diameter. In the experimental unit it equals to 1 mm. In addition, 163 Приборы и методы измерений 2019. – Т. 10, № 2. – С. 160–168 M.A. Bezuglyi et al. Devices and Methods of Measurements 2019, vol. 10, no. 2, pp. 160–168 M.A. Bezuglyi et al. Result and discussion The angular photometry by ellipsoidal reflector results (Figure 1) contain groups of photometric images (Figures 5, 6) for three wavelengths under different incident angles with the step 2.5°. Methods and tools (1.a) (1.b) (1.a) The characteristic value for radiation flux distribution in each region of analysis is relative illuminance of the zone EA. It calculates as the ratio of total pixel brightness Ipix to the area of corresponding zone: (1.b) 164 Devices and Methods of Measurements 2019, vol. 10, no. 2, pp. 160–168 M.A. Bezuglyi et al. Приборы и методы измерений 2019. – Т. 10, № 2. – С. 160–168 M.A. Bezuglyi et al. properties (Table) of which were determined similarly [20]. The tissue was separated perpendicularly to muscle tissues for providing of minimal divergences of anisotropy factor [33]. Further samples were preserved under normal conditions in 0.9 % solution of NaCl during 30 min. E I A A pix = . (3) The area of the zone can be expressed by metric value as in [17, 22] or can be represented by total sum of pixels, which forms corresponding zone. Result and discussion 10, no. 2, pp. 160–168 M.A. Bezuglyi et al. Приборы и методы измерений 2019. – Т. 10, № 2. – С. 160–168 M.A. Bezuglyi et al. Devices and Methods of Measurements 2019, vol. 10, no. 2, pp. 160–168 M.A. Bezuglyi et al. Приборы и методы измерений 2019. – Т. 10, № 2. – С. 160–168 M.A. Bezuglyi et al. Приборы и методы измерений 2019. – Т. 10, № 2. – С. 160–168 M.A. Bezuglyi et al. For the applied ellipsoidal reflector parameters (eccentricity e = 0.6, focal parameter p = 18 mm, and focal distance f = 16.5 mm) and laser beam of diameter 1 mm, the operational range of angle of incident ray equals to 12.5–62.5°. 405 nm and 532 nm in the range of incident angles 20–40°. Graphs for Type 2 tissues with the increasing of incidence angle continues to grow with different acceleration until reaching of definite angle. After increasing of that angle there occurs decline with different velocity. Authors explain this by the reaching of specific “critical” angle, under which in the ellipsoidal reflector there observes doubled reflection from the opposite side. Due to slot presence on this side, the radiation leaves the measuring core of photometer with ER. The absence of mentioned peak of illuminance for porcine muscle tissue indicates that it possibly present in the incident angles, which exceeds the working range of angles for mounted mirror ellipsoid of revolution (Figure 2). At the same time, characteristic view of graphs for both tissues proofs the relative interconnection between optical properties of muscle tissues in the defined wavelengths [7]. Note that in the current research there are specified results of angular photometry for thick muscle tissue samples. The efficiency of application of ellipsoidal reflector was proved only for maximum consolidation of reflected collimated flux. However, experiments proved the same efficiency for the sample thickness less than 1 mm. Also, we can receive the useful information from the component of external ring A2. Based on the received images for both samples of muscle tissues (porcine and chicken) at the wavelength 405 and 532 nm, the dynamic change of incident flux ellipse shape performs according to laws of geometric optics for reflecting surface. At the same time, the shape of spot of incident flux at the wavelength of 650 nm is close to elliptic only for big incident angles. Result and discussion This fact explains the dependency of backscattered radiation more from the optical properties (Table) inside the tissue itself then from the surface properties and sub-surface layers. Figure 7 – Illuminance of area of collimated reflection for Type 1 (a) and Type 2 (b) samples of muscle tissues for wavelength 405 nm (blue line), 532 nm (green line), and 650 nm (red line) b а b а а Conclusion The specificities of angular photometry by ellipsoidal reflectors was investigated in the current research. Experiments was done for samples of both type of muscle tissue at wavelengths 405 nm, 532 nm, and 650 nm. The investigated method allows qualitative estimation of influence of internal or surface layers of biologic tissues optical properties on the light scattering under variable angles of incident radiation by the shape of zone of incident light. Illuminance of collimated reflection area for various wavelengths mutually correlates with the optical properties of biologic tissues. The determination of properties of such correlation refers to the creation of adequate interaction model of optical radiation with layers of biologic tissues, and boundaries of its distribution with correct consideration of parameters of laser radiation and incident angle. b Figure 7 – Illuminance of area of collimated reflection for Type 1 (a) and Type 2 (b) samples of muscle tissues for wavelength 405 nm (blue line), 532 nm (green line), and 650 nm (red line) Analysis of Figure 7a represent that collimated reflection at the wavelength 650 nm generate smaller illuminance of corresponding zone for Type 1 muscle tissue under all investigated angles of incident light. From the other side, the illuminance of the same zone for the chicken muscle tissue samples is significantly bigger comparing to illuminance on the wavelengths Result and discussion As the object for investigation it was selected the muscle tissues of porcine (Type 1) and chicken (Type 2) of various thickness, optical Table Table Optical properties Muscle tissue / optical properties λ, nm μa μs g Type 1 405 2.06 ± 0.08 49.5 ± 3.5 0.964 ± 0.012 532 1.93 ± 0.1 61.4 ± 4.9 0.966 ± 0.012 650 1.88 ± 0.2 41.8 ± 4.7 0.973 ± 0.009 Type 2 405 1.01 ± 0.04 124.1 ± 10.2 0.958 ± 0.01 532 0.74 ± 0.22 186.1 ± 12.6 0.958 ± 0.012 650 0.78 ± 0.1 216.7 ± 16.1 0.965 ± 0.019 Figure 5 – Photometric images of backscattered light by Type 1 muscle tissue samples with the thickness 4.8 ± 0.21 mm at the wavelength 405 nm (a, d, g, k), 532 nm (b, e, h, m), 650 nm (c, f, i, n) under incident angles of laser beam: 30º (a, b, c), 40º (d, e, f), 50º (g, h, i), 60º (k, m, n) correspondingly Figure 6 – Photometric images of backscattered light by Type 2 muscle tissue samples with the thickness 4.6 ± 0.27 mm at the wavelength 405 nm (a, d, g, k), 532 nm (b, e, h, m), 650 nm (c, f, i, n) under incident angles of laser beam: 30º (a, b, c), 40º (d, e, f), 50º (g, h, i), 60º (k, m, n) correspondingly Optical properties Figure 5 – Photometric images of backscattered light by Type 1 muscle tissue samples with the thickness 4.8 ± 0.21 mm at the wavelength 405 nm (a, d, g, k), 532 nm (b, e, h, m), 650 nm (c, f, i, n) under incident angles of laser beam: 30º (a, b, c), 40º (d, e, f), 50º (g, h, i), 60º (k ) di l Figure 6 – Photometric images of backscattered light by Type 2 muscle tissue samples with the thickness 4.6 ± 0.27 mm at the wavelength 405 nm (a, d, g, k), 532 nm (b, e, h, m), 650 nm (c, f, i, n) under incident angles of laser beam: 30º (a, b, c), 40º (d, e, f), 50º (g, h, i), 60º (k ) di l Figure 5 – Photometric images of backscattered light by Type 1 muscle tissue samples with the thickness 4.8 ± 0.21 mm at the wavelength 405 nm (a, d, g, k), 532 nm (b, e, h, m), 650 nm (c, f, i, n) under incident angles of laser beam: 30º (a, b, c), 40º (d, e, f), 50º (g, h, i), 60º (k, m, n) correspondingly Figure 6 – Photometric images of backscattered light by Type 2 muscle tissue samples with the thickness 4.6 ± 0.27 mm at the wavelength 405 nm (a, d, g, k), 532 nm (b, e, h, m), 650 nm (c, f, i, n) under incident angles of laser beam: 30º (a, b, c), 40º (d, e, f), 50º (g, h, i), 60º (k, m, n) correspondingly 165 Devices and Methods of Measurements 2019, vol. 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https://openalex.org/W2015720733	https://literator.org.za/index.php/literator/article/download/245/218	English	null	Translating traces: Deconstruction and the practice of translation	Literator	2,004	cc-by	10,860	Translating traces: Deconstruction and the practice of translation J.L. Kruger School of Languages Vaal Triangle Campus North-West University VANDERBIJLPARK E-mail: engjlk@puk.ac.za Translating traces: Deconstruction and the practice of translation Translating traces: Deconstruction and the practice of translation In this article I attempt to show that deconstruction and its practices should not be read as intimations towards plurality or relativism in translation, but should rather be utilised as a powerful analytical tool, a way of reading and writing with heightened awareness. In order to arrive at this conclusion, I discuss différance and the play of the trace in the context of the cont(r)act between two texts that are in a relationship of translation. I further argue that plurality as contained in Derrida’s différance is not a directive, but that the translator has to be aware of the existence of plurality and to take into account that the reader also participates in and contributes to this plurality. The key to an application of Derrida’s theory is shown to be situated in the process rather than in the product of translation, and this process has to move beyond a hierarchical opposition of “original” and translation. I conclude that différance becomes not an obstacle or barrier to translation, but specifically that which, in making something untranslatable, creates the need for translation. 1 As will be explained later, this does not constitute a call for, or justification of, “free” play – i.e. arbitrary meaning. Opsomming Die vertaling van spore: dekonstruksie en die praktyk van vertaling g p p y g In hierdie artikel poog ek om te toon dat dekonstruksie en die gebruike daarvan nie gesien moet word as ’n oproep tot veelvoudige inter- pretasie of relativisme in vertaling nie, maar dat dit eerder ingespan moet word as ’n kragtige analitiese vaardigheid; ’n manier van lees en skryf met verdiepte aandag. Om tot hierdie gevolgtrekking te kom bespreek ek différance en die spel van spore in die konteks van die kont(r)ak tussen twee tekste wat in ’n verhouding van vertaling tot Literator 25(1) April 2004:47-71 47 ISSN 0258-2279 Translating traces: Deconstruction and the practice of translation mekaar staan. Verder voer ek aan dat veelvoudigheid soos vervat in Derrida se différance, nie ’n voorskrif is nie, maar dat die vertaler bewus moet wees van die bestaan van veelvuldigheid en in ag behoort te neem dat die leser ook deelneem aan en bydra tot hierdie veelvuldigheid. Die sleutel tot die toepassing van Derrida se teorie is gesetel in die proses eerder as die produk van vertaling, en hierdie proses moet verder gaan as die hiërargiese teenstelling van “oorspronklike” en vertaling. Ek kom tot die slotsom dat différance nie ’n struikelblok vir vertaling is nie, maar dat dit eerder die behoefte vir vertaling daarstel deur onvertaalbaarheid te identifiseer. 1. Introduction The primary aim of this article is to investigate the possibilities offered by deconstruction, and particularly the contributions of Jacques Derrida, for the practice of translation. Like Kathleen Davis’s Deconstruction and translation (2001), this article will focus primarily on Derrida’s contributions since Derrida is the one “who coined the term ‘deconstruction’ and who produced (and continues to produce) most of what have become its primary texts” (Davis, 2001:1). In addition, the importance of Derrida’s other term, différance, to translation theory provides a useful angle into this self- questioning discourse. Deconstruction destabilises a number of “safe” conceptions as- sociated with translation theory. In the words of Koskinen (1994: 446), “by denying the existence of Truth, origin and center, de- construction deprives us of the comfortable fallacy of living in a simple and understandable world. We lose security, but we gain endless possibilities, the unlimited play of meanings”.1 What has to be determined, however, is whether deconstruction actually contributes to the practice of translation. Does its questioning of conventional notions (such as equivalence and faithfulness) not render its insights so devastatingly relativist that the practising translator cannot afford to pay it more than a passing and slightly amused glance before returning to the serious task at hand? The usefulness of deconstruction to the practice of translation has often been questioned due to its essentially philosophical nature. Many translators would probably agree with Anthony Pym (1999) Literator 25(1) April 2004:47-71 48 ISSN 0258-2279 J.L. Kruger that philosophers (and by implication theorists like Derrida) “have no time for the rubbish that most of us have to improve when we translate”. Indeed, contemporary translation theory does seem to be moving towards a more functionalist integration of theory and practice, and in this spirit it is as important not to discard philosophy as it is to remain grounded in the “reality” of translation practice with its improbable deadlines, impossible clients and less-than-perfect source texts. The main problem deconstruction poses to the practice of translation is its seemingly relativist open-endedness and its plural perspectives on the process of signification. Furthermore, deconstruction affects conventional notions such as equivalence2 and faithfulness in translation, rendered powerless the moment we question concepts such as “truth”, “origin” and “centre”. Put simply, deconstruction removes equivalence from the skopos (cf. Vermeer, 2000 [1989]) or purpose of translation. This simple action of elimination, however, has serious consequences for the very identity of translation. 2 Derived from the mathematical term of equivalence, the term is generally taken to mean that the translation as derivative text is equal in value to, or does not deviate from, the origin(al) as prior text. Equivalence will be defined more clearly under paragraph 3. 3 According to Derrida (1974:158), “there is nothing outside the text”, which is interpreted by Davis (2001:9) as making the point that “meaning cannot be extracted from, and cannot exist before or outside of a specific context”. 1. Introduction As any practising translator knows, equivalence, regardless of how one chooses to define this elusive concept, remains the single most important criterion by which translations are judged. However, as this article will attempt to show, deconstruction and its practices should not be read as intimations towards plurality or relativism in translation, but should rather be utilised as powerful analytical tools, ways of reading and writing with heightened awareness. After all, good translators are in the first instance good readers, and by virtue of their mediating position in the cont(r)act between text and translation, they have to (at least) be aware of the plethora of gaps and traces in source and target texts as well as source and target languages and cultures. Indeed, it is precisely this power that derives from its acute awareness of context3 and subjectivity that makes deconstruction invaluable to the practising translator. 2 Derived from the mathematical term of equivalence, the term is generally taken to mean that the translation as derivative text is equal in value to, or does not deviate from, the origin(al) as prior text. Equivalence will be defined more clearly under paragraph 3. 3 According to Derrida (1974:158), “there is nothing outside the text”, which is interpreted by Davis (2001:9) as making the point that “meaning cannot be extracted from, and cannot exist before or outside of a specific context”. Literator 25(1) April 2004:47-71 49 ISSN 0258-2279 Translating traces: Deconstruction and the practice of translation 2. Jacques Derrida and translation theory Derrida has been writing on translation directly or indirectly for most of the latter part of the twentieth century. Although, according to Davis (2001:9), “all of Derrida’s texts concern translation in various ways”, he addresses translation perhaps more directly in texts such as Des tours de Babel (Derrida, 1985a), The ear of the other: Otobiography, transference, translation (Derrida, 1985b), and, more recently, What is a “relevant” translation? (Derrida, 2001). Derrida’s contribution to translation theory is located primarily in his reclaiming of the power of the word and everything it has the potential to signify: “At the beginning of translation is the word. Nothing is less innocent, pleonastic and natural, nothing is more historical than this proposition, even if it seems too obvious” (Derrida, 2001:180.) The importance of the word is particularly evident in his notion of différance, which is central to deconstruction, and which will also be shown in this article to impact on the very activity of translation, literary or otherwise. Therefore, before the implications of the questioning of hierarchical oppositions for translation theory can be investigated more fully, we have to take a closer look at the nuances contained in the term différance. 2.1 Différance What is written as différance, then, will be the playing move- ment that ‘produces’ – by means of something that is not simply an activity – these differences, these effects of difference. This does not mean that the différance that produces differences is somehow before them, in a simple and unmodified – in-different – present. Différance is the non-full, non-simple, structured and differentiating origin of differences. Thus the name ‘origin’ no longer suits it (Derrida, 1982:11). What is written as différance, then, will be the playing move- ment that ‘produces’ – by means of something that is not simply an activity – these differences, these effects of difference. This does not mean that the différance that produces differences is somehow before them, in a simple and unmodified – in-different – present. Différance is the non-full, non-simple, structured and differentiating origin of differences. Thus the name ‘origin’ no longer suits it (Derrida, 1982:11). Derrida’s perspectives on translation are closely related to his definition of différance. The process by which différance is ap- proached and by which it approaches becomes the process by which words and translation are approached and approach; not in terms of what these words and processes signify, but in terms of what they activate or “produce” by means of “the playing movement” through both temporal and spatial dimensions. The very activity of translation cannot be separated from this difference between signifier and signified and becomes part of the existence and production of the inevitable tension. Literator 25(1) April 2004:47-71 ISSN 0258-2279 50 J.L. Kruger In 1968 Derrida defined différance in an address before the Société Française de Philosophie, subsequently published in Margins of philosophy (1982). He began the address with the sentence, “I will speak, therefore, of a letter” (Derrida, 1982:3). This already signals that the significance of différance is situated in one letter that erases the trace of what would otherwise have been a “word” or a “concept” but which Derrida (1982:7) insists is neither. In 1968 Derrida defined différance in an address before the Société Française de Philosophie, subsequently published in Margins of philosophy (1982). He began the address with the sentence, “I will speak, therefore, of a letter” (Derrida, 1982:3). This already signals that the significance of différance is situated in one letter that erases the trace of what would otherwise have been a “word” or a “concept” but which Derrida (1982:7) insists is neither. 2.1 Différance Différance is based on the French verb différer (from the Latin verb differre). This verb has two distinct meanings in French which are represented by two separate words in English, namely to differ and to defer.4 The first and more common sense of the verb brought to différance, namely to differ, is related to a spatial horizon or spacing, which implies “to be not identical, to be other, discernible, etc.” and also refers both to different things and differences of opinion (Derrida, 1982:8). The second sense of the verb, namely to defer, is in turn related to a temporal horizon or temporisation by which term Derrida (1982:8) summarises concepts such as “the action of putting off until later, of taking into account, of taking account of time and of the forces of an operation that implies an economical calculation, a detour, a delay, a relay, a reserve, a representation”. It also implies “to temporize, to take recourse, consciously or unconsciously, in the temporal and temporizing mediation of a detour that suspends the accomplishment or fulfillment of ‘desire’ or ‘will,’ and equally effects this suspension in a mode that annuls or tempers its own effect” (Derrida, 1982:8). Therefore, Gentzler’s (1993:158-159) statement that différance refers “not to what is there (language), but what is not there, and thus calls into question any ontological approach that attempts to determine a notion of Being based on presence” sums up the significance of this not-word, not-concept for translation. Translation now becomes a transformation of potential instead of a passive transfer of meaning or ontological presence. In the words of Davis (2001:14), meaning “is an effect of language, not a prior presence merely expressed in language. It therefore cannot be simply extracted from language and transferred”. 4 The fact that the dual meaning in French is therefore untranslatable in English in spite of the proximity of the words, emphasises exactly what différance attempts to capture, namely that which cannot be named. Even a similar attempt like defference fails to activate similar traces in English. Literator 25(1) April 2004:47-71 51 ISSN 0258-2279 Translating traces: Deconstruction and the practice of translation Différance is not – it contains its own death in the trace of spatial difference (it can never “be” present), as well as its sur-vival in the trace of temporal deferral (it is always “becoming” present). 5 Without elaborating on the point needlessly, it is important to note that Derrida does not deny the referential effect of language, but rather implies that the potential presence of the signified (“referent”) has no effect on the structure of the signifier (“mark”). In the words of Clarkson (2003:38), “the referential effect can take place precisely because the sign (mark) is constructed on the basis that it is able to endure the absence of the referent, and still do the job”. Derrida (1982:25-26) deconstructs différance as follows: There is no essence of différance; it is that which not only could never be appropriated in the as such of its name or its appearing, but also that which threatens the authority of the as such in general, of the presence of the thing itself in its essence. That there is not a proper essence of différance at this point, implies that there is neither a Being nor truth of the play of writing such as it engages différance. In these words the full impact of différance becomes evident. It remains indefinable because of a continual differing and deferring and at the same time it posits that there can be no such thing as Being precisely because of this play of differences. Davis (2001:15) points out that “Derrida’s use of the word ‘play’ in this context is often misunderstood, most grievously when taken as an argument for complete ‘freeplay’ in language: that is, the suggestion that a signifier can ultimately mean just anything at all”. In approaching translation or translation theory we have to take cognisance of the unnameable, but we also have to be aware of the play constituted by gaps that allow movement. However, as will be shown in the following section, this very fact not only makes translation as rewriting possible, but also renders it essential as process if not as product. 2.1 Différance It constitutes a silent error that disappears as it is spoken, leaving a trace that can never be present. Reference becomes extremely important here in that the “middle voice”, as well as the notion of something that is absent, transcends the “here and now” but also the “there and then”, creating a space that is simultaneously impossible to ignore and impossible to account for. Although the formalist concept of defamiliarisation or Verfremdung is still present in the term (involving as it does the abstract recreation and revoicing of silenced modes), the unnameable also creates a palimpsest that affects/effects presence through absence. Différance hints at presence without providing that which would make it possible to inscribe or infer presence.5 Begam (1992:876) points out that différance moves … along two essentially opposed trajectories of meaning: on the one hand, it gestures towards presence or self-identity … on the other hand, it gestures toward absence or difference … This means that to think différance is to think what is simul- taneously same and other, what is simultaneously itself and its opposite. … along two essentially opposed trajectories of meaning: on the one hand, it gestures towards presence or self-identity … on the other hand, it gestures toward absence or difference … This means that to think différance is to think what is simul- taneously same and other, what is simultaneously itself and its opposite. Or, in the words of Derrida (1982:9), “the sign represents the presence in its absence. It takes the place of the present … The sign, in this sense, is deferred presence”. This aspect of différance is of particular importance to translation theory, since it touches on the essence of translation and the relation between “original” and “translation”. Since translation deals with a representation of this representation of the sign, presence remains deferred in the translating text or rewriting just as it is deferred in the “original”. After all, translation also deals with the traces left by the presence, but always in absence and never with a fixed meaning that can be transferred between languages (as traditional theories would sometimes have it). Literator 25(1) April 2004:47-71 52 ISSN 0258-2279 J.L. Kruger 2.2 Gaps and traces A translation is never quite ‘faithful’, always somewhat ‘free’, it never establishes an identity, always a lack and a supplement, and it can never be a transparent representation, only an interpretive transformation that exposes multiple and divided meanings, equally multiple and divided. (Venuti, 1992:8; my emphases – JLK.) Through the inevitable existence of gaps and traces in any text, translation exposes and uncovers, but also establishes lacks and supplements. Derrida’s project, according to Gentzler (1993:160), “is one of trying to unveil … a play of covered-up but subconsciously discernible traces without referring to some sort of deep underlying meaning”. According to Davis (2001:15), “Derrida usually speaks of the trace, rather than the signifier, partly to recall its sense of a ‘track’ or even a ‘spoor’”. As a result of the play of differences, any text becomes a footprint that contains in it traces of the past and future, but that can never be Literator 25(1) April 2004:47-71 53 ISSN 0258-2279 Translating traces: Deconstruction and the practice of translation pinned to an “essence” or “fixed meaning”. “It is because of différance,” Derrida (1982:13) maintains, … that the movement of signification is possible only if each so- called ‘present’ element, each element appearing on the scene of presence, is related to something other than itself, thereby keeping within itself the mark of its past element, and already letting itself be vitiated by the mark of its relation to the future element, this trace being related no less to what is called the future than to what is called the past, and constituting what is called the present by means of this very relation to what it is not: what it absolutely is not, not even a past or a future as a modified present (my emphases – JLK). Furthermore, Derrida (1982:21) cautions that “the concept of the trace is incompatible with the concept of retention of the becoming- past of what has been present. One cannot think the trace – and therefore, différance – on the basis of the present, or the presence of the present”. According to Davis (2001:15), “[t]hese relations to past and future are often called retentive and protentive characteristics, and the trace is where the retentive/protentive relationship with the other is marked”. It is important to note that Derrida does not deny the importance of the truth of Being. 6 In this as well as in the rest of Derrida’s explication of trace, we have to bear in mind that “trace” is both verb and noun, and at the same time neither, just as it is neither active nor passive, yet both. 7 See Venuti’s (2000) notion of “foreignizing”, Lewis’s (2000 [1985]) notion of abusive translation (derived from Derrida), and Berman’s (2000 [1985]) trial of the foreign. In each of these authors’ work the impact of the play of traces on translation can be seen in some form or another. 2.2 Gaps and traces In fact, he regards the passage through the truth of Being as remaining an “incessant necessity” (Derrida, 1982:22). However, it remains a “passage through”, leading Derrida (1982:23) to define the trace further by stating: “Always differing and deferring, the trace is never as it is in the presentation of itself.6 It erases itself in presenting itself, muffles itself in resonating, inscribing its pyramid in différance.” Derrida suggests that one should not look at the original message or its codification, “but the multiple forms and interconnections through which it must pass in order to speak, to refer at all”, a process which entails what he terms “a play of traces” (Derrida, 1982:15). By extension, according to Gentzler (1993:160), “one could also project a translation theory aimed at protecting differences, reinvigorating 6 Literator 25(1) April 2004:47-71 54 ISSN 0258-2279 J.L. Kruger language with lost etymological resonances, thereby opening up new avenues of thought”.7 language with lost etymological resonances, thereby opening up new avenues of thought”.7 This action of “opening up” or “gapping” is central to Derrida’s “notion” of différance. In the words of Leonard Cohen (1992) in his song, ‘Anthem’, “There are cracks, / there are cracks in everything. / That’s how the light gets in”. The boundary between texts in a contract of translation is also the opening that allows the traces to evoke meaning ‘effects’ through the play of differences (see also Davis, 2001:30). Although the above focus on différance and its gaps and traces may already point to the potential of deconstruction in translation, the deconstructionist notion of untranslatability still seems to go directly against the practice of translation. If we remove equivalence from the translation brief, where is the sense in translating? Why would anyone want to pay a translator for a piece of work that offers no clarity, no final answers? After all, untranslatability seems to imply that equivalence is impossible and also that the “original” is untouchable. In order to attempt to address these questions, this article will now turn to the origins of the concept of equivalence as well as the status of the translation before attempting to cross the seemingly insurmountable hurdle of untranslatability. 3.1 Equivalence The history of translation theory can in fact be imagined as a set of changing relationships between the relative autonomy of the translated text, or the translator’s actions, and two other concepts: equivalence and function. (Venuti, 2000:5; my emphases – JLK.) Translation theory over the centuries has been concerned primarily with interlingual translation (in Jakobson’s sense; 2000 [1959]:114), in other words the translation of a text from one linguistic system into another. It is therefore not surprising that some notion of equivalence informs most translation theories up to the 1980s, and still does in many ways. Gentzler (1993:144) distinguishes between Literator 25(1) April 2004:47-71 55 ISSN 0258-2279 Translating traces: Deconstruction and the practice of translation various forms of equivalence with different emphases, for example the notions of linguistic structural/dynamic equivalence in the “science” of translation, corresponding literary function in early Translation Studies, and similar formal correlation governed by social acceptability in the target culture in polysystem theory and the Translation Studies of the eighties. Venuti (2000:5) similarly lists a number of terms that have been associated with equivalence, namely “‘accuracy’, ‘adequacy’, ‘correctness’, ‘correspondence’, ‘fidelity’, or ‘identity’; [equivalence] is a variable notion of how the translation is connected to the foreign text”. In short, according to Pym (2000), debates over equivalence “concern beliefs that some aspect of a source-text unit can equal some aspect of a target-text unit”. Indeed, Derrida himself states that “to make legitimate use of the word translation … in the rigorous sense conferred on it over several centuries by a long and complex history in a given cultural situation … the translation must be quantitatively equivalent to the original” (Derrida, 2001:180). The above-mentioned theories and paradigms deal with translation primarily as interlingual translation, thereby legitimising their use of the term “equivalence”. However, we cannot afford to view trans- lation in this restrictive manner, in spite of Pym’s (2000) call to do so by not being afraid to “distinguish translation from non-translation”. Even if translation is functionalist in purpose, the categories of intra- lingual translation and even intersemiotic translation are activated to some degree, rendering Jakobson’s distinctions problematic if taken as discontinuous categories. This merging of Jakobson’s categories of translation is even more expressly true when we deal with texts that rely heavily on gaps, silences and traces such as political, philosophical, and advertising texts and obviously also literary texts. 3.1 Equivalence In the translation of narrative fiction, for example, the gaps and traces created in the narration through different narrative levels and focalisations make certain demands in terms of the involvement of the translator (also as reader) in the actualisation of the text that clearly transcend mere interlingual translation. In texts such as these, the translator also has to focus on those interpretative elements of intralingual translation as well as the imaginative elements inherent in intersemiotic trans- lation that exist in both source and target text or, perhaps more appropriately, in both texts that form the contract of translation as rewriting. The reason for the failure of Jakobson’s tripartite division as well as the conventional notion of equivalence when viewed from a Literator 25(1) April 2004:47-71 56 ISSN 0258-2279 J.L. Kruger J.L. Kruger deconstructionist perspective, can mainly be ascribed to the failure of the limits they attempt to set.8 The very premise of the category of intralingual translation, for example, is that it is possible to determine the form of the limits of a language. In the words of Davis (2001:20), the limit of a language, “is not ‘decidable’ or absolute (which would cleanly cut languages off from each other), but a boundary and a structural opening between languages, contexts”. The development of equivalence as a term to describe the relation between source and target text makes perfect sense if we regard translation as some form of transfer between two languages with clear limits. However, Derrida’s contribution to the field is part of a “movement” that calls the very notion of transfer into question. The deconstructionist view that signifier and signified are linked, with the result that you cannot change the one without affecting the other, has a significant impact on translation (Koskinen, 1994:448). No longer can translation be viewed as the transfer of a stable signified between two languages. Derrida’s preference for the term “regulated transformation” over that of “translation” (Gentzler, 1993:168) is significant here, in that it draws attention to the fact that equivalence as the transfer of “pure signifieds” between two languages is impossible. 8 Of course the division also fails because its sets up a dichotomy between interlingual translation, or “translation proper” on the one hand and intralingual and intersemiotic translation on the other, relegating the latter pair to figural status (Davis, 2001:28). As such, the division privileges interlingual translation in the same way oppositions such as formal/free, original/translation, proper/ improper etc. do. Literator 25(1) April 2004:47-71 3.1 Equivalence Or, in Derrida’s words, we have to substitute for the notion of translation “a notion of transformation of one language by another, of one text by another” (Derrida, 1981:21; see also Derrida, 1982:14) – not a transfer of one language into another or one text into another – an action that once again presupposes limits rather than boundaries and openings or gaps. Derrida does not promote a position aimed at removing purpose from translation. Instead, he merely suggests that the play of traces in the source text remains a play of traces in the target text (if not the same trace), and therefore cannot be fixed in a stable signifier. After all, it is impossible to limit or predict the interpretation of a translation by different readers. Furthermore, deconstruction is not only concerned with the analysis of the source text and original meaning, as Pym proclaims, but is also a useful tool for the interpretation of the target text or translation, and Derrida cannot be 8 Literator 25(1) April 2004:47-71 57 ISSN 0258-2279 Translating traces: Deconstruction and the practice of translation accused of being in favour of anything other than meticulous reading of either of these two texts. The central problem here is that Pym’s argument does not suf- ficiently take into account that Derrida and proponents of de- construction are not necessarily prescriptive. Rather, according to Venuti (1992:7), “poststructuralist textuality redefines the notion of equivalence in translation by assuming from the outset that the differential plurality in every text precludes a simple correspondence of meaning”. Plurality as contained in Derrida’s différance is not a directive, nor is it relativistic per se. Deconstruction merely requires the translator to be aware of the existence of plurality and to take into account that the reader also participates in and contributes to this plurality. This makes deconstruction particularly useful in dealing with gaps and traces that exist in both the texts in the translation contract. Derrida’s notion of différance and his pre-occupation with the word have an even more profound impact on translation when we consider translation relevance. In an elaborate argument on the relevance of translation, What is a “relevant” translation? In other words, as “transaction and as transfer” (Derrida, 2001:176). 3.1 Equivalence Derrida (2001:181) describes recent “so-called literal translation” as: a kind of translating that is not word-to-word, certainly, or word- for-word, but nonetheless stays as close as possible to the equivalence of ‘one word by one word’ and thereby respects verbal quantity as a quantity of words, each of which is an irreducible body, the indivisible unity of an acoustic form that incorporates or signifies the indivisible unity of a meaning or concept. Against this background he continues to emphasise the precarious position of the notion of translation: This is why, whenever several words occur in one or the same acoustic or graphic form, whenever a homophonic or homo- nymic effect occurs, translation in the strict, traditional, and dominant sense of the term9 encounters an insurmountable limit – and the beginning of its end, the figure of its ruin … A homonym or homophone is never translatable word-to-word. It is necessary either to resign oneself to losing the effect, the economy, the strategy (and this loss can be enormous) or to add a gloss … Wherever the unity of the word is threatened or 9 Literator 25(1) April 2004:47-71 ISSN 0258-2279 58 J.L. Kruger put into question, it is not only the operation of translation that finds itself compromised; it is also the concept, the definition, and the very axiomatics, the idea of translation that must be reconsidered (Derrida, 2001:181). This view raises a number of issues that are central to an understanding of Derrida’s concern with the word. The above passage emphasises that so-called literal translation deals with words that are significant as words on the basis of their bodies and acoustic form and that each word in turn incorporates and signifies the “indivisible unity of a meaning or concept”. In Derrida’s view, relevant translation as the transfer of an intact signifier is impossible. Similarly, according to Venuti (2001:171), … the fact is that any translating replaces the signifiers constituting the foreign text with another signifying chain, trying to fix a signified that can be no more than an interpretation according to the intelligibilities and interests of the receiving language and culture. Words are therefore not merely incidental signifiers but bring with them a plethora of connotations and voices that are at the same time impossible to translate and essential to translate: “At every moment, translation is as necessary as it is impossible” (Derrida, 2001:183). 3.1 Equivalence Derrida’s conception of relevance is directly linked to his notion of translation as economy between property and quantity. What therefore renders equivalence suspect in a Derridean approach is not the fact of a relation obtaining between an “original” or source- text unit and a translation or target-text unit, but rather the status of “original” versus translation. 3.2 “Original” and translation: towards a contract 3.2 “Original” and translation: towards a contract In order to exist as meaningful events, texts must carry within themselves traces of previous texts, and are, therefore, acts of citation. The source text for a translation is already a site of multiple meanings and intertextual crossings, and is only accessible through an act of reading that is in itself a translation. The division between ‘original’ and ‘translation’, then – as important as it is to translators and translation scholars today – is not something pre-existing that can be discovered or proven, but must be constructed and institutionalized. It is therefore always subject to revision (Davis, 2001:16). 3.2 Original and translation: towards a contract In order to exist as meaningful events, texts must carry within themselves traces of previous texts, and are, therefore, acts of citation. The source text for a translation is already a site of multiple meanings and intertextual crossings, and is only accessible through an act of reading that is in itself a translation. The division between ‘original’ and ‘translation’, then – as important as it is to translators and translation scholars today – is not something pre-existing that can be discovered or proven, but must be constructed and institutionalized. It is therefore always subject to revision (Davis, 2001:16). Literator 25(1) April 2004:47-71 59 ISSN 0258-2279 Translating traces: Deconstruction and the practice of translation Translation augments and modifies the original, which, insofar as it is living on, never ceases to be transformed and to grow. It modifies the original even as it also modifies the translating language. This process – transforming the original as well as the translation – is the translation contract between the original and the translating text (Derrida, 1985b:122). Translation deals with an “original” and a translation insofar as a cont(r)act (i.e. a contract forged by the contact and subsequent continual rewriting) exists between two texts. However, the contract resulting from the contact between the two texts should not be viewed as a dichotomy between two binary opposites, but rather as a continuation, a relationship of mutual transformation, a symbiosis. In describing this cont(r)act, it is nevertheless important to define the relationship that obtains. Translation is more than the mere transformation of an “original”. In Derrida’s view, the process of translation transforms both “original” and translation and thereby ensures the survival of the “original”. 3.2 “Original” and translation: towards a contract However, on the basis of this contract, the boundaries between “original” and translation are obscured as the survival of the “original” becomes intertwined with the survival of the translation. Through the translating text, the translation becomes an “original” itself, in the process also ensuring the survival of language. The palimpsest of the “original” in the body of the translation becomes a play of movement that also engenders a palimpsest in the “original”, ensuring a contract through contact in which a symbiosis is established. This symbiosis obviously extends to all translation and is not limited to the translation of “high literature”. In the case of, for example, the translation of the minutes of a meeting, the “original” (or the minutes in the language they were taken down in by the minuting secretary), is in a very real sense intertwined with the translation for the benefit of a member who does not have full access to the “original”. Like- wise, the “original” in the form of an operating manual for a micro- wave in, for example, English, is intertwined with the translation of this document into, for example, German or Swahili, since clarifi- cations or factual omissions in the translation will have a direct influence on the act of using the microwave which cannot be removed from the instructions intended by the manufacturer and will therefore have to be reflected in the “original”. Consequently, the violent oppositions or dichotomies that exist in translation theory and require deconstruction, such as word-for-word versus free translation, theory versus practice and “original” versus translated text are all overturned. Furthermore, what Koskinen Literator 25(1) April 2004:47-71 ISSN 0258-2279 60 J.L. Kruger (1994:446) identifies as the most paralysing dichotomy, namely the hierarchical opposition that privileges the “original” and its author above the translation and translator, is rendered powerless and obsolete by this contract. When we question this hierarchy, the notion of an “original” being inherently untouchable (in that its sanctity will be affected by any rewriting, which will simply violate this sacred text in one way or another), is no longer sustainable. This is the case because any reading of a text changes it and each change triggers subsequent changes, just as any initial choice in a translation influences and determines the rest of the translation. Furthermore, the endless chain of signification (as signified becomes signifier ad infinitum), renders any attempt at closure futile and “meaning-less”. 3.2 “Original” and translation: towards a contract Just as there is no transcendental signified for the deconstructionist, there are also “no extralinguistic meanings” (Koskinen, 1994:447). This aspect obviously complicates the process of translation as well as the analysis and description of translations infinitely. However, the influence of deconstruction on translation does not necessarily have to be regarded as an assault on the integrity of the “original”. In the words of Van den Broeck (1988:267), “decon- struction is not an act of destruction, but an act of displacement”. The différance that is translation, or that we approach in translation, creates the possibility to approach the hidden, unnamed or un- nameable properties in a text without attempting to silence them. This possibility is created through a process of instilling through the translating text a number of gaps or traces that become hidden, unnamed or unnameable without necessarily corresponding with what was hidden, unnamed or unnameable in the “original”, but which are informed by the “original” and in turn inform the “original”. Just as it is impossible to translate all aspects of any given signifier between the two language systems involved in the translation contract, it is impossible to translate all aspects of the gaps and traces, and new gaps and traces originate in this process just as new chains of signification originate. But what does this actually mean for translation practice? Does différance render equivalence obsolete in translation? We could perhaps argue that the impossibility of translation – which suggests the impossibility of equivalence – also depends on the possibility of translation and therefore of equivalence for its very existence. Since any theory informed by a notion of equivalence, irrespective of the emphasis, has to distinguish fairly rigidly “between original texts and their translations, distinctions which determine subsequent claims Literator 25(1) April 2004:47-71 61 ISSN 0258-2279 Translating traces: Deconstruction and the practice of translation about the nature of translation” (Gentzler, 1993:144), equivalence as a transfer between “original” and translation has to be questioned. In this lies the problem of equivalence. In a deconstructionist per- spective on translation, equivalence can no longer be regarded as a norm for translation practice, but rather becomes a disappearing trace. Nevertheless, it remains a trace and to the extent that its potential remains, it remains relevant for translation. 3.2 “Original” and translation: towards a contract If Derrida’s contribution to translation theory is to have any sig- nificance for translation practice, the impact of the trace has to be determined – the impact of the spatial and temporal dimensions contained in différance. The key to an application of Derrida’s theory has to be sought in the process rather than in the product of translation, and this process has to move beyond a hierarchical opposition of “original” and translation. Deconstruction radically changes this hierarchy by posing a number of questions that subvert conventional theories. Questioning the primacy of the “original” on the one hand, and more specifically the separate existence of “original” and translation on the other, changes the face of translation entirely, since the “original” and its translation are now regarded to be in a symbiotic relationship. In this regard, Gentzler (1993:146-7) calls attention to Derrida’s view that … what does exist, are different chains of signification – including the ‘original’ and its translations in a symbiotic relationship – mutually supplementing each other, defining and redefining a phantasm of sameness, which has never existed nor will exist as something fixed, graspable, known, or under- stood. This phantasm, produced by a desire for some essence or unity, represses the possibility that whatever may be there is always in motion, in flux, ‘at play’, escaping in the very process of trying to define it, talk about it, or make it present. Ironically, although Pym (1999) questions the usefulness of de- construction for translation practice, Derrida’s “phantasm of same- ness” is not that far removed from Pym’s (2000) identification of the gap between equivalence as “necessary and functional illusion” and linguistic equivalence. However, Pym regards this illusion as something that the translator has to negotiate and produce for the translation user, whereas Derrida could be seen to regard the phantasm as a product of the desire for unity which merely represses the elusive nature of the trace. Therefore, Pym’s more functionalist approach can benefit substantially from a recognition and awareness of the operation of différance. Ironically, although Pym (1999) questions the usefulness of de- construction for translation practice, Derrida’s “phantasm of same- ness” is not that far removed from Pym’s (2000) identification of the gap between equivalence as “necessary and functional illusion” and linguistic equivalence. 3.2 “Original” and translation: towards a contract However, Pym regards this illusion as something that the translator has to negotiate and produce for the translation user, whereas Derrida could be seen to regard the phantasm as a product of the desire for unity which merely represses the elusive nature of the trace. Therefore, Pym’s more functionalist approach can benefit substantially from a recognition and awareness of the operation of différance. Literator 25(1) April 2004:47-71 ISSN 0258-2279 62 J.L. Kruger J.L. Kruger In terms of the role of the “original”, Bannet (1993:586) expresses the opinion that the translation must pay its debt to the “original”, not only by taking its directives from the “original”, but also by being a moment in the growth of the “original”. Bannet then states that In terms of the role of the “original”, Bannet (1993:586) expresses the opinion that the translation must pay its debt to the “original”, not only by taking its directives from the “original”, but also by being a moment in the growth of the “original”. Bannet then states that ... this does not mean that original and translation resemble each other or that the translation may not depart from the original, for the translation has only to touch on the original at a few fugitive and ‘infinitely small points of sense’ … the trans- lation adds to the original by freeing itself from the original and taking its own course (Bannet, 1993:586). ... this does not mean that original and translation resemble each other or that the translation may not depart from the original, for the translation has only to touch on the original at a few fugitive and ‘infinitely small points of sense’ … the trans- lation adds to the original by freeing itself from the original and taking its own course (Bannet, 1993:586). This refers to Benjamin’s notion that a translation touches the original at an infinitely small point of meaning (Benjamin, 1992:81). However, Derrida (1985a:189) reacts to Benjamin’s statement with the questions: “What can an infinitely small point of meaning be? What is the measure to evaluate it?” According to Derrida (1985a: 188): If the translator neither restitutes nor copies an original, it is because the original lives on and transforms itself. 3.2 “Original” and translation: towards a contract The trans- lation will truly be a moment in the growth of the original, which will complete itself in enlarging itself … And if the original calls for a complement, it is because at the origin it was not there without fault, full, complete, total, identical to itself. A logical result of the shift from metaphysical and fixed meaning to gaps and traces that cannot be named and therefore silenced or contained, is that the text is “created anew in every reading and no ownership can be declared” (Koskinen, 1994:448). This dethrones the author and renders the hierarchical opposition between source and target text, or “original” and translation, obsolete. If we accept this view of texts, the Renaissance metaphor of translation as footprint of the “original” (Koskinen, 1994:449) can also be revised substantially. The very act of translation (re)activates the play of traces and by definition creates further plays in relation to the past-become-future through the present, but never in the present, of translation. Therein lies untranslatability in translatability – untranslatability because there is no fixed meaning to begin with and because it is also impossible to transform the différance from one system into another in fixed form. Yet this also contains translatability and the life of translation as process of trace and différance. The very un- translatability provides the tension which makes translation ne- Literator 25(1) April 2004:47-71 ISSN 0258-2279 63 Translating traces: Deconstruction and the practice of translation cessary as curative process (see Bannet, 1993) – always approach- ing the potential of the unattainable. cessary as curative process (see Bannet, 1993) – always approach- ing the potential of the unattainable. This point of view changes the hierarchical difference between “original” and translation in that, although the translation issues forth from the “original”, it does so because the “original” is indebted to the translation for its survival. The translation is therefore not a footprint left by the “original”, but a different set of footprints with a similar relation to past, present and future – in other words different from and at the same time similar to the “original” in that it has the same impulse. According to Venuti (1992:7), the poststructuralist concept of textuality compromises the originality of the foreign text in such a way that “neither the foreign text nor the translation is an original semantic unity; both are derivative and heterogeneous”. 10 This aspect is illustrated in his article, “What is a ‘relevant’ translation?” (Derrida, 2001), where he provides an elaborate and eloquent justification for his choice of the French verb “relève” to translate the English verb “seasons” in The Merchant of Venice. 3.2 “Original” and translation: towards a contract In neither text can the “meaning” be final, for each reading is part of the process of creation that postpones or defers meaning in its creation of another set of gaps and traces. In the words of Koskinen (1994:450), translation includes the idea of repetition, but just as signs when repeated are never similar [and therefore change or are modified with every reading], translations can never be identical replicas. Différance takes part in every repetition. Translation is not the same text as the source text, but it is not a different text either. It deconstructs the opposition between difference and sameness. Inevitably, this deconstruction raises questions about translatability. If signs do not even remain the same in repetition and in intralingual transformations, how can translation begin to claim to be able to name? How can the very notion of différance not result in un- translatability? Inevitably, this deconstruction raises questions about translatability. If signs do not even remain the same in repetition and in intralingual transformations, how can translation begin to claim to be able to name? How can the very notion of différance not result in un- translatability? 3.3 Translation and the unnameable, untranslatable A text lives only if it lives on, and it lives on only if it is at once translatable and untranslatable ... Totally translatable, it dis- appears as a text, as writing, as a body of language. Totally untranslatable, even within what is believed to be one lang- uage, it dies immediately (Derrida, 1979:102). As a matter of fact, I don’t believe that anything can ever be untranslatable – or, moreover, translatable (Derrida, 2001:178). When Derrida states that nothing is untranslatable and also th nothing is translatable he refers to a concept of translation based 3.3 Translation and the unnameable, untranslatable A text lives only if it lives on, and it lives on only if it is at once translatable and untranslatable ... Totally translatable, it dis- appears as a text, as writing, as a body of language. Totally untranslatable, even within what is believed to be one lang- uage, it dies immediately (Derrida, 1979:102). As a matter of fact, I don’t believe that anything can ever be untranslatable – or, moreover, translatable (Derrida, 2001:178). When Derrida states that nothing is untranslatable and also that nothing is translatable, he refers to a concept of translation based on Literator 25(1) April 2004:47-71 64 ISSN 0258-2279 J.L. 11 Referring to the title of Benjamin’s essay, Derrida (1985a:175) remarks that it “also says, from its first word, the task (Aufgabe), the mission to which one is destined (always by the other), the commitment, the duty, the debt, the responsibility. Already at stake is a law, an injunction for which the translator has to be responsible. He must also acquit himself, and of something that implies perhaps a fault, a fall, an error and perhaps a crime.” 3.2 “Original” and translation: towards a contract Rather, translation becomes more focused on the complex set of relations Literator 25(1) April 2004:47-71 65 ISSN 0258-2279 Translating traces: Deconstruction and the practice of translation between the two texts, without awarding a primary status to either and without claiming the ability to gauge the exact meaning beneath the surface structure of the source text or to encode it in the surface structure of the target text (cf. Nida, 1964). On the contrary, what becomes important also includes everything that is not evidenced in the surface structure of a text, which in turn includes all aspects that are activated in the writing or rewriting of the text (therefore also in its reading and translation). between the two texts, without awarding a primary status to either and without claiming the ability to gauge the exact meaning beneath the surface structure of the source text or to encode it in the surface structure of the target text (cf. Nida, 1964). On the contrary, what becomes important also includes everything that is not evidenced in the surface structure of a text, which in turn includes all aspects that are activated in the writing or rewriting of the text (therefore also in its reading and translation). Commenting on the consequence of the notion of untranslatability for translation, Bannet (1993:580-1) states: After De Man, we might be tempted to stop here: the translator, per definition, fails. The translator can never do what the original text did. Any translation is always second in relation to the original … But after Derrida, it becomes apparent that … the failure of translation and the wandering, errance, and exile of language envelop two possibilities, one lethal, the other curative. In Bannet’s view, De Man develops the lethal possibility and Derrida the curative in their respective treatments of Benjamin’s Die Aufgabe des Übersetzers (1972; see Benjamin 1992). De Man interprets Benjamin “lethally”, as saying that the task and the duty of the translator are to give up and that translations do not relate to the life of the “original”, but to its death. This results in a view that “translation is the resistance to translation; and the wandering, the erring of metaphors, the resistance to metaphor” (Bannet, 1993:582- 584). In contrast, “Derrida explores the curative possibility en- veloped in the wandering, errance, and exile of language and in the inevitable failure of translation”. 3.2 “Original” and translation: towards a contract Kruger J.L. Kruger “the condition of a certain economy that relates the translatable to the untranslatable, not as the same to the other, but as same to same or other to other” (Derrida, 2001:178). This economy further signifies both property (in the sense of what is proper or appropriate) and quantity (in other words calculable quantity). A relevant translation, according to Derrida (2001:179), is therefore “a translation whose economy, in these two senses, is the best possible, the most appropriating and the most appropriate possible”. He explains this further as an “economy of in-betweenness” in which “any given translation, whether the best or the worst, actually stands between the two, between absolute relevance, the most appropriate, adequate, univocal transparency, and the most aberrant and opaque irrelevance” (Derrida, 2001:179). What Derrida’s “economy” there- fore seems to signify, is that anything is translatable if the translator is not limited in terms of quantity,10 but that the law of “quantity” renders it impossible to translate fully in terms of the law of “property”. In translation an economy is therefore essential and will always be situated somewhere between absolute relevance and absolute irrelevance. If we leave Derrida at this point of the argument, it would seem that he does indeed pronounce translation, in the conventional use of the term, to be impossible. Yet, this does not mean that his ideas are only meaningful in the ambit of (philosophical) interpretation and useless for translation practice and application, as Pym argues in his Doubts about deconstruction as a general theory of translation (1999). Untranslatability in Derrida’s use of the term does not imply that translators should not translate. It simply implies that it is impossible to produce the plurality of the source text in a translation while obeying the law of quantity. As mentioned in the introduction, deconstruction upsets traditional views of translation by removing equivalence from the skopos or purpose of translation. From the perspective of deconstruction, it is no longer possible to reduce the aim of translation to creating a target text that is equivalent to the source text (regardless of which aspects are considered important in terms of equivalence). 3.2 “Original” and translation: towards a contract Therefore, according to Bannet, Derrida interprets Benjamin as saying that “the duty of the translator is the duty of an inheritor, the debt that of a survivor, and the responsibility that of an agent of survival” who has to ensure the survival and living on of the “original”11 (Bannet, 1993:585). Indeed, Derrida (1985a:179) states that “[t]here is life at the moment when ‘sur-vival’ (spirit, history, works) exceeds biological life and death”. This is precisely where the curative force of différance lies. Because Literator 25(1) April 2004:47-71 66 ISSN 0258-2279 J.L. Kruger différance implies that meaning is always postponed and can never be present, the potential lives on in the trace, also constituted in translation. And traces, according to Davis (2001:30), “are always repeatable or iterable”. différance implies that meaning is always postponed and can never be present, the potential lives on in the trace, also constituted in translation. And traces, according to Davis (2001:30), “are always repeatable or iterable”. According to Bannet (1993:585), in Derrida’s translation of Ben- jamin, “the relation between original and translation is based not on resemblance, but on difference. The alliance … between original and translation … depends on the difference of two parties or two texts that can only be joined through their differences”. These differences complete them and in their completion they are both changed. Untranslatability is therefore intimately connected to the cont(r)act between the translation and the “original”, a contract through the contact between two texts that are always becoming in a symbiotic relationship of rewriting. This relationship to a large extent depends on the creative power contained in the play of the trace. Translation therefore no longer fixes the same meaning, but creates new avenues for further difference. Although there is a common misconception that Derrida claims complete freeplay and undecidability in language, it should be clear from the preceding that this is not the case. According to Davis (2001:30), Derrida does not claim that there can be no stability of meaning”. In fact, “stability and instability … are mutually constitutive necessities” (Davis, 2001:32). 3.2 “Original” and translation: towards a contract Davis elaborates on the concept of stability, stressing that, although it gives us access to texts due to historical repetition, institutionalisation etcetera, it is also limited because there is always difference at the origin, each repetition is different from all the others, and neither a text’s author nor its enactment in one context can fully determine its repetition in another context (Davis, 2001:32). To return to Derrida’s statement cited at the beginning of this paragraph, that he does not believe that “anything can ever be untranslatable – or, moreover, translatable” (Derrida, 2001:178), we could say the following: Nothing can ever be untranslatable because every sign is repeatable, and nothing can be translatable because it can only be repeated in a different context which, according to Davis (2001:34), causes the possibilities for its meaning to remain open. Literator 25(1) April 2004:47-71 ISSN 0258-2279 67 Translating traces: Deconstruction and the practice of translation 4. Conclusion: translating outwards Translation is writing; that is, it is not translation only in the sense of transcription. It is a productive writing called forth by the original text. (Derrida, 1985b:153.) Derrida’s contribution to translation theory goes beyond the obvious implications of différance. To a significant extent, Derrida could be said to reclaim the productive potential of translation that emerges from his emphasis on the play of the trace. Calling translation “a productive writing” imbues it with the power to activate hidden traces, ensuring the survival of the “original” at the same time as the translation issues from it. However, Derrida contrasts the necessity for translation (contained in the “calling forth” of the translation by the “original”) to the desire for an intact kernel. In this vein he says that “the desire or the phantasm of the intact kernel is irreducible – despite the fact that there is no intact kernel” (Derrida, 1985a:115). Because there is no intact kernel, this desire can never be satisfied, yet it remains and produces the tension that makes translation productive. The unattainable nature of translation as well as the opposing necessity for translation therefore produces a vortex that ensures the life of the “original” in its transformation by the translation – a process that necessarily proceeds outward. Only by passing through the trial of undecidability can the decisions on which translation is premised be made. 3.2 “Original” and translation: towards a contract In the words of Davis (2001:90), “since translations can never perfectly transport an ‘original’ (there being no fully determined original in the first place), they require decisions, in the strong sense of that word”. With regard to the theory of translation, deconstruction forces us to break with conventional logocentric approaches to translation that are necessarily directed inward, towards the source text and some metaphysical notion of meaning. Instead it becomes imperative to direct our thinking and translation theory as well as practice outward, in the spirit of Cicero’s notions of explicare, reddere, and exprimere imitando, which are identified by Robinson (1997:184-185) as providing a decidedly outward impetus. Explicare, for example, “is to explicate, of course, to expound, to interpret, but specifically in the conflicted sense of both ‘ordering’ and ‘setting free’, both ‘making plain’ and ‘spreading out’” (Robinson, 1997:186). Even in taking us back to Cicero, and in deconstructing his terms, Robinson therefore confirms the necessary condition of opposition, difference and Literator 25(1) April 2004:47-71 ISSN 0258-2279 68 J.L. Kruger tension contained in translation. For, as Derrida (1992:258) suggests, it is specifically that which is untranslatable which calls for translation; and, “a translation never succeeds in the pure and absolute sense of the term. Rather, a translation succeeds in promising success, in promising reconciliation” (Derrida, 1985b: 123). Therefore, différance becomes not an obstacle or barrier to translation, but specifically that which, in making something un- translatable, creates the need for translation. For just as conflict is a condition for change, the fact that a text is at once translatable and untranslatable allows for its survival – because the terms are not binary oppositions but become conditions for the existence of each other. Nothing can be wholly untranslatable or wholly translatable. The translatability is premised on what is untranslatable and vice versa. It is in the gaps arising from this conflict that the traces that generate meaning emerge – perhaps not a closed meaning, but the possibility and promise of as well as a yearning for meaning. Just as untranslatability becomes a condition for translatability, ‘mis- translation’ is a condition for translation. This dynamic quality of Derrida’s thoughts on translation makes it possible to project a theory of translation that invites translators and readers alike to engage in the play of the trace. 3.2 “Original” and translation: towards a contract Although such a theory is concerned with the word and the love for the word, it also leaves “the other body intact but not without causing the other to appear” (Derrida, 2001:175). List of references Bannet, E.T. 1993. The scene of translation: After Jakobson, Benjamin, De Man, and Derrida. New Literary History, 24:577-595, Summer. Bannet, E.T. 1993. The scene of translation: After Jakobson, Benjamin, De Man, and Derrida. New Literary History, 24:577-595, Summer. y y Begam, R. 1992. Splitting the différance: Beckett, Derrida and the unnamable. Modern Fiction Studies, 38(4):873-892, Winter. Begam, R. 1992. Splitting the différance: Beckett, Derrida and the unnamable. Modern Fiction Studies, 38(4):873-892, Winter. Benjamin, W. 1992. The task of the translator. Trans. Harry Zohn. In: Schulte, R. & Biguenet, J. (eds.) Theories of translation: An anthology of essays from Dryden to Derrida. Chicago : University of Chicago Press. p. 71-82. Benjamin, W. 1992. The task of the translator. Trans. Harry Zohn. In: Schulte, R. & Biguenet, J. (eds.) Theories of translation: An anthology of essays from Dryden to Derrida. Chicago : University of Chicago Press. p. 71-82. Berman, A. 2000. Translation and the trials of the foreign. In: Venuti, L. (ed.) The translation studies reader. London : Routledge. p. 284-297. Berman, A. 2000. Translation and the trials of the foreign. In: Venuti, L. (ed.) The translation studies reader. London : Routledge. p. 284-297. Cohen, L. 1992. The Future. London : Sony. y Clarkson, C. 2003. “By any other name”: Kripke, Derrida and an ethics of naming. Journal of Literary Studies, 32:35-47. Clarkson, C. 2003. “By any other name”: Kripke, Derrida and an ethics of naming. Journal of Literary Studies, 32:35-47. g y Davis, K. 2001. Deconstruction and translation. Manchester : St. Jerom Derrida, J. 1974. Of grammatology. Trans. Gayatri Chakravorty Spivak. Lon- don : Johns Hopkins University Press. Derrida, J. 1974. Of grammatology. Trans. Gayatri Chakravorty Spivak. Lon- don : Johns Hopkins University Press. Literator 25(1) April 2004:47-71 ISSN 0258-2279 69 Translating traces: Deconstruction and the practice of translation Derrida, J. 1979. “Living on” and “Border Lines”. Trans. James Hulbert. In: Bloom, H., De Man, P., Derrida, J., Hartman, G. & Miller, J.H. Deconstruction and criticism. New York : Seabury Press. p. 75-176 . y p Derrida, J. 1981. Positions. Trans. Alan Bass. Chicago : University of Chicago Press. Derrida, J. 1982. Margins of philosophy. Trans. Alan Bass. Brighton : Harveste Derrida, J. 1985a. Des tours de Babel. Trans. Joseph F. Graham. In: Graham, J.F. (ed.) Difference in translation. Ithaca : Cornell University Press. p. 209-248. p Derrida, J. 1985b. The ear of the other: Otobiography, transference, translation. Trans. Peggy Kamuf. Literator 25(1) April 2004:47-71 List of references New York : Schocken. Derrida, J. 1992. Ulysses gramophone: Hear Say Yes in Joyce. Trans. Tina Kendall. In: Attridge, D. (ed.) Jacques Derrida: Acts of literature. New York : Routledge. p. 253-309. g p Derrida, J. 2001. What is a “relevant” translation? Critical Inquiry, 27(2):174- 200, Winter. , Gentzler, E. 1993. Contemporary translation theories. London : Routledge. Jakobson, R. 2000. On linguistic aspects of translation. In: Venuti, L. (ed.) The translation studies reader. London : Routledge. p. 113-118. Koskinen, K. 1994. (Mis)translating the untranslatable: The impact of decon- struction and post-structuralism on translation theory. Meta: Journal des Traducteurs/Translators’ Journal, 39(3):446-452. ( ) Lewis, P.E. 2000. The measure of translation effects. In: Venuti, L. (ed.) The translation studies reader. London : Routledge. p. 264-283. Nida, E. 1964. Towards a science of translating. Leiden : Brill. Pym, A. 1999. Doubts about deconstruction as a general theory of translation. [Web:] http://www.fut.es/~apym/on-line/decon.html [Date of access: 28 March 2001]. Pym, A. 2000. European translation studies, une science qui dérange, and why equivalence needn’t be a dirty word. [Web:] http://www.fut.es/~apym/ deranger.html [Date of access: 25 January 2001]. Robinson, D. 1997. What is translation? Centrifugal theories, critical interventions. Kent : Kent State University Press. Van den Broeck, R. 1988. Translation theory after deconstruction. Linguistica Antverpiensia, 22:266-88. Venuti, L. (ed.) 1992. Rethinking translation: Discourse, subjectivity, ideology. London : Routledge. g Venuti. L. (ed.) 2000. The translation studies reader. London : Routledge. Venuti, L. 2001. Introduction. Critical Inquiry, 27(2):169-173, Winter. Vermeer, H.J. 2000. Skopos and commission in translational action. Trans. Andrew Chesterman. In: Venuti, L. (ed.) The translation studies reader. London : Routledge. p. 221-232. Literator 25(1) April 2004:47-71 70 ISSN 0258-2279 J.L. Kruger Literator 25(1) April 2004:47-71 Kernbegrippe: dekonstruksie spel van die spoor différance vertaling Kernbegrippe: dekonstruksie spel van die spoor différance vertaling Literator 25(1) April 2004:47-71 ISSN 0258-2279 71 Translating traces: Deconstruction and the practice of translation Literator 25(1) April 2004:47-71 72 ISSN 0258-2279
https://openalex.org/W4287194028	https://zenodo.org/records/6579546/files/OHEJP-Annual-Report-2020_final-1.pdf	English	null	D1.13. Complete version of annual report for stakeholders number 3	Zenodo (CERN European Organization for Nuclear Research)	2,021	cc-by	33,022	ehealthejp.eu @OneHealthEJP ONE Health EJP This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement No. 773830 ual Report 2020 1 onehealthejp.eu @OneHealthEJP ONE Health EJP UCTION ON CTURE EMENTS OMES NATION ATION This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement No. 773830 Annual Report 2020 MEDIA 41 43 44 45 47 48 49 50 51 52 53 54 55 56 57 58 60 62 63 64 65 66 67 68 69 73 s Team Surrey CO N TEN TS EJP 3 4 5 6 7 8 9 10 11 12 14 16 18 19 20 22 23 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 FED-AMR 41 TOX-detect 43 TELE-VIR 44 MEmE 45 PARADISE 47 IDEMBRU 48 One Health EJP PhD Programme 49 ECO-HEN 50 LIN-RES 51 HME-AMR 52 KENTUCKY 53 METAPRO 54 PEMbo 55 MACE 56 DESIRE 57 UDoFRIC 58 WILBR 60 EnvDis 62 AptaTrich 63 VIMOGUT 64 ToxSauQMRA 65 TRACE 66 Codes4strains 67 SUSTAIN 68 OHEJP Dissemination Activities 69 OHEJP Education and Training 73 Report prepared by the OHEJP Communications Team at the University of Surrey Introduction 3 Our Objectives 4 One Health EJP Governance 5 One Health EJP Structure 6 Third Year Achievements 7 Coordination of the One Health EJP 8 Communicating Success 9 Translation of Science to Policy 10 Response to COVID-19 11 One Health EJP Scientific Outcomes 12 ORION 14 COHESIVE 16 CARE 18 OH-HARMONY-CAP 19 MATRIX 20 NOVA 22 LISTADAPT 23 METASTAVA 25 AIR SAMPLE 26 MoMIR 27 MedVetKlebs 28 DISCOVER 29 BIOPIGEE 30 TOXOSOURCES 31 ADONIS 32 BeONE 33 IMPART 34 ARDIG 35 RaDAR 36 FARMED 37 FULL-FORCE 38 WORLDCOM 39 FED-AMR TOX-detect TELE-VIR MEmE PARADISE IDEMBRU One Health EJP PhD Programme ECO-HEN LIN-RES HME-AMR KENTUCKY METAPRO PEMbo MACE DESIRE UDoFRIC WILBR EnvDis AptaTrich VIMOGUT ToxSauQMRA TRACE Codes4strains SUSTAIN OHEJP Dissemination Activities OHEJP Education and Training Report prepared by the OHEJP Communication Report prepared by the OHEJP Communications Team at the University of Surrey onehealthejp.eu @OneHealthEJP ONE Health EJP INTRODUCTION VISION STRUCTURE ACHIEVEMENTS OUTCOMES DISSEMINATION EDUCATION O U R O B JECTIV ES The overarching objective of the OHEJP is to develop a collaborative European network of public research organisations with reference laboratory functions. A key aim of the OHEJP is to integrate medical, veterinary and food scientists to address three key research topics: foodborne zoonoses, antimicrobial resistance and emerging infectious disease threats. Public health concerns of consumers and other stakeholders are also at the forefront of the consortium’s focus. The One Health European Joint Programme (OHEJP) is a landmark partnership between 44 public health, animal health and food organisations and the Med-Vet-Net Association, a European Network of Excellence for Zoonoses research, the OHEJP spans 22 countries across Europe. The One Health concept recognises that human health is tightly connected to the health of animals and the environment, therefore the study of infectious diseases that may cross species and environmental barriers is imperative. The main focus of the OHEJP is to reinforce collaboration between partners by enhancing collaboration and integration of activities by means of dedicated Joint Research Projects (JRPs), Joint Integrative Projects (JIPs) and through education and training in the fields of foodborne zoonoses, antimicrobial resistance and emerging infectious disease threats. Through the OHEJP, there are opportunities for harmonisation of approaches, methodologies, databases and procedures for the assessment and management of foodborne zoonoses, emerging infectious disease threats and antimicrobial resistance across Europe, which will improve the quality and compatibility of shared information for policy decision making. onehealthejp.eu @OneHealthEJP ONE Health EJP Key objectives: To bring together the major representatives of European scientific communities with expertise in foodborne zoonoses, antimicrobial resistance and emerging infectious disease threats. To implement scientific integrative and collaborative projects related to the prevention of foodborne zoonoses, antimicrobial resistance and emerging infectious disease threats. To stimulate scientific excellence by co-funding Joint Research Projects and Joint Integrative Projects that have the potential to enhance scientific knowledge and provide tools for disease surveillance at both the national and European level. To foster the harmonisation and standardisation of laboratory methods by bringing together scientific and technical expertise. To exchange and communicate with European and international stakeholders, first and foremost with the European Centre for Disease Control and Prevention (ECDC) and the European Food Safety Authority (EFSA). To exchange and communicate with European and international stakeholders, first and foremost with the European Centre for Disease Control and Prevention (ECDC) and the European Food Safety Authority (EFSA). onehealthejp.eu @OneHealthEJP ONE Health EJP INTRODUCTION VISION STRUCTURE ACHIEVEMENTS OUTCOMES DISSEMINATION EDUCATION O N E HEA L TH EJP GO V ERN A N CE ence to Policy Translation - Work Package 5 5 ensures best use of the scientific outcomes of the JRPs and JIPs ough dissemination activities and networking with OHEJP stakeholders. ucation and Training - Work Package 6 6 develops and delivers innovative training platforms with a specific focus One Health. stainability - Work Package 7 7 explores operational means to sustain long-term research and innovation yond the duration of the OHEJP. N E HEA L TH EJP S TRU CTU RE H U M A N S A NI M AL S F O O D O O D N T H U M A N S F O O D M E N T A N M E N T A NI M AL S N O O D E N VI R O N M E N T AL S F O O D H U M A N S I N T E R N A T I O N A L / N A T I O N A L S T A K E H O L D E R S P R O J E C T S WP3 JOINT SCIENTIFIC PROJECTS WP4 JOINT INTEGRATIVE PROJECTS WP6 EDUCATION AND TRAINING WP7 SUSTAINABILITY WP2 STRATEGIC RESEARCH AGENDA WP5 SCIENCE TO POLICY TRANSLATION W P 1 C O O R D I N A T I O N O N E HEA L TH EJP GO V ERN A N CE A governing and management system was established at the beginning of the OHEJP. The governing boards specific to the OHEJP include: The Project Management Team (PMT), Scientific Steering Board (SSB) and Programme Managers Committee (PMC). There are also important contributions from members outside of the OHEJP and these include: The Programme Owners Committee (POC), the External Scientific Advisory Board (ESAB), the Stakeholders Committee (SC), the Ethics Advisors and National Mirror Groups. The OHEJP Coordination Team are based at the French Agency for Food, Environmental and Occupational Health & Safety (ANSES), France. The OHEJP Scientific Coordinator resides at Sciensano, the Belgian Institute for Health. The Project Management Team consists of all the Work Package (WP) Leaders and Deputy Leaders. J A governing and management system was established at the beginning of the OHEJP. The governing boards specific to the OHEJP include: The Project Management Team (PMT), Scientific Steering Board (SSB) and Programme Managers Committee (PMC). There are also important contributions from members outside of the OHEJP and these include: The Programme Owners Committee (POC), the External Scientific Advisory Board (ESAB), the Stakeholders Committee (SC), the Ethics Advisors and National Mirror Groups. The OHEJP Coordination Team are based at the French Agency for Food, Environmental and Occupational Health & Safety (ANSES), France. The OHEJP Scientific Coordinator resides at Sciensano, the Belgian Institute for Health. onehealthejp.eu @OneHealthEJP ONE Health EJP INTRODUCTION VISION STRUCTURE ACHIEVEMENTS OUTCOMES DISSEMINATION EDUCATION e OHEJP consist of seven work packages (WP), all are targeted towards ecific overarching needs and objectives of the OHEJP. Each work package sures the alignment and integration in the implementation of the oject. Visit the One Health EJP website for more information about e different work packages. ordination - Work Package 1 1 enables the successful functioning of the OHEJP and maintains environment where scientists can effectively and actively aborate. ategic Research Agenda - Work Package 2 2 is responsible for the Integrative Strategic Research Agenda the OHEJP, which identifies research and integrative priority ics aligning to stakeholder needs. nt Research Projects (JRPs) - Work Package 3 3 mainly supports the Joint Research Projects which carry out ntly prioritised research projects and stimulate collaboration and rmonisation across the projects and partner institutes. nt Integrative Projects (JIPs) - Work Package 4 4 is responsible for organising selection, supervision and evaluation the Joint Integrative Projects and stimulating harmonisation across rtner institutes and with other ongoing EU initiatives. O N E HEA L TH EJP S TRU CTU RE The OHEJP consist of seven work packages (WP), all are targeted towards specific overarching needs and objectives of the OHEJP. Each work package ensures the alignment and integration in the implementation of the project. Visit the One Health EJP website for more information about the different work packages. R Coordination - Work Package 1 WP1 enables the successful functioning of the OHEJP and maintains the environment where scientists can effectively and actively collaborate. WP6 develops and delivers innovative training platforms with a specific focu on One Health. T H U M A N S Sustainability - Work Package 7 Sustainability - Work Package 7 WP7 explores operational means to sustain long-term research and innovation beyond the duration of the OHEJP. WP7 explores operational means to sustain long-term research and innovation beyond the duration of the OHEJP. onehealthejp.eu @OneHealthEJP ONE Health EJP onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:PIXABAY What has the One Health EJP achieved in year three? What has the One Health EJP achieved in year three? IMAGE:PIXABAY CO O RDIN A TIO N O F THE O N E HEA L TH EJP The coordination of the OHEJP involves overseeing the organisation (WP1), coordinating the Joint Research and Joint Integrative Projects (WP3 and WP4, respectively) and coordinating the Education and Training activities (WP6), in addition to carrying out all central communication activities (WP1). What were the key coordination activities in year three? Communicating and collaborating with the OHEJP governance bodies: Coordination Team, Project Management Team, Scientific Steering Board, Programme Managers Committee and Programme Owners Committee.i • Communicating and collaborating with the OHEJP governance bodies: Coordination Team, Project Management Team, Scientific Steering Board, Programme Managers Committee and Programme Owners Committee.i Successful and proactive monitoring of the OHEJP’s progress and reporting of scientific outcomes to the European Commission (EC) and Research Executive Agency (REA). Preparing to expand the OHEJP consortium to include six additional European partners to complete more public health/animal health partnerships within the EU member states. A coordinated response to the COVID-19 pandemic to inform internal and external audiences of the OHEJP’s role and the One Health approach to the pandemic. Ongoing support for the JRPs, JIPs, Education and Training activities and other OHEJP scientific events. Successful and proactive monitoring of the OHEJP’s progress and reporting of scientific outcomes to the European Commission (EC) and Research Executive Agency (REA). Preparing to expand the OHEJP consortium to include six additional European partners to complete more public health/animal health partnerships within the EU member states. A coordinated response to the COVID-19 pandemic to inform internal and external audiences of the OHEJP’s role and the One Health approach to the pandemic. Ongoing support for the JRPs, JIPs, Education and Training activities and other OHEJP scientific events. The internal calls to organise the 2021 OHEJP activities were launched: the Annual Scientific Meeting (ASM) and ASM Satellite Workshop, Summer School, CPD Module, and Short Term Missions were launched. These will be hosted by one of the consortium member institutes. Co-ordination and management of 36 overarching OHEJP deliverables were submitted to the REA. The Communications Team have developed and enhanced communication throughout the consortium to stakeholders, and external audiences. The OHEJP brand, website and social media platforms were used to inform all audiences of all joint successes. Improved dissemination and publication procedures for key outcomes were determined and implemented to ensure consortium members were supported in communicating their research and impact. onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:PIXNIO CO M M U N ICA TIN G S U CCES S The Communications Team sits centrally in the OHEJP Consortium in WP1, delivering communications and coordinating activities effectively to ensure the OHEJP achieves its goals and fulfils its potential. The OHEJP Communications Team achieved several successes in year three: TRA N S L A TIO N O F S CIEN CE TO P O L ICY A key aim of the OHEJP is to identify Stakeholders’ needs to inform the Strategic Research Agenda (WP2), which ensures the scientific outcomes are useful, and supports the sustainability of the consortium (WP7). The impact of the OHEJP’s outcomes is maximised by targeted dissemination and communication to OHEJP Stakeholders (WP5). The OHEJP Communications Team achieved several successes in year three: The OHEJP Communications Team achieved several successes in year three: • The OHEJP brand was strengthened by improving visibility across all platforms including, meetings, workshops, Education and Training events, the OHEJP website and social media. • Supported all OHEJP events and facilitated the migration of physical events to virtual events in response to the COVID-19 pandemic. • Developed communication and dissemination tools for consortium members to support the dissemination of scientific outcomes and demonstrate impact.i Developed communication and dissemination tools for consortium members to support the dissemination of scientific outcomes and demonstrate impact.i Created several interactive documents and case studies to showcase the OHEJP to scienti and non-scientific audiences across the globe. Created several interactive documents and case studies to showcase the OHEJP to scientific and non-scientific audiences across the globe. Presented at the OHEJP Communication and Media Workshop in October 2020, delivering training and expertise in digital communications, creative communications and branding when organising events. Attended the virtual World One Health Congress in November 2020 which enabled the OHEJP to be visible to a global audience. Supported stakeholders in their work and social media campaigns, which included EU-JAMRAI’s Antimicrobial Resistance Symbol and the European Commission’s #ScienceFromHome campaigns. Supported stakeholders in their work and social media campaigns, which included EU-JAMRAI’s Antimicrobial Resistance Symbol and the European Commission’s #ScienceFromHome campaigns. Maintained the OHEJP Zenodo account to ensure that publications and deliverables are open access. Maintained the OHEJP Zenodo account to ensure that publications and deliverables are open access. onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:FLICKR What were the key outcomes for year three? • Consolidated relationships with key EU stakeholders ECDC and EFSA, and other European and global stakeholders by expanding the stakeholder network: The European Medicine’s Agency (EMA), European Environment Agency (EEA), Food and Agriculture Organization of the United Nations (FAO), the World Organisation for Animal Health (OIE) and the World Health Organization regional office for Europe (WHO-EURO), all joined the OHEJP Stakeholders Committee. • Consolidated relationships with key EU stakeholders ECDC and EFSA, and other European and global stakeholders by expanding the stakeholder network: The European Medicine’s Agency (EMA), European Environment Agency (EEA), Food and Agriculture Organization of the United Nations (FAO), the World Organisation for Animal Health (OIE) and the World Health Organization regional office for Europe (WHO-EURO), all joined the OHEJP Stakeholders Committee. The OHEJP Outcome Inventory was further developed to document and share all outcomes from the OHEJP projects. This has been widely shared with stakeholders and internal and external audiences. Stakeholders’ needs were regularly monitored to ensure that the OHEJP is at the forefront of current and topical research interests, which enables quick responses from the consortium and high levels of engagement with needs across Europe and globally. Strategic interactions with EU projects and initiatives were further developed and shared with the OHEJP consortium. The new projects added include: VEO, SAFECONSUME, ZODIAC, GNA NOW, EVA-GLOBAL and ENOVAT. Three Cogwheel Workshops in 2020 were designed to strengthen relationships between the OHEJP and selected identified EU projects: InfAct, JPI-AMR and SAFECONSUME. A Strategic Research and Innovation Agenda is under development which aims to define research needs beyond the lifetime of the OHEJP. onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:PIXABAY RES P O N S E TO CO V ID- 1 9 The third year of the One Health EJP was significantly impacted by the COVID-19 pandemic and many of our partner institutes and research groups turned their attention to surveillance and management during the pandemic. As a result, many of the projects funded in the first round of funding in 2018 were extended into 2021 which will enable the research to be completed. The OHEJP Project Management Team and Communications Team responded to the pandemic in a timely manner and ensured that communication to internal and external audiences was clear and reflected the OHEJP’s response to the pandemic. The Communications Team published a COVID-19 newsletter in March 2020 to update audiences on how the consortium and its partner institutes were turning their efforts and using their collaborations to take a One Health approach to the pandemic. A Latest News page was also set up on the One Health EJP website to inform audiences in real time and to act as a hub for key information. An additional Joint Integrative Project, COVRIN, was proposed to use the strengths of the consortium to make a significant impact during the pandemic. This project will begin in March 2021. onehealthejp.eu @OneHealthEJP ONE Health EJP onehealthejp.eu @OneHealthEJP ONE Health EJP One Health EJP Scientific Outcomes IMAGE:WIKIMEDIA onehealthejp.eu @OneHealthEJP ONE Health EJP One Health EJP Scientific Outcomes IMAGE:WIKIMEDIA VISION IMAGE:MJ RICHARDSON O N E HEA L TH EJP S CIEN TIF IC O U TCO M ES To date, the One Health EJP has funded 24 Joint Research Projects (JPRs), 5 Joint Integrative Project (JIPs) and 17 PhD projects. In 2020, the Continuing Professional Development Module, Summer School and Communication and Media Workshop were all funded and hosted virtually, and in addition, 2 Short Term Missions were funded before travel restrictions were implemented. What were the key outcomes in the third year? ORION - year 3 The primary aim of the ORION project is to establish and strengthen collaboration and cross-disciplinary knowledge transfer to promote One Health disease surveillance. The ORION project consists of 13 partners from veterinary and public health institutes from seven European countries. Having successfully moved through the first two project phases of “improvements and new resources” and “evaluation”, the ORION project progressed into the third phase which encompassed the implementation of national One Health pilots in addition to the continuous improvement of resources developed during the second phase. One aim of the ORION project was to create the One Health Surveillance Codex- a high level framework for harmonised, cross-sector descriptions and categorisation of surveillance data covering all surveillance phases and all knowledge types. A first version of the One Health Surveillance Codex was publicly released, presented during ASM2020 and a corresponding publication was submitted to the One Health journal. A new tool was developed to maintain and exploit the OHEJP Glossary, enabling automatic matching of a user-defined document and a set of online glossaries. Also, the One Health Consensus Report Annotation Checklist (OH-CRAC) was extensively validated, improved and applied in several pilot applications. A new web-based service was created that supports the adoption of OH-CRAC by end-users. The ORION project also created the One Health Surveillance Knowledge Hub, a cross-domain inventory of currently available data sources, methods / algorithms / tools, that support One Health surveillance data generation, data analysis, modelling and decision support. The input into this hub is the combined efforts of the One Health knowledge bases EPI, Integration and NGS. The knowledge base EPI was significantly improved in Y3, from the content as well as from the technical side, with resource investment into the preparation and implementation of national pilots (with some pilots being delayed due to COVID-19). Shiny web applications were created to provide public access to the user-friendly surveillance systems inventory as well as the methods and tools inventory. Publications in 2020: Joensen, KG., Kiil, K., Gantzhorn, MR.,Nauerby, B., Engberg, J., Holt, HM., Nielsen, HL.,Petersen, AM., Kuhn, KG.,Sandø, G.,Ethelberg, S.,Nielsen, EM. (2020). Whole-Genome Sequencing to Detect Numerous Campylobacter jejuni Outbreaks and Match Patient Isolates to Sources, Denmark, 2015–2017. Emerging Infectious Diseases, 26(3), 523-532. DOI: https://dx.doi.org/10.3201/eid2603.190947. The content of the web-based community (knowledge base) NGS handbook continued to be enriched and new functional NGS data analysis pipelines in collaboration with the IRIDA project were created. What were the key outcomes in the third year? Support for extensions of some projects funded in 2018 was established and projects were granted a 6-month no cost extension. Information was collected regarding the synergies between OHEJP projects to encourage collaboration and integration across the multitude of different activities. Procedures were established and followed to ensure data and publications were open access. The following projects were completed in December 2020: IMPART, RaDAR, AIR SAMPLE and MedVetKlebs. Procedures were established and followed to ensure data and publications were open access. The following projects were completed in December 2020: IMPART, RaDAR, AIR SAMPLE and MedVetKlebs. A total of 52 peer reviewed publications were published. A call for an additional JIP was launched in light of the COVID-19 pandemic. The proposal “SARS-CoV2 Research Integration and Preparedness” (COVRIN) was completed and evaluated by the REA steering group. A call for an additional JIP was launched in light of the COVID-19 pandemic. The proposal “SARS-CoV2 Research Integration and Preparedness” (COVRIN) was completed and evaluated by the REA steering group. he Second One Health Annual Scientific Meeting attracted a record audience as an online onference. The Second One Health Annual Scientific Meeting attracted a record audience as an onlin conference. The first 3 Minute Thesis (3MT) competition for the One Health EJP PhD students was organised virtually at the Annual Scientific Meeting. The first 3 Minute Thesis (3MT) competition for the One Health EJP PhD students was organised virtually at the Annual Scientific Meeting. onehealthejp.eu @OneHealthEJP ONE Health EJP onehealthejp.eu @OneHealthEJP ONE Health EJP JO IN T IN TEGRA TIV E P RO JECTS JO IN T IN TEGRA TIV E P RO JECTS COHESIVE - year 3 ORION - year 3 Houe, H., Nielsen, SS., Nielsen, LR., Ethelberg, S., Mølbak, K. (2020). Opportunities for Improved Disease Surveillance and Control by Use of Integrated Data on Animal and Human Health. Frontiers in Veterinary Medicine, 6: 301, pp. 1-8. DOI: https://doi.org/10.3389/fvets.2019.00301. onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:PIXABAY JO IN T IN TEGRA TIV E P RO JECTS Also, the first cross-sector IRIDA NGS data analysis platform at the Norwegian Research and Education Cloud successfully implemented. Also, the first cross-sector IRIDA NGS data analysis platform at the Norwegian Research and Education Cloud successfully implemented. In January 2020 an ORION pilot workshop took place to synchronise the work done in the different country specific pilots under the overarching One Health Surveillance Codex framework. A generic template for evaluating impact and outcomes of each pilot was established, as well as a strategy for how lessons learned from the individual pilots can be captured and communicated. The national integration pilots contributed to improved One Health reporting, e.g. in the Salmonella chapter of the DANMAP 2019 report. Further development work focused on establishing the conceptual and technical foundations for innovative data interoperability solutions related to the linked open data concept. During the national pilot, this concept was promoted and new processes with improved collaboration among the national One Health sectors were established. This led to a significantly improved understanding on each sector’s surveillance activities and results. Specifically, new instructions and guidelines were produced that will be used in future reporting practices, which also pave the way for the application of data interoperability solutions developed by the ORION project. Dissemination continued with members of the ORION project presenting research results at the ASM2020 and the 6th World One Health Congress. Several scientific publications were submitted or are in the final preparation phase. ORION organised an OHEJP internal Cogwheel workshop and an online (knowledge base) NGS workshop. onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:FLICKR IMAGE:FLICKR JO IN T IN TEGRA TIV E P RO JECTS COHESIVE - year 3 The COHESIVE project aims to strengthen the human-veterinary-food collaboration, with the ultimate goal of improving risk assessment, communication and exchange of information and data, and bridging the gaps in risk-analysis in the public health, food safety and veterinary health sectors, by enhancing collaboration on all zoonotic threats. The project worked closely with the Tripartite Guide to Addressing Zoonotic Diseases in Countries to determine the national One Health approaches that could be established and/or strengthened in European countries. During the past year, the COHESIVE project has continued to gather information to further build on the creation of web-based European guidelines regarding the implementation of the Tripartite Guide. Several pilots have been planned in Belgium, Portugal, Norway and Italy to go through the first steps of the guidelines, with three countries established as a core group and a stakeholder analysis has been partly performed. However, due to COVID-19 the systems mapping workshops were all delayed. Another key aim of this project is to develop an online decision tool to help the user decide on the most appropriate method to use when tasked with conducting a risk assessment for a specific situation. Version 1 of the tool is complete and is live. The tool is designed so that it can be updated as new risk assessment resources emerge and tool improvements will continue to be made as they are identified through feedback in the project. IMAGE:FLICKR Interviews with professionals working within food safety, public health and veterinary medicine at central, regional or local levels were performed in six countries, with the aim of identifying factors that contribute to well-functioning systems or processes to share signals of zoonotic events within and between countries. The thematic analysis of the interview data has been finalised at the national level and a joint thematic analysis is ongoing. A follow-up of the horizon scanning pilot exercise regarding One Health that took place in November 2019 has been conducted; horizon scanning is a technique for detecting developments through examination of potential threats. The main drivers for One Health were political and decision-making behaviour, people and consumer behaviour, science and innovations, market behaviour, new and re-emerging pathogens or threats and climate change. onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:FLICKR IMAGE:FLICKR JO IN T IN TEGRA TIV E P RO JECTS A key aim of the COHESIVE project is to learn from past experiences with respect to zoonotic outbreaks. CARE - year 1 CARE - year 1 The CARE project aims to develop new One Health concepts for External Quality Assurance (EQA) schemes for laboratories, reference materials, and quality and availability of demographic data. The CARE project aims to develop new One Health concepts for External Quality Assurance (EQA) schemes for laboratories, reference materials, and quality and availability of demographic data. In the first year of the project, a mapping review was conducted, identifying available and existing EQAs offered to the National Reference Laboratories for zoonotic bacterial agents and antimicrobial resistance from both the public health and veterinary health sectors. Developing new EQA schemes that can be used cross-sectorally and thereby used to evaluate the capacity to manage foodborne problems from a One Health perspective, is an important part of the CARE project. This review served as guidance for discussions within the CARE project for setting up new pilot EQA schemes expanding to whole genome sequencing, which for many laboratories in the EU is still a novel technology. Progress has also been observed identifying the priority pathogens for which reference material should be collected. A list of nine pathogens, all known to be responsible for foodborne human infections, were agreed upon among partners. The target list will serve as a basis for which gaps will be identified in available reference material to cover all known serotypes, genotypes and generally pathogenic variants of the selected pathogens which are relevant for human health, also including AMR h i d i t d t d t A ti i di i t d t CARE t t i i Progress has also been observed identifying the priority pathogens for which reference material should be collected. A list of nine pathogens, all known to be responsible for foodborne human infections, were agreed upon among partners. The target list will serve as a basis for which gaps will be identified in available reference material to cover all known serotypes, genotypes and generally pathogenic variants of the selected pathogens which are relevant for human health, also including AMR mechanisms and associated metadata. A questionnaire was disseminated to CARE partners to inquire about available reference material. A large number of available reference materials was reported, and work will continue with this material. mechanisms and associated metadata. A questionnaire was disseminated to CARE partners to inquire about available reference material. COHESIVE - year 3 Hantavirus and Q-fever in the UK were chosen to be retrospectively analysed and all UK-government publications relating to Hantavirus and Q-fever have been compiled and their data-sources tracked and mapped. A general European-level Q-fever surveillance map has been made in a similar way, using input from consortium members and available online publications. Following an initial systems mapping, with a specific focus on Q-Fever, several potential improvements for surveillance in England have been identified and will be highlighted to animal health surveillance colleagues in the beginning of 2021. Building on the findings within the COHESIVE project, a pilot One Health Early Warning System has been started. The aim is to share low threshold potential signals for pathogens with One Health impacts across multiple countries within the consortium, building relationships between countries and starting a forum that can continue post-project. Unfortunately, due to the outbreak of highly pathogenic avian influenza across Northern Europe the first meeting has been further postponed until early 2021. The development of a tool for systematic cost-benefit analysis (CBA), with the aim of having a common method making CBA comparable across countries that is applicable to zoonotic pathogens, is underway with a literature review of CBA methods. As CBA can vary due to country specific conditions, affecting both costs and benefits, the aim is to produce a report focusing on the similarities, differences, strengths and weaknesses of different models and methods. The COHESIVE project also aims to develop a prototype information system at the national level, allowing different databases to be interoperable. A survey has been conducted to gather detailed information on existing databases and information systems for whole genome sequence data management and analysis adopted or available among countries. A demo version of the COHESIVE prototype information system (CIS) has been produced and available for Italy. The feasibility studies in The Netherlands, Italy and Norway are ongoing. The Italy feasibility study is almost finished, while the others have some delays. IMAGE:FLICKR The development of the tracing web portal - FoodChain-Lab Web – (FCL Web) has advanced further and is available in production mode. A JavaScript Object Notation-based data exchange format allows for analysing delivery data from a data collection mask developed in a national project in FCL Web. onehealthejp.eu @OneHealthEJP ONE Health EJP onehealthejp.eu @OneHealthEJP ONE Health EJP JO IN T IN TEGRA TIV E P RO JECTS IMAGE:FLICKR CARE - year 1 CARE - year 1 CARE - year 1 A large number of available reference materials was reported, and work will continue with this material. The final agreement and layout of the reference material database structure catalogue was delivered, followed by a synthesis document listing partner expectations, a list of existing software and technical choice. The planned risk assessment activities are progressing as planned, initially identifying whom to be surveyed including the target audience from the EFSA network for microbial risk assessment. A small literature review was conducted to establish criteria to assess the data quality and accessibility in the field of risk assessment. The target group of risk assessors was approached with a developed questionnaire to query criteria to assess the data quality and accessibility as well as what type of risk assessment data and associated data to record. The outcome allowed the CARE project to establish a roadmap for sharing information for pathogens and AMR useful for exposure/risk assessment with already established and existing other initiatives. onehealthejp.eu @OneHealthEJP ONE Health EJP JO IN T IN TEGRA TIV E P RO JECTS INTRODUCTION VISION STRUCTURE ACHIEVEMENTS OUTCOMES DISSEMINATION EDUCATION OH-HARMONY-CAP - year 1 JO IN T IN TEGRA TIV E P RO JECTS 20 INTRODUCTION VISION STRUCTURE ACHIEVEMENTS OUTCOMES DISSEMINATION EDUCATION IMAGE:SNAPPYGOAT MATRIX - year 1 OH-HARMONY-CAP - year 1 The OH-Harmony-CAP project aims to collect information on current capabilities, capacities and interoperability at both the National Reference Laboratory (NRL) and the primary diagnostic level. The quantitative description of current and best practices and the development of harmonised protocols will identify and possibly close knowledge gaps and suggest future studies of how best to detect and characterise foodborne pathogens across the One Health sectors. A pilot survey on current practices with regard to capabilities, capacities and interoperability has been conducted and a report summarising the results was compiled. These results will enable development of the benchmarking OHLabCap instrument, a Europe-wide in-depth survey on One Health laboratory interoperability, capacity, and performance. This instrument will be repeatable and sustainable at EU and national level. One new and important observation that emerged during the analysis of the OHLabCap pilot survey was related to adaptability i.e., the capacity and ability to adjust preparedness, methodology and organisation of each laboratory. In light of the COVID-19 pandemic, it became increasingly apparent that adaptability was more relevant than ever, and additional focus will be placed on adaptability in the development of the instrument in the coming years. The first technical report created by the OH-Harmony-CAP project provided descriptions of the current and best practices for sampling and testing for Shiga toxin-producing E. coli (STEC), Enterotoxigenic Escherichia coli (ETEC), Cryptosporidium and antimicrobial resistance (AMR) in Salmonella and Campylobacter. This information will contribute to the development of harmonised protocols across the different laboratories and different EU member states. The first step to producing recommendations on how to harmonise the methodology for the detection and typing of these model pathogens was undertaken with the collection of laboratory protocols which are in use in the EU/EEA laboratories. This will provide an overview of the protocols adopted for the detection, characterisation and typing of the selected pathogens and the outcome will be a list of protocols for the detection, characterisation and typing of the selected pathogens in the EU/EEA NRLs. onehealthejp.eu @OneHealthEJP ONE Health EJP JO IN T IN TEGRA TIV E P RO JECTS MATRIX - year 1 The MATRIX project aims to advance the implementation of One Health Surveillance (OHS) in practice, by building on existing resources, adding value to them and creating synergies across all pillars of One Health. The MATRIX project acknowledges that each country has different infrastructural and economic realities with respect to setting up functional OHS, and all outputs from the MATRIX project will take this into consideration. The project was met with a few challenges during the first year, especially during the first 6 months: the sudden and severe emergence of COVID-19 had a significant impact on the work planned within the MATRIX project. However, many institutes were able to continue after a short delay and as a result of the pandemic many have even strengthened national level communications by creating an oppor- tunity for laboratories and epidemiologists from the animal health and food safety sectors to help support the epidemic control work carried by the public health institutes in their country. To identify and describe the commonalities and differences of the various operational frameworks in animal health, public health and food safety, the MATRIX project collaborated with the ORION project in developing and populating an inventory of surveillance systems across the different sectors of project member’s countries. A report has been compiled that describes the commonalities and differences between the individual surveillance components across sectors. Responsibility for the future continuation of, collecting and creating an inventory of current surveillance frameworks developed in the ORION project was agreed. Mapping of food chain disease surveillance started through country-based case studies. This process took hazards and the associated food chains to analyse across at least two different countries. The case studies selected were Salmonella and the pork meat food chain, Listeria and dairy products, Campylobacter and the poultry meat food chain and Hepatitis E and the wild boar meat food chain. To collect information about the surveillance in place for these case studies online questionnaires were created and distributed across the human, animal and food sectors. A literature review of output-based surveillance studies was started. Several of the partners have completed internal inventories of methodologies or ongoing work that complement the work package in the area of case-finding and evaluation of existing surveillance systems for antimicrobial resistance (AMR). More activities are planned in the second half of the project. JO IN T RES EA RCH P RO JECTS : F O O DB O RN E ZO O N O S ES ( F B Z) JO IN T RES EA RCH P RO JECTS : F O O DB O RN E ZO O N O S ES ( F B Z) NOVA - year 3 NOVA - year 3 The NOVA project aims to develop new surveillance tools and methods, and to harmonise and optimise the use of the existing surveillance data. The project consists of 19 institutes across 10 countries. This collaborative structure makes it possible to help advance the use of modern surveillance principles across Europe, in addition to having cost-saving impacts on how surveillance of existing and emerging zoonotic diseases is being conducted across Europe. Studies of potential barriers and opportunities for surveillance across the food chain were ongoing in the third year, preparations to use the best methodologies to collect these data were carefully considered and interviews with disease surveillance experts is still ongoing. Additionally, a large dataset of food purchase data has been obtained, which will enable analyses of simulated outbreaks. An electronic web-based module, in which consumers can give consent for their purchase data to be used has also been prepared. Research related to food purchase data at an institute level for investigation into hospital foodborne outbreaks is ongoing and includes a literature review manuscript and a questionnaire study. Development of improved tools for food risk mapping are ongoing and are being integrated into the FoodChain-Lab, a state-of-the-art tool for tracing foodborne disease outbreaks. Improvement of surveillance systems is being investigated with the aim of improving outbreak detection in humans. For example, veterinary and public health institutes in Norway have come together to integrate veterinary data, such as incidence of Campylobacter in poultry, and environmental data, such as rainfall and temperature, to current public health surveillance systems for human gastrointestinal outbreaks. These data have then been used by the SVA to develop a surveillance system that processes these data to calculate the value of evidence for outbreaks on a weekly basis. Partners at ANSES are also working to develop detection algorithms that can process data from seven animal, food and human databases from multiple time series simultaneously, in order to predict human gastrointestinal outbreaks. Other algorithms that are being developed as part of the NOVA project include using machine learning to correlate Salmonella infection data with environmental drivers, with the aim of applying these algorithms to different scenarios to identify environmental risk factors for Salmonella outbreaks and the incidence of antimicrobial resistance (AMR). Publications in 2020: Rosendal, T., Widgren, S., Ståhl, K., Frössling, J. (2020). Modelling spread and surveillance of Mycobacterium avium subsp. paratuberculosis in the Swedish cattle trade network. Preventative Veterinary Medicine. MATRIX - year 1 onehealthejp.eu @OneHealthEJP ONE Health EJP onehealthejp.eu @OneHealthEJP ONE Health EJP JO IN T IN TEGRA TIV E P RO JECTS IMAGE:SNAPPYGOAT A preliminary comparative analysis of the criteria used in previously developed surveillance tools (ECOSUR, NEOH, and RISKSUR) was conducted to characterise the overlap and differences between those tools. This comparison aimed to identify a set of strategic criteria which would be included in EU-EpiCap tool. These criteria would consider governance and organisation of multi-sectoral collaborations, technical characteristics of the collaboration, functional characteristics, surveillance short and intermediate-term outcomes. Initial data collection has begun for the analysis for a One Health Surveillance Roadmap and the analysis of resources from other One Health EJP projects has started. In addition, the collection of requirements for the technical infrastructure of the Knowledge-Integration Platform (KIP) was performed and it was decided to extend the One Health Surveillance Codex developed by the ORION project. Developing this platform will link project outcomes and resources (e.g. tools/technologies/ features) as well as support knowledge exchange between different MATRIX partners and stakeholders. The work to facilitate the development of dashboards that enable data-driven surveillance to be carried out as an inter-sectorial activity has, thus far, been restricted to Sweden and Norway agencies. Focus will be given to cementing achievements of previous projects, such as the dashboards developed in the NOVA project and the open data publishing routines created in the ORION project. onehealthejp.eu @OneHealthEJP ONE Health EJP Publications in 2020: Rosendal, T., Widgren, S., Ståhl, K., Frössling, J. (2020). Modelling spread and surveillance of Mycobacterium avium subsp. paratuberculosis in the Swedish cattle trade network. Preventative Veterinary Medicine. 183, 105152. DOI: https:// doi.org/10.1016/j.prevetmed.2020.105152. Apenteng, OO., Arnold, ME., Vigre, H. (2020). Using stochastic dynamic modelling to estimate the sensitivity of current and alternative surveillance program of Salmonella in conventional broiler production. Scientific Reports, 10, pp. 19441. DOI: https://doi.org/10.1038/s41598-020-76514-3. Bosch, J., Iglesias, I., Martínez, M., de la Torre, A. (2020). Climatic and topographic tolerance limits of wild boar in Eurasia: implications for their expansion. Geography, Environment, Sustainability, 13(1), pp.107-114. DOI: https:// doi.org/10.24057/2071-9388-2019-52. IMAGE:PXFUEL LISTADAPT- year 3 The LISTADAPT project aims to decipher the molecular mechanisms of adaptation seen in Listeria monocytogenes to its various ecological niches by comparing both phenotypic and genotypic data from a large, balanced set of strains from human clinical cases, animals, food and environments in several European countries. Publications in 2020: Sévellec, Y., Torresi, M., Félix, B., Palma, F., Centorotola, G., Bilei, S., Senese, M., Terracciano, G., Leblanc, JC., Pomilio, F., Roussel S (2020) First report on the occurrence of Listeria With 21 partner institutes from public health, veterinary health, food and environment laboratories, the LISTADAPT project complied a dataset of 1575 L. monocytogenes strains and their genomes. These strains were collected from 20 European countries which ensured dataset was representative of the large number of clonal complexes occurring worldwide, and also covered many diverse habitats which was balanced between ecological niches and geographical regions. The aim of this dataset is to contribute to the improved understanding of L. monocytogenes and improve surveillance, therefore it was important to make all of this information available to the scientific community (it has been submitted to the European Nucleotide Archive (ENA)). Of the 1575 strains collected, a subset of 200 were selected from 34 clonal complexes with a balance between three ecological niches: environment, animal and food. Phenotypic tests were performed on these strains to investigate the strain’s ability to survive in soil and subsequently the strains were categorised into three groups depending on survival. Genotypic analysis was then performed by way of Genome Wide Association Studies (GWAS) to investigate genetic factors that may contribute to improved survival, of which there were many. A more in-depth investigation on strains from the same niche or clonal complex identified genomic variations in various transcriptional regulators, stress genes and (pro)phage related genes. The 200 strains in the main library showed the same growth kinetics at pH 7 while there was more variation at pH 5.4. The deviating strains belonged to different clonal complex groups and niches. An in-depth analysis is therefore needed to explore which DNA sequences that are related to the deviating growth kinetics. Antimicrobial susceptibility testing also played a significant role in the LISADAPT project with 11 antibiotics and 4 biocides being tested against the panel of L. monocytogenes isolates used in this project. JO IN T RES EA RCH P RO JECTS : F O O DB O RN E ZO O N O S ES ( F B Z) JO IN T RES EA RCH P RO JECTS : F O O DB O RN E ZO O N O S ES ( F B Z) LISTADAPT- year 3 NOVA - year 3 183, 105152. DOI: https:// doi.org/10.1016/j.prevetmed.2020.105152. Publications in 2020: Rosendal, T., Widgren, S., Ståhl, K., Frössling, J. (2020). Modelling spread and surveillance of Mycobacterium avium subsp. paratuberculosis in the Swedish cattle trade network. Preventative Veterinary Medicine. 183, 105152. DOI: https:// doi.org/10.1016/j.prevetmed.2020.105152. Apenteng, OO., Arnold, ME., Vigre, H. (2020). Using stochastic dynamic modelling to estimate the sensitivity of current and alternative surveillance program of Salmonella in conventional broiler production. Scientific Reports, 10, pp. 19441. DOI: https://doi.org/10.1038/s41598-020-76514-3. Apenteng, OO., Arnold, ME., Vigre, H. (2020). Using stochastic dynamic modelling to estimate the sensitivity of current and alternative surveillance program of Salmonella in conventional broiler production. Scientific Reports, 10, pp. 19441. DOI: https://doi.org/10.1038/s41598-020-76514-3. Bosch, J., Iglesias, I., Martínez, M., de la Torre, A. (2020). Climatic and topographic tolerance limits of wild boar in Eurasia: implications for their expansion. Geography, Environment, Sustainability, 13(1), pp.107-114. DOI: https:// doi.org/10.24057/2071-9388-2019-52. IMAGE:PXFUEL Research with different transmission models to investigate potential disease spread and compare surveillance strategies is in the final stages. Additionally, the coding of a simulation model to utilise metagenomics on samples from very large pools of data has been developed and aims to improve the ability to measure the occurrence of AMR in animal production at a national level. A model for estimating the cost-effectiveness of retail sampling in the prevention of human disease outbreaks has also been produced. Bosch, J., Iglesias, I., Martínez, M., de la Torre, A. (2020). Climatic and topographic tolerance limits of wild boar in Eurasia: implications for their expansion. Geography, Environment, Sustainability, 13(1), pp.107-114. DOI: https:// doi.org/10.24057/2071-9388-2019-52. onehealthejp.eu @OneHealthEJP ONE Health EJP Publications in 2020: Sévellec, Y., Torresi, M., Félix, B., Palma, F., Centorotola, G., Bilei, S., Senese, M., Terracciano, G., Leblanc, JC., Pomilio, F., Roussel, S. (2020). First report on the occurrence of Listeria monocytogenes ST121 strain in a dolphin brain. Pathogens. 9 (10) 802 DOI: https://doi org/10 3390/pathogens9100802 LISTADAPT- year 3 Overall, results revealed that strains isolated from food had overall higher minimum inhibitory concentrations (MICs) for the following biocides: quaternary ammonia compounds and peracetic acid compared to strains isolated from animal or the environment. Conversely, no significant differences were observed for MIC of antibiotics from strains from different niches. Publications in 2020: Sévellec, Y., Torresi, M., Félix, B., Palma, F., Centorotola, G., Bilei, S., Senese, M., Terracciano, G., Leblanc, JC., Pomilio, F., Roussel, S. (2020). First report on the occurrence of Listeria monocytogenes ST121 strain in a dolphin brain. Pathogens. 9 (10), 802. DOI: https://doi.org/10.3390/pathogens9100802. Publications in 2020: Sévellec, Y., Torresi, M., Félix, B., Palma, F., Centorotola, G., Bilei, S., Senese, M., Terracciano, G., Leblanc, JC., Pomilio, F., Roussel, S. (2020). First report on the occurrence of Listeria monocytogenes ST121 strain in a dolphin brain. Pathogens. 9 (10), 802. DOI: https://doi.org/10.3390/pathogens9100802. Interestingly, repeated exposure to quaternary ammonia compounds recurrently led to a decrease Interestingly, repeated exposure to quaternary ammonia compounds recurrently led to a d onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:DAIZY JOHN JO IN T RES EA RCH P RO JECTS : F O O DB O RN E ZO O N O S ES ( F B Z) Publications in 2020: Standardisation of methods for metagenomic data generation and analysis were completed in the third year of the project. Investigation of publicly available next generation sequencing raw data indicated that metagenomic sequencing data on pathogen/sample combinations relevant to the METASTAVA projects lacked sufficient sample and methodological metadata, thus highlighting the need to focus on harmonised datasets within this project. A general guidance document was created for the informed implementation of metagenomic methods for diagnostics. This document aimed to inform scientists and stakeholders on an abundance of published information about past and ongoing efforts in the field of metagenomics, in addition to documenting validation and quality control guidelines. Oude Munnink, BB., Nieuwenhuijse, DF., Stein, M., O’Toole, A., Haverkate, M., Mollers, M.,Kamga, SK., Schapendonk, C., Pronk, M., Lexmond, P., van der Linden, A., Bestebroer, T., Chestakova, I., Overmars, RJ., van Nieuwkoop, S., Molenkamp, R., van der Eijk, AA,. Geurtsvan Kessel, C., Vennema, H., Meijer, A., Rambaut, A., van Dissel, J., Sikkema, RS., Timen, A., Koopmans, M. (2020). Rapid SARS-CoV-2 whole-genome sequencing and analysis for informed public health decision- making in the Netherlands. Nature Medicine. 26, p 1405–1410. DOI: https://doi.org/10.1038/s41591-020-0997-y. Oude Munnink, BB., Nieuwenhuijse, DF., Stein, M., O’Toole, A., Haverkate, M., Mollers, M.,Kamga, SK., Schapendonk, C., Pronk, M., Lexmond, P., van der Linden, A., Bestebroer, T., Chestakova, I., Overmars, RJ., van Nieuwkoop, S., Molenkamp, R., van der Eijk, AA,. Geurtsvan Kessel, C., Vennema, H., Meijer, A., Rambaut, A., van Dissel, J., Sikkema, RS., Timen, A., Koopmans, M. (2020). Rapid SARS-CoV-2 whole-genome sequencing and analysis for informed public health decision- making in the Netherlands. Nature Medicine. 26, p 1405–1410. DOI: https://doi.org/10.1038/s41591-020-0997-y. Extensive investigations were carried out to ensure that metagenomic approaches could be applied to and used in diagnostic settings. For example, proficiency testing evaluated the interlaboratory reproducibility and robustness of methods for data generation and analysis of swine faecal samples naturally infected with several porcine astrovirus species. Laboratories were provided samples and used methods that had be standardised earlier on in the METASTAVA project. Van Borm, S., Fu, Q., Winand, R., Vanneste, K., Hakhverdyan, M., Höper, D., Vandenbussche, F. (2020) Evaluation of a commercial exogenous internal process control for diagnostic RNA virus metagenomics from different animal clinical samples. Journal of Virological Methods, 283. DOI: https://doi. org/10.1016/j.jviromet.2020.113916. JO IN T RES EA RCH P RO JECTS : F O O DB O RN E ZO O N O S ES ( F B Z) of susceptibility toward ciprofloxacin, a fluoroquinolone antibiotic, largely used in human and veterinary medicine and considered as a critically important antimicrobial. Additionally, these lower levels of susceptibility to ciprofloxacin remained stable in most strains even after subculture without biocide selection pressure, suggesting an adaptation involving modifications at the genetic level. Genomic analysis suggested that the accessory genome was associated with biocide tolerance, often linked to prophages and mobile genetic elements, thus demonstrating the adaptability of L. monocytogenes and the need to further characterise and understand these important organisms. onehealthejp.eu @OneHealthEJP ONE Health EJP le, C., ., mp, n, me on- 410. an, tic doi. BB., ne h 696- :FLICKR METASTAVA - ended in December 2020 METASTAVA - ended in December 2020 The METASTAVA project investigated the use of metagenomic high throughput sequencing methods for unbiased pathogen detection in public veterinary, medical and food laboratories. This project focussed on providing guidelines for informed metagenomic experimental design for diagnostic purposes. This included standardisation of methods for generating and analysing data, providing quality control metrics, tools and interlaboratory assessments, and assessing the analytical properties of metagenomics for pathogens and samples relevant to the One Health EJP. JO IN T RES EA RCH P RO JECTS : F O O DB O RN E ZO O N O S ES ( F B Z) JO IN T RES EA RCH P RO JECTS : F O O DB O RN E ZO O N O S ES ( F B Z) AIRSAMPLE - ended December 2020 AIRSAMPLE - ended December 2020 The AIRSAMPLE project aimed to develop and validate air sampling as a low-cost method for detection of Campylobacter in broiler production; air sampling was investigated as a method to replace analysis of faecal droppings and boot swabbing. The research in this project moved through four phases: Harmonisation -> Implementation -> Evaluation -> Validation. Harmonisation -> Implementation -> Evaluation -> Validation. Publications in 2020: In the third and final year of the project, the validation phase was the key focus, in addition to ensuring the methods used in this project were sustainable. In order to do this, the project focussed on producing publications, standard operating procedures and guidelines for the use of air sampling methods. Hoorfar, J., Koláčková, I., Johannessen, GS., Garofolo, G., Marotta, F., Wieczorek, K., Osek, J., Torp, M., Spilsberg, B., Sekse, C., Thornval, NR., Karpíšková, R. (2020). A multi -center proposal for a fast screening tool in biosecured chicken flocks. Foodborne Campylobacter. Applied and Environmental Microbiology, 86 (20) e01051-20. DOI: https:// doi.org/10.1128/AEM.01051-20. Hoorfar, J., Koláčková, I., Johannessen, GS., Garofolo, G., Marotta, F., Wieczorek, K., Osek, J., Torp, M., Spilsberg, B., Sekse, C., Thornval, NR., Karpíšková, R. (2020). A multi -center proposal for a fast screening tool in biosecured chicken flocks. Foodborne Campylobacter. Applied and Environmental Microbiology, 86 (20) e01051-20. DOI: https:// doi.org/10.1128/AEM.01051-20. An online video to educate the broiler industry was created and can be found here. Furthermore, the air sampling guidelines have been converted into user guidelines for wider communications to the public and stakeholders and can be found here. This research and subsequent communications have been disseminated to EU authorities and the European reference laboratory for Campylobacter (EURL- Campylobacter), and are predicted to make significant impact as the results showed that air sampling, when coupled with real-time PCR can improve detection of Campylobacter in commercial settings across Europe. The benefit of a European-wide approach to validation of these air sampling methods was that it showed that even for low-prevalence countries such as Norway, air sampling and real-time PCR were as effective as culture. Johannessen, GS., Garofolo, G., Di Serafino, G., Koláčková, I., Karpíšková, R., Wieczorek, K., Osek, J., Christensen, J., Torp, M., Hoorfar, J. (2020) Campylobacter in chicken- Critical parameters for international, multi-centre evaluation of air sampling and detection method. Food Microbiology, 90, 1-6. DOI: https://doi.org/10.1016/j.fm.2020.103455. Wieczorek, K., Wołkowicz, T., Osek, J. Publications in 2020: METASTAVA protocols, expertise and resources were applied across the different partners and research included investigation of the virome in farms where neonatal piglet diarrhoea was problematic, in addition to the characterisation of SARS-CoV-2 during the COVID-19 pandemic and characterisation of Norovirus variants. METASTAVA protocols were validated for characterisation for animal coronaviruses, in addition to investigating animal samples yielding false positive test results using the antigen detection lateral flow device for rapid detection of SARS-CoV-2. Izquierdo Lara, RW., Elsinga, G., Heijnen, L., Oude Munnink, BB., Schapendonk, CME., Nieuwenhuijse, D., Kon, M., Lu, L., Aarestrup, FM., Lycett, S., Medema, G., Koopmans, MPG., Miranda de Graaf, M. (2020) Monitoring SARS-CoV-2 circulation and diversity through community wastewater sequencing. medRxiv DOI: https://doi. The analytical properties of metagenomics for model pathogens and standardised procedures defined earlier in the project were investigated for detection of hepatitis E virus, Norovirus, animal zoonotic pox virus, Shiga toxin producing E. coli and antibiotic resistance genes. These investigations showed that different approaches are needed for different pathogen/sample matrix combinations and that metagenomic approaches may also need to be coupled with targeted sequencing and sample enrichment prior to metagenomic sequencing depending on the diagnostic or scientific question at hand. org/10.1101/2020.09.21.20198838 Van Borm S, Vanneste K, Fu Q, Maes D, Schoos A, Vallaey E, Vandenbussche F. (2020). Increased viral read counts and metagenomic full genome characterisation of porcine astrovirus 4 and Posavirus 1 in sows in a swine farm with unexplained neonatal piglet diarrhea. Virus Genes. 56(6):696- 704. DOI: https://doi.org/10.1007/s11262-020-01791-z onehealthejp.eu @OneHealthEJP ONE Health EJP JO IN T RES EA RCH P RO JECTS : F O O DB O RN E ZO O N O S ES ( F B Z) JO IN T RES EA RCH P RO JECTS : F O O DB O RN E ZO O N O S ES ( F B Z) INTRODUCTION VISION STRUCTURE ACHIEVEMENTS OUTCOMES DISSEMINATION EDUCATION MoMIR - year 3 The MoMIR project aims to develop new approaches to predict, identify and prevent the emergence of animal and human super-shedders based on immune responses and gut microbiota composition. In order to achieve this, the key focuses of the project were to define predictive markers that signal the risk of animals and humans becoming Salmonella super-shedders, to identify immune and microbiota biomarkers to detect super and low-shedders, to identify measures to prevent and control Salmonella super-shedders and to develop mathematical models for risk management. van Wagenberg, CPA., van Horne, P.L.M., van Asseldonk, M.A.P.M. (2020). Cost-effectiveness analysis of using probiotics, prebiotics, or synbiotics to control Campylobacter in broilers. Poultry Science. 99 (8), pp. 4077-4084. DOI: https:// doi.org/10.1016/j.psj.2020.05.003. Studies to identify predictive biomarkers for Salmonella super-shedders have been undertaken and allowed partners to identify biomarkers based on the composition of the chicken gut microbiota. However, further studies to identify these markers and immune biomarkers have been delayed due to the COVID-19 pandemic. A number of joint publications have been accepted or are in preparation to disseminate these results. Early investigation into other biomarkers which may indicate super or low Salmonella shedders in both chicken and pigs have suggested that the number of immune cells in the blood cannot be used to distinguish super vs low shedder animals. Additionally, the virulence of the Salmonella strain did not correlate to super or low shedders. Recruitment of participants in the human aspect of the project is now ongoing. Labarthe, S., Laroche, B., Nguyen, T. N. T., Polizzi, B., Patout, F., Ribot, M., Stegmaier, T. (2020). A Multi-Scale Epidemic Model of Salmonella infection with Heterogeneous Shedding. ESAIM: Proceedings and Surveys, 67, p. 261-284. DOI: https:// doi.org/10.1051/proc/202067015. Rebollada-Merino, A., Ugarte-Ruiz, M., Hernández, M., Miguela-Villoldo, P., Abad, D., Rodríguez-Lázaro, D., de Juan, L., Domínguez, L., Rodríguez-Bertos, A. (2020). Reduction of Salmonella Typhimurium cecal colonisation and improvement of intestinal health in broilers supplemented with fermented defatted ‘alperujo’, an olive oil by-product. Animals. 10(10), 1931. DOI: https://doi.org/10.3390/ani10101931. To investigate possible mitigation strategies for the control of Salmonella super-shedders, probiotics were investigated. Numerous probiotic strains were isolated and characterised by partner institutes, and two poultry derived strains and two porcine derived strains were selected for further investigation. The COVID-19 pandemic has delayed some of the in vivo studies, however the initial results indicate that the pre and probiotics are efficacious at reducing Salmonella shedding in pigs and chickens. JO IN T RES EA RCH P RO JECTS : F O O DB O RN E ZO O N O S ES ( F B Z) JO IN T RES EA RCH P RO JECTS : F O O DB O RN E ZO O N O S ES ( F B Z) MoMIR - year 3 AIRSAMPLE - ended December 2020 (2020) MLST-based genetic relatedness of Campylobacter jejuni isolated from chickens and humans in Poland. PLOS ONE, 15(1), e0226238. DOI: https://doi.org/10.1371/journal. pone.0226238. The studies for the AIRSAMPLE project demonstrated that the likelihood of detecting Campylobacter using air sampling and real-time PCR quadrupled compared to more traditional swab and culture methods. As a result, air sampling could be especially useful for farmers when assessing the cleanliness of their poultry houses before introducing new chicks for production. Wieczorek, K., Bocian, L., Osek, J. (2020) Prevalence and antimicrobial resistance of Campylobacter isolated from carcasses of chickens slaughtered in Poland – a retrospective study. Food Control, 112, 107159. DOI: https://doi. org/10.1016/j.foodcont.2020.107159. Wieczorek, K., Bocian, L., Osek, J. (2020) Prevalence and antimicrobial resistance of Campylobacter isolated from carcasses of chickens slaughtered in Poland – a retrospective study. Food Control, 112, 107159. DOI: https://doi. org/10.1016/j.foodcont.2020.107159. Marotta, F., Janowicz, A., Di Marcantonio, L., Ercole, C., Di Donato, G., Garofolo, G., Di Giannatale, E. (2020) Molecular characterisation and antimicrobial susceptibility of C. jejuni isolates from Italian wild bird populations. Pathogens, 9(4), 304. DOI: https://doi.org/10.3390/pathogens9040304. Marotta, F., Janowicz, A., Di Marcantonio, L., Ercole, C., Di Donato, G., Garofolo, G., Di Giannatale, E. (2020) Molecular characterisation and antimicrobial susceptibility of C. jejuni isolates from Italian wild bird populations. Pathogens, 9(4), 304. DOI: https://doi.org/10.3390/pathogens9040304. onehealthejp.eu @OneHealthEJP ONE Health EJP Publications in 2020: van Wagenberg, CPA., van Horne, P.L.M., van Asseldonk, M.A.P.M. (2020). Cost-effectiveness analysis of using probiotics, prebiotics, or synbiotics to control Campylobacter in broilers. Poultry Science. 99 (8), pp. 4077-4084. DOI: https:// doi.org/10.1016/j.psj.2020.05.003. JO IN T RES EA RCH P RO JECTS : F O O DB O RN E ZO O N O S ES ( F B Z) JO IN T RES EA RCH P RO JECTS : F O O DB O RN E ZO O N O S ES ( F B Z) 28 INTRODUCTION VISION STRUCTURE ACHIEVEMENTS OUTCOMES DISSEMINATION EDUCATION MedVetKlebs - ended December 2020 The MedVetKlebs project aimed to enhance Klebsiella pneumoniae research and surveillance by developing and harmonising study methods and by investigating its ecology and transmission across sources. The need for this research is increasingly important due to the emergence of multidrug resistance K. pneumoniae strains, which are an increasing concern for public health, animal health, environmental health and its ability to contaminate food. Chiarelli, A., Cabanel, N., Rosinski-Chupin, I., Zongo PD., Naas. T., Bonnin, RA., Glaser, P. (2020). Diversity of mucoid to non-mucoid switch among carbapenemase-producing Klebsiella pneumoniae. BMC Microbiology, 20, 325. DOI: https://doi.org/10.1186/s12866-020-02007-y. The MedVetKlebs project developed protocols for Klebsiella isolation from various sources including food, water and human and animal faeces. Molecular detection methods were also developed using a highly sensitive qPCR method known as the ZKIR assay, this assay detects K. pneumoniae and its closely related species from soil, food, faeces, and other complex matrices. During the project, novel taxonomic classifications of four new Klebsiella species and subspecies were identified, in addition to novel biomarkers and laboratory identification tools such as MALDI-TOF mass spectrometry. Protocols developed during the projects were widely disseminated through publications and the open platform: protocols.io. Further methodological developments such as development of a combined qPCR method for detecting and differentiating medically important Klebsiella species and creating a publicly available MALDI-TOF Klebsiella identification website will be finalised shortly. Barbier, E., Rodrigues, C., Depret, G., Passet, V., Gal, L., Piveteau, P., Brisse, S. (2020). The ZKIR Assay, a Real-Time PCR Method for the Detection of Klebsiella pneumoniae and Closely Related Species in Environmental Samples. Applied and Environmental Microbiology, 86(7), pp. e02711-19. DOI: https://doi.org/10.1128/AEM.02711-19. Huynh, BT., Passet, V., Rakotondrasoa, A.,. Diallo, T., Kerleguer, A., Hennart, M., Lauzanne, A., Herindrainy, P., Seck, A., Bercion, R., Borand, L., Pardos de la Gandara, M., Delarocque-Astagneau, E., Guillemot, D., Vray, M., Garin, B., Collard, JM., Rodrigues, C., Brisse, S. (2020). Klebsiella pneumoniae carriage in low-income countries: antimicrobial resistance, genomic diversity and risk factors. Gut Microbes, 11(5), pp. 1287-1299. DOI: https://doi.org/10.1080/1949097 6.2020.1748257. Using protocols harmonised in the MedVetKlebs project, nearly 4000 samples were collected from different sources including food, the environment and healthy humans and animals. MoMIR - year 3 Once they are complete, over 3000 samples will be sent for metagenomic studies to determine how pre- and probiotic interventions and Salmonella status influence the gut microbiome. Studies to determine the influence of commensal bacteria on Salmonella shedding status and immune responses have been completed and the data are under analysis. Rebollada-Merino, A., Ugarte-Ruiz, M., Hernández, M., Miguela-Villoldo, P., Abad, D., Cuesta-Álvaro, P., Rodríguez-Lázaro, D., de Juan, L., Domínguez, L., Rodríguez-Bertos, A. (2020). Dietary supplementation with fermented defatted “alperujo” induces modifications of the intestinal mucosa and cecal microbiota of broiler chickens. Poultry Science. DOI: https://doi.org/10.1016/j. psj.2020.07.015. Studies have revealed that a combination of probiotics were, in part, able to protect chickens from Salmonella colonisation. Additionally, chickens fed with fermented defatted ‘alperujo’ (a by-product of olive oil extraction), were also, in part, protected from Salmonella colonisation. Kempf, F., Menanteau, P., Rychlik, I., Kubasová, T., Trotereau, J., Virlogeux-Payant, I., Schaeffer, S., Schouler, C., Drumo, R., Guitton, E., Velge, P. (2020) Gut microbiota composition before infection determines the Salmonella super- and low-shedder phenotypes in chicken. Microbial Biotechnology. 13 (5), 1611– 1630. DOI: https://doi org/10.1111/1751-7915.13621. A first version of a mathematical model of the interplay between the gut microbiota, the pathogen and the host’s immune response has been developed. Finally, a draft inventory of relevant intervention strategies against Salmonella in laying hens has been developed and the cost effectiveness of probiotic intervention strategies has been calculated. Finally, a draft inventory of relevant intervention strategies against Salmonella in laying hens has been developed and the cost effectiveness of probiotic intervention strategies has been calculated. onehealthejp.eu @OneHealthEJP ONE Health EJP Publications in 2020: Chiarelli, A., Cabanel, N., Rosinski-Chupin, I., Zongo PD., Naas. T., Bonnin, RA., Glaser, P. (2020). Diversity of mucoid to non-mucoid switch among carbapenemase-producing Klebsiella pneumoniae. BMC Microbiology, 20, 325. DOI: https://doi.org/10.1186/s12866-020-02007-y. JO IN T RES EA RCH P RO JECTS : F O O DB O RN E ZO O N O S ES ( F B Z) INTRODUCTION VISION STRUCTURE ACHIEVEMENTS OUTCOMES DISSEMINATION EDUCATION DISCOVER - year 1 JO IN T RES EA RCH P RO JECTS : F O O DB O RN E ZO O N O S ES ( F B Z) These samples were investigated for Klebsiella contamination and it was found that among the food samples tested, chicken meat and ready to eat salads had the highest K. pneumoniae recovery rate: 30% and 20%, respectively. In the environment, all sources tested had a high prevalence of K. pneumoniae. As a result of this broad testing, more specific sources of potential relevance for One Health related transmission were investigated. This included chicken meat, ready to eat salads, onions, seawater, soil, and human carriage. Loncaric, I., Rosel, AC., Szostak, MP., Licka, TF., Allerberger, F., Ruppitsch, W., Spergser, J. Broad-Spectrum Cephalosporin -Resistant Klebsiella spp. Isolated from Diseased Horses in Austria (2020). Animals, 10(2), pp. 332. DOI: https://doi. org/10.3390/ani10020332. Whole genome sequencing of K. pneumoniae isolated from these identified sources was performed and there were found to be a very high level of genetic diversity between strains, but importantly, a low prevalence of antimicrobial resistance and virulence genes. Targeted metagenomic approaches are currently being used to explore the diversity and prevalence of K. pneumoniae in the human gut. Rebollada-Merino A., Bárcena C., Ugarte-Ruiz M., Por- ras-González N., Mayoral-Alegre F., Tome-Sánchez I., Domínguez L. and Rodríguez-Bertos A. (2020). Effects on Intestinal Mucosal Morphology, Productive Parameters and Microbiota Composition after Supplementation with Fermented Defatted Alperujo (FDA) in Laying Hens. Antibiotics, 8(4), pp. 215. DOI: https://doi.org/10.3390/anti- biotics8040215. Finally, mathematical modelling was explored to simulate the diversification and mutation rate of a K. pneumoniae lineage which contaminates food. This research will be used to define clusters of related K. pneumoniae strains that impact the food chain. This model can be generalised to all foodborne or environmental bacterial species and will help with defining short term transmission of pathogens to humans and animals. onehealthejp.eu @OneHealthEJP ONE Health EJP onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:PIXABAY JO IN T RES EA RCH P RO JECTS : F O O DB O RN E ZO O N O S ES ( F B Z) JO IN T RES EA RCH P RO JECTS : F O O DB O RN E ZO O N O S ES ( F B Z) JO IN T RES EA RCH P RO JECTS : F O O DB O RN E ZO O N O S ES ( F B Z) BIOPIGEE - year 1 DISCOVER - year 1 The DISCOVER project aims to address the challenges of source attribution using an interdisciplinary One Health approach. As there is no gold standard for source attribution, this project will take a comprehensive approach by applying several different methodologies and models in a comparative fashion. The project aims to map existing knowledge and recommend new studies and methods that may be required to fill knowledge gaps. The research into mapping existing knowledge gaps has begun and has used input from all partners in the DISCOVER project. A systematic literature search was completed with defined search criteria to search for literature relevant to the source attribution of Salmonella, Campylobacter and E. coli, in addition to searching for antimicrobial resistance in these bacterial species. Using the data collected from these literature searches, knowledge maps were created which showed the number of publications that were found for each method of source attribution for each bacterial species. Mapping of existing data for Salmonella, Campylobacter and E. coli and associated antimicrobial resistance profiles was completed. An inventory of information was compiled with the aim of providing an overview of strains and their genome sequences to pinpoint areas with limited data availability. The work to further expand this inventory is ongoing. Data has been collected from each of the partner institutes regarding Salmonella, Campylobacter and E. coli and antimicrobial resistance. Discussions have taken place to determine how these datasets could be improved throughout the project and to determine which source attribution approaches will be applied, further developed and compared during the project. onehealthejp.eu @OneHealthEJP ONE Health EJP onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:NEIL THEASBY TOXOSOURCES - year 1 The TOXOSOURCES project aims to answer the research question: what are the relative contributions of the different sources of Toxoplasma gondii infection? The project addresses this question using several multidisciplinary approaches and novel and improved methods to yield robust estimates that can inform risk managers and policy makers. Toxoplasma gondii is a highly prioritised foodborne pathogen that causes a high disease burden. The relative contributions of the different transmissible stages, sources and transmission pathways to the infection and disease in humans remain unknown, partly due to lack of appropriate methods, and as a consequence, systematic control of this zoonotic foodborne pathogen is lacking in Europe and globally. In the first year, the TOXOSOURCES project started a collection of data and the building of a quantitative microbiological risk assessment model for T. gondii. The quantitative microbiological risk assessment model will cover both meatborne and environmental exposure, and will be applied in a multi-country study. A questionnaire was developed for collecting food consumption data from the selection of countries across Europe. For the first time in Europe, a systematic literature review on T. gondii prevalence covering animal species raised and hunted for human consumption as well as cats, which are the definitive hosts of the parasite, was performed. An extensive literature review was performed, and practices and experiences were surveyed to support the selection of the most suitable molecular method to detect T. gondii oocysts in fresh produce, which will be applied in a multicentre study. Bioinformatic selection of promising protein candidates for novel serology method was finalised and recombinant expression of selected proteins was started, as part of exploring serology for detecting T. gondii infections caused by oocysts. An unprecedented effort of Whole Genome Sequencing of T. gondii isolates and DNAs was used to identify polymorphic marker regions, which will pave the way for a new typing method to be developed for detecting within-genotype variation. Publications: Klein, S., Stern, D., Seeber, F. Expression of in vivo biotinylated recombinant antigens SAG1 and SAG2A from Toxoplasma gondii for improved seroepidemiological bead-based multiplex assays. (2020). BMC Biotechnology. 20, 53. DOI: https://doi.org/10.1186/s12896-020-00646-7. Publications: Klein, S., Stern, D., Seeber, F. Expression of in vivo biotinylated recombinant antigens SAG1 and SAG2A from Toxoplasma gondii for improved seroepidemiological bead-based multiplex assays. (2020). BMC Biotechnology. 20, 53. DOI: https://doi.org/10.1186/s12896-020-00646-7. Klein, S., Stern, D., Seeber, F. JO IN T RES EA RCH P RO JECTS : F O O DB O RN E ZO O N O S ES ( F B Z) JO IN T RES EA RCH P RO JECTS : F O O DB O RN E ZO O N O S ES ( F B Z) TOXOSOURCES - year 1 BIOPIGEE - year 1 The BIOPIGEE project aims to identify cost-effective biosecurity best practices which will reduce the occurrence of Salmonella and hepatitis E virus in European pig production. The project will develop tools to limit pathogen load along the food chain, thus resulting in healthier animals and a safer food chain. This research will create a benchmark of biosecurity practices throughout different stages of pig production in several European countries. A biosecurity questionnaire for European pig farms was developed to assess the relevance of control measures for the prevalence of Salmonella and hepatitis E virus. Software was used to conduct the survey and to translate the questionnaire into the different languages of the participating countries. Protocols for farm recruitment, sampling and laboratory testing were designed, and farm recruitment is currently in progress, with 61 farm visits completed by the end of the first year of the project. Additionally, existing biosecurity protocols for slaughterhouses across all partner countries have been collated. Discussions are in progress to select a panel of Salmonella strains to compare methods for disinfectant effectivity testing, and to define harmonised methods for biofilm assays. Furthermore, work is ongoing to further develop a harmonised hepatitis E virus infectivity assay. An online catalogue of effective biosecurity measures against Salmonella and hepatitis E virus was created. A systematic literature review on the efficacy of biosecurity methods was conducted between the BIOPIGEE partners. Literature was screened, assessed and information was extracted and compiled for data to be prepared for meta-analyses. An expert panel of scientists was built to rate the biosecurity measures during development of the biosecurity questionnaire. This panel has been expanded with practicing veterinarians, advisors and inspectors in order to also identify factors that will be used to create the benchmark of biosecurity practices later in the project. Additionally, a BIOPIGEE flyer was created to promote the project. Additionally, a BIOPIGEE flyer was created to promote the project. onehealthejp.eu @OneHealthEJP ONE Health EJP onehealthejp.eu @OneHealthEJP ONE Health EJP JO IN T RES EA RCH P RO JECTS : F O O DB O RN E ZO O N O S ES ( F B Z) JO IN T RES EA RCH P RO JECTS : F O O DB O RN E ZO O N O S ES ( F B Z) ADONIS - year 1 TOXOSOURCES - year 1 Expression of in vivo biotinylated recombinant antigens SAG1 and SAG2A from Toxoplasma gondii for improved seroepidemiological bead-based multiplex assays. (2020). BMC Biotechnology. 20, 53. DOI: https://doi.org/10.1186/s12896-020-00646-7. Fernández-Escobar, M., Calero-Bernal, R., Benavides, J., Regidor-Cerrillo, J., Guerrero-Molina, M. C., Gutiérrez-Expósito, D., Collantes-Fernández, E., Ortega-Mora, L. M. Isolation and genetic characterization of Toxoplasma gondii in Spanish sheep flocks. (2020). Parasites and Vectors. DOI: https://doi. org/10.1186/s13071-020-04275-z. Fabian, B. T., Hedar, F., Koethe, M., Bangoura, B., Maksimov, P., Conraths, F. J., Villena, I., Aubert, D., Seeber, F., Schares, G. Fluorescent bead-based serological detection of Toxoplasma gondii infection in chickens. (2020). Parasites and Vectors. DOI: https://doi.org/10.1186/s13071-020-04244-6. onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:PIXABAY ADONIS - year 1 The ADONIS project aims to identify determinants responsible for the increasing incidence of Salmonella enteritidis in humans and poultry in the EU. A cross- sectorial One Health approach will be used to investigate factors that may be related to increasing incidence in primary poultry production, epidemiology and exposure to S. Enteritidis and also investigating the pathogen itself. In the first year, the ADONIS project focussed on preparation for data gathering and data analysis. This included gathering audit reports from Salmonella National Control Programmes, preparing a survey to collect data on the key information from the Salmonella National Control Programmes in laying hens at the country level, and the design of a study protocol for primary production in-field investigations. Currently, 232 recommendations were identified from 38 public audit reports from 24 countries and the surveys prepared are currently being shared among veterinary agencies in the partner countries of the ADONIS project. Countries were selected for evaluation of human surveillance systems and detailed epidemiological trend analysis for Salmonella. A document was prepared which highlighted the trends of each country, from which countries were selected depending on the identified trends in Salmonella: the UK and Spain were selected as countries with an increasing trend, the Netherlands and Belgium were selected as countries with a stable trend and Norway was selected as a country with a decreasing trend. Additionally, preparations for an inventory of available Salmonella sequence data and a pilot genome wide association study began. This research will investigate DNA markers for specific Salmonella phenotypes and determinants possibly associated with the increasing trend in Salmonella incidence and the possible interventions. onehealthejp.eu @OneHealthEJP ONE Health EJP onehealthejp.eu @OneHealthEJP ONE Health EJP r g IMAGE:FLICKR JO IN T RES EA RCH P RO JECTS : F O O DB O RN E ZO O N O S ES ( F B Z) JO IN T RES EA RCH P RO JECTS : A N TIM ICRO B IA L RES IS TA N CE ( A M R) JO IN T RES EA RCH P RO JECTS : A N TIM ICRO B IA L RES IS TA N CE ( A M R) Pauly, N., Hammerl, JA., Grobbel, M., Käsbohrer, A., Tenhagen, BA., Malorny, B., Schwarz, S., Meemken, D., Irrgang, A. (2020). Identification of a blaVIM-1-Carrying IncA/C2 Multi-resistance Plasmid in an Escherichia coli Isolate Recovered from the German Food Chain. Microorganisms. 9, 29. DOI: https://doi. org/10.3390/microorganisms9010029. IMPART - ended December 2020 The IMPART project aims to develop and harmonise laboratory methods for the detection of antimicrobial resistance. The optimisation and harmonisation of laboratory methods for detection of resistant bacteria is essential for antimicrobial resistance surveillance in food producing animal pathogens and determining the risk of transmission of resistance from animals to humans via food and the environment. Pauly, N., Hammerl, JA., Schwarz, S., Grobbel, M., Meemken, D., Malorny, B., Tenhagen, BA., Käsbohrer, A., Irrgang, A. (2020). Co-occurrence of the blaVIM-1 and blaSHV-12 genes on an IncHI2 plasmid of an Escherichia coli isolate recovered from German livestock. Journal of Antimicrobial Chemotherapy. 76(2), 531–533. DOI: https://doi.org/10.1093/jac/dkaa436. The results of the final ring trial investigating standardised laboratory methods for detection of colistin-resistant Enterobacteriaceae using a combination of commercial selective media and PCR were collated and sent to all participants. Results showed that PCR detection of mcr- genes showed 100% specificity and one of the three media tested demonstrated higher sensitivity for selecting colistin-resistant, mcr-positive strains. Additionally, it showed that screening for colistin-resistant strains was more successful from caecal samples, compared the meat samples under experimental conditions. Diaconu, EL., Carfora, V., Alba, P., Di Matteo, P., Stravino, F., Buccella, C., Dell’Aira, E., Onorati, R., Sorbara, L., Battisti, A., Franco, A. (2020). Novel IncFII plasmid harbouring blaNDM-4 in a carbapenem-resistant Escherichia coli of pig origin, Italy. Journal of Antimicrobial Chemotherapy. 75(12), 3475–3479. DOI: https://doi.org/10.1093/jac/dkaa374. Diaconu, EL., Carfora, V., Alba, P., Di Matteo, P., Stravino, F., Buccella, C., Dell’Aira, E., Onorati, R., Sorbara, L., Battisti, A., Franco, A. (2020). Novel IncFII plasmid harbouring blaNDM-4 in a carbapenem-resistant Escherichia coli of pig origin, Italy. Journal of Antimicrobial Chemotherapy. 75(12), 3475–3479. DOI: https://doi.org/10.1093/jac/dkaa374. The final ring trial for detection of carbapenemase-producing Enterobacteriaceae was also completed, and results were distributed to participants near the end of the third year. JO IN T RES EA RCH P RO JECTS : A N TIM ICRO B IA L RES IS TA N CE ( A M R) The main outcome indicated that some of the commercially available selective agar media are not sensitive or selective enough to detect bacteria expressing low levels of carbapenemase production. Some bacteria harbouring specific genes that circulate in European livestock populations may be missed using protocols currently set by the EU. Therefore, more studies are required to test additional culture techniques. Irrgang, A., Tausch SH., Pauly, N., Grobbel, M., Kaesbohrer, A,. Hammerl, JA. (2020). First Detection of GES-5-Producing Escherichia coli from Livestock—An Increasing Diversity of Carbapenemases Recognized from German Pig Production. Microorganisms. 8 (10), 159. DOI: https://doi.org/10.3390/ microorganisms8101593. The production and collection of minimum inhibitory concentrations (MICs) from over 2800 bacterial strains from 17 different species for 34 different antimicrobials was completed and uploaded to the EUCAST website. To date, new epidemiological cut off values have been set for Staphylococcus pseudintermedius, S. hyicus, Pasteurella multocida and Mannheimia haemolytica (https://mic.eucast.org/). Microorganisms. 8 (10), 159. DOI: https://doi.org/10.3390/ microorganisms8101593. Pauly, N., Hammerl, JA., Grobbel, M., Tenhagen, BA., Käsbohrer, A., Bisenius, S., Fuchs, J., Horlacher, S., Lingstädt, H., Mauermann, U., Mitro, S., Müller, M., Rohrmann, S., Schiffmann, AP., Stührenberg, B., Zimmermann, P., Schwarz, S., Meemken, D., Irrgang, A. (2020). ChromID® CARBA Agar Fails to Detect Carbapenem-Resistant Enterobacteriaceae With Slightly Reduced Susceptibility to Carbapenems. Frontiers in Microbiology. 11, 1678. DOI: https://doi. org/10.3390/microorganisms9010029. Irrgang, A., Pauly, N., Tenhagen, BA., Grobbel, M., Kaesbohr- er, A., Hammerl, JA. (2020). Spill-Over from Public Health? First Detection of an OXA-48-Producing Escherichia coli in a German Pig Farm. Microorganisms. 8(6), 855. DOI: https://doi. org/10.3390/microorganisms8060855. IMAGE:FLICKR Pauly, N., Hammerl, JA., Grobbel, M., Tenhagen, BA., Käsbohrer, A., Bisenius, S., Fuchs, J., Horlacher, S., Lingstädt, H., Mauermann, U., Mitro, S., Müller, M., Rohrmann, S., Schiffmann, AP., Stührenberg, B., Zimmermann, P., Schwarz, S., Meemken, D., Irrgang, A. (2020). ChromID® CARBA Agar Fails to Detect Carbapenem-Resistant Enterobacteriaceae With Slightly Reduced Susceptibility to Carbapenems. Frontiers in Microbiology. 11, 1678. DOI: https://doi. org/10.3390/microorganisms9010029. Pauly, N., Hammerl, JA., Grobbel, M., Tenhagen, BA., Käsbohrer, A., Bisenius, S., Fuchs, J., Horlacher, S., Lingstädt, H., Mauermann, U., Mitro, S., Müller, M., Rohrmann, S., Schiffmann, AP., Stührenberg, B., Zimmermann, P., Schwarz, S., Meemken, D., Irrgang, A. (2020). ChromID® CARBA Agar Fails to Detect Carbapenem-Resistant Enterobacteriaceae With Slightly Reduced Susceptibility to Carbapenems. Frontiers in Microbiology. 11, 1678. DOI: https://doi. A disk diffusion method as an alternative method for antimicrobial susceptibility testing of Clostridioides difficile was optimised and validated using a collection of 527 well-characterised C. BeOne - year 1 The BeOne project aims to develop an integrated surveillance dashboard in which molecular and epidemiologically data for foodborne pathogens can be analysed, visualised and interpreted interactively by experts across different disciplines and sectors. Surveillance of foodborne infections and outbreak detection is primarily handled at the national or regional level and often spans different sectors and disciplines. This means that data are increasingly complex and not interoperable, therefore, highlighting the need for tools that can allow the integration of various types of data and facilitate their analysis and interpretation. In the first year of the project, it was decided that the BeOne project would build upon the work achieved in the One Health EJP ORION project. This work began with discussing the advantages and disadvantages of centralised vs decentralised approaches to surveillance and investigating the currently available surveillance platforms with regard to software, data management and analysis pipelines. A survey was conducted between the BeOne project partners to define their whole genome sequence metadata contribution and to create harmonised guidelines for whole genome sequence data and metadata collection. A database system was decided within the project, this system can be built upon; for example, compatible data structures can be built to capture the complexity of intersectoral surveillance data, while maintaining focus on the usability of the data. The BeOne project aims to link genomics and epidemiology by building knowledge and algorithms for outbreak detection. In the first year, a summary of the existing knowledge on epidemiology of targeted pathogens was created. After which, the BeOne project outlined detection issues and outlined a conceptual model for the biological and epidemiological factors impacting outbreak detection. This model aims to serve as a first step in developing and evaluating new algorithms for the detection of pathogens and outbreaks. The project aims to ensure that its findings are sustainable and therefore the base code created during the project is available on Github, with full support and documentation. onehealthejp.eu @OneHealthEJP ONE Health EJP onehealthejp.eu @OneHealthEJP ONE Health EJP JO IN T RES EA RCH P RO JECTS : A N TIM ICRO B IA L RES IS TA N CE ( A M R) JO IN T RES EA RCH P RO JECTS : A N TIM ICRO B IA L RES IS TA N CE ( A M R) ARDIG - final year JO IN T RES EA RCH P RO JECTS : A N TIM ICRO B IA L RES IS TA N CE ( A M R) difficile strains. A robust protocol for the disk diffusion was established and inhibition zone diameter distributions were determined for eight antimicrobials using the entire strain collection. A ring trial to finalise the validation of this method was completed in the third year. Overall the optimised method proved to be reliable for most of the tested antimicrobials and highly reproducible. Irrgang, A., Pauly, N., Tenhagen, BA., Grobbel, M., Kaesbohr- er, A., Hammerl, JA. (2020). Spill-Over from Public Health? First Detection of an OXA-48-Producing Escherichia coli in a German Pig Farm. Microorganisms. 8(6), 855. DOI: https://doi. org/10.3390/microorganisms8060855. onehealthejp.eu @OneHealthEJP ONE Health EJP ARDIG - final year The ARDIG project aims to examine the dynamics of antibiotic administration and antimicrobial resistance (AMR) in humans, animals, food and the environment across six countries in Europe, using a One Health approach. This will provide a better understanding of the types of resistances, their prevalence and their variation in different populations over time, with the hope that this can contribute to the control of multi-drug resistant superbugs. Using a One Health approach will help to overcome the limitations in comparability between data from different sectors and countries. Publications in 2020 Brouwer, MSM., Goodman, RN., Kant, A., Mevius, D., Newire, E., Roberts, AP., Veldman, KT. (2020). Mobile colistin resistance gene mcr-1 detected on an IncI1 plasmid in Escherichia coli from meat. Journal of Global Antimicrobial Resistance. 23, 145- 148. DOI: https://doi.org/10.1016/j.jgar.2020.08.018. Antimicrobial resistance and antimicrobial usage data was obtained from healthy and diseased animals from different partners and countries across Europe. Preliminary analysis of these data has shown that due to the difference in methodology and interpretation criteria, direct comparisons of minimum inhibitory concentrations (MIC) and antibiotic disk diffusions data will be difficult- hence the importance of improving the interoperability of data. Due to these difficulties, the ARDIG project has explored statistical methods to set epidemiological cut-off (ECOFF) values for each antimicrobial. By using a harmonised method to calculate the ECOFF value, a more detailed analysis of the trends in clinical data sets could be made across countries, in addition to enabling comparisons between clinical and non-clinical datasets. Duggett, N., AbuOun, M., Randall, L., Horton, R., Lemma, F., Rogers, J., Crook, D., Teale, C., Anjum, MF. (2020). The importance of using whole genome sequencing and extended spectrum beta-lactamase selective media when monitoring antimicrobial resistance. Scientific Reports. 10, 19880. DOI: https://doi.org/10.1038/s41598-020-76877-7. Both public health and animal health partners collected clinically important E. coli strains from farm and hospital studies over 12 months. Many partners have characterised E. coli isolated through national surveillance programmes. These became part of a whole genome sequencing project to investigate the persistence of AMR in E. coli. During a whole genome sequencing workshop approximately 450 genome sequences submitted from partner institutes were subjected to five different pipelines used to investigate AMR: APHA SeqFinder/ Abricate, PHE GeneFinder, WBVR, Ariba and ResFinder/PointFinder. In addition to the genotypic studies, AMR phenotypes were investigated for these strains using the EFSA panel of antimicrobials with the aim of comparing AMR genotype to corresponding phenotype. Massot, M., Haenni, M., Nguyen, TT., Madec, JY., Mentré, F., Denamur, E. (2020). Temporal dynamics of the fecal microbiota in veal calves in a 6-month field trial. Animal Microbiome. 2(32). DOI: https://doi.org/10.1186/s42523-020-00052-6. Rodriguez-Rubio, L., Serna, C., Ares-Arroyo, M., Matamoros, BR., Delgado-Blas, JF., Montero, N., Bernabe-Balas, C., Wedel, EF., Mendez, IS., Muniesa, M., Gonzalez-Zorn, B. (2020). Extensive antimicrobial resistance mobilization via multicopy plasmid encapsidation mediated by temperate phages. Journal of Antimicrobial Chemotherapy, 75 (11), pp. 3173–3180. DOI: https://doi.org/10.1093/jac/dkaa311. JO IN T RES EA RCH P RO JECTS : A N TIM ICRO B IA L RES IS TA N CE ( A M R) JO IN T RES EA RCH P RO JECTS : A N TIM ICRO B IA L RES IS TA N CE ( A M R) RADAR - ended December 2020 The RaDAR project aims to improve and harmonise modelling methods for better understanding of risks, sources and disease burden by developing risk assessment models to quantify the on-farm development of antimicrobial resistance (AMR) and the subsequent spread to humans. AMR threatens the effective prevention and treatment of an ever-increasing range of infections and is a global threat to public, animal and environmental health. Assessment of the importance of different transmission routes and quantifying public health effects (i.e. disease burden) associated with AMR represent major knowledge gaps. The RaDAR project contributed significantly to these identified knowledge gaps by producing and harmonising modelling methods and frameworks specifically for AMR-related problems using a cross-discipline approach across all pillars of One Health. A large-scale database of AMR plasmids from a range of different bacterial species and sources was curated, which gave an overview of plasmid classification and diversity. The novel resource aims to help researchers to understand the genetic plasticity and transmission route of plasmids, which are crucial in the fight against antimicrobial resistant pathogens. The database is available here. Harmonising risk assessment tools was also an important component of the RaDAR project. Infrastructure for exchanging and annotating risk assessment models was developed in an exchangeable and reproducible file format called FSK, which means that this infrastructure can be used across all pillars of One Health, by risk assessors worldwide. Additionally, state of the art AMR risk assessment models for different food chains were produced using a generic framework. Using generic frameworks may be cruder, but allows for combining risks in different categories, thus may help to create a more complete picture of AMR issues. Machine learning methods were also applied to AMR risk to identify risk factors from data with many variables. A mathematical model was designed to estimate the excess disease burden of AMR, this model considers the mortality and morbidity associated with AMR which is over and above the mortality and morbidity associated with the same, antibiotic sensitive infection. A One Health source attribution model was also produced which can estimate the contribution of reservoirs of AMR and transmission routes. Finally, metagenomic approaches were applied to AMR surveillance, in general, these data correlated at both the phenotypic and genotypic level. Publications in 2020 Rodriguez-Rubio, L., Serna, C., Ares-Arroyo, M., Matamoros, BR., Delgado-Blas, JF., Montero, N., Bernabe-Balas, C., Wedel, EF., Mendez, IS., Muniesa, M., Gonzalez-Zorn, B. (2020). Extensive antimicrobial resistance mobilization via multicopy plasmid encapsidation mediated by temperate phages. Journal of Antimicrobial Chemotherapy, 75 (11), pp. 3173–3180. DOI: https://doi.org/10.1093/jac/dkaa311. The E. coli strains collected were characterised to investigate AMR genes, plasmids and mobile genetic elements using whole genome sequencing as well as other molecular techniques. Phylogenetic analysis was performed on datasets, which helped to identify transmission events or epidemiological links between isolates of particular sequence types or clones of E. coli collected from different hospital or farm settings. AMR pipeline comparison work was also carried out to assist in the harmonisation of in silico AMR gene prediction. Analysis of all the phenotypic and genotypic data is in progress. Patiño-Navarrete, R., Rosinski-Chupin, I., Cabanel, N., Gauthier, L., Takissian, J., Madec, JY., Hamze, M., Bonnin, RA., Naas, T., Glaser, P. (2020). Stepwise evolution and convergent recombination underlie the global dissemination of carbapenemase-producing Escherichia coli. Genome Medicine, 12(10). DOI: https://doi.org/10.1186/s13073-019-0699-6. onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:PIXABAY Publications in 2020 Douarre, P E, Mallet, L, Radomski, N, Felten, A, Mistou, M. (2020). Analysis of COMPASS, a New Comprehensive Plasmid Database Revealed Prevalence of Multireplicon and Extensive Diversity of IncF Plasmids. Frontiers in Microbiology, 11, pp. 1-15. DOI: https://doi.org/10.3389/ fmicb.2020.00483. JO IN T RES EA RCH P RO JECTS : A N TIM ICRO B IA L RES IS TA N CE ( A M R) JO IN T RES EA RCH P RO JECTS : A N TIM ICRO B IA L RES IS TA N CE ( A M R) Publications in 2020 Douarre, P E, Mallet, L, Radomski, N, Felten, A, Mistou, M. (2020). Analysis of COMPASS, a New Comprehensive Plasmid Database Revealed Prevalence of Multireplicon and Extensive Diversity of IncF Plasmids. Frontiers in Microbiology, 11, pp. 1-15. DOI: https://doi.org/10.3389/ fmicb.2020.00483. Douarre, P E, Mallet, L, Radomski, N, Felten, A, Mistou, M. (2020). Analysis of COMPASS, a New Comprehensive Plasmid Database Revealed Prevalence of Multireplicon and Extensive Diversity of IncF Plasmids. Frontiers in Microbiology, 11, pp. 1-15. DOI: https://doi.org/10.3389/ fmicb.2020.00483. onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:FLICKR IMAGE:FLICKR IMAGE:FLICKR FARMED - year 1 FARMED - year 1 Antimicrobial resistance (AMR) threatens global health and current AMR pathogen detection methods are reliant on classic culture techniques. The development of new tools for rapid, real-time detection of pathogens and their resistance profile is much needed, but requires robust protocols using minimal technical equipment that can be used outside a laboratory environment. Additionally, metagenomic techniques could be an invaluable tool for diagnosis and assessment of microbial communities for potential pathogens and AMR or virulence genes. However, metagenomics cannot accurately associate individual genes in a community to specific organisms, which is a limitation for detection of AMR in pathogens. The FARMED project aims to address these issues by assessing the feasibility of long-read metagenome sequencing using the MinION from Oxford Nanopore Technology (ONT), for use as a diagnostic tool to detect pathogens and AMR or virulence genes. To begin this work, in the first year of the project, a survey of the FARMED project consortium was used to identify the different methods and experiences of each of the institutes. This included experience with both short- and long-read sequencing technologies, and highlighted experience with different matrices from a variety of human, animal, environmental sources, DNA extraction methods and bioinformatics tools used to detect bacterial species and/or AMR. Based on the survey results, sample matrices were selected, these included simple matrices such as water and saliva and complex matrices such as human/animal faeces, feed additives and boot swabs. To test the effectiveness of the different laboratory-based DNA extraction methods used by each institute, a defined microbial community will be used to inoculate a simple (water) and complex (animal faeces) matrix. Work is underway to compare the output from each of the partner institutes, DNA extraction methods, and sequencing technology, to identify and optimise a metagenome sequencing workflow. The bioinformatics analysis tool, KMA (DTU), is being used and outputs optimised to characterise the metagenome as well as identify the bacterial species included in the defined microbial community. The FARMED project has undertaken a literature review of commonly used metagenome DNA extractions methods, considered the key requirements for onsite metagenome DNA extraction and long read sequencing and summary of equipment with the potential for onsite DNA extraction and sequencing (planned to be trialled in during the project). As much of the FARMED project is laboratory-based work, the COVID-19 pandemic has severely affected progress. FARMED - year 1 onehealthejp.eu @OneHealthEJP ONE Health EJP Publications in 2020: Diaconu, EL., Carfora, V., Alba, P., Di Matteo, P., Stravino, F., Buccella, C., Dell’Aira, E., Onorati, R., Sorbara, L., Battisti, A., Franco, A. (2020). Novel IncFII plasmid harbouring blaNDM-4 in a carbapenem-resistant Escherichia coli of pig origin, Italy. Journal of Antimicrobial Chemotherapy. 75(12), 3475–3479. DOI: https://doi.org/10.1093/jac/dkaa374. IMAGE:PIXABAY JO IN T RES EA RCH P RO JECTS : A N TIM ICRO B IA L RES IS TA N CE ( A M R) JO IN T RES EA RCH P RO JECTS : A N TIM ICRO B IA L RES IS TA N CE ( A M R) WORLDCOM - year 1 WORLDCOM - year 1 The WORLDCOM project aims to develop on-site diagnostic tools, linked to mobile referencing technology, for the detection of antimicrobial resistance (AMR) genes in E. coli, Salmonella and Campylobacter in agricultural and environmental settings. These rapid on-site detection tools will be applicable to all pillars of One Health and have the potential to detect the presence of AMR in various environments at earlier stages than is currently possible. Furthermore, with the development of machine learning algorithms, will enhance assay utility and enable predictions of AMR in the environment to be made, thus improving surveillance and possible intervention. IMAGE:PIXABAY All E. coli, Salmonella, Klebsiella and Acinetobacter genomes were extracted from the NCBI Pathogens database. From this database of genomes, all types and sub-types of Extended Spectrum β-Lactamases (ESBLs, enzymes that break down β-lactam ring containing compounds), carbapenemases and plasmid-mediated colistin resistance genes were analysed for their frequency among E. coli, Salmonella, Klebsiella and Acinetobacter. High frequency resistance gene subtypes were highlighted for further sequence analysis to illustrate geographic distribution and geography specified single nucleotide polymorphisms (SNPs). Results can be found here. Whole genome sequencing has been undertaken on a number of strains known to harbour antimicrobial resistance genes. This includes: Whole genome sequencing has been undertaken on a number of strains known to harbour antimicrobia resistance genes. This includes: De novo sequencing and genome assembly of E. coli strains. De novo sequencing and genome assembly of E. coli strains. 88 Campylobacter strains underwent whole genome sequencing, assembly and MLST typing. Aminoglycoside and β-lactam resistance genes encoded by Enterobacteria from companion animals were analysed, which led to the identification of new epidemic plasmids that will be studied by the WORLCOM project. 50 plazmocin resistant Enterobacteria isolated from animals, sewage, wastewater, humans and food were sequenced. 50 plazmocin resistant Enterobacteria isolated from animals, sewage, wastewater, humans and food were sequenced. 30 E. coli strains, resistant to 3rd and 4th generation cephalosporins, were isolated from swine and alpaca hosts, characterised with respect to phylogeny, antimicrobial resistance patterns and plasmid profile, and whole genome sequenced. 30 E. coli strains, resistant to 3rd and 4th generation cephalosporins, were isolated from swine and alpaca hosts, characterised with respect to phylogeny, antimicrobial resistance patterns and plasmid profile, and whole genome sequenced. E. coli strains with the CTX-M gene were whole genome sequenced from environmental samples. E. JO IN T RES EA RCH P RO JECTS : A N TIM ICRO B IA L RES IS TA N CE ( A M R) JO IN T RES EA RCH P RO JECTS : A N TIM ICRO B IA L RES IS TA N CE ( A M R) FULL-FORCE - year 1 FULL-FORCE - year 1 The FULL-FORCE project aims to supply 17 EU partners with a technological toolbox and hands-on training in Single-Molecule Real-Time (SMRT) sequencing, and to apply this knowledge to six case studies and applications in metagenomic and AMR transmission models. This technology allows real-time sequencing of long sequence reads, providing additional information of where the read should be placed in the genome. Using this state-of-the-art technology, public health and veterinary laboratories will have the ability to use full-length sequencing and gain detailed insights into mobile genetic elements (MGEs) which carry antimicrobial resistance and virulence genes within, and across species. Currently, European monitoring systems for antimicrobial resistance falls short in distinguishing clona from horizontal transmission of AMR genes and the FULL-FORCE project aims to improve this technology. Progress of this project has been significantly hindered by the COVID-19 pandemic, and therefore many of the activities have been delayed. Despite these delays, the FULL-FORCE project made progress on many aspects of the project. A final consensus protocol for SMRT sequencing was agreed and distributed between partners, alongside instructions for a proficiency test. The SMRT sequencing workshop was initially postponed as it was designed to be an onsite event, however, was rescheduled as a virtual event in September 2020. During this workshop the FULL-FORCE Plasmid Assembler was created and is currently undergoing refinement. The focus of the FULL-FORCE AMR genome studies was determined in the first year to investigate mobile genetic element evolution across data sets shared from other One Health EJP projects, other EU funded projects and National Surveillance programmes. Furthermore, the project has made progress on developing, evaluating and creating a database of mobile genetic elements from single isolates and metagenomic databases. This database currently contains approximately 4450 mobile genetic element sequences from approximately 1050 different bacterial species, and several types of mobile genetic element have been identified. Finally, the design of a transmission spread model of plasmid AMR transmission has begun. Publications in 2020: Diaconu, EL., Carfora, V., Alba, P., Di Matteo, P., Stravino, F., Buccella, C., Dell’Aira, E., Onorati, R., Sorbara, L., Battisti, A., Franco, A. (2020). Novel IncFII plasmid harbouring blaNDM-4 in a carbapenem-resistant Escherichia coli of pig origin, Italy. Journal of Antimicrobial Chemotherapy. 75(12), 3475–3479. DOI: https://doi.org/10.1093/jac/dkaa374. onehealthejp.eu @OneHealthEJP ONE Health EJP onehealthejp.eu @OneHealthEJP ONE Health EJP WORLDCOM - year 1 coli strains resistant to 3rd and 4th generation cephalosporins and/or carbapenems and/or colistin from human and animal origin were also sequenced by WGS. onehealthejp.eu @OneHealthEJP ONE Health EJP FED-AMR - year 1 FED-AMR - year 1 The FED-AMR project aims to investigate the relevance of extracellular free DNA in transferring antimicrobial resistance (AMR) via horizontal antimicrobial resistance gene transfer on free extracellular DNA over ecosystem boundaries relative to bacterial conjugation. Free extracellular DNA is abundant in natural environments and is sufficiently stable to constitute an important reservoir for AMR genes. AMR gene concentrations, diversity, variability, mobility and bacterial biodiversity will be determined in an annual longitudinal study covering a crop’s growing period. Different fertilisation and land management techniques will be investigated, in addition to different environmental compartments, for example: pig farm > manure > soil > crop/food/feed > ground surface water > pig farm. Movement of AMR genes across these different environments will be inferred by sequence comparisons and linkages between human and non-human reservoirs of AMR will be investigated. Zoonotic vs anthropogenic antimicrobial resistance transmission over ecosystem boundaries will be investigated with C. difficile as a model organism. The prevailing selection pressure in each tested habitat during the longitudinal study is determined by quantifying antimicrobials, herbicides and trace elements in the tested compartments. Environmental conditions which may induce or inhibit the expression of competence genes which are necessary to enable the uptake of free extracellular DNA by bacteria will be identified in soil microcosms and in a pig gut model. Experimental data obtained during the project will be used to feed and tune probabilistic modelling of the emergence of AMR in target bacteria and to delineate the relative contribution of transformation and conjugation to antimicrobial resistance gene acquisition in soil environments. Mechanistic models will address key questions regarding the spatio-temporal changes observed in microbial communities and shall support the identification of critical control points for intervention to reduce the spread of AMR from environmental sources. In the first year, the longitudinal study to investigate different crop growing techniques began, with 11 different environmental compartments currently being studied. This study aims to analyse microbial biodiversity and AMR genes across food/feed chains, evaluate the relevance of free extracellular DNA on horizontal gene transfer across ecosystem boundaries, identify points for intervention to reduce the spread of AMR genes, compare geographical differences in trends in AMR and antimicrobials in natural environments and focus on multidrug and emerging resistances. JO IN T RES EA RCH P RO JECTS : A N TIM ICRO B IA L RES IS TA N CE ( A M R) JO IN T RES EA RCH P RO JECTS : A N TIM ICRO B IA L RES IS TA N CE ( A M R) JO IN T RES EA RCH P RO JECTS : A N TIM ICRO B IA L RES IS TA N CE ( A M R) JO IN T RES EA RCH P RO JECTS : A N TIM ICRO B IA L RES IS TA N CE ( A M R) 40 INTRODUCTION VISION STRUCTURE ACHIEVEMENTS OUTCOMES DISSEMINATION EDUCATION The development of Loop-mediated Isothermal Amplification (LAMP) assays has made significant progress in the first year of the project. Development and validation of assays to detect CTX-M resistance genes in E. coli strains has been ongoing and protocols have been shared with partners within the project for further testing and validation using veterinary samples. LAMP assays to detect colistin, carbapenem and β- lactamase resistance genes have been developed and validated and can detect certain resistance genes in 3-5 minutes. IMAGE:PIXABAY Furthermore, good progress has been made on the development and evaluation of rapid DNA extraction protocols for use in the field, including detection of AMR genes in water samples. LAMP assays targeting AMR genes coupled with optimised sample-preparation methods will be crucial for on-site detection of AMR in different settings. onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:PIXABAY FED-AMR - year 1 Overall, the FED-AMR project has been able to coordinate all the different sampling campaigns across four EU regions and generate novel sampling protocols for all the compartments included in the onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:PIXABAY JO IN T RES EA RCH P RO JECTS : A N TIM ICRO B IA L RES IS TA N CE ( A M R) JO IN T RES EA RCH P RO JECTS : A N TIM ICRO B IA L RES IS TA N CE ( A M R) longitudinal study. Bacterial culture protocols, in addition to protocols for analysis of antimicrobials and herbicides in environmental samples have been created. Furthermore, guidelines for sample distribution, transportation and conservation have been established. Protocols for extracellular DNA extraction in the various different compartments (e.g. soil, water, manure, faeces and crops) were compiled, harmonised and finalised across partner institutes. One of the main developments in the first year of the project was the consensual replacement of the qPCRs by a target enrichment approach, which will significantly help to address the aims of the project. In addition, the first preliminary results from all aspects of the project have already been obtained. onehealthejp.eu @OneHealthEJP ONE Health EJP ng the oxigenic s and r B. cereus ve been Content A Damage, and their B. cereus and CR assays uding ped and on between . A global ther analysis oped red and IMAGE:FLICKR Publications in 2020: Lima, DB., Dupré, M., Santos, MDM., Carvalho, PC., Chamot-Rooke, J., (2020). Development, validation and application of an LC-MS/MS method for SEN quantification. Journal of the American Society of Mass Spectrometry. Accepted. JO IN T RES EA RCH P RO JECTS : EM ERGIN G THREA TS ( ET) INTRODUCTION VISION STRUCTURE ACHIEVEMENTS OUTCOMES DISSEMINATION EDUCATION TOX-detect - year 3 The TOX-Detect project aims to contribute to increased consumer health protection by filling the critical knowledge gaps in the detection of bacterial toxins and characterising foodborne toxigenic bacteria. The TOX-Detect project compiled a strain collection of S. aureus, B. cereus and C. perfringens and exchange of these strains between the partner institutes is planned. Culture conditions for B. cereus and C. perfringens were optimised and defined in standard operating procedures which have been shared between partner institutes. Additionally, methods for cytotoxicity testing and High Content Analysis tests were developed. High Content Analysis assays will investigate apoptosis, DNA Damage, mitochondrial membrane potential and the pro-inflammatory responses to these bacteria and their toxins. RNA extraction protocols were developed and optimised in various culture conditions for B. cereus and C. perfringens. These protocols ensured that sufficient RNA was extracted to develop qRT-PCR assays and to send for RNA depletion and sequencing. Bioinformatic analysis was performed including differential gene expression analyses. For both organisms, the methods have been developed and validated, and the pipeline for data analysis is functional. These results will allow correlation between gene expression and strain patterns to be studied. Analytical development and analyses of Staphylococcus enterotoxins M, N & O is underway. A global method based on an “on filter” digestion was finalised and the selection of peptides for further analysis has been done based on the theoretical protein sequence. Maldi-Tof library and methods to detect toxins B. cereus and C. perfringens have been developed and shared with the partners of the TOX-Detect project in order for the methods to be shared and inter-laboratory testing to be performed for validation of standard operating procedures. TELE-Vir - year 1 The TELE-Vir project aims to develop a fast point-of-evidence toolbox for identification and characterisation of emerging virus threats for human and/or domestic animals and wildlife. The TELE-Vir project is combining a suitable field-deployable point-of-care approach, and a direct upload of genomic, phenotypic and epidemiological data into a user-friendly bioinformatics toolkit for fast identification and characterisation of new emerging virus threats. Existing point-of-care methods and tools are being developed, adapted and expanded to create harmonised protocols for field analysis. These tools are being designed such that they only require minimal laboratory equipment and are being designed to be compatible with MinION sequencing technology. Moreover, the project aims to combine and integrate in the point-of-care toolbox phenotypic and epidemiological data to aid risk assessment and management. The toolbox will be made available to other interested national and international stakeholders and shared with established networks. The TELE-Vir project used coronaviruses and influenza A virus as model viruses for the proof-of-principle studies. A literature review was performed to identify coronavirus phenotypes of relevance to tropism, emergence, and clinical disease, and any data available for their prediction based on genotype. A summary was prepared for circulation to partner institutes and reference laboratories for expert opinion, and a similar exercise is underway for influenza A virus. Many of the TELE-Vir partners have been involved in SARS-CoV-2 diagnostics and due to the shortage of reagents for nucleic acid extraction, the partners have been forced to develop alternative methods of extraction. This work aligns with the aim of developing a field-based protocol for MinION sequencing using a minimum of laboratory equipment (the point-of-care toolbox). In addition, surveillance programs for SARS-CoV-2 are based on sequencing of the virus, which has resulted in an upgraded version of the INSaFLU software. A survey was created and circulated to TELE-Vir partners, other One Health EJP partner institutes and associated virologists, to obtain the views of virologists (potential end users of the TELE-Vir toolbox) on coronavirus phenotype prediction and variant monitoring activities that they would like to see in a genomic surveillance toolbox; and to obtain test datasets for further development of the toolkit. Analysis of these responses is ongoing. Publications in 2020: Publications in 2020: Lima, DB., Dupré, M., Santos, MDM., Carvalho, PC., Chamot-Rooke, J., (2020). Development, validation and application of an LC-MS/MS method for SEN quantification. Journal of the American Society of Mass Spectrometry. Accepted. onehealthejp.eu @OneHealthEJP ONE Health EJP onehealthejp.eu @OneHealthEJP ONE Health EJP JO IN T RES EA RCH P RO JECTS : EM ERGIN G THREA TS ( ET) Publications: Fomsgaard, AS., Rosenstierne, MW. (2020). An alternative kfl f l l d i f SARS C V 2 TELE-Vir - year 1 MEmE - year 1 Skrzypek, K., Karamon, J., Samorek-Pieróg, M., Dąbrowska, J., Kochanowski, M., Sroka, J., Bilska-Zając, E., Cencek Tomasz. (2020). Comparison of Two DNA Extraction Methods and Two PCRs for Detection of Echinococcus multilocularis in the Stool Samples of Naturally Infected Red Foxes. Animals. 10, 2381. DOI: https://doi.org/10.3390/ani10122381. The MEmE project is a multicentre collaborative international project which aims to fill relevant research gaps highlighted by international agencies (EFSA, ECDC, WHO) for the detection and control of cystic and alveolar echinococcosis. The MEmE project focusses on the standardisation, harmonisation and validation of existing parasitological and molecular methods, in addition to the development and comparative assessment of innovative molecular tools and biomarkers to detect Echinococcus multilocularis and Echinococcus granulosus s.l. along the food chain. Production of epidemiological data on the presence of Echinococcus multilocularis and Echinococcus granulosus s.l. eggs in the food chain focuses on vegetables for human consumption as well as canine faeces in selected endemic countries. The MEmE project includes integrative activities to harmonise procedures and to improve detection of Echinococcus multilocularis and Echinococcus granulosus s.l. Santucciu, C., Bonelli, P., Peruzzu, A., Fancellu, A., Marras, V., Carta, A., Mastrandrea, S., Bagella, G., Piseddu, T., Profili, S., Porcu, A., Masala, G. (2020). Cystic Echinococcosis: Clinical, Immunological, and Biomolecular Evaluation of Patients from Sardinia (Italy). Pathogens. 9(11), 907. DOI: https://doi. org/10.3390/pathogens9110907. Santucciu, C., Bonelli, P., Peruzzu, A., Fancellu, A., Marras, V., Carta, A., Mastrandrea, S., Bagella, G., Piseddu, T., Profili, S., Porcu, A., Masala, G. (2020). Cystic Echinococcosis: Clinical, Immunological, and Biomolecular Evaluation of Patients from Sardinia (Italy). Pathogens. 9(11), 907. DOI: https://doi. org/10.3390/pathogens9110907. Santolamazza, F., Santoro, A., Possenti, A., Cacciò, S M., Casulli, A. (2020). A validated method to identify Echinococcus granulosus sensu lato at species level. Infection, Genetics and Evolution. 85, 104575. DOI: https://doi.org/10.1016/j.meegid.2020.104575. During its first year, MEmE produced standard operating procedures (SOPs) for sampling different matrices for detection of Echinococcus spp. The collection of samples started in the field. Parasite sample collection from red foxes, arctic foxes, dogs, pigs, sheep and intermediate hosts is ongoing. Bonelli , P., Loi, F., Cancedda, MG., Peruzzu, A., Antuofermo, E., Pintore, E., Piseddu, T., Garippa, G., Masalam G. (2020). Bayesian Analysis of Three Methods for Diagnosis of Cystic Echinococcosis in Sheep. Pathogens. 9(10), 796. DOI: https://doi. org/10.3390/pathogens9100796. Furthermore, organisation for the collection of faecal samples from dogs for an epidemiological study began. TELE-Vir - year 1 Publications: Overall, the COVID-19 crisis has had a positive impact on the TELE-Vir project and many of the experiences and problems encountered during the crisis have been used or translated to further develop the TELE-Vir point-of-care toolbox, which aims to help control future outbreaks of new emerging viruses at national, regional, European and even global levels. Fomsgaard, AS., Rosenstierne, MW. (2020). An alternative workflow for molecular detection of SARS-CoV-2 – escape from the NA extraction kit-shortage, Copenhagen, Denmark, March 2020. Eurosurveillance. 25(14), pp: 2000398. DOI: https://doi.org/10.2807/1560-7917. ES.2020.25.14.2000398. Fomsgaard, AS., Rosenstierne, MW. (2020). An alternative workflow for molecular detection of SARS-CoV-2 – escape from the NA extraction kit-shortage, Copenhagen, Denmark, March 2020. Eurosurveillance. 25(14), pp: 2000398. DOI: https://doi.org/10.2807/1560-7917. ES.2020.25.14.2000398. onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:PIXABAY JO IN T RES EA RCH P RO JECTS : EM ERGIN G THREA TS ( ET) 45 INTRODUCTION VISION STRUCTURE ACHIEVEMENTS OUTCOMES DISSEMINATION EDUCATION Bonelli, P., Dei Giudici, S., Peruzzu, A., Mura, L., Santucciu, C., Maestrale, C., Masala, G. (2021). Identification of Echinococcus granulosus Genotypes G1 and G3 by SNPs Genotyping Assays. Pathogens. 10, 125. DOI: https://doi.org/10.3390/pathogens10020125. us ys. 0125. a, asz. Two ool 1. V., S., l, oi. asulli, ulosus . 85, 75. o, . ic ://doi. ction . DOI: hi, S., (2020). ulosus rns of ns, 0444. The 214- GE:FLICKR MEmE - year 1 MEmE - year 1 Participants were sent information and collection kits for owners and veterinary practises in rural areas of highly endemic regions. Currently, three hundred and eighty faecal samples from dogs were collected in this way. Additionally, hundreds of dog faecal samples from the environment were collected as a potential matrix for validation. Maksimov, P., Bergmann, H., Wassermann, M., Romig, T., Gottstein, B., Casulli, A., Conraths, FJ. (2020). Species Detection within the Echinococcus granulosus sensu lato Complex by Novel Probe-Based Real-Time PCRs. Pathogens, 9(10), 791. DOI: https://doi.org/10.3390/pathogens9100791. Maksimov, P., Bergmann, H., Wassermann, M., Romig, T., Gottstein, B., Casulli, A., Conraths, FJ. (2020). Species Detection within the Echinococcus granulosus sensu lato Complex by Novel Probe-Based Real-Time PCRs. Pathogens, 9(10), 791. DOI: https://doi.org/10.3390/pathogens9100791. The target group for dogs included in the epidemiological study are those originating from highly endemic areas (high prevalence of E. multilocularis in foxes as a proxy), or from regions with relatively high prevalence of E. granulosus in sheep. A questionnaire was compiled and sent to owners and vets, with questions concerning data which will be used in epidemiological analysis. M’rad, S., Oudni-M’rad, M., Bastid, V., Bournez, L., Mosbahi, S., Nouri, A., Babba, H., Grenouillet, F., Boué, F., Umhang, G. (2020). Microsatellite Investigations of Multiple Echinococcus granulosus Sensu Stricto Cysts in Single Hosts Reveal Different Patterns of Infection Events between Livestock and Humans. Pathogens, 9(6), pp. 444. DOI: https://doi.org/10.3390/pathogens9060444. An additional multicentre study for the detection of Echinococcus eggs in fresh vegetables (lettuces) for human consumption is ongoing. Protocols for washing, filtration and molecular identification of Echinococcus were established and sampling will start soon. Another core activity conducted during the first year of the MEmE project was the validation of established parasitological and novel molecular diagnostic procedures. This work aims to detect Casulli, A, (2020), Recognising the substantial burden of neglected pandemics cystic and alveolar echinococcosis, The Lancet, 8 (4):PE470-E471. DOI: https://doi.org/10.1016/S2214- 109X(20)30066-8. onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:FLICKR JO IN T RES EA RCH P RO JECTS : EM ERGIN G THREA TS ( ET) JO IN T RES EA RCH P RO JECTS : EM ERGIN G THREA TS ( ET) 46 INTRODUCTION VISION STRUCTURE ACHIEVEMENTS OUTCOMES DISSEMINATION EDUCATION JO IN T RES EA RCH P RO JECTS : EM ERGIN G THREA TS ( ET) JO IN T RES EA RCH P RO JECTS : EM ERGIN G THREA TS ( ET) multilocularis and E. granulosus s.l. genotype/species in different matrices along the food cha A key aim of the MEmE project is to generate new innovative tools for rapid detection, differential diagnosis, and tracking of infection, both at large and small-scale settings. Work has begun towards the development and validation of the following new tools: new molecular markers for Echinococcus species from rapid diagnostics to source attribution; new multiplex qPCR for detection and discrimination of E. multilocularis and E. granulosus s.l and E. granulosus s.l genotypes; sequencing using Region-Specific Extraction (RSE) and Next Generation Sequencing for the detection of E. multilocularis and E. granulosus s.l in complex samples. onehealthejp.eu @OneHealthEJP ONE Health EJP Publications: Lopez, T., Müller, L., Vestergaard, LS., Christoffersen, M., Andersen, A., Jokelainen, P., Agerholm, JS., Stensvold, CR. Veterinary students have a higher risk of contracting Cryptosporidiosis when calves with high fecal Cryptosporidium loads are used for fetotomy exercises. (2020). Applied and Environmental Microbiology DOI: https://doi PARADISE - year 1 PARADISE - year 1 The PARADISE project aims to deliver informative typing schemes and innovative detection strategies applicable to food matrices for both Cryptosporidium and Giardia, two foodborne parasites that are major contributors to the global burden of gastrointestinal disease. Using Next Generation Sequencing technologies (genomics and metagenomics), the project will generate much needed data that will enrich the understanding of the epidemiology and genomics of these organisms and provide the basis on which improved strain-typing schemes will be developed and rigorously tested. In parallel, strategies to enrich for the target pathogens in different matrices will also be developed and tested. During the first year of the project, many partners in the PARADISE project collected samples, which allowed the project to sequence 24 novel Giardia duodenalis and 44 novel Cryptosporidium parvum genomes. Based on genome wide analysis of the available genomes, a first selection of C. parvum and G. duodenalis assemblage B variable genomic regions was achieved. This resulted in the identification of genomic regions suitable for inclusion in novel typing schemes. An inventory of the parasite samples (genomic DNA, faeces, and other relevant matrices) available at each partner institute was compiled; samples will now be used to test the identified markers. In silico metagenomics was performed to confirm the presence of parasite sequences in public metagenomes generated from various matrices. In addition, the amplicon-based sequencing approach was extensively tested using new primers for the detection of flagellates (including Giardia). Reference material (parasite cysts) for spiking experiments was also produced, and experiments have been planned to evaluate the limit of detection, the specificity and sensitivity, which are essential parameters to understand the applicability of amplicon-based and shotgun metagenomics as a platform for foodborne parasite detection. As per the enrichment strategies, the work on nanobodies has progressed and potential binders have been obtained using whole cyst and oocyst antigens as prey. Two capture systems for DNA fishing were designed for Cryptosporidium and one for Giardia. High specificity and sensitivity demonstrated for Cryptosporidium. Lopez, T., Müller, L., Vestergaard, LS., Christoffersen, M., Andersen, A., Jokelainen, P., Agerholm, JS., Stensvold, CR. Veterinary students have a higher risk of contracting Cryptosporidiosis when calves with high fecal Cryptosporidium loads are used for fetotomy exercises. (2020). Applied and Environmental Microbiology. DOI: https://doi. org/10.1128/AEM.01250-20. PARADISE - year 1 onehealthejp.eu @OneHealthEJP ONE Health EJP onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:PXHERE JO IN T RES EA RCH P RO JECTS : EM ERGIN G THREA TS ( ET) IDEMBRU - year 1 IDEMBRU - year 1 The IDEMBRU project aims to create a toolkit for the identification of emerging Brucella species and their reservoirs, this will ensure the rapid detection, identification and characterisation of Brucella. The project will investigate the detection and investigation of these pathogens from different sources and locations, phenotypically and genotypically characterise emerging Brucella species, understand the virulence and zoonotic potential of these isolates and develop a toolkit for the integration of data across Europe and a guide to characterisation of Brucella. In the first year, the IDEMBRU project created a common database for all samples collected and treated before and during the project. In 2021, each partner will include their sample collection of atypical Brucella spp. originating from multiple animal species and environment. As further animal and environmental sampling had to be postponed due to the COVID-19 pandemic, it was decided to use the existing collections from previous projects in partner institutions, to generate initial data on which sampling strategy can be based. A sampling and analytical strategy list of new targets, according to previous epidemiological information from the different partners was finalised. Three environments from which Brucella has been isolated will be targeted: forest, freshwater habitats and coastal regions; animal species to target and number of samples will be defined by each partner. An epidemiological questionnaire and associated standard operating procedures for sample collection and identification were prepared for sample collection to ensure harmonisation of approaches. One key aim for IDEMBRU is to develop a diagnostic toolkit for emerging Brucella in humans. A survey will be created for different networks to ask how they would diagnose emerging Brucella and how they would differentiate classical from atypic species cases. To further understand infection in humans a study will be conducted to test potential antigens in humans. Testing thus far has highlighted the need to design new serological tests more adapted to atypical and emerging Brucella species. IDEMBRU partner institutes have contributed to a survey describing DNA extraction protocols currently applied to relevant samples their laboratories; harmonisation and optimisation of these methods is ongoing. Research into standardised protocols which will be used for phenotypic and genotypic characterisation of novel emerging Brucella spp., is ongoing and will be adapted, as necessary during the project work. onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:PXHERE P hD P RO JECTS One Health EJP PhD Programme Progress ECO-HEN The ECO-HEN project commenced in February 2019, and the project studies Escherichia coli (E. coli), which contributes to antimicrobial resistance (AMR) spread by clonal strains being able to survive on the food chain and by its ability for horizontal transfer of genetic platforms (such as plasmids and integrons) containing AMR genes across bacteria. The presence of AMR E. coli in animal intestinal microbiota such as pigs and broilers are well documented, however the dynamics of AMR E. coli populations in commercial table eggs production has been scarcely studied. The main goal of the PhD is to fill the knowledge gap on the transmission dynamics of AMR E. coli in commercial laying hen production and to determine to what extent this animal production poses a public health risk through food and/or environment contamination. The knowledge obtained from this PhD will reveal the extent to which the table egg production system represents a risk for spread of AMR to humans and the environment as genomic data will be shared across electronic platforms available for the larger community of clinical and environmental microbiologists for comparative analyses. The results will also reveal what the effect is of reduced antimicrobial use on the AMR bacteria initially present in the day-one chicks. In 2020 significant progress was made on the reconstruction of plasmids which spread AMR genes from animal isolates to eggshell isolates. The objective of this work was to reconstruct the plasmids responsible for dissemination of AMR genes across isolates from different sources. It is widely recognised that the epidemiology of certain AMR genes (e.g. those conferring resistance to critically important antimicrobials in human medicine such as third-generation cephalosporins and colistin) is linked mainly to AMR gene spread via plasmids rather than via bacterial clones therefore knowledge on the AMR plasmids is essential to describe the flow of AMR in different ecological niches. Analysing relationships between AMR bacteria of laying hens and eggs was a key aim in 2020 and the research focused on the in deep analysis of the surroundings of the trimethoprim resistance gene dfrA36, which was identified as the gene responsible for trimethoprim resistance in some isolates of E. coli obtained from day-old chicks. In particular, the large plasmid harbouring this gene is under analysis. In addition, the in deep characterisation of the isolates obtained in selective media (with cefotaxime and with ciprofloxacin, respectively) is also in progress. P hD P RO JECTS m . d IMAGE:PIXABAY One Health EJP PhD Programme Progress Between 2018 and 2019, 16 PhDs were co-funded by the One Health EJP Education and Training activities as part of Work Package 6. The research focus of the individual PhD projects falls within at least one of the three research domains of the OHEJP: foodborne zoonoses, antimicrobial resistance and emerging threats. An additional PhD was funded in 2019 which focuses on sustainability and lies in the field of social sciences and public health. The PhD projects provide opportunities to explore and share skills, expertise and knowledge from the OHEJP consortium, therefore accelerating both the rate and quality of research in addition to developing the One Health scientific leaders of the future contributing to the sustainability of the One Health approach. There is significant scope for inter-disciplinary networking among OHEJP partners in addition to the interaction with the JRPs and JIPs. The JRPs and JIPs have expertise that can support the PhD students, and provide opportunities to explore and share skills and knowledge, accelerating both the rate and quality of the research. These interactions help to bring the physical, biological, and social sciences together, and allow greater flexibility in the PhD projects to ensure innovative hypothesis driven research. Despite the challenges caused by the COVID-19 pandemic, our PhD students continued to advance their scientific research and work towards achieving their key deliverables and milestones. The students also participated at the Annual Scientific Meeting in May 2020, where they presented their projects and research through presentation of their scientific poster and an oral presentation at the three-minute thesis (3MT) competition, to an audience of over 750 scientists and professionals across the globe. To find more information about the PhD students, click here. You can read their 12-month reports describing their progress between January to December 2020 here. onehealthejp.eu @OneHealthEJP ONE Health EJP onehealthejp.eu @OneHealthEJP ONE Health EJP P hD P RO JECTS LIN-RES The LIN-RES project commenced in January 2019, and focuses on the antimicrobial Linezolid, one of the last resort drugs used to fight human infections caused by multi-resistant Gram-positive bacteria such as Streptococci, Staphylococci and Enterococci. It is commercially available since 2000 and has not been licensed for use in animals. In 2008, the first instance of transferable resistance to Linezolid caused by the 23SrRNA methylase Cfr (Chloramphenicol Florfenicol Resistance) was reported in US Staphylococcal isolates from human infections. A second gene, optrA, conferring resistance to Linezolid and Phenicols whose sequence was first reported in 2015, is an ABC-type membrane transporter and works as an efflux pump. After its initial finding in China, it recently emerged in animal and human Enterococci/Staphylococci on both the American and European continents. animal and human Enterococci/Staphylococci on both the American and European continents. This project aims to investigate the molecular basis, origin, transferability, and risk factors associated with Linezolid-resistance emergence in Gram-positive bacteria of both human and animal origin. The following tasks for this project were successfully completed in 2020: • Sampling and collection of bacteria This project aims to investigate the molecular basis, origin, transferability, and risk factors associated with Linezolid-resistance emergence in Gram-positive bacteria of both human and animal origin. The following tasks for this project were successfully completed in 2020: • Sampling and collection of bacteria • Next-Generation Sequencing (NGS) resistance analysis of all isolates • NGS subtyping of the strains and associated host specificity • Investigation of the genetic organisation of the contigs carrying LIN-RES genes and incompatibility groups From early 2019 to early 2020, 1325 faeces samples (from cattle, pigs or poultry) and 148 nasal swabs samples (from pigs) were collected in Belgium for the official monitoring of antimicrobial resistance and were analysed on blood agar supplemented with linezolid to select resistant strains (a process called selective monitoring). In 2020, 40 nasal swab samples and 392 faecal samples collected were screened for linezolid resistant bacteria and analysed by mass spectrometry (MALDI-TOF). The 155 linezolid resistant isolates were all sequenced by whole genome sequencing (WGS), assembled and analysed. A core genome Multilocus Sequence Typing (cgMLST) analysis was conducted to study the relatedness of these isolates and compare them with published sequences of linezolid-resistant isolates. Three different resistance genes, cfr, optrA and poxtA, were found in this collection as well as mutations in the 23SrRNA gene conferring resistance to linezolid. ECO-HEN The aim is to check if the absence of AMR use along the rearing and production periods affects their persistence on the farm throughout the production cycle, as well as the characterisation of possible mobile genetic elements for these AMR determinants. onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:PXHERE P hD P RO JECTS LIN-RES The project will focus on the NGS analysis of all isolates and the investigation of putative risk factors associated with the numerous positive farms from which these isolates came from. The next task in this project has started, which is to conduct laboratory experiments to demonstrate transferability of linezolid resistance genes and estimate transfer rates. The establishment and testing of the protocol and a first conjugation experiment was performed to assess the transferability of linezolid resistance genes. onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:PIXABAY KENTUCKY Salmonella enterica serovar Kentucky (S. Kentucky) is a common causative agent of gastroenteritis in humans. It is one of most notorious Salmonella serotypes, as it is strongly associated with antimicrobial resistance (AMR). Ciprofloxacin-resistant S. Kentucky (CIPR S. Kentucky) belongs to a single sequence type (ST198), which acquired a variant of the Salmonella genomic island 1 (SGI1) conferring resistance to first-line antimicrobials (b-lactams, aminoglycosides, sulphonamides, tetracyclines). In addition to CIPR, S. Kentucky is able to gain additional antibiotic resistance determinants through the acquisition of locally circulating plasmid-borne genes, such as ESBLs, AmpC and/or carbapenemase. Most recently, the situation has worsened, as ECDC launched an Urgent Inquiry (UI-464) on a CIPR S. Kentucky ST198 strain carrying a chromosomally integrated blaCTX-M-14b gene encoding for cephalosporin resistance. The insertion event was traced back to Malta, but the strain has already spread to Belgium, UK, The Netherlands and five other EU countries. To date, this clone is only reported in humans, as opposed to (for example) the CipS S. Kentucky ST152 clone widely found in poultry in the USA but rarely reported in humans. The KENTUCKY PhD project will investigate (i) what explains the evolutionary success of the multidrug resistant S. Kentucky ST198 clone, and (ii) what is the mechanism of the integration (and potential further transfer) of the ESBL gene in its chromosome. IMAGE:PIXABAY The PhD student was recruited in January 2020. In the first year of study, the project focused on the genetic environment of the integrated blaCTX-M-14b gene. By hybrid sequencing, the entire genomes of four clinical S. Kentucky strains with this genotype were reconstituted. The ISEcp1B transposase, which is part of the IS1380 family, was detected in this region adjacent to the ESBL gene and was hypothesised to catalyse the transfer. This finding was investigated further by initiating an in silico database mining to correlate insertion sequences with AMR genes among clinical S. enterica, Klebsiella pneumoniae and Escherichia coli isolates. The results are expected by April 2021. Meanwhile, the transfer of the resistance gene from plasmid to chromosome using in vivo transposition dynamics via time-lapse microscopy is being investigated. The aim is to track and quantify the (inducers of) genetic hopping using fluorescence microscopy. In 2021, the influence of serotype, species and antibiotics and other stressors on the chromosomal transfer will be explored, and therefore will be able to perform a risk assessment on this dangerous genotype. HME-AMR A key element in managing antimicrobial resistance (AMR) in the One Health paradigm is to reduce the spread of resistance genes between microorganisms in the agri-food environment. Heavy metals occur ubiquitously in the agri-food environment and sometimes in high concentrations in soil. In food animal production, heavy metals such as zinc and copper are frequently added to animal feed to promote growth and health. Such heavy metals may not be fully absorbed from the animal gut and are excreted in faeces into the environment. It is recognised that a One Health approach is required to tackle AMR, which includes the role of the environment, and the food production environment in particular. Very limited information is available regarding the impact that selective pressures such as heavy metals may have on the mobilisation of AMR and its potential transfer into the food chain. There is a clear need for more data on the impact of heavy metal concentrations in food production settings, and their potential impact on the co-selection and dissemination of AMR in the environment and food chain, and this is therefore the driver of this project. The HME-AMR PhD project is investigating the role of heavy metals in the environment as a selective pressure for the dissemination of antimicrobial resistance. There has been a substantial delay to the commencement of this project due to recruitment issues, followed by the COVID-19 pandemic. Prior to the pandemic, there were significant challenges during the recruitment process which led to two further recruitment rounds. In March 2020, a suitable candidate was identified and offered he position, but they were unable to procure a study visa for Ireland by October 2020. The position was advertised once again, and a candidate has now accepted the position and has commenced work in February 2021. Tighter sampling schedules will be employed to address project delays to date. Furthermore, Dr Burgess and Dr Morris have spoken with Geological Survey Ireland (GSI) regarding the identification of suitable sampling sites to ensure the project sampling campaign started at the same time the student started the position and are also collaborating with a nationally funded project to procure control samples in areas of high zinc application. The project has also been highlighted in internal workshops within Teagasc to encourage cross programme linkages. onehealthejp.eu @OneHealthEJP ONE Health EJP onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:PIXABAY P hD P RO JECTS KENTUCKY onehealthejp.eu @OneHealthEJP ONE Health EJP cs and genomic approaches for the animals, and the environment. rinary tract infections (UTI) caused by ever, the expression of acquired 16S rRNA to plazomicin. of the COVID-19 pandemic. The student is h became part of a network to diagnose The PhD student was actively involved in o progress could be made in his PhD strictions added further difficulties in pecifically in relation to the task of he team decided to start the analysis with was involved in. Preliminary bioinformatic nging to different pig farms across Spain n detected, including some of low he assembly-based analysis did not yield Illumina (short reads). A DNA re-extraction d long-read technologies is foreseen in ntact was established with a few potential beginning of 2021. There are still several at this stage, however it is hoped that in be found. IMAGE:PIXABAY IMAGE:PIXABAY P hD P RO JECTS PEMbo Bovine tuberculosis (bTB), mainly caused by Mycobacterium bovis, is a zoonotic disease intimately and historically associated to cattle rearing. Although developing countries suffer the most from bTB, this disease remains a major problem in some developed countries. When cattle breeding developed into an established industry, strong control strategies were initially setup in Europe and other developed countries. In France, this resulted in a rapid decline in the number of infected herds, and in 2000, France obtained the official bTB free status from the EU. Despite considerable financial and social efforts against this disease, bTB continues to slowly but continuously rise and persist at regional levels. obtained the official bTB free status from the EU. Despite considerable financial and social efforts against this disease, bTB continues to slowly but continuously rise and persist at regional levels. The aim of the PEMbo project, a collaborative study between ANSES and INRA, two French One Health EJP partners, is to better understand the complex biology of M. bovis through the study of the complete genomes of a large panel of isolates. The first task, culture, cloning and extraction of M. bovis strains of work package 1 (WP1) of this project (Establishment of reference sequences from the main French clonal groups) was completed in 2020. Ten representative M. bovis genotypes were selected from strains circulating in France. These were selected and cultured, which took between 3-6 months given that field M. bovis isolates are very slow h f h i l d f l d i l d i The aim of the PEMbo project, a collaborative study between ANSES and INRA, two French One Health EJP partners, is to better understand the complex biology of M. bovis through the study of the complete genomes of a large panel of isolates. The first task, culture, cloning and extraction of M. bovis strains of work package 1 (WP1) of this project (Establishment of reference sequences from the main French clonal groups) was completed in 2020. Ten representative M. bovis genotypes were selected from strains circulating in France. These were selected and cultured, which took between 3-6 months given that field M. bovis isolates are very slow growers. The DNA from these isolates was extracted for long-read sequencing. As long-read sequencing requires a high DNA quantity and quality, the extraction process was optimised so that appropriate concentrations of high-quality DNA were obtained. METAPRO The METAPRO project is investigating the use of metagenomics and genomic approaches for the prevention of the spread of plazomicin resistance in humans, animals, and the environment. The METAPRO project is investigating the use of metagenomics and genomic approaches for the prevention of the spread of plazomicin resistance in humans, animals, and the environment. Plazomicin is used as a last resort antibiotic in complicated urinary tract infections (UTI) caused by multidrug resistant Gram-negative bacteria in humans. However, the expression of acquired 16S rRNA methyltranferases by bacteria results in complete resistance to plazomicin. The PhD candidate was recruited in March 2020, at the start of the COVID-19 pandemic. The student is based at a lab in the Complutense University of Madrid, which became part of a network to diagnose COVID-19 in elderly homes in Madrid at the start of the PhD. The PhD student was actively involved in this activity and due to all the restrictions applied in Spain, no progress could be made in his PhD project until June 2020. In addition, the national COVID-19 restrictions added further difficulties in advancing the research between June and September 2020, specifically in relation to the task of establishing the potential sampling points in Spain. Therefore, the plans for the PhD project were adapted, and the team decided to start the analysis with samples that were collected from previous projects that the lab was involved in. Preliminary bioinformatic analyses have been made with metagenomic sequences belonging to different pig farms across Spain and several acquired 16S rRNA methyltransferases have been detected, including some of low worldwide prevalence when using a read-based approach. The assembly-based analysis did not yield concluding results since the samples were only sequenced via Illumina (short reads). A DNA re-extraction and sequencing of the same faecal samples via both short and long-read technologies is foreseen in January 2021. In addition, in the period of November to December 2020, contact was established with a few potential sampling points to start sampling as soon as possible in the beginning of 2021. There are still several ecological niches that were intended to be sampled missing at this stage, however it is hoped that in early 2021, the situation will allow suitable sampling spots to be found. onehealthejp.eu @OneHealthEJP ONE Health EJP onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:PIXABAY P hD P RO JECTS PEMbo DNA genotypes were sent to for quality checking and DNA purification protocol validation. This methodology has been validated for the first strains tested, and the quality control of the remaining DNA samples is underway. Once the DNA quality control is completed the following tasks will begin: the second task which is sequencing and de novo assembly, and the first task, sequencing of supplementary strains, will start. The progress of the second task, Genomic markers analysis, is ahead of schedule. Genomic analyses on previously available genomes were performed. The first steps in bioinformatics focused on the presence and distribution of an insertion sequence, IS6110, in the genomes of M. bovis. IS6110 is a very useful genetic marker of the Mycobacterium tuberculosis complex employed for TB direct detection by PCR and for genotyping. The bioinformatics scripts were adapted to find the number and localisation of IS6110 in the bacterial genomes using contigs from reads of Illumina sequencing. A wide diversity in the copy number and the localisation of this sequence in the different genetic families of M. bovis in France was highlighted. These multicopy strains were grouped on specific nodes. These genotypes are mostly found in French bTB endemic regions and another study shows the strong stability of IS6110 number and genomic location in these groups over time. At present, in silico analyses is being performed to determine if these insertions can lead to phenotypic behaviours, which could explain a better fitness or transmissibility and thus a potential epidemiological success. nehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:FLICKR P hD P RO JECTS MACE Cystic echinococcosis is a zoonotic parasite disease of significant public health concern in many parts of the world, with over 5000 new cystic echinococcosis cases reported each year in South America. The burden, extending to include economic impacts, is mostly felt in subsistence livestock keepers and other rural and peri-urban populations where other health competing interests persist. Under reporting of the condition is large and the evidence to inform efficient surveillance and control approaches is scarce. The comparison of evidence is further complicated due to the large range of surveillance and control measures across all hosts, and their varied application across geographies. The MACE project aims to inform the most efficient portfolio of surveillance options and interventions towards cystic echinococcosis control and elimination, accounting for the various risks, disease control capacities, and risk preferences across geographies. The project is novel in the joint application of mathematical modelling and economic evaluation, and in the active elicitation of risk attitudes towards cystic echinococcosis and related control measures to formally model their impact on the uptake of interventions and their efficacy. In this project, two highly cystic echinococcosis endemic areas will be targeted- Argentina (high incidence) and Albania (low incidence). In January 2020, the PhD student was recruited, and work commenced. The following tasks were progressed in this project: • An elicitation questionnaire was developed to capture the attitude to investment into disease surveillance sensitivity by stakeholders. This is in planning to be conducted in a representative sample audience of stakeholders. • A spatio-temporal model was developed to estimate the risk and prevalence of cystic echinococcosis in dogs across all regions of Uruguay (excluding Montevideo). This framework will be extended and adapted to support current planning of a screening programme across the country. A spatio-temporal model was developed to estimate the risk and prevalence of cystic echinococcosis in dogs across all regions of Uruguay (excluding Montevideo). This framework will be extended and adapted to support current planning of a screening programme across the country. A review of cystic echinococcosis surveillance and control methods, as well as historical mathematical modelling approaches used, has been drafted in preparation for a report due 15 months after the start of the PhD (Confirmation report). onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:FLICKR P hD P RO JECTS UDoFRIC Since 2005, Campylobacter has been the most reported gastrointestinal bacterial pathogen for people (campylobacteriosis), with an EU notification rate of 64.8 per 100,000 population in 2017. Since 2005, Campylobacter has been the most reported gastrointestinal bacterial pathogen for people (campylobacteriosis), with an EU notification rate of 64.8 per 100,000 population in 2017. Across European member states, antimicrobial resistance (AMR) monitoring in clinical Campylobacter isolates has reported increasingly high levels of ciprofloxacin resistance. Ciprofloxacin is an antibiotic of the fluroquinolone class and has been declared as a major public health concern by the World Health Organisation. Campylobacter originating from poultry are considered a main source of campylobacteriosis in people. The use of ciprofloxacin in broiler flocks has been linked to the development of resistance in Campylobacter that can persist after use has ceased and is a potential source of resistance in human campylobacteriosis. In the EU, a ban of routine use of feed supplemented with antibiotics was implemented in 2006, however therapeutic use of fluroquinolones in poultry remains an option. The UDoFRIC project aims to exploit the archives of Campylobacter and associated information from surveillance and research across the food-chain to investigate temporal trends in the development and diversity of fluroquinolone resistance in UK and French broiler flocks. The project will examine the relationship between fluroquinolone use in poultry and development of resistance, assess fitness benefits/costs of acquired resistance and determine if any specific fluroquinolone resistant variants found in poultry are more or less likely to persist and cause disease in people. The data from this project would feed into the risk assessment for ongoing use of fluroquinolone in poultry and consequent risks of fluroquinolone resistance in clinical cases. The PhD student was recruited in March 2020 and has been making progress in the project’s objectives. There has been advancement in the writing of a literature review focusing on the background and previous research conducted on fluroquinolone resistance in Campylobacter. Data analysis has been carried out on a subset of data available to this project, obtained by previous UK national research and surveillance studies in poultry. The project has access to six distinct datasets over a 24-year period. Campylobacter samples were obtained by sampling either caecal contents of broilers at slaughter or from broiler carcasses after processing. There has been progress with analysing the trends of fluroquinolone resistance over time and production factors associated with fluroquinolone resistance (e.g. P hD P RO JECTS 58 INTRODUCTION VISION STRUCTURE ACHIEVEMENTS OUTCOMES DISSEMINATION EDUCATION DESIRE Brown and black rats carry a multitude of pathogens with public and veterinary health importance. Their potential to rapidly reach high population numbers creates unpredictable situations of high pathogen transmission risks. Rat populations are heavily affected by environmental changes such as urbanisation and climate change. A new phenomenon known as ‘greening’ and ‘blueing’ refers to improving living conditions and biodiversity in the city, and to combat heat; however, the consequences of these on rat-borne diseases is unknown. Sustainable intervention is required to be directed to situations where the risk of transmission of pathogens creates human or veterinary risks. To perform risk assessment and mitigate the risks, a surveillance system is needed which has information both about the pathogen distribution as well as rat population developments. The overarching aim of the DESIRE project is to design and test an effective surveillance system for rat-borne diseases, using the Netherlands as a test case. This project will provide evidence-based insights in four key elements of this surveillance system- monitoring of populations, monitoring of pathogens, risk assessment and intervention. The project will build onto existing surveillance activities and extend these by collaboration with international institutes. The focus for the PhD candidate in the first year was on the field study, comprising of several tasks. The work started with interviews of various stakeholders, including municipal employees who oversee urban ecology and/or pest management. This resulted in a better problem definition and helped the design of the field study. The field study commenced in May and ran till October 2020, leaving little time for other activities. It comprised of two cities, one of which, unfortunately did not result in many captured rats. Therefore, to ensure enough power to perform the proposed analyses, another city will be added to the project in 2021. After collection of the samples the first set of diagnostic analyses were performed for zoonotic pathogens. However, this was disrupted shortly afterwards due to lab restrictions at the RIVM due to COVID-19. Furthermore, the PhD candidate has started on the second task, which is Next-Generation Sequencing analysis of the bacterial pathobiome, using rat samples from the biobank. It was decided to extend this with the analysis of the viral pathobiome, which will be done in the first half of 2021. onehealthejp.eu @OneHealthEJP ONE Health EJP P hD P RO JECTS UDoFRIC bird age, farming method, bird weight). Work has also been conducted identifying Campylobacter lineages and aims to determine if there is a link between MLST or whole genome sequence information and fluroquinolone resistance. onehealthejp.eu @OneHealthEJP ONE Health EJP WILBR The propagation and spread of microorganisms resistant to antimicrobials is a global phenomenon that is affecting both human and animal health. The propagation and spread of microorganisms resistant to antimicrobials is a global phenomenon that is affecting both human and animal health. Through the One Health agenda, the risks posed by the medical and veterinary sectors are being assessed and addressed through national and global initiatives and programmes. However, there has been limited focus both in Europe and globally on the role of the environment in propagating resistant microorganisms through inadequate treatment of contaminated/wastewater, or medical, biological and food waste, which may be dispersed further through wildlife such as wild birds. Migratory birds, which represent ~40% of total birds in the world, can fly many thousands of kilometres often overwintering in Africa and Eurasia and returning to the northern hemisphere in spring. These birds add another level of complexity to identifying and controlling the routes for spread of antimicrobial resistance, as they often overwinter in countries or areas where they may be little information of resistance trends due to limited surveillance and diagnostic capacity, with the burden of AMR unknown. To help provide an assessment of the environmental risk posed by AMR and identify management options with clear indicators of effectiveness, the WILBR project aims to understand the contribution of factors such as wild birds in the spread of AMR in the environment in general, and on livestock farms in particular. The PhD student was successfully recruited to the WILBR project in February 2020. Although, initial plans were made for desk, laboratory and farm-based work to be performed in the first year of the PhD, due to the COVID-19 pandemic, only desk and lab-based tasks have commenced. In 2020, a literature review was undertaken on the role of wild birds in spread and persistence of AMR in the farm environment. The review includes sections on identifying the current situation regarding AMR in different environments; drivers for AMR; the role of vectors and environment in persistence and dissemination of AMR; the role of AMR surveillance; and evaluation of different methodologies for identifying AMR by phenotype and genotype, in bacteria. Some historical E. P hD P RO JECTS 59 INTRODUCTION VISION STRUCTURE ACHIEVEMENTS OUTCOMES DISSEMINATION EDUCATION A collection of historic isolates and information relating to them has also been collated and used to identify gaps in the data collected. To create uniformity in the information available in each dataset for comparison, work has begun on historic isolates. These historic isolates have been recovered from the Campylobacter national reference laboratory archives, their species classification has been confirmed and the DNA of these isolates has been extracted and sequenced ready for further analysis. onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:WIKIMEDIA P hD P RO JECTS WILBR coli isolated from gull faeces, collected from an outdoor pig farm, during a longitudinal study in the One Health EJP JRP project ARDIG are being utilised in this PhD project because of the delays to farm based work caused by COVID-19. Over 200 previously unused isolates have undergone whole genome sequencing, and downstream bioinformatics is currently taking place. An outdoor pig farm, known to have wild birds persistently present on farm, had been recruited for a longitudinal onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:WIKIMEDIA EnvDis Foodborne diseases remain an important cause of morbidity, mortality, and healthcare costs worldwide. The problem is expected to be exacerbated by population growth and the rising of antibiotic resistance. Furthermore, anthropogenic activities are constantly changing the environment, for example climate change, land use and socio-economic factors, which are a well-recognised driver of diseases. The environment can affect pathogen abundance, survival, and virulence, host susceptibility to infection as well as human behaviour. It is thought that the accelerating rate of global climate and other environmental change will impact the distribution, frequency, and patterns of established diseases as well as the emergence and re-emergence of new and old ones. Foodborne diseases remain an important cause of morbidity, mortality, and healthcare costs worldwide. The problem is expected to be exacerbated by population growth and the rising of antibiotic resistance. Furthermore, anthropogenic activities are constantly changing the environment, for example climate change, land use and socio-economic factors, which are a well-recognised driver of diseases. The environment can affect pathogen abundance, survival, and virulence, host susceptibility to infection as well as human behaviour. It is thought that the accelerating rate of global climate and other environmental change will impact the distribution, frequency, and patterns of established diseases as well as the emergence and re-emergence of new and old ones. The EnvDis project aims to develop a tool to assess the public health risk of foodborne zoonoses based on information of relevant environmental factors. This will be done using Salmonella, for which the mechanism of transmission is relatively well-known, and will help validate the approach. The literature review carried out by the PhD student at the start of 2020 showed that salmonellosis continues to be the second most reported foodborne disease in humans in Europe, with a seasonal pattern of incidence focused on the warmest weeks of the year. Amongst the food sources of infection, eggs and chicken meat were identified as relevant sources of contamination. As a first step in the project, an equation was developed linking the growth of Salmonella on eggs and chicken meat to temperature. Assuming that the probability for a human getting infected is proportional to the temperature-dependent number of bacteria, a simplistic predictive model was created using the high-resolution meteorological records for the past 20 years provided by the Met Office. P hD P RO JECTS study, but due to COVID-19 restrictions we were unable to undertake any farm visit to collect environmental samples, faecal samples from pigs, and caecal samples from corvids present on farm. Although this farm is now unlikely to be sampled during the course this project, an opportunity to examine the spread of AMR in wild bird populations across the UK for a 12-month period through the APHA’s wild bird scanning surveillance programme is currently being pursued. study, but due to COVID-19 restrictions we were unable to undertake any farm visit to collect environmental samples, faecal samples from pigs, and caecal samples from corvids present on farm. Although this farm is now unlikely to be sampled during the course this project, an opportunity to examine the spread of AMR in wild bird populations across the UK for a 12-month period through the APHA’s wild bird scanning surveillance programme is currently being pursued. onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:PIXABAY P hD P RO JECTS EnvDis To validate the model, the predictions were compared to the incidence of salmonellosis in England and Wales provided by PHE for the same period. Both curves have the same pattern albeit some minor differences, suggesting that the three parameters considered (eggs, chicken and temperature) play a big role on the seasonality of salmonellosis. For this work, different exploratory charts were created to find further trends and causality, such as a geographical effect by comparing different areas of England and Wales, to hypothesise different contribution in the risk factor associated with eggs versus chicken, and to create a model to investigate the effect of an average increasing of temperatures in the incidence of the disease. onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:FLICKR IMAGE:FLICKR P hD P RO JECTS VIMOGUT Antimicrobial resistance (AMR) is a major public health concern, and many factors contribute to this, including the use of antimicrobials as growth promoters in livestock. It is essential to reduce prevalence of AMR in livestock to reduce the likelihood of antibiotic resistant bacteria passing through the food chain and to retain effective therapeutic treatment of the livestock itself. Preliminary results show that the microbiome of chickens colonised early in life by extended-spectrum beta-lactamase (ESBL) producing E. coli is less diverse than those of flock mates that are not colonised. This is supported by in vivo studies that have shown that competitive exclusion through probiotics is currently the most effective prevention strategy for colonisation by organisms harbouring ESBLs. However, this strategy has been tested with limited attention for chick age. Furthermore, in practice, probiotics are considered too expensive to use throughout the whole production cycle. An in vitro chicken gut model was developed at the APHA, UK as an alternative to study bacterial interactions in complex communities such as the microbiome. This system allows the evaluation of new treatment interventions at different stages of the microbiome development, without the ethical concerns and high cost of in vivo experiments. The VIMOGUT PhD project investigates the chicken microbiome development of chickens on farms to determine if the reported microbial progression is reproducible between different production rounds and farms. By screening these samples for the presence of ESBL E. coli, the significance of the reduced diversity of early colonised chickens will be determined. The in vitro chicken gut model has been set up to test strategies for the reduction of ESBL E. coli and compare these with published data from in vivo studies. When the model can efficiently reproduce these in vivo studies, it can be used for further study of new ESBL E. coli colonisation prevention strategies. A dataset was generated of the microbiome of broiler chickens on a single farm which complements a preliminary dataset from the same flock to generate additional statistical power for the analysis. The datasets were merged and analysed. The outcome of the analysis is currently prepared in a manuscript for publication. Preparations to start sampling new broiler chicken farms in the Netherlands will commence shortly, and additional farms will be visited in 2021 to collect data from broiler flocks (if it is possible during the COVID-19 pandemic). AptaTrich Trichinellosis is a zoonosis caused by the consumption of raw or undercooked meat of animals (mainly pigs, wild boars, horses) infected with the nematode, Trichinella spp.. To date, Trichinella remains in the top three of prioritised foodborne parasites in Europe and this parasite is still of major public health and economic importance at international level. Due to the very low Trichinella prevalence in pigs, a test specificity bordering 100% is needed, as false positive samples would need to be retested with a second serological method. Such tests can only be performed by specialised laboratories, making the testing logistics more complicated and expensive. Therefore, new diagnostic methods with higher specificity and earlier detection are needed for prevention and to improve human disease detection. One such method is the use of aptamers. Aptamers are synthetic nucleic acids that fold into unique 3D conformations capable of binding pathogen antigens with remarkable affinity and specificity, thus, combining the ease of serological sampling, and the direct detection of the presence of the pathogen. Aptamers have successfully been used for the detection of parasites in fresh produce, including in aptamer-based biosensors. The AptaTrich project is investigating the development of an aptamer-based detection system for Trichinella which would bypass the caveats associated with serological testing and enable specific and early detection in both human diagnostics and Trichinella monitoring programmes in pigs. The technique can also be combined with aptamers designed against other pig diseases (e.g., Toxoplasma, Salmonella, etc) making a wider future application possible. In January to December 2020, two protocols for Trichinella spiralis-specific aptamers selection were planned. The first one is against whole muscle larvae, the infective stage of this nematode. The second one is against protein(s) considered as potential biomarker(s) from the excretory or secretory whole muscle larvae products. The PhD student was trained at McGill institute for the Systematic Evolution of Ligands by Enrichment methods (SELEX) methods. For both protocols, much work has been accomplished in producing single-stranded DNA sequences from double-stranded DNA PCR products, an essential step in the successful isolation of target specific aptamers. Furthermore, the protocols to adapt the SELEX method to T. spiralis whole muscle larvae have been established and optimised at ANSES. onehealthejp.eu @OneHealthEJP ONE Health EJP onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:FLICKR P hD P RO JECTS VIMOGUT This research aims to confirm if the differences in microbiome composition that were measured in the first dataset can be found in additional flocks. Setup of an in vitro model of the broiler caecum was designed and completed using a continuous culture fermenter system. The model was seeded with caecal content from broiler chickens to create a stable microbial community. This model will be used to test the effects of feed interventions and to study the effect of substances on the transfer of AMR encoding plasmids between bacteria in the established microbial community. Initial runs have been performed to test the effects of two phytochemicals for which microbiome analysis is currently carried out. onehealthejp.eu @OneHealthEJP ONE Health EJP onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:FLICKR IMAGE:FLICKR ToxSauQMRA Toxoplasma gondii is an intracellular parasite and one of the most successful parasites worldwide. Humans, as intermediate hosts, can become infected with T. gondii through ingestion of oocysts (e.g. when handling soil or cat litter, or on unwashed vegetables) or tissue cysts in raw or undercooked meat. Pigs, like other livestock, can harbour tissue cysts following the ingestion of oocysts. Products such as raw cured meats are a possible source of T. gondii and pose a major issue to public health. For example, in 2015, France produced just over 108,000 tonnes of sausages and dry sausages, representing ~9% of the total tonnage of all sausages (FICT data, 2016), therefore monitoring the prevalence of T. gondii is essential. The high prevalence of T. gondii infection in France in humans and the fact that the main mode of contamination is foodborne, justifies the conduct of a quantitative microbiological risk assessment (QMRA). In France, T. gondii seroprevalence estimates in various animal species (ovine, bovine, pig, horses) are available, enabling such an analysis, however knowledge gaps include the quantitative estimates concerning the distribution of parasites in the various parts (muscles) of a carcass and the reduction of the parasite load according to the cooking, preparation, or preservation, as identified by an expert group of ANSES in 2005. The ToxSauQMRA project aims to answer the scientific question- “What is the attribution of the traditional raw pork products in the human Toxoplasma gondii infection?” based on three areas: (i) A thorough investigation of the preferential sites for T. gondii in experimentally infested pig carcasses with two different stages (tissue-cyst versus oocyst).f The ToxSauQMRA project aims to answer the scientific question- “What is the attribution The ToxSauQMRA project aims to answer the scientific question- “What is the attribution of the traditional raw pork products in the human Toxoplasma gondii infection?” based on three areas: (i) A thorough investigation of the preferential sites for T. gondii in experimentally infested pig carcasses with two different stages (tissue-cyst versus oocyst).f (ii) Evaluation of the impact of the manufacturing process (including different incorporation rates of nitrites and NaCl) and the conservation of dry sausage on the viability of T. gondii. (iii) A quantitative microbiological risk assessment analysis to be conducted for the various raw pork products (dry sausage, dry ham, etc.). ToxSauQMRA The key activities, results, and achievements in 2020 are the following: Successful experimental infection of pigs with oocysts and tissue cysts. onehealthejp.eu @OneHealthEJP ONE Health EJP IMAGE:HIPPO PX IMAGE:HIPPO PX P hD P RO JECTS Codes4strains Whole genome sequencing allows the tracking of pathogenic strains and informs infection control, diagnostics and sometimes treatment strategies. To track strains globally, and as they spread between the environment, food, animals and humans, universal strain nomenclatures are necessary. The core genome Multilocus Sequence Typing (cgMLST) approach is an accurate, reproducible and portable strain genotyping method that underlies widely used strain nomenclatures, in which groups are generally determined by single-linkage clustering. However, cgMLST groups are unstable due to the possibility of group fusion upon subsequent sampling. Recently, a new coding approach named LIN (Life Identification Number) was introduced by Marakeby et al (2014). It provides a numerical code for each genome based on its similarity (estimated using the Average Nucleotide Identity, ANI) to the closest genome already encoded. As LIN codes are attributed to genome rather than groups, they are stable. The aim of the Codes4strains project is to develop a novel genome-based genotyping approach taking the best of the two above classification approaches, i.e., combining the advantages of cgMLST discrimination, standardisation) with those of the LIN code approach (complete stability). Thus, the aim is to develop and explore the strain classification utility of cgMLST-based LIN code (cgLINcodes) systems, and compare the cgLINcodes approach with other existing classification approaches: the SNP address and multi-level single-linkage classifications. The two important pathogens Klebsiella pneumoniae and E.coli are being used to develop and evaluate our approach. s: , Panunzi, LG., Rodrigues, C., Gaday, Q., Baines, In 2020, the Codes4strains project developed procedures (bioinformatics and algorithmic) to for a cgMLST-based LIN code system. cgMLST profiles from genomes were generated using defined schemes, resulting in the cgMLST schemes being finalised. For K. pneumoniae, 629 gene loci were defined for the cgMLST scheme from a previously published data. For E. coli, the cgMLST scheme from EnteroBase was utilised, which included 2513 gene loci. As a second task, the development of the cgMLST-based LIN code algorithm was defined and a bioinformatics implementation was developed in Python. A set of bioinformatics tools and metrics were used to define thresholds, considering the phylogenetic structure of the species, and with the view to maximise its usefulness in population biology and epidemiology. For K. pneumoniae, a large dataset of 7060 high-quality genomes was used to define the overall structure and diversity of the population and to finalise a cgLINcode system comprising 11 thresholds. Codes4strains For E.coli, a genome dataset that was previously published by Public Health England (PHE) was utilised. This allowed a database of cgLINcodes aligned with other approaches to be created. Finally, a comparison between the different classification systems (HierCC, cgLINcodes and SNP address) was carried out for E.coli. For K. pneumoniae, only cgLINcodes and multi-level Single Linkage clustering (equivalent to HierCC) were compared, as the SNP database required for the SNPaddress approach remains to be constructed. TRACE Hepatitis E virus is a zoonotic virus responsible of acute hepatitis E in human in Western countries. The main route of HEV transmission in Europe is through consumption of raw or undercooked contaminated pork, which have been associated to clinical Hepatitis E. Molecular analyses showed that hepatitis E virus strains detected in pigs and humans in the same geographical region present high genetic identity, indicating that swine are the main source of infection for humans. Since 2014, an increase in clinical cases was observed in many EU member states including the Netherlands. During the summer of 2017 and 2018 in the Netherlands, a temporal drop was observed in acute hepatitis E virus clinical infections, after which the incidence went back up to similar numbers as had been observed before. These events have remained unexplained to this date. To explain the predominance of certain hepatitis E virus variants and to explain differences in virulence, the TRACE project aims to characterise the hepatitis E virus variants and attempt to identify virulence factors in hepatitis E virus strains detected in clinical patients, in the general population and in domestic swine. Given the high genetic diversity of hepatitis E virus it is critical to obtain higher resolution genomic data (i.e. whole genome sequences) in order to gain more understanding on its molecular epidemiology and possible variations in its adaptive traits. The PhD student was recruited in January 2020, and the timing of the COVID-19 crisis did impact the progress of the PhD. However, some progress was made on establishing optimisation of sample (pre) processing to make whole genome sequencing (WGS) possible. Different methods were used, and DNA depletion treatments were explored. The messenger RNA of 18S (host) and 16S (bacterial) origin was successfully removed. The hepatitis E virus whole genome sequences were successfully generated using above enrichment methods and specific primers. Work is currently ongoing to analyse the sensitivity. Hepatitis E virus culture preceding sequencing may be considered to increase the amount of hepatitis E virus (the work on a culture method is part of BIOPIGEE project). onehealthejp.eu @OneHealthEJP ONE Health EJP ublications: ennart, M., Panunzi, LG., Rodrigues, C., Gaday, Q., Baines, L., Barros-Pinkelnig, M., Carmi-Leroy, A., Dazas, M., Wehenkel, AM., Didelot, X., Toubiana, J., Badell, E., Brisse, S. 020). Population genomics and antimicrobial resistance in orynebacterium diphtheriae. Genome Medicine. 12, p 1-18. OI: https://doi.org/10.1186/s13073-020-00805-7. TRACE Publications: Hennart, M., Panunzi, LG., Rodrigues, C., Gaday, Q., Baines, SL., Barros-Pinkelnig, M., Carmi-Leroy, A., Dazas, M., Wehenkel, AM., Didelot, X., Toubiana, J., Badell, E., Brisse, S. (2020). Population genomics and antimicrobial resistance in Corynebacterium diphtheriae. Genome Medicine. 12, p 1-18. DOI: https://doi.org/10.1186/s13073-020-00805-7. SUSTAIN The SUSTAIN project lies at the interface of social sciences and public health and aims to understand the political drivers and constraints for increased transboundary integration and institutionalisation of the One Health approach across EU member states. The challenges for implementing the One Health approach are complex political and institutional structures. Complex political structures emerge through various levels on which politics can be discussed, such as local, regional, national, and international levels. Within and across these institutions, information infrastructure, collaboration and relationships can pose obstacles for implementing a One Health approach. Understanding the barriers and facilitators to change policy processes, and how these differ across sectors and across EU member states is important information for the future of One Health in the EU. The first step of this project was a literature search to inform subsequent studies. Hereafter, quantitative studies of databases and of a survey addressed to institutions working on One Health topics will follow. A qualitative study will be conducted which will include interviews and observations. The studies will inform different aspects, such as the current state of One Health institutionalisation in the EU as well as on a national level, and the One Health networks’ interactions and relationships. In 2020, data gathering has started as planned for the PhD project. This included conducting interviews with experts from National Public Health, Veterinary, Food and Environment agencies in Sweden. Initially, it was planned to travel to Sweden to conduct the interviews in person. Due to the COVID-19 pandemic, the interviews were conducted online via videoconferencing. A one month trip to the Istituto Superiore di Sanità (ISS) in Italy was completed in autumn 2020, during this trip the PhD student was integrated into the everyday work of the institute and was able to conduct interviews with experts working in different departments (public health, veterinary health, environment, nutrition, and food) at ISS. Most of the data from interviews with experts have been gathered, but a few interviews are left to be conducted with Italian experts, especially those working in the veterinary (Istituto Zooprofilattico Sperimentale) and environmental institutes (Istituto Superiore per la Protezione e la Ricerca Ambientale). Data from the interviews in Sweden was analysed in NVivo and a first-authored article was prepared for publication, submitted, and accepted in December 2020. The data analysis from the Italian interviews began in November 2020 and will be ongoing in 2021. Publications: Hennart, M., Panunzi, LG., Rodrigues, C., Gaday, Q., Baines, SL., Barros-Pinkelnig, M., Carmi-Leroy, A., Dazas, M., Wehenkel, AM., Didelot, X., Toubiana, J., Badell, E., Brisse, S. (2020). Population genomics and antimicrobial resistance in Corynebacterium diphtheriae. Genome Medicine. 12, p 1-18. DOI: https://doi.org/10.1186/s13073-020-00805-7. Hennart, M., Panunzi, LG., Rodrigues, C., Gaday, Q., Baines, SL., Barros-Pinkelnig, M., Carmi-Leroy, A., Dazas, M., Wehenkel, AM., Didelot, X., Toubiana, J., Badell, E., Brisse, S. (2020). Population genomics and antimicrobial resistance in Corynebacterium diphtheriae. Genome Medicine. 12, p 1-18. DOI: https://doi.org/10.1186/s13073-020-00805-7. onehealthejp.eu @OneHealthEJP ONE Health EJP Publications: Sarah Humboldt-Dachroeden, S., Olivier, R., Frid-Nielsen, SS. (2020). The state of One Health research across disciplines and sectors – a bibliometric analysis. One Health, 10, pp. 100146. DOI: 10.1016/j.onehlt.2020.100146. IMAGE:LIBRESHOT P hD P RO JECTS SUSTAIN The process will be completed when all interviews have been conducted and analysed. Thereafter, a publication will be prepared. Publications: Additionally, preparation of a survey to address ministries, EU institutions and high-level members of staff in public health, veterinary health, environment and food agencies, has started and is planned for dissemination in spring 2021. This survey will aim to collect data about the understanding of One Health and how One Health is put into practice. Sarah Humboldt-Dachroeden, S., Olivier, R., Frid-Nielsen, SS. (2020). The state of One Health research across disciplines and sectors – a bibliometric analysis. One Health, 10, pp. 100146. DOI: 10.1016/j.onehlt.2020.100146. onehealthejp.eu @OneHealthEJP ONE Health EJP onehealthejp.eu @OneHealthEJP ONE Health EJP onehealthejp.eu @OneHealthEJP ONE Health EJP One Health EJP Dissemination Activities IMAGE:PIXABAY ehealthejp.eu @OneHealthEJP ONE Health EJP One Health EJP Dissemination Activities IMAGE:PIXABAY O N E HEA L TH EJP DIS S EM IN A TIO N A CTIV ITIES The One Health EJP is committed to sharing results and knowledge from all activities. Every OHEJP consortium member plays a key role in dissemination, and there are several ways this was achieved in 2020: OHEJP outcomes were regularly disseminated at key governance meetings such as Scientific Steering Board meeting, Stakeholders Committee meeting, the Programme Management – Programme Owners Committee meeting. OHEJP outcomes were regularly disseminated at key governance meetings such as Scientific Steering Board meeting, Stakeholders Committee meeting, the Programme Management – Programme Owners Committee meeting. wo targeted reports were created to ensure that key EU Stakeholders are informed of the k cientific and integrative outcomes from the OHEJP. To highlight the responsiveness and timeliness of the OHEJP, two additional reports were created including “Links between COVID-19 related needs of stakeholders and One Health EJP activities” and “One Health EJP Thematic Report on AMR”. The OHEJP Outcome Inventory was updated and acts as a repository for all OHEJP outcomes and is a hub for information for both national and international stakeholders and both internal and external audiences. The Data Management Plan was further developed, and an abundance of support has been provided to the Project Leaders of OHEJP projects to ensure that data generated in their projects are FAIR (findable, accessible, interoperable and reusable). Case studies for several OHEJP project were created and disseminated to a global audienc raise awareness of results and their impact. Case studies for several OHEJP project were created and disseminated to a global audience to raise awareness of results and their impact. The OHEJP website and social media platforms served as central platforms to share public events, news, research outcomes and key information to a global audience.i The OHEJP issued regular newsletters which contained highlights and links to scientific and collaborative activities, these newsletters were an important tool to disseminate news on a regular basis. The OHEJP Annual Scientific Meeting provided a central platform for communication and dissemination of the scientific outcomes from the Joint Research and Joint Integrative activities, in addition to the OHEJP PhD projects. This event also facilitated collaboration with One Health experts both internal and external to our consortium. The establishment of excellent relationships with key EU and international stakeholders facilitates translation of science to policy, in addition to ensuring there is no duplication of research between major European organisations. O N E HEA L TH EJP DIS S EM IN A TIO N A CTIV ITIES onehealthejp.eu @OneHealthEJP ONE Health EJP a regular basis to the European nsuring exchange of information and ects such as EU- JAMRAI, JPIAMR and l expertise in the scientific community. n training the next generation of rt consortium members with alth Congress in November 2020 platform to demonstrate the impact IMAGE:PIXABAY The OHEJP disseminated important scientific results on a regular basis to the European Commission and the Research Executive Agency (REA), ensuring exchange of information and knowledge and maximising impact. The OHEJP disseminated important scientific results on a regular basis to the European Commission and the Research Executive Agency (REA), ensuring exchange of information and knowledge and maximising impact. Close relationships were established with other EU projects such as EU- JAMRAI, JPIAMR and AVANT with the aim of exchanging scientific and technical expertise in the scientific community. Organisation of Education and Training activities to aid in training the next generation of One Health researchers. A Dissemination Information Pack was created to support consortium members with dissemination of their outcomes. Attending global conferences such as the World One Health Congress in November 2020 extended the reach to global audiences and provided a platform to demonstrate the impact that the OHEJP has in One Health research. onehealthejp.eu @OneHealthEJP ONE Health EJP INTRODUCTION VISION STRUCTURE ACHIEVEMENTS OUTCOMES DISSEMINATION EDUCATION The second One Health EJP Annual Scientific Meeting The second One Health EJP Annual Scientific Meeting Organising institutes: the National Institute of Public Health and the Veterinary Research Institute, (Czech Republic) in collaboration with University of Surrey (UK) Location: Online Dates: 27th-29th May 2020 Streamed live to over 750 participants worldwide, the second One Health EJP Annual Scientific Meeting was a huge success. The event showcased a wide variety of One Health research with 5 Keynote speakers, 48 oral presentations and 110 poster presentations, focussing on the themes of foodborne zoonoses, antimicrobial resistance and emerging threats. The second One Health EJP Annual Scientific Meeting included the first 3 Minute Thesis competition of the One Health EJP PhD students. This event gave each of the 17 PhD students the opportunity to showcase their work to a global audience. The Keynote speakers brought a wealth of experience from across the globe and included Stef Bronzwaer from EFSA, Jaroslav Hrabák from University Hospital Pilsen, Czech Republic, Monika Dolejská from VFU Brno, Czech Republic, Mieke Uyttendaele from Ghent University, Belgium and Elizabeth Mumford from the WHO. The event also included a significant social media presence with excellent interaction with stakeholders and scientists worldwide. onehealthejp.eu @OneHealthEJP ONE Health EJP onehealthejp.eu @OneHealthEJP ONE Health EJP Educating the next generation of One Health researchers IMAGE:PXHERE EDU CA TIO N A N D TRA IN IN G The Education and Training activities (WP6) develop and deliver innovative training platforms and materials with a specific focus on One Health. Our Education and Training activities uniquely bring together students, early-career researchers, and key experts with diverse expertise in the human, animal, and environmental health fields. These activities reinforce collaboration across multiple disciplines in these health fields, and integration between the consortium member institutes and stakeholders (and beyond) bringing these individuals together so that One Health knowledge and experiences can be shared, and collaborative engagements and future relationships can be formed for One Health activities. Thanks for you and One Health EJP provide a such good chance to study! And I am very happy to study with everyone. I break out a lot of my stereotypes!” Boxuan Wang, China Agricultural University “ In 2020, as global travel was significantly impacted by the COVID-19 pandemic, WP6 pivoted to deliver our training events online until safe travel resumes to providing unique opportunities for training in One Health and encouraging collaboration and exchange of knowledge and experiences amongst scientists across the globe. healthejp.eu @OneHealthEJP ONE Health EJP Educating the next generation of One Health researchers IMAGE:PXHERE IMAGE:PXHERE Thanks for you and One Health EJP provide a such good chance to study! And I am very happy to study with everyone. I break out a lot of my stereotypes!” Boxuan Wang, China Agricultural University “ O N E HEA L TH EJP S U M M ER S CHO O L 2 0 2 0 Thanks for you and One Health EJP provide a such good chance to study! And I am very happy to study with everyone. I break out a lot of my stereotypes!” Boxuan Wang, China Agricultural University “ Thanks for you and One Health EJP provide a such good chance to study! And I am very happy to study with everyone. I break out a lot of my stereotypes!” Boxuan Wang, China Agricultural University Our annual summer schools are an important component of the One Health EJP, as they provide One Health training opportunities for the next generation of One Health scientists from across Europe and worldwide. It was a very interesting, enhan fantastic experience. Thank you s this terrific opportunity! Good lu I hope to keep in touch. Giorgia Baiocchi, University of Bolo “ s a very interesting enhan Organising institutes: Wageningen Bioveterinary Research (the Netherlands) in collaboration with Wageningen Institute of Animal Sciences, Netherlands Centre for One Health, Agreenium (France), INRA (France,) and University of Surrey (UK). Organising institutes: Wageningen Bioveterinary Research (the Netherlands) in collaboration with Wageningen Institute of Animal Sciences, Netherlands Centre for One Health, Agreenium (France), INRA (France,) and University of Surrey (UK). Theme: Global One Health- From Research to Practice Location: Online Dates: 17th-28th August 2020 Collaborative interactions: The programme was delivered by leading EU and international experts in public health, animal health and environmental health who face today’s challenges to implement One Health strategies in the different health fields. The collaborative interactions between these experts and 35 delegates from 22 countries across the globe provided opportunities for knowledge, skills, and competencies to be shared amongst individuals with different perspectives and experiences. onehealthejp.eu @OneHealthEJP ONE Health EJP Thanks for you and One Health EJP provide a such good chance to study! And I am very happy to study with everyone. I break out a lot of my stereotypes!” Boxuan Wang, China Agricultural University “ INTRODUCTION VISION STRUCTURE ACHIEVEMENTS OUTCOMES DISSEMINATION EDUCATION Thanks for you and One Health EJP provide a such good chance to study! And I am very happy to study with everyone. I break out a lot of my stereotypes!” Boxuan Wang, China Agricultural University “ Participants attended from several countries within the EU such as Denmark, Germany, France, Spain, and Romania, but also from countries beyond the EU such as Ghana, India, Ukraine, Mexico, USA, China, Canada and many more. O N E HEA L TH EJP CO N TIN U IN G P RO F ES S IO N A L DEV EL O P M EN T M O DU L E A really well organized, informative and interactive workshop. I felt very well and friendly supervised and motivated for my future work.” Jennie Fischer, BfR, Germany “ Continuing Professional Development (CPD) is the process of recording and reflecting on the skills, knowledge, and experience you gain as you work. CPD includes formal or informal learning beyond any initial qualification or training you have undertaken. The OHEJP’s CPD modules cover several themes in One Heath and are targeted at Early Career Researchers who can apply the training they receive in their future careers and in the training of future junior researchers. Organising institutes: National Institute of Public Health and the Environment, RIVM (the Netherlands) Theme: Outbreak Preparedness Location: Online Dates: 16th and 17th November 2020 A really well organized, informative and interactive workshop. I felt very well and friendly supervised and motivated for my future work.” Jennie Fischer, BfR, Germany Collaborative interactions: The module consisted of sessions delivered by leading European experts in the fields of national outbreak preparedness, risk analysis, risk communication and response in the One Health domains of human health, animal health and environmental health. The module provided an opportunity to bring experts and 20 delegates from our consortium partner institutes, stakeholders and OHEJP alumni together to share knowledge and experiences from countries such as France, the Netherlands, Italy, Norway, Switzerland, Sweden, Poland, UK, Germany and more. The diversity in ed- ucational background, experience and countries significantly enhanced the quality of the module and the experience of all those that participated. Delegates: This training event was attended by PhD students and Early Career Researchers from across our consortium partner network, each with some experience in a related health field. Participants from our stakeholders EFSA, also participated. Bringing together people from across the health disciplines made this module truly cross- disciplinary and highlighted the advantages of a One Health approach. The delegate’s educational backgrounds were diverse across the One Health fields and included biological sciences, veterinary medicine, social sciences, and public health, which helped to facilitate the sharing of One Health knowledge and expertise. Programme: The module delivered a platform to share experiences, knowledge and lessons learned from past outbreaks. Outbreaks involving new or re-emerging zoonoses can occur anytime and everywhere, something that the entire world has experienced with the COVID-19 pandemic. Strengthening the human-veterinary collaboration is essential to prevent, detect and respond to zoonotic threats. O N E HEA L TH EJP S U M M ER S CHO O L 2 0 2 0 This diversity significantly enriched the experience of all those that participated and added value to the course. Delegates and lecturers had the opportunity to network and socialise with each other at the virtual social events arranged on the first and last day of the summer school. Some delegates have attended the OHEJP’s other training events since the summer school, and so this training event directly contributed to building a future consortium to support the sustainability of the OHEJP, and to extend the OHEJP outside the EU in alignment with our global initiatives. Delegates: Bringing together delegates from a range of education levels and interdisciplinary backgrounds brought a diverse pool of experience and knowledge which facilitated unique multi-disciplinary and collaborative interactions. Delegates represented various stages of their career including bachelors, master’s students, PhD students, early career post-doctoral researchers and teaching lecturers. They belonged to multiple disciplines across the One Health domains including veterinary medicine, human medicine, biological sciences, infectious diseases, disease surveillance, microbiology, mathematical modelling, economics, social sciences, sustainability sciences, public health, and virology. Thanks for you and One Health EJP provide a such good chance to study! And I am very happy to study with everyone. I break out a lot of my stereotypes!” Boxuan Wang, China Agricultural University “ Programme: The global One Health concept emphasises the interdependence of human health with the health of animals, plants and sustainable ecosystems from a global perspective. This summer school programme aimed to understand and learn how to operationalise a global One Health approach to improve the health of people, animals and plants within a sustainable ecosystem. The programme delivered an introduction to global One Health basics, prediction approaches, analyses of integrated disease surveillance, outcomes research, risk management, and decision quality. Delegates had opportunities to present their own experiences and ideas in the global One Health field which were subsequently discussed with the key experts delivering the course. s a very interesting, enhan It was a very interesting, enhan fantastic experience. Thank you s this terrific opportunity! Good lu I hope to keep in touch. Giorgia Baiocchi, University of Bolo “ To read more about this event, view the programme and blog post on our website click here. View the full report here. O N E HEA L TH EJP S U M M ER S CHO O L 2 0 2 0 onehealthejp.eu @OneHealthEJP ONE Health EJP I very much enjoyed this event knowledge and insights that to use in my future work with In addition, the event provid in collaboration and communi important part in outbreak pr Nadja Karamehmedovic, FoH “ A really well organized, informative and interactive workshop. I felt very well and friendly supervised and motivated for my future work.” Jennie Fischer, BfR, Germany “ 76 INTRODUCTION VISION STRUCTURE ACHIEVEMENTS OUTCOMES DISSEMINATION EDUCATION O N E HEA L TH EJP CO N TIN U IN G P RO F ES S IO N A L DEV EL O P M EN T M O DU L E Yet, implementation and operationalisation of the One Health concept often remains onehealthejp.eu @OneHealthEJP ONE Health EJP I very much enjoyed this event knowledge and insights that to use in my future work with In addition, the event provid in collaboration and communi important part in outbreak pr Nadja Karamehmedovic, FoH “ A really well organized, informative and interactive workshop. I felt very well and friendly supervised and motivated for my future work.” Jennie Fischer, BfR, Germany “ A really well organized, informative and interactive workshop. I felt very well and friendly supervised and motivated for my future work.” Jennie Fischer, BfR, Germany “ a challenge. In addition, countries differ globally in their approaches to One Health issues. Sharing best practices and experiences of One Health approaches stood at the centre of this module, providing a learning platform for knowledge integration, and strengthening of One Health collaboration across Europe. The outbreak preparedness module consisted of lectures and interactive working group sessions, and a simulation exercise where participants were able to learn several technical skills. a challenge. In addition, countries differ globally in their approaches to One Health issues. Sharing best practices and experiences of One Health approaches stood at the centre of this module, providing a learning platform for knowledge integration, and strengthening of One Health collaboration across Europe. The outbreak preparedness module consisted of lectures and interactive working group sessions, and a simulation exercise where participants were able to learn several technical skills. To read more about this event, view the programme and blog post on our website click here. View the full report here. A really well organized, informative and interactive workshop. I felt very well and friendly supervised and motivated for my future work.” Jennie Fischer, BfR, Germany onehealthejp.eu @OneHealthEJP ONE Health EJP I “ A really well organized, info and interactive workshop very well and friendly supe and motivated for my futur Jennie Fischer, BfR, Germ “ I really enjoy the breakout workshop where we have learnt from our experiences and created a presentation that reflects our different contexts.” Gerome Sambou, Ministry of livestock and animal production of Senagal, Senegal “ INTRODUCTION VISION STRUCTURE ACHIEVEMENTS OUTCOMES DISSEMINATION EDUCATION I really enjoy the breakout workshop where we have learnt from our experiences and created a presentation that reflects our different contexts.” “ N E HEA L TH EJP CO M M U N ICA TIO N A N D M EDIA W O RKS HO P Communication of science is one of the most challenging skills to acquire and yet is crucial to realising the impact of research. A comprehensive communication and media workshop was organised in 2020, to develop the communication skills of the young researchers to equip them to drive the research forward and generating maximum impact for their target audiences. Organising institutes: Bulgarian Food Safety Agency, BFSA (Bulgaria) in collaboration with University of Surrey (UK). Theme: How science achievements reach people and contribute to a better life Location: Online Dates: 5th and 6th October 2020 Theme: How science achievements reach people and contribute to a better life Collaborative interactions: The training programme was delivered by communication experts in public health with a specific focus on food safety and risk assessment, the OHEJP Communications Team, and included training by a former TV News broadcaster, presenter and reporter who is an expert on media performance and media interviews. This programme brought together the experiences of our key experts and 35 delegates from 18 countries across the globe on the challenges of communicating scientific research to defined target audiences, and an assessment of the risk communication practices and roles of public and private media in this process in different countries. Delegates attended from across the globe, including Sweden, France, Spain, the UK, Nigeria, India, Greece, USA, Nepal. In addition, our stakeholders, World Organisation for Animal Health (OIE) also participated in this event. This diversity in experience and background significantly enhanced the experience of all those that participated. Some delegates from this workshop also attended other OHEJP training events, which further contributes to building a future consortium to support the sustainability of the OHEJP. A really well organized, info and interactive workshop very well and friendly supe and motivated for my futur Jennie Fischer, BfR, Germ “ Delegates: Delegates represented various career stages, including bachelor’s students, master’s students, PhD students, early career post-doctoral researchers and Doctor of Human (MD) and Veterinary medicine (DVM). They also represented all pillars of One Health: veterinary medicine, human medicine, biological sciences, environmental sciences, pharmaceutical sciences, public health, infectious diseases, microbiology, mathematics, social sciences, economics, and communications. Bringing together people from across disciplines made this workshop truly cross-disciplinary and highlighted the advantages of a One Health approach. N E HEA L TH EJP CO M M U N ICA TIO N A N D M EDIA W O RKS HO P onehealthejp.eu @OneHealthEJP ONE Health EJP I “ A really well organized, info and interactive workshop very well and friendly supe and motivated for my futur Jennie Fischer, BfR, Germ “ I really enjoy the breakout workshop where we have learnt from our experiences and created a presentation that reflects our different contexts.” Gerome Sambou, Ministry of livestock and animal production of Senagal, Senegal “ 79 INTRODUCTION VISION STRUCTURE ACHIEVEMENTS OUTCOMES DISSEMINATION EDUCATION I really enjoy the breakout workshop where we have learnt from our experiences and created a presentation that reflects our different contexts.” “ Programme: The comprehensive workshop programme provided delegates with the opportunity to learn from experts about risk communication, EU communication campaigns, and the coordination of communication activities and strategies related to public health and food safety risks. Delegates also learned techniques and tools to communicate their research more effectively through the creation of a communication strategy, brand awareness and the use of social media. To ensure the workshop was interactive despite being online, delegates participated in group-work presentation exercises, and had the opportunity to attend and participate in exemplary ‘live’ public media mock interviews on their topical research area, teaching them how to answer the questions of journalists and how to deliver their key messages effectively. Gerome Sambou, Ministry of livestock and animal production of Senagal, Senegal To read more about this event, view the programme and blog post on our website click here. View the full report here. onehealthejp.eu @OneHealthEJP ONE Health EJP INTRODUCTION VISION STRUCTURE ACHIEVEMENTS OUTCOMES DISSEMINATION EDUCATION For further details, read the full report here. N E HEA L TH EJP F U N DED 5 S HO RT TERM M IS S IO N S IN 2 0 2 0 The onset of the pandemic affected the success of these missions considerably due to the disruption to travel plans. Two of five STMs took place before travel was disrupted in March 2020. Of the remaining three STMs, although one mission was cancelled, the remaining two missions and training have been postponed until when safe travel resumes. STM 1: Study of the interactions between STEC and human gut microbiota in the ARtificial COLon (ARCOL) model Theme: Skills Development Missions Home Institute: Istituto Superiore di Sanità (ISS), Italy Home Institute: Istituto Superiore di Sanità (ISS), Italy Mission Hosting Institute: Université d’Auvergne Clermont, France Background and Aim of mission: Shiga toxin-producing E. coli (STEC) are zoonotic pathogens, that may cause severe symptoms in humans, including uncomplicated diarrhoea, haemorrhagic colitis, up to the life-threatening haemolytic uraemic syndrome. The different clinical demonstrations appear to be linked to several factors, counting the age of the patient, the host immune status and the interaction of the infecting strains with the intestinal microbiota. Indeed, bacterial species inhabiting the human intestine can interfere with STEC in colonising the gastrointestinal tract. On the other hand, commensal bacteria may influence the severity of disease and amplify the production and release of the Shiga toxin by offering a susceptible bacterial population for the Stx-phage amplification. The aim of this study was to investigate the interactions between STEC and human intestinal microbiota, using the ARtificial COLon model (ARCOL), which simulates the human large intestine functionality. In detail, the aim of the STM was to perform in vitro infection experiments with STEC in presence of a normal human intestinal microbiota using this innovative model to understand the role played by the human intestinal microflora upon STEC infection, and to investigate changes occurring in the gut microbiota composition in presence of infecting STEC strains. For further details, read the full report here. onehealthejp.eu @OneHealthEJP ONE Health EJP onehealthejp.eu @OneHealthEJP ONE Health EJP INTRODUCTION VISION STRUCTURE ACHIEVEMENTS OUTCOMES DISSEMINATION EDUCATION For further details, read the full report here. STM 2: Cross-domain and cross-country collaboration to develop multivariate syndromic surveillance Theme: Skills Development Missions Home Institute: Swedish National Veterinary Institute (SVA), Sweden Mission Hosting Institute: Norwegian Institute of Public Health (NIPH), Norway STM 2: Cross-domain and cross-country collaboration to develop multivariate syndromic surveillance Theme: Skills Development Missions Home Institute: Swedish National Veterinary Institute (SVA), Sweden Mission Hosting Institute: Norwegian Institute of Public Health (NIPH), Norway Duration of mission: 2 weeks p Home Institute: Swedish National Veterinary Institute (SVA), Sweden Mission Hosting Institute: Norwegian Institute of Public Health (NIPH), Norway Duration of mission: 2 weeks Aim and background of mission: The aim of the STM was collaboration and knowledge exchange between Norway and Sweden in the development of One Health systems for multivariate syndromic surveillance of veterinary and public health data combined. Two separate systems are in development in Sweden at the SVA and Norway at the NIPH. The Swedish system is explanatory, aiming to combine several data sources to explain outbreaks and improve the accuracy of detection. Norway, on the other hand, is developing a predictive system, which aims to use some source(s) of data to predict the outcome of others. As a test case, both systems are being evaluated on the surveillance and outbreak detection of Campylobacter in humans, using data from public health, broiler chicken and weather. For further details, read the full report here. onehealthejp.eu @OneHealthEJP ONE Health EJP hejp eu @OneHealthEJP ONE Health EJP IMAGE: PXHERE P REV EN T DETECT RES P O N D l l onehealthejp.eu @OneHealthEJP ONE Health EJP h l h j O l h O l h P REV EN T DETECT RES P O N D l l onehealthejp.eu @OneHealthEJP ONE Health EJP
https://openalex.org/W4394695556	https://resar.elpub.ru/jour/article/download/341/237	Russian	null	Modeling of ontogenetic changes in the shell cross section of the most ancient (Late Permian) representatives of <i>Otoceras</i> (Ammonoidea)	Prirodnye resursy Arktiki i Subarktiki	2,024	cc-by	7,082	Природные ресурсы Арктики и Субарктики / Arctic and Subarctic Natural Resources. 2024;29(1):48–59 Природные ресурсы Арктики и Субарктики / Arctic and Subarctic Natural Resources. 2024;29(1):48–59 Природные ресурсы Арктики и Субарктики / Arctic and Subarctic Natural Resources. 2024;29(1):48–59 УДК 564.533.3:551.736.3 https://doi.org/10.31242/2618-9712-2024-29-1-48-59 Оригинальная статья Моделирование онтогенетических изменений поперечного сечения раковин древнейших (позднепермских) представителей рода Otoceras (Ammonoidea) Р. В. Кутыгин, А. Н. Килясов Институт геологии алмаза и благородных металлов СО РАН, Якутск, Российская Федерация rkutygin@mail.ru Институт геологии алмаза и благородных металлов СО РАН, Якутск, Российская Федерация rkutygin@mail.ru Аннотация Впервые для древнейшего (позднепермского) представителя рода Otoceras сделаны поперечные сечения рако- вин. По этим сечениям реконструированы онтогенетические изменения формы раковины O. concavum Tozer на стадиях от мельчайших до очень крупных размеров. При мельчайших размерах умеренно широкая ракови- на, обладающая умеренно узким умбиликом, интенсивно сужается, становясь узкой. На стадии очень мелких размеров раковина расширяется, вновь превращаясь в умеренно широкую, а умбилик незначительно сужает- ся, по-прежнему оставаясь умеренно узким. При мелких размерах изменение указанных характеристик проис- ходит в том же направлении, но с усилившейся интенсивностью. Важные изменения наблюдаются на стадии средних размеров, когда расширение раковины прекращается, а умбилик становится узким. В конце изученно- го онтогенеза морфологическое развитие моллюска направлено на формирование умеренно узкой формы с очень узким умбиликом. Из ранее установленного у аммоноидей многообразия форм раковины, насчитываю- щего 35 типов, представители вида O. concavum на протяжении изученного онтогенеза приобретают только три: субдискокон, тумарикон и пахикон. Построенная онтогенетическая модель наглядно демонстрирует осо- бенности развития поперечного сечения раковин O. concavum в процессе их роста, подчеркивая вышерассмо- тренные онтогенетические тренды изменения важнейших показателей раковины. Выявленные преобразова- ния формы раковины способствуют диагностике мелкорослых отоцерасов и может послужить основой для последующей реконструкции морфогенетического развития семейства Otoceratidae. лючевые слова: поздняя пермь, чансинский век, цератиты, Otoceras, онтогенез, форма раковин ерхоянье Финансирование. Исследования выполнены за счет Российского научного фонда и Республики Саха (Яку- тия) (грант № 22-24-20112, https://rscf.ru/project/22-24-20112/). Для цитирования: Кутыгин Р.В., Килясов А.Н. Моделирование онтогенетических изменений поперечного сечения раковин древнейших (позднепермских) представителей рода Otoceras (Ammonoidea). Природные ре сурсы Арктики и Субарктики. 2024;29(1):48–59. https://doi.org/10.31242/2618-9712-2024-29-1-48-59 Modeling of ontogenetic changes in the shell cross section of the most ancient (Late Permian) representatives of Otoceras (Ammonoidea) Ruslan V. Kutygin, Afanasy N. Kilyasov Ruslan V. Kutygin, Afanasy N. Kilyasov Diamond and Precious Metal Geology Institute, Siberian Branch of the Russian Academy of Sciences, Yakutsk, Russian Federation rkutygin@mail.ru Original article Modeling of ontogenetic changes in the shell cross section of the most ancient (Late Permian) representatives of Otoceras (Ammonoidea) Abstracti Abstract For the first time, shell cross-sections were made for the most ancient (Late Permian) representatives of the genus Otoceras. From these cross-sections, ontogenetic changes in the shell shape of O. concavum Tozer were reconstructed at size stages ranging from tiny to very large. A moderately wide shell with a moderately narrow umbilicus narrows © Кутыгин Р. В., Килясов А. Н., 2024 48 R. V. Kutygin, A. N. Kilyasov  Modeling of ontogenetic changes in the shell... intensely to its tiny size, becoming a narrow shell. At the stage of very small size, the shell expands, again becoming moderately wide, and the umbilicus narrows slightly, remaining moderately narrow. However, at small sizes, the change in these characteristics occurs in the same direction, but with increased intensity. Moreover, important chang- es are observed at the medium-sized stage, when the expansion of the shell stops and the umbilicus becomes narrow. At the end of the studied ontogenesis, the morphological development of the mollusk was aimed at the formation of a moderately narrow shape with a very narrow umbilicus. Of the variety of shell shapes previously established among ammonoids, numbering 35 types, representatives of the species O. concavum throughout the studied ontogenesis had only three: subdiscocone, tumaricone, and pachycone. Finally, the constructed ontogenetic model clearly demon- strated the features of the development of the cross-sectional shape of O. concavum shells during their growth, empha- sizing ontogenetic trends in changes in the most important parameters of the shell. We conclude that the identified transformations of the shell shape contribute to the diagnosis of small-sized Otoceras, and can serve as the basis for the subsequent reconstruction of the morphogenetic development of the family Otoceratidae. p g p y e Permian, Changhsingian, ceratitids, Otoceras, ontogeny, shell shape, Southern Verkhoyanie q p g p y Keywords: Late Permian, Changhsingian, ceratitids, Otoceras, ontogeny, shell shape, Southern Verkhoyanie Funding This study was supported by the Russian Science Foundation and the Republic of Sakha (Yakutia) (grant q p g p y Keywords: Late Permian, Changhsingian, ceratitids, Otoceras, ontogeny, shell shape, Southern Verkhoyanie Funding. This study was supported by the Russian Science Foundation and the Republic of Sakha (Yakutia) (grant No. 22-24-20112, https://rscf.ru/en/project/22-24-20112/). For citation: Kutygin R.V., Kilyasov A.N. Modeling of ontogenetic changes in the shell cross section of the most ancient (Late Permian) representatives of Otoceras (Ammonoidea). Arctic and Subarctic Natural Resources. 2024;29(1):48–59. Abstracti (In Russ.); https://doi.org/10.31242/2618-9712-2024-29-1-48-59 For citation: Kutygin R.V., Kilyasov A.N. Modeling of ontogenetic changes in the shell cross section of the most ancient (Late Permian) representatives of Otoceras (Ammonoidea). Arctic and Subarctic Natural Resources. 2024;29(1):48–59. (In Russ.); https://doi.org/10.31242/2618-9712-2024-29-1-48-59 Введение и южнее Сеторымского разреза – в верховьях р. Дыба [15, 16]. Все это свидетельствует о более широком распространении нижнего биострато- на отоцерасовых слоев в Верхоянье, чем счита- лось ранее. В прошлом столетии с отоцерасами связыва- лось начало триасового периода, а вмещающие их отложения (Otoceras bed) относились к ба- зальной части индского яруса, триасовой систе- мы и всего мезозоя [1–6; и др.]. В настоящее вре- мя граница между пермью и триасом стала про- водиться выше – внутри отоцерасовых слоев [7], что существенно усложнило обоснование ниж- ней границы триаса по аммоноидеям и потребо- вало дополнительных детальных морфологиче- ских исследований на представительном и хоро- шо привязанном к разрезу материале. р В процессе изучения мелкорослых раковин отоцерасов из разреза Тирях-Кобюме [17] мы столкнулись с серьезной проблемой их диагно- стики, поскольку для наиболее древних видов рода Otoceras какие-либо данные об онтогенезе раковины отсутствовали, а отличительные при- знаки ранее были приведены лишь для очень крупных экземпляров [8, 18]. В связи с необхо- димостью уточнения зонального деления отоце- расовых слоев в Южном Верхоянье и выявления различий между видами рода Otoceras на ран- них и средних оборотах впервые для его древ- нейшего представителя (вида Otoceras concavum Tozer) выполнены детальные онтогенетические и морфометрические исследования, результаты которых приведены ниже. В Арктической Канаде [8] и Верхоянье [9] отоцерасовые слои разделены на две последова- тельные зоны – Otoceras concavum и O. boreale. Ранее считалось, что в южной части Верхоянья зона concavum выделяется лишь в басс. р. Сето- рым [10] Аллах-Юньской структурно-фациаль- ной зоны (СФЗ) [11], тогда как вышезалегаю- щий биостратон в указанной СФЗ распростра- нен шире и к тому же вскрывается в верховьях р. Томпо Томпонской СФЗ. Существовало обосно- ванное мнение, что во всех направлениях р. Се- торым вначале выпадает зона concavum, а затем и зона boreale [12]. Однако недавнее детальное изучение разреза на р. Кобюме [13] позволило не только установить присутствие отоцерасовых слоев в Кобюминской СФЗ, но и выявить в осно- вании некучанской свиты зону concavum, которая перекрывается зоной boreale [14]. Присутствие зоны concavum недавно нами было установлено Arctic and Subarctic Natural Resources. 2024;29(1):48–59 Изученные экземпляры хранятся в Институте геологии алмаза и благородных металлов (ИГАБМ) СО РАН. Рис. 1. Местонахождения аммоноидей Otoceras conca vum Tozer c изученными поперечными сечениями раковин: 1 – руч. Уступный, обн. 17R1, 2 – руч. Правый Суол, обн. PS, 3 – руч. Левый Суол, обн. LS Материал и методика исследований В последние годы мы вели целенаправлен- ные поиски аммоноидей в пограничных пермско- триасовых отложениях басс. р. Сеторым. Собран обширный материал [19, 20], позволяющий про- водить онтогенетическое изучение видов-индек- сов обоих зональных стратонов. На первом этапе исследований монографически обработаны кол- лекции аммоноидей из зоны concavum, относя- щейся к чансинскому ярусу верхней перми [21]. Основные сборы раковин O. concavum сделаны Arctic and Subarctic Natural Resources. 2024;29(1):48–59 49 ыгин, А. Н. Килясов  Моделирование онтогенетических изменений поперечного сечения раковин... Р. В. Кутыгин, А. Н. Килясов  Моделирование онтогенетических изменений поперечного сечения раковин... нетических изменений поперечного сечения раковин... Рис. 2. Основные измерения аммоноидей: D – диаметр раковины; W, H и M – ширина, высота и медиальная высота оборота; U – диаметр умбилика у , р Рис. 1. Местонахождения аммоноидей Otoceras conca vum Tozer c изученными поперечными сечениями раковин: 1 – руч. Уступный, обн. 17R1, 2 – руч. Правый Суол, обн. PS, 3 – руч. Левый Суол, обн. LS Рис. 2. Основные измерения аммоноидей: D – диаметр раковины; W, H и M – ширина, высота и медиальная высота оборота; U – диаметр умбилика Рис. 2. Основные измерения аммоноидей: D – диаметр раковины; W, H и M – ширина, высота и медиальная высота оборота; U – диаметр умбилика Fig. 2. Main measurements of ammonoids: D – shell diam- eter; W, H and M – whorl width, height and medial height; U – umbilical diameter Изученные экземпляры хранятся в Институте геологии алмаза и благородных металлов (ИГАБМ) СО РАН. Результаты и обсуждение Левый Суол, обр. 16LS-3-2p, некучанская свита, в 2 м выше ее подошвы, верхнечан- синский подъярус, зона Otoceras concavum. Длина масштабного отрезка 1 см Рис. 3. Otoceras aff. concavum Tozer, экз. № 234/208: а – с вентральной стороны, б – сбоку; в, г – поперечное сечение раковины и его реконструкция. Руч. Левый Суол, обр. 16LS-3-2p, некучанская свита, в 2 м выше ее подошвы, верхнечан- синский подъярус, зона Otoceras concavum. Длина масштабного отрезка 1 см Fig. 3. Otoceras aff. concavum Tozer, specimen no. 234/208: а – ventral view, б – lateral view, в, г – shell cross-section and its reconstruction. Levyi Suol Creek, sample 16LS-3-2p, Nekuchan Formation, 2 m above its base, Upper Changhsingian, Otoceras concavum Zone. Scale bar 1 cm Fig. 3. Otoceras aff. concavum Tozer, specimen no. 234/208: а – ventral view, б – lateral view, в, г – shell cross-section and its reconstruction. Levyi Suol Creek, sample 16LS-3-2p, Nekuchan Formation, 2 m above its base, Upper Changhsingian, Otoceras concavum Zone. Scale bar 1 cm большое внимание ранее уделялось показателю Д.М. Раупа [27, 28], равному отношению квадра- тов большого и малого радиусов раковины за по- луоборот и называемому whorl expansion rate (скорость возрастания оборотов). Для анализа изменения этого показателя в онтогенезе необхо- димо наличие в замеряемом экземпляре прото- конха. Поскольку последний в поперечных сече- ниях раковин O. concavum не сохранился, мы не имеем возможности использовать этот показатель в морфометрических исследованиях. сохранили лишь два изученных образца (рис. 4), для которых и была выполнена реконструкция поперечных сечений (рис. 3), позволившая выяс- нить особенности онтогенетического развития древнейших представителей рода Otoceras. Наи- более ранние стадии онтогенеза можно наблюдать в экземпляре с руч. Правый Суол (см. рис. 4, г–ж, рис. 5, б), но и у него, к сожалению, прото- конх и первые обороты не сохранились. Второй экземпляр, обнаруженный в разрезе руч. Уступ- ный (см. 4, а–в, рис. 5, а), наращивает онтогенез предыдущего. По этим двум раковинам нам удалось составить общую модель поперечного сечения (рис. 5, в), представляющую преобразо- вания в их форме при размерах от 3,2 до 122 мм (рис. 5, г). сохранили лишь два изученных образца (рис. 4), для которых и была выполнена реконструкция поперечных сечений (рис. 3), позволившая выяс- нить особенности онтогенетического развития древнейших представителей рода Otoceras. Наи- более ранние стадии онтогенеза можно наблюдать в экземпляре с руч. Правый Суол (см. рис. 4, г–ж, рис. 5, б), но и у него, к сожалению, прото- конх и первые обороты не сохранились. Результаты и обсуждение В коллекции аммоноидей из зоны Otoceras concavum басс. р. Сеторым преобладают уме- ренно широкие экземпляры, удовлетворяющие диагностическим признакам вида-индекса зоны. Реже встречаются раковины с узкой уплощенной формой и менее выраженной оттянутостью ум- биликального края. Отличия экземпляров этой немногочисленной группы от O. concavum по- зволили отнести их к отдельному виду, предва- рительно обозначенному как O. aff. concavum Tozer. Однако выполненное поперечное сечение лучшего экземпляра (рис. 3), выявило серьезную деформацию внутренних оборотов и, вероятно, пластичное смятие внешнего оборота, произо- шедшее после жизни моллюска. В связи с этим мы не можем исключать того, что отличительные признаки O. aff. concavum возникли не в резуль- тате морфогенеза отоцерасов, а в процессе лито- генеза вмещающей породы. Fig. 1. Locations of the ammonoid Otoceras concavum Tozer with studied shell cross-section: 1 – Ustupnyi Creek, out- crop 17R1, 2 – Pravyi Suol Creek, PS outcrop, 3 – Levyi Suol Creek, LS outcrop на ручьях Суол и Уступный правобережья р. Се- торым (рис. 1). Для изучения онтогенеза аммоноидей инфор- мативными являются поперечные сечения ра- ковин [22], позволяющие проследить историю индивидуального развития внешней формы и конфигурации оборотов. Выполненные сечения пришлифовывались на стекле с использованием абразивных порошков. Затем они сканировались на планшетном сканере и фотографировались под микроскопом. Измерения (рис. 2) проводились на компьютере в графическом редакторе. Чтобы выяснить закономерности индивиду- ального развития формы раковины аммоноидей, проведено моделирование изменения поперечных сечений в онтогенезе по ранее рассмотренной методике [23, 24]. Несмотря на внешне хорошую сохранность, ряд разрезанных экземпляров O. concavum также оказались деформированными. Форму оборотов Природные ресурсы Арктики и Субарктики. 2024;29(1):48–59 50 R. V. Kutygin, A. N. Kilyasov  Modeling of ontogenetic changes in the shell... R. V. Kutygin, A. N. Kilyasov  Modeling of ontogenetic changes in the shell... Рис. 3. Otoceras aff. concavum Tozer, экз. № 234/208: а – с вентральной стороны, б – сбоку; в, г – поперечное сечение раковины и его реконструкция. Руч. Левый Суол, обр. 16LS-3-2p, некучанская свита, в 2 м выше ее подошвы, верхнечан- синский подъярус, зона Otoceras concavum. Длина масштабного отрезка 1 см Fig. 3. Otoceras aff. concavum Tozer, specimen no. 234/208: а – ventral view, б – lateral view, в, г – shell cross-section and its reconstruction. Levyi Suol Creek, sample 16LS-3-2p, Nekuchan Formation, 2 m above its base, Upper Changhsingian, Otoceras concavum Zone. Scale bar 1 cm Рис. 3. Otoceras aff. concavum Tozer, экз. № 234/208: а – с вентральной стороны, б – сбоку; в, г – поперечное сечение раковины и его реконструкция. Руч. Arctic and Subarctic Natural Resources. 2024;29(1):48–59 Fig. 4. Otoceras concavum Tozer. а–в – specimen no. 234/241-1: a, в – lateral views, б – ventral view; Ustupnyi Creek, sample 17R1-71-1.1p; Nekuchan Formation, 1.1 m above its base. г–ж – specimen no. 234/370-1: г, ж – lateral views, д, е – ventral views; Pravyi Suol Creek, sample 22PS-3-0.5p; Nekuchan Formation, 0.5 m above its base. Upper Permian, Changhsingian Stage, Upper Changhsingian Substage Природные ресурсы Арктики и Субарктики. 2024;29(1):48–59 Рис. 4. Otoceras concavum Tozer. а–в – экз. № 234/241-1: a, в – сбоку, б – с вентральной стороны; руч. Уступный, обр. 17R1-71-1,1p, некучанская свита, в 1,1 м выше ее подошвы. г–ж – экз. № 234/370-1: г, ж – сбоку, д, е – с вентральной стороны; руч. Правый Суол, обр. 22PS-3-0.5p, некучанская свита, в 0,5 м выше ее подошвы. Верхняя пермь, чансинский ярус, верхний подъярус Результаты и обсуждение Второй экземпляр, обнаруженный в разрезе руч. Уступ- ный (см. 4, а–в, рис. 5, а), наращивает онтогенез предыдущего. По этим двум раковинам нам удалось составить общую модель поперечного сечения (рис. 5, в), представляющую преобразо- вания в их форме при размерах от 3,2 до 122 мм (рис. 5, г). Взамен сложно определяемого показателя Д.М. Раупа исследователями предложен одно именный показатель WER, равный (D/(D–M))2 [29, 30]. Как отмечено одним из авторов настоящей статьи, существует прямая зависимость между WER и M/D, а диаграммы изменения этих пока- зателей в онтогенезе почти идентичны [31]. Ис- ходя из этого, для изучения формы навивания оборотов мы ограничимся анализом изменения возрастания спирали (M/D). Выполненные измерения (см. таблицу) по- луоборотов в поперечных сечениях экземпляров (рис. 5, а, б ) и в их модели (рис. 5, в) показывают отчетливые тенденции в изменении основных показателей, что является важным шагом к мор- фогенетической увязке с ранее установленными данными онтогенетического развития более мо- лодого вида (O. boreale) [25, 26]. Преобразования формы медиальной спирали (рис. 6, а) на внутренних оборотах не имеют хо- рошо выраженного однонаправленного тренда – показатель M/D колеблется в интервале от 0,31 до 0,33. На средних оборотах у O. concavum про- Для выяснения геометрии раковин аммонои- дей и определения экологических морфотипов 51 Arctic and Subarctic Natural Resources. 2024;29(1):48–59 Р. В. Кутыгин, А. Н. Килясов  Моделирование онтогенетических изменений поперечного сечения раковин... . Otoceras concavum Tozer. а–в – экз. № 234/241-1: a, в – сбоку, б – с вентральной стороны; руч. Усту ,1p, некучанская свита, в 1,1 м выше ее подошвы. г–ж – экз. № 234/370-1: г, ж – сбоку, д, е – с ве руч. Правый Суол, обр. 22PS-3-0.5p, некучанская свита, в 0,5 м выше ее подошвы. Верхняя пермь, ч ний подъярус Otoceras concavum Tozer. а–в – specimen no. 234/241-1: a, в – lateral views, б – ventral view; Ustupnyi Cr .1p; Nekuchan Formation, 1.1 m above its base. г–ж – specimen no. 234/370-1: г, ж – lateral views, д, i S l C k l 22PS 3 0 5 N k h F ti 0 5 b it b U P i Ch h i Рис. 4. Otoceras concavum Tozer. а–в – экз. № 234/241-1: a, в – сбоку, б – с вентральной стороны; руч. Уступный, обр. 17R1-71-1,1p, некучанская свита, в 1,1 м выше ее подошвы. г–ж – экз. № 234/370-1: г, ж – сбоку, д, е – с вентральной стороны; руч. Правый Суол, обр. Результаты и обсуждение 234/241-1 at D = 122 mm, Ustupnyi Creek, sample 17R1- 71-1.1p; б – specimen no. 234/370-1 at D = 41 mm, Pravyi Suol Creek, sample 22PS-3-0.5p; в, г – a model at D = 122 mm and the sequence of its half-whorls Shell diameter in millimeters is shown on the cross-section Рис. 5. Поперечные сечения раковин Otoceras concavum: а – экз. № 234/241-1 при D = 122 мм; б – экз. № 234/370-1 при D = 41 мм; в, г – модель при D = 122 мм (в) и последовательность ее полуоборотов (г). На сечениях указаны диаметры раковин в мм Рис. 5. Поперечные сечения раковин Otoceras concavum: а – экз. № 234/241-1 при D = 122 мм; б – экз. № 234/370-1 при D = 41 мм; в, г – модель при D = 122 мм (в) и последовательность ее полуоборотов (г). На сечениях указаны диаметры раковин в мм Fig. 5. Shell cross-sections of Otoceras concavum: а – specimen no. 234/241-1 at D = 122 mm, Ustupnyi Creek, sample 17R1- 71-1.1p; б – specimen no. 234/370-1 at D = 41 mm, Pravyi Suol Creek, sample 22PS-3-0.5p; в, г – a model at D = 122 mm and the sequence of its half-whorls. Shell diameter in millimeters is shown on the cross-section Fig. 5. Shell cross-sections of Otoceras concavum: а – specimen no. 234/241-1 at D = 122 mm, Ustupnyi Creek, sample 17R1- 71-1.1p; б – specimen no. 234/370-1 at D = 41 mm, Pravyi Suol Creek, sample 22PS-3-0.5p; в, г – a model at D = 122 mm and the sequence of its half-whorls. Shell diameter in millimeters is shown on the cross-section 20–30 мм. На этом промежутке онтогенеза, обо- значаемом как фаза максимального расширения, W/D достигает 0,64–0,67. В дальнейшем, до кон- ца существования особи, вероятно, происходит не- прерывное сужение раковины (уменьшение W/D). Изменения показателей H/D и U/D у нормально навивающихся аммоноидей обычно взаимозави- симы и противоположны по направлению. Для представителей O. concavum характерны увели- чение относительной высоты оборота и умень- шение размера умбилика (рис. 6, в, г). Отно- шение ширины оборота к его высоте (рис. 6, д) практически повторяет направление и интенсив- ность изменений вышерассмотренного показа- 20–30 мм. На этом промежутке онтогенеза, обо- значаемом как фаза максимального расширения, W/D достигает 0,64–0,67. В дальнейшем, до кон- ца существования особи, вероятно, происходит не- прерывное сужение раковины (уменьшение W/D). Arctic and Subarctic Natural Resources. 2024;29(1):48–59 Результаты и обсуждение 22PS-3-0.5p, некучанская свита, в 0,5 м выше ее подошвы. Верхняя пермь, чансинский ярус, верхний подъярус Рис. 4. Otoceras concavum Tozer. а–в – экз. № 234/241-1: a, в – сбоку, б – с вентральной стороны; руч. Уступный, обр. 17R1-71-1,1p, некучанская свита, в 1,1 м выше ее подошвы. г–ж – экз. № 234/370-1: г, ж – сбоку, д, е – с вентральной стороны; руч. Правый Суол, обр. 22PS-3-0.5p, некучанская свита, в 0,5 м выше ее подошвы. Верхняя пермь, чансинский ярус, верхний подъярус Fig. 4. Otoceras concavum Tozer. а–в – specimen no. 234/241-1: a, в – lateral views, б – ventral view; Ustupnyi Creek, sample 17R1-71-1.1p; Nekuchan Formation, 1.1 m above its base. г–ж – specimen no. 234/370-1: г, ж – lateral views, д, е – ventral views; Pravyi Suol Creek, sample 22PS-3-0.5p; Nekuchan Formation, 0.5 m above its base. Upper Permian, Changhsingian Stage, Upper Changhsingian Substage Fig. 4. Otoceras concavum Tozer. а–в – specimen no. 234/241-1: a, в – lateral views, б – ventral view; Ustupnyi Creek, sample 17R1-71-1.1p; Nekuchan Formation, 1.1 m above its base. г–ж – specimen no. 234/370-1: г, ж – lateral views, д, е – ventral views; Pravyi Suol Creek, sample 22PS-3-0.5p; Nekuchan Formation, 0.5 m above its base. Upper Permian, Changhsingian Stage, Upper Changhsingian Substage Природные ресурсы Арктики и Субарктики. 2024;29(1):48–59 52 R. V. Kutygin, A. N. Kilyasov  Modeling of ontogenetic changes in the shell... Рис. 5. Поперечные сечения раковин Otoceras concavum: а – экз. № 234/241-1 при D = 122 мм; б – экз. № 234/370-1 при D = 41 мм; в, г – модель при D = 122 мм (в) и последовательность ее полуоборотов (г). На сечениях указаны диаметры раковин в мм Fig. 5. Shell cross-sections of Otoceras concavum: а – specimen no. 234/241-1 at D = 122 mm, Ustupnyi Creek, sample 17R1- 71-1.1p; б – specimen no. 234/370-1 at D = 41 mm, Pravyi Suol Creek, sample 22PS-3-0.5p; в, г – a model at D = 122 mm and the sequence of its half-whorls. Shell diameter in millimeters is shown on the cross-section Рис. 5. Поперечные сечения раковин Otoceras concavum: а – экз. № 234/241-1 при D = 122 мм; б – экз. № 234/370-1 при D = 41 мм; в, г – модель при D = 122 мм (в) и последовательность ее полуоборотов (г). На сечениях указаны диаметры раковин в мм Fig. 5. Shell cross-sections of Otoceras concavum: а – specimen no. Результаты и обсуждение Изменения показателей H/D и U/D у нормально навивающихся аммоноидей обычно взаимозави- симы и противоположны по направлению. Для представителей O. concavum характерны увели- чение относительной высоты оборота и умень- шение размера умбилика (рис. 6, в, г). Отно- шение ширины оборота к его высоте (рис. 6, д) практически повторяет направление и интенсив- ность изменений вышерассмотренного показа- исходит интенсивное растяжение спирали, при этом максимальные значения M/D превышают 0,36. На внешних оборотах спираль интенсивно сжимается, приобретая форму навивания, близ- кую к мелкоразмерным раковинам. В завершаю- щей фазе онтогенеза показатель M/D становится меньше 0,33. исходит интенсивное растяжение спирали, при этом максимальные значения M/D превышают 0,36. На внешних оборотах спираль интенсивно сжимается, приобретая форму навивания, близ- кую к мелкоразмерным раковинам. В завершаю- щей фазе онтогенеза показатель M/D становится меньше 0,33. Как видно из диаграммы (рис. 6, б ), при очень мелких размерах (D 5,7 мм) раковина достигает наиболее узкой формы (W/D = 0,44), что явля- ется фазой максимального сужения раковины. В дальнейшем происходит интенсивное расши- рение раковины (увеличение показателя W/D). Наиболее широкими раковины становятся при D 53 Arctic and Subarctic Natural Resources. 2024;29(1):48–59 Р. В. Кутыгин, А. Н. Килясов  Моделирование онтогенетических изменений поперечного сечения раковин... . . утыгин, . . илясов оделирование онтогенетичес их изменений поперечного сечения ра овин... Размеры (мм) раковин O. concavum Tozer Dimensions (in mm) of O. concavum Tozer Экз. Результаты и обсуждение No П/о D W H U M W/D H/D U/D W/H M/D 234/241-1 1 15,2 9,10 7,10 3,40 0,599 0,467 0,224 1,282 * 20,0 12,93 9,69 4,05 0,646 0,485 0,202 1,333 2 22,6 15,00 11,10 4,40 7,4 0,664 0,491 0,195 1,351 0,327 3 33,4 21,70 16,70 5,60 10,8 0,650 0,500 0,168 1,299 0,323 * 50,0 31,69 26,52 7,51 0,634 0,530 0,150 1,195 4 52,5 33,20 28,00 7,80 19,1 0,632 0,533 0,149 1,186 0,364 5 82 48,50 43,50 10,50 29,5 0,591 0,530 0,128 1,115 0,360 * 100 56,74 52,95 12,08 0,567 0,530 0,121 1,071 6 122 66,80 64,50 14,00 40,0 0,548 0,529 0,115 1,036 0,328 234/370-1 1 2,6 1,30 1,10 0,500 0,423 1,182 2 3,8 1,90 1,60 1,10 1,2 0,500 0,421 0,289 1,188 0,316 * 5,0 2,28 2,14 1,45 0,456 0,427 0,289 1,067 3 5,7 2,50 2,45 1,65 1,9 0,439 0,430 0,289 1,020 0,333 4 8,4 4,30 3,75 2,20 2,7 0,512 0,446 0,262 1,147 0,321 * 10,0 5,18 4,49 2,52 0,518 0,449 0,252 1,155 5 12,2 6,40 5,50 2,95 3,8 0,525 0,451 0,242 1,164 0,311 6 18,1 11,00 8,60 4,00 5,9 0,608 0,475 0,221 1,279 0,326 * 20,0 12,33 9,62 4,20 0,616 0,481 0,210 1,282 7 26,7 17,00 13,20 4,90 8,6 0,637 0,494 0,184 1,288 0,322 8 41,0 25,00 21,30 6,50 14,3 0,610 0,520 0,159 1,174 0,349 Модель 1 2,2 1,15 0,95 0,523 0,432 1,211 2 3,2 1,60 1,35 0,90 1,0 0,500 0,422 0,281 1,185 0,313 3 4,7 2,20 2,02 1,33 1,5 0,468 0,430 0,283 1,089 0,319 5,0 2,35 2,16 1,40 0,470 0,432 0,281 1,089 4 7,0 3,35 3,08 1,90 2,3 0,479 0,440 0,271 1,088 0,329 * 10,0 5,21 4,51 2,51 0,521 0,451 0,251 1,157 5 10,3 5,40 4,65 2,57 3,3 0,524 0,451 0,250 1,161 0,320 6 15,1 9,00 7,05 3,40 4,8 0,596 0,467 0,225 1,277 0,318 * 20,0 12,74 9,60 4,11 0,637 0,480 0,206 1,327 7 22,3 14,50 10,80 4,45 7,2 0,650 0,484 0,200 1,343 0,323 8 33,4 21,70 16,80 5,80 11,1 0,650 0,503 0,174 1,292 0,332 * 50,0 31,69 26,36 7,63 0,634 0,527 0,153 1,202 9 52,5 33,20 27,80 7,90 19,1 0,632 0,530 0,150 1,194 0,364 10 82 48,50 43,50 10,70 29,5 0,591 0,530 0,130 1,115 0,360 * 100 56,74 52,95 12,19 0,567 0,530 0,122 1,071 11 122 66,80 64,50 14,00 40,0 0,548 0,529 0,115 1,036 0,328 Примечание. П/о – полуоборот, * линейная интерполяция показателей. теля W/D. Примечание. П/о – полуоборот, * линейная интерполяция показателей. Note. П/o – half-turn, * linear interpolation of indicators. Природные ресурсы Арктики и Субарктики. 2024;29(1):48–59 Результаты и обсуждение В фазу максимального сужения ра- ковины (D 5,7 мм) значения высоты и ширины оборота становятся близкими, тогда как в осталь- ные периоды индивидуального развития, исклю- чая внешние обороты, ширина существенно пре- восходит высоту, причем в фазу максимального Природные ресурсы Арктики и Субарктики. 2024;29(1):48–59 54 R. V. Kutygin, A. N. Kilyasov  Modeling of ontogenetic changes in the shell... R. V. Kutygin, A. N. Kilyasov  Modeling of ontogenetic changes in the shell... g f g g Рис. 6. Диаграммы изменения M/D (а), W/D (б ), H/D (в), U/D (г) и W/H (д) в онтогенезе Otoceras concavum. Tiny shells – мельчайшие раковины, Very small shell – очень мелкие ракови- ны, Small shells – мелкие раковины, Medium-sized shells – сред- неразмерные раковины, Large and very large shells – крупные и очень крупные раковины, Spiral expansion – растяжение спира- ли, Spiral compression – сжатие спирали, Shell narrowing – су- жение раковины, Shell expansion – расширение раковины, Slow whorl-height expansion – слабое расширение сечения оборота, Umbilicus narrowing – сужение умбилика, Whorl narrowing – сужение оборота, Whorl expansion – расширение оборота Fig. 6. Diagrams of changes of M/D (а), W/D (б ), H/D (в), U/D (г) and W/H (д) in the ontogeny of Otoceras concavum расширения раковины (D 20–30 мм) W/H может превышать 1,3. На внешних оборотах значения W и H вновь уравниваются. При изучении морфологии нормально нави- вающихся аммоноидей ранее было показано, что взаимоотношение размера умбилика (U/D) и относительной ширины оборота (W/D) во многом отражает внешнюю форму их раковины [23, 32]. Но куда важнее выяснить не само взаимоотно- шение отношение U/D и W/D, а их изменение в процессе роста раковины, которое у отдельных видов являются неповторимым [23], что делает этот признак крайне важным для разграничения и диагностики таксонов. Для рассмотрения этапности изменения фор- мы раковины онтогенез разбит на ряд размер- ных стадий [23, 24] (рис. 7). Учитывая субло- гарифмический характер навивания раковины (медиальной спирали), выделенные размерные ста- дии являются соразмерными по длине оборотов. р р р На фоне выдержанных значений U/D мель- чайшая раковина (рис. 7, а) в онтогенезе сужает- ся, превращаясь из умеренно широкой в узкую. При очень мелких размерах (рис. 7, б ) раковина расширяется, вновь становясь умеренно широ- кой. На этой стадии незначительно сужается ум- билик, оставаясь по-прежнему умеренно узким. Изменение обсуждаемых показателей (W/D и U/D) на стадии мелких размеров (рис. 7, в) продол- жается в том же направлении, но его интенсив- ность усиливается. Arctic and Subarctic Natural Resources. 2024;29(1):48–59 Р. В. Кутыгин, А. Н. Килясов  Моделирование онтогенетических изменений поперечного сечения раковин... Р. В. Кутыгин, А. Н. Килясов  Моделирование онтогенетических изменений поперечного сечения раковин... Р. В. Кутыгин, А. Н. Килясов  Моделирование онтогенетических изменений поперечного сечения раковин... Рис. 7. Изменение формы раковин Otoceras concavum на отдельных размерных стадиях онтогенеза (а–е). На сечения зле маркеров показаны диаметры раковин в мм. Ост. пояснения см. рис. 6. Fig 7 Ontogenetic changes in the shell shape of Ot at different si e stages ( ) Shell diameter in millimeter Рис. 7. Изменение формы раковин Otoceras concavum на отдельных размерных стадиях онтогенеза (а–е). На сечениях и возле маркеров показаны диаметры раковин в мм. Ост. пояснения см. рис. 6. Fig. 7. Ontogenetic changes in the shell shape of Otoceras concavum at different size stages (а–е). Shell diameter in millimeters is shown on the cross-section and near the markers. For other explanations, see Fig. 6 Fig. 7. Ontogenetic changes in the shell shape of Otoceras concavum at different size stages (а–е). Shell diameter in millimeters s shown on the cross-section and near the markers. For other explanations, see Fig. 6 общей формы сечения оборота и его относитель- ной ширины носит цикличный характер. На ста- дии мельчайших размеров происходит сужение раковины, которое при очень мелких и мелких размерах сменяется интенсивным расширением. После периода стабилизации W/H и W/D, наблю- даемой при средних размерах, до окончания жизни моллюска происходит уменьшение этих показа- телей – сужение раковины и сечения оборота. Отсутствие подобных детальных исследований изменения формы раковины в онтогенезе дру- гих представителей рода Otoceras существенно cavum приобретают только три, в следующей последовательности: тумарикон → субдиско- кон → тумарикон → пахикон. Результаты и обсуждение Важные преобразования в онтогенезе наблюдаются при средних размерах раковины (рис. 7, г), когда расширение (увеличе- ние W/D) прекращается, а умбилик становится узким. На завершающих стадиях изученного он- тогенеза (рис. 7, д, е), при медленном сокраще- нии размера умбилика, раковина сужается. Здесь мы наблюдаем онтогенетический тренд, направ- ленный на формирование умеренно узкой формы с очень узким умбиликом (субоппеликон по клас- сификации [23]). Рис. 6. Диаграммы изменения M/D (а), W/D (б ), H/D (в), U/D (г) и W/H (д) в онтогенезе Otoceras concavum. Tiny shells – мельчайшие раковины, Very small shell – очень мелкие ракови- ны, Small shells – мелкие раковины, Medium-sized shells – сред- неразмерные раковины, Large and very large shells – крупные и очень крупные раковины, Spiral expansion – растяжение спира- ли, Spiral compression – сжатие спирали, Shell narrowing – су- жение раковины, Shell expansion – расширение раковины, Slow whorl-height expansion – слабое расширение сечения оборота, Umbilicus narrowing – сужение умбилика, Whorl narrowing – сужение оборота, Whorl expansion – расширение оборота Построенная модель (рис. 8) наглядно демон- стрирует особенности развития формы попереч- ного сечения раковин Otoceras concavum в про- цессе их роста, подчеркивая рассмотренные выше онтогенетические тренды изменения размера ум- билика и относительной ширины раковины. При этом из установленного ранее много образия U/D–W/D форм, насчитывающих 35 ти- пов [23], изученные представители вида O. con Fig. 6. Diagrams of changes of M/D (а), W/D (б ), H/D (в), U/D (г) and W/H (д) in the ontogeny of Otoceras concavum Fig. 6. Diagrams of changes of M/D (а), W/D (б ), H/D (в), U/D (г) and W/H (д) in the ontogeny of Otoceras concavum 55 Arctic and Subarctic Natural Resources. 2024;29(1):48–59 Заключение Triassic stratigraphy and faunas, Queen Elizabeth Island Arctic Archpilago. Memoirs of the Geo logical Survey of Canada. 1961;(316):1–116. усложняет решение сложной проблемы проис- хождения и морфогенетического развития семей- ства Otoceratidae. усложняет решение сложной проблемы проис- хождения и морфогенетического развития семей- ства Otoceratidae. g y f ( ) 5. Шевырев А.А. Аммоноидеи и хронострати графия триаса. М.: Наука; 1990. 178 с. Ermakova S.P. Lower boundary of the Triassic. Ote chestvennaya geologiya. 1999;(4):33–34. (In Russ.) 5. Шевырев А.А. Аммоноидеи и хронострати графия триаса. М.: Наука; 1990. 178 с. Заключение По изученным поперечным сечениям построе- ны модели онтогенетического изменения формы раковин вида Otoceras concavum Tozer на стадиях от мельчайших до очень крупных размеров. Вы- явлено однонаправленное уменьшение размера умбилика и увеличение относительной высоты оборота в процессе роста раковины. Изменение Природные ресурсы Арктики и Субарктики. 2024;29(1):48–59 56 R. V. Kutygin, A. N. Kilyasov  Modeling of ontogenetic changes in the shell... Рис. 8. Модель развития формы раковин Otoceras concavum в онтогенезе На сечениях и маркерах показаны ди R. V. Kutygin, A. N. Kilyasov  Modeling of ontogenetic changes in the shell... R. V. Kutygin, A. N. Kilyasov  Modeling of ontogenetic changes in the shell... R. V. Kutygin, A. N. Kilyasov  Modeling of ontogenetic changes in the shell... Рис. 8. Модель развития формы раковин Otoceras concavum в онтогенезе. На сечениях и маркерах показаны диаметры раковин в мм. Narrow shells – узкие раковины, Moderately wide shells – умеренно широкие раковины, Wide shells – широкие раковины, Medium-sized umbilicus – умбилик среднего размера, Moderately narrow umbilicus – умеренно узкий умбилик, Narrow umbilicus – узкий умбилик, Very narrow umbilicus – очень узкий умбилик, Subdiscocone – субдискокон, Tumari- cone – тумарикон, Pachycone – пахикон, Ontogeny – онтогенез Рис. 8. Model depicting the ontogeny of the shell shape of Otoceras concavum. Shell diameter in mm is shown on the cross- Рис. 8. Модель развития формы раковин Otoceras concavum в онтогенезе. На сечениях и маркерах показаны диаметры раковин в мм. Narrow shells – узкие раковины, Moderately wide shells – умеренно широкие раковины, Wide shells – широкие раковины, Medium-sized umbilicus – умбилик среднего размера, Moderately narrow umbilicus – умеренно узкий умбилик, Narrow umbilicus – узкий умбилик, Very narrow umbilicus – очень узкий умбилик, Subdiscocone – субдискокон, Tumari- cone – тумарикон, Pachycone – пахикон, Ontogeny – онтогенез Рис. 8. Модель развития формы раковин Otoceras concavum в онтогенезе. На сечениях и маркерах показаны диаметры раковин в мм. Narrow shells – узкие раковины, Moderately wide shells – умеренно широкие раковины, Wide shells – широкие раковины, Medium-sized umbilicus – умбилик среднего размера, Moderately narrow umbilicus – умеренно узкий умбилик, Narrow umbilicus – узкий умбилик, Very narrow umbilicus – очень узкий умбилик, Subdiscocone – субдискокон, Tumari- cone – тумарикон, Pachycone – пахикон, Ontogeny – онтогенез Рис. 8. Model depicting the ontogeny of the shell shape of Otoceras concavum. 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Paleontologicheskij zhurnal. 1971;(3):50–59. (In Russ.) 26. Zakharov Yu.D. Ammonoid succession of Seto- rym River (Verkhoyansk Area) and problem of Permian- Triassic Boundary in Boreal Realm. Journal of China University of Geosciences. 2002;13(2):107–123. 16. Kutygin R.V., Kilyasov A.N., Biakov A.S. The first record of the goniatite genus Paramexicoceras in the Changhsingian deposits of the Upper Permian in North- eastern Asia. Doklady Earth Sciences. 2023;509(1):144– 147. https://doi.org/10.1134/S1028334X22601936 27. Raup D.M. Geometric analysis of shell coiling: Coiling in ammonoids. Journal of Paleontology. 1967; 41(1):43–65. 28. Barskov I.S., Boiko M.S., Konovalova V.A., et al. Cephalopods in the Marine Ecosystems of the Paleozoic. Paleontological Journal. 2008;42(11):1167–1284. https:// doi.org/10.1134/S0031030108110014 17. Кутыгин Р.В., Будников И.В., Бяков А.С. и др. Первые находки цератитов рода Otoceras в Кобюмин- ской зоне Южного Верхоянья, Северо-Восток России. Ученые записки Казанского университета. Серия 58 Природные ресурсы Арктики и Субарктики. 2024;29(1):48–59 About the authors KUTYGIN, Ruslan Vladimirovich, Cand. Sci. (Geol. and Mineral.), Head of Laboratory, https://orcid. org/0000-0003-4115-5976, ResearcherID: J-3318-2018, Scopus Author ID: 9277169500, SPIN: 2547-9417, e-mail: rkutygin@mail.ru KILYASOV, Afanasy Nikolaevich, Junior Researcher, https://orcid.org/0000-0001-7469-2132, ResearcherID: AAH-6016-2021, SPIN: 7741-4120, e-mail: kilyasov1993@mail.ru Поступила в редакцию / Submitted 27.11.2023 Поступила после рецензирования / Revised 21.12.2023 Принята к публикации / Accepted 17.01.2024 KILYASOV, Afanasy Nikolaevich, Junior Researcher, https://orcid.org/0000-0001-7469-2132, ResearcherID: AAH-6016-2021, SPIN: 7741-4120, e-mail: kilyasov1993@mail.ru Поступила в редакцию / Submitted 27.11.2023 Поступила после рецензирования / Revised 21.12.2023 Принята к публикации / Accepted 17.01.2024 59 59 Arctic and Subarctic Natural Resources. 2024;29(1):48–59 59
https://openalex.org/W1562933393	http://telkomnika.uad.ac.id/index.php/TELKOMNIKA/article/download/1336/925	Indonesian	null	ANALISIS SPEKTRUM FREKUENSI NON-LINEAR SINYAL TUTUR DENGAN ALIH RAGAM FOURIER CEPAT	Telkomnika	2,007	cc-by-sa	4,629	ISSN: 1693-6930 ISSN: 1693-6930 51  ANALISIS SPEKTRUM FREKUENSI NON-LINEAR SINYAL TUTUR DENGAN ALIH RAGAM FOURIER CEPAT Salman Abd. Cadum1, Prayoto2, Adhi Susanto3, Kirbani Sri Brotopuspito4 1,3Jurusan Teknik Elektro Fakultas Teknik Universitas Gadjah Mada e-mail: salman_abd_2002@yahoo.com 2Jurusan Fisika Fakultas MIPA Universitas Gadjah Mada 4Jurusan Geofisika Fakultas MIPA Universitas Gadjah Mada Abstrak Pada penelitian ini akan diteliti analisis spektrum frekuensi non-linear sinyal tutur dengan menggunakan alih ragam Fourier cepat (Fast Fourier Transform, FFT). Hasil penelitian menunjukkan bahwa: suatu skala logaritmis akan memperluas daerah frekuensi yang rendah dari spektrum dan mempersempit daerah frekuensi yang tinggi pada tampilan, dibutuhkan suatu FFT yang jauh lebih besar guna mendapatkan resolusi frekuensi yang sangat tinggi pada frekuensi yang rendah, penerapan fungsi berbagai window terhadap data dapat membantu mengurangi efek kebocoran yang terjadi pada spektrum frekuensi, metode ini berjalan lebih cepat jika jumlah point data merupakan kelipatan dua (128, 256, 1024, 2048, atau 4096, dan seterusnya) dan memilih suatu resolusi frekuensi yang tepat serta resolusi waktu yang sesuai menjadi suatu kesesuaian antara kebutuhan untuk mengamati detail frekuensi yang baik dalam spektrum dengan kebutuhan untuk mengamati variasi waktu yang cepat dalam spektrum. Kata kunci : spektrum frekuensi non-linear, sinyal tutur, FFT 1. PENDAHULUAN Spektrum menggunakan suatu transform Fourier cepat (Fast Fourier Transform, FFT) matematis untuk melakukan analisis frekuensi. FFT biasanya dinyatakan dengan jumlah point data masukan yang digunakan dalam setiap perhitungan yang selalu berupa kelipatan dua (128, 256, 512, 1024, 2048, atau 4096). Resolusi frekuensi spektrum selalu merupakan nilai cuplikan digital sinyal audio/tutur yang dibagi dengan jumlah point data FFT. Semakin besar jumlah point data FFT, semakin baik resolusi frekuensi spektrum. Frekuensi maksimum yang dihitung oleh FFT dan batas frekuensi tertinggi spektrum adalah setengah nilai cuplikan digital [1]. [ ] Dalam setiap proses analisis spektrum, resolusi waktu dan resolusi frekuensi memiliki hubungan terbalik, resolusi frekuensi yang sangat bagus berkaitan dengan resolusi waktu yang buruk, sebaliknya resolusi waktu yang sangat bagus berkaitan dengan resolusi frekuensi yang buruk. Hubungan antara resolusi waktu (Time Resolution, TR) dalam detik dan resolusi frekuensi (Frequency Resolution, FR) dalam Hz adalah: (1) (1) TR = 1/FR TR = 1/FR TR = 1/FR Akibat logis prinsip ketidakpastian ini adalah bahwa memilih resolusi frekuensi yang sangat bagus akan menutupi detail waktu dalam spektrum sinyal audio/tutur. Memilih suatu resolusi frekuensi yang tepat dan resolusi waktu yang sesuai menjadi suatu kesesuaian antara kebutuhan untuk mengamati detail frekuensi yang baik dalam spektrum dengan kebutuhan untuk mengamati variasi waktu yang cepat dalam spektrum [2]. Teori Fourier menyatakan bahwa bentuk gelombang (waveform) periodis dapat dipecah ke dalam serangkaian sinusoid yang memiliki frekuensi, amplitude, dan fase yang berbeda. Dalam sintesis wavetable, amplitude parsialnya tetap, dan sementara hal ini umumnya cukup untuk memberikan kesan (impression) tipe warna nada (timbre) tertentu, keterbatasannya mencegahnya menerima hasil yang lebih tahan dalam kelangsungan hidup (life-like), hal ini Analisis Spektrum Frekuensi Non-Linear Sinyal Tutur ……(Salman Abd. Cadum)  52 ISSN: 1693-6930 terutama karena dua alasan: a) di alam, frekuensi parsial jarang merupakan perkalian bilangan bulat yang tepat (exact), dan b) amplitude parsial individu hampir selalu berbeda-beda relatif terhadap satu sama lain selama suatu nada (tone). Kedua efek ini mustahil dicapai dengan sintesis wavetable, dan sintesis aditif, oleh karena itu, dengan hubungannya yang jelas dengan analisis Fourier, maka Fourier menawarkan solusi masalah ini. terutama karena dua alasan: a) di alam, frekuensi parsial jarang merupakan perkalian bilangan bulat yang tepat (exact), dan b) amplitude parsial individu hampir selalu berbeda-beda relatif terhadap satu sama lain selama suatu nada (tone). Kedua efek ini mustahil dicapai dengan sintesis wavetable, dan sintesis aditif, oleh karena itu, dengan hubungannya yang jelas dengan analisis Fourier, maka Fourier menawarkan solusi masalah ini. Penganalisa FFT mentransformasikan data dari domain waktu ke domain frekuen dengan menghitung FFT. Hal ini didasarkan pada integral Fourier persamaan 2 berikut. dt e)t( x y ft 2j        (2) dt e)t( x y ft 2j        (2) Namun ini merupakan suatu bentuk yang dapat dihitung secara numeris. Integral ini mensyaratkan bahwa suatu sinyal kontinyu diintegralkan selama waktu yang tak terhingga, tentu saja, diinginkan hasil dalam waktu yang terhingga. Dan karena komputer berhubungan dengan angka, maka diperlukan digitize (men-digital-kan) bentuk gelombang, yang dapat membuat waktu bersifat diskrit. Kedua perubahan terhadap sinyal ini mengakibatkan kesalahan dalam spektrum frekuensi yang dihitung. Cuplikan sinyal pada waktu diskrit dapat menyebabkan aliasing (yang dapat terlihat sebagai sinyal bayangan (phantom) pada tampilan). TR = 1/FR Pengubahan batas integral panjang tak terhingga menjadi panjang terhingga dapat menyebabkan kesalahan yang disebut kebocoran (yang muncul sebagai energi dari titik tertentu dalam spektrum terbaur (smeared) naik dan turun melintasi spektrum). Karena ketidakmungkinan mengukur suatu sinyal untuk waktu yang tak terhingga, maka penganalisa mengubah batas integral ke panjang waktu yang dibutuhkan untuk mengumpulkan blok cuplikan. Blok cuplikan disebut time record. FFT mensyaratkan bahwa sinyal dalam time record diulang terus-menerus sepanjang waktu. Jika time record yang diulang secara aktual tampak seperti sinyal asli, maka tidak akan terjadi kebocoran. Jika, pada sisi lain, tak terlihat seperti sinyal aslinya, maka terjadi kebocoran. Penerapan fungsi window terhadap data yang ada akan dapat membantu mengurangi efek kebocoran dalam domain frekeunsi [3]. Namun ini merupakan suatu bentuk yang dapat dihitung secara numeris. Integral ini mensyaratkan bahwa suatu sinyal kontinyu diintegralkan selama waktu yang tak terhingga, tentu saja, diinginkan hasil dalam waktu yang terhingga. Dan karena komputer berhubungan dengan angka, maka diperlukan digitize (men-digital-kan) bentuk gelombang, yang dapat membuat waktu bersifat diskrit. Kedua perubahan terhadap sinyal ini mengakibatkan kesalahan dalam spektrum frekuensi yang dihitung. Cuplikan sinyal pada waktu diskrit dapat menyebabkan aliasing (yang dapat terlihat sebagai sinyal bayangan (phantom) pada tampilan). Pengubahan batas integral panjang tak terhingga menjadi panjang terhingga dapat menyebabkan kesalahan yang disebut kebocoran (yang muncul sebagai energi dari titik tertentu dalam spektrum terbaur (smeared) naik dan turun melintasi spektrum). Karena ketidakmungkinan mengukur suatu sinyal untuk waktu yang tak terhingga, maka penganalisa mengubah batas integral ke panjang waktu yang dibutuhkan untuk mengumpulkan blok cuplikan. Blok cuplikan disebut time record. FFT mensyaratkan bahwa sinyal dalam time record diulang terus-menerus sepanjang waktu. Jika time record yang diulang secara aktual tampak seperti sinyal asli, maka tidak akan terjadi kebocoran. Jika, pada sisi lain, tak terlihat seperti sinyal aslinya, maka terjadi kebocoran. Penerapan fungsi window terhadap data yang ada akan dapat membantu mengurangi efek kebocoran dalam domain frekeunsi [3]. Guna menghitung spektrum y dari sinyal tutur X dapat digunakan FFT dengan menggunakan persamaan berikut: N ikm 2 1 N 0 k k m e. X y     (3) dengan m = 0 … N-1, dan k = 0 … N-1. N ikm 2 1 N 0 k k m e. X y     (3) dengan m = 0 … N-1, dan k = 0 … N-1. (3) dengan m = 0 … N-1, dan k = 0 … N-1. TR = 1/FR dengan m = 0 … N-1, dan k = 0 … N-1. Demikian juga untuk menghitung sinyal X dari spektrum y dapat digunakan invers transform Fourier cepat (IFFT) dengan menggunakan persamaan berikut:       1 N 0 m N ikm 2 m k e. y N 1 X (4) dengan m = 0 … N-1, dan k = 0 … N-1.       1 N 0 m N ikm 2 m k e. y N 1 X (4) dengan m = 0 … N-1, dan k = 0 … N-1.       1 N 0 m N ikm 2 m k e. y N 1 X (4) dengan m = 0 … N-1, dan k = 0 … N-1.       1 N 0 m N ikm 2 m k e. y N 1 X dengan m = 0 … N-1, dan k = 0 … N-1.       1 N 0 m N ikm 2 m k e. y N 1 X (4) dengan m = 0 … N-1, dan k = 0 … N-1. Pememilihan suatu data window biasanya dilakukan melalui suatu kompromi. Setiap window ‘mengotori’ (smear) spektrum frekuensi. Ini berarti bahwa suatu ‘puncak yang runcing’ yang berkaitan dengan suatu gelombang sinus dalam sinyal semakin melebar. Di sisi lain kebocoran palsu (spurious) ke dalam frekuensi di sekitarnya akan berkurang. Fungsi window disajikan pada Gambar 1. TELKOMNIKA Vol. 5, No.1, April 2007 : 51 - 60 53 ■ 53 ISSN: 1693-6930 TELKOMNIKA Gambar 1. Gambar fungsi window fungsi window data fungsi window Gambar 1. Gambar fungsi window Analisis Frekuensi untuk suatu sinyal dengan tingkat tekanan suara yang konstan pada semua frekuensi seperti derau putih, suatu jumlah energi yang sama terkandung dalam setiap pita pengukuran. Suatu penganalisa spektrum yang dikalibrasikan secara tepat dengan masukan derau putih akan menunjukkan suatu spektrum, ketika menggunakan skala frekuensi logaritmis, lebar setiap pita pengukuran akan meningkat bersama dengan frekuensi. Tingkat tekanan suara yang diukur dalam setiap pita akan meningkat dengan nilai kenaikan sebesar 3dB per oktaf dalam frekuensi. 2. METODE PENELITIAN Ukuran FFT dapat dihitung pada spektrum dengan menggunakan FFT pada point (128, 256, 512, 1024, 2048, atau 4096). Resolusi frekuensi paling tinggi pada spektrum adalah nilai cuplikan digital yang dibagi dengan ukuran FFT. Jika digunakan FFT yang lebih besar hanya untuk analisi resolusi yang tinggi atau dengan skala frekuensi logaritmis. FFT dengan resolusi yang lebih tinggi membutuh waktu lebih banyak untuk menghitung spektrum. Untuk alasan ini kadang-kadang sebaiknya mengurangi nilai cuplikan ketika merekam data audio/tutur, jika resolusi frekuensi yang tinggi dibutuhkan, dari pada menggunakan suatu FFT dengan resolusi yang lebih tinggi. Algoritma dasar FFT berikut digunakan untuk menghitung spektrum y dari sinyal X ebagai berikut: M l i 1. Mulai; 2. Konstanta sinyal  Larik TFFTFloat; y 3. Variabel: spektrum  Larik TFFTComplex; 4. Isikanlah data dengan nilai nol hingga ke kelipatan 2 berikutnya dapat dijadikan suatu pilihan bila kecepatan dianggap penting. p gg p p g 5. Untuk m  0..N-1, dan k  0..N-1; 5. Untuk m  0..N-1, dan k  0..N-1; 6. Hitunglah spektrum y sinyal x: N ikm 2 1 N 0 k k m e. X y     0 k 7. Larik spektrum dan larik sinyal harus memiliki panjang yang sama N. 7. Larik spektrum dan larik sinyal harus memiliki panjang yang sama N 7. Larik spektrum dan larik sinyal harus memiliki panjang yang sama N. 8. Algoritma ini berfungsi untuk setiap jumlah N, tetapi algoritma ini akan sangat cepat jika adalah kelipatan 2, juga bekerja lebih cepat untuk N genap. 9. Karena sinyal masukan bernilai riil, spektrum memiliki simetri ini untuk m  1..(N-1) div yang mungkin berguna untuk menyederhanakan beberapa kalkulasi: 9. Karena sinyal masukan bernilai riil, spektrum memiliki simetri ini untuk m  1..(N-1) div 2, yang mungkin berguna untuk menyederhanakan beberapa kalkulasi: * y y m m N   y p ( ) yang mungkin berguna untuk menyederhanakan beberapa kalkulasi: * y y m m N   10. Separuh bagian atas adalah konjugasi kompleks separuh bagian bawah. 11. Selesai. 10. Separuh bagian atas adalah konjugasi kompleks separuh bagian bawah. 11 S l i Diagram alir algoritma FFT dapat dilihat pada Gambar 2. Diagram alir algoritma FFT dapat dilihat pada Gambar 2. Diagram alir algoritma FFT dapat dilihat pada Gambar 2. Analisis Spektrum Frekuensi Non-Linear Sinyal Tutur ……(Salman Abd. Cadum)  54 ISSN: 1693-6930 Gambar 2. 2. METODE PENELITIAN Diagram alir algoritma dasar FFT mulai N ikm 2 1 N 0 k k m e. X y     N = {0 = kelipatan 2, atau genap } karena sinyal bernilai riil, maka spektrum memeliki simetri untuk m = 1 ke (N-1) div 2, untuk menyederhanakan kalkulasi * y y m m N   separuh bagian atas = konjugasi kompleks separuh bagian bawah selesai panjang larik spektrum = panjang larik sinyal = N untuk m = 0 ke N-1 k = 0 ke N-1 konstanta: sinyal = larik TFFTFloat variabel: spektrum = larik TFFTComplex mulai variabel: spektrum = larik TFFTComplex N = {0 = kelipatan 2, atau genap } Gambar 2. Diagram alir algoritma dasar FFT Algoritma dasar IFFT dapat menghitung sinyal X dari spektrum y sebagai berikut: 1. Mulai; 5. sinyal  Larik TFFTFloat; y 6. Algoritma ini berfungsi untuk setiap jumlah N, namun sangat cepat jika N adalah kelipatan juga bekerja lebih cepat bahkan untuk N genap. p j g j p g p 7. Untuk m  0..N-1, dan k  0..N-1, p j g j p 7. Untuk m  0..N-1, dan k  0..N-1, p j g j p g p 7. Untuk m  0..N-1, dan k  0..N-1, 8. Hitunglah sinyal x spektrum y: N ikm 2 1 N 0 m m k e . y N 1 X      9. Larik spektrum dan larik sinyal harus memiliki panjang yang sama N. 9. Larik spektrum dan larik sinyal harus memiliki panjang yang sama N. 10. Algoritma dasar FFT menghasilkan suatu sinyal bernilai kompleks tetapi hanya bagian riil yang disalin ke keluaran larik sinyal, sementara bagian imajiner diabaikan. * 10. Algoritma dasar FFT menghasilkan suatu sinyal bernilai kompleks tetapi hanya bagian riil yang disalin ke keluaran larik sinyal, sementara bagian imajiner diabaikan. 11. Terdapat nol dimana spektrum memiliki simetri untuk m  1..(N-1) div 2: * y y m m N   12. Separuh bagian atas adalah konjugasi kompleks separuh bagian bawah. 12. Separuh bagian atas adalah konjugasi kompleks separuh bagian bawah. 13 Selesai TELKOMNIKA Vol. 5, No.1, April 2007 : 51 - 60 55 ■ 55 ISSN: 1693-6930 TELKOMNIKA Diagram alir algoritma IFFT dapat disajikan pada Gambar 3. Gambar 3. 2. METODE PENELITIAN Diagram alir algoritma dasar IFFT mulai N = {0 = kelipatan 2, atau genap } spektrum memeliki simetri untuk m = 1 ke (N-1) div 2, untuk menyederhanakan kalkulasi * y y m m N   separuh bagian atas = konjugasi kompleks separuh bagian bawah selesai larik spektrum = larik sinyal = N untuk m = 0 ke N-1 k = 0 ke N-1 variabel: sinyal = larik TFFTFloat konstanta: spektrum = larik TFFTComplex N ikm 2 1 N 0 m m k e . y N 1 X      sinyal = riil + imajiner; {imajiner diabaikan} mulai larik spektrum = larik sinyal = N Gambar 3. Diagram alir algoritma dasar IFFT Skala frekuensi pada spektrum ini menggunakan skala frekuensi logaritmis untuk menghitung suatu spektrum. Suatu skala logaritmis akan memperluas daerah frekuensi rendah dari spektrum tersebut dan mempersempit daerah frekuensi yang tinggi pada tampilan. Skala frekuensi pada spektrum ini menggunakan skala frekuensi logaritmis untuk menghitung suatu spektrum. Suatu skala logaritmis akan memperluas daerah frekuensi rendah dari spektrum tersebut dan mempersempit daerah frekuensi yang tinggi pada tampilan. Spektrum menyediakan suatu kapabilitas logging data otomatis untuk tujuan ini, baru kemudian ditentukan tingkat tekanan suaranya. Tingkat tekanan suara dinyatakan dalam desibel (dB) yang merujuk pada amplitude minimum suatu sinyal audio/tutur 16 bit yang dihasilkan oleh kartu suara. Jika amplitude suatu komponen frekuensi adalah A, maka tingkat sinyal dalam desibel ditetapkan sebagai S = 20log (A). Hal ini menghasilkan suatu tingkat sinyal maksimum sebesar 90 dB yang berkaitan dengan nilai amplitude 16 bit maksimum sebesar 32767. Nilai ini sama dengan tingkat tekanan suara absolut pada setiap frekuensi plus suatu faktor desibel konstan yang sama dengan tingkat hasil (gain) yang digunakan oleh mikrofon, kartu suara komputer, dan penapis analisis spektrum yang dilakukan dalam perangkat lunak. Tampilan spektrum menampilkan data dengan skala frekuensi logaritmis. Skala logaritma membagi sumbu frekuensi menjadi interval frekuensi logaritmis yang sama. Skala frekuensi logaritmis memberikan keunggulan lebih besar pada frekuensi yang rendah dengan memperluas ruang tampilan untuk frekuensi rendah dengan mengorbankan frekuensi yang tinggi. Tampilan bidang (1/3) oktaf adalah suatu kasus khusus pada suatu tampilan skala Analisis Spektrum Frekuensi Non-Linear Sinyal Tutur ……(Salman Abd. Cadum)  56 ISSN: 1693-6930 frekuensi logaritmis di mana spektrum tingkat tekanan suara pada pita frekuensi yang berdekatan dengan lebar sepertiga oktaf direncanakan. frekuensi logaritmis di mana spektrum tingkat tekanan suara pada pita frekuensi yang berdekatan dengan lebar sepertiga oktaf direncanakan. 2. METODE PENELITIAN Skala logaritmis membutuhkan resolusi frekuensi yang sangat tinggi pada frekuensi yang rendah. Akibatnya adalah bahwa penggunaan suatu skala log membutuhkan suatu FFT yang jauh lebih besar guna mendapatkan resolusi frekuensi yang dibutuhkan. Mengingat muatan komputasional suatu FFT yang besar, skan terhadap berkas gelombang atau masukan audio/tutur dapat berjalan lebih lambat ketika menggunakan suatu skala frekuensi logaritmis. Ketika menggunakan suatu skala frekuensi logaritmis, dipilih FFT terkecil yang memberikan resolusi frekuensi yang tepat pada spektrum atau bidang pada frekuensi yang rendah. Jika daerah frekuensi yang rendah pada suatu tampilan frekuensi logaritmis kelihatannya terhalang (blocky), dinaikkan resolusi frekuensi rendah dengan dipilihnya suatu ukuran FFT yang lebih besar. Penting untuk memahami bagaimana pilihan terhadap suatu skala frekuensi logaritmis mempengaruhi tampilan spektrum tingkat tekanan suara. Spektrum mengukur tingkat tekanan suara pada pita frekuensi sempit diskrit di sepanjang spektrum. Suatu sinyal akan menghasilkan ketajaman, karena rutin FFT menganggap masukan sebagai suatu sinyal periodis yang dapat mencacat (distort) spektrum. Untuk mengurangi masalah tersebut suatu window data pertama-tama dapat diterapkan pada sinyal. Tabel 1. 2. METODE PENELITIAN Cadu 0 50 100 150 200 250 300 350 400 450 500 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Jendela Hamming n Magnitude 0 50 100 150 200 250 300 350 400 450 500 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Jendela Hanning n Magnitude 0 50 100 150 200 250 300 350 400 450 500 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Jendela Segitiga (Bartlett) n Magnitude 0 50 100 150 200 250 300 350 400 450 500 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Jendela Blackman n Magnitude 0 50 100 150 200 250 300 350 400 450 500 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Jendela Nuttall n Magnitude 0 50 100 150 200 250 300 350 400 450 500 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Jendela Welch n Magnitude 0 50 100 150 200 250 300 350 400 450 500 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 Jendela FlatTop n Magnitude 0 50 100 150 200 250 300 350 400 450 500 -0.5 -0.45 -0.4 -0.35 -0.3 -0.25 -0.2 -0.15 -0.1 -0.05 0 Jendela Segiempat n Magnitude Gambar 4. 2. METODE PENELITIAN Persamaan window yang diterapkan pada spektrum No Tipe Nilai cuplikan Kisaran k 1 Segiempat    0 1 w k 1 N k 0    else 2 Segitiga Bartlett , atau Parzen 1 N k 2 1 1 w k     1 N k 0    3 Hanning           1 N k 2 cos 1 2 1 w k 1 N k 0    4 Hamming 1 N k 2 cos 46 .0 54 .0 w k     1 N k 0    5 Blackman 1 N k 4 cos 08 .0 1 N k 2 cos 5.0 42 .0 w k        1 N k 0    6 Welch   2 0 x 1 ,x Wlech                x else 7 FlatTop    x 2 cos 1980389663 .0 x cos * 520897135 .0 2810638602 .0 w k     1 N k 2 x    1 N k 0    8 Nuttall      1 N k 2 x 1 n x 2 cos * 125 .0 ) 1 n x cos( * 5.0 375 .0 w k             1 N k 0    TELKOMNIKA Vol. 5, No.1, April 2007 : 51 - 60 TELKOMNIKA Vol. 5, No.1, April 2007 : 51 - 60 ■ 57 ISSN: 1693-6930 ■ 57 TELKOMNIKA 0 50 100 150 200 250 300 350 400 450 500 -0.5 -0.45 -0.4 -0.35 -0.3 -0.25 -0.2 -0.15 -0.1 -0.05 0 Jendela Segiempat n Magnitude (a) Jendela Segiempat 0 50 100 150 200 250 300 350 400 450 500 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Jendela Blackman n Magnitude (b) Jendela Blackman Analisis Spektrum Frekuensi Non-Linear Sinyal Tutur ……(Salman Abd. 2. METODE PENELITIAN Berbagai jendela yang diterapkan pada analisis spektrum (c) Jendela Hamming (d) Jendela Nuttall (e) Jendela Hanning (f) Jendela Welch (g) Jendela Segitiga (Bartlett) (h) Jendela FlatTop (b) Jendela Blackman (a) Jendela Segiempat (a) Jendela Segiempat (b) Jendela Blackman (b) Jendela Blackman 0 50 100 150 200 250 300 350 400 450 500 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Jendela Nuttall n Magnitude (d) Jendela Nuttall ( ) 0 50 100 150 200 250 300 350 400 450 500 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Jendela Hamming n Magnitude (c) Jendela Hamming (d) Jendela Nuttall (c) Jendela Hamming 0 50 100 150 200 250 300 350 400 450 500 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Jendela Hanning n Magnitude (c) Jendela Hamming (e) Jendela Hanning 0 50 100 150 200 250 300 350 400 450 500 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Jendela Welch n Magnitude (f) Jendela Welch (f) Jendela Welch (e) Jendela Hanning (e) Jendela Hanning 0 50 100 150 200 250 300 350 400 450 500 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Jendela Segitiga (Bartlett) n Magnitude (e) Jendela Hanning (g) Jendela Segitiga (Bartlett) 0 50 100 150 200 250 300 350 400 450 500 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 Jendela FlatTop n Magnitude (h) Jendela FlatTop (h) Jendela FlatTop (g) Jendela Segitiga (Bartlett) Gambar 4. Berbagai jendela yang diterapkan pada analisis spektrum Analisis Spektrum Frekuensi Non-Linear Sinyal Tutur ……(Salman Abd. Cadum) ISSN: 1693-6930  58 3. HASIL DAN PEMBAHASAN 3. HASIL DAN PEMBAHASAN Spektrum dengan skala logaritmis dapat disajikan pada Gambar 5 TELKOMNIKA Vol. 5, No.1, April 2007 : 51 - 60 (a) Spektrum skala logaritmis dengan non- window (b) Spektrum skala logaritmis dengan window Blackman (c) Spektrum skala logaritmis dengan window Hanning (d) Spektrum skala logaritmis dengan window Nuttal (f) Spektrum skala logaritmis dengan window Flattop (e) Spektrum skala logaritmis dengan window Hamming Spektrum dengan skala logaritmis dapat disajikan pada Gambar 5 (b) Spektrum skala logaritmis dengan window Blackman (a) Spektrum skala logaritmis dengan non- window (a) Spektrum skala logaritmis dengan non- window (b) Spektrum skala logaritmis dengan window Blackman window (c) Spektrum skala logaritmis dengan window Hanning (c) Spektrum skala logaritmis dengan window Hanning (d) Spektrum skala logaritmis dengan window Nuttal (f) Spektrum skala logaritmis dengan window Flattop (c) Spektrum skala logaritmis dengan window Hanning (e) Spektrum skala logaritmis dengan window Hamming (d) Spektrum skala logaritmis dengan window Nuttal (e) Spektrum skala logaritmis dengan window Hamming (f) Spektrum skala logaritmis dengan window Flattop TELKOMNIKA Vol. 5, No.1, April 2007 : 51 - 60 TELKOMNIKA Vol. 5, No.1, April 2007 : 51 - 60 TELKOMNIKA Vol. 5, No.1, April 2007 : 51 - 60 ISSN: 1693-6930 TELKOMNIKA 59 Pada Tabel 2 berikut ditunjukkan perbandingan efek pemilihan window terhadap spektrum sinyal tutur. Gambar 5. Tampilan spektrum skala logaritmis dengan menggunakan berbagai window. (g) Spektrum skala logaritmis dengan window Taylor 60 dB (h) Spektrum skala logaritmis dengan window Triangle (i) Spektrum skala logaritmis dengan window Taylor 80 dB (j) Spektrum skala logaritmis dengan window Welch (h) Spektrum skala logaritmis dengan window Triangle (g) Spektrum skala logaritmis dengan window Taylor 60 dB (g) Spektrum skala logaritmis dengan window Taylor 60 dB (h) Spektrum skala logaritmis dengan window Triangle (h) Spektrum skala logaritmis dengan window Triangle window Triangle (j) Spektrum skala logaritmis dengan window Welch y (i) Spektrum skala logaritmis dengan window Taylor 80 dB (i) Spektrum skala logaritmis dengan window Taylor 80 dB (i) Spektrum skala logaritmis dengan window Taylor 80 dB (j) Spektrum skala logaritmis dengan window Welch Gambar 5. Tampilan spektrum skala logaritmis dengan menggunakan berbagai window. Pada Tabel 2 berikut ditunjukkan perbandingan efek pemilihan window terhadap spektrum sinyal tutur. Analisis Spektrum Frekuensi Non-Linear Sinyal Tutur ……(Salman Abd. Cadum) Analisis Spektrum Frekuensi Non-Linear Sinyal Tutur ……(Salman Abd. Cadum) ISSN: 1693-6930  60 Tabel 2. Perbandingan pemilihan window terhadap analisis spektrum frekuensi non-linear No. Nama window Tingkat sidelobe tertinggi Sidelobe fall-off Keterangan 1. 4. SIMPULAN 4. SIMPULAN Berdasarkan hasil dan pembahasan dapat ditarik kesimpulan sebagai berikut: 4. SIMPULAN Berdasarkan hasil dan pembahasan dapat ditarik kesimpulan sebagai berikut: MPULAN Berdasarkan hasil dan pembahasan dapat ditarik kesimpulan sebagai berikut: p p p g 1. Suatu skala logaritmis akan memperluas daerah frekuensi yang rendah dari spektrum, dan mempersempit daerah frekuensi yang tinggi pada tampilan. 2. Skala frekuensi logaritmis membutuhkan resolusi frekuensi yang sangat tinggi pada frekuensi yang rendah, oleh karena itu dibutuhkan suatu FFT yang jauh lebih besar guna mendapatkan resolusi frekuensi yang dibutuhkan. 2. Skala frekuensi logaritmis membutuhkan resolusi frekuensi yang sangat tinggi pada frekuensi yang rendah, oleh karena itu dibutuhkan suatu FFT yang jauh lebih besar guna mendapatkan resolusi frekuensi yang dibutuhkan. p y g 3. Penerapan fungsi berbagai window terhadap data dapat membantu mengurangi efek kebocoran yang terjadi pada spektrum frekuensi. p y g 3. Penerapan fungsi berbagai window terhadap data dapat membantu mengurangi efek kebocoran yang terjadi pada spektrum frekuensi. y g j p p 4. Metode ini berjalan lebih cepat jika point data merupakan kelipatan dua (128, 256, 1024, 2048, atau 4096, dan seterusnya). 4. Metode ini berjalan lebih cepat jika point data merupakan kelipatan dua (128, 256, 1024, 2048, atau 4096, dan seterusnya). y ) 5. Memilih suatu resolusi frekuensi yang tepat dan resolusi waktu yang sesuai menjadi suatu kesesuaian antara kebutuhan untuk mengamati detail frekuensi yang baik dalam spektrum dengan kebutuhan untuk mengamati variasi waktu yang cepat dalam spektrum. y ) 5. Memilih suatu resolusi frekuensi yang tepat dan resolusi waktu yang sesuai menjadi suatu kesesuaian antara kebutuhan untuk mengamati detail frekuensi yang baik dalam spektrum dengan kebutuhan untuk mengamati variasi waktu yang cepat dalam spektrum. 3. HASIL DAN PEMBAHASAN (tanpa window) 13.3dB 6dB/Oct Lobe utama yang mungkin yang paling sempit, tetapi terjadi kebocoran yang cukup besar. 2. Bartlett (Triangle) 26.6dB 12dB/Oct Welch memiliki suatu lobe utama yang sedikit lebih sempit dibanding Bartlett, tetapi sidelobe-nya agak lebih tinggi. 3. Welch (parabola) ? 12dB/Oct Welch memiliki suatu lobe utama yang sedikit lebih sempit dibanding Bartlett, tetapi sidelobe-nya agak lebih tinggi. 4. Hanning 31.5dB 18dB/Oct Lobe utama yang agak sempit, reduksi kebocoran jauh asimptotik yang bagus. 5. Hamming 43dB 6dB/Oct Lobe utama yang agak sempit, reduksi kebocoran dekat yang bagus. 6. Blackman 58.11dB 18dB/Oct Lobe utama yang lebih luas dibanding Hanning dan Hamming, reduksi kebocoran dekat dan jauh yang bagus. 7. Nuttall 93.3dB 18dB/Oct Reduksi kebocoran dekat yang lebih bagus daripada window Blackman tetapi lobe utama yang lebih luas. 8. FlatTop 43.2dB 6dB/Oct Window Flattop digunakan ketika menghitung amplitude spektrum suatu puncak dengan energi spektrum mendekati aslinya dan menyediakan akurasi amplitude yang bagus. Window Flattop memiliki suatu lobe utama yang luas dengan suatu ‘flat top’ melewati dua garis spektrum. Ini berarti bahwa setiap frekuensi sinyal yang berada di antara dua garis spektrum akan terlihat pada garis spektrum yang terdekat tanpa kehilangan amplitude. Window FlatTop digunakan ketika pengukuran komponen spektrum amplitude dari frekuensi arbitrer dianggap penting. 4. SIMPULAN DAFTAR PUSTAKA [1]. Rabiner, L., R., and R. W. Schafer, “Digital Processing of Speech Signals”. Prentic Hall, 1978. [1]. Rabiner, L., R., and R. W. Schafer, “Digital Processing of Speech Signals”. Prentice Hall, 1978. [2]. Sugamura, N., and F. Itakura, “Speech Analysis and Synthesis Methodes Developed at ECL in NTT- from LPC to LSP”, Speech Communication, 5:199-215, 1986. [3]. Fallside, F., and W.A. Woods, ”Computer Speech Processing”, Cambridge University Engineering Department, Printice Hall, 1985. TELKOMNIKA Vol. 5, No.1, April 2007 : 51 - 60
https://openalex.org/W4307420705	https://zenodo.org/records/7256946/files/29%20EIIRJ%20VOL-IX%20Ishu%20Sept-Oct%202020.pdf	Marathi (Marāṭhī)	null	ANNABHAU SATHE AANI MATANG SAMAJ	Zenodo (CERN European Organization for Nuclear Research)	2,020	cc-by	4,171	अण्णा भाऊ साठे आणण मातंग समाज श्री. बा. रा. वाघमारे, संस्थापक, अण्णा भाऊ साठे पीपल्स फोसस अण्णा भाऊ साठे आणण मातंग समाज असं शिर्सक वापरुन मी काही अण्णा भाऊ साठेंना जातीत बंदीस्त करू इणछित नाही. तसं कोणी करतो म्हटलं तरी त्याला ते िक्य नाही. कारण अण्णा भाऊ साठे म्हणजे सातासमुद्रापशलकडे गेलेलं एक महान व्यक्तिमत्व आहे. त्यामुळे जातीिी त्यांचा संबंध दिसवणं म्हणजे एक प्रकारे त्यांछया क्तवचारापासून दूर जाणे होय. अण्णा भाऊ साठेंनी कधीही जातीचं भांडवल केलं नाही. त्यांनी काही मातंग समाजासाठीच शलहहलं नाही. तर ते एक थोर लेखक आहेत. त्यांनी जगभरातल्या दशलत – िोक्तर्त - पीहडत – वंशचत - कामगार लोकांसाठी शलहहलं आहे. दुसरं म्हणजे इथला कोणताही राजकीय पक्ष मातंग समाजाछया माणसाला कधीही मोठं होवू देत नाही. एखाद्या गावासाठी हकंवा एखाद्या पक्षासाठी मातंग समाजाछया कायसकत्याांनं आपला सारा जीव आटवला असला तरीही त्याछया बाबतीत तो त्या गावातला हकंवा त्या पाटीतला एक मोठा कायसकतास आहे. असं मात्र कधीच म्हटलं जात नाही. उलट त्याची ओळख करून देताना मातंग समाजाचा कायसकतास असं म्हणूनच करून हदली जाते. आणण शतकडे मात्र त्यांछयातला बेअकली जरी कायसकतास असला तरीही गावातला एक प्रशतक्तित कायसकतास म्हणून त्याची ओळख शनमासण करून हदली जात असते. त्यामुळं त्याछया नावासमोर त्याची जात जोडण्याची त्यांना कोणतीही गरज नसते. हा रोग तसा फार पूवीपासूनच या देिात आहे. गोरा कुंभार - सावता माळी - चोख्या महार असं म्हटलं जातं. परंतू ज्ञानोबा ब्राह्मण असं मात्र कधीही म्हटलं जात नाही. उलट त्याला माऊली म्हटलं जातं. गोरोबा माऊली, चोखोबा माऊली असं म्हटलं तर यांछया बापाचं काही क्तबघडणार आहे काय? पण तसं ते कधीही म्हणणार नाहीत. या अण्णा भाऊ साठे आणण मातंग समाज असं शिर्सक वापरुन मी काही अण्णा भाऊ साठेंना जातीत बंदीस्त करू इणछित नाही. तसं कोणी करतो म्हटलं तरी त्याला ते िक्य नाही. कारण अण्णा भाऊ साठे म्हणजे सातासमुद्रापशलकडे गेलेलं एक महान व्यक्तिमत्व आहे. त्यामुळे जातीिी त्यांचा संबंध दिसवणं म्हणजे एक प्रकारे त्यांछया क्तवचारापासून दूर जाणे होय. अण्णा भाऊ साठेंनी कधीही जातीचं भांडवल केलं नाही. त्यांनी काही मातंग समाजासाठीच शलहहलं नाही. तर ते एक थोर लेखक आहेत. त्यांनी जगभरातल्या दशलत – िोक्तर्त - पीहडत – वंशचत - कामगार लोकांसाठी शलहहलं आहे. दुसरं म्हणजे इथला कोणताही राजकीय पक्ष मातंग समाजाछया माणसाला कधीही मोठं होवू देत नाही. एखाद्या गावासाठी हकंवा एखाद्या पक्षासाठी मातंग समाजाछया कायसकत्याांनं आपला सारा जीव आटवला असला तरीही त्याछया बाबतीत तो त्या गावातला हकंवा त्या पाटीतला एक मोठा कायसकतास आहे. असं मात्र कधीच म्हटलं जात नाही. उलट त्याची ओळख करून देताना मातंग समाजाचा कायसकतास असं म्हणूनच करून हदली जाते. EIIRJ Volume–IX Issues–V ISSN–2277- 8721 Sept – Oct. 2020 EIIRJ Volume–IX Issues–V ISSN–2277- 8721 Sept – Oct. 2020 अण्णा भाऊ साठे आणण मातंग समाज आणण शतकडे मात्र त्यांछयातला बेअकली जरी कायसकतास असला तरीही गावातला एक प्रशतक्तित कायसकतास म्हणून त्याची ओळख शनमासण करून हदली जात असते. त्यामुळं त्याछया नावासमोर त्याची जात जोडण्याची त्यांना कोणतीही गरज नसते. हा रोग तसा फार पूवीपासूनच या देिात आहे. गोरा कुंभार - सावता माळी - चोख्या महार असं म्हटलं जातं. परंतू ज्ञानोबा ब्राह्मण असं मात्र कधीही म्हटलं जात नाही. उलट त्याला माऊली म्हटलं जातं. गोरोबा माऊली, चोखोबा माऊली असं म्हटलं तर यांछया बापाचं काही क्तबघडणार आहे काय? पण तसं ते कधीही म्हणणार नाहीत. या Electronic International Interdisciplinary Research Journal (EIIRJ) Page 144 SJIF Impact Factor: 6.21 Peer Reviewed Refereed Journal Electronic International Interdisciplinary Research Journal (EIIRJ) Electronic International Interdisciplinary Research Journal (EIIRJ) Page 144 Volume–IX SJIF Impact Factor: 6.21 Peer Reviewed Refereed Journal EIIRJ Volume–IX Issues–V ISSN–2277- 8721 Sept – Oct. 2020 देिात डॉ. बाबासाहेब आंबेडकर यांना देखील दशलतांचे नेते म्हणण्या मागचा हाच उद्देि होता. अण्णा भाऊ साठे यांछया बाबतीत देखील त्यांचं तेच धोरण आहे. त्यामुळे अण्णा भाऊंना जातीत बंहदस्त करणं म्हणजे जातीयवाद्यांना मदत करणे होय. त्यामुळे मी काही ते पाप करणार नाही. परंतू दुसरी िोकांशतका ही आहे की, इथ प्रत्येकाला आपली जात प्यारी झाली आहे. रक्तवदासांची जयंती चमसकार लोकंच करतात. शिवाजी महाराजांची जयंती मराठा जातीचे लोकंच करतात. अण्णा भाऊंची जयंती मातंग समाजाचे लोकंच साजरी करतात. डॉ. बाबासाहेब आंबेडकर यांछया बाबतीत मात्र थोडसं वेगळं आहे. कारण त्यांची जयंती देिातल्या प्रत्येक राज्यात साजरी होत असते. परदेिात साजरी होत असते. शतथं काही बाबासाहेबांछया जातीचे लोकं नसतात. बहूजन चळवळीला हदलासा देणारी ही एक फार मोठी गोष्ट आहे. त्यामुळे कोणती जात कोणत्या महापुरुर्ाला मानते. हा क्तवर्य काही महत्त्वाचा नाही. तर त्या-त्या महापुरुर्ाछया क्तवचारावर ती ती जात उभं राहणं जास्त महत्वाचं आहे. म्हणून अण्णा भाऊ साठे आणण मातंग समाज असं जे काही लेखाच शिर्सक आपण वापरलं आहे. ते काही फारसं चुकीचं आहे असं शनदान मला तरी वाटत नाही . मातंग समाज अण्णा भाऊ साठे यांची जयंती साजरी करतो. ही गोष्ट जेवढी खरी आहे, तेवढीचं ही गोष्ट खरी आहे की, मातंग समाज हा अण्णा भाऊ साठेंचा क्तवचारच आचरणात आणत नाही. म्हणून मग अण्णा भाऊ साठे यांचा क्तवचार मातंग समाजाने नाकारला का?... अणजबात नाही. म्हणून जसा तो नाकारला नाही तसा तो क्तवचार स्वीकारला देखील नाही. फुले-आंबेडकर तर दूरच राहहले, परंतु त्यांछयाही आधी या देिात मातंग समाजाचे लहुजी साळवे होऊन गेले मुिा साळवे होऊन गेली. त्यांचं देखील या समाजानं कधी ऐकलं नाही. मग मातंग समाज असं का वागतो?.... तो वेडा आहे काय?.... अण्णा भाऊ साठे आणण मातंग समाज त्याला जर मी वेडा म्हणू तर कोणतीही गोष्ट त्याला सांगा, त्याछया पुढची तो दुसरी एखादी भलतीच गोष्ट सांगून टाकतो. एखाद्याचा िहाणा म्हणून या क्तवर्यी काही सल्ला माशगतला तर तो शतसरेच काहीतरी सांगून त्यात आणखी गुंताच शनमासण करेल, परंतु आपण एकत्र बसून त्यातून EIIRJ देिात डॉ. बाबासाहेब आंबेडकर यांना देखील दशलतांचे नेते म्हणण्या मागचा हाच उद्देि होता. अण्णा भाऊ साठे यांछया बाबतीत देखील त्यांचं तेच धोरण आहे. त्यामुळे अण्णा भाऊंना जातीत बंहदस्त करणं म्हणजे जातीयवाद्यांना मदत करणे होय. त्यामुळे मी काही ते पाप करणार नाही. परंतू दुसरी िोकांशतका ही आहे की, इथ प्रत्येकाला आपली जात प्यारी झाली आहे. रक्तवदासांची जयंती चमसकार लोकंच करतात. शिवाजी महाराजांची जयंती मराठा जातीचे लोकंच करतात. अण्णा भाऊंची जयंती मातंग समाजाचे लोकंच साजरी करतात. डॉ. बाबासाहेब आंबेडकर यांछया बाबतीत मात्र थोडसं वेगळं आहे. कारण त्यांची जयंती देिातल्या प्रत्येक राज्यात साजरी होत असते. परदेिात साजरी होत असते. शतथं काही बाबासाहेबांछया जातीचे लोकं नसतात. बहूजन चळवळीला हदलासा देणारी ही एक फार मोठी गोष्ट आहे. त्यामुळे कोणती जात कोणत्या महापुरुर्ाला मानते. हा क्तवर्य काही महत्त्वाचा नाही. तर त्या-त्या महापुरुर्ाछया क्तवचारावर ती ती जात उभं राहणं जास्त महत्वाचं आहे. म्हणून अण्णा भाऊ साठे आणण मातंग समाज असं जे काही लेखाच शिर्सक आपण वापरलं आहे. ते काही फारसं चुकीचं आहे असं शनदान मला तरी वाटत नाही . Electronic International Interdisciplinary Research Journal (EIIRJ) Page 145 SJIF Impact Factor: 6.21 Peer Reviewed Refereed Journal मातंग समाज अण्णा भाऊ साठे यांची जयंती साजरी करतो. ही गोष्ट जेवढी खरी आहे, तेवढीचं ही गोष्ट खरी आहे की, मातंग समाज हा अण्णा भाऊ साठेंचा क्तवचारच आचरणात आणत नाही. म्हणून मग अण्णा भाऊ साठे यांचा क्तवचार मातंग समाजाने नाकारला का?... अणजबात नाही. म्हणून जसा तो नाकारला नाही तसा तो क्तवचार स्वीकारला देखील नाही. फुले-आंबेडकर तर दूरच राहहले, परंतु त्यांछयाही आधी या देिात मातंग समाजाचे लहुजी साळवे होऊन गेले मुिा साळवे होऊन गेली. त्यांचं देखील या समाजानं कधी ऐकलं नाही. मग मातंग समाज असं का वागतो?.... तो वेडा आहे काय?.... त्याला जर मी वेडा म्हणू तर कोणतीही गोष्ट त्याला सांगा, त्याछया पुढची तो दुसरी एखादी भलतीच गोष्ट सांगून टाकतो. एखाद्याचा िहाणा म्हणून या क्तवर्यी काही सल्ला माशगतला तर तो शतसरेच काहीतरी सांगून त्यात आणखी गुंताच शनमासण करेल, परंतु आपण एकत्र बसून त्यातून Page 145 Volume–IX p काही मागस काढू. असं मात्र तो कधीच म्हणणार नाही. चौथा एखादा मध्येच येतो आणण इथं असं झालं पाहहजे होतं आणण शतथं तसं झालं पाहहजे होतं असं उगीचच काहीतरी सांगत असतो. बरं तो एवढ्यावरच थांबत नाही. तर चक्क सवसच कायसकत्याांना तो मूखासत काढत असतो. अण्णा भाऊ साठे आणण मातंग समाज सब घोडे बारा टक्के याच नजरेनं तो प्रत्येकाकडं बघत असतो. समाजात वावरत असताना प्रत्यक्षात कायसकत्याांसमोर काही अडचणी असू िकतात, याचा मात्र तो कधीच क्तवचार करत नाही. म्हणून कोणत्या वेळी कोणता मांग काय म्हणेल, याचा काही नेम नसतो. तो आपला मांग मसळा कधीच सरू देत नसतो. िहाणाही तसाच आणण वेडा ही तसाच. त्यामुळे या समाजाचं सगळं वाटोळं होऊन गेलं आहे. हा समाज 100% अण्णा भाऊ साठेंचा अनुयायी आहे, त्याबद्दल शतळमात्र िंका नाही. परंतु िोकांशतका ही की, हा अनुयायी 100% आंधळा आहे. एक आंधळा दुसऱ्या आंधळ्याला जसा रस्ता दाखवू िकत नाही, नेमकं तसंच काहीसं हे झालं आहे. अण्णा भाऊ साठे यांछया जयंती मध्ये कोणत्या राजकीय पाटीचे लोकं बोलवायचे यावरून समाजात गावोगावी गटं तटं पडले आहेत. ही गोष्ट समाजातल्या क्तवचारवंतांना हदसत नाही काय ?... या गतेतून समाजाला बाहेर काढण्याची त्यांची काही जबाबदारी नाही काय? परंतु ही लोकं कोणतीच ररस्क घेऊ िकत नाहीत. ह्ांना कोणी फासावर चढवणार आहे काय?... भर सभेत अण्णा भाऊ साठेंची हे लोकं कुतरओढ करीत असतात. कोणी अण्णा भाऊ साठे माक्ससवादी होते असं सांगतात, तर कोणी ते आंबेडकर वादी होते असे सांगतात. पण या वादाचा समाजावर काय पररणाम होतो माहीत नाही. परंतु या वादावादीमुळ गावातला जो कोणी पाटीलकीछया थाटातला माणूस त्या सभेचा अध्यक्ष असतो, त्याची मात्र खूप मोठी करमणूक होत असते. कायसक्रम घेणार् याबदल काय बोलावं ?... कायसक्रम घेऊन त्याचा तो मोकळा झालेला असतो. ना तो माक्ससवादी असतो, ना तो आंबेडकर वादी असतो. तो तर हहंदुत्ववादी असतो. जयंतीत तो काय कमावतो आणण काय गमावतो?.... हे त्याच त्यालाच माहीत. परंतु पररवतसनवादी क्तवचार घेऊन चालणारा जो कोणी कायसकतास असतो, त्याछया हातात मात्र जयंतीचा कारभार हा कधीच जाऊ देत नाही. Electronic International Interdisciplinary Research Journal (EIIRJ) Page 146 SJIF Impact Factor: 6.21 Peer Reviewed Refereed Journal SJIF Impact Factor: 6.21 SJIF Impact Factor: 6.21 Electronic International Interdisciplinary Research Journal (EIIRJ) Electronic International Interdisciplinary Research Journal (EIIRJ) Page 146 EIIRJ Volume–IX Issues–V ISSN–2277- 8721 Sept – Oct. 2020 खरं सांगू का? ज्याचे गुडघे फुटतात तेच खरे बाप असतात. ज्याचे गुडघे कधी फुटत नसतात त्यानं बाप म्हणून शमरवू नये, या शनशमत्यानं माझं एवढंच एक म्हणणं आहे. मातंग समाजातला प्रत्येक कायसकतास हा कोणत्या ना कोणत्या राजकीय पक्षांमध्ये काम करत असतो. परंतु िोकांशतका ही आहे की, एक राजकीय कायसकतास म्हणून त्याछयाकडं कोणतंही राजकीय धोरण नाही. केवळ एक रोजगाराचं साधन म्हणून अथवा त्यांची ती एक व्यक्तिगत गरज म्हणून तो राजकारण करत असतो. गाई मागून वासरू, घोड्या मागून शिंगरु जसं चालतं नेमकं तसंच काहीसं याचं असतं. त्याछया राजकारणाचं स्वरूप म्हणजे ‘मान ना मान मै तेरा मेहमान’ असंच असतं. अण्णा भाऊ साठे आणण मातंग समाज पक्षपातळीवर त्याछयाकडे कोणतंही महत्वाचं पद नसतं. शनणसय प्रहक्रयेत त्याला कवडीचीही हकंमत नसते. मग तो कााँग्रेसमध्ये काम करणारा राहो की, भाजप - सेनेमध्ये काम करणार राहो. एक राजकारण म्हणून तो कधीही इथल्या संसदीय िासन प्रणाली वर बोलताना हदसत नाही. या देिात लहुजी साळवे यांनी जे काही स्वाशभमानाचं राजकारण केलं यावर तो कोणतंही भाष्य करीत नाही. केवळ साहेब, साहेबाची बायको, साहेबाची गाडी, आणण साहेबाची घोडी एवढ्याच काय तो राजकारण म्हणून त्याछयाकडे चचेचा क्तवर्य असतो. इकडं हे अस आहे तर शतकडं बीएसपी हकंवा वंशचत आघाडीत काम करणाऱ्या मांगाची एक वेगळीच भानगड आहे. कािीरामांनी हे केलं आणण बाळासाहेबांनी ते केलं. असं सांगून ते सहज मोकळं होतात. परंतु त्या पक्षांमध्ये असं एक एकटं जाण्यापेक्षा अख्ख्या समाजालाचं घेऊन जावं, असं मात्र ते कधीच म्हणणार नाहीत. कदाशचत हे काम तेवढं सोपं नाही. हे त्यांना कळून चुकलं असावं. काहीजण तर धड इकडचेही नाहीत ना धड शतकडचेही नाहीत. त्यामुळं जसं जमलं तसं ते दोन्ही कडूनही वाजवतात. मी काही राजकीय कायसकतास नाही. म्हणून इतरांनीही राजकारण करू नये, असं मात्र मी कधीच म्हणणार नाही. इथं आपल्या राजकीय कायसकत्याांना अनेक जण स्वाथी म्हणतात, लाचार म्हणतात. कोणी कोणी तर त्यांना चक्क चमचे म्हणतात. तसं मी काही Electronic International Interdisciplinary Research Journal (EIIRJ) Page 147 SJIF Impact Factor: 6.21 Peer Reviewed Refereed Journal SJIF Impact Factor: 6.21 SJIF Impact Factor: 6.21 Electronic International Interdisciplinary Research Journal (EIIRJ) Electronic International Interdisciplinary Research Journal (EIIRJ) Page 147 EIIRJ Volume–IX Issues–V ISSN–2277- 8721 Sept – Oct. 2020 Volume–IX त्यांछया या वागण्यामुळे समाजाचं फार मोठं नुकसान झालेलं आहे. याची नुकसान भरपाई मी कोणाकडे मागू? ..... सध्या महाराष्ट्रात तीन पार्टयासचं सरकार आहे. त्या सरकारमध्ये बौद्ध समाजाचे तीन माणसं मंत्री आहेत. शतथं मातंग समाजाचा एकही मंत्री नाही. केंद्र सरकार मध्ये देखील मातंग समाजाचा कोणीही मंत्री नाही. समाजाचा एक घटक म्हणून हा प्रश्न मी कोणाला क्तवचारावा?.... इथल्या कोणत्याही राजकीय पक्षानं आतापयांत मातंग समाजाचा एकही माणूस राज्यसभेवर घेतला नाही कोण्या क्तवधान पररर्देवर घेतला नाही. एकही मांग या देिात राष्ट्रपती शनयुि खासदार झाला नाही हकंवा राजपाल शनयुि एखादा आमदार झाला नाही याचा जाब मी कोणाला क्तवचारू?.... हा एक फार मोठा अवघड प्रश्न आहे. अण्णा भाऊ साठे पीपल्स फोससछया माध्यमातून समाजात वावरताना काही अनुभव माझ्या वार्टयाला आलेले आहेत. त्यातला एक सांगण्यासारखा अनुभव म्हणजे, मातंग समाज काही सुधारणार नाही, असं अनेकजण म्हणू लागले आहेत. त्यांना मी एवढंच म्हणतो की, हा समाज सुधारला पाहहजे याछयासाठी तुम्ही काय केलं ते सांगा?..... यावर मात्र त्यांछयाकडे कोणतंही उत्तर नाही. रोज एका गावात ध्या हदवसा मांगाछया माणसाला मारलं जात आहे. मग काय?.... अण्णा भाऊ साठे आणण मातंग समाज मार खाण्याची फार हौस आहे म्हणून मातंग समाजाचा माणूस रोज एका गावात मार खातो काय?..... परंतु खरं दुखणं हे आहे की, समाजानं आपल्या पाठीिी राहहलं पाहहजे, असं अनेकांना वाटत असतं. परंतु समाज पाठीिी राहायला पाहहजे, यासाठी जे काही कष्ट घ्यावे घ्यावे लागतात, त्यासाठी मात्र त्याची कोणतीही तयारी नसते. कायसकतास होण्याची लायकी असो अथवा नसो, याचा मात्र त्यांना थोडासाही क्तवचार नसतो. काही शमळत असेल तर समाजाचं काम कराव. आणण जर का मग काही शमळत नसेल तर मग समाजाचं काम काय म्हणून करावं?... हा प्रश्न देखील तोच उपणस्थत करतो. त्याला आणखी शचडवले तर मग तो म्हणतो की, समाजासाठी आपल्या लेकरा-बाळांना उपािी मारावं काय? मग काय तोडून खावं काय?.... Electronic International Interdisciplinary Research Journal (EIIRJ) Page 148 SJIF Impact Factor: 6.21 Peer Reviewed Refereed Journal SJIF Impact Factor: 6.21 Peer Reviewed Refereed Journal Electronic International Interdisciplinary Research Journal (EIIRJ) Page 148 Volume–IX EIIRJ Volume–IX Issues–V ISSN–2277- 8721 Sept – Oct. 2020 खरं तरं मातंग समाज हा अनेकांना मदत करीत आला आहे. त्यामुळे समाज आता कोणावर क्तवश्वास ठेवायला तयार नाही. समाज माझ्याकडून काय अपेक्षा करतो आहे माहहत नाही परंतु एक कायसकतास म्हणून जी जी गोष्ट मी समाजाला सांगतो आहे. ती ती गोष्ट समाज माझ ऐकतो आहे. खरंच आपलं देखील काही अणस्तत्व या देिात असलं पाहहजे, असं म्हणतो आहे. ध्येय तर माझं फार मोठं भव्यहदव्य आहे. परंतु डॉ. बाबासाहेब आंबेडकर म्हणतात की, “चळवळीत कुठलाही िॉटसकट नसतो,” म्हणून चळवळीसाठी जेवढा काही लागणारा नैसशगसक वेळ असतो, तेवढा तो लागणारच असतो, माझ्याआधी समाजात जे काही काम करणारे कायसकते होते. त्या काळात मांगवाड्यात घरापरत देवकरण्या होत्या. त्यांचं हे देवपण समाजातून शनघून जावं. यासाठी त्यावेळछया कायसकत्याांनी मनावर घेतलं, त्या कामाचा ध्यास घेतला. त्याचा पररणाम असा झाला की, समाजात आज नवीन मुलींमध्ये िोधून देखील एकही देवकररण सापडणार नाही. मांगानी देव – धमस सोडून देणं ही काय मामुली गोष्ट होती काय?... परंतु िोकांशतका ही की, मातंग चळवळीला कोणी भाष्यकार शमळाला नाही. त्यामुळे हा घडून आलेला बदल म्हणावा तेवढा क्रांशतकारक वाटला नाही. परंतु मातंग समाज देखील बदलू िकतो. देवधमस फेकून देऊ िकतो, हे एक नवं सत्य समोर आलं हे देखील काही कमी नाही. काहींनी सांशगतलं मांग बाजा बंद करा. आणण तो बंद झाला. तर मी सांगत होतो की, समाज माझं ऐकतो आहे. आणण हे बघून काहीजण भयंकर जळू लागले आहेत. आणण शचमटे घेऊन हळूच एखाद्याला सांगू लागले आहेत की, ‘माणसानं आधी आपलं आपलं सुधारलं पाहहजे, त्याचं बघून मग समाज सुधरत असतो.’ मग यावर लोककवी वामनदादा कडसक यांछया भार्ेत मी त्यांना जाहीरपणे क्तवचारतो की, “बार मध्ये रोजच खातोस कोंबड्याचे पाय, पण समाजाचं काय?... अण्णा भाऊ साठे आणण मातंग समाज तुमचे धंदे तरी काय आहेत ते सांगा ना?... शनदान ते धंदे मी समाजाला सांगू िकेल. कोणाला श्रीमंती नको आहे?.... तुमछयाकडे हे सगळं काही कुठून येतं?... यावर मग मात्र त्यांछयाकडे ्त्् SJIF Impact Factor: 6.21 Electronic International Interdisciplinary Research Journal (EIIRJ) Electronic International Interdisciplinary Research Journal (EIIRJ) Page 149 EIIRJ Volume–IX Issues–V ISSN–2277- 8721 Sept – Oct. 2020 घेणं बंद करा, मातंग समाज हा भोळा भाबडा समाज आहे, गरीब समाज आहे, अडाणी समाज आहे. ज्याला कायसकतास व्हायचे आहे, त्याला या समाजात काम करण्याची फार मोठी संधी आहे. परंतु हे ज्याला कळत नाही त्याला मी काय करू िकतो?.... माझ्यापेक्षा समाजात शिकलेले लोकं खूप आहेत. पैसे वाले देखील खूप आहेत. घेऊन जा ना समाज पाठीमागं, माझ्या बापाचं काय जाणार आहे.. जर तसा कोणी हदसला तर त्याछया चरणावर माथा टेकवणारा पहहला माणूस मीचं असेल. Electronic International Interdisciplinary Research Journal (EIIRJ) Page 150 SJIF Impact Factor: 6.21 Peer Reviewed Refereed Journal ह ू समाजावर कुठ अत्याचार झाला असेल तर त्या हठकाणी मी काही फारसं धावून जात नाही. हकती हठकाणी म्हणून धावत जाऊ?.... एक कायसकतास म्हणून समाजात काम करण्याची माझी पद्धत थोडीिी वेगळी आहे. अन्याय झाला शतथं धावून जाण्यापेक्षा या समाजावर कोणी अन्यायच करणार नाही. याछयासाठी मी काम करतो आहे. समाजातील माझे एक प्राध्यापक शमत्र आहेत त्यांनी मला एकदा सांशगतलं की, “कोणत्यातरी राजकीय पाटीचं पाठबळ घेतल्याशिवाय कोणतंही संघटन वाढूच िकत नाही. त्यामुळे अण्णा भाऊ साठे पीपल्स फोससला पाठबळ देणारी एखादी पाटी शनवडा” त्यांना मी म्हटलं की, अण्णा भाऊ साठे पीपल्स फोसस नाही वाढली तरी चालेल, परंतु मी कोणत्याही राजकीय पक्षाचं पाठबळ घेणार नाही. एक प्राध्यापक म्हणून एवढंच करा की, अण्णा भाऊ साठे पीपल्स फोसस ही संघटना कोणत्याही राजकीय पक्षािी बांधील नाही, असं समाजासमोर जाऊन सांगा. परंतु त्यांनी आणखी तसं काही केलं नाही. इथं प्रत्येकाला राजकीय स्वातंत्र आहे. मग माझ्यावर कोण बंधनं घालणार आहे. परंतु माझी मीच माझ्यावर काही बंधनं घालून घेतलेली आहेत. कालछया लोकसभेछया शनवडणुकीछया वेळेस बी एम पी कडून मला शनवडणूक लढवण्याची एका कायसकत्यासकडून मला ऑफर आली होती. तरीही मी लोकसभेची शनवडणूक लढवली नाही. नांदेडछया पीपल्स कॉलेजमध्ये चमसकार समाज समन्वय सशमतीछया वतीने मागे एक कायसक्रम ठेवण्यात आला होता. शतथला प्रत्येक विा हा कांिीरामांचं स्वप्न साकार करण्याछया संदभासत बोलत होता. शतथं बोलताना मी सांशगतलं की, “कांिीरामांचं स्वप्न SJIF Impact Factor: 6.21 Peer Reviewed Refereed Journal समाजावर कुठ अत्याचार झाला असेल तर त्या हठकाणी मी काही फारसं धावून जात नाही. हकती हठकाणी म्हणून धावत जाऊ?.... एक कायसकतास म्हणून समाजात काम करण्याची माझी पद्धत थोडीिी वेगळी आहे. अन्याय झाला शतथं धावून जाण्यापेक्षा या समाजावर कोणी अन्यायच करणार नाही. अण्णा भाऊ साठे आणण मातंग समाज याछयासाठी मी काम करतो आहे. समाजातील माझे एक प्राध्यापक शमत्र आहेत त्यांनी मला एकदा सांशगतलं की, “कोणत्यातरी राजकीय पाटीचं पाठबळ घेतल्याशिवाय कोणतंही संघटन वाढूच िकत नाही. त्यामुळे अण्णा भाऊ साठे पीपल्स फोससला पाठबळ देणारी एखादी पाटी शनवडा” त्यांना मी म्हटलं की, अण्णा भाऊ साठे पीपल्स फोसस नाही वाढली तरी चालेल, परंतु मी कोणत्याही राजकीय पक्षाचं पाठबळ घेणार नाही. एक प्राध्यापक म्हणून एवढंच करा की, अण्णा भाऊ साठे पीपल्स फोसस ही संघटना कोणत्याही राजकीय पक्षािी बांधील नाही, असं समाजासमोर जाऊन सांगा. परंतु त्यांनी आणखी तसं काही केलं नाही. इथं प्रत्येकाला राजकीय स्वातंत्र आहे. मग माझ्यावर कोण बंधनं घालणार आहे. परंतु माझी मीच माझ्यावर काही बंधनं घालून घेतलेली आहेत. कालछया लोकसभेछया शनवडणुकीछया वेळेस बी एम पी कडून मला शनवडणूक लढवण्याची एका कायसकत्यासकडून मला ऑफर आली होती. तरीही मी लोकसभेची शनवडणूक लढवली नाही. नांदेडछया पीपल्स कॉलेजमध्ये चमसकार समाज समन्वय सशमतीछया वतीने मागे एक कायसक्रम ठेवण्यात आला होता. शतथला प्रत्येक विा हा कांिीरामांचं स्वप्न साकार करण्याछया संदभासत बोलत होता. शतथं बोलताना मी सांशगतलं की, “कांिीरामांचं स्वप्न Electronic International Interdisciplinary Research Journal (EIIRJ) Page 150 EIIRJ Volume–IX Issues–V ISSN–2277- 8721 Sept – Oct. 2020 साकार करण्यासाठी एक प्रमुख कायसकतास लागतो. तो कसा असला पाहहजे ते आधी मला सांगा, ती लायकी माझ्यात नसेल तर नैशतक दृष्र्टया बोलण्याचा मला अशधकारच राहणार नाही. आणण जर का मग ती लायकी माझ्यात असेल तर चळवळीसाठी लागणारी साधनं मला द्या, मी बघतो कांिीरामांचं स्वप्न महाराष्ट्रात कसं साकार होणार नाही ते” यावर स्टेज वरछया सवस मान्यवरांनी, संयोजकांनी या प्रस्तावावर शनणितच क्तवचार करू असं सांशगतलं. परंतु नंतर त्यांचा काय क्तवचार ठरला, तो अजूनही माहहती नाही. तर असो, मी राजकारण तर करणार नाहीच आणण यदाकदाशचत राजकीय भूशमका घेतलीच तर मग माझं राजकरण कुठल्या हदिेने जाणार आहे . ही गोष्ट इथं आपल्या ध्यानात नक्कीच आली असेल. मुंबई क्तवद्यापीठास अण्णा भाऊ साठे यांचे नाव देण्यात यावे, या मागणीछया संदभासत जनजागृती करत मराठवाड्यात मी हफरलो आहे. कोणत्याही गावात जा, एकही माणूस तुम्हाला हदसणार नाही. ऊस तोडणीछया कामाला, वीट भर्टटीछया कामाला, समाज पोटासाठी भटकत आहे. त्यांछया मुलांछया शिक्षणाचं काय?.... यावर क्तवचार करायला कोणाकडे वेळ आहे काय?.... दीक्षाभूमीवर हकंवा चैतन्य भूमी वर बौद्ध लोक लाखोंछया संख्येने एकत्र जमतात. तसं कुठेतरी मातंग समाज एकत्र येऊन, आपली ििी जगाला दाखवणार आहे का?.... याचा आम्हाला कुठेतरी क्तवचार करावा लागणार आहे. प्रा. अिोक जाधव सरांनी या पुस्तकाछया माध्यमातून ही जी काही क्तवचार मांडण्याची संधी उपलब्ध करून हदली आहे. आणण प्रा. डॉ. नामदेव वाघमारे यांछया सांगण्यावरून मनातल्या भावना व्यि करता आल्या, त्यामुळे या उभयतांचे आभार मानल्यावाचून राहवत नाही. अण्णा भाऊ साठे आणण मातंग समाज खरंच अण्णा भाऊ साठे जन्म िताब्दी वर्स म्हणजे मातंग समाजासाठी एक भाग्यिाली वर्स आहे. कारण िंभर वर्ासत जेवढं प्रबोधन झालं नाही, तेवढं समाज प्रबोधन या वर्सभरात घडणार आहे. आंबेडकरी चळवळीचे भाष्यकार राजा झाले यांचे शनवासण झालं. त्यांनी दोन वर्ाांपूवी मला सांशगतलं Volume–IX EIIRJ Volume–IX Issues–V ISSN–2277- 8721 Sept – Oct. 2020 साकार करण्यासाठी एक प्रमुख कायसकतास लागतो. तो कसा असला पाहहजे ते आधी मला सांगा, ती लायकी माझ्यात नसेल तर नैशतक दृष्र्टया बोलण्याचा मला अशधकारच राहणार नाही. आणण जर का मग ती लायकी माझ्यात असेल तर चळवळीसाठी लागणारी साधनं मला द्या, मी बघतो कांिीरामांचं स्वप्न महाराष्ट्रात कसं साकार होणार नाही ते” यावर स्टेज वरछया सवस मान्यवरांनी, संयोजकांनी या प्रस्तावावर शनणितच क्तवचार करू असं सांशगतलं. परंतु नंतर त्यांचा काय क्तवचार ठरला, तो अजूनही माहहती नाही. तर असो, मी राजकारण तर करणार नाहीच आणण यदाकदाशचत राजकीय भूशमका घेतलीच तर मग माझं राजकरण कुठल्या हदिेने जाणार आहे . ही गोष्ट इथं आपल्या ध्यानात नक्कीच आली असेल. SJIF Impact Factor: 6.21 Peer Reviewed Refereed Journal Electronic International Interdisciplinary Research Journal (EIIRJ) Electronic International Interdisciplinary Research Journal (EIIRJ) Page 151 EIIRJ Volume–IX Issues–V ISSN–2277- 8721 Sept – Oct. 2020 मग सरकार देखील तेवढ्याच मोठ्या प्रमाणात हे जन्म िताब्दी वर्स साजरं करेल. परंतु राजा ढाले यांछया अपेक्षेप्रमाणे आम्हाला प्रयत्न करता आले नाहीत. याची खंत मला अजन्म राहणार आहे. या जन्मिताब्दी वर्ासछया शनशमत्ताने कायसकत्याांनी आपल्या मनातला द्वेर् काढून टाकला पाहहजे. एक आंबा म्हणतो, तर एक शचंच म्हणतो, हे कुठंतरी थांबलं पाहहजे. कोणाछयाही मागे जाऊन काही शमळणार नाही. क्तबलवरी या कथेतील अण्णा भाऊ साठेंची नाशयका तमािाचा फड चालवून 200 माणसांना जगवत होती काही मातब्बर शतला वाडे बांधून द्यायला तयार होते , काहींनी जशमनीची बक्षीस पत्र तयार ठेवली होती. परंतु क्तबलवरी त्या मोहाला बळी पडली नाही. कुठल्याही पररणस्थतीत शतने आपला स्वाशभमान क्तवकला नाही. यातून आम्ही काही शिकणार आहोत की नाही?.... िाहीर अमर िेख हा अण्णा भाऊ साठेंचा एक सछचा दोस्त होता, तसा एखादा सछचा मुसलमान दोस्त आम्हाला शमळेल काय?.... सत्तू भोसले फहकराचा दोस्त होता. तसा एखादा दोस्त मराठा समाजातून आम्हाला कोणी शमळेल काय?.... क्तवष्णू पंत कुलकणी सारखा एखादा ब्राम्हण आम्हाला शमळेल काय ?.... याचा मी िोध घेतो आहे. आणण या कामी मला समाजातील प्रत्येक माणसाची मला गरज आहे. SJIF Impact Factor: 6.21 Peer Reviewed Refereed Journal मग सरकार देखील तेवढ्याच मोठ्या प्रमाणात हे जन्म िताब्दी वर्स साजरं करेल. परंतु राजा ढाले यांछया अपेक्षेप्रमाणे आम्हाला प्रयत्न करता आले नाहीत. याची खंत मला अजन्म राहणार आहे. या जन्मिताब्दी वर्ासछया शनशमत्ताने कायसकत्याांनी आपल्या मनातला द्वेर् काढून टाकला पाहहजे. अण्णा भाऊ साठे आणण मातंग समाज एक आंबा म्हणतो, तर एक शचंच म्हणतो, हे कुठंतरी थांबलं पाहहजे. कोणाछयाही मागे जाऊन काही शमळणार नाही. क्तबलवरी या कथेतील अण्णा भाऊ साठेंची नाशयका तमािाचा फड चालवून 200 माणसांना जगवत होती काही मातब्बर शतला वाडे बांधून द्यायला तयार होते , काहींनी जशमनीची बक्षीस पत्र तयार ठेवली होती. परंतु क्तबलवरी त्या मोहाला बळी पडली नाही. कुठल्याही पररणस्थतीत शतने आपला स्वाशभमान क्तवकला नाही. यातून आम्ही काही शिकणार आहोत की नाही?.... िाहीर अमर िेख हा अण्णा भाऊ साठेंचा एक सछचा दोस्त होता, तसा एखादा सछचा मुसलमान दोस्त आम्हाला शमळेल काय?.... सत्तू भोसले फहकराचा दोस्त होता. तसा एखादा दोस्त मराठा समाजातून आम्हाला कोणी शमळेल काय?.... क्तवष्णू पंत कुलकणी सारखा एखादा ब्राम्हण आम्हाला शमळेल काय ?.... याचा मी िोध घेतो आहे. आणण या कामी मला समाजातील प्रत्येक माणसाची मला गरज आहे. लहुजी साळवेंनी ज्या पद्धतीनं उमाजी नाईकांछया छया माध्यमातून कोळी, शभल्ल, रामोिी, मांग, महार, चांभार, लोहार, सुतार, यांना सोबत घेऊन एक राजकीय लढा उभा केला. नेमकं तसंच मी काही करू पाहतो आहे. अण्णा भाऊ साठेनी देणखल सांगून ठेवलं आहे की, “बोलवा त्या सकलांना, साऱ्या जहालांना, सोिाशलस्टाना, िेकापवाल्यांना, दशलतांछया क्तवक्तवध पक्षांना, आणण त्याचबरोबर साऱ्या लोकिाहीवाद्याना या साऱ्यांछया एकजुटीचा रथ करा आणण त्या रथावर स्वार होऊन चालत राहा.” अण्णा भाऊ साठेंचा हा संदेि ऐकून कोणी उतावीळ होऊ नये, एवढंच माझं म्हणणं आहे. नाही तर आपल्या समाजातल्या लोकांना एक फार वंगळी सवय लागलेली आहे. लातूरछया एका सभेत मी सहजच बोललो होतो की, “मातंग समाज भक्तवष्यात सत्ताधारी बनू िकतो,” तर लगेच एकानं सत्ता संपादन मेळावा Electronic International Interdisciplinary Research Journal (EIIRJ) Page 152 SJIF Impact Factor: 6.21 Peer Reviewed Refereed Journal लहुजी साळवेंनी ज्या पद्धतीनं उमाजी नाईकांछया छया माध्यमातून कोळी, शभल्ल, रामोिी, मांग, महार, चांभार, लोहार, सुतार, यांना सोबत घेऊन एक राजकीय लढा उभा केला. नेमकं तसंच मी काही करू पाहतो आहे. अण्णा भाऊ साठेनी देणखल सांगून ठेवलं आहे की, “बोलवा त्या सकलांना, साऱ्या जहालांना, सोिाशलस्टाना, िेकापवाल्यांना, दशलतांछया क्तवक्तवध पक्षांना, आणण त्याचबरोबर साऱ्या लोकिाहीवाद्याना या साऱ्यांछया एकजुटीचा रथ करा आणण त्या रथावर स्वार होऊन चालत राहा.” अण्णा भाऊ साठेंचा हा संदेि ऐकून कोणी उतावीळ होऊ नये, एवढंच माझं म्हणणं आहे. नाही तर आपल्या समाजातल्या लोकांना एक फार वंगळी सवय लागलेली आहे. लातूरछया एका सभेत मी सहजच बोललो होतो की, “मातंग समाज भक्तवष्यात सत्ताधारी बनू िकतो,” तर लगेच एकानं सत्ता संपादन मेळावा SJIF Impact Factor: 6.21 Electronic International Interdisciplinary Research Journal (EIIRJ) Page 152 SJIF Impact Factor: 6.21 Peer Reviewed Refereed Journal Electronic International Interdisciplinary Research Journal (EIIRJ) Page 152 EIIRJ Volume–IX Issues–V ISSN–2277- 8721 Sept – Oct. अण्णा भाऊ साठे आणण मातंग समाज 2020 घेऊन, स्वतःचं हसं करून घेतलं, शसडकोछया एका कायसक्रमात मी बोललो होतो की, “मातंग समाजातील नवीन पोरांना आता आपणही या देिात बंद पाळावा असं वाटू लागलं आहे,” मी असं बोलल्या बरोबर काही हौिी लोकांनी भारत बंदची हाकं देऊन स्वत:चं सोंग करून घेतलं. म्हणून असं वागणं समाजासाठी घातक ठरणार आहे. याचं भान आम्ही ठेवलं पाहीजे, एवढंच या हठकाणी सांगून थांबतो. EIIRJ Volume–IX Issues–V ISSN–2277- 8721 Sept – Oct. 2020 घेऊन, स्वतःचं हसं करून घेतलं, शसडकोछया एका कायसक्रमात मी बोललो होतो की, “मातंग समाजातील नवीन पोरांना आता आपणही या देिात बंद पाळावा असं वाटू लागलं आहे,” मी असं बोलल्या बरोबर काही हौिी लोकांनी भारत बंदची हाकं देऊन स्वत:चं सोंग करून घेतलं. म्हणून असं वागणं समाजासाठी घातक ठरणार आहे. याचं भान आम्ही ठेवलं पाहीजे, एवढंच या हठकाणी सांगून थांबतो. SJIF Impact Factor: 6.21 Peer Reviewed Refereed Journal Electronic International Interdisciplinary Research Journal (EIIRJ) Page 153
https://openalex.org/W2069318699	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0041978&type=printable	English	null	Rapid and Sensitive Detection of Plesiomonas shigelloides by Loop-Mediated Isothermal Amplification of the hugA Gene	PloS one	2,012	cc-by	4,941	Abstract Plesiomonas shigelloides is one of the causative agents of human gastroenteritis, with increasing number of reports describing such infections in recent years. In this study, the hugA gene was chosen as the target to design loop-mediated isothermal amplification (LAMP) assays for the rapid, specific, and sensitive detection of P. shigelloides. The performance of the assay with reference plasmids and spiked human stools as samples was evaluated and compared with those of quantitative PCR (qPCR). No false-positive results were observed for the 32 non-P. shigelloides strains used to evaluate assay specificity. The limit of detection for P. shigelloides was approximately 20 copies per reaction in reference plasmids and 56103 CFU per gram in spiked human stool, which were more sensitive than the results of qPCR. When applied in human stool samples spiked with 2 low levels of P. shigelloides, the LAMP assays achieved accurate detection after 6-h enrichment. In conclusion, the LAMP assay developed in this study is a valuable method for rapid, cost-effective, and simple detection of P. shigelloides in basic clinical and field laboratories in the rural areas of China. Citation: Meng S, Xu J, Xiong Y, Ye C (2012) Rapid and Sensitive Detection of Plesiomonas shigelloides by Loop-Mediated Isothermal Amplification of the hugA Gene. PLoS ONE 7(10): e41978. doi:10.1371/journal.pone.0041978 Editor: Stefan Bereswill, Charite´-University Medicine Berlin, Germany R i d M 30 2012 A t d J 27 2012 P bli h d O t b 15 2012 Citation: Meng S, Xu J, Xiong Y, Ye C (2012) Rapid and Sensitive Detection of Plesiomonas shigelloides by Loop-Mediated Isothermal Amplification of the hugA Gene. PLoS ONE 7(10): e41978. doi:10.1371/journal.pone.0041978 di f ll h ´ d l Editor: Stefan Bereswill, Charite´-University Medicine Berlin, Germany Received May 30, 2012; Accepted June 27, 2012; Published October 15, 2012 Received May 30, 2012; Accepted June 27, 2012; Published October 15, 2012 ng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. Copyright: 2012 Meng et al. This is an open-access article distributed under the terms of the Creative Commons Attributi unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Science and Technology Key Project on Major Infectious Diseases (2008ZX10004-001, 2009ZX10004-101, 2011ZX10004-001). Rapid and Sensitive Detection of Plesiomonas shigelloides by Loop-Mediated Isothermal Amplification of the hugA Gene Shuang Meng, Jianguo Xu, Yanwen Xiong, Changyun Ye* State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping, Beijing, People’s Republic of China Shuang Meng, Jianguo Xu, Yanwen Xiong, Changyun Ye* g g g g gy State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping, Beijing, People’s Republic of China Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Beijing, People’s Republic of China Abstract The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: yechangyun@icdc.cn Competing Interests: The authors have declared that no competing interests exist. * E-mail: yechangyun@icdc.cn Several methods such as culture studies and biochemical assays have been developed for detection and identification of P. shigelloides. Despite their effectiveness and accuracy, these assays are time consuming, usually requiring up to 5 days to complete. The isolation of P. shigelloides from clinical samples has often been unsuccessful owing to the fastidious nature of the organism and the low level of transient bacteremia associated with the disease process. Rapid, specific, and sensitive nucleic acid amplification tests (NAATs) such as standard and real-time PCR have been developed to detect P. shigelloides by targeting genes encoding for major virulence factors [5,13–14]. The major limitation to the widespread use of these assays is the fact that a sophisticated thermal cycler is an indispensable requirement of such tests, thereby limiting their wide applicability. Bacterial strains and culture conditions A total of 52 strains (20 P. shigelloides and 32 non-P. shigelloides strains, as described in Table 1) was used for specificity testing. The bacterial load of the strains used for specificity evaluation was 105 pg/mL, which is high enough to avoid the false-negative amplification. Strain ATCC 51903 was used for the assay optimization , sensitivity evaluation, and simulating human stool samples. P. shigelloides and Enterobacteriaceae were cultured at 37uC overnight on brain heart infusion agar (BHI; BD Diagnostic Systems, Sparks, MD, USA). Non-Enterobacteriaceae strains were grown on blood agar, except for Vibrio strains, for which trypticase soy agar (TSA) supplemented with 2% NaCl was used. Campylo- bacter strains were grown under microaerophilic conditions (85% N2, 10% CO2, and 5% O2). LAMP Detection of Plesiomonas shigelloides LAMP Detection of Plesiomonas shigelloides Table 1. Strains used in this study. Latin name Strain Strains number Plesiomonas shigelloides ATCC51903 1 Isolated strains 19 Enteropathogenic E. coli Isolated strain 1 Enterotoxigenic E. coli Isolated strain 1 Enteroinvasive E. coli Isolated strain 1 Enterohemorrhagic E. coli EDL933 1 Enteroaggregative E. coli Isolated strain 1 Salmonella enteric ATCC14028 1 Shigella flexneri Isolated strain 1 Shigella sonnei ATCC25931 1 Salmonella typhi H98125 1 Klebsiella pneumoniae ATCC700603 1 Proteus vulgaris Isolated strain 1 Aeromonas veronii 1.2205 1 Clostridium perfringens Isolated strain 1 Enterobacter cloacae Isolated strain 1 Serratia marcescens Isolated strain 1 Vibrio parahaemolyticus ATCC17802 1 Staphylococcus aureus ATCC6538 1 Streptococcus pneumoniae Isolated strain 1 Streptococcus pyogenes Isolated strain 1 Streptococcus sanguis Isolated strain 1 Streptococcus salivarius Isolated strain 1 Streptococcus bovis Isolated strain 1 Enterococcus faecalis ATCC35667 1 Yersinia enterocolitica ATCC23715 1 Pseudomonas aeruginosa ATCC15442 1 Aeromonas hydrophila ATCC7966 1 Listeria monocytogenes 54003 2 Enterobacter sakazakii ATCC51329 1 Campylobacter jejuni ATCC33291 1 Vibrio minicus Isolated strain 1 Vibrio vulnificus Isolated strain 1 Vibrio fluvialis Isolated strain 1 doi:10.1371/journal.pone.0041978.t001 Table 1. Strains used in this study. Ethics statement Feces samples were acquired with the written informed consent from a healthy donor. This study was reviewed and approved by the ethics committee of the National Institute for Communicable Disease Control and Prevention, China CDC, according to the medical research regulations of the Ministry of Health, China. LAMP primers and reaction conditions p A set of 6 primers targeted toward the hugA gene of the species P. shigelloides were designed using PrimerExplorer V4 software (Eiken Chemical Co. Ltd., Tokyo, Japan) based on the conserved sequences determined by the alignment of the hugA gene sequences obtained from GenBank. The primers shown in Table 2 were synthesized by Sangon Biotech (Shanghai, China). The primer sequences and their positions in the expression site of the hugA gene are shown in Fig. 1. All LAMP reactions were performed with the Loopamp Kit (Eiken Chemical Co. Ltd., Tokyo, Japan) in a 25-mL mixture containing 1.6 mM FIP and BIP primers (each), 0.8 mM LF and LB primers (each), 0.2 mM F3 and B3 primers (each), 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 8 mM MgSO4, 10 mM (NH4)2SO4, 0.1% Tween 20, 0.8 M betaine, 1.4 mM deoxynu- cleoside triphosphates (dNTPs; each), and 1 mL of Bst DNA polymerase (8 U/mL). The reaction mixture was incubated in a real-time turbidimeter LA320 (Teramecs, Tokyo, Japan) at 65uC for 60 min, followed by 80uC for 5 min to terminate the reaction. Positive and negative samples were distinguished from one another by a turbidity cutoff value of 0.1. After amplification, the LAMP products were detected by electrophoresis on 2% agarose gels with ethidium bromide staining or were determined by visual inspection after adding 1 mL of 1,0006 SYBR green I. Introduction Plesiomonas shigelloides is a motile, oxidase-positive, facultatively anaerobic, gram-negative rod bacterium, which is presently classified in the family Vibrionaceae [1]. P. shigelloides has been isolated from a variety of environmental sources, primarily aquatic [2–4], and is distributed worldwide. Moreover, P. shigelloides has been associated with seafood-associated outbreaks [5]. P. shigelloides has been implicated as an agent of human gastroenteritis for many years, with an increasing number of reports describing such infections during the recent years [6]. This bacterium is also of considerable clinical importance as the etiological agent respon- sible for different types of opportunistic infections [7]. Although extra-intestinal infections such as septicemia, cellulitis, and meningitis caused by P. shigelloides are rarely reported, it has been associated with secondary infections in immunocompromised patients [8–10]. Salerno et al [12] also described an infection of P. shigelloides with a fatal outcome in a newborn. Since most laboratories concentrate on recovery of Salmonella, Shigella, E. coli and other classical enteropathogens, P. shigelloides may be overlooked during routine culture of stool samples. The lack of routine analysis for P. shigelloides in cases of gastroenteritis leads to only sporadic and occasional identification of this bacterium [11]. However, the greatest challenge to clinicians and epidemiologist is the lack of a rapid, early, and accurate diagnostic method for the detection of P. shigelloides as an emerging infectious disease in China. Recently, a novel NAAT technology termed loop-mediated isothermal amplification (LAMP) has attracted a great deal of attention as a rapid, accurate, and cost-effective method for detection of pathogens in clinical diagnostics [15,16]. LAMP employs 4–6 specially designed primers and a strand-displacing Bst DNA polymerase (isolated from Bacillus stearothermophilus) to amplify up to 109 target DNA copies under isothermal conditions (60uC– 65uC) within an hour [15], making LAMP a potentially rapid and simple diagnostic tool for detection of P. shigelloides infection. In this study, we aimed to develop a rapid, sensitive, and highly specific LAMP assay to detect P. shigelloides and evaluate the assay performance with pathogen-simulated human stool. 1 October 2012 \| Volume 7 \| Issue 10 \| e41978 PLOS ONE \| www.plosone.org Figure 1. Names and locations of target sequences used as primers for the expression site of hugA LAMP. doi:10.1371/journal.pone.0041978.g001 were homogenized with buffered peptone water (BPW; BD Diagnostic Systems) supplemented with 50 mg/mL ampicillin (Sigma-Aldrich, St. Louis, MO, USA), followed by incubation at 37uC for up to 10 h. Aliquots (1 mL) of the enrichment broth were removed at 4, 6, 8, and 10 h, and processed similarly by QIAamp DNA stool mini kit (QIAGEN). Two microliters of the sample DNA extracts were subjected to both LAMP and qPCR assays, which were repeated twice for each sample. Genomic DNA of the 32 non-P. shigelloides strains were detected by LAMP to determine the specificity of the hugA LAMP assay. All detection assays were performed in triplicate. A set of 3 reference plasmids with varying concentrations (106, 104, and 102 copies/mL) was amplified in two ways (10 times on 1 day and once on each of 10 days) to evaluate the reproducibility of the LAMP assay. The intra-assay coefficient of variation (CVi) and inter-assay coefficient of variation (CVo) were analyzed at the time of peak precipitation, as measured by turbidity on a real-time turbidimeter. Statistical analyses were conducted using SAS software version 9.1. LAMP application in simulated human stools The specificity of the LAMP assay targeting hugA gene was tested with 52 bacterial strains (Table 1). Positive amplifications were observed in 20 P. shigelloides strains within a 60-min incubation period. By contrast, 32 non-P. shigelloides strains were not amplified after a 60-min incubation period. This result indicates that no false-positive amplifications were observed with these heterologous species in the LAMP assay. Human stool specimens were obtained from a healthy donor and immediately processed. We have screened the human stool specimens by culture assay and PCR, no P. shigelloides strain was detected and no positive amplification of P. shigelloides DNA was observed. To determine the detection limit of LAMP in human stool, serial 10-fold dilutions of a mid-log-phase culture of P. shigelloides grown in BHI were prepared in phosphate-buffered saline (PBS) and quantified using the standard plating method, resulting in spiking levels between 56107 and 56101 CFU/g stool. Aliquots (0.2 g) of the stools were removed for DNA extraction with a QIAamp DNA stool mini kit (QIAGEN, Venlo, Nether- lands). This experiment was independently repeated 3 times, and the supernatants (2 mL) were used for both LAMP and qPCR. In addition, the capability of the LAMP assay to detect low levels of P. shigelloides in human stool was evaluated. For this application, simulated human stool samples were spiked with P. shigelloides cultures at 2 levels: 1 to 2 and 10 to 20 CFU/0.5 g. The samples Reference plasmid To compare the sensitivities of the LAMP assay and quantita- tive PCR (qPCR), the serially diluted reference plasmids (at concentrations of 16106, 105, 104, 103, 102, 101, and 100 copies/ mL) containing the target DNA were used to define the limit of detection. The qPCR assay was performed with the primers and probe in Table 2. qPCR amplification was performed in a 20-mL reaction volume containing 0.25 mM primer (each), 0.18 mM probe, 16Premix (Takara Bio, Inc., Otsu, Japan) Ex TaqTM, and 2 mL of DNA template. The assays were conducted using the PCR settings of pre-denaturation at 95uC for 30 s, 40 cycles of denaturation at 94uC for 5 s, and extension at 60uC for 34 s in an ABI PRISM system (Applied Biosystems, Carlsbad, CA, US). Fluorescence readings were acquired using the 6-carboxyfluor- escein (FAM) channel. To determine the sensitivity of the LAMP assay, a recombinant plasmid containing the target sequence of the hugA gene from the P. shigelloides strain (ATCC 51903) was constructed as follows: 1) A pair of primers was designed to span the sequences between the F3 and B3 primers; forward primer hugA-F (59- GCGGTCTCCGGTTTCAAAT-39) and reverse primer hugA- R (59-GTTACCGGGTCTGCGTTATG-39); 2) the PCR prod- ucts (259 bp) were cloned into the pEASY-T1 vector using the pEASY-T1 Cloning Kit (Transgen, Beijing, China); 3) the recombinant plasmid was quantified with a NanoPhotometer (Implen, Munich, Germany) and was serially diluted (to concen- trations of 16106, 105, 104, 103, 102, 101, and 100 copies/mL) in order to evaluate the limit of detection and the reproducibility of the LAMP assay. October 2012 \| Volume 7 \| Issue 10 \| e41978 PLOS ONE \| www.plosone.org 2 LAMP Detection of Plesiomonas shigelloides Sensitivity of LAMP assay The limit of detection of LAMP (Fig. 2A) and qPCR for the hugA gene were 20 and 200 copies/reaction, respectively. This result indicates that the LAMP assay is more sensitive than qPCR for detecting P. shigelloides DNA. The LAMP products could also be detected by electrophoresis (Fig. 2C) and visual inspection after adding 1 mL of 1,0006 SYBR green I (Fig. 2D). Table 2. LAMP and qPCR primers used in this study to detect P. shigelloides. Assay type Primer/Probe name Sequence (59-39) Position hugA-LAMP F3 AACACGTTGCAGCCCATC 3776–3973 B3 ACTTTACCGCCGAAGACAAG 3958–3977 FIP CGTTACGACGAAGCGTTCCGTGAAGTGAGTACCGGTGGTGT 3846–3867, 3806–3824 BIP GTCAGCCAATCAGTCGCCGCAATATCGCCGGCTCCGAG 3878–3897, 3940–3957 LF ACCGAGCATGGAAGAGATGT 3825–3844 LB GCGACAGGTGATCTTCGCTAC 3918–3938 hugA-qPCR F GGAATATCGGCCTGTACAT 4022–4040 R TATGGCGGCGATATTTA 4121–4137 Probe FAM-CCCCAGACTTTGCTGCGACCATCGG-BHQ-1 4046–4070 doi:10.1371/journal.pone.0041978.t002 Table 2. LAMP and qPCR primers used in this study to detect P. shigelloides. Table 2. LAMP and qPCR primers used in this study to detect P. shigelloides. doi:10.1371/journal.pone.0041978.t002 3 Figure 2. Real-time sensitivity and detection limit of hugA-LAMP. (A) Real-time sensitivity of hugA-LAMP as monitored by the measurement of turbidity (optimal density at 650 nm). A turbidity of .0.1 was considered to be positive for hugA-LAMP. The detection limit was 20 copies/reaction. (B) The relation between the threshold time (Tt) of each sample and the log copies/reaction. The standard curve was drawn on the basis of 3 independent repeats and the linear relationship R2 =0.9787. (C) Sensitivities of electrophoretic analysis of hugA-LAMP amplified products. Lane M: DL2000 marker; lane 1: 26106 copies/reaction; lane 2: 26105 copies/reaction; lane 3: 26104 copies/reaction; lane 4: 26103 copies/reaction; lane 5: 26102 copies/reaction; lane 6: 26101 copies/reaction; lane 7: 26100 copies/reaction; lane 8: no template. (D) SYBR green I fluorescent dye-mediated monitoring of hugA-LAMP assay amplification. The original orange color of the SYBR Green I changed to green in case of positive amplification, whereas the original orange color was retained for a negative control with no amplification. doi:10.1371/journal.pone.0041978.g002 LAMP Detection of Plesiomonas shigelloides LAMP Detection of Plesiomonas shigelloides Figure 2. Real-time sensitivity and detection limit of hugA-LAMP. (A) Real-time sensitivity of hugA-LAMP as monitored by the measurement of turbidity (optimal density at 650 nm). A turbidity of .0.1 was considered to be positive for hugA-LAMP. The detection limit was 20 copies/reaction. (B) The relation between the threshold time (Tt) of each sample and the log copies/reaction. The standard curve was drawn on the basis of 3 independent repeats and the linear relationship R2 =0.9787. Sensitivity of LAMP assay (C) Sensitivities of electrophoretic analysis of hugA-LAMP amplified products. Lane M: DL2000 marker; lane 1: 26106 copies/reaction; lane 2: 26105 copies/reaction; lane 3: 26104 copies/reaction; lane 4: 26103 copies/reaction; lane 5: 26102 copies/reaction; lane 6: 26101 copies/reaction; lane 7: 26100 copies/reaction; lane 8: no template. (D) SYBR green I fluorescent dye-mediated monitoring of hugA-LAMP assay amplification. The original orange color of the SYBR Green I changed to green in case of positive amplification, whereas the original orange color was retained for a negative control with no amplification. doi:10.1371/journal.pone.0041978.g002 LAMP Detection of Plesiomonas shigelloides A typical LAMP judgment graph generated for human stool enrichment samples is shown in Fig. 3. Regardless of spiking levels, none of the 4-h-enrichment samples tested positive for P. shigelloides by either LAMP or qPCR. We observed positive results with LAMP at 6 h with significantly higher threshold time (Tt) values, while for samples enriched for 8, 10, 12, and 24 h, lower and stable Tt values were observed. A similar trend of detection was observed for qPCR (Table 3). In addition, qPCR results were presented by cycles, which were approximately 1 min/cycle. Therefore, an additional 20–40 min of amplification time was needed for qPCR when testing the same enrichment sample. summarizes LAMP and qPCR results in human stool samples spiked with 2 low levels (1 to 2 and 10 to 20 CFU/0.5 g) of P. shigelloides strains after various enrichment periods. A typical LAMP judgment graph generated for human stool enrichment samples is shown in Fig. 3. Regardless of spiking levels, none of the 4-h-enrichment samples tested positive for P. shigelloides by either LAMP or qPCR. We observed positive results with LAMP at 6 h with significantly higher threshold time (Tt) values, while for samples enriched for 8, 10, 12, and 24 h, lower and stable Tt values were observed. A similar trend of detection was observed for qPCR (Table 3). In addition, qPCR results were presented by cycles, which were approximately 1 min/cycle. Therefore, an additional 20–40 min of amplification time was needed for qPCR when testing the same enrichment sample. To the best of our knowledge, this is the first study applying the novel LAMP technology for the detection of P. shigelloides in human stool. Previously, spiked samples were usually enriched overnight without characterizing the effects of different enrichment times on the detection outcomes [23,24]. In this study, P. shigelloides strain ATCC 51903 was used in experiments with simulated human stool samples, with the LAMP assays having a detection limit of 56103 CFU/g stool. Positive detection occurred after a 6-h period of enrichment, and consistently thereafter, for the human stool samples spiked with 2 low levels (1 to 2 and 10 to 20 CFU/0.5 g) of ATCC 51903. We observed that the LAMP assay performed better than qPCR with respect to detection limit and assay speed in spiked human stool. LAMP Detection of Plesiomonas shigelloides In general, molecular level-based detection methods such as PCR and LAMP are subjected to a variety of inhibitors present in clinical samples. Some researchers have reported that the Bst polymerase in LAMP is less sensitive to the presence of inhibitors than the Taq polymerase used in classic PCR [25,26]. Our results showed that the LAMP assay is more accurate and sensitive than qPCR methods using simulated human stool samples, and proved markedly faster than qPCR by at least 20 min, thereby significantly shortening the total assay time. LAMP Detection of Plesiomonas shigelloides Figure 3. A typical LAMP amplification graph generated when testing human stool samples spiked with the low level of P. shigelloides strain after various enrichment periods (4, 6, 8, 10, 12, and 24 h). In this graph, the human stool sample was spiked with 1.3 CFU of Strain ATCC 51903. doi:10.1371/journal.pone.0041978.g003 lengthy. Modified PCR techniques such as nested PCR and real- time PCR are complicated and require a high-precision thermal cycler, and therefore, they are not adapted to diagnosing P. shigelloides in basic clinical and field laboratories in rural areas. In contrast, the LAMP assay reported in this study is advantageous because of the following 3 features: rapid reaction, simple operation, and easy detection. The LAMP assay does not require sophisticated and expensive equipment, maintaining a constant temperature of 60uC–65uC for 1 h is sufficient for the reaction [16]. These features demonstrate that the LAMP assay is suitable for the detection of P. shigelloides in basic clinical and field laboratories in rural areas. Although the pathogenesis of P. shigelloides-associated gastroen- teritis has not yet been elucidated, a number of potential virulence factors have been described [19,20]. Acquisition of iron is known to be involved in the virulence of a variety of bacterial pathogens [21,22]. Heme is the most abundant source of iron in the body, and many pathogenic bacteria possess heme transport systems. The hugA gene, one of the characterized genes encoded in the heme iron utilization system of P. shigelloides, encodes an outer membrane receptor that is required for heme iron utilization. Figure 3. A typical LAMP amplification graph generated when testing human stool samples spiked with the low level of P. shigelloides strain after various enrichment periods (4, 6, 8, 10, 12, and 24 h). In this graph, the human stool sample was spiked with 1.3 CFU of Strain ATCC 51903. doi:10.1371/journal.pone.0041978.g003 In this study, all hugA gene sequences of P. shigelloides recorded in the GeneBank were aligned, and the LAMP primers were designed on the basis of the conserved regions. We tested 32 non-P. shigelloides strains to evaluate the specificity of the hugA LAMP assay for the bacteria, with the results showing that the specificity of the LAMP assay was 100%. summarizes LAMP and qPCR results in human stool samples spiked with 2 low levels (1 to 2 and 10 to 20 CFU/0.5 g) of P. shigelloides strains after various enrichment periods. Evaluation of LAMP assay in simulated human stool Evaluation of LAMP assay in simulated human stool Evaluation of LAMP assay in simulated human stool The detection limit of LAMP in simulated human stool was also examined. The LAMP assays detected the presence of P. shigelloides strains down to as little as 56103 CFU/g. By comparison, the qPCR assays had a detection limit of 56104 CFU/g for hugA gene in simulated human stool samples (data not shown). Table 3 The CVi was assessed by testing 3 reference plasmids with varying concentrations (106, 104, and 102 copies/mL), 10 times in a single run, whereas the CVo was assessed by testing the same plasmids 10 times in 10 separate runs. The CVi ranged from 1.21% to 1.54%, while the CVo ranged from 2.17% to 3.23%. PLOS ONE \| www.plosone.org October 2012 \| Volume 7 \| Issue 10 \| e41978 4 References 1. Garrity GM, Bell JA, Lilburn TG (2003) Taxonomic outline of the procaryotes. Bergey’s Manual of Systematic Bacteriology, 2nd edn. Release 4.0 Available: http://141.150.157.80/bergeysoutline/main.htm. 14. Gu W, Levin RE (2006) Quantitative detection of Plesiomonas shigelloides in clam and oyster tissue by PCR. Int J Food Microbiol 111:81–86. 15. Mori Y, Notomi T (2009) Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. J Infect Chemother 15:62–69. p g y 2. Krovacek K, Eriksson LM, Gonza´lez-Rey C (2000) Isolation, biochemical and serological characterisation of Plesiomonas shigelloides from freshwater in Northern Europe. Comp Immunol Microbiol Infect Dis 23:45–51. 16. Notomi T, Okayama H, Masubuchi H (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28:E63. 3. Reinhardt JF, George WL (1985) Plesiomonas shigelloides-associated diarrhea. JAMA 253:3294–3295. p 17. Paul R, Siitonen A, Ka¨rkka¨inen P (1990) Plesiomonas shigelloides b 17. Paul R, Siitonen A, Ka¨rkka¨inen P (1990) Plesiomonas shigelloides bacteremia in a healthy girl with mild gastroenteritis. J Clin Microbiol 28:1445–1446. 4. Escobar JC, Bhavnani D, Trueba G (2012) Plesiomonas shigelloides infection, Ecuador, 2004–2008. Emerg Infect Dis 18:322–324. healthy girl with mild gastroenteritis. J Clin Microbiol 28:1445–1446. 18. Lee AC, Yuen KY, Ha SY (1996) Plesiomonas shigelloides sept 18. Lee AC, Yuen KY, Ha SY (1996) Plesiomonas shigelloides septicemia: ca and literature review. Pediatr Hematol Oncol 13:265–269. 5. Gonza´lez-Rey C, Svenson SB, Bravo L (2000) Specific detection of Plesiomonas shigelloides isolated from aquatic environments, animals and human diarrhoeal cases by PCR based on 23S rRNA gene. FEMS Immunol Med Microbiol 29:107–113. , , ( ) g p and literature review. Pediatr Hematol Oncol 13:265–269. 19. Janda JM, Abbott SL (1993) Expression of hemolytic activity by Plesiomonas shigelloides. J Clin Microbiol 31:1206–1208. 20. Santos JA, Gonza´lez CJ, Lo´pez TM (1999) Hemolytic and elastolytic activities influenced by iron in Plesiomonas shigelloides. J Food Prot 62:1475–1477. 6. Wouafo M, Pouillot R, Kwetche PF (2006) An acute foodborne outbreak due to Plesiomonas shigelloides in Yaounde, Cameroon. Foodborne Pathog Dis 3:209–211. 21. Villarreal DM, Phillips CL, Kelley AM (2008) Enhancement of recombinant hemoglobin production in Escherichia coli BL21(DE3) containing the Plesiomonas shigelloides heme transport system. Appl Environ Microbiol 74:5854–5856. 7. Miller WA, Miller MA, Gardner IA (2006) Salmonella spp., Vibrio spp., Clostridium perfringens, and Plesiomonas shigelloides in marine and freshwater invertebrates from coastal California ecosystems. Microb Ecol 52:198–206. 22. LAMP Detection of Plesiomonas shigelloides clinical samples. We proved that the LAMP assay demonstrated superior performance to qPCR in simulated human stool samples, and may facilitate rapid and reliable diagnosis of P. shigelloides infections in basic clinical and field laboratories in rural areas. Author Contributions Conceived and designed the experiments: SM CY JX. Performed the experiments: SM. Analyzed the data: SM YX. Contributed reagents/ materials/analysis tools: SM YX. Wrote the paper: SM CY. Conceived and designed the experiments: SM CY JX. Performed the experiments: SM. Analyzed the data: SM YX. Contributed reagents/ materials/analysis tools: SM YX. Wrote the paper: SM CY. Discussion Some reports have suggested that P. shigelloides may cause enteric diseases in normal hosts [5,17]. Moreover, septicemia, cellulitis, meningitis, and cholecystitis due to P. shigelloides have also been documented among immunocompromised patients or patients with other underlying conditions [8–10]. The mortality rate associated with Plesiomonas-induced septicemia is high [18]. Individuals with serious infections are faced with the lack of a rapid and sensitive diagnostic method and inappropriate antimi- crobial therapy, and therefore, they, often, cannot receive timely treatment, leading to diseases and fatal outcomes. In conclusion, the LAMP assay was successfully validated in this study for rapidity, sensitivity, specificity, and robustness; thus, this assay may serve as an effective means for screening P. shigelloides in It is well known that the bacteriological methods available for the isolation and identification of P. shigelloides are tedious and Table 3. Comparison of LAMP and qPCR assays in human stool samples spiked with low levels of P. shigelloides. Cell level (no. of CFU/ 0.5 g) LAMP Tt (min) after enrichment qPCR CT (cycles) after enrichment 4 h 6 h 8 h 10 h 12 h 24 h 4 h 6 h 8 h 10 h 12 h 24 h 1–2 Not available 30.3 27.1 25.2 22.7 22.4 Not available 36.5 33.2 30.8 29.6 29.1 10–20 Not available 28.5 25.3 23.8 21.5 21.3 Not available 33.7 29.1 27.7 26.3 25.8 doi:10.1371/journal.pone.0041978.t003 PLOS ONE \| www.plosone.org 5 October 2012 \| Volume 7 \| Issue 10 \| e41978 Table 3. Comparison of LAMP and qPCR assays in human stool samples spiked with low levels of P. sh 5 LAMP Detection of Plesiomonas shigelloides References Oldham AL, Wood TA, Henderson DP (2008) Plesiomonas shigelloides hugZ encodes an iron-regulated heme binding protein required for heme iron utilization. Can J Microbiol 54:97–102. y 8. Schneider F, Lang N, Reibke R (2009) Plesiomonas shigelloides pneumonia. Med Mal Infect 39:397–400. 9. Ozdemir O, Sari S, Terzioglu S (2010) Plesiomonas shigelloides sepsis and meningoencephalitis in a surviving neonate. J Microbiol Immunol Infect 43:344–346. 23. Ohtsuka K, Tanaka M, Ohtsuka T (2010) Comparison of detection methods for Escherichia coli O157 in beef livers and carcasses. Foodborne Pathog Dis 7:1563– 1567. 10. Auxiliadora-Martins M, Bellissimo-Rodrigues F, Viana JM (2010) Septic shock caused by Plesiomonas shigelloides in a patient with sickle beta-zero thalassemia. Heart Lung 39:335–339. 24. Hara-Kudo Y, Niizuma J, Goto I (2008) Surveillance of Shiga toxin-producing Escherichia coli in beef with effective procedures, independent of serotype. Foodborne Pathog Dis 5:97–103. 11. Chan SS, Ng KC, Lyon DJ (2003) Acute bacterial gastroenteritis: a study of adult patients with positive stool cultures treated in the emergency department. Emerg Med J 20:335–338. 25. Kaneko H, Kawana T, Fukushima E (2007) Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances. J Biochem Biophys Methods 70:499–501. 12. Salerno A, Cizˇna´r I, Krovacek K (2010) Phenotypic characterization and putative virulence factors of human, animal and environmental isolates of Plesiomonas shigelloides. Folia Microbiol (Praha) 55:641–647. 26. Okada K, Chantaroj S, Taniguchi T (2010) A rapid, simple, and sensitive loop- mediated isothermal amplification method to detect toxigenic Vibrio cholerae in rectal swab samples. Diagn Microbiol Infect Dis 66:135–139. g ( ) 13. Herrera FC, Santos JA, Otero A (2006) Occurrence of Plesiomonas shigelloides in displayed portions of saltwater fish determined by a PCR assay based on the hugA gene. Int J Food Microbiol 108:233–238. PLOS ONE \| www.plosone.org October 2012 \| Volume 7 \| Issue 10 \| e41978 6
https://openalex.org/W4205939211	https://www.ejournal.warmadewa.ac.id/index.php/paduraksa/article/view/2895/3007	Indonesian	null	PEMANFAATAN INFRASTRUKTUR DAN RUANG PASCA PEMBANGUNAN INFRASTRUKTUR DI DESA KERTA, PAYANGAN, GIANYAR	Paduraksa : Jurnal Teknik Sipil Universitas Warmadewa	2,021	cc-by	6,242	297 297 DOI: 10.22225/pd.10.2.2895.297-316 PEMANFAATAN INFRASTRUKTUR DAN RUANG PASCA PEMBANGUNAN INFRASTRUKTUR DI DESA KERTA, PAYANGAN, GIANYAR Desak Putu Korpiyoni1) Desak Putu Korpiyoni1) 1) Magister Arsitektur, Universitas Udayana, Denpasar, Bali desakkorpiyoni@gmail.com Desak Putu Korpiyoni1) 1) Magister Arsitektur, Universitas Udayana, Denpasar, Bali 1) Magister Arsitektur, Universitas Udayana, Denpasar, Bali desakkorpiyoni@gmail.com PADURAKSA: Volume 10 Nomor 2, Desember 2021 ABSTRACT Indonesia consists of urban areas and rural areas. The problems also can be find it’s not only urban areas have many problems, in rural areas have many problems too. Some problems in rural areas which is not complete is lack of infrastructure, which is to support accessibility and productivity distribution of agriculture product, and it has been hampering economic growth in rural areas. In several things needed for support villagers agriculture activities in rural area is design of infrastructure should be compatible with society and public needs in rural area, other than that, the potential in rural area can be visible. Kerta Village located in Payangan District, Gianyar Regency. The consideration of Kerta Village was chosen as an object of research because infrastructure built with massive and followed with space development during six years clearly visible so that until now village tourism has been built. It need requires an analysis can planning and development according to the needs of Kerta Villagers. The objective of this research is to discover the impact of the planning and development of infrastructure in Kerta Village. This research used qualitative method with case study approach. The result of the research is the analysis of planning and development infrastructure in Kerta Village during six years started 2014 until 2019 and also space utilization since the infrastructure development in Kerta Village. Keywords: rural areas, infrastructure development, spatial utilization P-ISSN: 2303-2693 E-ISSN: 2581-2939 PADURAKSA: Volume 10 Nomor 2, Desember 2021 DOI: 10.22225/pd.10.2.2895.297-316 298 PADURAKSA: Volume 10 Nomor 2, Desember 2021 PENDAHULUAN memiliki kewenangan secara penuh untuk dapat mengatur dirinya sendiri. Menurut Asnudin (2009) Pembangunan infrastruktur perdesaan tersebut, dengan pelibatan secara penuh masyarakat setempat dalam setiap tahapan (tahap perencanaan s/d tahap operasional dan pemeliharaan). Pelibatan masyarakat perdesaan dalam pembangunan infrastruktur perdesaan akan memberikan beberapa dampak, antara lain: 1) kualitas pekerjaan yang dihasilkan; 2) keberlangsungan operasional dan pemeliharaan infrastruktur tersebut; 3) kemampuan masyarakat dalam membangun suatu kemitraan dengan berbagai pihak, serta 4) penguatan kapasitas masyarakat untuk mampu mandiri memfasilitasi kegiatan masyarakat dalam wilayahnya. Hal ini berdampak pada perencanaan desa yang akan cenderung tepat sasaran dan sesuai dengan kebutuhan masyarakat desa. Salah satunya adalah kebutuhan terhadap infrastruktur. Infrastruktur pada kawasan perdesaan menjadi kebutuhan dasar bagi masyarakat, untuk dapat memperlancar aksesibilitas dan produktifitas dalam mendistribusikan hasil pertanian. memiliki kewenangan secara penuh untuk dapat mengatur dirinya sendiri. Menurut Asnudin (2009) Pembangunan infrastruktur perdesaan tersebut, dengan pelibatan secara penuh masyarakat setempat dalam setiap tahapan (tahap perencanaan s/d tahap operasional dan pemeliharaan). Pelibatan masyarakat perdesaan dalam pembangunan infrastruktur perdesaan akan memberikan beberapa dampak, antara lain: 1) kualitas pekerjaan yang dihasilkan; 2) keberlangsungan operasional dan pemeliharaan infrastruktur tersebut; 3) kemampuan masyarakat dalam membangun suatu kemitraan dengan berbagai pihak, serta 4) penguatan kapasitas masyarakat untuk mampu mandiri memfasilitasi kegiatan masyarakat dalam wilayahnya. Hal ini berdampak pada perencanaan desa yang akan cenderung tepat sasaran dan sesuai dengan kebutuhan masyarakat desa. Salah satunya adalah kebutuhan terhadap infrastruktur. Infrastruktur pada kawasan perdesaan menjadi kebutuhan dasar bagi masyarakat, untuk dapat memperlancar aksesibilitas dan produktifitas dalam mendistribusikan hasil pertanian. Kawasan perdesaan dengan kondisi Kawasan perdesaan di Indonesia memiliki isu banyaknya kemiskinan. Selain kemiskinan, kurangnya infrastruktur dan tidak mendukungnya keberadaan infrastruktur di kawasan perdesaan, dalam mendukung mobilitas dan produktifitas masyarakat desa juga menjadi isu pada kawasan perdesaan. Menurut Ellis & Freeman (2004) dalam Prastyanti (2015) penyebab dari banyaknya kantong-kantong kemiskinan di pedesaan adalah rendahnya pendapatan keluarga di pedesaan di seluruh negara yang berkaitan dengan sempitnya kepemilikan tanah dan ternak dan ketergantungan yang tinggi masyarakat pedesaan pada pertanian khusunya tanaman pangan. Indonesia memiliki program pembangunan nasional yang tidak saja menyasar kawasan perkotaan saat ini, tetapi kawasan perdesaan juga menjadi prioritas utama dalam pembangunan nasional. Salah satunya adalah tercermin dari Nawacita pemerintah pusat saat ini, yaitu membangun Indonesia dari pinggiran dengan memperkuat daerah-daerah dan desa dalam kerangka negara kesatuan. Hal ini diperkuat dengan terbitnya Undang- undang Nomor 6 Tahun 2014 tentang Desa. ABSTRAK Indonesia terdiri dari kawasan perkotaan dan kawasan perdesaan. Tidak hanya kawasan perkotaan yang ditemukan permasalahan, kawasan perdesaan juga ditemukan permasalahan. Beberapa permasalahan di kawasan perdesaan yang belum bisa di tuntaskan adalah kurangnya infrastruktur yang bisa mendukung aksesibilitas, dan produktifitas distribusi hasil pertanian, hal ini menjadi penghambat pertumbuhan ekonomi kawasan perdesaan. Beberapa hal yang dibutuhkan untuk mendukung aktivitas pertanian masyarakat di kawasan perdesaan adalah perencanaan infrastruktur yang sesuai dengan kebutuhan masyarakat kawasan perdesaan, selain itu potensi dari kawasan perdesaan tersebut mulai terlihat. Desa Kerta terletak di Kecamatan Payangan Kabupaten Gianyar. Dijadikannya Desa Kerta sebagai objek penelitian dikarenakan terbangunnya infrastruktur secara masif dan diikuti perkembangan ruang selama enam tahun terlihat jelas sehingga sampai saat ini terbangun wisata desa. Hal ini membutuhkan analisa di setiap tahap perencanaan pada proses pembangunannya, sehingga Desa Kerta selama kurun waktu enam tahun dapat melakukan perencanaan dan pembangunan sesuai kebutuhan masyarakat Desa Kerta. Tujuan dari penelitian ini adalah untuk mengetahui perkembangan ruang yang terjadi akibat proses perencanaan dan pembangunan infrastruktur di Desa Kerta. Metode yang digunakan dalam penelitian ini adalah metode kualitatif dengan pendekatan beberapa studi kasus. Hasil penelitian ini berupa analisa perencanaan dan pembangunan infrastruktur di Desa Kerta dalam kurun waktu enam tahun pada tahun 2014 hingga tahun 2019 serta kondisi pemanfaatan ruang pasca pembangunan infrastruktur yang terjadi di Desa Kerta. Kata kunci: kawasan perdesaan, pembangunan infrastruktur, pemanfaatan ruang P-ISSN: 2303-2693 E-ISSN: 2581-2939 DOI: 10.22225/pd.10.2.2895.297-316 299 PENDAHULUAN Desa mendapatkan perhatian lebih dari pemerintah, dalam hal ini desa Kawasan perdesaan dengan kondisi infrastruktur yang tidak dalam kondisi baik, membuat distribusi hasil pertanian P-ISSN: 2303-2693 E-ISSN: 2581-2939 PADURAKSA: Volume 10 Nomor 2, Desember 2021 DOI: 10.22225/pd.10.2.2895.297-316 300 menjadi kurang maksimal. Jalur dengan kondisi masih berupa tanah tanpa perkerasan membuat mobilitas kendaraan menjadi sangat sulit. Namun saat infrastruktur telah terbangun dan sesuai dengan kebutuhan masyarakat, dampak positif terlihat di kawasan perdesaan tersebut dan masyarakat desa, tidak saja dari kemudahan dari aksesibilitas masyarakat dalam menjangkau suatu tempat dan produktifitas dalam kegiatan pertanian hingga distribusi hasil pertanian, selain itu juga akan berkembangnya potensi yang ada di kawasan perdesaan tersebut. dukung tersebut menyebabkan lemahnya peran kawasan agropolitan terhadap pengembangan wilayah di Kabupaten Kulonprogo, serta lemahnya peran sektor- sektor industri pertanian yang ada pada kawasan agropolitan yang belum memberikan trickle down effect terhadap sektor-sektor yang ada disekitarnya. Sehingga penelitian tersebut dilakukan untuk dapat mengkaji dari keberhasilan pengembangan kawasan agropolitan di Kabupaten Kulonprogo. Adapun hasil dari penelitian tersebut berupa, pembangunan kawasan agropolitan tersebut belum memberikan dampak yang signifikan terhadap pembangunan perdesaan di Kabupaten Kulonprogo. Faktor yang mempengaruhi adalah kurangnya ketersediaan sarana dan prasarana agribisnis hulu-hilir, tidak didukung oleh SDM yang handal maka perlu peningkatan kapasitas dalam pengelolaan sarana dan prasarana sub sistem agribisnis, serta inovasi teknologi pertanian untuk mewujudkan penerapan kawasan agropolitan. Keberadaan dan kebutuhan infrastruktur pada kawasan perdesaan memang menjadi topik yang paling sering dalam penelitian kawasan perdesaan. Sebelumnya telah terdapat penelitian sejenis mengenai kawasan perdesaan. Penelitian Suroyo (2014) menyusun penelitian mengenai Pengembangan Kawasan Agropolitan di Kabupaten Kulonprogo, Daerah Istimewa Yogyakarta. Penelitian tersebut membahas mengenai ditetapkannya Kabupaten Kulonprogo menjadi kawasan agropolitan pada tahun 2010. Pemerintah daerah belum dapat secara signifikan memberikan dorongan terhadap ketersediaan berupa sarana dan prasarana agribisnis. Minimnya daya Penelitian lain yang sejenis adalah jurnal Pranoto et al. (2006) mengenai Pembangunan Perdesaan Berkelanjutan Melalui Model Pengembangan Agropolitan Penelitian tersebut menguraikan mengenai mengembangkan P-ISSN: 2303-2693 E-ISSN: 2581-2939 PADURAKSA: Volume 10 Nomor 2, Desember 2021 DOI: 10.22225/pd.10.2.2895.297-316 301 konsep pembangunan perdesaan berkelanjutan, melalui model pengembangan agropolitan pada beberapa tipologi dan tingkat perkembangan kawasan agropolitan. PENDAHULUAN Hasil dari penelitian tersebut yang dapat dijadikan sebagai referensi dengan penelitian saat ini adalah pengembangan agropolitan sebagai pendekatan pembangunan perdesaan yang berkelanjutan, dapat tercapai ketika peningkatan produksi pertanian dan peningkatan saran dan prasarana permukiman, transportasi dan pemasaran disertai dengan peningakatan konservasi sumber daya alam, pengembangan agrobisnis dan pembangunan agroindustry, dibarengi dengan perbaikan pemasaran secara berkesinambungan, perencanaan dan pelaksanaan program dibarengi dengan peningkatan peran dan kinerja kelembagaan yang ada. Selain itu penelitian Saraswaty (2013) mengenai strategi pengembangan infrastruktur kawasan agropolitan, menganalisis strategi prioritas dengan penyediaan infrastruktur, dan diharapkan dapat berfungsi sebagai penggerak utama dalam pengembangan kawasan agaropolitan. Dikaitkan dengan penelitian saat ini, strategi dalam penataan ruang kawasan perdesaan dengan infrastruktur yang telah dibangun dan dianalisis mengenai strategi keberlanjutannya. Mengenai obyek penelitian yang serupa, menggunakan penelitian Rudita (2012) mengenai potensi obyek wisata dan keterpaduan dalam pengembangan kawasan agropolitan. Penelitian tersebut mengindikasi bahwa, sektor pertanian dan sektor pariwisata berjalan sendiri-sendiri dan keterlibatan masyarakat masih sangat kurang. Dalam penelitian yang dilakukaan saat ini, akan melihat bagaimana potensi alam dan potensi buatan akan dapat dikembangkan di kawasan perdesaan Kerta dan menganalisis strategi keberlanjutan sektor pertanian dan sektor pariwisata dapat seimbang. Objek penelitian saat ini terdapat di Desa Kerta. Desa Kerta terletak di Kecamatan Payangan, Kabupaten Gianyar. Desa Kerta merupakan kawasan perdesaan yang pada Rencana Tata Ruang Wilayah (RTRW) Tahun 2012-2032 Kabupaten Gianyar merupakan kawasan perdesaan dan kawasan agrowisata. Dijadikan Desa Kerta menjadi objek penelitian ini adalah terbangunnya infrastruktur secara masif dalam kurun waktu enam tahun, dengan diiringi berkembangnya ruang dengan potensi yang ada menjadi wisata desa, sehingga perlu dianalisa yang mengawali hal tersebut mulai dari perencanaan dan pembangunan infrastruktur. Tujuan P-ISSN: 2303-2693 E-ISSN: 2581-2939 PADURAKSA: Volume 10 Nomor 2, Desember 2021 E-ISSN: 2581-2939 DOI: 10.22225/pd.10.2.2895.297-316 302 penelitian ini adalah untuk mengetahui perkembangan ruang yang terjadi, akibat pembangunan infrastruktur kawasan perdesaan Kerta dalam kurun waktu enam tahun, mulai dari perencanaan hingga pembangunan. Sehingga hal yang diperlukan adalah mengenai proses perencanaan dan identifikasi pembangunan infrastruktur yang terjadi selama kurun waktu enam tahun, yaitu tahun 2014 hingga tahun 2019 di Desa Kerta dan menganalisa ruang yang berkembang disekitar infrastruktur yang terbangun pada rentan waktu tersebut, dengan didukung potensi yang ada disekitarnya dan wisata desa terbangun. kawasan perdesaan Kerta dan masyarakat Desa Kerta. Kajian pustaka yang digunakan dalam penelitian ini berupa penataan ruang, pembangunan desa, kawasan perdesaan, infrastruktur, pembangunan kawasan perdesaan. Penataan Ruang Menurut Wahid (2014) perencanaan dalam arti yang luas merupakan suatu proses yang berkelanjutan dalam merumuskan dan melaksanakan satu matriks multidimensi dan keputusan yang saing berhubungan yang diarahkan untuk mencapai tujuan-tujuan pembangunan dalam satu jangka dan urutan waktu yang ditentukan dengan cara-cara yang optimal. Tata ruang sebagai wujud penataan ruang pada intinya merupakan sarana untuk mewujudkan pembangunan berkelanjutan. Adapun pembangunan yang berkelanjutan adalah pembangunan yang memasukkan perlimbangan lingkungan hidap dalam kebijaksanaan pembangunan sehingga pembangunan itu tidak saja untuk memecahkan masalah peningkatan kesejahteraan masa sekarang tetapi juga peningkatan kesejahteraan jangka panjang. Konsep pembangunan berkelanjutan mengakomodasikan tujuan pertumbuhan Perbedaan penelitian yang dilakukan saat ini dengan penelitian yang telah dilakukan sebelumnya oleh Suroyo (2014) dan Pranoto et al. (2006) adalah Desa Kerta merupakan kawasan perdesaan agrowisata, sehingga pertanian dan pariwisata tergabung dalam satu kawasan perdesaan. Perencanaan infrastruktur dan ruang selain untuk dapat mendukung kegiatan pertanian yang ada di Desa Kerta juga dapat mengembangkan potensi berupa wisata desa yang dapat dikembangkan di Desa Kerta. Penelitian ini juga menunjukkan mengenai tahapan perencanaan infrastruktur dan dampak dari infrastruktur yang telah terbangun terhadap P-ISSN: 2303-2693 E-ISSN: 2581-2939 PADURAKSA: Volume 10 Nomor 2, Desember 2021 E-ISSN: 2581-2939 DOI: 10.22225/pd.10.2.2895.297-316 303 ekonomi, tujuan pengentasan kemiskinan dan pengelolaan sumber alam dan lingkungan dalam rangka pemenuhan kebutuhan jangka panjang dan tujuan tersebut konsisten satu sama lainnya. Dalam hubungan tersebut dapat dipahami pentingnya peranan perencanaan tata ruang wilayah dalam konsep pembangunan keberlanjutan sebagai kebijaksanaan pembangunan dalam alokasi sumber alam dan pengelolaan lingkungan hidup untuk menopang pertumbuhan dan pengentasan kemiskinan yang bersifat jangka panjang. penggunaan lahan adalah zona konservasi dan fungsi lindung. Beberapa tujuan pemanfaatan ruang kawasan pedesaan adalah: 1. Pemanfaatan ruang di kawasan pedesaan diatur untuk membantu meningkatkan kesejahteraan rakyat dan sebagai bentuk penanggulangan dampak negatif pada lingkungan buatan dan sosial. 1. Pemanfaatan ruang di kawasan pedesaan diatur untuk membantu meningkatkan kesejahteraan rakyat dan sebagai bentuk penanggulangan dampak negatif pada lingkungan buatan dan sosial. 2. Fungsi kawasan pedesaan ditingkatkan agar menciptakan keseimbangan dalam perkembangan lingkungan dan cara hidup masyarakat. Menurut Gai et al. (2020) pedesaan memiliki sumberdaya sebagai penyangga kehidupan perekonomian masyarakat yaitu pertanian dan lingkungan hidup. Desa juga berfungsi sebagai penyangga interaksi sosial, meningkatkan kesejahteraan masyarakat dan sebagai penyeimbang ekosistem lingkungan. Sumber daya alam dan lingkungan hidup seringkali dianggap sebagai halangan dalam pengembangan pertanian, namun dapat pula dikondisikan dengan memanfaatkan kearifan lokal dan pendekatan lingkungan yang berkelanjutan demi kelestarian alam. PADURAKSA: Volume 10 Nomor 2, Desember 2021 Penataan Ruang Pada tujuan pengembangan pedesaan, pola penggunaan lahan ruang pada desa diutamakan untuk zona konservasi dan fungsi lindung. Hal ini dapat dilihat pada peruntukan ruang kawasan pedesaan, dimana kecenderungan 3. Tata ruang pedesaaan perlu dicapai untuk menciptakan pengembangan kehidupan manusia yang optimal, serasi, selaras dan seimbang. 4. Dinamika pembangunan dan kawasan pedesaan perlu didorong agar mencapai kehidupan pedesaan yang berkeadilan dan tetap melestarikan budaya. 5. Membentuk hubungan fungsional antar kawasan pedesaan. 4. Dinamika pembangunan dan kawasan pedesaan perlu didorong agar mencapai kehidupan pedesaan yang berkeadilan dan tetap melestarikan budaya. 5. Membentuk hubungan fungsional antar kawasan pedesaan. P-ISSN: 2303-2693 E-ISSN: 2581-2939 PADURAKSA: Volume 10 Nomor 2, Desember 2021 DOI: 10.22225/pd.10.2.2895.297-316 304 mudah dan lain-lain. Selain itu menurut Rondinelli (1985) dalam Kasikoen (2011) dalam pembangunan spasial jenis-jenis keterkaitan yang utama dapat dikelompokkan dalam 7 (tujuh) tipe, antara lain keterkaitan fisik, keterkaitan ekonomi, keterkaitan pergerakan penduduk, keterkaitan teknologi, keterkaitan interaksi sosial, keterkaitan pelayanan, keterkaitan politik administrasi dan organisasi 6. Mengendalikan peralihan penggunaan lahan secara besar- besaran. 7. Mencegah terjadinya kerusakan lingkungan. 8. Peningkatan manfaat pada sumber daya alam dan sumber daya lainnya. 9. Menciptakan lingkungan perumahan dan permukiman yang layak huni. Perencanaan Pembangunan Desa 10. Menciptakan peningkatan dalam kondisi ekonomi masyarakat pedesaan Conyers & Hills (1990) dalam Solekhan (2014) berpendapat bahwa “Define planning as acontinuous process which involves decisiona, or choices, about alternative ways of using available resources, with the aim of achieving particular goals at some time in the future”. (Perencanaan didefinisikan sebagai suatu proses yang berkesinambungan yang mencakup keputusan-keputusan atau pilihan-pilihan berbagai alternative penggunaan sumberdaya untuk mencapai tujuan-tujuan pada masa yang akan datang). Menurut Aldillah (2020) teori pembangunan desa ditujukan guna meningkatkan produktifitas dan potensi wilayah desa dengan teori pembangunan desa. Menurut Rondinelli (1985) dalam Aldillah (2020) yang memprioritaskan integrasi desa kota, sangat terkait dengan pelaksanaan program pembangunan perdesaan, selain itu pemanfaatan hasil pembangunan fisik desa yaitu dengan membangun atau memperbaiki prasarana jalan desa akan menciptakan atau memperbaiki kehidupan masyarakat desa. Dengan adanya pembangunan prasarana jalam, masyarakat dapat menggunakan jalan tersebut dengan berbagai kebutuhan yang mereka perlukan, seperti malaku-kan mobilitas, pemasaran hasil pertaniannya, mangangkut hasil pertanian agar lebih Menurut Salam (2002) dalam Solekhan (2014) berpendapat bahwa “perencanaan adalah usaha membuat suatu pilihan tindakan dari berbagai alternatif yang mungkin dapat tersedia yang meliputi strategi, kebijakan, program, proyek, dan prosedur dalam rangka mencapai tujuan P-ISSN: 2303-2693 E-ISSN: 2581-2939 PADURAKSA: Volume 10 Nomor 2, Desember 2021 DOI: 10.22225/pd.10.2.2895.297-316 305 Yang artinya bahwa sistem infrastruktur merupakan hal yang selalu berkaitan dengan kehidupan masyarakat baik di dalam sistem sosial maupun sistem ekonomi guna untuk memenuhi kebutuhan dasar manusia secara fisik dengan menyediakan transportasi, pengairan, drainase, bangunan-bangunan dan fasilitas publik lainnya yang digunakan untuk kepentingan masyarakat. Selain itu menurut Kodoatie (2003) dalam Aldillah (2020) infrastruktur dapat juga didefinisikan sebagai fasilitas-fasilitas fisik yang dikembangkan atau dibutuhkan oleh agen-agen publik untuk fungsi-fungsi pemerintahan dalam penyediaan air, tenaga listrik, pembuangan limbah, transportasi dan pelayanan-pelayanan similar untuk memfasilitasi tujuan-tujuan ekonomi dan sosial. organisasi”. Menurut Siagian (2000) mendefinisikan planning sebagai keseluruhan proses pemikiran dan penentuan secara matang daripada hal-hal yang akan dikerjakan dimasa yang akan datang dalam rangka mencapai tujuan yang ditentukan (Solekhan 2014). Pembangunan desa agropolitan menjadi konsep dalam pembangunan desa. Menurut Rondinelli (1983) dalam Holis (2017) konsep desentralisasi pengembangan wilayah perdesaan atau Development from below (DFB), yaitu dalam hal ini agropolitan atau minapolitan untuk menghadirkan keseimbangan dalam sebuah pembangunan desa, alasan munculnya strategi agropolitan atau tipe- tipe pembangunan dari bawah. Infrastruktur Menurut Grigg dalam Kodoatie (2005) dalam Aldillah (2020), infrastruktur merujuk pada sistem fisik yang menyediakan transportasi, pengairan, drainase, bangunan-bangunan gedung, dan fasilitas publik yang lain yang dibutuhkan untuk memenuhi kebutuhan dasar manusia dalam lingkup sosial dan ekonomi. Pengertian ini merujuk pada infrastruktur sebagai suatu sistem. Dimana infrastruktur dalam sebuah sistem adalah bagian-bagian berupa sarana dan prasarana (jaringan) yang tidak terpisahkan satu sama lain. Menurut Grigg (1988) dalam Mustar (2019) infrastruktur dibagi kedalam enam kategori dasar bersar infrastruktur yaitu kelompok jalan (jalan, jalan raya, jembatan), kelompok pelayanan transportasi (transit, jalan rel, pelabuhan, Bandar udara), kelompok air (air bersih, air kotor, semua sistem air, termasuk jalan air), kelompok manajemen limbah (sistem manajemen limbah padat), kelompok bangunan dan fasilitas olahraga luar, Kelompok produksi dan distribusi energy P-ISSN: 2303-2693 E-ISSN: 2581-2939 PADURAKSA: Volume 10 Nomor 2, Desember 2021 E-ISSN: 2581-2939 DOI: 10.22225/pd.10.2.2895.297-316 306 (listrik dan gas). Menurut Mustar (2019) untuk menunjang pembangunan desa secara maksimal diperlukan dukungan infrastruktur perdesaan untuk memberikan kemudahan bagi pelaku usaha dalam menjalankan kegiatan ekonominya dan memudahkan masyarakat dalam mengakses fasilitas kebutuhan dasar lainnya. Isu dan permasalahan yang dihadapi pemerintah terhadap hasil pembangunan infrastruktur perdesaan adalah belum maksimalnya pemanfaatan dan atau tidak berkelanjutannya sarana dan prasarana yang telah dibangun. Untuk dapat menjadikan sarana dan prasarana yang dibangun berkelanjutan diperlukan dukungan dari beberapa aspek seperti teknis, kelembagaan, keuangan, peran serta masyarakat dan aspek lingkungan. pembangunan harus mencerminkan perubahan total suatu masyarakat atau penyesuaian sistem sosial secara keseluruhan, tanpa mengabaikan keragaman kebutuhan dasar dan keinginan individual maupun kelompok-kelompok sosial yang ada di dalamnya, untuk bergerak maju menuju suatu kondisi yang lebih baik, secara material maupun spiritual. Menurut Anonim (2002) kawasan perdesaan adalah kawasan yang mempunyai kegiatan utama pertanian termasuk pengelolaan sumber daya alam dengan susunan fungsi kawasan sebagai tempat permukiman perdesaan, pelayanan jasa pemerintahan, pelayanan sosial dan kegiatan ekonomi. Menurut Hardianti et al. (2017) keberhasilan pembangunan desa yang berlangsung di desa disamping di tentukan oleh partisipasi masyarakat juga nilai-nilai tradisional yang mendasari keterlibatan masyarakat sebagai potensi yang dapat digerakan dalam pembangunan melalui strategi manajemen yang sesuai. Untuk mewujudkan pembangunan berkelanjutan di suatu daerah, diperlukan komponen penduduk yang berkualitas. Karena dari penduduk yang berkualitas itulah memungkinkan untuk bisa mengolah dan mengelola potensi sumber daya alam Pembangunan Perdesaan Pembangunan menurut Todaro (2000) dalam Sigalingging (2014) diartikan sebagai suatu proses perubahan sosial dengan partisipatori yang luas dalam suatu masyarakat yang dimaksudkan untuk mencapai kemajuan sosial dan material (termasuk bertambah besarnya keadilan, kebebasan dan kualitas lainnya yang dihargai) untuk mayoritas rakyat melalui kontrol yang lebih besar terhadap lingkungan mereka. Pada hakekatnya PADURAKSA: Volume 10 Nomor 2, Desember 2021 P-ISSN: 2303-2693 E-ISSN: 2581-2939 P-ISSN: 2303-2693 E-ISSN: 2581-2939 DOI: 10.22225/pd.10.2.2895.297-316 307 penelitian ini dilakukan dengan wawancara dan observasi lapangan. Observasi yang gunakan adalah observasi non partisipan yang dimana peneliti tidak terlibat dan hanya sebagai pengamat independen. Sedangkan teknik wawancara yang dilakukan adalah dengan teknik wawancara tidak terstruktur, yang bertujuan agar wawancara bersifat terbuka. Dalam penelitian ini proses analisis data kualitatif menggunakan analisis data selama dilapangan model Miles dan Huberman. Menurut Miles & Huberman (1984) dalam Sugiyono (2017) mengemukakan bahwa aktivitas dalam analisis data kualitatif dilakukan secara interaktif dan berlangsung secara terus menerus sampai tuntas, sehingga datanya sudah jenuh. Aktifitas dalam analisis data yaitu reduksi data (data reduction, penyajian data (data display), dan conclusion drawing/verification. dengan baik, tepat, efesien dan maksimal dengan tetap menjaga kelestarian lingkungan. METODE PENELITIAN Metode penelitian ini menggunakan metode penelitian kualitatif dengan pendekatan studi kasus. Digunakannya penelitian kualitatif dengan pendekatan studi kasus karena penelitian ini membutuhkan eksplorasi, observasi secara mendalam pada setiap tahapan perencanaan pembangunan di Desa Kerta. Sasaran dan target penelitian ini adalah Pemerintah Desa Kerta, masyarakat Desa Kerta dan kelembagaan, hal ini dikarenakan tiga komponen ini yang menjadi penggerak utama disetiap tahapan pembanguann Desa Kerta mulai dari perencanaan, pemanfaatan dan pengendalian. Teknik penetuan informan dalam penelitian ini menggunakan teknik sampling purposive sampling dan snowball sampling. Informan yang terpilih nantinya adalah yang mengetahui tahapan dalam pembangunan desa mulai dari tahapan perencanaaan. Informan yang terpilih adalah Perbekel Desa Kerta, perangkat desa, masyarakat Desa Kerta dan kelompok sadar wisata (Pokdarwis) Desa Kerta. Teknik pengumpulan data pada HASIL DAN PEMBAHASAN Desa Kerta merupakan desa yang terletak di Kecamatan Payangan, kabupaten Gianyar. Desa Kerta ditetapkan menjadi desa definitif pada tahun 1958. Secara administratif Desa Kerta terdiri dari delapan dusun dan delapan desa adat. Desa Kerta merupakan kawasan perdesaan dan juga menjadi kawasan agropolitan P-ISSN: 2303-2693 E-ISSN: 2581-2939 PADURAKSA: Volume 10 Nomor 2, Desember 2021 DOI: 10.22225/pd.10.2.2895.297-316 308 Kabupaten Gianyar dan kawasan agrowisata. Secara keseluruhan luas wilayah Desa Kerta adalah 1442.3 Ha yang terbagi menjadi delapan dusun yaitu Dusun Mawang, Dusun Penyabangan, Dusun Kerta, Dusun Margatengah, Dusun Saren, Dusun Seming, Dusun Bunteh, Dusun Pilan. menjadi awal dari seluruh perencanaan yang akan dilakukan selama enam tahun kedepan sejak tahun 2014. Kondisi infrastruktur kawasan perdesaan di Desa Kerta sebelum tahun 2014 belum adanya perkerasan atau masih berupa tanah pada jalan desa dan jalan usaha tani yang ada di Desa Kerta. Hal ini menjadi penghambat utama dalam distribusi hasil pertanian, karena masyarakat kesulitan mengakses langsung tegalan dengan roda empat untuk dapat memindahkan hasil pertanian, hanya dengan roda dua tegalan dapat dijangkau, sehingga hal ini membutuhkan waktu yang lumayan lama untuk distribusi hasil pertanian. Gambar 1. Peta Administrasi Desa Kerta (Sumber: Profil Desa Kerta, 2018) Adapun permasalahan infrastruktur yang terjadi di Desa Kerta yang menyebabkan terhambatnya kegiatan masyarakat Desa Kerta dalam pendistribusian hasil pertanian adalah jalan usaha tani, jalan desa dan drainase. Permasalahan jalan usaha tani dan jalan lingkungan terlihat pada belum adanya perkerasan jalan, dan kondisi sudah terdapat perkerasan namun rusak. Permasalahan drainase terlihat pada kondisi drainase yang rusak dan belum adanya drainase. Kondisi permasalahan infrastruktur ini terjadi di seluruh dusun yang ada di Desa Kerta sebagai berikut pada Tabel 1. Gambar 1. Peta Administrasi Desa Kerta (Sumber: Profil Desa Kerta, 2018) Identifikasi perkembangan ruang di Desa Kerta mengacu pada saat awal penyusunan Rencana Pembangunan Jangka Menengah Desa Kerta (RPJMDes) Kerta. Pada saat penyusunan RPJMDes ini identifikasi terhadap permasalahan yang dialami di Desa Kerta terutama pada permasalah infrastruktur. RPJMDes ini P-ISSN: 2303-2693 E-ISSN: 2581-2939 PADURAKSA: Volume 10 Nomor 2, Desember 2021 DOI: 10.22225/pd.10.2.2895.297-316 309 Tabel 1. Permasalah Infrastruktur Disetiap Dusun No Dusun Jalan usaha tani Jalan desa Drainase Tanpa perkerasan Perkerasan dan rusak Tanpa Perkerasan Perkerasan dan rusak Ada, Tidak berfungsi Ada, rusak 1 Penyabangan ● ● ● 2 Kerta ● ● ● 3 Mawang ● ● ● 4 Margatengah ● ● ● 5 Pilan ● ● ● 6 Saren ● ● ● 7 Bunteh ● ● ● 8 Seming ● ● ● Tabel 1. HASIL DAN PEMBAHASAN Permasalah Infrastruktur Disetiap Dusun No Dusun Jalan usaha tani Jalan desa Drainase Tanpa perkerasan Perkerasan dan rusak Tanpa Perkerasan Perkerasan dan rusak Ada, Tidak berfungsi Ada, rusak 1 Penyabangan ● ● ● 2 Kerta ● ● ● 3 Mawang ● ● ● 4 Margatengah ● ● ● 5 Pilan ● ● ● 6 Saren ● ● ● 7 Bunteh ● ● ● 8 Seming ● ● ● Tabel 1. Permasalah Infrastruktur Disetiap Dusun Dokumen RPJMDes ini nantinya akan menjadi acuan program kegiatan setiap tahunnya yang dapat dilaksanakan sesuai dengan kebutuhan infrastruktur. Sehingga pada Tahun 2015 awal pembangunan infrastruktur pun dilaksanakan hingga tahun 2019 (Tabel 2). Identifikasi permasalahan infrastruktur seperti Tabel 1 menjadi awal dalam penyusunan RPJMDes Kerta Tahun 2014-2020. Pemerintah Desa Kerta dapat menyusun skala prioritas dalam penanganan permasalahan, dan melakukan perencanaan terhadap kebutuhan masyarakat di bidang infrastruktur. Tabel 2. Pembangunan Infrastruktur di Desa Kerta Tahun 2015-2019 No Jenis Infrastruktur Kegiatan Lokasi (Dusun) Tahun 1 Jalan usaha tani Rabat beton jalan akses lahan Tempekan Sengkiding Kerta 2017 2 Jalan usaha tani Rabat beton jalan akses lahan Padang Puuk Mawang 2017 3 Jalan usaha tani Rabat beton jalan usaha tani Saren 2017 4 Jalan usaha tani Pemeliharaan jalan usaha tani Bunteh 2019 5 Jalan desa Rabat beton balan di Br. Bunteh Bunteh 2015 6 Jalan desa Pembukaan jalan di Br. Penyabangan Penyabangan 2015 7 Jalan desa Rabat beton dan penahan tanah di Br. Saren . Saren 2015 8 Jalan desa Peningkatan jalan jurusan Penyabangan-Mawang Penyabangan- Mawang 2016 9 Jalan desa Perbaikan jalan jurusan Penyabangan-Pilan Penyabangan- Pilan 2016 10 Jalan desa Peningkatan jalan dengan rabat beton di Banjar Dinas Saren Saren 2016 11 Jalan desa Rabat beton jalan lingkungan Br. Saren Saren 2017 12 Jalan desa Rabat beton jalan lingkungan Sema Bangsel-Kancasidem Kerta 2017 13 Jalan desa Rabat beton jalan Petiratan Bhujangga Margatengah 2019 14 Drainase Drainase senderan jalan di Br. Margatengah Timur Margatengah 2015 15 Drainase Pembuatan drainase di Banjar Dinas Kerta Kerta 2016 16 Drainase Drainase Br. Bunteh Bunteh 2017 Tabel 2. Pembangunan Infrastruktur di Desa Kerta Tahun 2015-2019 P-ISSN: 2303-2693 E-ISSN: 2581-2939 PADURAKSA: Volume 10 Nomor 2, Desember 2021 DOI: 10.22225/pd.10.2.2895.297-316 DOI: 10.22225/pd.10.2.2895.297-316 310 Kegiatan pembangunan infrastruktur berupa jalan usaha tani, jalan lingkungan, drainase menjadi prioritas kegiatan yang dilakukan sejak tahun 2015 hingga 2019, baik pembangunan maupun pemeliharaan. 2. Dusun Seming Infrastruktur yang terbangun berupa jalan desa dengan rabat beton. Pemanfaatan infrastruktur jalan desa menjadi jalur cycling. Sedangkan pemanfaatan ruang yang ada disekitar infrastruktur yang telah terbangun masih belum terlihat dan dimanfaatkan. Dari identifikasi potensi yang dilakukan oleh Pemerintah Desa Kerta dan kelembagaan masyarakat, dan pembangunan infrastruktur yang telah dimanfaatkan oleh masyarakat Desa Kerta. Pemanfaatan ruang dan pemanfaatan infrastruktur dimasing-masing dusun yang ada di Desa Kerta disesuaikan dengan potensi dan kondisi dari infrastruktur yang telah terbangun. Adapun uraian dari pemanfaatan ruang dan pemanfaatan 1. Dusun Margatengah 2. Dusun dengan potensi perkebunan campuran terdiri dari Dusun Mawang, Dusun Penyabangan, Dusun Bunteh dan Dusun Saren. Infrastruktur berupa jalan usaha tani yang telah terbangun di Dusun Margatengah dimanfaatkan menjadi jalur trekking mengelilingi perkebunan jeruk yang ada. Sedangkan pemanfaatan ruang yang ada disekitar infrastruktur yang telah terbangun masih belum terlihat dalam upaya pemanfaatan ruangnya. Setelah dilakukan identifikasi kelompok dusun selanjutnya dipetakan potensi-potensi yang ada di masing-masing dusun. Pemetaan potensi ini menjadi tahapan setelah dilakukan pembangunan infrastruktur. Selain bertujuan untuk identifikasi potensi juga bertujuan untuk pemanfaatan ruang menjadi wisata desa. Pada Gambar 2 pemetaan potensi Desa Kerta yang merupakan hasil kajian dari pemerintah desa dan kelembagaan masyarakat. HASIL DAN PEMBAHASAN Masyarakat dan Pemerintah Desa Kerta fokus pada tahun tersebut untuk dapat merampungkan kegiatan pembangunan infrastruktur agar masyarakat tidak lagi menemukan permasalahan pada akses transportasi dan distribusi kegiatan pertanian. Upaya yang dilakukan oleh Pemerintah Desa Kerta dan kelembagaan masyarakat yang ada di Desa Kerta selain mempermudah akses distribusi hasil pertanian juga agar mewadahi potensi yang ada yaitu salah satunya dengan pemenuhan infrastruktur, dikarenakan saat ini potensi yang ada tidak dapat diperkenalkan secara maksimal terkendala dari akses untuk dapat menikmati potensi yang ada. dari perkembangan ruang baru yang ada di Desa Kerta. Setelah infrastrukur terbangun, masyarakat memulai untuk memanfaatkan infrastruktur yang telah terbangun, masyarakat Desa Kerta memulai untuk memanfaatkan infrastruktur yang ada dengan kegiatan- kegiatan yang rutin dilakukan oleh masyarakat Desa Kerta. Jalan usaha tani dimanfaatkan untuk pendistribuasi kegiatan pertanian. Jalan desa dimanfaatkan untuk akses menghubungkan antara dusun. Drainase dimanfaatka untuk dapat mengaliri air agar sesuai dengan alirannya. Masyarakat Desa Kerta sangat memanfaatkan dengan baik infrastruktur yang telah dibangun. Selanjutnya upaya yang dilakukan Pemerintah Desa Kerta, masyarakat dan kelembagaan berupa kelompok sadar wisata Desa Kerta (Pokdarwis) adalah menggali potensi yang ulai terlihat pasca pembangunan infrastruktur. Hal yang terkait dengan potensi yang dimiliki oleh Desa Kerta dimanfaatkan menjadi daya tarik wisata. Pemerintah Desa Kerta dan Pokdarwis mengidentifikasi potensi masing-masing dusun yang ada, dan dikelompokkan berupa: Setelah pembangunan infrastruktur dilakukan di Desa Kerta selama kurun waktu lima tahun sejak tahun 2015 hingga tahun 2019, yang menjadi prioritas pembangunan adalah jalan usaha tani, jalan desa dan drainase. Terbangunnya infrastruktur yang sesuai dengan kebutuhan masyarakat, maka masyarakat dapat melakukan kegiatan pendistribusian hasil pertanian dengan baik. Permbangunan infrastruktur menjadi awal 1. Dusun dengan potensi perkebunan jeruk dan pertanian holtikultura yang terdiri dari P-ISSN: 2303-2693 E-ISSN: 2581-2939 PADURAKSA: Volume 10 Nomor 2, Desember 2021 DOI: 10.22225/pd.10.2.2895.297-316 311 Dusun Margatengah, Dusun Pilan, Dusun Kerta dan Dusun Saren. infrastruktur yang telah berkembang sampai saat ini adalah sebagai berikut: 4. Dusun Pilan. Infrastruktur yang terbangun di Dusun Penyabangan berupa jalan desa dan jalan usaha tani. Pemanfaatan infrastruktur jalan desa dan jalan usaha tani dijadikan jalur cycling. Sedangkan untuk pemanfaatan ruang masih belum dimanfaatkan ruang sekitar pembangunan infrastruktur. Infrastruktur yang terbangun di Dusun Pilan berupa jalan usaha tani. Pembangunan jalan usaha tani di Dusun Pilan ini dimanfaatkan menjadi trekking. Selain itu pembukaan jalan yang menghubungkan Dusun 7. Dusun Mawang Infrastruktur yang terbangun di Dusun Mawang berupa jalan desa. Pemanfaatan infrastruktur jalan desa ini dimanfaatkan menjadi jalur cycling. Sedangkan untuk pemanfaatan ruang disekitar pembangunan infrastruktur yang telah terbangun masih belum dimanfaatkan. 3. Dusun Saren Infrastruktur yang terbangun di Dusun Saren berupa jalan desa dan jalan usaha tani dengan perkerasan beton. Pemanfaatan infrastruktur jalan usaha tani dan jalan desa di Desa Kerta ini menjadi jalur cycling. Pemanfaatan ruang yang ada disekitar jalan usaha tani dan jalan desa masih belum dimanfaatkan. P-ISSN: 2303-2693 E-ISSN: 2581-2939 PADURAKSA: Volume 10 Nomor 2, Desember 2021 P-ISSN: 2303-2693 E-ISSN: 2581-2939 E-ISSN: 2581-2939 DOI: 10.22225/pd.10.2.2895.297-316 312 PADURAKSA: Volume 10 Nomor 2, Desember 2021 P-ISSN: 2303-2693 E-ISSN: 2581-2939 Gambar 2. Pemetaan Potensi Desa Kerta Kajian Pemerintah Desa Kerta dan Kelembagaan Masyarakat (Sumber: Dokumen Desa Kerta, 2019) Gambar 2. Pemetaan Potensi Desa Kerta Kajian Pemerintah Desa Kerta dan Kelembagaan Masyarakat (Sumber: Dokumen Desa Kerta, 2019) P-ISSN: 2303-2693 E-ISSN: 2581-2939 PADURAKSA: Volume 10 Nomor 2, Desember 2021 DOI: 10.22225/pd.10.2.2895.297-316 313 4. Dusun Pilan. Infrastruktur yang terbangun di Dusun Pilan berupa jalan usaha tani. Pembangunan jalan usaha tani di Dusun Pilan ini dimanfaatkan menjadi trekking. Selain itu pembukaan jalan yang menghubungkan Dusun Penyabangan dan Dusun Pilan menjadi salah satu pembangunan infastruktur yang ada di Dusun Pilan. Pemanfaatan ruang di setelah pembangunan infrastruktur di Dusun Pilan terlihat dengan berkembangnya ruang yang semula merupakan lahan kosong milik desa adat dikembangkan menjadi bumi perkemahan Puncak Sari, dikarenakan akses menuju tempat ini telah dipermudah karena sudah terdapat perkerasan menuju bumi perkemahan dan pembukaan jalan terlah dilaksanakan. 8. Dusun Kerta Infrastruktur yang terbangun di Dusun Kerta berupa pembangunan drainase dan senderan jalan. Sedangkan untuk jalan desa di Dusun Kerta sudah dalam kondisi baik. Pemanfaatan infrastruktur di Dusun Kerta berupa pembangunan senderan jalan dan drainase dilanjutkan dengan pemanfaatan ruang disekitar drainase untuk ditanami bunga potong. Sehingga pemanfaatan ruang sekitar drainase 5. Dusun Bunteh Infrastruktur yang terbangun di Dusun Bunteh berupa jalan desa. Pemanfaatan infrastruktur jalan desa ini dijadikan jalur cycling. Sedangkan pemanfaatan ruang yang ada disekeliling pembangunan infrastruktur belum dimanfaatkan. 6. Dusun Penyabangan 6. Dusun Penyabangan 6. Dusun Penyabangan 6. Dusun Penyabangan P-ISSN: 2303-2693 E-ISSN: 2581-2939 PADURAKSA: Volume 10 Nomor 2, Desember 2021 DOI: 10.22225/pd.10.2.2895.297-316 314 menjadi perkebunan bunga potong dan pembibitan bunga potong. Hal ini tentunya menciptakan ruang baru untuk wisata desa berupa agrowisata bunga potong. Berdasarkan teori pembangunan desa yang dikemukakan oleh Rondinelli (1982) dalam bahwa pemanfaatan hasil pembangunan fisik desa yaitu dengan membangun atau memperbaiki prasarana jalan desa akan menciptakan atau memperbaiki kehidupan masyarakat desa. Dengan adanya pembangunan prasarana jalan, masyarakat dapat menggunakan jalan tersebut dengan berbagai kebutuhan yang mereka perlukan, seperti melakukan mobilitas, pemasaran hasil pertaniannya, mangangkut hasil pertanian agar lebih mudah dan lain-lain. Jika dikaitkan dengan yang ada di Desa Kerta pembangunan Desa Kerta memang berawal dari pembangunan fisik infrastruktur desa, yaitu dengan memperbaiki dan membangun infrastruktur yang mendukung seluruh aktivitas utama bagi masyarakat Desa Kerta yang didominasi bergerak dibidang pertanian yaitu berupa membangun atau memperbaiki jalan usaha tani, jalan desa dan drainase yang ada. Sampai saat ini tahun 2019 infrastruktur yang ada di Desa Kerta sangat mengalami perubahan dan membantu masyarakat Desa Kerta dalam melakukan mobilisasi mengangkut hasil pertanian. Teori pengembangan wilayah yang Berdasarkan teori pembangunan desa yang dikemukakan oleh Rondinelli (1982) dalam bahwa pemanfaatan hasil pembangunan fisik desa yaitu dengan membangun atau memperbaiki prasarana jalan desa akan menciptakan atau memperbaiki kehidupan masyarakat desa. Dengan adanya pembangunan prasarana jalan, masyarakat dapat menggunakan jalan tersebut dengan berbagai kebutuhan yang mereka perlukan, seperti melakukan mobilitas, pemasaran hasil pertaniannya, mangangkut hasil pertanian agar lebih mudah dan lain-lain. Jika dikaitkan dengan yang ada di Desa Kerta pembangunan Desa Kerta memang berawal dari pembangunan fisik infrastruktur desa, yaitu dengan memperbaiki dan membangun infrastruktur yang mendukung seluruh aktivitas utama bagi masyarakat Desa Kerta yang didominasi bergerak dibidang pertanian yaitu berupa membangun atau memperbaiki jalan usaha tani, jalan desa dan drainase yang ada. Sampai saat ini tahun 2019 infrastruktur yang ada di Desa Kerta sangat mengalami perubahan dan membantu masyarakat Desa Kerta dalam melakukan mobilisasi mengangkut hasil pertanian. Pada uraian perkembangan ruang dan infrastruktur dari masing-masing dusun diatas memperlihatkan bahwa setiap dusun yang ada di Desa Kerta sejak tahun 2014 hingga tahun 2019 seluruh dusun melakukan perencanaan dengan pengajuan usulan yang berasal dari masyarakat masing-masing dusun pada saat penyusunan RPJMDes Tahun 2014 dan pembangunan di realisasikan mulai tahun 2015 hingga 2019. Terlihat dari delapan dusun yang ada di Desa Kerta perkembangan infrastrukturnya mengarah pada pembangunan jalan desa dan jalan usaha tani, dan pembangunan drainase. 6. Dusun Penyabangan Setelah adanya pembangunan infrastruktur pada delapan dusun, terlihat perkembangan yang terjadi pada dua tahun terakhir yaitu tahun 2018 dan 2019, bahwa dusun-dusun yang mulai berkembang dan memanfaatkan infrastruktur yang ada ditandai dengan memanfaatkan potensi yang ada dan menjadikan wisata desa. Dua dusun yaitu Dusun Pilan dan Dusun Kerta yang dapat melakukan pemanfaatan infrastruktur dan pemanfaatan ruang dengan beriringan. Pada uraian perkembangan ruang dan infrastruktur dari masing-masing dusun diatas memperlihatkan bahwa setiap dusun yang ada di Desa Kerta sejak tahun 2014 hingga tahun 2019 seluruh dusun melakukan perencanaan dengan pengajuan usulan yang berasal dari masyarakat masing-masing dusun pada saat penyusunan RPJMDes Tahun 2014 dan pembangunan di realisasikan mulai tahun 2015 hingga 2019. Terlihat dari delapan dusun yang ada di Desa Kerta perkembangan infrastrukturnya mengarah pada pembangunan jalan desa dan jalan usaha tani, dan pembangunan drainase. Setelah adanya pembangunan infrastruktur pada delapan dusun, terlihat perkembangan yang terjadi pada dua tahun terakhir yaitu tahun 2018 dan 2019, bahwa dusun-dusun yang mulai berkembang dan memanfaatkan infrastruktur yang ada ditandai dengan memanfaatkan potensi yang ada dan menjadikan wisata desa. Dua dusun yaitu Dusun Pilan dan Dusun Kerta yang dapat melakukan pemanfaatan infrastruktur dan pemanfaatan ruang dengan beriringan. Teori pengembangan wilayah, yang salah satunya adalah konsep agropolitan dikembangkan oleh Friedman dan Douglas P-ISSN: 2303-2693 E-ISSN: 2581-2939 PADURAKSA: Volume 10 Nomor 2, Desember 2021 PADURAKSA: Volume 10 Nomor 2, Desember 2021 DOI: 10.22225/pd.10.2.2895.297-316 315 (1975) menekankan pentingnya pendekatan agropolitan dalam pengembangan perdesaan di kawasan Asia dan Afrika. Pembangunan infrastruktur yang intensif untuk mendukung pemanfaatan potensi sumber daya alam akan mampu mempercepat pengembangan wilayah tersebut. Dalam konteks ini Pemerintah Desa Kerta belum menggunakan konsep Agropolitan menjadi salah satu pendekatan dalam pengembangan wilayah di Desa Kerta. Masyarakat dan Pemerintah Desa Kerta hanya beranggapan pembangunan infrastruktur akan menjadi awal dari sebuah wilayah akan berkembang. Dengan adanya infrastruktur akan membuat ruang- ruang yang ada di Desa Kerta lebih bermanfaat secara maksimal. dimanfaatkan untuk ditanami bunga potong dan menjadi wisata desa dengan pertanian bunga potong. Pemanfaatan infrastruktur dan ruang pasca pembangunan infrastruktur ini terlihat dengan maksimal dikarenakan potensi secara maksimal telah tergali di dua dusun tersebut. Sedangkan enam dusun lainnya pemanfaatan infrastruktur saja yang sudah optimal namun pemanfaatan ruang sekitar infrastruktur yang terbangun masih belum terlihat berkembang dan dimanfaatkan menjadi wisata desa. Pembangunan infrastruktur menjadi awal dari sebuah wilayah akan dapat berkembang dengan menggali potensi yang dimiliki. 6. Dusun Penyabangan Hal ini jika dilakukan sesuai dengan perencanaan maka enam dusun lainnya di Desa Kerta akan dapat secara maksimal memanfaatkan ruang yang ada disekeliling infrastruktur yang telah terbangun dengan baik menjadi wisata desa. SIMPULAN DAN SARAN Pemanfaatan infrastruktur dan ruang yang ada di Desa Kerta secara beriringan dalam kurun waktu lima tahun yaitu tahun 2015 hingga tahun 2019 terlihat di Dusun Pilan dan Dusun Kerta. Pemanfaatan infrastruktur di Dusun Pilan dimaksimalkan untuk membuka akses menuju lahan kosong miliki desa adat dan dijadikan bumi perkemahan. Sedangkan di Dusun Kerta pasca pembangunan drainase dan senderan jalan, area dipinggir jalan Penelitian ini dapat menjadi acuan bagi desa yang melakukan perencanaan dan pembangunan infrastruktur di kawasan perdesaan, yang memiliki potensi alam maupun potensi buatan yang dapat dikembangkan menjadi wisata desa. Dengan mengacu pada kebutuhan infrastruktur oleh masyarakat kawasan perdesaan, yang selanjutnya dilakukan perencanaan infrastruktur, pembangunan P-ISSN: 2303-2693 E-ISSN: 2581-2939 PADURAKSA: Volume 10 Nomor 2, Desember 2021 E-ISSN: 2581-2939 DOI: 10.22225/pd.10.2.2895.297-316 316 infrastruktur, pemanfaatan infrastruktur dan pemanfaatan ruang yang ada disekitar infrastruktur yang telah terbangun. Mustar, S. (2019). Panduan Sederhana Pembangunan Infrastruktur Untuk Perdesaan Perencanaan Infrastruktur Perdesaan. Jakarta: PT. Mediatama Saptakarya. Pranoto, S. M., Syamsul, M., Surjonoo, H. S., & Hermanto, S. (2006). Pembangunan Perdesaan Berkelanjutan Melalui Model Pengembangan Agropolitan. Jurnal Manajemen Agribisnis, 3 (1). DAFTAR PUSTAKA Aldillah, D. (2020). Fungsi Infrastruktur Jembatan Bagi Perubahan Masyarakat Kelurahan Lempake Kecamatan Samarinda Utara. Journal Sosiatri-Sosiologi, 8 (1), 72- 86. Prastyanti, S. (2015). Pendekatan Pembangunan dan Pengentasan Kemiskinan di Pedesaan. 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Partisipasi Masyarakat Dalam Partisipasi Pembangunan (Studi Kasus Pada Kecamatan Sidikalang Kabupaten Dairi). Jurnal Administrasi Publik, 2 (2). Holis, Y. M. (2017). Local Economic Linkages Pada Kota Kecil/Menengah Sebagai Instrumen Konsep Keterkaitan Desa-Kota (Rural-Urban Linkage). Institut Teknologi Bandung: http://pasca.unand.ac.id/id/prosiding- seminar-nasional-perencanaan- pembangunan-inklusif-desa-kota. Solekhan, M. (2014). Penyelenggaraan Pemerintahan Desa Berbasis Partisipasi Masyarakat. Malang: Setara Press. Suroyo, B. T. (2014). Pengembangan Kawasan Agropolitan di Kabupaten Kulonprogo, Daerah Istimewa Yogyakarta. Jurnal Perencanaan Wilayah dan Kota, 25 (3). Kasikoen, K. M. (2011). Keterkaitan Antar Wilayah Studi Kasus Kabupaten Cilacap. Jurnal Teknik Planologi, 2 (2). Wahid, Y. (2014). Pengantar Hukum Tata Ruang. Jakarta: Prenadamedia Group. P-ISSN: 2303-2693 E-ISSN: 2581-2939 PADURAKSA: Volume 10 Nomor 2, Desember 2021
https://openalex.org/W4297749002	https://zenodo.org/record/6451682/files/1006-1008.pdf	English	null	pσ Relationship in the Oxidation of Substituted Toluenes by CeIV	Zenodo (CERN European Organization for Nuclear Research)	1,970	cc-by	889	[ jorur. "ladian Chem. Soc .• Vol. 47. No. 10, 1970 ] [ jorur. "ladian Chem. Soc .• Vol. 47. No. 10, 1970 ] pa Relationship in the Oxidation of Substituted Toluenes by CeiV P. S. Radhakrishnunurti and Mahendra K. Mahanti ln om· previous communication!, we reported the oxidation of toluene and substituted toluenes mainly with electron withdrM~ing substituents. It was reported that the reaction is total second order and the p va]ur- is -1·7 and evidt-nc~·s were presented to show that it it' mainly a radical process. This communication deals with the oxidation of toluenes with electron uouating groups. There is a remarkable change of oxidation course and it appears that these oxidations with electron donating groups follow a different mechanism, involving a two electron oxidation under these conditions. These reactions are also total second order and the products are also similar as found earlier. The 1·atc const.ants for xylenes, methoxy toluenes and toluenes with electron withdrawing snbstituents are given in Tablr-s J, H and III respectively. TABLE 1 Second Order Rate OonBtants (litre mol-1 min-1) for tke oxidation of xyletM38 at 35°, in the presence of H OlO 1 Solvent % HOAc-H20 vfv HC104(M) para 1neta orthn 5(1 J.O 3.046 0.8973 1.556 60 1.0 7.964 2.125 :~. 071) 70 1.0 13.9 5.176 8.363 50 1.25 5.828 1.674 :3.027 50 1.50 11.74 3.365 6.04tJ TABLE II Second Order Rate Constants (litt·e mol-1 mi'n-1) fm· the oxidation of toluene and metkoxy toluenes at 35°, in the presence of O.lOM HOl04 Solvent Toluene p-methoxy m-methoxy o-methoxy % HOAc-HaO vfv tolueno toluene toluene- 50 0.0169 a3.57 :l.131 6.681 60 0.0277 73.04 6.910 12.710 70 0.0568 106.50 11.390 22.850 1. P. 8. Radha.krishnamurti and Subas 0. Pa.ti, Ohem. and Ind., 1967, 702. pa RELATION~HlP TN THE OXIDATION OF SUBSTITUTED TOLUENES BY CEtv 1007 TABLE III 8econ.d Order Rate Gonstnnt8 (litre mol-1 min-1) Jot· the oxidation of to 81tb8tituted tol7lene8 at 35°, in the presence of nazo. 8econ.d Order Rate Gonstnnt8 (litre mol-1 min-1) Jot· the oxidation of toluene and 81tb8tituted tol7lene8 at 35°, in the presence of nazo. tlolvent HC104(M) 'rolnen<> p-nitro m-nitro m-chloro % HOAc-H,.O v/,- toluenp toluene toluene 50 1.0 II. 32411 n.OI60 0.0284 0 0639 60 1.0 0.4772 0.0761 0.0812 0.1437 70 1.0 0.8706 0. 160!! 0.1777 0.3282 /)1) 1.25 0.61;55 o-.Ot-127 0.056:! 0.1284 1)0 1.50 1.2640 0.0656 0.1133 0.2774 'fho most important point noted is the p value of -4·3 for xylem•s and -4·5 for methoxy i.olueneA, ns against -1·7 noted earlier for toluenes with electron withdrawing groups. A combined plot (Figure 1) is given, indicating the abrupt. 3. E. S. Gould, "1\'Iecha.nism and Structure in Organio Chemistry", Holt, Reinhart and Winston, New York, 1965, p. 219. pa Relationship in the Oxidation of Substituted Toluenes by CeiV P. S. Radhakrishnunurti and Mahendra K. Mahanti change in the p value suggesting a change in mechanism from electron withdrawing substituents t.o electron releasing groups in these oxidations. This indicates that an ionic process2 is operating when electron donating substituents arP present, and a onP electron transfer takes place when f'lectron wit.hdrawing substihtf'>nts are presf'nt. (If Cl. r NITROTOI.u[OIE .f,. .,_. NoTJ(oTO<.IJENo c -....-c.I4LOI!orowttNE o:l. fOLUfNE e.. m-'KY<CN£ f p-~YI.EN£ ~'-¥----.. -~ -- 0 - - .. -o~,--·-,..,~,-~+-:-:OC,--J-..,.•~q a-.r-- Fig. 1. 2. H. C. Rrown and V. Oka.oto, J. Or(/. Ohern." 1957, 22. 185; J. Am.er, Ohem. Soc., 1958, 80, 4980. 2. H. C. Rrown and V. Oka.oto, J. Or(/. Ohern." 1957, 22. 185; J. Am.er, Ohem. Soc., wn and V. Oka.oto, J. Or(/. Ohern." 1957, 22. 185; J. Am.er, Ohem. Soc., 1958, 80, 498 2. H. C. Rrown and V. Oka.oto, J. Or(/. Ohern." 1957, 22. 185; J. Am.er, Ohem. Soc., 1958, 80, 4980. 008 P 8 RADHAKRISHN 1008 Department of Chemistry, K.hallikote College, Berhampur (Ga.njam), Orissa. :P. 8: RADHAKRISHNAMURTI AND MAHENDRA K. MAHANTI :P. 8: RADHAKRISHNAMURTI AND MAHENDRA K. MAHANT It will be seen from Table II that the meta methoxy group is also accelerating the reaction much against its u+ value ( +0·047). It is presumed that probably the electron withdrawing nature of meta methoxy group as seen by its o+ value is true for reactions which require electron recession at the site of the reaction. But from the nature of p value for xylenes and methoxy toluenes, it seems that these reactions are ionic processes which require electron accession. It is clear in view of the above that the o+ value for meta methoxy is not applicable for reactions which require electron accession like the reaction under considera- tion. It may be made clear that the methoxy group at ortho and para positions involves resonance effect, and the same effect probably operates from meta position, otherwise the rates cannot be rationalized as para > ortho > meta > unsubstituted in the case of methoxy toluenes. Resonance effect at meta position has to be invoked, as suggested by Goulda : "When the substituent and the reaction centre lie meta to each other, here due to the alternating nature of the atoms in a conjugated system, resonance effect becomes less, although they do not, as might be supposed, entirely disappear." Department of Chemistry, K.hallikote College, Berhampur (Ga.njam), Orissa. Received April 1, 1970 Received April 1, 1970 Received April 1, 1970 3. E. S. Gould, "1\'Iecha.nism and Structure in Organio Chemistry", Holt, Reinhart and Winston, New York, 1965, p. 219.
https://openalex.org/W2132703128	https://www.scielo.br/j/bjb/a/nYZyxR8VsHpzqTR94MXMgSR/?lang=en&format=pdf	English	null	Diversity and biomass of Chironomidae (Diptera) larvae in an impacted coastal lagoon in Rio de Janeiro, Brazil	Brazilian Journal of Biology	2,002	cc-by	4,149	CALLISTO, M.,1 MORENO, P.,1 GONÇALVES, J. F. Jr.,2 1Universidade Federal de Minas Gerais, ICB, Depto. Biologia Geral, Lab. Limnologia/Ecologia de Bentos, C.P. 486, CEP 30161-970, Belo Horizonte, MG, Brazil 1Universidade Federal de Minas Gerais, ICB, Depto. Biologia Geral, Lab. Limnologia/Ecologia de Bentos, C.P. 486 CEP 30161-970, Belo Horizonte, MG, Brazil 2Universidade Federal do Rio de Janeiro, CCS, IB, Depto. Ecologia, Lab. Limnologia, CEP 21941-540, Rio de Janeiro, RJ, Brazil Correspondence to: Marcos Callisto, Universidade Federal de Minas Gerais, ICB, Depto. Biologia Geral, Laboratório de Limnologia/Ecologia de Bentos, C.P. 486, CEP 30161-970, Belo Horizonte, MG, Brazil, e-mail: callisto@mono.icb.ufmg.br Correspondence to: Marcos Callisto, Universidade Federal de Minas Gerais, ICB, Depto. Biologia Geral, Laboratório de Limnologia/Ecologia de Bentos, C.P. 486, CEP 30161-970, Belo Horizonte, MG, Brazil, e-mail: callisto@mono.icb.ufmg.br Received October 9, 2000 – Accepted January 8, 2001 – Distributed February 28, 2002 Received October 9, 2000 – Accepted January 8, 2001 – Distributed February 28, 2002 ABSTRACT Diversity and biomass of Chironomidae larvae were studied between January-November 1993 and March- November 1994 in an impacted lagoon in Rio de Janeiro, Southeastern Brazil, in an attempt to estab- lish the ecological consequences of anthropogenic eutrophication processes. Nine hundred and seventy- five organisms belonging to the Tanypodinae, Orthocladiinae, and Chironominae were collected. Polypedilum (62%) and Chironomus (58%) were the most common genera found in the limnetic and littoral zones. Key words: biodiversity, eutrophication, chironomids, bioindicators, coastal lagoon. Diversidade e biomassa de larvas de Chironomidae (Diptera) em uma lagoa costeira impactada no Estado do Rio de Janeiro, Brasil Diversidade e biomassa de larvas de Chironomidae (Diptera) em uma lagoa costeira impactada no Estado do Rio de Janeiro, Brasil Foram estudadas a diversidade e a biomassa de larvas de Chironomidae entre janeiro e novembro de 1993 e março-novembro de 1994 em uma lagoa costeira no Estado do Rio de Janeiro, Sudeste do Brasil, com o objetivo de identificar as conseqüências ecológicas do processo de eutrofização arti- ficial. Ao todo, 975 larvas de Tanypodinae, Orthocladiinae e Chironominae foram coletadas e Polypedilum (62%) e Chironomus (58%) foram os taxa mais comuns nas regiões litorânea e limnética. alavras-chave: biodiversidade, eutrofização, Chironomidae, bioindicadores, lagoa costeira Palavras-chave: biodiversidade, eutrofização, Chironomidae, bioindicadores INTRODUCTION and aperiodic openings of the sandbar separating it from the ocean (Faria et al., 1998; Fernandes, 1998; Gonçalves et al., 1998a; Albertoni et al., 1999). The most frequent consequences are pre- dominance of fine particles (silts and clay), increase in organic matter accumulation on the sediment due to enhanced detritus production, and increased production of toxic gases such as H2S and CH4 with consequent decrease in dissolved oxygen concen- tration at the bottom, near the sediment-water in- terface (Esteves, 1998; Gonçalves et al., 1998b). Taxonomic diversity (including taxonomic ri- chness and evenness) and biomass are fundamen- tal in describing the structure and distribution of communities (Begon et al., 1996). This information is an important ecological tool for biological studies of pollution level indicators in freshwater ecosystems, in well-balanced monitoring programs involving physical, chemical, and biological measurements. The Imboassica Lagoon has suffered mul- tiple anthropogenic impacts. Among these are urbanization of its margins; landfills; and frequent The benthic macroinvertebrate community in Imboassica Lagoon is dominated by Gastropoda Braz. J. Biol., 62(1): 77-84, 2002 CALLISTO, M. et al. 78 (Heleobia australis), Polychaeta-Nereidae, and Chironomidae larvae. Together they represent ca. 95% of the fauna (Callisto et al., 1998b; Gonçalves et al., 1998a). Chironomidae are broadly distributed worldwide and frequently are the most abundant insects in many freshwater ecosystems. Certain species show ecological adaptations, in ecosys- tems at different trophic levels, to extreme environ- mental situations related to high temperature, pH, organic matter content in the sediment, and low dissolved oxygen in the water-sediment interface (Cranston, 1995). High diversity of chironomids (estimated at more than 15,000) is better known taxonomically in temperate Northern Hemisphere countries. In the Neotropics, especially Brazil, which is known as a megabiodiversity country (Bar- bosa et al., 1998; Myers et al., 2000), it is estimated that more than 98% of the chironomid species are still undescribed. A shortage of specialists and a lack of basic information at the species level, limit taxonomic identification of the genera, especially in ecological studies of immature forms of Brazilian chironomids (Nolte, 1989; Nessimian & Sanse- verino, 1995; Callisto, 1997). 05’W), and is separated from the sea by a sand bar approximately 50 meters wide. The lagoon is situated on the coastal plain (restinga) where the climate is an AW type (according to Köppen’s classification), with hot and humid characteristics. Temperatures oscillate between 18.7oC and 27.4oC. INTRODUCTION This ecosystem is situated in the Macaé County urban zone and has an estimated area of 3.26 km2, with a mean depth of 1.1 m (Panosso et al., 1998). Littoral zone urbanization and do- mestic waste discharge are important anthropogenic impacts on the lagoon. Artificial openings through the sandbar cause exchange with marine water, resulting in salinity elevation of 0o/oo to 35o/oo (Gon- çalves et al., 1998a; Callisto et al., 1998b). The littoral zone is colonized by Typha domingensis (Typhaceae), grasses, Salvinia auriculata (Sal- vineaceae), and Eichhornia crassipes (Pontede- riaceae). The limnetic zone includes large banks of Chara spp. (Characeae). Sampling of chironomids Chironomidae larvae play an important ecolo- gical role in the bioturbation process at the sediment- water interface. In eutrophic environments, they do so in nitrogen remobilization for the primary producers (Fukuhara & Sakamoto, 1988; Svensson & Leonardson, 1996; Svensson, 1997). In lacustrine ecosystems, these organisms participate in two webs: (a) by the detritus chain, ingesting organic fragments and associated microorganisms, (b) by the food-web, by eating smaller organisms and being consumed by other insects, alevins, aquatic birds, and benthophagous fishes (Callisto et al., 1996; Branco et al., 1997; Galdean et al., 1997; Aguiaro & Caramaschi, 1998). Therefore, in lake environments, they are important components nee- ding further study. Water and sediment samples were collected during January-November 1993 and March-No- vember 1994, in the limnetic and littoral zones (2 meters from a domestic sewage channel). In some months (February, March, and July 1993, June, August, and September 1994 at the littoral zone; January and February 1993, June and July 1994, at the limnetic zone) no samples were taken due to lagoon emptying after sandbar opening. Sediment and benthic macroinvertebrates samples were collected using a “corer” sampler (modified from Ambühl & Bührer, 1975), with 8 cm diameter. The studied fraction was 0-10 cm. Five samples were collected from each sam- pling station (0.025 m2 in total area). In the labo- ratory, sediment samples were washed in two sieves (1.00 and 0.50 mm mesh) and sorted under a stereo- microscope. The aim of this study was to evaluate the diversity of Chironomidae larvae, their distribution and biomass, and to estimate the impacts of the anthropic eutrophication process on this com- munity, by comparing the littoral (close to an un- treated sewage channel) and the limnetic zones of Imboassica Lagoon. Chironomidae larvae were mounted in lac- tophenol solution 10% and identified under the microscope, using 400x magnification. The sam- pled individuals are deposited in the Benthic Macroinvertebrate Collection of the Laboratory of Ecology of Benthos, Institute of Biological Sciences, Federal University of Minas Gerais, following methodology described in Callisto et al. (1998a). Data analysis For larval biomass calculation, the log-linear equation was used: ln W = ln a + b ln l. The maximum densities were found in June, August, and September 1993, and February 1994. In the limnetic zone, Polypedilum larvae represented 62% of the fauna, followed by Ablabesmyia (3%), and Goeldichironomus (2%). Maximum densities were found in May and July 1993. We used the equations proposed by Smock (1980), Sephton & Paterson (1986), and Nolte (1990): l Polypedilum: ln a = –7.308 and b = 2.603 r = 0.964 Biomass value distribution followed the pattern observed for density values. In the littoral zone, Chironomus larvae represented 70% to 100% of the organism biomass, except in March 1994, when Polypedilum larvae reached maximum values (ap- proximately 70%). As for genera richness, a diffe- rent variation was observed while comparing the two sampling stations. l Other Chironominae and Orthocladiinae: ln a = –5.279 and b = 2.32 r = 0.94 l Tanypodinae: ln a = –5.573 and b = 2.603 r = 0.92 l Tanypodinae: DIVERSITY AND BIOMASS OF CHIRONOMIDAE IN A LAGOON 79 of the fauna, followed by Polypedilum (27%), and Tanypus (7%). Study area Imboassica Lagoon is located in northern Rio de Janeiro State (22o15’-22o30’S, 41o30’-42o Braz. J. Biol., 62(1): 77-84, 2002 RESULTS Eleven taxa of chironomids in the littoral zone, and fourteen taxa in the limnetic zone were found in Imboassica Lagoon (Table 1). Tanypodinae larvae, predominantly carni- vorous, were scarce throughout this study. All larvae were small. In the littoral zone, Chironomus larvae presented almost uniform body size distri- bution among the studied size-classes (Table 2). Among the taxa found in the littoral zone, larvae of the genus Chironomus represented 58% Braz. J. Biol., 62(1): 77-84, 2002 Braz. J. Biol., 62(1): 77-84, 2002 TABLE 1 Taxa Littoral Limnetic Tanypodinae Ablabesmyia 22 176 Djalmabatista – 20 Labrundinia 22 40 Tanypus 168 – Orthocladiinae Cricotopus – 65 Nanocladius – 34 Chironominae Asheum – 34 Beardius – 34 Chironomus 1448 1448 Cladopelma 22 56 Goeldichironomus 22 128 Harnischia 22 24 Nimbocera paulensis 22 16 Parachironomus 22 16 Paracladopelma 22 – Polypedilum 696 3560 Total 2488 5651 Composition and density (ind/m2) of the larvae of Chironomidae in the littoral and limnetic zones of the Imboassica Lagoon. Composition and density (ind/m2) of the larvae of Chironomidae in the littoral and limnetic zones of the Imboassica Lagoon. Braz. J. Biol., 62(1): 77-84, 2002 80 CALLISTO, M. et al. CALLISTO, M. et al. 1993 1994 Taxa Jan Apr May Jun Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jul Oct Nov Dec Tanypodinae Ablabesmyia – – 0.24 – – – – – – – – – – – – – – – Labrundinia – – 0.16 0.35 0.14 – – – – – – – – – – – – – Tanypus – – – 0.07 – 0.36 0.61 – 0.22 0.47 – – 0.14 – – 0.24 0.48 0.32 Chironominae Chironomus – 0.82 1.69 34.07 27.21 22.05 8.73 2.68 4.39 7.06 2.95 0.15 6.26 5.94 0.62 0.49 7.31 – Cladopelma – – – 0.25 – – 0.08 0.29 – 0.11 0.05 0.28 – – – – – – Goeldichironomus – – – – 0.05 – – – – – – – – – 0.50 – – – Harnischia – 0.08 – 0.08 – – – – – – – – – – – – – – Nimbocera paulensis – – – – 0.07 – – – – – – – – – – – – – Parachironomus – – – – 0.17 – – – – – – – – – – – – – Paracladopelma – – – 0.36 – – – – – – – – – – – – – – Polypedilum 0.03 – – – 0.75 0.01 – 0.06 0.11 0.42 0.98 0.74 0.08 – – – – – Total 0.03 0.90 2.09 35.18 28.39 22.42 9.42 3.03 4.72 8.06 3.98 1.17 6.48 5.94 1.12 0.73 7.79 0.32 TABLE 2 (a) Biomass of chironomids (mg/m2) along 1993-1994 sample periods in the littoral zone of the Imboassica Lagoon. Braz. J. DISCUSSION The biomass distribution data can be related to two different situations. The first might be that the larvae were not developing a body size larger than 0.3 mm, probably due to selective predator pressure (Tanypodinae larvae with other chironomids in the gut content, benthophagous fishes, and ale- vins). The second might be related to intrinsic spe- cies aspects with Polypedilum present in this lagoon area. Larvae of some Polypedilum species might naturally have small body-size. Imboassica Lagoon offers a great diversity of trophic resources, such as aquatic macrophytes, periphyton, plant detritus (notably in the littoral zone), and fine particulate organic matter (Furtado et al., 1997). The high organic matter content in the sediment suggests food limitation neither for Chironomus larvae nor for Polypedilum in the littoral and limnetic zones, respectively. The results obtained in this research showed that anthropic eutrophication in the Imboassica Lagoon has influenced the structure of the benthic community (prevalence of detritivorous organisms, such as Chironomus and Polypedilum), resulting in high biomass and low taxonomic diversity, when compared to other studies developed on coastal ecosystems in Rio de Janeiro State (Nessimian, 1995; Nessimian & Sanseverino, 1995). We believe that this information can be useful in making more evident to local communities and politicians the urgent need for measures to minimize the anthropic impacts observed in Imboassica Lagoon. Ecological information on coastal lagoons subject to anthropic influence is necessary in these areas to define and limit human activities, as well as for establishing the management techniques to be applied in the future. In the Imboassica Lagoon case, new studies should be developed to define species composition and benthic community struc- tures (and their functional role), to better characterize habitats and evaluate impact levels and subsequent aquatic biodiversity loss. Moreover, such results supply fundamental information on the diversity and biomass of one of the benthic macroinverte- brate main groups, besides suggesting their role as environmental quality bioindicators. Tanypodinae are located at the top of the food web in benthic communities. The abundance of Tanypodinae is regulated directly by available food items (which may be other Chironomidae larvae, Rotifera, Protozoa, Oligochaeta, etc.) (Epler, 1995). TABLE 1 Biol., 62(1): 77-84, 2002 DIVERSITY AND BIOMASS OF CHIRONOMIDAE IN A LAGOON 81 1993 1994 Taxa Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Aug Sep Oct Nov Dec Tanypodinae Ablabesmyia 0.30 – – 0.80 0.03 – 0.03 – – 0.02 0.27 1.07 0.62 – – – – – 0.04 – Djalmabatista – – 0.01 – – – – – – – – – – – – – – – – – Labrundinia – – – 0.18 – 0.05 – 0.09 0.09 0.11 – 0.08 – – – – – – – 0.03 Orthocladiinae Cricotopus – 0.11 – – – – – – – – – 0.08 0.05 – – – – – 0.40 0.21 Nanocladius – – – – – – – 0.16 – – – – – – – – – – – – Chironominae Asheum – – – 0.24 – – – 0.10 – – – – – – – – – – – – Beardius – – – – – – – – – – – – – – – – – – – – Chironomus – – – – – – – – – – – – – – – 0.16 .050 2.17 – – Cladopelma – – – – – – – – – – – – 0.16 – – – – – – – Goeldichironomus – – – – – 0.54 0.07 0.27 – – 0.06 0.18 – – – – – – – – Harnischia – – – – 0.18 – – – – – – – – – – – – – – – Nimbocera paulensis – – – – – – – – – – 0.05 0.47 – – – – – – – – Parachironomus – 0.63 – 0.09 – – – 0.18 – – – – – – – – – – – – Polypedilum 0.91 0.02 6.27 0.15 2.73 – 1.08 – – – 0.18 0.04 – 0.02 0.10 – – – 0.01 – Total 1.21 0.76 6.28 1.46 2.94 0.59 1.18 0.80 0.09 0.13 0.56 1.92 0.83 0.02 0.10 0.16 0.50 2.17 0.45 0.24 TABLE 2 (b) Biomass of chironomids (mg/m2) along 1993-1994 sample periods in the limnetic zone of the Imboassica Lagoon. Braz. J. Biol., 62(1): 77-84, 2002 82 CALLISTO, M. et al. probably be related to domestic sewage release and even to heavy metal presence. TABLE 1 Deformities have been used in environmental impact evaluation studies on freshwater ecosystems (Janssens De Bisthoven et al., 1998; Diggins & Stewart, 1998), and suggested as a biological parameter to be inclu- ded in long-term water-quality monitoring programs (Callisto et al., 2000). On the other hand, in the limnetic zone the majority of Polypedilum larvae showed body size reduction (smaller than 0.3 mm). DISCUSSION The high abundance of Chironomus and Polypedilum larvae, predominantly detritivorous, is probably related to the high abundance of organic detritus in the Imboassica Lagoon sediment, as is corroborated by the high content of total and dis- solved nutrients in the water and great concen- trations in the sediment (Petrucio & Furtado, 1998; Furtado et al., 1997), a typical situation in lacustrine ecosystems subjected to artificial eutrophication. Acknowledgments — Most special thanks to Professor Fran- cisco A. R. Barbosa, Dr. Claudia Jacobi, and Dr. Geraldo Wilson Fernandes for their valuable comments on a previous manuscript draft. We are also indebted to our colleagues from the Laboratório de Limnologia/UFRJ for valuable help with the samples, and to those from the Laboratório de Limnologia/ Ecologia de Benthos/UFMG for the identification phase and biomass evaluation. Financial support was received from PETROBRAS and CNPq. Some Chironomus and Polypedilum larvae (about 5% of the studied material) found presented deformities on mouth parts (mentum). Several stu- dies have shown the relationship between the oc- currence of structural deformities in Chironomidae larvae and degraded environmental conditions, resulting from contamination by heavy metals, her- bicides, fungicides, and insecticides (Dermott, 1991; Warwick, 1985). According to Janssens De Bisthoven et al. (1992), these deformities can be interpreted as a sign of sublethal pollutant concen- trations which in the Imboassica Lagoon would Braz. J. Biol., 62(1): 77-84, 2002 DIVERSITY AND BIOMASS OF CHIRONOMIDAE IN A LAGOON 83 AMBÜHL, H. & BÜHRER, H., 1975, Zur Technik der Entnahme ungestörter Gossproben von Seesedimenten: ein verbessertes Bohrlot. Schweiz Z. Hydrol., 37: 175-186. EPLER, J. H., 1995, Identification manual for the larval Chironomidae (Diptera) of Florida. Department of Envi- ronmental Regulation, State of Florida, 427p. ESTEVES, F. 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https://openalex.org/W2025314640	https://journals.plos.org/plosntds/article/file?id=10.1371/journal.pntd.0000786&type=printable	English	null	A Nuclear Family A DNA Polymerase from Entamoeba histolytica Bypasses Thymine Glycol	PLoS neglected tropical diseases	2,010	cc-by	11,337	Guillermo Pastor-Palacios1, Elisa Azuara-Liceaga2, Luis G. Brieba1* 1 Laboratorio Nacional de Geno´mica para la Biodiversidad, CINVESTAV, Irapuato, Me´xico, 2 Posgrado en Ciencias Geno´micas, Universidad Auto´noma de la Ciudad d Me´xico, Me´xico Distrito Federal, Me´xico para la Biodiversidad, CINVESTAV, Irapuato, Me´xico, 2 Posgrado en Ciencias Geno´micas, Universidad Auto´noma de la Ciudad de i Abstract Motif B is located at the fingers subdomain and is involved in positioning the template strand into the polymerase active site [15]. In eukaryotes, family A polymerases are involved in the replication of mitochondrial and chloroplast genomes [17,18]. The archetypical Abstract histolytica contains five DNA polymerases which share high sequence homology with DNA polymerases from autonomous replicating elements found in other protozoa and with the well-characterized DNA polymerase from bacteriophage w29[12]. E. histolytica also contain one family A DNA polymerases in its genome. Family A DNA polymerases are modular enzymes consisting of three independent domains: a N- terminal 59-39 exonuclease domain, a 39-59 exonuclease domain, and a C-terminal polymerase domain [1,13,14]. Crystal structures of family A DNA polymerases revealed a modular organization of the polymerase domain and its division into three subdomains: palm, fingers, and thumb, which together form a cleft that binds the primer-template [15]. Family A DNA polymerases contain three conserved motifs: A, B, and C in the polymerization domain [16]. Motifs A and C are located at the palm subdomain and contain two carboxylates involved in the coordination of two metal ions involved in the nucleophilic attack of the incoming deoxynucleotide to the 39 OH of the primer strand [13]. Motif B is located at the fingers subdomain and is involved in positioning the template strand into the polymerase active site [15]. In eukaryotes, family A polymerases are involved in the replication of mitochondrial and chloroplast genomes [17,18]. The archetypical proteins, that compose the principal DNA polymerase involved in translesion synthesis of thymine dimers: DNA pol f [10,11]. In addition, the genome of E. histolytica contains five DNA polymerases which share high sequence homology with DNA polymerases from autonomous replicating elements found in other protozoa and with the well-characterized DNA polymerase from bacteriophage w29[12]. E. histolytica also contain one family A DNA polymerases in its genome. Family A DNA polymerases are modular enzymes consisting of three independent domains: a N- terminal 59-39 exonuclease domain, a 39-59 exonuclease domain, and a C-terminal polymerase domain [1,13,14]. Crystal structures of family A DNA polymerases revealed a modular organization of the polymerase domain and its division into three subdomains: palm, fingers, and thumb, which together form a cleft that binds the primer-template [15]. Family A DNA polymerases contain three conserved motifs: A, B, and C in the polymerization domain [16]. Motifs A and C are located at the palm subdomain and contain two carboxylates involved in the coordination of two metal ions involved in the nucleophilic attack of the incoming deoxynucleotide to the 39 OH of the primer strand [13]. Abstract Background: Eukaryotic family A DNA polymerases are involved in mitochondrial DNA replication or translesion DNA synthesis. Here, we present evidence that the sole family A DNA polymerase from the parasite protozoan E. histolytica (EhDNApolA) localizes to the nucleus and that its biochemical properties indicate that this DNA polymerase may be involved in translesion DNA synthesis. Methodology and Results: EhDNApolA is the sole family A DNA polymerase in E. histolytica. An in silico analysis places family A DNA polymerases from the genus Entamoeba in a separate branch of a family A DNA polymerases phylogenetic tree. Biochemical studies of a purified recombinant EhDNApolA demonstrated that this polymerase is active in primer elongation, is poorly processive, displays moderate strand displacement, and does not contain 39–59 exonuclease or editing activity. Importantly, EhDNApolA bypasses thymine glycol lesions with high fidelity, and confocal microscopy demonstrates that this polymerase is translocated into the nucleus. These data suggest a putative role of EhDNApolA in translesion DNA synthesis in E. histolytica. Conclusion: This is the first report of the biochemical characterization of a DNA polymerase from E. histolytica. EhDNApolA is a family A DNA polymerase that is grouped into a new subfamily of DNA polymerases with translesion DNA synthesis capabilities similar to DNA polymerases from subfamily n. Citation: Pastor-Palacios G, Azuara-Liceaga E, Brieba LG (2010) A Nuclear Family A DNA Polymerase from Entamoeba histolytica Bypasses Thymine Glycol. PLoS Negl Trop Dis 4(8): e786. doi:10.1371/journal.pntd.0000786 Editor: Anuradha Lohia, Bose Institute, India Received January 19, 2010; Accepted July 12, 2010; Published August 10, 2010 Received January 19, 2010; Accepted July 12, 2010; Published August 10, 2010 Copyright: 2010 Pastor-Palacios et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by intramural funds from Langebio-Cinvestav (http://www.ira.cinvestav.mx/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: lgbrieba@ira.cinvestav.mx proteins, that compose the principal DNA polymerase involved in translesion synthesis of thymine dimers: DNA pol f [10,11]. In addition, the genome of E. www.plosntds.org www.plosntds.org Phylogenetic analysis and structural modeling of EhDNApolA We tested the antibodies for their response and specificity in total extracts of E. histolytica strain HM1:IMSS and against recombinantly induced EhDNApolA. For Western blot assays, we used total, nuclear, and cytoplasmic extracts from E. histolytica strain HM1:IMSS prepared as previously described [29]. Protein extracts were separated using a 15% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The membranes were incubated with a 1 to 2000 dilution of the purified immune sera and an anti-actin antibody [30] in 1% nonfat dry milk, 0.05% Tween-20 in PBS 7.4 for 2 hours. The reactivity was detected using peroxidase conjugated secondary antibodies (1 to 2000 dilution) with the ECL Plus detection kit (GE Healthcare). As a control, we used antibodies against actin and CBP-B previously characterized. To identify putative family A DNA polymerases in E. histolytica, we initially used the amino acid sequence of the Klenow fragment of E. coli (Protein Data Bank accession code: 1KFS) to blast the Pathema database (http://pathema.jcvi.org/Pathema/). The phylogenetic tree was constructed using the amino acid sequences of family A DNA polymerases of representative mitochondrial DNA polymerases, bacteriophage DNA polymer- ases, DNA polymerases n, and bacterial DNA polymerases. The amino acid sequences of these proteins were aligned using the program ClustalW [25]. The catalytic amino acids of motifs A, B, and C, were conserved through the alignment. This sequence alignment was used to construct a dendogram with the Neighbor- Joining method of the Molecular Evolutionary Genetic Analysis (MEGA) software [26]. The robustness of the dendogram was assessed by bootstrap analysis of 1000 replicates. Production of anti-EhDNApolA antibodies Seven Balb/c mice were bled and tested for their response to total protein extracts of E. histolytica. Five mice did not present any response and were inoculated with a peptide corresponding to residues 286 to 297 of the thumb subdomain of EhDNApolA (HKIEMETKKIIG). The mice were immunized with 150 mg of the peptide combined with Freund’s adjuvant. Six weekly bursts were applied and the reactivity of each mouse was assessed using recombinant EhDNApolA. After six weeks of immunization, the immune sera was collected, purified, and stored at 220uC. In this work, we report the initial characterization of the sole family A DNA polymerase from E. histolytica (EhDNApolA). We propose a role of this DNA polymerase in translesion DNA synthesis of oxidative lesions like 8-oxo guanosine and thymine glycol. These lesions may be generated by the oxidative environment of the colonic tissue and the constant insult of the reactive oxygen species produced by phagocytes during E. histolytica pathogenesis. All animal work was conducted according to the legislation enforced in Me´xico (NOM-062-ZOO-1999) and by CINVES- TAV’s committee for animal care and use. The Mexican legislation is based on the Guide for the Care and Use of Laboratory Animals, NRC. All animal work was conducted according to the legislation enforced in Me´xico (NOM-062-ZOO-1999) and by CINVES- TAV’s committee for animal care and use. The Mexican legislation is based on the Guide for the Care and Use of Laboratory Animals, NRC. Introduction DNA replication and translesion DNA synthesis in eukaryotes is accomplished by a battery of DNA polymerases. For instance, the genome of Homo sapiens contains 15 DNA polymerases divided into four families: A, B, X, and Y according to their amino acid sequence homology [1–3]. Nuclear replicative DNA polymerases d and e? belong to family B, whereas DNA polymerases involved in translesion DNA synthesis are present in all four families. y p Entamoeba histolytica is a parasitic protozoa which causes amebic dysentery and liver abscess [4]. In comparison to eukaryotes that contain DNA in organelles like mitochondria or chloroplasts. E. histolytica is an early branching eukaryote in which its mitochondria diverged to form an organelle with no detectable DNA. This organelle is dubbed mitosome [5,6], and although its function is not definitively established, experimental evidence suggests a role in sulfate activation [7] and oxygen detoxification [8]. Thus, the 24 Mbp genome of E. histolytica is exclusively nuclear and it encodes several putative DNA polymerases (Table S1) [9]. As an eukaryotic organism, the genome of E. histolytica is expected to be replicated by DNA polymerases d and e. Although a gene encoding DNA polymerase e is not present in the current genome annotation of E. histolytica, a gene encoding DNA polymerase d is present. E. histolytica contains homologs of Rev 1 and Rev 3 1 August 2010 \| Volume 4 \| Issue 8 \| e786 Entamoeba histolytica Family A DNA Polymerase (Protein Data Bank accession code: 1KFS) [27], using the program Molecular Operating Environment (MOE). As Klenow Fragment contains 605 amino acids and EhDNApolA has 657, the gaps between the two aligned proteins were built according to the peptide library present in the MOE database. Twenty models were generated and each model was minimized using the CHARMM27 force field. Author Summary Author Summary Genotoxic agents like ultraviolet radiation, alkylating compounds and reactive oxidative species have the potential to originate DNA lesions that are not bypassed by replicative DNA polymerases. Eukaryotic organisms contain a specialized subset of DNA polymerases capable of translesion DNA synthesis. These DNA polymerases belong to DNA polymerases from families A, B, and Y. In this work, we characterized the sole family A DNA polymerase of the parasitic protozoa E. histolytica, EhDNApolA. The biochemical characterization of recombi- nant EhDNApolA indicates that this protein is an active DNA polymerase able to primer extension and moderate strand displacement. The ability of EhDNApolA to faithfully incorporate dATP opposite thymine glycol, and its nuclear localization indicates that this polymerase may have a role in translesion DNA synthesis. E. histolytica is exposed to oxidative stress during tissue invasion by phagocytes. Understanding DNA metabolism in E. histolytica is important because this parasite has shaped some meta- bolic pathways by horizontal gene transfer, infects approximately 50 million people annually, and is the second leading cause of death among protozoan diseases. E. histolytica cultures Trophozoites of HM1:IMSS strain were axenically cultured in TYI-S-33 medium supplemented with 15% of bovine serum [28] at 37uC and used in logarithmic growth phase for all experiments. Cloning of EhDNApolA gene The open reading frame of EhDNApolA was amplified by PCR from genomic DNA of E. histolytica strain HM1:IMSS. To allow directional cloning, the sense oligonucleotide (59-ggttgg ggatcc atg gaa aaa aca cca aga aat tct-39) contained a BamH I restriction site (underlined) and the antisense oligonucleotide (59-ggttgg aagctt tta att caa gtt gta agg atg aag-39) contained a Hind III restriction site (underlined). PCR was carried out using 150 ng of genomic DNA, 25 pmol of each oligonucleotide, and 125 mM of each dNTP. The amplified product was simultaneously digested with BamH I and Hind III and ligated into the pCOLD I vector (Takara). The ligation mixtures were transformed into an E. coli DH5a strain. Plasmidic DNA was analyzed using restriction mapping and confirmed by DNA sequencing. Cloning of the open reading frame of EhDNApolA in the pCOLD I vector confers a 6-His tag at the N terminus of the recombinant protein. DNA polymerase in eukaryotes is DNA polymerase c, which is the replicative mitochondrial DNA polymerase. Besides DNA polymerase c, vertebrates contain two other family A DNA polymerases: DNA polymerase n and DNA polymerase h. In contrast to DNA polymerase c, the localization of these poly- merases is nuclear. Human DNA polymerases n and h are capable of translesion DNA synthesis and they have a role in DNA repair [19–24]. www.plosntds.org Polymerization substrates cDNA and 15 pmol of each specific oligonucleotide combination. The segment corresponding to motif A was amplified using the sense oligonucleotide 59-agagacttattattacacat3-’ and antisense oligonucleotide, 59-attctttttaagccaatgtgc-39. Motif C was amplified using the sense oligonucleotide; 59-ttacattcaagttgggtaggt-39 and antisense oligonucleotide 59-aacagtaactacaacaggaac-39. The actin control was amplified with the sense oligonucleotide 59-aag ctg cat caa gca gtg aa-39 and antisense 59-gga atg atg gtt gga aga gg -39. RT-PCR products were separated by gel electrophoresis in 1.5% agarose gels, stained with ethidium bromide, and visualized with a standard UV transilluminator. cDNA and 15 pmol of each specific oligonucleotide combination. The segment corresponding to motif A was amplified using the sense oligonucleotide 59-agagacttattattacacat3-’ and antisense oligonucleotide, 59-attctttttaagccaatgtgc-39. Motif C was amplified using the sense oligonucleotide; 59-ttacattcaagttgggtaggt-39 and antisense oligonucleotide 59-aacagtaactacaacaggaac-39. The actin control was amplified with the sense oligonucleotide 59-aag ctg cat caa gca gtg aa-39 and antisense 59-gga atg atg gtt gga aga gg -39. RT-PCR products were separated by gel electrophoresis in 1.5% agarose gels, stained with ethidium bromide, and visualized with a standard UV transilluminator. The following oligonucleotides were used to generate double stranded polymerization substrates: a) 45mer template (59-cct tgg cac tag cgc agg gcc agt tag gtg ggc agg tgg gct gcg-39) b) 24mer primer (59-cgc agc cca cct gcc cac cta act-39); c) 18mer primer (59- cgc agc cca cct gcc cac-39); and d) 21mer non-template (59-ggc cct gcg ctagtgccaagg-39).100 nmols of the 24mer primer or the 18mer primer were 59end labeled using T4 Kinase with c-[32]ATP. The probes were purified using the nucleotide removal kit (Qiagen) according to the manufacturer instructions. The polymerization substrates were annealed to a final concentration of 10 nM in 20 mM Tris pH 7.5, 150 mM NaCl. Semi-quantitative RT-PCR assays were performed using total cellular RNA isolated from Entamoeba histolytica grown in basal culture conditions using SV Total RNA Isolation System (Promega Madison, WI, USA). The amount of total or messenger RNA isolated from the cells was quantified using an ND-1000 spectrophotometer (NanoDrop, Fisher Thermo, Wilmington, DE, USA). cDNA was synthesized using gene-specific primers. 1 mg of total RNA was added to a reaction containing 625 mM EhDNApolA motif A antisense oligonucleotide or actin antisense oligonucleotide, 0.5 mM of the deoxynucleotide triphosphates, 1 unit of RNasin Ribonuclease Inhibitor, 1 ml of ImProm-IITM Reverse Transcriptase (Promega Madison, WI, USA) and RNase- free water to 20 ml. Kinetic analysis For steady-state kinetic analysis, DNA polymerase activity assays were performed using 2 pmol of duplex DNA incubated with 10 fmol of EhDNApolA and varying dNTP concentrations. The reactions were incubated for 10 minutes at 37uC. Four different DNA duplexes were used to determine the kinetic parameters of each nucleotide opposite to its cognate base. To assure linearity, less than 20% of the substrate was converted to product. Translesion DNA synthesis Templates containing 8-oxo guanosine and abasic site were purchased from Oligos Etc. Templates containing 5 S-6R thymine glycol, 5R-6S thymine glycol, cis-syn cyclobutane pyrimidine dimer, and 6-4 photo product were synthesized by Professor Shigenori Iwai’s group as previously described [31]. A specific 59 c-[32]ATP labeled primer was annealed to each template, so the first template base corresponds to each specific lesion. 60, 120 and 240 fmol of EhDNApolA were incubated with 100 fmol of each primer-template at 37uC for 2.5 minutes with 100 mM of each dNTP. Reactions were stopped by adding an equal volume of gel stop/loading buffer. The reactions were run on a 16% denaturing 8 M urea polyacrylamide gel. DNA binding A radiolabeled DNA substrate consisting of the 45mer template annealed to the 24mer primer was incubated with increasing concentrations of EhDNApolA (from 0 to 180 nM) in a buffer containing 50 mM NaCl, 10 mM Tris-HCl pH 7.5, 2.5 mM MgCl2, 1 mM dithiothreitol (DTT), 1 mg/ml BSA, and 5% glycerol. DNA-protein complexes were resolved through a 6% non-denaturing polyacrylamide gel (PAGE) and electrophoresed at 80 V for 2 h at room temperature in 0.5x TBE buffer. Gels were vacuum-dried and radioactive complexes were detected in a Phosphor Imager apparatus and analyzed using the ImageQuant software (BioRad). Polymerization reactions 20 ml polymerization reactions consisted of 20 mM Tris-HCl pH 7.5, 2.5 mM MgCl2, 1 mM DTT, 1 mg/ml BSA, 200 fmol primer-template, 60 fmol EhDNApolA. Reactions were stopped with a buffer containing 95% formamide, 1mM EDTA, 0.01% xylene cyanol. Samples were resolved on a 16% polyacrylamide, 8M urea denaturing gels. Quantification of the polymerization products was carried out in a Phosphorimager using ImageQuant software. Protein expression and purification The pCOLDI-EhDNApolA construct was transformed into an E. coli BL21 DE3-Rosseta II strain. Transformants were inoculated in 100 ml of LB supplemented with 100 mg/ml of ampicilin and 35 mg/ml of chloramphenicol and used to inoculate 2 liters of LB. This culture was grown at 37uC until it reached an OD600 of 0.6. The culture was incubated in ice for 30 minutes and IPTG was added to a final concentration of 0.5 mM. Induction proceeded for 16 hours at 16uC. The cell pellet was harvested by centrifugation at 6,500 rpm. Cell lysis was carried out using a French press in a buffer containing 50 mM potassium phosphate pH 8, 300 mM NaCl, and 1 mM PMSF. The lysate was centrifuged at 17,000 rpm for 30 minutes at 4uC. The soluble fraction was filtrated and the recombinant EhDNApolA was purified using a Ni2+-NTA affinity chromatography in a previously equilibrated Hi-Trap Column (GE Healthcare). The initial wash consisted of 50 ml of lysis buffer supplemented with 35 mM imidazol and the second wash consisted of 100 ml of lysis buffer supplemented with 50 mM imidazol. Protein elution was carried out in lysis buffer supplemented with 500 mM imidazol. The eluate was dialyzed in a buffer containing 50 mM potasium phosphate pH 7.0, 5 mM b-mercaptoethanol (BME), 50 mM NaCl, 2 mM EDTA and 5% glycerol. To further purify EhDNApolA, the dialyzed protein was loaded into a phospho- cellulose column and eluted with a NaCl gradient (100 to 1500 mM). EhDNApolA eluted between 600 to 650 mM of NaCl. The collected fractions were dialyzed in 50 mM potasium phosphate pH 7.0, 1 mM b-mercaptoethanol, 150 mM NaCl and 1 mM EDTA and stored at 4uC. Protein samples were run on a 10% SDS-PAGE and stained with Coomassie Brilliant Blue R-250. Polymerization substrates Reactions were incubated at 25uC for 5 min, then at 42u for 60 min followed by 75uC for 15 min, to inactivate the reverse transcriptase. PCR was performed using EhDNApolA or actin specific sprimers to amplify cDNA segments of 168 or 192 bp in length respectively, with the estimated primer melting temperature of 61.5 or 52uC. RT-PCR products were separated by gel electrophoresis in 1% agarose gels, stained with ethidium bromide, and visualized with a standard UV transilluminator. RT-PCR assays To build the structural model of EhDNApolA, the amino acid sequence of EhDNApolA was structurally aligned with the amino acid sequence present in the crystal structure of Klenow fragment cDNA was synthesized using 1 mg of total E. histolytica RNA with an oligo(dT) adaptor. The RT-PCR reactions contained 0.5 ml of August 2010 \| Volume 4 \| Issue 8 \| e786 2 Entamoeba histolytica Family A DNA Polymerase Entamoeba histolytica Family A DNA Polymerase Confocal microscopy Trophozoites of E. histolytica grown in basal cell culture condition were transferred to glass coverslips. Cells were fixed www.p August 2010 \| Volume 4 \| Issue 8 \| e786 3 www.plosntds.org Entamoeba histolytica Family A DNA Polymerase putative polymerase EhDNApolA. The predicted amino acid sequence of EhDNApolA was used as query to search for homologous proteins in the genomes of E. invadens and E. dispar. We found that locus EIN_094210 of E. invadens and locus EDI_083910 of E. dispar also code for putative family A DNA polymerases with 50% and 88% amino acid sequence identity to EhDNApolA respectively. The lack of a conserved 39-59 exonuclease active site in the DNA polymerases of the genus Entamoeba indicates that these polymerases are not related to mitochondrial DNA polymerases. A phylogenetic analysis of 37 DNA polymerases (Table S2) positions the DNA polymerases from the genus Entamoeba in a separate branch with respect to other subfamily A DNA polymerases. In this division, family A DNA polymerases are grouped into five separate branches or subfamilies (Figure 1A). The high bootstrap value of each branch validates this division. (Figure 1A). Family A DNA polymerase from Entamoeba have a clear conservation of the catalytic motifs present in the polymerization domain. (Figure 1B). The disappearance of the exonuclease domain is a common feature in some family A DNA polymerases, including DNA polymerase n, DNA polymerase h, and several bacterial polymerases. The crystal structure of Klenow fragment bound to duplex DNA in its exonuclease domain was used as template to build a homology model of EhDNApolA [32]. The structural model of EhDNApolA depicts the modular organization present in family A polymerases. In this model, EhDNApolA adopts a structure that resembles a cupped right hand in which the three with 4% paraformaldehyde for 1 hour at 37uC, washed with PBS pH 6.8, permeabilized with 0.5% (v/v) Triton X-100 at 37uC for 60 min, and blocked with 50 mM glycine for 1 h at 37uC and with 1% fetal bovine serum for 15 min. Finally, they were incubated with anti-EhDNApolA antibodies (1 to 75) overnight at 4uC. The cells were washed and conjugated with fluorescein labelled secondary antibodies (Jackson Immuno Research) at 1:500 dilution. The nucleic acids were stained with DAPI (49,69- diamidino-2-phenylindole) washed, and mounted with Vectashield solution (Vector Lab. Burlingame, CA). Confocal microscopy Light optical sections were obtained through a Nikon inverted microscope attached to a laser confocal scanning system (Leica Microsystems) and analyzed by Confocal Assistant software Image. putative polymerase EhDNApolA. The predicted amino acid sequence of EhDNApolA was used as query to search for homologous proteins in the genomes of E. invadens and E. dispar. We found that locus EIN_094210 of E. invadens and locus EDI_083910 of E. dispar also code for putative family A DNA polymerases with 50% and 88% amino acid sequence identity to EhDNApolA respectively. The lack of a conserved 39-59 exonuclease active site in the DNA polymerases of the genus Entamoeba indicates that these polymerases are not related to mitochondrial DNA polymerases. A phylogenetic analysis of 37 DNA polymerases (Table S2) positions the DNA polymerases from the genus Entamoeba in a separate branch with respect to other subfamily A DNA polymerases. In this division, family A DNA polymerases are grouped into five separate branches or subfamilies (Figure 1A). The high bootstrap value of each branch validates this division. (Figure 1A). Family A DNA polymerase from Entamoeba have a clear conservation of the catalytic motifs present in the polymerization domain. (Figure 1B). The disappearance of the exonuclease domain is a common feature in some family A DNA polymerases, including DNA polymerase n, DNA polymerase h, and several bacterial polymerases. The crystal structure of Klenow fragment bound to duplex DNA in its exonuclease domain was used as template to build a homology model of EhDNApolA [32]. The structural model of EhDNApolA depicts the modular organization present in family A polymerases. In this model, EhDNApolA adopts a structure that resembles a cupped right hand in which the three www.plosntds.org www.plosntds.org Identification of a family A DNA polymerase in E. histolytica Heterologous expression and purification of E. histolytica family A DNA polymerase. (A) Coomassie blue stained SDS-PAGE (10%) gel showing the expression and purification of EhDNApolA. Lane1, uninduced pCOLD-EhDNApolA construct; lane 2, IPTG induced sample; lane 3, insoluble fraction; lane 4, soluble lysate; lane 5, nickel agarose flow-through; lane 6, 35 mM imidazol wash; lane 7, 50mM imidazol wash; lane 8, nickel agarose column eluate; lane 9, phosphocelulose column eluate; lane 10, molecular weight standards. (B) Detection of recombinant and endogenous EhDNApolA.Recombi- nant EhDNApolA and total extracts of E. histolytica were resolved by SDS-PAGE (15%), electroblotted onto nitrocellulose membrane, and immunoblotted with diverse antibodies. Lane 1, recombinant EhDNA- polA treated with commercial anti-6 histidines antibody; lane 2, recombinant EhDNApolA treated with mouse antibodies raised against an epitope of EhDNApolA; lane 3, total protein extracts of E. histolytica treated with preimmune serum; lane 4, total protein extracts of E. histolytica treated with specific mouse antibodies raised an epitope of EhDNApolA. doi:10 1371/journal pntd 0000786 g002 E. coli strain BL21- Rosseta II (Figure 2A, lanes 1 and 2, and data not shown). The recombinant EhDNApolA was soluble (Figure 2A, lane 4) and purified nearly to homogeneity using Ni2+-NTA affinity chromatography as a first chromatographic step (Figure 2A, lane 8). To assure the purity of the recombinant protein and avoid a possible contamination with endogenous DNA polymerases, we performed a second chromatographic step using a phosphocellu- lose chromatography. After this step, the recombinant protein was more than 95% pure (Figure 2A, lane 9). Our structural model of EhDNApolA was used to design epitopes to raise polyclonal antibodies. The best epitope candidate was a peptide located at the thumb subdomain (residues 286 to 297) of EhDNApolA. The pre- immune serum did not unveil any reactivity against total extracts of E. histolytica (Figure 2B, lane 3) and recombinant expressed polymerase (data not shown). The raised antibodies recognized a single band of 75 kDa in bacterial extracts expressing recombinant EhDNApolA and in total extracts from E. histolytica (Figure 2B, lanes 2 and 4). As observed in Figure 2B, the raised polyclonal antibodies were highly specific for EhDNApolA and did not present any cross reactivity that could compromise the localization of EhDNApolA in vivo. Figure 2. Heterologous expression and purification of E. histolytica family A DNA polymerase. (A) Coomassie blue stained SDS-PAGE (10%) gel showing the expression and purification of EhDNApolA. Identification of a family A DNA polymerase in E. histolytica A survey of E.histolytica genome with the amino acid sequences of Klenow Fragment and representative family A DNA polymerases revealed that this parasite contains a single open reading frame that codes for a putative family A DNA polymerase. This open reading frame is located at locus EHI_073640 and codes for a protein of 657 amino acids with GenBank accession number XP_653960 and 25% amino acid identity to Klenow fragment. In this work we dubbed this Figure 1. Phylogenetic analysis and structural modeling of E. histolytica family A DNA Polymerase. (A) Phylogenetic relationship of family A polymerases from different organisms. ClustalW multiple sequence alignment of diverse DNA polymerases were used to construct a phylogenetic tree using the neighbor joining algorithm present in the MEGA program. GenBank accession numbers of each protein are indicated in Table S2. The significance of each branch of the phylogenetic tree is indicated by a bootstrap percentage (B) Domain organization of EhDNApolA. EhDNApolA lacks the 39-59 exonuclease domain present in Klenow fragment. The polymerization domain of EhDNApolA is organized into three subdomains: thumb, palm, and fingers (C) Structural model of EhDNApolA. The N-terminal domain is shown in orange. The polymerization subdomains: thumb, palm, and fingers are colored in green, red, and cyan respectively. The model was constructed using the crystal structure of Klenow Fragment as a template (PDB ID: 1KFS). doi:10 1371/journal pntd 0000786 g001 Figure 1. Phylogenetic analysis and structural modeling of E. histolytica family A DNA Polymerase. (A) Phylogenetic relationship of family A polymerases from different organisms. ClustalW multiple sequence alignment of diverse DNA polymerases were used to construct a phylogenetic tree using the neighbor joining algorithm present in the MEGA program. GenBank accession numbers of each protein are indicated in Table S2. The significance of each branch of the phylogenetic tree is indicated by a bootstrap percentage (B) Domain organization of EhDNApolA. EhDNApolA lacks the 39-59 exonuclease domain present in Klenow fragment. The polymerization domain of EhDNApolA is organized into three subdomains: thumb, palm, and fingers (C) Structural model of EhDNApolA. The N-terminal domain is shown in orange. The polymerization subdomains: thumb, palm, and fingers are colored in green, red, and cyan respectively. The model was constructed using the crystal structure of Klenow Fragment as a template (PDB ID: 1KFS). doi:10.1371/journal.pntd.0000786.g001 August 2010 \| Volume 4 \| Issue 8 \| e786 4 Entamoeba histolytica Family A DNA Polymerase Figure 2. Over expression and purification of recombinant In order to test the biochemical properties of EhDNApolA, its open reading frame was cloned into the pCOLD I vector (Takara). Heterologous protein expression was enhanced with the use of the In order to test if recombinant EhDNApolA displays polymer- ization activity, we measured its ability to incorporate deoxynu- Figure 3. Biochemical characterization of EhDNApolA. (A) EhDNApolA binds to double stranded DNA. Increasing concentrations of recombinant EhDNApolA (from 0 to 180 nM) were incubated with a fixed amount of double stranded primer-template. The binding of the EhDNApolA to the primer-template is observed by the formation of a slower migrating complex on a native 6% polyacryalmide gel. (B) EhDNApolA is an active DNA polymerase. DNA polymerase activity was measured by the extension of 24mer primer annealed to a 45mer template. Lanes 1 and 4 contained reactions with no added polymerase, EhDNApolA (lanes 2 and 3) or Kf (exo-) (lanes 5 and 6). Reactions in lanes 2 and 5 were incubated with dTTP,dGTP and ddATP. Reactions in lanes 3 and 6 were incubated with all four dNTPs. Incorporation of ddATP results in extension to a 36mer and incubation with all four dNTPs results in a 45mer product. (C) Strand displacement activity of EhDNApolA. Strand displacement was determined using a six-nucleotide substrate gap depicted below Figure3D. The reactions contained 25 units of w 29 DNA polymerase (lane 2), 4 units of Taq DNA polymerase (lane 3), 5 units of T7 DNA polymerase (lane 4), and 45 fmol of EhDNApolA (lane 5). The 18mer primer can be freely extended to a 24mer. Further extension is only possible if the DNA polymerase displaced the annealed 21mer. doi:10.1371/journal.pntd.0000786.g003 Figure 3. Biochemical characterization of EhDNApolA. (A) EhDNApolA binds to double stranded DNA. Increasing concentrations of recombinant EhDNApolA (from 0 to 180 nM) were incubated with a fixed amount of double stranded primer-template. The binding of the EhDNApolA to the primer-template is observed by the formation of a slower migrating complex on a native 6% polyacryalmide gel. (B) EhDNApolA is an active DNA polymerase. DNA polymerase activity was measured by the extension of 24mer primer annealed to a 45mer template. Lanes 1 and 4 contained reactions with no added polymerase, EhDNApolA (lanes 2 and 3) or Kf (exo-) (lanes 5 and 6). Reactions in lanes 2 and 5 were incubated with dTTP,dGTP and ddATP. Reactions in lanes 3 and 6 were incubated with all four dNTPs. Identification of a family A DNA polymerase in E. histolytica Lane1, uninduced pCOLD-EhDNApolA construct; lane 2, IPTG induced sample; lane 3, insoluble fraction; lane 4, soluble lysate; lane 5, nickel agarose flow-through; lane 6, 35 mM imidazol wash; lane 7, 50mM imidazol wash; lane 8, nickel agarose column eluate; lane 9, phosphocelulose column eluate; lane 10, molecular weight standards. (B) Detection of recombinant and endogenous EhDNApolA.Recombi- nant EhDNApolA and total extracts of E. histolytica were resolved by SDS-PAGE (15%), electroblotted onto nitrocellulose membrane, and immunoblotted with diverse antibodies. Lane 1, recombinant EhDNA- polA treated with commercial anti-6 histidines antibody; lane 2, recombinant EhDNApolA treated with mouse antibodies raised against an epitope of EhDNApolA; lane 3, total protein extracts of E. histolytica treated with preimmune serum; lane 4, total protein extracts of E. histolytica treated with specific mouse antibodies raised an epitope of EhDNApolA. d i 10 1371/j l d 0000786 002 EhDNApol A is a functional DNA polymerase with moderate strand displacement and no exonuclease activity EhDNApol A is a functional DNA polymerase with moderate strand displacement and no exonuclease activity We tested the ability of EhDNApolA to shift a fixed amount of primer-template (3 nM) by increasing the EhDNApolA concen- tration from equimolar amounts to 60 fold excess (Figure 3A, lanes 2 to 7). The appearance of a major retarded band that increased in intensity according to the amount of added recombinant protein indicates that EhDNApolA is able to recognize a primer-template substrate. A few minor bands were also detected, however they had low abundance in comparison to the more abundant complex. It is possible that these bands resulted from some alternate binding mode of EhDNApolA to the primer-template, for instance a binding that resembled an editing complex [32,33]. doi:10.1371/journal.pntd.0000786.g002 subdomains of the polymerization domain (fingers, palm, and thumb) form a DNA binding cleft (Figure 1C). Although this structural model depicts high degree of conservation in the polymerization domain, it should not be interpreted as an experimental structure. www.plosntds.org EhDNApolA incorporates dNTPs with high selectivity EhDNApolA incorporates dNTPs with high selectivity EhDNApolA incorporates dNTPs with high selectivity Family A DNA polymerases are highly variable in their DNA replication accuracy. Polymerases from bacteriophages, bacteria, and mitochondria are high fidelity polymerases. In contrast, human DNA polymerases h and n are low fidelity polymerases. For instance, human DNA polymerase n misincorporates thymine across from a guanine template with a frequency of 0.45 [20]. To test the fidelity of EhDNApolA, we used a set of primer-templates in which the first template base is different from the following templated base (Figure 4). EhDNApolA selectively incorporated the incoming nucleotide according to the Watson-Crick rules at all four template bases (Figures 4A, 4B, 4C, and 4D). EhDNApol does not extensively misincorporate at canonical templates. This is in contrast to DNA polymerases of subfamilies n and h that are low fidelity polymerases. Although an extensive kinetic analysis is needed to quantify the fidelity of EhDNApol, it is evident that EhDNApol follows the Watson-Crick rules during nucleotide incorporation at canonical templates. Some family A DNA polymerases, like DNA polymerase c and DNA polymerase n are capable of strand displacement. We tested the strand displacement capabilities of EhDNApolA in comparison to other DNA polymerases. The strand displacement activity of EhDNApolA was measured in a primer-template substrate containing a gap of six nucleotides and this activity corresponds to the appearance of primer elongation products longer than 24nt (Figure 3C). w29 DNA polymerase has strong strand displacement capabilities and is a highly processive polymerase. According to these characteristics, w29 DNA polymerase is not halted at position 24 (Figure 3C, lane 2). Taq DNA polymerase and T7 DNA polymerase are DNA polymerases with moderate strand displacement, as some polymerase’s population are blocked at positions 24 and 23 (Figure 3D, lanes 3 and 4). We found that EhDNApolA was able to perform strand displacement at similar levels that Taq DNA polymerase (Figure 3C, lanes 3 and 5). However, in contrast to Taq and T7 DNA polymerases, EhDNApolA has weak primer-template affinity during strand displacement, as evidenced by the apparition of bands from 25 to 30 nt (Figure 3C lane 5).We tested the ability of the purified EhDNApolA to degrade a labeled primer-template. No detectable 39-59 exonuclease activity was observed even after 8 minutes of incubation with EhDNApolA (data not shown). Translesion DNA synthesis by EhDNApolA y y DNA lesions can be classified as non-blocking and strong blocking. DNA lesions like 8-oxo guanosine are readily bypassed by the majority of family A DNA polymerases. On the other hand, DNA lesions like thymine glycol, abasic site, and thymine dimers are strong blocks to replication. Seeming exceptions are the cases of DNA polymerase n that efficiently bypasses 5S-thymine glycol and DNA polymerase h that bypasses abasic sites [19,23]. To measure translesion DNA synthesis by EhDNApolA, we tested increasing amounts of the polymerase in a control template thymine and in several DNA lesions. To permit the relative extension comparison, less than 50% of the control thymine template was extended at the lower polymerase concentration. EhDNApolA extended a thymine template to the final 45mer product with an efficiency of 42% at the higher polymerase concentration (Figure 5, lane 4). EhDNApolA efficiently bypasses 8-oxo guanosine, as 26% of the labeled primer was extended to the final 45mer product at the higher polymerase concentration (Figure 5, lanes 5 to 8). EhDNApolA bypasses 5S, 6R thymine glycol with an efficiency of 6%, this efficiency is low in comparison to the thymine template, but is significantly larger than other DNA polymerases, like RB69 that which is completely blocked by this lesion [20,39]. The stalled 17mer product constitutes 80% of the total labeled DNA in the reaction (Figure 5, lanes 9 to 12). Similar results have been observed for an exonuclease deficient Klenow fragment [20,40]. EhDNApolA bypasses the 5R, 6S thymine glycol with an efficiency of 4% (Figure 5, lanes 13 to 16). As in the case of the 5S, 6R thymine glycol lesion, this efficiency was low in comparison to the control thymine but is more efficient than DNA Over expression and purification of recombinant Incorporation of ddATP results in extension to a 36mer and incubation with all four dNTPs results in a 45mer product. (C) Strand displacement activity of EhDNApolA. Strand displacement was determined using a six-nucleotide substrate gap depicted below Figure3D. The reactions contained 25 units of w 29 DNA polymerase (lane 2), 4 units of Taq DNA polymerase (lane 3), 5 units of T7 DNA polymerase (lane 4), and 45 fmol of EhDNApolA (lane 5). The 18mer primer can be freely extended to a 24mer. Further extension is only possible if the DNA polymerase displaced the annealed 21mer. doi:10.1371/journal.pntd.0000786.g003 August 2010 \| Volume 4 \| Issue 8 \| e786 5 www.plosntds.org Entamoeba histolytica Family A DNA Polymerase cleotides to an annealed primer-template. The presence of elongation products indicates that the recombinant EhDNApolA is a functional DNA polymerase (Figure 3B, lanes 2 and 3). The experimental setup placed the first template thymine at position 36 (Figure 3B). Thus, if dGTP, dCTP and ddATP were added as the only nucleotides in the reaction mixture, it is expected that elongation would stop at position 36. EhDNApolA readily incorporates ddATP and it is halted at position 36 (Figure 3B, lane 2). This is in contrast to Klenow fragment that did not efficiently incorporate ddATP, and replicates beyond the first thymine template (Figure 3B lane 5). Mutagenesis studies demonstrated that residue F762 of Klenow fragment is responsible for ddNTPs selectivity [34]. DNA polymerases with a tyrosine in the corresponding position incorporate ddNTP efficiently because the hydroxyl group of the tyrosine compensates for the missing 39 OH of the ddNTPs [34]. The corresponding residue of Klenow fragment’s F762 in EhDNApolA is a tyrosine (residue Y485). Thus, as it is observed, EhDNApolA efficiently incorporates ddNTPs during primer extension (Figure 3B lane 2). Several bands of lower molecular weight were observed during primer extension reactions. These bands may indicate that, like Klenow Fragment, EhDNApolA is a poorly processive DNA polymerase (Figure 3B, lanes 2–3 and 5–6). concentration was 2.5 mM (Figure S1B). This metal concentration was similar to the optimal concentration of Thermus aquaticus and Klenow Fragment DNA polymerases. The optimal pH for polymerization activity is 7.5. EhDNApolA has approximately 80% of activity between pH 7 and 8 (Figure S1C). As expected for an enzyme from a mesophilic organism, the optimal temperature for EhDNApolA activity was 37uC (Figure S1D). www.plosntds.org EhDNApolA incorporates dNTPs with high selectivity This is in agreement of our in silico prediction which indicates that EhDNApolA does not contain the motifs needed for 39-59 exonuclease activity [35]. Over expression and purification of recombinant Using the optimal buffers, we determined the kinetic parameters for EhDNApolA activity using steady-state kinetics. The Km of the incoming nucleotide varied from 1.49 to 2.3 mM and the Vmax varied between 2.9 to 3.3 nMol/min (Table S3). The kinetic constants of EhDNApolA were similar to several family A DNA polymerases including the DNA polymerase from Bacillus stereothermophilus, Klenow Fragment and human DNA polymerase n [20,37,38]. The Km of the incoming nucleotide varied from 1.49 to 2.3 mM and the Vmax varied between 2.9 to 3.3 nMol/min (Table S3). The kinetic constants of EhDNApolA were similar to several family A DNA polymerases including the DNA polymerase from Bacillus stereothermophilus, Klenow Fragment and human DNA polymerase n [20,37,38]. www.plosntds.org Kinetics parameters for EhDNApolA nucleotide incorporation An important step to measure kinetic parameters is to determine the optimal reaction conditions. Thus, we determined the optimal salt concentration, pH, MgCl2 concentration, and temperature for EhDNApolA activity. EhDNApolA is strongly inhibited by NaCl. The optimal NaCl concentration for EhDNApolA activity is from 0 mM to 50 mM NaCl (Figure S1A, lanes 2 to 5). Increasing the NaCl concentration to 100mM only permitted the incorporation of a single nucleotide (Figure S1A, lane 6). EhDNApolA was not active at 200mM NaCl, a concentration that is similar to physiological concentrations. In this respect, EhDNApolA resem- bles Klenow fragment which has decreased activity at concentra- tions higher than 50 mM NaCl [36]. The optimal MgCl2 August 2010 \| Volume 4 \| Issue 8 \| e786 6 Entamoeba histolytica Family A DNA Polymerase Figure 4. Nucleotide insertion fidelity of EhDNApolA. 16% denaturing polyacriylamide gel showing a primer-template extension by EhDNApolA in the presence of 100 mM of the indicated nucleotide. The first templated base is denoted with an X as depicted in figures A, B, C, and D. The identity of each dNTP in each reaction is indicated. The 24mer substrates and the 25mer products are indicated by an arrow. (A) Nucleotide fidelity for templated adenine (lanes 1 to 4). (B) Nucleotide fidelity for templated thymine (lanes 5 to 8). (C) Nucleotide fidelity for templated cytidine (lanes 9 to 12). (D) Nucleotide fidelity for templated guanine (lanes 13 to 16). doi:10.1371/journal.pntd.0000786.g004 Figure 4. Nucleotide insertion fidelity of EhDNApolA. 16% denaturing polyacriylamide gel showing a primer-template extension by EhDNApolA in the presence of 100 mM of the indicated nucleotide. The first templated base is denoted with an X as depicted in figures A, B, C, and D. The identity of each dNTP in each reaction is indicated. The 24mer substrates and the 25mer products are indicated by an arrow. (A) Nucleotide fidelity for templated adenine (lanes 1 to 4). (B) Nucleotide fidelity for templated thymine (lanes 5 to 8). (C) Nucleotide fidelity for templated cytidine (lanes 9 to 12). (D) Nucleotide fidelity for templated guanine (lanes 13 to 16). doi:10.1371/journal.pntd.0000786.g004 polymerase n [20] or any other family A DNA polymerase characterized to date. The stalled 17mer product represents 33% of the product (Figure 5, lane 16). EhDNApolA is unable to bypass the CPD and the 6-4 photoproduct (Figure 5, lanes 17 to 24). Kinetics parameters for EhDNApolA nucleotide incorporation EhDNApolA incorporates only one nucleotide opposite an abasic site (Figure 5, lanes 25 to 28) and some bypass occurs at higher polymerase concentrations (data not shown). EhDNApolA incorporated dATP across from 8-oxoguanosine more efficiently than dCTP (Figure 6A, lanes 2 and 5). The rationale for this incorporation resides in the nature of a specific residue at the fingers subdomain. A bulky residue like K635 in T7 DNApol dictates the incorporation of dCTP [41] whereas a glycine residue in B. stearothermophilus DNA polymerase may dictate the incorporation of dATP [38,42]. EhDNApolA contains a serine in the position corresponding to residue K635 of T7 DNA polymerase, thus the preferential incorporation of dATP is predicted. Family A DNA polymerases preferentially insert dATP across from an abasic site, a phenomena known a as the ‘‘A-rule’’ [43]. EhDNApolA incorporates preferentially dATP (Figure 6B, lane 3) and dGTP (Figure 6B, lane 6) opposite abasic sites. EhDNApolA only incorporates a purine across from the lesion, but it does not extend from the lesion (Figure 6B). This characteristic is conserved with other family A DNA polymerases, like Klenow fragment [44] or DNA polymerase n [20]. However, DNA polymerase h is able to bypass abasic sites [23] www.plosntds.org www.plosntds.org EhDNApolA bypasses 8-oxoguanosine with low fidelity and follows the ‘‘A rule’’ during dNTP incorporation across from an abasic site EhDNApolA was able to bypass 8-oxoguanosine and to incorporate across from an abasic site (Figure 5, lanes 6 to 8 and 26 to 28). In order to test the fidelity of lesion bypass, we tested the incorporation of each deoxyribonucleotide across from each lesion. 8-oxoguanosine is a dual code lesion that can template for dCTP and dATP. The syn conformation of 8-oxoguanosine mimics a thymine template that allows dATP incorporation[41]. Figure 5. Translesion DNA synthesis by EhDNApolA. 16% denaturing polyacrylamide gel electrophoresis showing translesion bypass of EhDNApolA. The first templated base is denoted with an X. The structure of each templated lesion and a templated thymine are depicted for comparison. The reactions were incubated with increased amounts of EhDNApolA (0, 60, 120, and 240 fmol) and 20pM of each substrate. Thymine (lanes 1–4); 8-oxo guanosine (lanes 5–8,); 5 S-6R thymine glycol (lanes 9–12); 5R-6S thymine glycol (lanes 13–16,); cis-syn cyclobutane pyrimidine dimer (lanes 17–20); 6-4 photo product (lanes 21–24); abasic site (lanes 25–29). The bottom arrow indicates the substrate length and the top arrow indicates the expected full-length products. doi:10.1371/journal.pntd.0000786.g005 Figure 5. Translesion DNA synthesis by EhDNApolA. 16% denaturing polyacrylamide gel electrophoresis showing translesion bypass of EhDNApolA. The first templated base is denoted with an X. The structure of each templated lesion and a templated thymine are depicted for comparison. The reactions were incubated with increased amounts of EhDNApolA (0, 60, 120, and 240 fmol) and 20pM of each substrate. Thymine (lanes 1–4); 8-oxo guanosine (lanes 5–8,); 5 S-6R thymine glycol (lanes 9–12); 5R-6S thymine glycol (lanes 13–16,); cis-syn cyclobutane pyrimidine dimer (lanes 17–20); 6-4 photo product (lanes 21–24); abasic site (lanes 25–29). The bottom arrow indicates the substrate length and the top arrow indicates the expected full-length products. doi:10.1371/journal.pntd.0000786.g005 August 2010 \| Volume 4 \| Issue 8 \| e786 7 www.plosntds.org Entamoeba histolytica Family A DNA Polymerase Figure 6. Fidelity of translesion DNA synthesis by EhDNApolA. 16% denaturing polyacrylamide gel electrophoresis showing translesion bypass fidelity of EhDNApolA. 0.2 pmol of EhDNApolA were incubated with a set of substrates containing several DNA lesions. The reactions were carried out with four dNTPs or single dNTP addition. (A) 8-oxo guanosine (lanes 1 to 6). (B) abasic site (lanes 1 to 6). (C) 5 S-6R thymine glycol (lanes 1 to 6). (D) 5R-6S thymine glycol (lanes 1 to 6). The identity of each nucleotide is indicated in the figure. Nuclear localization of EhDNApolA In order to verify that the gene of EhDNApolA is transcribed in vivo, we carried out a RT-PCR using specific oligonucleotides that amplified the two conserved motifs A and C of the EhDNApolA gene. The oligonucleotides were designed to amplify a region of 168 bp corresponding to motif A and a region of 156 bp corresponding to motif C of the EhDNApolA gene (Figure 7A, lanes 3 and 4 respectively). The RT-PCR control product of the actin gene control corresponds to 192 bp (Figure 7A, lane 2) and the no RT reaction showed no appearance of a new band (data not shown). The RT-PCR reaction produced the expected products, thus confirming that the EhDNApolA gene is tran- scribed under basal conditions in E. histolytica. In order to quantify the abundance of the EhDNApolA transcript, we compared the relative transcript under basal conditions in comparison to actin. The average intensity of the EhDNApolA transcript is approxi- mately 70% of the intensity of the actin transcript (Figure S2). Thus, the EhDNApol A gene is expressed at similar levels than the actin gene in basal cell culture conditions. To determine the localization of EhDNApolA in E. histolytica, we carried out Western blot analyses of fractionated cytoplasm and nuclear extracts using the anti-peptide EhDNApol A antibody and anti-actin and anti- C/EBPb antibodies as controls. The appearance of a single protein band of 75kDa in the nuclear and cytoplasmic fractions using the anti-peptide EhDNApol A antibody indicates that a population of EhDNApolA is translocated from the cytoplasm into the nucleus (Figure 7B, lanes 1 and 2). The same patter is observed with the anti-actin antibody, as actin is a protein with cytoplasmic and nuclear localization [30]. Because nuclear fractions are often contaminated with cytosolic fractions, we used the identification of C/EBPb, as a control of the nuclear extract purification protocol. The antibody against this protein identifies a double band of approximately 65 kDa in Western blot assays; however this recognition occurs predominantly in nuclear extracts and not in the cytoplasmic fraction [45]. The data indicates that a population of EhDNApolA is imported from the cytoplasm into the nucleus. Confocal microscopy of E.histolytica trophozoites stained with antibodies against the peptide of EhDNApolA corroborates that EhDNApolA is translocated into the nucleus (Figure 7). EhDNApolA bypasses thymine glycol with high fidelity In contrast to replicative DNA polymerases, like DNA polymerase RB69, that stall at thymine glycol lesion, EhDNApol is able to bypass this lesion. Family A DNA polymerases, like an exonuclease deficient Klenow fragment bypasses the 5S, 6R thymine glycol lesion, but are halted at the 5R, 6S-thymine glycol lesion [20]. Although EhDNApol readily incorporates across from a 5S, 6R thymine glycol lesion, it is severely hampered during its elongation. The 5R, 6S thymine glycol lesion is also bypassed by EhDNApol, although with different properties than the 5S, 6R thymine glycol lesion. The accumulation of the first incorporated nucleotide occurs less efficiently than in the 5S, 6R lesion. Structural studies suggest that thymine glycol prevents primer extension by obstructing the next 59 templated base to stack against it [39]. EhDNApolA is able to accurately bypass thymine glycol (Figures 6C and 6D). EhDNApolA inserts dATP opposite 5S, 6R thymine glycol (Figure 6C, lane 3) and 5R, 6S thymine glycol (Figure 6D, lane 3). EhDNApolA did not incorporate any other nucleotide opposite 5S, 6R or 5R, 6S thymine glycol (Figure 6C, lanes 4 to 6 and Figure 6D, lanes 4 to 6). EhDNApolA incorporates dATP at the 5S, 6R thymine glycol lesion and in this context misincorporates dATP opposite template dCTP. A similar phenomenon occurs in human DNA polymerase k [40] indicating that a subtle DNA distortion originated by the lesion may influence nucleotide incorporation fidelity by these DNA polymerases, as observed by the apparent low fidelity of EhDNApolA. This is in contrast to the high fidelity opposite template dCTP in a canonical template (Figure 4C). EhDNApolA bypasses 8-oxoguanosine with low fidelity and follows the ‘‘A rule’’ during dNTP incorporation across from an abasic site The length of the substrates, single nucleotide extensions and full-length products are indicated by arrows. d i 10 1371/j l td 0000786 006 www.plosntds.org Nuclear localization of EhDNApolA DAPI staining indicates the localization of nuclear double-stranded DNA in the parasite (Figure 7D) and immunofluorescence analysis using anti-EhDNApolA antibodies shown a possible nuclear localization (Figure 7E). Merged field indicated that EhDNApol A colocalizes with DAPI staining of the nuclear DNA of E. histolytica (Figure 7 F). An analysis of the EhDNApolA amino acid sequence using the pSORT program (http://psort.ims.u-tokyo.ac.jp/) predicted the presence of several nuclear localization signals. DNA polymeriza- tion in E. histolytica is inhibited by aphidicolin, which is an inhibitor of family B DNA polymerases and is weakly inhibited by ddNTPs [46,47]. As EhDNApolA readily incorporates ddNTPs (Figure 3B) and family A DNA polymerase are not inhibited by aphidicolin, EhDNApolA should not play a preponderant role in DNA replication of E. histolytica’s genome. Figure 6. Fidelity of translesion DNA synthesis by EhDNApolA. Figure 6. Fidelity of translesion DNA synthesis by EhDNApolA. 16% denaturing polyacrylamide gel electrophoresis showing translesion bypass fidelity of EhDNApolA. 0.2 pmol of EhDNApolA were incubated with a set of substrates containing several DNA lesions. The reactions were carried out with four dNTPs or single dNTP addition. (A) 8-oxo guanosine (lanes 1 to 6). (B) abasic site (lanes 1 to 6). (C) 5 S-6R thymine glycol (lanes 1 to 6). (D) 5R-6S thymine glycol (lanes 1 to 6). The identity of each nucleotide is indicated in the figure. The length of the substrates, single nucleotide extensions and full-length products are indicated by arrows. doi:10.1371/journal.pntd.0000786.g006 Discussion In this work we report the cloning and biochemical character- ization of a family A DNA polymerase present in E. histolytica. Although E. histolytica contains a mitocondrial remnant organelle dubbed mitosome, this organelle does not contain DNA. Further- more, the genome of E. histolytica does not contains a phage-type RNA polymerase and DNA helicase involved in transcription and replication of mitochondrial DNA [9]. EhDNApolA may have evolved from the ancestral mitochondrial DNA polymerase c or was August 2010 \| Volume 4 \| Issue 8 \| e786 8 Entamoeba histolytica Family A DNA Polymerase Figure 7. Cellular identification and localization of EhDNApolA. (A) RT-PCR using total RNA from E. histolytica trophozoites grown in basal culture conditions 1% Agarose gel stained with ethidium bromide showing the RT-PCR products of motifs A (lane 3) and C (lane 4) of the EhDNApolA gene. The RT-PCR product of the actin gene control is shown in lane 2. (B) Immunodetection of EhDNApolA. Western blot assays using, cytoplasmic extracts (lane 1), and nuclear extracts (lane 2) of E. histolytica’s trozophoites against mouse polyclonal anti- EhDNApolA antibodies (upper pannel). Control using mouse polyclonal anti-actin antibodies (middle panel) and anti- C/EBPb antibodies (lower panel) (C–F) Cellular localization of EhDNApolA analyzed by confocal immunofluorescence microscopy. (C) Nomarsky image of a single E. histolytica cell stained with 49,6-diamidino-2- phenylindole (DAPI) and incubated with anti-EhDNApolA antibodies treated with FITC-labeled secondary antibodies (D) fluorescence signal generated by DAPI (E) fluorescence signal generated by the binding of the FITC-conjugated antibody to anti-EhDNApol A antibody (F) Merge image of the fluorescence emitted by DAPI and FITC. doi:10.1371/journal.pntd.0000786.g007 Entamoeba histolytica Family A DNA Polymerase acquired by horizontal gene transfer from a bacterial family A DNA polymerase. The fact that EhDNApolA is biochemically related to those DNA poly Excision Repair in Figure 7. Cellular identification and localization of EhDNApolA. (A) RT-PCR using total culture conditions 1% Agarose gel stained with ethidium bromide showing the RT-PCR products gene. The RT-PCR product of the actin gene control is shown in lane 2. (B) Immunodetection of extracts (lane 1), and nuclear extracts (lane 2) of E. histolytica’s trozophoites against mouse pol Control using mouse polyclonal anti-actin antibodies (middle panel) and anti- C/EBPb anti EhDNApolA analyzed by confocal immunofluorescence microscopy. www.plosntds.org www.plosntds.org References 19. Marini F, Kim N, Schuffert A, Wood RD (2003) POLN, a nuclear PolA family DNA polymerase homologous to the DNA cross-link sensitivity protein Mus308. J Biol Chem 278: 32014–32019. 1. Bebenek K, Kunkel TA (2004) Functions of DNA polymerases. Adv Protein Chem 69: 137–165. 2. Shcherbakova PV, Bebenek K, Kunkel TA (2003) Functions of eukaryotic DNA polymerases. Sci Aging Knowledge Environ 2003: RE3. 20. Takata K, Shimizu T, Iwai S, Wood RD (2006) Human DNA polymerase N (POLN) is a low fidelity enzyme capable of error-free bypass of 5S-thymine glycol. J Biol Chem 281: 23445–23455. 3. Pavlov YI, Shcherbakova PV, Rogozin IB (2006) Roles of DNA polymerases in replication, repair, and recombination in eukaryotes. Int Rev Cytol 255: 41–132. p p y y 4. Stanley SL Jr (2003) Amoebiasis. Lancet 361: 1025–1034. 21. Arana ME, Takata K, Garcia-Diaz M, Wood RD, Kunkel TA (2007) A unique error signature for human DNA polymerase nu. DNA Repair (Amst) 6: 213–223. 5. Embley TM, Martin W (2006) Eukaryotic evolution, changes and challenges. Nature 440: 623–630. 22. Arana ME, Seki M, Wood RD, Rogozin IB, Kunkel TA (2008) Low-fidelity DNA synthesis by human DNA polymerase theta. Nucleic Acids Res 36: 3847–3856. 6. van der Giezen M, Tovar J (2005) Degenerate mitochondria. EMBO Rep 6: 525–530. 7. Mi-ichi F, Abu Yousuf M, Nakada-Tsukui K, Nozaki T (2009) Mitosomes in Entamoeba histolytica contain a sulfate activation pathway. Proc Natl Acad Sci U S A 106: 21731–21736. 23. Seki M, Masutani C, Yang LW, Schuffert A, Iwai S, et al. (2004) High-efficiency bypass of DNA damage by human DNA polymerase Q. EMBO J 23: 4484–4494. 8. Maralikova B, Ali V, Nakada-Tsukui K, Nozaki T, van der Giezen M, et al. (2010) Bacterial-type oxygen detoxification and iron-sulfur cluster assembly in amoebal relict mitochondria. Cell Microbiol 12: 331–342. 24. Seki M, Marini F, Wood RD (2003) POLQ (Pol theta), a DNA polymerase and DNA-dependent ATPase in human cells. Nucleic Acids Res 31: 6117–6126. 9. Loftus B, Anderson I, Davies R, Alsmark UC, Samuelson J, et al. (2005) The genome of the protist parasite Entamoeba histolytica. Nature 433: 865– 868. 25. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673–4680. 10. Supporting Information Figure S1 Optimal activity conditions of EhDNApolA. To determine the optimal conditions for DNA polymerization, 60 fmol of EhDNApolA were incubated with 200 fmol of nicked substrate for 10 min under varying experimental conditions. DNA polymerase activity was measured by the extension of a 24 mer primer to a 45mer product (A) Effect of salt on DNA poly- merization activity. Lane1 corresponds to negative control with no polymerase, lane 2 no added salt salt, lanes 3 to 8 NaCl concentrations from 12.5 mM to 400 mM (B) MgCl2 dependence of EhDNApolA activity. The values were normalized to 100% as the higher polymerization value and the subsequent values were calculated as a relative percentage. The solid bars represent the relative percentage of polymerization. (C) pH influence on DNA polymerization activity (D) Temperature dependence of EhDNA- polA activity. Table S1 Putative DNA polymerases present in the genome of E. histolytica. This table presents a comparison between the putative DNA polymerases present in the genome of E. histolytica and homologous DNA polymerases. Found at: doi:10.1371/journal.pntd.0000786.s003 (0.05 MB DOC) Table S2 GenBank accession numbers of the DNA polymerases used in the phylogenetic tree. This table contains the GenBank accession numbers of the DNA polymerases used to build the phylogenetic tree. Found at: doi:10.1371/journal.pntd.0000786.s004 (0.07 MB DOC) Table S3 Kinetic parameters for nucleotide incorporation by EhDNApolA. This table contains the kinetics parameters (Km and Vmax) for nucleoide incorporation of EhDNApolA. Found at: doi:10.1371/journal.pntd.0000786.s001 (0.14 MB TIF) Found at: doi:10.1371/journal.pntd.0000786.s001 (0.14 MB TIF) Figure S2 mRNA expression profiles of the EhDNApolA gene. RT-PCR analysis of EhDNApolA (Upper panel) in comparison to actin (lower panel). Amplification products using isolated RNA treated with Reverse Transcriptase (RT +) (lanes 1 to 3) or with- out Reverse Transcriptase (RT -) (data not shown) were run on a 1% agarose gel and stained with ethidium bromide. The densitometric analysis of the RT-PCR product corresponding to the actin gene was designated as 100%. The EhDNApolA gene is Found at: doi:10.1371/journal.pntd.0000786.s005 (0.03 MB DOC) References Szuts D, Marcus AP, Himoto M, Iwai S, Sale JE (2008) REV1 restrains DNA polymerase zeta to ensure frame fidelity during translesion synthesis of UV photoproducts in vivo. Nucleic Acids Res 36: 6767–6780. 26. Kumar S, Tamura K, Nei M (2004) MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief Bioinform 5: 150–163. 11. Yoon JH, Prakash L, Prakash S (2010) Error-free replicative bypass of (6-4) photoproducts by DNA polymerase zeta in mouse and human cells. Genes Dev 24: 123–128. 27. Brautigam CA, Steitz TA (1998) Structural principles for the inhibition of the 39- 59 exonuclease activity of Escherichia coli DNA polymerase I by phosphor- othioates. J Mol Biol 277: 363–377. 12. Herrera-Aguirre ME, Luna-Arias JP, Labra-Barrios ML, Orozco E (2010) Identification of four Entamoeba histolytica organellar DNA polymerases of the family B and cellular localization of the Ehodp1 gene and EhODP1 protein. J Biomed Biotechnol 2010: 734898. 28. Diamond LS, Harlow DR, Cunnick CC (1978) A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba. Trans R Soc Trop Med Hyg 72: 431–432. 29. Schreiber E, Matthias P, Muller MM, Schaffner W (1989) Rapid detection of octamer binding proteins with ‘mini-extracts’, prepared from a small number of cells. Nucleic Acids Res 17: 6419. 13. Steitz TA (1999) DNA polymerases: structural diversity and common mechanisms. J Biol Chem 274: 17395–17398. 14. Burgers PM, Koonin EV, Bruford E, Blanco L, Burtis KC, et al. (2001) Eukaryotic DNA polymerases: proposal for a revised nomenclature. J Biol Chem 276: 43487–43490. 30. Diaz-Barriga F, Carrizalez L, Yan˜ez L, Hernandez JM, et al. (1989) Interaction of cadmiun with actin microfilamnets in vitro. Toxicology in vitro 3: 277–284. 31. Iwai S (2001) Synthesis and thermodynamic studies of oligonucleotides containing the two isomers of thymine glycol. Chemistry 7: 4343–4351. 15. Doublie S, Tabor S, Long AM, Richardson CC, Ellenberger T (1998) Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 A resolution. Nature 391: 251–258. 32. Beese LS, Derbyshire V, Steitz TA (1993) Structure of DNA polymerase I Klenow fragment bound to duplex DNA. Science 260: 352–355. 16. Delarue M, Poch O, Tordo N, Moras D, Argos P (1990) An attempt to unify the structure of polymerases. Protein Eng 3: 461–467. 33. Beese LS, Friedman JM, Steitz TA (1993) Crystal structures of the Klenow fragment of DNA polymerase I complexed with deoxynucleoside triphosphate and pyrophosphate. Biochemistry 32: 14095–14101. 17. Discussion August 2010 \| Volume 4 \| Issue 8 \| e786 9 Entamoeba histolytica Family A DNA Polymerase expressed 71% of the actin control. Standard deviations were calculated based on three independent experiments. Found at: doi:10.1371/journal.pntd.0000786.s002 (0.49 MB TIF) expressed 71% of the actin control. Standard deviations were calculated based on three independent experiments. Found at: doi:10.1371/journal.pntd.0000786.s002 (0.49 MB TIF) Author Contributions Conceived and designed the experiments: GPP LGB. Performed the experiments: GPP EAL. Analyzed the data: GPP EAL LGB. Wrote the paper: GPP LGB. Conceived and designed the experiments: GPP LGB. Performed the experiments: GPP EAL. Analyzed the data: GPP EAL LGB. Wrote the paper: GPP LGB. Discussion (C) Nomarsky image of a si phenylindole (DAPI) and incubated with anti-EhDNApolA antibodies treated with FITC-labe generated by DAPI (E) fluorescence signal generated by the binding of the FITC-conjugated anti of the fluorescence emitted by DAPI and FITC. doi:10.1371/journal.pntd.0000786.g007 Figure 7. Cellular identification and localization of EhDNApolA. (A) RT-PCR using total RNA from E. histolytica trophozoites grown in basal culture conditions 1% Agarose gel stained with ethidium bromide showing the RT-PCR products of motifs A (lane 3) and C (lane 4) of the EhDNApolA gene. The RT-PCR product of the actin gene control is shown in lane 2. (B) Immunodetection of EhDNApolA. Western blot assays using, cytoplasmic extracts (lane 1), and nuclear extracts (lane 2) of E. histolytica’s trozophoites against mouse polyclonal anti- EhDNApolA antibodies (upper pannel). Control using mouse polyclonal anti-actin antibodies (middle panel) and anti- C/EBPb antibodies (lower panel) (C–F) Cellular localization of EhDNApolA analyzed by confocal immunofluorescence microscopy. (C) Nomarsky image of a single E. histolytica cell stained with 49,6-diamidino-2- phenylindole (DAPI) and incubated with anti-EhDNApolA antibodies treated with FITC-labeled secondary antibodies (D) fluorescence signal generated by DAPI (E) fluorescence signal generated by the binding of the FITC-conjugated antibody to anti-EhDNApol A antibody (F) Merge image of the fluorescence emitted by DAPI and FITC. doi:10.1371/journal.pntd.0000786.g007 acquired by horizontal gene transfer from a bacterial family A DNA polymerase. The fact that EhDNApolA is biochemically related to DNA polymerase n may be a case of convergent evolution as DNA polymerases of subfamily N are only present in vertebrates [19,48]. Thymine glycol is a DNA lesion formed by chemical oxidation and ionizing radiation [49]. E. histolytica is subject to reactive oxygen species produced at the colonic tissue and by phagocyte release [4,50]. In eukaryotic organisms, thymine glycol can be bypassed by DNA polymerases k and g [40,51]. However, E. histolytica lacks those DNA polymerases. E. histolytica contains genes for Base Excision Repair including functional 8-oxo guanosine and thymine glycol glycosylases (Garcia et al, manuscript in preparation). Although, the in vivo function of EhDNApolA is unknown, its abilities to bypass thymine glycol and nuclear localization suggest a possible role of this enzyme in translesion DNA synthesis. This role is reminiscent of family A DNA polymerases of Arabidopsis thaliana postulated to be involved in DNA repair at the chloroplast [52] and eukaryotic family A DNA polymerases n and h [19,24]. Entamoeba histolytica Family A DNA Polymerase Entamoeba histolytica Family A DNA Polymerase 35. Bernad A, Blanco L, Lazaro JM, Martin G, Salas M (1989) A conserved 39----59 exonuclease active site in prokaryotic and eukaryotic DNA polymerases. Cell 59: 219–228. 43. Shibutani S, Takeshita M, Grollman AP (1997) Translesional synthesis on DNA templates containing a single abasic site. A mechanistic study of the ‘‘A rule’’. J Biol Chem 272: 13916–13922. J 44. Sheriff A, Motea E, Lee I, Berdis AJ (2008) Mechanism and dynamics of translesion DNA synthesis catalyzed by the Escherichia coli Klenow fragment. Biochemistry 47: 8527–8537. 36. Lopes DO, Regis-da-Silva CG, Machado-Silva A, Macedo AM, Franco GR, et al. (2007) Analysis of DNA polymerase activity in vitro using non-radioactive primer extension assay in an automated DNA sequencer. Genet Mol Res 6: 150–155. 45. Marchat LA, Gomez C, Perez DG, Paz F, Mendoza L, et al. (2002) Two CCAAT/enhancer binding protein sites are cis-activator elements of the Entamoeba histolytica EhPgp1 (mdr-like) gene expression. Cell Microbiol 4: 725–737. 37. Polesky AH, Steitz TA, Grindley ND, Joyce CM (1990) Identification of residues critical for the polymerase activity of the Klenow fragment of DNA polymerase I from Escherichia coli. J Biol Chem 265: 14579–14591. 46. Makioka A, Kobayashi S, Takeuchi T (1996) Detection and characterization of DNA polymerase activity in Entamoeba histolytica. Parasitol Res 82: 87–89. 38. Hsu GW, Ober M, Carell T, Beese LS (2004) Error-prone replication of oxidatively damaged DNA by a high-fidelity DNA polymerase. Nature 431: 217–221. 47. Makioka A, Kumagai M, Ohtomo H, Kobayashi S, Takeuchi T (1999) DNA polymerase activity in encysting Entamoeba invadens. Parasitol Res 85: 604–606. 39. Aller P, Rould MA, Hogg M, Wallace SS, Doublie S (2007) A structural rationale for stalling of a replicative DNA polymerase at the most common oxidative thymine lesion, thymine glycol. Proc Natl Acad Sci U S A 104: 814–818. 48. Engelhardt BE, Jordan MI, Muratore KE, Brenner SE (2005) Protein molecular function prediction by Bayesian phylogenomics. PLoS Comput Biol 1: e45. 49. Cathcart R, Schwiers E, Saul RL, Ames BN (1984) Thymine glycol and thymidine glycol in human and rat urine: a possible assay for oxidative DNA damage. Proc Natl Acad Sci U S A 81: 5633–5637. 40. Fischhaber PL, Gerlach VL, Feaver WJ, Hatahet Z, Wallace SS, et al. (2002) Human DNA polymerase kappa bypasses and extends beyond thymine glycols during translesion synthesis in vitro, preferentially incorporating correct nucleotides. J Biol Chem 277: 37604–37611. References Filee J, Forterre P (2005) Viral proteins functioning in organelles: a cryptic origin? Trends Microbiol 13: 510–513. 34. Astatke M, Grindley ND, Joyce CM (1998) How E. coli DNA polymerase I (Klenow fragment) distinguishes between deoxy- and dideoxynucleotides. J Mol Biol 278: 147–165. 18. Filee J, Forterre P, Sen-Lin T, Laurent J (2002) Evolution of DNA polymerase families: evidences for multiple gene exchange between cellular and viral proteins. J Mol Evol 54: 763–773. August 2010 \| Volume 4 \| Issue 8 \| e786 August 2010 \| Volume 4 \| Issue 8 \| e786 10 www.plosntds.org Entamoeba histolytica Family A DNA Polymerase g 50. Bogdan C, Rollinghoff M, Diefenbach A (2000) Reactive oxygen and reactive nitrogen intermediates in innate and specific immunity. Curr Opin Immunol 12: 64–76. 41. Brieba LG, Eichman BF, Kokoska RJ, Doublie S, Kunkel TA, et al. (2004) Structural basis for the dual coding potential of 8-oxoguanosine by a high-fidelity DNA polymerase. EMBO J 23: 3452–3461. 51. Kusumoto R, Masutani C, Iwai S, Hanaoka F (2002) Translesion synthesis by human DNA polymerase eta across thymine glycol lesions. Biochemistry 41: 6090–6099. p y J 42. 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https://openalex.org/W2617231285	https://www.mdpi.com/2072-4292/9/6/536/pdf?version=1495947604	English	null	Quantifying the Spatiotemporal Trends of Canopy Layer Heat Island (CLHI) and Its Driving Factors over Wuhan, China with Satellite Remote Sensing	Remote sensing	2,017	cc-by	14,338	Quantifying the Spatiotemporal Trends of Canopy Layer Heat Island (CLHI) and Its Driving Factors over Wuhan, China with Satellite Remote Sensing Long Li 1, Xin Huang 2,3,, Jiayi Li 2, and Dawei Wen 3 1 Jiangsu Center for Collaborative Innovation in Geographical Information Resource Development and Application, Key Laboratory of Virtual Geographic Environment of Ministry of Education, College of Geographic Science, Nanjing Normal University, Nanjing 210023, China; li_miany@163.com 2 School of Remote Sensing and Information Engineering, Wuhan University, Wuhan 430079, China 3 State Key Laboratory of Information Engineering in Surveying, Mapping and Remote Sensing, Wuhan University, Wuhan 430079, China; daweiwen@whu.edu.cn * Correspondence: xhuang@whu.edu.cn (X.H.); zjjerica@whu.edu.cn (J.L.) Academic Editors: Dale A. Quattrochi and Prasad S. Thenkabail Received: 25 March 2017; Accepted: 24 May 2017; Published: 27 May 2017 1 Jiangsu Center for Collaborative Innovation in Geographical Information Resource Development and Application, Key Laboratory of Virtual Geographic Environment of Ministry of Education, College of Geographic Science, Nanjing Normal University, Nanjing 210023, China; li_miany@163.com 2 School of Remote Sensing and Information Engineering, Wuhan University, Wuhan 430079, China 3 State Key Laboratory of Information Engineering in Surveying, Mapping and Remote Sensing, Wuhan University, Wuhan 430079, China; daweiwen@whu.edu.cn * Correspondence: xhuang@whu.edu.cn (X.H.); zjjerica@whu.edu.cn (J.L.) Academic Editors: Dale A. Quattrochi and Prasad S. Thenkabail Received: 25 March 2017; Accepted: 24 May 2017; Published: 27 May 2017 Abstract: Canopy layer heat islands (CLHIs) in urban areas are a growing problem. In recent decades, the key issues have been how to monitor CLHIs at a large scale, and how to optimize the urban landscape to mitigate CLHIs. Taking the city of Wuhan as a case study, we examine the spatiotemporal trends of the CLHI along urban-rural gradients, including the intensity and footprint, based on satellite observations and ground weather station data. The results show that CLHI intensity (CLHII) decays exponentially and signiﬁcantly along the urban-rural gradients, and the CLHI footprint varies substantially and especially in winter. Quantifying the Spatiotemporal Trends of Canopy Layer Heat Island (CLHI) and Its Driving Factors over Wuhan, China with Satellite Remote Sensing We then quantify the driving factors of the CLHI by establishing multiple linear regression (MLR) models with the assistance of ZY-3 satellite data (with a spatial resolution of 2.5 m), and obtain ﬁve main ﬁndings: (1) built-up area had a signiﬁcant positive effect on daily mean CLHII in summer and a negative effect in winter; (2) vegetation had signiﬁcant inhibiting effects on daily mean CLHII in both summer and winter; (3) absolute humidity has a signiﬁcant inhibiting effect on daily mean CLHII in summer and a positive effect in winter; (4) anthropogenic heat emissions exacerbated the daily mean CLHII by about 0.19 ◦C (90% conﬁdence interval −0.06–0.44 ◦C) on 17 September 2013 and by about 0.06 ◦C (−0.06–0.19 ◦C) on 23 January 2014; and (5) if most of the urban area is transformed into roads (i.e., an extreme case), we estimate that the daily mean CLHII would reach 1.41 ◦C (0.38–2.44 ◦C) on 17 September 2013 and 0.14 ◦C (0.08–0.2 ◦C) on 23 January 2014 in Wuhan metropolitan area. Overall, the results provide new insights into quantifying the CLHI and its driving factors, to enhance our understanding of urban heat islands. Keywords: urban heat island (UHI); surface air temperature; spatiotemporal trend; NDVI; NDBI; absolute humidity and nighttime lights remote sensing remote sensing www.mdpi.com/journal/remotesensing 2. Study Area anthropo Wuhan, situated in central China (as shown in Figure 1), covers an area of 8594 km2 and has a population of over 10 million, with the mean annual temperature ranging from 15.8 ◦C to 17.5 ◦C [44,45]. As the capital of Hubei province, it is recognized as the industrial base, scientiﬁc education base, and transportation center of central China. Its metropolitan area is divided into three parts by the conﬂuence of the Yangtze River and Han River. It has a water area of about 2217 km2, which covers a quarter of the total area. Over the last three decades, Wuhan has experienced rapid urbanization that is similar to that experienced in Shanghai, Beijing, and Guangzhou [46]. Therefore, research into the CLHI is of special signiﬁcance for this city. 2. Study Area Wuhan, situated in central China (as shown in Figure 1), covers an area of 8594 km2 and has a population of over 10 million, with the mean annual temperature ranging from 15.8 °C to 17.5 °C [44,45]. As the capital of Hubei province, it is recognized as the industrial base, scientific education base, and transportation center of central China. Its metropolitan area is divided into three parts by the confluence of the Yangtze River and Han River. It has a water area of about 2217 km2, which covers a quarter of the total area. Over the last three decades, Wuhan has experienced rapid urbanization that is similar to that experienced in Shanghai, Beijing, and Guangzhou [46]. Therefore, research into the CLHI is of special significance for this city. Figure 1. Study area, showing the location of the city of Wuhan in central China and the locations of the 436 weather stations. All of the weather stations were used for mapping the SAT; the 28 weather stations recording vapour pressure and SAT data were used to predict the absolute humidity. The acquisition times of the multi-temporal satellite observations and ground weather station data are 17 September 2013, and 23 January 2014. Figure 1. Study area, showing the location of the city of Wuhan in central China and the locations of the 436 weather stations. All of the weather stations were used for mapping the SAT; the 28 weather stations recording vapour pressure and SAT data were used to predict the absolute humidity. 1. Introduction Urbanization, which refers to a major anthropogenic inﬂuence on the earth’s surface, is taking place at an unprecedented rate in China [1]. Concurrently, the world urban population increased from 1.35 billion in 1970 to 3.63 billion in 2011, and is expected to reach 5 billion by 2030 and 6.4 billion by 2050 [2,3]. As a result of the urbanization process, the global area of impervious surfaces is increasing rapidly [4], triggering a series of negative environmental impacts [5]. Among these impacts, the urban heat island (UHI) effect, i.e., the phenomenon of urban areas having higher air/surface temperatures Remote Sens. 2017, 9, 536; doi:10.3390/rs9060536 www.mdpi.com/journal/remotesensing 2 of 18 Remote Sens. 2017, 9, 536 than rural areas, can affect not only local and regional climate [6], but also air quality, water supplies, vegetation phenology, and human health [7–10]. At present, two main strands of research have evolved for quantifying UHIs: canopy layer heat islands (CLHIs) and surface UHIs (SUHIs). A CLHI is determined by measuring surface air temperature (SAT, usually 2 m) above ground, and a SUHI is determined by land surface temperature (LST) derived from remote sensing data [11]. Generally speaking, most of the existing studies have focused on SUHI analysis [12–20], and few studies have addressed SAT retrieval from space for CLHI analysis [21–23]. Several studies have reported that SAT can have an impact on human health as a result of affecting human sensible temperature and increasing disease transmission [24–26]. Thus, there is a strong impetus to understand CLHI effects. Remote Sens. 2017, 9, 536 2 of 18 strands of research have evolved for quantifying UHIs: canopy layer heat islands (CLHIs) and surface UHIs (SUHIs). A CLHI is determined by measuring surface air temperature (SAT, usually 2 m) above ground, and a SUHI is determined by land surface temperature (LST) derived from remote sensing data [11]. Generally speaking, most of the existing studies have focused on SUHI analysis [12–20], and few studies have addressed SAT retrieval from space for CLHI analysis [21–23]. Several di h d h SAT h i h h l h l f ff i h In contrast to rural areas, several studies have analyzed data obtained from ground weather stations and concluded that urban areas have become signiﬁcantly warmer (i.e., the CLHI effect) in recent decades [27–32]. However, these studies have been limited by the sparse distribution of weather stations [33,34]. 1. Introduction Satellite imagery can help to overcome the spatial discontinuity issue of ground weather stations, and has been used to derive LST in many studies [35–39]. Unfortunately, the application of LST to quantify the SUHI effect in large-scale areas is impacted by the limited satellite time-series data and cloud and mist disturbance [40–42]. Ho et al. combined satellite observations with ground weather station data to map maximum temperature distributions in urban areas [43], but they did not focus on the studies of CLHI, such as its spatiotemporal trends and driving factors. studies have reported that SAT can have an impact on human health as a result of affecting human sensible temperature and increasing disease transmission [24–26]. Thus, there is a strong impetus to understand CLHI effects. In contrast to rural areas, several studies have analyzed data obtained from ground weather stations and concluded that urban areas have become significantly warmer (i.e., the CLHI effect) in recent decades [27–32]. However, these studies have been limited by the sparse distribution of weather stations [33,34]. Satellite imagery can help to overcome the spatial discontinuity issue of ground weather stations, and has been used to derive LST in many studies [35–39]. Unfortunately, the application of LST to quantify the SUHI effect in large-scale areas is impacted by the limited satellite time-series data and cloud and mist disturbance [40–42]. Ho et al. combined satellite observations with ground weather station data to map maximum temperature distributions in urban areas [43], but they did not focus on As the first attempt, based on meteorological data and satellite remote sensing products, the main objectives of our study are: (1) to examine the spatiotemporal trends of the CLHI, including intensity and footprint; (2) to compare the CLHI and SUHI with regard to intensity, footprint, and spatiotemporal continuity, and to confirm the effectiveness of real-time monitoring of CLHI; and (3) to quantify the driving factors of the CLHI (e.g., absolute humidity, built-up land, vegetation, and anthropogenic heat emissions). station data to map maximum temperature distributions in urban areas [43], but they did not focus on the studies of CLHI, such as its spatiotemporal trends and driving factors. 1. Introduction As the first attempt, based on meteorological data and satellite remote sensing products, the main objectives of our study are: (1) to examine the spatiotemporal trends of the CLHI, including intensity and footprint; (2) to compare the CLHI and SUHI with regard to intensity, footprint, and spatiotemporal continuity, and to confirm the effectiveness of real-time monitoring of CLHI; and (3) to quantify the driving factors of the CLHI (e.g., absolute humidity, built-up land, vegetation, and 2. Study Area anthropo The acquisition times of the multi-temporal satellite observations and ground weather station data are 17 September 2013, and 23 January 2014. Figure 1. Study area, showing the location of the city of Wuhan in central China and the locations of the 436 weather stations. All of the weather stations were used for mapping the SAT; the 28 weather stations recording vapour pressure and SAT data were used to predict the absolute humidity. The acquisition times of the multi-temporal satellite observations and ground weather station data are 17 September 2013, and 23 January 2014. Figure 1. Study area, showing the location of the city of Wuhan in central China and the locations of the 436 weather stations. All of the weather stations were used for mapping the SAT; the 28 weather stations recording vapour pressure and SAT data were used to predict the absolute humidity. The acquisition times of the multi-temporal satellite observations and ground weather station data are 17 September 2013, and 23 January 2014. 3 of 18 Remote Sens. 2017, 9, 536 3.2. Predicting SAT and Absolute Humidity In the study conducted by Ho et al. of Greater Vancouver, British Columbia, Canada, it was found that the random forest model could produce a more reasonable map of SAT distribution than ordinary least squares (OLS) regression and support vector machine [43]. Therefore, in our study, the random forest model was used to supplement meteorological monitoring network by estimating the SAT and absolute humidity of 100,000 random points in the study area. Based on supplementary monitoring network, inverse distance weighted method was used to map SAT and absolute humidity. A number of different data layers (14 predictive variables) were derived from the Landsat-8 and DEM data as predictive variables in the random forest model: bands 1–7, bands 10–11, the normalized difference vegetation index (NDVI), the normalized difference built-up index (NDBI), the normalized difference water index (NDWI), the DEM, and the LST. All of the 436 weather stations were used for the calibration and validation of the regression for predicting SAT, and these weather stations were divided into two groups: training (50%) and validation (50%), respectively. Moreover, absolute humidity (α) was determined (in g/m3) using the well-known equation of state for water vapour [52]: (1) α = e Rw·T (1) where e represents vapour pressure (hPa), and T represents air temperature (Kelvin), and Rw equal to 0.4615 Jg−1 K−1. As the same with the mapping of SAT, absolute humidity was divided into two groups including training (70%) and validation (30%), and the spatial pattern was generated by random forest model and inverse distance weighted method. All the layers, except for the elevation, were derived from the Landsat-8 data. The NDVI is constructed by exploiting the great difference in the spectral reﬂectance in the near-infrared (which is strongly reﬂected by vegetation) and red bands (which is absorbed by vegetation): NDVI = (NIR −R) (NIR + R) (2) (2) where NIR is reﬂectance in the near-infrared (OLI 5) band, and R is reﬂectance in the red (OLI 4) band. The NDBI is designed [53], using the reﬂectance in the near-infrared and short-wave bands for exploiting the built-up areas: (SWIR NIR) where NIR is reﬂectance in the near-infrared (OLI 5) band, and R is reﬂectance in the red (OLI 4) band. 3.1. Data Preparation In this study, data for a total of 436 regional automatic weather stations were obtained from the Meteorological Bureau of Hubei Province [47]. The type of automatic weather stations includes ZQZ-A, HY-361, DZZ4 and CAWS600-RT. Temperature and humidity sensors from theses weather stations are installed in the window-shades for preventing from the inﬂuence of strong winds, rain and snow. Meteorological data from these stations have measuring accuracies in 0.1 ◦C and 0.1 hpa for air temperature and vapour pressure, respectively. Among these weather stations, 137 weather stations were situated in the study area (red rectangle in Figure 1), and both vapour pressure and air temperature were recorded by a total of 28 weather stations. Landsat-8 images were obtained for 17 September 2013, and 23 January 2014 [48], with a spatial resolution of 30 m. A digital elevation model (DEM) was obtained with a spatial resolution of approximately 100 m [49]. A “nighttime lights” time series product for 2013 was obtained at a 30 arc second grid [50]. The latest Version-5 MODIS-Terra LST product (MOD11A1) monitored at 10:30 h (daytime) and 22:30 h (nighttime) local solar time with an approximately 1-km spatial resolution was also obtained [51]. 3.2. Predicting SAT and Absolute Humidity The NDBI is designed [53], using the reﬂectance in the near-infrared and short-wave bands for exploiting the built-up areas: (SWIR NIR) NDBI = (SWIR −NIR) (SWIR + NIR) (3) (3) where SWIR is reﬂectance in the short-wave infrared (OLI 6) band, and NIR is reﬂectance in the near-infrared (OLI 5) band. where SWIR is reﬂectance in the short-wave infrared (OLI 6) band, and NIR is reﬂectance in the near-infrared (OLI 5) band. Remote Sens. 2017, 9, 536 4 of 18 The NDWI is an index designed to quantify vegetation water status [54], by using a weak liquid water absorption band (shortwave-infrared band) and the liquid water intensive band (near-infrared band): Remote Sens. 2017, 9, 536 4 of 18 The NDWI is an index designed to quantify vegetation water status [54], by using a weak liquid NDWI = (NIR −SWIR) (NIR + SWIR) (4) q y g [ ], y g q -infrared band) and the liquid water intensive band (near-infrared (4) where NIR is reﬂectance in the near-infrared (OLI 5) band, and SWIR is reﬂectance in the short-wave infrared (OLI 6) band. NDWI = ሺNIR −SWIRሻ ሺNIR + SWIRሻ (4) LST was derived by the atmospheric correction method [55], i.e., radiative transfer equation (RTE). A ﬂowchart of the methodology used in this study is given in Figure 2. Next, a regression calibration approach was used to reduce the deviation when quantifying the CLHI owing to the tendency of random forest to “regress towards the mean”. It can make the ﬁtted curve of the scatter diagram of observed versus predicted values become a diagonal line. Finally, the RMSE was used to quantify the magnitude of error. The formulas are as follows: where NIR is reflectance in the near-infrared (OLI 5) band, and SWIR is reflectance in the short-wave infrared (OLI 6) band. LST was derived by the atmospheric correction method [55], i.e., radiative transfer equation (RTE). A flowchart of the methodology used in this study is given in Figure 2. Next, a regression calibration approach was used to reduce the deviation when quantifying the CLHI owing to the tendency of random forest to “regress towards the mean”. It can make the fitted curve of the scatter diagram of observed versus predicted values become a diagonal line. Finally, the RMSE was used RMSE = s ∑n i=1(Xobs,i −Xmodel,i)2 n (5) f error. 3.2. Predicting SAT and Absolute Humidity The formulas are as follows: RMSE = ඨ∑ ൫X୭ୠୱ,୧−X୫୭ୢୣ୪,୧൯ ଶ ୬ ୧ୀଵ n (5) (5) n −X where Xobs,i represents the observed value, Xmodel,i represents the predicted value for the weather station, n represents the total number of weather stations. n where X୭ୠୱ,୧ represents the observed value, X୫୭ୢୣ୪,୧ represents the predicted value for the weather station, n represents the total number of weather stations. Figure 2. Flowchart showing the methodology implemented in this study. Figure 2. Flowchart showing the methodology implemented in this study. Figure 2. Flowchart showing the methodology implemented in this study. Figure 2. Flowchart showing the methodology implemented in this study. 3.3. Quantifying the CLHII and Footprint In this study to examine the CLH 3.3. Quantifying the CLHII and Footprint 3.3. Quantifying the CLHII and Footprint In this study to examine the CLH 3.3. Quantifying the CLHII and Footprint The formula is ∆Tn = A × e−S×n (7) (7) ∆T୬= A ൈeିୗൈ୬ (7) where A indicates the maximum SAT difference (close to the CLHII); n represents the numbers of urban zones within the buffern; S represents the decay rate of the CLHII along the urban-rural where A indicates the maximum SAT difference (close to the CLHII); n represents the numbers of urban zones within the buffern; S represents the decay rate of the CLHII along the urban-rural gradients, and a larger S indicates a smaller footprint of CLHI [58]. gradients, and a larger S indicates a smaller footprint of CLHI [58]. However, there is no universally accepted method for defining the SUHI footprint since the ∆T varies substantially over time and space [31,42,56,58]. Previous studies have focused on the binary classification of measurement sites as “urban” or “rural”, thereby defining the CLHII [27,28,59], but have not focused on the CLHI footprint. Zhou et al. assumed that the SUHI footprint was the continuous extent emanating outward from the urban center to the rural areas with a ∆T୬ statistically larger than zero [58]. However, this method fails to calculate the CLHI footprint because SAT is significantly affected by wind, relative to the SUHI footprint. Therefore, we quantified the CLHI footprint as the continuous extent emanating outward from the urban center to the rural areas with the CLHI showing an inverse trend in certain buffer zones (i.e., CLHI: ∆T଴൐0, ∆T୬൏∆T୬ାଵ, ∆T୬൏∆T୬ାଶ, CLCI: ∆T଴൏0, ∆T୬൐∆T୬ାଵ, ∆T୬൐∆T୬ାଶ, where n represents the CLHI footprint, unit: number of the buffer zone). Batty introduced a graphical representation termed the However, there is no universally accepted method for deﬁning the SUHI footprint since the ∆T varies substantially over time and space [31,42,56,58]. Previous studies have focused on the binary classiﬁcation of measurement sites as “urban” or “rural”, thereby deﬁning the CLHII [27,28,59], but have not focused on the CLHI footprint. Zhou et al. assumed that the SUHI footprint was the continuous extent emanating outward from the urban center to the rural areas with a ∆Tn statistically larger than zero [58]. However, this method fails to calculate the CLHI footprint because SAT is signiﬁcantly affected by wind, relative to the SUHI footprint. 3.3. Quantifying the CLHII and Footprint In this study to examine the CLH 3.3. Quantifying the CLHII and Footprint The red border represents the manually delineated urban area, and the white borders are nine buffer zones that each cover the same area of land as the urban area. Figure 3. Delineation of the Wuhan metropolitan area and nine buffer zones (a). Daily mean SAT in summer (b). Daily mean SAT in winter (c). The red border represents the manually delineated urban area, and the white borders are nine buffer zones that each cover the same area of land as the urban Figure 3. Delineation of the Wuhan metropolitan area and nine buffer zones (a). Daily mean SAT in summer (b). Daily mean SAT in winter (c). The red border represents the manually delineated urban area, and the white borders are nine buffer zones that each cover the same area of land as the urban area. The intensity represents the magnitude of the CLHI (i.e., the CLHII). In this study, we considered the median of the SAT in the three outermost buffer zones as the rural SAT (i.e., the background temperature), because this was able to reduce the bias caused by the possible outliers among the three outermost buffer zones [58]. We calculated the SAT difference (∆T୬) in each buffer zone and the rural SAT as follows: The intensity represents the magnitude of the CLHI (i.e., the CLHII). In this study, we considered the median of the SAT in the three outermost buffer zones as the rural SAT (i.e., the background temperature), because this was able to reduce the bias caused by the possible outliers among the three outermost buffer zones [58]. We calculated the SAT difference (∆Tn) in each buffer zone and the rural SAT as follows: (6) (6) ∆T T T (6) ∆Tn = Tn −Trural (6) ∆T = T T ∆Tn = Tn −Trural ∆T୬= T୬−T୰୳୰ୟ୪ (6) where ∆T୬ represents the difference between the SAT in buffer୬ ሺ0 ൑n ൑9ሻ and the rural SAT, T୬ represents the SAT in buffer୬ ሺ0 ൑n ൑9ሻ, and T୰୳୰ୟ୪ represents the SAT in the rural area (i.e., the background SAT). Among them, ∆T଴ indicates the CLHII in the urban area. A positive value of ∆T଴ indicates a CLHI effect, and the opposite indicates a “canopy layer cool island” (CLCI) effect. 3.3. Quantifying the CLHII and Footprint In this study to examine the CLH 3.3. Quantifying the CLHII and Footprint Therefore, the CLHII is loosely defined as the temperature difference between the urban and rural where ∆Tn represents the difference between the SAT in buffern (0 ≤n ≤9) and the rural SAT, Tn represents the SAT in buffern (0 ≤n ≤9), and Trural represents the SAT in the rural area (i.e., the background SAT). Among them, ∆T0 indicates the CLHII in the urban area. A positive value of ∆T0 indicates a CLHI effect, and the opposite indicates a “canopy layer cool island” (CLCI) effect. Therefore, the CLHII is loosely defined as the temperature difference between the urban and rural areas (i.e., ∆T0). areas (i.e., ∆T଴). The footprint represents the areas significantly affected by the CLHI (i.e., the extent of the CLHI). Previous studies have reported that the strength of the SUHI influence decays exponentially with distance from the perimeter of the urban land cover [9]. However, to our knowledge, few studies have focused on the footprint of CLHI, based on the spatial distribution of SAT in the whole region. Thus, an exponential decay model was applied to assess the trends of ∆T୬ in each buffer f i l d t i t l f 1 h i diff t ( d i t ) Th f l i The footprint represents the areas significantly affected by the CLHI (i.e., the extent of the CLHI). Previous studies have reported that the strength of the SUHI influence decays exponentially with distance from the perimeter of the urban land cover [9]. However, to our knowledge, few studies have focused on the footprint of CLHI, based on the spatial distribution of SAT in the whole region. Thus, an exponential decay model was applied to assess the trends of ∆Tn in each buffer zone for a single day at an interval of 1 h, in different seasons (summer and winter). The formula is expressed as follows: h, in different seasons (summer and winter). 3.3. Quantifying the CLHII and Footprint In this study to examine the CLH 3.3. Quantifying the CLHII and Footprint In this study, to examine the CLHI with regard to the spatiotemporal trends of intensity and footprint, we first defined the urban area and the surrounding buffer zones. A three-step method was used to define the urban area [56]. In view of the concentrated distribution of built-up areas in Wuhan, we extracted the urban border based on visual interpretation. The urban area covered a total of 743 km2 in 2013. Emanating outwards from the urban border to the rural areas, nine buffer zones were generated in ArcGIS 10.2. The area of each buffer zone was equal to the urban area (as shown in Figure 3; the scope of Figure 3 is the “study area” outlined in red in Figure 1). Water bodies and pixels with a relatively higher elevation (more than 50 m) in the urban area were excluded from this analysis because these pixels may have overshadowed the effect of the urbanization on SAT [57]. In this study, to examine the CLHI with regard to the spatiotemporal trends of intensity and footprint, we ﬁrst deﬁned the urban area and the surrounding buffer zones. A three-step method was used to deﬁne the urban area [56]. In view of the concentrated distribution of built-up areas in Wuhan, we extracted the urban border based on visual interpretation. The urban area covered a total of 743 km2 in 2013. Emanating outwards from the urban border to the rural areas, nine buffer zones were generated in ArcGIS 10.2. The area of each buffer zone was equal to the urban area (as shown in Figure 3; the scope of Figure 3 is the “study area” outlined in red in Figure 1). Water bodies and pixels with a relatively higher elevation (more than 50 m) in the urban area were excluded from this analysis because these pixels may have overshadowed the effect of the urbanization on SAT [57]. 5 of 18 Remote Sens. 2017, 9, 536 Figure 3. Delineation of the Wuhan metropolitan area and nine buffer zones (a). Daily mean SAT in summer (b). Daily mean SAT in winter (c). The red border represents the manually delineated urban area, and the white borders are nine buffer zones that each cover the same area of land as the urban area Figure 3. Delineation of the Wuhan metropolitan area and nine buffer zones (a). Daily mean SAT in summer (b). Daily mean SAT in winter (c). 3.4. Quantifying Potential Drivers of the CLHI The OLS model has been widely used in the analysis of the SUHI effect [17,61,62]. However, few studies have focused on the drivers of CLHI. The OLS model was thus used to analyze the CLHI effect. Considering the interacting effects of the driving factors of daily mean CLHII, we quantiﬁed the relationships between daily mean CLHII and its driving factors (i.e., absolute humidity, built-up land, vegetation, and anthropogenic heat emissions), to gain a better understanding of the CLHI by establishing the multiple linear regression (MLR) model. In this study, the mean values of the driving factors in every buffer zone were used to quantify the relationships with daily mean CLHII. The driving factors of built-up land, vegetation, absolute humidity, and anthropogenic heat emissions were considered as the explanatory variables, and the daily mean CLHII was considered as the response variable to quantify. Speciﬁcally, nighttime lights were used as a proxy of anthropogenic heat release (e.g., buildings, industry, and vehicles) in both the urban area and suburbs [42,56,63]. The NDBI was able to describe built-up areas [53]. The NDVI was used to describe the vegetation abundance. p g In order to quantify the potential CLHII in different conditions (e.g., a built-up area covered with sparse vegetation) based on the MLR model, we extracted the land-cover types, including bare land, woodland, herbage, building, road, river, and lake, to determine the NDBI, the NDVI, and the absolute humidity of the land cover. We extracted the land cover using the maximum likelihood method and a post-classification process (including majority analysis, clump classes, and sieve classes) implemented in ENVI 5.1. The nighttime lights product was used to extract urban impervious surfaces (threshold set as 20). In the urban area, the classes of road/building and woodland/herbage were distinguished with the assistance of high-resolution remote sensing images (i.e., ZY-3 satellite images with a spatial resolution of 2.5 m) [64,65]. Particularly, land cover map obtained from ZY-3 was used to extract parameter values in the urban area, and based on the land cover map obtained from Landsat-8, we extract the parameter values in the rural area. NDBI and NDVI parameters for building, road, woodland, and herbage are calculated in the urban area to indicate the difference index of reflectance of ground objects. 3.3. Quantifying the CLHII and Footprint In this study to examine the CLH 3.3. Quantifying the CLHII and Footprint Therefore, we quantiﬁed the CLHI footprint as the continuous extent emanating outward from the urban center to the rural areas with the CLHI showing an inverse trend in certain buffer zones (i.e., CLHI: ∆T0 > 0, ∆Tn < ∆Tn+1, ∆Tn < ∆Tn+2, CLCI: ∆T0< 0, ∆Tn >∆Tn+1, ∆Tn > ∆Tn+2, where n represents the CLHI footprint, unit: number of the buffer zone). Batty introduced a graphical representation termed the “rank clock” to examine the trajectories of cities in US [60]. The “rank clock” was used to describe the spatial and Remote Sens. 2017, 9, 536 6 of 18 temporal variations of size of cities. Based on “rank clock”, we examined the dynamics of CLHI footprint. The circumference represents the time series range from 0:00 to 23:00, and the distance away from the center represents the CLHI footprint. 3.4. Quantifying Potential Drivers of the CLHI Considering the interaction effect in space, humidity parameters for woodland and herbage are calculated only in rural areas because they can be underestimated when built-up area is covered by dense vegetation. In addition, humidity parameters for building and road are calculated only in urban areas because they can be overestimated when built-up area is covered with sparse vegetation. The parameter values of the NDBI, the NDVI, the absolute humidity, and the digital number (DN) of the nighttime lights were substituted into the equation of the MLR model to quantify potential CLHII. 4.2. CLHI and SUHI D il SAT 4.2. CLHI and SUHI 4.2. CLHI and SUHI Daily mean SAT is shown in Figure 7. LST derived from Landsat-8 and MODIS-terra is shown in Figure 8. Although the obvious differences that high LST existed in the western part for the LST from MODIS-terra in summer, it would not affect the quantification of SUHI, because the study area mainly covers the eastern part, where the city SUHI is well established. Exponential decay trends of daily mean CLHII and SUHII along the urban-rural (buffer zones 0–9) gradients are given in Figure 9. Particularly, MODIS (Terra) and Landsat-8 OLI/TIRS were used to derive the LST, but the LST obtained from the MOD11A1 product was not available during both daytime and nighttime in winter, because of cloud cover. In addition, exponential decay trends of the CLHI at an interval of 2 hour are also given in Figure 10. Based on estimated and observed temperature, the change trends of daily SAT (maximum, mean, and minimum value) are given in Figure 11. The temporal trends of the CLHII were successfully quantified (as shown in Figure 12a). Furthermore, the CLHI footprint was Daily mean SAT is shown in Figure 7. LST derived from Landsat-8 and MODIS-terra is shown in Figure 8. Although the obvious differences that high LST existed in the western part for the LST from MODIS-terra in summer, it would not affect the quantification of SUHI, because the study area mainly covers the eastern part, where the city SUHI is well established. Exponential decay trends of daily mean CLHII and SUHII along the urban-rural (buffer zones 0–9) gradients are given in Figure 9. Particularly, MODIS (Terra) and Landsat-8 OLI/TIRS were used to derive the LST, but the LST obtained from the MOD11A1 product was not available during both daytime and nighttime in winter, because of cloud cover. In addition, exponential decay trends of the CLHI at an interval of 2 hour are also given in Figure 10. Based on estimated and observed temperature, the change trends of daily SAT (maximum, mean, and minimum value) are given in Figure 11. The temporal trends of the CLHII were successfully quantified (as shown in Figure 12a) Furthermore the CLHI footprint was Daily mean SAT is shown in Figure 7. LST derived from Landsat-8 and MODIS-terra is shown in Figure 8. 4.1. Data Processing Performance We used a regression calibration approach to reduce the deviation when quantifying the CLHI. This approach makes the ﬁtted curve of the scatter diagram of observed versus predicted values become a diagonal line (an example is shown in Figure 4). The determination coefﬁcients (R2 = 0.53) in this study were superior to the previous study [43]. However, the other roughly 50 % of variance could be affected by many potential factors, such as wind and clouds. The prediction errors (RMSE) at an interval of 1 h are displayed in Figure 5. The daily mean RMSEs were 1.70 ◦C (SAT in summer), 2.32 ◦C (SAT in winter), 1.36 g/m3 (absolute humidity in summer) and 0.71 g/m3 (absolute humidity in winter) after correction. The maximum likelihood method (overall accuracy = 88.04%, Kappa coefﬁcient = 0.84) and a post-classiﬁcation process were used to extract the land-use types in the study area. The classes of road/building and woodland/farmland were distinguished based on high-resolution remote sensing images (i.e., ZY-3 satellite data with a spatial resolution of 2.5 m) in the 7 of 18 7 of 18 Remote Sens. 2017, 9, 536 urban area, and woodland/farmland was distinguished based on Landsat-8 images in the rural area (Figure 6). the urban area, and woodland/farmland was distinguished based on Landsat-8 images in the rural area (Figure 6). the urban area, and woodland/farmland was distinguished based on Landsat-8 images in the rural area (Figure 6). Figure 4. Scatter plot of the observed versus predicted SAT values obtained using the random forest model. No correction (black); Correction (red) based on the regression calibration approach. Figure 4. Scatter plot of the observed versus predicted SAT values obtained using the random forest model. No correction (black); Correction (red) based on the regression calibration approach. Figure 4. Scatter plot of the observed versus predicted SAT values obtained using the random forest model. No correction (black); Correction (red) based on the regression calibration approach. Figure 4. Scatter plot of the observed versus predicted SAT values obtained using the random forest model. No correction (black); Correction (red) based on the regression calibration approach. Figure 4. Scatter plot of the observed versus predicted SAT values obtained using the random forest model. No correction (black); Correction (red) based on the regression calibration approach. Figure 4. Scatter plot of the observed versus predicted SAT values obtained using the random forest model. 4.1. Data Processing Performance No correction (black); Correction (red) based on the regression calibration approach. Figure 5. The prediction errors (RMSE) of SAT at an interval of 1 hour obtained using the random forest model. Correction in summer = “C in S”; correction in winter = “C in W”; non-correction in summer = “NC in S”; non-correction in winter = “NC in W”. “Correction” indicates that the predicted values estimated from the random forest model were stretched based on the regression calibration approach. “Non-correction” represents the predicted values based on the random forest model. CLHI and SUHI Figure 5. The prediction errors (RMSE) of SAT at an interval of 1 hour obtained using the random forest model. Correction in summer = “C in S”; correction in winter = “C in W”; non-correction in summer = “NC in S”; non-correction in winter = “NC in W”. “Correction” indicates that the predicted values estimated from the random forest model were stretched based on the regression calibration approach. “Non-correction” represents the predicted values based on the random forest model. Figure 5. The prediction errors (RMSE) of SAT at an interval of 1 h obtained using the random forest model. Correction in summer = “C in S”; correction in winter = “C in W”; non-correction in summer = “NC in S”; non-correction in winter = “NC in W”. “Correction” indicates that the predicted values estimated from the random forest model were stretched based on the regression calibration approach. “Non-correction” represents the predicted values based on the random forest model. Figure 5. The prediction errors (RMSE) of SAT at an interval of 1 hour obtained using the random forest model. Correction in summer = “C in S”; correction in winter = “C in W”; non-correction in summer = “NC in S”; non-correction in winter = “NC in W”. “Correction” indicates that the predicted values estimated from the random forest model were stretched based on the regression calibration approach. “Non-correction” represents the predicted values based on the random forest model. Figure 5. The prediction errors (RMSE) of SAT at an interval of 1 hour obtained using the random forest model. Correction in summer = “C in S”; correction in winter = “C in W”; non-correction in summer = “NC in S”; non-correction in winter = “NC in W”. “Correction” indicates that the predicted values estimated from the random forest model were stretched based on the regression calibration approach. 4.1. Data Processing Performance “Non-correction” represents the predicted values based on the random forest model. Figure 5. The prediction errors (RMSE) of SAT at an interval of 1 h obtained using the random forest model. Correction in summer = “C in S”; correction in winter = “C in W”; non-correction in summer = “NC in S”; non-correction in winter = “NC in W”. “Correction” indicates that the predicted values estimated from the random forest model were stretched based on the regression calibration approach. “Non-correction” represents the predicted values based on the random forest model. 4.2. CLHI and SUHI D il SAT 4.2. CLHI and SUHI 4.2. CLHI and SUHI Although the obvious differences that high LST existed in the western part for the LST from MODIS-terra in summer, it would not affect the quantiﬁcation of SUHI, because the study area mainly covers the eastern part, where the city SUHI is well established. Exponential decay trends of daily mean CLHII and SUHII along the urban-rural (buffer zones 0–9) gradients are given in Figure 9. Particularly, MODIS (Terra) and Landsat-8 OLI/TIRS were used to derive the LST, but the LST obtained from the MOD11A1 product was not available during both daytime and nighttime in winter, because of cloud cover. In addition, exponential decay trends of the CLHI at an interval of 2 h are also given in Figure 10. Based on estimated and observed temperature, the change trends of daily SAT (maximum, mean, and minimum value) are given in Figure 11. The temporal trends of the CLHII were successfully Remote Sens. 2017, 9, 536 8 of 18 8 of 18 quantiﬁed (as shown in Figure 12a). Furthermore, the CLHI footprint was successfully revealed by the rank clock (as shown in Figure 12b). Remote Sens. 2017, 9, 536 8 of 18 Remote Sens 2017 9 536 8 of 18 Figure 6. Land-cover maps obtained from Landsat-8 data for the city of Wuhan, China, and building/road and woodland/grassland derived from ZY-3 satellite data (with a spatial resolution of 2.5 m) for the Wuhan metropolitan area. Figure 6. Land-cover maps obtained from Landsat-8 data for the city of Wuhan, China, and building/road and woodland/grassland derived from ZY-3 satellite data (with a spatial resolution of 2.5 m) for the Wuhan metropolitan area. Figure 6. Land-cover maps obtained from Landsat-8 data for the city of Wuhan, China, and building/road and woodland/grassland derived from ZY-3 satellite data (with a spatial resolution of 2.5 m) for the Wuhan metropolitan area. Figure 6. Land-cover maps obtained from Landsat-8 data for the city of Wuhan, China, and building/road and woodland/grassland derived from ZY-3 satellite data (with a spatial resolution of 2.5 m) for the Wuhan metropolitan area. Figure 6. Land-cover maps obtained from Landsat-8 data for the city of Wuhan, China, and building/road and woodland/grassland derived from ZY-3 satellite data (with a spatial resolution of 2.5 m) for the Wuhan metropolitan area. Figure 6. 4.2. CLHI and SUHI D il SAT 4.2. CLHI and SUHI 4.2. CLHI and SUHI Land-cover maps obtained from Landsat-8 data for the city of Wuhan, China, and building/road and woodland/grassland derived from ZY-3 satellite data (with a spatial resolution of 2.5 m) for the Wuhan metropolitan area. Figure 7. Daily mean SAT in (a) summer and (b) winter. Figure 7. Daily mean SAT in (a) summer and (b) winter. Figure 7. Daily mean SAT in (a) summer and (b) winter. Figure 7. Daily mean SAT in (a) summer and (b) winter. Figure 7. Daily mean SAT in (a) summer and (b) winter. Figure 7. Daily mean SAT in (a) summer and (b) winter. 9 of 18 Remote Sens. 2017, 9, 536 Figure 8. LSTs derived from Landsat-8 and MODIS-terra. Figure 8. LSTs derived from Landsat-8 and MODIS-terra. Figure 8. LSTs derived from Landsat-8 and MODIS-terra. Figure 8. LSTs derived from Landsat-8 and MODIS-terra. Figure 8. LSTs derived from Landsat-8 and MODIS-terra. Figure 8. LSTs derived from Landsat-8 and MODIS-terra. Figure 9. Exponential decay trends of (a) the daily mean CLHII and (b) the SUHII along the urban-rural (buffer zones 0–9) gradients in summer and winter. The LST products, as shown in Figure 8, were derived from MODIS-Terra (MOD11A1) and Landsat-8 imageries. The hollow black circles indicate missing data caused by cloud or mist disturbance; 0 represents the metropolitan area, and 1–9 represents the distance of buffer zone to the boundary of metropolitan area (1–3.9 km, 2–8.1 km, 3–12 km, 4–15.5 km, 5–19 km, 6–22 km, 7–25 km, 8–27.6 km, 9–30 km). Figure 9. Exponential decay trends of (a) the daily mean CLHII and (b) the SUHII along the urban-rural (buffer zones 0–9) gradients in summer and winter. The LST products, as shown in Figure 8, were derived from MODIS-Terra (MOD11A1) and Landsat-8 imageries. The hollow black circles indicate missing data caused by cloud or mist disturbance; 0 represents the metropolitan area, and 1–9 represents the distance of buffer zone to the boundary of metropolitan area (1–3.9 km, 2–8.1 km, 3–12 km, 4–15.5 km, 5–19 km, 6–22 km, 7–25 km, 8–27.6 km, 9–30 km). Figure 9. Exponential decay trends of (a) the daily mean CLHII and (b) the SUHII along the urban-rural (buffer zones 0–9) gradients in summer and winter. The LST products, as shown in Figure 8, were derived from MODIS-Terra (MOD11A1) and Landsat-8 imageries. 4.2. CLHI and SUHI D il SAT 4.2. CLHI and SUHI 4.2. CLHI and SUHI Exponential decay trends of the CLHI along the urban-rural (buffer zones 0–9) gradients at an interval of 2 hour in summer and winter; 0 represents the metropolitan area, and 1–9 represents the distance of buffer zone to the boundary of metropolitan area (1–3.9 km, 2–8.1 km, 3–12 km, 4– 15.5 km, 5–19 km, 6–22 km, 7–25 km, 8–27.6 km, 9–30 km). Figure 10. Exponential decay trends of the CLHI along the urban-rural (buffer zones 0–9) gradients at an interval of 2 h in summer and winter; 0 represents the metropolitan area, and 1–9 represents the distance of buffer zone to the boundary of metropolitan area (1–3.9 km, 2–8.1 km, 3–12 km, 4–15.5 km, 5–19 km, 6–22 km, 7–25 km, 8–27.6 km, 9–30 km). Figure 10. Exponential decay trends of the CLHI along the urban-rural (buffer zones 0–9) gradients at an interval of 2 hour in summer and winter; 0 represents the metropolitan area, and 1–9 represents the distance of buffer zone to the boundary of metropolitan area (1–3.9 km, 2–8.1 km, 3–12 km, 4– 15.5 km, 5–19 km, 6–22 km, 7–25 km, 8–27.6 km, 9–30 km). Figure 10. Exponential decay trends of the CLHI along the urban-rural (buffer zones 0–9) gradients at an interval of 2 hour in summer and winter; 0 represents the metropolitan area, and 1–9 represents Figure 10. Exponential decay trends of the CLHI along the urban-rural (buffer zones 0–9) gradients at an interval of 2 h in summer and winter; 0 represents the metropolitan area, and 1–9 represents the Figure 10. Exponential decay trends of the CLHI along the urban-rural (buffer zones 0–9) gradients at an interval of 2 hour in summer and winter; 0 represents the metropolitan area, and 1–9 represents Figure 10. Exponential decay trends of the CLHI along the urban-rural (buffer zones 0–9) gradients at an interval of 2 hour in summer and winter; 0 represents the metropolitan area, and 1–9 represents the distance of buffer zone to the boundary of metropolitan area (1–3.9 km, 2–8.1 km, 3–12 km, 4– 15.5 km, 5–19 km, 6–22 km, 7–25 km, 8–27.6 km, 9–30 km). Figure 10. 4.2. CLHI and SUHI D il SAT 4.2. CLHI and SUHI 4.2. CLHI and SUHI Exponential decay trends of the CLHI along the urban-rural (buffer zones 0–9) gradients at an interval of 2 h in summer and winter; 0 represents the metropolitan area, and 1–9 represents the distance of buffer zone to the boundary of metropolitan area (1–3.9 km, 2–8.1 km, 3–12 km, 4–15.5 km, 5–19 km, 6–22 km, 7–25 km, 8–27.6 km, 9–30 km). Figure 10. Exponential decay trends of the CLHI along the urban-rural (buffer zones 0–9) gradients at an interval of 2 hour in summer and winter; 0 represents the metropolitan area, and 1–9 represents the distance of buffer zone to the boundary of metropolitan area (1–3.9 km, 2–8.1 km, 3–12 km, 4– 15.5 km, 5–19 km, 6–22 km, 7–25 km, 8–27.6 km, 9–30 km). Figure 10. Exponential decay trends of the CLHI along the urban-rural (buffer zones 0–9) gradients at an interval of 2 hour in summer and winter; 0 represents the metropolitan area, and 1–9 represents the distance of buffer zone to the boundary of metropolitan area (1–3.9 km, 2–8.1 km, 3–12 km, 4– 15.5 km, 5–19 km, 6–22 km, 7–25 km, 8–27.6 km, 9–30 km). Figure 10. Exponential decay trends of the CLHI along the urban-rural (buffer zones 0–9) gradients at an interval of 2 h in summer and winter; 0 represents the metropolitan area, and 1–9 represents the distance of buffer zone to the boundary of metropolitan area (1–3.9 km, 2–8.1 km, 3–12 km, 4–15.5 km, 5–19 km, 6–22 km, 7–25 km, 8–27.6 km, 9–30 km). Figure 10. Exponential decay trends of the CLHI along the urban-rural (buffer zones 0–9) gradients at an interval of 2 hour in summer and winter; 0 represents the metropolitan area, and 1–9 represents the distance of buffer zone to the boundary of metropolitan area (1–3.9 km, 2–8.1 km, 3–12 km, 4– 15.5 km, 5–19 km, 6–22 km, 7–25 km, 8–27.6 km, 9–30 km). Figure 11. The change trends of daily SAT (maximum, mean, and minimum value) obtained the using the random forest model and regression calibration approach, and observed daily mean values (black line) for the Wuhan metropolitan area: (a) SAT in summer; and (b) SAT in winter. The red shading and green shading depict the mean ± one standard deviation of the SAT in summer and winter, respectively. Figure 11. 4.2. CLHI and SUHI D il SAT 4.2. CLHI and SUHI 4.2. CLHI and SUHI The hollow black circles indicate missing data caused by cloud or mist disturbance; 0 represents the metropolitan area, and 1–9 represents the distance of buffer zone to the boundary of metropolitan area (1–3.9 km, 2–8.1 km, 3–12 km, 4–15.5 km, 5–19 km, 6–22 km, 7–25 km, 8–27.6 km, 9–30 km). Figure 9 Exponential decay trends of (a) the aily mean CLHII and (b) the SUHII along the urban-rural (buffer zones 0–9) gradients in summer and winter. The LST products, as shown in Figure 8, were derived from MODIS-Terra (MOD11A1) and Landsat-8 imageries. The hollow black circles indicate missing data caused by cloud or mist disturbance; 0 represents the metropolitan area, and 1–9 represents the distance of buffer zone to the boundary of metropolitan area (1–3.9 km, 2–8.1 km, 3–12 km, 4–15.5 km, 5–19 km, 6–22 km, 7–25 km, 8–27.6 km, 9–30 km). Figure 9. Exponential decay trends of (a) the daily mean CLHII and (b) the SUHII along the urban-rural (buffer zones 0–9) gradients in summer and winter. The LST products, as shown in Figure 8, were derived from MODIS-Terra (MOD11A1) and Landsat-8 imageries. The hollow black circles indicate missing data caused by cloud or mist disturbance; 0 represents the metropolitan area, and 1–9 represents the distance of buffer zone to the boundary of metropolitan area (1–3.9 km, 2–8.1 km, 3–12 km, 4–15.5 km, 5–19 km, 6–22 km, 7–25 km, 8–27.6 km, 9–30 km). Figure 9. Exponential decay trends of (a) the daily mean CLHII and (b) the SUHII along the urban-rural (buffer zones 0–9) gradients in summer and winter. The LST products, as shown in Figure 8, were derived from MODIS-Terra (MOD11A1) and Landsat-8 imageries. The hollow black circles indicate missing data caused by cloud or mist disturbance; 0 represents the metropolitan area, and 1–9 represents the distance of buffer zone to the boundary of metropolitan area (1–3.9 km, 2–8.1 km, 3–12 km, 4–15.5 km, 5–19 km, 6–22 km, 7–25 km, 8–27.6 km, 9–30 km). Remote Sens. 2017, 9, 536 Remote Sens. 2017, 9, 536 R t S 2017 9 536 10 of 18 10 of 18 10 f 18 Figure 10. 4.2. CLHI and SUHI D il SAT 4.2. CLHI and SUHI 4.2. CLHI and SUHI The change trends of daily SAT (maximum, mean, and minimum value) obtained the using the random forest model and regression calibration approach, and observed daily mean values (black line) for the Wuhan metropolitan area: (a) SAT in summer; and (b) SAT in winter. The red shading and green shading depict the mean ± one standard deviation of the SAT in summer and winter, respectively. Figure 11. The change trends of daily SAT (maximum, mean, and minimum value) obtained the using the random forest model and regression calibration approach, and observed daily mean values (black line) for the Wuhan metropolitan area: (a) SAT in summer; and (b) SAT in winter. The red shading and green shading depict the mean ± one standard deviation of the SAT in summer and winter, respectively. Figure 11. The change trends of daily SAT (maximum, mean, and minimum value) obtained the using the random forest model and regression calibration approach, and observed daily mean values (black line) for the Wuhan metropolitan area: (a) SAT in summer; and (b) SAT in winter. The red shading and green shading depict the mean ± one standard deviation of the SAT in summer and winter, respectively. Figure 11. The change trends of daily SAT (maximum, mean, and minimum value) obtained the using the random forest model and regression calibration approach, and observed daily mean values (black line) for the Wuhan metropolitan area: (a) SAT in summer; and (b) SAT in winter. The red shading and green shading depict the mean ± one standard deviation of the SAT in summer and winter, respectively. Figure 11. The change trends of daily SAT (maximum, mean, and minimum value) obtained the using the random forest model and regression calibration approach, and observed daily mean values (black line) for the Wuhan metropolitan area: (a) SAT in summer; and (b) SAT in winter. The red shading and green shading depict the mean ± one standard deviation of the SAT in summer and winter, respectively. 11 of 18 Remote Sens. 2017, 9, 536 Figure 12. The spatiotemporal trends of the CLHI, including (a) the CLHII; and (b) the CLHI footprint for 17 September 2013 (summer), and 23 January 2014 (winter), in the Wuhan metropolitan area. The circumference represents the time series range from 0:00 to 23:00; the distance away from the center represents the CLHI footprint. Figure 12. 4.3. Relationships between CLHI and Potential Drivers 4.3. Relationships between CLHI and Potential Drivers p We analyzed the relationships between CLHI and potential drivers based on the mean values of the driving factors and CLHII in every buffer zone. Figure 13 shows single-level relationships between daily mean CLHII and its driving factors quantified by the OLS model. We tried to gain a better understanding of the CLHI by establishing the MLR model. The mean values of the metrics used for quantifying the daily mean CLHII in the different land-cover types are given in Table 1. Estimated correlation coefficients and intervals for every driving factor are given in Table 2. Buffer residuals along the urban-rural (buffer zones 0–9) gradients are shown in Figure 14. We analyzed the relationships between CLHI and potential drivers based on the mean values of the driving factors and CLHII in every buffer zone. Figure 13 shows single-level relationships between daily mean CLHII and its driving factors quantiﬁed by the OLS model. We tried to gain a better understanding of the CLHI by establishing the MLR model. The mean values of the metrics used for quantifying the daily mean CLHII in the different land-cover types are given in Table 1. Estimated correlation coefﬁcients and intervals for every driving factor are given in Table 2. Buffer residuals along the urban-rural (buffer zones 0–9) gradients are shown in Figure 14. Table 1. The mean values of the normalized difference built-up index (NDBI), the normalized difference vegetation index (NDVI), and absolute humidity for the different land-cover types in summer and winter Table 1. The mean values of the normalized difference built-up index (NDBI), the normalized difference vegetation index (NDVI), and absolute humidity for the different land-cover types in summer and winter. Table 1. The mean values of the normalized difference built-up index (NDBI), the normalized difference vegetation index (NDVI), and absolute humidity for the different land-cover types in summer and winter Table 1. The mean values of the normalized difference built-up index (NDBI), the normalized difference vegetation index (NDVI), and absolute humidity for the different land-cover types in summer and winter. summer and winter. Metric NDBI NDVI Absolute Humidity (g/m3) Summer Winter Summer Winter Summer Winter Building −0.04 −0.02 0.13 0.03 17.25 3.75 Road −0.05 −0.01 0.13 0.03 17.25 3.75 Woodland −0.14 −0.05 0.26 0.08 21.34 4.09 Herbage −0.16 −0.02 0.31 0.07 20.84 3.79 Table 2. MLR model: estimated correlation coefficients and interval between daily mean CLHII and its metrics in summer and winter. 4.2. CLHI and SUHI D il SAT 4.2. CLHI and SUHI 4.2. CLHI and SUHI The spatiotemporal trends of the CLHI, including (a) the CLHII; and (b) the CLHI footprint for 17 September 2013 (summer), and 23 January 2014 (winter), in the Wuhan metropolitan area. The circumference represents the time series range from 0:00 to 23:00; the distance away from the center represents the CLHI footprint. Figure 12. The spatiotemporal trends of the CLHI, including (a) the CLHII; and (b) the CLHI footprint for 17 September 2013 (summer), and 23 January 2014 (winter), in the Wuhan metropolitan area. The circumference represents the time series range from 0:00 to 23:00; the distance away from the center represents the CLHI footprint Figure 12. The spatiotemporal trends of the CLHI, including (a) the CLHII; and (b) the CLHI footprint for 17 September 2013 (summer), and 23 January 2014 (winter), in the Wuhan metropolitan area. The circumference represents the time series range from 0:00 to 23:00; the distance away from the center represents the CLHI footprint. 4.3. Relationships between CLHI and Potential Drivers 4.3. Relationships between CLHI and Potential Drivers Summer: p < 0.01, R2 = 0.99; winter: p = 0.01064, R2 = 0.89; alpha = 0.1 (90% confidence interval). Metric NDBI NDVI Absolute Humidity (g/m3) Summer Winter Summer Winter Summer Winter Building −0.04 −0.02 0.13 0.03 17.25 3.75 Road −0.05 −0.01 0.13 0.03 17.25 3.75 Woodland −0.14 −0.05 0.26 0.08 21.34 4.09 Herbage −0.16 −0.02 0.31 0.07 20.84 3.79 Table 2. MLR model: estimated correlation coefﬁcients and interval between daily mean CLHII and its metrics in summer and winter. Summer: p < 0.01, R2 = 0.99; winter: p = 0.01064, R2 = 0.89; alpha = 0.1 (90% conﬁdence interval). Table 2. MLR model: estimated correlation coefficients and interval between daily mean CLHII and its metrics in summer and winter. Summer: p < 0.01, R2 = 0.99; winter: p = 0.01064, R2 = 0.89; alpha = 0.1 (90% confidence interval). Table 2. MLR model: estimated correlation coefﬁcients and interval between daily mean CLHII and its metrics in summer and winter. Summer: p < 0.01, R2 = 0.99; winter: p = 0.01064, R2 = 0.89; alpha = 0.1 (90% conﬁdence interval). Metric Constant Absolute Humidity Summer Winter Summer Winter Coefficients 6.19 −3.21 −0.26 0.90 Confidence Interval (90%) [−2.75, 15.13] [−5.55, −0.87] [−0.71, 0.19] [0.23, 1.57] NDBI NDVI DN of Nighttime Lights Summer Winter Summer Winter Summer Winter −10.83 3.04 −8.13 −4.56 0.003 0.001 [−22.98, 1.32] [−10.06, 3.98] [−13.26, −3.00] [−9.67, 0.55] [−0.001, 0.007] [−0.001, 0.003] Metric Constant Absolute Humidity Summer Winter Summer Winter Coefﬁcients 6.19 −3.21 −0.26 0.90 Conﬁdence Interval (90%) [−2.75, 15.13] [−5.55, −0.87] [−0.71, 0.19] [0.23, 1.57] NDBI NDVI DN of Nighttime Lights Summer Winter Summer Winter Summer Winter −10.83 3.04 −8.13 −4.56 0.003 0.001 [−22.98, 1.32] [−10.06, 3.98] [−13.26, −3.00] [−9.67, 0.55] [−0.001, 0.007] [−0.001, 0.003] 12 of 18 12 of 18 12 of 18 Remote Sens. 2017, 9, 536 Remote Sens. 2017, 9, 536 Remote Sens 2017 9 536 Figure 13. Single-level relationships between daily mean CLHII and its driving factors quantified b the OLS model. The red lines represent summer; the green lines represent winter. Figure 13. Single-level relationships between daily mean CLHII and its driving factors quantiﬁed by the OLS model. The red lines represent summer; the green lines represent winter. Figure 13. Single-level relationships between daily mean CLHII and its driving factors quantified by the OLS model. The red lines represent summer; the green lines represent winter. Figure 13. 5.1. Comparison between SUHII and CLHII 5.1. Comparison between SUHII and CLHII 5.1. Comparison between SUHII and CLHII SAT were measured in the built-up area (i.e., the red border in Figure 3a) for 17 September 2013, and 23 January 2014. Results showed a difference between observed daily mean values and predicted daily mean values, and we also found large amplitude for predicted values in comparison with observed values, which indicated the limitation of sparse weather stations on monitoring CLHI. A comparison between SUHII and CLHII is given in Table 3. Results indicated that extreme high temperature accompanied by strong CLHI occurred during 14:00–15:00, and low temperature accompanied by CLHI effect at 7:00 in summer, and the CLHI and CLCI effects alternately appeared in winter over Wuhan built-up area. In addition, the surface urban cool island (SUCI) effect was not detected based on the satellite data, and the SUHII measured at 10:30, 10:58, and 22:30 was greater than the maximum CLHII (1.42 ◦C at 14:00). Results indicated obvious difference between SUHII and CLHII for quantifying the magnitude of UHI effect, since the former represents the SAT difference, but the latter is the LST difference between urban and rural areas. Comparatively speaking, the analysis of the CLHII helps with the understanding of global temperature change in different cities, especially where the metropolis area has the strongest CLHII accounting for global warming. Table 3. Comparison between SUHII and CLHII (~represents NULL). Summer (17 September 2013) Winter (23 January 2014) Event Time Magnitude Event Time Magnitude Maximum CLCI effect 7:00 −0.39 ◦C Maximum CLCI effect 1:00 −0.95 ◦C Lowest SAT 7:00 24.88 ◦C Maximum CLHI effect 5:00 1.15 ◦C Maximum CLHI effect 14:00 1.42 ◦C Lowest SAT 6:00 −4.52 ◦C Mean SAT 14:00 35.69 ◦C Highest mean SAT 16:00 20.84 ◦C Highest SAT 15:00 42.27 ◦C Highest SAT 16:00 22.77 ◦C MODIS-SUHI 10:30 3.3 ◦C ~ ~ ~ Landsat 8-SUHI 10:58 4.3 ◦C Landsat 8-SUHI 10:58 −0.19 ◦C MODIS-SUHI 22:30 2.2 ◦C ~ ~ ~ 5.2. Spatiotemporal Trends of the CLHI Table 3. Comparison between SUHII and CLHII (~represents NULL). 5.2. Spatiotemporal Trends of the CLHI We found that not only did the SUHI footprint show exponential decay and signiﬁcantly, but also the CLHI footprint, for both summer and winter, signifying the difference between the radiative energy absorbed at the surface and the anthropogenic heating released in urban areas. 5.1. Comparison between SUHII and CLHII It hence caused uneven incoming energy radiated into the atmosphere accompanied by CLHI (or CLCI) effect occurring. CLHI showed increasing trends in 17 September, 2013 and decreasing trends in 23 January 2014. In summer, the CLHI footprint was stable and varied from −1 to 9 times the urban area (standard deviation (SD) = 2.62, where −1 indicates a CLCI). The mean CLHI footprint was 5.08 times the urban area, and the mean footprint was 7.55 times the urban area from 8:00 to 18:00, as a result of solar radiation. In winter, the CLHI footprint varied substantially and ranged from −8 to 9 times the urban area (SD = 6.43). The mean CLHI footprint in winter was only 0.58 times the urban area. In contrast, the CLHI effect tended to occur in summer. Overall, the time-series analysis at an interval of 1 h allows us to continuously monitor the CLHI effect, and helps with the understanding of the spatiotemporal trends of the CLHI. 4.3. Relationships between CLHI and Potential Drivers 4.3. Relationships between CLHI and Potential Drivers Single-level relationships between daily mean CLHII and its driving factors quantified by the OLS model. The red lines represent summer; the green lines represent winter. Figure 13. Single-level relationships between daily mean CLHII and its driving factors quantiﬁed by the OLS model. The red lines represent summer; the green lines represent winter. Figure 13. Single-level relationships between daily mean CLHII and its driving factors quantified by the OLS model. The red lines represent summer; the green lines represent winter. Figure 14. Buffer residuals along the urban-rural (buffer zones 0–9) gradients based on the MLR model in (a) summer and (b) winter. Alpha = 0.1 (90% confidence interval). Figure 14. Buffer residuals along the urban-rural (buffer zones 0–9) gradients based on the MLR model in (a) summer and (b) winter. Alpha = 0.1 (90% confidence interval). Figure 14. Buffer residuals along the urban-rural (buffer zones 0–9) gradients based on the MLR model in (a) summer and (b) winter. Alpha = 0.1 (90% conﬁdence interval). Figure 14. Buffer residuals along the urban-rural (buffer zones 0–9) gradients based on the MLR model in (a) summer and (b) winter. Alpha = 0.1 (90% confidence interval). Figure 14. Buffer residuals along the urban-rural (buffer zones 0–9) gradients based on the MLR model in (a) summer and (b) winter. Alpha = 0.1 (90% confidence interval). Figure 14. Buffer residuals along the urban-rural (buffer zones 0–9) gradients based on the MLR model in (a) summer and (b) winter. Alpha = 0.1 (90% conﬁdence interval). 13 of 18 13 of 18 Remote Sens. 2017, 9, 536 5.4. Interacting Effects Quantiﬁed with the MLR Model Absolute humidity had a signiﬁcant inhibiting effect on daily mean CLHII in summer (estimated coefﬁcients = −0.26) and a positive effect in winter (estimated coefﬁcients = 0.9). Fountain is suggested to mitigate the CLHI in summer because of vast area of waters. Vegetation had signiﬁcant inhibiting effects on daily mean CLHII in both summer (estimated coefﬁcients = −8.13) and winter (estimated coefﬁcients = −4.56). Built-up area had a signiﬁcant positive effect on daily mean CLHII in summer (estimated coefﬁcients = −10.83) and a negative effect in winter (estimated coefﬁcients = 3.04). Anthropogenic heat emissions (e.g., buildings, industry, and vehicles) have positive effects in both summer (estimated coefﬁcients = 0.003) and winter (estimated coefﬁcients = 0.001). The factories producing high heat emissions are suggested to be pushed to the outskirts, and public transport is promoted in metropolitan area for mitigating the CLHI in Wuhan, because it is regarded as the industrial base and transportation center of central China. Particularly, based on MLR model, the correlation with daily mean CLHII for NDBI and absolute humidity was contrary to the results of OLS model in winter. However, the result of MLR model was signiﬁcant (R2 = 0.89, p = 0.011) and OLS models were not signiﬁcant for NDBI (R2 = 0.31, p > 0.05) and absolute humidity (R2 = 0.29, p > 0.05) in winter. Therefore, the results underscores the importance of quantifying the interacting effects of the driving factors of the CLHI based on the MLR model rather than the OLS model. We calculated the daily mean CLHII in different conditions to understand the contribution of the different driving factors to CLHI. In summer, when we set absolute humidity as 17.25 g/m3, the NDBI as −0.04, the NDVI as 0.13, and the DN of nighttime lights as 63 (i.e., most of the land use was physically transformed into buildings in the urban area; refer to Table 1 for the parameter values and Table 2 for the estimated correlation coefﬁcients), the daily mean CLHII was expected to reach 1.30 ◦C (90% conﬁdence interval 0.38–2.22 ◦C). Comparably, when most of the land use was transformed into roads, the daily mean CLHII was expected to reach 1.41 ◦C (0.38–2.44 ◦C). Conversely, the daily mean CLHII was expected to reduce to 0.27 ◦C (−0.04–0.57 ◦C) for the urban area covered by grassland. 5.3. Single-Level Relationships Quantiﬁed with the OLS Model The results of the OLS model signiﬁed that NDBI (summer: R2 = 0.94, p < 0.01; winter: R2 = 0.31, p = 0.093) and nighttime lights (summer: R2 = 0.94, p < 0.01; winter: R2 = 0.60, p < 0.01) consistently exhibited a positive effect on the daily mean CLHII, and NDVI (summer: R2 = 0.96, p < 0.01; winter: R2 = 0.62, p < 0.01), and absolute humidity (summer: R2 = 0.94, p < 0.01; winter: R2 = 0.29, p = 0.11) 14 of 18 14 of 18 Remote Sens. 2017, 9, 536 consistently showed a negative effect in both summer and winter. We found that these driving factors were signiﬁcantly correlated with the daily mean CLHII in summer (R2 > 0.90). Conversely, the results showed a decreased correlation in winter (R2 < 0.65), which is mostly due to the substantial variation of SAT over time and space in winter. Especially for the contribution of NDBI and absolute humidity to CLHI, insigniﬁcant correlations existed in summer and winter, respectively. According to the single-level relationships quantiﬁed with the OLS model, we thus concluded that anthropogenic heat emissions (e.g., buildings, industry, and vehicles) have signiﬁcant positive effects on CLHI, and vegetation has signiﬁcant negative effects in both summer and winter. Built-up area has signiﬁcant positive effects on CLHI, and absolute humidity has signiﬁcant negative effects in summer. However, both the contribution of built-up area and absolute humidity were not signiﬁcant in winter based on the analysis of OLS model. 6. Conclusions Quantifying SUHI is signiﬁcantly impacted by the limited satellite time-series data and cloud and mist disturbance, and quantifying CLHI is signiﬁcantly limited by sparse meteorological monitoring network. The ﬁndings of this study indicate that quantifying the CLHI effects based on satellite observations as well as ground weather station data can enable us to overcome the issues of spatial discontinuity and the low temporal resolution, and also conﬁrm the effectiveness of real-time monitoring of CLHI. The CLHI effect decays exponentially and signiﬁcantly along urban-rural gradients. Regarding hourly CLHII, the trends of the exponential decay are more signiﬁcant in winter. Conversely, the daily mean CLHII decays exponentially, but it is more signiﬁcant in summer than in winter, which is mostly attributed to the substantial variation over time and space (i.e., CLHI and CLCI effects alternating) in winter, signifying the difference in the radiative energy absorbed at the surface and anthropogenic heating released in urban areas compared to rural areas. This study underscores the importance of quantifying the interacting effects of the driving factors of the CLHI based on the MLR model rather than the OLS model. Additionally, the ZY-3 satellite data enable us to obtain more subtle information. We concluded that vegetation had signiﬁcant inhibiting effects on daily mean CLHII in both summer and winter. Absolute humidity has a signiﬁcant inhibiting effect on daily mean CLHII in summer and a positive effect in winter. Built-up area had a signiﬁcant positive effect on daily mean CLHII in summer and a negative effect in winter. Anthropogenic heat emissions (e.g., buildings, industry, and vehicles) have positive effects in both summer and winter. Therefore, planting trees and fountain, and reducing the emissions from industry and vehicle (e.g., the factories producing high heat emissions are suggested to be pushed to the outskirts, and public transport is promoted in metropolitan area) are suggested to mitigate the CLHI in the city of Wuhan. Acknowledgments: This work was supported by the China National Science Fund for Excellent Young Scholars under grant 41522110, by the National Key Research and Development Plan under grant 2016YFB0501403, and by the Foundation for the Author of National Excellent Doctoral Dissertation, China, under grant 201348. 5.4. Interacting Effects Quantiﬁed with the MLR Model In addition, when the urban area was covered by woodlands, the daily mean CLHII was expected to reduce to 0.21 ◦C (0.03–0.38 ◦C). In winter, the daily mean CLHII was expected to reach 0.17 ◦C (0.04–0.3 ◦C), 0.14 ◦C (0.08–0.2 ◦C), 0.03 ◦C (−0.04–0.09 ◦C), and 0.34 ◦C (0.05–0.64 ◦C) in the above conditions, respectively. Therefore, the contribution of roads to the CLHI is greater than buildings, and the mitigation of the CLHI through afforestation is superior to planting grass in summer. In winter, the contribution of roads to the CLHI is less than buildings, and the mitigation of the extreme low-temperature weather through afforestation is superior to planting grass. Therefore, planting trees and avoiding the construction of wide roads are suggested to mitigate the CLHI. In addition, it was found that anthropogenic heat emissions exacerbated the daily mean CLHII by about 0.19 ◦C (−0.06–0.44 ◦C) on 17 September 2013. However, anthropogenic heat emissions exacerbated daily mean CLHII 0.06 ◦C (−0.06–0.19 ◦C) on 23 January 2014. Remote Sens. 2017, 9, 536 15 of 18 6. Conclusions Author Contributions: Long Li and Xin Huang Conceived and designed the experiments; Long Li Collected, prepared, processed, analyzed data, interpreted and discussed the results, and wrote the manuscript; Xin Huang Supplied climatological data and contributed in the results interpretation, manuscript editing, and supervised all the work that has been done by the ﬁrst author; Jiayi Li Provided suggestion and comments for results interpretation and manuscript editing; Dawei Wen Supplied ZY-3 satellite data and contributed with manuscript editing before submission. All authors reviewed the manuscript. Conﬂicts of Interest: The authors declare that there is no conﬂict of interest. References . Bai, X.; Shi, P.; Liu, Y. Realizing China’s urban dream. Nature 2014, 509, 158–160. [CrossRef] [PubMed] 2. Angel, S.; Parent, J.; Civco, D.L.; Blei, A.; Potere, D. The dimensions of global urban expansion: Estimates d j ti f ll t i 2000 2050 P Pl 2011 75 53 107 [C R f] 2. 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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2789464058	https://www.frontiersin.org/articles/10.3389/fneur.2018.00234/pdf	English	null	Association between peripheral inflammation and DATSCAN data of the striatal nuclei in different motor subtypes of Parkinson Disease	bioRxiv (Cold Spring Harbor Laboratory)	2,018	cc-by	8,219	Original Research published: 16 April 2018 doi: 10.3389/fneur.2018.00234 A Hossein Sanjari Moghaddam†, Farzaneh Ghazi Sherbaf†, Mahtab Mojtahed Zadeh, Amir Ashraf-Ganjouei and Mohammad Hadi Aarabi* Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran The interplay between peripheral and central inflammation has a significant role in dopami- nergic neural death in nigrostriatal pathway, although no direct assessment of inflammation has been performed in relation to dopaminergic neuronal loss in striatal nuclei. In this study, the correlation of neutrophil to lymphocyte ratio (NLR) as a marker of peripheral inflammation to striatal binding ratios (SBRs) of DAT SPECT images in bilateral caudate and putamen nuclei was calculated in 388 drug-naïve early PD patients [288 tremor dominant (TD), 73 postural instability and gait difficulty (PIGD), and 27 indeterminate] and 148 controls. NLR was significantly higher in PD patients than in age- and sex-matched healthy controls, and showed a negative correlation to SBR in bilateral putamen and ipsilateral caudate in all PD subjects. Among our three subgroups, only TD patients showed remarkable results. A positive association between NLR and motor severity was observed in TD subgroup. Besides, NLR could negatively predict the SBR in ipsilateral and contralateral putamen and caudate nuclei in tremulous phenotype. Nonetheless, we found no significant association between NLR and other clinical and imaging findings in PIGD and indeterminate sub- groups, supporting the presence of distinct underlying pathologic mechanisms between tremor and non-tremor predominant PD at early stages of the disease. Edited by: Massimiliano Caiazzo, Utrecht University, Netherlands Reviewed by: Bo Zhou, Stanford University, United States Xuping Li, Houston Methodist Research Institute, United States *Correspondence: Farzaneh Ghazi Sherbaf farzanehghazi@gmail.com; Mahtab Mojtahed Zadeh mahtabmojtahedzadeh@gmail.com; Mohammad Hadi Aarabi mohammadhadiarabi@gmail.com †These authors have contributed equally to this work. Specialty section: This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neurology Keywords: Parkinson disease, DATScan data, peripheral inflammation, motor subtypes, postural instability and gait difficulty, tremor-dominant, neutrophil to lymphocyte ratio INTRODUCTION Progressive dopaminergic demise in the nigrostriatal system has long been considered the patho- genic mainstay of motor features in Parkinson’s disease (PD). Lots of ergot mechanisms have been enumerated to be responsible for the neural loss in PD, of which inflammation, both central and peripheral, has been explained in the initiation and progression of PD.lt Received: 27 October 2017 Accepted: 26 March 2018 Published: 16 April 2018 Edited by: Massimiliano Caiazzo, Utrecht University, Netherlands Citation: Inflammatory processes, which once were believed to be just the afterclap of neural death, are now considered as an etiological factor, which are also presented in early stages of PD. Several PD susceptibility genes are documented to be involved in the functions of immunity components like microglia (1–3). The result from polymorphism studies determined several pro-inflammatory genes such as IL-1β, TNF-α, HLA-DBQ1, HLA-DRA, and HLA-DRB1 to be associated with an increased susceptibility to PD (4, 5). It is also proved that nonsteroidal anti-inflammatory drugs can in the long term diminish the risk of PD (6). In essence, the authors exhibited that dopaminergic neurons are selectively susceptible to oxidative stress and inflammation (7, 8). Freshly, a neuroimaging study using positron emission tomography detected activated microglia in the SN and putamen of 1-year duration Sanjari Moghaddam H, Ghazi Sherbaf F, Mojtahed Zadeh M, Ashraf-Ganjouei A and Aarabi MH (2018) Association Between Peripheral Inflammation and DATSCAN Data of the Striatal Nuclei in Different Motor Subtypes of Parkinson Disease. Front. Neurol. 9:234. doi: 10.3389/fneur.2018.00234 April 2018 \| Volume 9 \| Article 234 1 Frontiers in Neurology \| www.frontiersin.org Association Between Peripheral Inflammation and DATSCAN in PD Sanjari Moghaddam et al. PD patients (9), implying that there is an active inflammatory condition in the initial phases of PD. Furthermore, the authors suggested that inflammatory circumstances, both central and peripheral, are present in the prodromal PD (10, 11). Altogether, these immense lines of evidence highly signify the importance of inflammation in the pathophysiology of PD. before study enrollment. The study was performed in accordance with relevant guidelines and regulations. Only baseline visit data are analyzed in this research. PD status was confirmed by Movement Disorder Society-Unified Parkinson’s Disease Rating Scale (MDS- UPDRS), and dopamine transporter deficit was observed on DAT scans. All PD patients at baseline were drug-naïve, non-demented, at Hoehn and Yahr (H&Y) staging I or II, and were confirmed negative for any medical or psychiatric disorders apart from PD. l A robust body of evidence has espoused the involvement of peripheral inflammatory conditions in the pathophysiology of PD. In this context, markers of peripheral inflammatory pro- cesses including, but not limited to, TNF-α and its receptors and IL-1β have been exhibited to be elevated in individuals with PD in the peripheral blood (12–16), cerebrospinal fluid, and postmor- tem brain tissues (12, 17–19). Citation: Related data showed that IL-6 is associated with an increased risk of the development of PD (20). Besides the peripheral immune markers, peripheral immune cells undergo some quantitative alterations in PD patients. The most prominent of these changes is the lower total number of lymphocytes in individuals with PD compared to controls mainly due to a decrement in the percentage of CD3+ and CD19+ lym- phocytes (21–24). Furthermore, decreased counts of T helper cells and increased/unchanged counts of cytotoxic T cells have been documented in PD patients (25). Dopaminergic Imaging Despite lots of evidence on the role of inflammation in dopamin- ergic neural loss, no direct assessment has been performed in this regard. This work was designed to investigate peripheral inflamma- tion in terms of NLR concerning the loss of dopaminergic uptake in caudate and putamen on Dopamine Transporter Single Photon Emission Computed Tomography (SPECT) (DaTscan) images, which signifies the dopaminergic activity, among treatment-naïve early PD patients with different motor phenotypes and severities. As a preventive measure, all females with the potential of pregnancy were confirmed negative for urine pregnancy test prior to receiving the 123Ioflupane SPECT injection. None of the subjects had received any of the drugs that would interfere with DAT SPECT imaging within 6 months of screening (full PPMI study protocol is provided at http://www.ppmi-info.org/study-design/research-documents- and-sops/). DAT imaging at baseline visit from all participating sites was centrally reconstructed and was attenuation corrected, followed by spatial normalization for consistent orientation by experienced nuclear medicine experts. Standard volume of interest template was applied on caudate, putamen, and occipital regions. Striatal binding ratios (SBRs) for left and right putamen and caudate were finally calculated, using the formula: (SBR) = (striatal region)/(occipital) −1, where occipital lobe DAT count is the reference (36). Regarding motor dominancy, SBRs of ipsilateral and contralateral putamen and caudate were compared between PD and HC and within PD Clinical Assessment and Motor Classification Motor and non-motor symptoms were evaluated among subjects with clinical tests at baseline visits at each participating site. The most common PD rating scales, i.e., UPDRS, H&Y staging, and the Schwab and England rating of activities of daily living (Modified Schwab & England ADL) were assessed (34). Tremor score and postural insta- bility and gait difficulty (PIGD) score were identified for PD subjects with MDS-UPDRS. Then, PD subjects were classified into three groups of tremor dominant (TD), PIGD, and indeterminate based on the ratio of tremor score/PIGD score. If ratio > = 1.15 or PIGD = 0 and tremor >0, then the subject was TD. If ratio < = 0.9, the subject was classified as PIGD. If ratio >0.9 and <1.15, or tremor score and PIGD score = 0, the subject was tagged as indeterminate (35). Non-motor symptoms were investigated by the Montreal Cognitive Assessment (MoCA) for mild cognitive impairment, 15-item geriatric depression scale for depressive symptoms, The University of Pennsylvania Smell Identification Test for olfaction function, and independent validation of the scales for outcomes in Parkinson’s disease-autonomic (SCOPA-AUT) for autonomic dysfunction. Owing to the presence of blood–brain barrier (BBB), central nervous system is an immune-privileged organ. However, BBB dysfunction has been postulated to play a key role in the patho- genesis of neurodegenerative diseases like PD (26, 27). It has been shown that endothelium residing in substantia nigra pars compacta (SNpc) of patients with PD undergoes radical pathologic mor- phological changes (28, 29), and CD4+ and CD8+ T-lymphocytes have been reported to enter and invade the SNpc of idiopathic PD patients (30). Using a mouse model involving the injection of vas- cular endothelial growth factor (a detrimental protein to BBB) into the mice SNpc, researchers demonstrated a significant association between BBB dysfunction and dopaminergic cell death (31, 32). Furthermore, inflammatory cytokines can induce the activation of microglia and therefore the dopaminergic cell death. In this context, one of the valid indicators for peripheral inflammatory processes is the neutrophil to lymphocyte ratio (NLR) (33), representing the active inflammation (neutrophils) merged with immunological regulatory processes (lymphocytes).l Blood Sample Collection As it is pronounced in the PPMI biologics manual (http://www. ppmi-info.org/), baseline blood sample collection was completed at each study site. PAXgene blood RNA tubes were utilized for the collection of blood samples following the study protocol. The number of neutrophils and lymphocytes was calculated by an autoanalyzer device on participants’ whole blood samples, right after the collection. NLR was simply calculated by dividing neutrophil to lymphocyte count. Participants Parkinson’s disease patients and healthy control (HC) subjects enrolled in this study were recruited from the Parkinson Progression Markers Initiative (PPMI, http://www.ppmi-info.org/). The study was approved by the institutional review board of all participating sites. Written informed consent was obtained from all participants Parkinson’s disease patients and healthy control (HC) subjects enrolled in this study were recruited from the Parkinson Progression h April 2018 \| Volume 9 \| Article 234 Frontiers in Neurology \| www.frontiersin.org 2 Association Between Peripheral Inflammation and DATSCAN in PD Sanjari Moghaddam et al. motor subtypes. The calculated average of SBRs for the left and the right nuclei is presented for HCs or symmetrical motor involvement in PD individuals. was used to test the association between NLR and other charac- teristics in patients with PD. Finally, P-values less than 0.05 were considered to be statistically significant. Between Group Comparisons Neutrophil to lymphocyte ratio and DAT scan data were provided for 388 de novo PD patients and 148 HC on baseline visits. Baseline clinical characteristics and detailed demographic data of PD and HC subjects are demonstrated in Table 1. Healthy participants Table 1 \| Demographic information and comparison of clinical outcomes between HCs and patients with PD. Characteristic HCs (n = 148) PD (n = 388) P-value Age, mean (SD) (95% CI), years 61.1 (11.3) (59.3–62.9) 61.7 (9.6) (60.7–62.6) 0.775a Female/male, No. (%male) 49/99 (66.9) 134/254 (65.4) 0.755b Left-handed/right-handed, No. (%right-handed)c 22/119 (80.4) 33/346 (89.2) 0.029b Education, mean (SD) (95% CI), years 16.1 (3.0) (15.7–16.6) 15.5 (3.0) (15.2–15.8) 0.034a Disease duration, median (range), m – 4.0 (0–36) – NLR, mean (SD) (95% CI) 2.2 (0.8) (2.1–2.3) 2.5 (0.9) (2.4–2.6) <0.001a SBR score in caudate nucleus, mean (SD) (95% CI) 3.0 (0.6) (2.9–3.1) contralaterald: 1.8 (0.6) (1.8–1.9) ipsilateral: 2.2 (0.6) (2.1–2.2) <0.001a SBR score in putamen nucleus mean (SD) (95% CI) 2.1 (0.5) (2.0–2.2) contralateral: 0.7 (0.3) (0.6–0.7) ipsilateral: 1.0 (0.4) (0.9–1.0) <0.001a Hoehn and Yahr stage, mean (SD) – 1.6 (0.5) – MDS-UPDRS part I score, mean (SD) 3.0 (2.7) 5.6 (4.0) <0.001a MDS-UPDRS part II score, mean (SD) 0.4 (1.0) 6.0 (4.2) <0.001a MDS-UPDRS part III score, mean (SD) 1.2 (2.1) 20.4 (8.8) <0.001a Modified Schwab & England ADL, mean (SD) – 93.2 (6.0) – MoCA score, mean (SD) 28.2 (1.1) 27.1 (2.3) <0.001a GDS score, mean (SD) 1.1 (1.5) 2.3 (2.4) <0.001a UPSIT score, mean (SD) 34.4 (4.5) 22.3 (8.2) <0.001a SCOPA-AUT score, mean (SD) 5.7 (3.5) 9.4 (6.0) <0.001a HCs, healthy controls; PD, Parkinson disease; NLR, neutrophil to lymphocyte ratio; SBR, striatal binding ratios; MDS-UPDRS, Movement Disorder Society-sponsored revision of the Unified Parkinson’s Disease Rating Scale; MoCA Montreal cognitive assessment; GDS Geriatric depression scale; Modified Schwab & England ADL overall activities of daily living; UPSIT score, mean (SD) SCOPA-AUT score, mean (SD) HCs, healthy controls; PD, Parkinson disease; NLR, neutrophil to lymphocyte ratio; SBR, striatal binding ratios; MDS-UPDRS, Movement Disorder Society-sponsored revision of the Unified Parkinson’s Disease Rating Scale; MoCA, Montreal cognitive assessment; GDS, Geriatric depression scale; Modified Schwab & England ADL, overall activities of daily living; UPSIT, University of Pennsylvania Smell Identification Test; SCOPA-AUT, Scales for Outcomes in Parkinson’s disease. aBased on Mann–Whitney U-test. HCs, healthy controls; PD, Parkinson disease; NLR, neutrophil to lymphocyte ratio; SBR, striatal binding ratios; MDS-UPDRS, Movement Disorder Society-sponsored revision of the Unified Parkinson’s Disease Rating Scale; MoCA, Montreal cognitive assessment; GDS, Geriatric depression scale; Modified Schwab & England ADL, overall activities of daily living; UPSIT, University of Pennsylvania Smell Identification Test; SCOPA-AUT, Scales for Outcomes in Parkinson’s disease. aBased on Mann–Whitney U-test. cOthers were mixed-handed. Table 2 \| Demographic data and comparison of certain characteristics between patients with Parkinson’s disease (PD) based on tremor/postural instability and gait difficulty (PIGD) score ratio. Characteristic PIGDa (n = 73) Indeterminate (n = 27) Tremor dominant (n = 288) P-value Age, mean (SD) (95% CI), years 60.8 (9.5) (58.6–63.0) 62.8 (10.0) (58.8–66.8) 61.8 (9.7) (60.7–62.9) 0.744b Female/male, No. (%male) 29/44 (60.0) 6/21 (77.7) 99/189 (65.6) 0.261c MDS-UPDRS part III score, mean (SD) (95% CI) 19.2 (8.7) (17.1–21.2) 18.1 (7.7) (15.1–21.2) 20.4 (9.0) (19.9–22.0) 0.195b NLR, mean (SD) (95% CI) 2.5 (0.8) (2.3–2.6) 2.6 (1.0) (2.2–3.0) 2.5 (1.0) (2.4–2.6) 0.952b SBR in contralateral nuclei, mean (SD) (95% CI) Caudate 1.8 (0.6) (1.6–1.9) 1.8 (0.5) (1.6–2.0) 1.8 (0.6) (1.8–1.9) 0.631b Putamen 0.7 (0.3) (0.6–0.8) 0.6 (0.2) (0.5–0.7) 0.7 (0.3) (0.6–0.7) 0.163b SBR in ipsilateral nuclei, mean (SD) (95% CI) Caudate 2.1 (0.7) (2.0–2.3) 2.1 (0.6) (1.9–2.4) 2.2 (0.6) (2.1–2.2) 0.813b Putamen 1.0 (0.4) (0.8–1.0) 1.0 (0.4) (0.8–1.2) 1.0 (0.4) (0.9–1.0) 0.713b NLR, neutrophil to lymphocyte ratio; SBR, striatal binding ratios; MDS-UPDRS, Movement Disorder Society-sponsored revision of the Unified Parkinson’s Disease Rating Scale. aThe ratio of tremor and PIGD score in MDS-UPDRS. PD patients with a ratio of ≥1.15 were classified as TD, ratio ≤0.90 were classified as PIGD, and 0.90 < ratio < 1.15 were classified as indeterminate. bBased on Kruskal–Wallis test. cBased on χ2-test. Table 2 \| Demographic data and comparison of certain characteristics between patients with Parkinson’s disease (PD) based on tremor/postural instability and gait difficulty (PIGD) score ratio comparison of certain characteristics between patients with Parkinson’s disease (PD) based on tremor/postural instability and gait RESULTS The statistical analysis was performed using SPSS version 22 (BM Corp., Armonk, NY, USA). Pearson’s chi-square was used to assess nominal variables across groups. Mann–Whitney U-test was used to assess differences between HCs and PD patients, and Kruskal–Wallis test was used for multiple comparisons in three groups of PD motor subtypes. Spearman’s rank-order correlation Between Group Comparisons i n MDS-UPDRS. PD patients with a ratio of ≥1.15 were classified as TD, ratio ≤0.90 were classified as PIGD, and 0.90 < ratio < 1.15 were phocyte ratio; SBR, striatal binding ratios; MDS-UPDRS, Movement Disorder Society-sponsored revision of the Unified Parkinson’s Disease R HCs, healthy controls; PD, Parkinson disease; NLR, neutrophil to lymphocyte ratio; SBR, striatal binding ratios; MDS-UPDRS, Movement Disorder Society-sponsored revision of the Unified Parkinson’s Disease Rating Scale; MoCA, Montreal cognitive assessment; GDS, Geriatric depression scale; Modified Schwab & England ADL, overall activities of daily living; UPSIT, University of Pennsylvania Smell Identification Test; SCOPA-AUT, Scales for Outcomes in Parkinson’s disease. aBased on Mann Whitney U test Characteristic As shown in Table 2, there was no difference between age, gender, UPDRS-III as well as NLR and SBRs in both ipsilateral and contralateral striatal nuclei among PD motor subtypes. Characteristic Age Disease duration Tremor score PIGD score UPDRS part III score Modified Schwab & England ADL UPSIT score MoCA score SCOPA-AUT score SBR score in caudate (contralateral) SBR score in putamen (contralateral) SBR score in caudate (ipsilateral) SBR score in putamen (ipsilateral) NLR, P-value (rho) HCs (n = 148) 0.405 (0.069) – – – 0.388 (−0.072) – 0.133 (−0.124) 0.704 (−0.031) 0.480 (−0.059) 0.590 (−0.045) 0.702 (−0.032) – – All PD (n = 388) 0.011 (0.129) 0.495 (0.035) 0.066 (0.093) 0.662 (0.022) 0.058 (0.096) 0.015 (−0.123) 0.744 (−0.017) 0.891 (−0.007) 0.185 (0.067) 0.199 (−0.066) 0.026 (−0.114) 0.014 (-0.126) 0.002 (−0.158) PIGDa (n = 73) 0.991 (0.001) 0.834 (0.025) 0.706 (−0.045) 0.667 (0.051) 0.947 (−0.008) 0.695 (0.047) 0.732 (−0.041) 0.232 (0.142) 0.581 (0.066) 0.947 (0.008) 0.780 (−0.033) 0.936 (0.010) 0.942 (0.009) Indeterminate (n = 27) 0.813 (−0.048) 0.488 (−0.139) 0.867 (0.034) 0.921 (0.020) 0.374 (−0.178) 0.058 (−0.370) 0.926 (0.019) 0.524 (0.128) 0.720 (0.072) 0.97 (0.008) 0.287 (−0.213) 0.953 (0.012) 0.659 (−0.089) TD (n = 288) 0.003 (0.172) 0.371 (0.053) 0.050 (0.116) 0.603 (0.031) 0.009 (0.155) 0.015 (−0.144) 0.685 (−0.024) 0.426 (−0.047) 0.214 (0.073) 0.106 (−0.096) 0.041 (−0.120) 0.005 (−0.168) <0.001 (−0.208) HCs, healthy controls; PD, Parkinson’s disease; PIGD, postural instability–gait difficulty; NLR, neutrophil to lymphocyte ratio; UPDRS, Unified Parkinson’s Disease Rating Scale; MoCA, Montreal cognitive assessment; Modified Schwab & England ADL, overall activities of daily living; UPSIT, University of Pennsylvania Smell Identification Test; SCOPA-AUT, Scales for Outcomes in Parkinson’s disease; SBR, striatal binding ratios. 4.0 (1–35) 4.0 (0–36) 0 2.4 (0.9) 2.7 (1.0) 0 1.9 (0.6) 1.8 (0.5) 0 0.7 (0.3) 0.7 (0.2) 0 2.3 (0.6) 2.1 (0.5) 0 1.1 (0.4) 0.9 (0.3) <0 0.5 (0.2) 0.6 (0.3) 0 0.1 (0.1) 0.2 (0.2) 0 15.1 (5.4) 25.4 (8.5) <0 95.2 (5.1) 92.0 (5.8) <0 27.3 (2.2) 26.8 (2.4) 0 2.1 (2.5) 2.4 (2.3) 0 23.7 (8.1) 21.0 (8.0) 0 9.0 (6.4) 10.3 (6.1) 0 to lymphocyte ratio; SBR, striatal bin MDS-UPDRS, Movement Disorder kinson’s Disease Rating Scale; MoC c depression scale; Modified Schwa UPSIT, University of Pennsylvania Sm utcomes in Parkinson’s disease. were age- and sex-matched to PD patients, while scored higher on all motor and non-motor tests. As expected, PD patients had significantly lower SBRs in striatal nuclei. NLR was significantly higher in PD cohort. Characteristic April 2018 \| Volume 9 \| Article 234 3 Association Between Peripheral Inflammation and DATSCAN in PD Sanjari Moghaddam et al. Table 4 \| Demographic information and comparison of clinical outcomes in patients with Parkinson’s disease (PD) based on H&Y stage. Characteristic H&Y stage I (n = 124) H&Y stage II (n = 164) P-value Age, mean (SD), years 59.0 (9.9) 63.9 (8.9) <0.001a Disease duration, median (range), m 4.0 (1–35) 4.0 (0–36) 0.864a NLR, mean (SD) 2.4 (0.9) 2.7 (1.0) 0.003a SBR score in caudate nucleus (contralateral), mean (SD) 1.9 (0.6) 1.8 (0.5) 0.166a SBR score in putamen nucleus (contralateral), mean (SD) 0.7 (0.3) 0.7 (0.2) 0.032a SBR score in caudate nucleus (ipsilateral), mean (SD) 2.3 (0.6) 2.1 (0.5) 0.004a SBR score in putamen nucleus (ipsilateral), mean (SD) 1.1 (0.4) 0.9 (0.3) <0.001a Tremor score, mean (SD) 0.5 (0.2) 0.6 (0.3) 0.017a PIGD score, mean (SD) 0.1 (0.1) 0.2 (0.2) 0.056a MDS-UPDRS part III score, mean (SD) 15.1 (5.4) 25.4 (8.5) <0.001a Modified Schwab & England ADL, mean (SD) 95.2 (5.1) 92.0 (5.8) <0.001a MoCA score, mean (SD) 27.3 (2.2) 26.8 (2.4) 0.074a GDS score, mean (SD) 2.1 (2.5) 2.4 (2.3) 0.081a UPSIT score, mean (SD) 23.7 (8.1) 21.0 (8.0) 0.006a SCOPA-AUT score, mean (SD) 9.0 (6.4) 10.3 (6.1) 0.051a H&Y, Hoehn and Yahr stage; NLR, neutrophil to lymphocyte ratio; SBR, striatal binding ratios; PIGD, postural instability–gait difficulty; MDS-UPDRS, Movement Disorder Society-sponsored revision of the Unified Parkinson’s Disease Rating Scale; MoCA, Montreal cognitive assessment; GDS, Geriatric depression scale; Modified Schwab & England ADL, Overall activities of daily living; UPSIT, University of Pennsylvania Smell Identification Test; SCOPA-AUT, Scales for Outcomes in Parkinson’s disease. aBased on Mann–Whitney U-test. Table 4 \| Demographic information and comparison of clinical outcomes in patients with Parkinson’s disease (PD) based on H&Y stage. Table 3 \| The correlation (Spearman) between NLR and other characteristics in HCs and patients with PD based on tremor/PIGD score ratio. Frontiers in Neurology \| www.frontiersin.org DISCUSSION In search of possible correlation between peripheral inflammation and striatal DAT availability in a large sample of drug-naïve early PD patients, we found that (1) NLR is significantly higher in PD patients than in age- and sex-matched HCs. (2) Although NLR and striatal SBR did not differ between subtypes of PD in the early stages of the disease, only TD phenotype and more specifically at H&Y stage II confirmed predictive value of NLR to dopaminergic loss in bilateral striatal nuclei. (3) Worse motor severity was asso- ciated with higher NLR values in only TD phenotype. ciated with higher NLR values in only TD phenotype. Tremor is a cardinal motor feature of PD that has always exhibited unalike clinical course to other motor phenotypes. In contrast to TD, non-TD subtypes, especially PIGD, predict worse and rapid disease progression with higher rates of non-motor features such as cognitive decline, mood disturbances, REM sleep behavior disorder, and olfaction dysfunction (37–42). A longitudinal study has revealed that TD patients will not develop dementia until irreversibly converted to PIGD phenotype (43). Differed clinical pattern is also reflected on structural and functional assays. Autopsy of TD-PD patients has exposed less severe dopaminergic loss in SN than other PD subtypes (44, 45). This is in concordance with a slower progression of symptoms in this group (37, 46) and is replicated on a recent probabilistic tractography study, which has revealed normal structural con- nectivity in nigro-pallidal and fronto-striatal circuits in TD patients, which was altered in non-TD (47). Postmortem studies have also demonstrated distinct striatal pathology among differ- ent PD motor subtypes. While akinesia and rigidity evolve from cell loss in ventrolateral SNpc which project to the posterior putamen, degeneration in TD is most prominent in dorsomedial SNpc projecting to the caudate and anterior putamen and to the retrorubral field with subsequent projections to the dorsolateral striatum and ventromedial thalamus (48). In support of this model, Eggers et al. showed lesions in caudate and lateral puta- men (eagle-wing shape) on DAT scans of TD patients, compared to lesions in dorsal putamen with an egg-shape pattern of dopa- minergic loss in akinetic-rigid phenotype (49). This pattern is consistent with our finding of NLR correlation to lower dopamine uptake in both putamen and caudate in TD subgroup, while it was linked to a lower DAT binding in contralateral putamen and not contralateral caudate in all PD patients. Correlations Between NLR and Other Clinical and Imaging Metrics Contralaterala nuclei Ipsilateral nuclei Age Disease duration Tremor score PIGD score UPDRS part III score Modified Schwab & England ADL UPSIT score MoCA score SCOPA-AUT score SBR score in caudate SBR score in putamen SBR score in caudate SBR score in putamen NLR, P-value (rho) H&Y stage I (n = 124) 0.902 (−0.011) 0.548 (−0.055) 0.563 (0.052) 0.242 (−0.106) 0.486 (0.063) 0.064 (−0.167) 0.263 (0.101) 0.754 (0.028) 0.980 (0.002) 0.738 (−0.030) 0.947 (−0.006) 0.640 (−0.043) 0.541 (−0.056) H&Y stage II (n = 164) 0.002 (0.240) 0.088 (0.134) 0.140 (0.116) 0.217 (0.097) 0.278 (0.085) 0.352 (−0.073) 0.292 (−0.083) 0.338 (−0.075) 0.241 (0.092) 0.040 (−0.162) 0.017 (−0.188) 0.003 (−0.221) 0.019 (−0.185) H&Y, Hoehn and Yahr stage; NLR, neutrophil to lymphocyte ratio; UPDRS, Unified Parkinson’s Disease Rating Scale; MoCA, Montreal cognitive assessment; Modified Schwab & England ADL, overall activities of daily living; UPSIT, University of Pennsylvania Smell Identification Test; SCOPA-AUT, Scales for Outcomes in Parkinson’s disease; SBR, striatal binding ratios. aNumbers for all SBRs are representative of added partial correlation between NLR and SBR in putamen and caudate nuclei, controlling for age. Significant correlations are bolded. Correlations Between NLR and Other Clinical and Imaging Metrics Spearman’s rank-order correlation was performed in HCs and three subgroups of PD patients (Table 3). NLR had a strong posi- tive correlation with age and UPDRS-III score and a negative cor- relation with Modified Schwab & England ADL score only in TD subgroup of PD patients. Moreover, TD patients showed a negative correlation between NLR values and SBR in contralateral putamen (p-value of 0.041) and ipsilateral putamen and caudate regarding motor dominancy (P-values of <0.001 and 0.005, respectively). No significant correlation was observed between NLR and other features in two other motor phenotypes. Results did not differ after controlling for the effect of age (data not shown). f Eventually, TD patients were tested for possible correlations between NLR and other characteristics, based on their H&Y stage. In 164 TD-PD patients at H&Y stage II, NLR had a positive April 2018 \| Volume 9 \| Article 234 Frontiers in Neurology \| www.frontiersin.org 4 Association Between Peripheral Inflammation and DATSCAN in PD Sanjari Moghaddam et al. correlation with age. As TD patients at H&Y stage II were sig- nificantly older than TD patients at stage I (Table 4), we carried partial correlation between NLR and SBR in putamen and caudate nuclei, controlling for age. Lower SBRs in bilateral putamen and caudate nuclei were significantly associated with higher NLRs in only the subgroup in stage II (Table 5). No significant correlation was found in 124 TD patients with H&Y stage I. Table 5 \| The correlation (Spearman) between NLR and other characteristics in patients with tremor-dominant (TD) Parkinson’s disease (PD) based on H&Y stage. Frontiers in Neurology \| www.frontiersin.org DISCUSSION In fact, there is consistent report of a higher extent of dopamine depletion in putamen than caudate in PD (50). Moreover, it is proposed that tremor severity may have a different mechanism than other motor features, as in contrast to bradykinesia and rigidity, does not depend on the degree of reduced striatal dopamine uptake (50–53), but rather on the relative contribution of a reduced dopamine in putamen and caudate, indicative of a more severe caudate involvement April 2018 \| Volume 9 \| Article 234 5 5 Association Between Peripheral Inflammation and DATSCAN in PD Sanjari Moghaddam et al. (51). Therefore, a strong correlation of NLR to SBR in caudate of TD patients, but a weak association with putamen (Table 5), agrees with these sightings. In this study, we did not find any dif- ference between SBR values in ipsilateral or contralateral caudate and putamen nuclei between three examined subtypes with similar motor stage and severity. However, early drug-naïve TD patients of another study have shown higher DAT availability on bilateral putamen but no difference on caudate nuclei compared to akinetic-rigid PD patients with higher UPDRS-III and H&Y (54). This may point to the possibly greater impact of putamen on motor severity in non-TD phenotype. In this study, we did not investigate other neurotransmitters than dopamine which are conferred regarding tremorgenesis, and its severity is shown to be mediated by reductions of serotonin transporter availability in the raphe nuclei more than that of dopamine in putamen (55, 56), or mediated by neurotransmitter imbalance between reduced GABAergic inhibitory influx from putamen confronted to intact dopaminergic input from SN to the internal pallidum (57). of distinct neural circuits outside the nigrostriatal region strongly suggest the different underlying pathophysiology of PD tremor from that of other motor features (66). This study is the first that has captured the different pathogenesis of the dopaminergic neuronal loss in terms of higher NLR interrelated to lower striatal dopamine in TD but not in non-TD PD. NLR is an easy accessible marker of peripheral inflammation, and is identified as a predictor of worse outcome in multiple chronic clinical and neurological disorders, which are more prevalent in geriatric population. This marker also positively correlates to age in normal population (67). Only two previous studies have investigated the association of NLR in idi- opathic PD with opposite results. Akil et al. DISCUSSION have found higher NLR in 51 PD (NLR = 3.1 ± 1.3) compared to 50 HC (NLR = 2.1 ± 10.3) (68), while Uçar et al. did not find any difference comparing 46 PD (NLR = 2.66 ± 1.05) and 60 HC (NLR = 2.46 ± 1.04) (69). The later study also did not find any difference between TD and akinetic- rigid subtypes. These studies have assessed relatively small sample sizes of PD patients already on dopaminergic replacement therapy, which is shown to recover changes on T-lymphocytes (70). In this study, we surveyed a larger sample of 388 drug-naïve PD patients who showed higher levels of NLR (2.5 ± 0.9) compared to 148 age-matched HCs (NLR = 2.2 ± 0.8), although there was no such difference within PD subtypes. NLR is also shown to be attributed to a lower connectivity in several white matter tracts implicated in early or prodromal PD pathology (71). p g p p On the other hand, we found surprisingly much more substan- tial reduction in DAT availability in ipsilateral putamen and caudate and a weak reduction in contralateral nuclei in TD patients at H&Y stage II compared to TD at stage I. This is also true for the strong cor- relation of NLR to ipsilateral caudate (p-value = 0.003) and a weak relationship with contralateral caudate nucleus (p-value = 0.04). This is the same for ipsilateral versus contralateral putamen among all PD patients as well as all individuals tagged in TD subtype. Motor symptoms in PD are considered to evolve from dopaminergic depletion in contralateral nigrostriatal system. However, there are several recent reports of motor dominancy ipsilateral to the side of predominant dopaminergic deficit, especially in TD patients (58–60). This is also true for medication-naïve TD patients from PPMI cohort on early stages of the disease (61). Another study on PPMI cohort (56) disclosed that a proportion of TD group at baseline progressed to bilateral limb involvement over 2 years of follow-up, which may be due to the observed ipsilateral striatal dopamine depletion in this study. It is of particular note that TD patients showed negative association between NLR and ipsilateral caudate, and while splitting them based on H&Y stage, contralateral caudate emerged to have such an association only in the group at stage II. More studies are needed to further elucidate the contribu- tion of ipsilateral basal ganglia motor circuits to the tremorgenesis. DISCUSSION Several studies have manifested that PD progression does not follow a linear model, and a decline in striatal uptake observed on DAT scans is more rapid in early stages of the disease followed by a much more slower progression in advanced stages (72–74). Therefore, it seems that early stages of the disease without the inter- ference of dopaminergic replacement therapy are the best point during the disease course to evaluate the contribution of inflam- mation to dopaminergic loss. A growing body of evidence suggests that presynaptic terminals in dorsal striatum undergo degeneration even prior to the SN, and axonal degeneration should be the pri- mary target for neuroprotective therapies (75). This is supported by in vivo DAT SPECT (76) and confirms that lower SBRs in puta- men and caudate nuclei truly reflect the nigrostriatal pathology in early stages of PD. However, study on early stages imposes some limitations that should be considered when interpreting our results. First, motor classification tends to change during the course of the disease, and our subtype classification was only based on screen- ing visits. Second, tremor predominant PD patients recognize the symptoms and seek medical advice earlier and owing to the more benign course they more readily cooperate with PPMI project that includes patients not receiving any parkinsonian treatment for 6 months. Therefore, a higher number of cases tagged in TD subgroup and relatively fewer cases in PIGD and indeterminate in PPMI cohort may have contributed to the false-negative results in non-tremor predominant groups. More studies are clearly needed to investigate the generatability of these solid findings. Outside the nigrostriatal tract, imaging studies have imposed the increased functional and metabolic activity of cerebel- lothalamocortical pathway in emerging parkinsonian tremor, which largely drives by depleted dopaminergic input from basal ganglia (62–64). In a recent diffusion tensor imaging study, Wen et al. have revealed that drug-naïve early TD patients recruited from PPMI project have a greater white matter integrity and a lesser neural degeneration, while they observed a widespread white matter dehiscence in PIGD group with similar motor stage and severity. The authors have discussed this finding as a neural compensation for nigrostriatal dopaminergic loss leading to more benign disease course in TD subtype. 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Fox Foundation for Parkinsons Research, AbbVie, Avid Radiopharmaceuticals, Biogen, Bristol-Myers Squibb, Covance, GE Healthcare, Genentech, GlaxoSmithKline (GSK), Eli Lilly and Company, Lundbeck, Merck, Meso Scale Discovery (MSD), Pfizer, Piramal Imaging, Roche, Servier, and UCB (www.ppmi-info.org/fundingpartners). All relevant data are available in the Parkinson’s Progression Markers initiative (PPMI) database (http://www.ppmi-info.org/data). AUTHOR CONTRIBUTIONS HSM, FGS, MMZ, and MA contributed to the conception and design of the study. AA-G, and MA contributed to data collection and analysis. HSM, FGS, MMZ, and MA contributed to writing the manuscript. FGS contributed to revising of the final draft. CONCLUSION In this research, we investigated the association between immune and nervous system. PD patients were divided into three groups based on tremor/PIGD score, being TD, PIGD, and indeterminate Overall, the different pattern of clinical course and dopamin- ergic loss along with the consistent findings of the involvement April 2018 \| Volume 9 \| Article 234 6 Sanjari Moghaddam et al. Association Between Peripheral Inflammation and DATSCAN in PD its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study. subgroups. We demonstrated that NLR can negatively predict SBR in bilateral putamen and caudate nuclei of TD group and more specifically at H&Y stage II. Not observing such association in non-tremor predominant PD patients points to the different pathophysiology mechanisms between tremor and other motor symptoms in PD. 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https://openalex.org/W1980545091	https://europepmc.org/articles/pmc2500003?pdf=render	English	null	The effectiveness of self help technologies for emotional problems in adolescents: a systematic review	Child and adolescent psychiatry and mental health	2,008	cc-by	8,551	Open Acc Review The effectiveness of self help technologies for emotional problems in adolescents: a systematic review Muna Ahmead1 and Peter Bower2 ddress: 1School of Public Health, Al-Quds University, Jerusalem, Israel and 2NPCRDC, 5th Floor, Williamson Buildin M13 9PL, UK Email: Muna Ahmead - munaahmead@yahoo.com; Peter Bower - peter.bower@manchester.ac.uk * Corresponding author Published: 23 July 2008 Child and Adolescent Psychiatry and Mental Health 2008, 2:20 doi:10.1186/1753-2000-2-20 Received: 23 January 2008 Accepted: 23 July 2008 This article is available from: http://www.capmh.com/content/2/1/20 © 2008 Ahmead and Bower; 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. Received: 23 January 2008 Accepted: 23 July 2008 © 2008 Ahmead and Bower; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativ which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cit © 2008 Ahmead and Bower; 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. Child and Adolescent Psychiatry and Mental Health BioMed Central Open Access Interventions Self help materials were interventions delivered through information technology (e.g. web-based or stand alone computer programs); paper-based delivery (i.e. biblio- therapy); audiotapes or videotapes. For inclusion, the materials had to be used by the participants with no or minimal individual contact with a health professional or researcher. Populations Adolescence was defined as age between 12–25 years to cover the wide variation in the definition of adolescence in the literature [30-34]. Abstract Background: Adolescence is a transition period that involves physiological, psychological, and social changes. Emotional problems such as symptoms of anxiety and depression may develop due to these changes. Although many of these problems may not meet diagnostic thresholds, they may develop into more severe disorders and may impact on functioning. However, there are barriers that may make it difficult for adolescents to receive help from health professionals for such problems, one of which is the limited availability of formal psychological therapy. One way of increasing access to help for such problems is through self help technology (i.e. delivery of psychological help through information technology or paper based formats). Although there is a significant evidence base concerning self help in adults, the evidence base is much weaker in adolescents. This study aims to examine the effectiveness of self help technology for the treatment of emotional problems in adolescents by conducting a systematic review of randomized and quasi- experimental evidence. Methods: Five major electronic databases were searched: Medline, PsycInfo, Embase, Cochrane Controlled Trials Register and CINAHL. In addition, nine journals were handsearched and the reference lists of all studies were examined for any additional studies. Fourteen studies were identified. Effect sizes were calculated across 3 outcome measures: attitude towards self (e.g. self esteem); social cognition (e.g. self efficacy); and emotional symptoms (i.e. depression and anxiety symptoms). Results: Meta analysis showed small, non-significant effect size for attitude towards self (ES = -0.14, 95% CI = -0.72 to 0.43), a medium, non-significant effect size for social cognition (ES = -0.49, 95% CI = -1.23 to 0.25) and a medium, non-significant effect size for emotional symptoms (ES = -0.47, 95% CI = -1.00 to 0.07). However, these findings must be considered preliminary, because of the small number of studies, their heterogeneity, and the relatively poor quality of the studies. Conclusion: At present, the adoption of self help technology for adolescents with emotional problems in routine clinical practice cannot be recommended. There is a need to conduct high quality randomised trials in clearly defined populations to further develop the evidence base before implementation. Page 1 of 12 (page number not for citation purposes) Page 1 of 12 (page number not for citation purposes) Child and Adolescent Psychiatry and Mental Health 2008, 2:20 Child and Adolescent Psychiatry and Mental Health 2008, 2:20 Child and Adolescent Psychiatry and Mental Health 2008, 2:20 http://www.capmh.com/content/2/1/20 Inclusion and exclusion criteria Study design Randomized controlled trials (RCT) and quasi-experi- mental studies which used a control group (e.g. usual care, placebo controls, waiting list controls, or no treat- ment controls) were eligible for the review. Similarly, anxiety problems range from presence of symp- toms to clinical conditions such as separation anxiety, social phobia, generalized anxiety disorder, obsessive compulsive disorder, panic disorder and phobias [4,8]. Prevalence rates for having at least one childhood anxiety disorder vary, with 12 month estimates in the United States and internationally from 8.6% to 20.9% [9]. Ado- lescents with elevated but subsyndromal levels of anxiety symptoms report significant levels of functional impair- ment [10,11]. Background One option is the use of self help treatments. In mental health, self help is seen as 'the manualization of evidence based treatment' [23]. This involves taking aspects of proven treatments and providing them through technol- ogy, such as information technology and written paper- based formats. g Adolescence is considered a challenging stage of life. It is a transition period from childhood to adulthood that involves physiological changes, developments in cogni- tion and emotion, changes in social roles with peers and the opposite sex, and considerations of school and career. It involves the development of identity, independence from family and adaptation to peer groups [1]. If children and adolescents struggle to cope with these changes, they may develop emotional disorders, such as anxiety, depres- sion and obsessions [2,3]. Current clinical guidelines in the United Kingdom suggest guided self help technology could be a useful treatment for some emotional problems in adolescents [24]. A number of randomized controlled trials and systematic reviews have indicated that self help technologies are helpful for adult patients [25-29]. However, the evidence base is far weaker in relation to adolescents. Depression covers a range of personal moods from a mild case of the 'blues' to clinical conditions that are character- ized by severe symptoms and functional impairments [4]. Data collected for The Youth Risk Behavior Surveillance System found that in the United States, during the 12 months preceding the survey, 28.5% of students had felt so sad or hopeless almost every day for more than 2 weeks in a row that they stopped doing some usual activities [5]. Prevalence of depression reaching diagnostic thresholds is estimated at around 0.4–8% in adolescents over 12 months [6,7]. This study aimed to determine the effectiveness of self help technology for the treatment of emotional problems in adolescents using systematic review methods. Disorders Emotional symptoms including depression [35] and anx- iety [35,36] were included. These could be symptoms or disorders severe enough to reach diagnostic thresholds. Two other outcomes were also included: attitude towards self, including self concept [37] and self esteem [38]; and social cognition, such as self efficacy and locus of control [39]. Both of these concepts may be important causes or consequences of emotional problems [40-45]. Depression and anxiety in children and adolescents have a large number of potential consequences including aca- demic failure, poor peer relationships, behavioural prob- lems, conflict with parents, substance abuse [12] recurrent anxiety or depressive disorders [13] and suicide attempts [14]. There are a number of studies that show the effectiveness of cognitive behaviour therapy for adolescents with clini- cal depression [15-19], although combination treatment with medication may be optimal [20]. CBT also has an important role in the management of anxiety [21]. How- ever access to psychological therapy is limited, and is appropriately targeted at those with more severe disor- ders. This raises the importance of alternative solutions, especially for those who have early symptoms of anxiety, depression or emotional distress (such as poor peer rela- tions, low self-esteem, withdrawal or behavioural prob- lems) but do not reach formal diagnostic thresholds [22]. Search strategy h i The strategy involved searching five major electronic data- bases: Medline (1966 onwards), PsycInfo (1967 onwards), Embase (1980 onwards), CINAHL (1982 Page 2 of 12 (page number not for citation purposes) Page 2 of 12 (page number not for citation purposes) http://www.capmh.com/content/2/1/20 http://www.capmh.com/content/2/1/20 Child and Adolescent Psychiatry and Mental Health 2008, 2:20 1. Adolescents exp adolescent/ or exp child/ or infant/ exp Minors/ exp Students/ adolescen$.mp. pre-adolescen$.mp. youth$.mp. teen$.mp. (young$ adj (people$ or person$ or adult$)).mp. 2. Technology bibliotherap$.mp. written material$.mp. manual$.mp. (printed adj5 communicat$).mp. exp Bibliotherapy/ Teaching Materials/ Computer Systems/ Programmed Instruction/ 3. Self help self care.mp. self help.mp. self manage$.mp. self treat$.mp. Self Care/ exp Self help Groups/ exp Self Administration/ 4. Mental health treatment cogniti$ therap$.mp. behavio?r$ therap$.mp. cogniti$ behavio?r$.mp. behavio?r$ modif$.mp. exp Cognitive Therapy/ Behavior Therapy/ exp Psychotherapy/ exp Counseling/ The search combined these four search sets so that st 1. Adolescents 1. Adolescents exp adolescent/ or exp child/ or infant/ exp adolescent/ or exp child/ or infant/ exp adolescent/ or exp child/ or infant/ exp Minors/ exp Students/ adolescen$.mp. pre-adolescen$.mp. youth$.mp. teen$.mp. (young$ adj (people$ or person$ or adult$)).mp. exp Minors/ exp Minors/ St d t / exp Students/ adolescen$.mp. pre-adolescen$.mp. The search combined these four search sets so that studies were identified that included adolescents and technology, and in addition had terms relating to either self help or mental health treatment. Academy of Child and Adolescent Psychiatry Academy of Child and Adolescent Psychiatry Adolescence Journal of Affective Disorder Journal of Depression and Anxiety Journal of Medical Internet Research Journal of Mental Health British Journal of Psychiatry Archives of General Psychiatry Journal of Clinical Psychology Journal of Clinical Psychology Data analysis: Computation of effect size The meta analysis used the standardized mean difference estimate of effect size. Effect size for each study was calcu- lated by subtracting the control group mean from the experimental group mean, and dividing by the pooled standard deviation (SD) [49]. Where relevant statistics (e.g. standard deviation) were not available, effect size was calculated from other indices using published meth- ods [49]. The main comparison was between self help technology versus no treatment or delayed treatment control group. Only one outcome measure was selected from each study according to its relevance to the 3 major outcomes (emo- tional symptoms, attitude towards self and social cogni- tion). Both fixed effects and random effects model were used in calculating the overall effect size using the metan routine within Stata. The former assume that the effect of treat- ment is the same across studies and that any difference between their results is due to sampling error, while the latter assume that the effect of treatment is not the same across studies and that variation in treatment effect between studies occurs as a result of factors other than sampling error [49,50]. Both models give the same results when there is no significant heterogeneity. When marked heterogeneity is present, the random effects model pro- duces wider confidence intervals and may produce a dif- ferent estimate of effect [51,52]. Heterogeneity was assessed using the I2 statistic [53]. Both fixed effects and random effects model were used in calculating the overall effect size using the metan routine within Stata. The former assume that the effect of treat- ment is the same across studies and that any difference between their results is due to sampling error, while the latter assume that the effect of treatment is not the same across studies and that variation in treatment effect between studies occurs as a result of factors other than sampling error [49,50]. Both models give the same results when there is no significant heterogeneity. When marked heterogeneity is present, the random effects model pro- duces wider confidence intervals and may produce a dif- ferent estimate of effect [51,52]. Heterogeneity was assessed using the I2 statistic [53]. Nine journals which were identified on the basis of scop- ing searches as publishing papers on self help and related topics were hand searched from 1995 to March-May 2005 (see Figure 1). Data extraction A data extraction sheet was developed and data were extracted by two independent reviewers, with disagree- ments resolved by discussion or contact with the authors. The data extraction sheet included the following groups of variables: study data variables, methodological variables, population variables, and intervention and outcome vari- ables. Data analysis: Computation of effect size The reference lists of all studies were exam- ined for any additional studies, and Google Scholar was searched regularly for any relevant studies from 2004 to 2006. Only studies reported in the English language were included because of the lack of a budget for translation. The first author checked the titles and abstracts of all stud- ies identified by the searches. Any studies that were judged potentially eligible were set aside for discussion by both authors before a final decision about inclusion was made. Example s Figure 1 Example search strategy (MEDLINE) and list of handsearched journals Figure 1 Example search strategy (MEDLINE) and list of handsearched journals. The search was structured to search for studies with four key characteristics: adolescents, technology, self help and mental health treatment. Each search used a combi- nation of free text and subject headings. Examples are given below, and the entire search strategy is available from the authors. The search combined these four search sets so that studies were identified that included adolescents and technology, and in addition had terms relating to either self help or mental health treatment. The listed journals were hand searched from first issue 1995 to March-May 2005. Page 3 of 12 (page number not for citation purposes) Page 3 of 12 (page number not for citation purposes) Page 3 of 12 (page number not for citation purposes) Child and Adolescent Psychiatry and Mental Health 2008, 2:20 http://www.capmh.com/content/2/1/20 onwards) and the Cochrane Controlled Trials Register. These databases were searched initially in late 2004 and early 2005, and then updated in April 2006. Specific searches were developed for each database to maximise the effectiveness of each search. The search terms were grouped into those concerning the adolescent population (e.g. Adolescent, Minors), those related to the technology (e.g. Bibliotherapy, Internet) and those related to the intervention, either self help (e.g. Self Care, Self Adminis- tration) or mental health treatment (e.g. Cognitive Ther- apy, Counselling). Further details are provided in Figure 1. The search strategy was chosen to maximise sensitivity at some loss of specificity. For example, study design terms were not included in the search strategy because some quasi-experimental studies may not be correctly indexed. Similarly, terms related to problems or disorders such as depression and anxiety were also excluded from the search strategy, and a decision was made to identify all possible studies on the basis of population, technology and intervention only. Relevant studies were then included or excluded on the basis of study design or dis- orders after reading titles and abstracts. pants' and 'treatment side effects') [47]. The reliability of the QRS ratings was assessed by measuring inter-observer agreement using the kappa statistic [48]. Results A total of 55,480 studies were identified (figure 2). As noted above, the searches were designed to maximise sen- sitivity over specificity, and many studies identified in the initial search were excluded when scanning the abstracts because they failed to meet the criteria for study design and disorder. Fourteen studies eventually met the inclu- sion criteria. The review found six studies (listed in table 1) involving anxiety (including test and dating anxiety), 2 studies involving depression, and six involving other problems related to anxiety and depression (e.g. self esteem, problem solving skills). Thirteen studies were conducted in the USA and one in Australia. Quality assessment In this review, quality assessment including internal valid- ity (i.e. study design, conduct and analysis) and external validity (i.e. recruitment of the population) were assessed using the Quality Rating Scale (QRS) rated by two researchers independently [46]. The QRS consists of 23 items. Each item is scored 0, 1, or 2 and the total score ranges from 0–46. In this study, two items were excluded because they were not applicable ('blinding of partici- Characteristics of the participants Students were the target populations in most studies (n = 12). Studies used volunteer participants who were recruited using advertisements, letters, and contact with school personnel or through course entry. Effectiveness of self help technology h k h The systematic review is a key methodology in evidence based practice and the gold standard for the assessment of the effectiveness of interventions [51]. Overall, this review reveals a number of interesting findings. Despite the high prevalence of depression among adolescents, only two studies that involved self help technology for symptoms of depression were identified [54,56]. The bulk of the studies (n = 6) involved anxiety symptoms and six con- cerned other outcomes theoretically or empirically related to depression. Despite the explosion in the use of the internet, nearly half the studies involved written biblio- therapy. The low quality score of the studies included in this meta analysis occurred as a result of a wide range of methodo- of depression were identified [54,56]. The bulk of t studies (n = 6) involved anxiety symptoms and six co cerned other outcomes theoretically or empirically relat to depression. Despite the explosion in the use of t internet, nearly half the studies involved written bibli therapy. In this review, effect sizes were calculated across 3 ou come measures: attitude towards self, social cognitio and emotional symptoms. The analyses indicat medium effect sizes for social cognition and emotion symptoms and a small effect size on attitudes towards se However, all three meta analyses were statistically no significant, although the effects on emotional symptom approached significance (ES = -0.47, 95% CI = -1.00 0.07). Many of the studies did not use samples from clinic populations. It is possible that concerns about the appr priateness of self help in adolescents has meant th researchers have tended to pilot these interventions Review search flowchart Figure 2 Review search flowchart. Studies excluded after examining title and abstract (n=55439) Potentially eligible studies (n=41) Initial search (n=55480) Studies included in review (n=14) Studies excluded (n=27) Did not meet criteria for age, outcomes etc Studies excluded from meta analysis (n=3) (no intervention control group, missing data to calculate effect size) Studies included in meta analyses (n=11) Review search flowchart Figure 2 Review search flowchart. Characteristics of the participants Only two stud- Page 4 of 12 (page number not for citation purposes) Target population and outcomes N Age Sex Follow up (weeks) Attrition QRS score Adolescents with elevated depression symptoms (10+ on the Child Depression Inventory and 10+ on the Hamilton Rating Scale for Depression) 22 Mean 15 years and 11 months M: 36% 4 weeks follow up 26.6% 21 Students with test anxiety 84 17–21 M: 25% 7 weeks 24% 14 Students with test anxiety (scoring above the 60th percentile on the Test Anxiety nventory) 50 18–22 M: 58% Post treatment, but minimum of 6 weeks 28% 17 Students with spider fear (scoring in the upper 20th percentile of a spider fear nventory and failing a behavioural avoidance test) 70 18–21 M: 10% Not clear (week following last therapy session) Not clear 13 Male students with dating anxiety 50 18–21 M: 100% 4 weeks and 8 months follow up 8% 18 Self concept in students with earning disability and emotional handicap 96 12–14 M: 83% Not clear (post treatment) Not clear 11 Students with depression symptoms 78 15–16 M: 100% 8–10 weeks and 16 weeks follow up 41% 23 Stress management training in students 132 18 – 23+ M: 47% 4 weeks and 4 weeks follow up 12% 16 Students with test anxiety (meeting Test Anxiety Inventory criterion cut off score of 50) 121 Mean 18.6 M: 31% Treatment completion, 4 week follow up 8% 16 Health behaviour change in graduate and undergraduate students 952 18–25 Not clear 16 weeks 23% 16 Self esteem and locus of control n high school students 121 14–17 M: 46% 2 weeks 13% 12 Problem solving in adolescents iving with single parent mothers who had been separated or divorced from fathers for approximately 6–48 months 100 13 – 17 M: 37% 4 weeks post intervention and 4 weeks follow up 3% 24 Prevention of problems in youth of changing families 48 13–15 M: 48% Not clear Not clear 13 Competence building in adolescents in church youth and schools 139 13–18 M: 40% 2 months 22% 15 rget population and comes N Age Sex Follow up (weeks) Attrition QRS score olescents with elevated ression symptoms (10+ on Child Depression Inventory 10+ on the Hamilton Rating e for Depression) 22 Mean 15 years and 11 months M: 36% 4 weeks follow up 26.6% 21 dents with test anxiety 84 17–21 M: 25% 7 weeks 24% 14 dents with test anxiety oring above the 60th centile on the Test Anxiety ntory) 50 18–22 M: 58% Post treatment, but minimum of 6 weeks 28% 17 dents with spider fear oring in the upper 20th centile of a spider fear ntory and failing a avioural avoidance test) 70 18–21 M: 10% Not clear (week following last therapy session) Not clear 13 e students with dating ety 50 18–21 M: 100% 4 weeks and 8 months follow up 8% 18 concept in students with ning disability and emotional dicap 96 12–14 M: 83% Not clear (post treatment) Not clear 11 dents with depression ptoms 78 15–16 M: 100% 8–10 weeks and 16 weeks follow up 41% 23 ss management training in dents 132 18 – 23+ M: 47% 4 weeks and 4 weeks follow up 12% 16 dents with test anxiety eting Test Anxiety Inventory erion cut off score of 50) 121 Mean 18.6 M: 31% Treatment completion, 4 week follow up 8% 16 lth behaviour change in duate and undergraduate dents 952 18–25 Not clear 16 weeks 23% 16 esteem and locus of control igh school students 121 14–17 M: 46% 2 weeks 13% 12 blem solving in adolescents g with single parent mothers o had been separated or orced from fathers for roximately 6–48 months 100 13 – 17 M: 37% 4 weeks post intervention and 4 weeks follow up 3% 24 vention of problems in youth hanging families 48 13–15 M: 48% Not clear Not clear 13 mpetence building in lescents in church youth and ools 139 13–18 M: 40% 2 months 22% 15 http://www.capmh.com/content/2/1/20 Child and Adolescent Psychiatry and Mental Health 2008, 2:20 http://www.capmh.com/content/2/1/20 logical weaknesses. Quality assessment of included studies Six studies were RCTs [37,54,58,61,64,66] and eight stud- ies were quasi-experiments [55,56,59,62-65]. Inter- observer agreement on quality ratings assessed using the kappa statistic was 'substantial' (0.77). The mean quality score of all included studies was low (16.4 out of 42). Table 1 shows the overall quality score of each study. Characteristics of the participants For example, studies generally had small sample sizes (less than 50 participants per group); none reported concealment of allocation; few reported power calculations; and all had follow up periods of less than 6 months. ies reported the inclusion of clinical participants [54,55], and only two studies reported the number of the eligible participants who did not participate in the study to judge their representativeness [56,57]. Most of the studies included both genders, but with more females than males, although two studies had males only [56,58]. Description of the interventions Meta analysis was used to determine the overall effective- ness of self help technology. The analyses indicated signif- icant statistical heterogeneity for attitude towards self (chi-squared = 23.39, d.f. = 4, p = 0.000, I2= 82.9%), emo- tional symptoms (chi-squared = 25.38, d.f. = 6, p = 0.000, I2= 76.4%) and social cognition (chi-squared = 16.63, d.f. = 2, p = 0.000, I2 = 88.0%). Therefore, analyses were con- ducted using random effects models. Details of the interventions are shown in Table 2 [see Additional file 1]. Four studies used computer interven- tions, 8 studies used bibliotherapy and 2 studies used vid- eotaped interventions. Seven studies delivered interventions through specific group sessions (duration between 30 to 60 minutes per session) in which the par- ticipants worked alone on self help interventions or with minimal instructions from the therapists or researchers [55,56,59-63]. In the other seven studies, the participants read the materials alone at home and they either had at least one telephone call per week during the intervention period [37,54,64,65], were sent a newsletter [66] or received two letters and one telephone call as a reminder [58]. One study had no contact [57]. The random effects models showed a small effect size [67] with confidence intervals that included zero (ES = -0.14, 95% CI = -0.72 to 0.43, n = 5, figure 3) for the effect of self help technology on attitudes towards self, a medium effect size with confidence intervals that included zero (ES = -0.49, 95% CI = -1.23 to 0.25, n = 3, figure 4) for social cognition, and a medium effect size with confidence inter- vals that included zero (ES = -0.47, 95% CI = -1.00 to 0.07, n = 7, figure 5) for emotional symptoms. Standardised Mean Difference (95% CI) Standardised Mean Difference -3 -2.5 -2 -1.5 -1 -.5 0 .5 1 1.5 2 2.5 3 Standardised Mean Difference Analysis of attitude towards self, random effects Figure 3 Analysis of attitude towards self, random effects. studies; empirical evidence indicates that studies with positive results are more likely to be published [68,68]. However, the issue of including unpublished studies in systematic reviews is still controversial [68]. Some researchers argue against their inclusion, as such studies may have serious limitations that have prevented their publication [69]. Egger found in one meta analysis of 60 studies that unpublished studies had lower methodologi- cal quality than published trials and that they did not report important quality criteria such as concealment of allocation or blinding [70]. The funnel plot is a test that is used to examine publication bias in systematic reviews, but was not applicable in this review, due to the small number of studies [51]. groups with more minor or circumscribed problems. As noted in the introduction, self help treatments may be especially useful for adolescents with early symptoms of anxiety, depression or emotional distress that do not reach diagnostic thresholds [22]. Further research using clinical populations may be required if these treatments are going to have more general utility. In those cases where interventions are ineffective, this may reflect the lack of theoretical basis to the intervention, or poor uptake amongst clients. However, the descriptions of the theoretical basis of the interventions and their uptake were very limited, which made it difficult to examine the relationships between these factors and outcomes. Effectiveness of self help technology h k h Studies excluded after examining title and abstract (n=55439) Potentially eligible studies (n=41) Initial search (n=55480) Studies included in review (n=14) Studies excluded (n=27) Did not meet criteria for age, outcomes etc Studies excluded from meta analysis (n=3) (no intervention control group, missing data to calculate effect size) Studies included in meta analyses (n=11) Studies excluded after examining title and abstract (n=55439) In this review, effect sizes were calculated across 3 out- come measures: attitude towards self, social cognition, and emotional symptoms. The analyses indicated medium effect sizes for social cognition and emotional symptoms and a small effect size on attitudes towards self. However, all three meta analyses were statistically non- significant, although the effects on emotional symptoms approached significance (ES = -0.47, 95% CI = -1.00 to 0.07). Studies excluded from meta analysis (n=3) (no intervention control group, missing data to calculate effect size) Many of the studies did not use samples from clinical populations. It is possible that concerns about the appro- priateness of self help in adolescents has meant that researchers have tended to pilot these interventions in Review search flowchart Figure 2 Review search flowchart. Review search flowchart Figure 2 Review search flowchart Review sea Figure 2 Review se Figure 2 g Review search flowchart. g Review search flowchart. Page 6 of 12 (page number not for citation purposes) Page 6 of 12 (page number not for citation purposes) Child and Adolescent Psychiatry and Mental Health 2008, 2:20 http://www.capmh.com/content/2/1/20 Standardised Mean Difference -3 -2.5 -2 -1.5 -1 -.5 0 .5 1 1.5 2 Study Sheridan Standardised Mean Difference (95% CI) 0.10 (-0.59, 0.78) Sandor 0.17 (-0.22, 0.56) Walker -0.57 (-1.10,-0.04) Okearney 0.65 ( 0.06, 1.24) Salt -1.04 (-1.57,-0.51) Overall 2.5 3 -0.14 (-0.72, 0.43) Page 7 of 12 (page number not for citation purposes) Comparisons with other studies The effectiveness of self help technology for emotional symptoms in the present analysis is similar to the analysis of 8 studies in adults with depression and anxiety in pri- mary care, which reported a mean effect size of 0.41 [73], and a more recent review of 12 randomized controlled studies for internet-based cognitive behaviour therapy programs for symptoms of depression and anxiety [29] which reported a mean effect size of 0.40. However, in general, the effect size estimates from the current study were lower than the values that were reported by other meta analyses of the effectiveness of self help technology in adults. For example, Gould meta analysed 40 studies of self help for a wide range of problems (including depres- sion, fear, headache, sleep and behavioural problems) and reported an overall effect size of 0.76, with an effect size of 0.74 in depression [27]. Cuijpers reviewed seven studies in unipolar depression and reported an overall effect size of 0.82 [26], while Marrs, in his review of 70 One meta analysis investigated the effectiveness of bibilo- therapy for depression among patients in three age groups: adult, adolescents and elderly. Based on five ado- lescent studies, the review found an effect size of 1.32 (95% CI = 0.90 to 1.73) [74]. However, this discrepancy may reflect a number of differences, including the defini- tion of bibliotherapy, the amount of therapist contact, the review methodology (the Gregory study included ran- domised trials and pre and post single treatment group studies) and inclusion criteria (the Gregory review was restricted to depression). Methodological issues The inclusion of only English language studies is another limitation of this study. Excluding trials reported in lan- guages other than English may introduce bias and reduce the precision of results [68,71]. However, Egger found that meta analyses based exclusively on English language studies produced estimates close to those without any lan- guage restriction [72]. An extensive search strategy was used, involving the searching of five electronic databases, hand-searching nine journals, checking the reference lists of identified studies, contact with authors and regular updating of searches. However, the search had a number of limita- tions. First, grey literature was not searched systematically, which increased the possibility of missing unpublished Page 7 of 12 (page number not for citation purposes) Page 7 of 12 (page number not for citation purposes) Child and Adolescent Psychiatry and Mental Health 2008, 2:20 http://www.capmh.com/content/2/1/20 Standardised Mean Difference -3 -2.5 -2 -1.5 -1 -.5 0 .5 1 Study Sandor Standardised Mean Difference (95% CI) -0.28 (-0.67, 0.11) Ramsey 0.08 (-0.33, 0.48) Salt -1.33 (-1.88,-0.78) Overall 1.5 2 2.5 3 -0.49 (-1.23, 0.25) Sandor Ramsey Overall Standardised Mean Difference Analysis of social cognition, random effects Figure 4 Analysis of social cognition, random effects. Analysis of social cognition, random effects Figure 4 Analysis of social cognition, random effects. studies of a wide range of problems such as anxiety, depression, weight loss, and smoking, and reported a mean effect size of 0.57 for depression and 0.95 for anxi- ety [25]. A large number of studies were identified because the search focussed on sensitivity rather than specificity. The initial check of titles and abstracts were undertaken by the first author alone. It would have been preferable to have these checked by both authors to ensure reliability, but time and resource limitations meant that this was not pos- sible. These higher effect sizes in adults may be due to the differ- ence between adolescent and adult populations (in terms of motivation and compliance with treatment) or meth- odological issues, such as differences in inclusion criteria (many of these meta analyses covered a wide range of problems) or the quality of the studies included. Standardised Mean Difference (95% CI) Standardised Mean Difference -3 -2.5 -2 -1.5 -1 -.5 0 .5 1 1.5 2 2.5 3 Standardised Mean Difference 1.5 1 .5 0 .5 1 1.5 Analysis of emotional symptoms, random effects Figure 5 Analysis of emotional symptoms, random effects. all mean quality score of the studies included was low (16.4 out of 42). Therefore the results of the review can only be considered preliminary until the completion of more rigorous studies. studies reported the inclusion of clinical participants [54,55]. The participants included in the review may differ from those found in routine clinical settings in the severity of their problems, their willingness to participate in research, their motivation and adherence to treatment. However, it is also possible that the nature of self help treatments means that they will generally be used only with a proportion of adolescents who are willing and able to use them. As noted above, a number of studies included groups with circumscribed mental health issues (such as test, spider and dating anxiety) and the results may not generalise to clinical samples with more complex problems. In addition to the rating of the quality of the studies, gen- eralising the results of the review must be done with cau- tion as there are significant limits to the external validity of the study findings. Few studies reported the number of eligible participants who took part in the study. Thirteen out of 14 studies were published in the USA and one in Australia. There are many factors that may affect the use of self help technology in other countries: the degree to which these technologies are acceptable to adolescents; the structure of the health and education systems; skills training of professionals in providing these interventions; education and skills of adolescents; socio-cultural issues such as stigma; and the quality of care in control groups. Furthermore, the review used an inclusive age range for adolescents, and the acceptability and effectiveness of treatments for students aged 18–25 may be very different for adolescents aged between 12 and 17. As noted above, all studies reported volunteer participants and only two Page 9 of 12 (page number not for citation purposes) Quality assessment In this study, quality assessment was done using the QRS [47] by two researchers working independently. The over- Page 8 of 12 (page number not for citation purposes) Page 8 of 12 (page number not for citation purposes) Child and Adolescent Psychiatry and Mental Health 2008, 2:20 http://www.capmh.com/content/2/1/20 Standardised Mean Difference -3 -2.5 -2 -1.5 -1 -.5 0 .5 1 1.5 2 Study Denny Standardised Mean Differenc (95% CI) -0.65 (-1.55, 0.24) Ackerson -2.57 (-3.69,-1.46) Grossman 0.12 (-0.72, 0.96) Register -0.48 (-0.99, 0.03) Ramsey 0.26 (-0.15, 0.67) O'Kearney -0.19 (-0.76, 0.38) Allen -0.48 (-1.44, 0.48) Overall 2.5 3 -0.47 (-1.00, 0.07) Implications for research 4. Association AP: Diagnostic and Statistical Manual of Mental Disorders 4th, text version edition. Washington DC, American Psychiatric Association; 2000. p The findings indicated weak evidence for the use of self help technology in the management of emotional prob- lems in adolescents. There is a need for randomized trials to provide rigorous evidence of the effectiveness and cost effectiveness of self help technology for adolescents with emotional problems, compared to usual care in order to measure the effectiveness of these technologies in decreas- ing emotional symptoms. Also there is need for rand- omized trials to compare the effectiveness of different types of self help technology (e.g. bibliotherapy and infor- mation technology) to find out which is more effective and acceptable. Further randomized trials are needed to evaluate the effectiveness of self help technology in the long term. Most of the randomized studies identified in the current review report short follow up periods of less than 6 months. Finally none of the included studies inves- tigate the effectiveness of self help technology in relation to age, gender or other characteristics of the participants. Further research is needed to investigate important mod- erators of treatment effect [75,76]. 5. Centers for Disease Control and Prevention: Youth risk behavior surveillance—United States, 2005. 2006 [http://www.cdc.gov/ mmwr/preview/mmwrhtml/ss5505a1.htm]. Accessed April 17th 2008 6 Birmaher B Ryan N Williamson D Brent D Kaufman J Dahl R Perel 6. Birmaher B, Ryan N, Williamson D, Brent D, Kaufman J, Dahl R, Perel J, Nelson B: Childhood and adolescent depression: a review of the past ten years. Part I. J Am Acad Child Adolesc Psychiatry 1996, 35:1427-1439. 7. Harrington R: Depressive disorders. In Child and Adolescent Mental Health Services: strategy, planning, delivery and evaluation Edited by: Wil- liams R and Kerfoot M. New York, Oxford University Press; 2005:203-214. 8. Bradley SJ: Anxiety and mood disorders in children and adoles- cents: a practice update. Paediatric and Child Health 2001, 6:459-463. 9. Costelllo E, Egger H, Angold A: Developmental epidemiology of anxiety disorders. In Phobic and Anxiety Disorders in Children and Adolescents Edited by: Ollendick T and March J. New York, Oxford University Press; 2004:61-91. y 10. Evans D, Foa E, Gur R, Hendin H, O'Brien C, Seligman M, Walsh B: Treating and preventing adolescent mental health disorders: what we know and what we don't know New York, Oxford University Press; 2008. y 11. Additional file 1 Additional file 1 Table 2. Click here for file [http://www.biomedcentral.com/content/supplementary/1753- 2000-2-20-S1.doc] 18. Lewinsohn PM, Clarke GN, Hops H, Andrew J: Cognitive-behav- iour treatment for depressed adolescents. Behavior Therapy 1990, 21:385-401. 19. Weisz JR, Thurber CA, Sweeney L, Proffitt VD, LeGagnoux GL: Brief treatment of mild-to-moderate child depression using pri- mary and secondary control enhancement training. Journal of Consulting and Clinical Psychology 1997, 65:703-707. 20. Treatment for Adolescents With Depression Study (TADS) Team: Fluoxetine, cognitive-behavioral therapy, and their combi- nation for adolescents with depression: treatment for ado- lescents with depression study (TADS) randomized controlled trial. JAMA 2004, 292:807-820. The implications for practice The implementation of self help technology for adoles- cents with emotional problems would be premature until further high quality randomized controlled studies are conducted. The potential benefits of self help technology (increasing access, decreasing costs) should be weighed against the possible risk of implementing these technolo- gies without strong evidence of effectiveness. Page 9 of 12 (page number not for citation purposes) Page 9 of 12 (page number not for citation purposes) Child and Adolescent Psychiatry and Mental Health 2008, 2:20 Child and Adolescent Psychiatry and Mental Health 2008, 2:20 http://www.capmh.com/content/2/1/20 Acknowledgements The authors would like to acknowledge the assistance of Rosalind McNally in developing the search strategies. MA was funded by a studentship from the Ford Foundation. PB is funded by the Department of Health, United Kingdom. J 21. JAACAP: Practice Parameter for the Assessment and Treat- ment of Children and Adolescents With Anxiety Disorders. J Am Acad Child Adolesc Psychiatry 2007, 46:267-283. J y y 22. National Institute for Health and Clinical Excellence: Promoting children's social and emotional wellbeing in primary educa- tion (NICE public health guidance 12). 2008 [http:// www.nice.org.uk/guidance/index.jsp?action=byID&o=11948]. 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https://openalex.org/W2903264166	https://uscholar.univie.ac.at/detail/o:931221.pdf	English	null	Quantitative Phosphoproteomic and System-Level Analysis of TOR Inhibition Unravel Distinct Organellar Acclimation in Chlamydomonas reinhardtii	Frontiers in plant science	2,018	cc-by	16,546	ORIGINAL RESEARCH published: 28 November 2018 doi: 10.3389/fpls.2018.01590 Quantitative Phosphoproteomic and System-Level Analysis of TOR Inhibition Unravel Distinct Organellar Acclimation in Chlamydomonas reinhardtii Valentin Roustan1 and Wolfram Weckwerth1,2* 1 Department of Ecogenomics and Systems Biology, University of Vienna, Vienna, Austria, 2 Vienna Metabolomics Center (VIME), University of Vienna, Vienna, Austria Rapamycin is an inhibitor of the evolutionary conserved Target of Rapamycin (TOR) kinase which promotes and coordinates translation with cell growth and division. In heterotrophic organisms, TOR regulation is based on intra- and extracellular stimuli such as amino acids level and insulin perception. However, how plant TOR pathways have evolved to integrate plastid endosymbiosis is a remaining question. Despite the close association of the TOR signaling with the coordination between protein turn-over and growth, proteome and phosphoproteome acclimation to a rapamycin treatment have not yet been thoroughly investigated in Chlamydomonas reinhardtii. In this study, we have used in vivo label-free phospho-proteomic analysis to proﬁle both protein and phosphorylation changes at 0, 24, and 48 h in Chlamydomonas cells treated with rapamycin. Using multivariate statistics we highlight the impact of TOR inhibition on both the proteome and the phosphoproteome. Two-way ANOVA distinguished differential levels of proteins and phosphoproteins in response either to culture duration and rapamycin treatment or combined effects. Finally, protein– protein interaction networks and functional enrichment analysis underlined the relation between plastid and mitochondrial metabolism. Prominent changes of proteins involved in sulfur, cysteine, and methionine as well as nucleotide metabolism on the one hand, and changes in the TCA cycle on the other highlight the interplay of chloroplast and mitochondria metabolism. Furthermore, TOR inhibition revealed changes in the endomembrane trafﬁcking system. Phosphoproteomics data, on the other hand, highlighted speciﬁc differentially regulated phosphorylation sites for calcium-regulated protein kinases as well as ATG7, S6K, and PP2C. To conclude we provide a ﬁrst combined Chlamydomonas proteomics and phosphoproteomics dataset in response to TOR inhibition, which will support further investigations. Keywords: TOR, rapamycin, proteomics, phosphoproteomics, energy signaling, plant systems biology, biofuels Reviewed by: Weimin Ma, Reviewed by: Weimin Ma, Shanghai Normal University, China Luis Valledor, Universidad de Oviedo, Spain Shanghai Normal University, China Luis Valledor, Correspondence: Wolfram Weckwerth wolfram.weckwerth@univie.ac.at Specialty section: This article was submitted to Plant Proteomics, a section of the journal Frontiers in Plant Science Received: 28 April 2018 Accepted: 15 October 2018 Published: 28 November 2018 Citation: Roustan V and Weckwerth W (2018) Quantitative Phosphoproteomic and System-Level Analysis of TOR Inhibition Unravel Distinct Organellar Acclimation in Chlamydomonas reinhardtii. Front. Plant Sci. 9:1590. doi: 10.3389/fpls.2018.01590 Specialty section: This article was submitted to Plant Proteomics, a section of the journal Frontiers in Plant Science Received: 28 April 2018 Accepted: 15 October 2018 Published: 28 November 2018 Received: 28 April 2018 Accepted: 15 October 2018 Published: 28 November 2018 Edited by: Silvia Mazzuca, Università della Calabria, Italy Università della Calabria, Italy Reviewed by: Weimin Ma, Shanghai Normal University, China Luis Valledor, Universidad de Oviedo, Spain Correspondence: Wolfram Weckwerth wolfram.weckwerth@univie.ac.at INTRODUCTION While in human and yeast the TOR kinase can form two diﬀerent complexes (TORC1 and TORC2), only TORC1 is conserved in plants and algae like Chlamydomonas (van Dam et al., 2011; Roustan et al., 2016). TOR associated proteins; LST8 and RAPTOR have been identiﬁed in Chlamydomonas and are conserved along the viridiplantae lineage (Crespo et al., 2005; Díaz-Troya et al., 2008a,b; Shemi et al., 2015). It was found that rapamycin was able to inhibit TOR activity in yeast and mammals and Chlamydomonas but not in Arabidopsis (Heitman et al., 1991; Vézina and Kudelski, 1975; Xu et al., 1998; Crespo et al., 2005; Sormani et al., 2007; Xiong and Sheen, 2012; Aylett et al., 2016). Mechanistically, TOR is inhibited by the complex formed between FKBP12 (a prolyl-cis,trans- isomerase protein) and rapamycin which in-turn impairs the TOR kinase domain and its targeted substrates. Consequently, a rapamycin treatment in Chlamydomonas induces a growth inhibition as well as a vacuolarization of the cell (Crespo et al., 2005). In plants, recent publications have highlighted the potential relationship between nutrient status and TOR activation. More particularly, those studies have studied the connection between TOR signaling, nutrient deprivation (such as nitrogen depletion), sulfur and inositol metabolisms (Pérez- Pérez and Crespo, 2010; Perez-Perez et al., 2010; Imamura et al., 2015, 2016; Couso et al., 2016; Dong et al., 2017; Roustan et al., 2017). While the knowledge about TOR signaling in yeast and mammals has a solid background the related signaling networks in plants and in particular in Chlamydomonas are rather underexplored. Nevertheless, some components regulated by TORC1 complex in Chlamydomonas have been highlighted with the use of rapamycin treatment. Indeed, it appears that TOR is involved in the control of protein synthesis and especially in the activity of the endoplasmic reticulum (ER) (Diaz-Troya et al., 2011). However, unlike in higher plants, yeast, and mammals, no direct link between TOR activation and the promotion of rRNA translation via the phosphorylation cascade involving the S6 Kinase protein and the ribosomal protein S6 were found in Chlamydomonas (Chung et al., 1992; De Virgilio and Loewith, 2006; Ma and Blenis, 2009; Schepetilnikov et al., 2011; Xiong and Sheen, 2012; Dobrenel et al., 2016b). While TOR is activating the protein synthesis under nutrient availability, several studies have pointed out the negative regulation of autophagy processes by TOR signaling in Chlamydomonas. Citation: November 2018 \| Volume 9 \| Article 1590 1 Frontiers in Plant Science \| www.frontiersin.org Rapamycin Induced Chlamydomonas Phosphoproteome Changes Roustan and Weckwerth INTRODUCTION data support the activation of autophagy during rapamycin treatment and nutrient deprivation via the ATG8 accumulation to the autophagosome in Chlamydomonas (Díaz-Troya et al., 2008b; Pérez-Pérez and Crespo, 2010; Perez-Perez et al., 2010; Perez-Perez and Crespo, 2014; Shemi et al., 2015). Additionally, several studies have analyzed the metabolic acclimation of Chlamydomonas to rapamycin treatment which also reveals similarity to nitrogen stress adaptation. A striking example, reported by several authors, indicate a tri-acyl-glycerol (TAG) accumulation during rapamycin treatment in green algae, including Chlamydomonas (Imamura et al., 2015; Couso et al., 2016; Mukaida et al., 2016) but also in distant red algae (Imamura et al., 2016) during nitrogen depletion (Breuer et al., 2013; Goodenough et al., 2014; Yang et al., 2015). TAG accumulation is of a major interest since it can be used for lipid and biodiesel production (Merchant et al., 2012; Liu and Benning, 2013). More dramatically, rapamycin treatment aﬀects directly the central primary metabolism. Further, metabolite proﬁling unraveled important changes for intermediates of the TCA cycle and amino acid metabolism, especially for methionine and cysteine metabolism deeply interconnected with the redox homeostasis (Foyer and Noctor, 2011; Ren et al., 2012; Caldana et al., 2013; Lee and Fiehn, 2013; Kleessen et al., 2015; Juppner et al., 2017). Finally, transcriptomic analysis has shed light on the role of TOR on the transcriptome regulation and its coordination with the metabolome (Kleessen et al., 2015). Indeed, Chlamydomonas transcriptomic analysis has shown that TOR inhibition up-regulated genes involved in tetrapyrrole synthesis, vacuolar function, amino acid metabolism and transport as well as folding and chaperonin related genes. On the other hand, genes involved in nucleotide metabolism, cell cycle and DNA replication and repair were down- regulated. Living organisms need to continuously adjust their cell growth to their energy status. All known organisms store energy in form of ATP via nutrient metabolization. Therefore, it is crucial to sense the nutritional input to coordinate the organisms growth. In yeast, nutrient availability is sensed by the evolutionary conserved Target Of Rapamycin complex (TORC) (Dobrenel et al., 2016a; Roustan et al., 2016). TORC, as well as its signaling pathway, is conserved in most of the lower and higher eukaryotes present in the three eukaryotic kingdoms (van Dam et al., 2011; Laplante and Sabatini, 2012; Xiong and Sheen, 2015; Couso et al., 2016; Roustan et al., 2016). RESULTS synthesis (Diaz-Troya et al., 2011). Furthermore, photosynthetic activity was measured as well as chlorophyll content. In both cases, PSII eﬃciency (Fv/Fm) and total chlorophyll content per milligram fresh weight, presented no signiﬁcant diﬀerence between control and rapamycin-treated samples, indicating that photosynthetic activity is not altered by TOR inhibition (Supplementary Figure S1). In this context, maintenance of photosynthetic activity while protein synthesis decrease should induce an accumulation of carbon storage compounds. Several studies have correlated cell growth inhibition to carbon storage molecules accumulation like starch and lipids (Merchant et al., 2012; Juergens et al., 2016). To decipher how the carbon pool is preferentially stored, starch and lipids were extracted as described in materials and methods; starch content was determined by an enzymatic assay and total lipid content was weighted (see Materials and Methods). Unlike in nitrogen depletion, lipid content was found to be stable during rapamycin treatment (Figure 1F). However, the starch content increased up to 50% at 48 h, compared to control samples (Figures 1E,F). Complementary, we investigated whether some lipid classes, like Tri-Acyl-Glycerol (TAG), could accumulate during TOR inhibition, as in nitrogen depletion. To investigate this possibility, Characterization of Chlamydomonas reinhardtii Growth and Physiological Parameter During Rapamycin Treatment Support for a speciﬁc TOR regulation under nitrogen depletion and recovery was recently published (Roustan et al., 2017). To allow data comparison, we applied similar growth condition in the present study (see Materials and Methods). Additionally, cells were treated with 500 nM rapamycin or drug vehicle and sampled at 0, 24, and 48 h to measure multiple physiological parameters. Remnant growth was still observed using both cell number and fresh weight (FW) level in rapamycin-treated samples (Figures 1A,B). Indeed Chlamydomonas growth is not fully arrested by rapamycin treatment even with 10 µM rapamycin as previously reported (Crespo et al., 2005; Juppner et al., 2017). Protein content per milliliter was assessed with Bradford assay (see Materials and Methods). Measured protein concentration per milligram fresh weight decreased by 40% (Figures 1C,D). Protein content result is in line with the previously measured [14C]Arg incorporation in de novo protein FIGURE 1 \| Phenotyping of Chlamydomonas reinhardtii cells, treated with 500 nM rapamycin (orange) or with drug vehicle (blue). A time course (0, 24, and 48 h) was sampled and cell number (A), fresh weight (B) and total protein per ml (C) were measured. INTRODUCTION Conserved autophagy-related genes (ATG) have been found in photosynthetic organisms and molecular Considering these results, we conducted a proteomics and phosphoproteomics survey to better understand the molecular response of Chlamydomonas to rapamycin-induced TOR inhibition. We analyzed the inﬂuence of rapamycin treatment on cell physiology, proteome, and phosphoproteome using a cell wall deﬁcient strain CC-503 during a 0, 24, and 48 h TOR time course under mixotrophic growth conditions and continuous light. The results were compared to transcriptomics and metabolomics studies. Rapamycin treatment induced cell growth inhibition, starch, and TAG accumulation. Proteomics and phosphoproteomics data were subjected to statistical analysis and functional annotation to provide a system-level investigation of stress perception and transduction. Signiﬁcant changes in both protein and phosphorylation pattern were further investigated with the help of STRING/protein–protein interaction networks (von Mering et al., 2005). Altogether, our data shed light on the plastidial interconnection between nucleotide synthesis, sulfur, serine, methionine and cysteine metabolism. As well, our data suggest an uncoupling between plastid and mitochondria metabolism in response to translation inhibition. Eventually, in vivo quantitative phosphoproteomics data highlight direct consequences of TOR signaling with speciﬁc changes of phosphorylations sites in TOR targets S6K and ATG7 proteins as well as the upstream regulators such as PP2C. November 2018 \| Volume 9 \| Article 1590 Frontiers in Plant Science \| www.frontiersin.org 2 Rapamycin Induced Chlamydomonas Phosphoproteome Changes Roustan and Weckwerth Proteome and Phosphoproteome Analysis of Rapamycin-Treated Chlamydomonas Cells in Comparison With Control Samples Unsupervised PCA (Figure 2) showed that principal component 1 (29.2% total explained variance) explained both the time course and rapamycin treatment highlighting also the strong eﬀect of culture duration. Those results are in line with metabolomics ﬁndings (Juppner et al., 2017). The culture duration eﬀect on the proteome is further strengthened by the HCA as indicated by the samples clustering (Figure 3). Protein cluster analysis indicates that growth inhibition induced by TOR treatment does not require a complete remodeling of the proteome but rather speciﬁc processes. While no signiﬁcant enrichment was obtained for cluster 1, cluster 2 was enriched in proteins related to mTOR signaling pathway, insulin pathway, and ribosomes. Cluster 3 regroups proteins with a time-dependent up-regulation pattern. Enriched categories in cluster 3 were related to fatty acid elongation in mitochondria and peroxisome. Cluster 4 and 5 encompass proteins function predominantly related to photosynthesis, as well as primary and secondary metabolism (Figure 3). Proteome and phosphoproteome were analyzed by an LC- MS shotgun technique as previously described (Wienkoop et al., 2010; Valledor et al., 2013, 2014; Roustan et al., 2017). Proteins were extracted from ﬁve biological samples (three biological samples for phosphoproteomics) treated with 500 nM rapamycin or with the drug vehicle and harvested at 0, 24, and 48 h time points. Proteome datasets were deﬁned based on proteins which were quantiﬁed in at least 4 biological replicates, in at least one class of samples (Supplementary Table S1). A total of 916 proteins were relatively quantiﬁed. To generate the phosphopeptide data matrix, the similar workﬂow was applied as in the recently published phosphoproteomic dataset obtained for nitrogen stress and recovery in Chlamydomonas (Roustan et al., 2017). Only peptides that had a phosphorylation localization probability > 0.75 and score diﬀerence > 5 were kept (Supplementary Table S2) resulting in 5283 identiﬁed phosphosites. Additionally, identiﬁed and quantiﬁed phosphopeptides were ﬁltered according to the total protein dataset. After ﬁltering, a data matrix of 1311 phosphopeptides mapped to 684 proteins was obtained (Supplementary Table S2). 1282, 25, 4 phosphopeptides that had single, double, triple phosphorylation sites, respectively, were detected. The distributions of phosphorylated Ser, Thr and Tyr residues were 1084, 185, and 42 respectively. Those distributions are comparable to previous studies (Wang et al., 2014; Roustan et al., 2017). RESULTS Further, protein (D), starch (in µM of glucose/mg FW) (E) and total lipid content (F) were measured and normalized by the fresh weight. (n = 4, p < 0.05). FIGURE 1 \| Phenotyping of Chlamydomonas reinhardtii cells, treated with 500 nM rapamycin (orange) or with drug vehicle (blue). A time course (0, 24, and 48 h) was sampled and cell number (A), fresh weight (B) and total protein per ml (C) were measured. Further, protein (D), starch (in µM of glucose/mg FW) (E) and total lipid content (F) were measured and normalized by the fresh weight. (n = 4, p < 0.05). November 2018 \| Volume 9 \| Article 1590 Frontiers in Plant Science \| www.frontiersin.org 3 Rapamycin Induced Chlamydomonas Phosphoproteome Changes Roustan and Weckwerth time-distribution of lipid class was analyzed by thin-layer- chromatography (Fuchs et al., 2011). We found that TAG accumulates during TOR inhibition (Supplementary Figures S1D,E). Taken together, our results indicate, that unlike nitrogen stress, only the proportion of TAG content accumulates in TOR inhibited cell and not the total lipid content. Additionally, we could observe a negative correlation between protein concentration and starch/TAG amount suggesting that TOR inhibition aﬀect carbon ﬂux. et al., 2017) whereas proteome adaptation to TOR inhibition remained unknown. To understand the global proteome acclimation of Chlamydomonas during rapamycin treatment, in vivo label-free shotgun proteomics was used to generate a proteome dataset. Protein changes between rapamycin treated samples and control samples were analyzed with principal component analysis (PCA) and hierarchical clustering (HCA) (Figures 2, 3). Identiﬁed protein clusters from HCA were used to perform a KEGG pathways enrichment analysis with the Algal Functional Annotation Tool (AFAT) (Lopez et al., 2011, 2014). Finally, the STRING database was used to understand the protein-protein interaction between the proteins identiﬁed as strictly aﬀected by the two-way ANOVA treatment factor results (Table 1). Proteome and Phosphoproteome Analysis of Rapamycin-Treated Chlamydomonas Cells in Comparison With Control Samples According to (Valledor et al., 2014), the mercator online tool was used for protein and phosphoprotein annotation based on various organism-speciﬁc databases (Lohse et al., 2014). FIGURE 2 \| Principal component analysis (PCA) of protein abundance in Chlamydomonas cells treated with rapamycin or drug vehicle during a time course experiment (0, 24, and 48 h). The PCA includes all quantiﬁed proteins. Dots are the biological replicates (n = 5–6), control samples are colored in blue and rapamycin samples are colored in orange. Proteome Differences Between Rapamycin and Control-Treated Samples The heterotrimeric TOR complex is known to regulate translational activity and autophagy. To this end, TOR interacts with ribosomal S6 Kinase, and activates, by a phosphorylation cascade, RPS6 proteins and eukaryotic initiation factor 4B (Dobrenel et al., 2016a; Roustan et al., 2016; Perez-Perez et al., 2017). On the other hand, TOR complex phosphorylates ATG1 and inhibits autophagy. Orthologs for TOR, S6K, RPS6, and ATG1 proteins were previously found in Chlamydomonas (Roustan et al., 2016). A recent study in Chlamydomonas highlighted the nitrogen and concanamycin (translation inhibitor) eﬀects on ATG8 and RPS6 protein content (Couso FIGURE 2 \| Principal component analysis (PCA) of protein abundance in Chlamydomonas cells treated with rapamycin or drug vehicle during a time course experiment (0, 24, and 48 h). The PCA includes all quantiﬁed proteins. Dots are the biological replicates (n = 5–6), control samples are colored in blue and rapamycin samples are colored in orange. FIGURE 2 \| Principal component analysis (PCA) of protein abundance in Chlamydomonas cells treated with rapamycin or drug vehicle during a time course experiment (0, 24, and 48 h). The PCA includes all quantiﬁed proteins. Dots are the biological replicates (n = 5–6), control samples are colored in blue and rapamycin samples are colored in orange. Frontiers in Plant Science \| www.frontiersin.org November 2018 \| Volume 9 \| Article 1590 4 Rapamycin Induced Chlamydomonas Phosphoproteome Changes Roustan and Weckwerth FIGURE 3 \| Hierarchical cluster analysis of quantiﬁed proteins. Clustering of proteins and samples was done based on Euclidian correlation and average linkage with Perseus. Clusters were exported in Supplementary Table S1 and KEGG enrichment analysis was done with the Algal functional annotation tool (Lopez et al., 2011) for each cluster as referred in Supplementary Table S1. FIGURE 3 \| Hierarchical cluster analysis of quantiﬁed proteins. Clustering of proteins and samples was done based on Euclidian correlation and average linkage with Perseus. Clusters were exported in Supplementary Table S1 and KEGG enrichment analysis was done with the Algal functional annotation tool (Lopez et al., 2011) FIGURE 3 \| Hierarchical cluster analysis of quantiﬁed proteins. Clustering of proteins and samples was done based on Euclidian correlation and average linkage with Perseus. Clusters were exported in Supplementary Table S1 and KEGG enrichment analysis was done with the Algal functional annotation tool (Lopez et al., 2011) for each cluster as referred in Supplementary Table S1. Proteome Differences Between Rapamycin and Control-Treated Samples November 2018 \| Volume 9 \| Article 1590 5 Frontiers in Plant Science \| www.frontiersin.org Rapamycin Induced Chlamydomonas Phosphoproteome Changes Roustan and Weckwerth TABLE 1 \| Summary of the 61 proteins identiﬁed as strictly regulated by rapamycin trea Protein IDs Control Rapamycin Two-way ANOV A8JH37 0.00688051 A8I263 0.00326497 A8J506 0.028858 A8JBI1 0.0491331 A8J841 0.021587 A8HVU9 0.0060891 A8JDA7 0.000628257 A8JGF4 0.00232198 A8HXT4 0.00511534 A8JEF7 0.00698897 Q9LLL6 0.0389221 A8IQU3 0.0220131 A8JFK6 0.0298598 A8IX80 0.000320047 A8J2E9 0.00189678 Q75NZ1 0.000406907 A8JBB4 0.000696152 A8J311 0.00141633 A8IAE5 0.00205656 A 8J EU3 0.00793963 A8HWZ8 0.00961465 A8HNG8 0.0121003 Q6Y682 0.0135182 A8IW72 0.0136876 A8JC51 0.0186181 A8J5B8 0.0217927 A8JHN0 0.0252416 A8IGN6 0.0252812 A8IYP3 0.0301636 A8HMX2 0.0315043 A8J090 0.0346941 A8J4I5 0.0434525 A8J8K4 0.0487621 A8JGY3 0.000562731 A8HPI1 0.0411809 TABLE 1 \| Summary of the 61 proteins identiﬁed as strictly regulated by rapamycin treatment. Protein IDs Control Rapamycin Two-way ANOVA p value treatment Mercator annotation A8JH37 0.00688051 Amino acid metabolism.synthesis.aspartate family.methionine A8I263 0.00326497 Amino acid metabolism.synthesis.central amino acid metabolism.aspartate.aspartate aminotransferase A8J506 0.028858 Amino acid metabolism.synthesis.glutamate family.arginine.arginosuccinate synthase A8JBI1 0.0491331 Cell.organization A8J841 0.021587 Co-factor and vitamine metabolism.thiamine A8HVU9 0.0060891 Glycolysis.cytosolic branch.phosphoglycerate mutase A8JDA7 0.000628257 Lipid metabolism.FA synthesis and FA elongation.Acetyl CoA Carboxylation.heteromeric Complex.Biotin Carboxyl Carrier Protein A8JGF4 0.00232198 Lipid metabolism.FA synthesis and FA elongation.Acetyl CoA Carboxylation.heteromeric Complex.Biotin Carboxylase A8HXT4 0.00511534 Lipid metabolism.FA synthesis and FA elongation.Acetyl CoA Carboxylation.heteromeric Complex.Biotin Carboxylase A8JEF7 0.00698897 Lipid metabolism.FA synthesis and FA elongation.ketoacyl ACP synthase Q9LLL6 0.0389221 Major CHO metabolism.synthesis.starch.AGPase A8IQU3 0.0220131 Mitochondrial electron transport / ATP synthesis.F1-ATPase A8JFK6 0.0298598 Mitochondrial electron transport/ATP synthesis.NADH-DH.complex I.carbonic anhydrase A8IX80 0.000320047 Not assigned.no ontology A8J2E9 0.00189678 Not assigned.no ontology Q75NZ1 0.000406907 Not assigned.unknown A8JBB4 0.000696152 Not assigned.unknown A8J311 0.00141633 Not assigned.unknown A8IAE5 0.00205656 Not assigned.unknown A 8J EU3 0.00793963 Not assigned.unknown A8HWZ8 0.00961465 Not assigned.unknown A8HNG8 0.0121003 Not assigned.unknown Q6Y682 0.0135182 Not assigned.unknown A8IW72 0.0136876 Not assigned.unknown A8JC51 0.0186181 Not assigned.unknown A8J5B8 0.0217927 Not assigned.unknown A8JHN0 0.0252416 Not assigned.unknown A8IGN6 0.0252812 Not assigned.unknown A8IYP3 0.0301636 Not assigned.unknown A8HMX2 0.0315043 Not assigned.unknown A8J090 0.0346941 Not assigned.unknown A8J4I5 0.0434525 Not assigned.unknown A8J8K4 0 0487621 Not assigned unknown TABLE 1 \| Summary of the 61 proteins identiﬁed as strictly regulated by rapamycin treatment. Proteome Differences Between Rapamycin and Control-Treated Samples Mercator annotation Amino acid metabolism.synthesis.aspartate family.methionine Amino acid metabolism.synthesis.central amin acid metabolism.aspartate.aspartate aminotransferase Amino acid metabolism.synthesis.glutamate family.arginine.arginosuccinate synthase Cell.organization Co-factor and vitamine metabolism.thiamine Glycolysis.cytosolic branch.phosphoglycerate mutase Lipid metabolism.FA synthesis and FA elongation.Acetyl CoA Carboxylation.heteromeric Complex.Biotin Carboxyl Carrier Protein Lipid metabolism.FA synthesis and FA elongation.Acetyl CoA Carboxylation.heteromeric Complex.Biotin Carboxylase Lipid metabolism.FA synthesis and FA elongation.Acetyl CoA Carboxylation.heteromeric Complex.Biotin Carboxylase Lipid metabolism.FA synthesis and FA elongation.ketoacyl ACP synthase Major CHO metabolism.synthesis.starch.AGPase Mitochondrial electron transport / ATP synthesis.F1-ATPase Mitochondrial electron transport/ATP synthesis.NADH-DH.complex I.carbonic anhydrase Not assigned.no ontology Not assigned.no ontology Not assigned.unknown Not assigned.unknown Not assigned.unknown Not assigned.unknown Not assigned.unknown Not assigned.unknown Not assigned.unknown Not assigned.unknown Not assigned.unknown Not assigned.unknown Not assigned.unknown Not assigned.unknown Not assigned.unknown Not assigned.unknown Not assigned.unknown Not assigned.unknown Not assigned.unknown Not assigned.unknown Nucleotide metabolism.phosphotransfer and pyrophosphatases.adenylate kinase Nucleotide metabolism.phosphotransfer and pyrophosphatases.misc Continue A8JGF4 0.00232198 Continued Continued November 2018 \| Volume 9 \| Article 1590 Frontiers in Plant Science \| www.frontiersin.org 6 Rapamycin Induced Chlamydomonas Phosphoproteome Changes Roustan and Weckwerth TABLE 1 \| Continued Protein IDs Control Rapamycin Two-way ANOVA p value treatment Mercator annotation A8IXD1 0.0193976 Nucleotide metabolism.salvage.nucleoside kinases.adenosine kinase A8IJJ8 8.46E-10 Nucleotide metabolism.synthesis.purine.AIR carboxylase A8JG58 0.0220205 Protein.aa activation.glycine-tRNA ligase A8IL08 0.000640739 Protein.degradation. metal loprotease A8J6C7 0.0371907 Protein.degradation. metal loprotease A8IL21 0.00331655 Protein.degradation.serine protease A8HQ77 2.58E-05 Protein.degradation.ubiquitin.E2 A 8IXU7 0.0258162 Protein.postranslational modiﬁcation A8JHX9 0.000303051 Protein.synthesis.elongation A8IA39 0.0229822 Protein.synthesis.elongation A8IW20 0.00122988 Protein.synthesis. initiation A8J503 0.0445697 Protein.synthesis.ribosomal protein.prokaryotic.chloroplast.50S subunit.L6 A8ISZ1 0.00519999 Protein.synthesis.ribosome biogenesis.export from nucleus A8HP84 0.0288901 PS.calvin cycle.GAP A8IRK4 0.0258706 PS.calvin cycle.seduheptulose bisphosphatase Q5S7Y5 0.000227069 PS.calvin cycle.TPI A8IVM9 0.0110128 PS.photorespiration.glycine cleavage.P subunit A8I2V3 0.0351867 Redox.peroxiredoxin.BASl A8I074 0.0236832 RNA. processing A8J4I2 0.0263681 RN A. processing.splicing A8IXQ5 0.0139685 RNA.regulation of transcription.unclassiﬁed A8J3Q6 0.0014942 S-assimilation.AKN A8J6A7 0.0222006 S-assimilation.APR A8JEV9 0.00294033 Secondary metabolism.isoprenoids.non-mevalonate pathway.HDR A8JGV6 0.00272543 Signaling.14-3-3 proteins Q7X7A7 0.00319663 Signaling.14-3-3 proteins A8IPQ9 0.000173987 Signaling.calcium A8J3P5 0.0318879 Stress.abiotic.heat A8J1V5 0.0334733 TCA/org transformation.TCA.pyruvate DH.E2 A8J4K4 1.15E-05 Tetrapyrrole synthesis.uroporphyrinogen III synthase A81164 0.0441812 Transport.p- and v-ATPases A8IA45 0.0209673 Transport.p- and v-ATPases.H+-transporting two-sector ATPase.subunit B In order to assess the synergistic eﬀect of multiple variables on Chlamydomonas proteome (time, treatment and time × treatment); two-way analysis of variance (ANOVA) was used. Two-way ANOVA tests the eﬀect of two independent factors (hours of culture vs. treatment) on the proteome. Thus, two-way ANOVA distinguishes the inﬂuence of each independent factor on the proteome but also determines if they signiﬁcantly interact. Frontiers in Plant Science \| www.frontiersin.org November 2018 \| Volume 9 \| Article 1590 Proteome Differences Between Rapamycin and Control-Treated Samples In total, 260 proteins were signiﬁcantly aﬀected by the rapamycine treatment, 587 proteins were signiﬁcantly changed across the time course and 172 proteins were found to be signiﬁcantly aﬀected by the interaction of both factors (Figure 4A). Temporal eﬀects on proteome, independent of the rapamycin treatment, were ﬁrst investigated. To capture a trend in which quantiﬁed proteins showed a predominant eﬀect of time dependency and a minimal eﬀect of treatment; proteins with the highest p-value for both interaction and rapamycin treatment and the lowest p-value for time factor were selected. Most of the identiﬁed proteins through this screen were related to chloroplast metabolism (Supplementary Table S1), conﬁrming the absence of signiﬁcant changes observed in the photosynthetic apparatus and total chlorophyll content between the two treatments (Supplementary Figure S1). Furthermore, the two- way ANOVA established that some proteins were signiﬁcantly changed by both, treatment and time factors (Figure 4A). Only 26 out of the 172 proteins were not overlapping with time or treatment factors. All the 26 proteins present inverse pattern between rapamycin and control treatment. Among them, a phosphoglucan water dikinase (A8J6C3) involved in starch degradation decreases up to 2-fold over the rapamycin treatment in contrast to the control cells. Further, abundance pattern of an isocitrate lyase (A8J244) involved in the gluconeogenesis declines in control sample, while it shows a 2.5-fold increase during the rapamycin treatment. Isocitrate lyase and phosphoglucan water dikinase protein changes corroborate with induced starch accumulation observed in rapamycin-treated cells (Figure 1E). While 260 proteins are signiﬁcantly diﬀerent between control and rapamycin-treated samples, only 61 proteins were speciﬁc to the treatment factor as summarized in Figure 4A. Interestingly, enrichment analysis of these 61 proteins points to sulfur, cysteine and methionine metabolic functions also recently shown to be involved in November 2018 \| Volume 9 \| Article 1590 Frontiers in Plant Science \| www.frontiersin.org 7 Rapamycin Induced Chlamydomonas Phosphoproteome Changes Roustan and Weckwerth IGURE 4 \| Graphic display of enriching fraction of KEGG pathways and protein–protein interaction network from proteins signiﬁcantly regulated by the rapamycin. Proteome Differences Between Rapamycin and Control-Treated Samples Phosphopeptides changes were analyzed by PCA and two-way ANOVA using the dataset for control and rapamycin-treated samples. Phosphoproteome PCA analysis shows similar results as for the proteome. In both cases, we could diﬀerentiate treatment at each time point (Figure 5), however, both growth conditions presented similar trajectory, indicating the time-dependent eﬀect on the data (all loading values can be found in Supplementary Table S2). In total two-way ANOVA identiﬁed 202 phosphopeptides signiﬁcantly aﬀected by treatment, 646 phosphopeptides signiﬁcantly changed across the time course, and 187 phosphopeptides found to be signiﬁcantly aﬀected by the interaction of both factors (Figure 6A). Table 2 present the 60 phosphopeptides speciﬁc to the treatment factor. Among them, A8J8X3 encodes an Aurora- kinase which is a member of a family of Ser/Thr kinases. Arabidopsis Aurora-kinase homolog is involved in the control of cell cycle, through its interaction with plant- speciﬁc cytoskeletal structures and its activities peak during cell division (Demidov et al., 2014). A8J8X3 phosphorylation site at Ser 244 is inhibited by about 15 % during the rapamycin treatment (Figure 6C). In line with cell cycle regulation, A8IZM6, and A8IFQ9 presented 2-fold lower phosphorylation level compared to control samples (Table 2). A8IZM6 encodes a homolog of human Lysine-Speciﬁc Demethylase1 involved in H3K4 methylation of ﬂowering time loci (FLC and FWA) and A8IFQ9 a DNA binding protein with a nuclease activity which is involved in response to singlet oxygen in Arabidopsis (Jiang et al., 2007; Chen Finally, we also investigated the 199 other proteins presenting a signiﬁcant diﬀerence in treatment factor, but also with time and interaction factors. Previously published transcriptomics data corroborate a signiﬁcant decrease of proteins involved in pyrimidine biosynthesis (A8IMN5 and A8JIR0) (Ramundo et al., 2014). Moreover, mitochondrial chaperonin proteins were found to be up-regulated during rapamycin treatment (A8IMK1 and A8JES1). Proteome Differences Between Rapamycin and Control-Treated Samples A) V di ith th t ANOVA lt (B) KEGG i h t l i d ith AFAT f th 61 t i iﬁ ll l t d b i FIGURE 4 \| Graphic display of enriching fraction of KEGG pathways and protein–protein interaction network from proteins signiﬁcantly regulated by the rapamycin A) Venn diagram with the two-way ANOVA results (B) KEGG enrichment analysis done with AFAT for the 61 proteins speciﬁcally regulated by rapamycin C) STRING protein–protein interaction network performed with signiﬁcantly changed 61 protein subset. Protein-Protein interaction network is based on evidences threshold based on medium conﬁdence score). For details, see Experimental Procedures, as well as Supplementary Table S1. FIGURE 4 \| Graphic display of enriching fraction of KEGG pathways and protein–protein interaction network from proteins signiﬁcantly regulated by the rapamycin. (A) Venn diagram with the two-way ANOVA results (B) KEGG enrichment analysis done with AFAT for the 61 proteins speciﬁcally regulated by rapamycin (C) STRING protein–protein interaction network performed with signiﬁcantly changed 61 protein subset. Protein-Protein interaction network is based on evidences (threshold based on medium conﬁdence score). For details, see Experimental Procedures, as well as Supplementary Table S1. November 2018 \| Volume 9 \| Article 1590 Frontiers in Plant Science \| www.frontiersin.org 8 Rapamycin Induced Chlamydomonas Phosphoproteome Changes Roustan and Weckwerth FIGURE 5 \| Principal component analysis (PCA) of phosphopeptides abundance in Chlamydomonas cells treated with rapamycin or drug vehicle during a time course experiment (0, 24, and 48 h). The PCA includes all phosphopeptides. Dots are the biological replicates (n = 3), rapamycin treated samples are colored in orange while control samples are colored in blue. TOR signaling or aﬀected by TOR inhibition in Arabidopsis (Figure 4B) (Dong et al., 2017). Similarly, proteins related to primary metabolism were enriched, such as amino-acid (A8I263 and A8J506), pyruvate (A8J1V5, A8HMX2, A8HXT4) and lipid metabolism (A8JDA7, A8JGF4, A8HXT4, A8JEF7) (Figure 4B). Moreover, PPI-network with protein sequences of each of the 61 proteins discussed above was generated with STRING database (Figure 4C) (Franceschini et al., 2013). PPI-network analysis highlighted additional relevant proteins. In line with transcriptomics data (Ramundo et al., 2014; Kleessen et al., 2015), tetrapyrrole metabolism related protein such as uroporphyrinogen-III synthase involved in tetrapyrrole biosynthesis (A8J4K4) showed a 2-fold increase during rapamycin treatment compared to control cells. Proteome Differences Between Rapamycin and Control-Treated Samples In contrast, proteins related to Calvin-cycle and photorespiration, such as A8HP84, A8IRK4, Q5S7Y5, and A8IVM9, were found to be down-regulated during rapamycin treatment. As well, several proteases were found to be diﬀerentially regulated during the rapamycin treatment. For instance, metalloprotease (A8IL08 and A8J6C7) predicted to be localized at the thylakoid membrane was down-regulated, while a serine protease (A8IL21) showed up to 2-fold accumulation during rapamycin treatment. Further, cell cycle-related protein CRB3 (Q6Y682) involved in circadian rhythm was 50% decreased compared to control samples. Interestingly, proteins related to the endomembrane systems such as the vacuole and the endoplasmatic reticulum (ER) were also aﬀected by rapamycin treatment. For example, vacuolar ATP synthase subunits (A8I164 and A8IA45) were found to be signiﬁcantly downregulated during rapamycin treatment compared to control cells. As well, 14-3-3 proteins (A8JGV6 andQ7X7A7) or CDPK2 homologs (A8IPQ9) have been shown to be located in the ER and present a signiﬁcant downregulation during rapamycin treatment. As well, proteins related to mitochondria such as ATP synthase (A8IQU3), gamma carbonic anhydrase (A8JFK6) and a (D)-2-hydroxyglutarate dehydrogenase (A8J2E9) were down-regulated. Nevertheless, a mitochondrial ubiquinol- cytochrome c oxidoreductase subunit (A8JC51) increased over rapamycin treatment. Similarly, proteins related to purine metabolism were found to be signiﬁcantly up-regulated during rapamycin treatment (A8JGY3, A8HPI1, A8IXD1, and A8IJJ8). FIGURE 5 \| Principal component analysis (PCA) of phosphopeptides abundance in Chlamydomonas cells treated with rapamycin or drug vehicle during a time course experiment (0, 24, and 48 h). The PCA includes all phosphopeptides. Dots are the biological replicates (n = 3), rapamycin treated samples are colored in orange while control samples are colored in blue. FIGURE 5 \| Principal component analysis (PCA) of phosphopeptides abundance in Chlamydomonas cells treated with rapamycin or drug vehicle during a time course experiment (0, 24, and 48 h). The PCA includes all phosphopeptides. Dots are the biological replicates (n = 3), rapamycin treated samples are colored in orange while control samples are colored in blue. Chlamydomonas growth. Analysis of proteomics data revealed that TOR inhibition elicits speciﬁc changes in a core set of proteins (Figure 4C). How TOR inhibition triggers those speciﬁcs change, is a remaining question. Since it was shown in mammals that rapamycin treatment inhibits TOR kinase activity (Jacinto et al., 2004), we take advantages of phosphoproteomics methods to decipher which signaling and regulatory mechanism could be controlled directly or indirectly by TOR kinase. Phosphoproteome Proﬁling of Rapamycin vs. Control-Treated Chlamydomonas Samples Proteomics, metabolomics and transcriptomics data show the central role of TOR signaling in the regulation of November 2018 \| Volume 9 \| Article 1590 Frontiers in Plant Science \| www.frontiersin.org 9 Rapamycin Induced Chlamydomonas Phosphoproteome Changes Roustan and Weckwerth FIGURE 6 \| Graphic display of proteins signiﬁcantly regulated by the rapamycin and overrepresented phosphorylation motif. (A) Venn diagram with the two-way ANOVA results (B) Motif enrichment analysis was done with Motif-x software with settings: >20 occurrences, signiﬁcance threshold was set to 0.000001 and background proteome was Chlamydomonas UniProt database centered on Serine residues. (C) Relative abundance of TOR pathway-related phosphopeptides in control and rapamycin-treated cells sampled at 0, 24, and 48 h. Values represent means of three biological replicates and standard deviation. FIGURE 6 \| Graphic display of proteins signiﬁcantly regulated by the rapamycin and overrepresented phosphorylation motif. (A) Venn diagram with the two-way ANOVA results (B) Motif enrichment analysis was done with Motif-x software with settings: >20 occurrences, signiﬁcance threshold was set to 0.000001 and background proteome was Chlamydomonas UniProt database centered on Serine residues. (C) Relative abundance of TOR pathway-related phosphopeptides in control and rapamycin-treated cells sampled at 0, 24, and 48 h. Values represent means of three biological replicates and standard deviation. et al., 2015). Subsequently, RNA processing related proteins such as A8HYQ9, an RNA binding protein involved in photoperiodism and ﬂowering in Arabidopsis, showed about 20% de-phosphorylation, at its phosphosite Thr 246, during TOR inhibition (Table 2) (Kim et al., 2016). Finally, several proteins related to cell signaling like a protein phosphatase 2C family protein (A8J7H2) and a calmodulin-like domain protein kinase (A8HSJ3) showed decreased phosphorylation level (Table 2). down to 20% in rapamycin-treated cells compared to control samples (Figure 6C). As well, a homolog of ABI1 (A8J0N4) Protein phosphatase 2C-like which regulates the activation of the Snf1-related kinase OST1 via abscisic acid presented signiﬁcantly increased phosphorylation level on Ser 532 (20% higher than control at 48 h) (Sridharamurthy et al., 2014). Lastly, an ortholog of human AUTOPHAGY 7 (A8IFG0), an activator of AUTOPHAGY 8, was found to be rapidly dephosphorylated at Tyr 40, with a phosphorylation level oscillating between 6 and 40% of the control samples. Interestingly, the Tyr 40 phosphosite is conserved between plants and mammals, however, its function is still unknown (Tanida et al., 2001). Further investigation was extended to TOR related proteins. Frontiers in Plant Science \| www.frontiersin.org Phosphoproteome Proﬁling of Rapamycin vs. Control-Treated Chlamydomonas Samples For instance, A8JDQ7 encodes a protein-serine kinase, homolog to AtS6k that phosphorylates ribosomal protein in vitro. S6K is involved in translational up-regulation of ribosomal proteins (Mahfouz et al., 2006; Tzeng et al., 2009; Son et al., 2016). Chlamydomonas S6K homolog protein presents a phosphorylation at Thr 23 which signiﬁcantly decreased Finally, to identify putative signaling pathways involved in TOR inhibition acclimation, we searched for over- represented sequence motifs in the 191 signiﬁcantly November 2018 \| Volume 9 \| Article 1590 Frontiers in Plant Science \| www.frontiersin.org 10 Rapamycin Induced Chlamydomonas Phosphoproteome Changes Roustan and Weckwerth TABLE 2 \| Summary of the 60 phosphosites identiﬁed as strictly regulated by rapamycin treatment. Phosphoproteome Proﬁling of Rapamycin vs. Control-Treated Chlamydomonas Samples Unique ID Control Rapamycin N: -Log Two-way ANOVA p value treatment Phosphosite Position Mercator annotation A8ITH3 1.56796 S 155 Cell.organization.cytoskeleton.actin.actin depolymerizing facto A8IPC9 1.46344 T 11 Co-factor and vitamine metabolism.riboﬂavin.riboﬂavin syntha A8IPC9 1.93558 T 3 Co-factor and vitamine metabolism.riboﬂavin.riboﬂavin syntha A8IZM6 1.67179 T 544 Misc.oxidases - copper, ﬂavone etc A8JE97 1.56225 S 410 Not assigned.unknown A8IFQ9 1.34204 T 435 Not assigned.unknown A8ILG0 1.3164 T 114 Not assigned.unknown A8IEN5 1.53897 S 263 Not assigned.unknown A8J8I8 1.46263 S 56 Not assigned.unknown A8J225 1.93544 S 20 Not assigned.unknown A8J526 1.79737 T 1344 Not assigned.unknown A8IGD9 1.30841 S 109 Not assigned.unknown A8IUB5 1.30183 S 5 Not assigned.unknown A8HPR1 3.18591 S 742 Not assigned.unknown A8J225 1.52739 S 22 Not assigned.unknown A8HQA6 2.9762 T 98 Not assigned.unknown A8I3H9 1.78541 S 85 Not assigned.unknown A8J7H2 1.58417 S 1127 Not assigned.unknown A8JBY2 1.6125 S 253 Not assigned.unknown A8JHN0 1.49047 S 46 Not assigned.unknown A8IZM8 2.27356 S 248 Not assigned.unknown A8IW11 2.38926 S 848 Not assigned.unknown A8J526 1.49352 S 1134 Not assigned.unknown A8J9K6 1.54472 S 86 Not assigned.unknown A8JBY2 1.628 S 243 Not assigned.unknown A8IYS5 2.46104 S 76 Not assigned.unknown A8J0B1 1.62396 S 204 Not assigned.unknown A8J7H2 1.53772 T 848 Not assigned.unknown A8IA11 1.56081 S 2 Not assigned.unknown A8IUQ2 1.75414 T 317 Not assigned.unknown A8JHV8 2.45841 S 217 Not assigned.unknown A8J225 2.6345 S 577 Not assigned.unknown A8IQS4 1.43096 S 590 Not assigned.unknown A8JI72 2.19246 T 16 Not assigned.unknown A8J431 1.30279 T 33 Not assigned.unknown A8JI39 2.0363 S 25 Not assigned.unknown A8IFZ1 2.72904 T 764 Not assigned.unknown A8IPC1 2.47052 Y 1181 Not assigned.unknown A8J225 1.57916 S 92 Not assigned.unknown A8JEZ5 2.02337 S 819 Not assigned.unknown A8J9K6 2.70466 S 211 Not assigned.unknown A8HNT4 1.96608 S 122 Not assigned.unknown A8JDP7 1.5779 S 101 Not assigned.unknown A8IYQ7 1.65023 T 101 Not assigned.unknown A8HNT4 2.44366 S 120 Not assigned.unknown A8IVD5 1.81326 T 336 Not assigned.unknown A8HPX0 1.48213 S 53 Not assigned.unknown A8HNI1 2.00991 S 38 Protein.degradation.ubiquitin.E3.RING A8J8X3 1.32031 S 244 Protein.postranslational modiﬁcation (Continue TABLE 2 \| Summary of the 60 phosphosites identiﬁed as strictly regulated by rapamycin treatment. Phosphoproteome Proﬁling of Rapamycin vs. Control-Treated Chlamydomonas Samples Unique ID Control Rapamycin N: -Log Two-way ANOVA p value treatment Phosphosite Position M A8ITH3 1.56796 S 155 C A8IPC9 1.46344 T 11 C A8IPC9 1.93558 T 3 C A8IZM6 1.67179 T 544 M A8JE97 1.56225 S 410 N A8IFQ9 1.34204 T 435 N A8ILG0 1.3164 T 114 N A8IEN5 1.53897 S 263 N A8J8I8 1.46263 S 56 N A8J225 1.93544 S 20 N A8J526 1.79737 T 1344 N A8IGD9 1.30841 S 109 N A8IUB5 1.30183 S 5 N A8HPR1 3.18591 S 742 N A8J225 1.52739 S 22 N A8HQA6 2.9762 T 98 N A8I3H9 1.78541 S 85 N A8J7H2 1.58417 S 1127 N A8JBY2 1.6125 S 253 N A8JHN0 1.49047 S 46 N A8IZM8 2.27356 S 248 N A8IW11 2.38926 S 848 N A8J526 1.49352 S 1134 N A8J9K6 1.54472 S 86 N A8JBY2 1.628 S 243 N A8IYS5 2.46104 S 76 N A8J0B1 1.62396 S 204 N A8J7H2 1.53772 T 848 N A8IA11 1.56081 S 2 N A8IUQ2 1.75414 T 317 N A8JHV8 2.45841 S 217 N A8J225 2.6345 S 577 N A8IQS4 1.43096 S 590 N A8JI72 2.19246 T 16 N A8J431 1.30279 T 33 N A8JI39 2.0363 S 25 N A8IFZ1 2.72904 T 764 N A8IPC1 2.47052 Y 1181 N A8J225 1.57916 S 92 N A8JEZ5 2.02337 S 819 N A8J9K6 2.70466 S 211 N A8HNT4 1.96608 S 122 N A8JDP7 1.5779 S 101 N A8IYQ7 1.65023 T 101 N A8HNT4 2.44366 S 120 N A8IVD5 1.81326 T 336 N A8HPX0 1.48213 S 53 N A8HNI1 2.00991 S 38 P A8J8X3 1.32031 S 244 P TABLE 2 \| Summary of the 60 phosphosites identiﬁed as strictly regulated by rapamycin treatment. (Continued) November 2018 \| Volume 9 \| Article 1590 11 Frontiers in Plant Science \| www.frontiersin.org Rapamycin Induced Chlamydomonas Phosphoproteome Changes Roustan and Weckwerth TABLE 2 \| Continued Unique ID Control Rapamycin N: -Log Two-way ANOVA p value treatment Phosphosite Position Mercator annotation Q6SA05 1.77006 S 53 PS.calvin cycle.rubisco interacting Q6SA05 1.81124 S 45 PS.calvin cycle.rubisco interacting Q00914 1.41437 S 71 PS.lightreaction.photosystem I.PSI polypeptide subunits A8J6D1 2.13894 S 103 PS.lightreaction.photosystem II.LHC-II Q93WL4 2.29714 T 24 PS.lightreaction.photosystem II.LHC-II A8J6D1 1.43077 T 27 PS.lightreaction.photosystem II.LHC-II A8HYQ9 2.20105 T 246 RNA.processing.splicing A8HQ72 1.68548 S 329 RNA.processing.splicing A8HSJ3 1.30175 S 448 Signaling.calcium A8HQY2 2.00138 S 106 Transport.ABC transporters and multidrug resistance systems Q93Z22 2.0816 S 1041 Transport.p- and v-ATPases processes at the protein level involved in TOR inhibition acclimation. Phosphoproteome Proﬁling of Rapamycin vs. Control-Treated Chlamydomonas Samples To gain further understanding, physiological parameter such as growth rate, photosynthesis, protein content and carbon storage (starch and lipids) were measured along the proteome and phosphoproteome. From all detected proteins we quantify 916 protein and 1312 phosphopeptides across all time point. We used multivariate statistics to investigate the dynamic acclimation of both proteome and phosphoproteome to rapamycin treatment. Both a strong time eﬀect as well as a signiﬁcant eﬀect of rapamycin treatment were observed in the data (Figure PCA and two-way ANOVA). These results are summarized in a proposed model of Chlamydomonas acclimation to rapamycin treatment as shown in Figure 7. changed phosphopeptides, from the treatment factor, by performing a motif-x analysis1. One motif was signiﬁcantly enriched and present in 60 phosphopeptides, representing 42 proteins (Figure 6B). RSXS motif presents a 9.33- fold increase (Figure 6B) and has a serine at position 0, a basic arginine at position −3 followed by a serine at position −2. This motif is known to be targeted by 14-3-3 proteins (Johnson et al., 2010). At the proteome, both 14-3-3 proteins were found to be inhibited by rapamycin treatment (Table 1). 1http://motif-x.med.harvard.edu/motif-x.html DISCUSSION In human, yeast, and Arabidopsis, TOR kinase forms a heterotrimeric complex with LST8 and RAPTOR which plays a central role in energy sensing and signaling. Indeed, upon suﬃcient level of energy and nutrients the TOR pathway stimulates cell growth through translation and transcription stimulation while it is inhibiting autophagy process (Roustan et al., 2016). In Arabidopsis, data strongly support a speciﬁc acclimation of TOR and its counterpart AMPK signaling pathway (Nukarinen et al., 2016). It was previously shown that Chlamydomonas cells treated with rapamycin present growth inhibition, which was abolished in a fkbp12 mutant line (Crespo et al., 2005). As well, several phylogenetic studies have identiﬁed orthologs of TOR and AMPK pathways, in Chlamydomonas (Roustan et al., 2016). However, a similar loss of genes, as for Arabidopsis, point toward a speciﬁc regulation of the energy sensing and signaling in plant kingdom (Roustan et al., 2016). Unlike Arabidopsis, fewer investigations have been conducted in Chlamydomonas to unravel TOR function and none of them have performed an in vivo label-free phosphoproteomics analysis. In this context, our work aimed to compare the proteome and the phosphoproteome of Chlamydomonas cells treated with rapamycin or drug vehicle (control) at 0, 24, and 48 h in continuous light to identify metabolic Rapamycin Treatment Concomitantly Inhibits Cell Growth and Induced Changes in Cell Cycle Related Proteins Here, we measured the phenotypic acclimation of Chlamydomonas in response to 500 nM of rapamycin. We found that this rapamycin concentration was suﬃcient to inhibit cell growth, but to a lesser extent that it was previously reported (Crespo et al., 2005). According to this observation, recent studies on Chlamydomonas acclimation to rapamycin, have used a higher concentration of rapamycin (Juppner et al., 2017). Consequently, most of the signiﬁcant changes occurring at the physiological level were observed at 48 h after the treatment, even if a clear trend could be observed in earlier time points. As already reported we did not found a total inhibition of the cell growth during rapamycin treatment at both cell number and fresh weight level. Previously, cell- cycle of Chlamydomonas cells treated with rapamycin was found to present a delay compared to the control (Juppner et al., 2017). Those results indicated that TOR could also be involved in cell cycle regulation. Coincidently, CRB3, involved in the coordination of the cell cycle with the circadian rhythm was downregulated in our data (Niwa et al., 2013). Similarly, other proteins involved in regulation of ﬂowering time loci or in cytoskeletons organization presented de-phosphorylation November 2018 \| Volume 9 \| Article 1590 Frontiers in Plant Science \| www.frontiersin.org 12 Rapamycin Induced Chlamydomonas Phosphoproteome Changes Roustan and Weckwerth FIGURE 7 \| Model for regulation of rapamycin treatment response. Intrestingly, our data suggest that chloroplastic function are not inhibited but rather adapted. Especially, plastid localized, Calvin cycle, sulfur, cystein and methionine related proteins are down regulated. Instead, starch accumulate and purine metabolsime is stimulated. Those observation are line with previous reports indicating a connection of sulfur metabolism with TOR signaling (Dong et al., 2017). Together with our esults, recent metabolic data suggest also a metabolic uncoupling between the plastid and mitochondria, associate with a decrease of mitochondrial metabolic activity (Juppner et al., 2017). In line with plastid maintenance, ribosomes didn’t present clear regulation pattern, instead EIF factors were down regulated. Reduced cytosolic translation activity is in line with S6K phosphorylation previously shown to be responsible for translation regulation (Dobrenel et al., 2016b). This reduced cytosolic translation activity could explain amino acid accumulation previously detected (Juppner et al., 2017). Further, protein associated to circadian cycle and owering time regulation were together decreasing in protein abundance or phosphorylation level. Rapamycin Treatment Signiﬁcantly Changes Phosphoprotein and Protein Dynamics of Evolutionary Conserved TOR – Targets Among the proteins and phosphosites measured in our approach, we could ﬁnd proteins related to TOR signaling. For instance, the evolutionary conserved S6K protein was identiﬁed in our phosphoproteomics screen. Together with TOR, S6K is known to be involved in the regulation of protein translation. In line with this hypothesis we found a signiﬁcant decline of the Thr 23 phosphosite of the S6K which is a TOR target (Figure 7). There is, however, no conservation of this particular phosphosite found between mammals, yeast and plants. y p Other proteins potentially involved in TOR downstream translational control and targets of S6K are RPS6 proteins (Nukarinen et al., 2016). While we did not detect phosphorylation levels of RPS6 protein, our proteomic data showed that RPS6 protein level are stable over the treatment. These results are in line with a recent investigation of the early response of the Chlamydomonas phosphoproteome to rapamycin treatment (Werth et al., 2018). More precisely, this study investigated the RPS6 phosphorylation at the conserved Ser242 site but could not detect any change in the phosphorylation level (Werth et al., 2018). This suggests that S6K protein is targeting other RPS6 phosphorylation sites or other ribosomal proteins in Chlamydomonas or functions via diﬀerent mechanisms At the proteome level, rapamycin treatment seems to aﬀect eukaryotic initiation factors rather than cytosolic ribosome proportion (Supplementary Table S1). Similarly, eukaryotic initiation factors were found to be aﬀected by nitrogen availability (Valledor et al., 2014; Roustan et al., 2017). Also metabolic enzymes related to starch metabolism showed a complex pattern. A8J6C3, a Phosphoglucan water dikinase (PWD1) involved in starch degradation showed decreased levels in rapamycin treated cells compared to control samples. As for lipids starch might be also a storage compound balancing the over ﬂow of reduced carbon under stress conditions. While proteins of the photosystems PSII and PSI are stable over treatment (Supplementary Table S1), we also observed that Calvin cycle-related proteins were found to be downregulated during the rapamycin treatment (Figure 7). Such observation could indicate that chloroplast to nucleus retrograde signaling is adjusting the import of plastid related proteins in order to reduce the energy impute and avoid a ROS burst. In line with this hypothesis, a Protein disulﬁde isomerase (A8JBH7) and a Glutathione peroxidase (O22448) showed increased levels under rapamycin treatment compared to control samples. Rapamycin Treatment Concomitantly Inhibits Cell Growth and Induced Changes in Cell Cycle Related Proteins Finally, phosphorylation of ATG7 is in line with previously detected autophagy induction and cell vacuolarization (Crespo et al., 2005; Couso et al., 2017). For example, the red- arrow between TOR and SK6 indicate that TOR complex directly and positively regulates RPS6 by phosphorylation. PS is photosystem; LHCII is Light-Harvesting complex-II; e- represents electron from the electro ransfer chain; TAG stands for tri-acy-glycerol; Cys for cysteine; Met for methionine; for protein accession number and name refer to results part. FIGURE 7 \| Model for regulation of rapamycin treatment response. Intrestingly, our data suggest that chloroplastic function are not inhibited but rather adapted. Especially, plastid localized, Calvin cycle, sulfur, cystein and methionine related proteins are down regulated. Instead, starch accumulate and purine metabolsime is stimulated. Those observation are line with previous reports indicating a connection of sulfur metabolism with TOR signaling (Dong et al., 2017). Together with our results, recent metabolic data suggest also a metabolic uncoupling between the plastid and mitochondria, associate with a decrease of mitochondrial metabolic activity (Juppner et al., 2017). In line with plastid maintenance, ribosomes didn’t present clear regulation pattern, instead EIF factors were down regulated. Reduced cytosolic translation activity is in line with S6K phosphorylation previously shown to be responsible for translation regulation (Dobrenel et al., 2016b). This reduced cytosolic translation activity could explain amino acid accumulation previously detected (Juppner et al., 2017). Further, protein associated to circadian cycle and ﬂowering time regulation were together decreasing in protein abundance or phosphorylation level. Finally, phosphorylation of ATG7 is in line with previously detected autophagy induction and cell vacuolarization (Crespo et al., 2005; Couso et al., 2017). For example, the red- arrow between TOR and SK6 indicate that TOR complex directly and positively regulates RPS6 by phosphorylation. PS is photosystem; LHCII is Light-Harvesting complex-II; e- represents electron from the electron transfer chain; TAG stands for tri-acy-glycerol; Cys for cysteine; Met for methionine; for protein accession number and name refer to results part. November 2018 \| Volume 9 \| Article 1590 Frontiers in Plant Science \| www.frontiersin.org 13 Rapamycin Induced Chlamydomonas Phosphoproteome Changes Roustan and Weckwerth publications (Imamura et al., 2015, 2016; Couso et al., 2016; Mukaida et al., 2016; Juppner et al., 2017). How TAG accumulates during rapamycin treatment is of importance since TAG molecules are of a major interest for general lipid and biodiesel production (Merchant et al., 2012; Liu and Benning, 2013; Furuhashi et al., 2016). Analysis of Proteins Related to Carbohydrate and Lipid Storage Pool Metabolism Suggest That Starch and TAG Accumulation Is Related to Carbon Flux Re-rooting Rather Than de novo Biosynthesis According to our and others observations rapamycin treatment in Chlamydomonas aﬀects protein translation via TOR inhibition. Since translation accounts for more than 60% of energy consumption, it is necessary to re-route energy toward storage compound synthesis (see Figure 1). In our study, decreasing protein content coincides with the increase of starch by approximatively 50%, while the lipid content stays stable over the time. However it appears that the lipid composition is changing. Investigation of the lipid composition by thin- layer-chromatography revealed a higher TAG amount during rapamycin treatment (Supplementary Figure S1). Lipid, starch and protein content are therefore in line with previous Rapamycin Treatment Signiﬁcantly Changes Phosphoprotein and Protein Dynamics of Evolutionary Conserved TOR – Targets Altogether, those results correlate with the recent observation made in tor Arabidopsis mutant which showed a strongly aﬀected photosynthesis (Dong et al., 2015). Analysis of Proteins Related to Carbohydrate and Lipid Storage Pool Metabolism Suggest That Starch and TAG Accumulation Is Related to Carbon Flux Re-rooting Rather Than de novo Biosynthesis Rapamycin Treatment Concomitantly Inhibits Cell Growth and Induced Changes in Cell Cycle Related Proteins At the proteome level we observed a complex dynamic pattern. Among the signiﬁcantly aﬀected proteins, three are part of the acetyl coenzyme A carboxylase complex involved in the lipid biosynthesis. Those proteins are A8JDA7, a Biotin Carboxyl Carrier Protein (BCC1), A8JGF4, a Biotin Carboxylase and A8HXT4 a Pyruvate carboxylase (PYC1). As well two other chloroplastic proteins, A8JEF7 3- ketoacyl-CoA-synthase (KAS1) and A8IQB8, a Plastid acyl-ACP desaturase (FAB2), involved in lipid elongation and desaturation. Whereas the proteins of fatty acid synthesis showed a slight but signiﬁcantly decreased level FAB2 had increased levels (Supplementary Tabel S1). FAB2 has been shown to be involved in the alteration of the chloroplast membrane in response to copper deﬁciency increasing the level of desaturation (Castruita et al., 2011). Altogether the data indicate that TAG accumulation is rather linked to a carbon pool reallocation than to de novo synthesis. While recent studies have highlighted the central role of ER in TAG synthesis (Li-Beisson et al., 2013; Couso et al., 2017; Kim et al., 2018), those identiﬁed proteins in our study are predicted to be localized in the chloroplast or in the cytosol (Supplementary Table S1). (Figure 7). Altogether our data suggest that TOR kinase coordinate the progression of the cell cycle with nutrient availability and protein translation (Dong et al., 2017; Werth et al., 2018). Cellular and Organellar Acclimation in Response to TOR Inhibition Transcriptomic data investigating the TOR role for transcriptome regulation and its coordination with the metabolome were previously published (Kleessen et al., 2015). Those studies have indicated that TOR inhibition up- regulated genes involved in tetrapyrrole synthesis, vacuolar function, amino acids metabolism and transport as well as folding and chaperonin related genes. On the other hand, genes involved in nucleotide metabolism, cell cycle and DNA November 2018 \| Volume 9 \| Article 1590 Frontiers in Plant Science \| www.frontiersin.org 14 Rapamycin Induced Chlamydomonas Phosphoproteome Changes Roustan and Weckwerth replication and repair, were down-regulated (Ramundo et al., 2014; Kleessen et al., 2015). In line with those studies, we have found similar changes in the proportion of proteins related to those functional groups. For instance, we have found increased amounts of tetrapyrrole related proteins, being in line with a slight increase of chlorophyll a/b ratio content during rapamycin treatment (Supplementary Figure S1B). It would be interesting to investigate the hypothesis that in absence of translation a part of the nitrogen pool is re-routed partially toward chlorophyll biosynthesis. Further, most of Calvin cycle-related proteins were found to be downregulated during the rapamycin treatment (Figure 7) indicating a potential adjustment of retrograde signaling in order to reduce the energy impute but also maintain nitrogen availability. Accordingly, we have observed a clear distinction between chloroplast acclimation during nitrogen depletion in our recent study (Roustan et al., 2017) and rapamycin treatment (this study), especially with the focus on chlorophyll content. Under nitrogen starvation there is a signiﬁcant drop of chlorophyll in the ﬁrst 48 h in contrast to the rapamycin treatment (Figure 1 in Roustan et al., 2017, and Supplementary Figure S1 this study). This indicates very diﬀerent acclimation strategies of the chloroplast organelle to TOR inhibition either induced by rapamycin or nitrogen depletion. Future studies will address this question in more detail. upregulated than pyrimidine related ones (Juppner et al., 2017). Both, purine and sulfur metabolism are interconnected since the AKN protein is also involved in the purine metabolism (Herrmann et al., 2015). Concerning the nucleotide metabolism, we observed an increase of enzymes involved in the synthesis of de novo purine biosynthesis such as A8J3Y6 (PUR4 a formylglycinamidine ribonucleotide synthase), A8I6R4 (PUR5 a phosphoribosylformylglycinamidine cyclo-ligase), A8JBQ5 (PUR7 a SAICAR synthetase) A8IJJ8 (AIR carboxylase) A8IVF0 (AICAR transformylase). Cellular and Organellar Acclimation in Response to TOR Inhibition Additionally, we could conﬁrm the downregulation of pyrimidine metabolism represented by two carbamoyl phosphate synthetases (A8IMN5, A8JIR0) and a translation factor (A8HTK7) (Figure 7). Altogether, data suggest a strong interplay between TOR signaling, sulfur metabolism and nucleotide metabolism, especially de novo purine biosynthesis. Interestingly, all those signaling pathways are located in the chloroplast. Remarkably, it was previously observed in Chlamydomonas that cysteine and methionine metabolites were strongly aﬀected in line with transcriptomics data (Kleessen et al., 2015). Our results were concordant also with the observed downregulation of sulfur assimilation enzymes. In Arabidopsis tor mutants, sulfur-related metabolism was shown to be impaired (Ren et al., 2012; Caldana et al., 2013). More recently, it was shown in Arabidopsis, that sulfur availability regulates plant growth via the glucose-TOR signaling pathway (Xiong et al., 2013; Dong et al., 2017). Further, it was previously observed in animals and yeast that cell lines treated with AICAR metabolite presented enhanced AMPK activity (Corton et al., 1995; Sakamoto et al., 2004). It would be of interest, to test whether AICAR could also stimulate Chlamydomonas AMPK pathway. Rapamycin Treatment Induces Changes in Sulfur and Nucleotide Metabolism Several proteins related to amino acid metabolism were found as diﬀerentially regulated during the rapamycin treatment. Among them, proteins related to serine, cysteine and methionine metabolism as well as to sulfur metabolism attracted our attention. Indeed, those three amino acid biosynthetic pathways are located in the chloroplast and are interconnected with sulfur metabolism. Sulfur metabolism itself was recently related to TOR signaling (Dong et al., 2017). At the sulfur metabolic level, an adenosine-5-phosphosulfate-kinase (A8J3Q6 - AKN) was found to be 2-fold upregulated during the rapamycin treatment while an adenosine 5-phosphosulfate reductase (A8J6A7) was found to be downregulated. Further, A8IH03, a phosphoserine aminotransferase which is involved in serine biosynthesis in the chloroplast and operates via the phosphorylated pathway presents higher abundance in the rapamycin-treated cell than in the control. Serine is then metabolized into O-acetyl serine (OAS) which is a limiting precursor for sulfur assimilation. The enzyme involved in the interconversion of OAS to cysteine, the O-acetylserine (Thiol)-lyase (A8ISB0 - OAS-TL B) was stable during rapamycin treatment while it increased in control cells. Concomitantly, we observed that proteins related to methionine biosynthesis such as an S-adenosyl-l-homocysteine hydrolase 2 (SAHH2) (A8IXE0), a plastidic methionine synthase (A8JH37) as well as a methionine adenosyltransferase (A8HYU5) were down-regulated during the rapamycin treatment (Figure 7). Recently, a metabolic study has observed that increased levels of cysteine displayed signiﬁcantly lower fold upshifts compared to serine (Juppner et al., 2017). Additionally, in the same study authors have found that purine related metabolites are more Finally, data suggest that mitochondrial metabolism is responsive to TOR inhibition. Indeed, we observed an inhibition of carbonic anhydrases (A8IT01, A8J4Z8, and A8JFK6), TCA cycle-related enzymes (A8HMQ1, A8JHU0, A8J1V5, and A8IWJ8), and proteins involved in mitochondria electron transfer and ATP synthase (A8IVJ7 and A8IQU3). It is known that TCA cycle is at the crossroad between amino acid metabolism and energy production in mitochondria and is fueled in part by sugar and amino acid metabolism. For instance, carbonic anhydrases are low-CO2 responding protein and their downregulation is in line with changes observed at the chloroplast level. Together this suggests that sudden TOR inhibition triggers an over-accumulation of carbon and energy, which is reﬂected by downregulation of mitochondrial metabolism and TCA cycle (Figure 7) (Ramanathan and Schreiber, 2009). Frontiers in Plant Science \| www.frontiersin.org Is TOR Involved in Endomembrane System Regulation? Interestingly, several proteins involved in endomembrane system traﬃcking or function were found. Regarding rapamycin treatment, several studies have linked translation and lipid metabolism with ER, vacuole, and autophagy (Perez-Perez et al., 2017). In Chlamydomonas, a vacuolarization process was observed during rapamycin treatment, in line with an increase of autophagy process and ATG8 accumulation (Crespo et al., 2005; November 2018 \| Volume 9 \| Article 1590 Frontiers in Plant Science \| www.frontiersin.org 15 Rapamycin Induced Chlamydomonas Phosphoproteome Changes Roustan and Weckwerth Couso et al., 2017). In our dataset, vacuolar ATPase subunits were decreasing over the rapamycin treatment. Activation of autophagy is driven by dephosphorylation of the phosphosite Y40 of AUTOPHAGY 7, an activator of AUTOPHAGY 8, by about 40% compared to control samples. Mechanistically, it is known that TOR is inhibiting autophagy via protein phosphorylation of ATG1 in human (Kim et al., 2011). Additionally, the vacuole is known to be a central player of protein turn-over (Kim et al., 2001). In a recently published metabolomics dataset, the rapid accumulation of most amino acids were found to occur in the ﬁrst 30 min (Juppner et al., 2017). In this context, we observed that most of the enzymes related to amino-acid metabolism were down-regulated. Altogether these data point toward a role of TOR in the protein turn-over regulation. Lastly, several proteins localized and/or related to endoplasmatic reticulum metabolism were also downregulated during a rapamycin treatment. For example, 14-3-3 proteins and the CDPK 2 protein were already found to be located to the ER (Voigt et al., 2004). Further, PDIL protein was in contrast up-regulated as other Redox related enzymes (Supplementary Table S1), suggesting a redox unbalance. inhibition is aﬀecting the nucleotide pool as found previously (Figure 7) (Juppner et al., 2017). In turn, it seems that the subcellular chloroplast-mitochondria synergy is uncoupled to avoid an overproduction of energy compared to a slow down of translational and growth processes (Figure 7). In line with such organelle acclimation, we found an accumulation of proteins related to the endomembrane traﬃcking system such as ESCRT- III related proteins. These data and the comparison with nitrogen starvation and recovery studies on the phosphoproteome, proteome and metabolome (Valledor et al., 2014; Roustan et al., 2017) force new research questions especially about organellar acclimation und subcellular compartmentation of metabolic changes, control of chloroplast-mitochondrium interaction and chloroplast nucleus retrograde signaling processes. Is TOR Involved in Endomembrane System Regulation? In future we will address these questions with non-aqueous subcellular fractionation combined with proteomics and metabolomics (incl. lipidomics, Furuhashi et al., 2016) as well as metabolic network modeling (Nägele and Weckwerth, 2013, 2014; Nägele et al., 2014, 2016; Fürtauer et al., 2016; Nukarinen et al., 2016). Physiological Measurements y g Growth parameter (cell number and fresh weight), starch, lipid and photosynthetic parameters (Fv/Fm, total chlorophyll content and chlorophyll a/b ratio) were measured as previously described (Valledor et al., 2014; Roustan et al., 2017). Total protein content was determined with Bradford assay using BSA as a protein standard. Tri-acyl-glycerol content was monitored by thin-layer- chromatography as described. Shortly 5 mg fresh weight were load and separated on silica gel 60 (EMD Chemicals) using hexane-diethyl ether-acetic acid (90:7.5:1 [v/v/v]) and visualized under UV light after spraying with primuline (Sigma). (1 mg in 100 ml of acetone/water, 80/20, v/v) (Li et al., 2008). MATERIALS AND METHODS In line with previously unraveled endomembrane dynamics under rapamycin treatment, we found here several proteins involved in the Endosomal Sorting Complex Required for Transport (ESCRT) machinery (VPS4, VPS60, and VPS46) to be signiﬁcantly upregulated by the rapamycin treatment. It would be of a major interest to further investigate the endomembrane system as well as the Endosomal Sorting Complex Required for Transport (ESCRT) in Chlamydomonas. Indeed, biochemical fractionation of Chlamydomonas cells suggests that TOR and LST8 are co-localized with membranous sites (Díaz-Troya et al., 2008a). Plants Material and Growth Conditions Chlamydomonas reinhardtii CC-503 cw92, mt+, agg1+, nit1, nit2 cultures were grown in HEPES-Acetate-Phosphate medium supplemented with 7 mM NH4Cl (HAP; TAP medium in which Tris was replaced by 5 mM HEPES) at 25◦C with shaking (120 rpm) in a 16:8 light:dark photoperiod (85 µmol m−2 s−1; Sylvania GroLux lamps). To start the experiments, cultures were pelleted down by centrifugation at the end of the night, washed two times with fresh HAP medium and re-suspended in HAP media treated with 500 nM rapamycin or with the drug vehicle to a ﬁnal density of 1–3 × 106 cells mL−1. From this point, cells were cultivated under continuous light. Cells were sampled at 0, 12, 24, and 48 h time points after transfer in the fresh medium supplemented or not with rapamycin for physiological parameter measurement. For proteomics and phosphoproteomics, cells were harvested at 0, 24, and 48 h. CONCLUSION Energy is at the heart of life and is the most important evolutionary driver. In order to continuously adjust the cell activity to the available energy, the TOR and AMPK pathways are regulating catabolic and anabolic processes. How plants TOR and AMPK pathways have speciﬁcally evolved to integrate plastid endosymbiosis is a remaining question (Roustan et al., 2016). Both TOR and AMPK are protein kinases. Therefore our main interest has been to investigate the dynamics of in vivo protein phosphorylation in response to TOR control. To address this question we have analyzed rapamycin treated Chlamydomonas reinhardtii cells with in vivo label-free shotgun phosphoproteomics in order to understand how single-cell green algae acclimate to TOR inhibition under continuous light. Our analysis points toward a central role of TOR in the control of the transcription and translation and therefore directly aﬀecting the carbon ﬂux and energy ﬂux (Figure 7). Indeed, the analysis of transcriptomic, proteomic and metabolic datasets indicate a negative correlation between the accumulation of amino acids and the down-regulation of the associated biosynthetic enzymes. As well, diﬀerential regulation between pyrimidine and purine metabolism were found suggesting that TOR Frontiers in Plant Science \| www.frontiersin.org For Phosphoproteomics Samples were dissolved in 11 µL of 2% ACN and 0.1% FA and 5 µL of the mixture was separated on an EASY-Spray PepMap RSLC 75 µm × 50 cm column (Thermo Fisher Scientiﬁc Inc., Waltham, MA, United States). Peptides were eluted using a 240 min linear gradient from 2 to 40% of mobile phase B (mobile phase A: 0.1% [v/v] formic acid (FA) in water; mobile phase B: 0.1% [v/v] FA in 90% [v/v] ACN) with 300 nL/min ﬂow rate generated with an UltiMate 3000 RSLCnano system. Peptides were measured with an LTQ-Orbitrap Elite (Thermo) using the following mass analyzer settings: ion transfer capillary temperature 275◦C, full scan range 350–1800 m/z, FTMS resolution 120000. Each FTMS full scan was followed by up to ten data dependent (DDA) CID tandem mass spectra (MS/MS spectra) in the linear triple quadrupole (LTQ) mass analyzer. Dynamic exclusion was enabled using list size 500 m/z values with exclusion width ± 10 ppm for 60 s. Charge state screening was enabled and unassigned and +1 charged ions were excluded from MS/MS acquisitions. For injection control automatic gain control (AGC) for full scan acquisition in the Orbitrap was set to 5 × 105 ion population, the maximum injection time (max IT) was set to 200 ms. Orbitrap online calibration using internal lock mass calibration on m/z 371.10123 from polydimethylcyclosiloxane was used. Multistage activation was enabled with neural losses of 24.49, 32.66, 48.999, 97.97, 195.94, and 293.91 Da for the 10 most intense precursor ions. Prediction of ion injection time was enabled and the trap was set to gather 5 × 103 ions for up to 50 ms. For Proteomics Samples were dissolved in 2% ACN and 0.1% FA to a ﬁnal concentration of 0.2 µg equivalent total protein per µL. 1 µg equivalent total protein was loaded into a one-dimensional (1D) nanoﬂow LC-MS/MS system equipped with a precolumn (Eksigent, Germany). Peptides were eluted using an Ascentis column (Ascentis Express, peptide ES-C18 HPLC column (SUPELCO Analytical, Bellefonte, PA, United States), dimension 15 cm × 100 µm, pore size 2.7 µm) during a 120 min gradient from 5% to 50% (v/v) acetonitrile, 0.1% (v/v) formic acid. MS analysis was performed on an Orbitrap LTQ XL mass spectrometer (Thermo, Germany) with a controlled ﬂow rate of 500 nL per minute. Speciﬁc tune settings for the MS were as follows: spray voltage was set to 1.8 kV; temperature of the heated transfer capillary was set to 180◦C, full scan range 350–1800 m/z, FTMS resolution 120000. Each full MS scan was followed by ten MS/MS scans, in which the ten most abundant peptide molecular ions were dynamically selected, with a dynamic exclusion window set to 60 s. Ions with a + 1 or unidentiﬁed charge state in the full MS were omitted from MS/MS analysis. For injection control automatic gain control Protein Extraction and Phosphopeptide Enrichments The protein pellets were washed twice with 0.1M ammonium acetate, one time with acetone and air dried at room temperature. The protein pellets were dissolved in 8M urea/100 mM ammonium bicarbonate (AmBic) supplemented with protease and phosphatase inhibitor cocktails as indicated by the supplier (Roche, Cat. No. 05 892 791 001 and Cat. No. 04 906 837 001). Protein concentration was determined using the Bio-Rad Bradford Assay with BSA as a standard. 200 µg (proteomic)/500 µg (phosphoproteomic) of total protein per sample was ﬁrst reduced with dithiothreitol (DTT) at a concentration of 5 mM at 37◦C for 45 min. Cysteine residues were alkylated with 10 mM iodoacetamide (IAA) in darkness at room temperature (RT) for 60 min. Alkylation was stopped by increasing DTT concentration to 10 mM and incubating the samples in the dark at RT for 15 min. Then the urea concentration was diluted to 2 M with 50 mM AmBic/10 % acetonitrile (ACN). CaCl2 was added to a ﬁnal concentration of 2 mM. Trypsin digestion (Poroszyme immobilized trypsin; 5:100 v:w) was performed at 37◦C overnight. Protein digests were desalted with C18 solid phase extraction (SPE) (Agilent Technologies, Santa Clara, CA, United States) and carbon graphite SPE as described by Furuhashi et al. (2014) and both fractions were subsequently pooled and dried before LC-MS measurement in the case of proteomics or before phosphopeptides enrichment for phosphoproteomics approach. 5 mg of TiO2 (Glygen Corp.) was used to enrich phosphopeptide as described previously (Bodenmiller et al., 2007; Chen et al., 2010) and dried in a vacuum concentrator. (AGC) for full scan acquisition in the Orbitrap was set to 5 × 105 ion population, the maximum injection time (max IT) was set to 500 ms. Orbitrap online calibration using internal lock mass calibration on m/z 371.10123 from polydimethylcyclosiloxane was used. Multistage activation was enabled with neural losses of 24.49, 32.66, 48.999, 97.97, 195.94, and 293.91 Da for the 10 most intense precursor ions. Prediction of ion injection time was enabled and the trap was set to gather 3 × 104 ions for up to 50 ms. 2http://www.maxquant.org Protein Extraction and Phosphopeptide Enrichments Total proteins from Chlamydomonas cell pellets were extracted by a phenol-phase extraction protocol as previously described November 2018 \| Volume 9 \| Article 1590 Frontiers in Plant Science \| www.frontiersin.org Frontiers in Plant Science \| www.frontiersin.org 16 Rapamycin Induced Chlamydomonas Phosphoproteome Changes Roustan and Weckwerth (Roustan et al., 2017). Protein precipitation was performed by mixing the phenol fraction with 2.5 volumes of 0.1M ammonium acetate in methanol. After a 16 h incubation period at −20◦C, the samples were centrifuged for 5 min at 5000 × g. The protein pellets were washed twice with 0.1M ammonium acetate, one time with acetone and air dried at room temperature. The protein pellets were dissolved in 8M urea/100 mM ammonium bicarbonate (AmBic) supplemented with protease and phosphatase inhibitor cocktails as indicated by the supplier (Roche, Cat. No. 05 892 791 001 and Cat. No. 04 906 837 001). Protein concentration was determined using the Bio-Rad Bradford Assay with BSA as a standard. 200 µg (proteomic)/500 µg (phosphoproteomic) of total protein per sample was ﬁrst reduced with dithiothreitol (DTT) at a concentration of 5 mM at 37◦C for 45 min. Cysteine residues were alkylated with 10 mM iodoacetamide (IAA) in darkness at room temperature (RT) for 60 min. Alkylation was stopped by increasing DTT concentration to 10 mM and incubating the samples in the dark at RT for 15 min. Then the urea concentration was diluted to 2 M with 50 mM AmBic/10 % acetonitrile (ACN). CaCl2 was added to a ﬁnal concentration of 2 mM. Trypsin digestion (Poroszyme immobilized trypsin; 5:100 v:w) was performed at 37◦C overnight. Protein digests were desalted with C18 solid phase extraction (SPE) (Agilent Technologies, Santa Clara, CA, United States) and carbon graphite SPE as described by Furuhashi et al. (2014) and both fractions were subsequently pooled and dried before LC-MS measurement in the case of proteomics or before phosphopeptides enrichment for phosphoproteomics approach. 5 mg of TiO2 (Glygen Corp.) was used to enrich phosphopeptide as described previously (Bodenmiller et al., 2007; Chen et al., 2010) and dried in a vacuum concentrator. (Roustan et al., 2017). Protein precipitation was performed by mixing the phenol fraction with 2.5 volumes of 0.1M ammonium acetate in methanol. After a 16 h incubation period at −20◦C, the samples were centrifuged for 5 min at 5000 × g. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018.01590/ full#supplementary-material FIGURE S1 \| Phenotyping of Chlamydomonas reinhardtii cells treated with 500 nM rapamycin (orange) or with drug vehicle (blue). (A) Chlorophyl content per ml, (B) Chl content per mg FW, (C) Fv/Fm. (D,E) Are TLC analysis of total lipid mixtures from Chlamydomonas control and rapamycin-treated samples at 0, 24, and 48 h. Lipid mixtures were extracted from and separated on a Silica gel 60 plates using the hexane: ethyl ether: acetic acid (90:7.5:1) solvent composition. Lipid class distribution was visualized under UV light after primuline spraying (Li et al., 2008). FIGURE S1 \| Phenotyping of Chlamydomonas reinhardtii cells treated with 500 nM rapamycin (orange) or with drug vehicle (blue). (A) Chlorophyl content per ml, (B) Chl content per mg FW, (C) Fv/Fm. (D,E) Are TLC analysis of total lipid mixtures from Chlamydomonas control and rapamycin-treated samples at 0, 24, and 48 h. Lipid mixtures were extracted from and separated on a Silica gel 60 plates using the hexane: ethyl ether: acetic acid (90:7.5:1) solvent composition. Lipid class distribution was visualized under UV light after primuline spraying (Li et al., 2008). TABLE S1 \| Proteins detected and quantiﬁed in this work and their abundance changes along two-way ANOVA results, as well as PCA loadings, HCA cluster and functional enrichment analysis for Figure 3. TABLE S2 \| Phosphopeptides detected and quantiﬁed in this work and their abundance changes along two-way ANOVA results, as well as PCA loadings. ACKNOWLEDGMENTS We would like to thank Dr. Verena Ibl, Dr. Michele Grieco, and Dr. Paul Roustan for critical reading of the manuscript. We would like to thank Dr. Verena Ibl, Dr. Michele Grieco, and Dr. Paul Roustan for critical reading of the manuscript. FUNDING This study was ﬁnanced by the Marie Curie ITN project CALIPSO (Grant Agreement Number: GA 2013-607-607). Data Analysis and Statistics The analysis was conducted on peptides which signiﬁcantly changed in treatment factor from the two-way ANOVA analysis. Two searches were performed with a serine residue or a threonine residue as central position. Background proteome was Chlamydomonas UniProt database centered on S, T and Y amino acids. Protein-protein interaction networks were created using the STRING database for Known and Predicted Protein-Protein Interactions with the standard setting4 (von Mering et al., 2005). AUTHOR CONTRIBUTIONS VR and WW conceived the study. VR performed measurements, analyzed the data, and wrote and revised the manuscript. WW analyzed the data and wrote and revised the manuscript. Data Analysis and Statistics Algal Functional Annotation tool (AFAT) was used to identify enriched functions from the protein cluster resulting from the HCA analysis or from the signiﬁcantly changed proteins (Lopez et al., 2011). The motif-x analysis was used to identify phosphorylation motifs that were over-represented in our dataset3 (Schwartz and Gygi, 2005). The analysis was conducted on peptides which signiﬁcantly changed in treatment factor from the two-way ANOVA analysis. Two searches were performed with a serine residue or a threonine residue as central position. Background proteome was Chlamydomonas UniProt database centered on S, T and Y amino acids. Protein-protein interaction networks were created using peptide quantiﬁcation was performed by peak integration at the MS1 level using Max Quant 1.5. For proteomics analysis the Label-free quantiﬁcation of proteins was done with a peptide ratio count 2 according to the Max Quant recommendations. The mass spectrometry (Phospho)proteomics data have been deposited to the ProteomeXchange Consortium (Deutsch et al., 2017) via the PRIDE (Vizcaíno et al., 2016) partner repository with the dataset identiﬁer PXD011489. Perseus 1.5 software, was used for further ﬁltering and data processing (Tyanova et al., 2016). Proteomics data set was obtained by ﬁltering the data matrix so that proteins that were identiﬁed in at least four biological replicates in at least one class of samples were included. Before PCA, values were log2 transformed and missing values were replaced by random numbers drawn from the normal distribution that represents low-abundance measurements of each sample. Phosphopeptide data were ﬁltered as the total proteomics data on the base of three biological samples per sample class. Additionally, only phosphopeptides that passed the class I criteria (phosphosite probability > 75% and score diﬀerence > 5) were included in the ﬁnal dataset (Olsen et al., 2006). Moreover, phosphopeptide abundance was normalized to the median of each sample, log2 transformed and missing values replaced using the same method as in total proteomics data. Further, PCA, HCA based on Spearman rank correlation and an average linkage of z-transformed phosphopeptide abundances, Analysis of variance (two-way ANOVA) was performed with Perseus software (Tyanova et al., 2016). Algal Functional Annotation tool (AFAT) was used to identify enriched functions from the protein cluster resulting from the HCA analysis or from the signiﬁcantly changed proteins (Lopez et al., 2011). The motif-x analysis was used to identify phosphorylation motifs that were over-represented in our dataset3 (Schwartz and Gygi, 2005). 3 http://string-db.org/ 4 http://motif-x.med.harvard.edu/motif-x.html 4 http://motif-x.med.harvard.edu/motif-x.html 3 http://string-db.org/ Data Analysis and Statistics MaxQuant 1.52 and the Andromeda search algorithm were used against the Chlamydomonas UniProt and JGI_236 version databases to perform peptide identiﬁcation, phosphorylation site mapping and protein and phosphopeptide quantiﬁcation (Cox and Mann, 2008; Cox et al., 2011). For both proteomics and phosphoproteomics data processing the following parameters were applied: Two and three missed cleavages were allowed respectively for the proteomics and phosphoproteomics analysis. Methionine oxidation and protein N-terminal acetylation were endorsed as dynamic modiﬁcations. For phosphoproteomics analysis, additionally, phosphorylation of serine, threonine and tyrosine residues was permitted to occur as dynamic modiﬁcations. Mass tolerance was set to 5 p.p.m. for parental ions and 0.8 Da for the MS/MS fragment. For both peptide and protein levels, false discovery rate was set to 1%. In both analysis November 2018 \| Volume 9 \| Article 1590 Frontiers in Plant Science \| www.frontiersin.org Frontiers in Plant Science \| www.frontiersin.org 17 Rapamycin Induced Chlamydomonas Phosphoproteome Changes Roustan and Weckwerth peptide quantiﬁcation was performed by peak integration at the MS1 level using Max Quant 1.5. For proteomics analysis the Label-free quantiﬁcation of proteins was done with a peptide ratio count 2 according to the Max Quant recommendations. The mass spectrometry (Phospho)proteomics data have been deposited to the ProteomeXchange Consortium (Deutsch et al., 2017) via the PRIDE (Vizcaíno et al., 2016) partner repository with the dataset identiﬁer PXD011489. Perseus 1.5 software, was used for further ﬁltering and data processing (Tyanova et al., 2016). Proteomics data set was obtained by ﬁltering the data matrix so that proteins that were identiﬁed in at least four biological replicates in at least one class of samples were included. Before PCA, values were log2 transformed and missing values were replaced by random numbers drawn from the normal distribution that represents low-abundance measurements of each sample. Phosphopeptide data were ﬁltered as the total proteomics data on the base of three biological samples per sample class. Additionally, only phosphopeptides that passed the class I criteria (phosphosite probability > 75% and score diﬀerence > 5) were included in the ﬁnal dataset (Olsen et al., 2006). Moreover, phosphopeptide abundance was normalized to the median of each sample, log2 transformed and missing values replaced using the same method as in total proteomics data. Further, PCA, HCA based on Spearman rank correlation and an average linkage of z-transformed phosphopeptide abundances, Analysis of variance (two-way ANOVA) was performed with Perseus software (Tyanova et al., 2016). REFERENCES Target of rapamycin (TOR) plays a critical role in triacylglycerol accumulation in microalgae. Plant Mol. Biol. 89, 309–318. doi: 10.1007/s11103- 015-0370-6 Demidov, D., Lermontova, I., Weiss, O., Fuchs, J., Rutten, T., Kumke, K., et al. (2014). Altered expression of Aurora kinases in Arabidopsis results in aneu- and polyploidization. Plant J. 80, 449–461. doi: 10.1111/tpj.12647 Deutsch, E. W., Csordas, A., Sun, Z., Jarnuczak, A., Perez-Riverol, Y., Ternent, T., et al. (2017). The ProteomeXchange consortium in 2017: supporting the cultural change in proteomics public data deposition. 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The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2018 Roustan and Weckwerth. 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. Wang, H., Gau, B., Slade, W. O., Juergens, M., Li, P., and Hicks, L. M. (2014). 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https://openalex.org/W4361255515	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Legend_from_CXC_Chemokine_Receptor_3_Expression_by_Activated_CD8_sup_sup_T_cells_Is_Associated_with_Survival_in_Melanoma_Patients_with_Stage_III_Disease/22363979/1/files/39808199.pdf	English	null	Supplementary Legend from CXC Chemokine Receptor 3 Expression by Activated CD8<sup>+</sup> T cells Is Associated with Survival in Melanoma Patients with Stage III Disease	null	2,023	cc-by	218	Supplemental Figure 1. CCR Evaluation Methodology. A) CD8+ T cells were enriched by negative immunomagnetic selection, then further subdivided into activated cells using immunomagnetic positive selection, based on expression of the activation marker CD45RO. Total RNA was prepared from this highly-pure CD8+CD45RO+ population for inclusion in RT-PCR protocols. B) cDNA was generated using oligo-dT primers and TAQ polymerase to reverse transcribe mRNA. CCR cDNA was amplified using gene-specific primers in multiplexed PCR reactions. C) Surface expression of CCRs was cross-validated by flow cytometery after staining with specific fluorochrome- conjugated monoclonal antibodies. Cells were co-stained with antibodies to CD8 and the activation marker CD45RO. Representative data shown in all panels. Supplemental Figure 1. CCR Evaluation Methodology. A) CD8+ T cells were enriched by negative immunomagnetic selection, then further subdivided into activated cells using immunomagnetic positive selection, based on expression of the activation marker CD45RO. Total RNA was prepared from this highly-pure CD8+CD45RO+ population for inclusion in RT-PCR protocols. B) cDNA was generated using oligo-dT primers and TAQ polymerase to reverse transcribe mRNA. CCR cDNA was amplified using gene-specific primers in multiplexed PCR reactions. C) Surface expression of CCRs was cross-validated by flow cytometery after staining with specific fluorochrome- conjugated monoclonal antibodies. Cells were co-stained with antibodies to CD8 and the activation marker CD45RO. Representative data shown in all panels.
https://openalex.org/W1966027500	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0116709&type=printable	English	null	Efficient and Anonymous Two-Factor User Authentication in Wireless Sensor Networks: Achieving User Anonymity with Lightweight Sensor Computation	PloS one	2,015	cc-by	13,788	RESEARCH ARTICLE Junghyun Nam1, Kim-Kwang Raymond Choo2, Sangchul Han1, Moonseong Kim3, Juryon Paik4, Dongho Won4 1 Department of Computer Engineering, Konkuk University, Chungju, Chungcheongbukdo, Korea, 2 School of Information Technology and Mathematical Sciences, University of South Australia, Mawson Lakes, South Australia, Australia, 3 Information Management Division, Korean Intellectual Property Office, Daejeon, Korea, 4 Department of Computer Engineering, Sungkyunkwan University, Suwon, Gyeonggido, Korea schan@kku.ac.kr Efficient and Anonymous Two-Factor User Authentication in Wireless Sensor Networks: Achieving User Anonymity with Lightweight Sensor Computation Sensor Computation Junghyun Nam1, Kim-Kwang Raymond Choo2, Sangchul Han1*, Moonseong Kim3, Juryon Paik4, Dongho Won4 Introduction The quest to understand real-world phenomena at a fine spatial-temporal resolution has led to a great increase in the interest in wireless sensor networks (WSNs). Where not already in place, a WSN is now being planned and deployed in various application settings such as wildlife mon- itoring, military surveillance, healthcare diagnostics, and vehicular tracking [1]. Providing an application service in a WSN environment introduces significant security challenges for the in- volved parties: sensors, users and gateways. One fundamental challenge is to establish a shared session key between a sensor and a user in an authenticated manner (known as authenticated key exchange) via a gateway, and thereby to prevent unauthorized access to sensitive sensor data and their transmissions. Since sensors have severe resource constraints and due to net- work characteristics such as unattended operation and unreliable communication channel, au- thenticated key exchange in WSNs is generally regarded as more challenging to achieve than in traditional networks with sufficient computing resources and pre-existing infrastructures. Achieving authenticated key exchange becomes even more difficult when user anonymity is de- sired. As the concern for privacy increases in our lives, user anonymity has become a vital secu- rity property in various WSN applications as well as in many other applications like location- Achieving authenticated key exchange becomes even more difficult when user anonymity is de- sired. As the concern for privacy increases in our lives, user anonymity has become a vital secu- rity property in various WSN applications as well as in many other applications like location- based services, e-voting, mobile roaming services, and anonymous web browsing. A smart-card-based user authentication scheme for WSNs (in short, a SCA-WSN scheme) al- lows a user holding its smart card issued by the gateway to achieve authenticated key exchange with a sensor, preferably in a way that its anonymity is preserved. Since the early work of Das [2], He et al. [3], Khan and Alghathbar [4] and Chen and Shih [5], all of which provide no key- exchange functionality, the design of SCA-WSN schemes has attracted much attention from re- searchers due to their potential to be widely deployed, and a number of proposals offering vari- ous levels of security and efficiency have been presented [6–20]. Some schemes consider only authenticated key exchange [6, 8, 9, 12, 20] while others attempt to additionally provide user anonymity [7, 10, 11, 13–19]. OPEN ACCESS 1 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0116709 April 7, 2015 Efficient, Anonymous User Authentication in Wireless Sensor Networks OPEN ACCESS A smart-card-based user authentication scheme for wireless sensor networks (hereafter re- ferred to as a SCA-WSN scheme) is designed to ensure that only users who possess both a smart card and the corresponding password are allowed to gain access to sensor data and their transmissions. Despite many research efforts in recent years, it remains a challenging task to design an efficient SCA-WSN scheme that achieves user anonymity. The majority of published SCA-WSN schemes use only lightweight cryptographic techniques (rather than public-key cryptographic techniques) for the sake of efficiency, and have been demonstrat- ed to suffer from the inability to provide user anonymity. Some schemes employ elliptic curve cryptography for better security but require sensors with strict resource constraints to perform computationally expensive scalar-point multiplications; despite the increased computational requirements, these schemes do not provide user anonymity. In this paper, we present a new SCA-WSN scheme that not only achieves user anonymity but also is effi- cient in terms of the computation loads for sensors. Our scheme employs elliptic curve cryp- tography but restricts its use only to anonymous user-to-gateway authentication, thereby allowing sensors to perform only lightweight cryptographic operations. Our scheme also en- joys provable security in a formal model extended from the widely accepted Bellare- Pointcheval-Rogaway (2000) model to capture the user anonymity property and various SCA-WSN specific attacks (e.g., stolen smart card attacks, node capture attacks, privileged insider attacks, and stolen verifier attacks). Citation: Nam J, Choo KKR, Han S, Kim M, Paik J, Won D (2015) Efficient and Anonymous Two-Factor User Authentication in Wireless Sensor Networks: Achieving User Anonymity with Lightweight Sensor Computation. PLoS ONE 10(4): e0116709. doi:10.1371/journal.pone.0116709 Academic Editor: Muhammad Khurram Khan, King Saud University, Kingdom of Saudi Arabia, SAUDI ARABIA Copyright: © 2015 Nam et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by Konkuk University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. Introduction Schemes such as the ones in [6, 12, 20] employ elliptic curve cryptography to provide perfect forward secrecy while most schemes [7–11, 13–19] use only lightweight cryptographic techniques, such as symmetric encryptions, message authentication codes and hash functions, to focus on improving the efficiency. One common security requirement for SCA-WSN schemes is to ensure that: PLOS ONE \| DOI:10.1371/journal.pone.0116709 April 7, 2015 only a user who is in possession of both a smart card and the corresponding password can be successfully authenticated (by the gateway) and access the sensor data. only a user who is in possession of both a smart card and the corresponding password can be successfully authenticated (by the gateway) and access the sensor data. only a user who is in possession of both a smart card and the corresponding password can be successfully authenticated (by the gateway) and access the sensor data. This requirement is commonly referred to as two-factor security [21–25] and is modelled via an adversary who is able to either extract all the information inside the smart card of a user or learn the password of the user, but not both. (Clearly, there is no means to prevent the adver- sary from impersonating a user if both the information in the smart card and the password of the user are disclosed.) The former requires physical access to the smart card and then mount- ing a side-channel attack [26, 27] on the (lost, misplaced or stolen) card, while the latter can be achieved with shoulder-surfing or by using a malicious card reader. Any attack exploiting the former ability is commonly called a stolen smart card attack and is considered practical under the assumption that users’ smart cards are non-tamper-resistant. Accordingly, SCA-WSN schemes should be designed to achieve their intended security properties, such as authenticated key exchange and user anonymity, against stolen smart card attacks. Despite the many research efforts to date, it remains a challenging task to design an efficient SCA-WSN scheme that provides user anonymity. The recent work of Wang and Wang [28, 29] shows that, under the non-tamper-resistance assumption of smart cards, no SCA-WSN scheme PLOS ONE \| DOI:10.1371/journal.pone.0116709 April 7, 2015 2 / 21 Efficient, Anonymous User Authentication in Wireless Sensor Networks can provide user anonymity without recourse to public key cryptography. This result is some- what surprising because it implies that all existing anonymous schemes using only lightweight cryptographic techniques [7, 10, 11, 13–19] fail to achieve user anonymity in the presence of an adversary who can mount a stolen smart card attack. As an example of such a failure, we here take the recent SCA-WSN scheme of Jiang et al. [19] which has been presented with a claim of user anonymity. To illustrate the failure, we only need to examine the user registration and login request phases of the scheme. Let MK be the master key of the gateway GW, and H be a cryptographic hash function. only a user who is in possession of both a smart card and the corresponding password can be successfully authenticated (by the gateway) and access the sensor data. Then, the two phases proceed as follow: User Registration. A user U registers with GW as follows: User Registration. A user U registers with GW as follows: User Registration. A user U registers with GW as follows: 1. U chooses its identity IDU and password PWU, generates a random number r, computes RPWU = H(rkPWU), and submits IDU and RPWU to GW via a secure channel. 2. If IDU is valid, GW generates a temporary identity for U, TIDU, and computes TCU = H (MKkIDUkTEU) and PTCU = TCURPWU, where TEU is the expiration time of TIDU. GW then stores (TIDU, IDU, TEU) in its verification table, and issues U a smart card con- taining {H(), TIDU, TEU, PTCU}. 3. U stores the random number r into the smart card, which then holds {H(), TIDU, TEU, PTCU, r}. Login Request. U inserts its smart card into a card reader, and inputs IDU and PWU. The smart card retrieves the current timestamp TU, selects a random key KU, and computes TCU = PTCUH(rkPWU), PKSU = KUH(TCUkTU) and CU = H(IDUkKUkTCUkTU). Then, U sends the login request message MU = hTIDU, CU, PKSU, TUi to GW. Assume an attacker A who has obtained the information {H(), TIDU, TEU, PTCU, r} stored on the smart card of user U. A eavesdrops and obtains the login request message MU = hTIDU, CU, PKSU, TUi, and mounts the following offline dictionary attack. Step 1. A makes a guess PW 0 U on the password PWU and computes TC0 U ¼ PTCU HðrkPW 0 UÞ and K0 U ¼ PKSU HðTC0 UkTUÞ. Step 1. A makes a guess PW 0 U on the password PWU and computes TC0 U ¼ PTCU HðrkPW 0 UÞ and K0 U ¼ PKSU HðTC0 UkTUÞ. Step 2. For each possible identity ID0 U, A computes C0 U ¼ HðID0 UkK0 UkTC0 UkTUÞ and verifies the correctness of PW 0 U and ID0 U by checking that C0 U is equal to CU. Note that, with an overwhelming probability, C0 U ¼ CU if and only if PW 0 U ¼ PWU and ID0 U ¼ IDU. Step 2. For each possible identity ID0 U, A computes C0 U ¼ HðID0 UkK0 UkTC0 UkTUÞ and verifies the correctness of PW 0 U and ID0 U by checking that C0 U is equal to CU. PLOS ONE \| DOI:10.1371/journal.pone.0116709 April 7, 2015 only a user who is in possession of both a smart card and the corresponding password can be successfully authenticated (by the gateway) and access the sensor data. Note that, with an overwhelming probability, C0 U ¼ CU if and only if PW 0 U ¼ PWU and ID0 U ¼ IDU. Step 3. A repeats Steps 1 and 2 until the correct password and identity are found. Step 3. A repeats Steps 1 and 2 until the correct password and identity are found. This dictionary attack works because the identity space is very limited in practice, being usu- ally even smaller than the password space [28, 29]. All other schemes using only lightweight cryptographic techniques are also vulnerable to similar dictionary attacks, as shown in [28, 29]. Note that simply using a symmetric encryption scheme cannot overcome the inherent failure. Although there are some published schemes that employ elliptic curve cryptography [6, 12, 20], these schemes were designed with no user anonymity in the first place and moreover, are not efficient in the sense that they impose expensive scalar-point multiplications on resource- constrained sensors. In this paper, we present an efficient and provably-anonymous SCA-WSN scheme that re- quires sensors to perform only lightweight cryptographic operations. Our scheme employs el- liptic curve cryptography but restricts its use to anonymous user-to-gateway authentication in order not to impose any (expensive) public-key operations, such as scalar-point multiplications and map-to-point operations, on sensors. We formally prove that our scheme achieves user an- onymity as well as authenticated key exchange in an extension of the widely accepted model of 3 / 21 Efficient, Anonymous User Authentication in Wireless Sensor Networks Bellare et al. [30]. In proving the security properties, we assume that the cryptographic hash functions used are random oracles and the elliptic curve computational Diffie-Hellman prob- lem is computationally hard. The extended model captures not only the notion of two-factor security but also standard attacks against SCA-WSN schemes like node capture attacks, privileged insider attacks, and stolen verifier attacks. The remainder of this paper is structured as follows. Section 2 describes an extended securi- ty model for the analysis of anonymous SCA-WSN schemes. Section 3 presents our proposed SCA- WSN scheme along with cryptographic primitives on which the security of the scheme relies. Section 4 provides proofs for the security properties of our proposed scheme in the extended security model. Section 5 concludes the paper with a comparative efficiency and security of our scheme and other SCA-WSN schemes. Protocol Executions A user U 2 may run multiple sessions of the authentication and key exchange protocol of a SCA-WSN scheme, either serially or concurrently, to establish a session key with a sensor SN 2 SN via assistance of the gateway GW. Therefore, at any given time, there could be multiple in- stances of the entities U, SN and GW. We use Pi E to denote instance i of entity E 2 E. Instances of U and SN are said to accept when they compute a session key in an execution of the protocol. We denote the session key of Pi E by ski E. We denote the session key of Pi E by ski E. Participants Let SN and U be the sets of all sensors and users, respectively, registered with the gateway GW. Let E = U[SN[{GW}. We identify each entity E 2 E by a string, and interchangeably use E and IDE to refer to this identifier string. To formally capture the user anonymity property, we as- sume that: (1) each user U 2 U has its pseudo identity PIDU in addition to the true identity IDU and (2) the adversary A is given only PIDU but not IDU. Long-Lived Keys During the initialization of the protocol, During the initialization of the protocol, • each U 2 U chooses its password PWU from a fixed dictionary D, and • each U 2 U chooses its password PWU from a fixed dictionary D, and • GW generates its master secret(s), issues a smart card to each U 2 U, and shares a crypto- graphic key with each SN 2 SN. • GW generates its master secret(s), issues a smart card to each U 2 U, and shares a crypto- graphic key with each SN 2 SN. A Security Model for Anonymous SCA-WSN Schemes This section describes a security model extended from the Bellare et al.’s model [30] to analyze authentication and key exchange protocols of anonymous SCA-WSN schemes. Our security model captures the notion of two-factor security as well as the resistance to node capture at- tacks, privileged insider attacks, stolen verifier attacks, and other common attacks. We provide two security definitions associated with the model, one for authenticated key exchange and one for user anonymity, which collectively define a secure, anonymous SCA-WSN scheme. Adversary Capabilities We assume there exists an adversary A running in a probabilistic polynomial time (PPT) in the security parameter κ, which represents the bit-length of session keys. We note that the size of the dictionary D is a fixed constant that is independent of the security parameter κ. The PPT ad- versary A has complete control of all communications between entities, can request for access to session keys and long-term keys, and can extract user’s information stored on the smart card. These capabilities of A are modeled via the following oracle queries which are allowed for A to make. • Execute(Pi U, Pj SN, Pk GW): This query models passive attacks against the protocol. It prompts an execution of the protocol between the instances Pi U, Pj SN and Pk GW, and outputs the transcript of the protocol execution to A. • Send(Pi E; m): This query sends a message m to an instance Pi E, modelling active attacks against the protocol. Upon receiving m, the instance Pi E proceeds according to the protocol specification. The message output by Pi E, if any, is returned to A. A query of the form Send(Pi U, start:hSN, GWi) prompts Pi U to initiate a protocol session with instances of SN and GW. • Reveal(Pi E): This query captures the notion of known key security. The instance Pi E, upon receiving the query and if it has accepted, returns the session key, ski E, back to A. • CorruptLLðUÞ=CorruptSCðUÞ: These queries together capture the notion of two-factor se- curity. The former returns the password of U while the latter returns the information stored in the smart card of U. • CorruptLL(SN): This query returns the long-lived secret(s) of the sensor SN, modelling node capture attacks. • CorruptLL(GW), modelling privileged insider attacks. • CorruptLL(GW), modelling privileged insider attacks. • CorruptVFR(GW): This query returns the password verifiers stored by GW, modelling sto- len verifier attacks. • TestAKE(Pi E): This query is used for determining whether the protocol achieves authenti- cated key exchange or not. If Pi E has accepted, then depending on a random bit b chosen by the oracle, A is given either the real session key ski E if b = 1 or a random key drawn from the session-key space if b = 0. • TestUA(U): This query is used for determining whether the protocol provides user anonym- ity or not. Partnering Informally, two instances are said to be partners of each other if they participate together in the same protocol session and as a result, compute the same session key. Formally, partnering be- tween instances is defined in terms of the notion of session identifier. A session identifier (sid) PLOS ONE \| DOI:10.1371/journal.pone.0116709 April 7, 2015 4 / 21 Efficient, Anonymous User Authentication in Wireless Sensor Networks is an identifier of a protocol session and is typically defined as a function of the messages ex- changed in the session. Let sidi E denote the sid of instance Pi E. We say that two instances, Pi U and Pj SN, are partners if (1) both the instances have accepted and (2) sidi U ¼ sid j SN. PLOS ONE \| DOI:10.1371/journal.pone.0116709 April 7, 2015 Adversary Capabilities Depending on a randomly chosen bit b, A is given either the identity actually used for U in the protocol sessions (when b = 1) or a random identity drawn from the identity space (when b = 0). 5 / 21 Efficient, Anonymous User Authentication in Wireless Sensor Networks CorruptLL queries all together also capture the notion of perfect forward secrecy. SN and GW are said to be corrupted when they are asked a CorruptLL query while U is considered as corrupted if it has been asked both CorruptLL and CorruptSC queries. CorruptLL queries all together also capture the notion of perfect forward secrecy. SN and GW are said to be corrupted when they are asked a CorruptLL query while U is considered as corrupted if it has been asked both CorruptLL and CorruptSC queries. Authenticated Key Exchange (AKE) Let SuccAKE0 be the event that A succeeds in the experiment ExpAKE0, and AdvAKE P ðAÞ denote the advantage of A in breaking the AKE security of protocol P. Then, we deﬁne AdvAKE P ðAÞ ¼ 2 PrP;A½SuccAKE0 1. Definition 2 (AKE Security). An authentication and key exchange protocol P is AKE-secure if AdvAKE P ðAÞ is negligible for any PPT adversary A. Definition 2 (AKE Security). An authentication and key exchange protocol P is AKE-secure if AdvAKE P ðAÞ is negligible for any PPT adversary A. Authenticated Key Exchange (AKE) The AKE security of an authentication and key exchange protocol P is defined via the notion of freshness. Intuitively, a fresh instance is one that holds a session key which should not be known to the adversary A, and an unfresh instance is one whose session key (or some informa- tion about the key) can be known by trivial means. A formal definition of freshness follows: Definition 1 (Freshness). An instance Pi E is fresh if none of the following occurs: Definition 1 (Freshness). An instance Pi E is fresh if none of the following occurs: 1. A queries RevealðPi EÞ or RevealðPj E0Þ, where Pj E0 is the partner of Pi E. 1. A queries RevealðPi EÞ or RevealðPj E0Þ, where Pj E0 is the partner of Pi E. A queries both CorruptLLðUÞ and CorruptSCðUÞ when U is E itself or the peer entity of E. 2. A queries both CorruptLLðUÞ and CorruptSCðUÞ when U is E itself or the peer entity of E. 3. A queries CorruptLLðSNÞ when SN is E itself or the peer entity of E. 4. A queries CorruptLLðGWÞ. Note that this definition of freshness is unable to capture the notion of perfect forward se- crecy. (As explained in the next section, the authentication and key exchange protocol of our scheme does not provide perfect forward secrecy.) The AKE security of protocol P is defined in the context of the following two-stage experiment: Experiment ExpAKE0: Experiment ExpAKE0: Experiment ExpAKE0: Stage 1. A makes any oracle queries at will, except that: 1. A is not allowed to make the TestAKE(Pi E) query if the instance Pi E is not fresh. 2. A is not allowed to make the Reveal(Pi E) query if it has already made a TestAKE query to Pi E or its partner instance. 3. A is not allowed to access to the TestUA oracle. 3. A is not allowed to access to the TestUA oracle. Stage 2. Once A decides that Stage 1 is over, it outputs a bit b0 as a guess on the hidden bit b chosen by the TestAKE oracle. A is said to succeed if b = b0. Let SuccAKE0 be the event that A succeeds in the experiment ExpAKE0, and AdvAKE P ðAÞ denote the advantage of A in breaking the AKE security of protocol P. Then, we deﬁne AdvAKE P ðAÞ ¼ 2 PrP;A½SuccAKE0 1. 1. A queries both CorruptLLðUÞ and CorruptSCðUÞ. User Anonymity An authentication and key exchange protocol that does not provide user anonymity may still be rendered AKE-secure. That is, the AKE security does not imply user anonymity. Therefore, a new, separate definition is necessary to capture the user anonymity property. Our definition of user anonymity is based on the notion of cleanness. Definition 3 (Cleanness). A user U 2 U is clean if none of the following occurs: 1. A queries both CorruptLLðUÞ and CorruptSCðUÞ. 2. A queries CorruptLLðGWÞ. PLOS ONE \| DOI:10.1371/journal.pone.0116709 April 7, 2015 6 / 21 Efficient, Anonymous User Authentication in Wireless Sensor Networks Note that the definition of cleanness does not impose any restriction on making CorruptLL queries to sensors. This reﬂects our objective to achieve user anonymity even against sensors. User anonymity is formalized in the context of the following two-stage experiment: Experiment ExpUA0: Experiment ExpUA0: Experiment ExpUA0: Stage 1. A makes any oracle queries at will, except that: Stage 1. A makes any oracle queries at will, except that: 1. A is not allowed to make the TestUA(U) query if the user U is not clean. 2. A is not allowed to corrupt GW and U if it has already made the TestUAðUÞ query. 2. A is not allowed to corrupt GW and U if it has already made the TestUAðUÞ query. 3. A is not allowed to access to the TestAKE oracle. 3. A is not allowed to access to the TestAKE oracle. 3. A is not allowed to access to the TestAKE oracle. Stage 2. Once A decides that Stage 1 is over, it outputs a bit b0 as a guess on the hidden bit b chosen by the TestUA oracle. A is said to succeed if b = b0. Let SuccUA0 be the event that A succeeds in the experiment ExpUA0, and AdvUA P ðAÞ de- note the advantage of A in attacking the user anonymity of protocol P. Then, we deﬁne AdvUA P ðAÞ ¼ 2 PrP;A½SuccUA0 1. Let SuccUA0 be the event that A succeeds in the experiment ExpUA0, and AdvUA P ðAÞ de- note the advantage of A in attacking the user anonymity of protocol P. Then, we deﬁne AdvUA P ðAÞ ¼ 2 PrP;A½SuccUA0 1. Definition 4 (User Anonymity). An authentication and key exchange protocol P provides user anonymity if AdvUA P ðAÞ is negligible for any PPT adversary A. User Anonymity Definition 4 (User Anonymity). An authentication and key exchange protocol P provides user anonymity if AdvUA P ðAÞ is negligible for any PPT adversary A. Our Proposed Scheme Our SCA-WSN scheme restricts the use of elliptic curve cryptography to anonymous user-to-gate- way authentication and thereby allows sensor nodes to perform only lightweight cryptographic operations such as symmetric encryption/decryption, MAC generation/verification, and hash function evaluation. We begin by describing the cryptographic building blocks on which the se- curity of our scheme depends. PLOS ONE \| DOI:10.1371/journal.pone.0116709 April 7, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0116709 April 7, 2015 Efficient, Anonymous User Authentication in Wireless Sensor Networks tag pair (m, σ) such that: (1) Verkðm; sÞ ¼ 1 and (2) σ has not been output by the oracle MackðÞ as a MAC on the message m. The, we say that the MAC scheme S is secure if AdvEFCMA S ðAÞ is negligible for every PPT adversary A. We use AdvEFCMA S ðtÞ to denote the maximum value of AdvEFCMA S ðAÞ over all adversaries A running in time at most t. tag pair (m, σ) such that: (1) Verkðm; sÞ ¼ 1 and (2) σ has not been output by the oracle MackðÞ as a MAC on the message m. The, we say that the MAC scheme S is secure if AdvEFCMA S ðAÞ is negligible for every PPT adversary A. We use AdvEFCMA S ðtÞ to denote the maximum value of AdvEFCMA S ðAÞ over all adversaries A running in time at most t. Cryptographic hash functions. Let κ be the bit-length of session keys, ‘ be as defined for S, and ω be the bit-length of EIDU (see the registration phase of our scheme described in the next section). Then, our scheme uses three cryptographic hash functions H:{0, 1} ! {0, 1}κ, J: {0, 1} ! {0, 1}‘, and I:{0, 1} ! {0, 1}ω. These hash functions are modelled as random oracles in our security proofs. Symmetric encryption schemes. A symmetric encryption scheme Δ is a pair of efficient algorithms (Enc, Dec) where: (1) the encryption algorithm Enc takes as input an ‘-bit key k and a plaintext message m, and outputs a ciphertext c; and (2) the decryption algorithm Dec takes as input a key k and a ciphertext c, and outputs a message m. For an eavesdropping adver- sary A against Δ, and for an integer n 1 and a random bit b 2 R{0, 1}, consider the following indistinguishability experiment where only a single encryption key is used: Experiment ExpINDSEK D ðA; n; bÞ Experiment ExpIND D Experiment ExpIN D k 2R {0, 1}‘ k 2R {0, 1}‘ for i = 1 to n (mi, 0, mi,1) A(Δ) ci Enckðmi;bÞ (mi, 0, mi,1) A(Δ) ci Enckðmi;bÞ b0 A, where b0 2 {0, 1} Building Blocks Elliptic curve computational Diffie-Hellman (ECCDH) problem. Let G be an elliptic curve group of prime order q. Typically, G will be a subgroup of the group of points on an ellip- tic curve over a finite field. Any elliptic curve and finite field recommended by NIST [31] can be used to instantiate the group G. The recent work of Choi et al. [20], for example, describes a typical elliptic curve group of a prime order. Let P be a generator of G. The ECCDH problem for G is to compute xyP 2 G when given two elements (xP,yP) 2 G2, where x; y2RZ q. We say that the ECCDH assumption holds for G if it is computationally infeasible to solve the ECCDH problem for G. Let AdvECCDH G ðAÞ be the advantage of an algorithm A in solving the ECCDH problem for G and be deﬁned as AdvECCDH G ðAÞ ¼ Pr½AðG; P; xP; yPÞ ¼ xyP. We assume that AdvECCDH G ðAÞ is negligible for all PPT algorithms A (i.e., the ECCDH assumption holds in G). We denote by AdvECCDH G ðtÞ the maximum value of AdvECCDH G ðAÞ over all algorithms A running in time at most t. Message authentication code schemes. A message authentication code (MAC) scheme S is a pair of efficient algorithms (Mac, Ver) where: (1) the MAC generation algorithm Mac takes as input an ‘-bit key k and a message m, and outputs a MAC σ; and (2) the MAC veriﬁca- tion algorithm Ver takes as input a key k, a message m, and a MAC σ, and outputs 1 if σ is valid for message m under the key k or outputs 0 if σ is invalid. We require that S should achieve the strong existential unforgeability against chosen message attacks. To formally deﬁne this re- quirement, let AdvEFCMA S ðAÞ be the probability that an adversary A, who mounts an adaptive chosen message attack against S with oracle access to MackðÞ and VerkðÞ, outputs a message/ PLOS ONE \| DOI:10.1371/journal.pone.0116709 April 7, 2015 7 / 21 b0 A, where b0 2 {0, 1} return b0 return b0 We use AdvINDSEK D ðAÞ to denote the advantage of A in violating the indistinguishability of Δ in experiment ExpINDSEK D ðA; n; bÞ, and deﬁne it as AdvINDSEK D ðAÞ ¼ jPr½ExpINDSEK D ðA; n; 0Þ ¼ 1 Pr½ExpINDSEK D ðA; n; 1Þ ¼ 1j: We say that the symmetric encryption scheme Δ is secure if AdvINDSEK D ðAÞ is negligible for every PPT eavesdropper A. Let AdvINDSEK D ðtÞ be the maximum value of AdvINDSEK D ðAÞ over all A running in time at most t. We now claim that if a symmetric encryption scheme is secure with respect to a single en- cryption key, then it is also secure with respect to multiple encryption keys. Now consider the following indistinguishability experiment where d encryption keys are used: Experiment ExpINDSEK D ðA; n; d; bÞ Experiment ExpINDSEK D ðA; n; d; bÞ for i = 1 to d ki 2R {0, 1}‘ for j = 1 to n (mi,j,0, mi,j,1) A(Δ) ci;j Enckiðmi;j;bÞ A(ci,j) b0 A, where b0 2 {0, 1} return b0 Experiment ExpINDSEK D ðA; n; d; bÞ for i = 1 to d ki 2R {0, 1}‘ for j = 1 to n j (mi,j,0, mi,j,1) A(Δ) ci;j Enckiðmi;j;bÞ A(ci,j) b0 A, where b0 2 {0, 1} return b0 (mi,j,0, mi,j,1) A(Δ) ci;j Enckiðmi;j;bÞ We define AdvINDMEK D ðAÞ and AdvINDMEK D ðtÞ respectively as AdvINDMEK D ðAÞ ¼ jPr½ExpINDMEK D ðA; n; d; 0Þ ¼ 1 Pr½ExpINDMEK D ðA; n; d; 1Þ ¼ 1j 8 / 21 Efficient, Anonymous User Authentication in Wireless Sensor Networks and and AdvINDMEK D ðtÞ ¼ max A fAdvINDMEK D ðAÞg; where the maximum is over all A running in time at most t. where the maximum is over all A running in time at most t. Lemma 1. For any symmetric encryption scheme Δ, AdvINDMEK D ðtÞ d AdvINDSEK D ðtÞ; where d is as deﬁned for experiment ExpINDMEK D ðA; n; d; bÞ. where d is as deﬁned for experiment ExpINDMEK D ðA; n; d; bÞ. where d is as deﬁned for experiment ExpINDMEK D ðA; n; d; bÞ. Proof. Assume an adversary A who attacks the indistinguishability of Δ in IND MEKð Þ Proof. Assume an adversary A who attacks the indistinguishability of Δ in ExpINDMEK D ðA; n; d; bÞ with time complexity t. b0 A, where b0 2 {0, 1} return b0 The proof proceeds with a standard hybrid ar- gument [32]. Consider a sequence of d + 1 hybrid experiments ExpINDMEK D;x ðA; n; d; bÞ, 0 ξ d, where each ExpINDMEK D;x ðA, n, d, b) is different from ExpINDMEK D ðA; n; d; bÞ only in that each ci,j is set as follows: ci;j Enckiðmi;j;1Þ if i x Enckiðmi;j;0Þ otherwise: 8 < : The experiments ExpINDMEK D;0 ðA; n; d; bÞ and ExpINDMEK D;d ðA; n; d; bÞ at the extremes of the se- quence are identical to the experiments ExpINDMEK D ðA; n; d; 0Þ and ExpINDMEK D ðA; n; d; 1Þ, respectively. As we move from ExpINDMEK D;x1 ðA; n; d; bÞ to ExpINDMEK D;x ðA; n; d; bÞ in the se- quence, we change the n ciphertexts cξ,1, . . ., cξ,n from encryptions of the ﬁrst plaintexts to en- cryptions of the second plaintexts. Since there are d such moves from ExpINDMEK D;0 ðA; n; d; bÞ to ExpINDMEK D;d ðA; n; d; bÞ, the inequality of the lemma follows immediately if we prove that The experiments ExpINDMEK D;0 ðA; n; d; bÞ and ExpINDMEK D;d ðA; n; d; bÞ at the extremes of the se- quence are identical to the experiments ExpINDMEK D ðA; n; d; 0Þ and ExpINDMEK D ðA; n; d; 1Þ, respectively. As we move from ExpINDMEK D;x1 ðA; n; d; bÞ to ExpINDMEK D;x ðA; n; d; bÞ in the se- quence, we change the n ciphertexts cξ,1, . . ., cξ,n from encryptions of the ﬁrst plaintexts to en- cryptions of the second plaintexts. Since there are d such moves from ExpINDMEK D;0 ðA; n; d; bÞ to ExpINDMEK D;d ðA; n; d; bÞ, the inequality of the lemma follows immediately if we prove that the difference between the probabilities that A outputs 1 in any two neighboring experiments ExpINDMEK D;x1 ðA; n; d; bÞ and ExpINDMEK D;x ðA; n; d; bÞ is at most AdvINDSEK D ðtÞ. That is, to com- plete the proof, it sufﬁces to show that for any 1 ξ d, The experiments ExpINDMEK D;0 ðA; n; d; bÞ and ExpINDMEK D;d ðA; n; d; bÞ at the extremes of the se- quence are identical to the experiments ExpINDMEK D ðA; n; d; 0Þ and ExpINDMEK D ðA; n; d; 1Þ, quence, we change the n ciphertexts cξ,1, . . b0 A, where b0 2 {0, 1} return b0 ., (mξ,n,0,mξ,n,1), Aξ outputs them a its own plaintext pairs in experiment ExpINDSEK D ðAx; n; bÞ, receives in return the cipher texts c1, . . ., cn, and sets cξ,1 = c1, . . ., cξ,n = cn. Then, it follows that: PLOS ONE \| DOI:10.1371/journal.pone.0116709 April 7, 2015 b0 A, where b0 2 {0, 1} return b0 ., cξ,n from encryptions of the ﬁrst plaintexts to en- cryptions of the second plaintexts. Since there are d such moves from ExpINDMEK D;0 ðA; n; d; bÞ to ExpINDMEK D;d ðA; n; d; bÞ, the inequality of the lemma follows immediately if we prove that the difference between the probabilities that A outputs 1 in any two neighboring experiments ExpINDMEK D;x1 ðA; n; d; bÞ and ExpINDMEK D;x ðA; n; d; bÞ is at most AdvINDSEK D ðtÞ. That is, to com- plete the proof, it sufﬁces to show that for any 1 ξ d, jPr½ExpINDMEK D;x1 ðA; n; d; bÞ ¼ 1 Pr½ExpINDMEK D;x ðA; n; d; bÞ ¼ 1j AdvINDSEK D ðtÞ: ð1Þ ð1Þ Let ¼j Pr½ExpINDMEK D;x1 ðA; n; d; bÞ ¼ 1 Pr½ExpINDMEK D;x ðA; n; d; bÞ ¼ 1 j. Then, to prove Equation 1, we will construct, from A, an adversary Aξ who attacks the indistinguishability of Δ in ExpINDSEK D ðA; n; bÞ with advantage . Let ¼j Pr½ExpINDMEK D;x1 ðA; n; d; bÞ ¼ 1 Pr½ExpINDMEK D;x ðA; n; d; bÞ ¼ 1 j. Then, to prove Equation 1, we will construct, from A, an adversary Aξ who attacks the indistinguishability of Δ in ExpINDSEK D ðA; n; bÞ with advantage . Let ¼j Pr½ExpINDMEK D;x1 ðA; n; d; bÞ ¼ 1 Pr½ExpINDMEK D;x ðA; n; d; bÞ ¼ 1 j. Then, to prove Equation 1, we will construct, from A, an adversary Aξ who attacks the indistinguishability of Δ in ExpINDSEK D ðA; n; bÞ with advantage . Aξ begins by invoking adversary A, then proceeds to simulate the indistinguishability ex- periment for A, and finally ends by outputting whatever bit A eventually outputs. In the simu- lated experiment, Aξ generates the ciphertexts exactly as in the hybrid experiment ExpINDMEK D;x ðA; b; nÞ except that it generates cξ,1, . . ., cξ,n as follows: When A outputs the n plaintext pairs (mξ,1,0,mξ,1,1), . . ., (mξ,n,0,mξ,n,1), Aξ outputs them as its own plaintext pairs in experiment ExpINDSEK D ðAx; n; bÞ, receives in return the cipher- texts c1, . . ., cn, and sets cξ,1 = c1, . . ., cξ,n = cn. When A outputs the n plaintext pairs (mξ,1,0,mξ,1,1), . . Description of the Scheme The scheme consists of three phases: the registration phase, the authentication and key ex- change phase, and the password update phase. During the system initialization, the gateway GW determines the following public parameters: (1) an elliptic curve group G with a generator P of prime order q, (2) a MAC scheme S ¼ ðMac; VerÞ, (3) a symmetric encryption scheme D ¼ ðEnc; DecÞ, and (4) three hash functions H, J and I. We assume that these parameters are known to all parties in the network including the adversary A. As part of the system initializa- tion, GW chooses two master secrets y 2 Z q and z 2 {0, 1}‘, computes its public key Y = yP, and shares a secret key kGS = J(IDSNkz) with each sensor SN. Registration phase. A user U should register itself with the gateway GW before it can ever gain access to the sensor network and data. The registration proceeds as follows: 1. U chooses its identity IDU and password PWU at will, and submits the identity IDU to GW via a secure channel. 1. U chooses its identity IDU and password PWU at will, and submits the identity IDU to GW via a secure channel. 2. GW computes EIDU ¼ EnczðIDUkIDGWÞ and issues U a smart card loaded with {EIDU, Y, IDGW, G, P, S, Δ, H, J, I}. (We assume that q is implicit in G.) U replaces EIDU with XEIDU = EIDUI(IDUkPWU). 3. U replaces EIDU with XEIDU = EIDUI(IDUkPWU). 3. U replaces EIDU with XEIDU = EIDUI(IDUkPWU). Authentication and key exchange phase. U needs to perform this phase with SN and GW whenever it wishes to access to the sensor network and data. The steps of the phase are depicted in Fig. 1 and are described as follows: Step 1. U inserts its smart card into a card reader and inputs its identity IDU and password PWU. Then, it follows that: • the probability that Aξ outputs 1 when the given ciphertexts are the encryptions of the first plaintexts is equal to the probability that A outputs 1 in the experiment ExpINDMEK D;x1 ðA; n; d; bÞ, and • the probability that Aξ outputs 1 when the given ciphertexts are the encryptions of the first plaintexts is equal to the probability that A outputs 1 in the experiment ExpINDMEK D;x1 ðA; n; d; bÞ, and PLOS ONE \| DOI:10.1371/journal.pone.0116709 April 7, 2015 9 / 21 Efficient, Anonymous User Authentication in Wireless Sensor Networks • the probability that Aξ outputs 1 when the given ciphertexts are the encryptions of the sec- ond plaintexts is equal to the probability that A outputs 1 in the experiment ExpINDMEK D;x ðA; n; d; bÞ. • the probability that Aξ outputs 1 when the given ciphertexts are the encryptions of the sec- ond plaintexts is equal to the probability that A outputs 1 in the experiment ExpINDMEK D;x ðA; n; d; bÞ. That is: AdvINDSEK D ðAxÞ ¼ jPr½ExpINDMEK D;x1 ðA; n; d; bÞ ¼ 1 Pr½ExpINDMEK D;x ðA; n; d; bÞ ¼ 1j: Since Aξ has time complexity t, it follows that AdvINDSEK D ðAxÞ AdvIND D Since Aξ has time complexity t, it follows that AdvINDSEK D ðAxÞ AdvINDSEK D ðtÞ by deﬁnition. h l h f f d h h f f This completes the proof of Equation 1 and hence the proof of Lemma 1. PLOS ONE \| DOI:10.1371/journal.pone.0116709 April 7, 2015 Description of the Scheme Then, the smart card retrieves the current timestamp TU, selects two random x 2 Z q and kUS 2 {0, 1}κ, and computes X ¼ xP; KUG ¼ xY; kUG ¼ JðTU k X k Y k KUGÞ; EIDU ¼ XEIDU IðIDU k PWUÞ; CU ¼ EnckUGðIDU k EIDU k kUSÞ; sU ¼ MackUGðIDGW k IDSN k TU k CUÞ: KUG ¼ xY; kUG ¼ JðTU k X k Y k KUGÞ; After the computations, the smart card sends the message M1 = hTU,IDSN,X,CU,σUi to the gateway GW. Step 2. GW rejects the message M1 (and aborts the session) if TU is not fresh. Otherwise, GW computes KUG = yX and kUG = J(TUkXkYkKUG), and checks if VerkUGðIDGWkIDSNk PLOS ONE \| DOI:10.1371/journal.pone.0116709 April 7, 2015 10 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0116709 April 7, 2015 doi:10.1371/journal.pone.0116709.g001 PLOS ONE \| DOI:10.1371/journal.pone.0116709 April 7, 2015 Then, SN sends the message M3 = hρSNi to the user U. Then, SN sends the message M3 = hρSNi to the user U. Step 4. With M3 in hand, U checks if ρSN is equal to H(kUSkIDSNkTU). U aborts the session if the check fails or otherwise computes the session key sk = H(kUSkTUkIDSN). Step 4. With M3 in hand, U checks if ρSN is equal to H(kUSkIDSNkTU). U aborts the session if the check fails or otherwise computes the session key sk = H(kUSkTUkIDSN). Password update phase. One of the recommended guidelines for achieving better pass- word security is to enforce regular password updates. In our scheme, users can change their passwords either non-interactively or interactively. The non-interactive password change pro- cedure proceeds as follows: 1. U inserts his smart card into a card reader and enters the identity IDU, the current password PWU, and the new password PW 0 U. 1. U inserts his smart card into a card reader and enters the identity IDU, the current password PWU, and the new password PW 0 U. 2. The smart card computes XEID0 U ¼ XEIDU IðIDUkPWUÞ IðIDUkPW 0 UÞ and replaces XEIDU with XEID0 U. 2. The smart card computes XEID0 U ¼ XEIDU IðIDUkPWUÞ IðIDUkPW 0 UÞ and replaces XEIDU with XEID0 U. 2. The smart card computes XEID0 U ¼ XEIDU IðIDUkPWUÞ IðIDUkPW 0 UÞ and replaces XEIDU with XEID0 U. Although this procedure is simple and non-interactive, it may render the smart card unus- able if the user enters a wrong password by mistake or an adversary intentionally inputs an ar- bitrary password after gaining temporary access to the smart card. When an invalid password is entered, subsequent login requests of the user will be rejected unless it reregisters with the gateway. This problem can be addressed by storing a password verifier on the smart card, which is used to check the correctness of the user-given password. However, as soon as the smart card contains a password verifier, the scheme becomes vulnerable to an offline dictionary attack under the non-tamper-resistance assumption of smart cards and, consequently, fails to achieve two-factor security. This is clearly unacceptable and, therefore, we suggest the follow- ing interactive password change procedure. 1. U inserts his smart card into a card reader and enters the identity IDU, the current password PWU, and the new password PW 0 U. 1. Efficient, Anonymous User Authentication in Wireless Sensor Networks Fig 1. Our proposed authentication and key exchange protocol for wireless sensor networks. PLOS ONE \| DOI:10.1371/journal.pone.0116709 April 7, 2015 11 / 21 Efficient, Anonymous User Authentication in Wireless Sensor Networks TUkCU,σU) = 1. If the check fails, GW aborts the session. Otherwise, GW decrypts CU with key kUG and then EIDU with key z, and checks if the decryption of EIDU yields the same IDU as produced through the decryption of CU. If only the two IDs match, GW retrieves the cur- rent timestamp TGW, computes TUkCU,σU) = 1. If the check fails, GW aborts the session. Otherwise, GW decrypts CU with key kUG and then EIDU with key z, and checks if the decryption of EIDU yields the same IDU as produced through the decryption of CU. If only the two IDs match, GW retrieves the cur- rent timestamp TGW, computes CGW ¼ EnckGSðkUSÞ; CGW ¼ EnckGSðkUSÞ; sGW ¼ MackGSðIDGW k IDSN k TGW k TU k CGWÞ; sGW ¼ MackGSðIDGW k IDSN k TGW k TU k CGWÞ; and sends the message M2 = hIDGW,TGW,TU,CGW,σGWi to the sensor SN. and sends the message M2 = hIDGW,TGW,TU,CGW,σGWi to the sensor SN. Step 3. Upon receiving M2, SN verifies that (1) TGW is fresh and (2) VerkGSðIDGWkIDSNkTGWk TUkCGW,σGW) = 1. If any of the verifications fails, SN aborts the session. Otherwise, SN de- crypts CGW to obtain kUS and computes the session key sk and the authenticator ρSN as follows: sk ¼ HðkUS k TU k IDSNÞ; rSN ¼ HðkUS k IDSN k TUÞ: sk ¼ HðkUS k TU k IDSNÞ; rSN ¼ HðkUS k IDSN k TUÞ: Then, SN sends the message M3 = hρSNi to the user U. The smart card sends a password update request hTU,X,CUi to the gateway GW. 3. GW rejects the request if TU is not fresh. Otherwise, GW computes KUG = yX and kUG = J (TUkXkYkKUG), decrypts CU with key kUG and then EIDU with key z, and checks whether the two decryptions return the same IDU. If the check succeeds, GW computes ρGW = H (IDGWkIDUkXkkUG) and sends it to the smart card. Otherwise, GW sends a failure message to the smart card. 4. The smart card aborts the password change procedure if it receives a failure message or ρGW is not equal to H(IDGWkIDUkXkkUG). Otherwise, it sets XEIDU ¼ EIDU IðIDUkPW 0 UÞ. This interactive password change procedure provides a secure yet practical way of updating user password, though it is more expensive than the non-interactive one. doi:10.1371/journal.pone.0116709.t001 Then, SN sends the message M3 = hρSNi to the user U. U inserts his smart card into a card reader and enters the identity IDU, the current password PWU, and the new password PW 0 U. 12 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0116709 April 7, 2015 Efficient, Anonymous User Authentication in Wireless Sensor Networks 2. The smart card retrieves the current timestamp TU, selects a random x 2 Z q, and computes 2. The smart card retrieves the current timestamp TU, selects a random x 2 Z q, and computes X ¼ xP; KUG ¼ xY; kUG ¼ JðTU k X k Y k KUGÞ; The smart card sends a password update request hTU,X,CUi to the gateway GW Efficient, Anonymous User Authentication in Wireless Sensor Networks Table 2. Crypto++ 5.6.0 benchmarks for SHA-1, HMAC and AES. Algorithm SHA-1 HMAC (SHA-1) AES (with 128-bit key) CTR CBC OFB CFB ECB Cycles Per Byte 11.4 11.9 12.6 16.0 16.9 16.1 16.0 doi:10.1371/journal.pone.0116709.t002 Table 2. Crypto++ 5.6.0 benchmarks for SHA-1, HMAC and AES. point multiplications on the resource-constrained sensor SN. Note that scalar-point multiplica- tion is much more expensive than the lightweight cryptographic operations considered in the table, such as symmetric encryption/decryption, MAC generation/verification, and hash func- tion evaluation. Moreover, these two schemes fail to achieve user anonymity despite their use of elliptic curve cryptography. The schemes presented in [10, 11, 13–19] are computationally efficient, but suffer from the inherent failure of user anonymity. To the best of our knowledge, all existing SCA-WSN schemes fall into one of the two classes. point multiplications on the resource-constrained sensor SN. Note that scalar-point multiplica- tion is much more expensive than the lightweight cryptographic operations considered in the table, such as symmetric encryption/decryption, MAC generation/verification, and hash func- tion evaluation. Moreover, these two schemes fail to achieve user anonymity despite their use of elliptic curve cryptography. The schemes presented in [10, 11, 13–19] are computationally efficient, but suffer from the inherent failure of user anonymity. To the best of our knowledge, all existing SCA-WSN schemes fall into one of the two classes. According to Crypto++ 5.6.0 benchmarks that ran on an Intel Core 2 1.83 GHz processor under Windows Vista in 32-bit mode, SHA-1 and HMAC take 11.4 and 11.9 cycles per byte re- spectively; while AES (with 128-bit key) takes 12.6 to 16.9 cycles per byte, depending on the op- eration mode used—see Table 2 and we refer interested readers to http://www.cryptopp.com/ benchmarks.html for Crypto++ benchmarks for commonly used cryptographic algorithms. Our scheme requires the sensor SN to perform 1E+1A+2H operations which amount to about 4.5H operations. Therefore, in terms of computational requirements for SN, our scheme is comparable with other SCA-WSN schemes [11, 13–16, 18, 19] using only lightweight crypto- graphic techniques. Although the schemes of Vaidya et al. [10] and Kim et al. [17] require SN to perform only 2 hash function evaluations, these schemes do not achieve user anonymity and are vulnerable to a stolen smart card attack. Under the non-tamper-resistance assumption of smart cards, our scheme is the only one that provides user anonymity and resists stolen smart card attacks. Performance and Security Comparison In Table 1, we provide a comparative summary between our scheme and other SCA-WSN schemes both in terms of computation and security. As shown in the table, our scheme requires the sensor SN to perform only lightweight cryptographic operations while enjoying provable anonymity in an extension of the widely accepted model of Bellare et al. [30]. While the recent schemes of Shi & Gong [12] and Choi et al. [20] provide forward secrecy, they impose 2 scalar- Table 1. A comparative summary of smart-card-based user authentication schemes for wireless sensor networks. Scheme Computation Security SN U+SN+GW SKS UA FS RSSC RNC Jiang et al. [19] 5H 22H Yes No No No Yes Khan & Kumari [18] 7H 3E+20H Yes No No No Yes Kim et al. [17] 2H 18H Yes No No No Yes Chi et al. [16] 2E+1A+1H 4E+1A+5H Yes No No No Yes He et al. [15] 2E+1H 10E+7H Yes No No No Yes Kumar et al. [14] 2E+1H 7E+8H Yes No No No Yes Li et al. [13] 6H 26H Yes No No No Yes Xue et al. [11] 6H 22H Yes No No No Yes Vaidya et al. [10] 2H 15H Yes No No No No [17] Our scheme 1E+1A+2H 3M+5E+4A+7H Yes Proven No Yes Yes Choi et al. [20] 2M+5H 6M+18H Yes No Yes No Yes Shi & Gong [12] 2M+4H 6M+15H No [20] No Yes No Yes Yeh et al. [6] 2M+1P+2H 8M+2P+9H No [33] No No [33] No Yes SKS: session key security; UA: user anonymity; FS: forward secrecy; RSSC: resistance to stolen smart card attacks; RNC: resistance to node capture attacks. M: scalar-point multiplication; P: map-to-point operation; E: symmetric encryption/decryption; A: MAC generation/veriﬁcation; H: hash function evaluation. comparative summary of smart-card-based user authentication schemes for wireless sensor networks. t-card-based user authentication schemes for wireless sensor networks. Table 1. A comparative summary of smart-card-based user authentication schemes for wireless sensor networks. Table 1. A comparative summary of smart-card-based user authentication schemes for wireless sen SKS: session key security; UA: user anonymity; FS: forward secrecy; RSSC: resistance to stolen smart card attacks; RNC: resistance to node capture attacks. M: scalar-point multiplication; P: map-to-point operation; E: symmetric encryption/decryption; A: MAC generation/veriﬁcation; H: hash function evaluation. PLOS ONE \| DOI:10.1371/journal.pone.0116709 April 7, 2015 13 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0116709 April 7, 2015 Security Proofs It is thus clear that privileged insider attacks cannot be mounted against our scheme. Stolen verifier attacks. In a stolen verifier attack, the adversary attempts to impersonate a legit- imate user by stealing the user’s password verifier stored on the gateway GW. However, in our scheme, GW does not store a password verifier of any kind but stores only two master secrets y and z which are selected independently of user passwords. Hence, our scheme is se- cure against stolen verifier attacks. User Anonymity Theorem 1. Our authentication and key exchange protocol, P, provides user anonymity in the random oracle model under the ECCDH assumption in G and the security of the symmetric en- cryption scheme Δ. Proof. Let A be a PPT adversary against the user anonymity property of protocol P. We prove the theorem by making a series of modifications to the original experiment ExpUA0, bounding the difference in the success probability of A between two consecutive experiments, and ending up with an experiment where A has a success probability of 1/2 (i.e., A has no advantage). Let SuccUAi denote the event that A correctly guesses the hidden bit b chosen by the TestUA or- acle in experiment ExpUAi. Let ti UA be the maximum time required to perform the experiment ExpUAi involving the adversary A. Experiment ExpUA1. In this experiment, we simulate the random oracle J as follows: Simulation of the J oracle: For each J query on a string str, the simulator first checks if an entry of the form (str,j) is in a list called JList which contains all the input-output pairs of J. If such an entry exists in JList, the simulator returns j as the output of the J query. Otherwise, the simulator chooses a random ‘-bit string j0, returns j0 in response to the query, and adds the entry (str,j0) to JList. For all other oracle queries of A, the simulator answers them as in the original experiment ExpUA0. Then, ExpUA1 is perfectly indistinguishable from ExpUA0 and therefore, Claim 1 holds. Claim 1. PrP;A½SuccUA1 ¼ PrP;A½SuccUA0. Claim 1. PrP;A½SuccUA1 ¼ PrP;A½SuccUA0. Experiment ExpUA2. Here, we modify the experiment so that X is computed as follows: The ExpUA2 modification: Experiment ExpUA2. Here, we modify the experiment so that X is computed as follows: The ExpUA2 modification: The ExpUA2 modification: • The simulator chooses a random exponent a 2 Z q and computes A = aP. • The simulator chooses a random exponent a 2 Z q and computes A = aP. • For each user instance, the simulator chooses a random r 2 Z q and sets X = rA. • For each user instance, the simulator chooses a random r 2 Z q and sets X = rA. As a result of the modification, each KUG is set to rayP for some random r 2 Z q. Security Proofs We now prove that the authentication and key exchange protocol of our scheme is AKE-secure (in the sense of Definition 2) and provides user anonymity (in the sense of Definition 4). Recall that the security model described in Section 2 captures various SCA-WSN specific attacks (such as stolen smart card attacks, node capture attacks, privileged insider attacks, and stolen verifier at- tacks) as well as other common attacks (like impersonation attacks, man-in-the-middle attacks, replay attacks, and known key attacks) [21, 23, 25, 34]. Before providing formal security proofs in the model, we briefly discuss the security of our scheme against SCA-WSN specific attacks. Stolen smart card attacks. Our scheme does not require a password verifier to be stored on the smart card of user U. Moreover, even if an adversary managed to obtain the ciphertext CU ¼ EnckUGðIDUkEIDUkkUSÞ, the adversary would be unable to exploit CU as a password verifier since, under the ECCDH assumption, it is infeasible to compute kUG = J (TUkXkYkKUG) from X and Y. Thus, our scheme is resistant against stolen smart card attacks. Stolen smart card attacks. Our scheme does not require a password verifier to be stored on the smart card of user U. Moreover, even if an adversary managed to obtain the ciphertext CU ¼ EnckUGðIDUkEIDUkkUSÞ, the adversary would be unable to exploit CU as a password verifier since, under the ECCDH assumption, it is infeasible to compute kUG = J (TUkXkYkKUG) from X and Y. Thus, our scheme is resistant against stolen smart card attacks. Node capture attacks. In our scheme, each sensor node SN holds its individual secret key kGS = J(IDSNkz) which is shared only with the gateway GW. In other words, different sensor nodes have different secret keys (with an overwhelming probability). Thus, the secret key kGS obtained by capturing a sensor node SN will be of no use in impersonating another PLOS ONE \| DOI:10.1371/journal.pone.0116709 April 7, 2015 14 / 21 Efficient, Anonymous User Authentication in Wireless Sensor Networks sensor node SN0 who holds a secret key other than kGS. Therefore, node capture attacks are not possible against our scheme. Privileged insider attacks. A privileged insider attack occurs when the gateway administrator can access a user’s password to impersonate the user. In our scheme, the gateway GW re- ceives no password-related information from the user U and does not manage any table for storing such information. PLOS ONE \| DOI:10.1371/journal.pone.0116709 April 7, 2015 User Anonymity Since the view of A is identical between ExpUA2 and ExpUA1, it follows that: Claim 2. PrP;A½SuccUA2 ¼ PrP;A½SuccUA1. As a result of the modification, each KUG is set to rayP for some random r 2 Z q. Since the view of A is identical between ExpUA2 and ExpUA1, it follows that: Claim 2. PrP;A½SuccUA2 ¼ PrP;A½SuccUA1. Experiment ExpUA3. We next modify the computations of X and Y as follows: The ExpUA3 modification: • The simulator chooses two random elements A, B 2 G and sets Y = B. PLOS ONE \| DOI:10.1371/journal.pone.0116709 April 7, 2015 15 / 21 Efficient, Anonymous User Authentication in Wireless Sensor Networks • For each instance of clean users, the simulator chooses a random r 2 Z q and sets X = rA. For other instances, the simulator computes X as in experiment ExpUA2. PLOS ONE \| DOI:10.1371/journal.pone.0116709 April 7, 2015 • For each instance of clean users, the simulator chooses a random r 2 Z q and sets X = rA. For other instances, the simulator computes X as in experiment ExpUA2. • For each instance of clean users, the simulator chooses a random r 2 Z q and sets X = rA. For other instances, the simulator computes X as in experiment ExpUA2. • For each instance of clean users, the simulator sets each kUG to a random ‘-bit string. For other instances, the simulator computes kUG as in experiment ExpUA2. • For each instance of clean users, the simulator sets each kUG to a random ‘-bit string. For other instances, the simulator computes kUG as in experiment ExpUA2. • For each instance of clean users, the simulator sets each kUG to a random ‘-bit string. For other instances, the simulator computes kUG as in experiment ExpUA2. Since kUG is set to a random ‘-bit string (for instances of clean users), the success probability of A may be different between ExpUA3 and ExpUA2 if it makes an J(TUkXkYkKUG) query. However, this difference is bounded by Claim 3. Claim 3. j PrP;A½SuccUA3 PrP;A½SuccUA2 j 1=qJ AdvECCDH G ðt3 UAÞ, where qJ is the number of queries made to the J oracle. Proof. We prove the claim via a reduction from the ECCDH problem which is believed to be hard. Assume that the success probability of A is non-negligibly different between ExpUA3 and ExpUA2. Then we construct an algorithm AECCDH that solves the ECCDH problem in G with a non-negligible advantage. The objective of AECCDH is to compute and output the value W = uvP 2 G when given an ECCDH-problem instance (U = uP, V = vP) 2 G2. AECCDH runs A as a subroutine while simulating all the oracles on its own. AECCDH handles all the oracle queries of A as specified in experiment ExpUA3 but using U and V in place of X and Y. When A outputs its guess b0, AECCDH chooses an entry of the form (TUkXkYkK,j) at random from JList and terminates outputting K/r. From the simulation, it is clear that AECCDH outputs the desired result W = uvP with probability at least 1/qJ if A makes a J(TUkXkYkKUG) query for some instance of a clean user U 2 U. This completes the proof of Claim 3. Experiment ExpUA4. We finally modify the experiment so that, for each clean user U 2 U, a random identity ID0 U drawn from the identity space is used in place of the true identity IDU in generating CU. • For each instance of clean users, the simulator chooses a random r 2 Z q and sets X = rA. For other instances, the simulator computes X as in experiment ExpUA2. Claim 4. j PrP;A½SuccUA4 PrP;A½SuccUA3 j AdvINDMEK D ðt4 UAÞ. Claim 4. j PrP;A½SuccUA4 PrP;A½SuccUA3 j AdvINDMEK D ðt4 UAÞ. Proof. We prove the claim by constructing an eavesdropping adversary AIND-MEK who attacks the indistinguishability of Δ in ExpINDMEK D ðA; n; d; bÞ with advantage equal to jPrP;A½SuccUA4 PrP;A½SuccUA3j (see Section 1 for details of experiment ExpINDMEK D ðA; n; d; bÞ). AIND-MEK begins by choosing a random bit b 2 {0, 1}. Then, AIND-MEK invokes the adver- sary A and answers all the oracle queries of A as in experiment ExpUA3 except that, for each clean user U 2 U, it generates CU by accessing its own encryption oracle as follows: AIND-MEK outputs ðIDUkEIDUkkUS; ID0 UkEIDUkkUSÞ as the first plaintext-pair in the indis- tinguishability experiment ExpINDMEK D . Let c1 be the ciphertext received in return for the first pair. AIND-MEK sets CU equal to the ciphertext c1. That is, AIND-MEK sets CU to the encryption of either IDUkEIDUkkUS or ID0 UkEIDUkkUS. Now when A terminates and outputs its guess b0, AIND-MEK outputs 1 if b = b0, and 0 otherwise. Then, it is clear that: That is, AIND-MEK sets CU to the encryption of either IDUkEIDUkkUS or ID0 UkEIDUkkUS. Now when A terminates and outputs its guess b0, AIND-MEK outputs 1 if b = b0, and 0 otherwise. Then, it is clear that: That is, AIND-MEK sets CU to the encryption of either IDUkEIDUkkUS or ID0 UkEIDUkkUS. Now when A terminates and outputs its guess b0, AIND-MEK outputs 1 if b = b0, and 0 otherwise. Then, it is clear that: • the probability that AIND-MEK outputs 1 when the first plaintexts are encrypted in the experi- ment ExpINDMEK D is equal to the probability that A succeeds in the experiment ExpUA3, and • the probability that AIND-MEK outputs 1 when the second plaintexts are encrypted in the ex- periment ExpINDMEK D is equal to the probability that A succeeds in the experiment ExpUA4. 16 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0116709 April 7, 2015 Efficient, Anonymous User Authentication in Wireless Sensor Networks That is, AdvINDMEK D ðAINDMEKÞ ¼j PrP;A½SuccUA4 PrP;A½SuccUA3 j. Note that in the sim- ulation, AIND-MEK eavesdrops at most qsend encryptions which is polynomial in the security pa- rameter ‘. This completes the proof of Claim 4. • For each instance of clean users, the simulator chooses a random r 2 Z q and sets X = rA. For other instances, the simulator computes X as in experiment ExpUA2. In the experiment ExpUA4, the adversary A gains no information on the hidden bit b cho- sen by the TestUA oracle because the identities of all clean users are chosen uniformly at ran- dom from the identity space. It, therefore, follows that PrP;A½SuccUA4 ¼ 1=2. This result combined with Claims 1–4 yields the statement of Theorem 1. AKE Security Theorem 2. As long as the MAC scheme S and the symmetric encryption scheme Δ are both se- cure, our authentication and key exchange protocol P is secure in the random oracle model under the ECCDH assumption in G. Proof. Fix a PPT adversary A against the security of the protocol P. To prove the theorem, we make a series of modifications to the original experiment ExpAKE0, bounding the effect of each change in the experiment on the success probability of A and ending up with an experi- ment where A has a success probability of 1/2. We use SuccAKEi to denote the event that A correctly guesses the hidden bit b chosen by the Test oracle in experiment ExpAKEi. Let ti AKE be the maximum time required to perform the experiment ExpAKEi involving the adversary A. y g y p p Let ti AKE be the maximum time required to perform the experiment ExpAKEi involving the adversary A. Experiment ExpAKE1. This experiment is different from ExpAKE0 in that the random oracle J is simulated as follows: Simulation of the J oracle: For each J query on a string str, the simulator first checks if an entry of the form (str,j) is in a list called JList which contains all the input-output pairs of J. If such an entry exists in JList, the simulator returns j as the output of the J query. Otherwise, the simulator chooses a random ‘-bit string j0, returns j0 in response to the query, and adds the entry (str,j0) to JList. The other oracle queries of A are answered as in the original experiment ExpAKE0. Then, since J is a random oracle, ExpAKE1 is perfectly indistinguishable from ExpAKE0, and Claim 5 immediately follows. y Claim 5. PrP;A½SuccAKE1 ¼ PrP;A½SuccAKE0. Claim 5. PrP;A½SuccAKE1 ¼ PrP;A½SuccAKE0. Claim 5. PrP;A½SuccAKE1 ¼ PrP;A½SuccAKE0. Experiment ExpAKE2. Here, we modify the experiment so that X is computed as follows: The ExpAKE2 modification: Experiment ExpAKE2. Here, we modify the experiment so that X is computed as follows: The ExpAKE2 modification: • The simulator chooses a random exponent a 2 Z q and computes A = aP. • For each instance of users, the simulator chooses a random r 2 Z q and sets X = rA. As a result, each KUG is set to rayP for some random r 2 Z q. PLOS ONE \| DOI:10.1371/journal.pone.0116709 April 7, 2015 AKE Security Since the view of A is identical between ExpAKE2 and ExpAKE1, it follows that: tween ExpAKE2 and ExpAKE1, it follows that: Claim 6. PrP;A½SuccAKE2 ¼ PrP;A½SuccAKE1. Experiment ExpAKE3. We further modify the experiment as follows: The ExpAKE3 modification: • The simulator chooses two random elements A,B 2 G and sets Y = B. • For each fresh instance, the simulator chooses a random r 2 Z q and sets X = rA. For other in- stances, the simulator computes X as in experiment ExpAKE2. • For each fresh instance, the simulator sets each kUG to a random ‘-bit string. For other in- stances, the simulator computes kUG as in experiment ExpAKE2. 17 / 21 Efficient, Anonymous User Authentication in Wireless Sensor Networks Since kUG is set to a random ‘-bit string (for fresh instances), the success probability of A may be different between ExpAKE2 and ExpAKE3 if it makes an J(TUkXkYkKUG) query. This difference is bounded by Claim 7. Claim 7 j PrP;A½SuccAKE3 PrP;A½SuccAKE2 j 1=qJ AdvECCDH G ðt3 AKEÞ, where qJ is the number of queries made to the J oracle. Proof. We prove the claim via a reduction from the ECCDH problem which is believed to be hard. Assume that the success probability of A is non-negligibly different between ExpAKE2 and ExpAKE3. Then we construct an algorithm AECCDH that solves the ECCDH problem in G with a non-negligible advantage. The objective of AECCDH is to compute and output the value W = uvP 2 G when given an ECCDH-problem instance (U = uP,V = vP) 2 G2. AECCDH runs A as a subroutine while simulating all the oracles on its own. AECCDH handles all the oracle queries of A as specified in experiment ExpAKE3 but using U and V in place of X and Y. When A outputs its guess b0, AECCDH chooses an entry of the form (TUkXkYkK,j) at random from JList and terminates outputting K/r. From the simulation, it is clear that AECCDH outputs the desired result W = uvP with probability at least 1/qJ if A makes a J(TUkXkYkKUG) query for some fresh instance of any U 2 U. This completes the proof of Claim 7. Experiment ExpAKE4. This experiment is different from ExpAKE3 in that it is aborted if the following event Forge occurs. Experiment ExpAKE4. AKE Security This experiment is different from ExpAKE3 in that it is aborted if the following event Forge occurs. g Forge: The event that the adversary A makes a Send query that contains a MAC forgery. Then we claim that: Forge: The event that the adversary A makes a Send query that contains a MAC forgery. Then we claim that: Then we claim that: Claim 8 j PrP;A½SuccAKE4 PrP;A½SuccAKE3 j qsend AdvEFCMA S ðt4 AKEÞ, where qsend is the number of queries made to the Send oracle. Claim 8 j PrP;A½SuccAKE4 PrP;A½SuccAKE3 j qsend AdvEFCMA S ðt4 AKEÞ, where qsend is the number of queries made to the Send oracle. Claim 8 j PrP;A½SuccAKE4 PrP;A½SuccAKE3 j qsend AdvEFCMA S ðt4 AKEÞ, where qsend is the number of queries made to the Send oracle. Proof. Assume that the event Forge occurs with a non-negligible probability. Then, we con- struct an algorithm AEF who generates, with a non-negligible probability, a forgery against the MAC scheme S. The algorithm AEF is is given access to the MackðÞ and VerkðÞ oracles. The goal of AEF is to produce a message/MAC pair (m,σ) such that: (1) Verkðm; sÞ ¼ 1 and (2) σ has not been output by the oracle MackðÞ on input m. Let n be the total number of MAC keys used in the sessions initiated via a Send query. AEF begins by choosing a random i 2 {1, . . ., n}. Let ki denote the ith key among all the n MAC keys, and Sendi be any Send query that is expected to be answered and/or veriﬁed using ki. AEF runs A as a subroutine and answers the oracle queries of A as in experiment ExpAKE3 except that: it answers all Sendi queries by accessing its MackðÞ and VerkðÞ oracles. As a result, the ith MAC key ki is not used during the simulation. If Forge occurs against an instance who holds AEF halts and outputs the message/MAC pair generated by A as its forgery. Otherwise, AEF terminates with a failure indication. If the guess i is correct, then the simulation is perfect and AEF achieves its goal. Namely, AdvEFCMA S ðAEFÞ ¼ Pr½Forge=n. AKE Security Since n qsend and AEF runs in time at most t4 AKE, we get Pr½Forge qsend AdvEFCMA S ðAEFÞ qsend AdvEFCMA S ðt4 AKEÞ: This completes the proof of Claim 8. This completes the proof of Claim 8. Experiment ExpAKE5. We next modify the way of answering queries to the H oracle as fol- lows:Simulation of the H oracle: For each H query on a string str, the simulator first checks if an entry of the form (str,h) is in a list called HList which is maintained to store input-output pairs of H. If it is, h is the answer to the hash query. Otherwise, the simulator chooses a random κ-bit string h0, answers the query with h0, and adds the entry (str,h0) to HList. PLOS ONE \| DOI:10.1371/journal.pone.0116709 April 7, 2015 18 / 21 Efficient, Anonymous User Authentication in Wireless Sensor Networks The other oracle queries of A are handled as in experiment ExpAKE4. Since ExpAKE5 is perfectly indistinguishable from ExpAKE4, it is clear that: Claim 9. PrP;A½SuccAKE5 ¼ PrP;A½SuccAKE4 Claim 9. PrP;A½SuccAKE5 ¼ PrP;A½SuccAKE4 Experiment ExpAKE6. We finally modify the experiment so that the session key sk is set to a random κ-bit string for each fresh instance and its partner. Accordingly, the success probabil- ity of A may be different between ExpAKE6 and ExpAKE5 if it asks an H query of the form H (kUSkTUkIDSN) for some uncorrupted U 2 U and SN 2 SN. But the difference is bounded by: a random κ-bit string for each fresh instance and its partner. Accordingly, the success probabil- ity of A may be different between ExpAKE6 and ExpAKE5 if it asks an H query of the form H (kUSkTUkIDSN) for some uncorrupted U 2 U and SN 2 SN. But the difference is bounded by: Claim 10 j PrP;A½SuccAKE6 PrP;A½SuccAKE5 j 1 qH AdvINDMEK D ðt6 AKEÞ, where qH is the number of queries made to the H oracle. Claim 10 j PrP;A½SuccAKE6 PrP;A½SuccAKE5 j 1 qH AdvINDMEK D ðt6 AKEÞ, where qH is the number of queries made to the H oracle. Proof. We prove the claim by constructing an eavesdropper AIND-MEK who attacks the indistinguishability of Δ in experiment ExpINDMEK D ðA; n; d; bÞ. AKE Security AIND-MEK invokes the adver- sary A and answers all the oracle queries of A as in experiment ExpAKE5 except that it gener- ates each CGW to be sent to a fresh sensor instance by accessing its own encryption oracle as follows: Let k0 US 6¼ kUS be a random string chosen from {0, 1}κ. AIND-MEK outputs ðkUS; k0 USÞ as a plaintext pair in the indistinguishability experiment ExpINDMEK D . Let c be the ciphertext received in return for the plaintext pair. AIND-MEK sets CGW equal to the ciphertext c. That is, each CGW is set to the encryption of either kUS or k0 US. Now when A terminates and outputs its guess b0, AIND-MEK selects an entry of the form (kUSkTUkIDSN,h) at random from HList and outputs 0 if k = kUS, and 1 otherwise. 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In WSNs, for example, designing a secure and effi- cient user authentication scheme without compromising user anonymity remains an area of active research. In this work, we have presented a SCA-WSN scheme, a smart-card-based user authentication scheme for wireless sensor networks, which achieves user anonymity without imposing (expen- sive) public key operations on sensors. Our result in this paper does not contradict the result of Wang and Wang [28, 29] but rather supports and clarifies it: in order for a SCA-WSN scheme to achieve user anonymity, the use of public key cryptography is inevitable but, if forward secrecy is not desired, can be avoided at least on the sensor side. 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https://openalex.org/W1593940196	https://europepmc.org/articles/pmc4495016?pdf=render	English	null	Silicone Implants with Smooth Surfaces Induce Thinner but Denser Fibrotic Capsules Compared to Those with Textured Surfaces in a Rodent Model	PloS one	2,015	cc-by	8,480	RESEARCH ARTICLE Purpose Citation: Fischer S, Hirche C, Reichenberger MA, Kiefer J, Diehm Y, Mukundan S, Jr., et al. (2015) Silicone Implants with Smooth Surfaces Induce Thinner but Denser Fibrotic Capsules Compared to Those with Textured Surfaces in a Rodent Model. PLoS ONE 10(7): e0132131. doi:10.1371/journal. pone.0132131 Capsular contracture is the most frequent long-term complication after implant-based breast reconstruction or augmentation. The aim of this study was to evaluate the impact of implant surface properties on fibrotic capsule formation in an animal model. Materials and Methods Twenty-four rats received 1 scaled down silicone implant each; 12 of the rats received implants with textured surfaces, and the other 12 received implants with smooth surfaces. After 60 and 120 days, rats in each group underwent 7-Tesla Magnetic Resonance Imaging (MRI) and high-resolution ultrasound (HR-US), and specimens of the capsules were acquired and used to measure capsule thickness through histology, collagen density through picro sirius red staining, and analyses of expression of pro-fibrotic and inflammatory genes (Collagen1-4, TGFb1, TGFb3, Smad3, IL4, IL10, IL13, CD68) through qRT-PCR. Furthermore, MRI data were processed to obtain capsule volume and implant surface area. Editor: Corrado Rubino, University of Salerno, ITALY Editor: Corrado Rubino, University of Salerno, ITALY Received: March 26, 2015 Accepted: June 10, 2015 Published: July 7, 2015 Copyright: © 2015 Fischer 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. * sfischer@hotmail.de Results On day 60, histology and HR-US showed that fibrotic capsules were significantly thicker in the textured implant group with respect to the smooth implant group (p<0.05). However, this difference did not persist on day 120 (p=0.56). Capsule thickness decreased significantly over the study period in both smooth and textured implant groups (p<0.05). Thickness mea- surements were substantiated by MRI analysis and volumes changed accordingly. Implant surface area did not vary between study dates, but it was different between implant types. Data Availability Statement: All relevant data are within the paper. Silicone Implants with Smooth Surfaces Induce Thinner but Denser Fibrotic Capsules Compared to Those with Textured Surfaces in a Rodent Model Sebastian Fischer1,2*, Christoph Hirche2, Matthias A. Reichenberger3, Jurij Kiefer2, Yannick Diehm2, Srinivasan Mukundan, Jr.4, Muayyad Alhefzi1, Ericka M. Bueno1, Ulrich Kneser2, Bohdan Pomahac1 1 Department of Surgery, Division of Plastic Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, United States of America, 2 Department of Hand-, Plastic and Reconstructive Surgery, BG Clinic Ludwigshafen, University of Heidelberg, Ludwigshafen, Germany, 3 Ethianum, Clinic for Plastic and Reconstructive Surgery, Aesthetic and Preventive Medicine at Heidelberg University Hospital, Heidelberg, Germany, 4 Department of Radiology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, United States of America a1111 Introduction According to the American society of plastic surgeons, about 400,000 women in the United States only undergo cosmetic or reconstructive breast augmentations annually, making inser- tion of silicone implants the most frequently performed intervention in plastic surgery [1]. Although no other biomaterial reveals comparable properties in terms of availability, adapt- ability and immunogenicity, silicone remains a foreign body that is prone to a physiological and obligatory foreign body reaction [2]. Thereby, the inserted silicone gets encapsulated by fibrotic tissue, which in spite of intending to protect the organism against potentially harmful materials, can actually lead to a harmful complication itself—namely capsular contracture [3]. Visible deformities, palpable hardness and progressive pain make capsular contracture clini- cally relevant in up to 30% of cosmetic and even 73% of reconstructive cases after radiation therapy, and thus capsular contracture is the most common long-term complication after sili- cone breast reconstruction and augmentation [4–6]. Proposed mechanisms include direct immunostimulation and sub-clinical infection, which are held mainly responsible for the initi- ation and maintenance of capsule formation [7, 8]. Both mechanisms are capable of inducing a chronic inflammatory reaction that stimulates proliferation and differentiation of fibroblasts and the ensuing synthesis of collagen and other extracellular matrix proteins. Whereas sterile working conditions and perioperative antibiotics reduce the risk of infection, modifications of the silicone implant, especially its surface, were invented to increase biocompatibility and thus decrease capsular fibrosis [9–11]. Today, smooth, textured and poly-urethane covered implants are available. Although the latter revealed good results in recent studies, removal can be pains- taking when necessary, and foremost within the context of the ongoing discussion about cancer induction, poly-urethane covered implants are the less preferred therapeutic option for most surgeons [4, 12]. In contrast smooth or textured implants are commonly used in clinical prac- tice for breast augmentation. Interestingly, the incidence of capsular fibrosis varies significantly in current literature and the choice of surface type relies mainly on the personal preference of the surgeon. However, in a recently published meta-analysis by Liu et al. only surface proper- ties and in particular smooth surfaces are more likely associated with capsular contracture [13]. Reports such as this demonstrate the impact of the implant’s surface on capsular fibrosis and the need for further studies that can explain these clinical findings. Conclusion Silicone implants with textured surfaces led to temporarily thicker but less dense fibrotic capsules compared with smooth surfaces. 7-Tesla MRI and HR-US are capable for non- invasive in-vivo assessment of capsular fibrosis in an animal model and can provide unique insights into the fibrotic process by 3D reconstruction and surface area measurement. Data Availability Statement: All relevant data are within the paper. Funding: The authors received no specific funding for this work. Competing Interests: The authors have declared that no competing interests exist. PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 1 / 21 Smooth versus Textured Silicone Implants On day 60, the density of collagen in the fibrotic capsules was significantly lower in the tex- tured implant group with respect to the smooth group (p<0.05), but again this difference did not persist on day 120 (p=0.67). Collagen 1 and CD68 were respectively over- and under expressed in the textured implant group on day 60. Significant differences in the expression of other genes were not observed. PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 Introduction In addition, animal models lack in non-invasive in-vivo assessments of capsule formation, and especially objective ways to assess contracture, which is mandatory for investigation of therapeutic applications in future studies. PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 2 / 21 Smooth versus Textured Silicone Implants Therefore, the aims of this study were to demonstrate the impact of smooth vs. textured sur- faces of silicone implants on capsule formation and to verify histological and biochemical find- ings by non-invasive in-vivo measurements. Animal Model After approval of the animal experimental protocol (AEP) by the IACUC committee of the Harvard Medical Area Standing Committee on Animals (AEP No.: 05086), twenty-four female Lewis rats (200-225gr./10-12wks.) were randomly divided into four groups of 6 rats each. Two groups (n = 12) received textured (t) silicone implants (Polytech Health and Aesthetic, Die- burg, Germany, gel filled, pore size range of 50–900 μm, Fig 1 left) and two groups received smooth (s) silicone implants (Polytech Health and Aesthetic, Dieburg, Germany, gel filled, 2cm in diameter, Fig 1 right). One sub-group (n = 6) within each implant type was euthanized at 60 days, and the second was euthanized at 120 days after insertion of the silicone device. According to Sengupta et al., one month of an adult rat’s life equates to 3 human years, provid- ing a total follow-up period of approximately 6 and 12 human years, respectively, which is rele- vant to the human population suffering from capsule contracture [14]. Insertion was performed as described before under sterile conditions through a 4 cm longitudinal paraverteb- ral incision [15]. The implant was placed on the opposite side of the spine above the scapula to prevent interference of capsule formation with scar tissue that might develop from the incision. During implantation particular attention was paid to hemostasis to avoid hematoma forma- tion. Absorbable 4–0 Vicryl sutures were used for wound closure. Explantation was performed at the given time point by resecting the implant with surround- ing capsule en-bloc. For further analysis we utilized the parietal part of the capsule between the implant and the skin. Fig 1. Gel-filled textured (left) and smooth (right) silicone implants (Polytech Health and Aesthetic, Dieburg, Germany). doi:10.1371/journal.pone.0132131.g001 Fig 1. Gel-filled textured (left) and smooth (right) silicone implants (Polytech Health and Aesthetic, Dieburg, Germany). d i 10 1371/j l 0132131 001 Fig 1. Gel-filled textured (left) and smooth (right) silicone implants (Polytech Health and Aesthetic, Dieburg, Germany). doi:10.1371/journal.pone.0132131.g001 doi:10.1371/journal.pone.0132131.g001 3 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 Smooth versus Textured Silicone Implants Magnetic Resonance Imaging Magnetic resonance imaging (MRI) was performed on a commercially available UltraHigh Field MRI preclinical scanner, 7.0T Bruker BioSpec (Billerica, MA, USA). Imaging was per- formed on 2 rats of each study group. Scans were run in the FLASH 3D mode using a volume transmit and receive coil with FOV of 30×30 mm2, TR/TE of 15/4.662 ms and a matrix size of 180×240×240. Isotropic spatial resolution of 0.150×0.150×0.150 mm/pixel was achieved in order to allow for accurate measurement in any geometric plane. The imaging parameters were kept identical for each animal and study group. Data were converted to DICOM format and analyzed via ImageJ (v. 1.46, NIH, USA) and 3D Volume measurement plug-in. Thickness measurements were performed in the axial plane, which was perpendicular to the fat-water- shift direction. The most visceral height of this plane was chosen and measurements were made at thickest part of each quadrant and from the outer margin of the implant to the outer side of the skin (Fig 2, red arrows). Volumetric measurements were made by VOLUMEST [16] plug-in for ImageJ (v. 1.46, NIH, USA). To assess the Volume of the capsule we subtracted the implant volume from the total cap- sule volume (Fig 3). To calculate surface area of the implant (A) we used the following formula: A ¼ ðSAE=2Þ þ p x y where SAE is the Knut Thomsen formula for calculation of the surface area of an ellipsoid [17]: SAE = 4π (((zx)1,6075+(zy)1,6075+(xy)1,6075)/3) 1/1,6075 p ¼ 3:14159 p ¼ 3:14159 X, y and z variables were measured from 3D reconstructed MR images with means of 3D viewer tool of ImageJ (v. 1.46, NIH, USA) (Fig 4). By dividing SAE by 2 and adding the surface area of the base of the implant, which is in the shape of an ellipse, we obtain an approximate surface area of the entire implant. All measurements were performed by two blinded observers and expressed as the mean ± standard deviation (SD). Histology After formalin fixation and paraffin embedding, hematoxylin eosin staining was utilized for capsule thickness assessment. The thickness of the capsules was measured between the cutis and the next tissue that does not belong to the capsule, namely fat or muscle. Measurements were performed with ImageJ (v. 1.46, NIH, USA) at the thickest part of the capsule. To assess collagen density we performed picrosirius red staining and measured pixel density with means of ImageJ (v. 1.46, NIH, USA) in two fields per section randomly chosen by two blinded inves- tigators. All measurements were performed by two blinded observers and expressed as the mean ± standard deviation (SD). High-Resolution Ultrasound High-resolution ultrasound (HR-US) was performed in each rat in a blinded fashion with means of a 48 MHz transducer (Accutome UBM plus, Accutome, Malvern, PA, USA) on day 60 and 120 after insertion, depending on the study group. Conventional transmission gel (Aquasonic 100, Parker Laboratories, Fairfield, NJ, USA) was applied to the shaved skin above the implant and a short video sequence was taken longitudinal across the implant and parallel to the spine. Measurements were performed on the height of the outer margin of the implant PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 4 / 21 Smooth versus Textured Silicone Implants Fig 2. Example picture of MR image in axial plane utilized for thickness measurements. Measurements were performed in each quadrant from the shell of the implant to the outer side of the skin (red arrows). Scale was set with means of the inner circle that had the standardized diameter of 0.8 centimeter. Fig 2. Example picture of MR image in axial plane utilized for thickness measurements. Measurements were performed in each quadrant from the shell of the implant to the outer side of the skin (red arrows). Scale was set with means of the inner circle that had the standardized diameter of 0.8 centimeter. axial plane utilized for thickness measurements. Measurements were performed in each quadrant from the shell red arrows). Scale was set with means of the inner circle that had the standardized diameter of 0.8 centimeter. Fig 2. Example picture of MR image in axial plane utilized for thickness measurements. Measurements were of the implant to the outer side of the skin (red arrows). Scale was set with means of the inner circle that had the stan doi:10.1371/journal.pone.0132131.g002 doi:10.1371/journal.pone.0132131.g002 doi:10.1371/journal.pone.0132131.g002 (Fig 5, yellow line). This measurement site equates to the measurement site of the MRI. All measurements were performed by two blinded observers and expressed as the mean ± standard deviation (SD). PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 Real-Time Quantitative PCR Total cellular RNA was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and reverse transcribed into cDNA by the ABI PRISM TaqMan reverse transcription method. Expression for genes of interest was analyzed in previously shock frozen tissue samples from capsules that were harvested at 60 or 120 days, depending on the study group. Primers were purchased from Life Technologies Corporation (Carlsbad, CA, USA) and are listed in Table 1. Quantitative real-time PCR was performed via ABI PRISM 7900HT Sequence Detection Sys- tem (Applied Biosystems, Carlsbad, CA) and data analyzed by ExpressionSuite Software v 1.0.4 (Life Technologies Corporation, Carlsbad, CA, USA). This software utilizes the comparative CT (ΔΔ CT) method to quantify relative gene expression. After normalization to the expression 5 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 Fig 3. Example pictures of MR images for volumetric measurements. From top to bottom: Blank slides, complete capsule volume (green), volume (red) and complete minus implant volume (green margin). Volume calculations were performed via VOLUMEST and the scale was adju automatically by the software. doi:10.1371/journal.pone.0132131.g003 Smooth versus Textured Sil Smooth versus Textured Silicone Implants Fig 3. Example pictures of MR images for volumetric measurements. From top to bottom: Blank slides, complete capsule volume (green), implant volume (red) and complete minus implant volume (green margin). Volume calculations were performed via VOLUMEST and the scale was adjusted automatically by the software. doi:10.1371/journal.pone.0132131.g003 6 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 Smooth versus Textured Silicone Implants Fig 4. Example picture of MR image after 3D reconstruction and displayed as multi-orthoslice. Measurements were performed at the x-, y- and z-ax (blue arrows). Scale was adjusted automatically by the software. doi:10.1371/journal.pone.0132131.g004 Fig 4. Example picture of MR image after 3D reconstruction and displayed as multi-orthoslice. Measurements were performed at the x-, y- and z-axis (blue arrows). Scale was adjusted automatically by the software. doi:10.1371/journal.pone.0132131.g004 of beta-2 microglobulin acting as housekeeping gene, relative gene expression among study groups are given in relative quantitation (RQ). of beta-2 microglobulin acting as housekeeping gene, relative gene expression among study groups are given in relative quantitation (RQ). Statistical Analysis The independent samples t-Test was utilized to analyze data related to capsular thickness and volume measurements, implant surface area measurements, collagen density and differences in expression of tested genes. Statistical significance level was set as p<0.05. PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 7 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 Smooth versus Textured Silicone Implants Fig 5. Example picture of HR-US image. Measurements were performed at the outer margin of the implant (yellow line). Scale was adjusted automatically by the software. age. Measurements were performed at the outer margin of the implant (yellow line). Scale was adjusted automatical Fig 5. Example picture of HR-US image. Measurements were performed at the outer margin of the implant (ye by the software. Fig 5. Example picture of HR-US image. Measurements were performed at the outer margin of the implant (yellow line). Scale was adjusted automatically by the software. doi:10.1371/journal.pone.0132131.g005 PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 High-Resolution Ultrasound Ultrasound measurements revealed significant differences in thickness of smooth vs. textured implants on day 60 (0.41±0.12mm vs. 0.73±0.12mm; p<0.05) and between textured implants on day 60 and smooth implants on day 120 (0.73±0.12mm vs. 0.31±0.3mm; p<0.05). On day 120, no significant differences related to implant surfaces were found (0.31±0.2mm vs. 0.51 ±0.1%; p = 0.07). Results are depicted in Fig 13. Histology Capsule thickness. The thickness of fibrotic capsules was significantly different between smooth and textured implants on day 60 (60s: 520.3±149μm vs. 60t: 1066.1±263.9μm; p<0.05) but not on day 120 (120s: 282.9±118μm vs. 120t: 361.6±52.4μm; p = 0.56). Sixty days after insertion of both smooth or textured implants, capsule thickness decreased significantly over time (60s vs. 120s; p<0.05 and 60t vs. 120t; p<0.005). Results are demonstrated in Fig 6. Collagen Density. Pixel density varied significantly between smooth and textured implants groups on day 60 (93.6±2.9% vs. 73.3±4.3%; p<0.05). In addition, significant differ- ences were seen between the textured group on day 60 and the smooth group on day 120 (94.3 ±3.6% vs. 73.3±4.3%; p<0.05). On day 120, we did not observe significant differences in colla- gen density related to implant surfaces (94.3±3.6% vs. 86±1.8%; p = 0.67). Results are depicted in Fig 7 and examples of histological pictures in Fig 8. 8 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 Smooth versus Textured Silicone Implants Table 1. List of Primers utilized for TaqMan quantitative real time PCR. Primer names and catalog num- bers are presented according to the vendor Life Technologies Corporation (Carlsbad, CA, USA). Primer name Catalog numbers Col1a1 Rn01463848_m1 Col2a1 Rn01533081_m1 Col3a1 Rn01437660_g1 Col4a1 Rn01482927_m1 TGFb1 Rn00572010_m1 TGFb3 Rn01517871_m1 Smad3 Rn01422009_m1 CD68 Rn01495634_g1 IL4 Rn01456866_m1 IL10 Rn00563409_m1 IL13 Rn00587615_m1 B2m (housekeeping gene) Rn00560865_m1 doi:10.1371/journal.pone.0132131.t001 Magnetic Resonance Imaging Capsules on day 60 appeared thicker and less dense when surrounding textured implants com- pared with those surrounding smooth implants, whereas apparent differences elapsed on day 120. Concomitant to thickness measurements, capsule volumes were higher in the textured (vs. smooth) implant group on day 60 and comparable across both surface groups on day 120 (Fig 9). Although the trend of each group correlated with histological and ultrasound measure- ments, differences in MRI were not significant between groups (Fig 10). Surface areas of implants were higher in the textured group compared to smooth surfaces after both 60 and 120 days. Within the same implant group, surface areas were comparable (Fig 11). Again, MRI- based measurements were not statistically significant between groups. Examples of 3D volume view images are demonstrated in Fig 12. PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 Real-Time Quantitative PCR Collagen 1 was significantly over expressed in the textured implant group on day 60 compared with both smooth implant groups on days 60 and 120 (RQ day 60 smooth vs. day 60 textured: 0.11±1.04, p<0.05; RQ day 120 smooth vs. day 60 textured: 0.16±0.95, p<0.05). Significant dif- ferences in the expression of collagen types 2, 3 and 4 were not detected among study groups. However, Collagen 2 trended towards highest expression in the smooth implant group on day 60 and decreased over the study period, that is, from day 60 to day 120 (Fig 14). In general, col- lagen subtypes appeared to be expressed less in smooth implant groups. With respect to inflammatory markers, significant differences were only detected for CD68; namely, there was lower expression in the smooth implant group on day 120 compared with the textured implant group on day 60 (RQ day 60 textured vs. day 120 smooth: 1.96±0.17; p<0.05) (Fig 15). Signifi- cant differences among study groups were not detected in any of the other investigated genes. PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 9 / 21 Smooth versus Textured Silicone Implants Fig 6. Mean (+/- standard deviation) capsule thickness measurements in histology sections of each study group. P<0.05 and p<0.005 were considered statistically significant and highly significant. Fig 6. Mean (+/- standard deviation) capsule thickness measurements in histology sections of each study group. P<0 05 and p<0 005 were Fig 6. Mean (+/- standard deviation) capsule thickness measurements in histology sections of each study group. P<0.05 and p<0.005 were considered statistically significant and highly significant. doi:10.1371/journal.pone.0132131.g006 Of note, inflammatory marker IL 10 was only present in the textured implant group on day 60 and not detectable anymore on day 120. In contrast, the expression of fibrotic markers, namely TGFbeta3 and 1, as well as Smad3 remained stable over the study period and unrelated to the implant type. Results are given in Fig 16 as heat map with increasing positive or negative delta CT values in brighter green or red colors, respectively. Black indicates that either the gene is not expressed or is below the limit of detection. PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 Discussion This study demonstrates significant differences in in-vivo capsule formation between smooth and textured silicone implants. Whereas smooth implant surfaces led to thinner capsules, colla- gen density was significantly higher compared to textured implant surfaces. These histological findings were substantiated by in-vivo imaging with means of 7-Tesla MRI and high-resolution ultrasound and associated with significant differences in gene expression of pro-fibrotic and 10 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 Smooth versus Textured Silicone Implants Fig 7. Mean (+/- standard deviation) collagen density in each study group. P<0.05 was considered statistically significant. d i 10 1371/j l 0132131 007 Fig 7 Mean (+/ standard deviation) collagen density in each study group P<0 05 was considered Fig 7. Mean (+/- standard deviation) collagen density in each study group. P<0.05 was considered statistically significant. doi:10.1371/journal.pone.0132131.g007 doi:10.1371/journal.pone.0132131.g007 Fig 8. Example pictures of picrosirius red staining under polarized light of both study groups on day 60. d i 10 1371/j l 0132131 008 Fig 8. Example pictures of picrosirius red staining under polarized light of both study groups on day 60. Fig 8. Example pictures of picrosirius red staining under polarized light of both study groups on day 60. doi:10.1371/journal.pone.0132131.g008 11 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 Smooth versus Textured Silicone Implants Fig 9. Mean (+/- standard deviation) capsule volume in MRI of each study group. Results were not statistically significant Fig 9. Mean (+/- standard deviation) capsule volume in MRI of each study group. Results were not statistically significant. doi:10.1371/journal.pone.0132131.g009 Fig 9. Mean (+/- standard deviation) capsule volume in MRI of each study group. Results were not statistically significant. Fig 9. Mean (+/- standard deviation) capsule volume in MRI of each study group. Results were not statistically significant. doi:10.1371/journal.pone.0132131.g009 doi:10.1371/journal.pone.0132131.g009 doi:10.1371/journal.pone.0132131.g009 Fig 10. Mean (+/- standard deviation) capsule thickness in MRI of each study group. Results were not statistically significant. doi:10.1371/journal.pone.0132131.g010 Fig 10. Mean (+/- standard deviation) capsule thickness in MRI of each study group. Results were not statistically significant. Fig 10. Mean (+/- standard deviation) capsule thickness in MRI of each study group. Results were not statistically significant. doi:10 1371/journal pone 0132131 g010 doi:10.1371/journal.pone.0132131.g010 PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 12 / 21 Smooth versus Textured Silicone Implants Fig 11. Mean (+/- standard deviation) implant surface area in MRI of each study group. Results were not statistically significant. d i 10 1371/j l 0132131 011 Fig 11. Mean (+/- standard deviation) implant surface area in MRI of each study group. Results were not statistically significant. doi:10 1371/journal pone 0132131 g011 doi:10.1371/journal.pone.0132131.g011 doi:10.1371/journal.pone.0132131.g011 doi:10.1371/journal.pone.0132131.g011 inflammatory markers. In the long-term, however, differences equalized and both textured and smooth implants revealed comparable results regarding thickness and density. The impact of implant surface properties on capsule formation is still controversially dis- cussed in current literature. Whereas most of the studies indicate a reduction of fibrous tissue deposition after surface texturing, some authors demonstrate no significant differences com- pared with smooth surfaces [11, 18, 19]. Clugston et al. investigated effects of surface texturing on capsular contracture in a rat model. Non-quantitative histological analysis revealed comparable findings within each group but with a tendency for thinner and less aligned collagen fibers after textured implants [18]. Recently, Minami et al. performed a detailed study of histologic alterations in capsule forma- tion after smooth and textured implants in a pig model [20], where comparison of both implant types did not show any significant differences at one specific time point, but capsule thickness varied significantly at different time points within the same study group. Whereas smooth implants provoked a constant increase in capsule thickness over the 270 days study period, textured surfaces led to a peak on day 180 after which the capsule declined again. In our study textured implants led to a comparable peak in capsule thickness in the mid-term. Furthermore, we were able to verify this histological aspect in-vivo by HR-US and 7-Tesla MRI. Especially the latter provides a unique tool to assess the morphology of the entire fibrotic capsule by 3D reconstruction and volumetric measurements. Concomitant to thickness, the volume of the fibrotic capsule was altered with higher volumes on day 60 in the textured implant group but comparable volumes across surface groups at the later time point. PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 Interest- ingly, surface areas did not vary between study dates, but they did between implant types. As contracture develops it has the effect of decreasing the surface area of the implant until the implant reaches a spherical shape, at which point further contraction is impossible because of PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 13 / 21 Smooth versus Textured Silicone Implants Fig 12. 3D reconstruction of 7-Tesla MRI scans of smooth implants on day 60 (60s, upper left), textured implants on day 60 (60t, upper right), smooth implants on day 120 (120s, lower left) and textured implants on day 120 (120t, lower right). Fig 12. 3D reconstruction of 7-Tesla MRI scans of smooth implants on day 60 (60s, upper left), textured implants on day 60 (60t, upper right), smooth implants on day 120 (120s, lower left) and textured implants on day 120 (120t, lower right). doi:10.1371/journal.pone.0132131.g012 doi:10.1371/journal.pone.0132131.g012 the non-compressibility of liquids [21]. In our opinion this is the most accurate method to define contracture as it reassembles the entire capsule and thus a complete overview of the con- tracture process. Our results indicate a more contracted capsule due to the smooth implant sur- face, as surface area was lower in this group. However, none of the MRI-based results were statistically significant what was most likely associated with still insufficient resolution capacity despite latest 7-tesla MRI technology as well as low sample size (n = 2). The latter might be of higher significance as thickness values did correlate with both histology and HR-US measure- ments. From an economical point of view HR-US is the preferred method for thickness mea- surements as it is remarkable capable to identify and assess the fibrotic capsule in-vivo. Of note, thickness measurements in MRI were thicker compared to both histology and HR-US, due to the fact that measurements were performed from the implant shell to the outer margin of the skin. The latter was necessary in MRI as the resolution in some samples was insufficient for proper identification of the sole capsule. the non-compressibility of liquids [21]. In our opinion this is the most accurate method to define contracture as it reassembles the entire capsule and thus a complete overview of the con- tracture process. Our results indicate a more contracted capsule due to the smooth implant sur- face, as surface area was lower in this group. PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 However, none of the MRI-based results were statistically significant what was most likely associated with still insufficient resolution capacity despite latest 7-tesla MRI technology as well as low sample size (n = 2). The latter might be of higher significance as thickness values did correlate with both histology and HR-US measure- ments. From an economical point of view HR-US is the preferred method for thickness mea- surements as it is remarkable capable to identify and assess the fibrotic capsule in-vivo. Of note, thickness measurements in MRI were thicker compared to both histology and HR-US, due to the fact that measurements were performed from the implant shell to the outer margin of the skin. The latter was necessary in MRI as the resolution in some samples was insufficient for proper identification of the sole capsule. PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 14 / 21 Smooth versus Textured Silicone Implants Fig 13. Mean (+/- standard deviation) capsule thickness measurements via high-resolution ultrasound of each study group. P<0.05 was considered statistically significant. doi:10.1371/journal.pone.0132131.g013 Fig 13. Mean (+/- standard deviation) capsule thickness measurements via high-resolution ultrasound of each study group. P<0.05 was considered statistically significant. doi:10.1371/journal.pone.0132131.g013 doi:10.1371/journal.pone.0132131.g013 With respect to collagen density, several studies demonstrated loose collagen structures related to textured surfaces. Smahel et al. revealed less compact capsule development 8 months after insertion of textured surfaces in rats [22]. In a clinical study of Wyatt et al. smooth implants more often had a dense collagenous capsule than textured implants after 5 years of follow-up [23]. Furthermore, the incidence of capsules with collagen fibers arranged parallel to the implant surface was significantly greater in the smooth group compared to the textured group. Our results are in accordance to these findings as capsules of textured implants were sig- nificantly less dense than those of smooth implants. Thus surface texturing led to thicker but less dense fibrotic capsules, leading to the question if total collagen deposition differs among both implant types. Bucky et al. compared smooth, textured and polyurethane covered implants in rabbits and found lowest Collagen 3 deposition around textured surfaces [24]. Minami et al. showed that over time thin collagen fibers were replaced by thick fibers indepen- dent of the implant type [20]. Thereby thin and thick fibers equate to collagen types 3 and 1, respectively [25]. PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 In our study, analysis of gene expression revealed a significant increase of Collagen 1 as well as a remarkable trend of Collagen 3 towards over expression both related to textured implants in the mid-term. However, despite mid-term up-regulation of collagen gene expression, collagen deposition revealed no significant differences in the long term. As fibrosis is evidentially related to inflammation, Meza-Brites et al. investigated peripros- thetic breast capsules and immunophenotypes of inflammatory cells in 80 breast implant cap- sules and found textured implants to induce a stronger local T-cell immune response [26, 27]. In our study, gene expression of CD68, a macrophage marker, was slightly increased on day 60 in the textured implant group when compared to the smooth group, and IL10 was detectable PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 15 / 21 PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 Smooth versus Textured Silicone Implants Fig 14. Results of quantitative real time PCR for Collagen 1 (upper left), 2 (upper right), 3 (lower left) and 4 (lower right) given in fold change compared to textured implant group of day 60 or 120, respectively. Asterisks and brackets indicate statistical significance and P<0.05 was considered statistically significant. Fig 14 Results of quantitative real time PCR for Collagen 1 (upper left) 2 (upper right) 3 (lower left) and 4 (lower right) given in fold change Fig 14. Results of quantitative real time PCR for Collagen 1 (upper left), 2 (upper right), 3 (lower left) and 4 (lower right) given in fold change compared to textured implant group of day 60 or 120, respectively. Asterisks and brackets indicate statistical significance and P<0.05 was considered statistically significant. doi:10.1371/journal.pone.0132131.g014 doi:10.1371/journal.pone.0132131.g014 textured but not in smooth surfaces. Interestingly, CD68 gene expression was significantly higher in the smooth implant group on day 120. Although this may indicate an increase of inflammation in the long run, significant upregulation of other inflammatory markers such as IL4 and IL13 was not observed at any time point. An alternative explanation was highlighted in recent studies, which found that especially in skin related tissues, CD68 is not macrophage- specific but also expressed by fibroblasts [27, 28]. Therefore, our findings may indicate an increase of fibroblast proliferation and thus stronger fibrotic reaction associated with smooth implant surfaces in the long term. However, no significant differences related to time or implant surface topography were observed for the expression of remaining fibrotic markers TGFbeta1 and 3, as well as Smad3. With regard to the clinical translation of our findings, several studies investigated on histo- logical criteria and their correlation to the clinical appearance of capsular fibrosis. Whereas most of the data in current literature indicate that capsule thickness correlates sig- nificantly with occurrence of capsular contracture, interestingly, none of these studies assessed PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 16 / 21 Smooth versus Textured Silicone Implants Fig 15. Results of quantitative real time PCR for CD68 given in fold change compared to textured implant group of day 60 or 120, respectively. Asterisks and brackets indicate statistical significance and P<0.05 was considered statistically significant. doi:10.1371/journal.pone.0132131.g015 Fig 15. Results of quantitative real time PCR for CD68 given in fold change compared to textured implant group of day 60 or 120, respectively. Asterisks and brackets indicate statistical significance and P<0.05 was considered statistically significant. Fig 15. Results of quantitative real time PCR for CD68 given in fold change compared to textured implant group of day 60 or 120, respectively. Asterisks and brackets indicate statistical significance and P<0.05 was considered statistically significant. doi:10.1371/journal.pone.0132131.g015 doi:10.1371/journal.pone.0132131.g015 the density of the fibrotic capsules [29–31]. Furthermore, there is contrary evidence that thick- ness of the capsule has no impact on clinical appearance, and does not correlate at all [32, 33]. In a recently published study about histological findings in capsule tissue specimens, Bui et al. assessed thickness and density and demonstrated that both criteria significantly correlate with symptoms [31]. In accordance to these findings Rubino et al. demonstrated via electron microscopy that contracted capsules of textured implants were not only thicker but also more compact compared to uncontracted capsules [34]. Of note, contracted capsules from textured implants still obtained a layer of irregular and woven appearance, whereas contracted capsules from smooth implants showed dense fibers throughout the entire capsule despite similar thick- ness. The latter highlights the impact of capsule density and that sole thickness of the capsule is insufficient for translation into clinical contracture. From a histomorphological point of view, contracture would rather come with an increase in density than thickness, which is even more likely to decrease due to the contracture process itself. the density of the fibrotic capsules [29–31]. Furthermore, there is contrary evidence that thick- ness of the capsule has no impact on clinical appearance, and does not correlate at all [32, 33]. In a recently published study about histological findings in capsule tissue specimens, Bui et al. assessed thickness and density and demonstrated that both criteria significantly correlate with symptoms [31]. In accordance to these findings Rubino et al. demonstrated via electron microscopy that contracted capsules of textured implants were not only thicker but also more compact compared to uncontracted capsules [34]. Of note, contracted capsules from textured implants still obtained a layer of irregular and woven appearance, whereas contracted capsules from smooth implants showed dense fibers throughout the entire capsule despite similar thick- ness. The latter highlights the impact of capsule density and that sole thickness of the capsule is insufficient for translation into clinical contracture. PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 From a histomorphological point of view, contracture would rather come with an increase in density than thickness, which is even more likely to decrease due to the contracture process itself. Therefore, our data indicates that textured implants are less prone to capsular contracture in the mid-term but comparable to smooth surfaces in the long-term. Furthermore, our results provide the histomorphological correlate to clinical studies demonstrating a delayed occur- rence of capsular contracture after textured implants [35, 36] as well as to those that state an absolute decrease of capsular contracture incidence irrespective of the follow-up period [13]. Another important aspect that needs to be addressed in the context of surface properties and capsule formation is the occurrence of implant-associated anaplastic large cell lymphoma (ALCL). Although the U.S. Food and Drug Administration stated that ALCL should not be of PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 17 / 21 Smooth versus Textured Silicone Implants Fig 16. Results of quantitative real time PCR for IL10, IL4, IL13, TGFb-1, TGFb-3 and Smad3 given in delta Ct (Ct gene—Ct housekeeping gene) for each study group and illustrated as heat map (Distance measure: Pearson’s Correlation; Clustering Method: Ave Linkage; Map Type: Global delta CT; Ct > 40,000 = not determined). Increasing positive or negative delta CT values are given in brighter green or red colors, respectively. Black indicates that either the gene is not expressed or is below the limit of detection. doi:10.1371/journal.pone.0132131.g016 Fig 16. Results of quantitative real time PCR for IL10, IL4, IL13, TGFb-1, TGFb-3 and Smad3 given in Fig 16. Results of quantitative real time PCR for IL10, IL4, IL13, TGFb-1, TGFb-3 and Smad3 given in delta Ct (Ct gene—Ct housekeeping gene) for each study group and illustrated as heat map (Distance measure: Pearson’s Correlation; Clustering Method: Ave Linkage; Map Type: Global delta CT; Ct > 40,000 = not determined). Increasing positive or negative delta CT values are given in brighter green or red colors, respectively. Black indicates that either the gene is not expressed or is below the limit of detection. doi:10.1371/journal.pone.0132131.g016 doi:10.1371/journal.pone.0132131.g016 major concern to patients considering silicone implants as the absolute risk remains very low, more and more cases recently appeared in current literature [37]. Interestingly, according to Brody et al. PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 all silicone implant associated ALCL reported thus far were diagnosed in patients, who had at least once in their past medical history an implant with a textured surface [38]. Although we did not find any clinical or histological signs for ALCL in this study (data not shown), the impact of this finding should not be overestimated. In contrast to the clinical con- jecture of rough surfaces as a cause for implant associated ALCL, Oppenheimer found that smooth surfaced foreign bodies implanted in animals were more prone to sarcoma formation, whereas rough or irregular surfaces did not generate tumors [39]. Further studies confirm Oppenheimer’s finding in laboratory animals and thus raising questions about the relevance of animal experiments for implant associated ALCL in general [40, 41]. There are several limitations to our study. Although considered as major contributor to the fibrotic process after silicone implant insertion, biofilm formation was not evaluated in this study. With respect to current literature, biofilm formation is only detectable in animal models if implant pockets were inoculated with bacteria previous to implant insertion [8, 42–44]. Thereby, Jacombs et al. could demonstrate that biofilm and capsule formation after smooth and textured implants revealed no significant differences among study groups and concluded that once a threshold of biofilm occurs, irrespective from surface properties, there seems to be PLOS ONE \| DOI:10.1371/journal.pone.0132131 July 7, 2015 18 / 21 Smooth versus Textured Silicone Implants an equal propensity to progress to capsular contracture [43]. These findings were substantiated by Pajkos et al., who demonstrated in a clinical study that biofilm formation was significantly associated with capsular contracture but its occurrence did not correlate with smooth or tex- tured implant surfaces [45]. Therefore we decided to exclude biofilm formation as outcome measurement as it is evidentially not related to surface properties relevant for capsule forma- tion. Furthermore, we wanted to prevent bacteria inoculation, which would provide significant bias rather than highlight the impact of implant surfaces on capsule formation. From a clinical point of view, capsular contracture is defined by its symptoms, namely palpable hardness, visi- ble deformities or pain, of which the latter is an absolute indication for surgical intervention. Therefore, capsule thickness, collagen density as well as gene expression of fibrotic or inflam- matory markers cannot mimic clinical symptoms and thus remain without therapeutic conse- quences. Acknowledgments The silicone implants were kindly provided by Polytech Health and Aesthetic (Dieburg, Germany). Conclusion Silicone implants with textured surfaces lead to temporarily thicker but less dense fibrotic cap- sules compared to smooth surfaces. This difference was apparent in histology, MRI and high- resolution ultrasound and substantiated by fibrotic and inflammatory gene expression. Differ- ences, however, equalized over time, revealing comparable results in the long-term and thus highlighting the impact of time on capsule evaluation. In addition, MR-based measurements provide unique options to quantify capsular fibrosis development and to objectify the occur- rence of contracture. Nevertheless, pain is difficult to evaluate, even in humans, and applanation tonometry or biochemical analysis of tissue compliance correlated well with histological find- ings in former studies [18, 20, 24]. Furthermore, macroscopic, histological and biochemical alterations in our study can explain the vast range of incidence of capsule contracture reported in clinical studies, which is apparently not only related to the implant surface but also to the time point of investigation [5]. Author Contributions Conceived and designed the experiments: SF UK BP SM EB. Performed the experiments: SF JK YD MA. Analyzed the data: SF JK YD MA. 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https://openalex.org/W4316035759	https://zenodo.org/records/7538305/files/VCS_article_98324.pdf	English	null	Ecological Indicator Values for Europe (EIVE) 1.0	Vegetation Classification and Survey	2,023	cc-by	21,693	Vegetation Classification and Survey 4: 7–29 doi: 10.3897/VCS.98324 VCS METHODS ECOINFORMATICS International Association for Vegetation Science (IAVS) Copyright Jürgen Dengler et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction occurrence of plant species with environmental conditions was introduced to vegetation ecology by Cajander (1926) and Iversen (1936). Subsequently, Ellenberg (1950a, 1950b, 1952) introduced the first explicitly quantitative approach within an agricultural context. Comprehensive EIV systems for the vascular plants of larger territories were then independently proposed by Ramensky et al. (1956) for the European part of the former USSR and El lenberg (1974) for Central Europe. Ramensky et al. (1956) published indicator values for grazing intensity, soil mois ture and a combination of soil fertility and salinity, while Ellenberg (1974; new edition by Ellenberg et al. 1991) cov ered seven ecological variables: light regime, temperature, continentality, moisture, reaction (pH), nutrient status and soil salinity. The high utility of these indicator values led to an expansion to other regions, with more than 30 EIV systems being published so far (Table 1). Some of the more recent EIV systems not only expanded the ap proach to new regions, but also added other taxonomic groups (e.g. bryophytes, lichens), other niche dimensions (e.g. mowing tolerance, hemeroby, CSR strategy, organic content of the soil, soil texture) or assessed niche width in addition to niche position. Very recently, new systems with a focus on Europe as a whole have been published: Hájek et al. (2020) published niche position, minimum and maximum for hydrological parameters for a compre Since the probability of species’ occurrence changes pre dictably along environmental gradients, plant community composition holds valuable information about local envi ronmental conditions. This basic notion, conceptualised as bioindication, has been a subject of research for a long time (see review by Diekmann 2003). In bioindication, individual plant species are assigned so-called ecological indicator values (EIVs) on ordinal scales based on the “optima” or “centres” of their realised ecological niches along given environmental gradients (niche dimensions) (Smart 2000; Diekmann 2003). “Realised niche” refers to the occurrence of species in plant communities under the influence of competition (or facilitation) of co-occurring species, as opposed to the “fundamental niche” describ ing the occurrence and performance in monoculture (Leibold 1995). In this paper, we use the term “niche po sition” to denote the central tendency of the distribution of a species even in the case of skewed or bimodal ecolog ical niches. Ecological Indicator Values for Europe (EIVE) 1.0 Jürgen Dengler1,2, Florian Jansen3, Olha Chusova1,4, Elisabeth Hüllbusch2,5, Michael P. Nobis6, Koenraad Van Meerbeek7, Irena Axmanová8, Hans Henrik Bruun9, Milan Chytrý8, Riccardo Guarino10, Gerhard Karrer11, Karlien Moeys7, Thomas Raus12, Manuel J. Steinbauer13, Lubomir Tichý8, Torbjörn Tyler14, Ketevan Batsatsashvili15, Claudia Bita-Nicolae16, Yakiv Didukh4, Martin Diekmann17, Thorsten Englisch18, Eduardo Fernández-Pascual19, Dieter Frank20, Ulrich Graf21, Michal Hájek8, Sven D. Jelaska22, Borja Jiménez-Alfaro23, Philippe Julve24, George Nakhutsrishvili25, Wim A. Ozinga26, Eszter-Karolina Ruprecht27, Urban Šilc28, Jean-Paul Theurillat29,30, François Gillet31 4 Department of Geobotany and Ecology, M.G. Kholodny Institute of Botany of the National Academy of Sciences of Ukraine, 5 Federal Agency for Nature Conservation (BfN), Bonn, Germany 6 Dynamic Macroecology Group, Land Change Science Unit, Swiss Federal Research Institute WSL, Birmensdorf, Switzerland 6 Dynamic Macroecology Group, Land Change Science Unit, Swiss F 7 Department of Earth and Environmental Sciences, KU Leuven, Leuven, Belgium 7 Department of Earth and Environmental Sciences, KU Leuven, Leuven, Belgium 8 Department of Botany and Zoology, Faculty of Science, Masaryk University, Brno, Czech Republic 8 Department of Botany and Zoology, Faculty of Science, Masaryk University, Brno, Czech Republic epartment of Biology, University of Copenhagen, Copenhagen, Denmark 10 Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), P 10 Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), Palermo University, Palermo, I 11 Institute of Botany, University of Natural Resources and Life Sciences Vienna, Vienna, Austria 11 Institute of Botany, University of Natural Resources and Life Sciences Vienna, Vienna, Austria 12 Botanischer Garten und Botanisches Museum Berlin, Freie Universität Berlin, Berlin, Germany 12 Botanischer Garten und Botanisches Museum Berlin, Freie Universität Berlin, Berlin, Germany 12 Botanischer Garten und Botanisches Museum Berlin, Freie Uni 13 Sport Ecology, University of Bayreuth, Bayreuth, Germany 14 Department of Biology, The Biological Museum, Lund University, Lund, Sweden 20 Environment Agency Saxony-Anhalt (LAU), Halle, Germany * Jürgen Dengler and Florian Jansen shall be considered as joint first authors. Copyright Jürgen Dengler et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Jürgen Dengler et al.: Ecological Indicator Values for Europe 1.0 8 Keywords bioindication, ecological indicator value, Ellenberg indicator value, Europe, light, moisture, niche position, niche width, nitrogen, pH, temperature, vascular plant Abstract Aims: To develop a consistent ecological indicator value system for Europe for five of the main plant niche dimensions: soil moisture (M), soil nitrogen (N), soil reaction (R), light (L) and temperature (T). Study area: Europe (and closely ad jacent regions). Methods: We identified 31 indicator value systems for vascular plants in Europe that contained assess ments on at least one of the five aforementioned niche dimensions. We rescaled the indicator values of each dimension to a continuous scale, in which 0 represents the minimum and 10 the maximum value present in Europe. Taxon names were harmonised to the Euro+Med Plantbase. For each of the five dimensions, we calculated European values for niche position and niche width by combining the values from the individual EIV systems. Using T values as an example, we externally validated our European indicator values against the median of bioclimatic conditions for global occurrence data of the taxa. Results: In total, we derived European indicator values of niche position and niche width for 14,835 taxa (14,714 for M, 13,748 for N, 14,254 for R, 14,054 for L, 14,496 for T). Relating the obtained values for temperature niche position to the bioclimatic data of species yielded a higher correlation than any of the original EIV systems (r = 0.859). The database: The newly developed Ecological Indicator Values for Europe (EIVE) 1.0, together with all source systems, is available in a flexible, harmonised open access database. Conclusions: EIVE is the most comprehensive ecological indicator value system for European vascular plants to date. The uniform interval scales for niche position and niche width provide new possibilities for ecological and macroecological analyses of vegetation patterns. The devel oped workflow and documentation will facilitate the future release of updated and expanded versions of EIVE, which may for example include the addition of further taxonomic groups, additional niche dimensions, external validation or regionalisation. Abbreviations: EIV = Ecological indicator value; EIVE = Ecological Indicator Values for Europe; EVA = European Vegetation Archive; GBIF = Global Biodiversity Information Facility; i = index for taxa; j = index for EIV systems; L = ecological indicator for light; M = ecological indicator for moisture; N = ecological indicator for nitrogen availability; R = ecological indicator for reaction; T = ecological indicator for temperature. Introduction To assess the site conditions of a vegetation plot or a plant community, the EIVs of all species present in that plot or community can be averaged for each niche dimension of interest.h The idea of using the presence of plants to assess site conditions by qualitatively matching the most probable 9 Vegetation Classification and Survey 9 Table 1. Overview of the 31 ecological indicator value systems (EIVs) used to derive the Ecological Indicator Values for Europe (EIVE) 1.0. Further details are provided in Suppl. material 1. EIVE name Country or region Reference(s) # of vascular plant taxa Habitat subset of species M min M max M amplitude coding N min N max N amplitude coding R min R max R amplitude coding L min L max L amplitude coding T min T max T amplitude coding Alps Switzerland + entire Alps Graf (unpubl.), updated and augmented from Landolt et al. (2010) 6470 All 1 5 I, II, x 1 5 I, II, x 1 5 I, II, x 1 5 I, II, x 1 5 I, II, x Austria Austria Englisch and Karrer (unpubl.), updated and aug mented from Englisch and Karrer (2001) 3253 All 1 12 I, x 1 9 I, x 1 9 I, x 1 9 I, II, x 1 9 I, x Austria_Panno nian Austria: wider sur roundings of Vienna Starmühlner and Ehrendorfer (1971) 954 All 1 6 range* 1 3 range* 1 5 range* 1 3 range* 1 3 range* British_Isles United Kingdom + Ireland Hill et al. (2004) 1867 All 1 11 NA 1 9 NA 1 9 NA 1 9 NA NA NA NA Czech_Republic Czech Republic Chytrý et al. (2018) 2972 All 1 11 I, II 1 9 I, II 1 9 I, II 1 9 I, II 1 9 I, II Czechoslova kia_Ambros Czech Republic + Slovakia Ambros (1986) 587 Forests 1 5 NA NA NA NA 1 5 NA 1 5 NA 1 5 NA Czechoslova kia_Jurko Czech Republic + Slovakia Jurko (1990) 2445 All 1 6 range 1 5 range 1 5 range NA NA NA NA NA NA Germany Germany + adjacent regions Ellenberg et al. Introduction (1991) 3405 All 1 11 I, II, x 1 9 I, II, x 1 9 I, II, x 1 9 I, II 1 9 I, II Germany_Dier schke Germany + adjacent regions Dierschke and Briemle (2002) 399 Grasslands 1 11 I, x 1 9 I, x 1 9 I, x NA NA NA NA NA NA Germany_GDR Germany: former GDR Frank and Klotz (1990) 1719 All 1 11 NA 1 9 NA 1 9 NA 1 9 NA 1 9 NA Spain_Asturias Spain: Asturias Mayor López (1999) 1842 All 1 5 I, x 1 5 I, x 1 5 I, x 1 5 NA 1 5 I, x Spain_Cantabria Spain: Cantabrian Mountains Jiménez-Alfaro et al. (2021) 1888 All 1 11 NA 1 9 NA 1 9 NA 1 9 NA 1 9 NA European_Mires Europe Hájek et al. (2020) 1771 Mires 1 11 range NA NA NA NA NA NA NA NA NA NA NA NA Faroer Faroe Islands Lawesson et al. (2003) 126 All 1 11 I, x 1 9 I, x 1 9 I, x NA NA NA NA NA NA France France: European part Julve (2022) 6166 All 1 11 NA 1 9 NA 1 9 NA 1 9 NA 1 9 NA Georgia Georgia: Kazbegi district Nakhutsrishvili and Batastsahvili (unpubl.), up dated and augmented from Sakhokia and Khut sishvili (1975) and Nakhutsrishvili et al. (2017) 1116 All 1 6 I, x 1 5 NA 1 5 NA 1 5 NA 1 5 NA Greece Greece: South Aegean region Böhling et al. (2002) 2400 All 1 11 I, II, x 1 9 I, x 1 9 #, I, x 1 9 I, x 1 9 #, I, II Hungary_Borhidi Hungary Borhidi (1995) 2088 All 1 11 NA 1 9 NA 1 9 NA 1 9 NA 0 8 NA Hungary_Soo Hungary Soó (1980) 2159 All 1 5 I, x 1 5 I, x 1 5 I, x NA NA NA 1 5 I, x Hungary_Zolyomi Hungary Zólyomi et al. (1967) 1243 All 0 11 NA NA NA NA 1 5 I, x NA NA NA 1 7 I, x Italy Italy Guarino (unpubl.), updated from Pignatti et al. (2005), Guarino et al. (2012), Domina et al. (2018) and Pignatti et al. Introduction However, the bioindication approach as such, and the wide use of EIVs, have also been criticised. One line of criticism holds that indicator values have been assigned to plant species mainly based on expert judgement, rath er than on accurate measurements (Wamelink et al. 2002; Diekmann 2003). Secondly, although large regional dif ferences in the niches of species have been demonstrated (Diekmann and Lawesson 1999; Pakeman et al. 2008), EIVs have often been applied outside the region for which they were developed (e.g. Hermy et al. 1999). This could potentially lead to misinterpretations (Godefroid and Dana 2007). Another line of critique has warned against averaging indicator values and subjecting them to para metric statistics, since they were defined on ordinal scales (e.g. Kowarik and Seidling 1989; Möller 1992). However, analysing mean EIVs does not lead to statistical issues, since the arithmetic means of values of any distribution per se follow a normal distribution (Central Limit Theorem; see Quinn and Keough 2002). Ewald (2003) demonstrated , p p Indicator values are widely applied in vegetation sci ence and global change studies. They are suitable to indi rectly assess environmental conditions and the drivers of observed vegetation differences in time or space (see re view by Diekmann 2003). Several factors can explain the success of their application. First, environmental variables may fluctuate strongly in time and space (e.g. Sercu et al. 2017), making one-time measurements scarcely repre sentative of average conditions or critically limiting ex tremes (Shipley et al. 2017). Thus, the appropriate assess ment of environmental variables often requires repeated measurements (not feasible in many projects) or is costly if to be done across numerous plots. Additionally, mea surements obtained at different times and with different techniques and equipment may not be directly compara ble. In contrast, the plant species composition of a site is an expression of the species’ responses to the prevailing environmental conditions integrated across the study area (e.g. a plot) over longer time periods (several months to several years). Therefore, bioindication using EIVs offers a less time-consuming and cheaper alternative to the direct measurement of local environmental variables (Englisch and Karrer 2001; Diekmann 2003; Didukh 2012; Zelený and Schaffers 2012; Marcenò and Guarino 2015). Finally, most historical vegetation data do not contain measure ments of environmental data (Dengler et al. 2011). The ability to reconstruct past environmental conditions from historical relevés (Pignatti et al. Introduction 2001; Van Calster et al. 2007; Diekmann et al. 2019) or floristic occurrence data (Finderup Nielsen et al. 2021; Hallman et al. 2022; Scher rer et al. 2022) can thus be very valuable in assessing trends in environmental change and their effects on biodiversity. Recent trends in ecoinformatics opened opportunities for continental-scale studies of plant community data in Europe. Important developments were the emergence of large-scale vegetation-plot databases like the European Vegetation Archive (EVA; Chytrý et al. 2016) and Grass Plot (Dengler et al. 2018), a European vegetation classifica tion system (EuroVegChecklist; Mucina et al. 2016) and an automated supervised habitat classification for vegetation plots in Europe (Chytrý et al. 2020; Bruelheide et al. 2021). Rapid environmental change over large regions increases the need to perform broad-scale analyses of changes and trends in community composition and environmental con ditions (Kempel et al. 2020; Leclère et al. 2020; Hallman et al. 2022). However, most of the large European vegeta tion-plot databases (e.g. Chytrý et al. 2016) do not contain in situ measured environmental data, at least not in easily accessible forms (but see Dengler et al. 2018). Thus, such analyses have to rely on approximate site conditions derived via plot coordinates from modelled geodata (e.g. CHELSA: Karger et al. 2017; SoilGrids: Poggio et al. 2021), but such modelled data are available only for larger grid cells (e.g. 250 m × 250 m for SoilGrids), while soil conditions can change dramatically within metres. Mean EIVs can con tribute to solving this challenge by easily linking the wealth of relevé data to abiotic conditions. Scherrer and Guisan (2019) showed that the application of mean EIVs instead of gridded environmental variables doubled the proportion of variance explained in species distribution models, with particularly strong improvements for light and soil condi tions. However, the more than 30 national and regional EIV systems lack consistency in scaling and coding of the eco logical indicators, as well as in plant nomenclature, imped ing analyses at the continental scale. These issues have part ly been solved by the recently published pan-European EIV systems (Hájek et al. 2020; Midolo et al. 2023; Tichý et al. 2023) but their coverage of indicators and taxa, respectively, is far from complete. Introduction (2023) derived a set of five disturbance indicators for more than 6,000 European vas cular plants. Recently, Tichý et al. (2023) presented a har monized dataset of six of the original Ellenberg indicator values for almost 9,000 European vascular plant taxa. with environmental measurements, even when species lists were incomplete. Finally, the use of environmental infor mation inferred from plant community composition to interpret vegetation patterns and dynamics has been criti cised for potential circularity (Zelený and Schaffers 2012). However, these authors and Wildi (2016) demonstrated proper ways to use EIVs in vegetation ecological studies. cular plants. Recently, Tichý et al. (2023) presented a har monized dataset of six of the original Ellenberg indicator values for almost 9,000 European vascular plant taxa. Indicator values are widely applied in vegetation sci ence and global change studies. They are suitable to indi rectly assess environmental conditions and the drivers of observed vegetation differences in time or space (see re view by Diekmann 2003). Several factors can explain the success of their application. First, environmental variables may fluctuate strongly in time and space (e.g. Sercu et al. 2017), making one-time measurements scarcely repre sentative of average conditions or critically limiting ex tremes (Shipley et al. 2017). Thus, the appropriate assess ment of environmental variables often requires repeated measurements (not feasible in many projects) or is costly if to be done across numerous plots. Additionally, mea surements obtained at different times and with different techniques and equipment may not be directly compara ble. In contrast, the plant species composition of a site is an expression of the species’ responses to the prevailing environmental conditions integrated across the study area (e.g. a plot) over longer time periods (several months to several years). Therefore, bioindication using EIVs offers a less time-consuming and cheaper alternative to the direct measurement of local environmental variables (Englisch and Karrer 2001; Diekmann 2003; Didukh 2012; Zelený and Schaffers 2012; Marcenò and Guarino 2015). Finally, most historical vegetation data do not contain measure ments of environmental data (Dengler et al. 2011). The ability to reconstruct past environmental conditions from historical relevés (Pignatti et al. 2001; Van Calster et al. 2007; Diekmann et al. 2019) or floristic occurrence data (Finderup Nielsen et al. 2021; Hallman et al. 2022; Scher rer et al. 2022) can thus be very valuable in assessing trends in environmental change and their effects on biodiversity. Introduction (2017‒2019) 5585 All 1 11 NA 1 9 NA 1 9 NA 1 9 NA 1 12 NA Netherlands Netherlands Netherlands Central Bureau of Statistics (1993) 1570 All 1 11 NA 1 9 NA 1 9 NA 1 9 NA 1 9 NA Poland Poland Zarzycki (1984), Zarzycki et al. (2002) 2209 All 1 6 range 1 5 range 1 5 range 1 5 range 1 5 range** Romania Romania Bita-Nicolae and Sanda (2011) 3620 All 1 6 NA NA NA NA 1 5 NA NA NA NA 1 5 NA Serbia Serbia Kojić et al. (1997) 2215 All 1 6 NA 1 5 NA 1 5 NA 1 5 NA 1 5 NA Sweden Sweden Tyler et al. (2021) 2422 All 1 12 NA 1 9 NA 1 8 NA 1 7 NA 18 1 NA Sweden_Diek mann Sweden: hemiboreal zone Diekmann (1995) 34 Forests 1 11 I, x NA NA NA 1 9 I, x 1 9 I, x NA NA NA Slovenia Slovenia Košir (1992) 683 Forests 1 11 NA NA NA NA NA NA NA NA NA NA NA NA NA Ukraine Ukraine Didukh (unpubl.), updated from Didukh (2011) 3326 All 1 23 range 1 11 range 1 15 range 1 9 range 1 17 range USSR_Ramensky Former USSR Ramensky et al. (1956) 1359 All 1 120 range NA NA NA NA NA NA NA NA NA NA NA NA USSR_Tsyganov Former USSR: hemi boreal zone Tsyganov (1983) 2122 All 1 23 range 1 11 range 1 13 range 1 9 range 1 17 range * In the source there are single values, ranges and “x”, all of which are now unified to ranges in the logic of the source; ** In the source there are single values intemediate values ranges and bimodal distributions which are now unified to ranges in the logic of the source Jürgen Dengler et al.: Ecological Indicator Values for Europe 1.0 10 hensive set of vascular plants and bryophytes occurring in mires, while Midolo et al. (2023) derived a set of five disturbance indicators for more than 6,000 European vas cular plants. Recently, Tichý et al. (2023) presented a har monized dataset of six of the original Ellenberg indicator values for almost 9,000 European vascular plant taxa. hensive set of vascular plants and bryophytes occurring in mires, while Midolo et al. Introduction Thus, there is still an urgent need for an integrated and comprehensive EIV system for Europe.i gf y However, the bioindication approach as such, and the wide use of EIVs, have also been criticised. One line of criticism holds that indicator values have been assigned to plant species mainly based on expert judgement, rath er than on accurate measurements (Wamelink et al. 2002; Diekmann 2003). Secondly, although large regional dif ferences in the niches of species have been demonstrated (Diekmann and Lawesson 1999; Pakeman et al. 2008), EIVs have often been applied outside the region for which they were developed (e.g. Hermy et al. 1999). This could potentially lead to misinterpretations (Godefroid and Dana 2007). Another line of critique has warned against averaging indicator values and subjecting them to para metric statistics, since they were defined on ordinal scales (e.g. Kowarik and Seidling 1989; Möller 1992). However, analysing mean EIVs does not lead to statistical issues, since the arithmetic means of values of any distribution per se follow a normal distribution (Central Limit Theorem; see Quinn and Keough 2002). Ewald (2003) demonstrated the robustness of the correlation of weighted mean of EIVs Here, we aim to fill this gap by developing the Ecolog ical Indicator Values for Europe (EIVE), a pan-European ecological indicator value system for the five niche di mensions most often included in the existing EIV systems and most frequently used in ecological analyses. These are the three main substrate variables moisture (M), ni trogen (N) and reaction (R), as well as light (L) and tem perature (T). We achieved this by numerically combining all available systems that contained these indicators into a “consensus system”. In doing so, we also implemented several novelties that should facilitate the future applica tion of EIVE: (i) all indicators were scaled from 0 to 10 on a continuous interval scale and (ii) for each indicator 11 Vegetation Classification and Survey Figure 1. Map of Europe showing the areas covered by ecological indicator value (EIV) systems that were used to de rive the Ecological Indicator Values for Europe (EIVE) 1.0. Colours indicate the number of EIV systems covering the complete vascular plant flora. Hatched and dotted areas refer to EIV systems that cover only a subset of specific habitats. Study area Our study covers Europe as a whole in the geographic sense, i.e. from the Atlantic Ocean to the Ural and Cau casus Mountains. We also included Georgia, whose place ment in either Europe or Asia is disputed, and kept the few species of the Asian part of the former Soviet Union that were included in Ramensky et al. (1956) and Tsyganov (1983). This means that according to Breckle (2002) the mediterranean (IV), oceanic (V), nemoral (VI), conti nental (VII), boreal (VIII) and arctic (IX) zonobiomes are included. However, given the availability of regional EIV systems (Figure 1), some regions of Europe are covered better than others. If several editions of the same EIV system existed (e.g. Landolt 1977 and Landolt et al. 2010), we used the most recent digitally available version with comprehensive in formation. In case of multiple independent systems for the same region by different authors (e.g. three EIV systems for Hungary), we included all. In total, we had 31 source sys tems for M, 24 for N, 28 for R, 33 for L and 23 for T. In Sup pl. material 2, we provide all EIV systems of vascular plants that we used with their original and harmonised plant no menclature (see below) and their original and rescaled val ues (see below) for the five dimensions considered. Introduction Please note that for several EIV systems we could only approximate the geographic scope, as they did not provide a map or precise verbal description. Two EIV systems refer to very small areas that are hardly visible on the European map: the Faroe Islands and the Kazbegi region of Georgia. 1 system 2 systems 3 systems Complete source systems Partial source systems European mires Czechoslovakia Ambros Germany Dierschke Slovenia Sweden Diekmann Figure 1. Map of Europe showing the areas covered by ecological indicator value (EIV) systems that were used to de rive the Ecological Indicator Values for Europe (EIVE) 1.0. Colours indicate the number of EIV systems covering the complete vascular plant flora. Hatched and dotted areas refer to EIV systems that cover only a subset of specific habitats. Please note that for several EIV systems we could only approximate the geographic scope, as they did not provide a map or precise verbal description. Two EIV systems refer to very small areas that are hardly visible on the European map: the Faroe Islands and the Kazbegi region of Georgia. gradients on numerical scales. Of those, we used the 31 EIV systems that included indicator values of vascular plants for at least one of the five most frequent indicators, namely moisture (M), reaction (R), nitrogen (N), temper ature (T) and light (L) (Table 1; further details in Suppl. material 1). We intentionally denote the N indicator as “nitrogen”, not as “nutrients” as in some EIV systems. The reason is that existing tests of correlations of mean N EIVs with measured environmental variables mostly reported significant relationships with nitrogen-related measures (C/N ratio, potential N mineralisation) (Ellenberg et al. 1991; Ewald 2003). Moreover, Tyler et al. (2021) defined separate indicators for nitrogen and phosphorus. we provide one value for niche position and one for niche width. In this paper, we describe the development of EIVE and release version 1.0 as an open access database to ini tiate a community-based approach for future updates and extensions. Harmonisation of plant taxonomy material 3: table S3.3): These belong mainly to three groups: (1) neophytes (of which only few are included in Euro+Med 2022), (2) recently described or locally endemic taxa not included in Euro+Med (2022) and (3) autonyms of polytypic species. The latter are cases where a taxon is treated as a species with several accepted subspe cies in Euro+Med (2022), but the typical subspe cies (the autonym) is missing for unknown reasons despite it is often the most widespread subspecies (e.g. Anthyllis vulneraria subsp. vulneraria and Che nopodium album subsp. album). Native European species not automatically matched to a Euro+Med taxon were only exceptionally accepted as separate taxa (these could be newly described species or lo cal endemics); we generally rather assumed that these were included in the concept of an accepted Euro+Med taxon, but not included in its list of syn onyms in Euro+Med (2022). (iii) Other additional taxa (Suppl. material 3: table S3.3): These belong mainly to three groups: (1) neophytes (of which only few are included in Euro+Med 2022), (2) recently described or locally endemic taxa not included in Euro+Med (2022) and (3) autonyms of polytypic species. The latter are cases where a taxon is treated as a species with several accepted subspe cies in Euro+Med (2022), but the typical subspe cies (the autonym) is missing for unknown reasons despite it is often the most widespread subspecies (e.g. Anthyllis vulneraria subsp. vulneraria and Che nopodium album subsp. album). Native European species not automatically matched to a Euro+Med taxon were only exceptionally accepted as separate taxa (these could be newly described species or lo cal endemics); we generally rather assumed that these were included in the concept of an accepted Euro+Med taxon, but not included in its list of syn onyms in Euro+Med (2022). Harmonisation of plant taxonomy We collected all indicator value systems known to us that contain assessments of plants regarding their niche posi tion (and potentially also niche width) along ecological We first split the original taxon names as they appeared in the 31 EIV systems into genus name, species epithet, Jürgen Dengler et al.: Ecological Indicator Values for Europe 1.0 12 of the EIV systems had been raised to species rank and, thus, there was no taxon concept available in Euro+Med (2022) to match the former polytypic species. Formal sections were given prevalence over informal aggregates; collective species and enumera tions of taxa (e.g. “Empetrum hermaphroditum + ni grum”) were replaced by aggregates. In cases where several different aggregates with different taxonom ic extent had been defined for the same species in different sources, we defined aggregates s.l., with at least one member being an aggregate itself. In con clusion, we accepted four types of supraspecific taxa on three hierarchy levels: (1) aggregates (aggr.), (2) aggregates s.l. (aggr. s.l.) and sections (sect.) and (3) enumeration of sections (one case: Rubus sect. Co rylifolii + Rubus). All of them were defined by listing their accepted member taxa of the next-lower rank. infraspecific epithets, rank-indicating abbreviations (such as subsp., var., aggr.) and taxonomic authorities. Genus names and species epithets were searched for typos and rank-indicating abbreviations standardised to “sect.”, “subg.”, “aggr.”, “subsp.”, “nothosubsp.”, “var.” and “×”. Ad ditions like “sensu lato” and “sensu stricto” were retained at this step, but harmonised in spelling to “s. l.” and “s. str.” to support name interpretation in the following steps. Taxonomic authorities were disregarded, as there is a huge variety of spelling variants and they rarely aid in the dis crimination of false vs. correct interpretations. This first step resulted in the assignment of a (preliminary) harmo nised original taxon name. In a second step, we retrieved the database underlying the Euro+Med Plantbase with accepted taxon names and all synonymy and parent-child relationships on 2022-03- 21 (Euro+Med 2022). When a harmonised original taxon name (not considering s. l. and s. str.) matched an accepted Euro+Med name, this name was assigned as our prelimi nary accepted name; when it matched a synonym in Eu ro+Med (2022), we assigned it to the accepted Euro+Med name. All other harmonised original taxon names were treated as “unresolved” at this step.h p (iii) Other additional taxa (Suppl. Harmonisation of plant taxonomy Third, the numerous “unresolved” names were all checked by experts to pinpoint reasons for mismatching and treated according to one of the following rules: (a) if the spelling harmonisation of step (1) had failed for some reason, the R code was adjusted; (b) if the reason for the non-match was a typo, such as “vemalis” instead of “ver nalis”, an epithet erroneously starting with a capital letter or a name field containing also the synonym (“Alchemi lla baltica=nebulosa”), the adjustments were made in the harmonised original taxon name; (c) if we, however, came to the conclusion that there was no spelling error, but the name was missing in Euro+Med (2022) completely (as ei ther accepted or synonymic name), we defined additions to Euro+Med (2022). (iv) Additional synonyms (Suppl. material 3: table S3.4): This file documents all additional synonymic rela tionships not included in Euro+Med (2022), includ ing those involving “aggregates”. Our additions fall into four categories and are compre hensively documented with explicit definition of content, taxonomic authorities and the source of the definition where applicable (Suppl. material 3): When making additions, we strived for consistency with Euro+Med (2022), respecting the species and genus concepts adopted in this source. For example, the origi nal hybrid taxon name Chamaecytisus ×versicolor in one of the EIV systems is treated as Cytisus ×versicolor be cause the genus Chamaecytisus in Euro+Med (2022) is included in the genus Cytisus. Therefore, this case led to entries both in the “additional synonyms” and the “hy brid definition” file. These four files with the taxonom ic additions were used to expand the “taxonomic back bone” of Euro+Med (2022) to something that we call “Euro+Med augmented”. (i) Hybrids (Suppl. material 3: table S3.1): All hybrids for which indicator values were given in at least one EIV system were accepted and defined by their parents. They were preferentially referred to by a bi nomen (or trinomen), and only if this was not avail able as a hybrid formula. (ii) “Aggregates” (here used as a generic term to refer to any formal or informal taxon between species and genus rank; Suppl. material 3: table S3.2): As a basis, we accepted the few aggregates, collective species (coll.) and sections that were accepted in Euro+Med (2022). To these we added all aggregates that occurred in any of the EIV systems and those that are widely used in vegetation science (e.g. Harmonisation of plant taxonomy from Ehrendorfer 1973; Wisskirchen and Haeupler 1998; Juillerat et al. 2017). We additionally defined ad-hoc aggregates when taxa treated as subspecies in one Further, we identified cases in which the same taxon name has been applied to different taxa by different EIV systems. Often the same correctly applied name might re fer to a concept of different width (subspecies vs. species, species vs. aggregate, aggregate vs. aggregate s.l.; see Jan sen and Dengler 2010). Rather rare are cases of names that 13 Vegetation Classification and Survey EIV systems “Austria” and “Hungary_Soo”, which differ entiated indicator values of woody taxa according to veg etation layer, we considered only the herb layer indicator. In the case of “USSR_Ramensky” and “Slovenia”, which contained different assessments depending on vegetation zone or soil type, we averaged the values per species. If a taxon was given as being “indifferent” (e.g. by the symbol “x”), we took the average indicator value across all taxa in the relevant EIV system as the most plausible assessment of its niche position. have been misapplied in a certain EIV system. When we identified such cases, we documented EIV-specific assign ments of the names (“concept synonyms”; Suppl. materi al 3: table S3.5). These assignments were then used at the end of the taxonomic workflow to overrule the prelimi nary assignments of accepted names in these cases.l We ran the whole automated workflow repeatedly over all combinations of original name and EIV system until only a small number of unresolved taxa remained and there were no evident mis-assignments. p Next, we scaled the raw indicator values of each EIV system (EIVori) linearly to a range of 0 to 10, with the idea that 0 should represent the lowest possible and 10 the high est possible value of the respective environmental variable in Europe (Figure 2, step 1). We applied an expert resca ling in case of T and for the dry end of M, when the defi nitions of the individual EIV system suggested that only part of the full European gradient was covered. For exam ple, in the case of T, this means that one must add three more levels to the classical scale of Ellenberg et al. (1991) to approximately reach the temperature conditions in the southern Mediterranean region, as proposed by Guarino et al. (2012). Likewise, Böhling et al. Harmonisation of plant taxonomy (2002) proposed that the driest sites in the southernmost Mediterranean areas are one level drier than the most xeric sites in Central Eu rope on the classical scale of Ellenberg et al. (1991). On the other hand, Böhling et al. (2002) assigned T = 1 to a mean annual temperature that roughly corresponds to the median of mean annual temperatures in Europe, not their minimum. Details of such adjusted “offsets” are doc umented in Suppl. material 1. By contrast, we assumed a priori that the scales in all source EIV systems cover the EIVEadj.mi = meanj(EIVadji,j) If an EIV system after our taxonomic harmonisation contained several taxa that correspond to the same tax on of “Euro+Med augmented”, we assigned the arithme tic mean of the indicator values to the latter. In cases of nested taxa (subspecies in species, species in aggregates) we derived EIV values for the superior level by averaging the EIV values of the member taxa of the next-lower level. This was only done if the taxon at the higher level did not have an EIV value assigned in the source. The resulting fit between EIVEadj and EIVadj was on average better (i.e. the slope was closer to 1) than be tween EIVEini and EIVini (Suppl. material 5). When we tried another round of iteration, this resulted in little to no further improvement. Thus, we retained EIVEadj for the remaining steps. However, the iteration gener ally caused a contraction, or very rarely an expansion, of the value range, so that EIVEadj did not cover the full intended range of 0 to 10 anymore. To remedy this, a final step of rescaling (Figure 2, step 5) was applied to EIVEadj to get EIVEres as the European indicator values of niche position: , = . + ( , − . ) . − . . − . , = . + ( , − . ) . − . . − . EIVadj.mi,j = ajmj + bjmj ∙ EIVinii,j . , = ( . , − . , ) ∙ EIVini.minj = lowest number scaled to the European range of 0 to 10 This numerical procedure standardised all regression lines for EIVEini vs. EIVadj to lie exactly on the 1:1 line. Subsequently, we created a new consensus system EIVE adj from the EIVadj values (Figure 2, step 4): ( ) − ( ) ( ) − ( ) EIVini.maxj = highest number scaled to the European range of 0 to 10 ( ) − ( ) ( ) − ( ) EIVini.maxj = highest number scaled to the European range of 0 to 10 Preparation of indicator values from the source EIV systems The 31 selected EIV systems were checked for entries that were not in accordance with their defined categories of the respective indicators and corrected if needed. Additional symbols, such as “~” for indication of fluctuating water table in the case of M, were removed. We merged indica tor values for moisture if they were defined by different growth forms under identical habitat conditions, such as M = 11 and M = 12 in Ellenberg et al. (1991), where 11 means “growing in permanent water, with leaves at or above the water surface” and 12 “growing in permanent water, with leaves below the surface”. For those systems that characterised the niche with minimum and maximum instead of a single number for niche position, we took the arithmetic mean of these two values as the metric of niche position. In four EIV systems, certain taxa had multiple assessments of their relevant indicators (Suppl. material 1). In the case of the Figure 2. Methodological workflow of deriving EIVE as a consensus system of the 31 EIV input systems. Orange and blue boxes refer to niche position and amplitude/niche width metrics, respectively. White letters refer to input and intermediate metrics, black letters describe the definitive metrics of EIVE 1.0. Numbers denote the steps which are described in more detail in the text. Figure 2. Methodological workflow of deriving EIVE as a consensus system of the 31 EIV input systems. Orange and blue boxes refer to niche position and amplitude/niche width metrics, respectively. White letters refer to input and intermediate metrics, black letters describe the definitive metrics of EIVE 1.0. Numbers denote the steps which are described in more detail in the text. 14 Jürgen Dengler et al.: Ecological Indicator Values for Europe 1.0 The initial indicator value of a taxon i of the European consensus system (EIVEini) was derived as follows from the scaled values in the individual EIV systems (EIVini) (Figure 2, step 2): same ranges for the dimensions R, N, L and the wet end of M (but note that, in some EIV systems, the full range of defined values is not actually covered by taxa). With these considerations in mind, the initial EIV value (EIVini) of taxon i in the individual EIV system j was derived from the EIV value on the original scale (EIVori) as follows: EIVEini.mi = meanj(EIVinii,j) EIVEini.mi = meanj(EIVinii,j) , = . + ( , − . ) . − . . − . with Using linear regression and correlation coefficients, we evaluated the results of EIVEini against all ex pert-scaled EIV systems (EIVini) for each of the five indicators (Suppl. material 5). While many regressions came close to the 1:1 line, most had a shallower slope (Suppl. material 5), meaning that the range of realised environmental conditions in a region was smaller than assumed in the expert-based scaling. Only in two cases (“Ukraine” and “USSR_Tsyganov” for M) the opposite was true (Suppl. material 5). We thus tried to remove the remaining major discrepancies in the concepts of the different EIV systems using an automated linear optimisation (Figure 2, step 3), with aj and bj being in tercept and slope, respectively, of the regression EIVEi ni vs. EIVini. In this way, the values of both EIVini and EIVEini were iteratively adjusted. . = 10 . − ( . ) EIVorii,j = original indicator value of taxon i in the respective EIV system j . = 10 . − ( . ) EIVorii,j = original indicator value of taxon i in the respective EIV system j ( . ) − ( . EIVinii,j = indicator value scaled to a European range of 0 to 10 = EIVori.minj = lowest number that is defined in the respective EIV system = EIVori.minj = lowest number that is defined in the respective EIV system 10 . − ( . ) ( . ) − ( . ) EIVori.maxj = highest number that is defined in the respective EIV system 10 . − ( . ) ( . ) − ( . ) EIVori.maxj = highest number that is defined in the respective EIV system EIVadj.mi,j = ajmj + bjmj ∙ EIVinii,j Deriving European niche width indicators to account for the fact that a niche width of zero does not exist. In case of EIV systems with categorical niche width coding, we assumed standard widths w for each of the four categories on the scale of 0 to 10, namely # → 1.25, I → 2.5, II → 5 and x → 7.5 (Figure 2, step 8). In absence of precise definitions (which was the case for most of the sources), we assume that these assignments should generally reflect the intended meaning of the authors, at least their relative relationships. The final amplitude of taxon i in EIV system j (EIV.ai,j) was calculated as follows (note that here EIVini and not EIVori had to be used as starting point): To establish a European indicator of niche width for each taxon in each of the five niche dimensions, we developed a separate workflow for the heterogeneous information in the various EIV systems. While some provided only a niche position, others provided niche width informa tion as a range (minimum and maximum values) or as amplitude classes with two to four levels. If a source EIV system contained categorical niche amplitude informa tion, we harmonised the coding. In Suppl. material 2, amplitude classes are stored as “#” for particularly nar row amplitude, “I” for normal amplitude, “II” for wide amplitude, but not indifferent, and “x” for “indifferent”. We considered uncertain information (coded by smaller font in Ellenberg et al. 1991) for the purpose of calcu lating mean indicator values of a plot as equivalent to a wide amplitude (II). , = . + ( , − . ) . − . . − . . = 10 . − ( . ) ( . ) − ( . EIV a w max EIVres min EIVres max EIVini min EIVini i j j j j j . , EIV a w max EIVres min EIVres max EIVini min EIVini i j j j j j . , To derive European indicators for niche width (Figure 3), we applied three different approaches to com bine the rescaled niche position and niche width indi cators of all EIV systems (Figure 2, step 9). They were constructed to meet the idea that the niche width at the European level is composed of intraregional and interre gional variability in the niches. Creating a consensus system of niche positions To derive European values of niche position, we applied three different approaches to combine the scaled EIVs of all systems in which the respective taxon was includ ed: (i) median; (ii) mean and (iii) weighted mean. With our “niche position” we aim to approximate the position on an ecological gradient which roughly separates equal halves of species occurrences. Therefore, niche position differs from realised niche optimum or mode (the envi ronmental conditions under which a species is most fre quent and/or reaches the highest cover values), particu larly in the case of skewed or bimodal distributions. In the following, we describe the “mean” variant, while the analogous calculations for “median” and “weighted mean” are explained in Suppl. material 4. . = 10 . − ( . ) ( . ) − ( . . = 10 . − ( . ) ( . ) − ( . The exact same rescaling was applied to EIVadj to get EIVres (Figure 2, step 6): . , = 10 . − ( . ) ( . ) − ( . ) . , = 10 . − ( . ) ( . ) − ( . ) . , = 10 . − ( . ) ( . ) − ( . ) . , = 10 . − ( . ) ( . ) − ( . ) Vegetation Classification and Survey 15 Comparing EIVE temperature indicators with bioclimate For one selected niche dimension, the temperature indica tor, we validated our three consensus approaches for niche position calculation (median, mean and weighted mean) by comparing their results for species with the bioclimat ic characteristics of these species globally. The T indicator was chosen since the temperature niche is relatively easy to calculate from readily available independent data. For this purpose, we correlated the T values of species (not consid ering other taxonomic ranks) with the temperature charac teristics derived from their geographic distributions. These were retrieved from the Global Biodiversity Information Facility portal (GBIF 2022; Suppl. material 6) for 9,446 spe cies (85% of the species in EIVE; if several EIVE species cor responded to the same GBIF species, they were not consid ered). The corresponding approx. 145 million distribution records were subsequently thinned to one coordinate per species and 30 arc second grid cell to reduce the bias of local oversampling. In addition, occurrences marked as managed in the GBIF database (field EstablishmentMeans) were not used for further analysis. From the remaining approx. 65.8 million coordinates, we extracted for each species nineteen bioclimatic variables from CHELSA V2.1 (Karger et al. 2017, 2021) with the same spatial resolution of 30 arc sec onds and calculated the median of each variable. Out of all nineteen bioclimatic variables, the variable bio10, i.e. mean daily mean air temperature of the warmest quarter, showed the highest Pearson correlation with our EIVE-T values (in each of the three variants, see above). Subsequently, we com pared the three variants to combine the rescaled individual EIV systems into a European consensus system with bio10 median values, defining the best-performing approach as EIVE 1.0. This system was then used for further compar isons, namely with the T values of the 23 source EIV sys tems that contained T. Moreover, we also compared EIVE 1.0 with the European T values recently proposed by Tichý et al. (2023). For all comparisons, Pearson correlations were calculated for the subset of species co-occurring in EIVE and the respective EIV system, and the most highly corre lated bioclimatic variable was determined for both EIVE and the EIV system. For the evaluation of our three variants to calculate EIVE niche width, we used the same CHEL SA bioclimatic data and GBIF coordinates but calculated the interquartile range (IQR) and the standard deviation of bio1 and bio10. Deriving European niche width indicators In the following, we de scribe the “nw3” variant, while the analogous calculations for “nw1” and “nw2” are explained in Suppl. material 4.h For the further calculations, we chose the final out comes of the EIVE niche position calculation, i.e. the rescaled values (EIVEres) of the best variant according to the external validation (see below). In EIV systems j with range-based niche width coding, we derived the ampli tude of taxon i (EIV.ai j) as follows (Figure 2, step 7): . , = 10 . − ( . ) ( . ) − ( . ) . , = ( . , − . , ) ∙ ( ) − ( ) ( ) − ( ) The nw3 indicator was calculated as the sum of the average amplitude of taxon i across EIV systems (intra regional variation) and twice the population standard de viation (σ) of the niche position (interregional variation), bounded to a maximum of 10: If, for a certain taxon in a range-based system, mini mum and maximum were the same (EIVori.maxi,j = EIVori. mini,j), we assigned to EIV.ai,j half of the minimum non-ze ro amplitude that occurred for other taxa in this system EIVE nw min EIV a EIVres i i i . , . 3 10 2σ EIVE nw min EIV a EIVres i i i . , . 3 10 2σ Figure 3. Combining interregional (based on niche position, red) and intraregional (based on niche amplitude, blue) information to derive a composite pan-European indicator of niche width (EIVE.nw, green), with three variants (grey). EIVres = regional ecological indicator value, rescaled, EIVEres = Ecological Indicator Value of Europe, rescaled. Figure 3. Combining interregional (based on niche position, red) and intraregional (based on niche amplitude, blue) information to derive a composite pan-European indicator of niche width (EIVE.nw, green), with three variants (grey). EIVres = regional ecological indicator value, rescaled, EIVEres = Ecological Indicator Value of Europe, rescaled. Jürgen Dengler et al.: Ecological Indicator Values for Europe 1.0 16 Comparing EIVE temperature indicators with bioclimate Comparing EIVE temperature indicators with bioclimate 6,470 (Alps) vascular plant taxa with at least one of the five niche dimensions assessed. The combined data com prised 77,795 rows of taxon name × EIV system combina tions, corresponding to 14,835 accepted taxa: 22 sections, 60 aggregates s.l., 664 aggregates, 11,148 species, 2,899 subspecies and 42 varieties. Comparing EIVE temperature indicators with bioclimate The comparison of the EIVE temperature indicator with bioclimate was performed in R (R Core Team 2022) using the R packages rgbif (Chamberlain et al. 2023) and terra (Hijmans 2022). Table 2. Accepted taxa, number of assessments (i.e. ac cepted taxa x EIV systems in which they were assessed) and mean number of assessments on which the consensus values in EIVE 1.0 was based. Indicator Accepted taxa Assessments Assessments / accepted taxa M – Moisture 14,714 74,640 5.1 N – Nitrogen 13,748 60,120 4.4 R – Reaction 14,254 65,281 4.6 L – Light 14,054 59,547 4.2 T – Temperature 14,496 63,889 4.4 The iterative workflow to derive EIVE 1.0 clearly im proved the congruence of the EIVE scaling to that of the individual EIV systems, as can be seen in an increase of the mean slope of the linear regressions from EIVEini vs. EIVini to EIVEres vs. EIVres (Suppl. material 5): the mean slope based on the mean variant for M improved from 0.872 to 0.878, for N from 0.756 to 0.775, for R from 0.709 to 0.722, for L from 0.755 to 0.761 and for T from 0.746 to 0.801 (Suppl. material 5: table S5.1). The iteration particularly brought those EIV systems closer to the 1:1 line that deviated strongest, as can be seen in the strong reduction of the absolute values of the extreme deviations to about one seventh to one half (columns “min diff.” and “max. diff.” in Suppl. material 5: table S5.1). The various steps of transformation (Figure 3) in most cases caused a contraction of the value ranges of individual EIV systems from EIVini to EIVres (after simple expert-based rescaling to 0…10; see Suppl. material 5). Performance of the consensus systems The Pearson correlation between EIVE-T values and me dian bio10 values was highest for the calculation variant “mean” (r = 0.859; see Figure 4) and showed minimal ly lower values for “median” (r = 0.857) and “weighted mean” (r = 0.857). Thus, we accepted the values of the consensus variant “mean” as the niche position indicators in EIVE 1.0 and used them for further comparisons with individual EIV systems. In addition to the bioclimatic Deriving European niche width indicators Of these, 13,017 were from Euro+Med (2022) while 1,819 were EIVE additions to the taxonomic backbone. Only 22 (0.03%) of all taxon name × EIV system combinations remained unresolved for the time being, meaning that we did not decide whether they are separate taxa or synonyms of other taxa. The European consensus system of the five niche dimensions contains between 13,748 and 14,714 accepted taxa, with an average of 4 and more assessments underlying each EIVE value (Table 2). Data processing Species occurring in at least four EIV systems are displayed in blue and the fitted regression line for this species subset is shown in red. Species which were covered by less than four EIV systems are in grey. Figure 4. Scatter plot of the temperature indicator T of EIVE 1.0 (mean approach) and median values of the CHELSA bioclimatic variable bio10 (mean daily mean air temperatures of the warmest quarter) at GBIF coordi nates of the species. The black line was fitted for all spe cies by least squares linear regression. Species occurring in at least four EIV systems are displayed in blue and the fitted regression line for this species subset is shown in red. Species which were covered by less than four EIV systems are in grey. Figure 5. Niche position (A) and niche width (B) distribu tion of the five niche dimensions in EIVE 1.0. The figure refers to the accepted calculation variants, i.e. “mean” in the case of niche position and “nw3” in the case of niche width. Equal-area violin plots are displayed with median (horizontal line), mean (point) and standard deviation (vertical error bar). The number of taxa for each niche dimension is indicated at the top of the upper plot. variable bio10, “mean annual air temperature” (bio1) was also frequently identified as the most highly correlated bi oclimatic variable for the restricted species subsets “medi an” (r = 0.857) and “weighted mean” (r = 0.857). Only in two cases did the original EIV-T values show higher cor relations with other bioclimatic variables: bio5, i.e. “mean daily maximum air temperature of the warmest month” for “Austria_Pannonian” and bio8, i.e. “mean daily mean air temperatures of the wettest quarter” in the case of “Greece”. Correlations of EIVE-T were in general higher than those of both the EIV-T values of the original EIV systems and the European Ellenberg-type indicator values (Tichý et al. 2023) (Table 3). and was less sensitive to extreme regional values. The Pearson correlation between EIVE-T niche width values and the standard deviation of bio1 and bio10 values was higher for variant “nw3” (r = 0.160 and 0.133, respec tively) than for the variants “nw2” (r = 0.143 and 0.106) and “nw1” (r = 0.087 and 0.030) (Suppl. material 7: fig ure S7.2). Data processing Thus, we decided to retain “nw3”, a composite niche width metric based on intraregional average am plitude and interregional dispersion of niche position, as the niche width indicator in EIVE 1.0. The distribution of interregional niche width met rics (position range and position standard deviation) was very skewed, with many 0 values, whereas the dis tribution of intraregional metrics (average amplitude) showed multimodality. However, the three variants of composite niche width metrics showed a more homo geneous distribution (Suppl. material 7: figure S7.1). Therefore, we decided to choose one of these three com posite metrics as consensus niche width. The first one (“nw1”), as the total range, generally had the highest val ues and was very sensitive to extreme position and am plitude values in some individual systems. The second one (“nw2”) is partly based on position range and may also be strongly influenced by extreme position values. The third one (“nw3”) generally had the lowest values Data processing After taxonomic harmonisation, the 31 source EIV sys tems contained between 34 (Sweden_Diekmann) and Vegetation Classification and Survey 17 Figure 4. Scatter plot of the temperature indicator T of EIVE 1.0 (mean approach) and median values of the CHELSA bioclimatic variable bio10 (mean daily mean air temperatures of the warmest quarter) at GBIF coordi nates of the species. The black line was fitted for all spe cies by least squares linear regression. Species occurring in at least four EIV systems are displayed in blue and the fitted regression line for this species subset is shown in red. Species which were covered by less than four EIV systems are in grey. Figure 5. Niche position (A) and niche width (B) dis tion of the five niche dimensions in EIVE 1.0. The refers to the accepted calculation variants, i.e. “me the case of niche position and “nw3” in the case of width. Equal-area violin plots are displayed with m (horizontal line), mean (point) and standard dev (vertical error bar). The number of taxa for each Figure 5. Niche position (A) and niche width (B) distribu tion of the five niche dimensions in EIVE 1.0. The figure refers to the accepted calculation variants, i.e. “mean” in the case of niche position and “nw3” in the case of niche width. Equal-area violin plots are displayed with median (horizontal line), mean (point) and standard deviation (vertical error bar). The number of taxa for each niche dimension is indicated at the top of the upper plot. Figure 4. Scatter plot of the temperature indicator T of EIVE 1.0 (mean approach) and median values of the CHELSA bioclimatic variable bio10 (mean daily mean air temperatures of the warmest quarter) at GBIF coordi nates of the species. The black line was fitted for all spe cies by least squares linear regression. Species occurring in at least four EIV systems are displayed in blue and the fitted regression line for this species subset is shown in red. Species which were covered by less than four EIV systems are in grey. Figure 4. Scatter plot of the temperature indicator T of EIVE 1.0 (mean approach) and median values of the CHELSA bioclimatic variable bio10 (mean daily mean air temperatures of the warmest quarter) at GBIF coordi nates of the species. The black line was fitted for all spe cies by least squares linear regression. Properties of EIVE 1.0 difference Alps 4253 BIO10 0.8611 0.8969 +0.0358 BIO10 0.8611 0.8969 +0.0358 Austria 2291 BIO10 0.8649 0.9152 +0.0503 BIO10 0.8649 0.9152 +0.0503 Austria_Pannonian 835 BIO5 0.5945 0.7864 +0.1919 BIO10 0.5933 0.7975 +0.2042 Czech_Republic 2024 BIO10 0.7859 0.8469 +0.0610 BIO10 0.7859 0.8469 +0.0610 Czechoslovakia_Ambros 364 BIO10 0.7157 0.8798 +0.1641 BIO10 0.7157 0.8798 +0.1641 France 3171 BIO1 0.8028 0.8756 +0.0728 BIO1 0.8028 0.8756 +0.0728 Georgia 897 BIO1 0.3603 0.6994 +0.3391 BIO1 0.3603 0.6994 +0.3391 Germany 2561 BIO10 0.8473 0.9014 +0.0541 BIO10 0.8473 0.9014 +0.0541 Germany_GDR 1089 BIO10 0.6982 0.8084 +0.1102 BIO10 0.6982 0.8084 +0.1102 Greece 1906 BIO8 0.5801 0.3972 -0.1829 BIO1 0.5067 0.8249 +0.3182 Hungary_Borhidi 1844 BIO10 0.704 0.7914 +0.0874 BIO10 0.704 0.7914 +0.0874 Hungary_Soo 1825 BIO10 0.5936 0.7991 +0.2055 BIO10 0.5936 0.7991 +0.2055 Hungary_Zolyomi 1038 BIO10 0.6176 0.8139 +0.1963 BIO10 0.6176 0.8139 +0.1963 Italy 4718 BIO1 0.8304 0.8916 +0.0612 BIO1 0.8304 0.8916 +0.0612 Netherlands 1128 BIO10 0.5837 0.7583 +0.1746 BIO1 0.5457 0.7619 +0.2162 Poland 1874 BIO10 0.7943 0.9042 +0.1099 BIO10 0.7943 0.9042 +0.1099 Romania 2856 BIO10 0.7225 0.8610 +0.1385 BIO10 0.7225 0.8610 +0.1385 Serbia 1947 BIO10 0.7114 0.8622 +0.1508 BIO10 0.7114 0.8622 +0.1508 Spain_Asturias 1596 BIO10 0.7091 0.8634 +0.1543 BIO1 0.7013 0.8700 +0.1687 Spain_Cantabria 1641 BIO1 0.5599 0.8623 +0.3024 BIO1 0.5599 0.8623 +0.3024 Sweden 2035 BIO1 0.8789 0.8811 +0.0022 BIO1 0.8789 0.8811 +0.0022 Ukraine 2520 BIO1 0.7731 0.8231 +0.0500 BIO10 0.7528 0.8506 +0.0978 USSR_Tsyganov 1815 BIO1 0.7187 0.7665 +0.0478 BIO10 0.7132 0.8078 +0.0946 Tichý et al. (2023) 6160 BIO1 0.8752 0.8839 +0.0087 BIO10 0.8516 0.8862 +0.0346 EIV system Species EIV based bioclimate selection EIVE based bioclimate selection best EIV cor. EIVE cor. difference best EIV cor. EIVE cor. Properties of EIVE 1.0 Finally, our documentation of all taxonomic deviations from Euro+Med (2022) forms part of the data base (Suppl. material 3). The selected EIVE niche width measure based on both intra- and interregional variation showed remarkably sim ilar patterns across the five niche dimensions (Figure 5b). All distributions were strongly skewed towards narrow niche width, with a pronounced main mode of the value distribution somewhere between 2.5 and 2.9 (Figure 5b). The potential value range up to 10 was (almost) covered only by a small number of taxa in all indicators. The mean EIVEnw values were lowest for moisture (3.2) and highest for nitrogen and reaction (3.8). The value distribution was smooth, as would be expected for an interval-scaled vari able. Only EIVEnw-M had a subordinate mode at 4.2 in addition to the main mode at 2.5 (Figure 5b). In addition to the online appendix of this paper, the EIVE 1.0 database is available at https://zenodo.org/re cord/7534792. The R code to derive EIVE from the source files and to produce the figures and statistics of this pa pers is available upon request from F.J. A source file to be used for the calculation of mean EIVEs in the software JUICE (Tichý 2002) is provided at: https://www.sci.muni. cz/botany/juice/?idm=10. An interactive tool to compare regional EIVs with the European variants for niche posi tion and niche width is available as a Shiny App at https:// data.loe.auf.uni-rostock.de/EIVE. Properties of EIVE 1.0 difference Alps 4253 BIO10 0.8611 0.8969 +0.0358 BIO10 0.8611 0.8969 +0.0358 Austria 2291 BIO10 0.8649 0.9152 +0.0503 BIO10 0.8649 0.9152 +0.0503 Austria_Pannonian 835 BIO5 0.5945 0.7864 +0.1919 BIO10 0.5933 0.7975 +0.2042 Czech_Republic 2024 BIO10 0.7859 0.8469 +0.0610 BIO10 0.7859 0.8469 +0.0610 Czechoslovakia_Ambros 364 BIO10 0.7157 0.8798 +0.1641 BIO10 0.7157 0.8798 +0.1641 France 3171 BIO1 0.8028 0.8756 +0.0728 BIO1 0.8028 0.8756 +0.0728 Georgia 897 BIO1 0.3603 0.6994 +0.3391 BIO1 0.3603 0.6994 +0.3391 Germany 2561 BIO10 0.8473 0.9014 +0.0541 BIO10 0.8473 0.9014 +0.0541 Germany_GDR 1089 BIO10 0.6982 0.8084 +0.1102 BIO10 0.6982 0.8084 +0.1102 Greece 1906 BIO8 0.5801 0.3972 -0.1829 BIO1 0.5067 0.8249 +0.3182 Hungary_Borhidi 1844 BIO10 0.704 0.7914 +0.0874 BIO10 0.704 0.7914 +0.0874 Hungary_Soo 1825 BIO10 0.5936 0.7991 +0.2055 BIO10 0.5936 0.7991 +0.2055 Hungary_Zolyomi 1038 BIO10 0.6176 0.8139 +0.1963 BIO10 0.6176 0.8139 +0.1963 Italy 4718 BIO1 0.8304 0.8916 +0.0612 BIO1 0.8304 0.8916 +0.0612 Netherlands 1128 BIO10 0.5837 0.7583 +0.1746 BIO1 0.5457 0.7619 +0.2162 Poland 1874 BIO10 0.7943 0.9042 +0.1099 BIO10 0.7943 0.9042 +0.1099 Romania 2856 BIO10 0.7225 0.8610 +0.1385 BIO10 0.7225 0.8610 +0.1385 Serbia 1947 BIO10 0.7114 0.8622 +0.1508 BIO10 0.7114 0.8622 +0.1508 Spain_Asturias 1596 BIO10 0.7091 0.8634 +0.1543 BIO1 0.7013 0.8700 +0.1687 Spain_Cantabria 1641 BIO1 0.5599 0.8623 +0.3024 BIO1 0.5599 0.8623 +0.3024 Sweden 2035 BIO1 0.8789 0.8811 +0.0022 BIO1 0.8789 0.8811 +0.0022 Ukraine 2520 BIO1 0.7731 0.8231 +0.0500 BIO10 0.7528 0.8506 +0.0978 USSR_Tsyganov 1815 BIO1 0.7187 0.7665 +0.0478 BIO10 0.7132 0.8078 +0.0946 Tichý et al. (2023) 6160 BIO1 0.8752 0.8839 +0.0087 BIO10 0.8516 0.8862 +0.0346 that the resulting value distribution of the EIVE indica tors was rather smooth, as would be expected for an inter val-scaled variable. Only EIVE-M had two main modes at 3.3 and 4.3, while EIVE-L had a main mode at 8.2 and a subordinate mode at 9.5, but even in these cases the modes were not clearly separated. source of the taxon concept (Euro+Med, EIVE addition, unresolved) and then for each of the five niche dimen sions (M, N, R, L, T) (4) the niche position value (e.g. EIVE-M for moisture), (5) the niche width indicator (e.g. EIVE-M.nw) and (6) the number of source EIV systems on which the consensus values were based (EIVE-M.n) (Suppl. material 8). Further, we provide all source EIV systems with their original and harmonised taxon names and the corresponding name in our taxonomic backbone, together with the original and rescaled niche position and, where available, niche width information (Suppl. material 2). Properties of EIVE 1.0 Per definition, the five EIVE indicators for niche posi tions cover the full range of 0 to 10. Plotting the number of plant species in the European species pool on the five niche dimensions revealed characteristic patterns (Figure 5a). EIVE-T showed an almost symmetric distribution of species centred around middle temperatures (EIVE-T ≈ 5), while the remaining four other indicators were asym metrically distributed. The EIVE distribution was skewed towards higher values in case of EIVE-R and EIVE-L but towards lower values in case of EIVE-M and EIVE-N (Figure 5a). Given that the source EIV systems were all ordinal with mostly only a few categories, it is interesting Jürgen Dengler et al.: Ecological Indicator Values for Europe 1.0 18 Table 3. Highest Pearson correlations between indicator values for temperature and the median values of 19 CHEL SA bioclimatic variables extracted at species occurrences (GBIF), comparing the source EIV systems with T values and the Ellenberg-type indicator values by Tichý et al. (2023) with EIVE. For a fair comparison, the bioclimatic vari ables with the highest correlations were determined separately for both EIV and EIVE indicator values (“best”), and correlations for EIVE (“EIVE cor.”) and the other EIV systems (“EIV cor.”) were calculated for the same number of species (“Species”) co-occurring in EIVE and the respective other system. For each comparison, the higher correla tion is indicated in bold, and the difference of the correlations is reported (for naming of EIV systems, see Table 1; BIO1, BIO5, BIO8, BIO10 are CHELSA bioclimatic variables). Table 3. Highest Pearson correlations between indicator values for temperature and the median values of 19 CHEL SA bioclimatic variables extracted at species occurrences (GBIF), comparing the source EIV systems with T values and the Ellenberg-type indicator values by Tichý et al. (2023) with EIVE. For a fair comparison, the bioclimatic vari ables with the highest correlations were determined separately for both EIV and EIVE indicator values (“best”), and correlations for EIVE (“EIVE cor.”) and the other EIV systems (“EIV cor.”) were calculated for the same number of species (“Species”) co-occurring in EIVE and the respective other system. For each comparison, the higher correla tion is indicated in bold, and the difference of the correlations is reported (for naming of EIV systems, see Table 1; BIO1, BIO5, BIO8, BIO10 are CHELSA bioclimatic variables). Properties of EIVE 1.0 that the resulting value distribution of the EIVE indica tors was rather smooth, as would be expected for an inter val-scaled variable. Only EIVE-M had two main modes at 3.3 and 4.3, while EIVE-L had a main mode at 8.2 and a subordinate mode at 9.5, but even in these cases the modes were not clearly separated. The selected EIVE niche width measure based on both intra- and interregional variation showed remarkably sim ilar patterns across the five niche dimensions (Figure 5b). All distributions were strongly skewed towards narrow niche width, with a pronounced main mode of the value distribution somewhere between 2.5 and 2.9 (Figure 5b). The potential alue range up to 10 as (almost) co ered source of the taxon concept (Euro+Med, EIVE addition, unresolved) and then for each of the five niche dimen sions (M, N, R, L, T) (4) the niche position value (e.g. EIVE-M for moisture), (5) the niche width indicator (e.g. EIVE-M.nw) and (6) the number of source EIV systems on which the consensus values were based (EIVE-M.n) (Suppl. material 8). Further, we provide all source EIV systems with their original and harmonised taxon names and the corresponding name in our taxonomic backbone, together with the original and rescaled niche position and, where available, niche width information (Suppl. material 2). Finally, our documentation of all taxonomic de iations from Euro+Med (2022) forms part of the data EIV system Species EIV based bioclimate selection EIVE based bioclimate selection best EIV cor. EIVE cor. difference best EIV cor. EIVE cor. Content of EIVE 1.0 EIVE 1.0 provides assessments of ecological niche position and niche width for a total of 14,835 vascular plant taxa, including 11,148 at species rank. In terms of taxa covered, EIVE 1.0 is thus the most comprehensive ecological indica tor value system published so far. In comparison, the most extensive source system of EIVE, Landolt et al. (2010), contains 6,471 vascular plant taxa. Compared to the su pranational Ellenberg-type indicator values developed in parallel by Tichý et al. (2023) with 8,908 accepted vascular plant taxa, EIVE has a 67% larger coverage (Table 4). While the exact number of vascular plant taxa occurring in Eu rope in the geographic sense is not known, we judge that a majority are included in EIVE 1.0, as we combined many national and regional EIV systems, most of them aiming at comprehensive coverage of the vascular plant flora of their focal territory. In comparison, Flora Europaea (Tutin et al. 1964–1980) enumerated 11,557 accepted species in Europe (but this number excludes most neophytes and any species described after the publication). Moreover, most of the countries lacking a dedicated EIV system host very few species that do not occur in neighbouring countries (e.g. Iceland, Norway, Finland, Denmark). We expect areas with non-negligible fractions of missing taxa to be concen trated in Mediterranean Iberia and the Balkan Peninsula.i p pl The majority of the source EIV systems had no in formation on niche width (e.g. Ambros 1986; Frank and Klotz 1990; Hill et al. 2004; Julve 2022), or distinguished only between species with definitive indicator values and “indifferent” species (e.g. Zólyomi et al. 1967; Mayor López 1999) (Table 1). However, a considerable number of EIV systems did actually provide information on niche ampli tudes by explicitly listing minima and maxima on the niche axes (e.g. Ramensky et al. 1956; Tsyganov 1983; Didukh 2011; Hájek et al. 2020) or systematic coding of three or four niche-width categories (Böhling et al. 2002; Landolt et al. 2010). However, EIVE is the first indicator value sys tem to provide a systematic and consistent assessment of niche width. Interestingly, our calculation approach “nw3” performed best in terms of external validation for the tem perature niche axis. Database The main part of the EIVE 1.0 database is a table with (1) the accepted taxon names, (2) the taxon rank, (3) the 19 Vegetation Classification and Survey Content of EIVE 1.0 (2023) EIVE 1.0 (this paper) Geographic coverage Focus on temperate Europe plus Italy; coverage varying between indicators Europe as a whole (in the geographic sense), extending slightly to adjacent areas Regional EIV systems used 12 (only those directly compatible with the original Ellenberg scales) 31 Number of accepted taxa 8,908* 14,835 Number of species 8,679* 11,148 Treatment of infraspecific taxa No Yes, as far as accepted in Euro+Med (2022) Indicators included M, N, R, S, L, T M, N, R, L, T Scaling of indicators Mostly 1‒9, but M and T 1‒12 and S 0‒9 All 0‒10 Values of indicators Interval scale, but prevalence of integers Interval scale Handling of indicator values that do not reflect the ecological niche but growth form or physiological niches Maintained as in Ellenberg et al. (1991) M values that differed only in growth form (such as 11 and 12 in Ellenberg et al. 1991) were merged Coding of niche width Not available Available for all indicator values and all species on an interval scale Calculation of European indicator values Mean of included EIV systems Mean of all available EIV systems after rescaling to the common 0-10 scale Use of species co-occurrence data from the European Vegetation Archive (EVA) EVA was used to add 431 species not covered in any of the included EIV systems Not used * According to M. Chytrý (pers. comm.) the number of 8,908 “species” given in Tichý et al. (2023) actually does not mean species but accepted taxa including aggregates. Table 4. Major differences between the two new ecological indicator value systems for Europe. Criterion Tichý et al. (2023) EIVE 1.0 (this paper) Geographic coverage Focus on temperate Europe plus Italy; coverage varying between indicators Europe as a whole (in the geographic sense), extending slightly to adjacent areas Regional EIV systems used 12 (only those directly compatible with the original Ellenberg scales) 31 Number of accepted taxa 8,908* 14,835 Number of species 8,679* 11,148 Treatment of infraspecific taxa No Yes, as far as accepted in Euro+Med (2022) Indicators included M, N, R, S, L, T M, N, R, L, T Scaling of indicators Mostly 1‒9, but M and T 1‒12 and S 0‒9 All 0‒10 Values of indicators Interval scale, but prevalence of integers Interval scale Handling of indicator values that do not reflect the ecological niche but growth form or physiological niches Maintained as in Ellenberg et al. Content of EIVE 1.0 This might be explained by the fact that using twice the standard deviation instead of the total range of niche positions across EIV systems (as in “nw1” and “nw2”) suppresses extreme outliers, which could be typos in the source systems (e.g. EIV-L = 9 for Abies alba on the 9-step scale in Frank and Klotz 1990). In addition to niche position, niche width is the second main parameter to describe an ecological niche, thus making the character isation of the ecological behaviour of species more com prehensive. Beyond that, we predict that by taking niche width into account when calculating mean ecological in dicator values, one could improve prediction at the plot scale. In the past, this has often been attempted using a yes/ no approach, i.e. disregarding species with the widest nich es (those assessed as indifferent or “x”), but we judge that a weighting approach accounting for continuous variation in niche width will improve predictions. There is hardly any We decided to include five indicators in EIVE 1.0, namely the three soil-related indicators, moisture, nitro gen and reaction, as well as light and temperature. We se lected these five because – apart from continentality and salinity – they have the highest coverage in the 31 avail able EIV systems addressing multiple particularly import ant dimensions of the ecological niches of plants at the same time. Continentality and salinity could be calculated with our approach relatively easily. However, we refrained from this step for the time being because we believe that, Table 4. Major differences between the two new ecological indicator value systems for Europe. Criterion Tichý et al. Discussion in their current form, these two indicators would not be compatible with the rest of the system. For continentality, Berg et al. (2017) highlighted the challenges of the current assessments and proposed an alternative approach, al though this is not yet available for a large fraction of the European vascular plant flora. Performance of EIVE 1.0 p Combining the EIVs of lower-rank taxa to obtain EIVs for species or species aggregates is another issue that may warrant some more work in future versions of EIVE, and which may be greatly facilitated by expected future results of ongoing projects such as the “Atlas Florae Europaeae” (Jalas and Suominen 1972 et seq.). Ideally, EIVs for such aggre gates, as well as for species comprising several subspecific taxa, should consider the relative geographic range and pop ulation size of the different included subordinate taxa, but since such sufficiently detailed and authoritative information is currently not available for all taxa, we decided to calculate EIVs for taxa of higher rank by simply averaging niche po sitions across all subordinate taxa. Although this method is admittedly suboptimal, it is simple and transparent, and it does not introduce any hidden errors caused by faulty bio geographic information. When considered appropriate for particular purposes (e.g. regional studies), the EIVs for these aggregate taxa can easily be re-calculated by individual us ers from the EIVs we provide for subordinate taxa. Further more, we only applied the aggregation when the source EIV system did not provide EIVs for the higher level itself. p 2 y One explanation for the good performance of EIVE might be that each of the included EIV systems is best understood as a single expert assessment, and every ex pert necessarily over- or underestimates niche positions of many species equivalent to “random measurement errors”. The more such independent assessments are combined, the closer they should get to reality, which is supported in our comparison (Figure 4) by a higher correlation for species occurring in at least four lists (r = 0.912 compared to the overall correlation r = 0.859). This effect might also explain why a combination of 23 systems (EIVE-T 1.0) performed better than that of only six systems (Tichý et al. 2023). An other explanation could be that by combining EIVs from a larger part of the geographic range of a species, we obtain a better estimate of the niche of the species as a whole, at least for temperature. While clearly advantageous for studies at broad geographic scales, the latter possible explanation may suggest that the regional EIV systems may still perform better within their individual geographic ranges. However, determining whether and when this is the case will require extensive testing (but see Moeys 2020). Performance of EIVE 1.0 We decided to follow the nomenclature and taxonomic concepts of the Euro+Med PlantBase (Euro+Med 2022) as much as possible, as this was considered the most com plete and authoritative taxonomic database covering all of Europe. However, most of our sources were based on na tional and older taxonomic references. Many discrepancies could be corrected automatically with our R code based on the lists of synonymous names included in the Euro+Med database. However, some species (mostly neophytes and hybrids) found in our source lists were absent from Eu ro+Med (2022) and thus had to be added manually using other sources. Such handling may introduce errors, but all of the taxa we added are fully documented (Suppl. material 3) and may thus easily be reassigned in future versions of EIVE if errors are encountered and reported. While it was beyond the scope of this paper to test the prediction accuracy of mean indicator values based on EIVE 1.0 for specific environmental variables, our exem plary validation using the temperature indicator showed a strong positive correlation between EIVE-T values and independent estimates of the temperature niche based on CHELSA bioclimate variables and GBIF occurrence records. Moreover, the correlation between our EIVE-T and bio10 or bio1 median values turned out to be better than that of any of the original EIV systems, albeit only slightly better than the system of Tichý et al. (2023), which also covers large parts of Europe (but was based on only six source systems for T, compared to 23 in the case of EIVE 1.0). The superior correlation of our combined sys tem might be unexpected at first glance, given the fact that ecological responses of species can shift along geographic gradients and regional indicator values may thus be ex pected to capture the regional species’ preferences better. While this pattern needs to be confirmed by testing the correlations of mean EIVE vs. mean EIV values against measured environmental factors of vegetation plots in re gional contexts, a test doing so with a “beta version” of EIVE, based on a much smaller number of source EIV systems than EIVE 1.0, found indeed a superiority of the EIVE approach over the regional EIV system in the ma jority of datasets with measured soil variables (volumetric water content, C/N ratio and pH in H2O; Moeys 2020). Content of EIVE 1.0 (1991) M values that differed only in growth form (such as 11 and 12 in Ellenberg et al. 1991) were merged Coding of niche width Not available Available for all indicator values and all species on an interval scale Calculation of European indicator values Mean of included EIV systems Mean of all available EIV systems after rescaling to the common 0-10 scale Use of species co-occurrence data from the European Vegetation Archive (EVA) EVA was used to add 431 species not covered in any of the included EIV systems Not used * According to M. Chytrý (pers. comm.) the number of 8,908 “species” given in Tichý et al. (2023) actually does not mean species but accepted taxa including aggregates. Table 4. Major differences between the two new ecological indicator value systems for Europe. Jürgen Dengler et al.: Ecological Indicator Values for Europe 1.0 20 experience on how such a weighting approach could best be applied, but Hájek et al. (2020) recently showed that it generally improves prediction. Hence, the niche width val ues reported in EIVE have potential to improve plot-level weighting of indicator values in the future. work essentially never can be completed, our documenta tion facilitates the detection of errors and inconsistencies by users, who are welcome to report such issues to the lead author for taxonomy (J.D.). In the forthcoming releases of EIVE, such issues can then be easily updated in our auto mated workflow in parallel to a continuous adjustment to up-to-date taxonomic concepts in Europe. Limitations of EIVE 1.0 We present a mathematically derived combination or “consensus system” of 31 individual EIV systems. One could thus argue that we are inheriting the limitations of the regional EIV systems, mainly being based on expert assessments rather than on statistical analyses of in situ measured environmental variables. While it was beyond the scope of this article to conduct comprehensive tests against measured environmental variables at the Euro pean scale, the often-demonstrated close relationship be tween mean regional EIVs and measured environmental variables (Ellenberg et al. 1991; Schaffers and Sýkora 2000; Ewald 2003; Lawesson 2003; Wamelink et al. 2022) and the indications that EIVE performs at least as well as the existing EIV systems (Moeys 2020; Table 3) support the general validity of the approach. Another limitation of EIVE is that, given the unequal spatial distribution of source EIV systems (Figure 1), the suitability and validity of EIVE will likely vary between re gions. On the one hand, Figure 4 suggests that EIVE indi cator values become more reliable if they are based on more source EIV systems. On the other hand, it is likely that in certain parts of Europe the average fraction of species per plot that have assigned values in EIVE 1.0 will be lower, and thus the predictions based on mean EIVE values might be less reliable (Ewald 2003). This potential limitation should mainly affect the Mediterranean parts of the Iberian Penin sula and the central parts of the Balkan Peninsula. g While the usefulness of EIVE at the continental scale is evident, EIVEcan also be meaningful for local to national studies. Despite the fact that we found 31 EIV systems for this study, country-specific EIV systems are still missing for most European countries. For some EIV systems, such as Poland (Zarzycki et al. 2002) and Slovenia (Košir 1992), the complexity of the coding/symbology largely prevented au tomated use for calculation of mean indicator values and, thus, researchers tended to prefer EIV systems from neigh bouring areas. Here, EIVE offers two solutions: one can ei ther use the European EIVE indicators or use the regional EIV indicators in their harmonised and ready-to-use edi tions also provided in the EIVE database (Suppl. material 2).h Lastly, while we are expanding the characterisation of the ecological niche of species to two parameters per niche dimension, i.e. Performance of EIVE 1.0 Instead of aiming at an unachievable “perfect” taxonom ic backbone, we developed decent and well-documented solution. With only 0.03% “unresolved” combinations of original taxon name × EIV systems, our rate is almost sure ly lower than that of pure taxonomic databases, such as Euro+Med (2022) or WFO (2022). We also worked inten sively on concept synonymy (Jansen and Dengler 2010), i.e. cases where the exactly same name refers to a different taxonomic content in different EIV systems. While typical taxonomic matching software is not able to address this crucial point, our experts overwrote the automatic assign ments with their expertise for 1,413 original taxon name × EIV system combinations (Suppl. material 3: table S3.5), leading to a content-wise much better match. While such a 21 Vegetation Classification and Survey developed and explored for other environmental variables, some of which may be expected to show more local vari ation. Therefore, the majority of continental-scale studies of vegetation in Europe, be it macroecological studies (e.g. Thuiller et al. 2005; Boonman et al. 2021; Dembicz et al. 2021) or characterisation of vegetation units (e.g. Marcenò et al 2018, 2019; Bonari et al 2021) restricted themselves to coarsely gridded climate data, despite extensive knowl edge that soil variables and disturbance regime are at least as decisive in shaping plant community composition. In one of the few attempts thus far to include gridded data of other environmental variables in a broad-scale analysis of vegetation-plot data, such as soil properties, Bruelheide et al. (2018) found a very low predictive power. This was probably driven by the fact that the global or continental geodatasets have modelled, not measured variables, and they are provided at resolutions of ca. 1 km for climate (Karger et al. 2017; but see Haesen et al. 2021) and 250 m for soil variables (Poggio et al. 2021), while microclimate (Pincebourde and Salle 2020) and soil conditions (Sercu et al. 2017) can change drastically within a few metres. Thus, EIVE can support the development of better models of vegetation properties – such as species richness or species composition – in Europe and might motivate vegetation ecologists on other continents to develop similar systems. As recently shown by Scherrer and Guisan (2019) in a re gional context, the predictive power of species distribution models improved considerably when fed with mean plot- based EIVs vs. modelled gridded environmental variables. Limitations of EIVE 1.0 niche position and niche width, and thus go beyond the majority of existing EIV systems, one could still consider this too simplistic. While these two parame ters can be statistically defined for species response curves along environmental gradients of any shape, they provide incomplete descriptions in case of skewed or bimodal distributions (Jansen and Oksanen 2013). Since the EIV source systems practically never contained more precise information on response curves (except very rare cases that indicated bimodality), our consensus approach did not allow to derive such information for EIVE 1.0 (but see below for future plans). p pp This taxonomic “backbone” is another central feature of EIVE and is provided open access to facilitate further improvements in a well-documented manner. While the EIVE backbone for vascular plants is based on Euro+Med (2022), the most comprehensive and up-to-date Europe an checklist currently available, it aims to overcome some shortcomings of the current Euro+Med Plantbase with the addition of taxa that are regularly recorded by vege tation ecologists. The most important are (a) formal and informal taxa between species and genus level (aggregates and sections), (b) hybrids and (c) neophytes. Beyond that, we also aimed at solving some apparent mistakes in Eu ro+Med (2022), e.g. when one aggregate member was not assigned to an aggregate or when the typical subspecies (autonym) was not listed despite Euro+Med (2022) os tensibly considering the species as polytypic, containing several other subspecies. We even implemented a solution Potential of EIVE 1.0 Our documented additions to Euro+Med (2022) are also an invitation to the team of the Euro+Med Plantbase to incorporate these taxa or a subset of these directly in future releases of their database. tion are known to often be particularly sensitive to envi ronmental conditions and thus suitable for bioindication (Cislaghi and Nimis 1997; Kirschbaum and Wirth 1997; Frahm 2001). Accordingly, terricolous bryophytes and lichens are included in several of the EIV systems used here (Ramensky et al. 1956; Tsyganov 1983; Ellenberg et al. 1991; Hill et al. 2004; Landolt et al. 2010; Didukh 2011; Hájek et al. 2020). In addition to these two groups, Julve (2020) also covers Charophyceae. Moreover, there are also specific EIV systems for bryophytes (Dierßen 2001; Sim mel et al. 2021) and lichens (Wirth 2010; Dingová Košu thová and Šibík 2013). Obvious candidates for additional ecological indicators are salinity (S) and continentality (C). Other potentially useful niche dimensions contained in one or several EIV systems and thus essentially accessible with our approach to derive a European consensus system are moisture variability (Ramensky et al. 1956; Tsyganov 1983; Landolt et al. 2010; Didukh 2011), soil phosphorus (Tyler et al. 2021), heavy metals in soil (Ellenberg et al. 1991; Landolt et al. 2010), soil humus content (Landolt et al. 2010), soil aeration (Landolt et al. 2010; Didukh 2011), air humidity (Tsyganov 1983; Didukh 2011), cryoclimate (Didukh 2011), snow layer duration (Odland and Munke jord 2008), mowing or grazing intensity (Dierschke and Briemle 2002; Landolt et al. 2010; Tyler et al. 2021), an thropogenic influence (or hemeroby) (Frank and Klotz 1990; Landolt et al. 2010) and CSR strategy (Frank and Klotz 1990; Thompson et al. 1993; Landolt et al. 2010). h p A second step would be to use the compositional data of the nearly two million vegetation plots from the European Vegetation Archive (EVA; Chytrý et al. 2016), combined with reciprocal averaging (e.g. Hill et al. 2000) or a sim ilar technique (Tichý et al. 2023), to increase the internal consistency of the current EIVE version and to add new taxa from EVA that are not included in any of the region al EIV systems. Combining EIVE with EVA would also allow the connection of ad hoc metrics of niche position and niche width to concrete statistical definitions. Potential of EIVE 1.0 The main motivation for the creation of EIVE was the de mand to have plot-based assessments of environmental conditions carry a broader set of meaningful predictors in macroecological studies of vegetation plots. Since the largest vegetation-plot databases globally, EVA (Chytrý et al. 2016) and sPlot (Bruelheide et al. 2019), do not provide in situ measured environmental variables (except slope as pect, inclination and elevation), researchers were hitherto forced to find “work-arounds”. The main approach was to use modelled environmental data for grid cells. This is well-established for climate data, e.g. WorldClim (Fick and Hijmans 2017) and CHELSA (Karger et al. 2017), but less Jürgen Dengler et al.: Ecological Indicator Values for Europe 1.0 22 for cases where the same name refers to different taxo nomic concepts in different EIV systems or countries, namely taxa of different width (see Jansen and Dengler 2010). In fact, we now provide something that comes close to what Dengler et al. (2012) proposed under the name “EuroSL”. There have been previous attempts to address the multitude of different names and taxonomic concepts of European plants in ways that can be incorporated into automated workflows, e.g. in JUICE (Tichý 2002) or R. Most prominently, Chytrý et al. (2020) presented a system that allows aggregating different taxon names into higher units within EUNIS-ESy, an expert system for the deter mination of habitat types from the species composition of vegetation plots. This system is now increasingly applied in European vegetation studies, including the European Ellenberg-type indicator values by Tichý et al. (2023). The advantage of this system is that it interprets individual names in a national or regional context, considering how they were largely used in vegetation sampling. However, this system does not differentiate between synonyms, sub ordinate taxa, taxon concepts of different width, misap plied names and typos. In contrast, our system separates all these different cases and documents them. We suggest that it can be used in European projects independent from the indicator values and, in combination with the region ally interpreted taxon names from the EUNIS-ESy, might help the big European vegetation-plot databases such as EVA (Chytrý et al. 2016) and GrassPlot (Dengler et al. 2018) to provide their content in a more harmonised way in the future. Potential of EIVE 1.0 Further, other attributes of the ecological niches of species, such as minimum, maximum, skewness or bimodality could be determined systematically. It would even be possible to derive complete response curves for each species along each niche dimension, allowing for the ecological charac terisation of a site not by averaging the EIVs of the occur ring species, but by multiplying these probability curves. Future plans for EIVE With this publication, the first version (1.0) of the Ecolog ical Indicator Values for Europe is released. At the same time, this is the start of an open-ended, community-based endeavour that calls for continuous future updates. All raw data and derived data of EIVE are published open access with a CC BY 4.0 licence, while the R code is available upon request, meaning that everyone is free to use, modify or ex pand the current system as long as proper credit is given to this publication. We plan to launch a website to host all these materials, possibly within the framework of the Euro pean Vegetation Survey (http://euroveg.org/). While every body is free to develop new systems based on EIVE 1.0, we plan to establish a committee whose responsibility will be to release future official versions of EIVE. Here, we envisage a workflow similar to the EVC Committee (http://euroveg. org/evc-committee) that releases official modifications of the EuroVegChecklist (Mucina et al. 2016) once they are approved by a majority (e.g. Biurrun and Willner 2020).i While for pan-European analyses, a single set of conti nent-wide indicator values appears to be the most practical solution, it should be acknowledged that the ecological nich es of species do change across large geographic distances. Some species might change their niche position (Diekmann 1995; Goedecke et al. 2019), while others decrease (Šilc et al. 2014) or increase their niche width from the centre to the margins of their distributional range (unpubl. observ. J.D.: various species appear to have a wider R niche in the hemi boreal than in the nemoral zone). However, up to now, there has been only limited empirical evidence and vague theoret ical expectations regarding the changes of ecological niches of species along geographic gradients. Here, EIVE, togeth A first and self-evident step is to expand the current consensus system to additional taxonomic groups and additional indicators. Non-vascular taxa in the vegeta Vegetation Classification and Survey 23 er with the vegetation-plot data from EVA, would offer the unique chance to explore how frequent such changes are, and whether there are prevailing patterns. Moreover, in a second step, one could complement the pan-European indicator values of EIVE with separate sets of indicator values for the major biogeographic regions in Europe, which then could be used with higher predictive power in regional studies. Author contributions The idea of the database and paper was conceived by J.D., the database was prepared by E.H., F.J., K.V.M. and J.D., while F.J. and F.G. set up the analytical procedures in R. F.J. implemented the automated taxonomic assign ment, which was refined by J.D., O.C., T.T., G.K., R.G. and T.R. M.N. ran the comparisons with GBIF and CHELSA data. J.D. led the writing, with major contributions from K.V.M., K.M., M.N., F.G., R.G., T.T, H.H.B. and M.J.S. The map was prepared by K.V.M. and the conceptual figures by F.G. Harmonised data from several regional EIV sys tems were contributed by I.A., M.C., L.T., G.K. and J.D., while all remaining co-authors contributed their nation al EIV system in digital format, partly with unpublished updates, and helped with their interpretation. All authors checked, improved and approved the manuscript. Future plans for EIVE With these qualities, we are convinced that EIVE 1.0 will open new analytical avenues and become an import ant tool for vegetation ecologists, conservation biologists, species distribution modellers and macroecologists work ing on the European vegetation and flora. The implemen tation of EIVE is facilitated by the fact that the system, its underlying data and R scripts are provided freely. EIVE is an open-source, community-based database that will be re leased with fixed version numbers following improvements Therefore, readers are invited to send to the lead authors in formation about overlooked, new or updated EIV systems of any taxonomic group of plants and any niche dimen sions, as well as any suggestions for further improvements. g p p g In this paper, we derived a European indicator value system without direct link to environmental variables – apart from the external validation of EIVE-T values with GBIF data. In the future, it would be important to conduct such validations with measured or at least inde pendently modelled environmental variables for the oth er indicators as well. For the light indicator (L), the EVA database might provide suitable proxies, such as slope, aspect and inclination and tree and shrub layer cover (for a possible approach, see Tichý et al. 2023). By contrast, for the soil indicators, EVA lacks well-curated and read ily available in situ measured environmental variables, such as pH value, C/N ratio or average depth of the wa ter table. However, other vegetation-plot databases that are specialised in this field, such as the Ecological Con ditions Database (Wamelink et al. 2012) and GrassPlot (Dengler et al. 2018), can provide the relevant data. Here, one could ask whether and how species cover should be used in the calculation of mean indicator values of a plot: not at all (i.e. only presence/absence), fully (i.e. weight ing by % cover) or an intermediate solution (e.g. square root transformed cover) (Käfer and Witte 2004). Finally, one can test which is the best approach to include the now available numeric niche-width information into the calculation of the mean indicator values of a plot: not at all, using a threshold, using inverse weighting or using an even more sophisticated approach. Conclusions and outlook In terms of geographical and taxonomic coverage, as well as number of included source systems, EIVE 1.0 is the most comprehensive system of ecological indicator values developed so far. While it was beyond the scope of this paper to test its link to measured environmental site conditions, the high correlation of our EIVE-T val ues with modelled temperature conditions over the spe cies distribution ranges indicates the general validity of the approach and shows that creating a consensus system from many source systems can even increase their perfor mance. Compared to many, if not all, previous indicator value systems in Europe or parts of Europe, EIVE comes with several methodological novelties that likely will in crease the utility of the system: (i) consistent range of 0 to 10 for all niche dimensions; (ii) interval (continuous) instead of ordinal (semi-quantitative) scaling; (iii); pro vision of both niche position and niche width and (iv) removal of logical inconsistencies, such as the fact that many systems assigned different M values for species that grow in the same habitat but have different morphology. Data availability The Ecological Indicator Values for Europe (EIVE) 1.0 and the data underlying their derivation are freely available in the Supporting material and in a permanent repository at https://www.doi.org/10.5281/zenodo.7534792. 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Acknowledgements We thank the Bayreuth Center of Ecology and Environ mental Research (BayCEER), University of Bayreuth, for a grant (“Anschubfinanzierung”) that supported the ini tial steps of this project. We thank Hartmut Dierschke (†), Nikolai Ermakov, Anna Maria Fosaa (†), Stephan M. Hennekens, Mark O. Hill and Niels Böhling for providing their regional EIV systems with relevant background in formation and the Euro+Med Secretariat Berlin for pro viding the Euro+Med database enabling us to use it as the basis for the taxonomic backbone of EIVE. Hallie Seiler provided thorough linguistic editing. We thank Wolfgang Willner as Subject Editor and three anonymous reviewers for a fast and careful review that allowed us to further im prove the manuscript. Jürgen Dengler et al.: Ecological Indicator Values for Europe 1.0 24 References Chytrý M, Tichý L, Dřevojan P, Sádlo J, Zelený D (2018) Ellenberg-type indicator values for the Czech flora. 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Fragmenta Botanica Musei His torico-Naturalis Hungarici 4: 101–142. 28 Jürgen Dengler et al.: Ecological Indicator Values for Europe 1.0 E-mail and ORCID Jürgen Dengler (Corresponding author, juergen.dengler@zhaw.ch), ORCID: https://orcid.org/0000-0003-3221-660X Florian Jansen (florian.jansen@uni-rostock.de), ORCID: https://orcid.org/0000-0002-0331-5185 Olha Chusova (olgachusova28@gmail.com), ORCID: https://orcid.org/0000-0002-8081-9918 Elisabeth Hüllbusch (elisabeth.huellbusch@bfn.de) Michael P. Nobis (michael.nobis@wsl.ch), ORCID: https://orcid.org/0000-0003-3285-1590 Koenraad Van Meerbeek (koenraad.vanmeerbeek@kuleuven.be), ORCID: https://orcid.org/0000-0002-9260-3815 Irena Axmanová (axmanovairena@gmail.com), ORCID: https://orcid.org/0000-0001-9440-7976 Hans Henrik Bruun (hhbruun@bio.ku.dk), ORCID: https://orcid.org/0000-0003-0674-2577 Milan Chytrý (chytry@sci.muni.cz), ORCID: https://orcid.org/0000-0002-8122-3075 Riccardo Guarino (riccardo.guarino@unipa.it), ORCID: https://orcid.org/0000-0003-0106-9416 Gerhard Karrer (gerhard.karrer@boku.ac.at), ORCID: https://orchid.org/0000-0001-5172-2319 Karlien Moeys (karlien.moeys@kuleuven.be), ORCID: https://orcid.org/0000-0001-7404-6034 Thomas Raus (t.raus@bo.berlin) ORCID: https://orcid.org/0000-0001-5778-4705 Manuel J. Steinbauer (steinbauer@uni-bayreuth.de), ORCID: https://orcid.org/0000-0002-7142-9272 Lubomir Tichý (tichy@sci.muni.cz), ORCID: https://orcid.org/0000-0001-8400-7741 Torbjörn Tyler (torbjorn.tyler@biol.lu.se), ORCID: https://orcid.org/0000-0002-7886-7603 Ketevan Batsatsashvili (ketevan_batsatsashvili@iliauni.edu.ge) Claudia Bita-Nicolae (claudia.bita@ibiol.ro), ORCID: https://orcid.org/0000-0003-3949-1989 Yakiv Didukh (ya.didukh@gmail.com), ORCID: https://orcid.org/0000-0002-5661-3944 Martin Diekmann (mdiekman@uni-bremen.de), ORCID: https://orcid.org/0000-0001-8482-0679 Thorsten Englisch (thorsten.englisch@v-p-n.at), ORCID: https://orcid.org/0000-0002-8616-6217 Eduardo Fernández-Pascual (fernandezpeduardo@uniovi.es), ORCID: https://orcid.org/0000-0002-4743-9577 Dieter Frank (dieter.frank@lau.mwu.sachsen-anhalt.de), ORCID: https://orcid.org/0000-0002-8602-9570 Ulrich Graf (ulrich.graf@wsl.ch), ORCID: https://orcid.org/0000-0001-6459-3034 Michal Hájek (hajek@sci.muni.cz), ORCID: https://orcid.org/0000-0002-5201-2682 Sven D. Jelaska (sven.jelaska@biol.pmf.hr), ORCID: https://orcid.org/0000-0003-3834-002X Borja Jiménez-Alfaro (jimenezalfaro@uniovi.es), ORCID: https://orcid.org/0000-0001-6601-9597 Philippe Julve (philippe.julve@univ-catholille.fr) George Nakhutsrishvili (nakgeorg@gmail.com) Wim A. Ozinga (wim.ozinga@wur.nl), ORCID: https://orcid.org/0000-0002-6369-7859 Eszter-Karolina Ruprecht (eszter.ruprecht@ubbcluj.ro), ORCID: https://orcid.org/0000-0003-0122-6282 Urban Šilc (urban.silc@zrc-sazu.si) ORCID: https://orcid.org/0000-0002-3052-699X Jean-Paul Theurillat (jean-paul.theurillat@unige.ch), ORCID: https://orcid.org/0000-0002-1843-5809 François Gillet (francois.gillet@univ-fcomte.fr), ORCID: https://orcid.org/0000-0002-3334-1069 Supplementary material Supplementary material 1 Detailed overview of the 31 ecological indicator value systems (EIVs) used to derive the Ecological Indicator Values for Europe (EIVE) 1.0 (.pdf). Link: https://doi org/10 3897/VCS 98324 suppl1 Supplementary material 1 Detailed overview of the 31 ecological indicator value systems (EIVs) used to derive the Ecological Indicator Values for Europe (EIVE) 1.0 (.pdf). Li k htt //d i /10 3897/VCS 98324 l1 Supplementary material 1 Detailed overview of the 31 ecological indicator value systems (EIVs) used to derive the Ecological Indicator Values for Europe (EIVE) 1.0 (.pdf). Link: https://doi.org/10.3897/VCS.98324.suppl1 Supplementary material 2 The analysed 31 EIV systems with original and harmonised plant nomenclature and original and rescaled indica tor values for M, N, R, L and T (.xlsx). Link: https://doi.org/10.3897/VCS.98324.suppl2 Supplementary material 3 Documentation of additions to and modifications of the taxonomic backbone from Euro+Med (2022) in EIVE 1.0 (.xslx). Link: https://doi.org/10.3897/VCS.98324.suppl3 Supplementary material 2 The analysed 31 EIV systems with original and harmonised plant nomenclature and original and rescaled indica tor values for M, N, R, L and T (.xlsx). Link: https://doi.org/10.3897/VCS.98324.suppl2 Supplementary material 3 Supplementary material 3 Documentation of additions to and modifications of the taxonomic backbone from Euro+Med (2022) in EIVE 1.0 (.xslx). Link: https://doi.org/10.3897/VCS.98324.suppl3 pp y Documentation of additions to and modifications of the taxonomic backbone from Euro+Med (2022) in EIVE 1.0 (.xslx). Link: https://doi org/10 3897/VCS 98324 suppl3 Link: https://doi.org/10.3897/VCS.98324.suppl3 29 Vegetation Classification and Survey Supplementary material 4 Methodological details of the calculations of the three variants of niche position (median, mean, weighted mean) and the three variants of niche width (nw1, nw2, nw3) (.pdf). Link: https://doi.org/10.3897/VCS.98324.suppl4 Supplementary material 5 Documentation of the stepwise approach to derive the European consensus system of niche positions EIVEres (EIVE 1.0) from the individual EIV source systems after initial rescaling (EIVini) (.pdf). Link: https://doi.org/10.3897/VCS.98324.suppl5 Supplementary material 6 Bioclimate statistics of the species used for EIVE evaluation, and the corresponding DOIs of the GBIF occurrence downloads (.xlsx). Link: https://doi.org/10.3897/VCS.98324.suppl6 Supplementary material 7 Comparison of different metrics of niche width regarding the resulting value distributions and the correlations with GBIF derived bioclimatic variables. (.pdf). Link: https://doi.org/10.3897/VCS.98324.suppl7 Supplementary material 8 EIVE 1.0 indicator values for niche position and niche width of M, N, R, L and T (.xlsx). Link: https://doi.org/10.3897/VCS.98324.suppl8 Supplementary material 4 Methodological details of the calculations of the three variants of niche position (median, mean, weighted mean) and the three variants of niche width (nw1, nw2, nw3) (.pdf). Link: https://doi.org/10.3897/VCS.98324.suppl4 Supplementary material 5 Documentation of the stepwise approach to derive the European consensus system of niche positions EIVEres (EIVE 1.0) from the individual EIV source systems after initial rescaling (EIVini) (.pdf). Link: https://doi.org/10.3897/VCS.98324.suppl5 Supplementary material 6 Bioclimate statistics of the species used for EIVE evaluation, and the corresponding DOIs of the GBIF occurrence downloads (.xlsx). Link: https://doi.org/10.3897/VCS.98324.suppl6 Supplementary material 7 Comparison of different metrics of niche width regarding the resulting value distributions and the correlations with GBIF derived bioclimatic variables. (.pdf). Link: https://doi.org/10.3897/VCS.98324.suppl7 Supplementary material 8 EIVE 1.0 indicator values for niche position and niche width of M, N, R, L and T (.xlsx). Link: https://doi.org/10.3897/VCS.98324.suppl8
https://openalex.org/W2546703798	https://europepmc.org/articles/pmc5126025?pdf=render	English	null	Angry, Scared, and Unsure: Mental Health Consequences of Contaminated Water in Flint, Michigan	Journal of urban health	2,016	cc-by	5,306	Journal of Urban Health: Bulletin of the New York Academy of Medicine, Vol. 93, No. 6 doi:10.1007/s11524-016-0089-y Journal of Urban Health: Bulletin of the New York Academy of Medicine, Vol. 93, No. 6 doi:10.1007/s11524-016-0089-y * 2016 The Author(s). This article is published with open access at Springerlink.com * 2016 The Author(s). This article is published with open access at Springerlink.com Cuthbertson, Newkirk, Ilardo, Loveridge, and Skidmore are with the Michigan State University, East Lansing, MI, USA. Correspondence: Courtney A. Cuthbertson, Michigan State University, East Lansing, MI, USA. (E-mail: cuthbe16@msu.edu) Angry, Scared, and Unsure: Mental Health Consequences of Contaminated Water in Flint, Michigan Courtney A. Cuthbertson, Cathy Newkirk, Joan Ilardo, Scott Loveridge, and Mark Skidmore ABSTRACT Natural and manmade crises impact community-level behavioral health, including mental health and substance use. This article shares ﬁndings from a larger project about community behavioral health, relevant to the ongoing water crisis in Flint, Michigan, using data from a larger study, involving monthly surveys of a panel of key informants from Genesee County. The data come from open-response questions and are analyzed as qualitative data using grounded theory techniques. Although respondents were not asked about the water issues in Flint, participants commented that the water situation was increasing stress, anxiety, and depression among the city’s population. Participants thought these mental health issues would affect the entire community but would be worse among low-income, African American populations in the city. Mental health consequences were related not only to the water contamination but to distrust of public ofﬁcials who are expected and have the authority to resolve the issues. The mental health effects of this public health crisis are signiﬁcant and have received inadequate attention in the literature. Public health response to situations similar to the water issues in Flint should include sustained attention mental health. KEYWORDS Water, Mental health, Substance use, Infrastructure management, Behavioral health, Flint KEYWORDS Water, Mental health, Substance use, Infrastructure management Behavioral health, Flint KEYWORDS Water, Mental health, Substance use, Infrastructure management, Behavioral health, Flint KEYWORDS Water, Mental health, Substance use, Infrastructure management, Behavioral health, Flint INTRODUCTION The built environment can affect mental health directly through factors such as housing quality and indirectly through factors such as a sense of personal control over one’s surroundings.1 For example, toxic waste landﬁlls are more likely to be sited near communities of color.2 People of low socioeconomic status and communities of color are more likely to be exposed to hazardous waste, with similar ﬁndings with regard to air and water pollution.3 Living in close proximity to industrial activity negatively impacts mental health, with disproportionately higher impacts on people of color.4 People who experience shocks from natural or manmade disasters have higher rates of mental illness, including major depression and posttraumatic stress disorder.5–7 Neighborhoods with higher collective efﬁcacy generally have better reported overall health8 and better mental health outcomes in the face of natural disasters.9, 10 899 900 CUTHBERTSON ET AL. While clinically diagnosable mental illnesses may be triggered or inﬂuenced by stressful events such as disasters and environmental injustices, we must point out that this differs from the psychological distress individuals may experience in similar or identical situations. Distress is often viewed as a natural reaction to stressful events or life conditions,11 as a psychosocial response to environmental disasters, based on direct and perceived individual impact and risk to health.12 Distress and disorder should be considered with relation to the experience of symptoms (severity, duration) relative to what one may expect based on the stressfulness of a particular situation,11 such that distress occurs in situations where experienced symptoms are less than or equal to what is expected based on stressful events or circumstances, and disorders occur when experienced symptoms are greater than what would be expected. Experiences and interpretations of distress vary by social positionality. In April 2014, the City of Flint began treating its own drinking water from the Flint River. Prior to that time, the city received treated water from the Detroit Water and Sewer Department (DWSD). Soon after the switch in water source, residents voiced their concerns that the water was discolored, had a bad smell and taste, and was causing health problems. State and city ofﬁcials initially dismissed many of the complaints as insigniﬁcant. Even when the water source was switched back to DWSD in October 2015 after state ofﬁcials publicly acknowledged problems with the water, some of those problems persisted. INTRODUCTION News accounts of citizens’ reactions to the situation note distrust of government because of the discounting of residents’ concerns about water and the length of time it took to act. Residents’ reactions include anger, disillusionment, abandonment, and feeling as though their govern- ment ofﬁcials cared little about them. Some physical health impacts have been described by residents and researchers, such as elevated blood lead levels,13 but the impact of the mental stress associated with the water crisis received substantially less attention to date. The media have compared the water situation in Flint to New Orleans after Hurricane Katrina, in terms of the disasters’ human health costs, the disproportionate impact on black and low-income residents, and the seemingly slow governmental response.14 15 Flint has long been a site of environmental injustice.15 Contemporary Flint is often characterized as a disadvantaged community, with a median income of just over $24,000,16 which is less than half the national average. In disadvantaged communities, social capital may mediate the negative effects of low income or income inequality on health status.17, 18 However, people with low social capital are more likely to experience mental illnesses.19 In Flint speciﬁcally, neighborhood social capital has been directly, negatively related to stress and depressive symptoms.20 The purpose of the current article is to investigate the mental health impacts of the problems with Flint’s water, as described by community leaders in Genesee County. METHODS The aim of the project was to create community monitoring or sentinel surveillance systems for mental health and substance use issues, piloting three methods across 17 different communities around the USA. The project was approved by the Institutional Review Board at Michigan State University. Partnering communities were selected through an open call for proposals and selected based on demonstrated capacity to successfully complete the project, as well as demographic and geographic diversity. For purposes of the current paper, only the results from the MENTAL HEALTH CONSEQUENCES OF CONTAMINATED WATER 901 Genesee County, Michigan, partnering community, which encompasses the city of Flint, are considered. Genesee County, Michigan, partnering community, which encompasses the city of Flint, are considered. Genesee County was part of a group of communities testing community monitoring via monthly surveys of a panel of 30 key informants. Panelist expertise included health services, substance use prevention, health-related non-governmental organizations, disability service organizations, schools, and researchers. Panelists were asked to complete a survey about changes in 30 behavioral health-related issues, including speciﬁc mental health and substance use disorders, each month for 1 year. The survey, developed by the project’s technical committee, was administered online via Qualtrics. Participants had 1 week to complete each monthly survey, with reminders sent in the middle of the survey period to any participant who had not yet responded. The survey asks panelists to identify any new behavioral health issues that were not present in the community before, as well as whether any community events occurred that may have impacted behavioral health. Responses to these open questions were not required for survey completion. Participants received $10 per survey completed. As shown in Fig. 1, panelists perceived stress to become much worse as the water crisis unfolded during the course of the surveys; panelists perceived a brief reduction of stress around the time of the switch back to DWSD, but that it rebounded as the long-term effects of lead exposure on individuals, families, and infrastructure became more widely known and as media attention increased. Data for this paper come from 12 monthly surveys completed by panelists in Genesee County. More speciﬁcally, the data come from the open-response questions about what new issues and community events emerged since the previous survey, as well as open-response comment ﬁelds where respondents could mention issues related to a subset of behavioral health problems. METHODS The data were analyzed as qualitative comments using grounded theory, including open and focused coding, and concept building techniques such as memoing.21–23 Within grounded theory, researchers start with the data and build theory from empirical ﬁndings to compare with existing knowledge. Each open-response comment represented one data point for coding. The open-response comment ﬁelds were coded using open coding to determine the most common themes from the data and again using focused coding to reﬁne themes and develop more detailed deﬁnitions. Concurrently with coding, FIG. 1 Participants’ perceptions of changes in stress in Genesee County. FIG. 1 Participants’ perceptions of changes in stress in Genesee County. 902 CUTHBERTSON ET AL. memoing aided in drawing connections between themes. All authors reviewed the data, one author completed coding and memoing, and all authors reviewed themes and details for accuracy. memoing aided in drawing connections between themes. All authors reviewed the data, one author completed coding and memoing, and all authors reviewed themes and details for accuracy. RESULTS Of the 263 completed open-response questions over 12 monthly surveys, 120 were about the contamination of water in Flint (45.6 %). The number of responses to the open-response questions and the number speciﬁcally about water varied over the course of the 12 surveys but notably increased starting at survey 6 which was administered in October 2015 after Michigan state ofﬁcials ﬁrst publicly acknowl- edged that there was a problem with the water in Flint (see Fig. 2). Four comments referred to the water problems either as disastrous or a disaster, and 58 comments referred to the situation as a crisis. Quotes included from the surveys are followed by the survey date in which those responses were given. Through the application of grounded theory coding and analysis techniques, the mental health concerns arising most clearly from the data are that the water situation has created and increased stress and anxiety for residents of Flint. From the data, stress is deﬁned as feeling nervous, scared, angry, frustrated, and distrustful, with a lack of conﬁdence in the political system or government. BEconomic and water situation in Flint, Michigan, continues to be a focus of concern and cause of stress. Majority of families we work with reside inside the city limits and have dealt with lead and other pollutants for over a year^ (January 2016). In the same survey, another panelist commented B[t]he water crisis in Flint is HUGE! Many people are very angry, scared, and unsure of the future.^ In March 2016, one panelist described the situation as an emergency, leading to Btoxic stress in the community.^ Stress was created by the potential and permanent physical health effects of lead exposure, the news coverage ﬁnding high levels of lead in the blood of Flint children, and lack of knowledge of where to ﬁnd lead testing for children. BParents are under more stress in seeking evaluations to identify impact of water-based lead poisoning of their children,^ one panelist commented (November 2015). As another panelist frankly stated, B[r]esidents are not able to drink the Flint water. This is causing stress, anxiety, ﬁnancial hardship, and community unrest.^ (October 2015). Stress FIG. 2 Percentage of Genesee County participants’ comments about water. FIG. 2 Percentage of Genesee County participants’ comments about water. MENTAL HEALTH CONSEQUENCES OF CONTAMINATED WATER 903 was perceived to increase not only because of the potential physical health effects of lead exposure but also because the solution and course of action were and remain unknown; additionally, panelists noted that residents faced extremely high water bills for unusable water. was perceived to increase not only because of the potential physical health effects of lead exposure but also because the solution and course of action were and remain unknown; additionally, panelists noted that residents faced extremely high water bills for unusable water. Panelists reported that the water issues affected the whole community but would have the greatest impact among African Americans and those of low socioeconomic status in the community. These two groups were identiﬁed as those who were disproportionately receiving contaminated water and those who had the least resources to cope with it. One panelist wrote, B[r]esidents of the city of Flint are paying huge water bills and they haven’t been able to use their water in some time. It is causing a ﬁnancial hardship on people that are already stretched to the max…All residents of the city of Flint are going through something because of the water issues, but it is having a devastating effect on lower income people^ (October 2015). The effects of stress were not limited to Flint residents and were perceived to have a contagion effect: BThe current Flint water crisis has people behaving differently due to the elevated stress level associated with living with poisoned water. Stress is through the roof for many that I come in contact with… Even those who are not directly affected are feeling stressed because they know people who are affected by it^ (April 2016). Relatedly, panelists identiﬁed anxiety and depression as increasing due to the Flint water issues for similar reasons. Panelists expressed that anxiety was elevated among community members because of the uncertainty of knowing whether they had been exposed to lead, and that the effects and severity of lead exposure would be unknown for some time. Depression was identiﬁed as an additional mental health effect of the water problems in Flint, although it was cited much less frequently than anxiety or stress. BThe water issue in our community is like…a natural disaster. BThere seems to be an ever increasing divide among Genesee County’s communities as everyone tries to distance themselves from Flint. You can see the ‘poor you’ look on the faces of people who ﬁnd out that you live in Flint^ (March 2016). Another panelist commented: BBecause of the continuing water problem in Flint, we are seeing increases in stress, substance use, and depression. Flint home property values, already down, are now in crisis mode. Some additional examples: Flint restaurants have lost business and have laid off workers, biological parents of foster children are demanding children not be placed in Flint foster homes, summer job programs for low-income county youth are being affected as families are asking for opportunities out of the city^ (March 2016). In the same month, another panelist shared that there was Bincreased anger from Flint homeowner residents due to [the] water crisis have identiﬁed stressful conversations due to home damage, decreased property values, illness, lack of funding to relocate, and concerns of affected family members.^ Several comments indicated a relationship between lack of conﬁdence in government, inability to trust authority ﬁgures, and mental health. On the Flint water issues, a panelist stated, B[i]t has added stress to an already vulnerable population and created even more distrust among city and state leadership^ (November 2015). Even in December 2015, after the water had been switched back to DWSD, one panelist responded, B[p]eople are unsure if the water is truly safe to drink now.^ In January 2016, a panelist commented that B[t]he mayor of Flint has declared a state of emergency due to lead and chemicals in the water going into homes throughout the city. There has been an increase in anxiety and stress related to trust issues with government ofﬁcials, families not being able to use or drink the water, and being required to pay a water bill for ‘bad’ water^ (January 2016). In the February 2016 survey, one panelist said, B[s]ince gaining national and worldwide attention in Flint due to water crisis, we are seeing an increase in stress factors and other problems in individuals, especially children and youth. Many are interpreting the increased attention as an indicator that something much worse is unfolding that they have no power over. It is very stressful and depressing to know that you have to pay for a commodity that you need but cannot use appropriately without potential harm to yourself and family… This increases the level of both stress and depression as it does not appear that any positive resolution is in sight^ (June 2015). BI believe it [the water crisis] has exacerbated depression and some PTSD patients^ (April 2016). The mental health effects of the water issues in Flint are perceived to have spillover or ripple effects into other areas of behavioral health, such as abuse and substance use. One panelist wrote, B[t]he higher level of anxiety and stress is impacting abuse^ (February 2016). Other panelists felt the water issues were causing increases in alcohol abuse, illicit drug use, and prescription misuse or abuse. BThere are signs on the bars that read ‘Drink beer not the water’. In addition, people are self-medicating to deal with the mental health issues experienced^ (February 2016). b h l d l ff l h l h h h Concerns about the water in Flint are indirectly affecting mental health through the effects of the situation on people living, working, and interacting in the city and county. BThe Flint water situation and the fact that it’s now become a topic of national concern. It was hard living here with all the negative reporting about crime and the economy. It’s even harder living here now… People look and sound defeated,^ one panelist wrote (January 2016). This individual felt that the Flint water issues were the cause of depression, stress, and hopelessness among city residents. Another panelist noted, B[t]he way people are expected to live is intolerable and weighing very heavy on people^ (February 2016). BThe residents seem to be on edge,^ one panelist wrote (February 2016). Beyond the perception of Flint residents experiencing anxiety and being Bon edge,^ there was a sense that there were mental health repercussions related to how people 904 CUTHBERTSON ET AL. from other communities have begun to treat Flint residents. BThere seems to be an ever increasing divide among Genesee County’s communities as everyone tries to distance themselves from Flint. You can see the ‘poor you’ look on the faces of people who ﬁnd out that you live in Flint^ (March 2016). Another panelist commented: from other communities have begun to treat Flint residents. DISCUSSION Genesee County panelists were in a unique position to assess the mental health impacts of the water issues in Flint and made astute observations about elevated levels of anxiety, stress, and depression among city residents. Panelists indicated that that the water problems were the root of increased stress, anxiety, and depression in Flint. Stress, especially prolonged or chronic stress, has the potential to lead to severe physical health outcomes such as cardiovascular disease,24, 25 increased blood pressure, and compro- mised immune systems.26 Stress worsens other mental health indicators, such as depressive symptoms,27 and in combination with low socioeconomic status may lead to greater risk of premature death.28 Lacking positive mental health increase the probability of mortality.29 Stress and depression both increase chances of major cardiac events.24 y p j Both the water contamination and the sense of distrust in government ofﬁcials appear to have mental health consequences for Flint residents, especially as residents have no control over what ﬂows through water service lines and into their homes. Generally, lower sense of control is associated with greater depressive symptoms30; in the face of disaster, decreased sense of control has been positively associated with acute stress disorder.31 In addition to the direct health effects of contaminated water, the mental health effects may also contribute to serious physical health reduction. Without intervention to reduce impacts, the stress effects of the water crisis and its aftermath may increase long-term health disparities faced by Flint’s predominantly poor and African American residents. Since 2002, the City of Flint has experienced numerous ﬁscal challenges, leading to two explicit interventions by state government since the early 2000s. An emergency ﬁnancial manager was appointed to manage Flint’s ﬁscal affairs from 2002 to 2004, and then again from 2012 to April of 2015.32 While the purpose of the intervention was to help restore the ﬁscal health and support the provision of basic public services, the decisions of the most recent emergency ﬁnancial manager were central to the emergence and continuation of the water crisis. It could be argued that the lack of democratic checks normally present in a functioning local government hindered action that might have resulted in response to citizens’ expressed concerns about the water quality problems. In this sense, the state economic intervention appears to have resulted increased budgetary costs as well as higher human physical and mental health tolls. They state they have lost trust in believing political ofﬁcials and community leaders and other than the band-aid ‘bottled water’ they are left with the consequences.^ This account illustrates that mental health effects are related to distrust of the ofﬁcials who should be providing solutions to the water issues, and that quick solutions are interpreted as inadequate. BLead in the water has caused not only physical issues but being misled by city and state ofﬁcials about the severity of the problem has caused stress, emotional, and mental issues, quality of life is down^ (March 2016). One panelist commented that it was not only a lack of conﬁdence in city and government ofﬁcials but a sense of abandonment that impacted mental health. One panelist responded, Bhow do you describe the feeling(s) of abandonment by those who you have put some degree of conﬁdence in to protect you. In addition to now, as a result, of the water crisis, being prey to the vultures who come to feed off of the pain of this issue and all of the repercussions resulting…[this affects] predominately Flint residents, who are educated and understand the broader perspective of the impact of all of this…All of us are affected and it is unfortunate that the voice of all, the entire community is not heard^ (March 2016). Part of the sense of abandonment is related to taking responsibility for the water problems in Flint: BThe overall water crisis, along with the poor attempts of those who created this man-made disaster to avoid accepting responsibility… [have increased] stress, depression, anger and the list goes on!^ (March 2016). Another panelist explained, B[m]ore information about the Flint water crisis is coming out as they are having legislative investigative MENTAL HEALTH CONSEQUENCES OF CONTAMINATED WATER 905 hearings… Trust issues have led to more anxiety and depression. Substances are often used to self-medicate^ (March 2016). hearings… Trust issues have led to more anxiety and depression. Substances are often used to self-medicate^ (March 2016). DISCUSSION Michigan Governor Rick Snyder agreed with Congress in recent legislative hearings that it would be a Bfair conclusion^ to say that Michigan’s emergency management system failed in this case;33 this failure has led to not only catastrophic physical health effects but potentially long-lasting and impactful negative mental health outcomes as well. The health and human services sector came together quickly to provide both information and interventions that address stress, anxiety, depression, and trauma brought about by the water crisis. The public mental health provider, Genesee Health System, convened the Flint Community Resilience Group (CRG) on February 2, 2016. The second meeting, held on February 16, was addressed by the US Surgeon General. At the writing of this paper, the CRG has six active workgroups: (1) planning and coordination, (2) data and gap analysis, (3) psychological ﬁrst aid, (4) stress management, (5) faith-based groups, and (6) vulnerable and underserved populations. These workgroups meet regularly with the overarching purpose of providing input regarding the needs within the community, assisting with planning, 906 CUTHBERTSON ET AL. and facilitating service delivery. For example, Psychological First Aid, an evidence- informed approach framed by the listen, protect, and connect approach was implemented with sessions speciﬁcally targeting adults and youth. By mid-April, 11 free-of-cost trainings were held and attended by approximately 225 individuals with an additional eight trainings scheduled between mid-April and mid-July. Data collection using the Community Assessment for Public Health Emergency Response (CASPER) was scheduled for mid-May 2016. Crisis counseling sites that connect residents to mental health services were established. The faith-based workgroup developed a presentation template that can be used at awareness summits, community conversations, workshops, and small group discussions to reduce stigma and encourage parishioners to seek mental health services. The water crisis served to draw professionals and community members together to support the Flint residents who are in need of behavioral health services. Under the leadership of the Genesee Health System, a Community Resilience Group (CRG) was formed in February 2016 to address the behavioral health issues rising from the crisis. A Community Response Team coordinated by the Red Cross is prioritizing mental health and coordinates its efforts with the CRG. The workgroups are planning and coordinating the following efforts: stress management/workforce health protection, assisting vulnerable and underserved populations, providing psychological ﬁrst aid, conducting data and gap analysis, and faith-based group activities. DISCUSSION The CRG has produced a draft Behavioral Health Recovery and Resilience Plan composed around a set of values: living-empowering individuals to thrive in the face of adversity; healing-connecting individuals to the care needed to restore health and wellness; trust-restoring conﬁdence in community resources and agencies; hope-instilling an expectation of a better future; connectedness- fostering relationships among community partners and residents; and knowledge- ensuring timely/accurate information. The plan addresses short-term, intermediate- term, and long-term goals for each of the aforementioned workgroups. 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https://openalex.org/W2727214129	https://pimi.bntu.by/jour/article/download/284/274	Russian	null	INFLUENCE OF SCATTERED NEUTRON RADIATION ON METROLOGICAL CHARACTERISTICS OF АТ140 NEUTRON CALIBRATION FACILITY	Pribory i metody izmerenij	2,017	cc-by	6,099	УДК 53.089.62; 519.245; 539.125.52 Влияние рассеянного нейтронного излучения на метрологические характеристики поверочной установки нейтронного излучения УПН-АТ140 Комар Д.И.1, Кутень С.А.2 1УП «АТОМТЕХ», ул. Гикало, 5, г. Минск 220005, Беларусь 2Институт ядерных проблем Белорусского государственного университета, ул. Бобруйская, 11, г. Минск 220030, Беларусь Поступила 13.01.2017 Принята к печати 14.02.2017 В метрологическом обеспечении средств измерений нейтронного излучения в качестве эталонов ши- рокое распространение получили установки с коллимированным полем излучения, создаваемым радио- нуклидными источниками нейтронов. Целью данной работы являлось исследование влияния рассеянного нейтронного излучения на такие метрологические характеристики поверочной установки нейтронного из- лучения УПН-АТ140, как плотность потока и амбиентный эквивалент мощности дозы с использованием метода Монте-Карло. При калибровке испытуемый прибор помещают в нейтронное поле с известной плотностью потока или амбиентным эквивалентом мощности дозы. Пучок нейтронов от радионуклидного источника быстрых нейтронов может быть сформирован в различных геометриях. В контейнере-коллиматоре установки пове- рочной нейтронного излучения УПН-АТ140 устанавливаются специальные вставки, отвечающие геометрии быстрых и геометрии тепловых нейтронов. Из-за наличия рассеянного излучения необходимо вносить по- правки на рассеянную компоненту, обусловленную рассеянием нейтронов в воздухе и стенами помещения. Измерение характеристик нейтронного поля установки могут быть проведены в ограниченном числе точек, а в остальных точках используется модельный метод. Контейнер-коллиматор установки со вставкой для геометрии тепловых нейтронов формирует пучок от радионуклидного источника нейтронов со значительной составляющей нейтронов тепловых энергий. Из полученной зависимости полного потока нейтронов от расстояния до источника нейтронов видно, что за счет рассеянного в коллиматоре излучения нарушается закон обратных квадратов. Вклад рассеянного стена- ми помещения излучения в полный поток нейтронов увеличивается при удалении от источника нейтронов и значительно влияет на характеристики поля нейтронов. При открытом источнике в геометрии теневого ко- нуса, спектр нейтронов имеет ярко выраженную тепловую компоненту, обусловленную рассеянием от стен. Рассмотрены основные типы геометрий для формирования поля эталонного нейтронного излучения с использованием радионуклидных источников. Разработана Монте-Карло модель 238Pu-Be-источника ней- тронов и контейнера-коллиматора установки УПН-АТ140. Показано, что наиболее существенным факто- ром, влияющим на энергетическое распределение нейтронов, является вклад излучения, рассеянного в помещении, где расположена установка. Это приводит к значительному изменению спектра нейтронного излучения при удалении от источника. При проектировании помещения и размещения в нем установки не- обходимо учитывать требования к качеству излучения. Ключевые слова: Монте-Карло моделирование, радионуклидный источник нейтронов, поле нейтронного излучения, коллиматор; метод теневого конуса. DOI: 10.21122/2220-9506-2017-8-1-23-31 Devices and Methods of Measurements 2017, vol. 8, no. 1, pp. 23–31 Komar D., Kutsen S. Devices and Methods of Measurements 2017, vol. 8, no. 1, pp. 23–31 Komar D., Kutsen S. Приборы и методы измерений 2017. – Т. 8, № 1. – С. 23–31 Комар Д.И., Кутень С.А. DOI: 10.21122/2220-9506-2017-8-1-23-31 Адрес для переписки: Комар Д.И. НПУП «АТОМТЕХ», ул. Гикало, 5, г. Минск 220005, Беларусь e-mail: damiankomar@yandex.ru Address for correspondence: Komar D. SPE «АТОМТЕХ», Gikalo str. 5 , Minsk 220005, Belarus e-mail: damiankomar@yandex.ru Для цитирования: Комар Д.И., Кутень С.А. Влияние рассеянного нейтронного излучения на метрологические характеристики поверочной установки нейтронного излучения УПН- АТ140. Приборы и методы измерений. 2017. – Т. 8, № 1. – С. 23–31. DOI: 10.21122/2220-9506-2017-8-1-23-31 For citation: For citation: Komar D., Kutsen S. [Influence of scattered neutron radiation on metrological characteristics of АТ140 Neutron Calibration Facility]. Pribory i metody izmerenii [Devices and Methods of Measurements]. 2017, vol. 8, no. 1, рр. 23–31(in Russian). DOI: 10.21122/2220-9506-2017-8-1-23-31 DOI: 10.21122/2220-9506-2017-8-1-23-31 Адрес для переписки: Комар Д.И. НПУП «АТОМТЕХ», ул. Гикало, 5, г. Минск 220005, Беларусь e-mail: damiankomar@yandex.ru Address for correspondence: Komar D. SPE «АТОМТЕХ», Gikalo str. 5 , Minsk 220005, Belarus e-mail: damiankomar@yandex.ru Для цитирования: Комар Д.И., Кутень С.А. Влияние рассеянного нейтронного излучения на метрологические характеристики поверочной установки нейтронного излучения УПН- АТ140. Приборы и методы измерений. 2017. – Т. 8, № 1. – С. 23–31. DOI: 10.21122/2220-9506-2017-8-1-23-31 For citation: For citation: Komar D., Kutsen S. [Influence of scattered neutron radiation on metrological characteristics of АТ140 Neutron Calibration Facility]. Pribory i metody izmerenii [Devices and Methods of Measurements]. 2017, vol. 8, no. 1, рр. 23–31(in Russian). DOI: 10.21122/2220-9506-2017-8-1-23-31 DOI: 10.21122/2220-9506-2017-8-1-23-31 Адрес для переписки: Комар Д.И. НПУП «АТОМТЕХ», ул. Гикало, 5, г. Минск 220005, Беларусь e-mail: damiankomar@yandex.ru Address for correspondence: Komar D. SPE «АТОМТЕХ», Gikalo str. 5 , Minsk 220005, Belarus e-mail: damiankomar@yandex.ru Для цитирования: Комар Д.И., Кутень С.А. Влияние рассеянного нейтронного излучения на метрологические характеристики поверочной установки нейтронного излучения УПН- АТ140. Приборы и методы измерений. 2017. – Т. 8, № 1. – С. 23–31. DOI: 10.21122/2220-9506-2017-8-1-23-31 For citation: For citation: Komar D., Kutsen S. [Influence of scattered neutron radiation on metrological characteristics of АТ140 Neutron Calibration Facility]. Pribory i metody izmerenii [Devices and Methods of Measurements]. 2017, vol. 8, no. 1, рр. 23–31(in Russian). DOI: 10.21122/2220-9506-2017-8-1-23-31 DOI: 10.21122/2220-9506-2017-8-1-23-31 Адрес для переписки: Комар Д.И. НПУП «АТОМТЕХ», ул. Гикало, 5, г. Минск 220005, Беларусь e-mail: damiankomar@yandex.ru 23 Devices and Methods of Measurements 2017, vol. 8, no. 1, pp. 23–31 Komar D., Kutsen S. Приборы и методы измерений 2017. – Т. 8, № 1. – С. 23–31 Комар Д.И., Кутень С.А. Influence of scattered neutron radiation on metrological characteristics of АТ140 Neutron Calibration Facility Komar D.1, Kutsen S.2 1SPE «АТОМТЕХ», Gikalo str., 5, , Minsk 220005, Belarus 2Institute of Nuclear Problems, Belarusian State University Bobruiskaya str., 11, Minsk 220030,, Belarus Received 13.01.2017 Accepted for publication 14.02.2017 Abstract Today facilities with collimated radiation field are widely used as reference in metrological support of devices for neutron radiation measurement. Neutron fields formed by radionuclide neutron sources. The aim of this research was to study characteristics of experimentally realized neutron fields geometries on АТ140 Neutron Calibration Facility using Monte Carlo method. il For calibration, we put a device into neutron field with known flux density or ambient equivalent dose rate. We can form neutron beam from radionuclide fast-neutron source in different geometries. In container- collimator of АТ140 Neutron Calibration Facility we can install special inserts to gather fast-neutron geo- metry or thermal-neutron geometry. We need to consider neutron scattering from air and room’s walls. We can conduct measurements of neutron field characteristics in several points and get the other using Monte Carlo method. i Thermal neutron collimator forms a beam from radionuclide source with a significant amount of neutrons with thermal energies. From found relationship between full neutron flux and distance to neutron source we see that inverse square law is violated. Scattered radiation contribution into total flux increases when we are moving away from neutron source and significantly influences neutron fields characteristics. While source is exposed in shadow-cone geometry neutron specter has pronounced thermal component from wall scattering. In this work, we examined main geometry types used to acquire reference neutron radiation using radio- nuclide sources. We developed Monte Carlo model for 238Pu-Be neutron source and АТ140 Neutron Calibra- tion Facility’s container-collimator. We have shown the most significant neutron energy distribution factor to be scattered radiation from room’s walls. It leads to significant changes of neutron radiation specter at a distance from the source. When planning location, and installing the facility we should consider radiation quality requirements. Keywords: Monte Carlo simulation, radionuclide neutron source, fields of neutron radiation, collimator, shadow cone method. DOI: 10.21122/2220-9506-2017-8-1-23-31 DOI: 10.21122/2220-9506-2017-8-1-23-31 DOI: 10.21122/2220-9506-2017-8-1-23-31 Адрес для переписки: Комар Д.И. НПУП «АТОМТЕХ», ул. Гикало, 5, г. Минск 220005, Беларусь e-mail: damiankomar@yandex.ru Address for correspondence: Komar D. SPE «АТОМТЕХ», Gikalo str. 5 , Minsk 220005, Belarus e-mail: damiankomar@yandex.ru Для цитирования: Комар Д.И., Кутень С.А. Влияние рассеянного нейтронного излучения на метрологические характеристики поверочной установки нейтронного излучения УПН- АТ140. Приборы и методы измерений. 2017. – Т. 8, № 1. – С. 23–31. DOI: 10.21122/2220-9506-2017-8-1-23-31 For citation: For citation: Komar D., Kutsen S. [Influence of scattered neutron radiation on metrological characteristics of АТ140 Neutron Calibration Facility]. Abstract Pribory i metody izmerenii [Devices and Methods of Measurements]. 2017, vol. 8, no. 1, рр. 23–31(in Russian). DOI: 10.21122/2220-9506-2017-8-1-23-31 Адрес для переписки: Комар Д.И. НПУП «АТОМТЕХ», ул. Гикало, 5, г. Минск 220005, Беларусь e-mail: damiankomar@yandex.ru Address for correspondence: Komar D. SPE «АТОМТЕХ», Gikalo str. 5 , Minsk 220005, Belarus e-mail: damiankomar@yandex.ru Для цитирования: Комар Д.И., Кутень С.А. Влияние рассеянного нейтронного излучения на метрологические характеристики поверочной установки нейтронного излучения УПН- АТ140. Приборы и методы измерений. 2017. – Т. 8, № 1. – С. 23–31. DOI: 10.21122/2220-9506-2017-8-1-23-31 For citation: For citation: Komar D., Kutsen S. [Influence of scattered neutron radiation on metrological characteristics of АТ140 Neutron Calibration Facility]. Pribory i metody izmerenii [Devices and Methods of Measurements]. 2017, vol. 8, no. 1, рр. 23–31(in Russian). DOI: 10.21122/2220-9506-2017-8-1-23-31 24 Devices and Methods of Measurements 2017, vol. 8, no. 1, pp. 23–31 Komar D., Kutsen S. Приборы и методы измерений 2017. – Т. 8, № 1. – С. 23–31 Комар Д.И., Кутень С.А. Введение ристики нейтронных полей в геометриях, реа- лизуемых экспериментально на установке пове- рочной нейтронного излучения УПН-АТ140, при помощи метода Монте-Карло; оценка вклада рас- сеянного излучения в пучок нейтронов в зависи- мости от геометрических размеров помещения; определение энергетических спектров нейтронов для контейнера-коллиматора со вставками для ге- ометрии быстрых и тепловых нейтронов, а также геометрии теневого конуса. При проведении испытаний средств изме- рений нейтронного излучения широко исполь- зуются радионуклидные источники быстрых нейтронов [1, 2]. В метрологическом обеспече- нии средств измерений нейтронного излучения в качестве эталонов широкое распространение получили установки с полем излучения, созда- ваемым радионуклидными источниками нейтро- нов. Обычно применяют установки, в которых нейтронное поле создается источником нейтро- нов, размещенном в контейнере-коллиматоре (коллимированная геометрия), и установки с от- сутствующими вблизи источника рассеивателями (открытая геометрия). Материалы и методы значение величины H на некотором расстоянии R от источ- ника определяется через значение величины H0 на некотором расстоянии R0 как: Рисунок 1 – Конструкция коллиматора, обеспечи- вающего геометрию узкого пучка: 1 – источник ней- тронов; 2 – контейнер-коллиматор; 3 – коллиматор; 4 – детектор нейтронов; 5 – полость блока-замедлите- ля; 6 – канал коллиматора Рисунок 1 – Конструкция коллиматора, обеспечи- вающего геометрию узкого пучка: 1 – источник ней- тронов; 2 – контейнер-коллиматор; 3 – коллиматор; 4 – детектор нейтронов; 5 – полость блока-замедлите- ля; 6 – канал коллиматора (1) H H R R = 0 0 2 ·( / ) . (1) H H R R = 0 0 2 ·( / ) . Материалы и методы Геометрия узкого пучка, или коллимирован- ная геометрия, характеризуется тем, что рассе- янные нейтроны не попадают в точку измерения. Обычно это достигается использованием специ- ально сконструированных коллиматоров, форма которых обеспечивает такие условия, при кото- рых нерассеянный пучок не попадает на внутрен- нюю поверхность выходной части коллиматора, а для рассеянных нейтронов вдоль пучка создают- ся ловушки. В геометрии широкого пучка детек- тор либо помещается в среду, либо располагается в непосредственной близости к рассеивателю. За счет эффектов обратного рассеяния нейтронов спектры в среде отличаются от спектров за ба- рьером. В точку измерения попадают как нерас- сеянные, так и значительное число рассеянных нейтронов [7]. Наиболее существенным фактором, влияю- щим на энергетическое распределение нейтрон- ного излучения, является рассеянное излучение, возникающее в помещении, где размещается установка [3–5], а также ослабление прямого и рассеянного нейтронного излучения воздухом [6]. В общем случае для коллимированной геоме- трии значение величины плотности потока или амбиентного эквивалента мощности дозы можно представить как сумму вкладов от трех источни- ков: от прямого излучения источника нейтронов, размещенного в начале координат (центр источ- ника); от источника, характеризующего рассеян- ное в контейнере-коллиматоре излучение; от рас- пределенного источника рассеянного в помеще- нии излучения [1, 7]. Измерения характеристик нейтронного поля установки могут быть прове- дены в ограниченном числе точек, а в остальных точках используется метод Монте-Карло [8]. На рисунке 1 показана типичная конструкция коллиматора, обеспечивающего геометрию узко- го пучка. Рисунок 1 – Конструкция коллиматора, обеспечи- вающего геометрию узкого пучка: 1 – источник ней- тронов; 2 – контейнер-коллиматор; 3 – коллиматор; 4 – детектор нейтронов; 5 – полость блока-замедлите- ля; 6 – канал коллиматора Figure 1 – Narrow beam geometry collimator design: 1 – neutron source; 2 – container-collimator; 3 – collimator; 4 – neutron detector; 5 – decelerator bloc cavity; 6 – collimator bore Контейнер-коллиматор изготавливается с применением материалов, которые будут миними- зировать вклад рассеянного коллиматором излуче- 5 В отсутствии ослабления и рассеяния излу- чения для точечного источника излучения выпол- няется закон обратных квадратов, т.е. H H R R = 0 0 2 ·( / ) . При наличии рассеянного излучения, в за- кон обратных квадратов необходимо вносить поправочные коэффициенты. Поправки будут зависеть от источника нейтронов, геометрии, размеров помещения и расстояния до центра ис- точника [9, 10]. Figure 1 – Narrow beam geometry collimator design: 1 – neutron source; 2 – container-collimator; 3 – collimator; 4 – neutron detector; 5 – decelerator bloc cavity; 6 – collimator bore Контейнер-коллиматор изготавливается с применением материалов, которые будут миними- зировать вклад рассеянного коллиматором излуче- Целью данной работы являлись: установле- ние влияния рассеянного излучения на характе- 25 Devices and Methods of Measurements 2017, vol. 8, no. 1, pp. 23–31 Komar D., Kutsen S. Приборы и методы измерений 2017. – Т. 8, № 1. – С. 23–31 Комар Д.И., Кутень С.А. Devices and Methods of Measurements 2017, vol. 8, no. 1, pp. 23–31 Komar D., Kutsen S. Приборы и методы измерений 2017. – Т. 8, № 1. – С. 23–31 Комар Д.И., Кутень С.А. ния. Обычно для этих целей применяется полиэ- тилен с добавлением бора и дополнительно уста- навливаются поглощающие кадмиевые экраны. ния. Обычно для этих целей применяется полиэ- тилен с добавлением бора и дополнительно уста- навливаются поглощающие кадмиевые экраны. цилиндрическими каналами для уменьшения альбедо нейтронов в направлении пучка. цилиндрическими каналами для уменьшения альбедо нейтронов в направлении пучка. Наиболее приемлемым расчетным методом является метод Монте-Карло. Преимущество ме- тода Монте-Карло перед другими методами опре- деляется возможностью рассмотрения переноса частиц в сколь угодно сложных по геометриче- ским условиям и по составу средах без необходи- мости существенного упрощения вычислитель- ного алгоритма [8]. На рисунке 2 изображена схема геометрии широкого пучка. р у Рисунок 2 – Геометрия широкого пучка: 1 – рассеива- тель; 2 – источник; 3 – слой поглотителя; 4 – детектор нейтронов Figure 2 – Wide beam geometry: 1 – scatterer; 2 – source; 3 – absorbent layer; 4 – neutron detector р у Монте-Карло моделирование проводилось с помощью кода MCNP (версия MCNP-4b). Раз- работана Монте-Карло модель контейнера-кол- лиматора установки УПН-АТ140 с геометрией быстрых и с геометрией тепловых нейтронов. На рисунке 3 изображена Монте-Карло модель кон- тейнера-коллиматора для обеих геометрий. Рисунок 2 – Геометрия широкого пучка: 1 – рассеива- тель; 2 – источник; 3 – слой поглотителя; 4 – детектор нейтронов a b Рисунок 3 – Монте-Карло модель контейнера-колли- матора с геометрией быстрых (a) и с геометрией те- пловых нейтронов (b): 1 – кожух из алюминия; 2 – кон- тейнер-коллиматор; 3 – 238Pu-Be-источник нейтронов ИБН-8-6; 4 – воздушные каналы; 5 – вставка для гео- метрии быстрых нейтронов; 6 – вставка для геометрии тепловых нейтронов b a В геометрии широкого пучка рассеивателем является среда расположенная в непосредствен- ной близости к источнику, либо источник может быть полностью в нее погружен (вода, парафин, полиэтилен). Поглотителем может быть как ве- щество, в которое помещен источник, так и спе- циальные экраны, такие как теневой конус, пла- стина кадмия, слой борированного полиэтилена и так далее [11]. Рисунок 3 – Монте-Карло модель контейнера-колли- матора с геометрией быстрых (a) и с геометрией те- пловых нейтронов (b): 1 – кожух из алюминия; 2 – кон- тейнер-коллиматор; 3 – 238Pu-Be-источник нейтронов ИБН-8-6; 4 – воздушные каналы; 5 – вставка для гео- метрии быстрых нейтронов; 6 – вставка для геометрии тепловых нейтронов Установка поверочная нейтронного излуче- ния УПН-АТ140, разработанная научно-произ- водственным унитарным предприятием «АТОМ- ТЕХ», предназначена для поверки дозиметров нейтронного излучения по амбиентному экви- валенту мощности дозы нейтронного излучения и радиометров нейтронного излучения по плот- ности потока быстрых нейтронов в стандартном поле нейтронного излучения источника 238Pu-Be (типа ИБН-8-6) и обеспечивает стабильные ха- рактеристики в диапазоне расстояний от 500 до 3000 мм. В этом диапазоне расстояний от центра источника с учетом поправок выполняется закон обратных квадратов (1). цилиндрическими каналами для уменьшения альбедо нейтронов в направлении пучка. Figure 3 – Monte-Carlo model of container-collimator with geometry for fast (a) and thermal-neutrons (b): 1 – aluminum casing; 2 – container-collimator; 3 – 238Pu-Be fast-neutron source (IBN-8-6); 4 – air channels; 5 – insert for fast-neutron geometry; 6 – insert for thermal-neutron geometry Источник типа ИБН-8-6 относится к радио- нуклидным источникам нейтронов, образую- щихся в результате взаимодействия α – частиц с легкими ядрами бериллия. Активная часть источ- ника представляет собой сплав диоксида плуто- ния-238 с порошком бериллия. Сплав помещен в стальную оболочку и герметизирован. Контейнер-коллиматор установки выполнен из борированного полиэтилена и размещает- ся в кожухе из алюминия с кадмиевой защитой. В контейнер-коллиматор устанавливаются специ- альные вставки, отвечающие геометрии быстрых или геометрии тепловых нейтронов. Вставка для геометрии быстрых нейтронов представляет со- бой коллиматор из борированного полиэтилена с каналом в форме конуса. Тепловая вставка вы- полнена целиком из полиэтилена с воздушными Для источника в MCNP необходимо задать энергетический спектр излучения [8]. Из работы [12] был взят энергетический спектр нейтронов для 238Pu-Be-источника, оцифрован и обработан для применения в MCNP (рисунок 4). Размеры нейтронного источника ИБН-8-6 были приняты 26 Devices and Methods of Measurements 2017, vol. 8, no. 1, pp. 23–31 Komar D., Kutsen S. Приборы и методы измерений в соответствии с технической документацией из- готовителя. в соответствии с технической документацией из- готовителя. в соответствии с технической документацией из- готовителя. На рисунке 5 видно, что спектр, сформиро- ванный «тепловой» геометрией, имеет значи- тельную составляющую тепловых нейтронов. В спектре от «быстрой» геометрии практически отсутствуют нейтроны с энергиями менее 1 эВ. готовителя. Рисунок 4 – Спектр нейтронов 238Pu-Be-источника нейтронов [12] За счет рассеянного в контейнере-коллима- торе излучения будет нарушаться закон обрат- ных квадратов. На рисунке 6 приведены значения полного потока нейтронов в зависимости от рас- стояния до центра источника нейтронов для раз- личных геометрий. Рисунок 4 – Спектр нейтронов 238Pu-Be-источника нейтронов [12] Figure 4 – Neutron specter for 238Pu-Be fast neutron source [12] Выход нейтронов был принят равным 1,91∙107 нейтр./с., что соответствует источнику ИБН-8-6. Результаты и их обсуждение Рисунок 6 – Зависимость значений полного потока нейтронов от расстояния до источника: 1 – «быстрая» геометрия; 2 – «тепловая» геометрия; 3 – «тепловая» геометрия с кадмиевым экраном; 4 – теоретическая кривая, описывающая закон обратных квадратов Рисунок 6 – Зависимость значений полного потока нейтронов от расстояния до источника: 1 – «быстрая» геометрия; 2 – «тепловая» геометрия; 3 – «тепловая» геометрия с кадмиевым экраном; 4 – теоретическая кривая, описывающая закон обратных квадратов Рисунок 6 – Зависимость значений полного потока нейтронов от расстояния до источника: 1 – «быстрая» геометрия; 2 – «тепловая» геометрия; 3 – «тепловая» геометрия с кадмиевым экраном; 4 – теоретическая кривая, описывающая закон обратных квадратов Приборы и методы измерений 2017. – Т. 8, № 1. – С. 23–31 Комар Д.И., Кутень С.А. влияние размеров помещения на количество рас- сеянных нейтронов [5]. На рисунке 7 приведены результаты расчетов зависимости вклада рассе- янного излучения от размеров помещения. влияние размеров помещения на количество рас- сеянных нейтронов [5]. На рисунке 7 приведены результаты расчетов зависимости вклада рассе- янного излучения от размеров помещения. рений через рассчитанные методами Монте-Кар- ло H и H0 [6, 10]. 0 В MCNP расчете использовался специальный детектор, так называемый точечный детектор [8], установленный на расстоянии R от источника. На рисунке 8 приведена полученная зависи- мость d от расстояния до центра источника для геометрии быстрых нейтронов в помещении 12 × 6 × 3 м. Рисунок 7 – Вклад рассеянного излучения в пол- ный поток нейтронов для геометрии с коллиматором: 1 – для помещения 12 × 6 × 3 м; 2 – для помещения 12 × 6 × 6 м; 3 – для помещения 12 × 10 × 10 м Figure 7 – Scattered radiation contribution into total neutron flux for collimated geometry: 1 – for 12 × 3 × 3 m room; 2 – for 12 × 6 × 6 m room; 3 – for 12 × 10 × 10 m room Figure 7 – Scattered radiation contribution into total neutron flux for collimated geometry: 1 – for 12 × 3 × 3 m room; 2 – for 12 × 6 × 6 m room; 3 – for 12 × 10 × 10 m room Рисунок 8 – Зависимость смещения эффективного центра источника d от расстояния до центра источни- ка: 1 – для полного потока нейтронов; 2 – для амбиент- ного эквивалента мощности дозы Из зависимости вклада рассеянного излуче- ния в значение величины полного потока ней- тронов и мощности дозы, можно сделать вывод, что в помещении присутствует поле рассеянного излучения, вклад которого в величину полного потока увеличивается с расстоянием. Вклад рас- сеянного излучения в полную мощность дозы будет меньше, чем в поток, так как отраженные нейтроны имеют меньшую энергию. Следова- тельно, увеличение размеров помещения и опти- мальное расположение установки позволяют до- биться снижения числа рассеянных нейтронов и улучшить её точностные характеристики. Рисунок 6 – Зависимость значений полного потока нейтронов от расстояния до источника: 1 – «быстрая» геометрия; 2 – «тепловая» геометрия; 3 – «тепловая» геометрия с кадмиевым экраном; 4 – теоретическая кривая, описывающая закон обратных квадратов Для оценки спектральных характеристик нейтронных полей, формируемых геометриями быстрых и тепловых нейтронов установки УПН- АТ140, были проведены расчеты потока нейтро- нов в энергетических интервалах с применением карты tally f4 [8]. Расчет проводился для точки на расстоянии 100 см от центра источника при отсут- ствии стен помещения. На рисунке 5 приведены полученные энергетические спектры нейтронов для обеих геометрий (источник ИБН-8-6). Figure 6 – Relationship between total neutron flux and distance to radiation source: 1 – fast geometry; 2 – thermal geometry; 3 – thermal geometry with Cd-shield; 4 – theoretical inverse square law curve Figure 6 – Relationship between total neutron flux and distance to radiation source: 1 – fast geometry; 2 – thermal geometry; 3 – thermal geometry with Cd-shield; 4 – theoretical inverse square law curve На рисунке 6 кривая, описывающая закон об- ратных квадратов (1), совмещена с кривой для те- пловой вставки в точке 100 см. При приближении к коллиматору значение полного потока больше теоретического (закона обратных квадратов) из- за вклада нейтронов, рассеянных коллиматором, а при удалении от коллиматора меньше из-за рас- сеяния нейтронов в воздухе [9]. д р ( ) Рисунок 5 – Энергетические спектры нейтронов: 1 – геометрия тепловых нейтронов; 2 – геометрия бы- стрых нейтронов Figure 5 – Neutron energy spectra: 1 – thermal-neutron geometry; 2 – fast-neutron geometry Для оценки вклада рассеянного излучения в пучок нейтронов была разработана модель помещения. Размеры помещения составляют 12 × 6 × 3 м с толщиной стен 100 см и потолка 150 см, пол – 15 см бетона на грунте. Контейнер- коллиматор установки c быстрой геометрией был размещен на расстоянии 2 м от задней стены по центру. При том же положении контейнера от- носительно передней и задней стен, расчет был проведен также для помещений 12 × 6 × 6 м и 12 × 10 × 10 м. Таким образом, можно установить Рисунок 5 – Энергетические спектры нейтронов: 1 – геометрия тепловых нейтронов; 2 – геометрия бы- стрых нейтронов Figure 5 – Neutron energy spectra: 1 – thermal-neutron geometry; 2 – fast-neutron geometry Рисунок 5 – Энергетические спектры нейтронов: 1 – геометрия тепловых нейтронов; 2 – геометрия бы- стрых нейтронов Рисунок 5 – Энергетические спектры нейтронов: 1 – геометрия тепловых нейтронов; 2 – геометрия бы- стрых нейтронов Figure 5 – Neutron energy spectra: 1 – thermal-neutron geometry; 2 – fast-neutron geometry 27 Devices and Methods of Measurements 2017, vol. 8, no. 1, pp. 23–31 Komar D., Kutsen S. Рисунок 6 – Зависимость значений полного потока нейтронов от расстояния до источника: 1 – «быстрая» геометрия; 2 – «тепловая» геометрия; 3 – «тепловая» геометрия с кадмиевым экраном; 4 – теоретическая кривая, описывающая закон обратных квадратов Рисунок 10 – Схема теневого конуса: 1 – источник нейтронов; 2 – секция из железа; 3 – секция из бори- рованного полиэтилена; 4 – детектор нейтронов Рисунок 10 – Схема теневого конуса: 1 – источник нейтронов; 2 – секция из железа; 3 – секция из бори- рованного полиэтилена; 4 – детектор нейтронов Рисунок 10 – Схема теневого конуса: 1 – источник нейтронов; 2 – секция из железа; 3 – секция из бори- рованного полиэтилена; 4 – детектор нейтронов Рисунок 9 – Зависимость поправочного коэффициен- та F от расстояния до центра источника: 1 – для полно- го потока нейтронов; 2 – для амбиентного эквивалента мощности дозы Figure 10 – Shadow cone design: 1 – neutron source; 2 – Fe-section; 3 – borated polyethylene section; 4 – neutron detector Figure 9 – Relationship between correction factor F and distance to radiation source: 1 – for total neutron flux; 2 – for ambient equivalent dose rate Пояснения к полученным зависимостям ана- логичны пояснениям по величине d. При работе в открытой геометрии коллима- тор отсутствует и в точку измерения попадает как прямое излучение от источника, так и рассе- янное от стен помещения. Для выделения из пол- ного потока нейтронов рассеянной компоненты, измерения проводят в два этапа. Сначала изме- ряется полный поток при отсутствии поглотите- ля во всем диапазоне расстояний, затем на оси пучка устанавливается поглощающий (теневой) конус. Конус экранирует детектор от прямых нейтронов и проводятся измерения потока от- раженных от стен частиц. По разнице показаний прибора в этих двух геометриях определяется прямое излучение. Рисунок 11 – Спектры нейтронов: 1 – открытая гео- метрия в помещении без конуса; 2 – после установки поглощающего конуса Figure 11 – Neutron spectra: 1 – geometry inside room without cone; 2 – after absorption cone installation Figure 11 – Neutron spectra: 1 – geometry inside room without cone; 2 – after absorption cone installation Теневой конус пропускает некоторое количе- ство быстрых нейтронов. При открытом источни- ке в помещении спектр имеет ярко выраженную тепловую компоненту, обусловленную рассеяни- ем от стен. Геометрия и материалы теневого конуса мо- гут изменятся как в зависимости от энергети- ческого спектра применяемого источника и его размеров, так и от формы детектора и методики проведения эксперимента. В случае открытой геометрии влияние те- невого конуса будет различным для каждой точ- ки. На небольших расстояниях между конусом и нейтронным источником количество рассеянных нейтронов будет незначительным, если конус эффективно поглощает большинство нейтронов, распространяющихся в переднюю полусферу по оси детектора [9]. Рисунок 6 – Зависимость значений полного потока нейтронов от расстояния до источника: 1 – «быстрая» геометрия; 2 – «тепловая» геометрия; 3 – «тепловая» геометрия с кадмиевым экраном; 4 – теоретическая кривая, описывающая закон обратных квадратов Figure 8 – Relationship between effective source center d and distance to radiation source: 1 – for total neutron flux; 2 – for ambient equivalent dose rate В данном примере, когда за реперную точку принята точка R0 = 100 см видно, что при R > R0 величина d принимает положительные значения, а при R < R0 отрицательные в некотором диапа- зоне расстояний. Это связано с распределением рассеянного излучения в помещении. При при- ближении к коллиматору установки вклад рассе- янного уменьшается из-за экранирования нейтро- нов, отраженных от стен, расположенных за кол- лиматором и не находящихся в прямой видимо- сти детектора. Дальнейшее уменьшение рассто- яния от источника приводит к увеличению числа нейтронов, рассеянных плоскостями коллимато- ра. По этой причине не рекомендуется проводить эксперименты на расстоянии менее 50 см. При проведении экспериментов в помещении значение величины полного потока либо мощно- сти дозы H на расстоянии R от центра источника представляется в виде суммы вкладов прямого излучения источника и рассеянного излучения коллиматором и стенами помещения. Для при- менения закона обратных квадратов на практике необходимо в формулу (1) добавить поправочные коэффициенты: Для определения величины H с применением закона обратных квадратов также можно исполь- зовать поправочный коэффициент F: (2) H H R R d = − 0 0 2 ·( / ) , H F H R R = · ·( / ) . 0 0 2 (2) H H R R d = − 0 0 2 ·( / ) , Монте-Карло моделирование и последую- щие расчеты проводились аналогично расчетам величины d. где d – смещение эффективного центра источника. Величины смещений d могут быть получены для всех расстояний в пределах диапазона изме- 28 Devices and Methods of Measurements 2017, vol. 8, no. 1, pp. 23–31 Komar D., Kutsen S. Приборы и методы измерений 2017. – Т. 8, № 1. – С. 23–31 Комар Д.И., Кутень С.А. Список использованных источников Figure 12 – M-factor dependency for shadow cone geometry: 1 – for total flux; 2 – for ambient equivalent dose rate 1. Eisenhauer, C.M. Calibration Techniques for Neu- tron Personal Dosymetry / C.M. Eisenhauer, J.B. Hunt, R.B. Schwartz // Radiation Protection Dosimetry. – 1985. – Vol. 10, iss. 1–4. – P. 43–57. 1. Eisenhauer, C.M. Calibration Techniques for Neu- tron Personal Dosymetry / C.M. Eisenhauer, J.B. Hunt, R.B. Schwartz // Radiation Protection Dosimetry. – 1985. – Vol. 10, iss. 1–4. – P. 43–57. При увеличении расстояния между конусом и детектором вклад рассеянного излучения уве- личивается и затем остается постоянным в неко- тором диапазоне расстояний. Дальнейшее удале- ние детектора приводит к быстрому увеличению показаний по рассеянному излучению, как толь- ко конус перестает экранировать нейтронный де- тектор от источника [9]. 2. Vega-Carrillo, H.R. Spectrometry and dosimetry of a neutron source / H.R. Vega-Carillo, E. Manzanares- Acuna, V.M. Hernandez-Davila // Radiation Effects and Defects in Solids. – 2009. – Vol. 164, iss. 4. – P. 218–223. 3. Vega-Carillo, H.R. Study of room-return neu- trons. / H.R. Vega-Carillo, E. Manzanares-Acuna, M.P. Ini- guez. // Radiation Measurements. – 2007. – Vol. 42, iss. 3. – P. 413–419. 3. Vega-Carillo, H.R. Study of room-return neu- trons. / H.R. Vega-Carillo, E. Manzanares-Acuna, M.P. Ini- guez. // Radiation Measurements. – 2007. – Vol. 42, iss. 3. – P. 413–419. Если же детектор располагается слишком близко к теневому конусу, последний экранирует детектор от нейтронов, рассеянных на стенах по- мещения. 4. McCall, R.C. Room scattered neutrons / R.C. Mc- Call, P.H. McGinley, K.E. Huffman // Medical Physics. – 1999. – Vol. 26, iss. 2. – P. 205–207. 4. McCall, R.C. Room scattered neutrons / R.C. Mc- Call, P.H. McGinley, K.E. Huffman // Medical Physics. – 1999. – Vol. 26, iss. 2. – P. 205–207. 5. Gallego, E. Characterictics of the Neutron Field of the Facility at DIN-UPM / E. Gallego, A. Lorente // Radiation Protection Dosimetry. – 2004. – Vol. 110, iss. 1–4. – P. 73–79. 5. Gallego, E. Characterictics of the Neutron Field of the Facility at DIN-UPM / E. Gallego, A. Lorente // Radiation Protection Dosimetry. – 2004. – Vol. 110, iss. 1–4. – P. 73–79. Рисунок 6 – Зависимость значений полного потока нейтронов от расстояния до источника: 1 – «быстрая» геометрия; 2 – «тепловая» геометрия; 3 – «тепловая» геометрия с кадмиевым экраном; 4 – теоретическая кривая, описывающая закон обратных квадратов Рисунок 12 – Зависимость поправочного коэффици- ента M для геометрии с теневым конусом: 1 – для пол- ного потока; 2 – для амбиентного эквивалента мощ- ности дозы Figure 12 – M-factor dependency for shadow cone t 1 f t t l fl 2 f bi t i l t у у Для геометрии быстрых нейтронов опреде- лены поправочные коэффициенты для расчета полного потока и амбиентного эквивалента мощ- ности дозы, обусловленные рассеянным излуче- нием. Теневой конус пропускает некоторое количе- ство быстрых нейтронов. При открытом источни- ке в помещении спектр нейтронов имеет ярко вы- раженную тепловую компоненту, обусловленную рассеянием от стен. Рисунок 12 – Зависимость поправочного коэффици- ента M для геометрии с теневым конусом: 1 – для пол- ного потока; 2 – для амбиентного эквивалента мощ- ности дозы Рисунок 12 – Зависимость поправочного коэффици- ента M для геометрии с теневым конусом: 1 – для пол- ного потока; 2 – для амбиентного эквивалента мощ- ности дозы Рисунок 6 – Зависимость значений полного потока нейтронов от расстояния до источника: 1 – «быстрая» геометрия; 2 – «тепловая» геометрия; 3 – «тепловая» геометрия с кадмиевым экраном; 4 – теоретическая кривая, описывающая закон обратных квадратов Для Pu-Be-источников используется конус, состоящий из двух секций: из железа длиной 20 см, и из борированного полиэтилена длиной 30 см [1, 9]. На рисунке 10 изображена схема применения теневого конуса. Для оценки изменений энергетических спектров нейтронов с учетом влияния теневого конуса был проведен Монте-Карло расчет. Ре- зультаты моделирования спектров приведены на рисунке 11. Для открытой геометрии с применением те- невого конуса закон обратных квадратов можно записать следующим образом: ( ) ( ) ( / ) , H H M H H R R o c oo co − = ⋅ − ⋅ 0 2 ( ) ( ) ( / ) , H H M H H R R o c oo co − = ⋅ − ⋅ 0 2 29 Devices and Methods of Measurements 2017, vol. 8, no. 1, pp. 23–31 Komar D., Kutsen S. Приборы и методы измерений 2017. – Т. 8, № 1. – С. 23–31 Комар Д.И., Кутень С.А. где Hо и Hc – значения величин, соответствующих расстояниям от центра источника Rо и R в откры- той геометрии; Hоo и Hco – значения величин, со- ответствующих расстояниям Rо и R, когда прямое излучение от источника поглощается, и значения этих величин обусловлены только рассеянным излучением; M – поправочные коэффициенты для каждого расстояния. излучения с использованием радионуклидных источников. Разработана Монте-Карло модель 238Pu-Be-источника нейтронов ИБН-8-6 и кон- тейнера-коллиматора установки УПН-АТ140. . С помощью моделирования методом Монте-Карло рассчитаны спектры нейтронов для геометрии быстрых и геометрии тепловых нейтронов. Наиболее существенным фактором, влияю- щим на энергетическое распределение нейтронов, является вклад рассеянного излучения, возникаю- щего в помещении, где расположена установка. Рассеянное стенами помещения нейтронное излу- чение приводит к значительному изменению энер- гетического спектра при удалении от источника и, таких метрологических характеристик установки, как плотность потока нейтронов и амбиентный эквивалент мощности дозы. Проведены оценки вклада рассеянного нейтронного излучения в зна- чение величины плотности потока нейтронов в зависимости от размеров помещения. Коэффициент M указывает на характер вли- яния теневого конуса в зависимости от расстоя- ния до детектора нейтронов и определяется через расчетные значения величин Hco, Hc и Ho, Hc. co c o c На рисунке 12 показана зависимость попра- вочного коэффициента M для геометрии теневого конуса. фф р конуса. Заключение Рассмотрены основные типы геометрий для формирования поля эталонного нейтронного 6. Eisenhauer, C.M. Effect of Air Scatter on Calibra- tion of Instruments for Detecting Neutrons / C.M. Eisen- 30 Devices and Methods of Measurements 2017, vol. 8, no. 1, pp. 23–31 Komar D., Kutsen S. Приборы и методы измерений 2017. – Т. 8, № 1. – С. 23–31 Комар Д.И., Кутень С.А. hauer, R.B. Schwartz, R.C. McCall // Radiation Protec- tion Dosimetry. – 1987. – Vol. 19, iss. 2. – P. 77–84. doi: 10.1016/j.radmeas.2007.01.036 7. Hunt, J.B. The Сalibration of Neutron Sensetive Spherical Devices / J.B. Hunt // Radiation Protection Do- simetry. – 1984. – Vol. 8, iss. 4. – P. 239–251. 4. McCall R.C., McGinley P.H., Huffman K.E. Room scattered neutrons. Medical Physics, 1999, vol. 26, iss. 2, pp. 205–207. doi: 10.1118/1.598505 8. Briesmeister, J.F. MCNP-A General Monte Carlo N-Paticle Transport Code, Version 4В. LA-12625-M./ J.F. Briesmeister; ed. Los Alamos National Laboratory. – Los Alamos, 1997, 736 с. 5. Gallego E., Lorente A. Characterictics of the Neutron Field of the Facility at DIN-UPM. Radiation Protection Dosimetry, 2004, vol. 110, iss. 1–4, pp. 73– 79. doi: 10.1093/rpd/nch199 9. Kim, S.I. Review of neutron scattering correc- tion for the calibration of neutron survey meters using the shadow-cone metod / S.I. Kim, B.H. Kim, J.L. Kim // Nuclear Engineering and Technology. – 2015. – Vol. 47, iss. 7. – P. 939–944. 6. Eisenhauer C.M., Schwartz R.B., McCall R.C. Effect of Air Scatter on Calibration of Instruments for Detecting Neutrons. Radiation Protection Dosimetry, 1987, vol. 19, iss. 2, pp. 77–84. doi: 10.1093/oxfordjournals.rpd.a079923 doi: 10.1093/oxfordjournals.rpd.a079923 10. Eisenhauer, C.M. Review of Scattering Correc- tions for Calibration of Neutron Instruments / C.M. Eisen- hauer // Radiation Protection Dosimetry. – 1989. – Vol. 28, iss. 4. – P. 253–262. 7. Hunt J.B. The Сalibration of Neutron Sensetive Spherical Devices. Radiation Protection Dosimetry, 1984, vol. 8, iss. 4, pp. 239–251. doi: 10.1093/oxfordjournals.rpd.a083078 11. Vega-Carillo, H.R. Low energy neutrons from a 239PuBe isotopic neutron source inserted in moderating media / H.R. Vega-Carillo, C.T. Muhech // Revista Mexi- cana de Fisica. – 2002. – Vol. 48, iss. 5. – P. 405–412. 8. Briestmeister J.F. ed. MCNP-A general Monte Carlo N-particle transport code, Version 4A. Report LA-12625-M, Los Alamos, NM: Los Alamos National Laboratory,1994, 736 pp. 12. Faghihi, F. Neutrons Flux Distributions of the Pu-Be Source and its Simulation by the MCNP-4B Code / F. Faghihi, S. Mehdizadeh, K. Заключение Hadad // International Journal of Modern Physics. – 2006. – Vol. 15, iss. 3. – P. 737–745. 9. Kim S.I., Kim B.H., Kim J.L. Review of neu- tron scattering correction for the calibration of neutron survey meters using the shadow-cone metod. Nuclear Engineering and Technology, 2015, vol. 47, iss. 7, pp. 939–944. doi: 10.1016/j.net.2015.07.005 References 10. Eisenhauer C.M. Review of Scattering Correc- tions for Calibration of Neutron Instruments. Radiation Protection Dosimetry, 1989, vol. 28, iss. 4, pp. 253–262. doi: 10.1093/oxfordjournals.rpd.a080514 1. Eisenhauer C.M., Hunt J.B., Schwartz R.B. Cali- bration Techniques for Neutron Personal Dosymetry. Radiation Protection Dosimetry, 1985, vol. 10, iss. 1–4, pp. 138–147. doi: 10.1093/oxfordjournals.rpd.a079410 11. Vega-Carillo H.R., Muhech C.T. 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https://openalex.org/W4241576875	http://turczaninowia.asu.ru/article/download/7190/5938	Russian	null	On the morphological variability and phylogenetic relationships of Crimean and Caucasian members of Lotus section Dorycnium	Turczaninowia	2,019	cc-by	9,806	Turczaninowia 22 (4): 87–103 (2019) DOI: 10.14258/turczaninowia.22.4.11 http://turczaninowia.asu.ru Turczaninowia 22 (4): 87–103 (2019) DOI: 10.14258/turczaninowia.22.4.11 http://turczaninowia.asu.ru Turczaninowia 22 (4): 87–103 (2019) DOI: 10.14258/turczaninowia.22.4.11 http://turczaninowia.asu.ru Turczaninowia 22 (4): 87–103 (2019) DOI: 10.14258/turczaninowia.22.4.11 http://turczaninowia.asu.ru ISSN 1560–7259 (print edition) TURCZANINOWIA ISSN 1560–7267 (online edition) УДК 582.736:581.9/.4+575.86 * автор для переписки Ключевые слова: география, систематика, филогения, ITS, Leguminosae, Loteae, Lotus dorycnium, Lotus grae cus, trnL интрон, trnL-F спейсер. Аннотация. В Крыму и на Кавказе секция Dorycnium (Mill.) D. D. Sokoloff рода Lotus L. s. l., относимая многими исследователями к роду Dorycnium Mill., представлена таксонами родства L. graecus L. (= Dorycnium graecum) и L. dorycnium L. (= D. pentaphyllum). Для уточнения видового состава, диагностических признаков и филогенетических связей представителей секции этих географических регионов были проанализированы семь крымских и пять кавказских выборок. Проведен морфологический анализ крымского и кавказского мате риала с использованием методов одномерной и многомерной (метод главных координат) статистики. Молеку лярно-филогенетический анализ отдельно по ITS1-5,8S-ITS2 ядерной рДНК и trnL-F хпДНК выполнен с при влечением репрезентативного набора видов рода Lotus s. l. Исследование выявило присутствие на территории Крыма и Кавказа двух видов: L. graecus и L. herbaceus (Vill.) Jauzein (представителя комплекса L. dorycnium), отличающихся по многим морфологическим признакам (размерам цветков, листьев, зубцов чашечки, харак теру опушения и наличию/отсутствию корневых отпрысков). Межвидовые гибриды не выявлены. В преде лах каждого вида географической дифференциации между последовательностями обоих изученных марке ров (ITS и trnL-F) не наблюдалось. Данное исследование подтвердило, что комплекс L. dorycnium (включая L. herbaceus) из секции Dorycnium по пластидным данным не отделен от L. hirsutus, вида из секции Bonjeanea (Rchb.) D. D. Sokoloff с широким средиземноморским ареалом, не доходящим до Крыма и Кавказа, что было ранее показано по другим маркерам хпДНК. О морфологической изменчивости и филогенетических связях представителей секции Dorycnium рода Lotus Крыма и Кавказа М. В. Лысова1, И. Г. Мещерский2, Т. Е. Крамина1* Московский государственный университет им. М. В. Ломоносова, Ленинские горы, 1, г. Москва, 119991, Росси E-mail: maya-kut@mail.ru, tkramina@yandex.ru Московский государственный университет им. М. В. Ломоносова, Ленинские горы, 1, г. Москва, 119991, Росси E-mail: maya-kut@mail.ru, tkramina@yandex.ru нститут проблем экологии и эволюции им. А. Н. Северцова Российской академии наук, Ленинский проспект, 3 г. Москва, 119071, Россия. E-mail: meschersky@rambler.ru 2 Институт проблем экологии и эволюции им. А. Н. Северцова Российской академии наук, Ленинский проспект, 33, г. Москва, 119071, Россия. E-mail: meschersky@rambler.ru Submitted 05.08.2019 Accepted 14.10.2019 Поступило в редакцию 05.08.2019 Принято к публикации 14.10.2019 Введение Rikli (1901) в его монографии. Rikli выделял в роде три секции: секцию Canaria Rikli с тремя видами: D. broussonetii Webb et Berthel., D. eri ophthalmum Webb et Berthel., D. spectabile Webb et Berthel.; секцию Bonjeanea Taubert с тремя видами: D. hirsutum (L.) Ser., D. rectum (L.) Ser., D. latifolium (L.) Ser. (синоним вида D. graecum Ser.); и секцию Eudorycnium Boiss. (современ ное название – секция Dorycnium) с видами: D. herbaceum Vill., D. jordanii Loret et Barr. (= D. gracile Jord.), D. suffruticosum Vill., D. germani cum Rouy, D. anatolicum Boiss., D. haussknechtii Boiss. Rikli (1901) в его монографии. Rikli выделял в роде три секции: секцию Canaria Rikli с тремя видами: D. broussonetii Webb et Berthel., D. eri ophthalmum Webb et Berthel., D. spectabile Webb et Berthel.; секцию Bonjeanea Taubert с тремя видами: D. hirsutum (L.) Ser., D. rectum (L.) Ser., D. latifolium (L.) Ser. (синоним вида D. graecum Ser.); и секцию Eudorycnium Boiss. (современ ное название – секция Dorycnium) с видами: D. herbaceum Vill., D. jordanii Loret et Barr. (= D. gracile Jord.), D. suffruticosum Vill., D. germani cum Rouy, D. anatolicum Boiss., D. haussknechtii Boiss. Проблема разделения родов Lotus и Doryc nium (Leguminosae, Loteae). К. Линней (Lin naeus, 1751) выделял род Dorycnium, но позднее включил его в род Lotus L., распространенный в Европе, Азии и Африке (Linnaeus, 1753). Ph. Miller (1754) восстановил род Dorycnium Mill. и включил в него два вида, в том числе вид, со ответствующий D. pentaphyllum Scop. (= Lotus dorycnium L.). y ) В ходе последующего изучения европей ских флор складывались представления о са мостоятельности родов Dorycnium и Lotus. Так, представители рода Dorycnium отличаются от типичных представителей рода Lotus обычно полукустарниковой, а не травянистой жизнен ной формой, почти полным отсутствием рахиса листа, более мелкими цветками в густых част ных соцветиях, белой или розовой, а не желтой окраской лепестков, слипающимися крыльями, несущими мешковидные полые поперечные вы росты, короткой и тупой лодочкой, гладким, а не папиллированным стилодием, короткими, обыч но односеменными плодами, створки которых при вскрывании не закручиваются (Kramina et al., 2016). Черты рода Dorycnium, отличающие его от Lotus, наиболее хорошо проявляются у представителей типовой секции Dorycnium. Однако ряд видов двух других секций (секции Canaria Rikli и секции Bonjeanea Taubert) несут мозаичное сочетание признаков типичных пред ставителей Lotus и Dorycnium. Граница между этими родами остается дискуссионной (Kramina et al., 2016). J. B. Gillett (1958) на основе морфологических данных исключил из рода Dorycnium секцию Ca naria. On the morphological variability and phylogenetic relationships of Crimean and Caucasian members of Lotus section Dorycnium M. V. Lysova1, I. G. Meschersky2, T. E. Kramina1 1 Lomonosov Moscow State University, Leninskie Gory, 1, Moscow, 119991, Russian Federation 2 A. N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Leninskiy pr. 33, 119071, Moscow, Russian Federation Keywords: geography, ITS, Leguminosae, Loteae, Lotus dorycnium, Lotus graecus, phylogeny, taxonomy, trnL intron, trnL-F spacer. Submitted 05.08.2019 Accepted 14.10.2019 Лысова М. В. и др. О морфологии и филогении Dorycnium Крыма и Кавказа 88 Summary. In the Crimea and the Caucasus, the section Dorycnium (Mill.) D. D. Sokoloff of the genus Lotus L. s. l., attributed by many researchers to the genus Dorycnium Mill., is represented by taxa related to L. graecus L. (= Do rycnium graecum) and L. dorycnium L. (= D. pentaphyllum). To clarify the species composition, diagnostic features, and phylogenetic relationships of the representatives of the section in these two geographical regions, seven Crimean and five Caucasian samples were analyzed. A morphological analysis of the Crimean and Caucasian material was carried out using the methods of one-dimensional and multidimensional (Principal Coordinate Analysis) statistics. Separate molecular phylogenetic analyses by ITS1-5.8S-ITS2 nrDNA and trnL-F cpDNA were performed using a representative set of species of the genus Lotus s. l. The study revealed the presence of two species in the Crimea and the Caucasus: L. graecus and L. herbaceus (Vill.) Jauzein (a representative of the L. dorycnium complex), which differ in many morphological characters (e.g., sizes of flowers, leaves, calyx teeth, and the type of pubescence and the pres ence/absence of root offspring). Interspecific hybrids were not identified. Within each species, the geographical dif ferentiation between the sequences of both studied markers (ITS and trnL-F) was not observed. The study confirmed that the L. dorycnium complex (including L. herbaceus) from Lotus section Dorycnium according to plastid data is not separated from L. hirsutus, a species from Lotus section Bonjeanea with a wide Mediterranean range that does not reach the Crimea and the Caucasus, as was previously shown by other cpDNA markers. Введение Молекулярные исследования последних лет (Degtjareva et al., 2006; Kramina et al., 2016) подтвердили этот вывод и показали, что секция Canaria не является близкой к другим секциям Dorycnium и относится к южной эволюционной ветви рода Lotus (Kramina et al., 2016). Объем секции Bonjeanea также был пере смотрен. На основе морфологического анали за и с опорой на работу P. Lassen (1986) в нее был включен Lotus strictus Fisch. et C. A. Mey., а D. graecum (= L. graecus) перенесен в секцию Dorycnium (Degtjareva et al., 2006). Молекуляр но-филогенетические исследования показали, что секция Bonjeanea не является естественной (как в понимании Rikli, так и в более поздней трактовке) (Degtjareva et al., 2006; Kramina et al., 2016). Состав секции Dorycnium трактуется по- разному. Согласно Degtjareva et al. (2006), в нее входят сложный видовой комплекс Lotus dorycnium L. s. l. (= Dorycnium pentaphyllum) и вид L. graecus L., а также родственные им виды (L. fulgurans (Porta) D. D. Sokoloff (= Dorycnium Система рода Dorycnium. Наиболее извест на система рода Dorycnium, предложенная M. Turczaninowia 22 (4): 87–103 (2019) 89 fulgurans (Porta) Lassen), L. sanguineus (Vural) D. D. Sokoloff (= D. sanguineum Vural) и L. axil liflorus (Hub.-Mor.) D. D. Sokoloff (= D. axilliflo rum Hub.-Mor.)). Виды секции имеют широкий средиземноморский ареал, доходящий до Цен тральной и Восточной Европы, Крыма и Кавка за. Во «Flora Iranica» K. H. Rechinger (1984) при водит только два вида: D. pentaphyllum Scop. с единственным представленным на террито рии флоры подвидом D. pentaphyllum subsp. haussknechtii (Boiss.) Gams и D. intermedium Ledeb. Greuter et al. (1989) в сводке по флоре Сре диземноморского региона представляют D. pen taphyllum aggr. в виде иерархической системы. Комплекс включает широко распространен ные виды D. herbaceum Vill. и D. pentaphyllum Scop. и локальные эндемики Турции (D. amani Zohary и D. axilliflorum Huber-Morath) и Балеар ских о-вов (D. fulgurans (Porta) Lassen). D. pen taphyllum, в свою очередь, включает пять под видов: Dorycnium pentaphyllum subsp. anatolicum (Boiss. et Heldr.) Gams, D. pentaphyllum subsp. germanicum (Gremli) Gams, D. pentaphyllum sub sp. haussknechtii (Boiss.) Gams, D. pentaphyllum subsp. pentaphyllum, D. pentaphyllum subsp. trans montanum Franco, а D. herbaceum – два подвида: D. herbaceum subsp. gracile (Jordan) Nyman и D. herbaceum subsp. herbaceum. Взгляды на Dorycnium в европейских и ази атских «Флорах» в XX и начале XXI вв. Введение Боль шинство авторов проанализированных нами источников рассматривают Dorycnium в ранге рода, отличного от рода Lotus (Ball, 1968; Demi riz, 1970; Rechinger, 1984; Greuter et al., 1989; Díaz Lifante, 2000). Lotus strictus относят либо к Lotus (Ball, 1968; Heyn, 1970; Chrtková-Žerto vá, 1984), либо к Dorycnium (Greuter et al., 1989). Как правило, морфологически хорошо обосо бленные виды, такие как D. hirsutum, D. rectum и D. graecum, трактуются разными авторами сход но, а наибольшие разногласия наблюдаются в трактовке комплекса D. pentaphyllum. Во «Flora Europaea» P. W. Ball (1968) выделяет в составе D. pentaphyllum четыре подвида: а) subsp. pen taphyllum, b) subsp. germanicum, c) subsp. grac ile, d) subsp. herbaceum. Автор указывает, что эти подвиды рассматриваются многими авторами как виды, но (а), (b) и (с) часто трудно разделить по морфологическим признакам, а подвид (d), как правило, хорошо отличается, но образует редкие переходные формы с другими подвида ми. На территории «Flora Iberica» Z. Díaz Li fante (2000) признает в составе секции Doryc nium виды D. pentaphyllum Scop., D. gracile Jord. (= D. herbaceum subsp. gracile (Jordan) Nyman) и D. fulgurans (Porta) Lassen. Типичный D. her baceum для Пиренейского п-ова автором не от мечен. Из истории изучения Dorycnium на терри тории бывшего СССР и России. На территории бывшего СССР представители рода Dorycnium распространены преимущественно в Крыму и на Кавказе (Steinberg, 1945), однако упоминается их произрастание также в Южной и Северной Бес сарабии (Молдавии) и Карпатах (Steinberg, 1945; Minjaev, 1987). В большинстве флор и опреде лителей признается распространение в Крыму и на Кавказе двух видов Dorycnium. Первый из них, Dorycnium graecum (L.) Ser. (syn. D. latifo lium Willd.), обычно не вызывает разногласий, являясь хорошо очерченным видом, отмечен в горах (кроме яйлы) и на Южном берегу Крыма, в Предкавказье, Дагестане, Зап. и Вост. Закавка зье (Grossheim, 1930; Steinberg, 1945; Chernova, Kryukova, 1972; Galushko, 1980; Minjaev, 1987; Greuter et al., 1989; Golubev, 1996; Yena, 2012). Полный ареал D. graecum включает северо-вос точное Средиземноморье и Причерноморье. Вто рой вид, приводимый для территории Крыма и Кавказа, относится к комплексу D. pentaphyllum Demiriz (1970) для флоры Турции приводит D. hirsutum, D. graecum, D. rectum, D. axilliflo rum, D. pentaphyllum subsp. herbaceum, D. pen- taphyllum subsp. anatolicum, D. pentaphyllum subsp. haussknechtii, D. amani Zohary. Çelebioğlu (1977) провела морфолого-анатомическое иссле дование вегетативных и генеративных органов всех представителей Dorycnium Турции, при водимых для ее флоры (Demiriz, 1970), кроме D. rectum и D. amani. Введение Ею было подробно описано их строение и выявлены различия между таксо нами. Несмотря на детальный морфолого-ана томический анализ видов Dorycnium, проведен ный Çelebioğlu, он основан на изучении только турецких представителей группы и выполнен на сравнительно небольших выборках, растения других частей ареала не были охвачены. В 1983 г. M. Vural описал новый эндемичный вид Dorycnium для Турции, D. sanguineum Vural (Vural, Tan, 1983), который Соколов перенес в секцию Dorycnium рода Lotus (Sokoloff, 2003). В 1983 г. M. Vural описал новый эндемичный вид Dorycnium для Турции, D. sanguineum Vural (Vural, Tan, 1983), который Соколов перенес в секцию Dorycnium рода Lotus (Sokoloff, 2003). 90 Лысова М. В. и др. О морфологии и филогении Dorycnium Крыма и Кавказа вающаяся пятью зубцами, более правильными, чем у D. suffruticosum (Villars, 1788). (= Lotus dorycnium). Общий ареал комплекса – широкий средиземноморский, доходящий на се вере до Ц. Европы, а на востоке – до Крыма и Кавказа. Таксономические границы между вида ми (или подвидами) комплекса нечеткие, о чем уже говорилось выше. На территории Крыма и Кавказа представителей этой группы трактуют по-разному: 1) как D. herbaceum Vill. (Grossheim, 1930; Greuter et al., 1989; Golubev, 1996) или D. pentaphyllum Scop. subsp. herbaceum (Vill.) Rouy (Yena; 2012); 2) как D. intermedium Ldb. (Steinberg, 1945; Chernova, Kryukova, 1972; Ga lushko, 1980). вающаяся пятью зубцами, более правильными, чем у D. suffruticosum (Villars, 1788). Ledebour (1820) описал D. intermedium, близ кий к D. herbaceum, но отличающийся острыми обратнояйцевидными листочками, треугольны ми зубцами чашечки, одревесневающими побе гами, а особенно более мохнатой поверхностью, остроконечными листочками, цветками белы ми, с лодочкой на конце пурпурно-черной, а не красноватой; трубкой чашечки с более коротки ми зубцами и отклоненными волосками, кото рые у D. herbaceum прижатые. Ледебур описал D. intermedium из Крыма (“Legimus in lapidosis cretaceis et schistosis Tauriae”), но отметил, что, по данным Биберштейна, он также, возможно, встречается в Иберии (Пиренейском п-ове) под названием D. monspeliensis (Ledebour, 1820). Общий ареал D. herbaceum Vill. включает также Ц. и ЮВ. Европу (Ball, 1968), С. и СЗ. Тур цию (Demiriz, 1970). Рикли (Rikli, 1901) в своей монографии рода Dorycnium считает, что D. in termedium и D. herbaceum не отделены четко друг от друга ни морфологически, ни географически. На это ссылается Штейнберг (Steinberg, 1945), указывая, что только D. intermedium, представ ляющий собой восточную расу D. herbaceum, встречается во флоре СССР. Она приводит точку зрения Рикли о D. herbaceum как сборном виде, находящемся в процессе формообразования. Если D. herbaceum и D. Введение Turczaninowia 22 (4): 87–103 (2019) 91 но-филогенетического анализа был использо ван репрезентативный материал по роду Lotus, охватывающий все секции рода, кроме секции Benedictella. В анализ были включены последо вательности ITS и trnL-F из Генбанка (см. табл. 1), подавляющее большинство которых было получено ранее в исследованиях при участии одного из авторов настоящей статьи и опублико вано в ряде работ (Degtjareva et al., 2003, 2006, 2008; Kramina et al., 2012, 2016, 2018), а также последовательность trnL-F, взятая из пластидно го генома Lotus miyakojimae Kramina, опублико ванная под названием L. japonicus (Regel) Larsen (Kato et al., 2000). Кроме того, в настоящей ра боте были получены новые последовательности trnL-F и, отчасти, ITS для образцов нескольких видов из секций Lotus (вида L. parviflorus Desf.), Dorycnium (видов L. herbaceus, L. axilliflorus, L. sanguineus) и Bonjeanea (вида L. hirsutus) из разных регионов (см. табл. 1). Целями настоящей работы являлись: иденти фицировать виды секции Dorycnium рода Lotus на территории Крыма и Кавказа; уточнить диа гностические признаки видов секции Dorycnium этих регионов; установить филогенетическое положение крымских и кавказских образцов сек ции Dorycnium внутри рода Lotus. Введение intermedium считать одним видом, то приоритетным будет название D. herbaceum. Молекулярно-филогенетические иссле дования Dorycnium и родственных таксонов. Филогенетические исследования трибы Loteae по маркеру nrITS показали, что род Lotus моно филетичен только при включении в его состав родов Dorycnium и Tetragonolobus Scop. (Allan et al., 2003; Degtjareva et al., 2006; Kramina et al., 2016). Молекулярно-филогенетический анализ рода Lotus s. l. (включая Dorycnium и Tetragonol obus), проведенный с использованием ядерных (nrITS, 5’ETS) и пластидных (psbA-trnH и ин трона rps16) маркеров выявил в эволюции рода раннее расщепление на «южную» и «северную» ветви. Южная ветвь включает большинство (де сять, согласно Degtjareva et al., 2006) секций рода Lotus и выделяется как по ядерным, так и по пластидным маркерам, а северная ветвь, объ единяющая членов только трех секций (Lotus, Dorycnium и Bonjeanea), обособляется только по пластидным данным. Филогенетические взаи моотношения внутри этой ветви наиболее про тиворечивы (Kramina et al., 2016). Н. А. Миняев (Minjaev, 1987) считал, что в европейской части СССР, помимо D. graecum, встречаются три отдельных вида: D. pentaphyl lum, D. herbaceum и D. intermedium. Во флоре Северно-Западного Кавказа А. С. Зернов (Zer nov, 2006) представил два вида Lotus секции Dorycnium (Miller) D. D. Sokoloff: L graecus L. (= Dorycnium graecum (L.) Ser.) и L. dorycnium L. s. l. (= Dorycnium herbaceum Vill.; D. interme dium Ledeb.). Вид D. herbaceum был описан D. Villars в 1779 г. как растение с прямостоячими травянистыми стеблями и многосеменными бобами. Позднее он описал другой вид этой же группы, D. suffruti cosum, и уточнил описание D. herbaceum (Villars, 1788). Он отметил, что D. herbaceum распростра нен в окрестностях Гренобля и Шамбери (Фран ция). В качестве черт, отличающих этот вид от D. suffruticosum, автор выделяет травянистые, а не полуодревесневшие побеги, разветвленные в верхней части, а не от основания. Также для D. herbaceum характерны пятилисточковые ли стья без черешка; с листочками продолговаты ми и тупыми, расширяющимися в верхней ча сти, с закругленным окончанием; мелкие белые цветки, в два раза более многочисленные, чем у D. suffruticosum, расположенные на верхушках цветоносов; чашечка более удлиненная, оканчи Таким образом, однозначного взгляда на со став и таксономическую структуру группы, ра нее выделяемой в качестве рода Dorycnium, а в последнее время относимой к двум секциям рода Lotus, не существует. Вызывают разногласия и трактовки этой группы на территории России, хотя здесь она представлена небольшим чис лом таксонов. Дополнительные исследования в Крыму и на Кавказе могут представлять интерес для систематики группы и понимания ее объема. Материалы и методы Полученные последовательности выравнивали с последовательностями из Генбанка, матрицу по ITS автоматически в программе MAFFT version 7.215 (Katoh et al., 2002; Katoh, Standley, 2013), а матрицу trnL-F – автоматически и вручную в программе BioEdit (Hall, 1999), ввиду наличия длинных инделей (Kramina et al., 2018). Поли морфизм ITS внутри образца определяли мето дом прямого секвенирования (без клонирования) по наличию двойных пиков на хроматограмме, проявляющихся при прямом и обратном прочте нии, и при высоте минорного пика не менее 20 % от главного. Число гапло- и риботипов оценива ли в программе DnaSP 6 (Rozas et al., 2017). При подсчете риботипов учитывали только главный сигнал (мажорный пик). ков в элементарном соцветии; 5–10) длину (мм), ширину (мм) и индекс листочков средних и верх них листьев; 11–16) длину коротких и длинных волосков на листьях, стебле и чашечке (мм); 17– 22) длину и ширину верхнего, бокового и нижне го зубцов чашечки (мм). Качественные признаки включали: 1–3) типы опушения на стебле, ли стьях и чашечке; 4–17) присутствие/отсутствие разных типов волосков (коротких прижатых, ко ротких курчавых, длинных прижатых, длинных курчавых и длинных отстоящих) на стебле, ли стьях и чашечке. Статистический анализ морфологических данных проводили методами одномерной стати стики в программе STATISTICA 7.1 for Windows (StatSoft Inc., 2006) и методом главных коорди нат (МГК) в программе Past3.15 (Hammer et al., 2001). В качестве одномерных методов сравне ния средних у двух видов по количественным признакам использовали тесты Стьюдента (для нормально распределенных признаков) и Ман на-Уитни (для признаков с распределением, от клоняющимся от нормального). При сравнении двух географических популяций (крымской и кавказской) в пределах каждого вида применяли тест Манна-Уитни ввиду малого объема одной из сравниваемых выборок. В МГК использовалась метрика Гоуэра (Gow er, 1971), позволяющая проводить ординацию по качественным и количественным признакам. Полученные в результате анализа координат ные оси отражают расстояния между образца ми. В анализ включены 33 образца (17 образцов L. herbaceus и 16 образцов L. graecus). Два об разца L. herbaceus, So4 и BL2, были исключены из-за отсутствующих значений некоторых при знаков. Анализ проводился в двух вариантах: 1) по 22 количественным и 6 качественным при знакам, 2) только по 22 количественным призна кам. Выделение, амплификация и секвенирова ние ДНК. Выделение геномной ДНК проводили с гербарных образцов набором NucleoSpin Plant II (Macherey-Nagel, Германия) согласно прото колу. Для молекулярно-филогенетического ана лиза были отобраны два участка ДНК: ITS1-2 ядерной рибосомальной ДНК и trnL-F спейсер и trnL интрон пластидной ДНК. Материалы и методы Подготовка к секвенированию включала в себя измерение концентрации очищенного амплификата с помо щью NanoPhotometer N60 (Implen) с последую щим разбавлением образца до концентрации 20 pmol/μl. На одну реакцию добавляли 3,2 pmol праймера и около 20 нг ДНК. Секвенировали прямые и обратные последовательности. Секве нирование производилось по Сэнгеру в лабора ториях ЦКП «Геном», ЗАО «Синтол» и кабинета методов молекулярной диагностики ИПЭЭ РАН. Полученные последовательности выравнивали с последовательностями из Генбанка, матрицу по ITS автоматически в программе MAFFT version 7.215 (Katoh et al., 2002; Katoh, Standley, 2013), а матрицу trnL-F – автоматически и вручную в программе BioEdit (Hall, 1999), ввиду наличия длинных инделей (Kramina et al., 2018). Поли морфизм ITS внутри образца определяли мето дом прямого секвенирования (без клонирования) по наличию двойных пиков на хроматограмме, проявляющихся при прямом и обратном прочте нии, и при высоте минорного пика не менее 20 % от главного. Число гапло- и риботипов оценива ли в программе DnaSP 6 (Rozas et al., 2017). При подсчете риботипов учитывали только главный сигнал (мажорный пик). ITS3 (White et al., 1990); для trnL-F – праймеры c, d, e и f (Taberlet et al., 1991). Полимеразные цепные реакции (ПЦР) проводились на ампли фикаторе T100TM Thermal Cycler (Bio-Rad) в реакционном объеме 20 μl, содержащем 4 μl го тового ПЦР-микса MasDDTaqMIX (200 μM каж дого dNTP, 1,5 mM MgCl2, 1,5 U SmarTaqDNA полимеразы и реакционный буфер (Диалат Лтд., Москва)), 15 μl деионизированной воды, 3,2 pmol каждого праймера и 1 μl матрицы ДНК не известной концентрации. ПЦР проводился по программе: предварительная денатурация 94 °С, 3:00, 30 циклов (денатурация 94 °С, 0:30, отжиг праймеров 57 °С, 0:40, элонгация 72 °С, 1:00), финальная денатурация 94 °С, 0:30, финальный отжиг праймеров 57 °С, 0:40, финальная элон гация 72 °С, 3:00. Электрофорез продуктов ПЦР проводился в 1,2 % агарозном геле 20 минут при мощности 120В. Очистка амплификата осу ществлялась колоночным способом с помощью набора Cleanup Mini (Евроген ЗАО). Подготовка к секвенированию включала в себя измерение концентрации очищенного амплификата с помо щью NanoPhotometer N60 (Implen) с последую щим разбавлением образца до концентрации 20 pmol/μl. На одну реакцию добавляли 3,2 pmol праймера и около 20 нг ДНК. Секвенировали прямые и обратные последовательности. Секве нирование производилось по Сэнгеру в лабора ториях ЦКП «Геном», ЗАО «Синтол» и кабинета методов молекулярной диагностики ИПЭЭ РАН. Материалы и методы Исследованный материал. Основным мате риалом для морфологического и молекулярного анализа служили собственные гербарные сборы 80 образцов представителей секции Dorycnium с семи точек на п-ове Крым (2015–2017 гг.) и пяти точек на Кавказе (2016–2017 гг.) (см. табл. 1 в приложении на сайте журнала; рис. 1). По сек циям Dorycnium и Bonjeanea изучен гербарный материал из ряда отечественных и зарубежных гербариев (ANK, ISTE, GAZI, H, MA, MHA, MW, LE, YALT). При проведении молекуляр Рис. 1. Места сбора образцов L. herbaceus и L. graecus в Крыму и на Кавказе. Обозначения популяций см. в табл. 1 в приложении на сайте журнала. Рис. 1. Места сбора образцов L. herbaceus и L. graecus в Крыму и на Кавказе. Обозначения популяций см. в табл. 1 в приложении на сайте журнала. собственных наблюдений. По количественным признакам проводили по три измерения каждо го признака на растении и вычисляли среднее значение. Количественные признаки включали: 1) длину трубки чашечки (мм); 2) длину цветка (мм); 3) длину цветоножки (мм); 4) число цвет собственных наблюдений. По количественным признакам проводили по три измерения каждо го признака на растении и вычисляли среднее значение. Количественные признаки включали: 1) длину трубки чашечки (мм); 2) длину цветка (мм); 3) длину цветоножки (мм); 4) число цвет Морфологический анализ. Морфометрия проводилась на выборке из 35 образцов растений (19 образцов L. herbaceus и 16 образцов L. grae cus) в состоянии цветения по 22 количественным и 17 качественным признакам. Отбор признаков проводился на основе литературных данных и 92 Лысова М. В. и др. О морфологии и филогении Dorycnium Крыма и Кавказа ITS3 (White et al., 1990); для trnL-F – праймеры c, d, e и f (Taberlet et al., 1991). Полимеразные цепные реакции (ПЦР) проводились на ампли фикаторе T100TM Thermal Cycler (Bio-Rad) в реакционном объеме 20 μl, содержащем 4 μl го тового ПЦР-микса MasDDTaqMIX (200 μM каж дого dNTP, 1,5 mM MgCl2, 1,5 U SmarTaqDNA полимеразы и реакционный буфер (Диалат Лтд., Москва)), 15 μl деионизированной воды, 3,2 pmol каждого праймера и 1 μl матрицы ДНК не известной концентрации. ПЦР проводился по программе: предварительная денатурация 94 °С, 3:00, 30 циклов (денатурация 94 °С, 0:30, отжиг праймеров 57 °С, 0:40, элонгация 72 °С, 1:00), финальная денатурация 94 °С, 0:30, финальный отжиг праймеров 57 °С, 0:40, финальная элон гация 72 °С, 3:00. Электрофорез продуктов ПЦР проводился в 1,2 % агарозном геле 20 минут при мощности 120В. Очистка амплификата осу ществлялась колоночным способом с помощью набора Cleanup Mini (Евроген ЗАО). Материалы и методы Для амплифи кации этих участков использовались прайме ры с известными последовательностями: для ITS1-2 – NNC-18S10 и С26А (Wen and Zimmer, 1996), а также универсальные праймеры ITS2 и Молекулярно-филогенетический анализ проводили отдельно по ITS1-5,8S-ITS2 и trnL-F в программе MEGAX (Kumar et al., 2008) мето дом максимального правдоподобия (ML). Мо дель эволюции выбирали на основе информаци онного критерия AICc. В обоих анализах (ITS и Turczaninowia 22 (4): 87–103 (2019) 93 trnL-F) была отобрана модель GTR + G (General Time Reversible model – частоты оснований не равны, 6 скоростей замен; G – параметр гамма- распределения, показывающий варьирование скоростей замен между сайтами). При постро ении филогенетических деревьев использовали метод bootstrap с числом шагов 500. Анализ по ITS включал 111 индивидуальных последова тельностей, по trnL-F – 87 индивидуальных по следовательностей (табл. 1). В качестве внешних групп в обоих анализах использовались предста вители ближайших к роду Lotus родов: Cytisopsis pseudocytisus (Boiss.) Fertig, Hammatolobium kre merianum (Coss.) K. Müll. и Tripodion tetraphyl lum (L.) Fourr. trnL-F) была отобрана модель GTR + G (General Time Reversible model – частоты оснований не равны, 6 скоростей замен; G – параметр гамма- распределения, показывающий варьирование скоростей замен между сайтами). При постро ении филогенетических деревьев использовали метод bootstrap с числом шагов 500. Анализ по ITS включал 111 индивидуальных последова тельностей, по trnL-F – 87 индивидуальных по следовательностей (табл. 1). В качестве внешних групп в обоих анализах использовались предста вители ближайших к роду Lotus родов: Cytisopsis pseudocytisus (Boiss.) Fertig, Hammatolobium kre merianum (Coss.) K. Müll. и Tripodion tetraphyl lum (L.) Fourr. Сравнение двух географических выборок L. herbaceus из Крыма и Кавказа не выявило до стоверных различий между ними по всем при знакам, кроме ширины бокового зубца чашеч ки, которая была несколько больше (p < 0,05) в крымской популяции. При сопоставлении двух выборок L. graecus было выявлено больше до стоверных (p < 0,01–0,05) отличий между гео графическими популяциями. Так, выборка с горы Кастель (Крым) отличалась по характеру опушения стебля (присутствовали только длин ные курчавые волоски и отсутствовали короткие курчавые и длинные отстоящие волоски, харак терные для выборок с Кавказа). Также крымская выборка характеризовалась более мелкими сред ними листьями и более широким боковым зуб цом чашечки, чем кавказские популяции. Результаты Ординация методом МГК по комплексу ка чественных и количественных признаков так же выявила четкие отличия между двумя ис следованными видами (рис. 2А). Все образцы L. herbaceus образовали единый кластер точек, в котором растения из Крыма и Кавказа пере мешаны и неразделимы. Образцы L. graecus сгруппировались в два отдельных кластера, со ответствующих выборкам из Кавказа и Крыма и хорошо обособленных как друг от друга, так и от кластера L. herbaceus. Анализ только по количественным данным выявил сходный ха рактер кластеризации, но расстояния между вы борками L. graecus с Кавказа и из Крыма были заметно меньше, что говорит о большом вкладе качественных признаков характера опушения в различия между географическими популяциями этого вида (рис. 2Б). Морфологический анализ. Тесты Манна- Уитни и Стьюдента по количественным данным показали достоверные отличия между выбор ками двух видов (L. graecus и L. herbaceus) по большинству признаков. Выявлен и ряд каче ственных отличий. Основные отличительные особенности двух видов на территории Крыма и Кавказа обобщены в таблице 2. Lotus graecus ха рактеризуется более крупными размерами цвет ков и листочков всех листьев и более длинными зубцами чашечки, а также характером опушения стеблей и листьев (рис. 5). Некоторые призна ки проявили сходный характер изменчивости у двух изученных видов, в частности: длина труб ки чашечки (около 1,5 мм) и типы ее опушения (наличие коротких (до 0,5 мм) прижатых и длин ных (0,5–1,7 мм) отстоящих волосков). Рис. 2. Результаты анализа МГК крымских и кавказских образцов L. herbaceus и L. graecus по морфологиче ским данным. В скобках по координатным осям указана доля объясненной дисперсии. А. Анализ по количе ственным и качественным признакам. Б. Анализ только по количественным признакам. Символы образцов — как на рис. 1, названия образцов см. в табл. 1 в приложении. Рис. 2. Результаты анализа МГК крымских и кавказских образцов L. herbaceus и L. graecus по морфологиче ским данным. В скобках по координатным осям указана доля объясненной дисперсии. А. Анализ по количе ственным и качественным признакам. Б. Анализ только по количественным признакам. Символы образцов — как на рис. 1, названия образцов см. в табл. 1 в приложении. 94 Лысова М. В. и др. Лысова М. В. и др. О морфологии и филогении Dorycnium Крыма и Кавказа Таблица 2 Отличительные признаки Lotus graecus и L. herbaceus в Крыму и на Кавказе L. graecus (N = 16) L. Результаты herbaceus (N = 19) Достоверность отличий (по p-значению) Тест Стьюдента Тест Манна-Уитни Длина цветка, мм 5,7 ± 0,2 (4,4–8,2) 3,7 ± 0,08 (3,1–4,4) * Длина листочка среднего листа, мм 24,1 ± 1,5 (16–37) 13,8 ± 0,5 (10–17) * Ширина листочка среднего листа, мм 11,3 ± 1 (7,7–19,4) 4,5 ± 0,2 (3,4–6,4) * Длина листочка верхнего листа, мм 15,3 ± 0,9 (11–26) 9,9 ± 0,3 (8–12) * Ширина листочка верхнего листа, мм 6,4 ± 0,6 (4,5–14,7) 2,7 ± 0,4 (2,1–3,5) * Длина зубцов ча шечки, мм (усред нено для верхнего, бокового и нижнего зубцов) 1,7 ± 0,07 (1,2–2,3) 0,7 ± 0,03 (0,4–1) * Ширина бокового зубца чашечки, мм 0,5 ± 0,02 (0,35–0,75) 0,4 ± 0,02 (0,3–0,55) * Волоски на стебле, длина (мм) и тип Короткие (0,1–0,3) курчавые. Длинные (0,7–2) отстоящие Короткие (0,1–0,3) прижатые. Длинные (0,8–1,5) отстоящие (есть/нет) Длина коротких волосков – Длина длинных волосков – (отличия только в типе коротких во лосков) Волоски на листьях, длина (мм) и тип Коротких нет. Длинные (0,9–1,6), прижатые и от стоящие Короткие (0,1–0,5) прижатые (есть/нет). Длинные (0,8–1,5) отстоящие Длина длинных волосков * Волоски на чашеч ке, длина (мм) и тип Длинные (0,7–1,7) отстоящие. Короткие (0,25– 0,5) прижатые Длинные (0,5–1,3) отстоящие. Короткие (0,1–0,4) прижатые Длина коротких волосков * Длина длинных волосков * Корневые отпрыски есть нет Табл Отличительные признаки Lotus graecus и L. herbaceus в Крыму и на Кавказе Примеч.: для количественных признаков приведены средняя арифметическая ± ошибка средней (в верхней строке) и минимальное, и максимальное значения (в нижней строке). Для длин волосков указан только размах изменчивости. Достоверность отличий средних в выборках двух видов по количественным признакам (слева – по критерию Стьюдента (для нормально распределенных признаков), справа – по критерию Манна-Уитни (для признаков с распределением, отклоняющимся от нормального)): отличия недостоверны «–», отличия до стоверны на уровне: * (p < 0,05), (p < 0,01), * (p < 0,001). Для качественных признаков (тип опушения, наличие корневых отпрысков) достоверность отличий не оценивалась. 230; 5,8S рРНК – 394; ITS2 – 420. У L. herbaceus полиморфизм выявлен в восьми сайтах, соот ветствующих позициям референсного сиквенса L. herbaceus D6 (Генбанк № KT250882): ITS1 – 74, 183, 191, 208; ITS2 – 444, 526, 587, 590. Ни один из сайтов полиморфизма L. herbaceus не со впал с сайтами полиморфизма у L. graecus. Генетический анализ. Число полученных для каждой выборки и каждого маркера инди видуальных и уникальных последовательностей представлено в табл. 3. Таблица 3 Таблица 3 Число нуклеотидных последовательностей, полученных для крымских и кавказских популяций Lotus graecus и L. herbaceus trnL-F ITS Lotus graecus Кавказ Крым Кавказ Крым Число последовательностей 4 2 11 4 Число гаплотипов/риботипов1 3 2 5 3 Lotus herbaceus Кавказ Крым Кавказ Крым Число последовательностей 3 4 4 17 Число гаплотипов/риботипов1 3 3 1 1 Примеч.: 1 – для ITS только по главному сигналу (без учета полиморфизма внутри образца). Таблица 3 Число нуклеотидных последовательностей, полученных для крымских и кавказских популяций Lotus graecus и L. herbaceus trnL-F ITS Lotus graecus Кавказ Крым Кавказ Крым Число последовательностей 4 2 11 4 Число гаплотипов/риботипов1 3 2 5 3 Lotus herbaceus Кавказ Крым Кавказ Крым Число последовательностей 3 4 4 17 Число гаплотипов/риботипов1 3 3 1 1 Примеч.: 1 – для ITS только по главному сигналу (без учета полиморфизма внутри образца). Число нуклеотидных последовательностей, полученных для крымских и кавказских популяций Lotus graecus и L. herbaceus Анализ методом ML по маркеру ITS (рис. 3) показал обособление южной клады рода Lo tus (бутстреп поддержка (БП) 79), в которую не вошел лишь L. glinoides (секция Chamaelotus Kramina et D. D. Sokoloff). Все члены северной эволюционной ветви Lotus сгруппировались в несколько клад, среди которых: большая клада секции Lotus (p.max.p. – БП 86), включающая большинство видов; клада секции Lotus груп пы L. parviflorus (БП 100); клада секции Bon jeanea с двумя видами (L. rectus и L. strictus) с низкой поддержкой (БП 61) и две клады секции Dorycnium, одна из которых включала также L. hirsutus из секции Bonjeanea. Первая из этих клад высоко поддержана (БП 98) и включает две субклады: а) все члены комплекса L. dorycnium (= D. pentaphyllum) и L. fulgurans, б) образ цы L. hirsutus. Вторая клада (БП 81) включает L. graecus и близкие к нему турецкие эндемики L. axilliflorus и L. sanguineus. Все изученные об разцы L. herbaceus из Крыма и Кавказа попали в общую кладу (хоть и неподдержанную) вместе с образцами L. herbaceus из Австрии и Греции. Все они надежно группируются с другими пред ставителями комплекса L. dorycnium. Все образ цы L. graecus, независимо от их географического происхождения (Крым, Кавказ, Турция, Греция), образовали общую высоко поддержанную кладу (БП 94). Анализ участка trnL-F методом ML (рис. 4) выявил южную и северную ветви рода Lotus, хоть и низко поддержанные. В состав южной клады входят те же представители, что и в анали зе ITS, но к ним добавился L. glinoides. Результаты У обоих видов в пределах индивидуальных образцов был отмечен полиморфизм последова тельностей ITS. У L. graecus полиморфизм был отмечен для семи сайтов, соответствующих по зициям референсного сиквенса L. graecus D9 (Генбанк № KT250876): ITS1 – 50, 105, 192, 193, Длина использованного в филогенетиче ском анализе обобщенного выравнивания ранее опубликованных и впервые представляемых Turczaninowia 22 (4): 87–103 (2019) 95 щенного выравнивания trnL-F составила 1021 п.о. (1–845 позиции сиквенса Lotus fulgurans 937 (= D. pentaphyllum subsp. fulgurans), Генбанк № MF314954), включая 122 вариабельных сайта и 56 парсимонно-информативных признаков. последовательностей ITS составила, соответ ственно: 668 п.о., и соответствовала 1–601 по зициям референсного сиквенса Lotus herbaceus D6 (Генбанк № KT250882). На данном участке было отмечено 272 вариабельных сайта, из них 204 – парсимонно-информативных. Длина обоб Таблица 3 О морфологии и филогении Dorycnium Крыма и Кавказа 2012), и межвидовых гибридов Dorycnium (в на шем понимании, секции Dorycnium рода Lotus) (Conesa et al., 2010). Большинство видов рода Lotus, напротив, имеет ITS последовательно сти, лишенные полиморфизмов внутри образца (Kramina et al., 2016). 2012), и межвидовых гибридов Dorycnium (в на шем понимании, секции Dorycnium рода Lotus) (Conesa et al., 2010). Большинство видов рода Lotus, напротив, имеет ITS последовательно сти, лишенные полиморфизмов внутри образца (Kramina et al., 2016). Учитывая последние обстоятельства, мы про вели дополнительный анализ литературных дан ных (Ball, 1968; Demiriz, 1970) и проанализиро вали более 200 гербарных образцов комплекса L. dorycnium и около 50 образцов вида L. hirsutus из гербарных коллекций ANK, GAZI, H, ISTE, LE, MHA, MW и MA, из чего следует, что, не смотря на некоторые черты сходства (короткие бобы с незакручивающимися при вскрывании створками, короткий рахис листа), различия между комплексом L. dorycnium и видом L. hir sutus по морфологии очень велики (см. рис. 5–6): размеры цветков (3–6 мм у L. dorycnium против 9–15 мм у L. hirsutus), характер лепестков (с по перечными мешковидными выростами у L. do rycnium и без таковых у L. hirsutus), размеры плодов (3–5 × 1,5–3 мм у L. dorycnium и 6–12 × 2–4 мм у L. hirsutus), число семян в плоде (обыч но 1 у L. dorycnium и 2–6 у L. hirsutus), длина и форма зубцов чашечки (короткие треугольные у L. dorycnium и длинные ланцетно-шиловидные у L. hirsutus), характер опушения (полуотстоящее или прижатое у L. dorycnium и сильноотстоящее у L. hirsutus). Гибридных образцов между этими видами по морфологическим признакам до сих пор выявлено не было. Полностью различные сайты полиморфизма ITS у видов L. graecus и L. herbaceus позволя ют предположить, что их возможное гибридное происхождение не связано. Однако изученных материалов пока недостаточно для обоснован ных предположений о родительских таксонах, которые могли участвовать в этих событиях ги бридизации. Возможны и другие причины полиморфизма в ITS. Так, для пионов лишь часть полиморфизмов поддерживала гипотезу о гибридном происхож дении от определенных видов, тогда как другая часть характеризовалась присутствием дополни тельных нуклеотидов и могла быть следствием внутривидовой генетической дифференциации (Efimov et al., 2018). Молекулярно-филогенетический анализ по ITS1-5,8S-ITS2 и trnL-F подтвердил основные направления филогении рода Lotus (включая Dorycnium и Tetragonolobus), полученные ранее (Degtjareva et al., 2006; Kramina et al., 2016). Таблица 3 Ре зультаты проведенного исследования поддержи вают выводы об отсутствии соответствия меж ду разделением бывших представителей рода Dorycnium на секции Dorycnium и Bonjeanea и молекулярно-генетическими данными (Kramina et al., 2016). Настоящее исследование поддер живает представления о том, что виды секции Bonjeanea (L. strictus, L. rectus и L. hirsutus) не являются монофилетической группой, а послед ний из них как по ядерным, так и по пластидным данным сближается скорее с видами секции Do rycnium, чем с видами своей секции (Degtjareva et al., 2006; Kramina et al., 2016). Филогенети ческие отношения внутри клады родства Lotus dorycnium разрешены лишь частично. Таблица 3 Северная ветвь дифференцирована нечетко, но включает ряд достаточно высоко поддержанных клад, та ких как: комплекс L. corniculatus (БП 98), группа родства L. conimbricensis (БП 87), группа родства L. graecus (БП 82), включающая помимо этого вида также L. axilliflorus и L. sanguineus, а также отдельные виды: L. parviflorus (БП 86), L. strictus (БП 95), L. rectus (БП 91). Выделение комплекса L. dorycnium секции Dorycnium не поддержано, последовательности представителей этого ком плекса (включая последовательности образцов L. herbaceus из Крыма и Кавказа) перемешаны с последовательностями L. hirsutus секции Bon jeanea, что не согласуется с морфологическими данными. L. hirsutus имеет обширный ареал, охватывающий Португалию и почти все страны Средиземноморского региона, за исключением Египта, Туниса и Марокко, и не доходящий до Крыма и Кавказа. 96 Лысова М. В. и др. О морфологии и филогении Dorycnium Крыма и Кавказа . Результаты филогенетического анализа рода Lotus (вкл. Dorycnium и Tetragonolobus) мето ку ITS1-5,8S-ITS2 ядерной рДНК с расширенной представленностью крымских и кавказских секции Dorycnium. Обозначение образцов – см. табл. 1. Приведены только значения бутстре лов, превышающие 50. Цветными значками выделены образцы видов: Lotus herbaceus (красн вказа, красный пустой квадрат – из Крыма), L. graecus (синий кружок – с Кавказа, синий пус рыма), L. hirsutus (зеленый кружок). Рис. 3. Результаты филогенетического анализа рода Lotus (вкл. Dorycnium и Tetragonolobus) методом ML по участку ITS1-5,8S-ITS2 ядерной рДНК с расширенной представленностью крымских и кавказских представи телей секции Dorycnium. Обозначение образцов – см. табл. 1. Приведены только значения бутстреп-поддерж ки узлов, превышающие 50. Цветными значками выделены образцы видов: Lotus herbaceus (красный кружок – с Кавказа, красный пустой квадрат – из Крыма), L. graecus (синий кружок – с Кавказа, синий пустой квадрат – из Крыма), L. hirsutus (зеленый кружок). Turczaninowia 22 (4): 87–103 (2019) 97 езультаты филогенетического анализа рода Lotus (вкл. Dorycnium и Tetragonolobus) методо rnL-F пластидной ДНК с расширенной представленностью крымских и кавказских предст Dorycnium. Обозначение образцов – см. табл. 1 в приложении. Приведены только значения б ки узлов, превышающие 50. Выделение образцов Lotus herbaceus, L. graecus и L. hirsutus Рис. 4. Результаты филогенетического анализа рода Lotus (вкл. Dorycnium и Tetragonolobus) методом ML по участку trnL-F пластидной ДНК с расширенной представленностью крымских и кавказских представителей секции Dorycnium. Обозначение образцов – см. табл. 1 в приложении. Приведены только значения бутстреп- поддержки узлов, превышающие 50. Выделение образцов Lotus herbaceus, L. graecus и L. hirsutus – как на рис. 4. 98 Лысова М. В. и др. Обсуждение Изучение отдельных морфологических при знаков и анализ МГК по морфологическим дан ным показали, что виды L. graecus и L. herbaceus хорошо различимы, морфологически переход ных образцов выявлено не было даже в местах совместного произрастания двух видов. При этом образцы из Кавказа и Крыма в пределах L. herbaceus не обособляются, а в пределах L. graecus можно различить крымскую и кавказ скую популяции. Выявление достаточно большого числа (7–8) полиморфных сайтов в последовательностях ITS обоих изученных видов может свидетельство вать о том, что оба таксона могли произойти в результате сравнительно недавней гибридиза ции, после которой ITS еще не успел стабилизи роваться. Подобные примеры описаны для пред ставителей разных семейств цветковых (Hřibová et al., 2011; Peruzzi et al., 2012; Punina et al., 2012; Hodač et al., 2014; Efimov et al., 2018). Полимор физм в ITS также был продемонстрирован и для некоторых видов Lotus, возникших в результате гибридизации (Sandral et al., 2010; Kramina et al., Как было ранее установлено в анализе спей сера psbA-trnH и интрона rps16 (Kramina et al., 2016), а в настоящем исследовании подтверж дено на примере участка trnL-F, по последова тельностям пластидной ДНК виды секций Lotus, Dorycnium и Bonjeanea образуют отдельную, «северную», ветвь рода Lotus, эволюционная судьба которой, по-видимому, включала события сетчатой эволюции, как отдаленной, так и совре менной, и требует дальнейшего изучения. Turczaninowia 22 (4): 87–103 (2019) 99 . Образцы Lotus graecus L. и L. herbaceus (Vill.) Jauzein. А, В – L. graecus, So5, «Краснодарский кра Сочи, Сочинский дендрарий, 43°34′ с. ш. 39°45′ в. д. 01 VI 2017. М. Кутурова s. n.» (MW); Б, Г – L. herba «Крым, 500 м от с. Верхнесадового, 44°41′ с. ш. 33°41′ в. д. 29 V 2015. П. В. Карпунина s. n.» (MW); А й вид гербарного образца (фото С. Р. Майорова), В, Г – участки побегов с соцветиями (фото Т. Е. Крами – внешний вид цветущих растений: Д – L. graecus, «Турция, Стамбул, окр. Синекли»; Е – L. herba ия, Сочи, между ж/д станциями Солоники и Лазаревская» (Д–Е, фото М. В. Лысовой). Рис. 5. Образцы Lotus graecus L. и L. herbaceus (Vill.) Jauzein. А, В – L. graecus, So5, «Краснодарский край, ку рорт Сочи, Сочинский дендрарий, 43°34′ с. ш. 39°45′ в. д. 01 VI 2017. М. Кутурова s. n.» (MW); Б, Г – L. herbaceus, VS1, «Крым, 500 м от с. Верхнесадового, 44°41′ с. ш. 33°41′ в. д. 29 V 2015. П. В. Карпунина s. Обсуждение n.» (MW); А, Б – общий вид гербарного образца (фото С. Р. Майорова), В, Г – участки побегов с соцветиями (фото Т. Е. Краминой); Д, Е – внешний вид цветущих растений: Д – L. graecus, «Турция, Стамбул, окр. Синекли»; Е – L. herbaceus, «Россия, Сочи, между ж/д станциями Солоники и Лазаревская» (Д–Е, фото М. В. Лысовой). 100 Лысова М. В. и др. Лысова М. В. и др. О морфологии и филогении Dorycnium Крыма и Кавказа др О морфологии и филогении Dorycnium Крыма и Кавказа р О морфологии и филогении Dorycnium Крыма и Кавказа Рис. 6. Гербарные образцы L. hirsutus L. A – “Croatia, Istria, Pula. 31 V 2010. J. Calvo and A. Uníó JC4730” (MA), побеги с соцветиями; Б – “Italia, Sicilia, Ragusa. 09 VI 2000. Álvarez et al. IA1784” (MA), образец HIRS2, плоды (фото Т. Е. Краминой). А Б Б А Рис. 6. Гербарные образцы L. hirsutus L. A – “Croatia, Istria, Pula. 31 V 2010. J. Calvo and A. Uníó JC4730” (MA), побеги с соцветиями; Б – “Italia, Sicilia, Ragusa. 09 VI 2000. Álvarez et al. IA1784” (MA), образец HIRS2, плоды (фото Т. Е. Краминой). Филогенетический анализ по четырем пла стидным маркерам (psbA-trnH, rps16 intron, trnL intron, trnL-F), проведенный для выборки 65 ви дов рода Lotus (включая Dorycnium), подтвердил обособление общей высоко поддержанной кла ды, в которой перемешаны образцы комплекса L. dorycnium и вида L. hirsutus (Kramina et al., 2019). Нам представляется маловероятным объ яснение близких филогенетических связей меж ду членами комплекса L. dorycnium, с одной сто роны, и L. hirsutus из секции Bonjeanea, с другой, современной гибридизацией этих двух таксонов. Потенциально гибридизация возможна, учиты вая пересекающиеся ареалы и одинаковое чис ло хромосом у этих таксонов, 2n = 14 (Goldblatt, Johnson, 1979), но гибридных образцов по мор фологии, как уже сказано, до сих пор выявлено не было. Возможно, мы имеем дело со скрытой гибридизацией (гибрид морфологически неот личим или слабо отличим от одного из родите лей) или с остатками древнего полиморфизма (т. е. неполной сортировкой линий), что требует более глубокого изучения с привлечением ту рецкого материала, т. к. именно Турция является центром разнообразия группы, трактуемой как род Dorycnium. вать весь комплекс L. dorycnium (= D. pentaphyl lum), то по данным ITS прослеживается тенден ция к географической дифференциации. Обсуждение В част ности, выделяются клада западноевропейских образцов (D2 из Португалии, D3 и PENT1 из Ис пании, D7 и D8 из Франции) и клада централь но-европейских и балканских образцов (D1 из Словении, D4 из Германии, D5 из Черногории). Изучение этой тенденции может стать целью бу дущих исследований, которые потребуют более репрезентативных выборок со всех частей ареа ла комплекса L. dorycnium. Благодарности Мы благодарим кураторов гербариев ANK, ISTE, GAZI, H, MA, MHA, MW, LE, YALT за предоставление возможности работать с матери алом по роду Lotus s. l., О. В. Юрцеву, П. В. Кар пунину, К. И. Фомичева и А. О. Виричеву за по мощь в сборе материала. Выражаем искреннюю признательность Д. Д. Соколову за ценные заме чания и советы и С. Р. Майорову и А. Д. Миль ко за помощь в подготовке иллюстраций. Моле кулярно-филогенетическая часть исследования выполнена при поддержке гранта РФФИ № 19- 04-00883, сборы материала в 2016–2017 гг. про ведены при поддержке гранта РНФ «Научные основы создания национального банка-депози тария живых систем» (грант № 14-50-00029). Мы предпочитаем использовать для предста вителей комплекса L. dorycnium из крымского и кавказского регионов название L. herbaceus как основанное на более раннем базиониме (D. her baceum Vill.), чем D. intermedium Ledeb., но счи таем, что для более обоснованного решения не обходимо изучить типовые материалы Вилларса и Ледебура по этим и близким видам, а также пределы варьирования видов этой группы на всем ареале. Выводы Морфологический и молекулярно-филогене тический анализ (с использованием маркеров ITS1-5,8S-ITS2 ядерной рибосомальной ДНК и trnL-F пластидной ДНК) крымских и кавказ ских выборок секции Dorycnium рода Lotus под твердил присутствие на этих территориях двух видов: L. graecus L. и L. herbaceus (Vill.) Jauzein (представителя комплекса L. dorycnium L.). L. graecus по сравнению с L. herbaceus харак теризуется более крупными размерами цветков (около 5,5 мм против 3,5 мм) и листочков всех листьев (15–24 × 6–11 мм против 10–14 × 3–5 мм), более длинными зубцами чашечки (1,2–2 мм против 0,4–1 мм), а также отличается харак тером опушения и наличием корневых отпры сков. Проведенное нами исследование показа ло, что нуклеотидные последовательности как L. graecus, так и L. herbaceus из Крыма и Кавказа не образуют отдельных, свойственных конкрет ному региону клад. Видимо, эти виды заселили данные территории, находящиеся на крайнем северо-восточном пределе их ареалов, сравни тельно недавно и географически разделенные популяции не приобрели существенных отличий в последовательностях. Однако, если рассматри В пределах каждого вида географические по пуляции из Крыма и Кавказа представлены сход ными последовательностями обоих изученных маркеров (ITS и trnL-F). 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https://openalex.org/W4389509218	https://link.springer.com/content/pdf/10.1007/s41020-023-00203-1.pdf	English	null	Interpreters as cultural experts? Asylum practices in Slovakia	Jindal global law review	2,023	cc-by	10,077	Jindal Global Law Review (2023) 14:241–257 https://doi.org/10.1007/s41020-023-00203-1 Jindal Global Law Review (2023) 14:241–257 https://doi.org/10.1007/s41020-023-00203-1 ARTICLE ARTICLE Helena Tužinská helena.tuzinska@uniba.sk Interpreters as cultural experts? Asylum practices in Slovakia Helena Tužinská1 Accepted: 9 October 2023 / Published online: 9 December 2023 © The Author(s) 2023 Accepted: 9 October 2023 / Published online: 9 December 2023 © The Author(s) 2023 1 Comenius University in Bratislava, Bratislava, Slovakia Keywords Cultural expertise · Interpreting · Asylum · Impartiality · Training Abstract Cultural expertise can be a meaningful part of judicial proceedings, including asy lum appeals. In countries where it is not yet common, the need to address questions of cultural identity might be communicated indirectly through interpreters. As a result, interpreters implicitly and explicitly respond to co-producing the Other. This paper uses empirical data from long-term ethnographic research on interpreting asylum hearings to trace their institutional conditions in Slovakia and the actors’ approaches to the culturally sensitive aspects of interpretation in these proceed ings. The perspectives of asylum applicants, their legal representatives, interpreters and decision-making authorities are analysed through the concepts of sayability and interpretability—the relationship between representations of legal, cultural, and linguistic identities. This article discusses how the research participants’ norms of interpreting, awareness of positionality, and procedural neutrality influence the spo ken and written file narrative. Legal terms and administrative jargon are often not translated into plain language, accessible to laypeople. The often unacknowledged misinterpretation also affects the flow of the proceedings. Thus, systemic train ing of interpreters is essential, particularly public service interpreters who actively shape their informal role as cultural experts. At a time of increased demand for cross-cultural understanding, this article provides new perspectives on exploring the challenges for interpreters as well as other public service professions in relation to the notions ascribed to cultural identities. Keywords Cultural expertise · Interpreting · Asylum · Impartiality · Training 1 3 3 Jindal Global Law Review (2023) 14:241–257 242 1 Introduction The problem of cultural translation in social sciences and humanities is particularly important where members of minority cultures are ascribed unequal decision-making opportunities, which also applies to refugees. Interpreting for asylum applicants pres ents the challenge of considering differences in legal cultures, institutionally estab lished standards, and social and cultural identities. Every act of interpretation transcends language boundaries and is the key to under standing its social context. Thus, the analytical focus cannot remain only on activity in the courtroom and situational inequality because they are part of wider social and political inequalities.1 This paper describes how the asylum field actors conceptualise their habits and normativise routines. I focus on how the spoken becomes written and the written becomes the fact. Notably, the previous experiences with which partici pants enter the courtroom co-create the communicative scenes of all the cases. Cultural expertise, in the format of independent expert witnessing such as country experts and social scientists appointed as experts, is very rare in court proceedings in Slovakia. In the case of asylum proceedings, this gap is implicitly, and in some cases explicitly, filled by interpreters. My aim is to address the visibility of inter preters’ performative power in constituting the Other. I focus on the negotiation of meanings within the following areas: (1) language identification and the flexibility of criteria for appointing interpreters; (2) notions of impartiality and ethical proce dures; (3) room for exegesis of socio-legal terminology and culture-specific terms, all discussed in corresponding sections. By analysing ethnographic observations of multicultural encounters in Slovakia, I show where the challenges of interpreting and cultural expertise intersect. 1 See Diana Eades, Courtroom Talk and Neocolonial Control (Walter de Gruyter 2008) 37; Susan Berk- Seligson, The Bilingual Courtroom: Court Interpreters in the Judicial Process (The University of Chi cago Press 2002) 25; Nick Gill and Anthony Good (eds), Asylum Determination in Europe: Ethnographic Perspectives (Palgrave 2019) 196. 3 Patrik Szicherle and Jana Kazaz, Perception of Ukrainian Refugees in the V4 (Globsec 2022) 7. Miroslava Hlinčíková and Martina Sekulová (eds), Globe in Motion 2: Multiple Shades of Migration: Regional Perspectives (Institute of Ethnology and Social Anthropology SAS 2019). 1 See Diana Eades, Courtroom Talk and Neocolonial Control (Walter de Gruyter 2008) 37; Susan Berk- Seligson, The Bilingual Courtroom: Court Interpreters in the Judicial Process (The University of Chi cago Press 2002) 25; Nick Gill and Anthony Good (eds), Asylum Determination in Europe: Ethnographic Perspectives (Palgrave 2019) 196. 2 Miroslava Hlinčíková and Martina Sekulová (eds), Globe in Motion 2: Multiple Shades of Migration: Regional Perspectives (Institute of Ethnology and Social Anthropology SAS 2019) 2 Miroslava Hlinčíková and Martina Sekulová (eds), Globe in Motion 2: Multiple Shades of Migration: Regional Perspectives (Institute of Ethnology and Social Anthropology SAS 2019). 3 Patrik Szicherle and Jana Kazaz, Perception of Ukrainian Refugees in the V4 (Globsec 2022) 7. 1.1 Research site and methods The asylum field as a research site in the Central European region is less studied than the number of asylum applicants would warrant. Slovakia lies on the Schen gen borders. A common narrative of refugees representing not only security but also an economic threat2 is in contrast with volunteering individuals, public initiatives and civic organisations in the so-called refugee crisis back in 2015. People have responded even more receptively when encountering refugees after the Russian invasion of Ukraine in 2022. Sentiments arising from the so-called cultural proxim ity of Slovaks and Ukrainians as Slavic people have declined within half a year, as refugee-related issues became critical again after the energy crisis, rising debt, and falling incomes among voters.3 Slovakia, with a rich history both in emigration and 1 3 Jindal Global Law Review (2023) 14:241–257 243 immigration,4 now acting as a country of asylum, is still in its own transformation of asylum’s infrastructural support. The same applies to interpreting for asylum appli cants: innovations get systemically fostered at a very slow pace. In Slovakia, as well as in its neighbouring countries—Poland, the Czech Republic and Hungary—the atti tude towards refugees is more cautious than in Western Europe.5 The negative media and political discourse about foreigners originating from outside the European Union (EU) has also been reflected in the distorted assumption of citizens that there are ten times more foreigners in Slovakia than there are in reality.6i immigration,4 now acting as a country of asylum, is still in its own transformation of asylum’s infrastructural support. The same applies to interpreting for asylum appli cants: innovations get systemically fostered at a very slow pace. In Slovakia, as well as in its neighbouring countries—Poland, the Czech Republic and Hungary—the atti tude towards refugees is more cautious than in Western Europe.5 The negative media and political discourse about foreigners originating from outside the European Union (EU) has also been reflected in the distorted assumption of citizens that there are ten times more foreigners in Slovakia than there are in reality.6i The presented research findings from 2017–2019, focusing on communication during trials, have followed my participation in the asylum field in 2005–2008 and 2010–2011. These earlier encounters were related to my ethnographic research in detention and reception centres as well as other asylum proceedings-related activities such as coordination meetings and trainings. 4 Ján Botík, An Ethnic History of Slovakia: Multi-ethnicity, Minorities, and Migrations (Stimul 2021). 5 ‘Asylum Seekers and Beneficiaries of International Protection in V4 Countries (Updated Report)’ (ReliefWeb, 26 June 2019). https://reliefweb.int/report/czechia/asylum-seekers-and-beneficiaries-inter national-protection-v4-countries-updated-report. Accessed 16 June 2023. 6 ibid 1.1 Research site and methods The participant observation of court hearings, judges, ethnographic interviews with asylum applicants, their legal rep resentatives, representatives of state institutions—policepersons and decision-mak ers, both court and ad hoc interpreters, became an integral part of my research,7 all first-hand. i Due to the lower number of long-lasting cases in Slovakia, all parties would be identifiable; hence the extracts are without a file number and the names are fictitious. Being aware of the vulnerability of the actors, I respect their privacy and maintain their anonymity. Verbal informed consent was obtained prior to all the interviews and the objectives were communicated as part of academic research.8 Extracts interpreted below come from the hearings at the Regional Court in Bratislava and related inter views with different actors. The interviews were held in Slovak or in English, while English was not the native language of either of us.9 Asylum seekers cited in this study (from the Middle East and Asia) persisted in appealing their asylum refusals and were eventually granted international protection. The other actors, including the interpreters quoted here, are Slovak citizens. The trajectories of asylum applicants are diverse, the conditions in which they are interpreted vary, and the professional backgrounds of all the participants are also manifold. Also, the typical coordinates of official contact are not the same in all the states of the EU. In general, if foreign ers cross the state border without a valid travel document and are unable to prove the necessary amount to cover the costs of their stay in the Slovak Republic, the relevant police department will ask them to provide an explanation. The police have 4 Ján Botík, An Ethnic History of Slovakia: Multi-ethnicity, Minorities, and Migrations (Stimul 2021). 5 ‘Asylum Seekers and Beneficiaries of International Protection in V4 Countries (Updated Report)’ (ReliefWeb, 26 June 2019). https://reliefweb.int/report/czechia/asylum-seekers-and-beneficiaries-inter national-protection-v4-countries-updated-report. Accessed 16 June 2023. 6 ibid. 7 Research participants are given fictional names starting with a capital letter referring to their position. 8 ‘Ethical Codex’ (Ethnographic Society of Slovakia, 2017). http://www.nss.sav.sk/o-nas/stanovy/. Accessed 21 February 2023. 9 Selected parts of this text were translated into English by John Minahane and Pavol Šveda, with the exceptions of the English originals. The readers might notice stylistic incongruences as some views are presented by non-natives. 9 Selected parts of this text were translated into English by John Minahane and Pavol Šveda, with the exceptions of the English originals. 4 Ján Botík, An Ethnic History of Slovakia: Multi-ethnicity, Minorities, and Migrations (Stimul 2021). 5 ‘Asylum Seekers and Beneficiaries of International Protection in V4 Countries (Updated Report)’ (ReliefWeb, 26 June 2019). https://reliefweb.int/report/czechia/asylum-seekers-and-beneficiaries-inter national-protection-v4-countries-updated-report. Accessed 16 June 2023. 6 ibid. 7 Research participants are given fictional names starting with a capital letter referring to their position. 8 ‘Ethical Codex’ (Ethnographic Society of Slovakia, 2017). http://www.nss.sav.sk/o-nas/stanovy/. Accessed 21 February 2023. 9 Selected parts of this text were translated into English by John Minahane and Pavol Šveda, with the exceptions of the English originals. The readers might notice stylistic incongruences as some views are presented by non-natives. 9 Selected parts of this text were translated into English by John Minahane and Pavol Šveda, with the exceptions of the English originals. The readers might notice stylistic incongruences as some views are presented by non-natives. 7 Research participants are given fictional names starting with a capital letter referring to their position 8 ‘Ethical Codex’ (Ethnographic Society of Slovakia, 2017). http://www.nss.sav.sk/o-nas/stanovy Accessed 21 February 2023. 7 Research participants are given fictional names starting with a capital letter referring to their position 10 Regulation (EU) 603/2013 of 26 June 2013 on the establishment of Eurodac for the comparison of f gerprints for the effective application of Regulation (EU) 604/2013 [2013] OJ L180/1. 14 For the state administration the remuneration is fixed according to Decree No. 491/2004 Coll. on Fees and Compensation of Costs and Lost Time for Experts, Interpreters and Translators (Slovakia).The statu tory price (20 Eur per hour) for interpreting at courts and for the police represents approximately half of the market price for languages with fewer interpreters, and one- third less in the case of world languages. Also, as some places are not easily accessible, and there is no compensation for the loss of time spent on the road (often long), the price is inadequate compared to the interpreter’s commercial remuneration. See Markéta Štefková, ‘Legal Translation and Legal Interpreting–from the Demands of Practice to the Educational Models’ in Martin Djovčoš and Pavol Šveda (eds), Translation and Interpreting Training in Slovakia (Stimul 2021) 127. 11 Act No. 404/2011 Coll. on Residence of Foreigners (Slovakia). 12 See ‘Statistics’ (Ministry of Interior, Slovak Republic). https://www.minv.sk/?statistiky-20. Accessed 21 February 2023. 13 Act No. 404/2011 Coll. (n 11). 15 Act No. 480/2002 Coll. on Asylum (Slovakia). See also, ‘Asylum in Slovakia’ (United Nations High Commissioner of Refugees). https://help.unhcr.org/slovakia/asylum/. Accessed 16 June 2023; ‘Frequently Asked Questions’ (Human Rights League). https://www.hrl.sk/en/frequently-asked-questions. Accessed 16 June 2023. 1.1 Research site and methods The readers might notice stylistic incongruences as some views are presented by non-natives. 1 3 Jindal Global Law Review (2023) 14:241–257 244 the right to take their fingerprints for identification in the system Eurodac,10 carry out a personal search, to withdraw the travel document, and return it once the asylum procedure is completed. A short interview is held to identify the foreigner and their family members, the route of arrival to the Slovak Republic as well as the reasons for leaving the country of origin.11 During my research, most asylum seekers wished to join their families or Mid dle Eastern and Asian communities in Western Europe and, therefore, hesitated to apply for asylum in Slovakia. Migration policy in the Slovak Republic has long been restrictive, and the success rate is very low. In 2017, there were 29 granted asylums out of 166 applications; in 2018, only 5/178; and in 2019, it was 9/232; subsidiary protection was given twice as often, for one year only, and could be prolonged. How ever, most of the proceedings were discontinued.12 The time limit for administrative expulsion in the case of unauthorised entry is 48 hours.13 According to my findings, in need of interpretation into scarcer languages, an ad hoc volunteer from the group of foreigners might be asked to interpret instead of a qualified interpreter, registered by the Ministry of Justice as a court interpreter.14 If the deportation is not carried out in time, the foreign nationals are transferred to detention camps. In case they apply for asylum or subsidiary protection, they must do so at the police asylum department and proceed to the reception camp on the border with Ukraine for a longer interview and questionnaire. This is held by the Migration Office of the Ministry of Interior (MO), the decisive body on not/granting international protection. After three/four weeks in a reception camp, refugees are placed in accommodation centres. They might be invited for a second or third interview with more detailed questions. In case international protection is granted, MO and other organisations provide integration assistance.15 If asylum or subsidiary protection has not been granted or the person has been unjustifiably detained, asylum applicants may appeal to the regional administrative courts in Bratislava or Košice. A lawsuit can be submitted within thirty days from 14 For the state administration the remuneration is fixed according to Decree No. 491/2004 Coll. 10 Regulation (EU) 603/2013 of 26 June 2013 on the establishment of Eurodac for the comparison of fin gerprints for the effective application of Regulation (EU) 604/2013 [2013] OJ L180/1. 11 Act No. 404/2011 Coll. on Residence of Foreigners (Slovakia). 12 See ‘Statistics’ (Ministry of Interior, Slovak Republic). https://www.minv.sk/?statistiky-20. Accessed 21 February 2023. 13 Act No. 404/2011 Coll. (n 11). 14 For the state administration the remuneration is fixed according to Decree No. 491/2004 Coll. on Fees and Compensation of Costs and Lost Time for Experts, Interpreters and Translators (Slovakia).The statu tory price (20 Eur per hour) for interpreting at courts and for the police represents approximately half of the market price for languages with fewer interpreters, and one- third less in the case of world languages. Also, as some places are not easily accessible, and there is no compensation for the loss of time spent on the road (often long), the price is inadequate compared to the interpreter’s commercial remuneration. See Markéta Štefková, ‘Legal Translation and Legal Interpreting–from the Demands of Practice to the Educational Models’ in Martin Djovčoš and Pavol Šveda (eds), Translation and Interpreting Training in Slovakia (Stimul 2021) 127. 15 Act No. 480/2002 Coll. on Asylum (Slovakia). See also, ‘Asylum in Slovakia’ (United Nations High Commissioner of Refugees). https://help.unhcr.org/slovakia/asylum/. Accessed 16 June 2023; ‘Frequently Asked Questions’ (Human Rights League). https://www.hrl.sk/en/frequently-asked-questions. Accessed 16 June 2023. 1.1 Research site and methods on Fees and Compensation of Costs and Lost Time for Experts, Interpreters and Translators (Slovakia).The statu tory price (20 Eur per hour) for interpreting at courts and for the police represents approximately half of the market price for languages with fewer interpreters, and one- third less in the case of world languages. Also, as some places are not easily accessible, and there is no compensation for the loss of time spent on the road (often long), the price is inadequate compared to the interpreter’s commercial remuneration. See Markéta Štefková, ‘Legal Translation and Legal Interpreting–from the Demands of Practice to the Educational Models’ in Martin Djovčoš and Pavol Šveda (eds), Translation and Interpreting Training in Slovakia (Stimul 2021) 127. 15 Act No. 480/2002 Coll. on Asylum (Slovakia). See also, ‘Asylum in Slovakia’ (United Nations High Commissioner of Refugees). https://help.unhcr.org/slovakia/asylum/. Accessed 16 June 2023; ‘Frequently Asked Questions’ (Human Rights League). https://www.hrl.sk/en/frequently-asked-questions. Accessed 16 June 2023. 1 3 Jindal Global Law Review (2023) 14:241–257 245 delivery of the decision. The court reviews compliance of the decision with the law, the sufficiency of its justification and the correctness of the proceedings. The lawsuit can be rejected if the court finds it unjustified. In case the objections of an asylum applicant are found justified, the decision of MO is cancelled and returned for re- examination. The courts might also specify binding instructions for the new proceed ings at MO. If the regional administrative courts reject the lawsuit, asylum seekers may bring the complaint to the Supreme Administrative Court. If the objections are considered to be well-founded, the Supreme Administrative Court returns it for re- assessment to MO.16 However, due to misinterpretation, mistranslation and lengthy investigation many have been pending in the system for much longer than they had expected. My aim here is not to derive some pure or true version of the stories being trans lated but rather to show the impact of the communication process on the decisions of the court. More importantly, there is no source text but only a co-produced dialogue. Although it was not always possible to apply ethnographic methods in the way that the individual cases would require, given that not all participants in the research could be repeatedly contacted, one can observe recurrent patterns. I have attempted to describe not only the answers to my research questions, but above all the questions that the participants themselves posed. 16 ibid. As of mid-2023, the newly established Regional Administrative Courts in Bratislava and Košice exercise competence in this proceeding. See Act No. 151/2022 Coll. on the Establishment of Administra tive Courts (Slovakia). 17 See generally Marketa Štefková, ‘Public Service Interpreting and Translation in Slovakia’ in Mar keta Štefková, Koen Kerremans, and Benjamin Bossaert (eds), Training Public Service Interpreters and Translators: A European Perspective (Comenius University of Bratislava 2020); Pavol Šveda, ‘Interpreter Training in Central Europe’ in Pavol Šveda (ed), Changing Paradigms and Approaches in Interpreter Training (Routledge 2021); Pavol Šveda and Martin Djovčoš (eds), Translation and Interpreting Training in Slovakia (n 14). 18 Willi M O’B Li i i E id L P d S i h C (A d i 1.1 Research site and methods The institutional encounters are analysed in the predominantly monolingual settings of the state and correspond to various forms of interpretation from trained and untrained interpreters.17 All selected examples cor respond to the institutional contexts in which interpreters are implicitly assigned the role of a cultural expert. 18 William M O’Barr, Linguistic Evidence: Language, Power, and Strategy in the Courtroom (Academ Press 1982) 122. 2 Language identification and contested meanings Most asylum applicants communicate in the first instance with the border police. This encounter is the primary reference point in their file, as their retold testimony is further investigated. Later in the asylum procedure, significant discrepancies might arise as the spoken becomes written and the written becomes the fact. The belief that ‘linguistic forms of variation are utterly consequential for the legal process’18 is not firmly established in such cases.i The first condition for the negotiation of meanings is to create a space for iden tifying the language which is to be chosen for all speakers, ideally the applicant’s 1 3 Jindal Global Law Review (2023) 14:241–257 246 mother tongue. The language of interpreting might be misidentified as the language varieties might not be differentiated by participants to a sufficient degree. Linguistic means are linked not only with diverse geographical regions but also with differing socioeconomic status and contextual usage of language. For the police, as Paula and Petra describe, however, the linguistic competencies of their staff are framed by their concept of the administrative language: We have Slovak as our administrative language, we speak Slovak, and naturally, that’s the linguistic equipment of our police. (...) There has genuinely been a change over time; we have young people who have language skills, though they aren’t required to speak to them because we don’t have any bonuses for that, but they’re willing to speak. A knowledge of languages isn’t a condition of acceptance by the Foreign Police. Our people know English—but maybe the foreigner doesn’t know English (...) it is not his native language; he begins to stammer and does not understand. If the language of interpreting is not one of the major world languages and an avail able ad hoc interpreter is relied on, they usually convey only an approximate mean ing. Some of them ascribe less importance to details in the varieties of one language or even to differences between languages (Urdu, Pashto, distinct Arabic dialects). On the other hand, applicants are aware of the diversity of registers within a single lan guage as Arne notes, ‘our language is complicated, like in courts, we use the formal language, but in everyday conversation, we use our dialect, which is very different from the formal’. 19 Katrijn Maryns, The Asylum Speaker: Language in the Belgian Asylum Procedure (St Jerome 2006) 323. 20 Susan Berk-Seligson, Coerced Confessions: The Discourse of Bilingual Police Interrogations (Walter de Gruyter 2009) 34–36. 21 Pavol Šveda and Marketa Štefková, Crisis Management: Developing PSIT Structures for Ukrainian Displaced Persons in Slovakia (2023) 10(1) FITISPos 85. 22 Rogers Brubaker, Ethnicity Without Groups (Harvard University Press 2006) 33; Thomas Hylland Erik sen, ‘We and Us: Two Modes of Group Identification’ (1995) 32(4) Journal of Peace Research 427. 23 Anthony Good, Anthropology and Expertise in the Asylum Courts (Routledge 2007) 164. 2 Language identification and contested meanings In accordance with Maryns,19 in practice, the procedural requirement of a mono lingual standard means oversimplification of linguistic variation within and between codes and disqualification of multilingual speaker repertoires. There are numerous widely spoken languages which have regional or national dialects so different from one another that they may be mutually incomprehensible. In the following example, the attorney Lucia describes his awareness of misapprehensions as the interpreter translated details for the applicant: It seems he understood something, but only a little—afterwards, they became aware of that (...) It’s as if you communicated with Slovaks in Polish, always you’ll make some false assumptions. And the worst part of it is that for the Police, it’s sufficient if they have entries just for those basic questions from the report. Anything else, let the applicants just sign it. It seems that the negotiation of meanings takes place either way, even if the swiftness of the proceedings is prioritised, but the number of inconsistencies in the file might be higher. Although appointing untrained people to provide interpreting services means instituting a weak link into the legal process, and the potential for a miscarriage of n Maryns, The Asylum Speaker: Language in the Belgian Asylum Procedure (St Jerome 2006) 1 3 Jindal Global Law Review (2023) 14:241–257 247 justice is high,20 the police may have no better than ad hoc interpreting at hand. Train ing for public service interpreters and their infrastructual support is not available, though first attempts have been noted.21l justice is high,20 the police may have no better than ad hoc interpreting at hand. Train ing for public service interpreters and their infrastructual support is not available, though first attempts have been noted.21l In reflections on the abovementioned problems, the state representatives also refer to asylum seekers’ ‘cultural disparity’. Noticeably, their reasoning does not go fur ther into distinctiveness stemming from a different class, educational or professional background, which may have more impact than those which are ascribed to ethnici ty.22 Nevertheless, the label of ethnicity with its language often remains a single hall mark of identity. Thus, interpreters are easily assigned the role of a cultural expert. The problem is that they might have lost direct contact with current political situa tions in their country of origin and the language terms connected with their contextu ality. 2 Language identification and contested meanings However, it is commonly encountered that even in the case of the same dialect of interpreter and appellant, comprehension problems occur because of distinctions in social status, generation, or gender.23 Interpreting has its own political and social context and intrinsically dialogic nature in each case. It is, therefore, not sufficient simply to determine the foreign language. I observed that if linguistic and social diversity is not recognised, commu nication becomes noise. The interpreter’s linguistic competence cannot be a single criterion for appointing interpreters. To understand the emergence of such break downs, I argue that the participants’ notions of what it means to be impartial limit the potential of dialogue. Only if both parties share a common ethic will the room for interpretation make sense. 3 Expertise, norms of interpreting, and impartiality Notions of interpreters’ impartiality are widespread among research participants. However, what exactly it means to be impartial depends on the control one exerts over the course of the procedure. On the one hand, examples of side-taking often include references to essentialist notions of culture. On the other hand, interpreters aware of the complexity of impartiality often mention the need for multiperspectivity. Not taking sides means that one acknowledges dialogue with any kind of expertise, its language register included. At the same time, no interpreter can be culturally intact but rather aware of their dilemmas and variable positions. Indeed, normative neutral ity is expected not only from interpreters but also from decision-makers and judges. According to Holden, it is crucial to distinguish between positionality and procedural neutrality. Providing cultural expertise in a procedurally neutral way means having the potential to empower vulnerable groups without contradicting the discipline’s Notions of interpreters’ impartiality are widespread among research participants. However, what exactly it means to be impartial depends on the control one exerts over the course of the procedure. On the one hand, examples of side-taking often include references to essentialist notions of culture. On the other hand, interpreters aware of the complexity of impartiality often mention the need for multiperspectivity. Not taking sides means that one acknowledges dialogue with any kind of expertise, its language register included. At the same time, no interpreter can be culturally intact but rather aware of their dilemmas and variable positions. Indeed, normative neutral ity is expected not only from interpreters but also from decision-makers and judges. According to Holden, it is crucial to distinguish between positionality and procedural neutrality. Providing cultural expertise in a procedurally neutral way means having the potential to empower vulnerable groups without contradicting the discipline’s 1 3 3 Jindal Global Law Review (2023) 14:241–257 248 ethics. In the whole process, all information given is subject to the scrutiny of the decision-making authorities.24 In the past, the monologic model of the intermediary25 was prevalent. The inter preter is, as it were, a medium of transmission, not a third party. 24 Livia Holden, ‘Anthropologists as Experts: Cultural Expertise, Colonialism, and Positionality’ (2022) 47(2) Law & Social Inquiry 686. 25 Cecilia Wadensjö, Interpreting as Interaction (Longman 1998). 26 Roxana Rycroft, ‘Hidden Penalties Faced by Non-English Speakers in the UK Criminal Justice System: An Interpreting Perspective’ in Anne Wagner and Le Cheng (eds), Exploring Courtroom Discourse: The Language of Power and Control (Routledge 2011) 224. 27 Act No. 382/2004 Coll. on Experts, Interpreters and Translators (Slovakia). 27 Act No. 382/2004 Coll. on Experts, Interpreters and Translators (Slovakia). Livia Holden, ‘Anthropologists as Experts: Cultural Expertise, Colonialism, and Positionality’ (2022) (2) Law & Social Inquiry 686. Roxana Rycroft, ‘Hidden Penalties Faced by Non-English Speakers in the UK Criminal Justice System: n Interpreting Perspective’ in Anne Wagner and Le Cheng (eds), Exploring Courtroom Discourse: The nguage of Power and Control (Routledge 2011) 224. 3 Expertise, norms of interpreting, and impartiality For example, the ad hoc interpreters may give information to the applicants without disclosing its contextualisation to the lawyers, as Lena notices: In the detention centre, I met with such behaviour from some interpreters, that, look, I’m here for the hundredth, two hundredth time and you’re here for the 1 3 Jindal Global Law Review (2023) 14:241–257 249 first time, so I’m going to talk a lot, and I’ll say an awful lot to the client, over and above what you say. Those interpreters have thrust themselves into the role of advisors, on the grounds of the proximity of their culture. first time, so I’m going to talk a lot, and I’ll say an awful lot to the client, over and above what you say. Those interpreters have thrust themselves into the role of advisors, on the grounds of the proximity of their culture. However, the lack of neutrality might be a result of unconscious routine. A member of the police, Patrik critically reflects on the interpreter’s position: In the preparatory proceedings, the police officer either takes on a court inter preter or appoints an ad hoc interpreter, and this person must be given instruc tions. The instruction is given formally. The interpreter, if he isn’t there for the first time, may not even read what is written, and just signs it. That would be OK if the police personnel were informed, those who work with the non-registered ones if those too had their training about why this is important. Because now all that is done as a formality. The first time, the second time, the interpreter takes the instructions, and afterwards, he approaches all that as routine. Sometimes we’re glad that he even turned up. (…) When certain things confirm to us, or you actually see – that the interpreter is making some sort of judgement, he’s minimising certain things, he’s evaluating, that’s not his proper business. After wards, we became aware of that, and we try not to cooperate with him. Also, in cases when interpreters were asked to express views on the accent of an asy lum seeker or comment on other markers of ethnicity, they were ascribed the role of expert linguists or anthropologists. They might even try to act in order to substitute for a systemic gap or just respond to the need to simplify the complexity of encoun ters with the Other. 28 Ethical Codex for Interpreters in Asylum Proceedings in Osobitosti tlmočenia v azylovom konaní (UNHCR 2010) 24–26. https://www.minv.sk/?osobitosti-tlmocenia-v-azylovom-konani&subor=428538. Accessed 21 February 2023. 3 Expertise, norms of interpreting, and impartiality From this perspec tive, the decision-maker Oleg describes a good interpreter: It is essential for us that a question be translated precisely and that an answer be translated precisely, because it isn’t the task of the interpreter to change anything or to take an initiative, or in any way to recast the answer or the ques tion. (…) Obviously, he’s making sense to applicants in that language; we put a question, he translates, the applicant gives his answer, he translates—and all this works. Such views are common, as if the interpreter could furnish motives and strategies exclusively from the point of view of the person speaking in a social vacuum. Inter preters may have dilemmas from the above-described expectations. The legal fiction of the conduit role of the interpreter is a form of bias against linguistically disadvan taged persons. Rycroft explains that the conduit role is a faulty conceptualisation of the work of the court and police interpreter, though this belief system is still sub scribed to for structural reasons. She emphasises the tension between the relativity of truth and the search for certainties in the legal process. Admission of the interpreter’s participatory presence seems like a wild card.26 Although ethical codes anticipate this unpredictability, they are not part of the legislation. In the Slovak Act on Experts, Interpreters and Translators,27 impartiality is mentioned without definition. General terms such as ‘ethical and moral principles’, ‘impartial and disinterested performance’ are also referred to in the uncertified inter preters’ oath at the court hearings as well as with the police. However, asylum seekers do not usually file a complaint against the misconduct of officially appointed inter preters because they do not want to sue the state from which they seek protection and which they see represented by the officially appointed interpreters. In certain institutional circumstances, interpreters lacking impartiality may take on the roles of all professions: decision-makers, legal representatives, members of the police, social workers, and psychologists. 3 Expertise, norms of interpreting, and impartiality For those who are interpreting asylum seekers in cooperation with UNHCR, there is a version of the ethical code available which has been used by the Slovak Migra tion Office28 as a recommendation. In practice, however, in situations in which the ethical code might have been expounded (police, office, courts), officials did not con front the interpreters with it. One of the respondents, a legal counsel Lea, mentions breaching the code of ethics in the first contact on the borders: They’re all frightfully pally with the police, they have them as friends on social networks, and this fellow brought them in cakes, and right when he arrived they were crying ‘hi!’ (…) They make friends of those foreigner-interpreters who are willing to cooperate with them and do what they tell them, without those interpreters adhering to any ethical principles or rules for their interpreting, about how they should interpret. In the view of a senior staff member Patrik at the detention centre, their reports do not play an essential role in evaluating the substantive merit of an application for asylum. The same attitude was held towards interpreting: 28 Ethical Codex for Interpreters in Asylum Proceedings in Osobitosti tlmočenia v azylovom konaní (UNHCR 2010) 24–26. https://www.minv.sk/?osobitosti-tlmocenia-v-azylovom-konani&subor=428538. Accessed 21 February 2023. 1 3 250 Jindal Global Law Review (2023) 14:241–257 As regards the reports from the Police Detention Units for Foreigners in rela tion to asylum proceedings, these reports are not crucial for decision-making. (…) Often at the beginning, the applicant is still ‘uninstructed’ and ‘uninflu enced’ by a third person who is not a police officer or Slovak Migration Office decision-maker, and as a general rule, he gives information about his motiva tions according to what he really feels. Many of the state’s representatives are convinced that the first police report is not fundamental and that the legal representatives and interpreters are already communi cating information about legal possibilities, and influencing the foreigner’s decision. In contrast, legal representatives Lucia and Linda say: Reports from the police may play a fundamental role. Because if the judge detects something procedurally dubious there, on this basis he can quash a decision. [The judge] discovered details of how the interview at the police premises was conducted, where they posed misleading questions. In some cases, if judges understood English or Russian during the hearings, they might recognise the discrepancies resulting from mistranslations on the spot. 29 Dn, dn refers not only to the sound of the keyboard, but also to the quick execution of an administrative duty. 30 See, generally, Sonja Pöllabauer, ‘Interpreting in Asylum Hearings: Issues of Role, Responsibility and Power’ (2004) 6(2) Interpreting 175; Julia Dahlvik, ‘Why Handling Power Responsibly Matters: The Active Interpreter Through the Sociological Lens’ in Gill and Good (eds), Asylum Determination in Europe (n 1) 133. 29 Dn, dn refers not only to the sound of the keyboard, but also to the quick execution of an administrativ duty. 30 See, generally, Sonja Pöllabauer, ‘Interpreting in Asylum Hearings: Issues of Role, Responsibili and Power’ (2004) 6(2) Interpreting 175; Julia Dahlvik, ‘Why Handling Power Responsibly Matter The Active Interpreter Through the Sociological Lens’ in Gill and Good (eds), Asylum Determination Europe (n 1) 133. 3 Expertise, norms of interpreting, and impartiality How ever, they could not verify the complaints about impartiality from preceding sessions if there was no recording. Although court hearings can already be recorded, for the first interviews at police stations this practice is not yet common, and the interpreter may stay as if they were invisible, remarked by Linda: For some of the interpreters who regularly go to interpret at the Migration Office or the foreign police, it is usual that they know the story of the proceedings (…), and they know the decision-maker expects to have down on report dn, dn, dn, dn.29 He knows what she wants to have recorded. He achieves that. Or that is conveyed, and he proactively takes that situation upon himself. On the other hand, interpreters might be treated as equal and visible participants in the hearings. They might intervene if they deem it necessary and provide explanations.30 Although, in general, the stakeholders indicated a rather textual model of speaking— verbatim translation with little room for interpretation—literalness in practice is not kept. The danger of misinterpretation is serious if ad hoc interpreters’ unaddressed visibility is not questioned, as the legal representative Lea notices: 1 3 Jindal Global Law Review (2023) 14:241–257 251 (Untrained interpreters) do not use the appropriate words, but some other words; they change the meanings. They do not fully translate some things, they summarise, and the judge has no means of checking that. Ultimately, in fact, the judge does not care much about it, because he too summarises statements when he dictates them into the report. So, there’s a kind of double summarisa tion, with a reformulation of somebody’s testimony. The final report of what the foreigner says may be diametrically opposed to what he said in his own language. That is equally so whether it is at a court hearing, at the police or at the migration offices. The stereotype of interpreters as impartial catalysts31 needs to be further problema tised. Presupposed invisibility of the interpreters might conceal the fact that interpret ers are active participants in multilingual and intercultural exchanges in the asylum field.32 Indeed, the interpreter’s control over proceedings may, in practice, be great. While the minima laid down by the ethical code are necessary, they are not sufficient. The interpreters play a key dialogic role, often in a similar vein as cultural experts, elucidating the previous context of applicants’ events. 31 P H Gulliver, Disputes and Negotiations. A Cross-Cultural Perspective (Academic Press 1979) cited in Cecilia Wadensjö, Interpreting as Interaction (Longman 1998) 64. 32 Robert Gibb and Anthony Good, ‘Interpretation, Translation and Intercultural Communication in Refu gee Status Determination Procedures in the UK and France’ (2014) 14(3) Language and Intercultural Communication 396. 31 P H Gulliver, Disputes and Negotiations. A Cross-Cultural Perspective (Academic Press 1979) cited in Cecilia Wadensjö, Interpreting as Interaction (Longman 1998) 64. 32 Robert Gibb and Anthony Good, ‘Interpretation, Translation and Intercultural Communication in Refu gee Status Determination Procedures in the UK and France’ (2014) 14(3) Language and Intercultural Communication 396 33 Jan Blommaert, ‘Language, Asylum, and the National Order’ (2009) 50(4) Current Anthropology 415; Anthony Good, ‘Cultural Evidence in Courts of Law’ (2008) 14(1) Journal of the Royal Anthropological Institute 47. 34 Livia Holden, ‘Cultural Expertise and Law: An Historical Overview’ (2020) 38(1) Law and History Review 29. 35 See also Julia Dahlvik, ‘Why Handling Power Responsibly Matters’ (n 30) 150. 3 Expertise, norms of interpreting, and impartiality The professional jargon and legal terminology of both countries are ideally part of the interpreters’ linguistic com petence. The following case represents the lack of interpreters’ impartiality, as the asylum seeker Arne describes how his interpreter from Midland chose terms that framed Arne’s narrative in the opposite meaning of what was intended. He discov ered this only when, after being detained, he contacted a legal representative and received her feedback on clarification questions. Since then, he has opted solely for English-language translators: At the Migration Office, I had that same interpreter, who I realised later that he wasn’t honest even in the entry interview at the police (…) I already had some information about his political direction. Both of us are from (Midland), and there are pro-regime and anti-regime people. You’re in a situation where you’re explaining why you cannot return because you’re against a really ter rorising regime, and the interpreter is like one of them, you know? I do not question the professionalism of the interpreter, but it’s just that I found that, for example, people whom I had spoken of as rebels in a positive sense revolts, like opposition fighters, while he interpreted as terrorists or outlaws, something that gives a negative image. I was talking about them in a positive way. I supported them, for me, they were opposition fighters, and for him, they were terrorists. The interpreter used the terminology of the government. He was not giving the image that I was trying to show. 1 3 252 Jindal Global Law Review (2023) 14:241–257 Applicants for asylum on political or religious grounds face the problem of ideologi cal partisanship of their interpreter-fellow citizen whom they do not always have an opportunity to replace even after detecting distortions in the interpreted text, as Arne remarks: Applicants for asylum on political or religious grounds face the problem of ideologi cal partisanship of their interpreter-fellow citizen whom they do not always have an opportunity to replace even after detecting distortions in the interpreted text, as Arne remarks: I was cautious regarding his possible collaboration with the government because it is highly probable that he is only paying back what he has received as benefits: the opportunity to travel abroad and to be in Czechoslovakia. (…) He travels freely, has his photo taken with government soldiers, and so on. 3 Expertise, norms of interpreting, and impartiality I think, in the current political context of (Midland) there is no person at all who could be impartial and thus professional. That is the reason why I asked for interpreting in English. The reasoning about the impartiality of the interpreters is bound to perspective-tak ing. Certainly, in practice, there are many examples where the linguistic incompe tence of the interpreter is sufficient to create inconsistency. According to the decision maker Oliver, asylum applicants are also making strategic moves. In terms of achiev ing their goal the easiest thing is to say: ‘I didn’t understand, and the interpreter translated it badly. When a contradiction arises, and it cannot be otherwise explained (…) Everyone is guarding his interests’. Most decision-making authorities do not ask interpreters to include either legal or cultural interpretation of terms during hearings,33 but some would consult them out of record regarding their interests: verification of a dialect and cultural practices of the claimants. 4 Interpreters as cultural experts? Holden defines cultural expertise as the special knowledge that enables experts to describe relevant facts considering the particular background of the litigants for the use of the court (or decision-making authority).34 In this paper, I have shown several examples in which cultural experts were not part of the proceedings, and this role was implicitly ascribed to interpreters instead. Indeed, there were many cases where authorities did not expect any kind of cultural mediation and had ‘just interpreters’. Interpreters themselves varied as well. Few of the interpreters reflected that in some cases it would be unethical for them not to intervene,35 and they acted with an extra contextual description. The sensitivity to the right intervention at the right time is part of professional ethics in general. 1 3 Jindal Global Law Review (2023) 14:241–257 253 For the applicants, the legal system often represents a riddle requiring an expert’s interpretation. Again, the interpreters have been asked as if they were already cul tural mediators. When interviewing policewomen about the ways in which their staff check applicants’ understanding, Paula emphasised that the responsibility was on the applicant’s side—both in knowing the rights and being proactive: I was always taught that ignorance of the law is no excuse and doesn’t excuse even me if I as a policewoman happen not to know something in the law. (...) After all, the foreigner himself, he can say, I don’t understand, explain that to me in some other way! Although the potential to intervene has been ascribed to asylum applicants, their assertive behaviour is highly context-dependent and, in my research, rarely clarified beforehand. The interpreter’s role in negotiating the communicative situation might also be proactive, in the sense of interventions which stem from the need to explain the context to any of the parties. In this vein, current approaches in community interpreting and linguistic media tion are described by a court interpreter Ivor, who avoided misunderstanding by ask ing each of the parties for an explanation or an alternative: When there’s a bad set-up and the conditions are only making the situation harder for that interpreter, he must speak up and protest. Or alternatively say, object to, whatever is hindering our work. Not to agree to everything. Because this also is the kind of situation where a person can slide into feeling that, after all, I’m just the medium and I have to adapt to everything. 36 See Sonja Pöllabauer, ‘The Interpreter’s Role’ in Handbook for Interpreters in Asylum Procedures (UNHCR 2017) 50–69. 37 See Ruth Morris, ‘The Moral Dilemmas of Court Interpreting’ (1995) 1(1) The Translator 25. 38 Good, Anthropology and Expertise in the Asylum Courts (n 23) 169. 4 Interpreters as cultural experts? (…) I spoke up on these lines: excuse me, I won’t be able to carry on like this. Or, I am sorry, but this is making my work harder, and to make understanding easier for both sides, I would suggest this. (…) So I would say, when I’m there things fall into place round me, and I think constantly of that phrase linguistic mediator. The above-described approach is part of the Dialogical Communication Method, which is recommended in communication with vulnerable groups and is applied more widely in ordinary interviews on asylum.36 Supplementing this method, inter preters also use an approach designated as cultural mediation or culturally sensitive translation. In principle, the mediators do not intervene in the interaction to give their own opinion on the correctness of the behaviour of, or of the information given by, the applicant. If they ask for clarification, it is always with the knowledge of all participants. Knowledge of the methodology of the approaches mentioned obviates the possibility that interpreters will take upon themselves the role of collaborators of the investigating parties and thus, in the final analysis, put their authority in doubt. The interpreters’ interactional stance would mean they could use their own strategies 36 See Sonja Pöllabauer, ‘The Interpreter’s Role’ in Handbook for Interpreters in Asylum Procedures (UNHCR 2017) 50–69. 1 3 Jindal Global Law Review (2023) 14:241–257 254 for identifying misunderstandings, elucidating context, investigating intention, and clarifying meaning.37i Culture-specific concepts are never self-explanatory. The interpreter Ivor ascer tains the participants’ precise meanings and is content to be guided by them. As a linguistic mediator, he verifies the contexts of what all the actors ‘whisper’ to him: Following, observing, it’s terribly important. My father, who’s a sports com mentator, told me once: see now how this player whispered to the other one. It’s a beautiful word. He had actually whispered by his movement how exactly he should play the ball to him, or whatever. And to my mind, that’s very important for the interpreter. To listen. To observe. Incessantly to observe, because after wards you’ll find that the applicant helps you greatly. He whispers to you, and when you use the word that he uses equally in that situation, when it’s suitable, needless to say, an experienced interpreter is capable of judging that accurately, in my view. He shouldn’t have any problem with that. So it will work out very well. 4 Interpreters as cultural experts? That’s how it should be, to my mind. From the interactional perspective, interpreting with insufficient sensitivity to whis pered contexts could not serve the purpose of asylum proceedings. Therefore, as demonstrated by above-discussed ‘rebels’, it is crucial to acknowledge that the diver sity of culture-specific concepts can be adopted by legal and political jargon. Any terminology connected with affinity with kin, class, caste, gender, religion, politi cal group, education, profession, and other identity markers—all refer to permutated social norms. However counterintuitive they might look, spatial coordinates are rarely static, such as the structuration of the place of residence, depiction of topogra phy, and various types of registration of postal addresses. Any labelling of territories alters with microcultural niche as well as political infrastructure. Many of these mentioned terms are regularly used in the interviews for asylum seekers. In the case of forms which are part of the applicant’s file, a standardised translation available in multiple languages could help address the issue. At the same time, the need to provide a culturally sensitive explanation and communicate the legal concepts in plain language remains. However, formally acknowledging and allowing translators to be considered as cultural experts would require considerable change in the law. Even if such amendments impacting the weight that the court could give to interpreters’ opinions are not on the horizon yet, awareness thereof can assist. Participants in a communication frame their statements, and the modes of fram ing change situationally, professionally, socially, and culturally. A mutual search for a wider contextual meaning is thus crucial. The interlocutors with decisive power may distinguish the essential semantic shifts and create communicative space within interpreting. Interpreters could never be noise-free channels of communication; on the contrary, they may have considerable influences on proceedings.38 However, this effect could be managed to a certain extent—if the status of interpreters is raised, and 1 3 Jindal Global Law Review (2023) 14:241–257 255 their capacity for cultural expertise is supported and acknowledged. The insistence on verbatim translation in the courtroom could be dropped. their capacity for cultural expertise is supported and acknowledged. The insistence on verbatim translation in the courtroom could be dropped. However, legal systems almost invariably impose the unrealistic and counter- productive requirement that interpreters shall be ‘verbatim’ as much as possible. 39 Act no. 382/2004 Coll. (n 27). This Act does not apply to ad hoc interpreters, they take an interpreters’ oath on the spot. 40 John L Austin, How to Do Things with Words (Clarendon 1975). 41 Helena Tužinská, ‘Doing things with Questions: Interpreting in Asylum Context’ (2019) 103 LUD 81–99. 42 See Maryns, The Asylum Speaker (n 19); Max Spotti, ‘It’s All About Naming Things Right: The Paradox of Web Truths in the Belgian Asylum-Seeking Procedure’ in Gill and Good (eds), Asylum Determination in Europe (n 1) 69–90. 43 See Susan Berk-Seligson, The Bilingual Courtroom: Court Interpreters in the Judicial Process (The University of Chicago Press 2002); Good, Anthropology and Expertise in the Asylum Courts (n 23); Katrijn Maryns ‘The Use of English as Ad Hoc Institutional Standard in the Belgian Asylum Interview’ 4 Interpreters as cultural experts? In practice, on the contrary, interpreters must simultaneously mediate between two often very different sets of cultural assumptions and convert technical terms from one distinctive legal system into the nearest equivalent terms in another. Interpreters often do so without specialised legal training, virtually no time to reflect and choose their words carefully, and without having in front of them the documents to which the legal actors are referring. Although the Slovak Act on Experts, Interpreters and Translators39 establishes verification of professional competence, in practice, diverse unexpected situations cannot possibly be covered. The interpreter has the demanding task of restraining from creating a contradiction by using a concept with a different contextual meaning. Veracious interpretation thus means to become dialogical, asking inspecting queries. The interpreters, together with representatives of the state, not only do things with words.40 Being interpretive means that both are acknowledged to do things with questions.41 See Susan Berk-Seligson, The Bilingual Courtroom: Court Interpreters in the Judicial Process (The niversity of Chicago Press 2002); Good, Anthropology and Expertise in the Asylum Courts (n 23); atrijn Maryns ‘The Use of English as Ad Hoc Institutional Standard in the Belgian Asylum Interview’ 42 See Maryns, The Asylum Speaker (n 19); Max Spotti, ‘It’s All About Naming Things Right: The Parado of Web Truths in the Belgian Asylum-Seeking Procedure’ in Gill and Good (eds), Asylum Determinatio in Europe (n 1) 69–90. 5 Conclusion In this article, I have presented some aspects of the institutional practice in the Slo vak asylum field, with a focus on interpreting, ethnographically investigated between 2017 and 2019. The negotiation of meanings in asylum proceedings starts with lan guage identification and the selection of interpreters. These first decisions are bound by notions of impartiality and ethical procedures of all participants. Both areas men tioned above provide room for a relevant exegesis of the socio-legal terminology and culture-specific terms. This study demonstrates the understanding of interpreters as actors who have the potential to bring cultural expertise to asylum proceedings in countries where it is not yet established. The findings may also serve as an inspiration for instituting ad hoc interpreter trainings. I have demonstrated how the routinised practices with a dominance of monolin gual standards result in the disqualification of multilingual speakers’ repertoires.42 In a legal setting, even within the same dialect, a (dis)respect for differences in gender, age, and social background has far-reaching consequences.43 My contribution is to 1 3 3 Jindal Global Law Review (2023) 14:241–257 256 question the kinds of strategies interpreters and other actors have for identifying mis understandings, elucidating context, investigating intentions, and clarifying meaning explicitly. I suggest that with the shortage of cultural experts and court interpreters, ad hoc interpreters, as well as other professions in public service, could incorporate cultural expertise into their training. Cultural concepts and professional jargon, all of which might be instruments of social control, may remain as intact as relics, accessible only to the initiated. On the other hand, both legal and culture-specific terminology can be faced with dialogical methods, mediating culturally sensitive information without erasure of context44 or upturn of hidden penalties.45 As it was aptly put by Good, however technically com petent the interpreters might be, the mere fact of their involvement converts the kinds of formalised, question-and-answer dialogues that characterise legal proceedings (and which normally serve to enhance the power of the legal actors) into trialogues, where the interpreter is every bit as much an active participant as the lawyer and the asylum applicant.46 Unless communication obviously and irretrievably breaks down, the other actors systematically overlook the interpreters’ key role in court. Interpreters are written out of the script, both literally and metaphorically. The court could be unaware of the moral dilemmas and technical difficulties they face. 44 Diana Eades, ‘Erasing Context in the Courtroom Construal of Consent’ in Susan Ehrlich, Diana Eades, and Janet Ainsworth (eds), Discursive Constructions of Consent in the Legal Process (Oxford University Press 2016) 71–92. 46 Interview with Anthony Good, Professor Emeritus, School of Social and Political Science, The Unive sity of Edinburgh (Edinburgh, 26 September 2019). 45 Roxana Rycroft, ‘Hidden Penalties Faced by Non-English Speakers in the UK Criminal Justice System: An Interpreting Perspective’ in Anne Wagner and Le Cheng (eds), Exploring Courtroom Discourse: The Language of Power and Control (Routledge 2011). (2017) 38(5) Applied Linguistics, 737; Helena Tužinská, Medzi riadkami: Etnografia tlmočenia azylovýc súdnych pojednávaní (Akamedia 2020). (2017) 38(5) Applied Linguistics, 737; Helena Tužinská, Medzi riadkami: Etnografia tlmočenia azylových súdnych pojednávaní (Akamedia 2020). 5 Conclusion This challenge can be met together with the interpreters, sensitised by training. More attention also needs to be paid to the linkage between the mode of posing questions and the communicative power of answers.47 The culture-specific context might be investigated and eluci dated with open ethnographic questions. In this vision of a communicative space, cultural expertise can be engaged in the understanding of the law, cross-cultural dif ferences, and resolution of conflicts, acting in a procedurally neutral way,48 not only for cultural experts but also for interpreters. (2017) 38(5) Applied Linguistics, 737; Helena Tužinská, Medzi riadkami: Etnografia tlmočenia azylových súdnych pojednávaní (Akamedia 2020). 44 Diana Eades, ‘Erasing Context in the Courtroom Construal of Consent’ in Susan Ehrlich, Diana Eades, and Janet Ainsworth (eds), Discursive Constructions of Consent in the Legal Process (Oxford University Press 2016) 71–92. 45 Roxana Rycroft, ‘Hidden Penalties Faced by Non-English Speakers in the UK Criminal Justice System: An Interpreting Perspective’ in Anne Wagner and Le Cheng (eds), Exploring Courtroom Discourse: The Language of Power and Control (Routledge 2011). 46 Interview with Anthony Good, Professor Emeritus, School of Social and Political Science, The Univer sity of Edinburgh (Edinburgh, 26 September 2019). 47 See generally Sandra Hale, Jane Goodman-Delahunty, and Natalie Martschuk, ‘Interpreter Performance in Police Interviews: Differences Between Trained Interpreters and Untrained Bilinguals’ (2018) 13(2) The Interpreter and Translator Trainer 107; Deborah Davis and Elisabeth Loftus, ‘Inconsistencies between Law and the Limits of Human Cognition’ in Lynn Nadel and Walter P Sinnott-Armstrong (eds), Memory and Law (Oxford University Press 2012) 29–58; Aneta Pavlenko, ‘Language Rights Versus Speakers’ Rights: On the Applicability of Western Language Rights Approaches in Eastern European Contexts’ (2011) 10(1) Language Policy 37; ‘Guidelines for Communicating Rights to Non-native Speakers of Eng lish in Australia, England and Wales, and the USA’ (American Association of Applied Linguists, 2016). https://www.aaal.org/guidelines-for-communication-rights. Accessed 21 February 2023. 48 See Sandra Hale, ‘Interpreting Culture: Dealing with Cross-cultural Issues in Court Interpreting’ (2014) 22(3) Perspectives: Studies in Translatology 321; Holden, ‘Cultural Expertise and Law’ (n 34); Ilenia Rug giu, Culture and the Judiciary: The Anthropologist Judge (Routledge 2019). 48 See Sandra Hale, ‘Interpreting Culture: Dealing with Cross-cultural Issues in Court Interpreting’ (2014) 22(3) Perspectives: Studies in Translatology 321; Holden, ‘Cultural Expertise and Law’ (n 34); Ilenia Rug giu, Culture and the Judiciary: The Anthropologist Judge (Routledge 2019). 5 Conclusion 1 3 257 Jindal Global Law Review (2023) 14:241–257 Acknowledgements I would like to express my gratitude to Anthony Good, for his aid and clarity. Also, my thanks belong to all participants for their helpful suggestions provided within the workshop at the Jindal Global Law School with the endorsement of ISRF Project Cultural Expertise and Litigation: Prac tices in South Asia and Europe. Funding Partial financial support was received from the Ministry of Education of the Slovak Republic, grant VEGA 1/0203/23, Moral Assessment of National, Ethnic, and Religious Groups in Situations of Threat. Open access funding provided by The Ministry of Education, Science, Research and Sport of the Slova Republic in cooperation with Centre for Scientific and Technical Information of the Slovak Republic. Open access funding provided by The Ministry of Education, Science, Research and Sport of the Slovak Republic in cooperation with Centre for Scientific and Technical Information of the Slovak Republic. Declarations Ethics approval and consent to participate This is an observational study. Verbal informed consent was obtained prior to all the interviews and observations. All data were anonymised, under the Ethical Codex of the Ethnographic Society of Slovakia (2017) which is based on the Code of Ethics of the American Anthropological Association, https://www.americananthro.org/ethics-and-methods. Accessed 21 February 2023. Competing interests The authors have no competing interests to declare that are relevant to the content of this article. Competing interests The authors have no competing interests to declare that are relevant to the content of this article. 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/. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 1 3 1 3 3
https://openalex.org/W3209180592	https://www.ijournalse.org/index.php/ESJ/article/download/706/pdf	English	null	Development and Prototyping of Jet Systems for Advanced Turbomachinery with Mesh Rotor	Emerging science journal	2,021	cc-by	15,832	Available online at www.ijournalse.org Available online at www.ijournalse.org Emerging Science Journal (ISSN: 2610-9182) Vol. 5, No. 5, October, 2021 Development and Prototyping of Jet Systems for Advanced Turbomachinery with Mesh Rotor In the course of numerical experiments, the extreme conditions of fluid and gas outflow through a nozzle equipped with a velocity vector control system, in the control range of adjustment of the velocity vector deflection angle from + 90o to -90o within a geometric hemisphere, have been considered for the first time. It was also shown that when using a dual-channel nozzle, there are possibilities to adjust the velocity vector angle (thrust vector) in the range of + 180o to -180o within the geometric sphere. Compared with the known variants, the control range of the velocity vector angle is increased by nine times. These calculated data are presented in addition to the previously published results of physical laboratory experiments. Preliminary results of numerical experiments show the possibility of creating a new theory in the field of mesh turbines and jet systems. Patents support the novelty of the developed technical solutions. Keywords: Energy Conversion; Energy Efficiency; Gas Dynamics; Hydrodynamics; Nozzle; Ejector; Mesh Turbine; Pump; Article History: Received: 25 July 2021 Revised: 18 September 2021 Accepted: 26 September 2021 Published: 01 October 2021 Abstract Abstract This article presents the research results that aim to develop promising mesh turbomachines equipped with jet control systems. The turbomachines operating in difficult conditions in oil and gas production are mainly considered. At the same time, some research results can be used in other production branches, including power engineering and transport. Three-dimensional models for computer simulation of net turbines and jet control systems were developed. Prototypes and micromodels were created to test the performance of mesh turbines and jet control systems using additive technologies. A methodological approach is proposed to create a classification of jet control systems considering their design and technological features. In the course of numerical experiments, the extreme conditions of fluid and gas outflow through a nozzle equipped with a velocity vector control system, in the control range of adjustment of the velocity vector deflection angle from + 90o to -90o within a geometric hemisphere, have been considered for the first time. It was also shown that when using a dual-channel nozzle, there are possibilities to adjust the velocity vector angle (thrust vector) in the range of + 180o to -180o within the geometric sphere. Compared with the known variants, the control range of the velocity vector angle is increased by nine times. These calculated data are presented in addition to the previously published results of physical laboratory experiments. Preliminary results of numerical experiments show the possibility of creating a new theory in the field of mesh turbines and jet systems. Patents support the novelty of the developed technical solutions. Keywords: Energy Conversion; Energy Efficiency; Gas Dynamics; Hydrodynamics; Nozzle; Ejector; Mesh Turbine; Pump; Article History: Received: 25 July 2021 Revised: 18 September 2021 Accepted: 26 September 2021 Published: 01 October 2021 Development and Prototyping of Jet Systems for Advanced Turbomachinery with Mesh Rotor Yuri Appolonievich Sazonov 1, Mikhail Albertovich Mokhov 2, Inna Vladimirovna Gryaznova 3, Victoria Vasilievna Voronova 4, Khoren Arturovich Tumanyan 5, Mikhail Alexandrovich Frankov 6, Nikolay Nikolaevich Balaka 7 1 Professor of Department of Machinery and Equipment for Oil and Gas Industry, Gubkin's National University of Oil and Gas, Moscow, Russian Federation 2 Professor of Department of Oil Field Development and Operation, Gubkin's National University of Oil and Gas, Moscow, Russian Federation 2 Professor of Department of Oil Field Development and Operation, Gubkin's National University of Oil and Gas, Moscow, Russian Federation 3 Senior teacher of Department of Gas and Gas Condensate Field Development and Operation, Gubkin's National University of Oil and Gas, Moscow, Russian Federation 3 Senior teacher of Department of Gas and Gas Condensate Field Development and Operation, Gubkin's National University of Oil and Gas, Moscow, Russian Federation 4 Associate Professor of Department of Gas and Gas Condensate Field Development and Operation, Gubkin's National University Gas, Moscow, Russian Federation Postgraduate of Department of Machinery and Equipment for Oil and Gas Industry, Gubkin's National University of Oil and Gas, M Russian Federation 6 Junior Researcher of Department of Machinery and Equipment for Oil and Gas Industry, Gubkin's National University of Oil and Gas, Moscow, Russian Federation 6 Junior Researcher of Department of Machinery and Equipment for Oil and Gas Industry, Gubkin's National University of Oil and Gas, Moscow, Russian Federation 7 Head of Department of Technological Support for Wells Construction, CJSC “Russian Company for Shelf Development”, Russian Federation 7 Head of Department of Technological Support for Wells Construction, CJSC “Russian Company for Shelf Development”, Russian Federation Abstract This article presents the research results that aim to develop promising mesh turbomachines equipped with jet control systems. The turbomachines operating in difficult conditions in oil and gas production are mainly considered. At the same time, some research results can be used in other production branches, including power engineering and transport. Three-dimensional models for computer simulation of net turbines and jet control systems were developed. Prototypes and micromodels were created to test the performance of mesh turbines and jet control systems using additive technologies. A methodological approach is proposed to create a classification of jet control systems considering their design and technological features. CONTACT: mikhal.mokhov@mail.ru DOI: http://dx.doi.org/10.28991/esj-2021-01311 © 2021 by the authors. Licensee ESJ, Italy. This is an open access article under the terms and conditions of the Creative Commons Attribution (CC-BY) license (https://creativecommons.org/licenses/by/4.0/). DOI: http://dx.doi.org/10.28991/esj-2021-01311 © 2021 by the authors. Licensee ESJ, Italy. This is an open access article under the terms and conditions of the Creative Commons Attribution (CC-BY) license (https://creativecommons.org/licenses/by/4.0/). * CONTACT: mikhal.mokhov@mail.ru DOI: http://dx.doi.org/10.28991/esj-2021-01311 © 2021 by the authors. Licensee ESJ, Italy. This is an open access article under the terms and conditions of the Creative Commons Attribution (CC-BY) license (https://creativecommons.org/licenses/by/4.0/). 1- Introduction In current conditions, the problem of reducing energy costs for the implementation of production processes remains relevant. In Gubkin Russian State University of Oil and Gas laboratories, a series of research works on creating new machines for working with gas-liquid mixtures—including pumps, compressors, turbines, and ejectors—have been DOI: http://dx.doi.org/10.28991/esj-2021-01311 © 2021 by the authors. Licensee ESJ, Italy. This is an open access article under the terms and conditions of the Creative Commons Attribution (CC-BY) license (https://creativecommons.org/licenses/by/4.0/). Page \| 775 Emerging Science Journal \| Vol. 5, No. 5 performed [1]. In these machines, flow channels have a mesh structure. In the conventional sense, a mesh represents a larger geometric area in smaller discrete cells. In a turbomachine, the larger blades are replaced by a set of smaller blades, which are interconnected to form flow channels in the form of a mesh structure. The results of studies of the mesh structure of flow channels in the development of lattice wings [2, 3], heat exchangers [4-6], and ejectors of various purposes are known [7]. In general, any ejector includes the following main parts: nozzle, mixing chamber, and diffuser. It is known that ejectors can perform the functions of a pump and a compressor; there are also known examples of using the ejector as an augmenter [8] for airborne reactive systems. The advantages of the ejector are often called the simplicity of design and the absence of moving parts. At the same time, the introduction of moving parts as part of the ejector can significantly expand the scope of such jet technology. Possible applications of ejectors in the development of turbines and hybrid turbomachinery are possible. For example, there are known jet turbines in which the working gas is fed through a fixed inlet nozzle, and the jet coming out of the nozzle is directed along the rotor rotation axis [9]. In the central part of the rotor, there is a rotating mixing chamber with a diffuser, which provides an increase in gas pressure. Jet outlet nozzles are located at the periphery of the turbine rotor. In such turbine designs, attention may be drawn to the increased pressure in the rotor channels, especially in the section from the central diffuser to the outlet nozzle. It will entail the increase in the thickness of walls in a rotor and increase the rotor mass, with possible restrictions of rotation speed. 1- Introduction It is also possible to note the issues of balancing axial and radial loads acting on the rotor. In addition, a jet-reactive turbine is known [10] in which the jet coming out of the nozzle is directed radially relative to the rotor rotation axis. Here, multiple mixing chambers combined into a mesh structure are used. The formed mesh structure is placed inside the turbine rotor. In this case, nozzles and mixing chambers of unconventional forms are used, and curvilinear surfaces of flow channels help to increase the efficiency of the energy conversion process in the turbomachine. The specificity of such turbomachines is connected with non-stationary or pulse processes in the flowing part of the ejector and turbine. The simplicity of design and correspondingly low price of such turbomachines indicate the prospects of their practical use in solving urgent problems in various industries. At the same time, attention should be paid to the complexity of gas-dynamic and hydrodynamic processes occurring in the channels of such turbomachines. For example, gas flows around a set of cylinders [11], which can form a grid, are thoroughly studied now. The workflow in a mesh turbine is similar to the workflow in a partial admission turbine [12-14]. The operating processes of turbines with rotating ejectors are still poorly studied. This area of science and technology seems promising as opportunities for creating highly effective hybrid turbomachines, combining properties of jet technique and properties of impeller machines, are opened. In this regard, research of gas-dynamic and hydrodynamic processes in channels with mesh structure can be fully attributed to the number of relevant works. In aviation engineering, many problems regarding selecting the optimal geometry for solid walls in the gas flow at different flowing regimes, at high speeds [15, 16] and low speeds, have been solved [17, 18]. Gas flow control systems are based on mechanical systems with certain limitations on the speed of commands after the control signals are applied [19, 20]. For improving the efficiency of technologies, it is advisable to search for new methods of gas flow control, which would differ by higher operating speeds in control systems. Prospects for the development of turbomachines are associated with a detailed study of gas flows in channels of various shapes. Increasing the efficiency of technological processes requires solving optimization problems in gas dynamics and hydrodynamics [21, 22]. 1- Introduction First, a concept was developed (see Concept Headings section) to study mesh turbomachinery equipped with jet control systems. Then the materials (see Materials section) for prototyping were selected, emphasizing the application of additive technologies. In the next Methods section, a flowchart and a software package were presented, which were used for computer simulations. Technical information about the developed mesh turbines was presented in the Results section. Prototypes of the new turbine and the new jet control system were created using additive technologies. The Discussion section reflects the discussion of the presented conceptual focus area, considering the new results obtained. A new methodological approach is proposed to create a classification of jet control systems considering their design and technological features. The results of computational experiments are presented and discussed. Demonstration experiments carried out to check the performance of the created prototypes are described. The prospects for developing scientific and design works in mesh turbomachines equipped with jet control systems are considered. The discussion made it possible to summarize the research results and draw the main conclusions presented in the Conclusion section. 2- Concept Headings The following hypothesis has been formulated: it is advisable to develop energy-saving technologies and equipment at the junction of two scientific and technical domains; the first domain is associated with mesh turbomachinery, the second domain is associated with jet control systems that allow controlling the velocity vector (thrust vector). The mesh structure of the turbomachine flow path ensures the rotor's structural strength and rigidity at a low weight. The jet control system is distinguished by high reliability (due to the small number of moving parts) at a low weight of the system as a Page \| 776 Page \| 776 Emerging Science Journal \| Vol. 5, No. 5 whole. In this research, an intermediate goal could be the development of a jet system prototype designed to create turbomachinery with a mesh rotor. Research results primarily focus on creating multi-mode turbomachines operating in difficult conditions (including in the conditions of offshore oil and gas fields). Simultaneously, the research results can also be used to solve practical problems in other industries, including increasing the efficiency of energy conversion processes to generate electrical energy. Our publications have already presented the results of physical experiments carried out in laboratory conditions. The question arises about the need for a partial transition from physical experiments to numerical studies, which will save time and material resources. It is important to confirm the possibility of jet diaphragm deflection by 90 degrees using computer simulations. In the course of a physical experiment, it was previously proved that the jet could undergo diaphragm deflection by 90 degrees [1, 23, 24]. This paper presents new research results and describes numerical experiments, confirming the possibility of jet deflection by 90 degrees. It is shown that numerical experiments can partially replace expensive physical experiments. The computer simulation results reflect the main flow patterns at the outlet of a nozzle equipped with a diaphragm. The main research tasks at this stage include 1) the development of gas flow control principles in the channels of the mesh turbine, 2) the development of a jet control system prototype for the mesh turbine, and 3) confirm the possibility of jet diaphragm deflection by 90 degrees using computer simulation. 3-1- Materials (a) Photo of 3D printers used to develop mesh turbines with jet control systems and (b) flowchart to explain the research methodology. 3-1- Materials 3D printers were used to manufacture prototypes (Figure 1a); the characteristics of these printers are given in Table 1. The purpose of the prototypes is marked in the flowchart (Figure 1b). Table 1. Printer characteristics. 3D Printer model ZAV-L Ultimaker 2 (RepRap) Manufacturing country Russia Russia Plot area (X×Y×Z), mm 180×200×180 200×200×200 Maximum print speed, mm/s 120 90 Nozzle diameter, mm 0.3 0.6 Extruder maximum temperature, °С 280 280 Maximum heated bed temperature, °С 150 150 Extruder type Bowden Bowden Positioning accuracy, mm 0.02 0.02 Printing materials PLA, ABS, SBS, PETG PLA, ABS, SBS, PETG Table 1. Printer characteristics. Table 1. Printer characteristics. (a) (a) Page \| 777 Emerging Science Journal \| Vol. 5, No. 5 Figure 1. (a) Photo of 3D printers used to develop mesh turbines with jet control systems and (b) flowchart to explain the research methodology. The main concept is related to the study of gas-dynamic and hydrodynamic processes in the pulse mode of flow through rotating and fixed channels with a mesh structure Development of three-dimensional models to study mesh turbines equipped with jet control systems Computer simulation of mesh turbines and jet control systems. Performing numerical experiments Development of plans for developing applied research and R&D works Developing prototypes and micro models to test the performance of mesh turbines and jet control systems. Performing physical experiments Analysis of the results of computer simulations and physical experiments Development of technical proposals for solving practical problems, including energy- saving technologies Patenting of new technical solutions designed to solve current production problems (b) The main concept is related to the study of gas-dynamic and hydrodynamic processes in the pulse mode of flow through rotating and fixed channels with a mesh structure Development of three-dimensional models to study mesh turbines equipped with jet control systems Developing prototypes and micro models to test the performance of mesh turbines and jet control systems. Performing physical experiments Computer simulation of mesh turbines and jet control systems. Performing numerical experiments Analysis of the results of computer simulations and physical experiments Analysis of the results of computer simulations and physical experiments Development of technical proposals for solving practical problems, including energy- saving technologies Patenting of new technical solutions designed to solve current production problems Development of plans for developing applied research and R&D works Figure 1. 3-2- Methods The software package Flow Simulation (FloEFD) was used for computer simulation and computational research. The 3D model was created using the SolidWorks 3D CAD system. In the process of modeling, a complete system of Navier- Stokes equations — described by mathematical expressions of the laws of mass, energy, and momentum conservation— was solved. The 𝑘−𝜀 turbulent viscosity model was used. CPU type: Intel(R) Core (TM) i5-6200U CPU@2.30GHz; CPU speed: 2401 MHz; RAM: 8065 MB; Operating system: Windows 10. The main concept is related to the study of gas-dynamic and hydrodynamic processes during pulsed flow through rotating and fixed channels with a mesh structure (Figure 1b). The development of three-dimensional models for the study of net turbines equipped with jet control systems is envisaged. Based on the three-dimensional models, computer simulations of the mesh turbines and jet control systems are performed. In parallel with the calculations, prototypes and micro models are created to test the performance of the mesh turbines and jet control systems. Additive technologies are used for this purpose. Based on the results of numerical and physical experiments, technical proposals are developed to Page \| 778 Emerging Science Journal \| Vol. 5, No. 5 solve practical problems, including the creation of energy-saving technologies. The research results are also used for creating new models and prototypes and as a basis for future applied research and R&D works. Separate developments are patented. solve practical problems, including the creation of energy-saving technologies. The research results are also used for creating new models and prototypes and as a basis for future applied research and R&D works. Separate developments are patented. 4- Results New approaches to developing special turbines, pumps, and compressors are being considered part of the ongoing comprehensive research. The peculiarity of such machines is using a mesh structure in profiling the rotor and stator flow parts in a hydraulic machine of the dynamic type. The possibilities for the practical use of the ejector are also taken into account, given the rotational motion of its separate parts. When developing a new turbine, known designs, including a patent [25], were considered. The disadvantage of this turbine is that a quick (or emergency) rotor stop will require correspondingly quick working flow overlapping that can provoke a hydraulic shock. The technical problem to be solved with the new turbine design is strengthening control over rotor speed under operating conditions (and under extreme conditions) by reducing time to stop the rotor (deceleration). That increases the turbine operation safety and expands the application area. The achievable technical result is the prevention of pressure pulsations and hydraulic shocks during rotor braking and stopping. It is achieved by flow control at the outlet of nozzle diaphragms. The essence of the developed turbine [26] is explained in the figures. Figure 2 shows a schematic of the mesh turbine, Figure 3 – its A−A cross-section with the open nozzle. Figure 4 presents its A−A cross-section in the rotor braking and stopping mode, Figure 5 – the same with the closed nozzle. Figure 6 shows a schematic of a control sleeve with through holes, and Figure 7 shows a B−B cross-section of the mesh turbine when using a rotary hydraulic cylinder. Figure 2. Schematic of the mesh turbine: 1- stator; 2- inlet nozzles; 3- rotor; 4- outer protrusions; 5- inner protrusions; 6- flow channels; 7- outlet channel; 8- rotation axis; 9- vortex chamber; 10- circulating annular channel; 11- movable control sleeve; 12 and 13- hydraulic chambers; 14 and 15- through sockets; 16- through-holes. Figure 2. Schematic of the mesh turbine: 1- stator; 2- inlet nozzles; 3- rotor; 4- outer protrusions; 5- inner protrusions; 6- flow channels; 7- outlet channel; 8- rotation axis; 9- vortex chamber; 10- circulating annular channel; 11- movable control sleeve; 12 and 13- hydraulic chambers; 14 and 15- through sockets; 16- through-holes. Figure 3. 4- Results Schematic of the mesh turbine, A−A cross-section with the open nozzle: 1- stator; 2- inlet nozzles; 4- outer protrusions; 5- inner protrusions; 6- flow channels; 7- outlet channel; 9- vortex chamber; 10- circulating annular channel; 11-movable control sleeve; 16- through-holes. Figure 2. Schematic of the mesh turbine: 1- stator; 2- inlet nozzles; 3- rotor; 4- outer protrusions; 5- inner protrusions; 6- flow channels; 7- outlet channel; 8- rotation axis; 9- vortex chamber; 10- circulating annular channel; 11- movable control sleeve; 12 and 13- hydraulic chambers; 14 and 15- through sockets; 16- through-holes. Figure 3. Schematic of the mesh turbine, A−A cross-section with the open nozzle: 1- stator; 2- inlet nozzles; 4- outer protrusions; 5- inner protrusions; 6- flow channels; 7- outlet channel; 9- vortex chamber; 10- circulating annular channel; 11-movable control sleeve; 16- through-holes. Figure 3. Schematic of the mesh turbine, A−A cross-section with the open nozzle: 1- stator; 2- inlet nozzles; 4- outer protrusions; 5- inner protrusions; 6- flow channels; 7- outlet channel; 9- vortex chamber; 10- circulating annular channel; 11-movable control sleeve; 16- through-holes. Page \| 779 Page \| 779 Emerging Science Journal \| Vol. 5, No. 5 Figure 4. Schematic of the mesh turbine, A−A cross-section in the rotor braking and stopping mode. Figure 4. Schematic of the mesh turbine, A−A cross-section in the rotor braking and stopping mode. Figure 5. Schematic of the mesh turbine, A−A cross-section with the closed nozzle. Figure 6. Schematic of the control sleeve (meshed diaphragm) with through holes: (а) and a prototype; (b) 11- movable control sleeve; 16- through-holes. Figure 6 shows a meshed diaphragm with holes on a cylindrical surface. Flat diaphragms [27] are known to contr he flow at the nozzle outlet. It is also possible to use a spherical surface to make a diaphragm with multiple holes f he passage of gas or liquid. The holes themselves may be a circular, square, rectangular, rhombus, or other geometr ncluding unique variants with unconventional geometry. The unconventional nozzle and methods of regulation of su ozzle diaphragms are still very poorly understood. (a) (b) Figure 4. Schematic of the mesh turbine, A−A cross-section in the rotor braking and stopping mode. Figure 5 Schematic of the mesh turbine A A cross section with the close Figure 5. Schematic of the mesh turbine, A−A cross-section with the closed nozzle. Figure 6. 4- Results B−B cross-section of the mesh turbine using a rotary hydraulic cylinder: 1- stator; 11- movable control sleeve; 12 and 13- hydraulic chambers; 14 and 15- through sockets. The developed turbine contains stator 1 with inlet nozzles 2 and is placed in it rotor 3 with outer protrusions 4. The turbine design can have one, two, or more nozzles. The nozzle diaphragm may have a mesh structure, where the surface of the nozzle has many holes. Rotor 3 is equipped with inner protrusions 5 and comprises a porous mesh structure consisting of protrusions 4 and 5 interconnected and having solid walls. Between inner 5 and outer 4 protrusions in rotor 3, there are flow channels 6, providing hydraulic connection of flow channels 6 in rotor 3 with inlet nozzles 2 in stator 1. Stator 1 is equipped with outlet channel 7. Given the turbine application conditions, inlet nozzles 2 and outlet channel 7 can be located at different distances from rotation axis 8 of rotor 3. Stator 1 contains vortex chamber 9 installed coaxially with the rotor. Inlet nozzles 2 are located in the central part of rotor 3, forming circulating annular channel 10 in the gap between inlet nozzles 2 and inner protrusions 5. In vortex chamber 9, movable control sleeve 11 is placed with the possibility of adjusting its adjustable rotary movement for partial or complete overlapping of inlet nozzles 2. The position of movable control sleeve 11 can be changed using known technologies, such as a hydraulic or electromagnetic drive. The figures show a variant with a hydraulic drive to rotate movable control sleeve 11. Hydraulic chambers 12 and 13 can be connected to the hydraulic control system through sockets 14 and 15 (the hydraulic control system is not shown in the figures). Control sleeve 11 has through- holes 16 through which inlet nozzles 2 communicate with flow channels 6 of rotor 3. Inflow channels 6 of rotor 3 can implement the ejector workflow, and flow channels 6 can act as a mixing chamber and ejector diffuser. The presented variant uses many mixing chambers combined into a mesh structure. The formed mesh structure is placed inside the turbine rotor. In this case, nozzles and mixing chambers of unconventional shape are used. The curvilinear surfaces of the flow channels help increase the efficiency of the energy conversion process in the turbomachine. 4- Results Schematic of the control sleeve (meshed diaphragm) with through holes: (а) and a prototype; (b) 11- movable control sleeve; 16- through-holes. Figure 6 shows a meshed diaphragm with holes on a cylindrical surface. Flat diaphragms [27] are known to control he flow at the nozzle outlet. It is also possible to use a spherical surface to make a diaphragm with multiple holes for he passage of gas or liquid. The holes themselves may be a circular, square, rectangular, rhombus, or other geometry, ncluding unique variants with unconventional geometry. The unconventional nozzle and methods of regulation of such ozzle diaphragms are still very poorly understood. (a) (b) Figure 5. Schematic of the mesh turbine, A−A cross-section with the closed nozzle. Figure 6. Schematic of the control sleeve (meshed diaphragm) with through holes: (а) and a prototype; (b) 11- movabl control sleeve; 16- through-holes (a) (b) (a) (b) Figure 6. Schematic of the control sleeve (meshed diaphragm) with through holes: (а) and a prototype; (b) 11- movable control sleeve; 16- through-holes. Figure 6 shows a meshed diaphragm with holes on a cylindrical surface. Flat diaphragms [27] are known to control the flow at the nozzle outlet. It is also possible to use a spherical surface to make a diaphragm with multiple holes for the passage of gas or liquid. The holes themselves may be a circular, square, rectangular, rhombus, or other geometry, including unique variants with unconventional geometry. The unconventional nozzle and methods of regulation of such nozzle diaphragms are still very poorly understood. Figure 6 shows a meshed diaphragm with holes on a cylindrical surface. Flat diaphragms [27] are known to control the flow at the nozzle outlet. It is also possible to use a spherical surface to make a diaphragm with multiple holes for the passage of gas or liquid. The holes themselves may be a circular, square, rectangular, rhombus, or other geometry, including unique variants with unconventional geometry. The unconventional nozzle and methods of regulation of such nozzle diaphragms are still very poorly understood. Page \| 780 Page \| 780 Emerging Science Journal \| Vol. 5, No. 5 Emerging Science Journal \| Vol. 5, No. 5 Figure 7. B−B cross-section of the mesh turbine using a rotary hydraulic cylinder: 1- stator; 11- movable control sleeve; 12 and 13- hydraulic chambers; 14 and 15- through sockets. Figure 7. 4- Results It is worth noting that the intermediate position of movable control sleeve 11 can be changed by modifying the volume of liquid contained in the two hydraulic chambers (which is a common approach in hydraulically-driven control systems based on a rotary hydraulic cylinder, as shown in Figure 7). Moreover, due to the angular movement of movable control sleeve 11, the flow rate at the outlet of nozzles 2 can be regulated through their partial or complete overlap, which would, in turn, modify the turbine working mode. In addition, by changing the position of through-holes 16 relative to nozzles 2, it is technically possible to reverse the rotation direction of rotor 3, as shown in Figure 4. Figure 3 shows the A−A cross-section of the turbine with nozzles 2 in the open position, with the flow direction at their outlet indicated by arrows. As can be seen from the image, turning movable control sleeve 11 in a clockwise direction can arrive at the position shown in Figure 4, thereby changing the flow direction at the outlet of nozzles 2 as indicated by the arrows. Due to the pressure-induced by medium flow, rotor 3 will gradually decelerate until it reaches a complete stop, even though the medium can flow towards outlet channel 7. In contrast to the turbine under the patent [25], the new turbine [26] has new design features and technical capabilities. Nonetheless, stopping rotor 3 will prevent pressure pulsations and hydraulic shocks when braking. In other words, rotor 3 will cease to move whether the inlet nozzles 2 are completely (Figure 5) or partially (Figure 4) closed. Therefore, the energy of the medium flow helps strengthen the control of the rotor speed when operating in extreme modes while reducing its stopping time (expediting its deceleration). This design feature increases the turbine operation safety and expands the range of its applications, including production facilities in the oil and gas industry. The developed operating principle of the turbine [26] was considered through an example, which does not limit the scope of application of the new technical solution. At the same time, turbine modifications are possible within the framework of the disclosed invention [26]. Based on the presented principle of operation, it is possible to create various single-stage and multi-stage impeller machinery with a mesh structure of flow channels. The jets can move in the radial and the axial direction. 4- Results The developed design of the turbine with a jet control system is patented in Russia [26]. The turbine works as follows: Stator 1 and inlet nozzles 2 form the flow (or several flows) of the fluid (medium) towards rotor 3 (Figure 2). The medium can be liquid, gas-liquid mixture, or gas (including steam or high-temperature combustion products of fuel-air mixture). The fluid flow impacts on inner protrusions 5 of rotor 3 and puts rotor 3 in motion. Thus, the kinetic energy of the fluid flow is converted into mechanical energy during the rotary motion of rotor 3. Rotor 3 can be connected with external mechanisms (not shown in the figures). In addition, the fluid flow through flow channel 6 penetrates the cavity of rotor 3, presented as a porous volumetric mesh structure. The fluid flow in this part of rotor 3 interacts with the inner 5 and outer 4 protrusions' solid walls, contributing to energy conversion. It is possible to implement both continuous and pulse fluid flow to the turbine rotor 3. The permeable mesh structure of rotor 3 provides energy conversion at different fluid properties, including gas-liquid mixtures different in density or viscosity. The fluid is removed through flow channel 6 in rotor 3 and outlet channel 7 out of stator 1. The position of movable control sleeve 11 and accordingly the one position of through-holes 16 can be changed. To close inlet nozzles 2, hydraulic chamber 12 is supplied with liquid under high pressure through socket 14. The liquid is displaced from hydraulic chamber 13 into the low-pressure line through nozzle 15. Under the differential pressure in the hydraulic chambers 12 and 13, movable control sleeve 11 turns around rotation axis 8 and closes nozzles 2. Page \| 781 Emerging Science Journal \| Vol. 5, No. 5 For opening inlet nozzles 2, the liquid is supplied into hydraulic chamber 13 under high pressure through socket 15. As a result, the fluid is displaced from hydraulic chamber 12 into the low-pressure line through socket 14. Owing to the pressure difference between hydraulic chambers 12 and 13, movable control sleeve 11 turns around the rotation axis 8, opening inlet nozzles 2. 4- Results An impeller, nozzle block, and control sleeve (diaphragm) prototype together with a disk axial turbine prototype. The turbine developed and presented in this article is similar to a partial admission turbine [12-14]. As a rule, a turbine is called a partial admission one when the nozzle block has blades only on the part of its circumference; in this connection, the gas is supplied to the impeller only within this part of the circumference. Figure 15 shows a typical schematic diagram of a partial admission turbine. Emerging Science Journal \| Vol. 5, No. 5 Figure 14. An impeller, nozzle block, and control sleeve (diaphragm) prototype together with a disk a An impeller, nozzle block, and control sleeve (diaphragm) prototype together with a disk axial turbine prototype The turbine developed and presented in this article is similar to a partial admission turbine [12-14]. As a rule, a turbine is called a partial admission one when the nozzle block has blades only on the part of its circumference; in this connection, the gas is supplied to the impeller only within this part of the circumference. Figure 15 shows a typical schematic diagram of a partial admission turbine. The turbine developed and presented in this article is similar to a partial admission turbine [12-14]. As a rule, a turbine is called a partial admission one when the nozzle block has blades only on the part of its circumference; in this connection, the gas is supplied to the impeller only within this part of the circumference. Figure 15 shows a typical schematic diagram of a partial admission turbine. Figure 15. Schematic of a partial admission turbine: 1- generator; 2- nozzle block; 3- working gas source; 4- casing; 5- impeller; 6- separation disc. Figure 15. Schematic of a partial admission turbine: 1- generator; 2- nozzle block; 3- working gas source; 4- casing; 5- impeller; 6- separation disc. In this case, the energy conversion process during the jet interaction with the solid walls of the impeller can be considered separately (Figure 15). When discussing the principle of operation, it is possible to use the following simplified formula for a partial admission turbine. (1) 𝑁0 = 𝑁𝑚+ 𝑑𝑁, where N0 is the working fluid flow rate with the mass flow rate Q0; Nm is mechanical power transmitted through the shaft connecting impeller 5 to the generator 1; 𝑑𝑁 is power loss in impeller 5. 4- Results For illustration, Figures 8-14 show photos of the developed and manufactured impeller prototypes for mesh centrifugal and axial turbines and a nozzle apparatus prototype operating according to the described principles under the patent [26]. Figure 8. Mesh turbine impeller prototypes: on the left in the background - a cylindrical centrifugal turbine, in the foreground in the center - an axial disk turbine, on the right in the background - a conical centrifugal turbine. esh turbine impeller prototypes: on the left in the background - a cylindrical centrifugal turbine, in the foregro in the center - an axial disk turbine, on the right in the background - a conical centrifugal turbine. Figure 8. Mesh turbine impeller prototypes: on the left in the background - a cylindrical centrifugal turbine, in the foreground in the center - an axial disk turbine, on the right in the background - a conical centrifugal turbine. Figure 9. A nozzle block prototype (shown on the left) and a control sleeve (diaphragm) prototype. Figure 9. A nozzle block prototype (shown on the left) and a control sleeve (diaphragm) prototype. Page \| 782 Page \| 782 Emerging Science Journal \| Vol. 5, No. 5 Figure 10. A nozzle block prototype (shown on the left) and a control sleeve (diaphragm) prototype in the working position. Emerging Science Journal \| Vol. 5, No. 5 Figure 10. A nozzle block prototype (shown on the left) and a control sleeve (diaphragm) prototyp A nozzle block prototype (shown on the left) and a control sleeve (diaphragm) prototype in the working position Figure 11. A nozzle block prototype (shown on the left) and a control sleeve (diaphragm) prototype in the position for reversible rotation of the turbine impeller. Figure 11. A nozzle block prototype (shown on the left) and a control sleeve (diaphragm) prototype in the position for reversible rotation of the turbine impeller. Figure 12. A nozzle block prototype (shown on the left) and a control sleeve (diaphragm) prototype in the closed position. Figure 12. A nozzle block prototype (shown on the left) and a control sleeve (diaphragm) prototype in the closed position. Figure 13. An impeller, nozzle, and control sleeve (diaphragm) prototype together with a conical centrifugal turbine prototype. Figure 13. An impeller, nozzle, and control sleeve (diaphragm) prototype together with a conical centrifugal turbine prototype. Page \| 783 Emerging Science Journal \| Vol. 5, No. 5 Figure 14. 4- Results The efficiency of such an energy conversion process for a partial turbine can be expressed using multiple valuation 𝜂𝑥. 𝜂𝑥= 𝑁𝑚 𝑁0 , (2) Suppose the separation disk 6 is removed from the partial admission turbine, and the nozzle block is equipped with a velocity vector (or thrust vector) control system. In that case, it is possible to pass on to a new class of impeller machinery. At the discussion stage, this new class of machines will be called hybrid mesh machinery. Figure 16 shows a schematic diagram of a hybrid mesh turbine. The velocity vector (thrust vector) control system can have various embodiments [25]. This paper considers a variant of controlling the velocity vector using a movable diaphragm installed at the nozzle outlet. A diaphragm with a round hole can be called a classic one. In the developed prototype and the patent [26], a diaphragm with several rectangular holes made on a cylindrical wall is used; all these diaphragm embodiments operate on the same principles. Page \| 784 Emerging Science Journal \| Vol. 5, No. 5 Emerging Science Journal \| Vol. 5, No. 5 Figure 16. A hybrid mesh turbine schematic: 1- generator; 2- nozzle block; 3- working gas source; 4- casing; 5- impeller; 7- velocity vector control system; 8, 9, and 10- annular flow channels in which the blades are located. Figure 16. A hybrid mesh turbine schematic: 1- generator; 2- nozzle block; 3- working gas source; 4- casing; 5- impeller; 7- velocity vector control system; 8, 9, and 10- annular flow channels in which the blades are located. The velocity vector control system 7 enables direct the working fluid flow into the annular channel 8, 9, or 10, solving the technical problem (view-A in Figure 16). In this case, it is possible to regulate the torque on the impeller shaft since the working radius changes (the distance from the impeller rotation axis to the point at which the working fluid flow interacts with the solid walls of the impeller). The blades in the annular flow channels 8, 9, and 10 can have different geometry and sizes, depending on the technical or technological problem. In a hybrid mesh turbine, two different workflows can run simultaneously. The first process is a turbine workflow, which is similar to the workflow taking place in the partial turbine channels. 4- Results The second workflow is a pumping process if the pumped fluid is a liquid. If the pumped fluid is a gas, the second workflow can be a compressor or fan process, depending on the pressure difference at the impeller inlet and outlet. A variant is also considered when the pumped fluid is a multiphase medium (gas-liquid mixture, for example). In the diagram, the part of the impeller located below the rotation axis participates in implementing the turbine workflow (or the first workflow). The kinetic energy of the working fluid flow Q0 is converted into mechanical energy, which is transferred to the impeller. In the diagram, the part of the impeller located above the rotation axis participates in implementing the second workflow. The impeller blades exert a force effect on the pumped fluid, transferring part of the energy to create (form) the flow Q1. A more detailed examination of the hybrid turbine requires considering separately the third working process, which takes place behind the impeller, in casing 4. Here, the ejector workflow will be implemented. The process of mixing the working fluid flow Q0 with the pumped medium flow Q1 can be fully considered as a separate process, namely, as an ejector workflow. Casing 4 serves as a mixing chamber within the ejector working process. Since impeller 5 is located in casing 4 between the nozzle block and the outlet channel, it can be stated that the flow channels of impeller 5 are part of the mixing chamber. The flow channels of impeller 5 make a rotational movement, activating the energy conversion process and activating the ejector workflow. Thus, it is necessary to suggest an ejector of an unconventional shape, in which the mixing chamber consists of at least two parts. In this case, one of these two parts of the mixing chamber makes a rotational movement. Such ejectors of unconventional shape can act as objects for independent scientific research. Considering the variety of applied (practical) tasks, we can discuss the expediency of developing this scientific domain. For example, certain tasks and operating conditions are known, in which the ejector operates with low efficiency, at the level of 15%. In the future, it will be possible to create an unconventional ejector with higher efficiency, at the level of 30% for these conditions. 4- Results The energy conversion process can be separately considered for a hybrid mesh turbine when the jet interacts with the solid walls of the impeller (Figure 16). At the stage of discussing the principle of operation, the following simplified formula can be applied: (3) 𝑁0 = 𝑁𝑚+ 𝑁1 + 𝑑𝑁, Where N1 is the flow rate of the pumped medium with the mass flow rate Q1. Good yield in such a simplified system can be estimated by the sum of two terms (Nm + N1). The efficiency of such an energy conversion process for a hybrid turbine can be expressed using multiple valuation ηx. 𝜂𝑥= 𝑁𝑚+ 𝑁1 𝑁0 , (4) 𝜂𝑥= 𝑁𝑚+ 𝑁1 𝑁0 , (4) Figure 17 shows a schematic diagram of a hybrid mesh turbine when switching between operating modes. The velocity vector control system enables to direct part of the working fluid or the entire flow 11 in the other direction, bypassing impeller 5. This switching may be associated with the need for the turbine emergency shutdown for safety reasons. Alternatively, such switching may be related to solving other problems within the framework of controlling the entire technological system, in which the hybrid turbine is considered a subsystem. Page \| 785 Emerging Science Journal \| Vol. 5, No. 5 Figure 17. Schematic of a hybrid mesh turbine when switching the operating mode: 1- generator; 2- nozzle block; 3- working gas source; 4- casing; 5- impeller; 7- velocity vector control system; 11- working fluid flow. Figure 17. Schematic of a hybrid mesh turbine when switching the operating mode: 1- generator; 2- nozzle block; 3- working gas source; 4- casing; 5- impeller; 7- velocity vector control system; 11- working fluid flow. The considered basic principles of a hybrid mesh turbine operation can be used to develop new technical and technological systems in various industries. For example, another machine can be used instead of generator 1 in Figure 16, such as a pump, a fan, or a compressor. Alternatively, another motor can be used instead of generator 1 in Figure 16: an electric motor or a turbofan gas turbine engine. In this case, impeller 5 of Figure 16 will operate as an afterburner for a temporary increase in power and thrust; such prospects are already visible for unmanned vehicles. 5- Discussion 5 According to the available evidence about known control systems, the deflection angle of the thrust vector (denoted ∝ in Figure 18) can vary from +20o to -20o [25, 29, 30]. However, laboratory test results ∝ can take any value from +90o to -90o in any direction (i.e., within a hemisphere) [1, 23]. According to the available evidence about known control systems, the deflection angle of the thrust vector (denoted ∝ in Figure 18) can vary from +20o to -20o [25, 29, 30]. However, laboratory test results ∝ can take any value from +90o to -90o in any direction (i.e., within a hemisphere) [1, 23]. These findings suggest that the control range of the thrust vector deflection angle can be significantly extended. Additionally, note that the control range can be extended even further with a more comprehensive approach to design. When designing multi-mode and universal turbine technologies, the goal to achieve an extremely large control range for changing ∝ from +180o to -180o in all directions have been set. Preliminary design studies and laboratory tests of the micromodels have shown that this goal is achievable and ∝ can vary from +180o to -180o in any direction within a full three-dimensional sphere, for example, in simultaneous regulating the mass rate of gas and ∝ and β values for two jets. More detailed information on this issue is planned to publish after conducting additional patent research. There is a good prospect for improving the technical performance of such control systems, and scientific and patent research should continue in this direction. Table 2 shows a schematic representation of the switchgear, indicating the flow direction for some working modes, given the similarity with the diagram in Figure 18. Table 2. Switchgear working modes. In Table 2, line “A” schematically shows the variants in which angle ∝=0. The gas flow rate through the upper channel Q∝ >0. Through the lower channel –Qβ>0. In this case, angle β can vary over a wide range of values (- 90)≤β≤(+90). Line “B” schematically shows some variants in which ∝ can vary over a wide range of values (- 90)≤∝≤(+90), the gas flow rate through the upper channel Q∝>0. Through the lower channel –Qβ>0. In this case, β can also vary in a wide range of values (-90)≤β≤(+90). Line “C” schematically shows some variants in which ∝=(+90), the gas flow rate through the upper channel Q∝>0. 5- Discussion Through the lower channel –Qβ>0. In this case, β can vary in a wide range of values: (-90)≤β≤(+90). Line “D” schematically shows some variants in which the gas flow rate through the upper channel Q∝=0. Through the lower channel –Qβ>0. In this case, β can vary in a wide range of values: (- 90)≤β≤(+90). By analogy, other variants can be considered when Q∝>0. And the gas flow rate through the lower channel Qβ=0.1. Fi 19 h h ti di f th it h i t t h ( l t ) th fl di ti th fl Table 2. Switchgear working modes. Table 2. Switchgear working modes. In Table 2, line “A” schematically shows the variants in which angle ∝=0. The gas flow rate through the upper channel Q∝ >0. Through the lower channel –Qβ>0. In this case, angle β can vary over a wide range of values (- 90)≤β≤(+90). Line “B” schematically shows some variants in which ∝ can vary over a wide range of values (- 90)≤∝≤(+90), the gas flow rate through the upper channel Q∝>0. Through the lower channel –Qβ>0. In this case, β can also vary in a wide range of values (-90)≤β≤(+90). Line “C” schematically shows some variants in which ∝=(+90), the gas flow rate through the upper channel Q∝>0. Through the lower channel –Qβ>0. In this case, β can vary in a wide range of values: (-90)≤β≤(+90). Line “D” schematically shows some variants in which the gas flow rate through the upper channel Q∝=0. Through the lower channel –Qβ>0. In this case, β can vary in a wide range of values: (- 90)≤β≤(+90). By analogy, other variants can be considered when Q∝>0. And the gas flow rate through the lower channel Qβ=0.1. Figure 19 shows a schematic diagram of the switchgear variant to change (regulate) the flow direction: the gas flow rate through the upper channel Q∝>0, through the lower channel –Qβ=0. In this example, the liquid or gas flow is supplied to switchgear 1 through inlet channel 2. At the outlet of switchgear 1, one jet 3 is formed, flowing out into the surrounding space. The diagram shows axial line 5 and angles ∝1 and ∝2 to quantify the jet direction. Jet 3 can interact with the solid wall of deflector 4, which can be stationary or movable. 5- Discussion When developing new jet systems in this study, research results from various industries, including aviation and space technologies, are utilized to expedite the process. In this regard, the study of jet technology has considered the control of the velocity vector or thrust vector, using aviation terminology. That is a novel approach to addressing this issue, as hydrodynamic and gas-dynamic systems' scientific and technical potential in controlling processes associated with changes in the fluid or gas flow direction, including the consideration of magneto-hydrodynamic control systems [28], has not been fully explored. As was shown in this work, the application area of new turbomachines can be greatly expanded by finding suitable means of changing the direction of fluid or gas flow. Figure 18 shows a schematic diagram of the switchgear variant with the ability to change (regulate) the direction of one or two flows. Additionally, a 3D model is shown for the switchgear variant, which explains the principles of flow distribution. In this example, a flow of liquid or gas is supplied to switchgear 1 through inlet channel 2. At the outlet of switchgear 1, one or two jets (denoted by 3 and 4) are formed, flowing into the surrounding space that can be filled with liquid or gas. The schematic diagram shows axial line 5 and angles ∝ and β denoting jet direction. This angular parameter can be called the deflection angle of the velocity vector (or the deflection angle of the thrust vector, by analogy with the term used in aviation technology). Figure 18. Switchgear diagram – variant No. 1 and a 3D model: 1- switchgear; 2- inlet channel; 3 and 4- gas jet; 5- axial line; 6- upper movable diaphragm; 7- the bottom movable diaphragm. (a) (b) Figure 18. Switchgear diagram – variant No. 1 and a 3D model: 1- switchgear; 2- inlet channel; 3 and 4- gas jet; 5- axial line; 6- upper movable diaphragm; 7- the bottom movable diaphragm. Radial displacement of diaphragms 6 and 7 changes the angles “∝” and “β”. In extreme positions, the diaphragm can completely block the flow channel. Radial displacement of diaphragms 6 and 7 changes the angles “∝” and “β”. In extreme positions, the diaphragm can completely block the flow channel. Page \| 786 Page \| 786 Emerging Science Journal \| Vol. 5, No. 5- Discussion This paper considers the variant in which deflector 4 can perform a rotational motion around rotation center 6. It may be noted that due to the complexity and insufficient study of gas-dynamic processes, the interest in studying various processes of interaction of a gas jet with a barrier is still strong [31-34]. Page \| 787 Page \| 787 Emerging Science Journal \| Vol. 5, No. 5 Emerging Science Journal \| Vol. 5, No. 5 Figure 19. Switchgear diagram – variant No. 2: 1- switchgear; 2- inlet channel; 3- gas jet; 4- deflector; 5- axial line; 6-rotation center. The presented scheme is the basis of the new turbine design. The developed turbine can be referred to as the group f pulse turbines [35-37]. Jet 3 can periodically interact with a solid wall of deflector 4. The spatial jet position can eriodically change, for example, due to the alternation of variants 2 and 3, according to Figures 19 and 20. Figure 19. Switchgear diagram – variant No. 2: 1- switchgear; 2- inlet channel; 3- gas jet; 4- deflector; 5- axial line; 6-rotation center. The presented scheme is the basis of the new turbine design. The developed turbine can be referred to as the group of pulse turbines [35-37]. Jet 3 can periodically interact with a solid wall of deflector 4. The spatial jet position can periodically change, for example, due to the alternation of variants 2 and 3, according to Figures 19 and 20. The presented scheme is the basis of the new turbine design. The developed turbine can be referred to as the group of pulse turbines [35-37]. Jet 3 can periodically interact with a solid wall of deflector 4. The spatial jet position can periodically change, for example, due to the alternation of variants 2 and 3, according to Figures 19 and 20. Figure 20. Switchgear diagram – variant No. 3. Figure 20. Switchgear diagram – variant No. 3. Figure 20. Switchgear diagram – variant No. 3. An increase in pressure (Figure 19) or a decrease in pressure (Figure 21), compared with the ambient pressure, can be observed at the contact zone of gas jet 3 with the solid wall of deflector 4. The reduction of gas (liquid) pressure near the surface of the solid wall may be related to the manifestation of the Coanda effect [38]. Figure 21. Switchgear diagram – variant No. 5- Discussion 4: 1- switchgear; 2- inlet channel; 3- gas jet; 4- deflector; 5- axial line. Figure 21. Switchgear diagram – variant No. 4: 1- switchgear; 2- inlet channel; 3- gas jet; 4- deflector; 5- axial line. Page \| 788 Page \| 788 Emerging Science Journal \| Vol. 5, No. 5 Deflector 4 may be in the form of a tube, as shown in variant No. 5 in Figure 22. The tube itself can be stationary or movable. For example, in the well-known Euler turbine, tubes are included in the rotating rotor. Deflector 4 may be in the form of a tube, as shown in variant No. 5 in Figure 22. The tube itself can be stationary or movable. For example, in the well-known Euler turbine, tubes are included in the rotating rotor. Figure 22. Switchgear diagram – variant No. 5: 1- switchgear; 2- inlet channel; 3- gas jet; 4- piped deflector. Figure 22. Switchgear diagram – variant No. 5: 1- switchgear; 2- inlet channel; 3- gas jet; 4- piped deflector At the stage of exploratory research, about the published information, it was decided to use the FloEFD software package for computer modeling, which allows studying impeller machinery and high-speed jet streams. Thus, computer modeling experts [39] note that comparing the results obtained in the FloEFD, ANSYS Fluent, and RC-VTOL software packages shows satisfactory and good compliance with the experiment in some modes. The implementation of sliding mesh technology for rotation modeling allowed FloEFD to dive into solving complex aerodynamic problems, such as modeling the main helicopter rotor and validate a single rotor blade flow together with the Central Institute of Aerohydrodynamics. In another research, it was concluded that the FloEFD software package could be used to study jet flows of a solid-propellant landing propulsion system, especially at the preliminary design stage, since it reproduces the general structure of the jet and gives satisfactory results for the near flow field with a small investment of time and computational resources [40]. Special diaphragms located at the nozzle outlet can be used to control the flow direction. The design of the adjustable nozzle using a deflector made in the form of a diaphragm is known from U.S. patent No. 3013494 [27]. One of such variants of the switchgear is shown in Figures 23 and 24. 5- Discussion In this case, nozzle 1 is equipped with movable diaphragm 2, according to the technical solution presented in the patent [27]. The figure does not show the parts for moving and fixing the diaphragm. As already noted, a round-hole diaphragm can be called classic. The developed prototype and the patent used a diaphragm with several rectangular holes made on a cylindrical wall [26]. All these diaphragm embodiments operate on the same principles. It was decided to start the research with the classic version, as the simplest variant. Figure 23. Structural model of a nozzle with a movable diaphragm: 1- nozzle; 2- the movable diaphragm. A 3D model of a nozzle with a movable diaphragm is shown in Figure 24. Figure 23. Structural model of a nozzle with a movable diaphragm: 1- nozzle; 2- the movable diaphragm. Figure 23. Structural model of a nozzle with a movable diaphragm: 1- nozzle; 2- the movable diaphragm. A 3D model of a nozzle with a movable diaphragm is shown in Figure 24. Figure 23. Structural model of a nozzle with a movable diaphragm: 1 nozzle; 2 the movable diaphragm. A 3D model of a nozzle with a movable diaphragm is shown in Figure 24. A 3D model of a nozzle with a movable diaphragm is shown in Figure 24. Page \| 789 Page \| 789 Emerging Science Journal \| Vol. 5, No. 5 Figure 24. 3D model of a nozzle with a movable diaphragm (view with a radial diaphragm displacement is shown). Nozzle dimensions: D11 = 8 mm; D1 = 6 mm; D2 = 8.34 mm; D22 = 13 mm; γ1 = 45 degrees; γ2 = 7 degrees. A eries of calculations were performed to assess the technical capabilities of the known nozzle equipped with a diaphragm. As an example, the conditions of air outflow through the nozzle with the inlet pressure of 1.0 MPa (temperature of 40 С) at ambient pressure of 0.1 MPa (temperature of 20 °С) are considered. When the nozzle and the diaphragm are rranged concentrically, as shown in Figure 25, the jet at the nozzle outlet extends along the longitudinal nozzle axis. Calculation parameters: total number of cells in the computational grid = 1153165; computation time = 29298 s; the umber of iterations = 1499. Emerging Science Journal \| Vol. 5, No. 5 Figure 24. 5- Discussion 3D model of a nozzle with a movable diaphragm (view with a radial diaphragm displacement is shown). ure 24. 3D model of a nozzle with a movable diaphragm (view with a radial diaphragm displacement is shown). Nozzle dimensions: D11 = 8 mm; D1 = 6 mm; D2 = 8.34 mm; D22 = 13 mm; γ1 = 45 degrees; γ2 = 7 degrees. A series of calculations were performed to assess the technical capabilities of the known nozzle equipped with a diaphragm. As an example, the conditions of air outflow through the nozzle with the inlet pressure of 1.0 MPa (temperature of 40 °С) at ambient pressure of 0.1 MPa (temperature of 20 °С) are considered. When the nozzle and the diaphragm are arranged concentrically, as shown in Figure 25, the jet at the nozzle outlet extends along the longitudinal nozzle axis. Calculation parameters: total number of cells in the computational grid = 1153165; computation time = 29298 s; the number of iterations = 1499. Nozzle dimensions: D11 = 8 mm; D1 = 6 mm; D2 = 8.34 mm; D22 = 13 mm; γ1 = 45 degrees; γ2 = 7 degrees. A series of calculations were performed to assess the technical capabilities of the known nozzle equipped with a diaphragm. As an example, the conditions of air outflow through the nozzle with the inlet pressure of 1.0 MPa (temperature of 40 °С) at ambient pressure of 0.1 MPa (temperature of 20 °С) are considered. When the nozzle and the diaphragm are arranged concentrically, as shown in Figure 25, the jet at the nozzle outlet extends along the longitudinal nozzle axis. Calculation parameters: total number of cells in the computational grid = 1153165; computation time = 29298 s; the number of iterations = 1499. (a) (b) Figure 25. Gas outflow pattern with a concentric arrangement of nozzle and diaphragm: (a) calculation data; (b) A fragment of the adapted computational grid; (с) A fragment of the report on the calculation data convergence. (a) (b) (c) (a) (a) (c) Figure 25. Gas outflow pattern with a concentric arrangement of nozzle and diaphragm: (a) calculation data; (b) A fragment of the adapted computational grid; (с) A fragment of the report on the calculation data convergence. Page \| 790 Emerging Science Journal \| Vol. 5, No. 5- Discussion 5 When the diaphragm is radially displaced relative to the nozzle, as shown in Figure 26, at the nozzle exit, the jet deviates from the longitudinal axis of the nozzle to a certain angle. Calculation parameters: total number of cells in the computational grid 1137689; computation time 27742 с; iterations 1447. When the diaphragm is radially displaced relative to the nozzle, as shown in Figure 26, at the nozzle exit, the jet deviates from the longitudinal axis of the nozzle to a certain angle. Calculation parameters: total number of cells in the computational grid 1137689; computation time 27742 с; iterations 1447. Figure 26. Gas outflow pattern when the diaphragm is radially displaced relative to the nozzle: (a) Calculation data; (b) A fragment of the adapted computational grid; (с) A fragment of the report on the calculation data convergence. Based on the calculations performed, it can be confirmed that using the known design of the expanding nozzle with a flat diaphragm, the largest jet deflection angle at the nozzle outlet can vary within a narrow range from +20o to -20o. The nozzle design was further modified when the cone angle γ2 was increased to 60o (according to the structural model (a) (b) (c) (a) (b) (a) (a) (a) (c) Figure 26. Gas outflow pattern when the diaphragm is radially displaced relative to the nozzle: (a) Calculation data; (b) A fragment of the adapted computational grid; (с) A fragment of the report on the calculation data convergence. Based on the calculations performed, it can be confirmed that using the known design of the expanding nozzle with a flat diaphragm, the largest jet deflection angle at the nozzle outlet can vary within a narrow range from +20o to -20o. The nozzle design was further modified when the cone angle γ2 was increased to 60o (according to the structural model in Figure 23). A series of calculations were performed to assess the technical capabilities of the modified nozzle equipped with a diaphragm. Calculations were performed for conditions using gaseous and liquid model media, and similar results were obtained in two different media. Different calculation results are presented graphically in Figures 27-29. Based on the calculations performed, it can be confirmed that using the known design of the expanding nozzle with a flat diaphragm, the largest jet deflection angle at the nozzle outlet can vary within a narrow range from +20o to -20o. 5- Discussion The nozzle design was further modified when the cone angle γ2 was increased to 60o (according to the structural model in Figure 23). A series of calculations were performed to assess the technical capabilities of the modified nozzle equipped with a diaphragm. Calculations were performed for conditions using gaseous and liquid model media, and similar results were obtained in two different media. Different calculation results are presented graphically in Figures 27-29. Nozzle dimensions: D11 = 12 mm; D1 = 6 mm; D2 = 6 mm; γ1 = 60 degrees; γ2 = 60 degrees The conditions of water flowing through the nozzle with the inlet pressure of 0.3 MPa at ambient pressure of 0.1 MPa are considered. Angle ∝ is marked in the inscription in Figure 18. Calculation parameters: total number of cells in the computational grid 334512; computation time 6974 с; the number of iterations 1189. Page \| 791 Page \| 791 Emerging Science Journal \| Vol. 5, No. 5 Emerging Science Journal \| Vol. 5, No. 5 Figure 27. Liquid outflow pattern from a nozzle with a diaphragm for conditions when ∝ =0o: (a) Calculation data; (b) A fragment of the adapted computational grid; (с) A fragment of the report on the calculation data convergence. Calculation parameters: total number of cells in the computational grid 320911; computation time 14404 с; the (a) (b) (c) (a) (b) (c) Figure 27. Liquid outflow pattern from a nozzle with a diaphragm for conditions when ∝ =0o: (a) Calculation data; (b) A fragment of the adapted computational grid; (с) A fragment of the report on the calculation data convergence. Calculation parameters: total number of cells in the computational grid 320911; computation time 14404 с; the number of iterations 1987. Calculation parameters: total number of cells in the computational grid 320911; computation time 14404 с; the number of iterations 1987. Page \| 792 (a) (b) (a) Page \| 792 Page \| 792 Emerging Science Journal \| Vol. 5, No. 5 Figure 28. Liquid outflow pattern from a nozzle with a diaphragm for conditions when ∝ is more than 0o but less than 90o: (a) Calculation data; (b) A fragment of the adapted computational grid; (с) A fragment of the report on the calculation data convergence. Calculation parameters: total number of cells in the computational grid 322292; computation time 2772 с; the number of iterations 610. (c) Figure 28. 5- Discussion Liquid outflow pattern from a nozzle with a diaphragm for conditions when ∝ is more than 0o but less than 90o: (a) Calculation data; (b) A fragment of the adapted computational grid; (с) A fragment of the report on the calculation data convergence. convergence. Calculation parameters: total number of cells in the computational grid 322292; computation time 2772 с; the number of iterations 610. Figure 29. Liquid outflow pattern from a nozzle with a diaphragm for conditions when ∝ = 90o: a) calculation data; b) a fragment of the adapted computational grid; с) a fragment of the report on the calculation data convergence. (c) (a) (b) Calculation parameters: total number of cells in the computational grid 322292; computation time 2772 с; the number of iterations 610. Calculation parameters: total number of cells in the computational grid 322292; computation time 2772 с; the number of iterations 610. (a) (b) (c) Figure 29. Liquid outflow pattern from a nozzle with a diaphragm for conditions when ∝ = 90o: a) calculation data; b) a fragment of the adapted computational grid; с) a fragment of the report on the calculation data convergence. Page \| 793 Page \| 793 Emerging Science Journal \| Vol. 5, No. 5 The computer simulation results (Figures 27 to 29) showed the possibility of the velocity vector deflection (at the nozzle outlet) at an angle ranging from +90o to -90o within the geometric hemisphere, and the velocity vector was controlled using a flat diaphragm. Earlier, similar results of the physical experiment [1, 23, 24] performed under laboratory conditions using a gaseous and a liquid model medium were obtained and published. Summarizing the results of the performed numerical experiments and the results of previously performed physical experiments, we can make an intermediate conclusion about the scientific novelty of the research as a whole: in the course of physical and numerical experiments, for the first time, the extreme conditions for the outflow of liquid and gas through a nozzle equipped with a velocity vector control system were considered, in the control range for the velocity vector deviation angle from +180o to -180o within the geometric sphere. Using computer simulation, by calculation, we have confirmed the possibility of deflecting the jet by 90 degrees with a control system equipped with a movable diaphragm. This result complies with the result of the physical experiment described earlier in our publications [1, 23]. 5- Discussion A previous paper [24] also noted that mesh turbines could be developed based on the Euler turbine. In this case, the curved tube is used as a mixing chamber for the ejector. A set of such mixing chambers is placed in the turbine rotor, and the ejector nozzle remains fixed. The pulse mode of gas (or liquid) flow is realized in the rotor flow channels with such a design. In this regard, the scheme of the jet system (Figure 22) can be considered a basis for developing special turbines (or jet systems) of this type. In inflow channels of such systems, two working processes are realized simultaneously: turbine and ejection working processes. As a result, we can talk about the prospect of developing hybrid turbomachines. Analysis of available jet control systems showed that the example with a movable diaphragm is only one of many ways to control gas flows at the nozzle outlet, so it is advisable to continue research in this direction. 5-1- Implication and Explanation of Findings Based on the computer simulation results, it is possible to partially confirm or repeat the conclusion of specialists and authors of [40]: the FloEFD software package used in the simulation reproduces the general flow structure and gives satisfactory results for the near flow field with low time and computing resource consumption. This paper considered only one particular case using a nozzle in which the cone angle γ2 was increased to 60o (according to the diagram in Figure 23). That helped to radically expand the control range for the deflection angle velocity vector. It seems that there are many other methods and possibilities to extend the control range for most jet parameters, including such a parameter as the deflection angle of the velocity vector (thrust vector). Therefore, there is a great prospect for improving the technical performance of such control systems, and scientific and patent research is advisable to continue in this direction. In physical and numerical experiments, it was observed that even an axisymmetric circular jet significantly changes its shape in the case of radial displacement of the regulating diaphragm (Figures 27-29). In this regard, an adjustable nozzle of this type can be called an unconventional nozzle. For this type of nozzle, computer simulation and special calculations are required to determine the Coriolis coefficient ∝k and the Boussinesq coefficient bk values. These coefficients will further be included in the momentum equation (Euler equation) when calculating jet devices of various designs, as part of the revision of the published calculation methodology [41] for ejector systems: ∝𝑘= 𝐹𝑘 −1 ∫𝑣̅3𝑑𝐹 𝐹𝑘 , (5) 𝑏𝑘= 𝐹𝑘 −1 ∫𝑣̅2𝑑𝐹, 𝐹𝑘 (6) ∝𝑘= 𝐹𝑘 −1 ∫𝑣̅3𝑑𝐹 𝐹𝑘 , (5) 𝑏𝑘= 𝐹𝑘 −1 ∫𝑣̅2𝑑𝐹, 𝐹𝑘 (6) ∝𝑘= 𝐹𝑘 −1 ∫𝑣̅3𝑑𝐹 𝐹𝑘 , 𝑏𝑘= 𝐹𝑘 −1 ∫𝑣̅2𝑑𝐹, 𝐹𝑘 (5) (6) where 𝑣̅ = 𝑣 𝑣𝑠 : relative flow velocity; vs: average flow velocity; Fk: an area of flow section; F: an area of channel section. In this case, the results of each such calculation will depend on the value of radial displacement of the regulating diaphragm. The parameters Fk and F will change their values depending on the location of the regulating diaphragm. Detailed study of effects with jet deformation is important for considering heat exchange processes and processes during jet interaction with solid walls of different types. 5-1- Implication and Explanation of Findings Studying the temperature distribution pattern along the length of this deformed jet (especially in the infrared spectrum) is interesting both from a scientific and practical point of view. Preliminary calculations have shown that the mesh turbine can have sufficiently high-efficiency indicators, which opens up the prospects for wide practical use of such equipment. Computer simulation and modern computational methods help to accelerate research work. However, as specialists note, a model of any level of complexity cannot claim to describe all properties of real turbulent flows adequately, and the problem of calculating turbulent flows will remain an important and topical problem of theoretical and computational fluid mechanics for a long time [31, 42]. In this regard, an important part of the research work is to conduct physical experiments in laboratory bench conditions, with the corresponding development of prototypes, models, and layouts. In Page \| 794 Emerging Science Journal \| Vol. 5, No. 5 Emerging Science Journal \| Vol. 5, No. 5 design and engineering work, the term prototype usually refers to a working model created to demonstrate and confirm the product's performance under development. The technology of quick prototyping provides a rapid realization of the basic functionality of the future product. Currently, rapid prototyping has become possible for a wide range of researchers and specialists through various additive technologies, including 3D printers. design and engineering work, the term prototype usually refers to a working model created to demonstrate and confirm the product's performance under development. The technology of quick prototyping provides a rapid realization of the basic functionality of the future product. Currently, rapid prototyping has become possible for a wide range of researchers and specialists through various additive technologies, including 3D printers. 5-2- Strengths and Limitations Figures 30-32 show photos of the developed prototypes manufactured using 3D printers. Also, the pneumatic and hydraulic tests' results confirmed the mesh turbine's suitability for its operation in conditions of changing the working medium density in a wide range from 1 to 1.000 kg per cubic meter. These results of pneumatic and hydraulic tests were described in [1, 23, 24]. Figure 30. Micromodels of a mesh turbine (left) and a vane pump wheel (right). Figure 30. Micromodels of a mesh turbine (left) and a vane pump wheel (right). In the figures below, arrows schematically show the working fluid flow Q0 and the pumped medium flow Q1. The turbopump casing is not shown in these figures. In the figures below, arrows schematically show the working fluid flow Q0 and the pumped medium flow Q1. The turbopump casing is not shown in these figures. In the figures below, arrows schematically show the working fluid flow Q0 and the pumped medium flow Q1. The turbopump casing is not shown in these figures. Figure 31. Rotor micromodel for a turbo-pump with radial mesh turbine. Figure 31. Rotor micromodel for a turbo-pump with radial mesh turbine. Figure 32. Micromodel of an experimental rotor with an axial mesh turbine. Figure 32. Micromodel of an experimental rotor with an axial mesh turbine. Page \| 795 Page \| 795 Emerging Science Journal \| Vol. 5, No. 5 In a further study of gas-dynamic processes in channels with a mesh structure, it will be necessary to consider choked flow conditions. One of the main features of the medium compressibility impact on its interaction with the permeable body is the choked flow conditions. In this case, the parameters on the windward side of the permeable body boundary cease to depend on the conditions on its leeward side. Then, any medium leakage laws linking the pressure drop at the permeable boundary with the parameters of the oncoming flow become unsuitable in principle, and there is a need to develop new experimental ways of determining the boundary relations [43-45]. It is known that the use of features of jet streams flowing from unconventional nozzles makes it possible to meet a variety of requirements arising in the design of technological processes [6, 46]. The use of a nozzle with a nonaxisymmetric outlet section allows for a significant increase or decrease in the force impact on a barrier [46]. 5-2- Strengths and Limitations Many researchers note that the three-dimensional gas-dynamic flow structure in nozzles with an unconventional shape of the critical section has not been sufficiently investigated [40]. The round, square, plane, and radial air (annular) jets have been investigated extensively [47]. Scientific publications present the results of research and development of new nozzle configurations of rocket engines, mainly annular (with internal and external flow expansion) for dense configurations of advanced missile stages and spacecraft [48]. The results of these studies have shown that known gas-dynamic methods of thrust vector control can be effectively used in expansion-deflection nozzles. At the same time, the existence of a relatively small-sized central body in this nozzle makes it possible to use new mechanical methods based on transverse displacement or oscillation of the central body. To clarify technical issues, Figures 33-36 show some materials obtained during the demonstration experiments. Some of these materials are supposed to be used in the educational process to familiarize students with the features of hybrid workflows in the theory of turbomachines. In one of the demonstration experiments, an aircraft model fan with a diameter of 55 mm was used as an impeller, Figure 33. Figure 33. The main elements of the experimental system are in a static state: 1- impeller; 2- nozzle. Figure 33. The main elements of the experimental system are in a static state: 1- impeller; 2- nozzle. , arrows schematically show the working fluid flow Q0 and the pumped medium flow Q1. In this case, own. In Figure 34, arrows schematically show the working fluid flow Q0 and the pumped medium flow Q1. In this case, air flows are shown. In Figure 34, arrows schematically show the working fluid flow Q0 and the pumped medium flow Q1. In this case, air flows are shown. Figure 34. The main elements of the experimental system in a dynamic state: 1- impeller; 2– nozzle; 3- fan operation zone; 4- turbine operation zone When air is supplied through nozzle 2 in impeller 1, two workflows run simultaneously. The first process is the turbine workflow. In the diagram, the part of the impeller located to the right of the rotation axis participates in implementing the turbine workflow. The airflow leaving nozzle 2 exerts a force effect on the blades of impeller 1. 5-2- Strengths and Limitations The kinetic energy of the working fluid flow Q0 is converted into mechanical energy, which is transferred to the impeller, and the impeller is involved in rotational motion. The operation area for the turbine process is schematically indicated in the figure as Figure 34. The main elements of the experimental system in a dynamic state: 1- impeller; 2– nozzle; 3- fan operation zone; 4- turbine operation zone When air is supplied through nozzle 2 in impeller 1, two workflows run simultaneously. The first process is the turbine workflow. In the diagram, the part of the impeller located to the right of the rotation axis participates in implementing the turbine workflow. The airflow leaving nozzle 2 exerts a force effect on the blades of impeller 1. The kinetic energy of the working fluid flow Q0 is converted into mechanical energy, which is transferred to the impeller, and the impeller is involved in rotational motion. The operation area for the turbine process is schematically indicated in the figure as zone 4. Page \| 796 Emerging Science Journal \| Vol. 5, No. 5 The second process is the fan workflow. In the diagram, the part of the impeller located to the left of the rotation axis participates in implementing the second workflow. The impeller blades exert a force effect on the pumped medium and create (form) the flow Q1. The ambient air is used as the pumped medium. The operation area for the fan workflow is schematically indicated in the figure as zone 3. Each blade of rotating impeller 1 passes through zone 3 and zone 4 periodically. In this case, a pulsed gas flow mode is implemented in the channels between the impeller blades. If the air supply to nozzle 2 is stopped, zone 4 will disappear. Moreover, all blades of impeller 1 will participate in the fan workflow implementation. When impeller 1 rotates, the stock of kinetic energy may be enough for impeller 1 to take off vertically upward, overcoming the action of gravity, as shown in Figure 35. This figure shows individual frames from the video recording, and the time in seconds is indicated under each frame, according to the timer readings on the camcorder. Figure 35. A set of frames from the video recording of the demonstration experiment. 6-4- Limitations and Future Studies At this stage, calculated studies are being carried out using either a gas or a liquid medium. Physical experiments have shown the possibility of working with gas-liquid mixtures, and subsequent work will focus on studying gas-liquid flows in channels with a mesh structure. As such, continued research will involve the study of turbomachinery and ejectors with a mesh structure in the flow channels. 6-1- Scientific Novelty During numerical experiments, the extreme conditions of liquid and gas outflow through a nozzle equipped with a velocity vector control system, in the control range for the velocity vector deflection angle from +90o to -90o within a geometric hemisphere, were considered for the first time. A prototype of a new mesh turbine equipped with a rotary cylindrical diaphragm (movable control sleeve) to control the velocity vector at the nozzle outlet has been developed and patented. The computer simulation results confirmed the possibility of developing a jet system that allows the deflection angle of the velocity vector (thrust vector) to be adjusted in the range from +90o to -90o within the geometric hemisphere for the conditions of one-outlet nozzle application. It was also shown that when using a two-output nozzle, there are possibilities for adjusting the deflection angle of the velocity vector (thrust vector) in the range from +180o to -180o within the geometric sphere. These calculated data are presented in addition to previously published results of physical laboratory experiments. 5-3- Summary The development of research will also relate to the study of acoustic characteristics of mesh turbines and nozzles. Modern calculation methods make it possible to solve such problems. In particular, the results of experimental studies of the acoustic characteristics of subsonic turbulent jets flowing from rectangular nozzles are known, and they show that the directivity of sound radiation and noise spectra in the far acoustic field of such jets are close to the corresponding characteristics of a jet flowing from a round nozzle [49]. The research results are mainly focused on the development of multimode mesh turbomachinery operating under difficult conditions. The work results can be used in various industries, including energy, transport, and robotics. In robotics, there is potential for the development of miniature turbomachines and jet engines with ejector augmenters. 5-2- Strengths and Limitations Similar demonstration experiments were also carried out using the prototype mesh turbines with a mesh rotor diameter of up to 120 mm, Figure 36. Figure 35. A set of frames from the video recording of the demonstration experiment. Similar demonstration experiments were also carried out using the prototype mesh turbines with a mesh rotor diameter of up to 120 mm, Figure 36. Similar demonstration experiments were also carried out using the prototype mesh turbines with a mesh rotor diameter of up to 120 mm, Figure 36. Similar demonstration experiments were also carried out using the prototype mesh turbines with a mesh rotor diameter of up to 120 mm Figure 36 Figure 36. Samples of mesh turbines with propeller screws were prepared for demonstration experiments. Figure 36. Samples of mesh turbines with propeller screws were prepared for demonstration experiments. Earlier, it was already noted in [41] that the turbine workflow could manifest itself in the channels of impeller pumps during some operating modes of the pumping unit. The expediency of scientific research of the relationship between the Earlier, it was already noted in [41] that the turbine workflow could manifest itself in the channels of impeller pumps during some operating modes of the pumping unit. The expediency of scientific research of the relationship between the Page \| 797 Emerging Science Journal \| Vol. 5, No. 5 Emerging Science Journal \| Vol. 5, No. 5 Emerging Science Journal \| Vol. 5, No. 5 pumping and the turbine workflows in the channels of one impeller was emphasized. The research development will relate to the study of the peculiarities of gas-dynamic and hydrodynamic processes in the pulsed flow of gases and liquids through channels with a mesh structure. Because of the variety of practical tasks and the significant amount of research work involved, the features of nozzles and ejectors with unconventional geometry can be studied in a separate scientific work. 7-1- Author Contributions 7-1- Author Contributions Conceptualization, Y.A.S. and M.A.M.; Data curation, V.V.V.; Formal analysis, Y.A.S.; Funding acquisition, M.A.M.; Investigation, M.A.M.; Methodology, I.V.G.; Project administration, M.A.M.; Resources, I.V.G.; Software, V.V.V.; Supervision, Y.A.S.; Validation, K.A.T., M.A.F. and N.N.B.; Visualization, N.N.B.; Writing – original draft, K.A.T.; Writing – review & editing, M.A.F. All authors have read and agreed to the published version of the manuscript. Conceptualization, Y.A.S. and M.A.M.; Data curation, V.V.V.; Formal analysis, Y.A.S.; Funding acquisition, M.A.M.; Investigation, M.A.M.; Methodology, I.V.G.; Project administration, M.A.M.; Resources, I.V.G.; Software, V.V.V.; Supervision, Y.A.S.; Validation, K.A.T., M.A.F. and N.N.B.; Visualization, N.N.B.; Writing – original draft, K.A.T.; Writing – review & editing, M.A.F. All authors have read and agreed to the published version of the manuscript. 6-3- Practical Importance The results of the ongoing research can be used to improve the energy efficiency of various technological systems, including systems for extracting and processing hydrocarbons. Some research results can be applied in robotic engineering and the development of small unmanned aerial vehicles. 6-2- Theoretical Contributions For the conditions of the other ejector work process in the rotor channels, a new direction for the theory of impeller machines has been proposed. There is a plan to develop underlying mathematical models to describe the ejector work process, considering the application of several mixing chambers with channels that form a mesh structure. Preliminary results of numerical experiments show the feasibility of creating a new theory. 7-2- Data Availability Statement The data presented in this study are available in article. The data presented in this study are available in article. Page \| 798 Page \| 798 Emerging Science Journal \| Vol. 5, No. 5 7-3- Funding The work has been performed with the financial support of the Ministry of Education and Science of the Russian Federation within the framework of the state contract in the area of scientific activities, topic number FSZE-2020-0006. 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https://openalex.org/W4311570826	https://zenodo.org/records/7442349/files/ILM.BOSHLAN%C4%A2ICH%20SINF%20MATEMATIKA%20DARSLARIDA%20O'QUVCHILARNI%20MANTIQIY%20FIKRLASHGA%20O'RGATISH.pdf	Tagalog	null	BOSHLANĢICH SINF MATEMATIKA DARSLARIDA O'QUVCHILARNI MANTIQIY FIKRLASHGA O'RGATISH	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	1,142	35-maktab Boshlanģich ta'lim o'qituvchisi Annotatsiya: Ushbu maqolada boshlang’ich sinf matematika darslarida o’quvchilarni mantiqiy va tanqidiy fikrlashga o’rgatailadigan topshiriqlarning ahamiyati va zarurati keltirib o’tilgan. O’quvchilarning mantiqiy fikrlashi bilan birgalikda muammolarga tanqidiy yondashishni o’rgatish usullari berilgan. Kalit so’zlar: tanqidiy, mantiqiy, mustaqil fikrlash, matematika, texnologiya, vazifa, masalalar. So’ngi yillarda ta’lim sohasining barcha bosqichlarini zamonaviy talablar asosida tashkil etish bo’yicha farmon va qarorlar qabul qilindi va bunga asosan deyarli barcha dastur hamda darsliklar asosiga o’zgartirishlar kiritildi. Bunga sabab bo’lgan eng katta omil shunda ediki, bizning milliy o’quv dasturimiz xalqaro \| 1 KONFERENSIYA \| 1 ILM – FAN TA’LIMDA INNOVATSION YONDASHUVLAR, MUAMMOLAR, TAKLIF VA YECHIMLAR ILM – FAN TA’LIMDA INNOVATSION YONDASHUVLAR, MUAMMOLAR, TAKLIF VA YECHIMLAR KONFERENSIYA \| 2 talablarga hamda chet elning ko’zga ko’ringan ta’lim jarayonlari natijalariga tenglasha olmasligi edi. Darsliklarda o’quvchilarrni mantiqiy va tanqidiy fikrlashga undaydigan misol va masalalar bilan boyitilish hozirda amalga oshirilayotgan jarayonlardan biri bo’lib kelmoqda. O’quvchilarning mantiqiy fikrlash qobilyatini shakllantirish masalasi bilan ularda togri, aniq, qisqa matematik nutqni o’stirish masalasi uzviy ravishda bo`glangan bo’ladi. Bu boshlang’ich ta’limning muhim vazifalaridan biri. Mantiqiy masalalarni yechish o’quvchilarda uzviy ravishda mustaqillikni o’stirish bilan birga o’qitish jarayonida qiziqishlarini oshiradi. Bunda tavsiya etilayotgan savol va topshiriqlar o’quvchilardan ma’lum darajada izlanish talab qilsa-da, u bola kuchi yetadigan darajada bo’lishi kerak. Matematik mashg’ulotlarda bunday topshiriqlarni tanlash katta ahamiyatga ega. Bu mashg’ulotlar o’quvchilardan mustaqil kuzatishlar o’tkazishni, qaralayotgan hodisalar narsalar va shu kabilarni taqqoslash, solishtirishni talab qiladi. Matematika fani boshlang’ich sinflardan boshlab inson hayotining mazmuni ekanligi ko’rsatib boriladi. Matеmatika haqiqiy olamning miqdoriy munosabatlari va fazoviy formalari haqidagi fandir. Matematikadan masalalar yechish o’quvchilarning fikrlash qobiliyatlarini rivojlantiradi.2 Darsda o’quvchilarni qiziqtiruvchi mantiqiy topshiriqlardan foydalanish katta ahamiyatga ega. Chunki o’quvchilar bunday topshiriqlarni katta qiziqish bilan tahlil qila oladilar. O’quvchilarni ko’proq faollashtirish uchun ko’pgina o’quvchilar foydalana oladigan, ular sevgan ertak qahramonlarini “jalb qilish” usuli katta ahamiyatga ega. 2 KONFERENSIYA \| ILM – FAN TA’LIMDA INNOVATSION YONDASHUVLAR, MUAMMOLAR, TAKLIF VA YECHIMLAR ILM – FAN TA’LIMDA INNOVATSION YONDASHUVLAR, MUAMMOLAR, TAKLIF VA YECHIMLAR O’quvchi mantiqiy topshiriqlarni bajarar ekan, topshiriq yuzasidan mushohada yuritib, ma’lum hukm va xulosalarni chiqaradi. Mulohazaning rеal (chin) yoki yolg’on ekanligini aniqlaydi, ya’ni ham mantiqiy ham tanqidiy fikr yuritadi. Shu o’rinda o’quvchilarni mantiqiy fikrlash qobiliyatini rivojlantirish bilan bir qatorda tanqidiy fikrlash qobiliyatini ham rivojlantirib borish muhim ekanligini aytib o’tish kerak. Tanqidiy fikrlashga o’rgatish o’quvchilarning muvaffaqiyatlarga erishishidagi asosiy omil bo’lib, boshlang’ich sinf matematika darslarida tanqidiy fikrlashni rivojlantirishda yangi pedagogik texnalogiyalar va didaktik o’yinlardan, muammoli savol va topshiriqlardan, turli xil rasm va boshqotirmalardan dars davomida oqilona hamda bolaning yosh va individual xususiyatlariga e’tibor bergan holda foydalanish o’qituvchining pedagogik mahoratiga bog’liq. Hozirgi kunda o’qituvchilarimizning raqamli texnologiyalardan dars mashg’ulotlarida to’g’ri foydalana olishi va o’quvchilarga bilim berishida raqamli ta’lim muhitini tashkil eta olishi kerak. Buning natijasida o’quvchilarning o’zlari mustaqil o’rganishi, shaxsiy o’rganishga moslashishi va o’zini ustida ishlashi kabi qobiliyatlari rivojlanadi. Zamonaviy inson uchun zarur bo'lgan kompetentsiyalarni tasniflashga urinayotgan xalqaro tashkilotlar raqamli, axborot va ilmiy savodxonlikning ahamiyati haqida gapirishadi. Ko'pincha bu turdagi savodxonlik bir -birini to'ldiradi. KONFERENSIYA \| 3 ILM – FAN TA’LIMDA INNOVATSION YONDASHUVLAR, MUAMMOLAR, TAKLIF VA YECHIMLAR ILM – FAN TA’LIMDA INNOVATSION YONDASHUVLAR, MUAMMOLAR, TAKLIF VA YECHIMLAR Tanqidiy fikrlash texnalogiyasining g’oyalari o’quvchilarni tabiatan izlanuvchanligi, dunyoni o’rganishga intilishi, jiddiy masalalarni ko’rib chiqish va o’ziga xos g’oyalarni ilgari sura olishidir. Bu borada o’qituvchining vazifasi o’quvchilarni tinimsiz o’rganishga undaydigan samarali fikrlash qobiliyatlarini rivojlantirishga yordam beradigan o’ychan yordamchi bo’lishdir. Tanqidiy fikrlashning muhim afzalligi shuki, u e’tiborsiz bildirilgan fikrlarni, noaniq tushunchalarini va yolg’on argumentlarni aniqlashtiradi va fosh etadi. Lekin uning kamchiligi yaratuvchi va konstruktiv kuchga ega emasligidir. Tanqidiy fikrlash g’oyamizni mustahkamlaydi, ammo konstruktiv, yaratuvchi fikr bilan ta’minlamaydi. Quyidagi mantiqiy topshiriqlarni I-IV sinf o’quvchilari bilan matеmatika darslarida bajarish tavsiya etiladi. Bunday turdagi mantiqiy topshiriqlar o’quvchilarni mantiqiy hamda tanqidiy fikrlash qobiliyatlarini rivojlantiradi. Tanqidiy fikrlash texnalogiyasining g’oyalari o’quvchilarni tabiatan izlanuvchanligi, dunyoni o’rganishga intilishi, jiddiy masalalarni ko’rib chiqish va o’ziga xos g’oyalarni ilgari sura olishidir. Bu borada o’qituvchining vazifasi o’quvchilarni tinimsiz o’rganishga undaydigan samarali fikrlash qobiliyatlarini rivojlantirishga yordam beradigan o’ychan yordamchi bo’lishdir. Tanqidiy fikrlashning muhim afzalligi shuki, u e’tiborsiz bildirilgan fikrlarni, noaniq tushunchalarini va yolg’on argumentlarni aniqlashtiradi va fosh etadi. Lekin uning kamchiligi yaratuvchi va konstruktiv kuchga ega emasligidir. Tanqidiy fikrlash g’oyamizni mustahkamlaydi, ammo konstruktiv, yaratuvchi fikr bilan ta’minlamaydi. Quyidagi mantiqiy topshiriqlarni I-IV sinf o’quvchilari bilan matеmatika darslarida bajarish tavsiya etiladi. Bunday turdagi mantiqiy topshiriqlar o’quvchilarni mantiqiy hamda tanqidiy fikrlash qobiliyatlarini rivojlantiradi. 1-topshiriq: Xo’roz tarozida ikki oyog’ida turgan holda o’lchanganda 3 kg chiqdi. Xo’roz bir oyoqda turgan holda o’lchanganda nеcha kilogramm kеladi? (Javob: 3 kg) kg) 2-topshiriq: Daraxt shoxida 9 ta chumchuq qator turibdi. To’rtinchi chumchuq uchib kеtdi. Daraxt shoxida nеchta chumchuq qoldi? (Javob: 8 ta) KONFERENSIYA \| 4 ILM – FAN TA’LIMDA INNOVATSION YONDASHUVLAR, MUAMMOLAR, TAKLIF VA YECHIMLAR ILM – FAN TA’LIMDA INNOVATSION YONDASHUVLAR, MUAMMOLAR, TAKLIF VA YECHIMLAR 3-topshiriq: 500 mеtr masofaga yugurish musobaqasida 5 ta sportchi qatnashdi. Har bir sportchi qancha masofaga yugurgan? (Javob: 500 m) 4-topshiriq: Stolda 70 dona qog’oz turibdi. Har 10 soniyada 10 ta qog’ozni sanash mumkin. Bu xolda 50 ta qog’oz sanab olish uchun necha soniya vaqt ketadi?(20 soniya. 10 soniyada birinchi o’ntasi, keyingi o’n soniyada ikkinchi o’ntasi sanaladi. Stolda esa 50 ta qog’oz qoladi.) 5-topshiriq: Bitta tayoqning 2ta uchi bo’lsa, bir yarimta tayoqning nechta uchi bo’ladi? (4ta) 6-topshiriq: Nodirning opa-singillari nеchta bo’lsa, shuncha akalari bor. Uning singlisi Umidaning akalari opalaridan uch marta ko’p. Oilada nеchta o’g’il va qiz bor? Ushbu ijodiy topshiriqni o’quvchi faqatgina mantiqan fikrlab yеcha oladi. Bunda o’quvchi quyidagicha mushohada qilishi mumkin: Nodirning opa-singillari soni uning akalari soniga tеng. Uning singlisi Umidaning akalari soni opasi sonidan uch marta ko’p. Dеmak, Umidaning 1 ta opasi va 3 ta akasi bor ekan. Oilada 3 ta o’g’il va 2 ta qiz bor. Xulosa qilib aytganda, hayotdan olingan turli mantiqiy masala- topshiriqlar o’quvchiga zavq bag’ishlaydi. O’quvchi topshiriqni yеchish yo’llarini qidiradi. Bunday topshiriqlar o’quvchining nafaqat matеmatik bilim va malakalarini mustahkamlaydi, balki uning mantiqiy tafakkurini o’stiradi, KONFERENSIYA \| 5 ILM – FAN TA’LIMDA INNOVATSION YONDASHUVLAR, MUAMMOLAR, TAKLIF VA YECHIMLAR ILM – FAN TA’LIMDA INNOVATSION YONDASHUVLAR, MUAMMOLAR, TAKLIF VA YECHIMLAR o’quvchini izlanishga, topqirlikka, maqsad sari intilishga da'vat etadi. Bunga oxshagan mantiqiy topshiriqlar darslikda kop uchraydi. Ular o`quvchilar diqqatini darsga qaratishga va ularning ijodiy faoliyatlarini oshirishda katta yordam beradi. Foydalanilgan adabiyotlar: o’quvchini izlanishga, topqirlikka, maqsad sari intilishga da'vat etadi. Bunga oxshagan mantiqiy topshiriqlar darslikda kop uchraydi. Ular o`quvchilar diqqatini darsga qaratishga va ularning ijodiy faoliyatlarini oshirishda katta yordam beradi. Foydalanilgan adabiyotlar: Foydalanilgan adabiyotlar: Akramjon O’ktamjonovich Mirzayev. O’quvchilarning raqamli dunyoda o’rganish konsepsiyasi bo’yicha savodxonligini rivojlantirish. Scientific Progress. Volume 2. ISSUE 8 ǀ 2021. Akramjon O’ktamjonovich Mirzayev. Mamlakatimiz ta’lim tizimida raqamlashtirish va raqamli dunyoda o’qitish va o’rganishning mavjud imkoniyatlari. Academic Research in Educational Sciences. VOLUME 2 \| ISSUE 12 \| 2021 Kazadayev, A., Sharopov, B., Hakimov, S., Umarov, I., Muxtoraliyeva, M., Dadaxanov, F., & Abdunazarov, A. (2022). MAMLAKATIMIZDA NEMIS TA’LIM TIZIMINI JORIY QILISHNING SAMARADORLIGI TAHLILI. Journal of new century innovations, 18(1), 124-129. Mukhtasar, M., Begyor, S., Aleksandr, K., Farrukh, D., Isroil, U., Sodiqjon, K., & Akbarjon, A. (2022). ANALYSIS OF THE EFFECTIVENESS OF THE DEVELOPMENT OF THE GERMAN EDUCATION SYSTEM IN OUR COUNTRY. Journal of new century innovations, 18(1), 168-173. KONFERENSIYA \| 6
https://openalex.org/W4317987618	https://zenodo.org/records/7567282/files/IJISRT23JAN789.pdf	English	null	How to Measure the Digital Economy in 5G Technologies and Digital Silk Road Era	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	2,174	How to Measure the Digital Economy in 5G Technologies and Digital Silk Road Era Dr. Davit Gondauri Professor of Business and Technology University, Tbilisi, Georgia and 0162 Dr. Otaviano Canuto Nonresident Senior Fellow in the Global Economy and Development Program at the Brookings Institution. Former World Bank Executive Director Dr. Nino Enukidze Rector of Business and Technology University Tbilisi, Georgia and 0162 Mikheil Batiashvili Professor, Chairman of Supervisory board Business and Technology University Tbilisi, Georgia and 0162 Abstract:- The current transformation of the society under the influence of digital technologies has no historical analogue in terms of its spread and the pace of development. The future will heavily depend on digital technologies. The role of the digital economy and 5G technologies is increasing day by day and for Georgia, - as a developing country, it is important not to fall behind the world trends. Measuring the digital economy and calculating its mathematical algorithm (gross virtual product) is important for the modern world, since, based on the world development trends, the digital economy plays an imperative role directly in the development of the economy. A non-existent, highly unusual and universal economic governance is being established - a corporate "digital dictatorship", with its benefits and risks, which will radically change human thinking, mentality, consumer behavior and economic activity. A new paradigm of employment is developing - Gigeconomy. A new model of economic relations is established - platform economy and many others. The Digital Silk Road significantly reduces the chances of a new global economic crisis and at the same time, offers many young people stable jobs. The growth rate of small business is 3 times higher if the company is led by digital 5G technologies. How and when will 5G affect the global economy? According to industry researchers, 5G will have its full economic impact worldwide by 2035. Among them, 13.2 trillion dollars will be directed to global economic products, 22.3 million dollars to the creation of new jobs and 2.1 trillion dollars to the growth of GDP. In addition, in order for Georgia's involvement in the Digital Silk Road project to be effective, it is necessary to investigate:  Unification of historical silk countries - cost of digital road (Internet traffic);  Where will digital hubs be located on the Digital Silk Road?  The purpose and the objectives of the research. It should be noted that one of the most populat topics in the modern world economy is the measurement of the digital economy. Volume 8, Issue 1, January – 2023 Volume 8, Issue 1, January – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Keywords:- 5G Technologies; Digital Silk Road; GVP Index, GVP, Index Divisor. I.  Economic and mathematical analysis of digital economy indices, on which ratings are constructed; Relevance and significance of the research. The existence of the single digital market of the Silk Road is the free movement of digital goods, services and capital, the growth of economic activities, which is the basis for the creation of new startups and laboratories. It will provide additional jobs and promote the introduction of innovative technologies. This road comes from Europe to Georgia, passes through Azerbaijan, continues under the Caspian Sea and joins the infrastructure in Kazakhstan, thus connecting Shanghai and Frankfurt, and Georgia itself is a part of this most important project. The amount of information in technologies is 140 zeta bytes. Information is the oil of the future. Data extraction is a project on the same level as oil pipelines were at one point.  Correlations between different indicators of digital inequality and the level of development of the digital economy;  Determination and analysis of correlation-mathematical connections of GVP-GDP. How to Measure the Digital Economy in 5G Technologies and Digital Silk Road Era The purpose of the present work is to research the issue. Based on the purpose of the research, we set the research objectives: IV. RESULTS AND ANALYSIS The product of the geometric mean of the digital indices of the subcategory of the digital economy on the index divisor presented in the methodology, allows us to obtain a mathematical model of the digital economy, which makes it possible to depict the absolute level of the digital economy. Another leading player of the "Digital Silk Road" is the Chinese B2B platform "DHgate", whose main goal is to simplify e-commerce and, at the same time, it aims to involve several developing countries in digital business. In addition, the company carries out foreign trade trainings in various universities of Turkey and Georgia, which are carried out within the framework of SREB (Southern Regional Education Board). (China Cross-Border E-Commerce (Export B2B) Report. 2017). INDEX(GVP)= √(N&(5g tech index)(ICT Development Index)(Global Connectivity Index)(EGDI)(DESI)(WDCI)(DEI) (1) The development of the communication costs and the development of the communication systems should be one of the driving forces for the spread of the Internet, the introduction of communication and telecommunication systems should be a contributing factor for the simplification of the communication and the use of the Internet and communication systems should be significantly simplified. (Digital Transformation Initiative Telecommunications Industry. 2017.) And, based on the digital index formula (1) presented above, the index divisor model is further used, from which, by multiplying the formulas (1) and (2) together, it is possible to move from the digital economy index to the absolute indicator of the digital economy. 𝐷(𝑡) = ∑ (𝑃𝑖,𝑡∗𝐹𝑖,𝑡∗𝑋𝑖,𝑡) 𝑛 𝑖=1 100 (2) 𝐷(𝑡) = ∑ (𝑃𝑖,𝑡∗𝐹𝑖,𝑡∗𝑋𝑖,𝑡) 𝑛 𝑖=1 100 (2) 𝐷(𝑡) = ∑ (𝑃𝑖,𝑡∗𝐹𝑖,𝑡∗𝑋𝑖,𝑡) 𝑛 𝑖=1 100 (2) 𝐷(𝑡) = ∑ (𝑃𝑖,𝑡∗𝐹𝑖,𝑡∗𝑋𝑖,𝑡) 𝑛 𝑖=1 100 (2) Gross Virtual Product (GVP) = INDEX (GVP) * D(t) = (1) * (2)  e-Government Development Index, EGDI  Telecommunication Infrastructure Index  Human Capital Index 𝐼𝑁𝐷𝐸𝑋(𝐺𝑉𝑃) = √𝐼𝑛𝑑𝑒𝑥1 ∗𝐼𝑛𝑑𝑒𝑥2 ∗𝐼𝑛𝑑𝑒𝑥3 ∗𝐼𝑛𝑑𝑒𝑥𝑁 𝑁 The "Digital Silk Road" is one of the priorities, which aims to reduce production costs while improving the global competitiveness of Chinese products and increasing trade among countries participating in the initiative. According to China, these goals are achievable with the digital economy, cloud technology, artificial intelligence, big data and other communication innovations. (Geissbauer , Schrauf, Koch & Kuge, 2014.)  After the calculated index, the regression (slope) correlation analysis of the above mentioned index with the overall economy model is carried out (GDP). III. RESEARCH METHODOLOGY Where, X(𝑖,t) - the number of index shares of the index component 𝑖 on the trading day; X(𝑖,t) - the number of index shares of the index component 𝑖 on the trading day; X(𝑖,t) - the number of index shares of the index component 𝑖 on the trading day; Various sub-category digital indices are used to evaluate the digital economy, and the geometric mean obtained from them shows a single-level digital economy index, which itself has a correlation with GDP. these are: g y; P(i,t) - price of index component 𝑖 on trading day; g y P(i,t) - price of index component 𝑖 on trading day; g y F(i,t) - Exchange rate for converting index component price 𝑖 on trading day 𝑡 into index currency In the end, we can get the absolute value of the digital economy by the following formula: In the end, we can get the absolute value of the digital economy by the following formula:  5g tech index  ICT Development Index  Global Connectivity Index – GCI II. One of the most interesting monographs on digital economy assessment and measurement is "Virtual Economy, Design and Analysis", in which the authors describe and argue why the world needs to determine the true scale of the digital economy. The theoretical aspects of the connection between GDP and the virtual economy are remarkably discussed (Lehdonvirta and Castronova, 2014.). Design and Analysis", in which the authors describe and argue why the world needs to determine the true scale of the digital economy. The theoretical aspects of the connection between GDP and the virtual economy are remarkably discussed (Lehdonvirta and Castronova, 2014.). IJISRT23JAN789 www.ijisrt.com 437 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 ch 𝐼𝑁𝐷𝐸𝑋(𝐺𝑉𝑃) = √𝐼𝑛𝑑𝑒𝑥1 ∗𝐼𝑛𝑑𝑒𝑥2 ∗𝐼𝑛𝑑𝑒𝑥3 ∗𝐼𝑛𝑑𝑒𝑥𝑁 𝑁 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 1, January – 2023 V. CONCLUSIONS AND RECOMMENDATIONS  Online Service Index The role of the digital economy and 5G technologies is increasing day by day, and for Georgia, as a developing country, it is important not to fall behind world trends. Based on the trends of the world development, the digital economy plays an important role directly in the development of the economy. The digital revolution has led to the existence of a large amount of various digital products in people's lives, therefore the study of these issues and the measurement of the digital economy are also important for the development of states. Many tech companies in the world play a major role in economic processes and their role is increasing day by day. All this affects the labor market and the specialization of people.  Digital Economy and Society Index, DESI  World Digital Competiveness Index – WDCI  Digital Evolution Index – DEI The research was planned with a triangulation approach, which implies the use of several research methods. The procedural plan of the research was drawn up as follows: 1. Data collection and processing; 2. Mathematical modeling; 3. Analysis/conclusion.  In order to obtain a single indicator (index) of the digital economy, each factor (index) affecting the digital economy is reduced to one indicator. The general indicator of the digital economy is obtained by the geometric mean of the indicators obtained from the factors, which can be represented by the following formula: Measuring the digital economy and calculating its mathematical algorithm is important for the modern world because:  In the nearest future, the digital economy will become the driver of the growth and development of the country`s economic system, as it has a number of advantages IJISRT23JAN789 www.ijisrt.com 438 Volume 8, Issue 1, January – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 [11]. Gondauri Davit, Bakhtadze Mamuka, Guramishvili Guram, Moistsrapishvili Manana & Mikheil Batiashvili, 2022. Georgian Railway Corridor`s Potential Analysis. https://www.generis- publishing.com//book.php?title=georgian-railway- corridor-s-potential-analysis-542 compared to the material (traditional) economy: the speed of delivery of goods and the immediacy of providing services, the low price of production and transaction performance, as well as the inexhaustibility of the production of digital goods;  A new integrated science and practical activity is developing - digital economy, as a positive correlation with the traditional branches of the economy and a higher level of their technological development; [12]. Lehdonvirta V., E.Castronova. V. CONCLUSIONS AND RECOMMENDATIONS 2014., Virtual Economies, designe and analysis. [13]. Andre Wheeler, 2020. China’s Digital Silk Road (DSR): the new frontier in the Digital Arms Race?. Silkroadbriefing.com  The content and meaning of the fundamental concepts: "time, "space, "labour activity, "high qualification, "profession, "consumer, "information and "knowledge" changes qualitatively;  The content and meaning of the fundamental concepts: "time, "space, "labour activity, "high qualification, "profession, "consumer, "information and "knowledge" changes qualitatively; [14]. https://www.silkroadbriefing.com/news/2020/02/19/chi nas-digital-silk-road-dsr-new-frontier-digital-arms-race/ [15]. Chan Jia Hao, 2019. China’s Digital Silk Road: A Game Changer for Asian Economies. 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https://openalex.org/W3103811804	https://hts.org.za/index.php/hts/article/download/6225/16530	English	null	Pastoral care with young people suffering from depression in the context of Soweto	HTS teologiese studies	2,020	cc-by	7,304	HTS Teologiese Studies/Theological Studies ISSN: (Online) 2072-8050, (Print) 0259-9422 Page 1 of 8 Original Research Original Research Page 1 of 8 Pastoral care with young people suffering from depression in the context of Soweto Authors: Mwansa C. Kimpinde1 Yolanda Dreyer1 Affiliations: 1Faculty of Theology and Religion, University of Pretoria, Pretoria, South Africa Research Project Registration: Project Leader: Yolanda Dreyer Project Number: 2546930 The situation and challenges of young people with depression in the urban African context of Soweto, Johannesburg, South Africa, are investigated from a pastoral care perspective. Depression is one of the more prevalent mental disorders. In African contexts it is often equated with demon possession. The aim of this article is to investigate the interplay between Western understandings of depression and African perspectives, and to come to a deeper understanding of the way in which support and healing are approached in this context. The article investigates the ways in which young South Africans in Soweto, their families and faith communities cope with and understand ‘depression’ on the one hand, and the culturally related phenomenon of ‘demon possession’ on the other. The article proposes a collaborative method of providing support which requires the cooperation of psychologists, psychiatrists, social workers, African spiritual healers and pastoral caregivers. Research Project Registration: Project Leader: Yolanda Dreyer Project Number: 2546930 Contribution: The article contributes to an understanding of the experience of depression among young people in a context where both Western psychological ideas and African cultural and religious beliefs with regard to demon possession, play a role. Professionals from the various relevant fields should collaborate in order to provide effective support. Description: This research is part of the project, ‘Gender Studies and Practical Theology Theory Formation’, directed by Prof. Dr Yolanda Dreyer, Department of Practical Theology, Faculty of Theology and Religion, University of Pretoria. Keywords: pastoral care; youth; depression; demonic possession; urban African context. Corresponding author: Yolanda Dreyer, yolanda.dreyer@up.ac.za The article proposes a pastoral care praxis, caring for young people in Soweto who suffer from depression or demon possession, depending on how their condition is diagnosed in medical terms, or designated in cultural terms. These two possible designations already indicate a clash of worldviews. Both worldviews can be found in their context. To provide adequate care for these young people, a holistic form of care should be found that effectively attends to the physical, psychological, social, cultural, mental and spiritual aspects of being human. How to cite this article: Kimpinde, M.C. & Dreyer, Y., 2020, ‘Pastoral care with young people suffering from depression in the context of Soweto.’, HTS Teologiese Studies/Theological Studies 76(3), a6225. https://doi. org/10.4102/hts.v76i3.6225 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. Dates: Received: 01 July 2020 Accepted: 21 Aug. 2020 Published: 10 Nov. 2020 Received: 01 July 2020 Accepted: 21 Aug. 2020 Published: 10 Nov. 2020 How to cite this article: Kimpinde, M.C. & Dreyer, Y., 2020, ‘Pastoral care with young people suffering from depression in the context of Soweto.’, HTS Teologiese Studies/Theological Studies 76(3), a6225. https://doi. org/10.4102/hts.v76i3.6225 Depression is the most prevalent form of psychotic behaviour and is characterised by a ‘breakdown of integrated personality function, withdrawal from reality, emotional blocking, distortion of thought and behaviour’ (Mohanty 1984:221). It is disabling and one of the most widespread behavioural disorders, also referred to as ‘mental illness’. According to Mojtabai, Olfson and Han (2016:48), depression is often accompanied by emotions such as a sense of loss, failure; a sense of injustice and ill health. Joy and sorrow, growth and decay, light and darkness are part of life and even the negative aspect of each of these pairs has a valid role to play in the greater scheme of things Assari et al. (2018:14). However, for those who are prone to depression it becomes increasingly difficult to sustain hope and to experience the positive side of life. Life’s ordinary struggles can become unbearable. If an already negative period in a person’s life is exacerbated by, for example, the emotional trauma of bereavement or severe financial loss, people can fear for their survival. The platitudes of well-meaning friends and loved ones do not alleviate their situation. They become increasingly convinced that no one understands what they are going through (Assari 2017:76). People who suffer from depression are not just ‘feeling sorry for themselves’. According to the American Psychiatric Association in its Diagnostic and Statistical Manual of Mental Disorders (DSM–5 2013) depression, as a recognised medical condition with specific signs and symptoms. It is the fastest growing epidemic in the developed world, as indicated by the World Health Organization (WHO), which ranks depression at number five in its top ten list of disabling illnesses. Copyright: © 2020. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. The context Over a period of 5 years the following statistics from the Psychiatric and Psychological Department Annual Report of 2019, indicate the prevalence of depression amongst patients in the Baragwanath Hospital in Soweto, Johannesburg. In 2014 there were 53 patients with depression out of 254, which is 36%; in 2015 there were 26 patients with depression out of 139 patients, which is 18%; in 2016 there were 12 patients with depression out of 74 patients, which is 19%; in 2017 there were 16 patients with depression out of 77, which is 18%; in 2018 there were six patients with depression out of 41 patients, which is 16%. This record, however, represents only the small number of families who dared to visit medical professionals with the problem of a family member with depression. There are probably many more who gave up hope without fully understanding the mental health issue at hand. Often, families are not equipped to deal with the problem of depression. Domestic violence, poverty and other socio-economic factors exacerbate the problems associated with depression – both with regard to contributing to the causes of the young people’s condition, and as an impediment to creating an optimal environment for their recovery. People with an African worldview depart from the cultural assumption that those who are ‘demon possessed’, or victims of witchcraft should be treated by a traditional healer. They could consult a prayer warrior, an exorcist, or can be taken by their family to a prayer home. Wiebe (2011:156–178) points out that symptoms of depression are often attributed to either demon possession or witchcraft. Individuals who start behaving strangely can be suspected of either. In such a cultural context it is rarely suggested that a medical doctor should be consulted. One of the recommended treatments would be for them to consult a prayer warrior. Another is to consult a sangoma who would perform rituals and incantations. The fees of such practitioners are rather high. Other ‘treatments can include whipping with tree branches’ (Halloy & Naumescu 2012:77). Clinical depression often sets in during early adulthood. In African contexts this is, at times, misdiagnosed as demon possession, or being bewitched. Relatives do what they can to help the sufferer. They take them to prayer warriors, sangomas or prayer homes, but as time passes without any signs of improvement, they lose hope and accept the malady as part of their life. Copyright: With the shift to modern and postmodern paradigms the idea of ‘demonic possession’ fell out of favour (Isaacs 2018:373). These ways of thinking have not presented answers to the phenomena http://www.hts.org.za Open Access Page 2 of 8 Page 2 of 8 Original Research Original Research associated with demonic possession, or developed appropriate methods of treatment for those who experience the signs and symptoms of classical possession. Though belief in possession is often rejected as ‘superstition’, the phenomena associated with demonic possessions do exist and must be dealt with. Crooks (2007:28, 45–76) defends the use of a traditional paradigm for understanding the phenomena associated with demonic possession. He suggests that demonic possession should be viewed as a valid phenomenon. He argues that denial, because of a shift in philosophical paradigms, does not negate the reality of an event. He does not find the recently adopted explanations of the natural sciences to be necessarily more convincing than traditional views. Using what he calls a ‘post-anecdotal’ method, Crooks (2007:45) shows that the ancient system of demonology is not only as adequate as the modern medical model, but more effectively describe the phenomena of possession states. Some see it as ‘mental illness’, whereas others do not know what this term signifies. Many are aware of the prevalence of depression amongst young people in Soweto, but even clinicians acknowledged in interviews that they sometimes fail to recognise the symptoms of depression in the initial stages. Generally, people in the Soweto context are not well equipped to deal with the reality of mental illness, including depression. In African contexts, mental disorders are often perceived as demonic possession, in other words as the result of some supernatural influence. Where a community places a strong emphasis on supernatural powers, it would be difficult to identify depression and provide people with the necessary help. In the context of Soweto, a variety of professionals are involved with matters of healing and fullness of life. These include psychiatrists, psychologists, social workers, pastors and traditional healers. The question is how these healers and caregivers can cooperate to provide effective holistic care to young people with depression in Soweto. Multicultural pastoral care and liberation African psychology psychological practice for oppressed peoples (eds. Montero & Sonn 2009:3). Martín-Baró (1994:27) outlined three basic tenets of liberation psychology, namely, a new goal, a new epistemology and a new praxis. The emerging field of African psychology is an important resource for an Africa-based, intercultural, pastoral theology and practice. In the contemporary context of Soweto, the ministry of pastoral care to young people who suffer from depression often do not yet utilise the useful theoretical perspectives and insights from this discipline. Although it is new and still evolving, it is increasingly making its presence felt in the broader area of African culture and religion (Awuah-Nyameke 2012:14). For the last two decades, African theologians have been pointing to the need for an in-depth focus on psychology and Africa. They have identified that strategies are needed to address the specific psychological problems and challenges of this cultural context with its tumultuous history. The most valuable goal of psychology, according to Martín- Baró (1994:29), is to focus its theoretical and practical energies on the ‘needs and sufferings of the majorities who are numbed by oppressive life circumstances’ (Martin-Baró 1994:28). He argues that there is a link between an individual person’s psychological problems and the wider social, political and economic contexts in which, the person’s life is embedded. He intends for psychology to gain clarity in understanding the relationship between psychic and social structures, ideologies and discourses that force people into ‘marginalised dependency’ and ‘oppressive misery’ by taking away their ability to define their own lives. Fragmentation and apathy reduce people to a state of submission and they no longer expect anything from life (Lester 2003:94). Liberation psychology is amenable to the communal, contextual and intercultural model of pastoral care that should be developed for African communities in general, and that will be of use with regard to the psychological struggles of young people who suffer from depression. Such an approach attends to people’s historical, cultural and social realities. From the perspective of African psychology, it is clear that a diverse range of psychological challenges face young people in Soweto who suffer from depression. Many of these are the direct result of the internalisation of oppressive messages and cultural behaviours regarding their condition (see Makhubela 2016:7). Psychological problems include low self- esteem, self-blame and internal conflict. On the cultural front they are burdened by misunderstandings about what depression is and how it manifests. The context However, not all people who are suspected of being possessed are in fact possessed. So also, not all people who are suspected of being victims of a curse, are suffering because of that (Mercer 2013:16). Demon possession is a common cultural and spiritual explanation for the psychological and biological condition of depression. This misconception then results in people not receiving the treatment they need. It is like when people are misdiagnosed and receive the wrong treatment in the medical world. Collaborative care which includes expertise from the various fields – medical, psychological, psychiatric, social work, pastoral and traditional-cultural – can contribute to avoiding misconceptions that can adversely affect the condition and life of persons who suffer and get them the treatment they need. Many people in this context have no in-depth knowledge of the classification and indicators of mental disorders. Some may have superficial knowledge of depression because they know people who suffer with it, or have heard of the condition. Many do not want to associate with individuals with depression for fear of stigmatisation. The slightest indication of a ‘tainted family lineage’ would mean social rejection in that community. This would, for example, affect marriage alliances between families and would in general impede social interaction. Those who are somewhat familiar with the term ‘depression’, often have a wide range of understanding of what it entails. Some see it as a ‘split personality’, others as a ‘curse’. http://www.hts.org.za Open Access Page 3 of 8 Page 3 of 8 Original Research Multicultural pastoral care and liberation African psychology They are often labelled and socially ostracised. For Martín-Baró (1994:27) the goals of liberation psychology include that oppressed people should gain knowledge and understanding of what may not yet be present in their current state of oppression, but will be present in the tomorrow of their liberty. Truth is not to be found, but made (Martín-Baró 1994:28). Truth is constructed, not as: Unlike many current psychological theories that tend to see the source of the problem inside the person, as is discussed by feminist theologian and clinician, Christie Cozad Neuger (2001:22), both feminist pastoral care and African psychology (Wasike 2006:18) have shown that the source of the problem is more often than not the oppressive structures of the prevalent system and its definitions of depression. The psychological problems that young African people experience are not ‘a disability’ or ‘a personality deficiency’ but are often survival adaptations, or harmful adjustments to prevailing unhealthy systems. They are generally not given room to understand and accommodate African cultural perspectives and African psychology, and embrace the alternative perspectives of traditional beliefs that can provide them with a different orientation. [A] matter of thinking for them or bringing them our ideas or solving their problems for them; it has to do with thinking and theorizing with them and for them. (Martín-Baró 1994:28) The communal, contextual and intercultural model of pastoral care that this article envisages has a similar goal. Oppressed people are subjects of history rather than mere objects of either history or pastoral care. Conventional models of pastoral care tended to not take oppressed peoples’ perspectives or experiences sufficiently seriously when devising pastoral care practices. This then deprives people of the subject status they deserve as human beings. In his quest to develop a new epistemology, Martín-Baró was influenced by Paulo Freire’s (2000) Pedagogy of the oppressed. Freire refers to ‘pedagogy of the oppressed’ and not for the oppressed – language that maintains their subject status. Ideas from liberation theology are evident. Liberation theology emphasises that the God of Jesus is to be found from the perspective of the poor. In the same vein a psychology of liberation teaches that from the oppressed existential truth can be discovered and built upon (Payne 2006:19). Martin-Baró (1994:29) proposes that existing knowledge be revised from the perspective of the poor and the oppressed. Psychology, Christianity and traditional cultures Despite the tremendous technological and material advancements, there are still areas where people are completely cut off from the rest of the world. They still practise traditional ways of living and surviving. Prior to development of modern medicine, treatment for the sick and suffering were often rather harsh (Lagacé-Seguin & d’Entremont 2010:37), especially the treatment of the mentally ill because ‘there were no effective methods for controlling their actions or helping them find relief from their inner torment’ (Collins 2007:648). Some of the more popular treatment practises for the mentally ill was that they were ‘whipped, starved, chained, seared with hot irons, dunked into freezing water’ (Collins 2007:648). Such actions were justified on the basis that the patients were demon possessed and the torture would make their body uninhabitable to the demon(s). Another method was to make holes in the patient’s scull to let the tormented spirits out. This was done without anaesthetics. Religious practitioners stood by as these methods were executed and exorcisms were done, ‘especially in treating disorders that had a strong physiological basis’ (Collins 2007:648). New Testament authors discredited physical and mental illnesses because of demon possession (Virkler 1988: 281–282). They distinguished between the state of being demon possessed and other illnesses. They also distinguish between ‘demonically caused and non-demonically caused illness in their discussion of healing’ (Virkler 1988:282). Both categories of people were healed unconditionally, but the mode of healing was different. The former was healed by casting out the demons out of the possessed, whereas the latter was healed by other means. However, it is often difficult to distinguish between whether a person is demon possessed or mentally ill, because the ‘symptoms arising from psychopathology and demonization overlap to a considerable extent’ (Virkler 1988:285). Alhough there was no knowledge of mental disorders and treatments known today, the Bible does provide insight into human nature, showing the human condition before God. It provides an understanding of human suffering and offers hope (Collins 2007:635). Though psychology, Christianity and traditional beliefs and practices are discussed together in this article, they represent quite different approaches. Psychology, like any scientific enterprise, consists of theories that are embedded in a scientific worldview and emanate in detailed findings (Watts 2002:74–75). Within each field there is also a variety of sometimes rather divergent approaches. In psychology there are, for instance, both a more introspective and a more behaviourist method. Page 4 of 8 Page 4 of 8 Original Research oppressing and possessing human beings (Virkler 1988:281). To distinguish between demon possession and other mental illnesses, some characteristics were identified by Meier et al. (2010:260). Demon possessed people have more physical strength than other people. Demon possessed people respond differently when the name of Jesus is spoken to people with psychological problems. There is a change of voice when the demon speaks. Demon possessed people can perform supernatural acts. People who are demon possessed speak rationally whereas individuals with psychological problems do not. Those who are possessed often have a history of occult activities such as participating in séances or Satan worship. Demon possessed individuals do not respond to therapy (Meier et al. 2010:260). Martín-Baró (1994:3) challenges psychology to a new praxis: ‘to place itself within the process alongside the dominated rather than alongside the dominator’. If such a new praxis that transforms people and reality is not developed, it will hamper the construction of a liberative psychological practice (Nylund & Nylund 2003:388). Psychologists should then take a stand for the oppressed people and make ethical choices. However, the call to ‘take sides’ can, for psychologists, social workers or clergy, create an unhealthy and harmful ‘us or them’ dynamic which will not contribute positively to the transformation of communities. Further, such a paradigm also has the potential to replicate the strategies of the dominant and not succeed in breaking the cycle of domination (Payne 2006:19). There is a need to rather, work in an integrative and dialogical manner. Oesterreich (1930:54–56) also identifies characteristic features of demon possession. For him, most striking characteristic features of demon possession is the change in the persons’ personality. They seem invaded and governed by a strange new soul. The second characteristic is their change in voice. The normal tone of the parent body is suppressed and transformed into a deep and heavy bass tone. The third characteristic feature is that the new voice does not speak according to the spirit of the normal personality but that of the new one. Psychology, Christianity and traditional cultures According to Watts (2002:75–76), it is ‘the more philosophical end of theology that intersects with scientific disciplines such as psychology’. Liberation psychology Although the idea of ‘liberation’ was adopted in a variety of fields, including theology, during the latter part of the 20th century, liberation psychology was conceived primarily by Ignacio Martín-Baró (1994:27), who was a priest as well as a trained social psychologist (eds. Montero & Sonn 2009:3). He formulated his ideas in an article titled, ‘Towards a psychology of liberation in the Bulletin of Psychology published by the Central American University in 1986. The article argues for the construction of a new transformative http://www.hts.org.za Open Access Page 4 of 8 Pastoral care perspectives Although ‘depression’ is understood differently in different cultures and ethnic groups, the condition is to be found in all walks of life (Davis & Epkins 2009:29). The condition can, however, be exacerbated by a difficult environment. Living with depression can be even more difficult for people who bear the emotional scars of a traumatic youth. It is the task of pastoral caregivers to provide effective care for all the individuals and communities they serve (Mojtabai et al. 2016:25). The aim is to guide people who suffer with depression to live their life with human dignity and regain In the Old Testament, there are references to Moses and the other prophets repeatedly warning against activities such as divination, sorcery and idol worship (Virkler 1988:281). Similarly, in the New Testament demons are regarded as personal, fallen, spiritual beings that are stronger than human beings, but weaker than God. They were capable of http://www.hts.org.za Open Access Original Research Original Research Page 5 of 8 Page 5 of 8 keep the young people who suffer to remain silent. Through conscientisation the young people can find solidarity in their communities and work towards the transformation of their social order to make it just and equal for all individuals and communities to exist in life-affirming ways. their confidence. Caregivers who journey with the young people who suffer from depression that is often equated to demonic possession in their culture should do so with respect and compassion. Kaplan and Benjamin (1991) describe the complexity of the work as follows: [T]he arena of pastoral work is multifaceted and full of surprises, unexpected problems and opportunities for profound insight into the human situation. It is an arena within which the pastor is privileged to be with people where they live and breathe, succeed and fail, relate intimately and experience alienation; it is the down-to-earth world of the human living. (p. 11) To achieve a liberating African pastoral care praxis, a narrative approach which makes use of the pastoral care methods of positive deconstruction (Pollard 1997:44) and reframing (Capps 1990:126), can be effective strategies for identifying, deconstructing and reframing dominant cultural narratives. For pastoral caregivers in Soweto, the church need not only be the object of their pastoral ministry but can also be their partner in pastoral ministry. The young people themselves should be mobilised to resist the forces of oppression, and work towards their own liberation. Pastoral care perspectives However, one of the most debilitating effects of the current situation is that the community is divided. They do not form a cohesive force to resist the oppression to which they have been subjected. The internal division sabotages efforts to raise their concerns with a unified voice in the public arena. Pastoral caregivers aim to provide guidance, support and encouragement to people in distress and to do so in a way that is liberating (Cobb, Kor & Miller 2015). Another challenge is to create a safe environment and foster a healthy and healing relationship. Healed people can contribute further to the healing and transformation of society (Kaplan & Benjamin 1991:11). The caregiver’s focus is on the empowerment of the person bringing the voiceless to voice and, if necessary, becoming the voice of the voiceless and their prophet to the powerful. The pastoral care method proposed in this article has the aim to empower young people who suffer from depression so that they can regain their dignity and develop confidence in themselves. The faith community should be a space that nurtures the growth and development of its members. It should challenge negative constructs, which have a life-limiting effect on people, and affirm positive and constructive perspectives. A pastor provides not only care but is also called to be a prophetic witness in culture and society when the need arises. Pastoral caregivers in Soweto can utilise the cultural capital represented by the young people and their families. For that, the church will have to develop and articulate a clear theological understanding of the relationship between the gospel and culture (Wasike 2006:12). Pastors can identify ways in which African cultural wisdom can be reclaimed and affirmed to become a medium through which the gospel message can be communicated effectively and experienced authentically (Cobb et al. 2015:69). The liberating and nourishing elements of the African culture can be utilised to meet the socio-cultural needs of the young people of Soweto. Another important strategy of pastoral ministry can be to collaborate with the authorities and with other religious movements, organisations and bodies, to address the many needs for care in the context of Soweto. Collaboration is essential if the needs of young people who suffer from depression are to be met. A new framework for a pastoral care praxis Pastoral care models that were developed in a Western context are often at a loss when it comes to the connection that is made in some cultures between the internal and external conditions of depression, and demonic possession (Matsaneng 2010:53–64). Often pastors do not have an adequate understanding of the condition of depression. Conventional pastoral care models generally focus on care and fail to include the two other critical needs of liberation and belonging (Gerkin 1997:70). The personal problems of the young people who suffer from depression and their families should be understood in the larger socio-cultural context. Both the internal and the external source of the depression should be addressed for the liberation and emancipation to be achieved. Education and conscientisation Education and creating awareness are about more than just teaching people about depression. It challenges the whole faith community to stand in solidarity before God, crying out for God’s help and liberation. It would also involve the training and empowerment of pastoral caregivers to provide effective guidance for young people who suffer from depression. iii Depression amongst the young people in Soweto affects not only themselves, but also their families, the broader community and the faith community in which they worship (Masoga 2005:98). Education is necessary if every member, also people who suffer from depression or those who are suspected of demon possession, is to be fully accepted and treated with love and care (Cotton et al. 2006:17). Some examples are workshops, sermons and a teaching series, Bible study sessions, liturgies and songs/hymns that are appropriate for the purpose (Johnson 2010:79). Educational programmes should be implemented with sensitivity. Information should be accurate and presented in a professional way. An attitude of love and compassion and a deep concern for the human rights and dignity of all people is needed. Educators should challenge those who act in hurtful ways towards people who suffer, affirm and support those who are derided, and persuade a lifestyle change in all people (Koole et al. 2010:53). They should examine conditions that promote hurtful behaviour and advocate for Christian values that call for behavioural change. Other crucial matters include gender and the empowerment of young people. The Church is called to examine critically ‘some of the cultural, customary and sexist practices that have been perpetuated in certain circles of society’ (Koole et al. 2010:54). Failure to do this would continue to render depressed young people vulnerable to their circumstances and in conflict with their families and surroundings. Creating a caring environment of trust In these testimonies there should also be space for lament regarding their experience of pain and trauma. Wimberley (2003:162) calls lament, the expression of the moans of the soul that arise from life’s struggles and losses. Lament is brought to speech in prayer. Liturgies can be designed for the benefit of those who suffer and find it difficult to speak. This gives them the opportunity to express their pain openly in worship. Creating a caring environment of trust As a pastoral caregiver in the context of Soweto, I found it a challenge to gain the trust of the young people. They were wary, not knowing what to expect. The challenge is to create trust, a ‘firm belief in the reliability, truth, ability, or strength of someone or something’ (Oxford Dictionary 2001:901) in order to be able ‘to put one’s confidence in a person or thing’ (Goyal et al. 2014). Trust is needed for them to share their stories openly without fear of suspicion and stigma. Communal resources of faith communities should be utilised to serve the needs of the young people with depression and their families. Mbiti (1990:56) points out that cultural resources should be valued and affirmed. Through culture- affirming practices from a liberation psychology perspective and intentional conscientisation, sufferers, their families and communities can gain insight into harmful ideologies and discourses they have internalised, and which have served to The distrust of the young people also extends to the faith community and the local community. People who suffer from depression, or have had experiences that are called demonic possession by others, do not feel safe to share their stories in the congregation for fear of being discriminated against, victimised and stigmatised. This exacerbates and perpetuates http://www.hts.org.za Page 6 of 8 Original Research Page 6 of 8 their suffering (Mercer 2013:17). The church should intentionally create an environment of trust where people can express their pain and struggles without fear of victimisation, discrimination and stigmatisation. To create such an environment, those in leadership will have to act with integrity when dealing with confidential matters. Empathetic care and a God-honouring, Christ-like disposition of the leaders can contribute to creating an environment of trust in the church (Mogoba 2011:170). Caregivers’ own lives should display the kind of character that is worth imitating by the rest of the community. For a faith community and community to be trustworthy and able to create a safe environment for people who suffer with depression, education and awareness are needed. live with their condition can give hope to those who are still struggling (Goldston et al. 2008:38). Those who have the courage, their testimonies can recoup a sense of strength, confidence and dignity in themselves. They can be empowered to go out and face their challenges with courage, knowing and feeling that they are supported by the community. Destigmatisation Stigma can be described as ‘a mark or sign of disgrace’ (Oxford Dictionary 2001:824). It is a sign of social unacceptability associated with a deep sense of shame. If communities and faith communities attach stigma to certain conditions and actions, those who suffer will not be able to come forward and share their stories and experiences for fear of being judged, rejected and discriminated against. Until stigma is eradicated, it will remain difficult for young people with depression to experience a sense of belonging and the acceptance they so desperately need from their faith community and the broader community. Destigmatisation would be to stop shaming and shunning people and to begin to treating them with dignity, love and compassion. Then they can feel fully human as people created in the image and likeness of God. They can then know and accept themselves as persons of worth and dignity, equal to all others in the community. This will communicate to them the strong message that they are still God’s own beloved children, who need not be ashamed of themselves because of a condition over which they have no control. Destigmatisation will convey to the sufferers that the message of the gospel is communicated in a manner that brings about healing and transformation. The faith community in its prophetic ministry should communicate a clear message that young people who suffer from depression should not be rejected, stigmatised, labelled and discriminated against in any way by fellow believers (Walker et al. 2014:105). The gospel’s message of love, care, acceptance, grace and compassion should be brought to those who live with depression, or demonic possession. Through education, the whole body of Christ is challenged to examine itself from a deep reflection on, and understanding of, how people are affected by depression, and the positive or negative role that families and the community can play. Collaborative support In general, depression is an often-misunderstood mental health condition in African cultures and contexts. From a pastoral perspective, a contribution can be made on various levels to improve the situation of the sufferers. The focus of this article was specifically on young people in the urban African context of Soweto. The contribution of pastoral caregivers can be to create awareness of the suffering of people with depression and the care they need from their support systems and communities. Another function, pastoral caregiver can fulfil is to educate both, the faith community and the broader community with regard to depression to foster a greater understanding of the realities and challenges of this particular mental health condition. With greater understanding harmful reactions and practices can be eliminated to a great extent. In African contexts specifically, where the condition of depression is often equated with demon possession, pastoral caregivers can also initiate and sustain cooperation amongst themselves and medical professionals, social workers and traditional healers to approach the problem holistically. The aim is that all the helping professions and the communities should work together to help and support young people who suffer, rather than foster misunderstandings of the condition and foment discord amongst the caregivers and support systems, exacerbating their suffering. The aim of the article was therefore to develop a contextual and collaborative pastoral care approach to depression amongst young people who suffer from depression, which takes seriously the interplay between Western understandings of depression and African perspectives in order to provide effective and holistic care and support. [T]o transform individuals into a real personal relationship with Jesus Christ through the baptism of the Spirit; to heal relationships and to build community – especially in the family and the neighbourhood community and, to transform society by healing relationships of injustice and oppression. (Goldston et al. 2008) Every local church in Soweto can be a centre of healing and support if it takes seriously its mission to those who suffer, including young people who suffer from depression who live in their communities and worship in their churches. Christian believers are called to proclaim the gospel of Jesus Christ for healing and transformation (Pollard 1997:43). The lack of support structures is, however, a problem throughout Africa. Pastors often feel ill-equipped to deal with the trauma that affects their congregants. Support Faith communities can begin support groups where people with experience of depression can come together to share their stories and encourage one another. Such groups can also be spaces where people whose behaviour is detrimental to those who suffer, are challenged to change their behaviour. This would give them the opportunity to make right where they harmed others through their actions. Support groups for Conscientisation can be achieved by means of sharing testimonies. The stories of those who have found a way to http://www.hts.org.za Open Access Open Access Original Research Page 7 of 8 Page 7 of 8 young people who suffer with depression and their families have the following aims: Competing interests The authors have declared that no competing interest exist. Authors’ contributions All authors contributed equally to this work. Collaborative support Masoga (2005:239) suggests that professional counselling centres be established with the aim to provide care for the clergy. Waruta and Kinoti (eds. 2005:230) emphasise that clergy care should pay specific attention to their personality and ministry. Such centres should be places of healing, restoration and transformation. Ethical consideration This article followed all ethical standards for a research without direct contact with human or animal subjects.’ Churches can establish recovery centres and partner with organisations that provide aid for those who suffer from depression. Together they can hold accountable those who do not live up to their mandate to deliver acceptable services to people who desperately need them. The body of Christ should follow his example: He challenged the causes of detrimental conditions in society. The Church’s responsibility: that of loving liberation rather than judgement (eds. Waruta & Kinoti 2005:133). Churches should be at the forefront of addressing the issue of depression with families and communities in order to fulfil its mandate to preach good news to the poor, proclaiming freedom to the captives, recovery of sight to the blind, releasing the oppressed and proclaiming the year of the Lord’s favour (Lk 4:18–19). Data availability statement Data sharing is not applicable. Funding information This research received no specific grant. Welfare, advocacy and collaboration Faith communities have the mission to be a visible sign of Christ’s presence in the world. According to Wasike (2006:35), the vitality of the Church depends to an extent on the health and integral well-being of the society and culture in which it operates. Therefore, it is the Church’s salvific mission to promote and foster, healthy and healing human relationships at a public level (eds. Waruta & Kinoti 2005:133). This means that the Church can and should partner with all other organisations that seek to advance the cause of those who suffer from depression. The Church must be the voice of the voiceless and the conscience of society. Its responsibility is to care for all who suffer. This includes young people who struggle with depression. Ministries can be developed with this group of people in mind. 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. http://www.hts.org.za Page 8 of 8 Page 8 of 8 Original Research Lester, A.D., 2003, The angry Christian, Westminster John Knox, London. 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https://openalex.org/W1582221131	https://www.intechopen.com/citation-pdf-url/45994	English	null	Chronic Toxicity of Weathered Oil-Contaminated Soil	InTech eBooks	2,014	cc-by	6,835	Chapter 3 Chapter 3 Chronic Toxicity of Weathered Oil-Contaminated Soil Dinora Vázquez-Luna Additional information is available at the end of the chapter http://dx.doi.org/10.5772/57253 http://dx.doi.org/10.5772/57253 distribution, and reproduction in any medium, provided the original work is properly cited. 1. Introduction Currently, the activities derived from the oil industry, such as extraction, transportation and processing of oil, have affected natural resources [1-3]. For decades, tropical lands have been contaminated by chronic oil spills, causing significant changes in physical and chemical characteristics of the soil, affecting plant development and reducing the growth of microor‐ ganisms [4]. Moreover, oil weathering and adaptation of some plants may hide the toxicity of high molecular weight hydrocarbons to other organisms. For this reason, it is necessary to incorporate aspects of chronic toxicity of weathered oil-contaminated soil in the study of “Soil Pollution”. This chapter aims to examine the chronic effects of old spills on soil, plants and beneficial microbes, in order to support the creation of new remediation technologies, focused on face the new challenges of soil contamination. ganisms [4]. Moreover, oil weathering and adaptation of some plants may hide the toxicity o high molecular weight hydrocarbons to other organisms. For this reason, it is necessary t incorporate aspects of chronic toxicity of weathered oil-contaminated soil in the study of “So Pollution”. This chapter aims to examine the chronic effects of old spills on soil, plants an beneficial microbes, in order to support the creation of new remediation technologies, focuse on face the new challenges of soil contamination. Oil pollution is a global problem of increasing importance [5], is estimated that every yea numerous spills affecting natural resources of Southeast Mexico, in 2011 were contamina ed more than 2,063 hectares as a result of 217 oil spills that affected soil and sea; of whic 85 were caused by uncontrolled illegal connections, number which increased 204% from 2009-2011 [6]. With respect to the damages to the ground, in 2012 reported an increase o 87% of leaks and spills, its main causes were corrosion damage and failure of materials i pipelines, in that year 30.07 hectares were contaminated in the Southern Region, joined thi there is lag in the care of cases from previous years. This year, the oil industry closed it operations with a total of 163.63 hectares waiting to be remedied, of which, 39.5% are locate in the southeast [7]. The environmental problems caused by oil spills, is not limited to visible pollution, becaus there are chronic effects that silently endanger ecosystems, biodiversity and environmenta © 2014 The Author(s). Licensee InTech. © 2014 The Author(s). Licensee InTech. 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. © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, 1. Introduction This chapter is distributed under the terms of the Creative Common 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. Oil pollution is a global problem of increasing importance [5], is estimated that every year numerous spills affecting natural resources of Southeast Mexico, in 2011 were contaminat‐ ed more than 2,063 hectares as a result of 217 oil spills that affected soil and sea; of which 85 were caused by uncontrolled illegal connections, number which increased 204% from 2009-2011 [6]. With respect to the damages to the ground, in 2012 reported an increase of 87% of leaks and spills, its main causes were corrosion damage and failure of materials in pipelines, in that year 30.07 hectares were contaminated in the Southern Region, joined this, there is lag in the care of cases from previous years. This year, the oil industry closed its operations with a total of 163.63 hectares waiting to be remedied, of which, 39.5% are located in the southeast [7]. The environmental problems caused by oil spills, is not limited to visible pollution, because there are chronic effects that silently endanger ecosystems, biodiversity and environmental Environmental Risk Assessment of Soil Contamination 88 balance, due to bioaccumulation, leaching and extension of contaminants into groundwater with potential effects on all living organisms [8]. In Mexico, current environmental regulations is the NOM-138-SEMARNAT/SS-2003 which establishes the maximum permissible limits of hydrocarbons in soil and specifications for its characterization and remediation [9], although in this standard are omitted criteria for assessing chronic effects on soil microorganisms and plants as a result of weathered oil contamination. However, the contributions of several studies show that pollution and waste generation caused by oil activities have deteriorated soil quality [10,11]; ie, the social responsibility of the industry and the government should not only attend acute environmental emergencies [12]. Crude oil is a complex mixture of thousands of compounds that when released to the envi‐ ronment, is subjected to physical, chemical and biological processes called weatherability [13]. This process includes adsorption, volatilization, dissolution, biodegradation, photolysis, oxidation, and hydrolysis. The effects of weathering are difficult to predict because it depends on many biotic and abiotic factors [14]. 1. Introduction Therefore, the mobility of hydrocarbons is also influenced by natural factors [15], which involve: a) chemical processes (hydrolysis, oxidation, reduction, photolysis), b) transport and physical processes (adsorption, advection, dispersion, diffusion, volatilization and dissolution), and c) biological processes (biodegradation, metab‐ olism and toxicity) [16, 17]. When an oil spill occurs, it covers the soil surface, but initially at high viscosity prevents penetration towards the subsoil. The oil is retained in the topsoil, during this phase, the light fraction is photo-oxidized and volatilized through the soil pore space and transported to the atmosphere, in this process involves the first n-alkanes (methane, ethane, propane and butane), which are evaporated in less than 24 hours in tropical climates [18]. The hydrocarbons which are not evaporated are incorporated into the soil to form a waterproofing layer that prevents the normal flow of water. This layer or film affects the structure, porosity, absorption and water penetration into the soil [19]. Subsequently, the oil soluble fraction diffuses into the soil solution through infiltration. The behavior of this fraction in the soil depends on the type of texture. The presence of fine texture allows the volatilization of some compounds (C5-C7), but when the texture is coarse, it can leach out and transport themselves to the groundwater, affecting other organisms [20], including the human [21-23]. Otherwise, clay soils rich in organic matter immobilize some compounds, reducing their toxicity and decreasing its spread and leaching through the soil profile [24, 25]. The most stable fraction of crude oil is composed with more than 18 carbons (Polycyclic Aromatic Hydrocarbons [PAHs] and polar compounds), which are adhered on the soil matrix decreasing the solubility and volatility [26], and increasing the capacity of adsorption on the mineral and organic fractions, owing to the high content of active surface of the soil (clay 2:1) and to the high molecular weight hydrocarbons [27]. At this stage, the development of bacteria and fungi have influence on mineralization as part of natural attenuation process, but it is not sufficient to remove all hydrocarbons weathered [28]. 2. Materials and methods In southeastern Mexico, the land has been affected as a result of extraction; handling and transportation of oil, there are also zones of oil discharges, which are deposited in the open, without any environmental protection measure [32]. The bad condition of the pipelines and the dispersion through surface runoff of rainwater, resulting from weather conditions become more complicated to calculate the chronic effects of soil contamination by hydrocar‐ bons [10]. Accordingly, this study was conducted in three stages, which were analyzed the chemical and physical properties of the soil, the toxic effects of weathered hydrocarbons on the growth and development of seedlings and after 150 and 240 days of exposure, and finally was evaluated the behavior of beneficial soil microorganisms in rhizospheric soil. 2.1. Effect on the physical and chemical properties of the soil The study was undertaken at the facilities the Colegio de Postgraduados, located in Tabasco, Mexico. The soil with weathered oil was collected within 2 km of the Petrochemical "La Venta" (18 ° 04 '54 "N and 94 ° 02' 31" W), Figure 1. The pollution-free soil according to NOM-138- SEMARNAT/SS-2003 was located in the community of Santa Teresa Arroyo Hondo. The objective was to identify the types of soils and their level of similarity. For both soils were determined the content of organic matter (Walkley and Black), pH (potentiometry), P and K exchangeable (by extraction with 1N ammonium acetate pH 7, quantification by atomic absorption and emission respectively), CEC (extraction 1N ammoni‐ um acetate pH 7, quantification by distillation and titration) and texture (Bouyoucos). The analytical methods used were those indicated in NOM-021-2000-RECNAT [33]. Total Petro‐ leum Hydrocarbons (TPH) were determined by the technique reported in the NOM-138- SEMARNAT/SS-2003 [9]. 1. Introduction Chronic Toxicity of Weathered Oil-Contaminated Soil http://dx.doi.org/10.5772/57253 89 89 Recent studies indicate that the major impediments to the biodegradation of hydrocarbons are the physical and chemical properties of the soil, the degree of contamination and the molecular weight of the compounds (C10-C40), but with Enhanced Natural Attenuation (ENA) may be observed a biodegradation of 26.4% [29] up to 60% with enriched amendments, although the n-alkanes are not removed completely [30]. Therefore, it is important to study the toxic potential, because there are reports indicating that can be bioaccumulated PAHs in vegetables such as C. pepo sp. [31]. 2.2. Effects on plants Bioassays to determine the phytotoxicity of weathered oil on two species of legumes (Crotalaria incana and Leucaena leucocephala) were carried out in the facilities of the Laboratory of Soil Microbiology, Colegio de Postgraduados, Tabasco, Mexico. Two states were studied phenological (plant and seedling), in two treatments: a) soil with 150 mg.kg-1 TPH (control treatment) and b) soil with 79.457 mg.kg-1 TPH weathered (this has been contaminated by over Environmental Risk Assessment of Soil Contamination 90 25 years). Both soils were characterized as Gleysols, with the same pedogenetic origin, as described in the previous section. Bioassays were established under a Completely Randomized Design (CRD) with three replications and two legumes, these were selected to be species that grow wild in oiled areas, but the former has tolerance, while the second shows sensitivity to high concentrations of crude oil [34]. In each bioassay was used 208 Protocol of the Organization for Economic Cooperation and Development (OECD) modified according to [35], which allows easily identify the symptoms of stress in the plant. Seedling bioassays: 50 seeds were sown C. incana and 25 L. leucocephala by repetition, respectively. Glass containers were used (32 x 22 x 5.5 cm). The number of seeds sown per plant species was calculated according to the size of the seed [36], the viability of the species [37] and the area of the container. Seeds were previously scarified to remove impermeable integument, which constitute a barrier for germination [38]. Scarification consisted of immersing the seeds in sulfuric acid for 15 minutes and washed with tap water subsequent to remove all acid residues [39]. Germination tests were performed to determine the initial seed viability, finding viability standard values in both blocks [40]. The test lasted 30 days and the variables evaluated were mortality, height, root length and dry matter accumulation aerial and root. Plant bioassays: Bioassays were established plants seedlings 30 days from uncontaminated soil. Subsequently, two of these were transplanted into containers, 15 days after a plant was removed from each container. Exposure of plants to pollutant lasted 150 days to C. incana and 240 days for L. leucocephala, because of its tolerance respective. The physiological variables were evaluated: height, root length, biomass (leaves and stems), root biomass and number of nodes, leaves and fruits. 2.3. Effect on soil microorganisms Quantification of microorganisms was determined by viable count method for serial dilutions [41] for Rhizobium extracts in nodules and Free Living Nitrogen Fixing Bacteria (FLNFB) in rhizospheric soil. Culture media were used combined carbon and yeast-mannitol agar [42]. 2.2. Effects on plants During both assays was provided with water to field capacity and were not supplied nutrients (N, P and K) to avoid interference on the growth of the specimens. The material was weighed on an analytical balance to obtain the values of DM. The multiple comparison of means was performed by Tukey test (a = 0.05). The numerical results were analyzed with SAS software version 9.1, using PROC GLM. 3.1. Effect on the physical and chemical properties of the soil The soil profile and laboratory results confirmed that both soils are Gleysols, with the same pedogenetic origin [43] (Table 1), but the contamination had abnormalities in their chemical characteristics (Table 2), the which have been reported in other studies undertaken in the South East (Table 3). Chronic Toxicity of Weathered Oil-Contaminated Soil http://dx.doi.org/10.5772/57253 91 Figure 1. Study zone Sampling 0-30 cm pH (H2O) OM P K CEC Clay Silt Sand Textural classification TPH (mg.kg-1) (%) mg.kg-1 Cmol(+)kg-1 (%) Soil † 6.3 10.2 23.1 0.35 45.2 61 21 18 Clayey 150 Soil †† 4.2 25.8 3.58 0.4 43.5 48 33 19 Clayey 79,457 * pH 1:2 (potentiometry), organic matter (Walkley and Black), P and K (extraction with 1N ammonium acetate pH 7 and quantification by atomic absorption and emission), CEC (Cation Exchange Capacity) (extraction with 1N ammonium acetate and quantification by distillation and titration), texture (Bouyoucos). † Control soil †† Soil with weathered oil (OM without previous removal of TPH) Table 1. Chemical properties and concentration of TPH in the soils studied. Fi 1 S d Figure 1. Study zone Sampling 0-30 cm pH (H2O) OM P K CEC Clay Silt Sand Textural classification TPH (mg.kg-1) (%) mg.kg-1 Cmol(+)kg-1 (%) Soil † 6.3 10.2 23.1 0.35 45.2 61 21 18 Clayey 150 Soil †† 4.2 25.8 3.58 0.4 43.5 48 33 19 Clayey 79,457 * pH 1:2 (potentiometry), organic matter (Walkley and Black), P and K (extraction with 1N ammonium acetate pH 7 and quantification by atomic absorption and emission), CEC (Cation Exchange Capacity) (extraction with 1N ammonium acetate and quantification by distillation and titration), texture (Bouyoucos). † Control soil Sampling 0-30 cm pH (H2O) OM P K CEC Clay Silt Sand Textural classification TPH (mg.kg-1) (%) mg.kg-1 Cmol(+)kg-1 (%) Soil † 6.3 10.2 23.1 0.35 45.2 61 21 18 Clayey 150 Soil †† 4.2 25.8 3.58 0.4 43.5 48 33 19 Clayey 79,457 * pH 1:2 (potentiometry), organic matter (Walkley and Black), P and K (extraction with 1N ammonium acetate pH 7 and quantification by atomic absorption and emission), CEC (Cation Exchange Capacity) (extraction with 1N ammonium acetate and quantification by distillation and titration), texture (Bouyoucos). * pH 1:2 (potentiometry), organic matter (Walkley and Black), P and K (extraction with 1N ammonium acetate pH 7 and quantification by atomic absorption and emission), CEC (Cation Exchange Capacity) (extraction with 1N ammonium acetate and quantification by distillation and titration), texture (Bouyoucos). 3.1. Effect on the physical and chemical properties of the soil * pH 1:2 (potentiometry), organic matter (Walkley and Black), P and K (extraction with 1N ammonium acetate pH 7 and quantification by atomic absorption and emission), CEC (Cation Exchange Capacity) (extraction with 1N ammonium acetate and quantification by distillation and titration), texture (Bouyoucos). †† Soil with weathered oil (OM without previous removal of TPH) Table 1. Chemical properties and concentration of TPH in the soils studied. Table 1. Chemical properties and concentration of TPH in the soils studied. Environmental Risk Assessment of Soil Contamination 92 92 Sampling 0-30cm pH OM P K CEC (%) mg.kg-1 C mol(+) kg-1 Control soil Moderately acid Medium High Medium Very High Polluted soil †† Strongly acidic Very high Low Medium Very High †† Soil with weathered oil (OM without previous removal of TPH) Table 2. Interpretation of the characteristics of the soils studied. The interpretation was based on the ranges indicated in NOM-021-2000-RECNAT. Alterations Effects High ratios of C / N and C / P Unfavorable microbial growth [44] Retaining TPH fractions in organic matter Alter the solubility of phosphorus [45] Increasing Na Limitations in the production of plants [11, 46] Decreasing pH Decrease microbionas populations [47] The electrical conductivity can be increased up to 5.6 times Salinity [48] Interruption in the interaction between cations Ca and K Reduction in capacity of soil to retain Ca and K [32] Table 3. Alterations reported in soils contaminated with TPH, in southeastern Mexico. Also, many physical properties of soil are altered, such as water retention capacity; this is because when an oil spill occurs, hydrocarbons bit compete with water filling the pores [49]. On the other hand, increasing the moisture content in the soil reduces the adsorp‐ tion of liquid hydrocarbons in the organic matter and clay and the filling of the pores and capillaries (Figure 2) [50]. Figure 2. Alteration in the retaining moisture of soil. Figure 2. Alteration in the retaining moisture of soil. The oil can form macroaggregates and macropores that increase water flow (Figure 3), and these changes inhibit the water retention in the soil, which is moistened only after a long period of contact [51], so that the plants may suffer water stress and die. Figure 2. Alteration in the retaining moisture of soil. 3.1. Effect on the physical and chemical properties of the soil The oil can form macroaggregates and macropores that increase water flow (Figure 3), and these changes inhibit the water retention in the soil, which is moistened only after a long period of contact [51], so that the plants may suffer water stress and die. The oil can form macroaggregates and macropores that increase water flow (Figure 3), and these changes inhibit the water retention in the soil, which is moistened only after a long period of contact [51], so that the plants may suffer water stress and die. The oil can form macroaggregates and macropores that increase water flow (Figure 3), and these changes inhibit the water retention in the soil, which is moistened only after a long period of contact [51], so that the plants may suffer water stress and die. Chronic Toxicity of Weathered Oil-Contaminated Soil http://dx.doi.org/10.5772/57253 93 Figure 3. Formation of macroaggregates in soil with 12,155 mg.kg-1 TPH weathered. Figure 3. Formation of macroaggregates in soil with 12,155 mg.kg-1 TPH weathered. 3.2. Effects on plants The results indicated that the lower height and root length were found in soil with 79,457 mg.kg-1 of weathered oil, this can be explained because of the limited development of these. Both legumes formed less biomass in soils with higher concentrations of oil, which was associated with lower production of biomass in leaves and stems, as a result of the presence of the contaminant in the soil (Table 4 and 5), which limits the entry of water to the plant. However, the hydrophobic effect also affects the early stages of germination as evidenced with a delay of up to five days in the emergency [52], even in high concentrations of TPH, germi‐ nation may be inhibited completely [53]. Some authors found that exposure to concentrations of 2 791, 9 025 and 79 457 mg.kg-1 of petroleum hydrocarbons in soil inhibited the vegetative growth and reduced plant biomass in seedlings of Echinochloa polystachya, Brachiaria mutica and Cyperus sp [10]. Others establish increased toxicity on the dry weight of rice seedlings (Oryza sativa) after 25 days of exposure to 90,000 mg.kg-1 weathered oil [54]. Biomass reduction is possibly due to widespread damage, which begins in the root system, hindering vegetative growth and therefore the accumulation of plant biomass. However, the first studies mentions that at low concentrations, oil could stimulate vegetative growth [55]. Crotalaria incana seedlings did not form nodules in soil contaminated with 79,457 mg.kg-1 of weathered oil after 30 days of exposure to oil. These results were similar to those obtained according to [34], who found that nodulation was completely inhibited in Crotalaria sp. and Mimosa pigra by concentrations above 50,000 mg.kg-1. Environmental Risk Assessment of Soil Contamination 94 In both plant species, growth decreased with higher concentration of hydrocarbons in soil (Table 5 and 6). This response may be related to decreased water absorption through the roots for the presence of the hydrophobic film formed by the oil added to the soil [56]. The lack of water absorbed decreases cell turgor, reduces or inhibits the processes of incorporation of nutrients and also affects vegetative growth [57, 58]. Water stress is related to the water potential gradient (which depends on the conditions present in the soil) and the membrane permeability to water (which depends on the species [59] therefore, Leucaena leucocephala shown to be more tolerant to water deficit). 3.2. Effects on plants Species Concentration mg.kg-1 Height Root length Mortality Biomass Leaves Stems Aerial Root Total cm (%) gr Leucaena 150 7.5a 19.0a 0.0a 3.3a 1.3a 4.6a 1.1a 5.7a 79,457 5.1b 5.1b 4.2a 1.5b 0.5b 2.0b 0.5b 2.5b Values with different letter are statistically different (Tukey, p = 0.05) Table 4. Response of Crotalaria incana seedlings to 30 days of weathered oil exposure. Species Concentration mg.kg-1 Height Root length Mortality Biomass Leaves Stems Aerial Root Total cm (%) gr Species Concentration mg.kg-1 Height Root length Mortality Biomass Leaves Stems Aerial Root Total cm (%) gr Leucaena 150 7.5a 19.0a 0.0a 3.3a 1.3a 4.6a 1.1a 5.7a 79,457 5.1b 5.1b 4.2a 1.5b 0.5b 2.0b 0.5b 2.5b Values with different letter are statistically different (Tukey, p = 0.05) f f Table 4. Response of Crotalaria incana seedlings to 30 days of weathered oil exposure. Table 4. Response of Crotalaria incana seedlings to 30 days of weathered oil exposure. Species Concentration mg.kg-1 Height Root length Mortality Biomass Leaves Stems Aerial Root Total cm (%) gr Crotalaria 150 9.8a 9.8a 2.6a 1.6a 1.0a 2.6a 0.6a 3.2a 79,457 2.9b 2.8b 28.6a 1.0a 0.5b 1.5b 0.4b 1.9b Values with different letter are statistically different (Tukey, p = 0.05) Table 5. Response of Leucaena leucocephala seedlings to 30 days of weathered oil exposure. ble 5. Response of Leucaena leucocephala seedlings to 30 days of weathered oil exposure. Crotaria incana plants showed a greater effect on biomass in response with exposure to the weathered oil (Figure 4), which may be related to increased toxicity of recalcitrants com‐ pounds. There are also some soil properties which allow the adsorption of pollutants [60]. Clay soils with high organic content and low pH may favor the persistence of toxic substances in the soil for a long time after the oil spill occurred [61, 34], due to the adhesiveness of organic matter [62]. Furthermore, fine texture allows the oil form a coarse structure on the outside and around the conglomerate making it waterproof [63], this has effects on root development, growth, and as a result will cause decrease in the accumulation biomass [59]. On the other hand, soil contam‐ ination by hydrocarbons can also modify some characteristics such as texture, bulk density, ratio of the particle size of the soil, reducing aeration and affecting the productive development of plants [64, 65]. Chronic Toxicity of Weathered Oil-Contaminated Soil http://dx.doi.org/10.5772/57253 95 a) b) c) d) Figure 4. 3.2. Effects on plants Toxicity on Leucaena leucocephala seedlings with: a) 150 mg.kg-1 y b) 79,457 mg.kg-1 TPH weathered, and plants with c) 150 mg.kg-1 y d) 79,457 mg.kg-1 TPH weathered. a) b) Figure 4. Toxicity on Leucaena leucocephala seedlings with: a) 150 mg.kg-1 y b) 79,457 mg.kg-1 TPH weathered, and plants with c) 150 mg.kg-1 y d) 79,457 mg.kg-1 TPH weathered. Species Concentration Height Root length Knot Leaves Fruits Biomass Aerial Root Leaves Stems Inflorescences Seeds Pods mg.kg-1 cm Number gr Crotalaria incana 150 91.7a 17.3a 16.0a 112.7a 54.0a 47.5a 5.6a 11.0a 14.7a 1.5a 9.6a 10.8a 79,457 32.3b 7.0b 15.0a 2.7b 0.0b 0.6b 0.2b 0.1b 0.6b 0.0b 0.0b 0.0b Values with different letter are statistically different (Tukey, p = 0.05) Table 6. Response of Crotalaria incana to 150 days of weathered oil exposure. Species Concentration Height Root length Knot Leaves Fruits Biomass Aerial Root Leaves Stems Inflorescences Seeds Pods mg.kg-1 cm Number gr Leucaena leucocephala 150 149.0a 107.7a 43.7a 19.1a -- 88.6a 34.0a 19.1a 69.5a -- -- -- 79,457 66.7b 7.3b 31.0b 4.6b -- 15.9b 6.9b 4.6b 11.3b -- -- -- Values with different letter are statistically different (Tukey, p = 0.05) Table 7. Response of Leucaena leucocephala to 240 days of weathered oil exposure. Table 7. Response of Leucaena leucocephala to 240 days of weathered oil exposure. Table 7. Response of Leucaena leucocephala to 240 days of weathered oil exposure. Environmental Risk Assessment of Soil Contamination 96 3.3. Effect on soil microorganisms On the other hand, several authors argue that changing Chronic Toxicity of Weathered Oil-Contaminated Soil http://dx.doi.org/10.5772/57253 97 the C: N: P, the water content and the water retention capacity of clay soils are crucial to obtain the highest rate of degradation of TPH [74, 75]. the C: N: P, the water content and the water retention capacity of clay soils are crucial to obtain the highest rate of degradation of TPH [74, 75]. Acknowledgements The author wants to especially thank Dr. Macario Vázquez Rivera, Irma Luna Capetillo, Mayra Vázquez Luna and Daniel Lara Rodríguez for their valuable support and the Technical Council of COLPOS for their scientific consultancy. Finally, the author wants to thank the Universidad Veracruzana for financing the publication of the present article. 4. Conclusion Soil is one of the most valuable resources that humanity has due to the variety of services offered and which depend for food. Currently, there is tremendous competition for land use, either for use as human settlements, commercial, industrial or produce raw materials (wood) and food, so weathered oil pollution is one of the most important challenges for scientists, because not only it is decontaminated, it comes to restoring the quality and safety of the soil, its properties and microbial component vital for production and proper plant development. Address all correspondence to: divazquez@uv.mx Universidad Veracruzana, México 3.3. Effect on soil microorganisms In plants Leucaena leucocephala high concentration of weathered oil did not affect populations of Rhizobium into nodules, as happened in the case of C. incana, in which both populations were significantly lower in a shorter time exposure (Figure 5). This is because the oil alters the physical and chemical characteristics of the soil, causing the blockage of gas exchange with the atmosphere and affecting microbial populations. Furthermore, the weathered oil is adsorbed in the ground, being less accessible and more difficult to degrade by microorganisms [66, 67]. This may bring a direct impact on rhizobia, because they are aerobic bacteria that remain in the soil as saprophytes, until they infect a radical hair. Some authors mention that soil conditions have a marked effect on rhizobia, because they can impact the survival and the infectivity of root hairs [68, 69]. However, there are many other factors that influence on effectiveness symbiosis such as specificity and virulence of the bacterium Rhizobium, nutrimental factors, soil temperature and pH [70], the latter is of utmost importance because has been reported to decrease significantly in contaminated soil [38]. Added to this there are other factors such as the accumulation of heavy metals and salts that affect soil microbiota [11, 4]. 0.0E+00 5.0E+05 1.0E+06 1.5E+06 2.0E+06 2.5E+06 3.0E+06 3.5E+06 4.0E+06 4.5E+06 150 79,457 Microorganism (CFU g-1) Concentration (mg.kg-1) Crotalaria incana 0.0E+00 5.0E+06 1.0E+07 1.5E+07 2.0E+07 2.5E+07 150 79,457 Concentration (mg.kg-1) Leucaena Leucocephala Rhizobium Free living nitrogen fixing bacteria Figure 5. Quantification of populations of Rhizobium extracts in nodules and Free Living Nitrogen Fixing Bacteria in rhizospheric soil. Leucaena Leucocephala Figure 5. Quantification of populations of Rhizobium extracts in nodules and Free Living Nitrogen Fixing Bacteria in rhizospheric soil. L. leucocephala almost doubled FLNFB populations with 79,457 mg.kg-1 of weathered oil. This can be explained because in tolerant species, some microorganisms can increase their popu‐ lations in the presence of hydrocarbons [71], allowing support microbial growth. 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https://openalex.org/W4293278426	https://eprints.whiterose.ac.uk/191230/1/HCCR_22_17911_Authorproof_1_.pdf	English	null	Assessment of Public-Private Partnership (PPP) models in health systems in least developed, low income and lower-middle-income countries and territories: A protocol for a Systematic Review	Research Square (Research Square)	2,022	cc-by	4,707	Article: Article: Naznin, Baby, Quayyum, Zahidul, Tajree, Jannatun et al. (16 more authors) (2022) Assessment of Public-Private Partnership (PPP) Models in Health Systems in Least Developed, Low Income and Lower-Middle-Income Countries and Territories:A Protocol for a Systematic Review. Health Care: Current Reviews. ISSN 2375-4273 https://doi.org/10.35248/2375-4273.22.10.306 https://doi.org/10.35248/2375-4273.22.10.306 Reuse This article is distributed under the terms of the Creative Commons Attribution (CC BY) licence. This licence allows you to distribute, remix, tweak, and build upon the work, even commercially, as long as you credit the authors for the original work. More information and the full terms of the licence here: https://creativecommons.org/licenses/ White Rose Research Online URL for this paper: https://eprints.whiterose.ac.uk/191230/ Version: Published Version Version: Published Version This is a repository copy of Assessment of Public-Private Partnership (PPP) Models in Health Systems in Least Developed, Low Income and Lower-Middle-Income Countries and Territories:A Protocol for a Systematic Review. White Rose Research Online URL for this paper: https://eprints.whiterose.ac.uk/191230/ Assessment of Public-Private Partnership (PPP) Models in Health Systems in Least Developed, Low Income and Lower-Middle-Income Countries and Territories: A Protocol for a Systematic Review Baby Naznin1*, Zahidul Quayyum1, Jannatun Tajree1, Su Golder2, Bassey Ebenso3, Deepa Barua4, Maisha Ahsan4, Faisal Kabir4, Deepak Joshi5, Sampurna Kakchapati5, Abena Engmann6, Pamela Adaobi Ogbozor7, Prince Agwu8, Chinyere Okeke9, Juliana Onuh10, Chukwuedozie Ajaero10, Aishwarya Vidyasagaran2, Helen Elsey2, Florence Sibeudu11 1Department of Public Health, BRAC University, Dhaka, Bangladesh;2Department of Health Sciences, University of York, York, England;3Department of Health Sciences, University of Leeds, Leeds, England; 4Department of Health, ARK Foundation, Dhaka, Bangladesh;5Department of Health, HERD International, Kathmandu, Nepal;6Department of Health, University of Ghana, Accra, Ghana; 7Department of Health Policy Research Group, University of Nigeria, Nsukka, Nigeria;8Department of Social Work and Health Policy Research Group, University of Nigeria, Nsukka, Nigeria;9Department of Community Medicine, University of Nigeria, Nsukka, Nigeria;10Department of Geography, University of Nigeria, Nsukka, Nigeria;11Deparment of Health, Nnamdi Azikiwe University, Nnewi, Nigeria Copyright: © 2022 Naznin B, 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. Citation: Naznin B, Quayyum Z, Tajree J, Golder S, Ebenso B, Barua D, et al. (2022) Assessment of Public-Private Partnership (PPP) Models in Health Systems in Least Developed, Low Income and Lower-Middle-Income Countries and Territories: A Protocol for a Systematic Review. Health Care Curr Rev. 10:306 Correspondence to: Baby Naznin, Department of Public Health, BRAC University, Dhaka, Bangladesh. E mail: baby.naznin@bracu.ac.bd Received: 30-Aug-2022, Manuscript No. HCCR-22-17911; Editor assigned: 02-Sep-2022, Pre QC No. HCCR-22-17911(PQ); Reviewed: 16-Sep-2022, QC No. HCCR-22-17911; Revised: 22-Sep-2022, Manuscript No. HCCR-22-17911(R); Published: 29-Sep-2022, DOI: 10.35248/2684-1320.22.10.306 Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request. eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ OPEN ACCESS Freely available online Book Reviews Assessment of Public-Private Partnership (PPP) Models in Health Systems in Least Developed, Low Income and Lower-Middle-Income Countries and Territories: A Protocol for a Systematic Review ABSTRACT Background: Private sectors play a significant role in health provision along with the public sector in both developed and developing countries. Given the limited resources of the public sector, public-private partnerships (PPPs) are considered as a good solution to address the growing public health challenges. But inadequate assessment of various health-related PPPs has resulted in a failure to gather knowledge and evidence that would facilitate the establishment of effective partnerships, sustain, and systematize them over time, as well as determine the role of PPPs in health system strengthening, particularly in terms of urban health provision. The objective of this research is to systematically review the effectiveness of PPPs on the utilization of urban health provision to achieve health outcomes in the urban contexts of least developed, low income, and lower-middle-income countries and territories. Methods: This systematic review will follow PRISMA-P guidelines for reporting. Relevant databases-EMBASE, MEDLINE, Health Management Information Consortium, Social Sciences Citation Index, Science Citation Index, Emerging Sources, CENTRAL, i.e., Database of disability and inclusion information resources, and WHO Library Database–will be searched for published articles in the urban context. Reference lists of relevant systematic reviews and commentaries and citations of key included studies will be checked for additional studies. Two reviewers will independently screen the studies in covidence following the exclusion and inclusion criteria. Data will be thematically analysed and narratively synthesized. Discussion: This review will comprehensively assess and appraise all the existing PPP models for urban health provision in the least developed, low income, and lower-middle-income countries and territories. The findings of the review will help to understand the modalities of the existing health related PPPs in urban areas, their functionalities, and their contribution in achieving health outcomes. Protocol registration: This protocol is registered with the International Prospective Register of Systematic Reviews, PROSPERO (ID-CRD42021289509, 23 November 2021). Keywords: Urban health care; Disease; Public-private partnership models; Territories Citation: Naznin B, Quayyum Z, Tajree J, Golder S, Ebenso B, Barua D, et al. (2022) Assessment of Public-Private Partnership (PPP) Models in Health Systems in Least Developed, Low Income and Lower-Middle-Income Countries and Territories: A Protocol for a Systematic Review. Health Care Curr Rev. 10:306 Copyright: © 2022 Naznin B, 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. Health Care: Curr Rev, Vol. Health Care: Curr Rev, Vol. 10 Iss. 8 No: 1000306 ABSTRACT 10 Iss. 8 No: 1000306 Health Care: Curr Rev, Vol. 10 Iss. 8 No: 1000306 1 1 aznin B, et al. Abbreviations: AIDS: Acquired Immunodeficiency Syndrome; ALG: Action Learning Group; CHORUS: Community-led Responsive and Effective Urban Health System; FCDO: Foreign, Commonwealth and Development Office; COPD: Chronic Obstructive Pulmonary Disease; DAC: Development Assistance Committee; HIV: Human Immunodeficiency Virus; HMIC: Health Management Information Consortium; LMIC: Lower-Middle-Income Countries; MMAT: Mixed Methods Appraisal Tool; OECD: Economic Co-operation and Development; PPP: Public Private Partnership; PRISMA: Preferred Reporting Items for Systematic Reviews and Meta-Analyses; SCI: Science Citation Index; SSCI: Social Sciences Citation Index; SWiM: Synthesis without meta-analysis; WHOLIS: WHO Library Database. Naznin B, et al. Abbreviations: AIDS: Acquired Immunodeficiency Syndrome; ALG: Action Learning Group; CHORUS: Community-led Responsive and Effective Urban Health System; FCDO: Foreign, Commonwealth and Development Office; COPD: Chronic Obstructive Pulmonary Disease; DAC: Development Assistance Committee; HIV: Human Immunodeficiency Virus; HMIC: Health Management Information Consortium; LMIC: Lower-Middle-Income Countries; MMAT: Mixed Methods Appraisal Tool; OECD: Economic Co-operation and Development; PPP: Public Private Partnership; PRISMA: Preferred Reporting Items for Systematic Reviews and Meta-Analyses; SCI: Science Citation Index; SSCI: Social Sciences Citation Index; SWiM: Synthesis without meta-analysis; WHOLIS: WHO Library Database. Abbreviations: AIDS: Acquired Immunodeficiency Syndrome; ALG: Action Learning Group; CHORUS: Community-led Responsive and Effective Urban Health System; FCDO: Foreign, Commonwealth and Development Office; COPD: Chronic Obstructive Pulmonary Disease; DAC: Development Assistance Committee; HIV: Human Immunodeficiency Virus; HMIC: Health Management Information Consortium; LMIC: Lower-Middle-Income Countries; MMAT: Mixed Methods Appraisal Tool; OECD: Economic Co-operation and Development; PPP: Public Private Partnership; PRISMA: Preferred Reporting Items for Systematic Reviews and Meta-Analyses; SCI: Science Citation Index; SSCI: Social Sciences Citation Index; SWiM: Synthesis without meta-analysis; WHOLIS: WHO Library Database. Defining Public Private Partnerships (PPP) Defining Public Private Partnerships (PPP) Overall, this study will deal with urban health provision in the context of PPPs. For the purpose of this review, we draw on the definitions of PPPs given by Tabrizi et al. [13] and by Hellowell [14], whereby PPP is any long term partnership (i.e. not a one-off event), including both formal and informal arrangements, where the public sector (government and other governmental entities) uses the capacity of the private sector (private companies, cooperatives, charities, non-governmental organizations and informal private providers) in order to improve and protect the health of populations. Besides, by the term ‘urban’, we refer to all the semi-urban, peri- urban, sub-urban, urban slum areas in least developed, low-income and lower middle-income countries. Health Care: Curr Rev, Vol. 10 Iss. 8 No: 1000306 INTRODUCTION sector, evidence lack on the effectiveness of different PPP models in achieving health outcomes in the context of growing urban system needs. The objective of this research is therefore to systematically review the existing PPP models in urban health systems in the context of least developed, low-income, and lower middle-income countries and to explore their contributions and effectiveness in bringing changes in urban health services and outcomes. Traditionally, it is the government that has been responsible for delivering health care to its population with support from tax and non-tax revenue [1]. In most of the Lower-Middle Income Countries (LMICs), health sector receives relatively lower funding compared to other development sectors [2]. This trend as well as underperformance of the public health sector resulted into gaps in health care service delivery, attracting private sector to play a role in the healthcare market [3]. Following this situation, private sector started to grow gradually and has become popular in health service delivery [4]. Since last two decades, private sector is playing a major role in health service provision along with public sector in both developed and developing countries. But private health providers are highly concentrated in urban areas due to rapid urbanization, larger scope compared to rural areas, and the need to make a profit from a relatively wealthier urban population. Besides, private health care is much more expensive than public health care because private providers have motives to make a profit out of health service provision. Thus, given the limited resources and inefficiencies of the public sector and the business approach of the private sector resulting in a higher level of out-of-pocket expenditure, it has been well understood that neither the public sector nor the private sector alone is in the best interest of any health system. In recent years, consequently, Public-Private Partnerships (PPPs) are progressively being considered as a straightforward mechanism to address the growing public health problems [5]. Governments from both developed and developing countries are increasingly trying to implement Public-Private Partnership (PPP) based health care models as a way to expand access and improved quality of health care [6]. Naznin B, et al. Review Action Learning Group (ALG) which aims to engage and develop capacity of researchers across CHORUS partners related to systematic review. Citation Index (SSCI), Science Citation Index (SCI), Emerging Sources, CENTRAL (via The Cochrane Library, includes EPOC search register), i.e., Database of disability and inclusion information resources, and WHO Library Database (WHOLIS). No language or date restrictions will be applied to the searches. Citation Index (SSCI), Science Citation Index (SCI), Emerging Sources, CENTRAL (via The Cochrane Library, includes EPOC search register), i.e., Database of disability and inclusion information resources, and WHO Library Database (WHOLIS). No language or date restrictions will be applied to the searches. Search strategy and data sources The search strategy will aim to identify studies examining different PPP models existing in urban health sectors in least developed, low income and lower middle-income countries and territories. A search strategy (Supplementary Table 3) has been developed by an information specialist based on the eligibility criteria indicated in Table 1. METHODOLOGY Besides, reference lists of relevant systematic reviews and commentaries will be checked for identifying any additional relevant research. In addition, citations of key included studies will be screened. Reporting of review findings This systematic review will use the reporting items for systematic reviews and meta-analyses protocols (PRISMA-P) guideline for reporting [15]. The PRISMA-P checklist 2020 is provided at Supplementary Table 1. Besides, a completed reporting checklist for the systematic review protocol has been added (Supplementary Table 2). Search terms Private public mix/, Public adj2 private adj2 partnership$, Public adj2 private adj2 mix, public sector$ and private sector$, Private adj2 public adj2 collaboration $, Private adj2 public adj2 cooperation $, Public-Private Sector Partnerships/, LMICs. Search strategy and data sources Objectives The study aims to assess the existing PPP models in urban health systems in least developed, low-income, and lower middle-income countries and to explore their contributions in bringing changes in urban health services and outcomes. The specific objectives are the following: 1. To investigate different types of PPPs (formal and informal contractual arrangements) that exist in the urban health context of least developed, low-income, and lower middle-income countries and territories. The World Health Organization (WHO) describes PPPs for health as “public sector programs with private sector participation” [7]. PPPs require the public and the private sector to share the risk, responsibility, benefits and to synchronize resources and expertise of both sectors [8]. In PPPs, usually, the role of government changes from the investor, implementer, and beneficiary to policymaker, regulator, and supervisor of the quality and quantity of services provided [9,10] while the private groups contribute to the process of service provision. Thus, various models of PPPs have been developed and trialled around the world in health service delivery. Some of them have produced highly encouraging results and some are challenging [11,12]. Nevertheless, inadequate assessment of these initiatives has resulted in failure to gather knowledge and necessary evidence that would facilitate the establishment of evidence-based effective models sustain and systematize them over time and facilitate transfer of successful programs [13]. Besides, there is lack of evidence on the role of PPPs in health system strengthening, particularly in terms of delivering urban primary and secondary health care services. Although several reviews have been taken place discussing the types and rationale of PPPs in health 2. To understand the specific roles of government and private sector or NGOs within the partnerships or contractual arrangements. 3. To understand the target populations being covered in the service provision of existing PPPs. 4. To study the level of access, coverage, and utilisation of services in existing PPPs. 5. To explore effectiveness of PPPs in increasing access/coverage/ utilization of urban health services and improving health outcomes. This systematic review will be conducted as part of the Community- led Responsive and Effective Urban Health System (CHORUS) Research Program Consortium, funded by Foreign, Commonwealth and Development Office (FCDO). The consortium aims to develop and evaluate health systems interventions to improve urban health in Bangladesh, Nepal, Nigeria, and Ghana. This review forms a part of the activities carried out by the CHORUS Systematic 2 Naznin B, et al. Eligibility criteria Data screening: Two reviewers will independently screen the studies, first by title and abstract, and then full-texts following the pathway of exclusion and inclusion indicated in Figure 1. The number of records identified, duplicates removed, titles and abstracts screened, studies retrieved and included in the review will be recorded in a PRISMA flow diagram for systematic reviews. Covidence will be used to manage the screening of all identified studies (Figure 1 and Table 1). Relevant published articles on health sector PPPs in the urban context of least developed, low income and lower middle-income countries and territories will be identified by searching a number of electronic databases-EMBASE, MEDLINE, Health Management Information Consortium (HMIC), Web of Science: Social Sciences Figure 1: Inclusion and exclusion pathway of the systematic review. Figure 1: Inclusion and exclusion pathway of the systematic review. Health Care: Curr Rev, Vol. 10 Iss. 8 No: 1000306 3 3 Naznin B, et al. Table 1: Inclusion and exclusion criteria [PICO]. Criteria Inclusion Exclusion Participants/population All urban populations in least developed, low income and lower middle- income countries and territories. All rural populations Intervention Urban health provision in the context of PPPs (i.e., primary/secondary/ tertiary healthcare provision, or diagnostic service provision, or provision of primary/secondary/tertiary prevention services) in any healthcare or non- healthcare setting. PPPs that do not deal with health provision/ services/ prevention relate Comparators General urban health provision systems (for studies showing comparison); Not applicable No comparison for descriptive or narrative studies on urban health related PPPs. Outcomes[1] Primary Outcome Not applicable ─ Access, coverage and utilization of PPP related urban health provision in relation in any of the secondary health outcomes as well as process outcomes Secondary Outcome Any health outcomes reported by the included studies (as long as they are for urban populations). These outcome(s) are likely to include the following health domains: · Sexual, reproductive and maternal health ─ Maternal deaths ─ Maternal mortality rate ─ Number or proportion of birth attended by skilled health personnel ─ Uptake of antenatal and postnatal care ─ Uptake of family planning ─ Total fertility rate · Child Health ─ Level and status of infant nutrition ─ Infant and child mortality rate ─ Level of Immunization coverage · Communicable diseases, e.g. Tuberculosis, Malaria, HIV/AIDS) · Non-communicable diseases and chronic conditions, e.g. AVAILABILITY OF DATA AND MATERIALS Not applicable. Risk of bias (Quality) assessment For quality assessment, we will use the Mixed Methods Appraisal Tool MMAT which covers mixed methods, qualitative and quantitative (subdivided into three sub-domains: Randomised controlled, non-randomised, and descriptive [16]. The MMAT facilitates critical appraisal of studies within systematic reviews by providing methodological quality criteria for different study designs/methods within a single tool. Two authors will conduct the quality appraisal independently and discrepancies will be identified and resolved by consensus between them, or where disagreements persist, by discussion with a third reviewer. CONCLUSION The findings of the review will help to understand the modalities of the existing health related PPPs in urban areas, their functionalities and their contribution in achieving health outcomes. Besides, the review will also explore the roles and responsibilities of different players (i.e., government, private sector, NGOs) within the partnerships and their collaboration mechanisms. In addition, this review is expected to help policymakers by informing the effectiveness of PPPs in increasing access/coverage/utilization of urban health services. Data synthesis Quantitative findings: We will present several ‘summary of findings’ tables based on extracted data, and an additional table of detailed quality appraisal. It is anticipated that included studies will have a variety of research designs and we will order studies by study design and within design type by quality/risk of bias. We anticipate grouping quantitative studies by: Type of PPP model, Health outcomes (e.g., maternal health, child health, etc.), process outcomes (e.g., cost, quality, staff competency, etc.). We may need to revise these groupings after extraction, noting any deviations from the protocol in our report. We will identify if we can calculate a standardised metric for different outcome groups, referring to methodological guidance (e.g., Cochrane Handbook) as appropriate. We will assess the level of heterogeneity across studies using the I² statistic to assess the appropriateness of conducting a meta-analysis. Given the wide variety of outcomes we consider a meta-analysis unlikely. We will report our narrative synthesis of quantitative findings using the SWiM Guidelines [17]. Eligibility criteria • Roles and responsibilities of various partners/actors (providers; purchasers/payers; financers) in the partnership • Roles and responsibilities of various partners/actors (providers; purchasers/payers; financers) in the partnership • Infrastructure, function and processes built for the partnership • Extent and types of investment of the partners • Regulatory and quality assurance/improvement measures Data collection Data collection was done with the use of a structured questionnaire with open and closed ended questions. The questionnaire was divided into four sections. Section A contained the demographic data, section B was the challenges face by HIV patients, section C contained sexual behavior and section D was the traditional beliefs of the participants. The questionnaires were inserted into envelopes with pens for confidentiality and were self-administered. FUNDING This systematic review will be conducted as part of the Community-led Responsive and Effective Urban Health System (CHORUS) Research Program Consortium, funded by Foreign, Commonwealth and Development Office (FCDO) with Grant Number: 301132 (Supplementary Table 4). RESULTS AND DISCUSSION • Any aspects of market failure in health care addressed In many cases, PPPs have been discussed as a better way to address the challenges of accessing health care services by providing cost- effective, efficient and quality healthcare. However, there is little clarity about the role of PPPs in achieving health outcomes in the context of urban health systems. This review will comprehensively assess and appraise all the existing health care models in the least developed, low income, and lower-middle-income countries and territories in the urban context. • Health service package delivered • Costs of care to each partner and any cost recovery measures (e.g., from health insurance etc.). • Costs of care to each partner and any cost recovery measures (e.g., from health insurance etc.). Eligibility criteria diabetes, cardiovascular disease, cancers, COPD, asthma, mental health, injuries · Geriatric care · Genetic disease care Process Outcomes · Effectiveness of partnership agreements, governance and contracting · Cost of care and health care financing · Quality of care · Patient satisfaction · Staff competence/motivation · Responsiveness of care · Targeting of services to urban poor Study design and publication type Published Journal Articles reporting primary research of any of the following types: qualitative and mixed-methods evaluations, cohort study, cross- sectional study, randomised control trial, observational study, retrospective study, intervention study Systematic or scoping reviews; lette editorials, conference abstracts Aspect of health care Primary/secondary/tertiary healthcare provision, or diagnostic service provision, or provision of primary/seconda tertiary prevention services Time period Since 1990 (year of inception of the databases) Language All languages (relying on the team’s own language skills/google translate) None Place of study Least developed, low income and lower middle income countries and territories following the defined DAC list of OECD 2021 Upper middle-income countries as OECD DAC list (https://www.oecd.org/dac/financing-sustainable-development/development- finance-standards/DAC-List-ODA-Recipients-for-reporting-2021-flows.pdf) Geographical limitation Urban (i.e., Semi-urban, peri-urban, sub-urban, urban slums) Rural or remote areas NOTE: [1] The effect measures of the outcomes will be specified through reporting of change in access, coverage and utilization of PPP related h l h / dd l / / l b b b k d h Table 1: Inclusion and exclusion criteria [PICO]. Health Care: Curr Rev, Vol. 10 Iss. 8 No: 1000306 4 Naznin B, et al. Data extraction and management Result-based convergent synthesis: Following the separate synthesis of qualitative and quantitative results, we will compare findings and explore the descriptions of context and models to help explain any differences in findings. This will allow us to present overall findings on the types of PPP models, the barriers and facilitators to their implementation and the resultant process and health outcomes [19]. Result-based convergent synthesis: Following the separate synthesis of qualitative and quantitative results, we will compare findings and explore the descriptions of context and models to help explain any differences in findings. This will allow us to present overall findings on the types of PPP models, the barriers and facilitators to their implementation and the resultant process and health outcomes [19]. DECLARATIONS AND ACKNOWLEDGEMENTS The mentors of CHORUS Systematic Review Action Learning Group (Zahidul Quayyum, Helen Elsey, Bassey Ebenso) have developed this protocol along with ALG members (the reviewers). One of the reviewers, Su Golder developed the search strategy following eligibility criteria and another reviewer, Aishwarya Vidyasagaran is coordinating the screening of the studies with the use of COVIDENCE. In addition, we would like to thank Tolib Mirzoev, Professor, Department of Global Health and Development, London School of Hygiene and Tropical Medicine for his guidance and suggestions in the study design. Health Care: Curr Rev, Vol. 10 Iss. 8 No: 1000306 CONSENT FOR PUBLICATION 6. World Bank Group. Public-Private Partnerships in Health, engagement in Health PPPs: An IEG Synthesis Report. 2016. All the authors of this systematic review have confirmed their interest for publishing this protocol. 7. WHO. Trade, foreign policy, diplomacy and health: Public–private partnerships for health. World Heal Organ. 2015 SUPPLEMENTARY FILES 15. Moher D, Shamseer L, Clarke M, Ghersi D, Liberati A, Petticrew M, et al. Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 statement. Systematic reviews. 2015; 4(1):1-9. Supplementary table 1: PRISMA P guideline. Supplementary table 1: PRISMA P guideline. Supplementary table 2: Reporting checklist for protocol of a systematic review. Supplementary table 2: Reporting checklist for protocol of a systematic review. 16. Pluye P, Gagnon MP, Griffiths F, Johnson-Lafleur J. A scoring system for appraising mixed methods research, and concomitantly appraising qualitative, quantitative and mixed methods primary studies in mixed studies reviews. Int J Nurs Stud. 2009; 4 6(4):529-546. Supplementary table 3: Search strategies. Supplementary table 3: Search strategies. Supplementary table 4: Funding related information. 17. Campbell M, McKenzie JE, Sowden A, Katikireddi SV, Brennan SE, Ellis S, et al. Synthesis without meta-analysis (SWiM) in systematic reviews: reporting guideline. bmj. 2020; 368. ETHICS APPROVAL AND CONSENT TO PARTICIPATE 8. Davies P. The role of the private sector in the context of aid effectiveness: consultative findings document, report prepared for the OECD Development Assistance Committee Working Party on Aid Effectiveness prior to the Fourth High-Level Forum on Aid Effectiveness . 2011. [Google Scholar] Not applicable. PROTOCOL REGISTRATION This protocol has been registered in the Prospective Register of Systematic Reviews international registry, PROSPERO (ID CRD42021289509, 23 November 2021) and available at: https://www.crd.york.ac.uk/prospero/display_record. php?RecordID=289509. 13. Tabrizi JS, Azami-Aghdash S, Gharaee H. Public-private partnership policy in primary health care: A scoping review. J Prim Care Community Health. 2020; 11:2150132720943769. 14. Hellowell M, Stafford A, Stapleton P. Austerity and hospitals in deficit: is PPP termination the answer?. Abacus. 2019; 55(3):535-556. [Crossref] [Google Scholar] DISSEMINATION 9. Zheng J, Roehrich JK, Lewis MA. The dynamics of contractual and relational governance: Evidence from long-term public–private procurement arrangements. Journal of purchasing and supply management. 2008; 14(1):43-54. We will disseminate the results of this review alongside the events organised by CHORUS RPC, such as project meetings, seminars, webinars and SR ALG sessions. Additionally, we will produce policy briefs including key results of the review, priority actions for policy-makers, stakeholders of CHORUS partner countries. Final manuscript will be submitted to an Open Access international peer reviewed journal to reach a larger audience and the results will also be presented in a relevant conference. 10. Vian T, McIntosh N, Grabowski A, Nkabane-Nkholongo EL, Jack BW. Hospital public–private partnerships in low resource settings: Perceptions of how the Lesotho PPP transformed management systems and performance. Health Syst Reform. 2015; 1(2):155-166. 11. Kosycarz EA, Nowakowska BA, Miko฀ajczyk MM. Evaluating opportunities for successful public–private partnership in the healthcare sector in Poland. J Public Health. 2019; 27(1):1–9. 12. Suchman L, Hart E, Montagu D. Public–private partnerships in practice: Collaborating to improve health finance policy in Ghana and Kenya. Health Policy Plan. 2018; 33(7):777-785. AUTHOR’S CONTRIBUTIONS Qualitative findings: Following the extraction of themes from qualitative papers, two reviewers will code the themes according to an adaptation of the RE-AIM framework: Facilitators and barriers to Reach, Effectiveness/impacts, Adoption by target staff, settings, systems and communities, Implementation, and Maintenance/ sustainment over time for the PPP models [18]. ZQ, HE, BE, AV will be part of the core group and are responsible for developing the main activities of the systematic review, such as screen and select the manuscripts, extract the data and data analysis and writing the manuscript and reports. The CHORUS Systematic Review ALG members BN, JT, DB, MA, FK, DJ, SK, FS, JO, CA, CO, PA, PAO, AE will participate in the whole review Health Care: Curr Rev, Vol. 10 Iss. 8 No: 1000306 5 Naznin B, et al. 3. Cruz CO, Marques RC. Infrastructure public-private partnerships_ decision, management and development. 2013. process under the guidance and mentorship of the participants from the core group. process under the guidance and mentorship of the participants from the core group. 4. Shaikh BT. Private sector in health care delivery: A reality and a challenge in Pakistan. J Ayub Med Coll Abbottabad. 2015; 27(2):496-498. CONFLICT OF INTERESTS 5. Whyle EB, Olivier J. Models of public-private engagement for health services delivery and financing in Southern Africa: a systematic review. Health policy and planning. 2016; 31(10):1515-1529. None declared. REFERENCES 18. Glasgow RE, Harden SM, Gaglio B, Rabin B, Smith ML, et al. RE-AIM planning and evaluation framework: adapting to new science and practice with a 20-year review. Frontiers in public health. 2019; 7:64. 1. Nduhura A, Nuwagaba I, Settumba JP, Molokwane T, Lukamba MT. Public private partnerships: Systematic review of available models for improving health care services. International Conference on Public Administration and Development Alternatives (IPADA). 19. Hong QN, Pluye P, Bujold M, Wassef M. Convergent and sequential synthesis designs: Implications for conducting and reporting systematic reviews of qualitative and quantitative evidence. Syst Rev. 2017 Dec; 6(1):1-4. 2. Mills A. Health care systems in low-and middle-income countries. N Engl J Med. 2014; 370(6):552-557. 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https://openalex.org/W3046586724	https://link.springer.com/content/pdf/10.1007/s00277-020-04204-1.pdf	English	null	Real-world efficacy of brentuximab vedotin plus bendamustine as a bridge to autologous hematopoietic stem cell transplantation in primary refractory or relapsed classical Hodgkin lymphoma	Annals of hematology	2,020	cc-by	5,984	* Zsófia Miltényi miltenyi.zsofia@med.unideb.hu Abstract Up to 30% of patients with classical Hodgkin lymphoma (cHL) are not responsive to frontline therapy or relapse after primary treatment. In these cases, autologous hematopoietic stem cell transplantation (AHSCT) is the standard of care. The combination of brentuximab vedotin and bendamustine (BV + B) is an effective salvage regimen in this challenging subpopulation. This nationwide multicenter study investigated the real-world efficacy and safety of the BV + B regimen as a bridge to AHSCT in patients with primary refractory or relapsed cHL. A total of 41 cHL patients underwent AHSCT after receiving at least 1 cycle of BV + B (with brentuximab vedotin given at 1.8 mg/kg on day 1 and bendamustine at 90 mg/m2 on days 1–2 every 4 weeks). After a median of 3 (1–6) cycles of BV + B, the objective response rate was 78%, with 29 (70.7%) patients achieving complete remission. Twelve (29.3%) patients relapsed after AHSCT, 2 (4.9%) of them died, while 2 (4.9%) patients are lost to follow-up. After a median of 17 months of follow-up, the estimated 2-year overall- and progression-free survival after AHSCT was 93 and 62%, respectively. Features of advanced disease at recurrence (p = 0.038) and the presence of stage IV cHL at relapse (p = 0.024) are strong predictor markers of unfavorable outcomes. Twenty-four (58.5%) patients experienced adverse events of any grade, while no grade IV toxicities were reported. BV + B is an effective salvage option with a manageable toxicity profile in cHL. The real-world safety and efficacy of this combination are similar to the observations made on the study population. Keywords Hodgkin lymphoma . Autologous hematopoietic stem cell transplantation . Brentuximab v Keywords Hodgkin lymphoma . Autologous hematopoietic stem cell transplantation . Brentuximab vedotin . Bendamustine . Survival Real-world efficacy of brentuximab vedotin plus bendamustine as a bridge to autologous hematopoietic stem cell transplantation in primary refractory or relapsed classical Hodgkin lymphoma László Imre Pinczés1,2 & Roxána Szabó1 & Árpád Illés1,2 & Dóra Földeák3 & Klára Piukovics3 & Árpád Szomor4 & László Gopcsa5 & Zsófia Miltényi1,2 Received: 27 January 2020 /Accepted: 27 July 2020 # The Author(s) 2020 / Published online: 3 August 2020 https://doi.org/10.1007/s00277-020-04204-1 Annals of Hematology (2020) 99:2385–2392 https://doi.org/10.1007/s00277-020-04204-1 Annals of Hematology (2020) 99:2385–2392 ORIGINAL ARTICLE 1 Division of Hematology, Department of Internal Medicine, Faculty of Medicine, University of Debrecen, Nagyerdei krt. 98, Debrecen 4032, Hungary * Zsófia Miltényi miltenyi.zsofia@med.unideb.hu Introduction With the new risk- and response-adapted treatment modalities, classical Hodgkin lymphoma (cHL) became a highly curable hematologic malignancy, with 80–90% of patients achieving long-term remission after standard first-line therapy [1, 2]. However, 20–30% of cHL patients have primary refractory disease or will experience recurrence. In these patients, an autologous hematopoietic stem cell transplantation (AHSCT) is the standard of care, despite the 50% relapse rate after transplantation in cHL [3]. Several prognostic factors associated with an increased risk of relapse following AHSCT include primary refractory cHL, stage IV disease at relapse, extranodal involvement, presence of B symptoms, and less than a complete remission (CR) to salvage therapy before AHSCT [4]. Achievement of CR by positron emission tomography/computed tomography (PET/CT) before AHSCT is a strong predictor for a favorable outcome [5–9]. 1 Division of Hematology, Department of Internal Medicine, Faculty of Medicine, University of Debrecen, Nagyerdei krt. 98, Debrecen 4032, Hungary 2 Doctoral School of Clinical Medicine, University of Debrecen, Debrecen, Hungary 3 Division of Hematology, 2nd Department of Internal Medicine, Faculty of Medicine, University of Szeged, Szeged, Hungary 4 Division of Hematology, 1st Department of Internal Medicine, Faculty of Medicine, University of Pécs, Pecs, Hungary 5 Department of Hematology and Stem Cell Transplantation, Central Hospital of Southern Pest National Institute of Hematology and Infectious Diseases, Budapest, Hungary 2 Doctoral School of Clinical Medicine, University of Debrecen, Debrecen, Hungary 3 Division of Hematology, 2nd Department of Internal Medicine, Faculty of Medicine, University of Szeged, Szeged, Hungary 5 Department of Hematology and Stem Cell Transplantation, Central Hospital of Southern Pest National Institute of Hematology and Infectious Diseases, Budapest, Hungary Ann Hematol (2020) 99:2385–2392 2386 before AHSCT, between January 01, 2016, and December 31, 2018, treated at the four national transplant centers: the University of Debrecen (Debrecen, Hungary), the University of Pecs (Pecs, Hungary), the University of Szeged (Szeged, Hungary), and the Central Hospital of Southern Pest National Institute of Hematology and Infectious Diseases (Budapest, Hungary). Patients were treated according to the evidence- and consensus-based practice guidelines of the Hungarian Society of Hematology and Transfusion (Fig. 1) [20]. Complete remission rates before AHSCT with convention- al salvage chemotherapy regimens, such as DHAP (cisplatin, cytarabine, and dexamethasone), ESHAP (etoposide, methyl- prednisolone, cytarabine, and cisplatin), IGEV (ifosfamide, gemcitabine, etoposide, and vinblastine), BeGEV (bendamustine, gemcitabine, and vinorelbine), and ICE (ifosfamide, carboplatin, and etoposide) vary from 17 to 76% [5, 6, 9–11]. Procedures and assessment Relapsed or refractory cHL patients received at least 2 cycles of a standard salvage chemotherapy regimen before the ad- ministration of BV + B combination therapy. Patients re- ceived 1 to 6 cycles of BV + B with a dose of 1.8 mg/kg brentuximab vedotin intravenously on day 1 and 90 mg/m2 of bendamustine intravenously on each of days 1 and 2 of a 21-day cycle. AHSCTs were performed with BEAM (carmustine, etoposide, cytarabine, and melphalan) condition- ing regimen. According to the European Medicines Agency (EMA), brentuximab vedotin is indicated for the treatment of adult cHL patients with relapsed or refractory cHL after AHSCT or following at least two prior therapies when AHSCT or multiagent chemotherapy is not a treatment option or for cHL patients at increased risk of relapse or progression fol- lowing AHSCT [19]. Therefore, in everyday practice, brentuximab vedotin can be used as a second salvage therapy for relapsed or refractory cHL patients as a sole agent, or even in combination. Response to the salvage therapies was assessed using the 2016 Refinement of the Lugano Classification Lymphoma Response Criteria [21]. Failure after at least one standard sal- vage chemotherapy regimen made relapsed or refractory cHL patients eligible for BV + B therapy. A dedicated PET/CT scan was performed after cycle 2 of BV + B combination ther- apy, and later as it was deemed necessary. PET-negative pa- tients (Deauville score 1–3) underwent AHSCT at any time after cycle 2, while PET-positive (Deauville score 4–5) pa- tients were administered further antitumor therapy. The purpose of this study was to evaluate the safety and efficacy of the BV + B combination therapy as a bridge to trans- plantation in relapsed or refractory cHL patients who previously received two or more multiagent chemotherapy regimens. Stem cell mobilization and collection and also the admin- istration of standard supporting treatment were performed ac- cording to institutional guidelines. Adverse events (AE) were monitored at every visit throughout treatment and follow-up. Introduction In recent years, novel therapies (brentuximab vedotin, anti-programmed cell death-1 (PD-1) inhibitors) became available to help improve transplant out- comes and also survival rate of patients relapsing after AHSCT. Eligible patients were aged 18 years or older and had a histologically confirmed diagnosis of classical cHL. Patients must have had relapsed or refractory disease following stan- dard first-line polychemotherapy. We included patients who received at least one cycle of BV + B regimen in guideline- based dose. No exclusion criteria were determined regarding marrow and other organ function, Eastern Cooperative Oncology Group (ECOG) performance status, or a total num- ber of previous therapies received. One of the novel combination therapies is brentuximab vedotin (BV) plus bendamustine (BV + B). BV is an antibody-drug conjugate, which consists of an anti-CD30 chi- meric monoclonal antibody and the microtubule-disrupting agent, monomethyl auristatin E. Bendamustine is a bifunctional molecule containing the alkylating agent nitrogen mustard and the purine analog fludarabine, causing intra- and inter-strand cross-links between DNA bases resulting in cell death. In heavi- ly pretreated cHL patients, BV used as monotherapy resulted in CR rates and overall response rates (ORR) of 27–35 and 72– 75%, respectively [12–14]. CR and OR rates associated with single-agent bendamustine therapy were 33 and 53% [15]. The combination of these two agents is outstanding in a practical way. The increased proportion of patients achieving CR (43– 74%), reduced toxicity burden compared with standard platinum-based salvage protocols, nonoverlapping toxicities of the combined agents, and the opportunity to treat patients in the outpatient setting highlight BV + B regimen, compared with other BV-based therapies [10, 16–18]. All patients provided written informed consent during en- rollment. Local research ethics committees of all participating centers approved the study, which was done according to the Declaration of Helsinki. Patient characteristics and treatment During the 3-year observational period, 41 cHL patients with relapsed or refractory cHL underwent AHSCT after receiving BV + B salvage therapy (Table 1). The majority (61%) of patients had nodular sclerosing cHL, and a marked male pre- dominance was present. Thirty-two (78%) patients had an advanced-stage disease at initial diagnosis. All patients had received ABVD as frontline therapy, in accordance with the national guidelines. Twenty-eight (68.3%) patients had prima- ry refractory disease. At relapse, 23 (56%) patients had stage III–IV disease, while 8 (19.5%) of them had extranodal involvement. The median number of prior salvage therapies preceding BV + B was 3 (range 1–6). Twenty (48.8%) patients received DHAP, while 4 (9.8%) patients received ifosfamide-based first salvage regimen. Seventeen (41.5%) patients received two or more salvage therapies before BV + B, including DHAP, ESHAP, IGEV, and PD-1 inhibitor. Patients received a median of 3 (range 1–6) cycles of BV + B. The last salvage regimen before AHSCT was BV + B. Outcomes We retrospectively analyzed the demographic data and clini- cal features of cHL patients receiving BV + B salvage therapy Our analysis focuses on the CR and OR rates of relapsed or refractory cHL patients treated with BV + B combination Ann Hematol (2020) 99:2385–2392 2387 Fig. 1 Practice guideline of the Hungarian Society of Hematology and Transfusion for the treatment of primary refractory or relapsed classical Hodgkin lymphoma. Abbreviations: PET, positron emission tomography; AHSCT, autologous hematopoietic stem cell transplantation tomography; AHSCT, autologous hematopoietic stem cell transplantation Fig. 1 Practice guideline of the Hungarian Society of Hematology and Transfusion for the treatment of primary refractory or relapsed classical Hodgkin lymphoma. Abbreviations: PET, positron emission tomography; AHSCT, autologous hematopoietic stem cell transplantation 14 patients with stage IV disease at cHL progression or re- lapse, the CR and ORR rates were 64.3 and 85.7%, respectively. therapy before AHSCT. Treatment response rates were also evaluated regarding PET/CT status according to the Lugano Classification. The overall survival (OS) was calculated from the day of AHSCT to the last follow-up visit or death. Progression-free survival (PFS) was defined as the time from AHSCT to disease progression, to relapse, or to death. Statistical analysis was performed via Fisher’s exact test, and survival data were calculated using the Kaplan-Meier method, with the SPSS 25.0 software. Table 1 Patient characteristics Patients % Men 25 61% Women 16 39% Histological subtypes MC 6 14.6% NS 25 61% LR 5 12.2% LD 1 2.4% ND 4 9.8% Stage at the diagnosis II 8 19.5% III 11 26.8% IV 21 51.2% Refractory 28 68.3% Relapse ≤12 months 9 22% Relapse > 12 months 4 9.7% Stage at relapse I 1 2.4% II 16 39% III 9 22% IV 14 34.1% Extranodal involvement 8 19.50% B symptoms 15 36.60% Number of salvage therapies 2 24 58.5% ≥3 17 41.5% PET −(before AHSCT) 29 70.7% PET + (before AHSCT) 12 22% Relapse after AHSCT 12 29.3% Alive 37 90.2% Dead 2 4.9% Lost to follow-up 2 4.9% MC mixed cellularity, NS nodular sclerosing, LR lymphocyte rich, LD lymphocyte depleted, ND not defined, PET positron emission tomogra- phy, AHSCT autologous hematopoietic stem cell transplantation Adverse events A total of 24 (58.5%) patients experienced treatment-related adverse events (AE) of any grade (Table 2). The most com- mon toxicities were neutropenia (17%), peripheral neuropathy (12.2%), and infusion-related reactions (IRR) with fever, chills, flushing, or pruritus (12.2%). No cases of anaphylaxis were recorded. There were no grade 4 toxicities, and only a total of 3 (7.3%) patients experienced grade 3 toxicities. Serious AEs were neutropenia in 2 (4.8%) patients and pe- ripheral neuropathy in 1 (2.4%) patient. One patient discontinued bendamustine due to severe, treatment-related neutropenia. Patients did not receive prophylactic corticoste- roids or growth factor support routinely. Thirty-seven patients who underwent AHSCT were at in- creased risk of relapse or progression, based on the EMA indication criteria. However, only 15 patients received addi- tional, posttransplant BV monotherapy. Eleven of the 12 PET- positive patients were candidates for single-agent BV after AHSCT, but only two of them were treated. Also, 13 of the Treatment response and long-term follow-up Of the 41 evaluable patients, 29 (70.7%) achieved CR with BV + B therapy before AHSCT. The ORR was 92.6% overall, with 9 (21.9%) patients having partial remission (PR). Twenty-nine (70.8%) patients were PET-negative, and 12 (29.2%) patients were PET-positive before AHSCT. Of the Ann Hematol (2020) 99:2385–2392 2388 Fig. 2 Median 2-year overall- and progression-free survival for all patients. Abbreviation: Tx, transplantation Fig. 2 Median 2-year overall- and progression-free survival for all patients. Abbreviation: Tx, transplantation -year overall- and progression-free survival for all patients. Abbreviation: Tx, transplantation Twelve (29.2%) patients relapsed after AHSCT, including 8 (19.5%) patients who underwent AHSCT with PET- negative cHL. With a median follow-up of 17 (range 2–40) months, 37 patients are alive, two patients died, and two have been lost to follow-up. One patient died of disease progression and one of septic shock. None of the deaths were considered treatment-related. The median 2-year OS and PFS were 93 and 62%, respectively (Fig. 2). Compared with patients with stage I–II cHL at relapse, patients with advanced disease fea- tures at recurrence had an inferior outcome (p = 0.038) (Fig. 3). Also, the presence of stage IV cHL at relapse is a strong predictor marker of unfavorable outcome (p = 0.024) (Fig. 4). It is noteworthy that the survival curves reach a pla- teau before 18 months of follow-up. No association was found between outcome and any of the following features: age, sex, B symptoms, or histological subtype. Also, patients who achieved PET negativity before AHSCT had no survival ben- efit compared with the PET-positive group. 26 available patients received BV maintenance in the PET- negative group. 26 available patients received BV maintenance in the PET- negative group. Discussion reported on 55 cHL patients who re- lapsed after first-line chemotherapy and were treated with BV + B within a multicenter, phase 2 trial [16]. The overall response and CR rates were 92.5 and 73.6%, respectively. Those 40 patients, who proceeded to AHSCT, had improved OR (95%) and CR (85%) rates, with a 2-year OS of 94.9% and a 2-year PFS of 69.8%. More than half (56.4%) of this patient population experienced grade 3–4 AEs, with lymphopenia, rash, and hypotension occurring most frequently. The inci- dence of an infusion-related reaction, defined as fever, chills, dyspnea, flushing, nausea, pruritus, hypotension, or the com- bination of these, was 60%, which is more than single-agent brentuximab vedotin or bendamustine caused alone (12–15%) [22, 23]. Peripheral neuropathy occurred in 54.4% of the evaluable patients. O’Connor et al. treated 37 patients with an ORR of 78% in a phase 2 study population [17]. Forty- three percent of these patients achieved a complete response, while the 2-year OS and PFS were 80 and 62%, respectively. The most common grade 3–4 AEs were neutropenia (35%) and lung infection (14%). Broccoli et al. also observed high remission rates (ORR 80%, CR 75%) and promising 3-year OS and PFS (88.1 and 67.3%, respectively) with BV + B in 40 cHL patients, who inadequately responded to standard induc- tion [18]. Martineau et al. administered BV + B combination Our results are similar to these data in terms of response rates, estimated survival, and toxicities (Table 3). Seemingly, cHL patients who do not respond to one or more traditional chemotherapy regimens could be effectively treated with BV + B salvage therapy and consolidated by AHSCT. However, it is important to note that, according to the national regulations on BV indication, we used BV + B combination as second salvage therapy. Notably, the majority of post-AHSCT relapses occur in the first year after transplantation. Around 18 months, the Kaplan-Meier PFS survival curve for all pa- tients began to plateau and extended to 38 months for the longest survival follow-up. Achievement of a negative PET/CT scan before AHSCT had no impact on PFS, which may be due to the low number of patients included in the analysis. Also, because of the expanse in therapeutic options, all cHL patients are transplanted in the deepest achievable metabolic remission. Positive PET/CT results predomi- nantly represent localized or non-widespread disease ac- tivity. Discussion also observed hig remission rates (ORR 80%, CR 75%) and promising 3-yea OS and PFS (88.1 and 67.3%, respectively) with BV + B in 4 cHL patients, who inadequately responded to standard induc tion [18]. Martineau et al. administered BV + B combinatio transplantation Table 2 Summary of treatment-related adverse events Adverse events Patients % Neutropenia 7 17% Peripheral neuropathy 5 12.2% Infusion-related reaction 5 12.2% Bronchitis, pneumonia 2 4.9% GI 2 4.9% Rash 1 2.4% CMV infection 1 2.4% Herpes zoster infection 1 2.4% CMV cytomegalovirus, GI gastrointestinal to 80 heavily pretreated, relapsed, or refractory cHL patients. They reported a CR in 49 (65%) of 76 patients evaluable for efficacy, with an estimated 2-year OS and PFS of 88.5 and 64%, respectively. Patients eligible to AHSCT had an im- proved posttransplant CR rate (81%), compared with patients in the group without AHSCT (49%). The most frequent (> 30%) toxicities were hematological and infectious [24]. patients. LaCasce et al. reported on 55 cHL patients who re- lapsed after first-line chemotherapy and were treated with BV + B within a multicenter, phase 2 trial [16]. The overall response and CR rates were 92.5 and 73.6%, respectively. Those 40 patients, who proceeded to AHSCT, had improved OR (95%) and CR (85%) rates, with a 2-year OS of 94.9% and a 2-year PFS of 69.8%. More than half (56.4%) of this patient population experienced grade 3–4 AEs, with lymphopenia, rash, and hypotension occurring most frequently. The inci- dence of an infusion-related reaction, defined as fever, chills, dyspnea, flushing, nausea, pruritus, hypotension, or the com- bination of these, was 60%, which is more than single-agent brentuximab vedotin or bendamustine caused alone (12–15%) [22, 23]. Peripheral neuropathy occurred in 54.4% of the evaluable patients. O’Connor et al. treated 37 patients with an ORR of 78% in a phase 2 study population [17]. Forty- three percent of these patients achieved a complete response, while the 2-year OS and PFS were 80 and 62%, respectively. The most common grade 3–4 AEs were neutropenia (35%) and lung infection (14%). Broccoli et al. also observed high remission rates (ORR 80%, CR 75%) and promising 3-year OS and PFS (88.1 and 67.3%, respectively) with BV + B in 40 cHL patients, who inadequately responded to standard induc- tion [18]. Martineau et al. administered BV + B combination patients. LaCasce et al. Discussion To date, three prior phase 1–2 studies and a retrospective analysis evaluated the combination of BV and bendamustine in relapsed or refractory cHL patients. BV + B regimen showed marked activity in a heavily pretreated population of Fig. 3 Median 2-year overall- and progression-free survival according to disease stage at recurrence (stage I–II vs. stage III–IV). Abbreviation: Tx, transplantation l- and progression-free survival according to disease stage at recurrence (stage I–II vs. stage III–IV). Abbreviation: Tx Fig. 3 Median 2-year overall- and progression-free survival according to disease stage at recurrence (stage I–II vs. stage III–IV). Abbreviation: Tx, transplantation Fig. 3 Median 2-year overall- and progression-free survival according to disease stage at recurrence (stage I–II transplantation Ann Hematol (2020) 99:2385–2392 2389 Fig. 4 Median 2-year overall- and progression-free survival according to disease stage at recurrence (stage I–III vs. stage IV). Abbreviation: Tx, transplantation disease stage at recurrence (stage I–III vs. stage IV). Abbreviation: Tx, Fig. 4 Median 2-year overall- and progression-free survival according to disease stage at recurrence (stage I–III vs. stage IV). Abbreviation: Tx, transplantation rding to disease stage at recurrence (stage I–III vs. stage IV). Abbreviation: Tx, ll- and progression-free survival according to disease stage at recurrence (stage I–III vs. stage IV). Abbreviation: Tx patients. LaCasce et al. reported on 55 cHL patients who re lapsed after first-line chemotherapy and were treated wit BV + B within a multicenter, phase 2 trial [16]. The overa response and CR rates were 92.5 and 73.6%, respectively Those 40 patients, who proceeded to AHSCT, had improve OR (95%) and CR (85%) rates, with a 2-year OS of 94.9% an a 2-year PFS of 69.8%. More than half (56.4%) of this patien population experienced grade 3–4 AEs, with lymphopenia rash, and hypotension occurring most frequently. The inc dence of an infusion-related reaction, defined as fever, chill dyspnea, flushing, nausea, pruritus, hypotension, or the com bination of these, was 60%, which is more than single-agen brentuximab vedotin or bendamustine caused alone (12–15% [22, 23]. Peripheral neuropathy occurred in 54.4% of th evaluable patients. O’Connor et al. treated 37 patients wit an ORR of 78% in a phase 2 study population [17]. Forty three percent of these patients achieved a complete response while the 2-year OS and PFS were 80 and 62%, respectively The most common grade 3–4 AEs were neutropenia (35% and lung infection (14%). Broccoli et al. Discussion In these cases, a high-dose conditioning regimen before AHSCT or posttransplant consolidation therapy can also be curative. Table 2 Summary of treatment-related adverse events Adverse events Patients % Neutropenia 7 17% Peripheral neuropathy 5 12.2% Infusion-related reaction 5 12.2% Bronchitis, pneumonia 2 4.9% GI 2 4.9% Rash 1 2.4% CMV infection 1 2.4% Herpes zoster infection 1 2.4% CMV cytomegalovirus, GI gastrointestinal Table 2 Summary of treatment-related adverse events The impact of BV maintenance therapy for patients with high risk for relapse after AHSCT is challenging to assess. According to the AETHERA trial, high-risk patients are the ones with primary refractory HL, relapsed HL with an initial remission duration of less than 12 months, or extranodal in- volvement at the start of pre-transplantation salvage chemo- therapy. While most patients were candidates according to this criteria, only 38% of them received BV consolidation, and there was no difference in either PFS or OS compared with Ann Hematol (2020) 99:2385–2392 2390 Table 3 Observations with brentuximab vedotin plus bendamustine combination in primary refractory or relapsed classical Hodgkin lymphoma Authors Reference no. No. of patients ORR (%) CR (%) 2-year PFS (%) LaCasce et al. 16 55 92.5 73.6 62.6 O’Connor et al. 17 37 78.0 43.0 62.0 Broccoli et al. 18 40 80.0 75.0 67.3 Martineau et al. 24 80 n/a 65.0 64.0 Pinczés et al. – 41 92.7 70.8 62.0 et al. et alia, no. number, ORR overall response rate, CR complete remission, PFS progression-free survival, n/a not available et al. et alia, no. number, ORR overall response rate, CR complete remission, PFS progression-free survival, n/a not available The treatment paradigm of relapsed and refractory cHL has changed with the availability of BV and checkpoint inhibitors. With the successful introduction of these novel agents into salvage therapy, there will be another shift in treatment, with these agents being incorporated into first-line regimens in the future. Also, the indication of radiation therapy has already been significantly reduced. The use of the BV + B regimen as a bridge to AHSCT in relapsed or refractory cHL patients can be an outstanding example of this process. BV + B is a prom- ising, highly active salvage option with a manageable toxicity profile and a potential for long-term disease control. Complemented by AHSCT, BV + B regimen has the potential to considerably improve the outcome of cHL patients progressing after first-line therapy. Code availability Not applicable. Authors’ contributions Zs.M., Á.I., and L.I.P. designed the analysis, interpreted the data, and wrote the initial draft of the manuscript. R.Sz., D.F., K.P., Á.Sz., and L.G. collected the data and performed the analyses of the data. All authors read and approved the final manuscript. Funding Information Open access funding provided by University of Debrecen. However, the results of this study suggest that patients with advanced stages, particularly with stage IV cHL at relapse, have inferior outcomes compared with relapsed or refractory cHL pa- tients with early-stage cHL at recurrence. These patients might be appropriate candidates for BV combined with traditional chemo- therapy regimens (e.g., augmented ICE) or other novel therapies (e.g., nivolumab) to achieve significantly improved PFS. Data availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reason- able request. Discussion The comparison of BV + B with other salvage regimens demands prospective analysis. low-risk cHL patients. The lack of proven survival benefit may also be the result of BV administration before AHSCT, as it provides better survival rates used as salvage therapy than conventional chemotherapy regimens. However, based on the results of the AETHERA study, consolidation treatment with BV is strongly recommended, as it significantly improved PFS compared with the placebo arm (5-year PFS of 59% vs. 41%, respectively) [25]. The low proportion of BV mainte- nance among the high-risk patients of this analysis is due to drug availability issues. In relapsed or refractory cHL patients, several standard sal- vage chemotherapy regimens (ICE, DHAP, ESHAP) and check- point inhibitors (nivolumab, pembrolizumab) were complemented with BV in early-phase studies, resulting in OR and CR rates ranging from 68 to 100% and 34–100%, respec- tively [11, 26]. Also, bendamustine-based BeGEV regimen reached OR and CR rates comparable with those achieved with BV + B combination and is considered a feasible candidate for first salvage in primary refractory or relapsed cHL. However, the possibility arises that the effectiveness of a subsequent treatment with BV + B would be impaired in BeGEV-resistant patients. Along with the achievement of a durable disease control and the favorable safety profile, the main advantage of the BV + B regimen, compared with the combinations mentioned above, is the benefit of the administration in the outpatient setting, resulting in the improvement of quality of life. Acknowledgments The authors acknowledge the clinicians and nurses at associated transplant centers who treated the patients and also the patients who participated in this study. Code availability Not applicable. References 1. 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Consent for publication Not applicable. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adap- tation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, pro- vide 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/. 8. Josting A, Rudolph C, Reiser M, Mapara M, Sieber M, Kirchner HH, Dörken B, Hossfeld DK, Diehl V, Engert A, Participating Centers (2002) Time-intensified dexamethasone/cisplatin/ cytarabine: an effective salvage therapy with low toxicity in patients with relapsed and refractory Hodgkin’s disease. Ann Oncol 13: 1628–1635. https://doi.org/10.1093/annonc/mdf221 9. Bartlett NL, Niedzwiecki D, Johnson JL, Friedberg JW, Johnson KB, van Besien K, Zelenetz AD, Cheson BD, Canellos GP (2007) Gemcitabine, vinorelbine, and pegylated liposomal doxorubicin (GVD), a salvage regimen in relapsed Hodgkin’s lymphoma: CALGB 59804. Ann Oncol 18:1071–1079. https://doi.org/10. 1093/annonc/mdm090 10. Vitolo U, Chiappella A (2018) Salvage regimens for Hodgkin’s lymphoma in the brentuximab vedotin era. Lancet Oncol 19:162– 163 11. Santoro A, Mazza R, Pulsoni A, Re A, Bonfichi M, Zilioli VR, Salvi F, Merli F, Anastasia A, Luminari S, Annechini G, Gotti M, Peli A, Liberati AM, di Renzo N, Castagna L, Giordano L, Carlo- Stella C (2016) Bendamustine in combination with gemcitabine and vinorelbine is an effective regimen as induction chemotherapy before autologous stem-cell transplantation for relapsed or refracto- ry Hodgkin lymphoma: final results of a multicenter phase II study. J Clin Oncol 34:3293–3299. https://doi.org/10.1200/JCO.2016.66. 4466 Compliance with ethical standards Potential limitations of our analysis include the retrospec- tive nature of data collection, limiting the ability to determine cause and effect. Also, due to the relatively low number of patients receiving consolidation, we were not able to assess the impact of BV maintenance therapy. However, we believe that the inclusion of patients from all age groups with no regard to co-morbidities represents real-world experience and can be considered the main strength of the current report. Conflict of interest The authors declare that they have no conflict of interest. Ethics approval Approval was obtained from the ethics committees of all participating centers. The procedures used in this study adhere to the tenets of the Declaration of Helsinki. Consent to participate Informed consent was obtained from all individ- ual participants included in the study. 2391 Ann Hematol (2020) 99:2385–2392 Blood 116:4934–4937. https://doi.org/10.1182/blood-2010-05- 282756 Publisher’s note Springer Nature remains neutral with regard to jurisdic- tional claims in published maps and institutional affiliations. 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Moskowitz CH, Walewski J, Nademanee A, Masszi T, Agura E, Holowiecki J, Abidi MH, Chen AI, Stiff P, Viviani S, Bachanova V, Sureda A, McClendon T, Lee C, Lisano J, Sweetenham J (2018) Five-year PFS from the AETHERA trial of brentuximab vedotin for Hodgkin lymphoma at high risk of progression or relapse. Blood 132:2639–2642. https://doi.org/10.1182/blood-2018-07-861641 22. Seattle Genetics Inc. (2020) ADCETRIS® (brentuximab vedotin) [package insert]. U.S. Food and Drug Administration website. https://www.accessdata.fda.gov/drugsatfda_docs/label/2014/ 125388_S056S078lbl.pdf. Accessed 26 Jan 2020 26. Herrera AF, Moskowitz AJ, Bartlett NL, Vose JM, Ramchandren R, Feldman TA, LaCasce AS, Ansell SM, Moskowitz CH, Fenton K, Ogden CA, Taft D, Zhang Q, Kato K, Campbell M, Advani RH (2018) Interim results of brentuximab vedotin in combination with nivolumab in patients with relapsed or refractory Hodgkin lympho- ma. Blood 131:1183–1194. https://doi.org/10.1182/blood-2017- 10-811224 23. Kahl BS, Bartlett NL, Leonard JP, Chen L, Ganjoo K, Williams ME, Czuczman MS, Robinson KS, Joyce R, van der Jagt RH, Cheson BD (2010) Bendamustine is effective therapy in patients with rituximab-refractory, indolent B-cell non-Hodgkin lymphoma: results from a multicenter study. Cancer 116:106–114. https://doi. org/10.1002/cncr.24714 Publisher’s note Springer Nature remains neutral with regard to jurisdic- tional claims in published maps and institutional affiliations. Publisher’s note Springer Nature remains neutral with regard to jurisdic- tional claims in published maps and institutional affiliations. 24. Martineau D, Sauvezie M, Oberic L et al (2018) Brentuximab- vedotin + bendamustine: a highly effective salvage treatment in refractory/relapsed patients with Hodgkin lymphoma.
https://openalex.org/W2897469144	https://rshare.library.torontomu.ca/articles/journal_contribution/Run-Time_Mitigation_of_Power_Budget_Variations_and_Hardware_Faults_by_Structural_Adaptation_of_FPGA-Based_Multi-Modal_SoPC/14640150/1/files/28116585.pdf	English	null	Run-Time Mitigation of Power Budget Variations and Hardware Faults by Structural Adaptation of FPGA-Based Multi-Modal SoPC	Computers	2,018	cc-by	19,871	Received: 13 July 2018; Accepted: 7 October 2018; Published: 11 October 2018 Abstract: Systems for application domains like robotics, aerospace, defense, autonomous vehicles, etc. are usually developed on System-on-Programmable Chip (SoPC) platforms, capable of supporting several multi-modal computation-intensive tasks on their FPGAs. Since such systems are mostly autonomous and mobile, they have rechargeable power sources and therefore, varying power budgets. They may also develop hardware faults due to radiation, thermal cycling, aging, etc. Systems must be able to sustain the performance requirements of their multi-task multi-modal workload in the presence of variations in available power or occurrence of hardware faults. This paper presents an approach for mitigating power budget variations and hardware faults (transient and permanent) by run-time structural adaptation of the SoPC. The proposed method is based on dynamically allocating, relocating and re-integrating task-speciﬁc processing circuits inside the partially reconﬁgurable FPGA to accommodate the available power budget, satisfy tasks’ performances and hardware resource constraints, and/or to restore task functionality affected by hardware faults. The proposed method has been experimentally implemented on the ARM Cortex-A9 processor of Xilinx Zynq XC7Z020 FPGA. Results have shown that structural adaptation can be done in units of milliseconds since the worst-case decision-making process does not exceed the reconﬁguration time of a partial bit-stream. Keywords: run-time structural adaptation; run-time design space exploration; multi-task multi-modal FPGA-based systems Dimple Sharma 1,, Lev Kirischian 1 and Valeri Kirischian 2 Dimple Sharma 1,, Lev Kirischian 1 and Valeri Kirischian 2 1 Electrical and Computer Engineering Department, Ryerson University, Toronto, ON M5B 2K3, Canada; lkirisch@ee.ryerson.ca 2 MDA Corporation, Brampton, ON L6S 0B6, Canada; valeri.kirischian@mdacorporation.com * Correspondence: dsharma@ee.ryerson.ca; Tel.: +1-647-401-1444 Dimple Sharma 1,, Lev Kirischian 1 and Valeri Kirischian 2 1 Electrical and Computer Engineering Department, Ryerson University, Toronto, ON M5B 2K3, Ca lkirisch@ee.ryerson.ca 2 MDA Corporation, Brampton, ON L6S 0B6, Canada; valeri.kirischian@mdacorporation.com Correspondence: dsharma@ee.ryerson.ca; Tel.: +1-647-401-1444 2 MDA Corporation, Brampton, ON L6S 0B6, Canada; valeri.kirischian@mdacorporation.com * Correspondence: dsharma@ee.ryerson.ca; Tel.: +1-647-401-1444 2 MDA Corporation, Brampton, ON L6S 0B6, Canada; valeri.kirischian@mdacorporation.com * Correspondence: dsharma@ee.ryerson.ca; Tel.: +1-647-401-1444 Computers 2018, 7, 52; doi:10.3390/computers7040052 www.mdpi.com/journal/computers Computers 2018, 7, 52 2 of 34 Computers 2018, 7, 52 Considering autonomous and mobile systems deployed for critical applications; they also face dynamic variations in the environmental conditions like available system power budget or hardware resources. Available power budget can vary as the power consumption of tasks in different modes varies. Power budget can also vary depending on the factors that affect the power sources like rechargeable batteries. Some of these factors are: the charging sources like wind or solar energy, operating temperature, number of charge/discharge cycles, etc. Available hardware resources can vary due to occurrence of transient or permanent hardware faults which may arise due to radiations, thermal cycling, vibration/acceleration intensity, hidden manufacturing defects, operation in hazardous environments or aging. Such dynamic environmental changes can impact the performance of the executing workload. Systems must be able to maintain the performance of their critical multi-modal tasks even in presence of dynamically varying environmental constraints. Since, in general, variations in both, workload and environmental conditions are unpredictable, complex autonomous and mobile systems must be able to adapt to the changing constraints at run-time! p y p g g Consider a space-borne system, say a satellite in an orbit. It operates on rechargeable batteries, which depend on solar energy. The set of tasks it will execute will depend on events like its position in orbit, intensity of solar storm, external requests (e.g., communication with mission center), etc. In such a case, it cannot be considered that power budget will change very slowly. For example, suppose the system is in a certain mode, performing a certain set of tasks. It encounters an unexpected event or receives a request for system mode change such that it is now required to run a new set of tasks which may have a signiﬁcantly different power consumption as compared to the previously executing set of tasks. In this case, the corresponding power budget change will occur in very short time (e.g., in range of milliseconds). Moreover, charging of the batteries, which depends on the available solar energy and the type of back-up power system used, could lead to changes in the power budget. The switching period to switch to the back-up power system and oscillations of the power transfer switch could also inﬂuence the power budget. Thus, for autonomous and mobile systems, there are many cases when their power budget changes quite rapidly. 1. Introduction Modern autonomous embedded systems are expected to be capable of high performance computing and also executing several such high performance tasks on a single platform. They are, therefore, mostly implemented using SoPC platforms due to the advantages they offer [1–4]. While processing of the algorithmically intensive tasks of the supported applications can be carried out on the sequential processors of the SoPC (hard-core processors), the computation-intensive tasks can execute as hardware tasks on FPGAs to provide the requisite high performance. This trend of high performance computing can be observed in several domains, from commercial applications like Global Positioning Systems (GPS), driver assistance, robotic systems etc. to critical military and aerospace applications. However, the increasing complexity and requirements of applications has resulted in the applications having multiple modes of operation, where a mode can be referred to as a set of tasks and their speciﬁcations that remain unchanged for a certain period of time. Changes in the functionality, number, priorities and/or performance speciﬁcations of the tasks imply a mode change for the application. Consequently, systems supporting multi-task workloads are now also expected to be able to support dynamic changes in their workload. Computers 2018, 7, 52; doi:10.3390/computers7040052 www.mdpi.com/journal/computers Computers 2018, 7, 52 Its practical application, however, has the following challenges: 1. In multi-task multi-modal systems, when the number of modes, tasks, and their ASP circuit variants increase, a large design space of system conﬁgurations is formed. For example, a system with a total of 16 tasks, 16 ASP circuit variants per task, 20 modes, and 5 tasks per mode will have a design space of 165 = 1,048,576 system conﬁgurations per mode. Since a solution must be found at run-time, within the permitted adaptation time, it may not possible to exhaustively evaluate each conﬁguration at run-time. As a result, there must be a Run-time Design Space Exploration (RT-DSE) method with a small execution-time overhead to select a suitable conﬁguration that satisﬁes the tasks’ performance speciﬁcations, DPC and hardware resource constraints. 2. 2. The RT-DSE method will need the DPC of candidate system conﬁgurations to decide the most suitable solution. It is practically not feasible to measure and store the DPC of all the possible system conﬁgurations in a Look-Up-Table (LUT). In the above example, this would mean measuring the DPC of 20 modes ×165 conﬁgurations per mode during system design phase and feeding these values in a large LUT. Furthermore, any addition or modiﬁcation of system modes, tasks, or their variants will imply re-doing the entire ofﬂine process all over again! Thus, it is necessary to have a run-time analytical model which can estimate the DPC of system conﬁgurations under evaluation during the run-time DSE process itself. 3. 3. Once a solution is provided by the run-time DSE method, the system needs to be dynamically reconﬁgured with the new chosen ASP circuit variants of the active tasks within the permitted adaptation time. It is to be noted that the permitted adaptation time is application speciﬁc. For a commercial video processing application, a loss of a couple frames can be permitted, but for a critical military application, loss of only one frame may be permitted for adaptation. A system must therefore have the infrastructure that allows a quick transformation to the new selected conﬁguration. ‘Multi-mode Adaptive Collaborative Reconﬁgurable self-Organized System’ (MACROS) framework has been developed for this purpose [6]. It permits reconﬁguration and automatic integration of ASP circuit variants with a very small time overhead in the order of only a couple clock cycles [7,8]. A brief description of MACROS is provided in Section 3. Computers 2018, 7, 52 There can also be simultaneous requirements for changes in system mode or occurrence of hardware faults. Systems need to be ready to adapt to all such dynamic changes at run-time to sustain the performance of their multi-task workloads and to avoid emergency shut-down. A potential solution to this challenging requirement is the use of Run-time Structural Adaptation (RT-SA); a method with which a system can dynamically modify its SoPC architecture to mitigate changes in workload, environmental constraints and possible occurrence of faults. In other words, run-time structural adaptation in SoPC means changing the set of components and/or links between the on-chip components during the execution of tasks to optimize system performance to the dynamic environment (e.g., variations in power budget, temperature ﬂuctuations, occurrence of faults, etc.) RT-SA enables: (a) dynamic selection of a suitable system architecture according to the existing set of constraints and (b) reconﬁguration of the selected architecture in run-time. For a given hardware task’s algorithm and its range of permitted performance speciﬁcations, several implementation variants can be obtained for that task, which exhibit different resource usage, operating frequency, performance, and hence power consumption [5]. They are referred as Application Speciﬁc Processing (ASP) circuit variants of that task. The ASP circuit variants of all system tasks can be stored in system memory as partial conﬁguration bit-streams (or bit-ﬁles), which can reconﬁgured in the Partially Reconﬁgurable Regions (PRRs) of the FPGA using Dynamic Partial Reconﬁguration (DPR). As conditions change, a suitable task variant which meets these conditions can be dynamically reconﬁgured to adapt to the situation. For example, in a low power budget scenario, a task variant occupying more hardware resources and operating at a reduced frequency can be reconﬁgured such that its performance is maintained and Dynamic Power Consumption (DPC) is reduced. Consider a system executing four tasks, each of which has ten ASP circuit variants. This results in a design space of 104 possible combinations of SoPC architectures. The system now has the ﬂexibility to select one suitable system conﬁguration, i.e., a combination of ASP circuit variants of the four tasks, depending on factors like 3 of 34 Computers 2018, 7, 52 current workload (mode) of the system, available power budget, available hardware resources etc. RT-SA can thus enable systems to dynamically sustain the performance of their tasks within the permitted range in presence of changing workload, environmental conditions, and faults. 2. Literature Review Most systems based on SoPC platforms incorporate a real-time operating system (RTOS) or a management system on the lines of an RTOS to adapt to dynamic workloads, power budget, performance, temperature and/or fault constraints. Research efforts have resulted in a generalized concept for RTOSs and their basic functions, which are time and resource management for optimized multi-tasking. These functions mainly include task scheduling, task mapping and allocation, inter-task communication, task to RTOS communication, task conﬁguration etc. [9,10]. Several RTOSs have been developed based on these concepts. They differ from each other based on the number and complexity of their functions, the mechanisms to carry out the functions, their implementation, all of which depend on the system structure that they are developed upon and the applications they are being used for. Refs [11–15] are some examples that support multi-tasking and workload management for hardware tasks, i.e., tasks executing on FPGAs. R3TOS [16,17], BORPH [18], CAP-OS [19], ReConOS [20] are RTOSs that serve systems with both hardware and software tasks (tasks executing on soft or hardcore processors). Some management systems [20–22] also support tasks that can have software and hardware versions of implementation. Such tasks can be dynamically relocated between software and hardware versions to ensure optimal resource management while maximum tasks are served and their deadlines are met. Several techniques have also been adopted for real-time power consumption and/or temperature management. A commonly used method for dynamic power management is power gating [23,24]; where portions of the conﬁgured circuits are turned off when they are not operating. Methods like Dynamic Voltage and Frequency Scaling (DVFS) and DFS are used to control power consumption [25,26] and temperature [27] and also to sustain task performances in presence of temperature variations [28]. Dynamic scheduling techniques [27,29–32] and dynamic mapping (or resource management) techniques [27,33–35] are other methods used to achieve power and/or thermal aware workload management. Dynamic scheduling and mapping techniques are also employed for fault mitigation [15,36,37]. To ensure reliability in mission-critical systems like space-borne systems, the emphasis is on protection against and recovery from transient or permanent faults due to radiation effects. Triple Modular Redundancy (TMR) and scrubbing [38–40] are the most common methods deployed for mitigating transient faults. Built-In Self-Test (BIST) procedures [41] or methods like device reprogramming to avoid damaged regions [42] are used to cater to permanent faults. Computers 2018, 7, 52 This paper is our effort in making progress towards practical deployment of RT-SA in autonomous and mobile FPGA-based systems. It has the following novel contributions: 1. It proposes a method for FPGA-based multi-task multi-modal systems for their run-time structural adaptation to an extensive set of possible situations of: (a) changing system modes, (b) changing power budgets, and (c) occurrence of hardware faults. It incorporates an RT-DSE mechanism which ﬁnds the most suitable system conﬁguration depending on the existing set of constraints, thus enabling RT-SA. 2. It proposes a method to derive the complete Dynamic Power Consumption Estimation Model (DPCEM) of an FPGA in terms of all its reconﬁgurable resources; clock frequency, Logic slices, Block RAM (BRAM) slices, and DSP slices. The DPCEM is used by the RT-DSE method to evaluate DPC of potential conﬁgurations. The paper is divided as follows: Section 2 discusses the current research in the ﬁeld of run-time adaptation and brings out the importance of run-time structural adaptation. Section 3 is a brief description of the MACROS framework’s architecture. Section 4 discusses the decision-making functionality of the run-time structural adaptation method. Section 5 presents the experimental setup for the DPCEM derivation method and outlines the derivation procedure using the example of Xilinx Zynq XC7Z020 and Kintex-7 XC7K325T FPGA devices. Section 6 shows how the run-time structural adaptation method uses the DPCEM to evaluate potential solutions during the adaptation process. 4 of 34 Computers 2018, 7, 52 Section 7 considers a wide variety of changing constraints and explains how a system can dynamically take decisions and adapt to each scenario using the run-time structural adaptation method. Section 8 analyzes the LUT-storage and time overhead of the method based on its implementation on the ARM Cortex-A9 processor of the Zynq XC7Z020 FPGA. Section 9 concludes the paper. 2. Literature Review Ref [47] proposes selection of a suitable combination of number of processor cores, the clock frequency and the placement of software threads based on the performance and power consumption constraints of the system. Ref [48] discusses the use of software task variants for massively parallel processor arrays. Based on temperature changes, a different conﬁguration of the same task, occupying a different number of processing elements is re-conﬁgured. The authors of [49] propose system adaptation using task variants varying from pure software to a mix of hardware and software implementations. The concept of using task variants is also gaining consideration in systems supporting purely hardware tasks. Ref [50] proposes the use of differently shaped variants for hardware tasks, which differ in performance and resource utilization. The focus of the algorithm, however, is only to improve FPGA resource utilization ratio and reduce task rejection ratio. It cannot therefore apply well to systems which have multiple varying constraints. The QoS-aware real-time management system presented in [51] uses tasks with implementation variants on different platforms like processors, FPGA, GPUs etc. Such a system can be useful speciﬁcally for heterogeneous systems only. Adaptation using variants of tasks and system conﬁgurations is discussed in [52,53] respectively. However, in both cases, the events that trigger the choice of a variant or a conﬁguration are ﬁxed. The system therefore cannot adapt to scenarios outside the pre-deﬁned scope of events. From a review of the literature, the following points can be noted: (a) Most of the dynamic adaptation methods mainly serve processor-centric systems. Even on FPGA-based SoPC platforms, the research focus is for task management on soft-core processors or tasks which execute as hardware accelerators for processors. (b) Most RTOSs are developed for tasks with deadlines, i.e., which run only for speciﬁc periods of time. (c) They are based on task attributes like arrival time, worst-case execution time, period, deadline, etc. The adaptation therefore becomes dependent on the nature of tasks. (d) In most power/thermal/fault-aware systems, the adaptation method caters to only one or two parameters and not all the constraints together. In practical scenarios, critical multi-task multi-modal systems usually run continuously executing stream processing hardware tasks and can face the conditions of varying power budgets, temperature, performance constraints, and hardware faults, all together! Thus, more research is required towards development of systems that can sustain their dynamic workload in varying environmental and fault conditions. 2. Literature Review Refs [7,8] propose a run-time relocation based mechanism with a very small time overhead to mitigate transient and permanent faults in FPGA-based systems deployed with the MACROS framework. The authors of [43] present a method for mitigating permanent faults in FPGA-based heterogeneous systems; several variants of the same task are stored such that they occupy different reconﬁgurable regions. The appropriate variant is conﬁgured when a fault is detected in a reconﬁgurable region. A method of relocating faulty computation or interconnection tiles to spare tiles to increase fault-tolerance is proposed in [44]. Ref [45] discusses a switching mechanism, where tasks are switched between their hardware/software versions in case of faults. Ref [46] presents a method using DPR to increase reliability in presence of faults in micro-processor based systems. Although signiﬁcant progress is seen in the development of run-time adaptive systems, the proposed solutions are not complete to support adaptation in dynamically varying environmental conditions like varying system power budget, temperature or occurrence of hardware faults. This is because the tasks that the systems manage have ﬁxed implementation circuits. It is due to this ﬁxed nature of tasks that systems can only re-schedule them (in time) and re-allocate or re-map them on different available resources (space). With this limited ﬂexibility in the dimensions of time and space, it may be possible to adapt to dynamically varying workloads, but it may not be completely possible 5 of 34 Computers 2018, 7, 52 to satisfy dynamically changing environmental constraints. This is because the adaptation techniques are applied to optimize a ﬁxed parameter in presence of a ﬁxed set of environmental constraints. For example, minimize power consumption with ﬁxed task-performance and resource constraints. However, in the case of mobile and autonomous systems operating in non-static environments, the environmental constraints themselves vary and hence even the parameter to be optimized. For example, if power budget drops, system power consumption must be minimized in accordance with the new power budget constraint by trading off task performances. If hardware faults occur, resource utilization must be minimized. If tasks being executed are critical, performance should be maximized. Such a ﬂexibility can only be achieved if a system is able to change its structure, i.e., use different task implementation circuits to accommodate dynamic variations in the set of constraints. Some research efforts have been observed in this direction. 2. Literature Review Our initial efforts resulted in a method for systems with static workloads to dynamically adapt to depleting power budgets and fault conditions [54]. This paper proposes a run-time structural adaptation method for systems with dynamically varying task-sets and their performance constraints. It allows run-time adaptation to the changing system modes, increasing or decreasing power budgets, and mitigating hardware faults. Adaptation to varying temperature conditions can also be easily incorporated in the proposed method. This method can be integrated with an RTOS to form a decision-making RTOS that supports run-time system adaptation to a wide range of dynamically varying constraints. 3. MACROS Framework Figure 1 shows the general architecture of the MACROS framework [6]. It is formed by: (a) several identical PRRs called “slots” on the FPGA, (b) a Distributed Communication and Control 6 of 34 Computers 2018, 7, 52 Infrastructure (DCCI), and (c) a Bit-stream and Conﬁguration Management system (BCM). A task variant’s ASP circuit may consist of multiple components, each occupying one slot on the FPGA. Identical slots reduce management of reconﬁguration of ASP circuit variants as their components can be reconﬁgured on any available slot. DCCI, a crossbar switch structure, permits seamless system communication. Inter-communication of task components to ﬁnd their up-stream and down-stream partners, their self-synchronization and inter-connection also happens over the DCCI [6]. The DCCI thus ensures dynamic and automatic integration of the task components to form the complete ASP circuit of a task. The BCM extracts the required bit-streams from the system memory and conﬁgures them on available slots. Figure 1. General architecture of MACROS framework. Figure 1. General architecture of MACROS framework. For a MACROS framework-based system, when a new conﬁguration is selected, the BCM conﬁgures the components of the chosen variant of every task in the available FPGA slots without any complex decision-making processes. The DCCI then dynamically interconnects the components that belong to the same task without the need for any additional time-consuming control processes. The new combination of task variants is ready to function with a very small time overhead [7,8], thus making the MACROS framework an essential element of run-time structural adaptation. 4.1. System Description The Explorer enables run-time structural adaptation for multi-task multi-modal systems. For such systems, let Tj, j = 0, 1, 2, . . . , l represent the system tasks and Mi, i = 0, 1, . . . , n represent the system’s mode number, where each mode is associated with a set of tasks that execute simultaneously while that mode is active. The number of tasks in a mode m is referred as Nm. Each task has the following attributes: 1. Priority of the task in a mode—P0 (highest priority), ..., Pk (lowest priority) 1. Priority of the task in a mode—P0 (highest priority), ..., Pk (lowest priority) 2. Range of performance available for this task in a mode, i.e., from hspec, the highest performance level (e.g., 240 frames per second (f ps) => 8) to lspec, the lowest performance level (e.g., 60 f ps => 2). These values are relative not absolute and thus, can be associated with different performance characteristics. 3. Existence condition, EC, a parameter that determines whether a task in a mode can be eliminated or not. The task can be terminated if its EC = 0; not if its EC = 1. 3. Existence condition, EC, a parameter that determines whether a task in a mode can be eliminated or not. The task can be terminated if its EC = 0; not if its EC = 1. The Explorer’s behavior is based on speciﬁc LUTs, namely, ‘Mode-speciﬁc LUT’ (‘MODE-LUT’) and ‘Variant-speciﬁc LUT’ (‘Variant-LUT’). This approach allows the fastest reaction on unpredicted events which may require minimum possible response time. Consider a multi-task multi-modal system having a total of 6 tasks, T0 to T5, and three modes, M0 to M2. Table 1 presents an example of MODE-LUT. It stores the tasks, their performance bounds and priorities for each mode. Task priority in the paper’s context means the task’s criticality, i.e., how important the functioning of a task is, with respect to the other tasks, when there is a need to either reduce some task’s performance or eliminate a task in situations like low power budget, limited available hardware resources etc. For example, as seen in Table 1, in mode M0 (N0 = 4), T5, the most critical task, has priority P0, and T4, the least critical task, has priority P3. This means adaptation to varying environmental conditions will begin with the least critical task T4. 4. Method for Run-Time Structural Adaptation to Varying System Modes, Power Budget, and Occurrence of Hardware Faults The proposed run-time structural adaptation method, termed as ‘Explorer’, is conceptually based on [54], which was an initial attempt to test a run-time adaptation mechanism on a static set of tasks. In real-life applications, mobile and autonomous systems supporting critical applications do not usually have a ﬁxed set of executing tasks. The set of tasks, number of tasks, their performance speciﬁcations, priorities etc. can change based on the occurrence of some events. For example, a system on-board a satellite might need to carry out some tasks when it gets enough sunlight. This means the applications, or the multiple tasks running on the system are multi-modal. Run-time adaptation to environmental conditions like varying power budget or hardware faults etc. is much more complex in the case of multi-modal tasks as compared to when the set of tasks is ﬁxed. Furthermore, in [54], the only scenario considered with respect to adaptation to power budget was when the power budget is depleting. However, in ﬁeld deployed systems, power budget can increase as well as decrease. For example, power budget of a space-borne system can increase when it is in the presence of sunlight, which allows charging up it’s solar-energy dependent batteries. On the other hand, if the system suddenly needs to run a set of tasks from its sleep mode, if will face a quick drop in its power budget. Adaptation to scenarios when the power budget can vary; i.e., increase or 7 of 34 Computers 2018, 7, 52 decrease, is different and more algorithmically intensive as compared to an adaptation mechanism only for depleting power budgets. Thus, in this paper we are proposing a novel run-time adaptation mechanism for multi-task systems which can have multiple modes, and which can face situations of increasing or decreasing power budgets, and/or hardware faults. The mechanism is a look-up-table based run-time design space exploration mechanism, that ﬁnds a close-to-optimum variant for each active task in the given system mode, such that the existing constraints of power budget (increased or decreased), performance speciﬁcations of the tasks, available hardware resources, etc., all are met. 4.1. System Description Since it has the least priority over the other tasks, its performance/functioning will be altered (within its range of speciﬁcations) ﬁrst to adapt to the new set of constraints. MODE-LUT also lists the range of performance speciﬁcations, i.e., hspec and lspec, for every task in each mode. For example, in Table 1, T0 has hspec = 8 and lspec = 2 in mode M0, whereas it is a critical task with strict performance constraints, i.e., hspec = lspec = 8 in mode M2. The hspec and lspec values in the Table 1 are a ratio with respect to the minimum performance instead of actual values. For example, if the tasks are video processing tasks, their performance will be in f ps. If minimum performance is 30 f ps, then in the above case, T0 will have hspec = 240 f ps and lspec = 60 f ps in mode M0, and hspec = lspec = 240 f ps in mode M2. Each of the six tasks has ten ASP circuit variants for run-time structural adaptation. Characteristics of the ASP circuit variant of all the tasks, i.e., their resource utilization, operating frequency, and performance are stored in the Variant-LUT, as shown in Table 2. In this table, only the task variants used for the discussion in Section 7 are listed. The Variant-LUT, comprising of all the variants of each of the six tasks, is shown in Table A1. A task with a particular performance can be implemented with different combinations of frequency and resource utilization. For example, in Table 2, variants T0 −0, T0 −1, and T0 −2 of task T0, all have a performance of 8. However, T0 −1 and T0 −2 have half and one fourth the operating frequency of T0 −0, and occupy twice and four times the number of slots 8 of 34 Computers 2018, 7, 52 as T0 −0 respectively. The choice of a variant depends on the existing set of constraints. Consider the example shown in Figure 2. The system frequency, Fsys, is initially at 240 MHz and T0 −0 has been conﬁgured. If the system’s power budget reduces, Fsys can be reduced to 120 MHz to reduce the system’s DPC. In order to maintain the performance of T0, T0 −1 can be reconﬁgured instead of T0 −0. 4.1. System Description Similarly, if power budget further reduces, Fsys can be reduced to 60 MHz and T0 −2 can be reconﬁgured. It must be noted that the set of tasks in a mode together result in the execution of the application supported by system, in that mode. As a result, all the tasks are assumed to execute at the same system frequency, i.e., Fsys. Thus, variants of tasks selected for adaptation are such that they all run at the Fsys selected at that point of time. For example, in the case of a video processing application, all the tasks will operate at the same Fsys to provide the same frame rate at a given resolution. Table 1. Mode-LUT. Mode # of Tasks (Nm) P0 P1 P2 P3 hspec lspec hspec lspec hspec lspec hspec lspec EC EC EC EC M0 N0 = 4 T5 T0 T2 T4 8 8 8 2 8 2 8 2 1 0 0 0 M1 N1 = 3 T2 T4 T1 8 8 8 2 8 2 1 0 0 M2 N1 = 3 T0 T1 T3 8 8 8 8 8 2 1 1 0 Table 2. Variant-LUT. Table 1. Mode-LUT. Mode # of Tasks (Nm) P0 P1 P2 P3 hspec lspec hspec lspec hspec lspec hspec lspec EC EC EC EC M0 N0 = 4 T5 T0 T2 T4 8 8 8 2 8 2 8 2 1 0 0 0 M1 N1 = 3 T2 T4 T1 8 8 8 2 8 2 1 0 0 M2 N1 = 3 T0 T1 T3 8 8 8 8 8 2 1 1 0 Table 2. Variant-LUT. Table 1. Mode-LUT. Table 2. Variant-LUT. Variant No. No. 4.1. System Description of Slots Fsys (MHz) Performance Logic Slices BRAM Slices DSP Slices T0 −0 1 240 8 3093 43 30 T0 −1 2 120 8 6062 86 60 T0 −2 4 60 8 11,877 172 120 T0 −3 2 60 4 6062 86 60 T0 −4 1 60 2 3093 43 30 T1 −0 1 240 8 2061 22 82 T1 −1 2 120 8 4040 44 164 T1 −2 4 60 8 7914 88 328 T2 −0 1 240 8 5003 27 24 T2 −1 2 120 8 9806 54 48 T2 −2 2 60 4 9806 54 48 T2 −3 1 60 2 5003 27 24 T3 −0 1 240 8 4009 16 46 T3 −1 2 120 8 7858 32 92 T4 −0 1 240 8 5088 39 51 T4 −1 2 120 8 9972 78 102 T4 −2 1 60 2 5088 39 51 T5 −0 1 240 8 2567 33 73 T5 −1 2 120 8 5031 66 146 T5 −2 4 60 8 9857 132 292 Computers 2018, 7, 52 9 of 34 Computers 2018, 7, 52 Computers 2018, 7, 52 Since each task has a range of performance speciﬁcations, it also has ASP circuit variants with different performance values. A variant with a lower performance will occupy lesser resources than another variant with a higher performance at the same Fsys. Thus, having a range of performance speciﬁcations enables continuous execution of the task instead of mere elimination if it is unable to ﬁt in the available space at a ﬁxed performance. For example, as seen in Table 1, in mode M0, T2 has hspec = 8 and lspec = 2. As seen in Figure 3, if its current performance is 4 and Fsys = 60 MHz, from Table 2, variant T2 −2, occupying 2 slots, needs to be conﬁgured. However, if only 1 slot is available in the FPGA, performance of T2 can be reduced to 2, so that variant T2 −3, occupying 1 slot, can be conﬁgured. This way, T2 can continue to execute at a reduced performance and its elimination due to lack of space is avoided. The current performance of a task is expressed by a parameter called Current Possible Performance (CPP). Since tasks can be eliminated to adapt to the existing constraints, the active number of tasks in a mode may not always be equal to Nm. 4.1. System Description A track of the active number of tasks is maintained by a parameter Na. Figure 2. Example of a task’s ASP circuit variants that have the same performance. Figure 2. Example of a task’s ASP circuit variants that have the same performance. Figure 2. Example of a task’s ASP circuit variants that have the same performance. 4.2. Decision-Making Run-Time Structural Adaptation Method The Explorer monitors the current power budget at regular intervals of time and is also invoked when there is a hardware fault or a need for a system mode change. It takes different decision paths depending on the system mode, power budget, hardware resource and task performance constraints to dynamically select a system conﬁguration which satisﬁes all the constraints. The system needs spare slots for adaptation; maintaining a task’s performance while reducing Fsys or relocating a task component due to a hardware fault requires free slots. The Explorer therefore, always selects a conﬁguration at the highest possible Fsys that meets the power budget to keep as many slots in reserve as possible. Mode_Change Flow: The Explorer uses this decision path when the system begins in its default mode or if there is a need to switch to another mode while the system is functioning. As shown in the left side of Figure 4, it extracts the characteristics of the tasks in the required mode, m, from the Mode-LUT and estimates the system’s Permitted DPC (PDPC) based on the available power budget. It sets Fsys to maximum, Na = Nm, and the current possible performance, CPP, of every task in the mode m to its respective hspec with the goal of ﬁnding a conﬁguration where every task will operate at 10 of 34 10 of 34 Computers 2018, 7, 52 its hspec at the highest possible Fsys. Once the Fsys is set, the Explorer scans the Variant-LUT of every active task to ﬁnd a suitable variant for it. It begins scanning the Variant-LUT of the task with highest priority P0, and then continues for all the Nm tasks in the mode m. If a variant for a task is not found, it checks if that was because a potential variant could not ﬁt in the available space. In that case, it goes to the Space_Adjustment Flow to cater to that issue. If that is not the case, there is an error. This can occur only if there is an error during the design phase of the system. Once variants for all the tasks are found, i.e., a candidate system conﬁguration is selected, the explorer goes to the Power_Analysis Flow for checking if the conﬁguration meets the power budget constraint. Figure 3. Example of a task’s ASP circuit variants that have different performances. Figure 3. Computers 2018, 7, 52 11 of 34 Computers 2018, 7, 52 Computers 2018, 7, 52 Computers 2018, 7, 52 When power budget increases as compared to the previous one, the Explorer sets Na = Nm and the CPP of every task in that mode to its respective hspec. This is because, with the increased budget, it may now become possible to execute all the Nm tasks at their hspec. If Fsys is not already maximum, the Explorer increases the Fsys by a step and ﬁnds suitable variants for all the tasks. If the new conﬁguration has a lower EDPC than the PDPC, Fsys is increased again and the process repeats. This continues until a conﬁguration is found whose EDPC exceeds the PDPC. The explorer now follows Reduce_System_DPC Flow and ﬁnally settles onto a conﬁguration which has lower EDPC than the PDPC. This process is needed to reach the highest possible Fsys for the available power budget. If the Explorer accepted a conﬁguration in the ﬁrst iteration itself, there could still be room for increasing some task’s performance or adding in an eliminated task and it would have been missed. During the iterations, if a conﬁguration has a lower EDPC than the PDPC even at maximum Fsys, it is accepted as a solution since the tasks are executing in their best possible conﬁguration. Reduce_System_DPC Flow: This decision ﬂow is shown in right side of Figure 5. Since the system’s DPC needs to be reduced due to a reduced power budget condition, the Explorer reduces Fsys by one step, ﬁnds a new conﬁguration for the active set of tasks at that Fsys, and proceeds to the Power_Analysis Flow. If Fsys is already at its minimum, it goes to Reduce_System_DPC_Minimum_Fsys Flow in an attempt to reduce the system’s DPC. Figure 4. Mode_Change and Power_Analysis Flows of the Explorer. Figure 4. Mode_Change and Power_Analysis Flows of the Explorer. Space_Adjustment Flow: This ﬂow is shown in Figure 6. When Fsys is reduced, the Explorer needs to select ASP circuit variants requiring more number of slots to maintain the performance of every task. Hence, it is possible that even after using the spare slots, all the tasks may not be accommodated. If a task with priority Ps does not ﬁt in the available space, the Explorer tries to reduce it’s CPP so that a variant which occupies lesser number of slots can be chosen. 4.2. Decision-Making Run-Time Structural Adaptation Method Example of a task’s ASP circuit variants that have different performances. Figure 3. Example of a task’s ASP circuit variants that have different performances. Power_Analysis Flow: This ﬂow is shown in the right side of Figure 4. The Explorer uses the run-time DPCEM discussed in Section 5 to estimate the DPC of the candidate conﬁguration. If it’s estimated DPC (EDPC) is lower than the PDPC, the set of ASP circuit variants is chosen as the new system conﬁguration and the Explorer waits for the next instance of power budget. Otherwise, it goes to Reduce_System_DPC Flow in an attempt to reduce the system’s DPC to adapt to the low power budget constraint. Power_Budget_Check Flow: This ﬂow, as shown in the left side of Figure 5, is used whenever it is time to check the system’s power budget. This means, after using the Mode_Change Flow either due to system start up or request for changing the system’s mode, the Explorer always comes to this ﬂow at regular intervals of time for adaptation to the new monitored power budget. At the time of new power budget check, the Explorer estimates the system’s PDPC. If the power budget has dropped with respect to the previous one, it checks the DPC of the current conﬁguration (CDPC). If it meets the PDPC constraint, the Explorer retains the conﬁguration and waits for the next instance of power budget. Otherwise, it goes to Reduce_System_DPC Flow in an attempt to reduce the system’s DPC to adapt to the low power budget constraint. Computers 2018, 7, 52 If it is already at it’s lspec, the Explorer saves the task priority Ps and then tries to reduce the CPP of a higher priority task to make 12 of 34 12 of 34 Computers 2018, 7, 52 room for the task under consideration. When successful, it comes back to the task with priority Ps in an attempt to accommodate it in the created space. However, if CPP of any task cannot be reduced even after reaching the task with highest priority P0, the Explorer eliminates the task with priority Ps if its EC = 0 and throws an error if its EC = 1. If the task with priority Ps is eliminated, the Explores moves on to the Power_Analysis Flow. If the task with priority Ps is accommodated and is the last task, i.e., the least priority task, the Explorer proceeds to the Power_Analysis Flow. If not, it moves on with ﬁnding variants of the remaining tasks. Figure 5. Power_Budget_Check and Reduce_System_DPC Flows of the Explorer. Figure 5. Power_Budget_Check and Reduce_System_DPC Flows of the Explorer. Reduce_System_DPC_Minimum_Fsys Flow: This ﬂow is shown in Figure 7. When Fsys is at it´s minimum, the Explorer tries to reduce the system DPC by reducing the CPP of a task, starting from the one with the least priority (PNa−1). If the task is already at its lspec, the Explorer tries to reduce the CPP of a higher priority task. If it is not possible to do so even after reaching the task with highest priority P0, the Explorer eliminates the least priority task after verifying its EC. It then proceeds to the Power_Analysis Flow. 13 of 34 13 of 34 Computers 2018, 7, 52 Computers 2018, 7, 52 Computers 2018, 7, 52 Hardware_Fault Flow: The Explorer chooses the same ﬂow as shown in Figure 7 when a hardware fault occurs in a slot. The fault mitigation method is based on the run-time component relocation method discussed in [7,8]. The basic idea is to relocate the affected task component to a spare slot. This process always takes the same time, which is the slot reconﬁguration time. The affected task component thus has a fast recovery. The faulty slot can then be tested for transient or permanent faults, while the affected task component is already up and functioning. Figure 6. Space_Adjustment Flow of the Explorer. Figure 6. Space_Adjustment Flow of the Explorer. For the kind of systems discussed here, a spare slot may not always be available for relocation in the case of a fault. In such a case, a spare slot will need to be created to cater to the situation. When a hardware fault is detected in a slot, the Explorer checks the availability of a spare slot. If found, it reconﬁgures the affected task component to that slot. If there is no spare slot, it tries to create one by reducing the CPP of a task, starting from the one with the least priority. If that is not possible even after reaching the task with highest priority P0, the Explorer eliminates the least priority task after verifying its EC and conﬁgures the affected task component in the new available spare slot. In the case of a transient fault, this adaptation is temporary; only until the affected slot is restored. However, when the fault is permanent, the available number of slots permanently reduce for the system. 4.3. Analyzing Cost of Run-Time Structural Adaptation 4.3. Analyzing Cost of Run-Time Structural Adaptation 4.3. Analyzing Cost of Run-Time Structural Adaptation Adaptation Time Overhead: From Section 4.2, it can be seen that the Explorer can dynamically maintain the performance of a system’s critical tasks by adjusting the performance values of its lower priority tasks, changing system frequency and/or relocating task components such that the system conﬁguration meets the existing power budget, hardware resource constraints, and the performance speciﬁcations of all the individual tasks. The cost of such a dynamic ﬂexibility is only a small period of adaptation time where some or all of the executing tasks are affected. This further depends on the availability of spare slots. For example, suppose that a variant of task needs to be reconﬁgured for adaptation. If spare slots are available, the new variant can be reconﬁgured to the spare slot(s) while the original task is executing. Once the new variant is reconﬁgured, the originally running task variant is stalled at an appropriate point in its execution cycle and the new one is integrated by the MACROS framework. The adaptation time in this case is only the time taken by MACORS to integrate the components of the newly conﬁgured task. However, if spare slots are not available, the original task variant is stalled, followed by reconﬁguration of the new task variant. In this case, the reconﬁguration time of the variant adds to the adaptation time. For a dynamic relocation example in [7], the adaptation time is in the order of milliseconds. This time frame can satisfy the permitted adaptation time for many applications; for example, multi-media/video/image processing applications, digital communication etc. The small time overhead of the method makes it suitable for supporting run-time adaptation. Figure 8. Find_System_Conﬁguration and Find_Task_Variant Flows of the Explorer. Figure 8. Find_System_Conﬁguration and Find_Task_Variant Flows of the Explorer. Storage Overhead and Exploration Time: Consider a system having 50 modes and 20 tasks. Each task has 16 ASP circuit variants. For simplicity, let there be 10 tasks in each mode. In this case, if system conﬁgurations need to be explored, for each mode, 1610 ≈1012 conﬁgurations will need to be stored and scanned. For a total of 50 modes, the Explorer would need to store 50 × 1610 ≈5 × 1013 conﬁgurations. It may not be feasible to store so many conﬁgurations in an LUT. Computers 2018, 7, 52 14 of 34 Computers 2018, 7, 52 14 of 34 Computers 2018, 7, 52 Find_System_Conﬁguration Flow: The Explorer comes to this ﬂow, shown in Figure 8, whenever there is a need to ﬁnd a candidate conﬁguration for the system, i.e., ﬁnd a combination of suitable variants of the active set of tasks. The Explorer starts with the task with highest priority P0. It ﬁnds a suitable variant for the task that satisﬁes the existing set of constraints, using the Find_Task_Variant Flow, and moves on to scan the LUT of the next lower priority task. This continues till the task with least priority. The selected variants of all the tasks form the candidate conﬁguration of the system. Figure 7. Reduce_System_DPC_Minimum_Fsys and Hardware_Fault Flows of the Explorer. Figure 7. Reduce_System_DPC_Minimum_Fsys and Hardware_Fault Flows of the Explorer. Find_Task_Variant Flow: The ﬂow to select a suitable variant for a task under consideration is shown in the expanded view in Figure 8. The explorer scans the Variant-LUT of the task to ﬁnd an ASP circuit variant whose frequency and performance are equal to Fsys and the CPP of that task, and which ﬁts in the available number of slots on the FPGA. If no variant is selected due to lack of space, it goes to the Space_Adjustment Flow in an attempt to accommodate the task. Otherwise, if there is no variant selected, there is a system design error. 15 of 34 15 of 34 Computers 2018, 7, 52 4.3. Analyzing Cost of Run-Time Structural Adaptation In such a case, if a new variant for a task needs to be reconﬁgured, it will be stalled only when a frame being processed is completed and the results are delivered. There is therefore, no loss of data within a frame. Depending on the time taken for reconﬁguration, as discussed above, in the ’Adaptation Time Overhead’ Section, a couple frames could be lost. Once the task is reconﬁgured, it begins operating from the new incoming frame. This means, for the kind of systems considered in this paper, adaptation does not require saving the states of tasks, as they process incoming streams, and they are stalled/initiated only at the completion/start of a new set of data (frame in this case).This is efﬁciently supported by the MACROS framework, which can auto-synchronize the set of tasks executing at that time. Thus, there is no time/storage overhead of the mechanism in terms of saving the tasks’ contexts. However, there is some loss of data, the cost of which depends on the nature of the application being supported by the system. For example, losing 2–3 frames in commercial applications does not matter since the loss of a few frames is not even visible to the human eye. Inﬂuence of Adaptation on Task Functioning: As discussed above, the systems considered for this paper are autonomous systems with tasks processing incoming streams of data. Such tasks mostly include BRAM-based buffers to store the incoming streams or the processed outputs. There is usually no interaction with external memory. External memory, in such systems, is mostly used to store the different bit-streams of the task variants and is accessed by the Bit-stream Manager while conﬁguring a selected task variant. When different variants of the ASP circuits of a task are conﬁgured for adaptation, they do not affect the functionality of the task. For example, consider an ASP circuit variant of an image processing task which occupies one slot, and operates at 120 MHz to give a performance of 120 f ps. The ASP circuit of this variant would include one buffer dealing with the entire image at 120 MHz. Suppose that the system needs to adapt to a low power budget condition and another variant which occupies two slots and operates at 60 MHz needs to be conﬁgured. 4.3. Analyzing Cost of Run-Time Structural Adaptation Additionally, even if such a large design space is stored in a memory system, exploring it would take a prohibitively large 16 of 34 16 of 34 Computers 2018, 7, 52 computation time to be used at run-time. However, as discussed in Section 4.2, the Explorer selects ASP circuit variants for all tasks individually, instead of selecting the entire system conﬁguration. Therefore, only ASP circuit variants of individual tasks need to be stored. For the above example, only 20 × 16 = 320 conﬁgurations will need to be stored. From these conﬁgurations, the Explorer will only explore 10 × 16 = 160 conﬁgurations for the existing mode. The number of conﬁgurations to be stored and explored reduce by a huge factor of (50 × 1610)/320 = 1.7 × 1011 and (1610)/160 = 6.9 × 109 respectively! The method thus not only reduces the memory requirements of the system, but also reduces the exploration time signiﬁcantly by reducing the number of conﬁgurations to be explored; a requisite for run-time adaptation. q p Reconﬁguration Power Consumption: While reconﬁguring a task component on a slot, the task component operating on that slot prior to reconﬁguration needs to be stalled. This means, during reconﬁguration, DPC is only due to the reconﬁguration process since there is no data processing during that time. Furthermore, DPR usually takes hundreds of micro-seconds to tens of milli-seconds depending on the size of the bit-stream and the medium used for reconﬁguration. It has been demonstrated that power consumption for this small frame of time is negligible as compared to any task operating on that slot [55]. In fact, since there is no data processing during DPR, the power budget improves for that period of DPR. This is because instead of power being consumed by an executing task, there is only a negligible power consumption of the DPR process. Thus, power consumed by DPR can easily be ignored from the power budget calculations. Data Loss during Adaptation: For the proposed run-time adaptation mechanism, it is important to consider the data/information lost as a result of the adaptation. As mentioned in Section 2, critical multi-task multi-modal systems usually run continuously executing stream processing hardware tasks. Consider a system executing tasks which process incoming video streams. 5. Method for Derivation of DPC Estimation Model From Section 4.2, it is seen that during the structural adaptation process, the Explorer uses the DPCEM of an FPGA to estimate the DPC of potential system conﬁgurations at run-time. This section therefore discusses the method to derive the DPCEM for an FPGA. During run-time structural adaptation, it is possible to reconﬁgure only the PRRs of the FPGA with the ASP circuits of the system tasks. This means run-time structural adaptation can result in a change only in the number and type of reconﬁgurable resources of the FPGA. Therefore, only the resources in the PRRs and hence only the system tasks contribute to DPC of the FPGA. Power consumption of any FPGA resource other than those in the PRRs become a part of the static FPGA power consumption. DPC of a system conﬁguration and hence the DPCEM can be therefore be expressed as the sum of the DPC of each type of reconﬁgurable resource used by the ASP circuits. Thus, the DPCEM of an FPGA must have the FPGA’s reconﬁgurable resources as its variable parameters. To derive the DPCEM, the power consumption behavior of each type of reconﬁgurable resource must be identiﬁed. From experiments performed on different FPGA devices based on the 28 nm CMOS technology (Xilinx 7-series and Zynq 7000 family), it was found that DPC of each type of reconﬁgurable resource in an ASP circuit has a linear relation with: (a) clock frequency and (b) utilization, i.e., number of slices of the resource deployed. This behavior is in line with DPC trend shown for the Xilinx FPGAs in [56]. Although the focus in [56] is not to observe the DPC behavior of FPGAs, the presented DPC results depict the linear behavior of the Stratix and Virtex-4 family of FPGAs. This can also be validated from the equation that represents the DPC of each resource in a FPGA [57]: (1) DPCresource = Cswitched × V2 × F (1 where Cswitched is the switched capacitance, V is the voltage supply, and F is the clock frequency of the resource. From this equation, it is clear that the DPC of any FPGA resource will have a linear relation with the clock frequency. Another point to be noted is that capacitance of the resource is inﬂuenced by its switching activity, i.e., number of transitions in a clock period [57]. 4.3. Analyzing Cost of Run-Time Structural Adaptation This variant would have two buffers, processing half the image simultaneously at 60 MHz, thus giving a frame rate of 120 f ps. Similarly, consider a situation where a variant with a reduced performance needs to be conﬁgured to adapt to a limited resource condition. If a variant which occupies one slot and operates at 60 MHz is conﬁgured, there would be one buffer processing the full image at 60 MHz, thus giving a Computers 2018, 7, 52 17 of 34 reduced performance of 60 f ps. This means adaptation does not affect the task functionality in any way. The variants of ASP circuits of the tasks are developed with the aim of run-time adaptation during their design phase. Furthermore, if external communication with the DDR memory is required, this is achieved through continuously executing static tasks, which operate at a ﬁxed frequency. Thus, irrespective of the changing operating frequencies of the conﬁgured task variants, their communication with the external DDR memory is not affected. It can thus be seen that the Explorer does not have any role to play in terms of the task functioning. It is solely responsible for selecting the appropriate system conﬁguration that meets the changing set of constraints. Run-time adaptation does not affect the functioning of the tasks and their communication with external memory. 5.1. Experimental Setup for DPCEM Derivation As discussed above, DPC of an FPGA strongly depends on its underlying micro-architecture and the switching activity factor of the circuit conﬁgured on it. As a result, the DPCEM of an FPGA corresponds to only the application for which it has been derived. If the application and/or the FPGA changes, a new set of DPCEM coefﬁcients will need to be established for that FPGA and application. However, the method of DPCEM derivation stays the same for any FPGA and any application. Thus, to be able to derive a DPCEM, it was necessary to develop an experimental setup, i.e., choose an FPGA platform and develop a test-circuit for which the DPCEM of that FPGA will be derived. Since the MACROS framework requires DPR to enable run-time structural adaptation, the latest Xilinx 7-series FPGA family based on 28 nm CMOS technology was selected. The ZedBoard [58], which houses the Zynq XC7Z020 device (Artix-7 equivalent) [59], and the KC705 evaluation board [60], which houses the Kintex-7 XC7K325T FPGA [61], were selected because they have different methods for DPC measurement. This way, both the methods can be tested for DPCEM derivation and their use in practical systems. Most modern FPGAs have three types of reconﬁgurable computing and memory resources, namely, Logic, BRAM and DSP slices [62,63]. A test-circuit was therefore developed such that it includes all the three types of resources and has a structure which permits variation of usage of each type of resource individually to isolate its behavior. It must be noted that in practice, for deriving the actual DPCEM of an FPGA, the application that will run on the FPGA should be implemented and tested instead of using the test-circuit discussed here. This aspect is brieﬂy discussed in the next Section. The developed test-circuit, as shown in Figure 9a, includes three modules, namely, Memory, Logic and DSP modules. The Memory Module mainly includes 2 buffers, formed using BRAM slices. One buffer acts as the input to the Logic and DSP Modules while the other is being written into, and they switch roles when the buffer is full. The buffers are continuously written into and read from to have some switching activity in the BRAM slices that will result in their DPC. The Logic and DSP modules are mainly formed using Logic and DSP slices respectively. Computers 2018, 7, 52 Computers 2018, 7, 52 18 of 34 Thus, DPCEM derivation essentially involves identifying the coefﬁcients representing the linear relationship of each type of reconﬁgurable resource with frequency and its utilization. These identiﬁed coefﬁcients will be dependent on the switching activity of the application. The DPCEM can therefore be represented as a simple linear equation. Such a model can have a very small execution time overhead and can hence be suitable for use at run-time. 5. Method for Derivation of DPC Estimation Model The switched capacitance of a resource can therefore be expressed as Cswitched = Ce f f × S, where Ce f f is the effective capacitance of the resource, that depends on the parasitic effects of the interconnection wires and transistors, and S is the switching activity of the resource. Thus, the total DPC of an FPGA resource with a utilization U can be written as: Total DPCresource = V2 × F × Ceff × U × S (2) From Equation (2), the DPC of the FPGA can be expressed as [57]: DPCFPGA = V2 × F × ∑Ceff_i × Ui × Si (3) Total DPCresource = V2 × F × Ceff × U × S (2) (2) om Equation (2), the DPC of the FPGA can be expressed as [57]: DPCFPGA = V2 × F × ∑Ceff_i × Ui × Si (3) (3) DPCFPGA = V2 × F × ∑Ceff_i × Ui × Si (3) where Ce f f_i, Ui, and Si are the effective capacitance, utilization, and switching activity of the resource i. 5.1. Experimental Setup for DPCEM Derivation They perform the same set of arithmetic functions, are fed with the same input from the Memory module and produce the same output. Their outputs are switched to form the circuit output to avoid the CAD tool from considering one of the modules redundant and eliminating it from the design. This circuit, consisting of Memory, Logic and DSP modules forms the base design for the experiments. From this design, several variants with individually varying Logic, BRAM and DSP slice utilization must be generated to identify the DPC behavior of each type of resource. This means, variants with increasing Logic slice utilization must be generated at multiple frequencies to observe the behavior of Logic slices alone. Similarly, another set of variants must be generated with increasing BRAM and DSP slice utilization at multiple frequencies to observe their individual behaviors. To be able to do so, each module is scaled multiple times and the outputs are logically ORed together to produce one single output. For example, design variants with varying Logic utilization can be generated by repeating only the Logic modules n times, where n = 1 to N; N is the number beyond which, if the module is repeated, the required logic utilization will become higher than the logic slices available in the FPGA. A combination of the scaled modules is termed as a ‘Bundle’. The scaled modules are logically ORed to produce a single output so that the design functionality is maintained in every variant. This means although every variant has a different Logic utilization, they all behave as if they have only one Logic module in their design. Any other method that can maintain the design 19 of 34 Computers 2018, 7, 52 functionality while varying resource utilization can also be used to generate the variants. Figure 9b shows a design variant where only Logic utilization is increased by scaling Logic Modules in the Logic bundle and the BRAM and DSP Modules remain untouched. While developing the base design, ﬁrst, the individual modules, i.e., the Memory, Logic, and DSP modules were designed. The attempt was that each module has a resource utilization between 15–25% so that at-least 4 variants of varying utilization of one reconﬁgurable resource could be generated by scaling the corresponding module (N = 4; n = 1 to 4). This way sufﬁcient data would be available for DPCEM derivation. 5.1. Experimental Setup for DPCEM Derivation Apart from this criteria, the resource utilization achieved for the base designs was arbitrary; purely dependent on the circuits involved in the design. Figure 9. (a) Memory Module; (b) A design-variant with increased Logic Utilization. Figure 9. (a) Memory Module; (b) A design-variant with increased Logic Utilization. The base design developed for the Zynq XC7Z020 device, which includes one Memory, Logic, and BRAM module, has a resource utilization of 3236, 35 and 22 Logic, DSP and BRAM slices, which is ≈25%, 16% and 16% respectively. These numbers are taken from the resource utilization ﬁle generated for the base design by Vivado. With this initial resource utilization, it was possible to generate 4 variants of varying Logic slice utilization and 5 variants of varying BRAM and DSP slice utilization. Similarly, the base design developed for the Kintex-7 XC7K325T device includes 11219, 140 and 70 Logic, DSP and BRAM slices, which is ≈22%, 17% and 16% respectively. This utilization permitted generation of 5 variants of varying utilization per resource. 5.2. Setup for DPCEM Derivation for Real Applications As discussed, a test-circuit has been developed in this paper to demonstrate the DPCEM derivation procedure. When deriving the DPCEM for real applications, the same concept that is used for developing the test-circuit must be applied. This means, the main idea is to be able to isolate the reconﬁgurable resources to identify their individual behaviors. The only assumption here is that the design/circuit for the application is modular. This will allow scaling the number of speciﬁc modules in the design to vary speciﬁc reconﬁgurable resources. For this purpose, modules that have a higher concentration of a particular resource must be identiﬁed, just like in the case of the test-circuit. For example, video/image processing applications usually have buffers to store the incoming frames. They also have modules that are responsible for data processing; such modules will usually have a higher percentage of logic slice utilization. DSP modules will have more DSP slice utilization, and so on. To generate variants that have varying utilization of a particular resource, only the corresponding module must be scaled multiple times, and not the entire circuit. For example, to generate variants with increasing logic utilization, only the data processing module can be scaled multiple times, with their outputs logically ORed together to produce one single output. This way, there is no change in the functioning of the module and its output interface. Similarly, to generate variants with increasing 20 of 34 Computers 2018, 7, 52 20 of 34 BRAM utilization, only the buffer modules can be scaled. The same applies for the DSP modules. Once this is done, the same method as discussed in this paper (Section 5.4) can be used to derive the DPCEM for the desired FPGA device. All applications may not use all the reconﬁgurable resources. Some may not need DSP modules, while some may not need BRAM modules. For such applications the model coefﬁcients will need to be derived only for the reconﬁgurable resources being used. The step in the derivation procedure, corresponding to the resource not being used, can be skipped. 5.3. Power Consumption Measurement Methods While deriving the DPCEM, DPC values of the FPGA for all the design variants are required in order to gather the initial data for model derivation. Software prediction tools like Xilinx Power Analyzer can be used for this purpose if accurate switching activity factors are available [64–66]. We have used the method of actual DPC measurement to maximize the accuracy of the model derivation process. This Section discusses the method of DPC measurement for the Zynq and Kintex devices housed on the ZedBoard and KC705 evaluation board respectively. Initially, the static power consumption (SPC) of both the boards is recorded. Total power consumption (TPC) of the boards is then measured for every design variant. SPC is subtracted from the TPC of every design variant to obtain the DPC of the respective FPGA for that design variant. The DPC corresponds to the FPGA and not to the board since the test design uses only the FPGA resources. The power consumed by the other on-board resources is a part of the board’s SPC. Measurement Method on ZedBoard: The ZedBoard has a 10 mΩsense resister in series with its 12 V power supply [58]. Power consumption of the board is obtained through the voltage recorded across the sense resistor using Agilent Technologies Digital Multimeter, U3401A. Measurement Method on KC705 Board: For the KC705 board, power supply is distributed in the form of individual rails, which can be monitored with the on-board Texas Instruments power controllers UCD9248PFC [60]. The individual rail voltages, currents, and thus power consumption can be read by I2C-based communication with the Power Management Bus (PMBus) connected to the controllers. Power consumption of the board is the sum of the power consumption of all rails. 5.4. DPCEM Derivation Process The Memory and DSP modules stay constant at one module for all the variants. The resource utilization ﬁle of the base design for the Zynq device shows that each Memory and DSP module consists of 22 BRAM and 35 DSP slices respectively (also discussed in Section 5.1). These BRAM and DSP slices remain constant for all the variants. For each of the four variants, ﬁve bit-streams are generated whose operating frequencies range from 30 MHz to 150 MHz, in multiples of 30 MHz. DPC of the FPGA is measured for each of the 20 bit-streams using the procedure described in Section 5.3. Since number of Logic slices and frequency are the only variable parameters in the design variants, variation in the measured DPC is also only due to variation in Logic slices and frequency. Therefore, the relation between DPC and number of Logic slices is observed at each of the ﬁve frequencies with the help of the graph shown in Figure 10a. Five linear equations are obtained, one for each frequency. The ‘Step 1’ tab of Table 3 lists the Coefﬁcients and Constants of these equations at each multiple of 30 MHz. The Coefﬁcient at a particular frequency corresponds to the slope of increase in DPC due to increase in number of Logic slices at that frequency. Similarly, the Constant at a particular frequency corresponds to the DPC of the constant resources in the design, including the constant BRAM and DSP slices (22 and 35 respectively in this case), at that frequency. It can be observed from the ‘Step 1’ tab of Table 3 that the Coefﬁcients and Constants linearly increase with frequency. Their values obtained at 30 MHz scale with the same factor as the frequency. This shows that the Logic slices, BRAM slices, DSP slices and other constant resources in the FPGA have a frequency dependent behavior. The set of ﬁve linear equations can, therefore, be encapsulated into a single frequency dependent equation as: DPC(Zynq7020) (mW) = Fcc(MHz) 30 × (0.013 × NLS + 55.17) = Fcc Fmin × (CLS × NLS + 55.17) (4) (4) where, Fcc is current operating clock frequency and Fmin is minimum operating frequency for the test design. CLS is the coefﬁcient relating the DPC to NLS; the number of Logic slices. 5.4. DPCEM Derivation Process This Section presents the procedure to derive the complete DPCEM of an FPGA in terms of all its reconﬁgurable resources, i.e., Logic, BRAM and DSP Slices. It is based on [67], which discusses a DPCEM derivation method for applications that use only Logic and BRAM slices. The DPCEM derivation procedure in this paper considers usage of DSP slices along with Logic and BRAM slices. It thus enables DPCEM derivation for an FPGA in terms of all its reconﬁgurable resources. The method of identifying the coefﬁcients for each resource is much simpler than the method in [67]. Since all the reconﬁgurable resources are considered, the test designs developed for the derivation are different as compared to the ones used in [67]. Furthermore, in [67], DPCEM results have been presented only for the ZedBoard. However, in this paper, DPCEM results have been presented for the ZedBoard and the KC705 evaluation board, i.e., for the Xilinx Zynq XC7Z020 and Xilinx Kintex 7 devices respectively. This is done to: (a) Validate the fact that the proposed procedure is generic and can be used to derive a DPCEM for any FPGA device or any FPGA-based board; as long as there is a mechanism to measure the power consumption of the FPGA/board, and (b) Analyze the DPC behavior of FPGA devices built on the same micro-architecture. Zynq XC7Z020 device, housed on the ZedBoard, is used as the experimental platform to demonstrate the derivation procedure. The DPCEM results for the KC705 board are presented next. It is to be noted that even though the Zynq device includes the ARM Cortex A9 core, we are interested in the DPCEM of only the FPGA. Since the test-circuits involve only the resources of the FPGA, the ARM processor has a static power consumption. It is therefore, a part of the SPC of the ZedBoard, which is eliminated from the TPC of the board to obtain the DPC of the FPGA. Thus, the ARM processor does C t 2018 7 52 21 f 34 21 of 34 Computers 2018, 7, 52 not inﬂuence the derived DPCEM of the FPGA, and thus does not affect Explorer’s decision while choosing a system conﬁguration. Step 1—Isolate Behavior of Logic Slices: In this step, four design variants with varying Logic utilization are generated from base design discussed in Section 5.1. The Logic modules in the Logic Bundle are scaled from n = 1 to 4. 5.4. DPCEM Derivation Process Fcc Fmin × 55.17 represents the frequency dependent behavior of the remaining constant resources in the design, including the constant BRAM and DSP slices. This means, in actual circuits, which will have a different BRAM and DSP slice utilization, their inﬂuence will be observed in the second term of Equation (4), i.e., Fcc Fmin × 55.17. The constant 55.17 will be replaced by a different value, which will depend on the BRAM and DSP slice utilization, and also on the switching activity factor of that hardware circuit. Step 2—Isolate Behavior of BRAM Slices: In this step, ﬁve design variants are generated by scaling the Memory modules in the Memory Bundle from 1 to 5. DSP utilization (35 slices) remains constant throughout this step. Frequency is varied from 30 MHz to 150 MHz for each variant, resulting in 25 bit-streams. DPC of Zynq is measured for each bit-stream. It must be noted that increasing BRAM utilization also increases Logic utilization. As a result, DPC of Logic slices must be eliminated to observe the behavior of BRAM slices alone. DPC of Logic slices in every design variant is calculated using the ﬁrst term of (4), i.e., Fcc(MHz) 30 × 0.013 × NLS and subtracted from the measured DPC values. Variation in DPC is now only due to variation in BRAM slices and frequency. The relation between DPC of BRAM slices is observed at each of the ﬁve frequencies with the help of the graph shown in Figure 10b. A set of ﬁve linear equations are obtained, whose Coefﬁcients and Constants are listed in the ‘Step 2’ tab of Table 3. The Coefﬁcient at a particular frequency corresponds to the slope of increase in DPC due to increase in number of BRAM slices at that frequency. Similarly, the Constant at a particular frequency corresponds to the DPC of the constant resources in the design at that frequency. The constant resources in this step include the DSP slices (35 in this case), but exclude the Logic slices as their power consumption has been eliminated from the measured power consumption. It can be observed from the ‘Step 2’ tab of Table 3 that these Coefﬁcients and Constants also linearly increase 22 of 34 Computers 2018, 7, 52 with frequency, like in Step 1. This again shows that the reconﬁgurable resources and other constant resources in the FPGA have a frequency dependent behavior. 5.4. DPCEM Derivation Process The set of equations can hence be combined into the following general equation: DPC(Zynq7020) (mW) = Fcc(MHz) 30 × (1.1 × NBS + 31) = Fcc Fmin × (CBS × NBS + 31) (5) (5) where, CBS is the coefﬁcient relating the DPC to NBS; the number of BRAM slices. Since DPC of Logic slices is eliminated, Fcc(MHz) 30 × 31 represents the frequency dependent behavior of the remaining constant resources in the design excluding the Logic slices. Figure 10. (a–c) Zynq DPC vs. Number of Logic, BRAM and DSP slices respectively. Figure 10. (a–c) Zynq DPC vs. Number of Logic, BRAM and DSP slices respectively. Table 3. Coefﬁcients and constants of linear equations obtained during DPCEM derivation. able 3. Coefﬁcients and constants of linear equations obtained during DPCEM derivation. Table 3. Coefﬁcients and constants of linear equations obtained during DPCEM derivation. F = Fcc (MHz) 30 Step 1 Step 2 Step 3 Coefﬁcient Constant Coefﬁcient Constant Coefﬁcient Constant 1 0.013 49.631 1.1 29.1 0.225 39.9 2 0.026 110.553 2.1 67.8 0.45 86.501 3 0.039 166.184 3.1 95.9 0.677 132.951 4 0.054 220.928 4.3 129.7 0.921 183.778 5 0.068 270.274 5.5 153.4 1.224 228.929 Approximation F × 0.013 F × 55.17 F × 1.1 F × 31 F × 0.226 F × 47 Step 3—Isolate Behavior of DSP Slices: In this step, ﬁve design variants are generated by scaling the DSP modules in the DSP Bundle from 1 to 5. BRAM utilization (22 slices) remains constant throughout this step. Frequency is varied from 30 MHz to 150 MHz for each variant, resulting in 25 bit-streams. DPC of Zynq is measured for each bit-stream. In this step too, scaling DSP modules also increases Logic slices. This is because the DSP module is made up of DSP slices and some Logic slices used as glue logic. Hence DPC of the Logic slices in every design variant is subtracted from the measured DPC values like in Step 2. The relation between DPC and DSP slices is observed at each of the ﬁve frequencies with the help of the graph shown in Figure 10c. A set of ﬁve linear equations Step 3—Isolate Behavior of DSP Slices: In this step, ﬁve design variants are generated by scaling the DSP modules in the DSP Bundle from 1 to 5. BRAM utilization (22 slices) remains constant throughout this step. 5.4. DPCEM Derivation Process Frequency is varied from 30 MHz to 150 MHz for each variant, resulting in 25 bit-streams. DPC of Zynq is measured for each bit-stream. In this step too, scaling DSP modules also increases Logic slices. This is because the DSP module is made up of DSP slices and some Logic slices used as glue logic. Hence DPC of the Logic slices in every design variant is subtracted from the measured DPC values like in Step 2. The relation between DPC and DSP slices is observed at each of the ﬁve frequencies with the help of the graph shown in Figure 10c. A set of ﬁve linear equations 23 of 34 Computers 2018, 7, 52 are obtained. Their Coefﬁcients and Constants are listed in the ‘Step 3’ tab of Table 3. The Coefﬁcient at a particular frequency corresponds to the slope of increase in DPC due to increase in number of DSP slices at that frequency. The Constant at a particular frequency corresponds to the DPC of the constant resources in the design at that frequency. The constant resources in this step include the BRAM slices (22 in this case), but exclude the Logic slices as their power consumption has been eliminated from the measured power consumption. Here too, the frequency dependent behavior of the FPGA resources is observed; the Coefﬁcients and Constants from the ‘Step 3’ tab of Table 3 scale with frequency. Therefore, the set of equations in this step also can be represented as the following general frequency dependent equation: DPC(Zynq7020) (mW) = Fcc(MHz) 30 × (0.226 × NDS + 47) = Fcc Fmin × (CDS × NDS + 47) (6) (6) where, CDS is the coefﬁcient relating the DPC to NDS; the number of DSP slices. Since DPC of Logic slices is eliminated, Fcc(MHz) 30 × 47 represents the frequency dependent behavior of the remaining constant resources in the design excluding the Logic slices. Step 4—Complete the DPCEM Equation: From (4)–(6) we have: CLS = 0.013, CBS = 1.1, and CDS = 0.226; the coefﬁcients which relate DPC to the number of Logic, BRAM and DSP slices respectively. Also, from Steps 1 to 3, it has been observed that the DPC of each type of resource linearly depends of frequency. A generic equation can thus be formed from (4)–(6), which represents the DPCEM of an FPGA. 5.4. DPCEM Derivation Process DPC(FPGA) (mW) = Fcc Fmin × (CLS × NLS + CBS × NBS + CDS × NDS + CF) (7) (7) where, CF is a frequency dependent coefﬁcient representing the behavior of the remaining constant resources in the design excluding the Logic, BRAM and DSP slices. g g g Comparing (4) from Step 1 with (7) from Step 4, we get the following relation: CBS × NBS + CDS × NDS + CF = 55.17 =⇒ 1.1 × 22 + 0.226 × 35 + CF = 55.17 =⇒ CF = 23.06 (8 (8) Similarly, comparing (5) and (6) with (7), the following relations are obtained: Similarly, comparing (5) and (6) with (7), the following relations are obtained: CDS × NDS + CF = 31 and CBS × NBS + CF = 47. (9) (9) The identiﬁed coefﬁcients CLS, CBS, CDS, and CF validate the relations in (9). Thus, the complete DPCEM for Zynq XC7Z020 involving the parameters of Logic slices, BRAM slices, DSP slices and frequency can be represented as: DPC(Zynq7020) (mW) = Fcc Fmin × (0.013 × NLS + 1.1 × NBS + 0.226 × NDS + 23.06) (10) (10) The maximum difference between the DPC values estimated using (10) and the measured DPC results is 30 mW. Thus, (10) is an accurate DPCEM for the Zynq XC7Z020 FPGA device, for the test design considered. It is clear that the BRAM slices highly inﬂuence the DPC of the FPGA as against the Logic slices (1.1/0.013 ≈85 times) and DSP slices (1.1/0.226 ≈5 times). Also, the following can be analyzed from the generic DPCEM Equation (7) obtained for an FPGA: Due to the presence of the frequency dependent coefﬁcient CF, it is possible to reduce power consumption by decreasing frequency alone. This means, a task variant V1 with double the resource utilization and half the operating frequency of another variant V2 will have lesser DPC as compared to V2. For example, consider the ﬁrst two variants, T0 −0 and T0 −1 listed in Table 2. T0 −0 has Fsys = 240 MHz and uses 1 slot on the FPGA. T0 −1 has Fsys = 120 MHz and uses 2 slots on the FPGA to give the same performance as T0 −0. 5.4. DPCEM Derivation Process If we assume that T0 −1 has double the number of resources 24 of 34 Computers 2018, 7, 52 used in T0 −0, it will still have lower power consumption as compared to T0 −0 due to the presence of the frequency dependent coefﬁcient CF in Equation (7). The value of the term Fcc Fmin × CF will be halved when the frequency reduces to 120 MHz. It must be clariﬁed that variant T0 −1 may not have exactly double the resources as that of T0 −0, even though it uses double the number of slots as compared to T0 −0. For example, T0 −0 could be carrying out some functions using more BRAM slices in a slot. On the other hand, T0 −1 could be carrying out the same function using more Logic slices in the two occupied slots instead of BRAM slices so that its DPC could further be reduced. To summarize, from the generic DPCEM Equation (7), it can be said that due to the frequency dependent coefﬁcient CF, the power consumption of a variant with double the resource utilization and half the operating frequency of another variant will be lower instead of being the same. Thus, conﬁguring a variant at a lower frequency will help reduce the system DPC. (Each system mode usually has only a few tasks whose performance need to be sustained at all times. The performance of other tasks may be reduced within the speciﬁed performance range, if needed, to accommodate the changing set of constraints. Thus, along with reduced Fsys, which helps reduce the system’s DPC, the lowered performance of non-critical tasks can further reduce the total DPC of the system.) y Since the DPCEM derivation procedure is generic, the same method has been followed to derive the DPCEM for the Kintex-7 XC7K325T device. For the steps 1, 2 and 3, the Logic, Memory and DSP modules are respectively scaled from 1 to 5. Frequency is varied from 30 to 210 MHz in steps of 30 MHz, resulting in 35 bit-streams per step. Figure 11a–c show the plots representing DPC vs. Logic, BRAM and DSP slices respectively. Based on the set of linear equations obtained, the following coefﬁcient values are derived: CLS = 0.012, CBS = 1, CDS = 0.22 and CF = 29.2 (11) (11) Figure 11. (a–c) Kintex-7 DPC vs. Number of Logic, BRAM and DSP slices respectively. Figure 11. Computers 2018, 7, 52 25 of 34 Computers 2018, 7, 52 The maximum difference between the DPC values estimated using (12) and the measured DPC results is 100 mW. (12) is an accurate DPCEM for the Kintex-7 FPGA, for the test design considered. It can be observed that the DPCEM for Kintex-7 has the same general form as (7); the one obtained while deriving the DPCEM for Zynq device. It thus validates the generic equation obtained as the DPCEM of an FPGA. Comparing (10) and (12) shows that the coefﬁcients obtained for Zynq and Kintex-7 devices are similar. This is because even though they belong to different FPGA families, they have the same fabrication technology and hence have similar power consumption behavior. The DPCEM results for the Zynq XC7Z020 and the Kintex-7 device also show that the proposed DPCEM derivation procedure is generic and can be applicable to any FPGA device. 6. DPCEM Usage during Run-Time Structural Adaptation This section shows how the Explorer identiﬁes the PDPC at every power budget check and how it uses the DPCEM to estimate the DPC of a conﬁguration-under-test. PDPC Calculation: Consider the system to be operating on a rechargeable battery with a capacity of I Ah at V volts. The battery capacity is thus Pavail(Wh) = V × I. If the system needs to function for H hours, the permitted total power consumption (PTPC) is given as: PTPC(W) = (Pavail/H). If the system’s SPC is Pstatic W, PDPC(W) = PTPC −Pstatic. At the time of a new power budget after an interval of Hnew hours, the battery capacity reduces by (Icon f ig × Hnew) Wh; where Icon f ig is the current being drawn by the present system conﬁguration in Amperes. Thus, Pavail(Wh) = Pavail −(Icon f ig × Hnew). The new PDPC of the system, based on the new available power budget can again be found using the above steps. DPC Estimation of a Conﬁguration-under-Test: Equation (7) is used to estimate the DPC of a candidate conﬁguration. The resource utilization for the conﬁguration can be obtained by accessing the resource utilization of the selected variants of all the tasks in the conﬁguration from the variant-LUT and summing them up. Equation (7) then becomes: EDPC (mW) = Fcc Fmin × (CLS × Nc−1 ∑ n=0 NLS + CBS × Nc−1 ∑ n=0 NBS + CDS × Nc−1 ∑ n=0 NDS + CF) (13) (13) where Nc is the number of tasks in the candidate conﬁguration. where Nc is the number of tasks in the candidate conﬁguration. 5.4. DPCEM Derivation Process (a–c) Kintex-7 DPC vs. Number of Logic, BRAM and DSP slices respectively. Figure 11. (a–c) Kintex-7 DPC vs. Number of Logic, BRAM and DSP slices respectively. The complete DPCEM for Kintex-7 involving the parameters of Logic slices, BRAM slices, DSP slices and frequency can be represented as: DPC(Kintex-7) (mW) = Fcc Fmin × (0.012 × NLS + 1 × NBS + 0.22 × NDS + 29.2) (12) (12) 7. Example of Run-Time Adaptation in Different Scenarios PDPC reduces to 3.09 W (Power_Budget_Check Flow). Since CDPC(3.195 W) ≥PDPC, the Explorer reduces Fsys to 120 MHz (Reduce_System_DPC Flow) and selects variant no. 1 for all the tasks to maintain their performance at hspec. EDPC of this conﬁguration, i.e., T5 −1, T0 −1, T2 −1 and T4 −1, as shown in Figure 12b, is 3.048 W. Since EDPC ≤PDPC, it is selected as the new system conﬁguration. Thus, due to availability of spare slots, the performance of all the tasks remains unaffected even when system adapts to a reduced power budget. The system can now run for 9.08 h, ≈16 min more than it would on the previous conﬁguration and ≈5 min more than required by the budget. Adaptation to a depleted power budget in absence of spare slots: After another hour, BC ≈80%. The system can function for 8.08 h. The new power budget estimates that since external conditions have not improved, the system will need to run for 9 more hours before a recharge is possible. PDPC drops to 2.56 W (Power_Budget_Check Flow). Since CDPC(3.048 W) ≥PDPC, the Explorer reduces Fsys to 60 MHz (Reduce_System_DPC Flow) and selects T5 −2, which occupies 4 slots, to maintain the performance of the critical task T5 at its hspec. Similarly, it chooses T0 −2 to maintain the performance of T0 at hspec = 8. Since there is no empty slot left for any other task, it reduces the CPP of T0 to 4 (Space_Adjustment Flow) and selects T0 −3. Following the same process, it selects variants T2 −3 and T4 −2. The EDPC of T5 −2, T0 −3, T2 −3 and T4 −2, shown in Figure 12c, is 1.44 W. Since EDPC ≤PDPC, it is therefore selected as the new system conﬁguration. In this case, the system can function for 12.22 h, 4.14 h more than the previous conﬁguration and 3.22 h more than that required by the power budget. This happens because performance of lower priority tasks is reduced due to lack of enough spare slots which substantially reduces system DPC. Mitigating hardware fault in absence of spare slots: While the system is running, there is a hardware fault in a slot where a component of T5 is executing. Since there are no spare slots available for relocation, the Explorer attempts to reduce the CPP of the least priority task, T4 (Hardware_Fault Flow). 7. Example of Run-Time Adaptation in Different Scenarios This section discusses an example of run-time structural adaptation of a system for different mode, power budget and fault conditions. Consider the same example of the multi-task multi-modal system having six tasks and three modes, described in Section 4.1. Tables 1 and 2 represent the ’mode-LUT’ and the ’Variant-LUT’ respectively. Consider this system to be developed on the Kintex-7 FPGA device which is deployed with the MACROS framework and has 8 slots. The system runs on a rechargeable 12 V battery with a capacity of 48 Wh. Fsys can take values of 30, 60, 120 and 240 MHz. Table 4 summarizes the ﬂow of events which are discussed next. Initial State: The default system mode is M0. On start-up, the battery capacity (BC) = 100%. Estimated time of next battery recharge is after 9 h. With a Pavail = 48 Wh and H = 9 h, PTPC = 48/9 = 5.33 W. Considering a Pstatic = 1.7 W, PDPC = 5.33 −1.7 = 3.63 W. The Explorer sets Fsys to 240 MHz and selects variant no. 0 for each of the tasks in M0, i.e., T5, T0, T2 and T4, to maintain their performance at maximum (Mode_Change Flow). Using Table 2, (11), and (13), EDPC of this combination can be calculated as: EDPC (mW) = 240 30 × {0.012 × 15751 + 1 × 142 + 0.24 × 178 + 26.4} = 3.195 W. Since EDPC ≤PDPC, T5 −0, T0 −0, T2 −0, T4 −0 is selected as the system conﬁguration, as seen in Figure 12a. The system can now function for 9.81 h, ≈48 min more than needed by the budget. 26 of 34 Computers 2018, 7, 52 Table 4. Flow of events discussed in Section 7. Time Elapsed (hours) System Mode Pavail (%) Current Lifetime (hours) Needed Lifetime PTPC (W) PDPC (W) Candi-Date EDPC (W) New Lifetime 0 M0 100.00 9.00 5.33 3.63 3.195 9.81 1 M0 89.80 8.79 9.00 4.79 3.09 3.048 9.08 1 M0 79.91 8.05 9.00 4.26 2.56 1.440 12.22 3.5 M2 57.02 8.67 8.00 3.42 1.72 1.251 9.27 0.5 M2 53.94 8.72 5.00 5.18 3.48 2.039 6.93 Adaptation to a depleted power budget in presence of spare slots: After one hour, BC ≈90%, which permits the system to function for 8.81 h. However, power budget shows that due to external conditions, battery recharge is possible after 9 h instead of the expected 8 h. 7. Example of Run-Time Adaptation in Different Scenarios T4 is already at its lspec and so is the higher priority task T2. It therefore reduces the CPP of T0 to 2 and selects T0 −4 which occupies only 1 slot. Thus, a free slot is created and the affected task component of T5 is relocated to that spare slot as shown in Figure 12d. Thus, the system can recover from a hardware fault and still maintain the performance of its critical task at hspec by adjusting the performance and resource utilization of the lower priority tasks. Adaptation to system mode change: For the next three hours, there are no changes in power budget, resource constraints or mode. So the system continues to have the same conﬁguration (Flow 6) as shown in Figure 12d. After half an hour, there is an interrupt to change the mode to M2 (Mode_Change Flow). Based on the new estimates, the system needs to be able to run for 8 h with BC ≈57%, PDPC = 1.72 W. Candidate conﬁgurations at 240 and 120 MHz do not satisfy the PDPC constraint. The Explorer sets Fsys to 60 MHz (Reduce_System_DPC Flow) and selects variant no. 2 for T0 and T1 to maintain their maximum performance. Since there are no more spare slots to accommodate T3 and it is not possible to reduce the CPP for T0 or T1, the Explorer removes task T3 after checking its EC = 0 (Space_Adjustment Flow). The EDPC of T0 −2, T1 −2 is 1.251 W. It is 27 of 34 27 of 34 Computers 2018, 7, 52 selected as the new conﬁguration for M2, as shown in Figure 12e. The system can run for 9.27 h, 1.27 h more than that required by the power budget. Figure 12. Different run-time structural adaptation scenarios. Figure 12. Different run-time structural adaptation scenarios. Adaptation to increased power budget: After half an hour, BC ≈54% and the system can run for 8.77 h. External conditions have improved and the battery recharge is now possible after 5 h. The power budget thus goes up to 3.47 W (Delta = 1, Power_Budget_Check Flow). The Explorer increases Fsys iteratively (Flow 6), until Fsys = 240 MHz. It selects T0 −0, T1 −0 and T3 −0, as shown in Figure 12f, which has an EDPC of 2.039 W. 7. Example of Run-Time Adaptation in Different Scenarios Since EDPC ≤PDPC, Fsys is maximum, and all the tasks are at their hspec, there is no need for further iterations. This combination becomes the new system conﬁguration. It lets the system run for 6.93 h, 1.93 h more than required. These scenarios demonstrate that given any set of conditions, the Explorer can ﬁnd a suitable system conﬁguration that satisﬁes the mode, performance, DPC and hardware resource constraints. As a by-product of the run-time structural adaptation, the system life-time can also increase; the extent of which depends on the relation between the power budget and DPC of the selected conﬁguration. Computers 2018, 7, 52 Computers 2018, 7, 52 28 of 34 28 of 34 Case 1—Initial state: When the system begins to function, it has a maximum power budget and a default mode of M0. In this case, the Explorer will be able to ﬁnd a conﬁguration at Fsys = 240 MHz itself. It takes ≈23 µs to select the conﬁguration shown in Figure 12a. Case 1—Initial state: When the system begins to function, it has a maximum power budget and a default mode of M0. In this case, the Explorer will be able to ﬁnd a conﬁguration at Fsys = 240 MHz itself. It takes ≈23 µs to select the conﬁguration shown in Figure 12a. Case 2—Worst-case depleted power budget: For adaptation to a worst-case power budget drop, the system conﬁguration consisting of tasks operating at the highest Fsys at their hspec would need to change to the one having only the critical tasks with their EC = 1 operating at the lowest Fsys at their lspec. So, in M0, if the initial conﬁguration is the one in Figure 12a, it would change to the one where only T5 is executing at 30 MHz occupying all the 8 slots. To reach this stage, the Explorer will need to evaluate conﬁgurations at 120 and 60 MHz and ﬁnally settle at the conﬁguration at 30 MHz. The Explorer takes ≈33.5 µs to reach this conclusion. Case 3—Worst-case increased power budget: The longest decision-making process in the case of an increased power budget needs to be evaluated for this case. In M0, suppose that the system conﬁguration consists of only T5 operating at 30 MHz. An increase in the power budget causes the Explorer to evaluate conﬁgurations at 60, 120 and 240 MHz. If EDPC > PDPC for a potential solution at 240 MHz, the Explorer will again reduce Fsys to 120 MHz and settle at a conﬁguration at 120 MHz. The Execution time for this case is ≈40 µs. It must be noted in the case of a maximum increase in power budget, the Explorer would settle at 240 MHz itself. It will not return to 120 MHz again for a solution. Hence the execution time would be less than 40 µs. Case 4—Worst-case mode change: Suppose while the system is functioning in a mode other than M0, it experiences the worst power budget drop, as described in Case 2. Computers 2018, 7, 52 In this situation, there is also an interrupt to change the to mode M0. In this case, the Explorer will need to evaluate conﬁgurations at 240, 120, 60 MHz and ﬁnally settle at the one where only the critical task T5 is executing at 30 MHz occupying all the 8 slots. Time recorded for this is ≈53 µs. This is also expected because it is a combination of Case 1, where the Explorer ﬁnds a conﬁguration at 240 MHz, and Case 3, where the Explorer settles onto the conﬁguration at 30 MHz from the one at 240 MHz. Case 5—Worst-case hardware fault: Consider the conﬁguration in Figure 12c. Assume for this example that all the tasks are at their lspecs. If a hardware fault occurs now, the Explorer will need to check the CPP of every task right from priority P3 to P0 and then ﬁnally decide that T4 (P3) needs to be removed. The time recorded for this is only ≈7 µs. From all the cases considered, the maximum execution time observed is 53 µs. A partial bit-stream of 395 KB takes a reconﬁguration time of 1 ms over the 32-bit ICAP port at a frequency of 100 MHz. The maximum execution time of the decision-making adaptation method is only 5% of the time taken to reconﬁgure a slot. The method thus proves to be suitable for use at run-time. 8. Experimental Results Storage Requirements: The Explorer has been implemented as a bare-metal C code on the ARM Cortex-A9 processor of the Zynq XC7Z020 device, operating at 666 MHz. The implementation covers the example discussed in Section 7. Since each of the six tasks in the example has ten ASP circuit variants, the Variant-LUT stores the operating frequency, performance, number of slots, Logic slices, BRAM slices and DSP slices used, for only 6 × 10 = 60 variants, irrespective of the modes. If a design space of system conﬁgurations was used, characteristics of 3 × 106 conﬁgurations would need to be stored in the Variant-LUT. The LUT size with the proposed method (8 bytes/variant × 60 variants = 480 bytes) is only 0.002% of the size when system conﬁgurations (8 bytes/variant × 3 ×106 variants ≈23 MB) are used. To reduce exploration time further, variants of each task are stored in ascending order of frequency. Thus, every time Fsys is reduced, the Explorer only needs to scan variants from the top of the LUT up to Fsys and can avoid the remaining ones at higher frequencies. Execution Time: Execution time of the code has been recorded for different scenarios of M0 since it has the maximum number of tasks among the other modes in the example. 9. Conclusions This paper proposes a method for mobile and autonomous, multi-task multi-modal FPGA-based embedded systems to be able to adapt structurally to unpredictable mode-change events and environmental conditions, and mitigate hardware faults. The decision-making capabilities of the method allow run-time selection of a suitable system conﬁguration for the existing system mode such that critical tasks are sustained at their maximum performance, individual task performances are within the speciﬁed range, system’s DPC is within the permitted DPC, and the conﬁguration ﬁts in the available resources. The paper also presents a generic procedure to derive a complete DPCEM of an FPGA in terms of all reconﬁgurable resources; a simple estimation tool used by the adaptation method to evaluate DPC of candidate conﬁgurations at run-time. The observed worst-case decision-making time of the method for the example considered in the paper is a very small fraction of the time taken to reconﬁgure a partial bit-stream on an FPGA slot. The small execution-time overhead validates the method to be suitable at run-time. Future research efforts will be directed towards ﬁnding a run-time optimization method to achieve the aforementioned adaption for very large systems where LUT-based search will not be able to satisfy the permitted adaptation time frames. 29 of 34 29 of 34 Computers 2018, 7, 52 Author Contributions: Conceptualization, D.S. and L.K. and V.K.; Methodology, D.S. and L.K. and V.K.; Data Curation, D.S.; Software, D.S.; Validation, D.S.; Project Administration, L.K.; Supervision, L.K.; Funding Acquisition, L.K., Resources, L.K.; Writing—Original Draft, D.S.; Writing—Review and Editing, L.K. and V.K. Funding: This research has been funded by external and internal sources: (a) The instrumentation and CAD software has been funded & provided by Canadian Microsystems Corporation (CMC); (b) Research and implementation of the MACROS framework (Section 3), the SoPC base for the presented research, has been funded by Ontario Centres of Excellence (OCE) and industrial partners; (c) The hardware platforms based on Xilinx Zynq-7020 FPGA and Kintex-7 FPGA have been purchased by internal (lab) funds. Conﬂicts of Interest: The authors declare no conﬂict of interest. Appendix A of Slots Fsys (MHz) Perfor-Mance Logic Slices BRAM Slices DSP Slices T0 −0 1 240 8 3093 43 30 T0 −1 2 120 8 6062 86 60 T0 −2 1 120 4 3093 43 30 T0 −3 4 60 8 11,877 172 120 T0 −4 2 60 4 6062 86 60 T0 −5 1 60 2 3093 43 30 T0 −6 8 30 8 23,259 344 240 T0 −7 4 30 4 11,877 172 120 T0 −8 2 30 2 6062 86 60 T0 −9 1 30 1 3093 43 30 T1 −0 1 240 8 2061 22 82 T1 −1 2 120 8 4040 44 164 T1 −2 1 120 4 2061 22 82 T1 −3 4 60 8 7914 88 328 T1 −4 2 60 4 4040 44 164 T1 −5 1 60 2 2061 22 82 T1 −6 8 30 8 15,499 176 656 T1 −7 4 30 4 7914 88 328 T1 −8 2 30 2 4040 44 164 T1 −9 1 30 1 2061 22 82 T2 −0 1 240 8 5003 27 24 T2 −1 2 120 8 9806 54 48 T2 −2 1 120 4 5003 27 24 T2 −3 4 60 8 19,212 108 96 T2 −4 2 60 4 9806 54 48 T2 −5 1 60 2 5003 27 24 T2 −6 8 30 8 37,623 216 192 T2 −7 4 30 4 19,212 108 96 T2 −8 2 30 2 9806 54 48 T2 −9 1 30 1 5003 27 24 T3 −0 1 240 8 4009 16 46 T3 −1 2 120 8 7858 32 92 T3 −2 1 120 4 4009 16 46 T3 −3 4 60 8 15,395 64 184 T3 −4 2 60 4 7858 32 92 T3 −5 1 60 2 4009 16 46 T3 −6 8 30 8 30,148 128 368 T3 −7 4 30 4 15,395 64 184 30 of 34 Computers 2018, 7, 52 Table A1. Cont. Variant No. No. Appendix A Table A1 is the Variant-LUT for the system example discussed in Section 4.1. It contains the characteristics of the ten variants of each of the six tasks T0 to T5. Table A1 is the Variant-LUT for the system example discussed in Section 4.1. It contains the characteristics of the ten variants of each of the six tasks T0 to T5. Table A1. Example Variant-LUT for six tasks. Table A1. Example Variant-LUT for six tasks. Variant No. No. Appendix A of Slots Fsys (MHz) Perfor-Mance Logic Slices BRAM Slices DSP Slices T3 −8 2 30 2 7858 32 92 T3 −9 1 30 1 4009 16 46 T4 −0 1 240 8 5088 39 51 T4 −1 2 120 8 9972 78 102 T4 −2 1 120 4 5088 39 51 T4 −3 4 60 8 19,538 156 204 T4 −4 2 60 4 9972 78 102 T4 −5 1 60 2 5088 39 51 T4 −6 8 30 8 38,262 312 408 T4 −7 4 30 4 19,538 156 204 T4 −8 2 30 2 9972 78 102 T4 −9 1 30 1 5088 39 51 T5 −0 1 240 8 2567 33 73 T5 −1 2 120 8 5031 66 146 T5 −2 1 120 4 2567 33 73 T5 −3 4 60 8 9857 132 292 T5 −4 2 60 4 5031 66 146 T5 −5 1 60 2 2567 33 73 T5 −6 8 30 8 19,304 264 584 T5 −7 4 30 4 9857 132 292 T5 −8 2 30 2 5031 66 146 T5 −9 1 30 1 2567 33 73 References 1. Kirischian, L. Reconﬁgurable Computing Systems Engineering: Virtualization of Computing Architecture; CRC Press: Boca Raton, FL, USA, 2016. 1. Kirischian, L. Reconﬁgurable Computing Systems Engineering: Virtualization of Computing Architecture; CRC Press: Boca Raton, FL, USA, 2016. 2. Architecture Brief—What Is an SOC FPGA? 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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2084010807	https://zenodo.org/records/1770336/files/article.pdf	English	null	SOME ACUTE DISEASES OF THE EAR; THEIR DIAGNOSIS AND TREATMENT.	JAMA	1,902	public-domain	4,126	PHILIP HAMMOND, M.D. It is my purpose in presenting this paper to call to your attention some of the more common conditions which confront us when we have occasion to examine the ear. The two most frequent symptoms which drive the ordinary person to seek medical attention for this organ are pain and deafness, and to treat either success- fully we must remember that we have to deal with symp- toms and not diseases. Take the pain, for instance, and we find that it may come from an abscess of the middle ear, from an ulcerated tooth, from a furuncle in the canal, or perhaps tonsillitis or rheumatism, or some other general disturbance is at the bottom of it. We learn to recognize that deafness, too, is but a symptom, and that it may come from one of many causes. Acute closure of the Eustachian tube is painless, and usually begins with a stuffy sensation in the head, fol- lowed by a more or less marked deafness. In the case of a child this may be attributed to stupidity. Sounds within the head appear to be very much louder than they really are, and to the patient his own voice sounds as if he were speaking with his head in a barrel. Inspec- tion of the drum shows a darkened, retracted membrane, without any injection of the vessels. If the disease has progressed so far that there is already fluid within the ear, we may be able to make out its upper level through the drum. In slight cases where there has been no exudation, the natural tendency is toward spontaneous recovery with the passing of the cold which caused the trouble. Re- peated attacks, however, can not but result in organic changes in the ear. which later in life become apparent as the familiar dry catarrh of the middle ear, with very considerable deafness. y y In examining a case of earache, we thoroughly inspect the drum, noting in the first place the presence or absence of normal appearances. If we detect any con- gestion we at once conclude that here is sufficient cause for pain and proceed to treat it. In the absence of any inflammation, we know that it is not possible for such an ear to give rise to pain, and we must look elsewhere for the cause. 50 994. 1 Chirurgie, 1896, o xxiii, p. 5 2. Rivista di Medicina y Cirurgia Pratica, 5 Jiullet, 1 C Chirurgie, 1896, xxiii, p 62. Rivista di Medicina y Cirurgia Pratica, 5 Jiullet, 1896, p. 24. 63. Archives Provinciales de Chirurgie, 1897. 62. Rivista di Medicina y Cirurgia Pratica, 5 Jiullet, 1896, p. 24. 63. Archives Provinciales de Chirurgie, 1897. G 6 y Cirurgia Pratica, 63. Archives Provinciales de Chirurgie, 1897. y Cirurgia Pratica, , , p 63. Archives Provinciales de Chirurgie, 1897. 63. c es Provinciales de Chirurgie, 1897. 64. Gaz. des H\l=o^\pitaux, April 1, 1897. de Chirurgie, 18 64. Gaz. des H\l=o^\pitaux, April 1, 1897. H\l o \pitaux, A 65. Lancet, Nov. 6, 1897. Lancet, 6, 1897. 66. La Loire M\l=e'\d.,Dec. 15, 1897. a \ e \d , ec 5, 67. Centralblatt f\l=u"\rChirurgie, 1897, vol. xxiv, p. 1301 Chirurgie, 1897, xxiv, p. 1 68. Med. Press and Circular, May 18, 1898. Cli i h 1898 g , , xxiv, p 68. Med. Press and Circular, May 18, 1898. 69 G D li 6 S d li d ll Cli i h 1898 N 20 Circular, May 18, 1898. 69. Gaz. Degli 6 Spedali e delle Cliniche, 1898, Circular, May 18, 69. Gaz. Degli 6 Spedali e delle Cliniche, 1898, Nov. 20. 70 Z it h ift f\l "\ Di t\l "\ti h d Ph ik li h Th i L i Degli Spedali C c e, 70. Zeitschrift f\l=u"\rDiat\l=a"\tischeund Physik g 1900. zig, 1900. 71. The Carolina Med. Jour., Feb., 1901. g, 1900. 71. The Carolina Med. Jour., Feb., 1901. zig, 1900. 71. The Carolina Med. Jour., Feb., 1901. Read before the Essex North District Medical Society, Jan. 1, 1902. 50 994. 1 51. Deutsche Med. Wochenschrift, Nov. 30, 1893-94. Deutsche Med. Wochenschrift, Nov. 30, 189 52. M\l=u"\nchenerMed. Wochenschrift, Jan. 23 1 Wochenschrift, Jan. 23, 53. Brit. Med. Jour May 4 1895. oc e sc t, 53. Brit. Med. Jour., May 4, 1895. , May , 54. II Policlinico, Rome, April, 1895. L S i M\l \di l A 7 189 54. II Policlinico, Rome, April, 1895. 55 La Semaine M\l=e'\dicale Aug 7 1895 Policlinico, Rome, April, 55. La Semaine M\l=e'\dicale,Aug. 7, 1895. 6 Policlinico, , April, 55. La Semaine M\l=e'\dicale,Aug. 7, 1895. 56 C di P titi 1895 vol 399 , p , 55. La Semaine M\l=e'\dicale,Aug. 7, 1895. 56. Canadian Practitioner 1895 vol. xx p 399. prohibited. Children are the most frequent sufferers from ear- ache, and perhaps the majority are subjected to all kinds of home treatment before the physician is .called. Their unusual susceptibility is due to the fact that there is a certain amount of adenoid tissue in the naso-pharynx of almost every child, and in some this is abnormally devel- oped, with the result that the child is continually getting head colds. With each cold nasal respiration is more or less blocked, the growth swelling so as to obstruct the naso-pharynx, and in this way the Eustachian tube becomes involved. As a direct result we have one of two things. The child may have severe pain with develop- ment of an abscess, or it may become deaf from closure of the Eustachian tube and subsequent exudation of serum into the ear cavity. The first is due to invasion of bacteria along the inflamed membrane, the second to the complete occlusion of the tube. We will take up the discussion of the latter condition first, leaving the sup- purative inflammation of the middle ear for a few min- utes. M\l e \dicale,Aug. 7, 56. Canadian Practitioner 1895, vol. xx, p. 399. Canadian Practitioner, 1895, xx, p. 399. 57. Wratsch, 1895, No. 46, p. 1299. Canadian Practitioner, 1895, 57. Wratsch 1895 No. 46 p 1299. Wratsch, 1895, 46, p. 1299. 58. Voiyeny Med. Jour., Nov., 1895. Voiyeny Jour., Nov., 59. University Med. Mag 1896-97, vol. ix, 9 266. U e s ty Mag., 896 9 , , 9, 60. Congr\l=e`\sde Chirurgie, Paris, Oct., 1896. Co g \ e \sde C u g e, a s, Oct , 61. Centralblatt f\l=u"\rChirurgie, 1896, vol. xxiii, p. 775. 42. Deutsche Med. Wochenschr., Leipzig, 1890, xvi, 1043. in extreme cases of a long, slim flaxseed poultice, curved about the ear in the shape of a horseshoe, have given the best results in the writer's practice. Incision should be resorted to only when the furuncle is pointing. Prema- ture opening apparently does no good, and many think that it favors the formation of more infected areas. The patient's general health should be attended to, and tonics given. Above all, any scratching or fingering of the ear should be prohibited 2. Wochenschr., Leipzig, 1890, xvi, 3. Lavori d. Cong di Med. Int., Milano, 1891, iv, 338 43. Cong. Int., Milano, 1891, iv, 338 341. 44. Internat. Jour. Surg., N. Y., 1892, v, 332. Ibid 43. Cong. Int., Milano, 1891, 44. Internat. Jour. Surg., N. Y., 1892, v, 332. 45. Ibid., page 86. g 45. Ibid., page 86. 46 M d N Phil l ii 436 5. Ibid., page 86. 6 Med N Phil 1893 l ii 436 45. Ibid., page 86. 46. Med. News, Phila., 1893 lxii, 436. e s, a , 1893, , 47. L'Union M\l=e'\d.,Aug. 5, 1893-94. M\l e \d.,Aug. 5, 1893 94. 48. Riforma Medica, vol. ix, 7., 105, 1893-94. ed ca, , , 05, 49. Gaz. des H\l=o^\pitaux Oct. 24, 1893-94. H\l=o \pitaux, 24, 1893 94. 50. Norsk Magazine for Laegevidenskaben, vol. viii, 1893-94, p. 94. Downloaded From: http://jama.jamanetwork.com/ by a University of Arizona Health Sciences Library User on 06/07/2015 PHILIP HAMMOND, M.D. The most common cause of earache, aside from actual inflammation of the ear itself, is due to the presence of a carious tooth, and this is usually found to be a lower molar on the affected side. In the severer forms where an exúdate has formed in the middle ear, treatment of the nasopharynx and infla- tion by means of Pulitzer's method will usually restore the hearing in a very short time. If the child is subject to these attacks, however, we must not look for a perma- nent cure until the adenoids, and particularly those around the Eustachian tubes, have been removed. The presence of a furuncle is usually easily demon- strated, as we have a sharply localized swelling in the canal which is extremely sensitive to the touch, and which renders examination rather difficult. In the early stages the drum can be seen, and the fact that it is not inflamed aids us in the diagnosis. Very rarely the fur- uncle will assume such proportions as to simulate a mas- toid swelling, the ear standing out from the side of the head, and there may even be fluctuation behind the ear. Diagnosis in these cases is often difficult and it may take a day or two to clear up the question. , This collection of fluid in the middle ear is by no means confined to children, and sometimes we haVe very much greater difficulty in securing absorption in adults. If after about ten days of careful treatment of the naso- pharynx, and inflations, the fluid has not disappeared, it may be advisable to perform paracentesis in the lower part of the drum, and then inflate, thus forcibly expell- ing the fluid. The hearing will be restored almost imme- diately to normal, and the incision will have healed by the end of twenty-four hours in the vast majority of cases. day p question. Treatment should be directed toward giving the patient comfort. Repeated hot applications, the use of a tight tampon of cotton, dipped in a 4 per cent, solu- tion of carbolic acid in glycerin, placed in the canal, and Acute suppuration of the middle ear, the cause of the earaches with which we are so familiar, presents an en- tirely different clinical aspect. The patient has almost g y p Read before the Essex North District Medical Society, Jan. 1, 1902. PHILIP HAMMOND, M.D. aded From: http://jama.jamanetwork.com/ by a University of Arizona Health Sciences Library User on 06/07/201 Downloaded From: http://jama.jamanetwork.com/ by a University of Arizona Health Sciences Library User o from the first a sharp pain in the ear, steadily growing worse. Inspection of the drum shows a congestion of the upper posterior part, which gradually increases until the whole upper part of the membrane is involved. Should the disease progress still further, there will appear a decided bulging of the upper posterior quardant, with marked deafness. in place for twenty-four hours, when it is replaced by a fresh one. Wicking is kept up as long as the discharge is thin enough to escape through the cotton. By means of this method of treatment we materially reduce the risk of infection from without, as must inevitably re- sult when we syringe the ear, and in a certain proportion of cases the ear will cease to discharge after one or two days, healing without having really suppurated. In the early stages, heat applied locally is of as much value as any one thing. A good way of using it is to douche the ear every hour or two with water as hot as can be tolerated, using from one to two quarts of fluid each time, in order to get a prolonged application. In severe cases it is well to also employ heat in the form of a hot water bottle, or hot salt bag, between the douch- ings. Free catharsis should be attended to, and it may be -necessary to give a little bromid, or phenacetin. In mild cases this treatment will cut short an otherwise painful illness, and the patient will escape with only a few -hours- of pain, and subsequently a feeling of fulness with slight -deafness. In the severer forms of infection the inflammation goes on rapidly to pus formation, with the Consequent bulging of the drum of which we have just spoken. y , g having really suppu ated When an ear is really suppurating actively, however, we have to resort to the use of the syringe as a measure of cleanliness. It is best used in the form of a douche, the heat being grateful to the patient, and tending to allay the inflammation. Leeches are sometimes of value where the congestion is very great. They are best ap- plied immediately in front of the ear. PHILIP HAMMOND, M.D. He knows that he is justified in using palliative treatment to a certain extent, but he also has in mind a wholesome dread of possible infection of the mastoid, or of the meninges, even, should the case be neglected. To allow the abscess to develop until it opens spontaneously is to needlessly expose the patient to these dangers. The question then naturally arises, when shall he perform paracentesis, and also, what risk is he running by so doing ? fi I running by doing In answer to the first I would say that whenever we have a bulging of the drum, with pain which is not easily controlled by the means we have mentioned, I should unhesitatingly open the membrane. In doing this the whole posterior half of the drum should be freely incised, a curved opening being preferable, as not so likely to close again too quickly. Regarding the dan- gers of this procedure, they refer mostly to the future hearing of the patient. Until one has had a little experi- ence, there is, of course, some likelihood of injuring the delicate mechanism of the middle ear, but hearing is of secondary importance as compared with bringing the patient safely out of the trouble. In practiced hands no evil result need be feared, as most of these cases recover their normal degree of hearing. The operation is an exceedingly painful one. and is best done under the influence of primary anesthesia. We must not expect the escape of a large quantity of pus immediately on opening the drum. There is usually a free bleeding at first, followed by a serous discharge for twelve or more hours, before we get pus. As has been repeatedly proven by cultures, the bacteria are there from the start, but have not sufficient opportunity to multiplv. To mini- mize this growth of bacteria, and to prevent the access of others from without, it is better before doing para- centesis to carefully cleanse the canal with a cotton stick dipped in an antiseptic, and then to allow a corrosive sublimate solution of a strength of 1 to 5000 to remain in the ear for at least five minutes. The paracentesis needle is, of course, sterilized, and after the bleeding has in a measure subsided, the canal is carefully cleared of blood clots, and a loose wick of sterilized absorbent cotton inserted. PHILIP HAMMOND, M.D. and over the mas- toid process. It is absolutely essential that the opening in the drum should be maintained during the progress of the disease, and this sometimes requires repeated openings. openings. It sometimes happens that the patient does not come to us for treatment until after the most severe of the inflammatory symptoms have, subsided. He may com- plain simply of deafness, or of the inconvenience of hav- ing a constant discharge from the ear. Examination after cleansing the canal of all secretion will usually show a red and edematous drum in which we may possibly see the perforation, or the latter may be so small as to be invisible except on performing Valsalvan inflation. When this is done, fluid or air will come out through the drum, making the location of the hole apparent. If we keep the canal cleared of the purulent discharge, the natural tendency in acute cases is toward recovery. Keeping in mind the fact that our abscess cavity is inside the drum, and that the perforation may be so small as to be invisible, we can readily see the futility of attempt- ing to bring about a cure by instilling the various solu- tions recommended for this trouble. It is probable that not one particle ever enters the tympanic cavity. Of course, this does not apply to cases with larger perfora- tions, but these do not usually appear in acute cases, and to discuss them would be to open up a larger field than we have time for to-day. When the discharge is profuse it is my custom to have the patient syringe the ear sev- eral times daily, allow it to dry as well as possible, and then blow in a small quantity of boracic acid. As the discharge diminishes, it is sometimes preferable to use the so-called dry treatment, mopping out the canal with a cotton stick, and then using the powder. We should keep track of the hearing during this time, for cessation of the -discharge alone might mean that the drum was closing prematurely, to cause mischief later on, but a diminished discharge with improved hearing would indi- cate recovery. j spoken. Here we have arrived at a stage which often puzzles the general practitioner. d From: http://jama.jamanetwork.com/ by a University of Arizona Health Sciences Library User on 06/07/2015 Professor of Obstetrics and Gynecology, Rush Medical College, the University of Chicago. CHICAGO. If the pain and tenderness of the mastoid persist for more than twenty-four hours after free paracentesis and the use of the cold, it would be advisable for most prac- titioners to consult with some one skilled in such mat- ters, as there is very great danger in delaying operation too long. No one can tell in which direction the pus will turn to seek relief, and practically the only notice we have of its penetration inwards to the lateral sinus or brain is a sharp chill, ushering in a condition of septi- cemia, and then it may be too late to interfere. Fig. 1.—Boldt table representing the elevation used in examining the female bladder. The author's shoulder support is shown, al- tached to the table-top. The leg-holders are also in position. y Carried out in competent hands the mastoid operation is devoid of danger, and in uncomplicated cases the pa- tients recover so rapidly that it is often advisable rather than submit to the tedious processes of spontaneous re- covery, which may take many weeks, and which gives us no assurance that the patient may not ultimately be obliged to submit to operation after weeks of delay. cently he has advocated the genupectoral posture as more advantageous. E h f h h d g Each of these methods has certain disadvantages. When the first posture is adopted, the examination is frequently unsatisfactory. The elevation necessarily leads to a flexing of the upper part of the abdomen on itself. This interferes with the free descent of the in- testines toward the diphragm, which is necessary to the complete distension of the bladder with air. The hips can not be raised much above twelve or fourteen inches without discomfort to the patient if she be not anes- thetized, and if an anesthetic be employed, the cramping of the abdomen interferes with free respiration. In women with tense or fat abdominal walls, it is often im- possible to obtain distension of the bladder by this method. Prescribing- by Druggists.—The Paris "Syndicat des Médecins" sued a pharmacist for illegal practice of medicine because he had made an analysis of the urine of an applicant and then prescribed for him on the ground of what he found in the urine. Professor of Obstetrics and Gynecology, Rush Medical College, the University of Chicago. CHICAGO. Professor of Obstetrics and Gynecology, Rush Medical College, the University of Chicago. CHICAGO. Professor of Obstetrics and Gynecology, Rush Medical College, the University of Chicago. CHICAGO. In his earlier papers describing his method of exam- ining the female bladder, Dr. Howard Kelly recom- mended that the necessary elevation of the pelvis be ob- tained by placing pillows under the hips as the patient lay on a flat table in the lithotomy position. More re- Fig. 1.—Boldt table representing the elevation used in examining the female bladder. The author's shoulder support is shown, al- tached to the table-top. The leg-holders are also in position. , although pus. Tenderness over the mastoid antrum is apt to be a bet- ter guide, but it must be remembered that this is often apparent only on deep pressure, and also that with some very thick bones we are not able to elicit tenderness. veryIn the absence of these two symptoms we are obliged to turn to an inspection of the condition of the middle ear. With an increasing collection of pus in the mastoid there will be a more pronounced bulging of the drum, and in many cases which give no evidence externally there will be almost a complete closure of the auditory canal from swelling of the wall nearest the mastoid. There is usually much pain with this con- dition, but in some cases, and especially with phthisical patients, there is none. The presence of this increased swelling of the drum and posterior canal wall is diag- nostic of mastoid trouble, however, and it should not be allowed to persist long without operation. persist long operation. Now as to the treatment which should be instituted once we have made the diagnosis. We must bear in m ¡lid the fact that mastoiditis is always secondary to a purulent inflammation of the middle ear, and use our best efforts to abort this trouble. Free drainage should be maintained through the drum, and at the first suspi- cion of tenderness or other sign of mastoid involvement we should apply cold by means of Leiter's coil. At this stage leeches to the mastoid are often of value, and the patient should, of course, be kept in bed. Active treat- ment should at the same time be continued for the sup- puration in the middle ear, as we have just indicated. PHILIP HAMMOND, M.D. Outside of this, still another piece of cotton is placed, for the purpose of receiving the capill- ary drainage of the wick. This latter piece is to be changed as often as it becomes soiled, the wick remaining y A point of great interest to the general practitioner is the recognition of the presence of infection of the mas- toid. Many times I have had the statement made to me by physicians that they have been in practice twenty years, we will say, and have never seen a case of mastoid- itis, and almost in the next breath ask : What are the symptoms? y p Here we have the whole matter in a nutshell. It is not that the disease is infrequent, but because the symp- toms are obscure, and in many cases to be made out only by inspection of the ear by means of the speculum and reflecting mirror, that it is not more often detected. Be- cause some years since mastoid abscesses were not com- monly opened until there was an exúdate of pus between the bone and periosteum, this is the picture most often before the minds of many of us at the mention of the AN IMPROVED METHOD OF EXAMINING THE FEMALE BLADDER. disease. We rather expect to see a certain amount of edema behind the ear, and perhaps fluctuation. , perhaps Fortunately for our patients, we are to-day enabled to detect the presence of extension to the mastoid long before it has reached this stage. Every patient who has an acute suppuration of the middle ear should be care- fully examined for evidences of mastoid involvement, as symptoms may appear as early as the second day. It is a well-known clinical fact that the absence of tem- perature is no guide whatever ; during the acute process in the middle ear we are apt to have some elevation, but this may entirely subside with free discharge from the ear, even although the mastoid be filled with pus. Professor of Obstetrics and Gynecology, Rush Medical College, the University of Chicago. CHICAGO. The court decided that pharmacists are not com- petent to draw conclusions from examination of the urine so as to prescribe understandingly, and consequently condemned the defendant to a tine of $10 and a similar sum to be paid to the syndicate. The genupectoral posture, while undoubtedly most nloaded From: http://jama.jamanetwork.com/ by a University of Arizona Health Sciences Library User on 06/07 Downloaded From: http://jama.jamanetwork.com/ by a University of Arizona Health Sciences Library User on 06
https://openalex.org/W4289801273	https://zenodo.org/record/3931281/files/BASICS%20OF%20THE%20THEORY%20OF%20CHEMICAL%20PROCESSES%20AND%20REACTORS.pdf	Russian	null	BASICS OF THE THEORY OF CHEMICAL PROCESSES AND REACTORS	Zenodo (CERN European Organization for Nuclear Research)	2,017	cc-by	73,398	Рекомендовано к печати редакционно-издательским советом НТУ «»ХПИ» (протокол №1 от 22 июня 2017 г.) О 75 Основы теории химических процессов и реакторов: монография / И. В. Питак, В. П. Шапорев, О. Я. Питак, А. О. Грубник, Б. Н. Комаристая. – Харьков: Технологический центр, 2017. – 192 с. О 75 Основы теории химических процессов и реакторов: монография / И. В. Питак, В. П. Шапорев, О. Я. Питак, А. О. Грубник, Б. Н. Комаристая. – Харьков: Технологический центр, 2017. – 192 с. , р , , ру , Б. Н. Комаристая. – Харьков: Технологический центр, 2017. – 192 Утверждено редакционно-издательским советом НТУ «»ХПИ», протокол № 1 от 22.06.2017 г. Харьков 2017 Харьков 2017 Рецензенты: Терешкин, О. Г. – доктор технических наук, профессор, заведующий кафедрой отельного и ресторанного бизнеса Харьковского государственного университета питания и торговли. Склабинский, В. В. − доктор технических наук, профессор, заведующий кафедрой процессы и оборудование химических и нефтехимических производств Сумского государственного университета. Рекомендовано к печати редакционно-издательским советом НТУ «»ХПИ» (протокол №1 от 22 июня 2017 г.) ISBN 978-617-7319-12-1 В монографии рассматриваются теоретические проблемы процессов химической технологии, которые исторически развивались на основе создания строительных материалов, металлургии, производства неорганических и органических соединений, а также переработки сельхоз продукции в пищевые продукты. Широко используя математические методы моделирования, метод теоретических обобщений, метод графов авторы дают новые системы уравнений статистики и кинетики процессов. Обсуждаются вопросы теории и методы расчета химических реакторов, которые считают основой «процессорной единицей» в технологическом производстве, а также рассматривают проблемы интенсификации и оптимизации химико- технологических процессов. УДК 66.021.3-253:532.5 © Питак И. В., Шапорев В. П., Питак О. Я., Грубник А. О., Комаристая Б. Н. ISBN 978-617-7319-12-1 Технологический Центр, 2017. ПРЕДИСЛОВИЕ В книге рассматриваются теоретические проблемы процессов химической технологии, которые исторически развивались на основе создания строительных материалов, металлургии, производства неорганических и органических соединений, а также переработки сельхоз продукции в пищевые продукты. Широко используя математические методы моделирования, метод теоретических обобщений, метод графов авторы дают новые системы уравнений статистики и кинетики процессов. На основе этого, авторы обсуждают вопросы теории и методы расчета химических реакторов, которые считают основой «процессорной единицей» в технологическом производстве, а также рассматривают проблемы интенсификации и оптимизации химико- технологических процессов. В книге достаточно внимания уделено методологии и организации научных исследований по разработке новых технологических процессов и реакторов для их реализации или их усовершенствованию, а также требованиям к содержанию и качеству исходных данных, выдаваемых исследователем для проектирования технологического процесса оборудования. Не менее важным является раздел книги «Инженерное оформление химико-технологических процессов», в котором авторы приводят несколько примеров недостаточно точно изученных процессов протекающих как в гомогенных, так и гетерогенных системах и которые считают перспективными в применении и развитие ряда отраслей химической технологии. Анализ указанных примеров базируется на теории химических реакций, математических методов моделирования процессов и методах теоретического обобщения и анализа. Материал книги охватывает важнейшие проблемы современной инженерной химии: приложение законов физической химии к решению инженерных задач, явления переноса массы, энергии и количества движения, вопросы теории подобия, теорию химических реакторов. Книга оригинальна по содержанию и может представлять большой интерес для научных работников, инженеров проектировщиков и производственников химической, нефтехимической и смежных с ними отраслей промышленности, а также студентов, которые являются соискателями степеней магистра и аспирантов высших учебных заведений. 3 ВВЕДЕНИЕ Инженерная химия (chemical engineering) есть наука, применяющая принципы естественных наук совместно с принципами экономики и социальных отношений к области, охватывающей непосредственно процессы и аппараты, в которых вещество обрабатывается с целью изменения его состояния, структуры, содержания энергии или состава. Данная наука базируется с одной стороны на практике (существующие рецепты, методы или правила), с другой стороны стремление управлять процессами и установить общие закономерности, присущие процессам аналогичного типа привело к необходимости абстрагирования от практического опыта и на основе использования общих законов (принципов) математики, физики, химии к созданию математических моделей, характеризующих отдельные процессы химической и перерабатывающей технологии, а также модели основных аппаратов (реакторов). Итак, из приведенного следует, что наука о процессах химической и перерабатывающей технологий и аппаратах (реакторах), в которых осуществляются процессы, имеет два первоисточника: промышленную практику, а также фундаментальные науки как математика, физика, химия, экология. Следует отметить, что фундаментальные науки физика, химия позволяют описать в той или иной мере эволюцию материи, то есть сформулировать законы микро и макромира [1]. Современные же взгляды на природу математики можно охарактеризовать следующими двумя тезисами [2]: А. В основе всей математики лежит чистая теория множеств. В. Специальные разделы математики занимаются структурами, принадлежащими к тем или иным специальным родам структур. Каждый род структур определяется соответствующей системой аксиом, выраженной на языке теории множеств. Математика интересуется только теми свойствами структур, которые вытекают из принятой системой аксиом, то есть, изучает структуры с точностью до изоморфизма. По существу мы имеем не одну математику, а две: «содержательно воспринимаемую» и «формализованную». Вторая, реализуется в виде символических исчислений, формулам которых не приписывается никакого смысла. Что касается содержательных утверждений об этих вычислениях, то они относятся к особой науке, которой Гильберт дал название метаматематики. Следует сразу подчеркнуть, что формализованная математика без метаматематики не представляет никакого интереса. Лишь метаматематика позволяет установить, каким формулам формализованной математики можно придать содержательное толкование, допускающее применение к изучению реального мира и в реальной человеческой практике. Законность применения этих видов математики для описания явлений полностью гарантируется при соблюдении двух условий: финитная часть математики должна быть содержательной и полной, а нефинитная, лишенная содержательной интерпретации и формализованная, − непротиворечивой. Математика является языком, благодаря которому постигают подлинною и глубокую красоту природы и она является языком, на котором разговаривает 4 природа. Как известно, Джинс сказал, что Великий Архитектор, который создал Вселенную, по-видимому, был математиком [3]. Математика – это язык, плюс рассуждения, это как бы язык и логика вместе. Математика – орудие для размышления. ВВЕДЕНИЕ Что касается экологии, то совершенно очевидно, предприятия на которых реализуются процессы химической и перерабатывающей технологии оказывают интенсивное антропогенное воздействие на окружающую среду. Это воздействие выдвигает в разряд наиболее актуальных научно-прикладных проблем экологии проблему устойчивости экологических систем и биосферы в целом. При трактовке понятия устойчивости, речь идет или об относительной неизменности экологической системы или, о ее способности восстанавливаться за приемлемое время. Развитие теории устойчивости экологических систем и ее практическое применение прямо связаны с совершенством технологических процессов, образования и возможности создания безотходных или малоотходных технологических циклов. Для выявления объективных законов развития химических и перерабатывающих производств человек должен собирать и накапливать производственный опыт, описывать его, систематизировать и с помощью фундаментальных наук анализировать. Совокупность полученных закономерностей и будет представлять собой основу для усовершенствования и создания новых технологических процессов и аппаратов. Сделанный вывод справедлив также и для других инженерных наук, например, строительная техника, отраслевое машиностроение. Химическая и пищевая технологии и др. нетрудно показать, что каждая из них является специальной областью знаний и пользуется своими характеристическими величинами, для них справедливы основные законы микро и макромира. В связи с этим далее целесообразно рассмотреть основные законы микро и макромира, а также принципы эволюции химических систем. 5 5 1 ОСНОВНЫЕ ЗАКОНЫ МИКРО И МАКРОМИРА. ПОНЯТИЕ СИСТЕМЫ. ЭВОЛЮЦИЯ ХИМИЧЕСКИХ СИСТЕМ Скандинавский эпос «Старшая Эдда» свидетельствует: «В начале времени не было в мире Ни песка, ни моря, ни волн холодных, Земли еще не было, и небосвода, Земли еще не было, и небосвода, Бездна зияла, трава не росла». При современных взглядах научного сообщества на Вселенную она бесконечна в пространстве и времени и неисчерпаема [7]. При современных взглядах научного сообщества на Вселенную она бесконечна в пространстве и времени и неисчерпаема [7]. Поэтому некоторые представления о наблюдаемой структуре Вселенной: «Квантово-полевой вакуум»; содержание темной энергии 60 – 62 %; содержание темной материи (32 – 34 %); содержание видимой материи, (звезды, Галактики и т.п. ~6 %) свидетельствует о различных видах фундаментальных взаимодействий в рассматриваемых областях, а так же по- видимому речь идет о непрерывном и бесконечном процессе преобразований и переходе материи от одной пространственно-временной области к другой. Человечество в основном живет и действует в пространственно-временной области, содержащей видимую материю, которая состоит из множества известных и экспериментально-установленных элементарных частиц: атомов, молекул и т.п. Этот важный факт является отдаленным следствием более или менее случайного сочетания множества законов микромира, описанных выше. Например, ядра, содержащие несколько элементарных частиц-прототипов и нейтронов, представляют собой чрезвычайно сложное объединение (систему). У них есть так называемые энергетические уровни, они могут находиться в различных сочетаниях или условиях, характеризуемых уровнем энергии, а эти уровни различны для различных ядер. Точное положение уровней, очевидно, является следствием невероятно сложных процессов, а поэтому, нет ничего особенно загадочного в том, что, например, у азота с его 15 элементарными частицами в ядре одному уровню соответствует энергия 2,4 МэВ, другому 7,1 МэВ и т.д. Но что, действительно, замечательно, так это то, что весь характер нашей вселенной в значительной степени зависит от точного значения одного энергетического уровня одного ядра. Как оказывается, один уровень ядра 12С равен 7,82 МэВ, и именно это делает нашу вселенную такой, какой она есть. Дело здесь вот в чем. Если начинать с водорода, то по мере сближения атомов водорода под действием сил притяжения и по мере повышения температуры водорода возникают условия для ядерных реакций, в результате которых может получиться гелий. Затем гелий может частично вступать в реакцию с водородом и образовывать несколько новых более тяжелых элементов. Но новые элементы немедленно распадаются вновь на гелий и водород. Таким образом, казалось что процессы, которые протекают в области видимой материи, не могут дать что-нибудь иное, кроме гелия и 5 – 6 других элементов, хотя на самом деле мы имеем множество химических элементов и их соединения (таблица Менделеева) и молекул. 1 ОСНОВНЫЕ ЗАКОНЫ МИКРО И МАКРОМИРА. ПОНЯТИЕ СИСТЕМЫ. ЭВОЛЮЦИЯ ХИМИЧЕСКИХ СИСТЕМ Современная наука о микромире – физика элементарных частиц – есть теория квантовых полей [4]. Квантовая теория поля вобрала в себя теорию относительности и квантовую механику. Основной физический объект этой теории – квантовое поле, оно синтезирует понятия классического поля типа электромагнитного поля Фарадея-Максвелла и поля вероятностей нерелятивистской квантовой механики. Концепция поля естественно возникает в теории относительности. Изменение движения определенной частицы не вызывает мгновенной реакции со стороны окружающих ее частиц, поскольку сигнал об этом не может распространяться быстрее чем скорость света. При этом, при изменении движения частицы, распространяется поле, которое переносит энергию, импульс и может передать их другим частицам. Обсуждаемая гипотетическая картина микромира на данном этапе выглядит следующим образом [5]. На расстоянии мене 10-33 см, мир имеет десять измерений (девять пространственных и одно временное) равноправных между собой. Единый фундаментальный объект в том мире релятивистская спиновая струна. Она обладает очень широкой симметрией, включающей в частности, группу общих координатных преобразований, а также расширенную группу суперсимметрии. Спектр возбуждений такой струны определяет набор существующих частиц. На расстоянии порядка 10−33 см происходит отщепление лишних шести пространственных измерений в результате компактификации. Топологические свойства пространства при этом очень существенны, ибо особенности симметрии компактного многообразия определяет в конечном счете группу симметрии эффективной низкоэнергетической теории. В результате компактификации из всего бесконечного спектра состояний замкнутой струны выживают лишь поля низких спинов, образующих мультиплеты гравитации. Расстояние порядка 10−33 см – это область господства теории супергравитации, спроектированной из десятимерного пространства в четырехмерное. Поля разного спина при этом еще неравноправны, но уже проявляется различие между полями материи и калибровочными полями. Все взаимодействия характеризуются единым зарядом. На расстоянии от 10−33 см до 10−30 см происходит отделение гравитации от остальных взаимодействий. На еще больших расстояниях происходит «развал» объединения на три ветви фундаментальных взаимодействий: сильное, электромагнитное и слабое. Согласно гипотезе объединения, достигаемое равенство зарядов различных взаимодействий элементарных частиц неслучайно и указывает на единый источник их возникновения. Дело в том, что заряды, рассматриваемые на больших масштабах, как постоянны, в действительности зависят от расстояния. Это характерное свойство квантовой теории поля, связанные с особенностями квантово-полевого вакуума. Вакуум в этой теории поля не есть физическая пустота. Она заполнена вакуумными флуктуациями – виртуальными частицами. Таким образом, согласно [6] квантово-полевой вакуум совпадает с идеей пустого пространства (темноты), в котором нет реальных частиц, то есть, 6 нет материи и нет фотонов, пустое пространство неизбежно содержит виртуальные частицы. Скандинавский эпос «Старшая Эдда» свидетельствует: нет материи и нет фотонов, пустое пространство неизбежно содержит виртуальные частицы. 1 ОСНОВНЫЕ ЗАКОНЫ МИКРО И МАКРОМИРА. ПОНЯТИЕ СИСТЕМЫ. ЭВОЛЮЦИЯ ХИМИЧЕСКИХ СИСТЕМ Столкнувшись с этой проблемой, ученые (Ф. Хойл и Солпитер Э. Э.) увидели только один выход – если в результате трех атомов гелия может образоваться атом углерода. Подсчитав 7 такую вероятность, было установлено, что она вообще равна нулю, если только у углерода не окажется энергетического уровня равного 7,82 МэВ. Если такой уровень есть, то три атома гелия соединившись в атом углерода, начнут снова распадаться и в результате значительно позже (медленнее), чем при отсутствии у атома углерода энергетического уровня в 7,82 МэВ. И тогда этого времени хватает для других преобразований для образования новых элементов. Поэтому, существование в нашем мире других элементов очень тесно связано с тем обстоятельством, что у углерода есть именно это значение энергетического уровня, который является случайным результатом очень сложного взаимодействия 12-ти сложных частиц. Мы можем анализировать явления нашего мира, выделяя в нем разные уровни, устанавливая некоторую иерархию понятий и представлений. Например, термин «теплота» − это результат хаотического движения атомов и когда называем что-нибудь горячим, подразумеваем, что имеется некоторая масса атомов в состоянии интенсивного хаотического движения. Другой пример того же рода, это пример с кристаллом твердого вещества. Если смотреть в корень, то эта система огромного числа протонов, нейтронов и элементов, но мы обходимся одним понятием кристаллическое твердое вещество, которое несет в себе целый образ совокупности элементарных взаимодействий. Точно такому же кругу идей принадлежит и понятие давления. Теперь, если еще сделать один шаг вверх и перейти на следующий уровень, мы столкнемся со свойствами веществ, которые характеризуются следующим. Например, коэффициентом преломления, определяющим, на какую величину отклоняется луч света, проходя через вещество; или коэффициентом поверхностного натяжения, объясняющим, почему вода (жидкость) имеет тенденцию оставаться в виде единого целого; или плотность вещества, объясняющая, почему объем той или иной субстанции имеет такую массу. Причем, и то и другое описывается определенным числом. Теперь, для того чтобы выяснить, что в конце концов все это сводится к взаимодействию атомов и молекул нам необходимо вернуться к самому началу на несколько ступеней вниз, пройдя ряд все менее сложных законов. Продолжая подъем по иерархической лестнице сложности, мы добираемся до таких вещей, как живой организм, сокращение мускулов или нервные импульсы – невероятно сложные явления физического мира, связанные с исключительно сложной организацией материи. Все дальше и дальше и вот уже перед нами понятие Человек, история, политическая целесообразность и другие понятия, которыми мы пользуемся для того чтобы разбираться в событиях на еще более высоком уровне. 1 ОСНОВНЫЕ ЗАКОНЫ МИКРО И МАКРОМИРА. ПОНЯТИЕ СИСТЕМЫ. ЭВОЛЮЦИЯ ХИМИЧЕСКИХ СИСТЕМ А затем, наступает черед таким вещам, как зло и красота, надежда и любовь … Следует особо остановиться на том, что общие законы и важнейшие свойства вещества (свойства газов, жидкостей, растворов и твердых тел) присущие некоторым коллективам элементарных частиц, которые принято называть системой или химической системой. От поведения коллектива частиц (взаимодействия, изменения, движения) с точки зрения возможности возникновения в них порядка, то есть самоорганизации и устойчивости проявляются те или иные характеристики системы. Различия между свойствами 8 коллектива частиц и индивидуальной частицы в наименьшей степени выражены у газов, по отношению к химическим свойствам. В жидкости, специфические особенности коллектива выражены более отчетливо вследствие явлений ассоциации, сольватации, комплексообразования и т.п. В твердых телах сохранение химической индивидуальности частиц коллектива существенно зависит от природы частиц и типов связи между ними. Слабые связи, обусловленные силами Ван-дер-Ваальса, способствуют доминированию индивидуальных признаков над коллективными. Сильные связи, возникающие, в металлах и полупроводниках, являются причиной больших различий между химическими свойствами отдельного атома и кристаллической решетки в целом. Однако, во всех этих случаях критериями устойчивого равновесия системы служат термодинамические критерии, к рассмотрению которых мы и переходим. Неравновесная система должна рано или поздно вернуться к состоянию равновесия. Происходящие при этом изменения составляют то, что называют процессом или движением. По Гегелю «движение есть изменение вообще». Марк Аврелий утверждая, что «поток изменения постоянно обновляет мир, как непрестанное течение времени постоянно сообщает юность беспредельной вечности». Вопрос о критериях равновесия классической термодинамики решается с помощью функции состояния, изменение которых не зависит от пути процесса и определяется только исходными и конечными значениями функций. К таким функциям в первую очередь относится энергия. Согласно существующих представлений суммарная энергия Вселенной постоянна [8], то есть, какие бы не шли процессы должен соблюдаться закон сохранения энергии. Однако сам закон сохранения энергии, несмотря на кажущуюся ясность и простоту в действительности нельзя считать ни простым, ни ясным. Этот закон выражает постоянство суммы четырех слагаемых: Этот закон выражает постоянство суммы четырех слагаемых: 1) кинетической энергии; 2) потенциальной энергии, зависящей от положения тела; 3) внутренней молекулярной энергии в атомах, тепловой, химической и электрической; 4) энергия, заключающаяся в самом веществе. При этом, как указывает Пуанкаре [9], такое выражение не представляло бы затруднений, если бы между указанными слагаемыми можно было провести строгое различие, и соответственно каждый вид энергии не зависел бы друг от друга. Пуанкаре показывает, что в этих условиях выбор функции, которую мы называем энергией, оказывается условным и, следовательно, единственно возможная формулировка закона сохранения энергии гласит: «Существует нечто, остающееся постоянным». 1 ОСНОВНЫЕ ЗАКОНЫ МИКРО И МАКРОМИРА. ПОНЯТИЕ СИСТЕМЫ. ЭВОЛЮЦИЯ ХИМИЧЕСКИХ СИСТЕМ Это замечание Пуанкаре становится еще более важным потому, что, согласно теории относительности само вещество должно рассматриваться как некая форма энергии. Действительно, согласно А. Эйнштейна, массе «m» соответствует энергия «Е» согласно формуле: 9 9 2 Е m с   где с − скорость света (3·108 м/с). Тогда m = 1 г соответствует энергия приблизительно равная 25·106 кВт·ч. Тогда m = 1 г соответствует энергия приблизительно равная 25·10 кВт·ч. Таким образом, формулировка «существует нечто, остающееся постоянным» охватывает как закон сохранения энергии, так и закон сохранения массы, причем, это «нечто» не является ни веществом, ни энергией, а представляет собой некоторую математическую функцию, физический смысл которой интуитивно не ясен. Однако, в рассматриваемых нами коллективах частиц (системах) тепловая и механическая энергии в достаточной степени разделимы и независимы друг от друга, а превращения массы в энергию проявляется только при проведении ядерных процессов, отсюда следует, что можно применять закон сохранения с соблюдением принципа эквивалентности теплоты и работы. Философы древности интуитивно пришли к выводу о законе сохранения и процессов превращения одного вида «нечто» в другой. Так Лукреций Кар в трактате «О природе вещей» писал: «Словом не гибнет нечто, что как будто совсем погибает. Так как природа всегда возрождает одно из другого и ничему не дает без смерти другого родиться». Первое начало термодинамики исключает для изолированной системы все те процессы, в которых внутренняя энергия систем изменяется; второе начало делает отбор более жестким – возможны лишь процессы, в которых энергия изолированной системы всей в целом возрастает. Максимум энтропии и есть в этом случае условием равновесия. Для систем, обменивающихся со средой энергией (но не массой) – закрытых по терминологии И. Пригожина – критерием равновесия служат также термодинамические функции. Предположим, что в системе постоянными являются давление и температура. Допустим, что система, находящаяся в некоторой среде, которую можно рассматривать как изолированную, получает от среды количество теплоты δQ. Энтропия закрытой системы возрастает на δQ/Т, кроме того в этой системе могут проходить и внутренние процессы (например диффузия), в результате которых энтропия также возрастает на величину diS. Общее изменение энтропии: Общее изменение энтропии: i Q dS d S T    или i du pdV dS d S T    (1.1) (1.1) где du − изменение внутренней энергии, в простейшем случае где du − изменение внутренней энергии, в простейшем случае pdV − работа (расширения). где du − изменение внутренней энергии, в простейшем случае pdV − работа (расширения). де du изменение внутренней энергии, в простейшем случае pdV − работа (расширения). 1 ОСНОВНЫЕ ЗАКОНЫ МИКРО И МАКРОМИРА. ПОНЯТИЕ СИСТЕМЫ. ЭВОЛЮЦИЯ ХИМИЧЕСКИХ СИСТЕМ pdV − работа (расширения). i du pdV TdS du pdV d S dS T T       . При указанных условиях (p, T − const) выражение ( TdS du pdV   ) является функцией состояния, то есть значение этой функции определяется только начальными и конечными состояниями системы. 10 ункция U TS pV G    называется энергией Гиббса. Следовательно: Функция U TS pV G    называется энергией Гиббса. Следовательно: Функция U TS pV G    называется энергией Гиббса. Следовательно: i dG d S T  . (1.2) (1.2) В замкнутой системе при постоянных p и T равновесие соответствует минимуму G и в самопроизвольном процессе эта функция должна уменьшаться. Если условия иные, например, постоянные температуры и объем, необходимо пользоваться другой функцией – функция A U TS F    . где F − энергия Гельмгольца. При решении вопроса, на сколько измениться энергия Гиббса при изменении количественного состава системы (например, при введении в систему «n» молей вещества) необходимо общую величину G разделить на число молей «n». Производная функции от G по числу молей называют химическим потенциалом : , , ... p T V T G A n n                     . (1.3) (1.3) G  Если система состоит из нескольких веществ, то производная G n   вычисляют, полагая постоянными p, T и концентрацией всех основных частей, кроме данного (i-го). Химический потенциал дает возможность легко решать задачи, связанные с равновесием, поскольку равновесное состояние характеризуется равенством химического потенциала данного вещества во всех фазах системы и можно записать: 0 i i V   , (1.4) 0 i i V   , (1.4) где Vi − стехиометрический коэффициент i-го компонента. где Vi − стехиометрический коэффициент i-го компонента. Как видно классическая термодинамика оперирует с континуумом, при этом, строго говоря, если говорить о структуре системы, как о пространственной упорядоченности коллектива частиц, неправомерно. Однако, если дополнить термодинамику молекулярно-кинетическими данными, то упорядоченность можно связать, например, с минимумом внутренней энергии и тогда кристаллическое состояние будет при низких температурах более устойчиво, чем жидкое или газообразное; это следует из равновесия. При постоянных объеме и температуре энергия Гельмгольца U−TS определяет равновесие и минимум U при малых T определяет и минимум A; при высоких температурах решающее значение оказывает член TS, поэтому, при низких температурах, устойчивой формой будет кристаллическая, а при высоких – жидкая или газообразная. Таким образом, рассматриваемые функции дают возможность судить о направлении процессов, как в изолированных, так и в 11 закрытых системах. Однако, каким путем система пришла к равновесию рассмотренные подходы не дают ответа, хотя для хода химической эволюции путь имеет первостепенное значение. Как видно, химическая система, состоящая из частиц, способных к взаимодействию, принципиально может перейти в качественно иное состояние, отвечающее большей термодинамической устойчивости, чем исходное. Процесс перехода представляет собой химическую реакцию. Опыт показывает, что скорость химической реакции пропорциональна произведению концентраций реагирующих веществ. Таким образом, в данном случае, выполняется закон действия масс, который уже более 100 лет служит надежной путеводной нитью в кинетических исследованиях. При этом, все индивидуальные особенности реагентов скрыты в коэффициентах пропорциональности, то есть в константах скоростей или в коэффициентах активности. Принципиальная возможность реакции, то есть знак изменения соответствующего термодинамического потенциала (ΔG, ΔA) еще не означает реального осуществления процесса. Огромное число термодинамических реакций не осуществляется вследствие активационных барьеров и других ограничений и запретов, способствующих длительному сохранению и развитию термодинамических неустойчивых состояний. Отчетливо выраженная зависимость скорости реакции от температуры была интерпретирована Аррениусом на основе представлений о существовании «активированных» молекул, имеющих некоторый избыток энергии сравнительно со средним уровнем – энергию активации. Из уравнения Аррениуса: lnk A T    или T k Ae    и закона распределения по энергиям Больцмана следует возможность истолкования экспоненциального множителя как доли молекул имеющих энергию «Е», минимально необходимую для того, чтобы столкнувшиеся молекулы могли вступить в реакцию. Поэтому, зависимость константы скорости от температуры стали представлять в виде: / E RT o k k e  , (1.5) где ko − отвечает значению константы при Е = 0. / E RT o k k e  , (1.5) нстанты при Е = 0. где Vi − стехиометрический коэффициент i-го компонента. / E RT o k k e  , (1.5) (1.5) o где ko − отвечает значению константы при Е = 0. отвечает значению константы при Е = 0. Энергия E в равновесной кинетике является полной энергией независимо от распределения ее по степеням свободы (колебательной, поступательной, вращательной). Термин «равновесная» означает, что в ходе реакции не нарушается равновесные распределения Максвела-Больцмана молекул по скорости и импульсам. Если же это распределение нарушается, то учитываются энергетические вклады в «Е» каждого из видов движения. Для равновесных реакций особо продуктивным методом оценки констант скоростей является метод переходного состояния [10]. В этом случае, реакцию можно рассматривать: А + ВС = АВ + С и согласно реакции перед тем как молекула ВС утрачивает свою индивидуальность образуется сочетание атомов 12 промежуточное между А…ВС и АВ….С. Это и есть переходное состояние. Именно оно обладает избыточной энергией «Е» и характеризуется непрерывным изменением расстояний между взаимодействующими атомами. Переходной комплекс соответствует такому состоянию химической системы, при котором потенциальная энергия на пути реакции достигает относительного максимума, а полная энергия активированного комплекса слагается из нулевой энергии всех образующих комплекс атомов и некоторой дополнительной энергии: 1 2 o i E hV E    . (1.6) (1.6) Истинная энергия активации – это та минимальная энергия, которую надо добавить к нулевой энергии исходных веществ, чтобы стало возможным химическое превращение, то есть, чтобы произошло превращение исходных веществ в активированный комплекс, а затем за счет распада комплекса образовали продукты реакции. Константа скорости химической реакции в рассматриваемом случае записывается уравнением: / / , S R H RT i kT k e e h    (1.7) (1.7) где h − постоянная планка, где h − постоянная планка, k − коэффициент Больцмана,  − коэффициент, близкий к величине 0,4÷0,5. Уравнение показывает, что константа скорости реакции ik зависит от двух множителей: / S R e и / H RT e  . Второй содержит тепловой эффект – энтальпию активации, а первый энтропию активации. Отрицательный знак перед показателем / H RТ  означает, что чем больше энтальпия активации, тем при прочих равных условиях реакция будет протекать медленнее. С другой стороны, положительный знак перед / S R  оказывает на обратное влияние энтропии активации. Чем больше ΔS, тем больше скорость реакции. Физический смысл большего значения скорости в том, что переходной комплекс по сравнению с исходными веществами менее компактен – движение в нем более беспорядочное, он представляет собой менее упорядоченную структуру. Практически, энтальпия активации очень близка к экспериментально определяемой энергии активации. 2 H H H   Энергия активации составляет 31 E  кДж/моль, а Р = 1; при проведении реакции С2Н4 + бутадиен = циклогексан С6Н10 энергия активации равна Е = 107 кДж/моль, а 5 4 10 Р   . В жидкостях средняя кинетическая энергия молекул меньше потенциальной энергии их взаимодействия, поэтому, движение частиц имеет иной характер сравнительно с движением в газах. Молекула жидкости, совершает колебательное движение в небольшой области, так называемой «клетке» − ближний порядок и переход из одной клетки ближнего порядка в другую, связан с преодолением активационного барьера; мерой его является / D E RT e , (1.9) узии. / D E RT e , (1.9) / D E RT e , (1.9) где ED − энергия активации диффузии. где ED − энергия активации диффузии. Приблизительно оценку амплитуды колебаний в клетке можно получить, взяв корень кубический из объема υ, приходящегося на одну молекулу жидкости, то есть, 1/3   1/3 / N  , (1.10)   1/3 / N  , (1.10) где Vi − стехиометрический коэффициент i-го компонента. Это значит, что реакция с большой энергией активации может протекать быстро, если для этой реакции энтропия активации достаточно велика. В большинстве случаев, как уже было сказано, энергия активации связана с кинетической энергией сталкивающихся частиц и их типом. Например, идеальный газ состоит из частиц принебрежительно малого объема, испытавающих упругие столкновения, связанные с обменом только кинетической энергией. Молекулы реальных газов при столкновениях могут обмениваться и другими видами энергии (то есть, кинетическая энергия может переходить во внутреннюю колебательную, 13 вращательную, электрическую). Реакция может произойти при столкновении частиц, если энергия столкнувшихся частиц равна или превышает энергию активации, и если выполнены некоторые геометрические условия, касающиеся взаимного расположения молекул (столкновение центр в центр, касательное столкновение и т.п.). Эти условия учитываются формально стерическим фактором «Р» и тогда, выражение для константы скорости имеет вид [11]: / , E RT o k z Pe  (1.8) / , E RT o k z Pe  (1.8) олкновений частиц / , E RT o k z Pe  (1.8) где zo − фактор чистоты столкновений частиц. (1.8) где zo − фактор чистоты столкновений частиц. где zo − фактор чистоты столкновений частиц. Фактор «Р» варьирует в широких пределах; с усложнением структуры реагирующих частиц и увеличения роли ограничений по симметрии орбиталей, фактор «Р» уменьшается. Так, например, при проведении газо-фазных реакций типа [12]: 2 H H H   где N − число молекул в объеме. где N − число молекул в объеме. Для константы скорости «k» реакции в жидкостях с учетом колебаний в клетках предложено уравнение [11]:    / / / E mT k P n E RT Pe      , (1.11) летке, окружающих молекулу, й ( 1012 1013 −1)    / / / E mT k P n E RT Pe      , (1.11)    / / / E mT k P n E RT Pe      , (1.11) где n − число соседей в клетке, окружающих молекулу,  − частота колебаний (имеет порядок 1012÷1013 с−1). (1.11) где n − число соседей в клетке, окружающих молекулу, 12 13 1  − частота колебаний (имеет порядок 1012÷1013 с−1). Вышерассмотренные закономерности оценки скорости процессов относятся к системам однородным по типу молекул-газовые или жидкие системы, то есть гомогенные. 14 В промышленной практике широко используется реакционные системы, в которых взаимодействие протекает между частицами, относящимися к различным фазам. Например, реакции между твердым веществом и газом или жидкостью; реакции между газом и жидкостью; реакции между твердым и газом; реакции между твердым и твердым; реакции между твердым, газом и жидкостью. По классификации, указанные реакции относятся к классу гетерогенных. Центральной задачей в большинстве случаев для этих реакций является природа промежуточных стадий, через которые происходит процесс, в том числе идентификация неустойчивых промежуточных продуктов, то есть переходного состояния. В этой области, речь идет о промежуточных состояниях систем, связанных с образованием поверхностных соединений адсорбированными атомами и молекулами. При этом между фазами возникает реакционная поверхность раздела, которую, по-видимому, можно трактовать как промежуточную стадию процесса. Например, в системах с участием твердого тела механизм взаимодействия, как показал Ф. Ф. Волькенштейн [13], существенно зависит от положения уровня Ферми (то есть, усредненного значения химического потенциала электрона в полупроводнике). Частицы на поверхности тела связаны с ней одноэлектронной или более прочной – двухэлектронной связью. Чем выше уровень Ферьми, тем больше доля частиц, несущих отрицательный заряд и тем меньше доля частиц, связанных с поверхностью донорной связью. Число нейтральных частиц при изменении уровня Ферьми проходит через максимум. Таким образом, свойства твердого тела определяется состоянием частиц на поверхности а, следовательно, и направление химических процессов между частицами твердого тела частицами газа или жидкости. Влияние дефектов или примесей также зависит от того, как отзывается их появление на положении уровня Ферьми. А. А. где N − число молекул в объеме. Баландин обратил особое внимание на геометрическое соответствие между строением и свойствами твердого тела и частицами жидкой или газообразной фаз. В работах Н. И. Кобозева, установлено, что небольшие скопления частиц (атомов) на поверхности твердого вещества по состоянию близкие к аморфным образованиям (ансамблям) способны проявлять высокую активность к взаимодействию с частицами другой фазы. Можно рассматривать с известным приближением и такие системы, как модели неизмеримо более гибких и пластинчатых природных систем, таких как ферментативный катализ. По-видимому, слишком строгое и неизменное следование кодовым правилам, определяемым жесткой геометрией взаимодействия частиц, несколько ограничивает возможности реакций, поэтому биологическая эволюция выдвинула на первый план именно белковые катализаторы (ферменты), обладающие огромным числом конформационных возможностей и связала их с такими субстратами, молекулы которых тоже в известной мере способны к деформациям. От этого кодовые требования стали менее строгими, а для ферментов открылись новые пути повышения активности и специфического действия. Таким образом, кинетические законы, которым следуют химические реакции на молекулярном уровне в гетерогенных системах по своей природе 15 аналогичны гомогенным системам и скорость химической реакции зависит от изменения концентрации вещества, энтальпии активации и энтропии активации. Формально формула для скорости химической реакции в любой из вышерассмотренных систем будет иметь вид: аналогичны гомогенным системам и скорость химической реакции зависит от изменения концентрации вещества, энтальпии активации и энтропии активации. Формально формула для скорости химической реакции в любой из вышерассмотренных систем будет иметь вид: n W k C   , (1.12) n W k C   , (1.12) где C − концентрация реагентов, n − показатель порядка реакции. Величина «k» и ее физический смысл определяются типом процесса (гомогенный, гетерогенный), свойствами частиц и их зарядами, величиной энергии активации. Выше в главе описаны основные термодинамические и кинетические законы, которым следуют химические реакции, причем, весь процесс рассматривается на молекулярном уровне. В тоже время в реальных условиях развитие химических систем привело к последовательному образованию множества сложных динамических структур, подготовивших переход химической эволюции в биологическую. Поэтому, проблема возникновения микро- и макроорганизации в неравновесных системах, получающих от внешней среды вещество и энергию исключительно важна. Трудность решения этой задачи обусловлена тем, что нет столь надежного признака устойчивости неравновесных систем, какими для равновесных систем являются экстремум соответствующего термодинамического потенциала. Поэтому, приходится прибегать к изучению кинетики процессов и в ней искать условия возникновения упорядоченности. В наиболее общей форме Тьюрингом [15] показано, что в результате развития химической реакции при постоянной температуре и диффузионном перемешивании концентрации промежуточных продуктов реакции могут распределяться в пространстве неравномерно, образуя зоны различной концентрации. где N − число молекул в объеме. Основываясь на этом термодинамика, неравновесных необратимых процессов, рассматривает потоки теплоты, энергии, зарядов, массы и др., возникающие под действием «обобщенных сил» [16]. В качестве таких сил фигурируют градиентны температуры, концентрации, химическое сродство. Поток теплоты представляет собой необратимое явление, причем в общем виде причиной потока случит не одна сила. Так, поток теплоты, возникающий под влиянием градиента температуры, вызывает появление градиента концентраций и как следствие поток вещества. Если система не слишком удалена от равновесия, то зависимость между потоками близка к линейной и коэффициенты пропорциональности (L) не зависят от размеров сил; для двух потоков 1I и 2 I можно записать: 1 11 1 12 2 2 21 1 22 2 I L X L X I L X L X     , (1.13) (1.13) где 1 X и 2 X − силы. [17] На основе принципа микроскопической обратимости Онсагерц [17] 16 удалось доказать важное соотношение: L12 = L21. Это соотношение справедливо для любого числа сил и потоков системы, подчиняющиеся линейным законам: i ik k I L X    . (1.14) (1.14) Это значит, что например, поток вещества (диффузия) действует на поток теплоты (теплопроводность) так же как и поток теплоты на поток вещества, то есть с тем же коэффициентом пропорциональности. Энтропия системы, в которой существует такой поток, непрерывно возрастает. Производную энтропии по времени (отнесенную к единице объема) называют производством энтропии(ζ). Анализ различных по своей природе потоков показывает, что всегда можно так выбрать силы, чтобы произведение сил и потоков давало бы производство энтропии. Для химической реакции, в которой в начальный момент времени было o in молей продукта реакции, по истечению времени появилось in молей, введем величину   Для химической реакции, в которой в начальный момент времени было o in молей продукта реакции, по истечению времени появилось in молей, введем величину   o i i i n n V    , (1.15) (1.15) iV i где iV − стехиометрический коэффициент, i где iV − стехиометрический коэффициент, где iV − стехиометрический коэффициент, где iV − стехиометрический коэффициент,  − степень полноты реакции, тогда, i dn d V  .  − степень полноты реакции, тогда, i dn d V  . i dn V . где N − число молекул в объеме. Из уравнений i dG d S T  и  , i Ri P T dG d   , путем последовательных подстановок и дифференцирования по времени получим: Из уравнений i dG d S T  и  , i Ri P T dG d   , путем последовательных подстановок и дифференцирования по времени получим: Из уравнений i dG d S T  и  , i Ri P T dG d   , путем последовательных подстановок и дифференцирования по времени получим: i i i d S d V d d       . (1.16) (1.16) Положив сумму i iV А    , эту сумму называют химическим сродством, это «обобщенная сила». Тогда, ζ будет равно: 1 1 id S d A V dT V d         . (1.17) (1.17) Производная 1 d V d    Производная 1 d V d    есть поток вещества, отнесенный к единице объема и единице времени, а в левой части уравнение производства энтропии. И. Пригожин [18] исследуя вопрос об изменении ζ в течении процесса, когда поток переходит от начального режима развития к устойчивому стационарному состоянию, пришел к выводу, что переход потока к стационарному состоянию характеризуется стремлением производства энтропии к минимуму: 17 min    . (1.18) (1.18) min    . Это условие позволяет предвидеть события в области неравновесных процессов и в известной мере, аналогично термодинамическим критериям равновесия. Однако, этот принцип справедлив только для линейных систем. Взяв вторую производную энтропии по времени. Пригожин и Николас [19] разделив эту величину на два слагаемых, одно, из которых содержит дифференциалы сил, а другое – дифференциалы потоков показали возможность разделения, соответственно и полного производства энтропии P dV   на две части x i d P d P dP d d d      . (1.19) (1.19) Первое слагаемое для ряда систем действительно стремиться к минимуму. Силы всегда изменяются так, что производство энтропии уменьшается. Уравнение dP d выражает критерий развития для диссипативных систем. В стационарном состоянии 0 x d P d , но относительно id P ничего сказать нельзя, поэтому знак dP d не определяется знаком x d P d. Термодинамика необратимых процессов намечает некоторые пути к пониманию законов возникновения временной и пространственной упорядоченности в диссипативной системе, но в рамках этой науки химические свойства отражаются лишь в константах кинетических реакций. где N − число молекул в объеме. р р Все сложные природные системы – от галактики до клетки, от циклона в атмосфере до водоворота в ручье – открытие и диссипативные, а большая их часть нелинейная. Это значит, что через границы системы происходит обмен веществом и энергией с другими системами, происходит рассеивание энергии, превращение одного вида энергии в другой, а также происходят и другие превращения, из-за которых процессы в таких системах оказываются необратимыми. В таких системах неизбежно должны происходить процессы самоорганизации. Процессы самоорганизации возникают за счет наложения на системы ограничений или «запретов» и это ведет к образованию организаций, проявляется способность существовать и сохранять устойчивость в некотором дискретном наборе состояний. Вполне понятно, что дискретность ряда устойчивых состояний означает и дискретные формы отношений, как со средой, так и с другими системами. Дискретность условий «разрешающих» данный процесс и исключающих другие, является основой развития химических систем и отчетливо проявляется в механизмах метаболизма и биологической эволюции. В общем, область научных исследований, связанная с вышеозначенными нелинейными системами, получила термин «синергетика» и она выявляет 18 общие закономерности в формировании структур и их устойчивости [20]. Среди основных принципов синергетики одним из самых важных является принцип подчинения. Принцип подчинения состоит в утверждении, что в самоорганизующихся системах у некоторых переменных время релаксации много больше чем у других. Медленно меняющиеся переменные как раз и описывают реальное поведение системы во времени, тогда как быстрые определяются их значениями. В этом случае число уравнений, которые нужно решать, резко уменьшается. Поведение системы становится когерентным: по определенным законам меняется во времени лишь небольшое число переменных – параметры порядка, другие же, почти мгновенно, приспосабливаются, просто следуя за ними. Другой важный принцип синергетики – принцип конкуренции мод. Вообще говоря, любое движение в пространстве можно представить в виде суперпозиции большого числа так называемых нормальных мод или волн с различными длинами или частотами, располагающихся вдоль координат. Разложение движения на моды представляет собой главное положение известного метода Фурье-анализа, который основан на том, что любую функцию можно разложить в ряд Фурье по другой системе функций. Оказалось, что в нелинейных системах некоторые моды усиливаются немного быстрее других, в то время как эти другие либо растут медленно, либо вообще сразу затухают. Важно то, что среди растущих, где самые быстрые оказываются наиболее долгоживущими и подчиняют себе все остальные, в том числе даже не устойчивые, но медленно растущие. Возникает упорядоченное поведение и формируется когерентные структуры из небольшого числа мод. Одним из ключевых понятий синергетики является понятие термодинамической ветви и диссипативной структуры. где N − число молекул в объеме. В случае релаксационных колебаний графики зависимости от времени переменно, потребляющей ресурс, и самого ресурса различаются – для переменной характерны острые пики, разделенные длительными интервалами низких значений, в то время как ресурс в эти длительные интервалы медленно растут, а в моменты пиков резко убывают. Конечно, в технике и природе встречаются совершенно разные колебательные режимы – от практически гармонических, синусоидальных до релаксационных, импульсных. Если в модели имеется два главных фактора самоорганизации – нелинейность и потоки через границу системы, то она может воспроизвести диссипативные структуры. Нелинейность обеспечивает действие принципов подчинения и конкуренции мод, а потоки энергии и вещества через границу создают отклонения от равновесия. Подобные модели называют вихреразрешающими. Важный результат самоорганизации, обнаруженный в расчетах по таким моделям, перестройка всей системы течений и принципиальная нестационарность новой структуры. Самым главным результатом действия вихрей можно считать интенсификацию струйных течений, происходящую из-за так называемого эффекта отрицательной вязкости, когда энергия крупномасштабного течения растет за счет энергии флуктуаций [23]. Очевидно, это частное проявление принципа конкуренции мод. Обозначая дискретные совокупности условий, которые необходимо соблюдать для перевода организации (системы) из одного параметры будут эволюционировать. Вообще говоря, самоорганизация иногда выражается в довольно сложной форме, приводя даже к стохастизации или хаотизации процессов. Состояние агрегированной системы в каждый момент времени обычно изображают точкой в фазовом пространстве – условном многомерном пространстве, по осям которого отложены значения характеризующих систему параметров, изменение ее состояния соответствует движению этой точки вдоль некоторой фазовой траектории [21]. Важно, что для большинства систем существуют аттракторы – выделенные фигуры в фазовом пространстве, к которым стремятся фазовые траектории. Если в агрегативной системе удается выделить, всего два параметра порядка (скажем количество вещества и температуры; кинетическая и потенциальная энергии; численность двух видов микроорганизмов и т.п.), то в таком двухмерном мире единственной формой самоорганизации, уводящий систему с термодинамической ветви, является переход в автоколебательный режим с простейшим циклом. Большая часть колебательных режимов в сильнонеравновесных системах – осцилляции Эль-Ниньо [22] имеют четко выраженный релаксационный характер. Это означает, что в какой-то момент времени система становится неустойчивой, и одна или несколько характеристик начинают быстро расти, черпая ресурсы для такого роста и других переменных. Например, если в результате реакции в химической системе содержание одного вещества растет, количество других реагирующих веществ должно убывать. Естественно, в таких условиях рост переменных ограничивается обеспечивающими этот рост запасами. Когда ресурсы заканчиваются, рост обрывается, переменная, вследствие диссипации принимает прежнее значение и начинается новая фаза накопления ресурсов. где N − число молекул в объеме. Уравнения модели некоторой системы могут давать при определенных значениях ее параметров стационарное устойчивое решение. В этом случае считают, что система находится на термодинамической ветви [20]. Однако, при превышении каких-то параметров критического значения, такое состояние может смениться совсем другим, при котором под влиянием тех же самых внешних воздействий образуются новые структуры в пространстве и времени, они могут снова привести систему на термодинамическую ветвь. Иными словами говоря, система со временем достигает стационарного состояния, как бы притягивается к нему. Точка на фазовой траектории, отвечающая этому режиму называют аттрактором (притягивать). Подобные процессы возможны только в открытых, диссипативных системах, поэтому сами эти структуры называют диссипативными. Сейчас понятие диссипативных структур расширилось: кроме стационарных говорят о нестационарных диссипативных структурах – спиральных автоволнах, волнах в транспортных потоках и других. Систему считают агрегативной, если ее можно описать сосредоточенными параметрами, едиными для всей системы (скажем, средней кинетической энергией или количеством вещества в реакторе). Такая система может находиться в термодинамической ветви, и тогда ее параметры остаются неизменными, а может произойти самоорганизация во времени, и эти 19 параметры будут эволюционировать. Вообще говоря, самоорганизация иногда выражается в довольно сложной форме, приводя даже к стохастизации или хаотизации процессов. Состояние агрегированной системы в каждый момент времени обычно изображают точкой в фазовом пространстве – условном многомерном пространстве, по осям которого отложены значения характеризующих систему параметров, изменение ее состояния соответствует движению этой точки вдоль некоторой фазовой траектории [21]. Важно, что для большинства систем существуют аттракторы – выделенные фигуры в фазовом пространстве, к которым стремятся фазовые траектории. Если в агрегативной системе удается выделить, всего два параметра порядка (скажем количество вещества и температуры; кинетическая и потенциальная энергии; численность двух видов микроорганизмов и т.п.), то в таком двухмерном мире единственной формой самоорганизации, уводящий систему с термодинамической ветви, является переход в автоколебательный режим с простейшим циклом. Большая часть колебательных режимов в сильнонеравновесных системах – осцилляции Эль-Ниньо [22] имеют четко выраженный релаксационный характер. Это означает, что в какой-то момент времени система становится неустойчивой, и одна или несколько характеристик начинают быстро расти, черпая ресурсы для такого роста и других переменных. Например, если в результате реакции в химической системе содержание одного вещества растет, количество других реагирующих веществ должно убывать. Естественно, в таких условиях рост переменных ограничивается обеспечивающими этот рост запасами. Когда ресурсы заканчиваются, рост обрывается, переменная, вследствие диссипации принимает прежнее значение и начинается новая фаза накопления ресурсов. где N − число молекул в объеме. В случае релаксационных колебаний графики зависимости от времени переменно, потребляющей ресурс, и самого ресурса различаются – для переменной характерны острые пики, разделенные длительными интервалами низких значений, в то время как ресурс в эти длительные интервалы медленно растут, а в моменты пиков резко убывают. Конечно, в технике и природе встречаются совершенно разные колебательные режимы – от практически гармонических, синусоидальных до релаксационных, импульсных. Если в модели имеется два главных фактора самоорганизации – нелинейность и потоки через границу системы, то она может воспроизвести диссипативные структуры. Нелинейность обеспечивает действие принципов подчинения и конкуренции мод, а потоки энергии и вещества через границу создают отклонения от равновесия. Подобные модели называют вихреразрешающими. Важный результат самоорганизации, обнаруженный в расчетах по таким моделям, перестройка всей системы течений и принципиальная нестационарность новой структуры. Самым главным результатом действия вихрей можно считать интенсификацию струйных течений, происходящую из-за так называемого фф б Самым главным результатом действия вихрей можно считать интенсификацию струйных течений, происходящую из-за так называемого эффекта отрицательной вязкости, когда энергия крупномасштабного течения растет за счет энергии флуктуаций [23]. Очевидно, это частное проявление принципа конкуренции мод. Обозначая дискретные совокупности условий, которые необходимо соблюдать для перевода организации (системы) из одного 20 состояния в другое, можно пользоваться термином «код». Для системы, состоящей из атомов и молекул, кодом могут быть наборы частот (мод) или квантовых чисел, отвечающих заданному переходу. Следует подчеркнуть, что во всех случаях такие взаимодействия, которые, не нарушая организацию, либо развивая ее структуру по определенному плану, переводят ее в возбужденное состояние, из которого она возвращается в исходное. Останавливаясь на вопросе устойчивости той или иной системы или состояния, отметим следующее. Переменные системы в общем случае являются функциями времени: 1 1 2 ( , ..... ) i n f t x x x    (1.20) (1.20) Если функция if непрерывна при t   (можно предположить 0  ), то устойчивость определяется по Ляпунову [23] для всего интервала: Если функция if непрерывна при t   (можно предположить 0  ), то устойчивость определяется по Ляпунову [23] для всего интервала: 1......., n x x   1......., n x x   Дискретный набор устойчивых соотношений обуславливает устойчивость организации, в ином случае ничтожно малые влияния внешней среды приводили бы к хаотизации системы. Для диссипативных дискретных систем варьируя путь изменения состояния, мы должны найти оптимальную траекторию «движение» системы, отвечающую наиболее выгодным формам связи. Выгодны те связи (коды), которые сочетают ряд признаков. Одним из таких признаков является процесс взаимодействия связанных осцилляторов. где N − число молекул в объеме. Если частота одного осциллятора приближается к частоте другого «сверху» (со стороны более высоких частот), то в результате второй осциллятор приобретает более высокую частоту; при приближении частоты «снизу» повышается частота первого осциллятора. То, что наблюдается в простейших системах, воспроизводится в главных чертах и в высших системах, так как в системах обоих классов общим является связь (код): система – система (осцилляторы) или система – среда. Для динамических диссипативных систем, постоянный кодовый обмен составляет необходимое условие устойчивости. Исключение этой связи со среды или с других систем означает и прекращение их организующих функций, и появление действия иных факторов, способных хаотизировать организацию. Следовательно, организованная система в обстановке постоянных изменений устойчива не только потому, что она способна отражать действия изменений, но и потому, что эти изменения существуют. Выводы к разделу 1 д р д у Химические системы – это коллектив частиц, между которыми действуют силы. В равновесных системах условия устойчивости выражены минимальными значениями соответствующих термодинамических потенциалов или максимальным значением энтропии, если рассматриваемая система изолирована. Для газа, эти условия означают и максимальную степень хаотизации. В жидкостях эффекты слабых межмолекулярных взаимодействий обуславливают 21 появление признаков структурированности. В твердых кристаллических телах степень упорядоченности достигает максимума в структурах имеющих минимальную потенциальную энергию. Применение методов статистической термодинамики к изучению равновесных состояний позволяет вычислять константы равновесия на основании знания сумм по состояниям. В неравновесных динамических системах диссипативного типа устойчивость связана с существованием стационарных состояний; если отклонение от равновесия невелико, то критерием устойчивости может служить производство энтропии, достигающее в стационарном состоянии минимального значения. Если система сильно отклонилась от равновесия, трудно указать критерии устойчивости; в отдельных случаях система способна «вращаться» вокруг стационарного состояния, периодически изменяясь. При этом могут возникать как временная, так и пространственная упорядоченности в исходно однородной системе. По мере усложнения диссипативных систем и перехода к предбиологическим и биологическим, энергетические критерии устойчивости утрачивают свое значение в том смысле, что потоки энергии и массы все в большей степени контролируются кодовыми механизмами. В неравновесных динамических системах диссипативного типа устойчивость связана с существованием стационарных состояний; если отклонение от равновесия невелико, то критерием устойчивости может служить производство энтропии, достигающее в стационарном состоянии минимального значения. Если система сильно отклонилась от равновесия, трудно указать критерии устойчивости; в отдельных случаях система способна «вращаться» вокруг стационарного состояния, периодически изменяясь. При этом могут возникать как временная, так и пространственная упорядоченности в исходно однородной системе. По мере усложнения диссипативных систем и перехода к предбиологическим и биологическим, энергетические критерии устойчивости утрачивают свое значение в том смысле, что потоки энергии и массы все в большей степени контролируются кодовыми механизмами. Кодовые механизмы, действующие в биологических системах, существуют за счет потоков энергии, то есть разностей химических потенциалов, однако эффективность действия кодовых сигналов по мере эволюции все в меньшей степени зависит от их энергетического эквивалента. Кодовые сигналы обладают двумя важнейшими свойствами: они способны повышать уровни организации системы и, вызывать распространение состояний и могут служить средством неэнергетической связи между системами. Связи такого типа открывают новые и практически безграничные возможности для стабилизации коллективов систем. 2 НЕКОТОРЫЕ НАУЧНЫЕ ОСНОВЫ ТЕХНОЛОГИЧЕСКИХ ПРОЦЕССОВ ХИМИЧЕСКОЙ И ПЕРЕРАБАТЫВАЮЩЕЙ ПРОМЫШЛЕННОСТИ Для указанных, а также ряда других отраслей промышленности характерного, что производится переработка материала, который называют исходным, в некоторое новое состояние со свойствами отличными от исходного, которое называют продуктом. Обработка исходного материала, как правило, производится последовательно с использованием на каждой стадии аппаратурно-процессорной единицы [24]. Аппаратурно-процессорная единица представляет собой определенный аппаратурный элемент с протекающим в нем процессом, причем осуществляемый процесс рассматривается во всем его многообразии. Указанный элемент рассматривается как система. Ряд аппаратурно- процессорных единиц, составляющих технологический процесс (схему), характеризуется результатом воздействия на исходный материал различных 22 условий, осуществляемых в процессорных единицах. При этом, в одной процессорной единице (химическом реакторе) достигается необходимая форма материала и его состояния, отвечающие форме и состоянию продуктов процесса, (эта процессорная единица является основной, а все относительные вспомогательные, обеспечивающие ее функционирование). «Теория химических реакторов и аппаратов является отраслью химической технологии, которая изучает вопросы промышленного оформления химических реакций, но не рассматривает процессы, протекающие в энергетических установках. Ее цель – разработка методов расчета и проектирования реакторов (аппаратов). Теория химических реакторов, вероятно, более чем какая-либо дисциплина дает право считать химическую технологию отдельной отраслью техники». Для каждой аппаратурно-процессорной единицы должно быть установлено определенное количество и тип величин необходимых и достаточных для описания состояний вещества в определенной точке (микрообласти) элемента процесса (чаще всего – на входе или выходе). Предварительно следует отметить, что между отдельными величинами может существовать внутренняя связь, недопускающая их выбора независимо друг от друга. Отсюда возникает требование – добиваться максимальной информации с минимальным числом величин. Таким образом, отбирается базовая система, состоящая из наиболее характерных (с производственно-технической точки зрения) свойств веществ. Эта проблема относится к термодинамике, основные законы и методы которой описаны в разделе I. По Гиббсу [25] для равновесных систем и систем, стремящихся к равновесию число степеней свободы системы (число независимо изменяемых параметров), описывается системой уравнений: (2.1) 2 G F k     , (2.1) где θ − число фаз (число уравнений), k − число компонентов. Согласно вышеприведенного уравнения систему можно считать определенной, если известны значения k+2−θ − переменных. Правило фаз Гиббса сформулировано с помощью интенсивных величин состояния и не содержат никаких данных о массе фаз. Из этого следует, что кроме k+2 интенсивных величин, характеризующих состояние, для каждой фазы может быть выбрана еще одна экстенсивная величина. В этом случае число степеней свободы выражается зависимостью: (2.2) E G F F    . (2.2) E G F F    . 3) теплоты или энтальпии; 2 НЕКОТОРЫЕ НАУЧНЫЕ ОСНОВЫ ТЕХНОЛОГИЧЕСКИХ ПРОЦЕССОВ ХИМИЧЕСКОЙ И ПЕРЕРАБАТЫВАЮЩЕЙ ПРОМЫШЛЕННОСТИ В общем случае исходя из термодинамики для характеристики любой системы достаточно четырех потоков: 1) массы; 2) компонента; 3) теплоты или энтальпии; 23 4) импульса. 4) импульса. 4) импульса. 4) импульса. Обобщенное количество, таким образом, можно заменить рядом конкретных величин. Поток массы, протекающий в единицу времени dm/dη или m/η кг/ч или кг/с. В определении потока массы химические свойства во внимание не принимаются. Поток компонента (поток вещества) является частным случаем потока массы. Термин относится только к массе выбранного i-того компонента, выражаемого в молях – моль/ч или кмоль/ч; моль/с или кмоль/с. Такой поток выражается в зависимости i dN d или N  . Поток теплоты иди энтальпии соответствует количеству переходящей энтальпии в единицу времени, отнесенное к единице массы и стандартному состоянию: ( ) p d C T dh d d    или p C h    − кДж/(кг·ч); вт/кг. Поток количества движения (импульса) характеризуется значением переходящего в единицу времени импульса ( ) d mW d или mW  − кг·м/ч2 ил кг·м/с. Поток количества движения (импульса) характеризуется значением переходящего в единицу времени импульса ( ) d mW d или mW  − кг·м/ч2 ил кг·м/с. Поток означает пространственное перемещение какой-либо величины, то есть множество частиц движется под действием сил из одного место в другое. Под этим надо понимать, что множество частиц с однозначно обобщенной плотностью «Г» передвигается по объему «V» в другое место пространства. Следовательно, элементарный поток выражается зависимостью: ( ) d ГV j d  . (2.3) (2.3) В общем виде, плотность проводящего потока пропорциональна обобщенной плотности «Г». (2.4) j grad Г    . (2.4) Согласно j при конвективном потоке плотность диффузионного потока пропорциональна градиенту концентрации: р р р у р іj D grad C    . (2.5) р р р у р іj D grad C    . (2.5) (2.5) іj D grad C    . (2.5) Плотность теплового потока: Плотность теплового потока: Плотность теплового потока: (2.6) ( ) q p j a grad C T      . (2.6) Аналогично для потока импульса: Аналогично для потока импульса: ( ) р p j grad grad           . (2.7) Аналогично для потока импульса: ( ) р p j grad grad           . (2.7) (2.7) ( ) р p j grad grad           . (2.7) В случае высокоскоростных потоков возникают турбулентные движения 24 частиц внутри потока фазы. В турбулентных потоках частицы движущейся субстанции движутся, по очень сложным траекториям. 4) импульса. Точное описание такого потока весьма затруднительно. Поэтому, в вышеприведенные уравнения вводят поправку к коэффициентам , , D a . Эта поправка называется коэффициентом проводимости турбулентного потока – «Н». Тогда, вышеприведенные уравнения примут следующую форму: ( ) ( ) ( ) і эф q p p эф р эф j D H grad C D grad j C a Н gradT C a gradT j Н grad grad                              . (2.8) (2.8) где эф D D H   − коэффициент турбулентной диффузии; эф a a H   − эффективный коэффициент температуропроводности; эф H     − эффективная кинематическая вязкость. где эф D D H   − коэффициент турбулентной диффузии; эф a a H   − эффективный коэффициент температуропроводности; эф H     − эффективная кинематическая вязкость. В случае турбулентного потока появляется, как уже было сказано, нерегулярный вихревой поток макроскопических неустановившихся скоплений частиц. Нерегулярное движение этих молекул (субстанций) подобно описываемому в кинетической теории газов движению отдельных молекул, а это значит, что частицы движутся вдоль характерного пути пробега 'l называемого путем смешения. Путь смешения играет в этом случае туже роль, что средняя длина свободного пробега молекул газа. Второй характерной для турбулентного потока величиной является среднее колебание скорости  u . В соответствии с этим значение «Н» будет представляться произведением двух величин: ' H l u  . (2.9) ' H l u  . (2.9) Все рассмотренные выше системы и потоки были свободны от источников или стоков, то есть были неразрывными. Однако, в определенных точках пространства могут существовать источники или стоки, вносящие изменения в систему. Так, в случае потока компонентов могут появиться новые компоненты, образующиеся при химической реакции или исчезнуть израсходованные компоненты. Могут произойти изменения в некоторых параметрах. р р В этом случае, общее уравнение потока должно содержать несколько членов: конвективный + основной + переходящий + источник = местные изменения. Приведем уравнение сначала в общей форм, а затем для потоков массы, компонентов, теплоты и импульса: 25         c для общего случая для потока массы Γ div× Г×v -div× δ×grad×Г +ω×ε×ΔГ+G=- τ ρ div× ρ×v -div× D ×grad×ρ +ω×β×Δρ+G=- τ         c для общего случая для потока массы Γ div× Г×v -div× δ×grad×Г +ω×ε×ΔГ+G=- τ ρ div× ρ×v -div× D ×grad×ρ +ω×β×Δρ+G=- τ         c для общего случая для потока массы Γ div× Г×v -div× δ×grad×Г +ω×ε×ΔГ+G=- τ ρ div× ρ×v -div× D ×grad×ρ +ω×β×Δρ+G=- τ     i i i i i i i p для потока компонентов c div× с ×v -di для потока тепло v× D × ты grad×c +ω×β ×Δc +v r=- τ div× ρc Tv -div× λ           p i для потока импул (ρc T) ×gradT +ω×αΔΤ+ν rΔΗ= τ (ρv) Div× ρv×v -Div× η×gradv +ω×γΔv+gradp= ьса τ (2.10) (2.10) Сокращение Diν обозначает не только дивергенцию, так как величины, стоящие внутри фигурных скобок, являются тензорами (второго порядка). Например, конвективная плотность потока импульса представляет собой произведение векторов v  и v . Таким образом, три составляющие (по трем осям) первого вектора должны быть рядами умножены на три составляющие второго вектора. где эф D D H   − коэффициент турбулентной диффузии; эф a a H   − эффективный коэффициент температуропроводности; эф H     − эффективная кинематическая вязкость. Следовательно, получим 3·3 = 9 составляющих. Теперь запишем это произведение: x x y x z x x y y y z y x z y z z z v v                        . (2.11) (2.11) Подобным же образом выражается и второе слагаемое четвертого уравнения системы, для которого сокращение Grad также начинается с большой буквы G, потому что оно обозначает не вектор градиента скалярного пространства, а тензор градиента векторного пространства (пространство скоростей). Решить это уравнение очень трудно, и общего пригодного для каждого случая решения дать нельзя. Однако, в инженерной практике обычно нет необходимости принимать во внимание все члены этого уравнения. В большинстве случаев достаточно только двух из них. Инженер должен учитывать это и знать (или уметь оценивать), какие члены равны нулю или ничтожно малы. Как видно из вышеприведенного в разделе структура математической модели любого процесса в аппаратурно-процессорной единице зависит от характера перемещения жидкости, газа, твердого тела или их смесей, и определяется прежде всего гидродинамическими параметрами. Отмеченное проявляется в характере распределения времени пребывания частиц потока в рассматриваемой системе (объеме процессорной единицы и ее конструкции). Этот характер распределения подвержен статистическим законам и находится по виду сигнала, проходящего через систему. В качестве такого 26 сигнала используется подача вещества (индикатора – абсолютно инертного и индеферентного вещества по отношению к частицам системы) на вход в систему в виде ступенчатого, импульсного или частотного возмущения. Функция распределения времени пребывания характеризует долю индикатора в выходном потоке, находившуюся в системе время, меньше «η». Функция распределения при импульсном возмущении (С-кривая) отвечает уравнению:  k o C C d     . (2.12) (2.12) Среднее время пребывания  определяется из соотношения время пребывания  определяется из соотношения   o o C d c c C d                , (2.13) (2.13) o где c − концентрация индикатора. Безразмерное время пребывания:    . (2.14) Безразмерное время пребывания: Безразмерное время пребывания:    . (2.14) (2.14) Поскольку величина C(η)dη характеризует долю индикатора в выходящем потоке с возрастанием к η + dη, то, умножив эту величину, на концентрацию индикатора СА остающуюся в элементе потока возраста η + dη, можно определить среднюю концентрацию СА индикатора, попадающего в реальную аппаратурно-процессорную единицу:  0 A A C C C d        . (2.15) (2.15) Связь между функциями распределения при ступенчатом и импульсном вводе индикатора дается соотношениями: ; o dF F Cd C d       . (2.16) (2.16) Указанные зависимости применимы только к линейным системам, для которых между входной и выходной величинами (сигналами) имеется прямая пропорциональность. Процессы неудовлетворяющие этим условиям являются нелинейными. Поэтому нелинейные системы необходимо анализировать в общей совокупности и их общее поведение нельзя предсказать зная точно каждую составляющую процесса. Поскольку, в диссипативных системах возможна реализация термодинамической ветви (то есть притягивающих 27 режимов), то для этих случаев для оценки гидродинамических режимов потоков через процессорную единицу допустимо использовать указанные функции распределения [26]. Таким образом, в зависимости от вида функции распределение все многообразие математических моделей потоков, возникающих в различных конструкциях процессорных единиц, может быть представлена в виде некоторых типовых моделей, которые представлены на рисунке 2.1. Не все реальные системы и структуры потоков можно описать при помощи представленных на рисунке 1 моделей. В частности, это относится к системам, имеющим байпасные и циркуляционные потоки, конструкциям аппаратов, не исключающих застойные зоны, несколько процессорных единиц соединенных последовательно или параллельно для осуществления в них одной и той же реакции и т.п. В указанных случаях используются комбинированные модели структуры потоков, которые предполагают, что процессорная единица состоит из отдельных зон, соединенных последовательно или параллельно, в которых наблюдаются различные структуры потоков. 28 28 29 Рисунок 2.1. Типовые модели структуры потоков в аппаратах химической те Типовые модели Схема потока Математическое описание ступенч Модель идеального вытеснения dc dc w d dx  Модель идеального смешения . время пребывания  определяется из соотношения ( ) вх dc Vc c с d V   Диффузионные модели вытеснения (однопараметрическая) 2 2 L dc dc d c w D d dx dx   Диффузионные модели вытеснения (двухпараметрическая) R dc dc D d dc R d dx R dR dR          Ячеечная модель 1 1 ( ) n i i dc c c m d      тах химической те описание ступенч c x )с  2 2 L d c D dx d dc R R dR       1 ) ic  тах хими )с  2 2 L d c D dx d dc R R dR     1 ) ic  тах химической )с  2 2 L d c D dx d dc R R dR       1 ) ic  ах хи 2 L dx d R d   ) ic  На рисунке 2.2 представлены выходные кривые (функции распределения) простейших комбинированных моделей при импульсном вводе индикатора. На рисунке 2.2 представлены выходные кривые (функции распределения) простейших комбинированных моделей при импульсном вводе индикатора. V, V1, V2 − объемные скорости Vr – объем реактора Рисунок 2.2. Импульсные выходные кривые V, V1, V2 − объемные скорости Vr – объем реактора Рисунок 2.2. Импульсные выходные кривые Именно по типу функций отклика определяется конструктивная особенность аппарата (реактора) или машины. Соответственно этим функциям проявляется определяющая схема потоков и математическая модель перемещения потока. Например, известные и широкоприменяемые аппараты переиодического действия и проточного типа с механическим перемешиванием мешалки различного типа, диффузоры – конфузоры, вращающиеся барабаны, устройства со встречными потоками и др.). Непрерывные ступенчатые реакторы (колонны), в которых на отдельных ступенях (ступенях равновесия) 30 осуществляется при противотоке реакционный обмен между движущимися фазами; непрерывные реакторы шахтного типа противоток между твердым или жидким и газом; непрерывные реакторы с неподвижной твердой фазой (катализатор) и проходящим через слой реакционным газом и др. Очевидно, из выше приведенного следует, что математическая модель передвижения среды (модель потока) будет влиять и на кинетику процессов превращения вещества и передачи энергии. Это подтверждается практикой и обоснованно в ряде фундаментальных научных исследований в этой области [27]. Таким образом, протекание химического процесса, учитывающее явление переноса вещества и теплоты, а также крупномасштабные потоки вещества и теплоты в объеме аппарата, относится к понятию макрокинетики процесса. Подводя итоги изложенному, приходим к следующему важнейшему выводу. время пребывания  определяется из соотношения Для расчета химического реактора необходимо знать: 2) функцию отклика системы, или кривую распределения времени пребывания; 2) функцию отклика системы, или кривую распределения времени пребывания; 3) свойства системы – принадлежность к макро- или микросистеме; 4) данные о смешении на ранней или поздней стадии. Классическая кинетика изучает химические превращения на микроуровне, и соответственно рассматриваемые системы являются микросистемами. Все, что было сказано ранее о расчете химических превращений различных моделей реакторов, относится к микросистемам. Рассмотрим теперь расчет химических реакторов на макроуровне для двух крайних моделей: идеального вытеснения и идеального смешения. Модель идеального вытеснения (трубчатые реакторы) Если в математической модели идеального вытеснения учесть источник изменения концентраций за счет химической реакции r , то модель реактора идеального вытеснения можно записать в виде: r с с x          . (2.17) (2.17) Для установившегося состояния 0 с    , и уравнение принимает вид: n r c kc x      . (2.18) Для установившегося состояния 0 с    , и уравнение принимает вид: n r c kc x      . (2.18) (2.18) Учитывая, что в установившемся состоянии при движении потока через трубу его линейная скорость  может быть выражена как: Учитывая, что в установившемся состоянии при движении потока через трубу его линейная скорость  может быть выражена как: x    . (2.19) x    . x    . (2.19) И производя соответствующую замену, окончательно получим: 31 n c kc     . n c kc     . Таким образом, приходим к выводу о том, что модель реактора идеального вытеснения соответствует уравнению формальной кинетики химической реакции. р Рассматривая реакцию общего типа А→ Продукт, протекающую в реакторе, описанном моделью идеального вытеснения, получим: n А А c kc      . Разделяя переменные и интегрируя, получим: 1 1 ( 1) A n n o Ao c A n A A A c o dc k d c c n K c             , (2.20) (2.20) где cAo, cA − соответственно начальная и конечная концентрации реагента А. где cAo, cA − соответственно начальная и конечная концентрации реагента А. Для реакции первого порядка n = 1 и соответственно будем иметь: ln o k l A A A A c c c k c     . Вводная степень превращения   o o A A A A c c x c   , получим уравнение   1 1 n o n А A А x kc x       . (2. Вводная степень превращения   o A A A A c c x c   , получим уравнение   1 1 n o n А A А x kc x       . Модель идеального вытеснения (трубчатые реакторы) (2.21) (2.21) После интегрирования получим время процесса η: После интегрирования получим время процесса η: − для реакции первого порядка (n = 1) − для реакции первого порядка (n = 1) ln(1 ) A x k    , (2.22) дка (n = 1) ln(1 ) A x k    , (2.22) − для реакции первого порядка (n = 1) ln(1 ) A x k    , (2.22) − для реакции других порядков 1 n      1 1 1 1 1 n o A n A c x n k            . (2.23) (2.23) Учитывая, что r c V V  , можно записать: Учитывая, что r c V V  , можно записать: Учитывая, что r c V V  , можно записать: 1 1 ( ) (1 ) A A A A n o A A o o c c x x k r A A n n A c r r A A A c c o o dc V dc dx dx c c V W W x k kc x           . (2.24) (2.24) Если время завершения реакции соответствует времени пребывания в реакторе, как это следует из модели вытеснения, то полученное уравнение модели реактора идеального вытеснения полностью соответствует интегралу 32 уравнений классической кинетики. Модели идеального вытеснения соответствуют проточные трубчатые реакторы при 100 T L d  , где dT − диаметр трубы при турбулентном режиме течения, так как развитая турбулентность создает выравненный профиль скоростей и концентраций. Реакторы периодического действия При периодическом проведении процесса исходные компоненты одновременно загружаются в реактор и находятся в нем заданное время, пока не будет достигнута необходимая конечная концентрация смеси. При этом реагенты перемешиваются мешалкой, нагреваются или охлаждаются, после чего продукты выгружаются и реактор подготавливается к следующей операции. В каждый момент концентрации компонентов одинаковы во всем объеме реакционной массы, но непрерывно меняются со временем до достижения заданной конечной концентрации или заданной степени превращения. Таким образом, в реакторе периодического действия, в каждый момент концентрация реакционной массы отличается от предыдущей, и в этом смысле для данного реактора пригодна модель идеального вытеснения вещества во времени. В этом случае идеализация реактора заключается в том, что допускается полная однородность концентрации в любом сечении его объема. Обозначим объем реакционной массы (объем реактора) через r V и число молей реагента A через NA. Процесс протекает во времени. Тогда по материальному балансу для исходного компонента «A» найдем: д д A r r dN V d     . (2.25) (2.25) Поскольку ( ) A A r dN d с V   , у ( ) A A r dN d с V   , Уравнение можно представить в виде Уравнение можно представить в виде ( ) A r r r d с V V d    . Отсюда A r dс d     , Уравнение можно представить в виде ( ) A r r r d с V V d    . Отсюда A r dс d     , Отсюда Отсюда или   1 1 n o n n А А А A А c x kc kc x            . (2.26)   1 1 n o n n А А А A А c x kc kc x            . (2.26) (2.26) Время пребывания в реакторе (в с) составляет: Время пребывания в реакторе (в с) составляет: Время пребывания в реакторе (в с) составляет: Время пребывания в реакторе (в с) составляет: 33 1 1 (1 ) A A n o Ao c x A A n n A A A c o dc dx c k kc x         . (2.27) (2.27) Таким образом, решения уравнений моделей реакторов периодического действия и идеального вытеснения относительно времени пребывания полностью совпадают. Однако следует иметь в виду, что модель периодически действующего реактора соответствует модели идеального вытеснения по координате времени, а не длины. Модель идеального смешения (проточные с мешалкой) С учетом химической реакции уравнение модели идеального смешения имеет вид С учетом химической реакции уравнение модели идеального смешения имеет вид     o c c r вх A A r r V V c с c с V V       . (2.28) (2.28) Отсюда для реагента А время пребывания в реакторе Отсюда для реагента А время пребывания в реакторе 1 (1 ) n o o o A A A A A A x r n n c r A A c c c c c V V kc k x            . (2.29) (2.29) Для реакции первого порядка уравнение принимает вид: Для реакции первого порядка уравнение принимает вид: 1 A A x k x  . (2.30) (2.30) Это соотношение может быть также из уравнения для передаточной функции инерционного звена Это соотношение может быть также из уравнения для передаточной функции инерционного звена , (0) , (0) 1 1 А вых А вх с с kT  . (2.31) (2.31) Подставляя в последнее уравнение степень превращения «х», выраженную как Подставляя в последнее уравнение степень превращения «х», выраженную как как как , , , А вх А вых А А вх с с х с   , и учитывая, что постоянная времени r c V T V    , приходим к соотношению 1 A A x kT k x    . (2.32 и учитывая, что постоянная времени r c V T V    , приходим к соотношению 1 A A x kT k x    . (2. 1 A A x kT k x    . (2.32) (2.32) Из последнего соотношения и уравнения следует, что величина kη может служить мерой эффективности процесса. 34 Диффузионная модель (трубчатые проточные реакторы с учетом смешения) для малых 35 значений L D L  , соотношение между реальным реактором ( , , r r r V L ) и реактором (V L ) значений L D L  , соотношение между реальным реактором ( , , r r r V L ) и реактором идеального вытеснения ( , , rВ rВ rВ V L  ) приводит к следующим соотношениям: значений L D L  , соотношение между реальным реактором ( , , r r r V L ) и реактором значений L D L  , соотношение между реальным реактором ( , , r r r V L ) и реактором ого вытеснения ( , , rВ rВ rВ V L  ) приводит к следующим соотношениям: 2 1 1 r r r L rВ rВ rВ L V DL kD k L V mL         , (2.37) (2.37) при одинаковых размерах 2 1 ( ) r В A L A c D k c L    . (2.38) при одинаковых размерах при одинаковых размерах 2 1 ( ) r В A L A c D k c L    . (2.38) (2.38) Графики зависимости отношения объемов реального реактора и реактора идеального вытеснения r rВ V V       от доли непревращенного продукта при различных значениях параметра L D L  для реакций первого и второго порядка представлены на рисунке 2.3. Оценку параметра L D L  для реакции дробного порядка можно получить интерполяцией данных графиков. Величину параметра L D L  , найденную по графику, представленному на рисунке 2.3, следует умножить на безразмерный комплекс T d L . Диффузионная модель (трубчатые проточные реакторы с учетом смешения) Диффузионная модель (трубчатые проточные реакторы с учетом шени ) Диффузионная модель (трубчатые проточные реакторы с учетом смешения) ) Для трубчатого проточного реактора получим: Для трубчатого проточного реактора получим: 2 2 ( ) R L r c c c D c D R x R R R x                         . (2.33) (2.33) Рисунок 2.3. Зависимость отношения объема реального реактора к объему реактора идеального вытеснения от доли непревращенного продукта для реакций первого и второго порядка Рисунок 2.3. Зависимость отношения объема реального реактора к объему реактора идеального вытеснения от доли непревращенного продукта для реакций первого и второго порядка Рисунок 2.3. Зависимость отношения объема реального реактора к объему реактора идеального вытеснения от доли непревращенного продукта для реакций первого и второго порядка При наличии только продольного перемешивания для установившегося режима будем иметь в безразмерном виде: 2 2 0 n L A dc D d c k c dz L dz        , (2.34) (2.34) Или с учетом степени превращения компонента A: 2 Или с учетом степени превращения компонента A: 2 1 2 (1 ) 0 n n A L A A A dx D d x k c x dz L dz          . (2.35) (2.35) Для реакции первого порядка решением уравнения является:   2 2 1 4 exp 2 1 (1 ) exp (1 ) exp 2 2 o L A A A L L L a D c x c a mL a mL a a D D                             , (2.36) где 1 4 L D a k L     . (2.36) При малых отклонениях от модели идеального вытеснения, т.е. Модель каскада реакторов идеального смешения В практике проведения химических реакций вследствие неполноты завершения процесса в одной ступени реактора идеального смешения и необходимости (по технологическим соображениям) обеспечить интенсивное перемешивание прибегают к использованию цепочки или каскада реакторов идеального смешения. Изменение концентраций в каскаде реакторов может быть представлено графически в следующем виде (рис. 2.4). Каждый реактор представляет одну ступень изменения концентраций, так как концентрации в каждом реакторе изменяются скачком. Соединяя вершины ступеней одной линией, получим кривую, аналогичную кривой реактора идеального вытеснения. С другой стороны, трубчатый реактор идеального вытеснения можно представить как каскад из большого числа проточных реакторов с мешалками, обладающий тем же самым суммарным объемом. 36 Рисунок 2.4. Схема каскада реакторов (а) и зависимость (б) концентрации «с» от числа ступеней «m» Рисунок 2.4. Схема каскада реакторов (а) и зависимость (б) концентрации «с» от числа ступеней «m» Рисунок 2.4. Схема каскада реакторов (а) и зависимость (б) концентрации «с» от числа ступеней «m» Ниже рассмотрены методы расчета каскада реакторов идеального смешения. Алгебраический метод расчета. Из уравнения для единичного реактора идеального смешения имеем: Аm А r С С      . (2.39) (2.39) Обозначим концентрацию реагента, выходящего из последнего аппарата, через CAm, где m − число реакторов. Тогда для уаскада реакторов равных объемов запишем: С С 1 Аm Аm m r С С      или 1 Аm m r Аm С С     Для реакции первого порядка   r Аm RС    и, следовательно: Для реакции первого порядка   r Аm RС    и, следовательно:   1 1 А A А А С RC С R С       Для реакции первого порядка   r Аm RС    и, следовательно:   1 1 Аm m Am Аm m Аm С RC С R С          1 1 Аm m Am Аm m Аm С RC С R С        0 1 1 1 А А С С R  . (2.40) откуда 1 1 Аm Аm m С С R   0 1 1 1 А А С С R  . (2.40)    0 1 2 2 1 2 1 1 1 А А А С С С R R R          откуда 1 1 Аm Аm m С С R   0 1 1 1 А А С С R  . 1 1 Аm Аm m С С R   0 1 1 1 А А С С R  . (2.40) (2.40)    0 1 2 2 1 2 1 1 1 А А А С С С R R R          Если время пребывания во всех реакторах одинаково, то Если время пребывания во всех реакторах одинаково, то С   0 1 А Аm m С С R   . (2.41) (2.41) Логарифмируя последнее уравнение, получим число реакторов: Логарифмируя последнее уравнение, получим число реакторов: 37   0 lg lg 1 m А А С С m R           . Алгебраический метод расчета. (2.42) (2.42) Для реакций высоких порядков выражение CAm через CA0 становится очень сложным, и поэтому применение алгебраического метода затруднительно. При полном объеме системы V, состоящей из m реакторов, время пребывания в системе η = V/Vc и среднее время пребывания для каждого реактора η/m. В этом случае степень превращения в каскаде реакторов может быть представлена в виде:   1 1 1 А m х R m        . (2.43) (2.43) По полученному уравнению построен график (рис. 2.5) Так как величина Rη прямо пропорциональна объему, то из представленных кривых следует, что для значений xA объем 1/Rη (отнесенный к одному реактору) уменьшается с увеличением числа реакторов и возрастанием степени превращения. Рисунок 2.5. Зависимость степени превращения «x» от величины «kη» для различного числа реакторов в каскаде Рисунок 2.5. Зависимость степени превращения «x» от величины «kη» для различного числа реакторов в каскаде Рисунок 2.5. Зависимость степени превращения «x» от величины «kη» для различного числа реакторов в каскаде На основании графика, представленного на рисунке 2.5, можно определить число реакторов и общий объем реакторной системы. Так, например, принимая входные и выходные концентрации для различного числа реакторов в системе одинаковыми, при x = 0,9 найдем значение kη для различного числа реакторов m: m kη 1 . . . . . . . . . 9 2 . . . . . . . . . 4,3 3 . . . . . . . . . 3,1 Полученные соотношения показывают, что при двух реакторах общий Полученные соотношения показывают, что при двух реакторах общий Полученные соотношения показывают, что при двух реакторах общий 38 объем можно уменьшить почти наполовину по сравнению с тем, который был при одном реакторе, а при трех реакторах – на треть. объем можно уменьшить почти наполовину по сравнению с тем, который был при одном реакторе, а при трех реакторах – на треть. Математические модели реакторов с учетом переноса тепла (2.51) / / o a E R T T х E RT o o k k e k e      . (2.51) (2.51) Данное уравнение не содержит температуры в качестве переменной. При прочих равных условиях константа скорости реакции является функцией только х , это позволяет сравнительно просто сопоставить различные реакции и модели реакторов, если параметры , , a E k T и o T или их комбинации выбраны соответствующим образом. Математические модели реакторов с учетом переноса тепла В этих аппаратах предусмотрен подвод или отвод тепла. Рассуждая аналогично, будем иметь ввиду: Политропические реакторы. В этих аппаратах предусмотрен подвод или отвод тепла. Рассуждая аналогично, будем иметь ввиду: у , у у − для реактора идеального смешения        − для реактора идеального смешения        c p k H c k H V k x V gc T T V c c K F T T       , (2.46) р д        c p k H c k H V k x V gc T T V c c K F T T       , (2.46) (2.46) − для реактора идеального вытеснения − для реактора идеального вытеснения         ' ' c p r V x c V x V gc dT f dl K F T T dl V dc K F T T dl            . (2.47) (2.47) При составлении математических моделей адиабатических реакторов обычно вводят понятие адиабатического изменения температуры, выражаемой отношением:    о а р с с с g     . (2.48) (2.48) где co, c − начальные и конечные концентрации реагентов.  о а р с с g    . (2.49) где co, c − начальные и конечные концентрации реагентов.  о а р с с g    . (2.49) ц р ц р  о а р с с g    . (2.49) (2.49) Примем, что при адиабатической реакции теплота реакции и теплоемкость смеси остаются практически постоянными. Тогда изменение температуры «T» для данной степени превращения х может быть выражено уравнением o aх  , (2.50) o aх  , (2.50 (2.50) где o  − начальная температура, де o  − начальная температура, a  − адиабатическая температура при полной степени превращения, «знак +» −для экзотермической реакции, «знак –» − для эндотермической реакции. знак –» − для эндотермической реакции. Используя уравнение Аррениуса, можно написать: / E R Используя уравнение Аррениуса, можно написать: / E R Используя уравнение Аррениуса, можно написать: / / o a E R T T х E RT o o k k e k e      . Математические модели реакторов с учетом переноса тепла В зависимости от тепловых и температурных режимов все реакторы делятся на изотермические адиабатические и политропические. Изотермические реакторы. Для сохранения постоянной температуры процесса в реакторах этого типа необходимо подводить или отводить тепло в соответствии с тепловым эффектом реакции. Однако изотермические реакторы сравнительно редко используются в крупномасштабных производствах: высокая стоимость оборудования для теплообмена делает процесс неэкономичным. Поэтому промышленные реакторы чаще проектируются как адиабатические или политропические. Адиабатический реактор. Реактор называют адиабатическим, если в рассматриваемой системе выделяемое в результате химической реакции тепло целиком идет на изменение энтальпии этой системы (теплообмен стремится к нулю). у ) Введем обозначения: r  − скорость реакции, кмоль·м−3·ч−1; r  − скорость реакции, кмоль·м−3·ч−1; c V − скорость реакционной массы, м3/ч; 3  − плотность, кг/м3; 2  − плотность, кг/м3; g − ускорение свободного падения, м/с2; p c − удельная теплоемкость при постоянном давлении, Дж·кг−1·К−1 (ккал·кг−1·оС−1);  − тепловой эффект реакции; знак плюс – для экзотермической реакции; знак минус – для эндотермической реакции, Дж/кмоль (ккал/кмоль); о  − тепловой эффект реакции; знак плюс – для экзотермической реакции; знак минус – для эндотермической реакции, Дж/кмоль (ккал/кмоль);  − температура реакционной массы, К(оС);  − температура реакционной массы, К(оС); х  − температура охлаждающего агента, К(оС); х  − температура охлаждающего агента, К(оС); 3 с – концентрация реагента, кмоль/м3; V  − коэффициент теплопередачи от реакционной массы к охлаждающему агенту, Вт·м-2·К−1 (ккал·м−2·С−1·ч−1); F − поверхность теплообмена, м2; ' 2 F − поверхность теплообмена, м2; ' F − поверхность теплообмена на единицу длины, м2/м ' F − поверхность теплообмена на единицу длины, м2/м; 2 ' F − поверхность теплообмена на единицу длины, м2/м; f − площадь поперечного сечения потока в реакторе, м2. F поверхность теплообмена на единицу длины, м /м; f − площадь поперечного сечения потока в реакторе, м2. f − площадь поперечного сечения потока в реакторе, м2. В зависимости от используемой модели справедливы следующие уравнения теплового баланса: − для реактора идеального смешения      c p k H c k H V gc T T V c c     , (2.44) − для реактора идеального смешения − для реактора идеального смешения      c p k H c k H V gc T T V c c     , (2.44) (2.44) − для реактора идеального вытеснения  (2.45)   c p r r V gc T dV     . (2.45) 39 Политропические реакторы. Псевдогомогенные модели Одномерная модель с поршневым потоком по подвижной фазе. 40 Материальный баланс по i-му компоненту: Материальный баланс по i-му компоненту: Материальный баланс по i-му компоненту:   , В i dc R с Т dl     1,....., i n  . (2.52) (2.52) Тепловой баланс:     4 p r CT R dT c T T dl d       . (2.53) (2.53) Начальные условия: 0 0 0; ; 1,....., ; i i l c i n T T     , 0 0 0; ; 1,....., ; i i l c i n T T     , где ω − линейная скорость подвижной фазы; где ω − линейная скорость подвижной фазы; В R и r  − соответственно скорости по веществу и химической реакции;  −коэффициент теплоотдачи;  В R и r  − соответственно скорости по веществу и химической реакци  −коэффициент теплоотдачи;  − тепловой эффект химической реакции;  − тепловой эффект химической реакции; cp − удельная теплоемкость; l − текущая длина реактора; TCT − температура стенки реактора; dR − диаметр реактора; n − число реагентов; c − вектор концентраций компонентов. c − вектор концентраций компонентов. c − вектор концентраций компонентов. Одномерная однопараметрическая модель. Материальный баланс по i-му компоненту: 2 2 0 1,...., эф B i i L i d c dc D R i n dl dl       . (2.54) (2.54) Тепловой баланс:     2 2 4 Lэф p r CT R d T dT c T T dl d dl         . (2.55) (2.55) Граничные условия: Граничные условия:   0 0 эф i L dc l c c D dl      ,   0 p эф dT l L c T T dl      где  − порозность слоя; где  − порозность слоя; где  − порозность слоя; эф L D −эффективный коэффициент (продольной) диффузии; Lэф  −эффективный коэффициент (продольной) теплопроводности; L − длина реактора. Lэф  −эффективный коэффициент (продольной) теплопроводности; L L − длина реактора. L − длина реактора. 41 Двумерная, однопараметрическая модель. Материальный баланс по i-му компоненту:   Двумерная, однопараметрическая модель. Материальный баланс по i-му компоненту: 2 2 1 0 1,...., эф B i i i r i c c c D R i n r r l r                       . (2.56) (2.56) 2 эф r i r r l r        вой баланс:   2 2 1 0 rэф p r T T dT c r r dl r                     . (2.57) Тепловой баланс: анс:   2 2 1 0 rэф p r T T dT c r r dl r                     . (2.57)   2 2 1 0 rэф p r T T dT c r r dl r                     . (2.57) (2.57) Начальные и граничные условия: Начальные и граничные условия: Начальные и граничные условия: 0 0; ; 1,....., ; i i o l c c i n T T     , 0; 0; 1,...., ; 0; 0 i i c c T r i n r R r r r             ,   1,...., ; эф CT r T i n T T r        , где эф r D и эф r  где эф r D и эф r  − соответственно эффективный коэффициент поперечной диффузии и теплопроводности; где эф r D и эф r  − соответственно эффективный коэффициент поперечной ф ф диффузии и теплопроводности; r − текущий радиус реактора. диффузии и теплопроводности; ффу р ; r − текущий радиус реактора. у r − текущий радиус реактора. Ячеечная модель с последовательно расположенными ячейками. где  − порозность слоя; Структура ячеечной модели: Ячеечная модель с последовательно расположенными ячейками. Структура ячеечной модели: Ячеечная модель с последовательно расположенными ячейками. Структура ячеечной модели: Ячеечная модель с последовательно расположенными ячейками. Структура ячеечной модели: Материальный баланс по i-му компоненту:     1 , 0 1,...., k k B i i k k k V c c V R c T i n      . (2.58) (2.58) Тепловой баланс: Тепловой баланс: Тепловой баланс:       1 , 0 p k k k r k k c T T V T c        , (2.59) (2.59) где V − объемная скорость; где V − объемная скорость;   , k k T c  − соответственно температура и вектор концентраций компонентов и k-той ячейки; чеечная модель с обратными потоками. Структура ячеечной модели: Ячеечная модель с обратными потоками. Структура ячеечной модели: 42 Материальный баланс по i-му компоненту: Материальный баланс по i-му компоненту: Материальный баланс по i-му компоненту:       1 1 , 0 1,...., k k k k B m i i m i i k k V c c g c c V R c T i n          . (2.60)       1 1 , 0 1,...., k k k k B m i i m i i k k V c c g c c V R c T i n          . где  − порозность слоя; (2.60) (2.60) Тепловой баланс: Тепловой баланс:         1 1 , 0 t p k k p t k k k r k k V c T T C g T T V T c             , (2.61) де вх V − объемная скорость на входе в реактор;         1 1 , 0 t p k k p t k k k r k k V c T T C g T T V T c             , (2.61) де вх V − объемная скорость на входе в реактор; бъемная скорость на входе в реактор; где вх V − объемная скорость на входе в реактор; tg , m g − величины обратных потоков для вещества и тепла. Двухфазные модели (2.67)        2 2 * * * 2 0 , 0 L p r d T dT c h T T dl dl h T T c T                . (2.67) Граничные условия:   0 0; ; 1,....., i L i i dc l D c c i n dl       ,   L p o T c T T l       , ; 0; 1,...., ; 0 ic T l L i n l l         .   0 0; ; 1,....., i L i i dc l D c c i n dl       ,   L p o T c T T l       , ; 0; 1,...., ; 0 ic T l L i n l l         . где , L L D  − соответственно коэффициенты продольной диффузии и теплопроводности для подвижной фазы. Одномерная однопараметрическая модель, учитывающая перенос тепла и массы внутри гранул катализатора и между фазами. Материальный баланс: 2 Одномерная однопараметрическая модель, учитывающая перенос тепла и массы внутри гранул катализатора и между фазами. Материальный баланс: 2 Одномерная однопараметрическая модель, учитывающая перенос тепла и массы внутри гранул катализатора и между фазами. Материальный баланс: 2     2 * 2 2 * * * * 2 0 2 , 0 1,...., эф i i L g i i B i i i d c dc D k c c dl dl c c D R c T i n r r r                          . Двухфазные модели Д у ф Одномерная модель, учитывающая межфазные градиенты по теплу и веществу. Материальный баланс по i-му компоненту: Одномерная модель, учитывающая межфазные градиенты по теплу и веществу. Материальный баланс по i-му компоненту:   * i g i i dc k c c dl     . (2.62) (2.62)     * * * 1 , 1,....., B g i i k c c R c T i n       . (2.63) (2.63) Тепловой баланс: Тепловой баланс:   * p dT с k T T dl     (2.64) (2.64)       * * * 2 , r k T T c T      , (2.65) (2.65) где g k , k − соответственно коэффициенты массо- и теплопередачи; где g k , k − соответственно коэффициенты массо- и теплопередачи;  − поверхность раздела фаз;  − поверхность раздела фаз; * * * * , ic T − соответственно концентрация i-го компонента и температура на поверхности раздела фаз; р р ф 1 , 2  − факторы эффективности. 1 , 2  − факторы эффективности. 1 , 2  − факторы эффективности. Одномерная однопараметрическая модель. Материальный баланс по i-му компоненту:   2 * 2 0 i i L g i i d c dc D k c c dl dl       . (2.66) (2.66) 43     * * * 1 , 1,....., B g i i k c c R c T i n      . Тепловой баланс: Тепловой баланс: Тепловой баланс:        2 * 2 * * * 2 0 , 0 L p r d T dT c h T T dl dl h T T c T                . (2.67)        2 * 2 * * * 2 0 , 0 L p r d T dT c h T T dl dl h T T c T                . Двухфазные модели (2.68) (2.68) r r r     Тепловой баланс: 2 d T dT   Тепловой баланс: 2 Тепловой баланс:       2 * 2 2 * * * * 2 0 2 , 0 L p эф r d T dT c h T T dl dl d T dT H c T r dr dr                         . (2.69)   2 * 2 0 L p d T dT c h T T dl dl         (2.69)  Граничные условия:  Граничные условия:   Граничные условия:   0 0; ; 1,....., ; i L i i dc l D c c i n dl         0 L p dT c T T dl        0 0; ; 1,....., ; i L i i dc l D c c i n dl         0 L p dT c T T dl      ; 0; 1,....., ; 0; i dc dT l L i n dl dl     * 0; 0; 1,....., ; 0; i dc dT r i n dr dr     ; 0; 1,....., ; 0; i dc dT l L i n dl dl     * 0; 0; 1,....., ; 0; i dc dT r i n dr dr     44   * * ; ; 1,....., ; i i эф i i dc r R D Kg c c i n dr       * эф dT T Т dr      , где i эф D , эф  − соответственно эффективные коэффициенты диффузии i-го компонента и теплопроводности в грануле катализатора; компонента и теплопроводности в грануле катализатора; р р у р R − радиус гранулы; R − радиус гранулы; р д у р у ;  − коэффициент теплоотдачи от потока реагентов к грануле катализатора.  − коэффициент теплоотдачи от потока реагентов к грануле катализатора. Ячеечная модель с последовательно расположенными ячейками, учитывающая межфазный перенос тепла и вещества. Двухфазные модели Структура ячеечной модели: Материальный баланс по i му  компоненту:         1 * * * * 1 0 , k k k k i i g i i k k в k k g i i i V c c k V c c k V c c VR c T          . (2.70) (2.70) Тепловой баланс: Тепловой баланс: Тепловой баланс:           * 1 * * * 2 0 , 0 k p k k k k k k k r V с T T haV T T haV T T V H c T            , (2.71) (2.71) где V − объемная скорость подачи сырья; где V − объемная скорость подачи сырья; V объем ячейки где V − объемная скорость подачи сырья; V − объем ячейки. V − объем ячейки. V − объем ячейки. V − объем ячейки. 45 Ячеечная модель с обратными потоками, учитывающая межфазовый перенос тепла и вещества. Структура ячеечной модели: Ячеечная модель с обратными потоками, учитывающая межфазовый перенос тепла и вещества. Структура ячеечной модели: 45 Материальный баланс по i-му компоненту: Материальный баланс по i-му компоненту: Материальный баланс по i-му компоненту: Материальный баланс по i-му компоненту:               1 1 * * * * 1 0 , 0 k k k k k k i i m i i g i i k k k k g i i i i V c c g c c k V c c k aV c c VR c T             . (2.72) (2.72) Тепловой баланс: Тепловой баланс:   k              1 1 * * * 2 0 , 0 , , k p k k T p k k k k k k k вх n V с T T g с T T haV T T haV T T V H с T V V g n m T                    . Двухфазные модели (2.73) (2.73) Ячеечная модель, учитывающая межфазовый перенос тепла и вещества, с неравным числом ячеек в подвижной и неподвижной фазах. Структура ячеечной модели: Материальный баланс по i-му компоненту: Материальный баланс по i-му компоненту: Материальный баланс по i-му компоненту: Материальный баланс по i-му компоненту:           1 * * * * 1 * * 1 0 , , 0 2 k k k k i i g i i k k в k k в k k g i i i i V c c k aV c c V k aV c c R c T R c T                (2.74) (2.74) Тепловой баланс: Тепловой баланс: Тепловой баланс:     1 * 1 2 0 2 k k p k k k V V с T T ha T T T               . (2.75) (2.75)         * 1 * * 1 * * * 2 2 , , 0 k k k k k k k haV T T T V H T с T с                . Приведенные выше типовые модели никоим образом не исчерпывают всего возможного многообразия математических моделей реакторов, отражающих различные возможности организации движения потоков, взаимодействия фаз и конструкций промышленных аппаратов. Они до некоторой степени являются типовыми в основном с точки зрения математического описания, так как более сложные физико-химические явления в реакторах могут быть отражены соответствующей совокупностью типовых моделей. В первую очередь это, например, относится к ячеечным моделям, 46 поскольку усложнение гидродинамической обстановки и увеличение числа фаз приводит к усложнению структуры. Но вид математического описания (система нелинейных алгебраических уравнений) останется прежним, хотя размерность уравнений, конечно, возрастает. Аналогичная зависимость часто справедлива и для моделей других типов. поскольку усложнение гидродинамической обстановки и увеличение числа фаз приводит к усложнению структуры. Но вид математического описания (система нелинейных алгебраических уравнений) останется прежним, хотя размерность уравнений, конечно, возрастает. Аналогичная зависимость часто справедлива и для моделей других типов. Сравнение неизотермических моделей реакторов. Количественную оценку моделей реакторов удобно провести, воспользовавшись методом сравнения наклонов кривых dx/dη в координатах x, η, дифференцируя уравнения материального баланса реактора в различных условиях теплообмена и принимая для адиабатических условий необходимые уравнения. Двухфазные модели Согласно уравнениям для модели идеального вытеснения тангенс угла наклона dx/dη составит:   1 0 1 n n dx с k x d    . (2.76) (2.76) Для реактора идеального смешения имеем: Для реактора идеального смешения имеем:   1 0 2 1 n х с k x     . (2.77) (2.77) Дифференцирование последнего уравнения при переменных x, η, k позволяет найти тангенс угла наклона кривых в координатах x, η.   Дифференцирование последнего уравнения при переменных x, η, k позволяет найти тангенс угла наклона кривых в координатах x, η.         1 0 1 1 1 1 1 1 n n dx x с k x dk d n x x x k dx                     . (2.78) (2.78) Сравнение уравнений показывает, что в координатах x, η наклоны кривых для реактора идеального смешения и идеального вытеснения будут отличаться один от другого на величину фактора:       1 1 1 1 1 x dk n x x x k dx                 . (2.79) (2.79) Для изотермических условий dk/dx = 0 и фактор приводится к виду: 1 x      ( Для изотермических условий dk/dx = 0 и фактор приводится к виду:   Для изотермических условий dk/dx = 0 и фактор приводится к виду:   1 1 1 x n x          . Для изотермических условий dk/dx = 0 и фактор приводится к виду:   1 1 1 x n x          . (2.80 Для изотермических условий dk/dx = 0 и фактор приводится к виду:     1 1 1 x n x          . (2.80) (2.80) Полученный фактор всегда меньше единицы, поэтому для реактора идеального смешения и наклона кривых всегда меньше, чем для реактора идеального вытеснения; следовательно, в изотермических условиях реактор 47 ого вытеснения всегда предпочтительнее реактора идеальног идеального вытеснения всегда предпочтительнее реактора идеального смешения. Для реактора идеального смешения имеем: Для эндотермических реакций получим:     Для эндотермических реакций получим: 0 0 0 2 0 0 exp 1 a a a T E E k RT T dk d R k dx dx T T x T x T                                  . (2.81) (2.81) Подставляя полученное в выражение и учитывая, что оно всегда меньше единицы, т. е. что для реактора идеального смешения наклон всегда меньше, чем для реактора идеального вытеснения, найдем: 2   2 1 1 a a o o o T T E n x x T RT T            . (2.82) (2.82) Левая часть выражения всегда отрицательна, а правая − всегда положительна, поэтому и фактор всегда меньше единицы. Следовательно, в адиабатическом реакторе идеального вытеснения для эндотермических реакций всегда достигается более высокая степень превращения на единицу объема. д д р р Для экзотермических реакций получим: д д р р щ д ц Для экзотермических реакций получим: р ц у 0 0 0 2 0 0 exp 1 a a a T E E k RT T dk d R k dx dx T T x T x T                                 . (2.83) (2.83) Графики для экзотермической реакции первого и второго порядков, а также для эндотермической реакции второго порядка построены при условиях: E Графики для экзотермической реакции первого и второго порядков, а также для эндотермической реакции второго порядка построены при условиях: E 0 E RT to o k k e   , 4 exp 1 0,1 to x k k x         , 0 E RT , 0 а Т T . (2.84) (2.84) Кривые, изображенные на рисунке 2.6, соответствуют различным порядкам «n» экзотермических реакций и уровням смешения реагентов. Сравнение графиков, приведенных на рисунке 2.6, позволяет сделать следующий вывод. 48 1 – вытеснение, 2 – смешение на макроуровне, 3 – смешение на микроуровне Рисунок 2.6. Для реактора идеального смешения имеем: Кривые степеней превращения для экзотермических реакций различных порядков при разных уровнях смешения 1 – вытеснение, 2 – смешение на макроуровне, 3 – смешение на микроуровне р ур Рисунок 2.6. Кривые степеней превращения для экзотермических реакций различных порядков при разных уровнях смешения Рисунок 2.6. Кривые степеней превращения для экзотермических реакций различных порядков при разных уровнях смешения При сравнительно малых и средних значениях «x»; кривые для реакторов идеального смешения в случае экзотермических реакций лежат несколько выше кривых для реакторов идеального вытеснения. Однако при больших значениях степени превращения наблюдается обратное явление, причем ход кривых для реакторов идеального смешения становится аналогичным их характеру для изотермических условий протекания экзотермических и эндотермических реакций. В случае эндотермических реакций в аппаратах идеального вытеснения достигается более высокая степень превращения, чем в аппаратах идеального смешения (рис. 2.7). 1 – вытеснение, 2 – смешение на макроуровне, 3 – смешение на микроуровне Рисунок 2.7. Кривые степеней превращения для эндотермических реакций различных порядков при разных уровнях смешения 1 – вытеснение, 2 – смешение на макроуровне, 3 – смешение на микроуровне 1 – вытеснение, 2 – смешение на макроуровне, 3 – смешение на микроуровне р ур Рисунок 2.7. Кривые степеней превращения для эндотермических реакций различных порядков при разных уровнях смешения 49 На рисунке 2.8 представлено изменение относительного времени пребывания, необходимого для достижения данной степени превращения в реакторах идеального смешения «ηm» и идеального вытеснения «ηb» в случае протекания эндотермических, изотермических и экзотермических реакций при ранее указанных значениях , , / ( ) to o k k E RT и   / a o T T . При изотермических условиях, и особенно при эндотермических реакциях наблюдается обратная картина − реактор идеального вытеснения предпочтительнее реактора идеального смешения. Графики, приведенные на рисунке 2.6, показано, что наклоны кривых для реактора идеального смешения выше наклонов кривых для реактора идеального вытеснения при низких значениях «х», равны при некоторой промежуточной степени превращения и ниже при высоких значениях «х». Рисунок 2.8. Зависимость относительного времени пребывания в реакторах идеального смешения «ηm» и идеального вытеснения «ηb» от степени превращения для различных реакций и разных значений порядков реакций. Рисунок 2.8. Зависимость относительного времени пребывания в реакторах идеального смешения «ηm» и идеального вытеснения «ηb» от степени превращения для различных реакций и разных значений порядков реакций. Рисунок 2.8. Зависимость относительного времени пребывания в реакторах идеального смешения «ηm» и идеального вытеснения «ηb» от степени превращения для различных реакций и разных значений порядков реакций. Для реактора идеального смешения имеем: Соответственно, получим величину фактора, который больше единицы при малых значениях «х» и меньше единицы при больших значениях «х»: 50       2 a o 2 a a o o o T >1при малых "х" 1-x × 1+ x T × =1при средних "х" T T E <1при больших "х" 1+ n-1 x× 1+ x -x 1-x × × T RT T                 , (2.85) o o o   или   2 1 1 a a o o o T T E n x x T RT T            . (2.86) или   2 1 1 a a o o o T T E n x x T RT T            . (2.86) (2.86) Из соотношения, приведенного выше видно, что при высокой степени превращения его правая часть приближается к нулю, когда «х» стремится к единице. При очень низкой конверсии (х≈0) из соотношениявыше получим: Из соотношения, приведенного выше видно, что при высокой степени превращения его правая часть приближается к нулю, когда «х» стремится к единице. При очень низкой конверсии (х≈0) из соотношениявыше получим: a o o T E n RT T   . (2.87) (2.87) Соотношение справедливо для большинства экзотермических систем в адиабатических условиях. Следует отметить, что, если это соотношение не выполняется, скорость реакции будет уменьшаться при увеличении степени превращения, и реактор идеального вытеснения окажется предпочтительнее реактора идеального смешения. Приведенная ранее оценка средних скоростей реакций позволяет объяснить эти выводы. Скорость эндотермических реакций уменьшается с увеличением степени превращения вследствие расходования реагентов и уменьшения температуры в системе. Средняя скорость реакции в аппарате идеального вытеснения (где она принимает среднее значение от максимального на входе и минимального на выходе) всегда выше средней скорости в реакторе идеального смешения; кроме того, высокая степень превращения достигается только при высоких средних скоростях реакций. Поэтому при теплообмене реактор идеального вытеснения всегда предпочтительнее реактора идеального смешения. Для большинства экзотермических процессов скорость увеличивается вначале (с возрастанием степени превращения) вследствие повышения температуры в системе, но снижается в конце (с уменьшением «х») за счет расходования реагентов. При сравнительно малых степенях превращения средняя скорость реакции в аппарате идеального смешения всегда выше, чем в аппарате идеального вытеснения. Для реактора идеального смешения имеем: Однако по достижении некоторого уровня «х» несколько большего, чем уровень, соответствующий максимуму скорости реакции, средние скорости для двух моделей реакторов становятся идентичными; после превышения указанного уровня реактор идеального вытеснения становится предпочтительнее. На степень превращения влияет также уровень сегрегации. На рисунках 2.6 и 2.7 представлены графики изменения степени превращения в зависимости от безразмерного времени пребывания для реакторов идеального вытеснения и идеального смешения на двух уровнях смешения соответственно для 51 эндотермических и экзотермических реакций. При графическом построении профилей использовалась температурная зависимость скорости реакций по Аррениусу. При этом температура исключалась путем составления теплового баланса для адиабатического реактора с последующим аналитическим или численным решением при следующих условиях: 40 o E RT  и 0,1 a o T T  . Сравнение состояния сегрегации с уровнем молекулярного смешения для эндотермических реакций (рис. 2.7) показывает, что наивысшая степень превращений достигается при сегрегированном состоянии для всех порядков реакций. Разница между этим результатом и результатом, рассмотренным при изотермических условиях, для которых существен порядок реакции, зависит от характера изменения скорости процесса. В эндотермической системе с увеличением степени превращения скорость уменьшается вследствие расходования реагентов и снижения температуры системы. Для описанных выше систем температурный эффект был большим, чем его компенсация за счет порядка реакции. Сравнение состояния сегрегации с уровнем молекулярного смешения для экзотермических реакций (рис. 2.6) приводит к противоположным выводам − наивысшая степень превращения, соответствует уровню молекулярного смешения. Это объясняется тем, что с возрастанием степени превращения в экзотермических системах начальная скорость увеличивается вследствие повышения температуры системы. Устойчивость тепловых режимов реакторов При оптимизации химических реакторов, в которых протекают экзотермические реакции, и управлении ими одним из основных вопросов является определение тепловой устойчивости реакторов. Понятие устойчивости системы впервые сформулировал А. М. Ляпунов: «Система называется устойчивой, если после наложения какого-либо возмущения она возвращается в прежнее состояние при снятии этого возмущения». Применительно же к химическим реакторам, в которых проходят экзотермические реакции, различные возмущения (изменение расходов поступающих компонентов, ухудшение теплоотвода, флуктуации состава исходной смеси и т. д.) могут настолько вывести процесс из стационарного состояния, что он уже не вернется к исходному режиму. Поэтому, возникает необходимость оценки, устойчивости стационарных режимов работы реактора. Рассмотрим этот вопрос на примере политропических реакторов. Основные положения теории устойчивости. Об устойчивости реактора судят по уравнениям нестационарного режима работы или его динамики:         ( ) ( ) o n A r c A A r o A n r p c p o V x r o A dc V V c c V k T c d dT gV c V gc T T K F T T V k T c d              , (2.88) (2.88) 52 линеаризованных в малой окрестности исследуемого режима. Линеаризация уравнений для реакции первого порядка (n = 1) дает:  линеаризованных в малой окрестности исследуемого режима. р р у р Линеаризация уравнений для реакции первого порядка (n = 1) дает:  2 2 2 1 ( ) ( ) ( ) 1 ( ) s s A o s A o s A A o s A p V o s A c p p d c E k T c k T c d RT d T k T c d c g K E k T c V c g c g RT                                              , (2.89) (2.89) где A c  − отклонение концентрации от ее значения в исследуемом режиме ( s A c ); где A c  − отклонение концентрации от ее значения в исследуемом режиме ( s A c ); A T  − отклонение температуры от ее значения в исследуемом режиме (Ts). Устойчивость тепловых режимов реакторов Значения cA и  определяются из уравнений, описывающих исследуемый режим в стационарном состоянии или в статике:        ( ) 0 ( ) 0 o c A A r o A c p o V x r o A V c c V k T c V gc T T K F T T V k T c H              . (2.90) (2.90) Систему можно записать в виде  Систему можно записать в виде  11 12 21 22 A А A А d c с d d Т с d                    , (2.91) (2.91)  где ik  − соответствующие коэффициенты системы.  где ik  − соответствующие коэффициенты системы.  где ik  − соответствующие коэффициенты системы. где ik  − соответствующие коэффициенты системы. Система будет устойчивой, и, следовательно, исследуемый режим будет устойчивым, если корни «λ» характеристического уравнения системы   11 12 2 11 22 11 22 21 12 21 22 0                     , (2.92)   11 12 2 11 22 11 22 21 12 21 22 0                     , (2.92) (2.92) являются отрицательными действительными числами или имеют отрицательные действительные части в случае комплексных корней. являются отрицательными действительными числами или имеют отрицательные действительные части в случае комплексных корней. являются отрицательными действительными числами или имеют отрицательные действительные части в случае комплексных корней. Устойчивость тепловых режимов реакторов Это возможно, если выполняются условия   Это возможно, если выполняются условия Это возможно, если выполняются условия   11 22 11 22 21 12 0 0          , (2.93) (2.93) которые с учетом значений ik  из уравнений системы имеют вид которые с учетом значений ik  из уравнений системы имеют вид 53       2 2 2 ( ) ( ) 0 1 ( ) 1 ( ) 0 V o s o s A c p p V o s o s A c p p E K F k T k T c V c g c g RT E K F k T k T c V c g c g RT                           . (2.94)     2 1 ( ) 1 ( ) 0 c p p V o s o s A c p p g g E K F k T k T c V c g c g RT                    . (2.94) (2.94) Физический смысл условия можно установить, если представить его так:   2 2 ( ) 1 ( ) o s o c p V r r o s c k T c E V gc K F V RT V k T V             . (2.95) (2.95) Левая часть неравенства представляет собой производную по температуре от величины теплоотвода из реактора: Левая часть неравенства представляет собой производную по температуре от величины теплоотвода из реактора:   отв c p V x Q V gc T K F T T     , (2.96) (2.96)   отв c p V x Q V gc T K F T T       отв c p V x Q V gc T K F T T     , (2.96) откуда отв c p V dQ V gc T K F dT    . (2.97) откуда откуда отв c p V dQ V gc T K F dT    . Устойчивость тепловых режимов реакторов (2.97) (2.97) Можно также показать, что правая часть неравенства является производной от количества тепла, выделяющегося в реакторе, так как   ( ) 1 ( ) Ao o s выд r c o r o s c k T c Q V V T V k T V           , (2.98) (2.98) и, следовательно и, следовательно и, следовательно   2 2 ( ) 1 ( ) Ao o s выд r r o s c k T c dQ E V dT RT V k T V          . (2.99)   2 2 ( ) 1 ( ) Ao o s выд r r o s c k T c dQ E V dT RT V k T V          . (2.99) (2.99) Таким образом, условия, приведенные выше эквивалентны следующему отв выд dQ dQ dT dT  , (2.100) (2.100) т.е. наклон линии расхода тепла в координатах Q-Т должен быть больше наклона линии прихода тепла. т.е. наклон линии расхода тепла в координатах Q-Т должен быть больше наклона линии прихода тепла. Таким образом, как показано выше, для того, чтобы режим работы реактора был устойчивым в общем случае, необходимо выполнение условий приведенных выше. Однако иногда об устойчивости режима работы реактора можно судить по выполнению лишь одного из условий. Комбинируя условия, можно показать, что если выполняется неравенство 54 1 c p V V gc x K F x      , 1 c p V V gc x K F x      , (2.101) (2.101) следовательно, неравенства являются необходимыми и достаточными условиями устойчивости режима работы реактора. При нарушении неравенства необходимым и достаточным условием устойчивости режима служит выполнение данного неравенства. Соотношение может быть наглядно истолковано с помощью S-образной диаграммы. На рисунке 2.9 представлен температурный профиль реактора идеального смешения, характеризующийся, как обычно, S-образной кривой с быстрым подъемом температуры при малой степени превращения и резким замедлением подъема при больших значениях «х» (быстрое выделение тепла в начальный момент и затухание при достижении большой степени превращения). р р щ ) Рисунок 2.9. Температурный профиль реактора идеального смешения Рисунок 2.9. Температурный профиль реактора идеального смешения Количество тепла, отводимого в адиабатических условиях, целиком определяется разностью теплот в потоке на входе в реактор и выходе из него. Устойчивость тепловых режимов реакторов Это можно изобразить в виде прямой, пересекающей абсциссу в точке Т0 (температура на входе). Для политропических условий линия теплоотвода проходит при более низкой температуре, отвечающей температуре охлаждающего агента. Из рисунка 2.9 следует, что может быть несколько точек пересечения линий прихода тепла и теплоотвода, или состояний, в которых расход тепла равен приходу, т.е. несколько стационарных состояний. Вообще же положение точек пересечения зависит от выбора рабочих условий процесса (температуры на входе, степени превращения, времени пребывания) и кинетики процесса (рис. 2.6, 2.7). Если линия теплоотвода проходит над линией прихода тепла, то выше точек пересечения существует устойчивое состояние работы реактора. Если же линия теплоотвода проходит ниже линий прихода тепла, то работа реактора неустойчива. 55 Реактор идеального смешения. Количество тепла, отводимого из системы, в политропическом реакторе идеального смешения составляет:     Реактор идеального смешения. Количество тепла, отводимого из системы, в политропическом реакторе идеального смешения составляет:     c p k o V V k x V gc T T K F T T     , (2.102) где FV − площадь охлаждения, м2/м3. системы, в политропическом реакторе идеального смешения составляет:     c p k o V V k x V gc T T K F T T     , (2.102) где FV − площадь охлаждения, м2/м3. (2.102)     c p k o V V k x V gc T T K F T T   , (2.102) где FV − площадь охлаждения, м2/м3. p где FV − площадь охлаждения, м2/м3. Вводя адиабатическую температуру, представим количество отводимого тепла в безразмерном виде   ' ' 1 V n V p расх k o a F K gc Q T T T      , (2.103) (2.103) где ' o T − кажущаяся температура реакционной смеси на входе в реактор: где ' o T − кажущаяся температура реакционной смеси на входе в реакт ' 0 1 V V n p o V V n x o p K F gc T T K F T T gc         . (2.104) (2.104) Из уравнений следует, что теплоотвод и температура на входе в реактор зависят от времени пребывания или скорости питания. Устойчивость тепловых режимов реакторов Рассмотрим параллельные реакции типа: мотрим параллельные реакции типа: 1 2 1 2 k k rR A rS A A R k c A S k c       И последовательные реакции типа 1 2 k k И последовательные реакции типа 1 2 1 1 2 2 k k rR A rR A R r R A R k c k c k c R S k c          Предположим, что R − целевой продукт, а S − побочный продукт. Для параллельной реакции тепловыделение составит: 1 2 1 1 2 2 k k rR A rR A R r R A R k c k c k c R S k c          Предположим, что R − целевой продукт, а S − побочный продукт. Для параллельной реакции тепловыделение составит: 1 2 1 1 2 2 k k rR A rR A R r R A R k c k c k c R S k c          Предположим, что R − целевой продукт, а S − побочный продукт. Для параллельной реакции тепловыделение составит:   1 1 2 2 A r H k H k c V   ,   1 1 2 2 A r H k H k c V   , (2.105) (2.105) где cA − концентрация A в реакторе идеального смешения равная.   1 2 1 Ao A n c с k k    . Устойчивость тепловых режимов реакторов (2.106) (2.106) Чтобы найти количество выделяемого тепла в безразмерном виде, действительно выделяемое тепло разделим на максимально возможное, выражаемое соотношениями: 2 0 1 2 1 0 1 2 A с A с H c V при H H H c V при H H         (2.107) (2.107) Тогда, вводя отношение 2 1 Н H H   получим при 1 Н :     1 2 1 2 1 n r n k k H Q k k H            Тогда, вводя отношение 2 1 Н H H   получим при 1 Н :     1 2 1 2 1 n r n k k H Q k k H            Тогда, вводя отношение 2 1 Н H H   получим при 1 Н :     1 2 1 2 1 n r n k k H Q k k H            56 при H<1:     1 2 1 2 1 n r n k k H Q k k      , (2.108)     1 2 1 2 1 n r n k k H Q k k      ,     1 2 1 2 1 n r n k k H Q k k      , (2.108) при H<1:     1 2 1 2 1 n r n k k H Q k k      , (2.108) при H<1: где r Q − тепло, подводимое за счет реакции. где r Q − тепло, подводимое за счет реакции. Для последовательных реакций тепловыделение составит: Для последовательных реакций тепловыделение составит:   1 1 2 2 r А R V H k с H k с    . (2.109) Для последовательных реакций тепловыделение составит:   1 1 2 2 r А R V H k с H k с    . (2.109) Д р   1 1 2 2 r А R V H k с H k с    . (2.109) (2.109) При установившемся состоянии имеем: При установившемся состоянии имеем:    1 1 1 2 1 1 1 Ao A n n Ao R n n c с k k c с k k               . (2.110) (2.110) Максимальное тепловыделение составит:   1 2 0 А с H H с V     Откуда:       1 2 1 2 1 1 1 1 1 n n r n n k H k Q k k H                     . (2.111) (2.111) Для последовательной реакции найдем:   1 1 Ao A p H c H Т g c        . (2.112) Для последовательной реакции найдем:   1 1 Ao A p H c H Т g c        . (2.112) Для последовательной реакции найдем:  реакции найдем:   1 1 Ao A p H c H Т g c        . (2.112) (2.112) Для сравнения характера осуществления двух рассматриваемых типов реакций введем температуру TR, при которой константы скорости желаемой (k1) и не желаемой (k2) реакций становятся равными величине RR:       1 2 1 2 1 1 1 2 1 2 1 exp exp 1 ln R R R b R R E E k k k RT RT E b k Т k k k R k                                  , (2.113) 1. 1 2 1 2 exp exp R R R E E k k k RT RT                 (2.113) где b = E2/E1. где b = E2/E1. где b = E2/E1. Отношение энергий активаций b является мерой изменения селективности с температурой. При этом под селективностью понимается отношение cR/(cR+cS). Чем больше b отличается от единицы, тем больше селективность изменяется с температурой: при b>1 селективность увеличивается с уменьшением температуры, при b<1 возрастает с ее повышением. 57 Введем теперь безразмерные отношения. Пусть 1 R k k k  − константа скорости первой реакции; 2 b R k k k  − константа скорости второй реакции; k R T T T  − температура в реакторе; R R c V k V   − время пребывания. Максимальное тепловыделение составит: Если b>1, реактор работает при температурном уровне T<1; если b<1 − при температурном уровне T>1 и высокой селективности. При выбранных значениях параметров b, 1 R RT E , H и п  для расчета расх Q и r Q можно использовать следующие безразмерные уравнения:   0 1 V V p R расх k a K F gc k Q T T T        . (2.114) (2.114) Для параллельных реакций имеем: Для параллельных реакций имеем: при 1 H  Для параллельных реакций имеем: 1 H при 1 H      0 1 1 A a p R b r b c H H T gc T K K H Q K K H             , (2.115) при 1 H      0 1 1 A a p R b r b c H T gc T K K H Q K K           . (2.116)     0 1 1 A a p R b r b c H H T gc T K K H Q K K H             , (2.115)     0 1 1 A a p R b r b c H H T gc T K K H Q K K H             , (2.115)     0 1 1 A a p R b r b c H H T gc T K K H Q K K H             , (2.115)     0 1 1 A a p R b r b c H T gc T K K H Q K K           . (2.116) (2.115) при 1 H  при 1 H      0 1 1 A a p R b r b c H T gc T K K H Q K K           . Максимальное тепловыделение составит: (2.116) (2.116) Для последовательных реакций находим:   Для последовательных реакций находим:   Для последовательных реакций находим:          0 1 1 1 1 1 1 1 A a p R b r b c H H T gc T K H K Q K K H                  . (2.117) (2.117) Из приведенных выше уравнений безразмерная температура в реакторе определяется соотношением: Из приведенных выше уравнений безразмерная температура в реакторе определяется соотношением: 2 1 1 1 1 1 1 ln 1 ln ln R T b RT K K k E k                 . (2.118) (2.118) 1   На рис. 2.10 и 2.11 (для параллельных реакций), а также на рис. 2.12 (для На рис. 2.10 и 2.11 (для параллельных реакций), а также на рис. 2.12 (для 58 последовательных реакций) представлен общий вид зависимостей ' r Q и   / R R s c c c  от безразмерной температуры в реакторе «Т» и безразмерного времени пребывания «». Как следует из указанных рисунков, с увеличением времени пребывания температурный уровень, при котором проходит реакция, уменьшается и селективность возрастает. Если b<1, то для достижения высокой селективности температурный уровень необходимо увеличить, а время пребывания уменьшить. Для высоких степеней превращения возникают два экстремума: один при низкой селективности (Q<1000, рис. 2.10, 2.11), дающий одну S-образную кривую, и другой − при высокой селективности (Q>1000 рис. 2.12), дающий двойную S-образную кривую. ), д щ д у р у р у Рисунок 2.10. Зависимость тепловыделения и выхода от времени пребывания и температуры в реакторе для параллельных реакций (H = 2; b = 2;   1 / 16,7 R E RT  ). Рисунок 2.10. Зависимость тепловыделения и выхода от времени пребывания и температуры в реакторе для параллельных реакций (H = 2; b = 2;   1 / 16,7 R E RT  ). Рисунок 2.10. Зависимость тепловыделения и выхода от времени пребывания и температуры в реакторе для параллельных реакций (H = 2; b = 2;   1 / 16,7 R E RT  ). Рисунок 2.11. Зависимость тепловыделения и выхода от времени пребывания и температуры в реакторе для параллельных реакций (H = 0,5; b = 2;   1 / 16,7 R E RT  ). Максимальное тепловыделение составит: Рисунок 2.11. Зависимость тепловыделения и выхода от времени пребывания и температуры в реакторе для параллельных реакций (H = 0,5; b = 2;   1 / 16,7 R E RT  ). 59 Рисунок 2.12. Зависимость тепловыделения и выхода от времени пребывания и температуры в реакторе для последовательных реакций (H = 2; b = 2;   1 / 16,7 R E RT  ). Рисунок 2.12. Зависимость тепловыделения и выхода от времени пребывания и температуры в реакторе для последовательных реакций (H = 2; b = 2;   1 / 16,7 R E RT  ). Рассмотрим теперь устойчивые состояния работы политропического реактора идеального смешения. Рассмотрим теперь устойчивые состояния работы политропического реактора идеального смешения. Рассмотрим теперь устойчивые состояния работы политропического реактора идеального смешения. Высокая степень превращения и низкая селективность. При высокой скорости превращения и низкой селективности общая форма кривой аналогична виду кривой для одной реакции, так как селективность настолько низка, что температуры протекания двух реакций практически одинаковы. На рисунке 2.13 приведен график устойчивости для параллельных реакций; Т0 − температура на входе; В − желаемая рабочая точка. В точке «В» имеется предпочтительное количество продукта R. При данных условиях в точке В и выше нее адиабатический режим невозможен, поскольку прямая, связывающая точки Т0 и В, дает устойчивое стационарное состояние только в точках То и Q, но в точке Q преимущественно получается продукт S. Рисунок 2.13. График устойчивости режима работы политропического реактора идеального смешения, в случае параллельных реакций (Q = 100; H = 2; b = 2;   1 / 16,7 R E RT  ). Рисунок 2.13. График устойчивости режима работы политропического реактора идеального смешения, в случае параллельных реакций (Q = 100; H = 2; b = 2;   1 / 16,7 R E RT  ). Таким образом, точка «В» характеризует неустойчивое стационарное состояние. Поэтому для достижения устойчивого стационарного состояния необходимо охлаждение (более крутой наклон прямой теплоотвода), 60 соответствующий точке С; линия ВС представляет собой граничный случай, когда достигается устойчивое состояние. Однако, такое решение имеет недостаток: температура охлаждающего агента должна быть выше температуры исходной смеси, вследствие чего теплоотвод становится зависимым от нагрузки реактора и, следовательно, времени пребывания . При изменении времени пребывания  (нагрузка) и температуры на входе концентрация реагента в питании или температура охлаждающего агента при сохранении выхода продукта должны изменяться более резко. На рисунке 2.14 показано влияние изменения нагрузки (времени пребывания ) на регулирование процесса для последовательных реакций. Принято, что при времени контакта =1 реактор работает в нижнем стационарном устойчивом режиме. При уменьшении скорости питания наполовину (0→2) образуется слишком много нежелательного продукта. При увеличении нагрузки вдвое (0→0,5) возникают три стационарных состояния. В зависимости от динамического поведения реактора возможны два устойчивых стационарных состояния: в точке А степень превращения слишком мала, а в точке В почти весь исходный продукт превращается в нежелательный. Рисунок 2.14. Влияние изменения нагрузки на устойчивые состояния реактора в случае последовательных реакций (H = 2; b = 2;   1 / 30 R E RT  ; 0,850 o T  ; 0,978 k T  ) Рисунок 2.14. Рассмотрим теперь устойчивые состояния работы политропического реактора идеального смешения. Влияние изменения нагрузки на устойчивые состояния реактора в случае последовательных реакций (H = 2; b = 2;   1 / 30 R E RT  ; 0,850 o T  ; 0,978 k T  ) Рисунок 2.14. Влияние изменения нагрузки на устойчивые состояния реактора в случае последовательных реакций (H = 2; b = 2;   1 / 30 R E RT  ; 0,850 o T  ; 0,978 k T  ) Высокая степень превращения и высокая селективность. На рис. 2.15 представлена зависимость ' r Q и ' расх Q от температуры в реакторе, из которой следует, что возможны пять стационарных состояний: Высокая степень превращения и высокая селективность. На рис. 2.15 представлена зависимость ' r Q и ' расх Q от температуры в реакторе, из которой следует, что возможны пять стационарных состояний: ойчивое (низкая степень превращения). 1. Устойчивое (низкая степень превращения). 2. Неустойчивое (затухание ведет к точке 1, а зажигание − к точке 3 3. Устойчивое (высокая степень превращения продукта R, низкая степень превращения продукта S). 4. Неустойчивое, ведущее к точке 3 или 5. 5. Устойчивое (высокая степень превращения продукта S, низкая степень превращения продукта R). Желательным режимом работы реактора являются условия, определяемые точкой 3. Если концентрацию реагента в питании постепенно увеличивать, 61 наклон линии А становится меньшим, точка 3 движется вправо, а точка 4 − влево. Когда линии а и А' совпадут, точки 3 и 4 сольются в точку 4', в которой понижение и повышение температуры приводит к тому, что приход тепла всегда выше теплоотвода. Возмущения по температуре при ее подъеме приведут (в конечном счете) к возникновению устойчивого состояния только в точке 5. Если концентрацию реагента уменьшить до значений, соответствующих линий А", то при достижении точки 2' произойдет полное затухание. затухание. Рисунок 2.15. Графическое определение пределов устойчивых режимов в адиабатическом реакторе в случае последовательных реакций (H = 2; b = 2;   1 / 16,7 R E RT  ; 6 10  ) у Рисунок 2.15. Графическое определение пределов устойчивых режимов в адиабатическом реакторе в случае последовательных реакций (H = 2; b = 2;   1 / 16,7 R E RT  ; 6 10  ) Рисунок 2.15. Графическое определение пределов устойчивых режимов в адиабатическом реакторе в случае последовательных реакций (H = 2; b = 2;   1 / 16,7 R E RT  ; 6 10  ) Трубчатый реактор. Рассмотрим теперь устойчивые состояния работы политропического реактора идеального смешения. Температурный профиль данного реактора определяется тем, что объем реактора, в котором в результате реакции выделяется тепло, пропорционален квадрату его диаметра, в то время как поверхность теплосъема пропорциональна диаметру в первой степени. Поэтому в трубах большого диаметра неустойчивость проявляется резче, чем в трубах малого диаметра. Следует иметь в виду, что, за исключением автокаталитических реакций с индукционным периодом, максимальной скоростью реакции при данной температуре является скорость в начальный период, т. е. при входе в реактор, где и возникает опасность резкого подъема температуры. Пусть по оси абсцисс отложена температура реакционной массы в реакторе Т, а по оси ординат − количество тепла на единицу объема реакционной массы в 1 ч; тогда для реактора идеального вытеснения получим график, изображенный на рисунок 2.16. 62 1, 2 – линии теплоотвода, Qz − теплота реакции (кривая прихода тепла) Рисунок 2.16. График устойчивости режима работы трубчатого реактора 1, 2 – линии теплоотвода, Qz − теплота реакции (кривая прихода тепла) Рисунок 2.16. График устойчивости режима работы трубчатого реактора Вначале наклон кривой прихода тепла увеличивается вследствие экспоненциального влияния температуры на скорость реакции. По достижении адиабатической температуры скорость реакции приближается к нулю. При постоянной температуре рубашки и постоянном коэффициенте теплопередачи линии теплоотвода имеют наклон, равный V K F , и пересекают ось абсцисс при температуре охлаждающего агента Тх. На рисунок 2.16 показано два случая теплопередачи. Прямая пересекает кривую прихода тепла в точке А при максимальной скорости реакции. Точка А характеризует устойчивость режима работы реактора, так как небольшое повышение температуры реакционной массы приводит к теплоотводу более быстрому, чем к приходу тепла. Небольшое снижение температуры также вызывает самокоррекцию. Прямая 2 пересекает линию прихода тепла в точках В С и А В точках В и D реактор устойчив а в точке С −неустойчив рр р р р В, С и А. В точках В и D реактор устойчив, а в точке С − неустойчив , р р у , у В точках А, В и D реактор устойчив потому, что тангенс угла наклона линии теплоотвода больше тангенса угла наклона линии прихода тепла, т. е. расх пр dQ dQ dT dT  . расх пр dQ dQ dT dT  . Условия работы в точке D не представляют практического интереса, так как реакция фактически завершается. Если бы точки В и С были близки одна к другой, реактор находился бы в потенциально неустойчивом состоянии. Быстрый подъем температуры в рубашке приведет к смешению линии теплоотвода вправо, и температура в реакторе может подняться от В до С. Рассмотрим теперь устойчивые состояния работы политропического реактора идеального смешения. Для иллюстрации вышеизложенного рассмотрим пример гипотетического процесса производства вещества С из вещества А и В: А + В → С + Q, Рассмотрим теперь устойчивые состояния работы политропического реактора идеального смешения. При восстановлении нормальной температуры рубашки это приведет к гораздо более высокой температуре. В данном случае мерой устойчивости режима работы реактора является разность температур, соответствующих точкам В и С. Эта разность, отражает флуктуацию температур в реакторе, вызываемую изменением потока хладагента или его температуры, концентрации реагентов или их расхода на входе в реактор. Основным вопросом и при тепловом расчете реактора является выбор температуры охлаждающего агента. При слишком низкой температуре реакция просто не пойдет, поэтому необходим детальный анализ с учетом тепловой устойчивости. 63 Что касается схемы производства, то в отличии от схемы потоков между аппаратурно-процессорными единицами и схем потоков в самых единицах схема процесса производства должна давать представление о конструкции аппаратов (реакторов), о применяемых машинах (по возможности в масштабе), о необходимых физических и химических параметрах, а также содержать сведения о веществах, входящих и выходящих из аппаратов и машин, и другие количественные данные. Таким образом, технологическая схема процесса включает в себя информацию: − о количестве и химическом составе основных, промежуточных, конечных и вспомогательных веществ, то есть содержит материальный баланс технологического процесса в целом; − о количестве и химическом составе основных, промежуточных, конечных и вспомогательных веществ, то есть содержит материальный баланс технологического процесса в целом; − о последовательности отдельных операций и связи основного процесса с побочными процессами в отдельных операциях; − о последовательности отдельных операций и связи основного процесса с побочными процессами в отдельных операциях; − о типе (конструкции), числе и габаритах применяемых машин и аппаратов (реакторов). В этом пункте особенно важным являются способы (методы) вводы и выводы из системы (аппараты) энергии (например, тепла), вещества (особенно, если вещество в различных фазах), а также способы передачи «V» объема энергии и массы между фазами и потоками. − о типе (конструкции), числе и габаритах применяемых машин и аппаратов (реакторов). В этом пункте особенно важным являются способы (методы) вводы и выводы из системы (аппараты) энергии (например, тепла), вещества (особенно, если вещество в различных фазах), а также способы передачи «V» объема энергии и массы между фазами и потоками. Технологический процесс работающего предприятия (установки) должен быть представлен на схеме в таком виде, чтобы можно было с ее помощью рассчитать, анализировать и оценивать потоки масс и энергии. Все аппараты, входящие в технологическую схему, могут быть разделены на две группы: к первой относятся аппараты, в которых происходят физические процессы (механические, гидро- и аэродинамические, тепловые и диффузионные), ко второй – аппараты, в которых происходят собственно химические процессы (реакторы). где Q – теплота энергии. Предположим, что химическая реакция происходит в жидкой фазе при температуре 150 °С и атмосферном давлении. Реакция экзотермическая, с выделением количества тепла Q, Дж на каждый моль прореагировавших веществ. Продуктом реакции является смесь веществ А, В и С, поскольку никогда исходные вещества не прореагируют полностью и не подаются в реактор в эквимолярном соотношении. Для выделения продукта С можно использовать его большую летучесть по сравнению с веществами В. Это означает, что при данном давлении С кипит при более низкой температуре, чем А и В. На рисунке 2.17, приведена технологическая схема рассматриваемого процесса, содержащая 4 аппарата и 3 емкости, которые непосредственно к процессу не относятся, но необходимы для хранения сырья и продукта. Компоненты сырья А и В, а также смесь этих же компонентов, возвращаемая из кипятильника «Ku», подаются в реактор «Pe». В реакторе происходит 64 перемешивание реакционной смеси и непосредственно химическая реакция. Для поддержания температуры реакционной смеси на нужном уровне реактор окружен охладительной рубашкой, в которой происходит испарение воды. перемешивание реакционной смеси и непосредственно химическая реакция. Для поддержания температуры реакционной смеси на нужном уровне реактор окружен охладительной рубашкой, в которой происходит испарение воды. Рисунок 2.17. Функциональная схема технологического производства вещества с элементами управления процессом Рисунок 2.17. Функциональная схема технологического производства вещества с элементами управления процессом Рисунок 2.17. Функциональная схема технологического производства вещества с элементами управления процессом В ректификационной колонне «Ko» продукты реакции разделяются в результате многократного противоточного парожидкостного контакта фаз. Для организации этого контакта в колонну встроены 20 тарелок, на которых жидкость (жидкая фаза) взаимодействует с поднимающимися через нее пузырями пара (паровая фаза). Восходящий поток пара образуется в кипятильнике «Ku» за счет тепла паров воды, подводимой через поверхность теплообмена. Дистиллят (практически чистый компонент С) конденсируется в теплообменнике «Kg», а часть его возвращается в колонну и образует нисходящий поток жидкости, который на своем пути обогащается менее летучими компонентами А и В. Жидкость в кипятильнике (кубовый остаток) практически содержит только реагенты А и В. Энергия подводится к процессу в виде тепловой энергии пара и электроэнергии для привода насосов. Насосы, служащие для организации потоков реагентов А и В в реактор, вывода излишка кубового остатка из кипятильника, а также подачи охлаждающей воды в теплообменник конденсатор и рубашку реактора, на рисунке 2.17 не показаны. Особенностью приведенной схемы процесса является наличие технологических обратных связей − рециклов, введенных для лучшего использования сырья и энергии. В результате рециркуляции сырьевых компонентов А и В из кипятильника в реактор происходит практически полное использование сырья и отсутствуют побочные продукты. где Q – теплота энергии. Теплота реакции же 65 используется как дополнительный источник энергии в кипятильнике. В ректификационной колонне рециркуляция введена для организации многократного противоточного контакта жидкой и паровой фазы с целью получения продукта нужной степени чистоты (содержания примесей А и В). Описанный процесс относится к непрерывному во времени. В принципе можно на том же оборудовании провести и периодический процесс. Тогда смесь реагентов сперва загружается в реактор выдерживается при температуре 150 °С до достижения нужной концентрации продукта, а затем перекачивается в кипятильник. Следует процесс ректификации до тех пор, пока содержание примесей А и В в дистилляте не превышает допустимого. На этом заканчивается один цикл производства. Периодическая организация процесса применяется при небольших объемах производства, а также при необходимости использовать одни и те же аппараты для поочередного проведения нескольких различных технологических процессов, что характерно для фармацевтической и лакокрасочной промышленности, а также бытовой химии и промышленности тонкого химического синтеза. Обобщенно любой химико-технологический процесс можно представить состоящим из трех стадий: 1. подготовка сырья, 2. химическое превращение, 3. разделение продуктов реакции. В нашем примере специальных аппаратов для проведения первой стадии нет. Химическое превращение осуществляется в реакторе совместно с перемешиванием реагентов и подогревом их до нужной температуры. Таким образом, реактор служит как для подготовки сырья, так и для проведения самого химического превращения. Все остальные аппараты относятся к стадии разделения продуктов реакции. Как видно, непрерывные технологические системы являются типичной сложной системой и состоит: 1) из большого числа подсистем (машин, аппаратов), функционирующих для достижения общей цели (выпуска продукции заданного качества); 1) из большого числа подсистем (машин, аппаратов), функционирующих для достижения общей цели (выпуска продукции заданного качества); 2) сложность поведения системы обусловлена сложностью взаимосвязей между подсистемами. В модельном представлении материальные потоки рассматриваются как обобщенные (векторные) потоки, составляющими которых являются потоки массы, энергии, компонентов и импульса (количества движения). Исходя из термодинамических соображений (правило фаз), можно утверждать, что для полного описания состояния каждой фазы потока необходимо иметь N + 2 переменные, где N − число компонентов. При описании потоков применяются два альтернативных комплекта переменных термодинамического состояния: р р р 1) N+2 экстенсивные переменные (потоки N компонентов, энергии и импульса), р р р 1) N+2 экстенсивные переменные (потоки N компонентов, энергии и импульса), 2) N+1 интенсивная и одна экстенсивная переменная (поток массы, N = 1 мольная, весовая или объемная доля компонентов, температура и давление). 2) N+1 интенсивная и одна экстенсивная переменная (поток массы, N = 1 мольная, весовая или объемная доля компонентов, температура и давление). где Q – теплота энергии. Таким образом, в модельном представлении материальный поток 66 описывается вектором переменных из множества 2, причем выбор этого набора не однозначен. Эту неоднозначность можно использовать для получения наиболее удобной формы задания отношения Ф – множество переменных. Интенсивными называют «точечные!» переменные термодинамического состояния, значения которых не зависят от размеров системы (температура, давление, концентрация и т. п.). Величина экстенсивной переменной пропорциональна объему (массе) системы. Учитывая векторный характер обобщенных потоков, структура непрерывной технологической системы описывается потоковым мультиграфом ( ) сх Q  , вершинами i c x x  которого являются подсистемы, а дугами ij q Q  − компоненты обобщенных потоков. В качестве частей этого графа выделяется массовый потоковый граф Гm энергетический потоковый граф Гn и потоковые графы компонентов 1...( 1) j j IV    . На рисунке 2.18 приведен технологический потоковый граф с  рассматриваемого процесса. Этот граф содержит две новые вершины Ф-1 и Ф- 2, соответствующие которым аппараты на технологической схеме процесса отсутствуют. В фиктивных емкостях Ф-1 и Ф-2 происходит смешение входящих потоков воды и пара. р Пар Ф-2 В Ки Конденсат Пар Вода С Е-3 Кд Ко Ре Е-1 Е-2 Qi xi Вода Ф-1 А Ф-1, Ф-2 – фиктивные емкости Рисунок 2.18. Технологический потоковый граф процесса производства вещества «С» Ф-1 В Ф-1, Ф-2 – фиктивные емкости Ф 1, Ф 2 фиктивные емкости Рисунок 2.18. Технологический потоковый граф процесса производства вещества «С» , ф Рисунок 2.18. Технологический потоковый граф процесса производства вещества «С» 67 Наряду с технологическими потоками между подсистемами имеются неорганизованные потоки вещества и энергии между НТС и окружающей средой. Понятие окружающей среды относительно и зависит от того, какую часть воображаемой метасистемы мы отнесем к НТС. Дополнение НТС до метасистемы и составляет окружающую среду. В некоторых случаях окружающую среду агрегируют в одну подсистему и рассматривают как элемент НТС. Объем любой подсистемы может содержать несколько взаимодействующих или невзаимодействующих фаз (газ, жидкость, твердая, дисперсная и т.п.). Фаза понимается в принятом в термодинамике смысле как совокупность гомогенных частей системы, обладающих одинаковыми термодинамическими свойствами. В строгом смысле слова о фазах можно говорить только при термодинамическом равновесии системы или подсистемы. Признаком взаимодействия фаз является наличие между ними потока вещества или энергии, проходящих через поверхность раздела, причем направление этого потока (или потоков) в общем, не определено. Например, на каждой тарелке ректификационной колонны. взаимодействуют паровая и жидкая фаза разделяемой смеси, состоящей из веществ (компонентов) А, В и С. Поверхностью раздела фаз в данном случае является поверхность пузырей пара в жидкости. где Q – теплота энергии. Каждая фаза в свою очередь состоит из компонентов. Компонентом назовем совокупность частиц фазы (или потока), отведенных на основании какого-либо физико-химического признака или комплекса признаков к одному классу эквивалентности. Чаще всего компонент отождествляется с химически индивидуальным веществом, а рассматриваемая частица его с молекулами. Обозначим мощность множества компонентов K через N = \|К\| \| В результате приведенного НТС можно представить в виде иерархической структуры (дерева), состоящей из уровней системы, подсистем, фаз и компонентов (рис. 2.19). 68 (р ) Компоненты Фазы Подсистемы НТС N 3 4 3 2 2 1 1 1 2 n Рисунок 2.19. Дерево НТС НТС Подсистемы Фазы Компоненты Компоненты Рисунок 2.19. Дерево НТС Рисунок 2.19. Дерево НТС 68 На рисунке 2.20 приведены потоковые графы гипотетического технологического процесса. На рисунке 2.20 приведены потоковые графы гипотетического технологического процесса. На рисунке 2.20 приведены потоковые графы гипотетического технологического процесса. х1 − источник реагента А; х2 − емкость реагента А; х3 − емкость реагента В; х4 − источник реагента В; х5 − реакционный объем реактора; х6 − ректификационная колонна; х7 − трубчатка конденсатора дистиллята, х8 − емкость продукта С; х9 − емкость кипятильника; х10 − сток продукта, х11 − сток конденсата пара; х12 − трубчатка кипятильника; х13 − фиктивная емкость греющего пара; х14 − источник 13-атм пара; х15 − рубашка реактора; х16 − фиктивная емкость воды; х17 − емкость конденсатора дистиллята, х18 − сток охлаждающей воды; х19 − источник охлаждающей воды исунок 2.20 Материальный потоковый граф производства вещества «С 69 Рисунок 2.21. Тепловой потоковый граф процесса производства » Х20С – источник компонента «С» Рисунок 2.21. Тепловой потоковый граф процесса производства вещества «С» С» Х20С – источник компонента «С» Х20С – источник компонента «С» Х20С – источник компонента «С» 70 Рисунок 2.22. Потоковые графы компонентов процесса производства вещества «С» 2 22 П ф Рисунок 2.22. Потоковые графы компонентов процесса производства вещества «С» Рисунок 2.22. Потоковые графы компонентов процесса производства вещества «С» По сравнению с технологией добавлены фиктивные вершины источников и стоков сырья, энергии и продукта. Все подсистемы, в которых происходит теплообмен между двумя фазами через поверхность теплообмена (реактор, конденсатор дистиллята и кипятильник) разбиты на две самостоятельные подсистемы. Штрихованные вершины и дуги с крестиком указывают на наличие связанных с соответствующей подсистемой или потоком измеряемых величин (расходов, температур, уровней). Ввиду отсутствия непосредственного взаимодействия между реакционной смесью и охлаждающей водой, а также между кубовым остатком и подогревающим паром, материальный потоковый граф Гm состоит из двух связных компонентов. где Q – теплота энергии. Потоковые графы не содержат петель и поэтому могут быть однозначно заданы своими матрицами инциденций А: i j ij 1, если из вершины x в вершину x движется обобщенный поток q     y j i ji i j a = -1 если из вершины x в вершину x движется обобщенный поток q 0 если между вершинами x и x обобщенный поток отсутствует     i j 0 если между вершинами x и x обобщенный поток отсутствует  В каждом столбце матрицы. А имеется ровно два ненулевых элемента. Таким образом, матрица инциденций одного из связных компонентов 71 материального потокового графа, порожденного вершинами 1 10 x x  , имеет следующий вид и для лучшей наглядности показано только распределение ненулевых элементов матрицы 1 m A . Am1= q1 q2 q3 q4 q5 q6 q7 q8 q9 q10 q11 q12 x1 −1 x2 1 −1 x3 −1 1 x4 −1 x5 1 −1 1 1 x6 1 −1 1 −1 1 x7 1 −1 −1 x8 1 −1 x9 −1 1 −1 x10 1 Am1= q1 q2 q3 q4 q5 q6 q7 q8 q9 q10 q11 q12 x1 −1 x2 1 −1 x3 −1 1 x4 −1 x5 1 −1 1 1 x6 1 −1 1 −1 1 x7 1 −1 −1 x8 1 −1 x9 −1 1 −1 x10 1 В рассматриваемой схеме в аппарате (Ре) протекание процессов характеризуется одновременным протеканием процессов физических (передача импульсов движения, тепла и массы) и химических, что заставляет прежде всего познавать законы изменения скорости реакции в зависимости от условий работы реактора. При проведении химико-технологического процесса необходимо получить ответ на два вопроса, касающихся химической реакции: − какая предельная полнота протекания процесса, или каковы условия равновесия; − как быстро можно достичь желаемого приближения к условиям равновесия. На указанные вопросы можно получить ответы, применяя методы термодинамически «кинетически» макрокинетически процессов, как уже отмечалось. Термодинамика дает представление о возможности осуществления, а также о направлении развития, как самой химической реакции, так и отдельных ее этапов. Кроме того, термодинамика позволяет получить данные, необходимые для определения степени превращения при равновесии, теплового эффекта реакции, зависимости константы равновесия от температуры и т.д. однако термодинамика не определяет время, необходимое для получения заданной степени превращения: этот процесс нельзя решить и на основе одной только кинетики химической реакции. Химическая кинетика – учение о скоростях химических реакций. Под кинетикой реакции понимают, зависимость скорости данной реакции от концентрации, температуры, давления, механизма реакции. где Q – теплота энергии. На этом уровне рассматривается протекание химических реакций как явлений химического взаимодействия между атомами, свободными радикалами, молекулами, ионами-радикалами и различными комплексами. Считается, что реакционная система полностью однородна и протекание реакции зависит исключительно от 72 свойств, взаимодействующих частиц. Скорость реакции определяется законами химической кинетики свойств, взаимодействующих частиц. Скорость реакции определяется законами химической кинетики. химической кинетики. Химия Химическая термодинамика Стехиометрия Кинетика химических реакций Физика Законы сохранения Процессы переноса Теория открытых систем Физические методы исследования Инженерные науки Аэрогидродинамика Конструкторские особенности Материаловедение Экономика Экология Математика Качественные методы исследования Теория дифференциальных уравнений Стохастические методы Вычислительная математика Математическое моделирование Кинетическое уравнение Влияние процессов переноса Определение оптимальных условий Модель реактора Анализ модели и определение оптимальных условий Согласование результатов вычислительного эксперимента с натуральным Модель для проектирования и управления В А Рисунок 2.23. Методология получения математической модели любого реактора В Химия Химическая термодинамика Стехиометрия Кинетика химических реакций А Кинетическое уравнение Влияние процессов переноса Определение оптимальных усл Модель реактора Анализ модели и определение оптимальных условий Согласование результатов вычислительного эксперимента с натуральным Модель для проектирования и управления Рисунок 2.23. Методология получения математической модели любого реактора На последующем уровне рассматривается протекание химического процесса на молекулярном уровне. При этом учитывается взаимосвязь химической реакции с сопутствующими ей физическими явлениями молекулярного переноса вещества и теплоты. Скорость химического процесса определяется законами макрокинетики. Макрокинетика также рассматривает протекание химического процесса в масштабе реактора как взаимосвязь и взаимодействие химического процесса и крупномасштабных потоков вещества и теплоты в объеме аппарата, а также в зависимости от его конструкции. Принципиальная схема методологии получения модели реактора или процесса приведена на рисунке 2.23. Таким образом, при разработке химической технологии проектирование (выбор) химических реакторов одна из важнейших и труднейших задач, с которыми встречается инженер-химик. Химический реактор, помимо чисто кинетических аспектов, одновременно является и теплообменником и массообменным аппаратом, и ему часто присущи некоторые черты устройств, для перемещения потоков и транспорта твердого материала. Приходится нередко обеспечивать контакт между твердой, жидкой и газовой фазами, применять мешалки и другие подобные устройства, а также вести реакцию в условиях высоких температур и давлений. Возникают серьезные проблемы, связанные с контролем процесса. Наконец, требуется самый тщательный экономический анализ, чтобы получить максимум 73 продукции нужного качества с минимальными производственными затратами. продукции нужного качества с минимальными производственными затратами. Сложность проблемы исключает возможность стандартного решения. Реальное проектное решение обычно является сочетанием научно- теоретического анализа с творческой интуицией и практическим опытом, когда используются эмпирические данные, полученные на действующих заводских или специально построенных опытных установках. где Q – теплота энергии. Тем не менее, на современном уровне, разработан общий принцип анализа любой сложной системы (системный подход). Очевидно, что процессы, протекающие в реакторах, обычно представляют собой сложную систему в том смысле, что она сформирована из большого количества отдельных элементов, связанных между собой различными переменными. Такие системы будут характеризоваться ансамблями или группами элементов, объединенных материальными, энергетическими и информационными потоками. Для детальной характеристики систем в каждой ее точки вводятся так называемые «переменные состояния» (температура, давление, скорость потока, концентрации и т.п.). Эти переменные разделяют на три группы: 1) входящие переменные, которые действуют на всю систему; 2) возмущающие параметры, которые могут фиксироваться в определенных пределах; 3) выходные переменные, которые являются результатом действия двух вышеперечисленных. 3) выходные переменные, которые являются результатом действия двух вышеперечисленных. На каждом этапе исследований возможно различное разделение всей системы на подсистемы, изучаемые раздельно. Но вместе с тем, такое разделение ограничено, поскольку подсистемы должны исследоваться независимо друг от друга. В общем случае две системы являются независимыми, если их связи существуют только в одном направлении (от j к i, т.е. входные переменные системы i равны выходным переменным системам j). Поскольку все эти процессы не протекают независимо друг от друга, то сложность проблемы, связана с учетом их взаимодействий. Так, процессы переноса существенно зависят от гидродинамической обстановки внутри аппарата. Это означает, что масштабы и размеры установки, в которой протекает процесс, оказывают различное влияние на отдельно рассматриваемые явления. В таблице 2.1 отмечено влияние размеров аппаратов отдельные составляющие протекающих в них сложных процессов. Таблица 2.1 − Влияние размеров аппаратов и отдельные составляющие протекающих сложных процессов Явление Основные переменные Влияние размеров аппарата Химическая кинетика Т, С, Р* нет Термодинамика Т, С, Р нет Теплопередача локальн. скорости Р, С косвенное Массопередача в жидкой фазе т, С, турбулентность косвенное Межфазный перенос относительные скорости движения фаз, с, Т косвенное ца 2.1 − Влияние размеров аппаратов и отдельные составляющие щих сложных процессов 74 Продолжение таблицы 2.1 Принудительная конвекция скорости потоков, геометрия сильное Естественная конвекция Р, С, Т, геометрия определяющее * I − температура, С − концентрация, Р − давление Продолжение таблицы 2.1 Принудительная конвекция скорости потоков, геометрия сильное Естественная конвекция Р, С, Т, геометрия определяющее * I − температура, С − концентрация, Р − давление Для анализа поведения анализируемой системы необходимо записать ее математическую модель, которая обычно включает фундаментальные уравнения сохранения массы, энергии, количества движения. где Q – теплота энергии. Учитываются характер движущегося потока, перенос вещества за счет турбулентной диффузии, перенос тепла путем турбулентной теплопроводности, химическая кинетика и генерация тепла вследствие протекания химической реакции, влияние давлениями сипы тяжести, нестационарность процесса. Математическая модель может быть упрощена, если рассматриваются стационарные условия протекания процесса. Ряд упрощений делается на основании геометрии анализируемой системы. Если процесс протекает в многофазной системе, то балансовые соотношения должны быть записаны для каждой из фаз и учтены массо- и теплообмен между фазами. В уравнения математического описания входят два члена, не зависящие от размеров аппарата − это скорость химической реакции и источник (сток) тепла за счет химической реакции. Остальные составляющие сильно зависят от геометрии системы и должны быть исследованы раздельно путем проведения специальных экспериментов с системами, в которых химические реакции не протекают. р В лабораторных экспериментах выполняются измерения, характеризующие явления, не зависящие от размеров аппарата. Это касается термодинамики и кинетики собственно химических превращений. Кроме того, на этом этапе измеряются зависимости физических переменных (удельный вес, вязкость, удельная теплоемкость, коэффициенты межфазного равновесия и т.п.) от условий проведения процесса. Пилотные испытания представляют собой важный этап в изучении химических процессов. Пилотная установка является агрегатом, соответствующим реализации процесса в промышленных масштабах. При этом ее геометрические размеры, так и режимные переменные должны быть представимы с помощью разработанной математической модели изучаемого объема. Пилотная установка позволяет одновременное изучение физических и химических явлений, и устанавливать степень их взаимодействий. Размеры пилотных установок зачастую могут быть невелики, поскольку не преследуется цель − собрать данные для масштабирования. Следовательно, их размеры могут выбираться в соответствии с материальными ограничениями и минимизацией общих затрат на осуществление процесса. В крупномасштабных установках (в натуральную величину) целесообразно анализировать явления наиболее чувствительные к размерам, такие как гидродинамическая обстановка. Такие аппараты часто называют «холодными» 75 моделями. Моделирование в промышленных масштабах проводят в «смягченных» условиях (температура окружающей среды и нормальное атмосферное давление) и с использованием сред, физические свойства которых близки к физическим свойствам реагирующих смесей. Моделирование здесь основано на теории подобия, что позволяет осуществлять недорогие эксперименты в аппаратах различных размеров, начиная от пилотных и кончая близкими к промышленным. Таким образом, эффективным средством решения задач расчета, проектирования и масштабирования реакторов служат математические модели изучаемых процессов и объектов. При этом в математическую модель не обязательно можно входить большое количество алгебраических или дифференциальных уравнений, в особенности, в частных производных, если это является допустимым. Поскольку следует учитывать, что более сложные модели требуют для своей проверки большего количества экспериментальных данных. Кроме того, в процессе постановки технологических и экономических требований возможно принятие компромиссных решений поставленных задач. где Q – теплота энергии. Общая последовательность решения задачи включает следующие этапы: Сбор и анализ основных данных Сбор и анализ основных данных Анализ механизмов явлений Выбор технологии Построение модели Пилотная установка Проверка модели Экстраполяция Промышленная установка Модель в натуральную величину Лабораторная установка Рисунок 2.24. Общая последовательность решения задачи Анализ механизмов явлений Выбор технологии Построение модели Модель в натуральную величину Модель в натуральную ве Проверка модели Экстраполяция Рисунок 2.24. Общая последовательность решения задачи Частным вопросам формирования системного анализа химических и других важнейших процессов химической, пищевой, биохимической и перерабатывающей технологии посвящено ряд монографий известных ученых: М. Г. Слинько, В. В. Кафаров, И. Н. Дорохов, Р. Смит, Л. Л. Товажнянский, С. Вейлас, П. Бенедек и А. Ласко, А. Г. Бондарь, Р. Арис, А. И. Черевко и др. [28 − 32]. Современное развитие химической, металлургической, пищевой и перерабатывающих отраслей промышленности обязано именно умелому применению вышеотмеченных методов, однако все сильнее отмечается потребность в знаниях общего характера, в понимании существа химических 76 реакций и работы химического реактора с использованием однофазных, двух и трехфазных систем. В заключении можно сказать, что все, в чем мы нуждаемся для выработки адекватного описания реактора − это выражение кинетического закона r (X, T, P), которое соответствовало бы действительности. При обосновании выбора определенного типа реактора для проведения того или иного процесса необходимо учитывать возможность конструктивного осуществления этого реактора. Поэтому, для характеристики конструкции выбрано два определяющих критерия: В заключении можно сказать, что все, в чем мы нуждаемся для выработки адекватного описания реактора − это выражение кинетического закона r (X, T, P), которое соответствовало бы действительности. При обосновании выбора определенного типа реактора для проведения того или иного процесса необходимо учитывать возможность конструктивного осуществления этого реактора. Поэтому, для характеристики конструкции выбрано два определяющих критерия: р р р − режим движения реакционной массы в реакторе; − вид поверхности теплообмена. Первая характеристика позволяет классифицировать реакционные аппараты в соответствии с известными идеальными типами реакторов, устанавливая, таким образом, связь между кинетическими закономерностями процессов, протекающих в реакторах, и конструкцией последних. Для каждого из идеальных типов реакторов конструкция будет зависеть также от того, нужна ли поверхность теплообмена (наружная, внутренняя) или нет. Классификация реакторов по конструктивным формам корпуса не имеет в основе научных классификационных критериев, но близка к заводским методам группирования реакционных аппаратов (котлы, печи, трубчатки, колонны, шахты и т.д.). где Q – теплота энергии. По конструктивным формам основные типы реакторов группируются следующим образом: По конструктивным формам основные типы реакторов группируются следующим образом: 1) трубчатые (реакторы типа теплообменника); 2) колонные (включая аппараты с неподвижным или движущимся слоем твердой фазы); 3) реакторы типа реакционной камеры (с механическим перемешиванием или без него); ) 4) другие широко используемые типы реакторов (например, печи). Для каждой группы сформированной на основе классификации химических реакций и существующих типов реакторов разработаны конкретные методы термодинамического, кинетического анализа и построения математической модели процесса в промышленном реакторе и ее анализа [33]. Для каждой группы сформированной на основе классификации химических реакций и существующих типов реакторов разработаны конкретные методы термодинамического, кинетического анализа и построения математической модели процесса в промышленном реакторе и ее анализа [33]. 3 МЕТОДОЛОГИЯ И ОРГАНИЗАЦИЯ НАУЧНЫХ ИССЛЕДОВАНИЙ В ИНЖЕНЕРНОЙ ХИМИИ В предыдущих главах были приведены научные основы инженерной химии, которые органически связывают положения о возможности, условиях (скорости, параметры) протекания того или иного процесса (химической реакции) и объема, геометрии (конструкции процессорной единицы), а также аппаратурных потоков (структуры и скорости). Без предварительного рассмотрения указанных научных основ и характеристики того или иного научного направления, а также значимости 77 этого направления для общества, то есть человека, вопросы связанные с методологией научных исследований для конкретной области знаний не рационально. Это связано со стремительным научным и технологическим развитием общества во многих областях и направлениях науки.. ныне человечесвто овладело мощными силами, сравнимыми по масштабу с геологическими. Научные исследования проникают в тончайшие механизмы наследственности, становится возможным управления живых систем, и это в принципе, может привести и к коренным изменениям хода жизненных процессов на нашей планете. Сегодня стали актуальными не только вопросы о направленности технологического развития и о грядущих изменениях материального базиса общества. Не менее важно и то, как научно-техническая революция воздействует на самого человека, на систему его ценностей, на качество и форму организации его жизни. Делает ли это жизнь интересней, совершенней и значимой. Ведь если нет развития человека, тогда и сами технологические преобразования теряют смысл. Развитие человека связывают с целесообразным действием в той ил иной сфере. Обычно понятие «действовать целесообразно» включает умение охранять себя от разрушающих внешних воздействий или, скажем способность содействовать увеличению производительных сил и, следовательно, потреблению или способствовать содействовать своему размножению, а также усовершенствованию личности [34]. Однако этим много не объяснить, и в первую очередь − сам прогресс в науках о природе. Научная деятельность − высокоразвитый и специализированный вид творческой деятельности. р Она связана с высшими духовными устремлениями человека. Несомненно, что прогресс в рассматриваемой нами области науки (и других областях) зависит от развития производства, но эта зависимость сложна и неоднозначна. Важнейшие достижения фундаментальных наук о природе не есть непосредственное выполнение заказов производства. Тем не менее, производство обуславливает развитие научного познания главным образом тем, что проводит отбор наиболее плодотворных (приспособленных) научных достижений и дает новые орудия для научных исследований. Сами причины и механизмы научных открытий носят достаточно независимый характер, особенно это относится к развитию фундаментальных наук. Первичная цель фундаментальной науки, утверждает В. Вайскопф [35] − заключается не в ее приложении, а в проникновении в сущность причин и законов, управляющих процессами. Тем не менее, она, прежде всего, выступает как теоретическая основа непрерывного совершенствования материально-производственной деятельности человека. Но это не единственное ее назначение. Развитие науки есть самоподдерживающийся, самоорганизовывающийся процесс. Наука, по выражению Л. 3 МЕТОДОЛОГИЯ И ОРГАНИЗАЦИЯ НАУЧНЫХ ИССЛЕДОВАНИЙ В ИНЖЕНЕРНОЙ ХИМИИ Де Бройля – непрерывно кует материальное и духовное оружие, позволяющее ей преодолевать встающие на пути ее развития трудности, открывать для исследования неразведанные области [36]. Как отмечалось, в нашу эпоху методы науки проникают во все области жизнедеятельности 78 человека – и в материальное производство, и в организацию социальных процессов, ив духовную жизнь. Тем самым, научное творчество выступает как необходимая предпосылка сил каждого человека, его целостного развития. Новая система ценностей, как отмечает Н. Н. Моисеев, должна основываться на фундаментальной роли творческих процессов в жизнедеятельности человека [37]. Творчество образует важнейшую компоненту труда в любой отрасли – основной ценности человека. А наука, как важнейший вид творческой деятельности может внести существенный вклад в наиболее совершенную организацию человеческого бытия. человека – и в материальное производство, и в организацию социальных процессов, ив духовную жизнь. Тем самым, научное творчество выступает как необходимая предпосылка сил каждого человека, его целостного развития. Новая система ценностей, как отмечает Н. Н. Моисеев, должна основываться на фундаментальной роли творческих процессов в жизнедеятельности человека [37]. Творчество образует важнейшую компоненту труда в любой отрасли – основной ценности человека. А наука, как важнейший вид творческой деятельности может внести существенный вклад в наиболее совершенную организацию человеческого бытия. Все это убедительно говорит о том, что прогресс в области инженерной химии невозможно понять и обосновывать без научных исследований. Поэтому, вопросы методологии и организации научных исследований являются очень важными. Определение объекта, цели и постановка задач научных й Как было показано в предыдущих главах, всякий технологический процесс, для его проведения, взаимодействует с некими, назовем их материальными объектами переработки (энергией, сырьем, заготовками, деталями, урожаем, созревшим в поле, грузом, находящимся в пункте «А») и человеком. Нам нужно, чтобы при проведении технологического процесса в аппарате или машине, транспортируя исходные «материальные потоки» в конечные продукты; в процессе работы аппараты и машины должны работать качественно, экономично и производительно. Эти требования сегодняшнего дня, и, видимо, представляя себе облик техники завтрашнего дня или, наверное, лучше сказать послезавтрашнего дня, мы вряд ли захотим думать, что и тогда указанные процессы и аппараты будут иметь такие же недостатки, как и ныне. Технологические процессы и аппараты будущего видятся нам как поставляющие много качественной продукции и потребляющие мало энергии и сырья. При этом очень важно прогнозировать не только то, как технологический процесс и аппаратура будут справляться со своими обязанностями, а как будет смотреться рядом с ними человек, сколько хлопот они ему будут доставлять. Сегодня взаимоотношение человека и устройства выглядит парадоксально. Он создает его, управляет им и …. обслуживает, причем часто выполняя гораздо менее квалифицированную работу, чем творение его собственных рук. Нас вполне устраивает наша первая роль (хотя и тут многое можно поручить автоматам), не всегда вторая (здесь поле деятельности для автоматов беспредельна) и никоим образом третья. Как же должны предстать перед нами технологические процессы и аппараты при взгляде в будущее? Ответить можно очень кратко: «Нам хотелось бы, чтобы они работали «сами», не требовали обслуживания (во всех смыслах) и не ломались». Или, оставаясь на реалистической почве, скажем так: чтобы вмешательство человека в работу (включая подсобные и прочие операции) было минимальным, чтобы 79 меньше времени нужно было тратить на их техническое обслуживание, наладку, профилактику, чтобы они были более надежными, а их ремонты менее частыми, да и не такими сложными. Стоит еще добавить, что они не должны наносить ущерб окружающей среде и человеку, а отходы производства должны либо отсутствовать вообще, либо использоваться в качестве сырья в другом технологическом процессе. Последнее, кстати, относится ко всем деталям и самой технологии в целом, когда их ресурс выработан. р ур р И хотя полная реализация такой программы выглядит все еще фантастичной, многое из того, о чем мы мечтаем, создается в наше время, а иногда уже создана и ждет лишь своего внедрения (имеется ввиду, конечно массовое внедрение). Определение объекта, цели и постановка задач научных й И хотя полная реализация такой программы выглядит все еще фантастичной, многое из того, о чем мы мечтаем, создается в наше время, а иногда уже создана и ждет лишь своего внедрения (имеется ввиду, конечно массовое внедрение). Вышеотмеченное, по-видимому, является основной целью научных исследований в рассматриваемом научном направлении. Таким образом, в качестве объекта исследований могут рассматриваться тот или иной технологический процесс производства в целом; технологический процесс в одной или нескольких процессных единицах, отвечающих за качество продукта или производительность; конструкция и геометрия процессорной единицы (машины, аппарата, реактора); методы управления и автоматизации вышеотмеченных объектов и т.п. Научное исследование объекта – это соответственно исследование определенного предмета или явления в выбранном объекте с помощью научных методов, которая преследует цель установления закономерностей его возникновения, развития и преобразования в интересах использования в практической деятельности людей. Основным отличием предмета исследований от выбранного объекта исследований есть то, что предмет исследования есть частью объекта. То есть под предметом исследования подразумеваются определяющие с теоретической или практической точки знания свойства, особенности или стороны объекта. В каждом объекте исследований может быть несколько предметов исследования и концентрация исследователя на одном из них означает, что другие предметы исследования остаются в стороне от интересов исследователя и с его точки зрения закономерности выбранного предмета исследований могут оказать небольшое влияние на достижение цели. При формировании цели, исследователь предполагает, какой результат он хочет получить, и каким он может быть. Цель соответственно конкретизируется в задачах исследовании, которые обязательны для большинства исследований: 1) экспериментальная или исследовательски-экспериментальная проверка сформулированных целей; создание модели, позволяющей выполнять прогнозирующие расчеты; предложение по оформлению технологического процесса и апробация их в лабораторных условиях и на пилотной установке. 2) разработка на основе полученных результатов конкретных рекомендаций и выдача исходных данных для проектирования опытно- промышленной или промышленной установки, технологии, реактора. 2) разработка на основе полученных результатов конкретных рекомендаций и выдача исходных данных для проектирования опытно- промышленной или промышленной установки, технологии, реактора. Определение объекта, цели и постановка задач научных й Что касается содержания разделов, рекомендаций или исходных данных Что касается содержания разделов, рекомендаций или исходных данных 80 для проектирования, то в общем случае они должны содержать 17 общих разделов [38]: р [ ] 1) общие сведения о проектируемой технологии; 2) характеристика выполненных научно-исследовательских и опытных работ, положенных в основу исходных данных (в том числе обзор литературных и патентных данных); 3) технико-экономическое обоснование рекомендуемого метода производства, перспективы производства и потребления; 4) патентный формуляр и определение уровня разработки (обзор и обобщение зарубежных патентов и патентов Украины); 5) технологическая характеристика исходного сырья вспомогательных материалов основных и конечных продуктов, целевое назначение и области применения основных продуктов; 6) физико-химические константы и свойства исходных, промежуточных и конечных продуктов; 7) химизм, физико-химические основы и принципиальная схема производства (подготовка сырья, получение продукта, выделение продукта и подготовка к потребителю); 8) рабочие технологические параметры производства периодических или непрерывных процессов; 9) материальные и тепловые балансы производства; 9) материальные и тепловые балансы производства; 10) технологические характеристик побочных продуктов и реализация отходов производства. Области их применения и методы утилизации; 10) технологические характеристик побочных продуктов и реализация отходов производства. Области их применения и методы утилизации; 11) математическое описание технологических процессов и аппа 12) данные для расчета, конструирования и выбора основного промышленного технологического оборудования и защиты строительных конструкций; 13) рекомендации для проектирования автоматизации производства и управления; ; алитический контроль производства; у р ; 14) аналитический контроль производства; 15) методы и технологические параметры очистки химических и механически загрязненных сточных вод, обезвреживание газовых выбросов и ликвидация вредных отходов; 16) мероприятия по технике безопасности, промсанитарии и противопожарной профилактики; 17) указатель отчета и рекомендуемой литературы по рассматриваемой технологии производства. Каждый из указанных разделов содержит перечень обязательных пунктов, которые необходимо изложить в конкретном виде. Из вышеприведенного очевидно следует, что для выдачи исходных данных, которые есть основой для практического использования, необходима формулировка задач исследования таким образом, чтобы получить ответы на все пункты. Это утверждение относится и для тех случаев, когда усовершенствуется какая-либо стадия технологического процесса или отдельный аппарат. Таким образом, задачи исследования дают представления о том, что 81 необходимо делать, чтобы достигнуть цели. Задачи исследования – это выбор путей и способов (методов) для достижения цели в соответствии с выдвинутым предположением (гипотезой). Р. Шовен отмечал, что «наука изучает только то, что можно измерить, это совершенно правильно. Однако, при этом необходимо выделить то, что заслуживает измерения». Р. Шовен отмечал, что «наука изучает только то, что можно измерить, это совершенно правильно. Однако, при этом необходимо выделить то, что заслуживает измерения». Определение объекта, цели и постановка задач научных й у р Решение поставленных задач осуществляется согласно общей схемы проведения исследований, которая, как правило, включает последовательность выполнения тех или иных работ при решении задач. План-схема, как правило, включает анализ научно-технической информации; выбор или разработка методик проведения эксперимента, особенно экспериментальных установок и методов измерений параметров и контроля методов химического и физико- химического анализов; алгоритмов обработки экспериментальных данных и методов оптимизации и ряд других особенностей, которые зависят от типа исследуемого объекта и поставленной задачи. Следовательно, в какой-то степени составление вышеотмеченного плана-схемы есть определение метода исследования. Метод исследований – это совокупность приемов, определяющий практическое или теоретическое освоение действительности, которая подчинена решению конкретной задачи. р р р О. Мирабо утверждал «порядок и последовательность нужнее великих дарований для того, что бы хорошо управлять». Фактически разница между методом и теорией представляет собой функциональный характер, формируясь как теоретический результат предыдущего исследования, метод выступает как исходный пункт и условие будущих исследований. р р р О. Мирабо утверждал «порядок и последовательность нужнее великих дарований для того, что бы хорошо управлять». Фактически разница между методом и теорией представляет собой функциональный характер, формируясь как теоретический результат предыдущего исследования, метод выступает как исходный пункт и условие будущих исследований. Содержательная часть вышеуказанной схемы проведения исследований зависит, как уже отмечалось, от поставленной цели. Исследователь- экспериментатор перед составлением схемы должен выбрать и обосновать тип экспериментальных работ или подходов. Имеется ввиду, что эксперименты по своему содержанию и подходу можно классифицировать как: натуральные, модульные, активные и пассивные. При натуральном эксперименте исследуется реальное явление или процесс. Эксперименты, как правило, имеют цель уточнить характеристики процесса, оборудования или объекта, определить надежность его функционирования в непредусмотренных или аварийных решениях, проверку правильности теоретических положений и расчетов. При этом учитываются все факторы, которые влияют на объект. Натурный эксперимент требует больших финансовых затрат. Модельный эксперимент, как уже упоминалось в предыдущем разделе, осуществляется на модельных установках и позволяет полностью определить объект и процессы, протекающие в нем. При этом, в отличии от натурного эксперимента, тут есть возможность провести исследования в «чистом» виде, то есть изолировать исследуемое явление или процесс от вторичных факторов, которые влияют на его протекание. Результаты модельных экспериментов служат основой для создания математических моделей и использования технических результатов. Модельный эксперимент дает возможность 82 многократно повторять ход исследуемого процесса в строго фиксированных условиях. Пассивный эксперимент предполагает интуитивный выбор замера и контроля за процессом в факторном пространстве переменных. То есть, выбор проводится бессистемно. Определение объекта, цели и постановка задач научных й Активный эксперимент предполагает разделение точек замера и контроля в факторном пространстве в соответствии с некоторым алгоритмом, что обеспечивает, в конце концов, уменьшение замеров в 5 – 10 раз по сравнению с традиционными. Как при пассивном, так и при активном эксперименте устанавливается корреляционная связь между каждой измеряемой величиной и соответствующим ей математическим ожиданием другой величины. Эта связь представляется в виде уравнения регрессии. В заключение этого раздела отметим следующее. Чтобы действительно знать объект (предмет), надо охватить, изучить все его стороны, все связи и «опосредования». По-видимому, мы никогда не достигнем этого полностью, но требования всесторонности предостерегает нас от ошибки. Исследование без размышлений бесполезно, но и размышления без подтверждения экспериментом опасно, особенно для практики. Закончим раздел известным изречением Аристотеля: «Благо везде и повсюду зависит от соблюдения двух условий: правильное установление конечной цели; отыскание соответствующих средств, ведущих к конечной цели». 4 ИНЖЕНЕРНОЕ ОФОРМЛЕНИЕ ХИМИЧЕСКИХ РЕАКТОР 4 ИНЖЕНЕРНОЕ ОФОРМЛЕНИЕ ХИМИЧЕСКИХ РЕАКТОРОВ Техническая революция, имевшая место в средине и конце 20 века, охватившая электронику, энергетику, вычислительную технику… почти не затронула основные производства, обеспечивающие цивилизацию металлом, энергоносителями, различными химическими продуктами, новыми материалами (конкретные примеры: производство кальцинированной соды, производство извести, многих неорганических и органических продуктов). Технология, применяемая в настоящее время в металлургии, химии и различных областях энергетики, а также областях, связанных с утилизацией и переработкой отходов производства, сформировалась на базе научных концепций 20 века. Дальнейшее развитие промышленной базы на этом пути, вероятно, нецелесообразно. Естественно, выходом из сложившейся ситуации, очевидно, должен быть переход на новые технологические решения, которые должны базироваться на современных методах подготовки и обработки сырья, а также глубокого исследования структуры химических систем и получении надежной информации об их поведении. Последнее подчеркивает определяющую значимость математического моделирования реакторов. Построение математического описания вносит порядок в исследования. Если строить модель, которая должна предсказывать протекание процесса, позволять найти оптимальные условия и давать рекомендации об управлении, то мы вынуждены вести свои работы по определенному плану в заданной 83 последовательности и делать то, что нужно, решая на каждом этапе соответствующую часть общей задачи создания реактора. Метод математического моделирования заставляет проводить эксперименты целенаправленно. Наведение порядка и установление определенной последовательности исследований даст огромную экономию в сроках, усилиях и затратах. Кроме того, наличие адекватной математической модели процесса в реакторе позволяет решать задачи усовершенствования промышленной технологии и конструкции реактора. Современные подходы к развитию промышленной базы базируется на инновациях (нововведениях), которые являются результатом творческой деятельности и финансовых усилий, направленных на разработку, создание и распространение новых видов технологических процессов, изделий (продуктов технологии) и внедрение новых организационных форм. Инновации с позиции общечеловеческих ценностей тесно взаимосвязаны с химической технологией и экологией. Создание новых товаров и услуг базируются на использовании (и порой, весьма нерациональном) природных ресурсов (материальных, минеральных и топливно-энергетических). Применение новых технологий, требует соблюдения экологических нормативов загрязнения окружающей среды (воздушного бассейна, земли, воды, недр и т.п.), а также оптимально малых капитальных затрат. В свою очередь, как уже отмечалось, это связано с разработкой, проектированием, созданием и обслуживанием соответствующих технологических процессов и оборудования. р ру В данной главе описаны некоторые современные и многообещающие методы переработки сырья, методы выбора, расчета и проектирования химических реакторов, которые до сих пор рассматривались только в технических журналах или информативных листах и не реализованы в промышленных масштабах. Классифицировать, охватить и проанализированть все технологические процессы и реакторы в вышеуказанных производствах трудно, да и авторы не ставят перед собой такую цель. 4 ИНЖЕНЕРНОЕ ОФОРМЛЕНИЕ ХИМИЧЕСКИХ РЕАКТОР Создание нового технологического процесса и конструкции реактора не поддается шаблону, и для создания процесса и его реализации можно предложить много разных подходов и конструкций реакторов. В поисках оптимальной конструкции не обязательно останавливаться на наиболее дешевой. Предлагаемые решения могу иметь низкую стоимость, однако дополнительная переработка полученных продуктов и утилизация отходов может стоить довольно дорого. Поэтому, при проектировании нужно учитывать экономичность процесса в целом. Исходя из вышеприведенного, в данном разделе рассмотрены некоторые перспективные подходы к конструкциям реакторов для проведения реакций в гомогенной газовой фазе; проведение некаталитических реакций в системе газ (жидкость)-твердое тело; газ-жидкость; проведение газофазных реакций в присутствии твердых катализаторов, биологической очистки водных систем от органических соединений. 84 4.1 Реакторы для проведения реакций в потоке гомогенной газовой фазы Исследование газофазных химических процессов (особенно неизотермических реакций в потоке, что является наиболее распространенным случаем химической технологии) требует выяснения влияния изменения начальных условий (температура, соотношение концентраций, гидродинамических процессов и т.п.) на решение системы дифференциальных уравнений, представляющих собой математическую модель процесса. При этом основополагающим является выбор конструкции реактора, которая обеспечивает ту или иную гидродинамическую обстановку, влияющую на распределение температур, концентраций и других параметров. Реактор в данных технологиях рассматривается как объект управления процессом. Известно, что для описания объекта управления необходимо установить связи между его выходными и входами с учетом модели потока движения среды в реакторе. В данном разделе на примере реакции пиролиза метана в плазменной струе приведено такое исследование. Эффективность процессов, использующих так называемую низкотемпературную плазму («низко» − это несколько тысяч градусов) хорошо доказана и широко известна [39]. Неравновесная плазма позволяет получать водород прямым разложением воды, хорошо получаются в ней: металлы из галогенидов, сера из сероводорода (а заодно и водород), превосходно идет синтез гидразина, формальдегида, метилового спирта, гидратов, карбонилов, ацетилена [39]. Однако, техническая реализация достигнутого далеко не всегда поспевает за результатами лабораторных исследований. Особенно это заметно, когда дело доходит до крупнотоннажных процессов. Существующие ныне плазматроны классифицированы на две группы: электродуговые и безэлектродные. К последним относят высокочастотные и сверхчастотные генераторы плазмы, а также устройства для получения тлеющего разряда. Безэлетродные плазматроны пока не обладают большой единичной мощностью, поэтому, не смотря на успешное их применение в различных малотоннажных производствах, технологи уделяют основное внимание наиболее традиционным электродуговым плазматронам. Дуговой генератор плазмы, достигающий мощности 0,5 МВт и более (до 10 МВт), стал ныне стандартным, довольно компактным устройством. Такой аппарат, подключенный к камере смешения реактора, подает в нее через сопло плазмообразующий газ, нагретый до высокой температуры, достаточной для самых разнообразных технологических процессов. Однако, струя истекающая из сопла, весьма неоднородна: в ней неизбежны резкие перепады температур и скорости потока. Кроме того, она «закручена» − таков результат действия устройств газодинамической или магнитной стабилизации плазмы, необходимых для устойчивого функционирования плазматрона. Чтобы справиться с неоднородностью, сделать режим работы камеры более управляемым, плазматроны подключают к ней не поодиночке, а, как правило, по три на каждую горячую зону. На такой основе, создается реактор, 85 представляющий собой, в сущности, высоко-эффективную печь, в которой «плазменная специфика» вынесена на периферию. Требования, предъявляемые к ней, те же, с которыми технолог подходит к любой печи: устойчивость и равномерность (или, наоборот, управляется неравномерность) нагрева, минимум потерь тепла и как меньшая металлоемкость. Большинству из них реакторы с плазменным смесителем удовлетворяют успешнее, чем печи обычных конструкций. 4.1 Реакторы для проведения реакций в потоке гомогенной газовой фазы Дополнительным преимуществом новой технологии нагрева оказывается возможность наращивать мощность аппарата (процессорной единицы), компонуя многоступенчатые (многомодульные) нагреватели с шестью или даже девятью плазмотронами, по три в каждой ступени. При этом, вполне достижимо и управление структурой плазменного потока, поступающего в смеситель с мощностью несложных конструктивных решений можно добиться такого распределения температуры, какое наиболее благоприятно для того или иного химического процесса. Достигают этого, выбирая оптимальную форму камеры (реактора) и способ ввода в нее газовой струи. Камера может иметь форму цилиндра, конуса или комбинации цилиндр- конус, а струи можно вводить в нее как перпендикулярно к оси, так и под углом. Несколько вариантов схемы реактора показаны на рисунке 4.1. в) 3 5 4 1 а) б) в) г) д) 1 2 3 4 5 3 3 4 5 5 4 1 1 − плазматрон, 2 − камера (реактор), 3 − патрубок ввода сырья, 4 − ось плазматрона, 5 − граница плазменной струи (профиль) Рисунок 4.1. Схемы камер смешения различной геометрии: а, б, в, − конические с радиальным входом плазмы (углы ввода β = 60о, 45о, 30о соответственно; г, д − цилиндрические с радиальным и тангенциальным вводом струй плазмы) б) 3 4 5 а) 3 4 5 б) 5 г) 1 2 д) 1 − плазматрон, 2 − камера (реактор), 3 − патрубок ввода сырья, 4 − ось плазматрона, 5 − граница плазменной струи (профиль) Рисунок 4.1. Схемы камер смешения различной геометрии: а, б, в, − конические с радиальным входом плазмы (углы ввода β = 60о, 45о, 30о соответственно; г, д − цилиндрические с радиальным и тангенциальным вводом струй плазмы) Рисунок 4.1. Схемы камер смешения различной геометрии: а, б, в, − конические с радиальным входом плазмы (углы ввода β = 60о, 45о, 30о соответственно; г, д − цилиндрические с радиальным и тангенциальным вводом струй плазмы) 86 Каждый из них имеет свои преимущества и недостатки. В цилиндрической камере с радиальной подачей струи температура плазменного потока распределяется по поперечному сечению наиболее равномерно, в центральной его части − в ядре она постоянна (центральная часть около 80 % площади сечения) однако вблизи стенок газ интенсивно остывает. Стенки, естественно, охлаждают − иначе никакой материал не выдержал бы; охлаждение производят либо водяное, либо для уменьшения потерь, двойное (комбинированное), сначала воздушное, потом водяное. Если камера (реактор) − конус, температурный профиль усложняется, на нем возникает максимум, лежащий на оси (когда сопла плазматронов «смотрят» прямо на нее). Два максимума по бокам появляются, когда плазматроны присоединены по касательной к цилиндрической камере. 4.1 Реакторы для проведения реакций в потоке гомогенной газовой фазы В последнем случае на оси реактора образуется «температурный провал». Вышеотмеченное показано на рисунке 4.2. Т, К Т, К Т, К в) б) а) а, б − цилиндрическая и коническая с радиальным вводом плазменных струй, в − цилиндрическая, с тангенциальным вводом струй плазмы Рисунок 4.2. Температурные профили характерные для камер смешения с разной геометрией Т, К Т, К Т, К в) б) а) а, б − цилиндрическая и коническая с радиальным вводом плазменных струй, в й а, б − цилиндрическая и коническая с радиальным вводом плазменны а, б − цилиндрическая и коническая с радиальным вводом плазменных струй, в − цилиндрическая, с тангенциальным вводом струй плазмы Рисунок 4.2. Температурные профили характерные для камер смешения с разной геометрией а, б − цилиндрическая и коническая с радиальным вводом плазменных струй, в − цилиндрическая, с тангенциальным вводом струй плазмы Рисунок 4.2. Температурные профили характерные для камер смешения с разной геометрией Собственно полный реактор, в который подаются исходные соединения (в виде газа, паров, капель или пыли) представляет собой цилиндрический или 87 перфорированный канал, присоединенный соосно к камере смешения. Таким образом, реализуется прямоточная схема, при которой плазменный поток и подлежащее обработке вещество подают в реактор вместе через камеру смешения. В принципе, возможна и противоточная схема со встречной подачей плазменного потока и вещества. На рисунке 4.3 приведены варианты схем плазменных реакторов на основе многоструйной камеры смешения. 2 4 3 1 а ) б) в ) г) 2 4 1 3 4 3 1 2 1 4 2 4 2 4 3 1 а ) б) в ) г) 2 4 1 3 4 3 1 2 1 4 2 4 а – прямоточная; б – противоточная; в – прямоточная двухступенчатая; г – противоточная двухступенчатая; 1 – плазматрон, 2 – камеры смешения, 3 – реактор, 4 – подача исходных реагентов Рисунок 4.3. Схемы плазменных реакторов на основе многоструйной камеры смешения б) а – прямоточная; б – противоточная; в – прямоточная двухступенчатая; г – противоточная двухступенчатая; а – прямоточная; б – противоточная; в – прямоточная двухступенчатая; г – противоточная двухступенчатая; р , р , р р, д д реагентов Рисунок 4.3. Схемы плазменных реакторов на основе многоструйной камеры смешения Для реализации реакции пиролиза метана в потоке водородной плазмы выберем цилиндрическую камеру смешения с радиальным вводом плазменных струй с равномерным распределением температуры в ядре потока (рис. 4.3). 4.1 Реакторы для проведения реакций в потоке гомогенной газовой фазы Целью исследования конверсии метана в ацетилен является определение влияния начальной температуры Т(0), начальной скорости плазменной струи υ(0) и начальной концентрации метана С1(0) на максимум концентрации ацетилена C2(zm), длину реактора L и другие величины. Процесс синтеза ацетилена описывается системой химических уравнений: 1 2 3 4 2 4 2 2 4 2 2 2 2 2 2 2 2 2 k k k СН C H H C H C H H C H C H       . (4.1) (4.1) Поскольку, процесс нестационарный, то совокупности констант скоростей Поскольку, процесс нестационарный, то совокупности констант скоростей 88 реакции k1, k2, k3 зависят от температуры: реакции k1, k2, k3 зависят от температуры:   реакции k1, k2, k3 зависят от температуры:   реакции k1, k2, k3 зависят от температуры: 1 1 1 2 2 2 3 3 3 ( ) exp ( ) exp ( ) exp o o o E k T k RT E k T k RT E k T k RT                         . (4.2) 2 2 2 3 3 3 ( ) exp ( ) exp o o E k T k RT E k T k RT                 . (4.2) (4.2) При этом 13 1 8 1 6 1 1 2 3 4,5 10 , 2,57 10 , 1,7 10 ; o o o k с k с k с          E1 = 372,6 кДж/моль; E2 = 167,6 кДж/моль; E3 = 125,7 кДж/моль. При этом 13 1 8 1 6 1 1 2 3 4,5 10 , 2,57 10 , 1,7 10 ; o o o k с k с k с          E1 = 372,6 кДж/моль; E2 = 167,6 кДж/моль; E3 = 125,7 кДж/моль. j  − мольная доля компонента ( 1,2,3,4,5 j  ) соответственно (СН4; С2Н5, С2Н2; С; Н2). С2Н2; С; Н2). Численное интегрирование приведенной системы уравнений на ЭВМ при заданной совокупности констант скоростей реакции k1(T); k2(T); k3(T) позволяет меняя начальные условия Т(0), υ(0), 1(0) С установить зависимости решений этой системы уравнений для любого z от Т(0), υ(0), 1(0) С , поэтому принимаем в качестве входов в реактор величины Т(0) υ(0) 1(0) С Численное интегрирование приведенной системы уравнений на ЭВМ при заданной совокупности констант скоростей реакции k1(T); k2(T); k3(T) позволяет Численное интегрирование приведенной системы уравнений на ЭВМ при заданной совокупности констант скоростей реакции k1(T); k2(T); k3(T) позволяет меняя начальные условия Т(0), υ(0), 1(0) С установить зависимости решений этой й б Т С Численное интегрирование приведенной системы уравнений на ЭВМ при заданной совокупности констант скоростей реакции k1(T); k2(T); k3(T) позволяет меняя начальные условия Т(0), υ(0), 1(0) С установить зависимости решений этой системы уравнений для любого z от Т(0), υ(0), 1(0) С , поэтому принимаем в качестве входов в реактор величины Т υ 1(0) С качестве входов в реактор величины Т(0), υ(0), 1(0) С . В качестве выходов будут рассматриваться следующие величины, определяемые в точке достижения максимума концентрации ацетилена (zm): а) длина отрезка от начала реактора z = 0 до z = zm. Этот отрезок, на котором разыгрывается в основном процесс превращения метана в ацетилен, обозначим через «L» и является эффективной длинной плазмохимического реактора; а) длина отрезка от начала реактора z = 0 до z = zm. Этот отрезок, на котором разыгрывается в основном процесс превращения метана в ацетилен, обозначим через «L» и является эффективной длинной плазмохимического реактора; б) максимум концентрации ацетилена 3( ) m z C ; в) степень разложения метана: 1(0) 1( ) 1(0) m z C C S C   . 1(0) C Каждый из выходов является функцией всех входов, а также параметрически зависит от констант скоростей реакций k1, k2, k3, которые являются коэффициентами в вышеуказанной системе уравнений, то есть:   1(0) 1 (0) 0 1 2 3 , , , , , L F T C k k k        , 3 1(0) 2 (0) 0 1 2 3 , , , , , C F T C k k k         , (4.4) 1(0) 3 (0) 0 1 2 3 , , , , , S F T C k k k        . 4.1 Реакторы для проведения реакций в потоке гомогенной газовой фазы Используя данные работы [40], введя определенные приближения, модель рассматриваемого плазмохимического процесса может быть представлена системой обыкновенных нелинейных дифференциальных уравнений первого порядка: порядка: 1( ) 1 1( ) ( ) 2( ) 2 1 1( ) 2 2( ) ( ) 1 3( ) 3 2 2( ) 3 3( ) ( ) 2 4( ) 4 3 3( ) ( ) 3 5( ) 5 5 1 1( ) ( ) 1 1 ( ) 1 1 ( ) ( ) 2 1 ( ) ( ) 1 2 ( ) 1 ( ) z z z z z z z z z z z z z z z z z dC k T C dz dC k T C k T C dz dC k T C k T C dz dC k T C dz dC k T C dz                                              5 2 2( ) 3 3( ) 2 3 5 5 ( ) ( ) ( ) ( ) 1 1 5 5 ( ) ( ) ( ) ( ) 1 1 ( ) ( ) 2 ( ) ( ) 2 z z j z j z z i z j j j j z z j z z i j j k T C k T C dC dC N h RT z M dz dz dT z dz dU dC N C R M dT                                                                      , (4.3) где ( ) 1 , z d dz    2 5 ( ) ( ) 1 ( ), 2 2 j z z i j z N N RT M M               ; , j j j C U M N k   − р д 1( ) 1 1( ) ( ) 2( ) 2 1 1( ) 2 2( ) ( ) 1 3( ) 3 2 2( ) 3 3( ) ( ) 2 4( ) 4 3 3( ) ( ) 3 5( ) 5 5 1 1( ) ( ) 1 1 ( ) 1 1 ( ) ( ) 2 1 ( ) ( ) 1 2 ( ) 1 ( ) z z z z z z z z z z z z z z z z z dC k T C dz dC k T C k T C dz dC k T C k T C dz dC k T C dz dC k T C dz                                              5 2 2( ) 3 3( ) 2 3 5 5 ( ) ( ) ( ) ( ) 1 1 5 5 ( ) ( ) ( ) ( ) 1 1 ( ) ( ) 2 ( ) ( ) 2 z z j z j z z i z j j j j z z j z z i j j k T C k T C dC dC N h RT z M dz dz dT z dz dU dC N C R M dT                                                                      , (4.3) где ( ) 1 , z d dz    2 5 ( ) ( ) 1 ( ), 2 2 j z z i j z N N RT M M               ; , j j j C U M N k   − (4.3) 89 89 89 константы, определяемые из начальных условий; константы, определяемые из начальных условий; константы, определяемые из начальных условий; j  − мольная доля компонента ( 1,2,3,4,5 j  ) соответственно (СН4; С2Н5, С2Н2; С; Н2). 4.1 Реакторы для проведения реакций в потоке гомогенной газовой фазы j  − мольная доля компонента ( 1,2,3,4,5 j  ) соответственно (СН4; С2Н5, С2Н2; С; Н2). j  − мольная доля компонента ( 1,2,3,4,5 j  ) соответственно (СН4; С2Н5, С2Н2; С; Н2). (4.4) Ввиду того, что система уравнений не может быть решение аналитически и невозможно непосредственно определить функциональные зависимости. Ввиду того, что система уравнений не может быть решение аналитически и невозможно непосредственно определить функциональные зависимости. Однако, численное интегрирование нашей системы уравнений на ЭВМ позволяет решать следующие задачи: Однако, численное интегрирование нашей системы уравнений на ЭВМ позволяет решать следующие задачи: 1) Построить приближенные зависимости выходов от входов при заданном наборе констант k1, k2, k3. Эти зависимости являются статистическими характеристиками плазмохимического реактора при изменении совокупности констант k1, k2, k3 и тем самым рассмотреть вопрос о влиянии неточности при определении констант скоростей реакций на выходы реактора. Различия в значениях выходов при фиксированных входах, обусловленные изменениями 1) Построить приближенные зависимости выходов от входов при заданном наборе констант k1, k2, k3. Эти зависимости являются статистическими характеристиками плазмохимического реактора при изменении совокупности констант k1, k2, k3 и тем самым рассмотреть вопрос о влиянии неточности при определении констант скоростей реакций на выходы реактора. Различия в значениях выходов при фиксированных входах, обусловленные изменениями 90 той или иной константы скорости реакции, служат мерой чувствительности математической модели плазмохимического реактора. Если допустить гипотезу о стационарности модели потока в реакторе при различных функционированных υ(0) (эта гипотеза базируется на том, что изменение υ(0) не влияет на кинетику химических реакций, а лишь изменяет пространственные масштабы процесса и обоснована в работе [41]), то систему уравнений можно преобразовать и упростить. При этом в качестве независимой переменной удобно выбрать время η, связанное с переменной z, фигурирующей в уравнениях системы простым соотношением ( ) 1 z d dz    . Осуществляя замену переменной z и η, исключаем из системы уравнений υ(z). Проведенные оценки слагаемых: мы уравнений υ(z). Проведенные оценки слагаемых: системы уравнений υ(z). Проведенные оценки слагаемых: 5 ( ) ( ) ( ) 1 j z z z j j C RT    и 5 ( ) ( ) 1 j z z j C R    ; (4.5) (4.5) В интервале температур (1000 − 3500) К показано, что указанные члены пренебрежимо малы (порядка 10−3÷10−4) по сравнению со слагаемыми, входящими в числитель и знаменатель шестого уравнения системы. Поэтому становится возможным сокращения числителя и знаменателя этого уравнения   на общий множитель ( ) 2 z N M         . j  − мольная доля компонента ( 1,2,3,4,5 j  ) соответственно (СН4; С2Н5, С2Н2; С; Н2). Нелинейные зависимости энтальпии jh от Т в этом уравнении могут быть приближенно представлены в виде суммы 8 0 i j ji j i h h T h     с табулированными коэффициентами [42]. Анализ нелинейных зависимостей по табличным данным [42] в указанном диапазоне температур, позволяет провести кусочно-линейную аппроксимацию с приемлемой для данных расчетов точностью ( 1%)   в этом уравнении могут быть приближенно представлены в виде суммы 8 ур у р р д д у 8 0 i j ji j i h h T h     с табулированными коэффициентами [42]. Анализ б [42] 8 0 i j ji j i h h T h     с табулированными коэффициентами [42]. Анализ нелинейных зависимостей по табличным данным [42] в указанном диапазоне температур, позволяет провести кусочно-линейную аппроксимацию с приемлемой для данных расчетов точностью ( 1%) ( ) ( ) j T j j h a T b   , (4.6) ( ( ) j T j j h a T b   , (4.6) (4.6) где j a , jb − коэффициенты линейной аппроксимации. где j a , jb − коэффициенты линейной аппроксимации. j  − мольная доля компонента ( 1,2,3,4,5 j  ) соответственно (СН4; С2Н5, С2Н2; С; Н2). При переходе производных концентраций и температуры по времени ( j C , Т  ). При переходе роизводных концентраций и температуры по времени ( j C , Т  ). При п от участка к участку конечные значения переменных (Т, j C ) для предыдущего участка являлись начальными значениями этих переменных для последующего участка. Это обстоятельство, а также то, что диапазон изменения концентраций в массовых долях лежит в пределах 0 – 1, а температуры в пределах (1000÷3500) К, дает возможность отображать эти переменные (в отличие от  производных j C , Т  ) на всех участках в едином масштабе. Поэтому, в момент  перехода от участка к участку, изменяются скачком лишь ( ) ik T , j C и Т  . j  − мольная доля компонента ( 1,2,3,4,5 j  ) соответственно (СН4; С2Н5, С2Н2; С; Н2). Введенные замены и упрощения позволяют вместо системы уравнений получить следующую систему уравнений, описывающую плазмохимический процесс образования ацетилена из метана: 91 1( ) 1 1( ) 2( ) 2 1 1( ) 2 2( ) 1 3( ) 3 2 2( ) 3 3( ) 2 4 4 3 3( ) 3 4 ( ) 5 1 5 ( ) 1 5 ( ) 1 ( ) 1 ( ) ( ) 2 ( ) ( ) 2 ( ) ( ; ) ( ) ( ) j j j j j j j j dC k T C d dC k T C k T C d dC k T C k T C d dC k T C d dC dC d d dC a T b d dT A T d C a                                                                           , (4.7) (4.7) где ( ) A T −скорость изменения температуры, характеризующая действие внешних отрицательных (положительных) источников тепла (функция закалки). где ( ) A T −скорость изменения температуры, характеризующая действие внешних отрицательных (положительных) источников тепла (функция закалки). Уравнение шесть в системе отличается от соответствующего уравнения системы, так как было использовано уравнение материального баланса: 5 ( ) 1 1 j j C     . (4.8) (4.8) Спецификой плазмохимического процесса являются большие скорости протекания химических реакций и большие перепады температур. В связи с  эти, коэффициенты ( ) ik T и производные концентраций j C изменяются в очень широком диапазоне (до 14 порядков). Ввиду этого, моделирование необходимо проводить путем разбиения всего времени процесса на участки. Величина каждого участка выбирается так, чтобы в его пределах константы скоростей ( ) ik T изменялись на два порядка за счет соответствующего изменения температуры Т. Таким образом, нелинейные зависимости ( ) ik T задавались переменными от участка к участку масштабами. С масштабами, меняющимися от одного временного участка к другому, моделировались величины 92 производных концентраций и температуры по времени ( j C , Т  ). 4.2 Результаты решений системы уравнений без учета закалки [ 4.2 Результаты решений системы уравнений без учета закалки [а(т)] 4.2 Результаты решений системы уравнений без учета закалки [а(т)] В таблице 4.1 приведены варианты решения системы уравнений, 4.2 Результаты решений системы уравнений без учета закалки [а(т)] В таблице 4.1 приведены варианты решения системы уравнений, приведенных выше. В таблице 4.1 приведены варианты решения системы уравнений, приведенных выше. Таблица 4.1 − Варианты решения системы уравнений Характеристика реактора Начальные параметры Номер варианта 1 2 3 4 Диаметр реакционного пространства – 0,01 м  0 , Т К 3150 3500 4000 4500 Количество газа, проходящего через реактор – 2800 м3/ч (0,8 м3/с) (0) j С , масс. доли 0,95 0,9 0,99 0,99 Давление в реакторе – 0,18 МПа для преодоления сопротивлений в реакторе и последующих установках 5(0) С , масс. доли 0,05 0,1 0,01 0,01 Таблица 4.1 − Варианты решения системы уравнений Для всех вариантов решения систем начальные значения 2 3 4 (0) (0) (0) 0 С С С   . Обработка результатов дает возможность построить зависимости   1 3 1 (0) , (0) , 0 j m m S С С С C              при фиксированной начальной температуре. При неизменной характеристике реактора указанные зависимости приведены на рисунках 4.5, 4.6, 4.7. 93 0 2 4 6 8 0.1 0.2 0.3 0.4 0.5 0.6 , iвес. доли t, 10-4 сек 2 1 3 4 5 6 7 8 9 1, 4, 9 − 0,9 вес. долей; 2, 5, 8 – 0,95 вес. долей; 3, 6, 7 – 0,99 вес. долей Рисунок 4.4. Зависимость концентраций ацетилена (1 − 3), углерода (4 − 6) метана (7 − 9) от времени при начальной температуре 3500 К и различных ачальных концентрациях метана 0 2 4 6 8 0.1 0.2 0.3 0.4 0.5 0.6 , iвес. доли t, 10-4 сек 2 1 3 4 5 6 7 8 9 0 8 t, 10-4 сек 1, 4, 9 − 0,9 вес. долей; 2, 5, 8 – 0,95 вес. долей; 3, 6, 7 – 0,99 ве 1, 4, 9 − 0,9 вес. долей; 2, 5, 8 – 0,95 вес. долей; 3, 6, 7 – 0,99 вес. долей Рисунок 4.4. Зависимость концентраций ацетилена (1 − 3), углерода (4 − 6) и метана (7 − 9) от времени при начальной температуре 3500 К и различных начальных концентрациях метана 1, 4, 9 0,9 вес. долей; 2, 5, 8 0,95 вес. 4.2 Результаты решений системы уравнений без учета закалки [ долей; 3, 6, 7 0,99 вес. долей Рисунок 4.4. Зависимость концентраций ацетилена (1 − 3), углерода (4 − 6) и метана (7 − 9) от времени при начальной температуре 3500 К и различных начальных концентрациях метана На рисунке 4.4 видно, как и следовало ожидать, что с ростом температуры Т(0) максимальная концентрация ацетилена и степень превращения метана возрастают тогда как время достижения максимальной концентрации ацетилена падает. Из рисунка 4.5 следует, что с ростом начальной концентрации метана максимальная концентрация ацетилена и степень конверсии метана медленно убывают, а время достижения максимальной концентрации ацетилена возрастает. Возрастание начальной концентрации метана при постоянной температуре приводит к уменьшению теплосодержания плазменной струи, чем и объясняется вид кривых на рисунке 4.6. 94 1 2 3 4 5 6 7 8 9 50 100 2,80 3,15 3,50 0,5 4 5 10  3 10 0,4 3 o T(0),10 K S, % m τ , сек 3m C , вес. доли 1, 4, 9 − 0,9 вес. долей; 2, 5, 8 – 0,95 0,9 вес. долей; 3, 6, 7 – 0,99 0,9 вес. долей; m τ , сек 1, 4, 9 − 0,9 вес. долей; 2, 5, 8 – 0,95 0,9 вес. долей; 3, 6, 7 – 0,99 0,9 вес. долей; Рисунок 4.5. Зависимость величин 3m С (1 – 3), S (4 −6), ηm (7 − 9) от начальной температуры при различных начальных концентрациях метана 1 2 3 4 5 6 7 8 9 50 100 0,90 0,95 0,99 0,5 4 5 10  3 10 0,4 1 C (0), вес. доли S, % m τ , сек 3m C , вес. доли 1, 4, 9 – 3500 К; 2, 5, 8 – 3150 К; 3, 6, 7 – 2800 К 4 6 Зависимость величин 3 С (1 3) S (4 6) η (7 9) от 1 2 3 4 5 6 7 8 9 50 100 0,90 0,95 0,99 0,5 4 5 10  3 10 0,4 1 C (0), вес. доли S, % m τ , сек 3m C , вес. доли 1, 4, 9 – 3500 К; 2, 5, 8 – 3150 К; 3, 6, 7 – 2800 К , 1 C (0), вес. доли 1( ), д 1, 4, 9 – 3500 К; 2, 5, 8 – 3150 К; 3, 6, 7 – 2800 К Рисунок 4.6. 4.2 Результаты решений системы уравнений без учета закалки [ Зависимость величин 3m С (1 – 3), S (4 −6), ηm (7 − 9) от начальной концентрации метана при различных начальных температурах/ Рисунок 4.6. Зависимость величин 3m С (1 – 3), S (4 −6), ηm (7 − 9) от начальной концентрации метана при различных начальных температурах/ 95 Одной из основных стадий плазмохимического процесса является закалка целевых продуктов, физическая модель процесса закалки позволяет получить зависимость скорости понижения температуры от параметров, характеризующих плазменную струю и метод охлаждения. Задача состоит в отыскании закона закалки [A(T)]. Величина [A(T)], входящая в систему уравнений аппроксимируется следующими относительно простыми функциями [42]: 1 1 ( ) ( ) 0 f f f Т T при Т T А Т при Т T        , (4.9) 2 2 2 ( ) ( ) 0 f f f Т T при Т T А Т при Т T         , 3 3 ( ) ( ) 0 f f f Т T при Т T Т А Т при Т T           , 0 4( ) 0 f f А const при Т T А Т при Т T       , (4.9) , де α1, α2, α3 − постоянные величины, являющиеся параметрами закалки Tf − температура, до которой следует проводить закалку [выбирается из условий, чтобы время разложения целевых продуктов (С2Н2) при этой температуре существенно превышало время, необходимое для дальнейшего охлаждения плазменной струи после выхода из закалочного устройства];  3 1 ( ) Т k T   − время релаксации по отношению к разложению ацетилена.  3 1 ( ) Т k T   − время релаксации по отношению к разложению ацетилена.  3 1 ( ) Т k T   − время релаксации по отношению к разложению ацетилена. В данном разделе при математическом описании процесса с помощью ЭВМ были изучены законы закалки в широком диапазоне параметров A0, α1, α2, α3 для различных моментов ее включения. Для этой цели решались уравнения мы при следующих начальных условиях 1 5 (0) 3500 , 0,9, 0,1 T К С С    На рисунке 4.9 приведены зависимости концентраций ацетилена и углерода, сохранившиеся после закалки при законах А1(Т) и А4(Т) от начальной скорости закалки (А0) для различных времен включения. Из этих графиков видно, сто постепенное увеличение А0 при различных моментах включения закалки для каждого из законов может приводить к качественно различным результатам. Если закалка при законе А4 включается слишком рано ηвкл = 1,35·10−4 сек по отношению к моменту образования максимума ацетилена без закалки ηm = 4,85·10−4 сек, то сохраненная концентрация ацетилена падает с ростом А0; при этом, падает и концентрация углерода. Подобное поведение можно объяснить тем, что слишком раннее включение достаточно интенсивной закалки приводит к быстрому понижению температуры, что замедляет процесс 96 ревращения этилена в ацетилен, так и образование углерода. превращения этилена в ацетилен, так и образование углерода. превращения этилена в ацетилен, так и образование углерода. превращения этилена в ацетилен, так и образование углерода. Сплошные линии – результаты при постоянном законе закалки; пунктирные – при линейном законе; 5, 6, 11, 12 – время включения закалки tвкл = 1,35·10−4 сек; 2, 3, 9, 10 − tвкл = 2,85·10−4 сек; 1, 4, 7, 8 − tвкл = 4,35·10−4 сек Рисунок 4.7. Зависимость концентраций ацетилена * 3 С (1 − 6) и углерода * 4 С (7 − 12) от скорости закалки (А0) при начальных условиях Т(0) – 3500 К и 1(0) С = 0,9 вес. доли р р р у р Сплошные линии – результаты при постоянном законе закалки; пунктирные – при линейном законе; 5, 6, 11, 12 – время включения закалки tвкл = 1,35·10−4 сек; 2, 3, 9, 10 − tвкл = 2,85·10−4 сек; 1, 4, 7, 8 − tвкл = 4,35·10−4 сек Рисунок 4.7. Зависимость концентраций ацетилена * 3 С (1 − 6) и углерода * 4 С (7 − 12) от скорости закалки (А0) при начальных условиях Т(0) – 3500 К и 1(0) С = 0,9 вес.  3 1 ( ) Т k T   − время релаксации по отношению к разложению ацетилена. доли Сплошные линии – результаты при постоянном законе закалки; пунктирные – при линейном законе; 5, 6, 11, 12 – время включения закалки tвкл = 1,35·10−4 сек; 2, 3, 9, 10 − tвкл = 2,85·10−4 сек; 1, 4, 7, 8 − tвкл = 4,35·10−4 сек * С Сплошные линии – результаты при постоянном законе закалки; пунктирные – при линейном законе; 5, 6, 11, 12 – время включения закалки tвкл = 1,35·10−4 сек; 2, 3, 9, 10 − tвкл = 2,85·10−4 сек; 1, 4, 7, 8 − tвкл = 4,35·10−4 сек * Рисунок 4.7. Зависимость концентраций ацетилена * 3 С (1 − 6) и углерода * 4 С (7 − 12) от скорости закалки (А0) при начальных условиях Т(0) – 3500 К и 1(0) С = 0,9 вес. доли Рисунок 4.7. Зависимость концентраций ацетилена 3 С (1 − 6) и углерода * 4 С (7 − 12) от скорости закалки (А0) при начальных условиях Т(0) – 3500 К и 1(0) С = 0,9 вес. доли 97 4 -4 вкл t ,10 ,сек 3 C , вес.доли 4 C , вес.доли Сплошные линии – постоянный закон закалки; пунктирные – линейный 4, 6, 7, 8 – А0 = 106 град/сек; 2, 5, 9, 10 − А0 = 3·106 град/сек; 1, 3, 11 − А0 = 107 град/сек * вкл Сплошные линии – постоянный закон закалки; пунктирные – линейный 4, 6, 7, 8 – А0 = 106 град/сек; 2, 5, 9, 10 − А0 = 3·106 град/сек; 1, 3, 11 − А0 = 107 град/сек Рисунок 4.8. Зависимость концентраций ацетилена * 3 С (1 − 6) и углерода * Рисунок 4.8. Зависимость концентраций ацетилена 3 С (1 − 6) и углерода * 4 С (7 − 11) от времени включения, закалки и при Т(0) – 3500 К и * 1(0) С = 0,9 вес. доли 4 4 С (7 − 11) от времени включения, закалки и при Т(0) – 3500 К и * 1(0) С = 0,9 вес. доли 98 5 10 5106 106 107 А0, град/сек Δη, 10-4, сек 1 – при линейном законе закалки, 2 – при законе (19) Δη Рисунок 4.9. Зависимость Δη от начальной скорости закалки (А0) при tвкл = 4,35·10−4 сек; Т(0) = 3500 К, 1(0) C = 0,9 вес. долей 107 А0, град/сек 0, р д 1 – при линейном законе закалки, 2 – при законе (19) Δη Рисунок 4.9.  3 1 ( ) Т k T   − время релаксации по отношению к разложению ацетилена. Зависимость Δη от начальной скорости закалки (А0) при tвкл = 4,35·10−4 сек; Т(0) = 3500 К, 1(0) C = 0,9 вес. долей 98 Сравнение результатов закалки с постоянной скоростью охлаждения А1, для различных значений параметра А0 показывает, что при времени включения закалки tвкл = 4,35 10−4 сек. концентрация сохранившегося ацетилена с увеличением А0 растет быстрее, чем при времени включения tвкл = 2,85 10−4 сек. Это объясняется тем, что теперь закалка в меньшей мере препятствует разложению этилена и, следовательно, образованию ацетилена, но в то же время сохраняется в значительной мере образовавшийся ацетилен. При этом образуется несколько больше углерода, так как в этом случае он в большем количестве успевает образоваться до закалки. На рисунке 4.6 представлены зависимости концентраций сохраненного ацетилена ( 3 с  ) и углерода ( 4* с  ) от времени включения при различных концентрациях А0 для закона закалки А4. Из этого рисунка видно, что для малой постоянной скорости понижения температуры при закалке (А0 = 106 град/сек) кривая зависимости сохраненной концентрации ацетилена от момента включения закалки носит немонотонный характер, достигая максимума в промежутке 1,35·10−4 < ηвкл < 4,35·10−4 сек. При больших А0 концентрациях сохраненного ацетилена монотонно возрастает с приближением момента включения закалки к моменту ηm, а концентрация сохраненного углерода при этом возрастает с ростом tвкл для всех А0. Из рисунка 4.6 и 4.7 видно, что линейный закон закалки А1 приводит к аналогичным зависимостям, вызывая лишь некоторые количесивенные изменения по сравнению с законом А4, которые обусловлены смягчением режима закалки. Влияние начальной скорости закалки (А0) на длительность охлаждения плазменной струи до 1200 К (Δη) показано на рисунке 4.9. Как и следовало ожидать, для постоянного закона закалки кривая лежит ниже, чем кривая, соответствующая линейному закону. При этом наибольшее расхождение между кривыми достигается при минимальном значении А0 = 106 град/сек. При больших значениях А0 = 107 град/сек эти различия несущественны. 5 Результаты расчета показали, что при значениях A0≤105 град/сек закалка практически не обеспечивается. Таким образом, проведенные расчеты позволили оценить интервал значений для нижней границы допустимых начальных скоростей А0 понижения температуры при закалке. Не приведенные здесь результаты рассмотрения квадратичного закона закалки А2 существенно не отличаются, и данных для линейного закона закалки А1. Как и следовало ожидать, худшие результаты при прочих равных условиях дает закон закалки А3. Полученные результаты указывают на пути оптимизации процессов синтеза закалки в плазмохимическом процессе. 4.3 Проведение некаталитических реакций в системе газ−твердое Эти реакции лежат в основе широкого класса производственных процессов, протекание которых обязано нагреву обрабатываемого материала (реакционной смеси) до заданных температур с последующей выдержкой и охлаждением. Наиболее широко известные из указанных процессов – это технологические процессы получения цементного клинкера, процессы термического разложения различных видов карбонатного сырья с получением оксидов кальция (извести), оксидов магния (периклаза), процессы основанные на твердофазном взаимодействии в сложных системах (смесях) различных видов сырья, например,   2 3 2 2 2 K CO nH O TiO nH O mKCl    или TiO2 − кислородсодержащие соединения Ba, Al или смесь кислородных соединений Ba, Cu. Как правило, при твердофазных взаимодействиях получают сложные соединения со специфическими свойствами и в виде нитевидных кристаллов или волокон. К специфическим свойствам относятся: пористость продукта, удельная поверхность – активность, дисперсность, проводимость и др. [43]. Исследование вышеперечисленных технологических процессов и их усовершенствование осуществляется многие десятилетия. Однако, по- прежнему, актуальными вопросами остаются: минимизация энергетических затрат на производство продукции и получении продукции, отвечающей все возрастающим требованиям к ее качеству; которое формируется все более жесткими требованиями в стандартах целевой продукции потребителей. р р р у р В настоящее время вышеотмеченное является основной целью проводимых исследований в данном направлении. р р р у р В настоящее время вышеотмеченное является основной целью проводимых исследований в данном направлении. р р В данном разделе рассмотрен технологический процесс обжига гидрокарбонатов магния во вращающихся и полочных муфельных печах с получением оксида магния, отвечающего стандартам для металлургической промышленности [44]. В данном разделе рассмотрен технологический процесс обжига гидрокарбонатов магния во вращающихся и полочных муфельных печах с получением оксида магния, отвечающего стандартам для металлургической промышленности [44]. Ассортимент и квалификация оксида и гидроксида магния, производимого в мировой практике, связан в основном с их областями применения: продукты для производства трансформаторной стали; продукт-наполнитель для резин, адгезивов, пластических масс; продукты фармацевтической и фармакопейной квалификации; продукты высокой чистоты для производства химикатов; продукты для производства периклаза (огнеупоры); плавленый оксид магния ядерной квалификации. В настоящее время наибольшие объемы использования оксида и гидроксида магния наблюдаются в производствах трансформаторной стали, наполнителей резины, адгезивов, пластических масс, а также огнеупоров.  3 1 ( ) Т k T   − время релаксации по отношению к разложению ацетилена. Применение такого подхода отнюдь не ограничивается конкретной задачей и позволяет при наличии полной системы математического описания явлений без натурного эксперимента оценить, и проанализировать и прогнозировать развитие исследуемого процесса. 99 4.3 Проведение некаталитических реакций в системе газ−твердое 4.3 Проведение некаталитических реакций в системе газ−твердое Продукту (оксид магния) предъявляются следующие показатели качества: удельная масса – 2,32 г/см; содержание MgO – более 95 %; примеси марганца и меди не более 0,0016 %; насыпная масса – 350 – 400 г/дм3, растворимость в разбавленных кислотах – полная; остаток при просеве через сито: 4900 отв./см2 < 0,1; 16900 отв./см2 не более 0,2; определяющий размер частиц 0,05 – 0,2 мкм – 95 %; удельная поверхность по БЭТ – 185 м2/г; йодное число > 100; 100 нерастворим в воде. Для некоторых производств требуется удельная поверхность < 20 м2/г, содержание MgO ≈ 99,9 %, размер частиц 0,05 − 0,1 мкм. Указанные показатели качества MgO соответствуют требованиям к продукту, которые используются в производствах трансформаторных сталей (для термостойкого покрытия электротехнической стали перед двухстадийным отжигом, последняя стадия в атмосфере водорода при 1073 и 1423 К, а затем программированное охлаждение), в производстве титановых заготовок (для создания двухслойного покрытия для предотвращения окисления поверхности заготовок), резинотехнической промышленности, производства композитов на основе пластмассовой матрицы (наполнитель) и др. Исходя из вышеизложенного, очевидно, что общие требования к качеству MgO для всех сфер применения это высокая чистота продукта и содержание основного вещества (> 95÷99 %масс.), а также определяющий размер частиц (0,05 ÷ 0,2 мкм). Одновременно при достижении указанных общих требований в различных сферах применения требуется различная химическая активность продукта, критерием которой является величина удельной поверхности по БЭТ или йодное число [45]. Анализ известных технологий и опубликованных работ [46] показывает, что только при получении оксида магния путем термического разложения кислородсодержащих соединений магния (карбонатов, гидрокарбонатов) можно обеспечить требуемую чистоту сырья и контролировать развитие реакционной поверхности обжигаемого материала и его степень превращения во времени. Именно эта особенность при проведении термического разложения указанных соединений отмечена в работах американских ученых Грегга, Паркера, Уитли [47], а также Дзисько [48], как технологическая основа приготовления оксида магния с желаемой степенью превращения исходного материала и удельной поверхностью продукта. Процесс термической диссоциации кислородсодержащих соединений магния и его интенсивность определяется механизмом и кинетикой процесса, а также типом печей (реакторов) в которых осуществляется процесс [49]. Таким образом, производство оксида магния для металлургической, для резинотехнической промышленности, а также для применения в качестве ингредиентов и наполнителей должно осуществляться на основе термического разложения кислородсодержащих соединений магния, поскольку в этом случае, возможно контролировать и управлять достигаемой степенью превращения и величиной удельной поверхности продукта. Анализ также показал, что наиболее распространенным сырьем для производства оксида магния с вышеотмеченными свойствами является синтетические карбонаты MgCO3∙3H2O; 3MgCO3∙Mg(OH)2∙3H2O, а также гидроксиды Mg: Mg(ОН)2, Mg(ОН)2∙nН2О. 4.3 Проведение некаталитических реакций в системе газ−твердое синтетические карбонаты диссоциируют на MgО, СО2 и Н2О в интервале температур 673 − 773 К, а гидроксид разлагается на MgО и Н2О при 973 К. При этом во всех случаях в процессе диссоциации удельная поверхность твердого остатка проходит через максимум (Sm), кривая изменения имеет колоколообразный вид, а кинетические кривые топохимического процесса диссоциации описываются сигмовидными кривыми. Таким образом, для 101 получения продукта MgО требуемого качества необходимо создать такие условия, чтобы максимально возможная степень разложения сырья совпадала с максимально или минимально возможной величиной удельной поверхности твердого остатка реакции. Как правило, в промышленных условиях для осуществления процесса разложения сырья наиболее распространены и используются печи: муфельная вращающаяся, муфельная полочная, вращающаяся с непосредственным контактом материала и топочных газов [50]. Характер изменения степени разложения (интенсивность диссоциации) и развития удельной поверхности твердого остатка разложения зависят от условий подвода тепла к обжигаемому материалу и отвода газообразных продуктов реакции. Скорость подъема температуры в слое материала (скорость нагрева) и однородность температурного слоя в слое играют определяющую роль при реализации процесса. Как правило, в реакторах (печах) для проведения термического процесса разложения твердого реагента последний подается в печь в виде массы, состоящей из отдельных частиц 75 − 150 мкм. Карбонатное сырье имеет свободную влажность в пределах 10 − 40 %масс. и как следует из литературных источников [51] практически не подвергаются грануляции без специальных добавок ни во влажном ни в обезвоженном виде. Используя известный результат исследований [52], в котором предлагается возможность получения карбонатного сырья в виде гранул с достаточной прочностью за счет введения в сырье 1 − 5 %масс. связки в виде геля Mg(ОН)2 (размер частиц ~30Ǻ, соотношение твердого к жидкому в массе геля 1:(2÷3) и последующей обработки смеси в шнековой прессующей машине). Средний размер гранул, который обеспечивается на выходе из прессующей машины, составляет ~(5÷10) мм. Таким образом, в печи материал представлен в виде пористого слоя или гранулята. В зависимости от типа (конструкции) печи зернистый (гранулированный) материал движется в печи в соответствии с определенной моделью потока, которая в значительной степени определяет условия тепломассообмена в слое и, следовательно, эффективность реализации рациональных режимов обжига. Термическую обработку материала в слое допустимо рассматривать в два этапа: − равномерный нагрев слоя материала с требуемой скоростью до требуемой температуры (тепло-масообмен при достижении условий ,которые обеспечат начало протекания химической реакции); − равномерный нагрев слоя материала с требуемой скоростью до требуемой температуры (тепло-масообмен при достижении условий ,которые обеспечат начало протекания химической реакции); − тепло-массообмен при протекании химической реакции, в течении времени при достигнутой температуре. 4.3 Проведение некаталитических реакций в системе газ−твердое − тепло-массообмен при протекании химической реакции, в течении времени при достигнутой температуре. Для типов печей непрерывного действия, которые используются в производстве оксида магния, недостаточно полно исследуемым является первый этап, а для реализации предложенной технологии этот этап определяющий, так как от эффективности этого этапа зависят многие условия (равномерность температурного слоя, скорость нагрева и т.п.). Поэтому, необходимо проанализировать протекание первого этапа в вышеотмеченных 102 типах печей. типах печей. Напомним, что для обжига карбонатов магния применяют вращающиеся печи с обогревом слоя материала через стенку, а также при непосредственном контакте теплоносителя и слоя материала, а также печи механические полочные с обогревом материала через стенки. Рассмотрим, прежде всего, качественную сторону процесса смешения и перемещения материала во вращающихся печах. Можно считать, что смешение материала в поперечном слое барабана происходит за счет перемещения частиц в радиальном и угловом направлениях. Перемещение в радиальном направлении происходит в основном за счет того, что толщина скатывающегося слоя (отрезок CN рис. 4.11) меньше толщины поднимающегося слоя (отрезок CM), и поэтому несколько частиц (1, 2, 3) поднимающегося слоя попадают в один подслой скатывающегося слоя (1’, 2’, 3’). При повторном попадании в поднимающийся слой частицы могут изменить свое положение на 1’’, 2’’, 3’’. Угловое смещение частиц происходит за счет того, что частицы 4, 5 ,первоначально находящегося в одном радиальном сечении поднимающегося слоя, не одновременно переходят в скатывающийся слой (положение 4’, 5’), в результате чего, после скатывая, они попадают в разные радиальные сечения поднимающегося слоя (4’’, 5’’). В предлагаемой модели считаем, что поднимающийся и скатывающийся слои состоят из «n» подслоев равной объемной производительностью. Рисунок 4.10. Схема движения частиц сыпучего материала в поперечном сечении гладкого вращающегося барабана Рисунок 4.10. Схема движения частиц сыпучего материала в поперечном сечении гладкого вращающегося барабана Рисунок 4.10. Схема движения частиц сыпучего материала в поперечном сечении гладкого вращающегося барабана Каждый подслой состоит из Ni элементарных объемов i (i − номер подслоя 1 ≤ i ≤ n). Принимаем, что за один оборот вокруг центра циркуляции частица может перейти только в близлежайший «верхний» или «нижний» элементарный объем. Именно за счет этих переходов, осуществляется перемещение частиц в 103 радиальном направлении. Угловое смещение происходит за счет того, что число элементарных объемов в каждом подслое различно, и слои «проскальзывают» один относительно другого. Следует отметить, что проскальзывание только при скатывании, равно как и переход из одного элементарного объема в другой. Отсюда время смешения материала  см z  пропорционально радиусу реактора в сечении Z−R и ω − скорости вращения. Таким образом, во вращающейся печи, модель движения материала является близкой к типовой комбинированной модели смешений-вытеснения. типах печей. ТСТ, ТЯ – температуры стенок материала; Х, Т – координаты; q – общий тепловой поток от нагревателя; qэ – тепловой поток к стенкам печи ограничивающим реакционную зону; qν – тепловой поток в слое материала Рисунок 4.11 Схема тепловых потоков на полке механической полочной печи ТСТ, ТЯ – температуры стенок материала; Х, Т – координаты; q – общий тепловой поток от нагревателя; qэ – тепловой поток к стенкам печи ограничивающим реакционную зону; qν – тепловой поток в слое материала Рисунок 4.11 Схема тепловых потоков на полке механической полочной печи ТСТ, ТЯ – температуры стенок материала; Х, Т – координаты; q – общий тепловой поток от нагревателя; qэ – тепловой поток к стенкам печи ограничивающим реакционную зону; qν – тепловой поток в слое материала Рисунок 4.11 Схема тепловых потоков на полке механической полочной печи ТСТ, ТЯ – температуры стенок материала; Х, Т – координаты; q – общий тепловой поток от нагревателя; qэ – тепловой поток к стенкам печи ограничивающим реакционную зону; qν – тепловой поток в слое материала Рисунок 4 11 Схема тепловых потоков на полке механической полочной ТСТ, ТЯ – температуры стенок материала; Х, Т – координаты; q – общий тепловой поток от нагревателя; qэ – тепловой поток к стенкам печи ограничивающим реакционную зону; qν – тепловой поток в слое материала Рисунок 4.11 Схема тепловых потоков на полке механической полочной печи Рисунок 4.11 Схема тепловых потоков на полке механической полочной печи Рисунок 4.11 Схема тепловых потоков на полке механической полочной печи То есть, гранулированный (зернистый) материал достаточно хорошо перемешивается в поперечном сечении и с относительно небольшой скоростью перемешанные объемы без продольного смешения перемещаются вдоль печи. Практически, аналогичная картина движения материала наблюдается на каждой полке механической полочной печи, что в обоих случаях характеризуется как стержневой поток. Следовательно, при расчете теплообмена можно рассматривать случай неподвижного потока, или что одно и тоже для слоя, движущегося в стержневом потоке [53] через стенку. Исходя из этого, можно представить для случая обогрева через стенку схему тепловых потоков в печах следующим образом (рис. 4.12). 104 С q qэ Т   qv Тсл Тсг d0/2 Рисунок 4.12 Схема тепловых потоков в сечении слоя материала вращающейся печи (обозначения те же что и на рис. 4.11) С Рисунок 4.12 Схема тепловых потоков в сечении слоя материала вращающейся печи (обозначения те же что и на рис. 4.11) Рисунок 4.12 Схема тепловых потоков в сечении слоя материала вращающейся печи (обозначения те же что и на рис. типах печей. l − 0,015 м, и частицы размером (0,75÷1,5)·10−6 м толщина слоя  − Начальные условия: η = 0, Т = Т0 = f(х, у, z) , 0 − Граничные условия третьего рода:     0 0 n СТ n k T T n               Удельный тепловой поток «q» определяется по уравнению Удельный тепловой поток «q» определяется по уравнению   0 2 СТ СЛ Q q c Т Т F        , где Тст − температура стенки, где Тст − температура стенки, Тсл − температура слоя материала, с − теплоемкость стенки реактора,  − плотность материала стенки,  − плотность материала стенки, δ − толщина стенки обогрева. δ − толщина стенки обогрева. Печь муфельная вращающаяся: с − 0,12 кДж/кг град; типах печей. 4.11) В случае использования печи вращающийся с непосредственным контактом теплоносителя и материала схема потоков представлена на рисунке 4.13. ТМ ν q  С qэ qэ q Т1 φ 0 d /2 j j+1 l  GM CMi GM CMi-1 j+1 j противоток Gr Cri Gr Cri+1 Gг − массовый расход топочных газов с Тг, Gм − массовый расход обжигаемого материала; q − тепловой поток от топочных газов к материалу; qv − тоже в слое; qэ − тепловой поток к стенкам печи; Тг, Gм − температуры топочных газов и материала. Рисунок 4.13. Элементы вращающейся печи ТМ ν q  С qэ qэ q Т1 φ 0 d /2 j j+1 l  GM CMi GM CMi-1 j+1 j противоток Gr Cri Gr Cri+1 С Gг − массовый расход топочных газов с Тг, Gм − массовый расход обжигаемого материала; q − тепловой поток от топочных газов к материалу; qv − тоже в слое; qэ − тепловой поток к стенкам печи; Тг, Gм − температуры топочных газов и материала. Рисунок 4.13. Элементы вращающейся печи р Рисунок 4.13. Элементы вращающейся печи Нагрев слоя частиц (гранул) в случае обогрева через стенку базируется на модели теплопроводности, которая включает дифференциальные уравнения теплопроводности при отсутствии внутренних источников тепла: 105 2 2 2 2 2 2 T T T a Y Z                      , (4.10) (4.10) где Т – температура, η − время, 2 η время, а – коэффициент температуропроводности, м2/с. р , а – коэффициент температуропроводности, м2/с. − Условия однозначности, задаваемые в виде физических параметров слоя: − Условия однозначности, задаваемые в виде физических параметров слоя: λ − коэффициент теплопроводности Вт/м·град ≈ 0,12 Вт/м·град; С − теплоемкость [Дж/(моль·град)]; кДж/кг·град = 0,25; С − теплоемкость [Дж/(моль·град)]; кДж/кг·град = 0,25; ρ − плотность 1,7 г/см , форма гранул − цилиндры: dт= 0,01 м, ρ плотность 1,7 г/см , форма гранул цилиндры: dт= 0,01 м, l − 0,015 м, и частицы размером (0,75÷1,5)·10−6 м толщина слоя . Печь полочная: Печь полочная: с − 0,22 кДж/кг·град;  − 1,716 г/см3; δ − 0,2 м; с − 0,22 кДж/кг·град;  − 1,716 г/см3; δ − 0,2 м; δ , ; F − поверхность реактора, через которую идет обогрев. F − поверхность реактора, через которую идет обогрев. F поверхность реактора, через которую идет обогрев. Приближение к вышеотмеченным начальным условиям будет при Приближение к вышеотмеченным начальным условиям будет при 0 0 dT d     и при характеристике перемешанности материала, обеспечивающей ΔТСЛ в пределах 2÷3 градуса. Характерное время смешения определяется как: 2 1 ( ) ( ) ( ) см эф z R z x     , (4.11) (4.11) где R(z) − радиус реактора (определяющий размер в сечении z по координате Т; ; λэф − эффективный коэффициент теплопроводности по координате х, то 106 есть в горизонтальном направлении. Коэффициент λэф определяется из соотношения: из     2 2 2 1 1 2 2 эф q x x T T       . (4.12) (4.12) Значения, изменяющиеся во времени коэффициентов теплообмена К, определяются как частные от деления потока на разность между температурой стенки и температурой слоя. При этом принимаем, что теплообмен в начальный период времени η ≥ 0 и при больших значениях времени η >>> 0 определяется различным сопротивлением. Так при η >>> 0 лимитирующим есть сопротивление проникновения потока qv вглубь слоя (сопротивление проницаемости слоя). При η ≥ 0 теплоперенос лимитируется сопротивлением контакта твердого материала с греющей стенкой. Для первого случая усредненный по времени коэффициент теплообмена Ксл, рассчитывается, используя теорию теплопроводности Фурье:   2 сл л с К        . (4.13) (4.13) При η ≥ 0 согласно Тсотсас и Шлюндеру, эффективное сопротивление определяется сопротивлением слоя газа в узком зазоре между греющей стенкой и первым слоем частиц по нормали (координата Т). Исходя их этого, коэффициент теплообмена при η ≥ 0 будет: 4 2 1 ln 1 1 2 g T r T T l d К d d l                          , (4.14) (4.14) где λg − коэффициент молекулярной теплопроводности газа, где λg − коэффициент молекулярной теплопроводности газа, l − длина свободного пробега молекулы газа. l − длина свободного пробега молекулы газа. l − длина свободного пробега молекулы газа. S − площадь поперечного сечения слоя дисперсного материала, ( /d ) S − площадь поперечного сечения слоя дисперсного материала, Комбинации уравнений, представленных выше, полностью описывает теплообмен при нагревании слоя, а ее решение позволит определить температуру стенки и время материала до заданной температуры. р уру р р р ур Для анализа и численного решения на основании уравнений введем безразмерные переменные в виде: ; ; ; ( ); ; СТ СЛ СТ СЛ i Ч Ч T T Т Т Х X a A f B Т Т d L d           , (4.18) (4.18) где L − ширина поверхности нагрева, где L − ширина поверхности нагрева, Тч − температура гранулы (частицы). Тч − температура гранулы (частицы). В безразмерном виде для рассматриваемого случая модель имеет вид:   2 2 CT CT Ч A         ,   2 2 0 Ч T CT Ч эф СТ d A q F Т            , (4.19) 0 0 0 0 1; T v СТ Ч F L CT d T T          ,     6 1 эф T а K d A f     2 2 CT CT Ч A         ,   2 2 0 Ч T CT Ч эф СТ d A q F Т            , (4.19) 0 0 0 0 1; T v СТ Ч F L CT d T T          , (4.19) где     0 2 6 1 ; ; эф cc T i i i T а K d A f B B F B d             , λ − коэффициент теплопроводности гранул (частиц), λ − коэффициент теплопроводности гранул (частиц), λ − коэффициент теплопроводности гранул (частиц), фф р р у ( ) αэф − эффективная температуро-проводность слоя. αэф − эффективная температуро-проводность слоя. Печь полочная: Тогда общий коэффициент теплообмена системы стенка-слой (с-с) будет определяться суммой: (4.15) сс сл r К К К     , (4.15) сс сл r  , ( ) где θ − коэффициент формы равен 0,8. Условие, когда сс сл К К  характеризуется критическим временем: сс сл r где θ − коэффициент формы равен 0,8. Условие, когда сс сл К К  характеризуется критическим временем:   р   4 2 кр сс с К         . (4.16) (4.16) Балансовое уравнение по высоте слоя для единицы высоты слоя будет иметь вид:   Балансовое уравнение по высоте слоя для единицы высоты слоя будет иметь вид: 2 3 1 4 4 / 3 r R q S R z r R                        , (4.17) (4.17) где λ − коэффициент теплопроводности твердых частиц, , ( ) где λ − коэффициент теплопроводности твердых частиц, 107 S − площадь поперечного сечения слоя дисперсного материала, ( /d ) 0 0 2 2 2 2 2 0 0 0 1 1; , СЛ T СЛ T эф СТ СЛ СЛ Ч F CT d d q F L Т T X T L                         . (4.20) (4.20) Анализ уравнений показывает, что при малых временах (η≥0); F0<0,1 и диаметрах гранул < 7 мм тепловые потоки и температуры слоя частиц непосредственно у стенки могут быть вычислены по уравнению, при (F0<0,1) можно использовать модель. Температура в ядре для любых F0 оценивается по модели. сно критерию Фурье (F0) в зависимости от θсл можно представить в Согласно критерию Фурье (F0) в зависимости от θсл можно представить в виде: 0 2 0 4 1 1 ln сл x F                , (4.21) (4.21) 108 учитывая, что 0 2 эф T а F d   уравнения можно записать в виде: 2 0 2 1 1 1 ln T ф сл x э d а                          . (4.22 учитывая, что 0 2 эф T а F d   уравнения можно записать в виде: T 2 0 2 1 1 1 ln T ф сл x э d а                          . (4.22) (4.22) По формуле представленной выше необходимое время нагрева слоя до заданной температуры. При принятых начальных условиях задача становится внутренней и процесс нагревания слоя определяется только размерами гранул, части материала и толщиной слоя и его физическими свойствами. При этом в силу интенсивного теплообмена температура на поверхности гранул принимает значение близкое к температуре стенки, критерий Bi в этом случае ≤100 и cc сл K K  . Из приведенного анализа следует, что при выполнении заданных условий, при определении усредненных значений параметров системы для ансамбля рассматриваемых частиц, задача может быть сведена к одиночной пробной гранулы, или частицы, находящейся в фиктивной среде, теплопроводность которой при удалении от частицы стремится к λэф. Краевые условия на поверхности частицы (гранулы) должны учитывать поверхностную теплопроводность и скачок температур в области контактов гранул. S − площадь поперечного сечения слоя дисперсного материала, ( /d ) Значения λэф будет зависеть от типа упаковки частиц, а как известно последняя определяется их формой. Этот вывод не противоречит выводам работ [55], в которых исследовалась эффективная теплопроводность зернистых засыпок. Для случая, когда теплопроводность среды (газа) в порах слоя существенно больше теплопроводности материала, то есть λ0<<< λM задача существенно упрощается и может быть сведена к процессу нагревания твердого тела в зависимости от его формы и размеров, которая корректно решена и проанализирована во многих классических работах [56]. Выше представленный подход к анализу процесса нагревания слоя был применен и к случаю, когда в печи реализуется непосредственный контакт теплоносителя и материала. В силу эквивалентности математической стороны задачи получены системы уравнений. Отличие заключается в том, что в уравнениях были использованы безразмерные температуры слоя – θ, газа – θГ, частиц – θч, а в параметр «А» вместо критерия iB введен критерий Nu, который характеризует теплообмен на границе «поверхность гранулы-газ» ( T d Nu     ; λ − коэффициент теплопроводности газа). Анализ полученных моделей позволит считать, что для любых F0 температуру в слое можно определить из уравнения: 109 0 0 2 2 2 2 0 0 1 2 0 1; , T T эф с Г Г л F Т x Т d d q F Т L L                         . (4.23) (4.23) Таким образом, в результате расчета теплообмена в промышленных печах необходимо определить при нагреве через стенку Тст и Ксс, при нагреве топочными газами Тг и α – коэффициент теплоотдачи от газа к поверхности гранул. Исследования по нагреву материала в печах и обжигу проводили на полупромышленных установках, принципиальная схема которых представлена на рисунке 4.14. 1 – бункер, 2 – шнек-питатель, 3 – вращающаяся печь, 4 – циклон, 5 – вентилятор, 6 – приемные камеры, 7 – двухпроводная горелка Рисунок 4.14. Схема опытно-промышленной установки вращающейся печи для обжига тригидрокарбоната магния 1 – бункер, 2 – шнек-питатель, 3 – вращающаяся печь, 4 – циклон, 5 – вентилятор, 6 – приемные камеры, 7 – двухпроводная горелка Рисунок 4.14. Схема опытно-промышленной установки вращающейся печи для обжига тригидрокарбоната магния Установка работала следующим образом. Исходный материал из бункера 1 по шнеку-питателю 2 поступал во вращающуюся печь 3. Барабан печи имел длину 8 м, диаметр в свету − 0,5 м и наружный диаметр − 1м. S − площадь поперечного сечения слоя дисперсного материала, ( /d ) Скорость вращения барабана регулировалась с помощью привода ПИСИ и во время опытов составляла 4,5 об/мин. Печь обогревалась природным газом, сжигаемым в двухпроводной горелке 7. Дымовые газы последовательно проходили через загрузочную камеру, циклон 4 и выбрасывались вентилятором 5 в атмосферу. Готовый продукт − оксид магния − из печи поступал в холодильник, а затем на затаривание. Степень заполнения барабана печи варьировали в пределах от 0,02 до 0,15. Максимальная температура топочных газов составляла 1000 °С, в печи реализуется противоток Gм − Gг, скорость вращения барабана 0,22 м/мин 110 (3,6·10−3 м/с), угол наклона к горизонту − 1 градус, линейная скорость газовой фазы (топочных газов) 1,8 ÷ 2,5 м/с, среднее время пребывания материала в печи от момента загрузки до момента выгрузки 60,5 мин (3630 с). При проведении процесса во вращающейся муфельной печи функционирование установки осуществлялось по аналогии с вышеописанным. Максимальная температура стенки печи составляла 900 °С, максимальная температура газового пространства в сечении печи свободном от материала 800 – 850 °С, в печи реализуется прямоток Gм − Gотг. Угол наклона печи к горизонту − 1 градус, скорость вращения печи 0,4 − 0,7 м/мин [(6,6 − 11,6)·10−3 м/с]. Диаметр печи в свету d − 0,37, длина рабочей зоны − 5м, степень заполнения барабана − 0,05. Среднее время пребывания материала в печи от момента загрузки до момента выгрузки 36 мин (2160 с). При исследовании процесса нагрева материала до температур протекания химической реакции МgСO3 → МgO+СO2 (температура начала интенсивного химического «кипения», то есть выделения СО2) учитывали процесс образования гранул с определением температуры Тсл при которой происходило полное обезвоживание (в том числе и от связанной воды). На рисунке 4.15 представлены кинетические кривые сушки и нагрева материала до заданных температур сушки и нагрева материала до заданных температур Тсл = 400 и Тсл = 600 оС в муфельной печи с электрообогревом. 111 111 А – Тсл(1), Тсл(2) кривые подъема температуры слоя, соответственно при Тст = 900 оС и Тст = 700 оС, m1, m2 – кривые потери массы гранул (сушка), соответственно ТСТ, В – мгновенные значения коэффициента теплообмена между стенкой и подвижным слоем. Р 4 15 К А – Тсл(1), Тсл(2) кривые подъема температуры слоя, соответственно при Тст = 900 оС и Тст = 700 оС, m1, m2 – кривые потери массы гранул (сушка), соответственно ТСТ, В – мгновенные значения коэффициента теплообмена между стенкой и подвижным слоем. S − площадь поперечного сечения слоя дисперсного материала, ( /d ) А – Тсл(1), Тсл(2) кривые подъема температуры слоя, соответственно при Тст = 900 оС и Тст = 700 оС, m1, m2 – кривые потери массы гранул (сушка), соответственно ТСТ, В – мгновенные значения коэффициента теплообмена между стенкой и подвижным слоем. у Рисунок 4.15. Кинетические кривые сушки и нагрева слоя гранул с вращающейся муфельной печи Кривые 1, 2 соответствуют условию при Тст(1) и Тст(2) Кривые 1, 2 соответствуют условию при Тст(1) и Тст(2) Кривые 1, 2 соответствуют условию при Тст(1) и Тст(2 Dr выражается как: ( ) 1 0 0 r m b r b D для m m D для m m        , (4.25) (4.25) где mb − промежуточное содержание влаги, период постоянной скорости заканчивается, когда поверхностная влага достигает mb. Во время падающей скорости материал разбивается на сухую и мокрую зоны. На их границе, плоскости испарения, вода изменяет свою фазу. Уравнение для мокрой зоны такое же, как для периода постоянной сушки. Граничное условие: b m m  для l n  и с    r b dm dn D F m dl d                для l n  и с    , (4.26) где F − параметр потока, 0 0 z m n l F D          ; b m m  для l n  и с    r b dm dn D F m dl d                для l n  и с    , (4.26)   (4.26) где F − параметр потока, 0 0 z m n l F D          ; где F − параметр потока, 0 0 z m n l F D          ; n − положение плоскости испарения от греющей поверхности; n − положение плоскости испарения от греющей поверхности; ηc − безразмерное время конца периода постоянной скорости сушки. Соотношение между скоростью сушки и положением плоскости испарения при условии сухой зоны имеет вид: 0 1 ( ) i F F B l n   , (4.27) где g i v lv K H B M D R T           − число Био массопередачи; 0 1 ( ) i F F B l n   , (4.27) (4.27) Kg− пленочный коэффициент массопередачи, кг (Па·см2); Kg− пленочный коэффициент массопередачи, кг (Па·см2); Kg− пленочный коэффициент массопередачи, кг (Па·см2); Mv − молекулярная масса пара, кг/моль; 2 Dlv −коэффициент диффузии пара, м2/с; R − универсальная газовая постоянная, Дж/моль·К; T − абсолютная температура, К. T − абсолютная температура, К. Сухая зона рассматривается как дополнительное сопротивление массопередачи к сопротивлению мокрой зоны. Кривые 1, 2 соответствуют условию при Тст(1) и Тст(2 (4.24) (4.24) С начальными условиями: m = m0 при 0  и 0≤1≤1; граничные условия: 0 dm dl  для l = 0 и 0  , где m − содержание влаги * m m           , ρm − массовая С начальными условиями: m = m0 при 0  и 0≤1≤1; граничные условия: 0 dm dl  для l = 0 и 0  , где m − содержание влаги * m m           , ρm − массовая С начальными условиями: m = m0 при 0  и 0≤1≤1; граничные условия: 0 dm dl  для l = 0 и 0  , где m − содержание влаги * m m           , ρm − массовая С начальными условиями: m = m0 при 0  и 0≤1≤1; граничные условия: 0 dm dl  для l = 0 и 0  , где m − содержание влаги * m m           , ρm − массовая концентрация, кг/м3, * m  − массовая концентрация при насыщении, кг/м3, l − безразмерное расстояние Z H       , Z − расстояние от плоскости нагрева, м, H − D фф фф 2/ D фф толщина слоя, м, Dr − коэффициент диффузии влаги, м2/с, Dz0 − коэффициент диффузии влаги в период постоянной скорости, м2/с, τ − безразмерное время толщина слоя, м, Dr − коэффициент диффузии влаги, м2/с, Dz0 − коэффициент диффузии влаги в период постоянной скорости, м2/с, τ − безразмерное время 112 0 2 z D H        , η − время, с, F0 − интенсивность сушки 0 0 0 z m H F D           , 0  − 0 2 z D H        , η − время, с, F0 − интенсивность сушки 0 0 0 z m H F D           , 0  − 2 массовый поток, скорость сушки, кг/м2·с. Кривые 1, 2 соответствуют условию при Тст(1) и Тст(2 При анализе кинетических кривых (рис. 4.15) предполагали, что материал изотропен и размеры гранул остаются неизменными. Мигрирующим компонентом является вода, то есть протекание реакции MgCO3→MgO+CO2 не наблюдается. В период постоянной скорости процесс сушки описывали следующим уравнением: ( ) 1 r m dm d dm D dl dl          . Кривые 1, 2 соответствуют условию при Тст(1) и Тст(2 На рисунке 4.15 приведены мгновенные значения коэффициента теплообмена между стенкой и слоем обрабатываемого материала. Как видно из графиков (рис. 4.15) в начальный момент η = 0 значение «К» постоянны, а затем начинают уменьшаться. Обработка экспериментальных данных показала, что общий коэффициент «К» находится через составляющие Ксс и Ксл по формуле проводимости. Если время 113 контакта гранул со стенкой меньше критического, то теплоперенос лимитируется сопротивлением контакта (1/ Ксс), если больше, то сопротивление проницаемости слоя (1/ Ксл), ηкр, согласно (рис. 4.15) «В» оценивается величиной 180 – 235 с (~1,2 – 4 мин). Уменьшение величины ηкр с повышением Тст обязано увеличению λэф и, следовательно, уменьшению параметра ( ) см z  , что уменьшает сопротивление проникновения теплового потока в слой и повышению экспериментально наблюдаемых значений «К». Как следует из рисунка 4.15, получены типичные кинетические кривые процесса сушки. Интенсивность процесса обезвоживания зависит от Тст и значение параметра ( ) см z  . От этих значений Тст и ( ) см z  зависит период постоянной скорости на кинетических кривых и время достижения критической точки, когда начинает подыматься температура слоя, то есть время перехода к периоду падающей скорости. Эти данные подтверждают то, что параметр «с» не является подгоняемым под эксперимент, а действительно характеризует степень перемешенности зернистого слоя. Из данных приведенных на рисунке 4.16 наблюдается определенная связь между температурой слоя, коэффициентами теплопередачи и процессом массопередачи при сушке гранул. При этом можно выделить небольшой период постоянной скорости сушки, период проницания (П) и период регулярного режима (РР). Возникновение этих режимов, их длительность, а   ; 500 s i i v lv k H B B M D R T             также зависимость числа Bi от температуры ; 500 s i i v lv k H B B M D R T              позволяет считать, что кинетика сушки и сопротивление теплопередачи (коэффициент теплопередачи) взаимосвязаны и определяются Тст и параметром ( ) см z  . Кривые 1, 2 соответствуют условию при Тст(1) и Тст(2 Анализ экспериментальных результатов и моделей, представленных выше, позволяет считать, что массовое количество влаги F0 удаляемое с 1 м2 поверхности материала в единицу времени может быть определено как:   0 1 1 СТ нас исп g F Т Т H K k     , (4.28) (4.28) где исп H − теплота испарения влаги, Дж/кг; где исп H − теплота испарения влаги, Дж/кг; нас Т − температура насыщенного пара, К. нас Т − температура насыщенного пара, К. Это соотношение отражает совместное влияние процессов тепломассопереноса на кинетику сушки в исследуемом случае. Время сушки материала при постоянной скорости можно определить как: р р   0 6 m исп эф н сл F Т Т           . (4.29) (4.29) 114 Сравнение расчетных данных η с наблюдаемыми в эксперименте 114 Сравнение расчетных данных η с наблюдаемыми в эксперименте 114 показывают расхождение 10 – 15 %, что как отмечается в работах М. В. Лыкова [57] для таких процессов не допустимо. [57] для таких процессов не допустимо. 1', 2' – кривые подъема температуры Тсл, соответственно при ТГ = 1000 оС и ТГ = 800 оС; 1, 2 – кривые потери массы, соответственно при ТГ = 1000 оС и ТГ = 800 оС Рисунок 4.16. Кинетические кривые сушки и нагрева слоя гидрокарабоната магния во вращающейся печи с контактом материала и топочных газов. [ ] д р ц д у 1', 2' – кривые подъема температуры Тсл, соответственно при ТГ = 1000 оС и ТГ = 800 оС; 1, 2 – кривые потери массы, соответственно при ТГ = 1000 оС и ТГ = 800 оС Рисунок 4.16. Кинетические кривые сушки и нагрева слоя гидрокарабоната 1', 2' – кривые подъема температуры Тсл, соответственно при ТГ = 1000 оС и ТГ = 800 оС; 1, 2 – кривые потери массы, соответственно при ТГ = 1000 оС и ТГ = 800 оС Рисунок 4.16. Кинетические кривые сушки и нагрева слоя гидрокарабоната магния во вращающейся печи с контактом материала и топочных газов. Рисунок 4.16. Кинетические кривые сушки и нагрева слоя гидрокарабоната магния во вращающейся печи с контактом материала и топочных газов. На рисунке 4.16 приведены кинетические кривые сушки и нагрева слоя материала во вращающейся печи с контактом материала и топочных газов. Как следует из рисунка 4.16, характер влияния TГ и ( ) см z  на ход кинетических кривых аналогичен влиянию Тст, ( ) см z  . Кривые 1, 2 соответствуют условию при Тст(1) и Тст(2 Общий коэффициент теплопередачи «К» не зависит от времени достигая значений 104 Вт/м2·К, при этом значение 1 сл К стремится к нулю. Это означает, что тепловой поток беспрепятственно 115 проникает в слой материала и процесс нагрева лимитируется коэффициентом теплоотдачи от газа к поверхности гранулы. На кинетических кривых отсутствует явно выраженный период постоянной скорости сушки (практически незначительный – кривые 1', 2') и период проницания (П), что, по видимому, обязано низкому значению 1/ 0 сл K  . Если допустить, что Dr − const, то интенсивность выделения влаги можно определить как:   2 4 r B F D m m      . (4.30) (4.30) Это означает, что физически наблюдается почти регулярный режим сушки, который мало зависит (в исследуемом интервале от TГ и ( ) см z  ). Так при Dr = 1, mb = 0,2, m = 1 в области падающей скорости Bi, характеризующее массопередачу равно 1000, ( 0) 0 0,9 l m m    . Эти значения близки для двух случаев, когда TГ = 1000 oC и TГ = 800 oC. Полученные результаты не противоречат теоретическим основам процессов сушки и теоретическим основам условий теплопередачи в подвижных зернистых слоях [58]. Таким образом, основными результатами, вышеприведенных исследований, являются: определение зон сушки и нагрева материала до температуры синтеза (разложение MgCO3) исследуемых печах; установление основных закономерностей процессов тепло-массопередачи в процессе нагрева в рассматриваемой зоне печи и подтверждение адекватности предложенных моделей процессов на данном этапе. Кроме того результаты исследований свидетельствуют о методически правильном подходе к интенсификации процессов тепломассопереноса в печных агрегатах за счет обжига гранулированного материала в пористом подвижном слое. После достижения в печи (в слое материала) температура синтеза, то есть тмепература при которой протекает химическая реакция разложения MgCO3, в печном агрегате в определенной зоне происходит образование продукта реакции, его стабилизация и далее выгрузка из печи в холодильник. В этой зоне печи кроме влияния температуры слоя Тсл, его структуры и условий смешения ( ) см z  существенное влияние будет оказывать состав газовой атмосферы над слоем и его порах. Моделирование процесса термического разложения при указанных условиях и возможность получены уравнения для прогнозированного расчета степени превращения при обжиге в этой зоне печи позволит провести расчеты различных режимов и их оптимизацию, выявить пути интенсификации процесса и т.п. В литературных источниках модель, учитывающая влияние всех вышеотмеченных факторов для рассматриваемых условий (модели): движение материала в печи отсутствует. Кривые 1, 2 соответствуют условию при Тст(1) и Тст(2 Имеются лишь указания [59], что модель превращения частицы (гранулы), реагирующей в атмосфере газовой смеси, должна учитывать все элементарные массообменные стадии реакции: сорбция – десорбция реагентов и продуктов реакции обеих фаз, их взаимодействие и диффузия в грануле. Поэтому ниже представлена математическая модель 116 процесса, которая позволяет проанализировать поведение процесса, уточнить рациональные условия технологического процесса. При построении модели полагали, что имеется N газов-реагентов и образуется соответственной N газообразных продуктов реакции. При этом химическая реакция взаимодействия твердого реагента с каждым газом- реагетом осуществляется значительно быстрее, чем процессы массопереноса, и фронт реакции разделяет области исходного реагента и твердого продукта реакции. Далее составляем уравнение кинетики всех стадий процесса. Определяя iS и iS , как доли площади поверхностного слоя, заполненные i-тым реагентом и соответствующим продуктом реакции, будем считать, что есть «свободная» поверхность ΔS, не заполненная молекулами реагентов и продуктов реакции. В предложении о независимой (легмюровской) адсорбции газов твердым реагентом уравнение кинетики заполнения поверхностного слоя имеют вид: i i i i i dS S S d         * * * , 1..... i i i i dS S S i N d        (4.31)   * 1 1 N i i i S S S     (4.31) где αi - константа скорости адсорбции из газовой фазы, где αi - константа скорости адсорбции из газовой фазы, где αi - константа скорости адсорбции из газовой фазы, βi - константа скорости адсорбции в газовую фазу, βi - константа скорости адсорбции в газовую фазу, η - время, р , δ - приращение соответствующих величин. Поверхностные концентрации i nC и * i nC реагентов и продуктов реакции равны 2 i i S l и * * i i S l соответственно. Здесь l - характерный линейный размер молекулы в поверхностном слое молекулы в поверхностном слое. молекулы в поверхностном слое. Уравнение кинетики обмена поверхностного слоя с твердой фазой: 2 o i i li i i i oi i i r R m C k m k l C S D r            * * * * * * * * 2 o i i li i i i oi i i r R m C k m k l C S D r             . (4.32) (4.32) * * ; ; 1....... Кривые 1, 2 соответствуют условию при Тст(1) и Тст(2 o o oi i r R oi i r R C C C C i N      Граничные условия на фронте реакции Граничные условия на фронте реакции  * * 0, , , 1.... i i i i i i C C C D D r R i N r r               где Ci - концентрация, где Ci - концентрация, 117 i  - соответствующие стехиометрические коэффициенты, i  - соответствующие стехиометрические коэффициенты, R R0 - радиус фронта реакции и частицы соответственно R, R0 - радиус фронта реакции и частицы соответственно, D - коэффициент диффузии, R, R0 - радиус фронта реакции и частицы соответственно, D - коэффициент диффузии, R, R0 - радиус фронта реакции и частицы соответственно, D фф фф k1, k2 - константы скорости адсорбции и десорбции в твердую фазу. Из условий материального баланса на фронте реакции имеем   1 N i i r R i dR C D d r            , (4.33) (4.33) где θ - концентрация реагента, присутствующего в твердой фазе, умноженная на сумму стехиометрических коэффициентов отдельных химических реакций. где θ - концентрация реагента, присутствующего в твердой фазе, умноженная на сумму стехиометрических коэффициентов отдельных химических реакций. Учитывая, что θ>>Coi, получаем, что времена релаксации концентрационных полей реагентов и продуктов реакции значительно меньше времени продвижения фронта реакции. Считая процесс квазистационарным, получим:       * * * , ; , i i oi i oi oi i D C C F r R C C C F r R D                 1 1 1 1 , , 1..... o F r R i N R r R R                 . Кривые 1, 2 соответствуют условию при Тст(1) и Тст(2 (4.34) (4.34) Введя безразмерные переменные с выделением газообразного реагента, давление которого в газовой смеси отлично от нуля (например, і = 1) в виде: R Введя безразмерные переменные с выделением газообразного реагента, давление которого в газовой смеси отлично от нуля (например, і = 1) в виде: 1 1 , , i i o R t R          2 2 1 1 , i i i i o o i i i b l Z b l                             1 2 1 , i i i o i i i k k l R K L D      (4.35) 2 1 , 1..... i o i M i N R l      . (4.35) Преобразуя систему уравнений с учетом Ki и Mi получаем: Преобразуя систему уравнений с учетом Ki и Mi получаем: 118         1 2 1 1 1 * * 1 1 1 1 1 1 1..... , 1 1 1 i i i i i i i N i i i i i N o i i i i i i i i i i dS K S b S S dt L S M K d S dt L S i N S S S dS Z K b S S dt L S                                                . (4.36) (4.36)   * * * 1 1 1 i i i i i i i dS Z K b S S dt L S          Начальные условия: * * 0, 1..... , 1, 0 i i o S S i N t t        * 0, 1..... , 1, 0 i i o S S i N t t        Проанализируем влияние состава газовой фазы на кинетику процесса. Кривые 1, 2 соответствуют условию при Тст(1) и Тст(2 Введем: 1 i i i i i i b b X L S k k     , , 1,2 i i i i i i i b Y L Z M i k       , (4.40) где ΔS определена по формуле 1 * 1 1 1 N i i i i i i i Z k b k b                          и, интегрируя 1 i i i i i i b b X L S k k     , , 1,2 i i i i i i i b Y L Z M i k       , (4.40) о формуле 1 * 1 1 1 N i i i i i i i Z k b k b                          и, интегрируя (4.40) где ΔS определена по формуле 1 * 1 1 1 N i i i i i i i Z k b k b                          и, интегрируя где ΔS определена по формуле 1 * 1 1 1 N i i i i i i i Z k b k b                          и, интегрируя последнее уравнение получаем уравнение движения фронта реакции:       2 2 3 3 1 2 1 2 2 2 1 2 2 1 2 2 1 1 2 2 1 1 1 1 1 3 2 X X X Y Z X Y Z X Z X Z X Z X Z                                  2 2 3 3 1 2 1 2 2 2 1 2 2 1 2 2 1 1 2 2 1 2 2 3 1 2 1 2 2 1 1 2 1 2 2 1 1 2 2 1 3 4 1 1 2 1 1 2 2 1 1 1 2 2 1 1 2 2 1 1 2 2 1 1 2 2 1 1 1 1 1 3 2 1 ln 1 o X X X Y Z X Y Z X Z X Z X Z X Z Z Z X Y X Y Z Z X Y X Y Z Y Z Y X Z X Z X Z X Z Z Y Z Y Z Y Z Y S t t X Z X Z X Z X Z                                              . Кривые 1, 2 соответствуют условию при Тст(1) и Тст(2 В начальный момент при малых «t» получим: * 2 1/3 2 1 , 2 3 1 2 i i i i i i N i i i i Z K S t S t M K t                                        . (4.37) (4.37) Здесь и далее рассматриваем степень превращения А = 1 − ξ. В результате получаем: Здесь и далее рассматриваем степень превращения А = 1 − ξ. В результате получаем: 2 2 2 1 1 2 1 3 3 2 2 3 N N li i i i i i i o i N li i o i k A M K t R l k dA d R l                                                   . (4.38) (4.38) Таким образом, на начальном этапе наблюдаем зависимость скорости процесса от особенности стадии адсорбции твердым реагентом газового реагента (от величины коэффициента скорости адсорбции и размера площади, занимаемой адсорбированными газовыми молекулами). Как известно, коэффициенты скорости адсорбции зависят от состояния поверхности твердого реагента и свойств адсорбционных молекул. Как вытекает из анализа и свойств газовых молекул определяются электронной структурой и способностью присоединять или отдавать электроны. Размер площади, занимаемой адсорбированной молекулой зависит от ее молекулярной массы и ее структуры, что согласуется с мультиплетной теорией катализа (Баландина). В простейшем случае (рассматриваемом в работе), когда адсорбция проходит без диссоциации 119 адсорбированных газовых молекул, αi - пропорционально парциальному давлению i-го реагента, следовательно, добавление газового реагента, являющегося более активным в процессе сорбции, при сохранении постоянным общего давления газовой фазы, позволяет существенно увеличить скорость термического разложения MgCO3 в начальный момент времени. На завершающих этапах процесса термического разложения твердого реагента при ξ−1>>1 * 0, 0 i i i i S S b S     . (4.39) 1 1 N i i i i i i M K d dt b L                   . Кривые 1, 2 соответствуют условию при Тст(1) и Тст(2 (4.39) Поскольку время завершения процесса определяется в основном продолжительностью завершающегося этапа, то для интенсификации требуется добавление газового реагента с оптимальной комбинацией из коэффициентов скоростей сорбции – десорбции и диффузии. Для определения зависимости координаты фронта реакции от времени под влиянием газообразной атмосферы, рассмотрим газовую смесь, состоящую из двух газов. Кривые 1, 2 соответствуют условию при Тст(1) и Тст(2 (4.41) (4.41) При наличии в атмосфере одного газового реагента α2 = λ2 = Z2 = 0, либо считая совпадающими кинетические характеристики газовых реагентов (x1 = x2, y2 = y1) можно получить обобщение в виде формулы Гинстлинга-Броунштейна [60], которую можно выразить в виде:   2/3 2/3 1 dG k G G d    , (4.42) (4.42) 120 120 где G – доля превращенного вещества. При замене количества прореагировавшего вещества на степень превращения и принятия в качестве переменной Тсл может быть трансформировано (приближено) к уравнению:   2/3 1 d K dT      . (4.43) (4.43) Это уравнение адекватно описывает кинетику разложения MgCO3 до α = 0,85. Таким образом, формула является более общим обобщением для случая взаимодействия твердой частицы с газовой фазой. Это свидетельствует об адекватности, представленной в данной работе математической модели. Оптимальный состав газовой смеси можно определить, решая задачу минимизации времени окончания процесса t* при соответствующих ограничениях (например, при сохранении неизменным давления газовой смеси). Численное исследование системы при оптимальном составе газовой смеси позволяет проанализировать влияние различных кинетических коэффициентов и внешних условий на процесс в частности размеров частиц твердого реагента и парциальных давлений реагирующих газов. Построенная модель не противоречит экспериментальным данным по процессу разложения MgCO3 3Н2О, объясняет поведение кинетических кривых процесса взаимодействия твердых веществ со смесями газов – реагентов и позволяет провести оптимизацию состава газовой смеси. На рисунке 4.17 приведены кинетические кривые изменения Тсл, СмMgO STB в горячей зоне муфельной вращающейся печи. 121 р уф р Рисунок 4.17 Кинетические кривые изменения температуры (Тсл), концентрации MgO (СмMgO), удельной поверхности твердого остатка реакции Рисунок 4.17 Кинетические кривые изменения температуры (Тсл), концентрации MgO (СмMgO), удельной поверхности твердого остатка реакции 121 (STB) в горячей зоне печи (зоне обжига). Печь муфельная, вращающаяся с электрообогревом. Номера кривых и характер (тип) экспериментальных точек соответствуют таковым для кривых Тсл. Как следует из конструктивных особенностей печи, механизм загрузки печи состоит из бункера и шнекового питателя, которые осуществляют дозировку и загрузку материала в барабан. Разгрузочный патрубок печи также имеет шнековую выгрузку, таким образом, уплотнение (изоляция камеры печи от окружающей среды) производится непосредственно обрабатываемым материалом. Реакционные газы отсасываются с выгрузной головки печи. Таким образом в момент начала термического разложения MgCO3 состав газовой атмосферы представляет смесь воздуха и водяных паров, образующихся в процессе сушки гранул. При этом количество паров воды в атмосфере достигает до 50%об. Как видно из данных приведенных на рисунке 4.17, кинетические кривые отражающие изменение концентрации СмMgO имеют типичный S-образный характер с отсутствием индукционного периода. Кривые 1, 2 соответствуют условию при Тст(1) и Тст(2 Обработка кинетических кривых позволила установить, что энергия активации процесса оценивается величиной 68,5 кДж/моль, что близко к значению, полученному при исследовании кинетики процесса в токе водяного пара (Е = 58,7 кДж/моль). Удельная поверхность твердого остатка реакции S1,2 проходит через максимум, который в зависимости от температуры слоя (Тсл) имеет значение 155 - 200 м2/г. максимум удельной поверхности твердого остатка «размыты», что свидетельствует о незначительных скоростях агломерации и спекания продукта, а кроме того максимум S коррелируется и совмещен с максимумом СмMgO. Последнее обеспечивает высокое качество продукта выходящего из печи. Зависимость S1,2 от Тсл закономерна и является следствием различия скоростей образования зародышей новой фазы и продвижения фронта реакции. Пологость («размытость») кривых S1,2 обязана тому, что после достижения Smax температурный режим Тсл и другие условия (состав газовой фазы) не обеспечивают интенсивного протекания процессов агломерации и спекания («лечения» дефектов кристаллической решетки MgO). На рисунке 4.18 приведены аналогичные кинетические зависимости для случая реализации процесса термического разложения MgСO3 во вращающейся печи с непосредственным контактом теплоносителя и материала. Примерный компонентный состав газовой фазы следующий (%об. на сухой газ): СО2 – 10 – 15; О2 – 0,2 – 0,5; СО – 0,1 – 0,2; Н2О – 15 – 20; остальное азот. Анализ кинетических кривых и их обработка свидетельствуют о том, что закономерности образования продукта обжига и величины его удельной поверхности аналогичны вышеописанным. Отличие заключается в том, что кривые S1,2, что свидетельствует о достаточно интенсивном протекании процессов агломерации и спекания (этому, по-видимому, способствует достаточно большое количество СО2). 122 Рисунок 4.18. Кинетические кривые изменения температуры (Тсл), концентрации MgO (СмMgO), удельной поверхности твердого остатка реакции (S) в горячей зоне печи (зоне обжига). Печь вращающаяся с непосредственным контактом теплоносителя и материала. Номер кривых и характер (тип) экспериментальных точек соответствуют таковым для кривых Тст. унок 4.18. Кинетические кривые изменения температуры (Тсл Рисунок 4.18. Кинетические кривые изменения температуры (Тсл), концентрации MgO (СмMgO), удельной поверхности твердого остатка реакции (S) в горячей зоне печи (зоне обжига). Печь вращающаяся с непосредственным контактом теплоносителя и материала. Номер кривых и характер (тип) экспериментальных точек соответствуют таковым для кривых Тст. Энергия активации процесса разложения составляет ~ 89,2 кДж/моль, что близко к значению, полученному при исследовании кинетики процесса в токе воздуха (Е = 87,5 кДж/моль). На выходе из печи удельная поверхность продукта в зависимости от Тсл, составляет 100 – 150 м2/г. Таким образом, можно утверждать, что кинетические закономерности процесса разложения гидрокарбонатов магния в промышленных условиях адекватны, кинетическая модель, описывающая движение фронта реакции, отображает реальные условия. Кривые 1, 2 соответствуют условию при Тст(1) и Тст(2 При указанных режимах обжига были наработаны опытно-промышленные партии активного оксида магния для металлургической промышленности. Основные результаты по эксплуатации печей при наработке приведены в таблице 4.2. Как следует из приведенного в таблицы 4.2 экспериментального материала, степень превращения ТГКМ и производительность печи возрастают при обжиге гранулята. Обжиг гранулята способствует не только повышению транспортных возможностей печи, но и интенсивному теплообмену в слое обжигаемого материала. Как и следовало ожидать, при обжиге в печи наблюдается зависимость степени превращения от размера гранул. Так в первом типе вращающейся печи максимальная степень вращения отмечалась при обжиге гранул размером 4,9 мм. При увеличении размера гранул до 7 мм и более наблюдается уменьшение содержания МgO в продукте. Этого можно 123 124 Таблица 4.2 − Результаты опытов по обжигу ТГКМ на полупромышл установившемся режиме работы (приведены средние показатели) Показатель Опыт печь с непосредственным контактом 1 2 3 4 5 6 7 Продолжительность опыта, 72 ч Паста ТГКМ Паста ТГКМ на подложке готового материала Сухая паста ТГКМ Паста ТГКМ гранулир ованная, диаметр гранул – 2,9 мм Паста ТГКМ гранулир ованная, диаметр гранул – 4,2 мм Паста ТГКМ гранулиро ванная, связка гель Mg(OH)2 диаметр гранул – 4,9 мм Паста ТГКМ грану ованн связк гель Mg(O диам грану 7 мм Влажность загружаемого материала, %(масс) 44,5 Паста ТГКМ 445 Содер жит крис- талл 42,8 43,5 44,5 44 Производительность по исходному материалу, кг/ч 140 155 148 200 300 420 42 Производительность по готовому продукту (100 %-й MgО), кг/ч 28,5 25,4 24,2 33,5 53 72 72 Расход, м3/ч − природного газа − первичного воздуха 9,9 30,5 10,2 32,0 10,2 30,7 11,5 35,4 14,8 35,0 15,2 35,2 15 35 Температура, оС − газовой среды в горячей головке печи 820 270 820 250 820 260 780 198 750 210 710 200 71 20 ус П оп В за м П 125 − отходящих газов − выгружаемого из печи материала (перед холодильником) 705 715 700 685 680 630 Разрежение в загрузочной камере, мм.вод.ст. Кривые 1, 2 соответствуют условию при Тст(1) и Тст(2 3 3 3 4 4 4 Химический состав готового продукта, % − MgO − CO2 − потери при прокаливании 90,3 2,6 6,0 91,5 1,8 5,0 92,0 1,9 4,2 91,0 2,1 5,2 93,0 1,2 3,8 96,9 0,5 1,2 Унос пыли (по балансу MgO), % (масс) 16,4 17,1 19,2 12,4 9,0 5,9 Удельный съем готового продукта (100 %-й MgO) с внутренней поверхности печи, кг/(м2·ч) 2,2 2,03 1,9 2,6 3,9 5,5 Удельный расход условного топлива на 1 т 100 %-го MgO, кг/т 427,4 502,8 527,9 377,4 338,4 251,4 Удельная поверхность готового продукта после холодильника, м2/ч 45 50 45 62,5 65 120,5 52,0 избежать, если при гранулировании исходный материал подвергается механо- химической обработке. Последняя обеспечивает не только равномерное распределение связки Mg(ОН)2 по объему гранулы MgCO3·3H2O, но и за счет особенностей процессов, протекающих в машине типа СН, обеспечивается активация смеси с увеличением количества потенциальных центров зародышеобразования новой фазы, что приводит к интенсификации процесса. Это наглядно следует из данных приведенных в таблице 4.2 (колонка 8). Рассмотрим особенности обжига гранул ТГКМ с добавкой в качестве связки Мg(ОН)2. Как следует из приведенных данных, при прочих равных условиях гранулы с добавкой геля Мg(ОН)2 обжигаются быстрее и достигаются более высокие степени превращения. Анализ наблюдаемого явления показал, что гель Мg(ОН)2 значительно влияет на процесс разложения ТГКМ и это влияние многофакторно. Во-первых, гель Мg(ОН)2 является хорошим связующим материалом. Это интенсифицирует процесс гранулирования, увеличивает прочность формовок и предотвращает их разрушение при термообработке в подвижном слое. Во-вторых, гранулы после зоны сушки в печи (200 – 300 °С) имеют развитую пористую структуру. Пористость гранул с добавкой Мg(ОН)2 составляла 48 % при среднем диаметре пор 16000 Å, в то время как без добавки Мg(ОН)2 пористость была 29 % при среднем диаметре пор 6000 Å. Высокая пористость гранул способствует увеличению интенсивности тепломассопереноса вглубь и изнутри на поверхность гранул. Пористость гранул повышается за счет дегидроксилации Mg(ОН)2 при температурах 200 – 300 °С, что способствует интенсивной миграции молекул воды изнутри на поверхность гранулы. По-видимому, эти же условия обусловливают не только получение пористой структуры гранул, но и более дефектной структуры карбоната магния. В-третьих, образовавшаяся из геля Мg(ОН)2 и равномерно распределенная по объему гранулы аморфный, оксид магния создает до начала разложения MgСO3 поверхность раздела фаз MgСO3 − МgO. Наличие такой поверхности, способствует значительному увеличению числа зародышей новой фазы за счет деформационного взаимодействия кристаллических решеток MgСО3 и MgO. Следовательно, добавка геля Mg(ОН)2 в гранулы влияет на физические, химические и макроскопические факторы процесса разложения ТГКМ. Кривые 1, 2 соответствуют условию при Тст(1) и Тст(2 Это обеспечивает значительное увеличение производительности печи, снижение расходных норм условного топлива на 1 т 100%-ного МgO и повышает реакционную способность продукта. Оценка показателей, полученных на опытно-промышленной установке, показала, что они превосходят аналогичные показатели наиболее эффективных промышленных печей в производстве оксида магния. Так, например, на одном из заводов работает муфельная механическая полочная печь диаметром 4,2 и высотой 7 м, имеющая 4 материальных свода с рабочей поверхностью 70 м. При начальной влажности исходного гидрокарбоната 50 – 55 %масс. производительность печи составляет 50 − 55 кг/ч, а удельный съем готового продукта не превышает 1 кг/м. При этом удельный расход топлива на 1 т 100%-ного превышает 2000 кг. Получаемый продукт содержит основного вещества не более 92 %. 126 Приведенные в таблице 4.2 результаты исследований свидетельствуют, что очень значительное влияние на процесс термического разложения ТГКМ может оказать состав газовой атмосферы в печи (табл. 4.2 колонки 9, 10, 11). Из приведенных данных следует, что обжиг гранулированного материала в атмосфере Н2О или NН3 позволяет при значительно более низких температурах достичь полного разложения МgСО3, существенно снизить энергозатраты на процесс и при этом получить продукт с удельной поверхностью более 190 м2/г. Механизм влияния этих газов на процесс разложения ТГКМ описан нами ранее, при построении кинетической модели зоны обжига, из результатов таблицы 4.2 можно сделать вывод об адекватности ранее представленного механизма, и математической модели процесса. Таким образом, результаты проведенных исследований свидетельствуют о перспективности применения вращающихся печей в производстве оксида − магния. Очевидно, что наиболее перспективен обжиг гранулированного материала на связке геля Мg(ОН)2. Это способствует улучшению всех показателей печи. В том числе снижению уноса пыли до 0,5 – 3 % по сравнению с уносом при обжиге не гранулированного материала (17 %) и уносом в промышленных печах (19 – 20 %). Для производства специальных сортов активного оксида магния с удельной поверхностью более 190 м2/г и содержанием основного вещества более 97 % целесообразно при гранулировании исходного материала осуществлять механохимическую активацию сырья и последующий обжиг в регулируемой газовой атмосфере. Приведенные в данном разделе подходы к анализу процесса термического разложения карбонатов, гидроксидов магния и математические модели процессов обладают некоторой универсальностью и могут быть использованы при исследовании процессов термического разложения по типу Т Т А В Г   для различного сырья, в печах с различным конструктивным оформлением (например, печах шахтного типа). 4.4 Реакторы для проведения каталитических реакций В настоящее время до 92 % процессов современной химической, перерабатывающей промышленности и почти все биохимические процессы являются областями применения катализа. Явление катализа может быть определено следующим образом: катализ – это избирательное ускорение одного из термодинамически возможных (в данных условиях) направлений реакции, осуществляющейся в присутствии катализатора по новому реакционному пути. Характерная особенность катализа связана с цикличностью процессов. Вступая в химическое взаимодействие с реагирующими веществами, катализатор выходит из него химически неизменным, благодаря чему многократно повторяет реакцию с новыми порциями веществ и не расходуется в процессе катализа. Дальнейшее развитие основных отраслей химической и нефте- перерабатывающей промышленности будет основываться на все более расширяющемся использовании и усовершенствовании каталитических 127 методов, а состояние научных исследований в области катализа будет в значительной мере определять технический уровень важнейших отраслей промышленности. Получение аммиака, серной и азотной кислот, каталитический крекинг, нефтехимический синтез и синтез мономеров, получение синтетического каучука и многих других полимеров, синтез целого ряда растворителей, а также полупродуктов красочной, пищевой и фармацевтической промышленности основаны на катализе. В биохимии органическими катализаторами высокого избирательного действия являются ферменты. Основываясь на фазовом состоянии катализатора и реагирующих веществ, можно выделить три типа процессов. 1. Гомогенный катализ − процессы, в которых катализатор и реагирующие вещества находятся в одном агрегатном состоянии, жидком или газообразном. Для гомогенного катализа характерно проявление активности отдельным ионом или молекулой катализатора. Поэтому, как правило, увеличение скорости реакции прямо пропорционально концентрации введенного катализатора. Процесс в гомогенном катализе развивается через образование лабильных нестойких промежуточных соединений стехиометрического состава. Эти промежуточные соединения могут быть выделены и исследованы. р у у 2. Гетерогенный катализ − процессы, в которых катализатор и реагирующие вещества находятся в различных агрегатных состояниях: катализатор − твердое вещество, реагирующие вещества − жидкость или газ. Гетерогенный катализ определяется наличием границы раздела фаз катализатор р р р р ф р − реагирующие вещества и тем, что одним из участников реакции является твердое тело. Поэтому в гетерогенном катализе процесс осуществляется через образование промежуточных поверхностных химических соединений нестехиометрического состава, характеризующихся широким набором энергий связи реагирующих атомов с атомами катализатора. р ру р 3. Ферментативный, или микрогетерогенный, катализ играет ведущую роль в химических превращениях в живой природе. Ферменты (называемые также энзимами) − катализаторы белковой природы, образующиеся в живых организмах. Ферменты осуществляют сложный комплекс химических превращений, обеспечивающих жизнедеятельность (дыхание, пищеварение, синтез белков и т. п.) живых организмов. По некоторым свойствам, в первую очередь высокой химической специфичности и колоссальной активности, ферменты значительно превосходят промышленные катализаторы. 4.4 Реакторы для проведения каталитических реакций ф р р р р При различии механизмов действия одним из общих эффектов, свойственным всем трем рассмотренным типам каталитических процессов, является увеличение скорости катализируемой реакции и возможность направить реакцию при помощи катализатора в одном, строго определенном направлении. Это свойство катализаторов называется избирательностью или селективностью действия. Избирательность, или селективность, − это способность катализатора избирательно осуществлять преимущественно одно из многих термодинамически возможных в данных условиях направлений реакции. Избирательность действия катализаторов приобретает решающее 128 значение в органическом катализе, где сложные органические молекулы в одних и тех же условиях способны реагировать одновременно в нескольких направлениях. Природа химического взаимодействия реагирующих веществ с катализатором весьма разнообразна. Многообразны механизмы каталитических реакций, меняющиеся с изменением природы катализатора и условий процесса. По характеру промежуточного взаимодействия можно выделить две основные группы каталитических процессов, свойственных как гомогенным, так и гетерогенным каталитическим реакциям. Это с одной стороны, процессы, осуществляющиеся по кислотно-основному механизму, с другой − по окислительно-восстановительному механизму. Во многих случаях кислотно- основное взаимодействие неотделимо от окислительно-восстановительного. Иногда осуществление каталитических реакций не связано с изменением заряда компонентов системы. Что касается процессорных единиц (реакторов) для проведения каталитических процессов, то для проведения гомогенного катализа конструкции реакторов аналогичны реакторам для проведения некаталитических гомогенных реакций. Принцип расчета этих реакторов также аналогичны и основаны на Арениусовской кинетике. Более сложное явление представляет собой гетерогенный катализ [61]. Следует только отметить, что развитие теории гетерогенного катализа приводит к установлению различных типов кинетических уравнений общего и частного характера. Следует только отметить, что развитие теории гетерогенного катализа приводит к установлению различных типов кинетических уравнений общего и частного характера. Законченное кинетическое уравнение каталитического гетерогенного процесса в системе газ – твердое тело должно учитывать: 1) перемещение реагентов путем конвекции из газового потока к его неподвижному (пограничному) слою, окружающему катализатор; 2) диффузию реагентов в этом слое и в порах катализатора; 3) химическую адсорбцию реагентов на поверхности катализатора; 4) химическую реакцию на поверхности катализатора; есорбцию продукта реакции; 5) десорбцию продукта реакции; 6) диффузию продукта реакции через неподвижный (пограничны 7) перемещение продукта реакции путем конвекции от внешней границы неподвижного слоя к потоку газа. Однако такое уравнение было бы весьма сложным. Обычно предполагают, что только один из этапов определяет скорость процесса. 4.4 Реакторы для проведения каталитических реакций Например, во внешнедиффузной области (1) скорость процесса определяется коэффициентом диффузии реагента (реагентов) к пограничному слою, окружающему катализатор по закону Фика: i э dC dG W D S d dz     , (4.44) (4.44) где G − количество компонента, перенесенное за время η в направлении z перпендикулярно к поверхности зерна катализатора при концентрации Ci диффундирующего компонента; 129 S − свободная поверхность катализатора; S − свободная поверхность катализатора; DЭ − эффективный коэффициент диффузии (совокупность молекулярной и турбулентной (конвективной) диффузии). Во внешне диффузионной области, как правило, используются катализаторы с очень высокой активностью, обеспечивающие большую скорость химических реакций и незначительное снижение энергии активации. Величину DЭ определяют в зависимости от соотношения размера пор в зерне катализатора (r) и длины свободного пробега молекул (λ). При λ<2r определяют DЭ как коэффициент молекулярной диффузии, а при λ>2r определяют по формуле Кнудсена: 2 8 3 э RT D r M   , (4.45) (4.45) где M − молекулярная масса диффундирующего вещества. де M − молекулярная масса диффундирующего вещества. где M − молекулярная масса диффундирующего вещества. Oбщее уравнение скорости каталитического процесса, протекающего в кинетической области, с учетом основных технологических параметров (концентрации реагентов в газовой фазу, температуры, давления и активности катализатора) может быть записано в виде: 0 1 exp n n dG E W k V CP d RT            , (4.46) (4.46) где n dG d − приращение продукта во времени; где n dG d − приращение продукта во времени; где n dG d − приращение продукта во времени; ko − предэкспонента; ko − предэкспонента; E − энергия активации химичкой реакции на данном катализаторе V − насыпной объем катализатора; ΔC − разность концентраций реагентов (движущая сила процесса) P1 − безразмерное давление (отношение действительного давления при протекании процесса к атмосферному); P1 − безразмерное давление (отношение действительного давления при протекании процесса к атмосферному); n − порядок реакции. n − порядок реакции. В кинетической области происходят процессы на малоактивных катализаторах, с мелкими размерами зерен и крупными порами при турбулентном режиме потока реагентов и при относительно низких температурах. Увеличение скорости катализа в кинетической области достигается повышением температуры. Таким образом, лимитирующую стадию процесса экспериментально и определяя ее скорость, легко определяются линейные размеры аппарата (реактора), а также по лимитирующей стадии и конкретные особенности аппарата (условия контакта, насадка и т.п.). Если аппаратурное оформление гомогенного катализа не требует специальной конструкции аппаратов, то аппаратура гетерогенного катализа, а особенно при взаимодействии газов на твердых катализаторах специфична и разнообразна. Конструкции контактных аппаратов различаются в зависимости 130 от способа контакта газов с катализатором, подвода или отвода теплоты и т.п. от способа контакта газов с катализатором, подвода или отвода теплоты и т.п. соба контакта газов с катализатором, подвода или отвода теплоты и т.п По данным признакам аппараты подразделяются на: 1) аппараты поверхностного контакта (катализ на стенках, трубках, катализаторных сетках); 1) аппараты поверхностного контакта (катализ на стенках, трубках, катализаторных сетках); 2) аппараты с фильтрующим слоем катализатора. При этом слой катализатора неподвижен, или движется плотным слоем вдоль реактора; 3) аппараты со взвешенным слоем катализатора. При этом слой псевдоожиженый; 4) аппараты типа пневмотранспорта мелкодисперсный катализатор распределен равномерно по объему движущегося газа, и эта система движется с условно одинаковой скоростью вдоль реактора. 4) аппараты типа пневмотранспорта мелкодисперсный катализатор распределен равномерно по объему движущегося газа, и эта система движется с условно одинаковой скоростью вдоль реактора. Выбор реактора зависит от типа химической реакции, протекающей на поверхности катализатора желаемых продуктов, которые необходимо получить (селективность) и в основном от физико-химических свойств твердого вещества, которое должно быть катализатором. где M − молекулярная масса диффундирующего вещества. Исходя из данного и теории катализа [62], катализатор должен обладать следующими характеристиками: 1) химическая природа катализатора должна соответствовать природе реагирующих веществ в смысле возможности хемосорбции; 1) химическая природа катализатора должна соответствовать природе реагирующих веществ в смысле возможности хемосорбции; 2) электронные свойства катализатора должны быть благоприятны для образования реакционно-способного адсорбционного слоя; 3) кристаллическая структура, параметры решеток и рельеф поверхности катализатора должны находиться в определенном соответствии с межатомными расстояниями и валентными углами реагирующих веществ, обеспечивая минимум необходимой деформации адсорбирующихся молекул; 4) энергия возникающей адсорбционной связи должна обеспечивать достаточную прочность поверхностных связей и максимум скорости адсорбции, чтобы образование и разложение поверхностных соединений не тормозили скорость суммарного процесса. Очевидно, что для выбора типа катализатора (твердого вещества) необходима теория и механизм катализа. Существующие теории не дают однозначного ответа [62]. Как уже упоминалось, в разделе I существуют различные взгляды на природу активности катализатора [63]. Ф. Ф. Волькенштейн связывал активность катализатора и механизм действия с определенным значением уровня Ферми (то есть усредненным значением химического потенциала электрона в полупроводнике). Чем выше уровень Ферми, тем большая доля частиц с отрицательным зарядом на поверхности и меньше с положительным зарядом. При объяснении механизма возникновения и природы относительной стабильности активных фаз используют теорию пересыщения С. З. Рогинского [63] и принцип ориентационного размерного соответствия Данкова- Конобеевского [63]. Пересыщение, то есть избыточная энергия, отличающаяся 1 моль вещества в данной степени от того же количества вещества в бесконечно большом куске формируется или обрабатывается в условиях максимально отличающихся от равновесных. Важным выводом выше отмеченных теорий является возможность накопления пересыщений за счет изменения условий 131 обработки твердого вещества (температуры, состава газовой фазы, концентрации растворов и методов осаждения из них твердой фазы, термообработки и т.п.). Особое внимание уделяют исследователи аморфному состоянию твердого вещества, которое характеризуется большим вкладом поверхностной энергии [64]. В разделах теории разупорядочения кристаллов твердого тела [65] считается, что аморфное состояние предшествует его плавлению. Н. И. Кобозов [64] используя понятие аморфное состояние сформулировал «теорию активных ансамблей» в которой активный центр на поверхности катализатора рассматривается как докристаллическое образование из нескольких атомов, n- атомный «ансамбль», закрепленный на поверхности носителя катализатора адсорбционными силами. Тем не менее, в настоящее время занимает ведущее положение мультиплетная теория теория сформулированная А. А. Баландиным. Это первая теория подбора катализаторов [66]. В основу теории легли принципы структурного и энергетического соответствия, в совокупности своей объединяющее влияние физических и химических факторов в катализе. где M − молекулярная масса диффундирующего вещества. Структурное соответствие заключается в том, что для протекания каталитической реакции пространственное расположение атомов реагирующих молекул и в катализаторе должно быть таким, чтобы молекулы своими реагирующими атомами (индексной группой) колебались с сохранением валентных углов на катализатор, соприкасаясь с его атомами (мультиплетом). Принцип энергетического соответствия требует, чтобы адсорбционный потенциал катализатора (сумма энергий связи реагирующих атомов с катализатором) для эндотермической реакции по возможности ближе подходил бы к половине суммы энергий реагирующих связей (среднее значение энергий разрываемых связей и вновь возникающих в газовой фазе), а энергетический барьер равнялся бы половине теплового эффекта реакции. При этом недопустимо раздельное применение обоих принципов мультиплетной теории для трактовки экспериментальных данных и решения вопроса подбора катализатора. До настоящего времени мультиплетная теория является единственной теорией, в которой конкретные каталитические реакции рассматриваются на атомном уровне, а именно с точки зрения конфигурации молекул и решеток твердых тел, а также длин и энергий химических связей. Исходная и основная предпосылка мультиплетной теории связана с представлением о том, что катализ − явление химическое. Поскольку радиус действия «валентно-химических» сил очень мал, то с достаточной степенью приближения можно считать, что атомы химически взаимодействуют только при соприкосновении, т. е. при своем сближении на расстоянии длины связи. Поэтому в каталитических реакциях должны участвовать не молекулы целиком, а отдельные входящие в них атомы. В реагирующей молекуле теория позволяет выделить группу атомов, между которыми при реакции происходит перераспределение химических связей. Эти атомы должны, согласно основному постулату, находиться на расстояниях, обеспечивающих 132 возможность образования связи между ними, и в определенный момент времени должны соприкасаться с атомами катализатора. Реальные размеры атомов таковы, что несколько реагирующих атомов молекулы при контакте с поверхностью не могут разместиться на одном атоме катализатора. Эти предпосылки приводят к выводу, что притяжение и адсорбция многоатомной молекулы должны осуществляться не одним атомом, а группой атомов катализатора − мультиплетом. р у В настоящее время можно наметить следующие пути расчета оптимального катализатора для заданной реакции. Прежде всего, необходимо решить вопрос о структурном соответствии и матричных функциях процесса в целом. Для заданного процесса и заданного катализатора нужно определить, какой механизм − электронный (ион-радикальный) или ковалентный (радикальный) − будет энергетически выгоднее в рассматриваемых условиях. Для ион-радикального механизма следует учитывать такие характеристики, как работа выхода электрона, ширина запрещенной зоны, сродство к электрону или протону реагирующих веществ и катализатора, а также их изменение в промежуточном взаимодействии. В настоящее время можно наметить следующие пути расчета оптимального катализатора для заданной реакции. Прежде всего, необходимо решить вопрос о структурном соответствии и матричных функциях процесса в целом. где M − молекулярная масса диффундирующего вещества. Для заданного процесса и заданного катализатора нужно определить, какой механизм − электронный (ион-радикальный) или ковалентный (радикальный) − будет энергетически выгоднее в рассматриваемых условиях. Для ион-радикального механизма следует учитывать такие характеристики, как работа выхода электрона, ширина запрещенной зоны, сродство к электрону или протону реагирующих веществ и катализатора, а также их изменение в промежуточном взаимодействии. Для радикального механизма определяются энергии разрывающихся и образующихся связей и энергии связей реагирующих атомов с атомами катализатора в процессе образования переходного комплекса реакции. При рассчитанном механизме необходимо далее учесть энергетическое соответствие, подобрать катализаторы с оптимальными энергиями связей. Принципиальной разницы между тремя основными типами каталитических процессов: ферментативным, гомогенным и гетерогенным катализом, по-видимому, нет. Во всех трех случаях каталитический процесс в зависимости от относительной реакционной способности реагирующих веществ и катализатора может развиваться как по радикальному (ковалентному), так и по ион-радикальному (электронному) механизму. Основные положения мультиплетной теории нельзя рассматривать в отрыве друг от друга. Лишь в отдельных случаях на первый план выступает та или иная ее сторона. Теория непрерывно развивается и в последнее время особенно в области энергетического соответствия. Энергетическое соответствие проявляется в очень узких пределах и при очень тонких различиях в строении реагирующих молекул, и различие в энергиях связи на 4 − 8 кДж/моль может иметь решающее значение. В этом отношении предвидение А. А. Баландина полностью оправдывается. Для дальнейшей интенсификации каталитических процессов необходимо больше внимания уделять возможности аккумуляции энергии реакции в системе. Применение специальных методов квантовомеханических расчетов указало на возможность захвата энергии катализатором на адсорбционной и реакционной стадиях процесса в виде колебательной энергии. Если колебания, перпендикулярные плоскости решетки, быстро рассеиваются в окружающую среду, то колебания в плоскости, параллельной решетке, могут длительное время сохраняться кристаллом. В результате на поверхности кристалла возникают «горячие» точки, на которых захваченная энергия может 133 передаваться молекулам реагирующих веществ опять-таки в виде колебательной энергии. Тем самым осуществляется дополнительная энергетическая активация реагирующих веществ. После выбора типа катализатора проблема проектирования каталитических реакторов заключается в применении уравнений скорости реакции и уравнений тепломассопереноса к конкретным промышленным случаям. Конечной целью при этом является создание экономичной и легкой в эксплуатации конструкции реактора с возможностью автоматизации и управления процессом вычислительной машиной при оптимальных условиях. Для нахождения размеров каталитического реактора нужно рассчитать объем катализатора, высоту и сечение слоя катализатора в реакторе или количество катализатора в объеме движущегося газа зависят от скорости газа. Наконец, форма реактора и особенно вид определяется размером теплообменной поверхности. где M − молекулярная масса диффундирующего вещества. Для реактора непрерывного действия зависимость между объемной скоростью и степенью превращения можно получить из уравнения: m c G dx wM  , (4.47) (4.47) m c G dx wM  , m c G dx wM  , (4.4 где Gm − массовый расход; где Gm − массовый расход; x − степень превращения; MC − масса катализатора. Проведя интегрирование получим:   Проведя интегрирование получим:   Проведя интегрирование получим:       2 1 1 1 1 ln 1 1 1 1 1 1 1 A B A B A c m k k k k k p k M G x c C k k k                                              1 1 k  , (4.48) где p − давление; где p − давление; kA − константа равновесия адсорбции компонента А; kB − константа равновесия адсорбции компонента В. n A C k k  , n A где k − константа скорости; где k − константа скорости; n − число активных центров. Уравнение можно интегрировать графически 134 1/wA XA 1/wA XA XA0 Vc/Qm dXA Высоту реактора можно определить из уравнения:    1 A A i A p f A m c G v f A A A A p p dp G Z M k a p p p p p              . (4.49) 1/wA XA 1/wA XA XA0 Vc/Qm dXA конструкции реактора. Ниже рассмотрим постановку и решение каждого вида задач. Расчет адиабатических реакторов с катализатором в слое используемых в процессах каталитического риформинга Цель каталитического риформинга бензинов первичной перегонки, получаемых из неочищенной нефти, или бензинов термического крекинга заключается в повышении их октанового числа. Бензины, полученные путем каталитического реформинга, имеют почти удвоенное октановое число по сравнению с алканами (предельными углеводородами нормального состояния). Между содержанием ароматических углеводородов в бензинах каталитического риформинга и октановым числом существует прямая связь. Химически действие каталитического риформинга можно представить как наложение одной на другую реакции изомеризации, дегидроциклизации и гидрогенизации, которые приводят в итоге к обогащению начальных бензинов ароматическими углеводородами и изоалкенами (предельными углеводородами изо строения). Углеводородный состав бензина прямой гонки %масс: ароматические − 12,0, нафтеновые − 38,0, парафиновые – 50,0. Формулы углеводородов: ароматические – CnH2H-6; нафтеновые − CnH2n; парафиновые − CnH2n+2. Соответственно, формулы для расчета молекулярной массы по углеводородному числу: Mn = 12n+1(2n − 6) = 14n – 6; Mn = 12n+1·2n = 14n; Mn = 12n+1(2n+2) = 14n+2, а числовое значение молекулярных масс углеводородов соответствовало: 101,8; 107,8; 109,8. При риформинге протекают следующие реакции: Углеводородный состав бензина прямой гонки %масс: ароматические − 12,0, нафтеновые − 38,0, парафиновые – 50,0. Формулы углеводородов: ароматические – CnH2H-6; нафтеновые − CnH2n; парафиновые − CnH2n+2. Соответственно, формулы для расчета молекулярной массы по углеводородному числу: Mn = 12n+1(2n − 6) = 14n – 6; Mn = 12n+1·2n = 14n; Mn = 12n+1(2n+2) = 14n+2, а числовое значение молекулярных масс углеводородов соответствовало: 101,8; 107,8; 109,8. XA Высоту реактора можно определить из уравнения: XA Высоту реактора можно определить из уравнения:    1 A A i Al p f A m c G v f A A A A p p dp G Z M k a p p p p p              . (4.49) (4.49) Первый член правой части уравнения, имеющий размерность длинны, называется высотой единицы переноса массы (ВЕП), а второй член, безразмерный, числом единицы переноса массы − Nm. Здесь kGA − коэффициент переноса массы компонента А; Здесь kGA − коэффициент переноса массы компонента А; pA − парциальное давление компонента А в потоке; pAi − парциальное давление компонента А у поверхности раздела фа av − поверхность единицы объема катализатора; δA − изменение числа молей газового потока вещества реакции. δA − изменение числа молей газового потока вещества реакции. Значение высоты единицы переноса массы может быть найдено: A р Значение высоты единицы переноса массы может быть найдено: (4.50)   ' ( ) Re Pr n m V a ВЕП A  , где Re , Pr m i G D      , μ − вязкость газовой фазы; μ − вязкость газовой фазы; ρ − плотность газовой фазы; D − коэффициент диффузии компонентов газовой фазы. D − коэффициент диффузии компонентов газовой фазы. При проектировании и расчете реакторов гетерогенного катализа в общем случае могут ставиться две задачи: − выбор типа катализатора, выбор типа реактора, его расчет с определением массы катализатора, основных конструктивных размеров, а также определение схемы потоков газовой фазы в реакторе; − разработка принципиальной реакционной схемы; − разработка принципиальной реакционной схемы − анализ эксплуатационного функционирования реактора гетерогенного катализа в промышленном процессе, разработка технических и технологических решений по интенсификации процесса и усовершенствованию 135 конструкции реактора. конструкции реактора. конструкции реактора. конструкции реактора. Расчет адиабатических реакторов с катализатором в слое используемых в процессах каталитического риформинга При риформинге протекают следующие реакции: − превращение нафтеновых углеводородов в ароматические 2 2 6 2 3 , 695 , 309 / n n n n C H C H H T K кДж м    − превращение нафтеновых углеводородов в ароматические 2 2 6 2 3 , 695 , 309 / n n n n C H C H H T K кДж моль     − превращение нафтеновых углеводородов в ароматические 2 2 6 2 2 3 , 695 , 309 / n n n n C H C H H T K кДж моль Н     − превращение нафтеновых углеводородов в парафиновые: 2 2 2, 741 , 199 / n n n n C H C H T K кДж моль    − превращение нафтеновых углеводородов в парафиновые: 2 2 2 2 , 741 , 199 / n n n n C H C H T K кДж моль Н     ; − превращение нафтеновых углеводородов в парафиновые: 2 2 2 2 , 741 , 199 / n n n n C H C H T K кДж моль Н     ; превращение нафтеновых углеводородов в парафиновые: 2 2 2 2 , 741 , 199 / n n n n C H C H T K кДж моль Н     ; − гидрокрекинг нафтеновых углеводородов:   2 2 4 2 6 3 8 4 10 5 12 , 3 15 n n n n C H H CH C H C H C H C H       2 773 , 251 / T K кДж моль Н    − гидрокрекинг парафиновых углеводородов: − гидрокрекинг парафиновых углеводородов:   2 2 2 4 2 6 3 8 4 10 5 12 3 , 3 15 n n n n C H H CH C H C H C H C H         2 810 , 230 / T K кДж моль Н    где n − углеродное число (число углеродных атомов в молекуле углев где n − углеродное число (число углеродных атомов в молекуле углеводорода). Расчет адиабатических реакторов с катализатором в слое используемых в процессах каталитического риформинга глеродное число (число углеродных атомов в молекуле углеводорода) Для указанных реакций можно записать четыре дифференциальных уравнения, описывающих уменьшение количества углеводородов в результате химических реакций: 136 2 1 2 2 3 1 1 1 2 3 4 H H A H R p H H H П R p H H R П H R dN k k p p p d k dN k k p p p d k dN p k d dN p k d                       , (4.51) (4.51) где NH, NП − для нафтеновых и парафиновых углеводородов, подвергнутых химическому превращению, кмоль/кмоль; где NH, NП − для нафтеновых и парафиновых углеводородов, подвергнутых химическому превращению, кмоль/кмоль; υR − величина, обратная объемной скорости питания газовой смеси (кг катализатора/(кмоль/ч сырья)); k1−4 − константы скоростей реакции, кмоль/ч·Па·кг катализатора. k1−4 − константы скоростей реакции, кмоль/ч·Па·кг катализатора. Несмотря на то, что реакции протекают в гетерогенной системе (твердый катализатор – газовая фаза), для расчета их скоростей можно предположить, что деструкция углеводородов происходит в гомогенной газовой фазе. В соответствии со сказанным выше для реакции вида: р аА вВ сС dD   , р аА вВ сС dD   , р аА вВ сС dD   , скорость реакции можно представить следующим образом: скорость реакции можно представить следующим образом:   ' 1 1 a b c d A A c A B c C D d c V dN r k c c k c c V d V d             , (4.52) (4.52) где NA −число молей компонента А, превращенных за время η. где NA −число молей компонента А, превращенных за время η. Если газообразные реагенты следуют законам идеальных газов, то N Если газообразные реагенты следуют законам идеальных газов, то Если газообразные реагенты следуют законам идеальных газов, то A A A N p c V R T    , (4.53) (4.53) и ' ' a b c d a b c d A c A B c C D A B C D r dN k c c k c c k p p k p p dV             . Расчет адиабатических реакторов с катализатором в слое используемых в процессах каталитического риформинга (4.54) ' ' a b c d a b c d A c A B c C D A B C D r dN k c c k c c k p p k p p dV             . (4.54) (4.54) Скорость основной реакции каталитического риформинга 1 моль циклоалкана 1 моль ароматического углеводорода будет равна Скорость основной реакции каталитического риформинга 1 моль циклоалкана 1 моль ароматического углеводорода будет равна 2 ' 3 N N Q H B dN r k p k p p d       . (4.55) (4.55) Здесь ηB − время константа, кг катализатора моль питания/ч . При равновесии имеем: Здесь ηB − время константа, кг катализатора моль питания/ч . При равновесии имеем: Здесь ηB − время константа, кг катализатора моль питания/ч . При равновесии имеем: Здесь ηB − время константа, 2 ' Q H p N p p k K p k   , (4.56) (4.56) 137 где Kp − константа равновесия. Принимая во внимание соотношение, получим уравнение общей скорости реакции в следующей форме: Принимая во внимание соотношение, получим уравнение общей скорости реакции в следующей форме:   2 3 Q H N N p B p N p p dN k r p K d K p                . (4.57) (4.57) Таким же образом для реакции Таким же образом для реакции Таким же образом для реакции 2 1моль циклоалкана+Н 1моль алкана константа равновесия дается соотношением k Таким же образом для реакции 2 1моль циклоалкана+Н 1моль алкана константа равновесия дается соотношением р р 2 1моль циклоалкана+Н 1моль алкана константа равновесия дается соотношением константа равновесия дается соотношением 2 ' p p N H p k K k p p    , (4.58) (4.58) а скорость реакции равна а скорость реакции равна р 2 2 p N N H p B p N H p dN k r p p K d K p p                 . (4.59) (4.59) Экспериментальные данные показывают, что в процессе гидрокрекинга метан, этан, пропан, бутан и пентан образуется в приблизительно равных количествах. Расчет адиабатических реакторов с катализатором в слое используемых в процессах каталитического риформинга Скорости реакций первого порядка будут, соответственно следую k g N p B g N N B k dN r p d P k dN r p d P         , (4.60) (4.60) где kg − константа скорости реакции гидрокрекинга; где kg − константа скорости реакции гидрокрекинга; P − общее давление в системе, ат. g P − общее давление в системе, ат. Анализ показывает, что реакции сопровождаются значительным увеличением объема, что требует, согласно правилу Ле-Шателье, повышения давления в реакторе (42 ат(4,2 МПа)), температуре от 442 оС до 537 оС, реакции протекают в кинетической области. Основные расчетные формулы для определения констант скорости и равновесия, а также скорости реакции приведены выше. По кинетическим данным определяем время контакта (продолжительность) каждой реакции. Одна из основных реакций риформинга – дегидрогенезация циклогексанов и его производных (−ΔH = 300 кДж/моль, E ≈ 150 кДж/моль) поэтому, как правило, выбор типа катализатора осуществляется по этой реакции, которая в структурном виде запишется: 138 СН2 СН2 СН2 СН2 СН2 СН2 +3Н2 и протекает надд металлическим катализатором. Для выбора катализатора используем мультиплетную теорию катализа описанную выше СН2 СН2 СН2 СН2 СН2 СН2 +3Н2 и протекает надд металлическим катализатором. Для выбора катализатора используем мультиплетную теорию катализа, описанную выше. и протекает надд металлическим катализатором. Для выбора катализатора используем мультиплетную теорию катализа, описанную выше. При дегидрировании циклогексана и его производных при плоскостном наложении молекулы на поверхность скорость реакции должна быть больше, чем при реберном наложении. Однако для осуществления плоской адсорбции молекулы циклогексана на шести атомах катализатора необходимо наличие общих элементов симметрии молекулы и поверхности катализатора Шестичленное кольцо налагается плоско на грань с расположением атомов в виде равносторонних треугольников. Продукт реакции − бензол имеет плоскую конфигурацию, и поэтому в какой-то момент времени его образования кольцо должно стать плоским. Согласно секстетной модели, это происходит при образовании активного комплекса дегидрогенизации циклогексана и обязано тому, что силы хемосорбции больше сил, стремящихся сохранить конформацию. Подобный же вид имеет модель обратной реакции − гидрирования бензола при более низких температурах. Адсорбированная плоско молекула циклогексана переходит в молекулу бензола при отрыве всех шести атомов водорода одновременно таким образом, что шестичленное кольцо не покидает поверхности до тех пор, пока оно не превратится в кольцо бензола. Поэтому в газовой фазе не обнаруживаются циклогексен и циклогексадиен. В процессе реакции активные центры А, В, С осуществляют отщепление и образование молекул водорода, а центры D, Е, F замыкают двойную связь между атомами углерода реакции дегидрирования цикланов. Расчет адиабатических реакторов с катализатором в слое используемых в процессах каталитического риформинга Секстетная модель позволяет предвидеть, что из элементов периодической системы катализаторами могут быть металлы, кристаллизующиеся в гранецентрированной кубической и гексагональной решетках. Более того кратчайшие межатомные расстояния металлов- катализаторов равные атомному диаметру d, должны лежать в определенных пределах, так как атомы Н в циклогексане будут или слишком удалены от притягивающих их атомов катализаторов, или кольцо не наложится плоско па грань решетки. Этим условиям отвечают металлы со значениями d, лежащими в пределах от 2,7746·10−8 до 2,4916·10−8 см (Ni). 139 Мультиплетной теорией предсказана активность трения для реакции дегидрирования циклогексана. Из металлов VIII группы только железо кристаллизующееся в объемноцентрированной кубической решетке, не включено в круг возможных катализаторов. В согласии с теорией циклогексан не может быть дегидрирован над марганцем, который обладает более сложной кристаллической структурой; олефиновая же связь над марганцем гидрируется. В реакциях гидрирования непредельных углеводородов необходимо исходить из принципа сохранения валентного угла. Поскольку активация реагирующей молекулы осуществляется в результате ее химического взаимодействия с катализатором, то необходимо считать, что любое дополнительное напряжение углеводородного скелета, вызывающее отклонение от валентного угла углерода, будет повышать энергетический барьер реакции, снижать ее скорость. Гидрирование непредельных связей осуществляется по дублетной схеме адсорбции. Раскрытие кратной углерод − углеродной связи при дублетной адсорбции может пройти или полностью, или только наметиться, что приводит к образованию промежуточного мультиплетного комплекса и рассматривается мультиплетной теорией. Скорость протекания этого процесса должна зависеть от пространственного соответствия между адсорбируемой молекулой и строением поверхности. Из схемы дублетной адсорбции на катализаторе: где с − межатомное расстояние С − С в углеводороде; де с − межатомное расстояние С − С в углеводороде; d − расстояние между атомами катализатора в кристаллической решетк b − длина связи углерод − катализатор; d − расстояние между атомами катализатора в кристаллической решетке; b − длина связи углерод − катализатор; b − длина связи углерод − катализатор; θ − валентный угол углерода, следует     cos 180 2 d c b     . Данное уравнение при известном валентном угле θ (θ = 109°) и известных величинах b и с позволяет рассчитать оптимальный для адсорбции и активации параметр кристаллической решетки катализатора, который оказался равным 2,74·10−8 см. Такие активные катализаторы гидрирования, как Pt и Pd, имеют межатомное расстояние плотнейшей упаковки, лежащее между 2,7·10−8 и 2,8·10−8 см, что близко к оптимальному параметру для адсорбции и активации 2,74·10−8 см. Таким образом, для проведения выше приведенных реакций наиболее целесообразно в качестве катализатора использовать платину, что действительно используется в нефтеперерабатывающей промышленности. Расчет адиабатических реакторов с катализатором в слое используемых в процессах каталитического риформинга 140 В промышленных условиях используется алюмоплатиновый катализатор (платина нанесена тонким слоем на носитель оксид алюминия или силикат алюминия, Pt – несколько атомних слоев, пористость носителя болем 40 %). Катализатор формируют в виде цилиндрических гранул размером в диаметре 2 ÷ 3 мм и высотой 4 ÷ 5 мм (d = 0,003 м, H = 0,005 м). Объем шара, эквивалентный объему частицы катализатора цилиндрической формы, м3 равен 2 / 4 ш d H     , сторона куба, описанного вокруг шара, равна эквивалентному диаметру 3 6 / э ш d    и соответственно, если используется слой катализатора (при допущении упорядоченного расположения) порозность слоя равна: / ш куб     , / ш куб     , (4.61) (4.61) / ш куб     , (4.61) где υкуб − объем куба, описанного вокруг шара, м3. где υкуб − объем куба, описанного вокруг шара, м3. Поскольку катализатор дорогостоящий и нанесен тонким слоем на носитель, то в качестве реакторов целесообразно применение реакторов со всзвешенным слоем или подобных им. 141 д 1 – корпус, 2 – катализатор, 3 – перфорированный стакан с сеткой, 4 – сетка, 5 – шарики фарфоровые, 6 – штуцер для эжекции газов, 7 – перфорированная трубка, 8 – штуцер для входа сырья, 9 – штуцер для выхода продуктов реакции, 10 − футеровка Рисунок 4.19. Схема к расчету основных размеров реактора 1 – корпус, 2 – катализатор, 3 – перфорированный стакан с сеткой, 4 – сетка, 5 – шарики фарфоровые, 6 – штуцер для эжекции газов, 7 – перфорированная трубка, 8 – штуцер для входа сырья, 9 – штуцер для выхода продуктов реакции, 10 − футеровка 1 – корпус, 2 – катализатор, 3 – перфорированный стакан с сеткой, 4 – сетка, 5 – шарики фарфоровые, 6 – штуцер для эжекции газов, 7 – перфорированная трубка, 8 – штуцер для входа сырья, 9 – штуцер для выхода продуктов реакции, 10 − футеровка 1 – корпус, 2 – катализатор, 3 – перфорированный стакан с сеткой, 4 – сетка, 5 – шарики фарфоровые, 6 – штуцер для эжекции газов, 7 – перфорированная трубка, 8 – штуцер для входа сырья, 9 – штуцер для выхода продуктов реакции, 10 − футеровка фу р Рисунок 4.19. Схема к расчету основных размеров реактора 141 На практике возможно применение реакторов, конструкции которых приведены на рисунках 4.19, 4.20 и 4.21. 1 – корпус, 2 – катализатор, 3 – шарики фарфоровые, 4 – люк для выгрузки катализатора, 5 – люк, 6 – штуцер для эжекции газов, 7 – штуцер для входа сырья, 8 – штуцер для выхода продуктов реакции, 9 − футеровка Рисунок 4.20. Схема к расчету основных размеров реактора 142 На практике возможно применение реакторов, конструкции которых приведены на рисунках 4.19, 4.20 и 4.21. 1 – корпус, 2 – катализатор, 3 – шарики фарфоровые, 4 – люк для выгрузки катализатора, 5 – люк, 6 – штуцер для эжекции газов, 7 – штуцер для входа сырья, 8 – штуцер для выхода продуктов реакции, 9 − футеровка Рисунок 4.20. Схема к расчету основных размеров реактора 1 – вход, 2 – цилиндрическиесетки, 3 – вход потока газа, 4 – выход катализатора, 5 – выход потока газа Рисунок 4.21. Реактор с движущимся слоем катализатора На практике возможно применение реакторов, конструкции которых приведены на рисунках 4.19, 4.20 и 4.21. На практике возможно применение реакторов, конструкции которых приведены на рисунках 4.19, 4.20 и 4.21. На практике возможно применение реакторов, конструкции которых приведены на рисунках 4.19, 4.20 и 4.21. 1 – корпус, 2 – катализатор, 3 – шарики фарфоровые, 4 – люк для выгрузки катализатора, 5 – люк, 6 – штуцер для эжекции газов, 7 – штуцер для входа сырья, 8 – штуцер для выхода продуктов реакции, 9 − футеровка Рисунок 4 20 Схема к расчету основных размеров реактора 1 – вход, 2 – цилиндрическиесетки, 3 – вход потока газа, 4 – выход катализатора, 5 – выход потока газа Рисунок 4.21. Реактор с движущимся слоем катализатора 1 – вход, 2 – цилиндрическиесетки, 3 – вход потока газа, 4 – выход катализатора, 5 – выход потока газа р , Рисунок 4.21. Реактор с движущимся слоем катализатора 142 Для расчета величины перепада давления в слое катализатора с целью, чтобы перепад не превышал допустимого значения [ΔПсл] при котором слой становится взвешенным можно воспользоваться формулой [67]: 0,35 1,35 2 0,29 255(1 ) сл см см э э П Н d d                , (4.62) 0,35 1,35 2 0,29 255(1 ) сл см см э э П Н d d                , (4.62) (4.62) где сл П Н  − потеря напора на 1 м толщины слоя катализатора в реакторе, Па/м; где сл П Н  − потеря напора на 1 м толщины слоя катализатора в реакторе, Па/м; λ − порозность слоя; λ − порозность слоя; ω − скорость фильтрования, м/с; 3 ρсм − плотность газов, кг/м3; νсм − кинематическая вязкость, м2/с; dЭ − эквивалентный диаметр частиц катализатора, м. Исходная смесь (бензин прямой гонки) при подаче в реактор нагревается до рабочей температуры, значение которой для каждой реакции приведено в системе уравнений. Очевидно, что исходя из типа реакции (см. схему) и значений температуры для каждой реакции риформинг бензиновых фракций необходимо осуществлять в блоке из четырех последовательно соединенных реакторов. Катализатор между реакторами распределили в соотношении 1:1:3:4. Если проанализировать конструкции реакторов и распределение потока газа в слое катализатора, то, повидимому, реактор на рисунке 4.21 более предпочтительный, поскольку режим (модель) движения газа через слой катализатора наиболее близка к модели идеального вытеснения. 142 На практике возможно применение реакторов, конструкции которых приведены на рисунках 4.19, 4.20 и 4.21. 1 – корпус, 2 – катализатор, 3 – шарики фарфоровые, 4 – люк для выгрузки катализатора, 5 – люк, 6 – штуцер для эжекции газов, 7 – штуцер для входа сырья, 8 – штуцер для выхода продуктов реакции, 9 − футеровка Рисунок 4.20. Схема к расчету основных размеров реактора 1 – вход, 2 – цилиндрическиесетки, 3 – вход потока газа, 4 – выход катализатора, 5 – выход потока газа Рисунок 4.21. Реактор с движущимся слоем катализатора Учитывая ранее принятое допущение, что реакции протекают в гомогенной фазе, то можно принять квазигомогенную модель протекания процесса в реакторах со следующими допущениями и записать в общем виде для любого реактора:     2 2 2 2 2 2 2 2 1 0 1 0 A A A A k A A e R p A A C C C C De D W W C R R l l R T T T T W gC HW C R R l l R                                                     . (4.63) (4.63) Граничные условия:   Граничные условия: Граничные условия:     0 . 0 . . 0 0 слоя кат A A A e p p e A сл кат l H C WC WC D l T W gC T W gC T l C T l H l l                             , (4.64) (4.64) 143 r (изменение от оси, при осевом) = 0 0 A C T R R       , (4.65) r (изменение от оси, при осевом) = 0 0 A C T R R       , (4.65) r (изменение от оси, при осевом) = 0 0 A C T R R       , (4.65) (4.65) r = R (размер от оси до внутренней стенки реактора)   1 0 A R T C R T k T T R         . (4.66) (4.66)   1 R T T k T T R     . (4.66) Схематично размеры представлены на рисунке 4.22 0,4D r R 0,2D L=0 D=2R l=L 2 / 4 к к сл М Н D    Катализатор Рисунок 4.22. 142 На практике возможно применение реакторов, конструкции которых приведены на рисунках 4.19, 4.20 и 4.21. 1 – корпус, 2 – катализатор, 3 – шарики фарфоровые, 4 – люк для выгрузки катализатора, 5 – люк, 6 – штуцер для эжекции газов, 7 – штуцер для входа сырья, 8 – штуцер для выхода продуктов реакции, 9 − футеровка Рисунок 4.20. Схема к расчету основных размеров реактора 1 – вход, 2 – цилиндрическиесетки, 3 – вход потока газа, 4 – выход катализатора, 5 – выход потока газа Рисунок 4.21. Реактор с движущимся слоем катализатора Схематичное изображение размеров реактора В выше приведенных уравнениях CA - концентрация i компонента в месях; De - коэффициент диффузии i компонента; DR - коэффициент адиальной диффузии i компонента; W - линейная скорость газа вдоль оординаты l (скорость фильтрации через слой катализатора); WA(CA) - скорость химической реакции превращения i компонента в целевой родукт; ρ - плотность газовой смеси; Cp - теплоемкость газовой смеси при остоянном давлении; λe, λR - соответственно, коэффициенты еплопроводности; ΔH - тепловой эффект реакции; D - диаметр реактора внутренний); R - радиус реактора (внутренний). Схематично размеры представлены на рисунке 4.22 Схематично размеры представлены на рисунке 4.22 Схематично размеры представлены на рисунке 4.22 0,4D r R 0,2D L=0 D=2R l=L 2 / 4 к к сл М Н D    Катализатор Рисунок 4.22. Схематичное изображение размеров реактора Катализатор Рисунок 4.22. Схематичное изображение размеров реактора В выше приведенных уравнениях CA - концентрация i компонента в смесях; De - коэффициент диффузии i компонента; DR - коэффициент радиальной диффузии i компонента; W - линейная скорость газа вдоль координаты l (скорость фильтрации через слой катализатора); WA(CA) - скорость химической реакции превращения i компонента в целевой продукт; ρ - плотность газовой смеси; Cp - теплоемкость газовой смеси при постоянном давлении; λe, λR - соответственно, коэффициенты теплопроводности; ΔH - тепловой эффект реакции; D - диаметр реактора (внутренний); R - радиус реактора (внутренний). Если допустить, что продольное перемешивание и поперечное 144 перемешивание в выбранной конструкции реактора незначительно, то систему основных уравнений можно представить в виде: перемешивание в выбранной конструкции реактора незначительно, то систему основных уравнений можно представить в виде:     2 / 4 0 0 к к сл A k A p A A М Н D C W W C R T W gC HW C l               . (4.67) (4.67) Тогда алгоритм (последовательность) расчета реакторного блока можно принять в следующем виде: 1. Принятие необходимых исходных величин и некоторые предварительные определения. 1. Принятие необходимых исходных величин и некоторые предварительные определения. 142 На практике возможно применение реакторов, конструкции которых приведены на рисунках 4.19, 4.20 и 4.21. 1 – корпус, 2 – катализатор, 3 – шарики фарфоровые, 4 – люк для выгрузки катализатора, 5 – люк, 6 – штуцер для эжекции газов, 7 – штуцер для входа сырья, 8 – штуцер для выхода продуктов реакции, 9 − футеровка Рисунок 4.20. Схема к расчету основных размеров реактора 1 – вход, 2 – цилиндрическиесетки, 3 – вход потока газа, 4 – выход катализатора, 5 – выход потока газа Рисунок 4.21. Реактор с движущимся слоем катализатора а) На основе промышленных и проектных данных принимаются: давление в начале процесса (первый реактор блока); температура, с которой сырье и циркулирующий газ подаются в первый реактор; объемная скорость подачи сырья; кратность циркуляции водородсодержащего газа и его состав, количество последовательно соединенных реакторов; а) На основе промышленных и проектных данных принимаются: давление в начале процесса (первый реактор блока); температура, с которой сырье и циркулирующий газ подаются в первый реактор; объемная скорость подачи сырья; кратность циркуляции водородсодержащего газа и его состав, количество последовательно соединенных реакторов; р р б) Рассчитывается мольный состав питания реактора и количества каждого компонента в циркулирующем газе. б) Рассчитывается мольный состав питания реактора и количества каждого компонента в циркулирующем газе. в) Рассчитывается необходимое количество алюмоплатинового катализатора с предварительным распределением его по реакторам. 2. Расчет первого реактора. а) Материальный баланс реактора рассчитывают по следующей схеме: определяют константу скорости реакции ароматизации; Расчет первого реактора. 2. Расчет первого реактора. а) Материальный баланс реактора рассчитывают по следующей схем − определяют константу скорости реакции ароматизации; ) Материальный баланс реактора рассчитывают по следующей схеме: а) Материальный баланс реактора рассчитывают по следующей схеме: − определяют константу скорости реакции ароматизации; − определяют константу скорости реакции ароматизации; − определяют константу химического равновесия реакции ароматизац − вычисляют уменьшение количества нафтеновых углеводородов в результате реакции ароматизации; − определяют константу скорости реакции превращения нафтеновых углеводородов в парафиновые; − определяют константу химического равновесия реакции превращения нафтеновых углеводородов в парафиновые; − вычисляют увеличение количества нафтеновых углеводородов результате реакции превращения парафиновых углеводородов в нафтеновые; − определяют константу скорости реакции гидрокрекинга нафтеновых углеводородов; − вычисляют уменьшение количества нафтеновых углеводородов в результате реакции гидрокрекинга; результате реакции гидрокрекинга; − вычисляют уменьшение количества парафиновых углеводородов в результате реакции гидрокрекинга; − рассчитывают материальный баланс реакций в реакторе; рассчитывают материальный баланс реакций в реакторе; − определяют состав газа, покидающего реактор; − определяют состав газа, покидающего реактор; − составляют материальный баланс реактора и определяют выходы продуктов риформинга. 145 б) Составляют тепловой баланс реактора с целью определения температуры выходящего из него потока. в) Принимают тип реактора (радиальный или аксиальный) и определяют его диаметр и высоту. 3. Расчет второго реактора. а) Выбирают температуру в начале процесса, имея в виду во втором реакторе практически полное превращение нафтеновых углеводородов. Давление в этом реакторе должно быть несколько ниже, чем в первом. б) Материальный баланс второго реактора рассчитывается в том же порядке, как и для первого реактора в) Расчет теплового баланса второго реактора аналогичен расчету для первого реактора. г) Определяются диаметр и высота реактора. 142 На практике возможно применение реакторов, конструкции которых приведены на рисунках 4.19, 4.20 и 4.21. 1 – корпус, 2 – катализатор, 3 – шарики фарфоровые, 4 – люк для выгрузки катализатора, 5 – люк, 6 – штуцер для эжекции газов, 7 – штуцер для входа сырья, 8 – штуцер для выхода продуктов реакции, 9 − футеровка Рисунок 4.20. Схема к расчету основных размеров реактора 1 – вход, 2 – цилиндрическиесетки, 3 – вход потока газа, 4 – выход катализатора, 5 – выход потока газа Рисунок 4.21. Реактор с движущимся слоем катализатора г) Определяются диаметр и высота реактора. 4. Расчет третьего реактора. а) Температуру в начале процесса принимают равной температуре потока, выходящего из второго реактора. б) Остальные расчеты ведутся так же, как и для первого и второго реакторов. Рассмотрим пример. Необходимо подвергнуть каталитическому риформингу в 4-х последовательно соединенных в ряд реакторах с неподвижным слоем платинового катализатора 11,56 м3 смеси углеводородов со следующими характеристиками: у р р Плотность, кг/дм3 1,05 Средняя молекулярная масса 114,8 Температура кипения, оС − начало кипения − 10 % − 50 % − 90 % − конец кипения 84 112 129 155 180 Плотность, кг/дм3 Средняя молекулярная масса Средняя молекулярная масса Температура кипения, оС − начало кипения − конец кипения Состав смеси приведен в таблице 4.3, условия работы – в таблице 4.4. Исходная смесь нагревается (в целом реакция эндотермична) перед каждым реактором до рабочей температуры. − конец кипения Таблица 4.3 – Состав смеси, подвергаемой каталитическому риформингу Компонент Состав смеси, %мол Молекулярная масса Количество кг моль Алканы 31,0 С8,2Н18,4 = 116,8 3830 32,9 Циклоалканы 58,3 С8,2Н16,4 = 114,8 7050 61,5 Ароматические углеводороды 10,7 С8,2Н10,4 = 108,8 1230 11,3 Всего 100,00 12110 105,7 146 Таблица 4.4 – Условия работы реакторов Таблица 4.4 – Условия работы реакторов Условия Реакторы I II III IV Соотношение катализатора в реакторах 1 1 2,75 3,5 Расход смеси, кг/(ч·кг катализатора) 22,69 22,69 8,25 6,48 Обратное время контакта 1/ηs, кмоль/(ч·кг катализатора) 0,198 0,198 0,072 0,056 Давление (абсолютное) в реакторе, ат 41,4 41,0 40,8 39,7 Температура на входе в реактор, оС 506 Соотношение циркулирующего потока и потока питания 69 Количество циркулирующего водорода, %мол 85,3 Количество циркулирующих молей 6,0 Давление (абсолютное) на входе в реактор, ат 36,5 Таблица 4.4 – Условия работы реакторов Условия Реакторы I II III IV Соотношение катализатора в реакторах 1 1 2,75 3,5 Расход смеси, кг/(ч·кг катализатора) 22,69 22,69 8,25 6,48 Обратное время контакта 1/ηs, кмоль/(ч·кг катализатора) 0,198 0,198 0,072 0,056 Давление (абсолютное) в реакторе, ат 41,4 41,0 40,8 39,7 Температура на входе в реактор, оС 506 Соотношение циркулирующего потока и потока питания 69 Количество циркулирующего водорода, %мол 85,3 Количество циркулирующих молей 6,0 Давление (абсолютное) на входе в реактор, ат 36,5 Таблица 4.5 – Расчетные результаты, полученные для каталитического риформинга 12,11 т углеводородов, и результаты, полученные на промышленных установках Характеристика Результаты, полученные при расчете реакторов Результаты, полученные в реакторах на промышленных установках I II III IV I II III IV Расход смеси, кг/(ч·кг катализатора) 22,69 22,69 8,25 6,48 22,69 22,69 8,25 6,48 Температура, оС: − на входе в реактор − на выходе из реактора 506 436 506 460 506 485 506 509 511 442 511 469 513 489 511 502 Понижение температуры, град 70 46 21 −3 69 42 24 9 Св+алкан, который вышел из реактора, кмоль 34,4 35,8 34,0 19,2 Св+циклоалкан, который вышел из реактора, кмоль 36,2 17,9 5,75 2,22 Ароматические углеводороды, которые вышли из реактора, кмоль 33,9 49,5 58,5 63,2 Всего 104,5 103,2 98,25 84,62 Общие результаты Понижение температуры в реакторе, град 134 144 Таблица 4.5 – Расчетные результаты, полученные для каталитического риформинга 12,11 т углеводородов, и результаты, полученные на промышленных установках Характеристика Результаты, полученные при расчете реакторов Результаты, полученные в реакторах на промышленных установках I II III IV I II III IV Расход смеси, кг/(ч·кг катализатора) 22,69 22,69 8,25 6,48 22,69 22,69 8,25 6,48 Температура, оС: − на входе в реактор − на выходе из реактора 506 436 506 460 506 485 506 509 511 442 511 469 513 489 511 502 Понижение температуры, град 70 46 21 −3 69 42 24 9 Св+алкан, который вышел из реактора, кмоль 34,4 35,8 34,0 19,2 Св+циклоалкан, который вышел из реактора, кмоль 36,2 17,9 5,75 2,22 Ароматические углеводороды, которые вышли из реактора, кмоль 33,9 49,5 58,5 63,2 Всего 104,5 103,2 98,25 84,62 Общие результаты Понижение температуры в реакторе, град 134 144 Количество компонентов, кмоль: − Н2 114 126 кмоль/12,11 т питания 147 − С1 − С2 − С3 − изо−С4 − изо−С5 − н−С5 − н−С6+алкан − циклоалкан −ароматические углеводороды 11,1 11,1 11,1 5,51 5,51 7,42 3,72 19,2 2,2 63,5 Всего, кмоль 254,36 Количество компонентов, %об − изо−С4 − изо−С6 − н−С6 − С6+алкан − циклоалкан −ароматические углеводороды − 0,625 0,312 2,24 0,242 5,75 0,287 0,287 0,184 2,46 0,23 5,92 Всего 9,17 9,368 Ароматические углеводороды в фракции С5, соответственно дистилату, %об 62,8 63,2 Полученный чистый октан, %об 95,4 95,5 Примечание. − конец кипения 1 кмоль изо−С4 является ½ фракции С4, а 1 кмоль изо−С5 является 3/2 фракции С5. В таблице 4.5 приведены результаты расчетов на ЭВМ и результаты, полученные при работе в промышленных условиях. I – IV – реакторы; 1 – 4 подогреватели; 5 – компрессор для рециркуляции газов I – IV – реакторы; 1 – 4 подогреватели; 5 – компрессор для рециркуляции газов Рисунок 4.23. Технологическая схема каталитического риформинга в установке из четырех реакторов 148 На рисунке 4.24 дана зависимость температуры в реакторах каталитического риформинга от ηs. На рисунке 4.24 дана зависимость температуры в реакторах каталитического риформинга от ηs. На основе полученных результатов можно сделать следующие выводы. Вычисленные температуры на входе в реактор намного меньше, чем температуры, полученные в промышленных установках при такой же степени превращения. Это несоответствие обусловливается тем, что константа скорости реакции и константа равновесия были рассчитаны для свежего катализатора. Вследствие дезактивации катализатора температура в промышленном реакторе должна быть более высокой. 448 472 496 424 520 Температура, оС 4 8 12 16 20 S 0,453 кг катализатора τ = моль питания/ч IV III II I Рисунок 4.24. Зависимость температур в реакторе каталитического риформинга от времени контакта ηs I – IV − номера реакторов моль питания/ч Рисунок 4.24. Зависимость температур в реакторе каталитического риформинга от времени контакта ηs I – IV − номера реакторов Рисунок 4.24. Зависимость температур в реакторе каталитического риформинга от времени контакта ηs I – IV − номера реакторов При понижении температуры дегидрогенизация становится преобладающей реакцией в первом и втором реакторе (реакция гидрогенизации эндотермична). В третьем реакторе, несмотря на понижение температуры по длине слоя катализатора, гидрокрекинг циклоалканов и алканов представлен правой частью температурной кривой. Гидрокрекинг происходит в наибольшей степени в четвертом реакторе. Этот метод расчета позволяет выбрать условия работы, число реакторов с внутренним теплообменом и толщину слоя катализатора в промышленных реакторах. Результаты расчета, проведенного по этому методу, совпадают с результатами, полученными в промышленных реакторах. Таким образом, представленный подход к инженерному расчету к конструктивному оформлению, а также математическое описание процесса (уравнения кинетики, материального и теплового баланса) позволяют проводить прогнозирующий расчет процесса и реактора, результаты которого могут быть использованы при проектировании установки. 149 5 АНАЛИЗ ЭКСПЛУАТАЦИОННОГО ФУНКЦИОНИРОВАНИЯ РЕАКТОРА ГЕТЕРОГЕННОГО КАТАЛИЗА В ПРОМЫШЛЕННЫХ УСЛОВИЯХ Любое научное исследование, проектирование или управление действующим производством сопровождается триадой: восприятием, переработкой и передачей информации. Применение ЭВМ значительно облегчает все вышеуказанные этапы, особенно на стадии решения математической модели процесса и ее анализа с целью оптимизации технологического процесса и конструкций реакторов, аппаратов и машин или создания новых технических решений. Математическая модель вторична и всегда основывается на физической и химической моделях. Такие модели в свою очередь являются идеальным отражением совокупности основных, существенных черт натурного объекта, будь то отдельно взятый реактор, аппарат, машина или технологическая операция и кончая производством в целом [68]. На рисунке 5.1 приведена схема этапов восприятия, переработки и передачи информации. РЕШЕНИЕ АНАЛИЗ РЕШЕНИЯ СБОР ИНФОРМАЦИЯ СОСТАВЛЕНИЕ ФИЗИЧЕСКОЙ, ХИМИЧЕСКОЙ МОДЕЛЕЙ МАТЕМАТИЧЕСКОЙ МОДЕЛИ Рисунок 5.1 Схема восприятия, переработки и передачи информации без применения ЭВМ (сплошная линия), при использовании ЭВМ (пунктирная линия) и комбинированная (штрихпунктирная линия) РЕШЕНИЕ АНАЛИЗ РЕШЕНИЯ СБОР ИНФОРМАЦИЯ СОСТАВЛЕНИЕ ФИЗИЧЕСКОЙ, ХИМИЧЕСКОЙ МОДЕЛЕЙ МАТЕМАТИЧЕСКОЙ МОДЕЛИ РЕШЕНИЕ АНАЛИЗ РЕШЕНИЯ СБОР ИНФОРМАЦИЯ СОСТАВЛЕНИЕ ФИЗИЧЕСКОЙ, ХИМИЧЕСКОЙ МОДЕЛЕЙ МАТЕМАТИЧЕСКОЙ МОДЕЛИ Рисунок 5.1 Схема восприятия, переработки и передачи информации без применения ЭВМ (сплошная линия), при использовании ЭВМ (пунктирная линия) и комбинированная (штрихпунктирная линия) Рисунок 5.1 Схема восприятия, переработки и передачи информации без применения ЭВМ (сплошная линия), при использовании ЭВМ (пунктирная линия) и комбинированная (штрихпунктирная линия) Большим подспорьем могут служить автоматизированные банки знаний и данных, включающие не только хорошо известные законы и эмпирические зависимости, но и открытия, эффекты и явления, полученные в самое последнее время. Применение их в комбинации с традиционными обычно приводит к созданию качественно новых технических решений способов проведения технологического процесса и конструкций машин и аппаратов. Математическое моделирование новых решений может быть непосредственным, когда анализ математической модели сразу подсказывает новые технические решения (рис. 5.2 а), и опосредственным, когда из физических и химических моделей логическими рассуждениями показывается принципиальная возможность нового технического решения и затем создается его математическая модель, позволяющая найти оптимальные количественные соотношения (рис. 5.2 б). Эффективна комбинированная схема математического моделирования изобретений, сочетающая непосредственный и опосредственный способы с одновременным анализом физической, химической и математической моделей (рис. 5.2 в). 5 АНАЛИЗ ЭКСПЛУАТАЦИОННОГО ФУНКЦИОНИРОВАНИЯ РЕАКТОРА ГЕТЕРОГЕННОГО КАТАЛИЗА В ПРОМЫШЛЕННЫХ УСЛОВИЯХ 150 Физическая или химическая модель Математическая модель и ее анализ Новое техническое решение а) Физическая или химическая модель Математическая модель и ее анализ Новое техническое решение б) Физическая или химическая модель Математическая модель и ее анализ Новое техническое решение в) а) непосредственная; б) опосредственная; в) комбинированная Рисунок 5.2 Схемы создания технических решений с использованием математического моделирования б) а) Физическая или химическая модель Математическая модель и ее анализ Математическая модель и ее анализ в) Физическая или химическая модель Математическая модель и ее анализ Метод математического моделирования новых технических решений особенно важно применять при получении ранее неизвестных в теории и практике результатов или физико-химических эффектов, позволяющих их использовать, прежде всего, в способах измерений и конструкций приборов, а затем и непосредственно в реакторах, аппаратах и технологических процессах. Вышеотмеченный тезис обосновывается тем, что многие исследователи публикуют полученные теоретические результаты, заявляя, что они касаются только выводов формул, и в них нет описания новых технических решений. Так как практически все периодические научные журналы и сборники научных трудов доступны для иностранных специалистов, то специалистам зарубежных фирм ничего не стоит провести анализ математической модели на предмет создания новых технологий и конструкций. ру Все рассмотренные выше алгоритмы получения новых технических решений на основе математической модели позволяют проводить анализ последней как без применения ЭВМ так и с применением ЭВМ, что обеспечивает значительное уменьшение затрат времени. Ниже на примере реакции гидрохлорирования ацетилена показаны возможности оптимизации техпроцесса и конструкции реактора при использовании ЭВМ лишь на стадии решения математической модели. Здесь творческие стадии: сбор информации, представление физической и химической картины процесса, составление уравнений, его описывающих, и особенно анализ решения выполняется исследователем, что способствует на наш взгляд лучшему пониманию рассматриваемого метода математического моделирования новых технических решений. 151 Анализ работы реакторов гидрохлорирования ацетилена показывает, что основной проблемой для них является крайняя неравномерность температуры реакционных газов по длине труб трубного пучка, из-за этого происходит термическая деструкция катализатора – (HgCl2) сулемы на активированном угле – и возникает необходимость его частой замены. Реакторы работают по японской лицензии, и каждый из них представляет собой кожухотрубный аппарат, где по трубам с твердым зернистым катализатором движется реакционная газовая смесь, а в мвжтрубном пространстве кипит вода, отводящая тепло реакции. Составление физической и математической моделей Реакция гидрохлорирования ацетилена k 2 2 2 k С Н HCl СН CHCl   2 2 2 k С Н HCl СН CHCl   Относится к газовым каталитическим экзотермическим реакциям с кинетическим уравнением dC Относится к газовым каталитическим экзотермическим реакциям с кинетическим уравнением A A dC kC dt  . (5.1) (5.1) Из уравнения Аррениуса 0 exp E k k RT         или после подставлении численных значений 1584 9,55exp k T         . Из уравнения Аррениуса 0 exp E k k RT         или после подставлении численных значений 1584 9,55exp k T         . Решение уравнения при замене dz dt v  после алгебраических преобразований A A dC k C dz   , (5.2) (5.2) z и в связи со степенью превращения и в связи со степенью превращения р р 0 1584 1 , 9,55 exp 1 А А А А А С d dz С T                 , приводится к виду 0 1584 1 exp 9,55 exp z А dz T                      . (5.3) Тогда 0 9,55 9,55 exp 1584 1584 exp exp z А dz d dz T T                                    . Составление физической и математической моделей (5.4) (5.3) (5.4) 152 153 Таблица 5.1 Технологические и физико-химические параметры реактора синтеза винилхлорида № Наименование параметра Обозначение Размерность Величина 1 Средняя скорость газа в трубе  м/с 0,16 2 Концентрация ацетилена САо кмоль/А/кмоль 0,085 3 Степень превращения А  доли 0,97 4 Расход ацетилена на входе в трубу A q кмоль А/с 1,1×10−5 5 Тепловой эффект реакции tq кДж/кмоль А 1,1×105 6 Константа скорости k С−1 9,55 7 Массовый расход реакционного газа в трубе m q Кг/с 3,75×10−3 8 Теплоемкость газов пиролиза нефти p c кДж/кг град 1,05 9 Давление в трубах p ата 6 – 7 10 Давление пара в межтрубном пространстве k p ата 1 11 Коэффициент теплопередачи tp вт/М2 град 42 12 Наружный диаметр трубы D м 0,08 13 Внутренний диаметр трубы В d м 0,075 14 Длина трубы l м 6,0 15 Температура реакционной массы на входе в трубу Т оС 100 16 Температура кипящей воды в межтрубном пространстве В Т оС 100 17 Энергия активации Е кДж/кмоль А 13140 18 Катализатор сулема на активированном угле доли 0,1 19 Газовая постоянная R кДж/кмоль А град 8,314 20 Константа скорости реакции при  o K 1/сек 9,55 qm qm qt dz z T Т dz z   ТВ Т z ; А А А С С С dz z   ; А А dz z      Рисунок 5.3 Схема тепловых и массообменных процессов в политропическом реакторе идеального вытеснения на элементе длины dz Т Рисунок 5.3 Схема тепловых и массообменных процессов политропическом реакторе идеального вытеснения на элементе длины dz Рисунок 5.3 Схема тепловых и массообменных процессов в политропическом реакторе идеального вытеснения на элементе длины dz в 153 Составим тепловой баланс для элемента длины трубы реактора. ( ) o m p t A t В q C dT q q d k d T T dz      , (5.5 (5.5) ру р р ( ) o m p t A t В q C dT q q d k d T T dz      , (5.5) где левая часть – приращение энергии в выделенном объеме; первый член в правой части – выделяющаяся энергия за счет теплопередачи через элементарную поверхность трубы к кипящей в межтрубном пространстве воде. элементарную поверхность трубы к кипящей в межтрубном пространстве воде. Составление физической и математической моделей После подставления в последнее выражение значения dχ получаем интегродифференциальную зависимость температуры от координаты длины z :   После подставления в последнее выражение значения dχ получаем интегродифференциальную зависимость температуры от координаты длины z : exp ( ) 1584 1584 exp exp o z o o m p t A t K o K K dz q C dT q q dz k d T T dz T T                                    , (5.6) Заменяя интегрирование суммированием и дифференциалы конечными разностями, придем к уравнению теплового баланса, удобного для расчета на ЭВМ Заменяя интегрирование суммированием и дифференциалы конечными разностями, придем к уравнению теплового баланса, удобного для расчета на ЭВМ 1 1 1 1 exp 1584 1584 exp exp o i t A o o i i m p j i j q q K K z z T T q C T T                                               . (5.7) (5.7) Основные допущения, сделанные при выводе уравнений следующие: температура по сечению трубы постоянна 0 dT dz  ; T = T(z); скорость потока постоянна как по сечению, так и по длине 0 dv dv dz dr   , то есть структура потока соответствует идеальному вытеснению. у у После подставления в уравнение числовых значений параметров получаем расчетную зависимость   1 1 1 1 1584 1584 exp exp 373 i i i i j j j Т T B a b T T T                              , (5.8) где 0 185 o t A m p q q K B z q C        , 0 0,6 K a z    , 0,01 z  . (5.8) Результаты расчетов приведены на рисунке 5.4 (кривая 1а) и практически совпадают с экспериментальной кривой распределения температур длине труб реактора в начальный период работы свежем катализаторе. Анализ решения р Как видно из графика (рис. 5.4), около полуметра высоты катализатора в 154 трубах имеют температуру больше 180 °С (на рис. 5.4 эта область заштрихована). По техническому регламенту температура больше 180 °С приводит к быстрой термической дезактивации катализатора, вследствие которой область максимальных, температур постепенно перемещается от входа к выходу, катализатор быстро выходит из строя, а степень превращения резко падает. Необходимо уменьшить максимальную температур реакции, чтобы предотвратить термическую дезактивацию катализатора. Рассмотрим возможные технологические варианты решения этой задачи, анализируя уравнение. Простой и хорошо известный технологический прием, сразу бросающийся в глаза, − уменьшение температуры исходных газов на входе в реактор. В программе изменение коснется лишь первой строчки: при tH = 60 °C 1 допустим х = 333°, р = 0. , р График полученной зависимости представлен рисунке 5.4, кривая 3а. 1 − tН = 100 °С (промышленный реактор); 2 − tН = 80 °С; 3 − tН = 60 °С; 4 − tН = 100 °С и = 80 °С (вакуум в межтрубном пространстве); 5 − два промышленных реактора, соединенных последовательно при увеличении скорости реакционных газов в 2 раза по сравнению с основным реактором Рисунок 5.4 Распределение температуры и концентрации ацетилена индексы а и б соответственно) по длине труб реактора синтеза винилхлорида 1 − tН = 100 °С (промышленный реактор); 2 − tН = 80 °С; 3 − tН = 60 °С; 4 − tН = 100 °С и = 80 °С (вакуум в межтрубном пространстве); 5 − два промышленных реактора, соединенных последовательно при увеличении скорости реакционных газов в 2 раза по сравнению с основным реактором Рисунок 5.4 Распределение температуры и концентрации ацетилена (индексы а и б соответственно) по длине труб реактора синтеза винилхлорида реакционных газов в 2 раза по сравнению с основным реактором Рисунок 5.4 Распределение температуры и концентрации ацетилена (индексы а и б соответственно) по длине труб реактора синтеза винилхлорида Как видно из графика, уменьшение температуры реакционных газов на входе со 100° до 60° уменьшает максимальную температуру до 180 °С, то есть соответствует максимально допустимой температуре по техрегламенту Серьезным недостатком этого способа является снижение температуры по всей высоте реактора и как следствие – уменьшение степени превращения (рис. 5.4, кривыѐ 1б, 2б, 3б). Несложен для анализа вариант уменьшения температуры кипящей в межтрубном пространстве воды. Анализ решения Поэтому можно увеличить скорость в 2 раза, если поставить два реактора, работать последовательно (сейчас одновременно работают шесть реакторов параллельно), поэтому уравнение приводится к виду: 156   1 1 1 1 1 1584 1584 92,5 exp 0,3 exp 0,0239 373 i i i i j j j Т T T T T                              . (5.12) Результаты расчета приведены на рисунке 5.4, кривая 5. С точки зрения выравнивания температуры по длине труб вариант хорош, но увеличение скорости в два раза приводит к увеличению энергозатрат на продавливание реакционных газов через трубы. Рассмотрим вариант увеличения скорости путем рецикла части прореагировавшего газа. Результаты расчетов приведены на рисунке 5.4 кривая 4. Как видно из графика, уже на 10 % рецикл уменьшает максимальную температуру до 177 оС. Патентная проработка показывает, что известны способы проведения каталитических гетерогенных процессов, предусматривающие возвращение части прореагировавших продуктов реакции на смешение с исходным сырьем. Очевидным недостатком этого способа является уменьшение времени пребывания реагирующей массы в трубах реактора и увеличение затрат энергии на продавливание газа через трубы. Основным же недостатком является уменьшение температуры по всей длине труб реактора. Очевидно, чтобы уменьшить максимальную температуру, нужен рецикл в области этих температур. Преимущество такого способа связаны с увеличением времени пребывания и степени превращения и снижением гидравлических сопротивлений. Практически на действующих производствах, чтобы не изменять конструкцию обычного кожухотрубного реактора, можно перед ним установить так называемый фор-реактор и проводить рецикл только в нем. Так как зона максимальных температур будет в этом фор-реакторе, то катализатор дезактивируется в нем сильнее, чем в основном реакторе. Но замена катализатора в маленьком фор-реакторе проще и может происходить без остановки основного реактора. Для этого достаточно на весь блок основных реакторов (6 штук) установить два − три фор-реактора. После пускового режима реактора (до 1 месяца) на свежем катализаторе его активность падает и можно в дальнейшем вести реакцию только в основном реакторе, а фор-реактор подключить к следующему основному реактору, работающему в пусковом режиме. Один из основных реакторов также, как и один фор-реактор, отключены – на них идет замена катализатора и ремонтно-профилактические работы. Для рассматриваемого варианта технологического процесса применение рецикла требует для своей реализации установки на линии дополнительного компрессора, чтобы компенсировать разницу в давлении газов на входе и выходе из реактора. Анализ решения 155 В основной программе это отразится в записи третьей строки, где температуру кипящей в межтрубном пространстве воды 373 К (или 100 °С) можно заменить, например, на 353 К (или 80 оC) Результаты расчетов приведены на рисунке 5.4 кривая 4. Максимальная температура реакционных газов стала меньше 180 оC, но она снизилась по всей высоте труб, а значит упала степень превращения. Кроме того, вариант технически сложен, так как для уменьшения температуры кипения воды нужно создавать в межтрубном пространстве вакуум. Таким образом, рассмотренные простые технологические решения не дают желаемого результата − снижения максимальной температуры и возрастания рабочих температур за точкой максимума. Дальнейший анализ уравнения показывает, что эффективным средством воздействия на температуру является скорость реакционных газов, причем эта зависимость носит обратный характер (во втором слагаемом в правой части скорость стоит в знаменателе, зависимость теплопередачи от скорости носит прямой характер, но третий член в правой части стоит со знаком минус). Как известно, коэффициент теплопередачи связан с коэффициентами теплоотдачи и теплопроводностью стенки уравнением 2 1 1 1 t i в i K         . (5.9) 2 1 1 1 t i в i K         . (5.9) (5.9) Коэффициент теплоотдачи к кипящей воде αв = 103÷104 вт/м2·град, термическое сопротивление стенки мало, поэтому практически 2 t K   , где α2 − коэффициент теплоотдачи от реакционных газов к стенке трубы. Из уравнения Лева для теплоотдачи в зернистом слое 0 813   0,9 3 0,813 Re exp 6 / в Nu d d  , (5.10) (5.10) где 2 2 2 , Re в в d d Nu         , где 2 2 2 , Re в в d d Nu         , где d3 − диаметр зерна катализатора. где d3 − диаметр зерна катализатора. Получаем: 0,9 1 1            . (5.11) (5.11) Простое увеличение скорости приводит к уменьшению времени пребывания. Анализ решения Увеличить скорость газов в фор-реакторе можно и без применения рецикла, а лишь уменьшением площади сечения труб, по которым движутся реакционные газы, из уравнения неразрывности: 157 2 4 m в q n d        . (5.13) 2 4 m в q n d        . (5.13) Так как массовый расход газов и их плотность – нереагируемые параметры, задаваемые техрегламентом, то увеличить скорость v в фоз- реакторе можно путем уменьшения числа труб n или их диаметра dв, или одновременным уменьшением обоих параметров. Этот вариант приводит к снижению времени пребывания и производительности реактора. р р р р р Задачу повышения скорости на выходе в реактор с ее постепенным снижением к выходу можно решить, используя конические трубы. у р , у Диаметр трубы изменяется по формуле: у р у ру Диаметр трубы изменяется по формуле: k H x H d d d d z l    . (5.14) (5.14) Чтобы время пребывания реакционной массы в конической трубе было не меньше времени пребывания в промышленном реакторе 6 37,5 0,16 l t с     . (5.15) (5.15) Найдем соотношение диаметров конической трубы на входе dН и на выходе dК, исходя из условия l 0 l z l d t   и 2 4q d    . 0 l z l d t   и 2 4q d    . Тогда 2 0 0 4 l k H z d d d t d z q l              . (5.16) 0 l z l d t   и 2 4q d    . Тогда 2 0 0 4 l k H z d d d t d z q l              . (5.16) Тогда 2 0 0 4 l k H z d d d t d z q l              . (5.16) (5.16) Или после преобразований Или после преобразований Или после преобразований   2 2 0 12 k k H t l d d d d q       . Анализ решения (5.17) (5.17) Если скорость изменяется в два раза, то 2 k H d d  следовательно   2 3 2 12 3 0,8246 1,166 3 2 H H k t l d q d d d d           . (5.18) Если скорость изменяется в два раза, то 2 k H d d  следовательно Если скорость изменяется в два раза, то 2 k H d d  следовательно   2 3 2 12 3 0,8246 1,166 3 2 H H k t l d q d d d d           . (5.18) (5.18) Скорость реакционной массы в произвольном сечении Скорость реакционной массы в произвольном сечении 158 2 4 1,47 1 0,414 k н н q z d d d z l l                      . (5.19) (5.19) Уравнение теплового баланса для произвольного сечения z на элементарной длине dz запишется в виде Уравнение теплового баланса для произвольного сечения z на элементарной длине dz запишется в виде   2 2 0,8 1 0,414 1 0,414 exp 1584 1584 1,47 exp 1,47 exp 1 0,824 0,344 o z t A o o m p o t k z z q q k k dz l l q C dt T T k d T T dz z l                                                                   . Анализ решения (5.20) Расчетная формула реактора с коническими трубами преобретает вид:   асчетная формула реактора с коническими трубами преобретает вид:   Расчетная формула реактора с коническими трубами преобретает вид:           2 2 1 1 1 1 0,8 1 0,824 0,057 0,824 0,057 exp 1584 1584 exp exp 0,824 0,057 i i i j i j i k B z z T T a T T A z T T                                             . (5.21) d . (5.21) Результаты расчета реактора с коническими трубами при 2 k н d d  представлены на рисунке 5.5 кривая 2. Трудности, связанные с изготовлением б Результаты расчета реактора с коническими трубами при 2 k н d d  представлены на рисунке 5.5 кривая 2. Трудности, связанные с изготовлением конических труб, можно компенсировать секционными цилиндрическими трубами с увеличением диаметра от секции к секции. Результаты расчета реактора с коническими трубами при 2 k н d d  н d представлены на рисунке 5.5 кривая 2. Трудности, связанные с изготовлением конических труб, можно компенсировать секционными цилиндрическими трубами с увеличением диаметра от секции к секции. представлены на рисунке 5.5 кривая 2. Трудности, связанные с изготовлением конических труб, можно компенсировать секционными цилиндрическими трубами с увеличением диаметра от секции к секции. у у При установке трехсекционных труб длиной lC по 2 м каждая и диаметром dC расчетная формула для каждой секции приобретает вид:   2 2 1 1 1 1 0,8 1 1584 1584 185 exp 0,6 exp 0,0257 373 c o i A c c i i A j i j c i q d d T T d q d T T d T d                                                   . Анализ решения (5.22) (5.22) где qAη = qAo для I секции;  где qAη = qAo для I секции;  где qAη = qAo для I секции;   − для II i c o o A A A A C q q C          ; 1 г o A A C p C  − доля непрореагировавшего ацетилена на выходе из первой секции; − для II i c o o A A A A C q q C          ; 1 г o A A C p C  − доля непрореагировавшего ацетилена на выходе из первой секции; − для II i c o o A A A A C q q C          ; 1 г o A A C p C  − доля непрореагировавшего ацетилена на выходе из первой секции; на выходе из первой секции; 159 − для III i c o o A A A A C q q C          ; 2 г o A A C p C  − доля непрореагировавшего ацетилена на выходе из второй секции. 1 − промышленный реактор; 2 – реактор с коническими трубами при 2 к н d d  ; 3 – реактор с секционными трубами при длине каждой секции 2 м и увеличении диаметра от секции в 1,5 раза; 4 – реактор с рециклом Rц = 0,1; 5 – реактор с рециклом Rц = 0,4. Рисунок 5 5 −Распределение температуры и концентрации ацетилена − для III i c o o A A A A C q q C          ; 2 г o A A C p C  − доля непрореагировавшего ацетилена на выходе из второй секции.   на выходе из второй секции. 1 − промышленный реактор; 2 – реактор с коническими трубами при 2 к н d d  ; 3 – реактор с секционными трубами при длине каждой секции 2 м и увеличении диаметра от секции в 1,5 раза; 4 – реактор с рециклом Rц = 0,1; 5 – реактор с рециклом Rц = 0,4. Анализ решения р р р ц Рисунок 5.5 − Распределение температуры и концентрации ацетилена (индексы а) и б) соответственно) по длине труб реактора синтеза винилхлорида Результаты приведены на рисунке 5.5 кривая 3. Анализ авторских свидетельств и патентов показал, что конструкции реакторов с коническими и секционными трубами обладают новизной, существенными отличительными признаками и положительным эффектом и является новыми техническими решающими признаками. Анализ авторских свидетельств и патентов показал, что конструкции реакторов с коническими и секционными трубами обладают новизной, существенными отличительными признаками и положительным эффектом и является новыми техническими решающими признаками. 1. Кожухотрубный реактор для проведения неизотермических реакций, состоящий из корпуса с пучком труб, закрепленных в трубных решетках, и патрубков для входа и выхода реакционной массы и теплоносителя, отличающийся тем, что с целью повьшения качества продуктов реакции за счет выравнивания профиля температур по длине реактора каждая труба трубного пучка выполнена из трех или более трубок равной длины, диаметр которых увеличивается по ходу потока реакционной массы. у у р 2. Реактор по пункту 1, отличающийся тем, что каждая труба трубного 2. Реактор по пункту 1, отличающийся тем, что каждая труба трубного 160 пучка выполнена конически расширяющейся по ходу потока реакциоиной массы. 3. Реактор по пункту 2, отличающийся тем, что каждая труба трубного пучка снабжена размещенными на ее наружной поверхности ребрами с высотой, уменьшающейся по ходу потока реакционной массы и наружным диаметром, равным диаметру трубы на выходе». 3. Реактор по пункту 2, отличающийся тем, что каждая труба трубного пучка снабжена размещенными на ее наружной поверхности ребрами с высотой, уменьшающейся по ходу потока реакционной массы и наружным диаметром, равным диаметру трубы на выходе». Последний, третий пункт позволяет нивелировать недостаток конических и секционных труб, связанный с уменыиением площади теплопередающей поверхности на входе, где тепловыделение или теплопоглощение за счет химической реакции наибольшее. Целесообразно применение таких оребрѐнных снаружи труб, когда лимитирующей стадией процесса теплопередачи является стадия теплоотдачи от стенки к хладоагенту в межтрубном пространстве. В рассматриваемом конкретном случае кожухотрубного реактора винилхлорида, как показано выше, лимитирующей стадией является теплоотдача от реакцяонных газов взернистом слое внутри трубы к стенкам, поэтому оребрение нецелесообразно. Общий вид кожухотрубного реактора с секционными трубами и конической трубы с оребрением показан на рисунке 5.6. конической трубы с оребрением показан на рисунке 5.6. Анализ решения Рисунок 5.6 Общий вид кожухотрубного реактора с секционными трубами (а) и конической оребренной снаружи трубы (б) Рисунок 5.6 Общий вид кожухотрубного реактора с секционными трубами (а) и конической оребренной снаружи трубы (б) Переменная площадь сечения ребер позволяет в области наибольшего тепловыделения или теплопоглощения на входе развить наибольшую площадь теплопередающей поверхности, которая уменьшается по мере продвижения 161 реакционной массы по трубе и снижения тепла реакции. реакционной массы по трубе и снижения тепла реакции. реакционной массы по трубе и снижения тепла реакции. Показанная возможность выравнивания профиля температуры по длине труб путем изменения скорости реакционных газов за счет изменения диаметра позволяет спроектировать реактор с постоянной температурой реакционной массы, то есть перейти от политропического реактора идеального вытеснения v = const, T = T(z) к изотермическому реактору реального вытеснения v = v(z), Т = const. Теоретическое решение уравнения должно быть таким, чтобы температура реакционных газов была постоянной, то есть 0 dT d . (5.23) 0 dT d . (5.23) Тогда   exp 1584 1584 exp exp o z t A o o t k m p o m p q q k k dz k d T T q C q C T T                                          . (5.24) (5.23) Тогда (5.24) Из уравнения Лева для теплоотдачи в зернистом слое получаем с учетом уравнения неразрывности потока в трубе переменного сечения Из уравнения Лева для теплоотдачи в зернистом слое получаем с учетом уравнения неразрывности потока в трубе переменного сечения 2,8 42 exp 0,24 1 в в t d d k d d                         , (5.25) (5.25) где dв − внутренний диаметр трубы промышленного реактора; d −текущий диаметр трубы промышленного реактора где dв − внутренний диаметр трубы промышленного реактора; где dв − внутренний диаметр трубы промышленного реактора; d − текущий диаметр трубы промышленного реактора. d − текущий диаметр трубы промышленного реактора. d − текущий диаметр трубы промышленного реактора. Анализ решения Тогда уравнение зависимости диаметра трубы переменного сечения от координаты длины, при которой сохраняется изотермический режим течения реакционных газов, принимает вид   2 0 3,8 exp 1584 1584 exp exp 42 exp 0,24 1 o z t A o o в в в в k q q k k d d d T T d d d T T d d                                                                       (5.26) (5.26) График этой зависимости приведен на рисунке 5.7, кривая 2а. на этом же рисунке (кривая 2б) показан теоретический профиль температуры по длине трубы переменного сечения 2а. здесь специально приведены расчеты для температуры t = 150 oC большей, чем средняя температура t = 132 oC в промышленном реакторе (рис. 5.7 кривая 1), так как нет необходимости опасаться локального скачка температуры на входе выше температуры термической стойкости катализатора. 162 160 100 120 140 180 1 2 3 4 Z, м t, oC l1 1 2 3 4 Z, м 50 1 2 4 3 1 2 3 1 а) б) d/2, мм допустимое 1 – промышленный реактор; 2 – изотермический реактор; 3 – реапктор с секционной трубой, первая секция которой коническая, а вторая – цилиндрическая, равная диаметру трубы dв промышленного реаткора; 4 – то же, что и 3, но при повышенной температуре кипящей в межтрубном пространстве воды dk = 119,6 oC, pk = 2 ата. Рисунок 5.7 Зависимость диаметра (а) и температуры (б) от координаты 160 100 120 140 180 1 2 3 4 Z, м t, oC l1 1 2 3 4 Z, м 50 1 2 4 3 1 2 3 1 а) б) d/2, мм допустимое Z, м Z, м 1 – промышленный реактор; 2 – изотермический реактор; 3 – реапктор с секционной трубой, первая секция которой коническая, а вторая – цилиндрическая, равная диаметру трубы dв промышленного реаткора; 4 – то же, что и 3, но при повышенной температуре кипящей в межтрубном пространстве воды dk = 119,6 oC, pk = 2 ата. Анализ решения р р p Рисунок 5.7 Зависимость диаметра (а) и температуры (б) от координаты длины в реакторе гидрохлорирования ацетилена. р р p Рисунок 5.7 Зависимость диаметра (а) и температуры (б) от координаты длины в реакторе гидрохлорирования ацетилена. Рисунок 5.7 Зависимость диаметра (а) и температуры (б) от координаты длины в реакторе гидрохлорирования ацетилена. Таким образом, уравнение показывает теоретическую возможность перехода от политропических трубчатых реакторов идеального вытеснения v const    ; T = T(z) к изотермическим реакторам реального вытеснения ν = ν(z); d = d(z); T = const. Конечно, практически не целесообразно делать трубы переменного сечения (рис. 5.7, кривая 2а). Однако, как видно из рисунка, входной участок такой трубы может быть с достаточной точностью заменен на коническую трубу l1 = 1,285 м и диаметром на входе dн = 0,7dв, а выходе имеющий диаметр, равный диаметру основной трубы промышленного реактора. Расчетная формула для определения профиля температуры в такой составной трубе, первая секция которой выполнена конической ,а вторая – цилиндрической, имеет вид, аналогичный следующей формуле: 1 2 1 0,7 0,7 1,285 , ; в в z в в в d d d d z l d d и d d при z l                          . (5.27) (5.27) 163 Результаты расчетов по последней формуле приведены на рисунке 5.7 кривая 3б. 5.7 Как видно из рисунка, установка конической секции на входе в основную трубу позволяет резко уменьшить скачок температуры и выровнять ее профиль по длине. Однако, желательно, уменьшив максимальную температуру, повысить ее на выходе из реактора, где она практически равна температуре кипящей в межтрубном пространстве воды. Таким образом, повысив температуру кипящей в межтрубном пространстве воды, можно увеличить температуру реакционных газов на выходе из реактора. р р р На рисунке 5.7 кривая 4 приведен профиль темепратур для реактора с конической секцией на входе в основную трубку, когда давление кипящей в межтрубном пространстве воды повышено с 1 до 2 ата, а температура со 100 оС до 119,6 оС. Как видно из рисунка, в конической секции максимум температуры в этом случае возрастает незначительно, зато средняя температура повышается на 20 оС за счет температуры реакционных газов на выходе из трубы. Анализ решения Этим же прием повышения температуры и давления кипящей в межьрубном пространстве воды можно применить по мере старения катализатора и потери им каталитических свойств в конце периода работы перед остановкой реактора и заменой отработанного катализаора на свежий. р р На действующих реакторах замена цилиндрических труб на конические или секционные невозможна. Здесь выручить может установка фор-реактора, площадь сечения трубного пространства в котором меньше, чем в основном реакторе, за счет меньшего диаметра труб или их числа. Дальнейший анализ математической модели показывает, что для уменьшения максимума температуры реакционных газов на входе нужно уменьшить количество прореагировавшего ацетилена. Известные решения предлагают в нижнюю часть труб засыпать отработанногои свежего катализатора или свежего катализатора с инертными гранулами. Например, на высоте l1 = 1,5 м при смешении свежего катализатора с инертными гранулами в отношении 1:1 формально можно считать, что константа скорости реакции падает в 2 раза. Поэтому для 1-й зоны математическая модель описывается уравнением, но В = 92,5; а = 0,3; b – без изменений. Для 2-й зоны 1 0 185 a a C B C           , где 1 0 1 a a C Р C  − доли непрореагировавшего Для 2-й зоны 1 0 185 a a C B C           , где 1 0 1 a a C Р C  − доли непрореагировавшего ацетилена на выходе из 1-й зоны, остальные коэффициенты а = 0,6; b = 0,0257. ацетилена на выходе из 1-й зоны, остальные коэффициенты а = 0,6; b = 0,0257. ацетилена на выходе из 1-й зоны, остальные коэффициенты а = 0,6; b = 0,0257. Известные решения связаны с дополнительными трудностями при ацетилена на выходе из 1-й зоны, остальные коэффициенты а = 0,6; b = 0,0257. Известные решения связаны с дополнительными трудностями при приготовлении катализатора и его засыпке в реактор. Известные решения связаны с дополнительными трудностями при приготовлении катализатора и его засыпке в реактор. Для уменьшения степени превращения на входе нужно, чтобы часть реакционных газов двигалась в трубе, не соприкасаясь с катализатором, то есть осесимметрично с основной трубой установить на входе трубку меньшего диаметра d1 (рис. 5.8) длиной l1. Например, если поток газа через эту трубку равен половине всего потока q1 = 0,5q, то контактировать с катализатором на 164 длине l1 будет в 2 раза меньше реакционных газов. Анализ решения При неизменной скорости  в трубе и вне ее в основной трубе  = 0,16 м/с, а qa1 = 0,5qa0. Подставляя численные значения параметров в уравнении получаем для первой зоны уравнение   1 1 1 1 1 1584 1584 185 exp 0,6 exp 0,0514 373 i i i i j i j T T T T T                                . (5.28) Для второй зоны l≥l1. Если l1 = 1м, то по результатам расчетов 1-й зоны 1 0 1 0,2807 a a C C  − доля непрореагировавшего ацетилена. Для второй зоны l≥l1. Если l1 = 1м, то по результатам расчетов 1-й зоны 1 1 0,2807 a a C C  − доля непрореагировавшего ацетилена. Материальный баланс по ацетилену на выходе из 1-й зоны     1 0 1 0 0 0 0 1 1 0,5 1 0,5 1,2807 0,64035 A A A A A A A A C q q C q q C q C C q q                      . (5.29) (5.29) Тогда расчетное уравнение для второй зоны будет аналогично, только коэффициент нужно заменить на 118,4 = 185∙0,6403, а Ті−1 = 400,3о – температуру на выходе из 1-й зоны. Результаты расчета реактора с центральной распределительной трубкой приведены на рисунке 5.8, криве 2 и 3. Вместо одного максимума кривая имеет два, но оба с температурой менше, чем в существующем реакторе. Анализ авторських свидетельств и патентов показал, что установка в трубы кожухотрубного реактора распределительных трубок обладает новизной, существенными отличительными признаками и положительным эффектом. Установка над каждой распределительной трубкой подвижной регулируемой крышки позволяет регулировать доли потока реакционных газов, поступающих внутрь трубки и между стенками основной трубы и трубки, то есть регулировать скорость и расход реакционных газов в каталитической зоне. При полностью закрытой крышке расход реагирующего компонента в каталитической зоне равен его расходу в промышленном реакторе, но скорости реакционных газов, а значит и теплоотдача увеличивается по пропорции 2 1 1 в d d         . Анализ решения 165 ным уст ой конц 166 1 – промышленный реактор; 2 – реактор с распределительным устр что и 2, но при 1 1 0,7; 1/ 6 d l d l         Рисунок 5.8 Распределение температуры (а) и относительной конце винилхлорида ом в 6 МЕХАНИЗМ И МАТЕМАТИЧЕСКАЯ МОДЕЛЬ ПРОЦЕССА БИОЛОГИЧЕСКОЙ ОЧИСТКИ СТОЧНЫХ ВОД О ТОРГАНИЧЕСКИХ СОЕДИНЕНИЙ В АЕРОТЕНКАХ Сточные воды являются сложными системами, как по составу, так и по химическим превращениям, происходящим в них. Одним из основных методов очистки хозяйственно-бытовых, а также промышленных сточных вод от установок переработки нефти, пищевой промышленности, белково-витаминных заводов, которые по классификации относятся к первой группе, является биологическая очистка. Как правило, процесс биологической очистки сточных вод осуществляется в аэротенках. Несмотря на то, что аэротенки применяются свыше пятидесяти лет, а процессы, происходящие в них, исследуются еще более длительное время, представления об основных его закономерностях недостаточны. Это приводит часто к неоправданному завышению строительного объема очистных сооружений и относительно низкой степени очистки. Выбор наиболее эффективных решений при проектировании очистных сооружений биологической очистки возможен лишь в результате изучения механизма и выявления основных химических закономерностей этого процесса и его математического описания. Этот подход широко распространен в химической технологии и базируется на методологии системного анализа. ру Чтобы реально подойти к оценке механизма и кинетики процесса, необходимо рассмотреть отличия биологической очистки сточных вод от микробиологических процессов, которые характерны при производстве кормовых дрожжей, антибиотиков, ферментов, аминокислот и т.п. Главной целью различных микробиологических процессов является достижение максимального выхода биомассы или продуктов обмена в единицу времени на единицу субстрата. Эти процессы ведутся при условиях, обеспечивающих возможно более высокую скорость роста микроорганизмов. Микробиологический процесс в каждой фазе состоит из комплекса многообразных биохимических реакций, конечным выражением которых является рост клеток, а началом потребления питательных веществ [69]. Математическое описание процесса для этого случая предложено Моно [70], который показал, что связь между концентрацией веществ «С», находящихся в минимальных количествах, и скоростью роста микроорганизмов Кможно выразить уравнением, аналогичным по форме уравнению Михаэлиса: max S С S С K S    , max S С S С K S    , (6.1) (6.1) где S − концентрация субстрата. Развитие модели Моно выполнено в работах Иерусалимского [71], в которой учтено влияние продуктов обмена на рост микроорганизмов. Однако обе модели описывают только рост клеток, в то время, как наряду с этим происходит уменьшение их массы. Последний фактор учитывается в модели Герберта [71]. Исчерпывающее решение задачи математического описания 167 процесса может быть получено путем применения кинетики ферментативных реакций. Для этих целей рекомендуется классическое уравнение Михаэлиса- Ментена [72]: max m V S С K S    , max m V S С K S    , (6.2) (6.2) где V − скорость реакции; где V − скорость реакции; − константа Михаэлиса-Ментена. Анализ уравнения показывает, что максимальная скорость реакции может быть получена, если весь энзим соединяется с субстратом. Величина Km является показателем специфичности энзима по отношению к субстрату. В отличии от вышеприведенного, цель биологической очистки − получение низких уровней концентрации субстрата в очищенных сточных водах. Это означает, что в большинстве случаев процесс идет в пределах концентраций субстрата, ограничивающих рост биомассы. Кроме того, начальные концентрации органических веществ в сточных водах значительно меньше, чем микробиологических реакторах. Поэтому в симбиозе активного ила возникает популяция клеток, в которых эндогенный метаболизм занимает большую часть общего метаболизма, чем в микробиологических процессах. Попытка описания процессов биологической очистки сточных вод с помощью вышеотмеченных моделей была сделана в работах [73, 74]. При анализе результатов, авторы [73, 74] пришли к выводу, что модели [71 − 72] не применимы для описания процесса разложения органических веществ при очистке сточных вод. Авторы [74] в своих работах также показали, что модели не отражают сущность процессов, происходящих в аэротенках и расчеты по моделям, не могут быть использованы даже как первое приближение. В работах [75, 76] предложена модель биологической очистки сточных вод с учетом активного ила. где S − концентрация субстрата. Автор предложенной модели Наканиси кроме закономерностей изменения субстрата:   2 1 1 1 2 3 01 01 4 dS x b a k S k S k S d a b k x               , (6.3) (6.3) предлагает учитывать закономерности изменения концентрации кислорода   предлагает учитывать закономерности изменения концентрации кислорода     2 02 2 1 2 3 4 02 x dС x a k S k S k S k L d a              , (6.4) (6.4) где x − концентрация активного ила; где x − концентрация активного ила; L − доля активного ила, способного окисляться при эндогенном дыхании. L − доля активного ила, способного окисляться при эндогенном ды Активность определяется в условиях выбранных заранее (а01) и в условиях, соответствующих исследуемому процессу очистки (а1) Уравнение, кроме свойств процесса, учитывает ингибирование субстратом, наступающее при его высоких концентрациях (k3S2), учитывается также БПК органических веществ, выделяемых единицей концентрации активного ила в результате обмена в окружающую среду (b01 условия, выбранные заранее; b1 168 условия, соответствующие исследуемому процессу), и пропорциональность общего количества выделяемых органических веществ концентрацией активного ила (k4). Уравнение показывает, что скорость потребления кислорода подчиняется тем же закономерностям, что и потребление субстрата. Скорость поглощения кислорода определяется в условиях а02 и в условиях а2 мг O2/(гила∙ч). Скорость поглощения O2 при эндогенном метаболизме пропорциональна ( ), концентрации активного ила (x) и доле активного ила (L), способного окисляться при эндогенном дыхании. ( ) р Данная модель представляет большие возможности при прогнозировании величины скорости поглощения кислорода, однако не дает информации об изменении концентрации активного ила по сухому веществу за счет роста, поскольку активность ила рассчитывается для постоянной величины его концентрации. Кроме того, как следует из проанализированных моделей, при подборе математической модели кинетики процесса биологической очистки предполагается, что этот процесс осуществляется одной микробной культурой. На самом деле активный ил представляет собой определенный симбиоз штаммов различных групп (бактерии, простейшие, грибы, коловратки и т. д.), между которыми существуют сложные физиологические связи. В работах [77 − 78] показано, что основную роль в процессе потребления органических соединений играют бактерии. Функциональная роль простейших заключается в осветлении очищенной воды за счет потребления разрушенных зооглейных скоплений. где S − концентрация субстрата. Для этих условий система кинетических уравнений записывается [84]: 1 1 1 1 1 1 1 0 N N i N N i N N i N N i dS k S d dS k S k S d dS k S d             , (6.7) (6.7) Si − субстраты, вступающие в реакцию расщепления на i-той стадии; где Si − субстраты, вступающие в реакцию расщепления на i-той стадии; Ф − соответствующие ферменты, катализирующие реакцию (i + k = N); N − число стадий; S где Si − субстраты, вступающие в реакцию расщепления на i-той стадии; Ф − соответствующие ферменты, катализирующие реакцию (i + k = N); N − число стадий; N − число стадий; SN − исходное соединение; S0 − конечный продукт; – константы cкорости реакций. Представления в работе [81], модель не дает количественных оценок о влиянии концентрации растворенного кислорода на процесс и характер его распределения по объему (размеры газовых пузырей, газонасыщение жидкости и т. д.). В тоже время, как известно из работ [85], последние факторы во многих случаях являются определяющими для интенсификации процессов и выбора конструкции аэротенков. Таким образом, отсутствует четкая формулировка и количественная оценка растворенного кислорода на кинетику биохимического разложения. В данной работе на основании анализа литературных данных и данных обследования водоочистных сооружений представлены: возможный механизм и математическая модель кинетики процесса биохимического окисления органических веществ в водных системах. где S − концентрация субстрата. Эти особенности процесса в определенной мере учитывает модель [79], в которой биологическая очистка представлена в виде цепочки трофической связи:   m s dS B S K B d y            , (6.5) m r s SB dB B fLBP d K B W      . (6.6)   m s dS B S K B d y            , (6.5) (6.5) m r s SB dB B fLBP d K B W      . (6.6) (6.6) В уравнении тем же способом, что и в предыдущей модели отражено потребление субстрата (S) бактериями активного ила (В). В уравнении учтено, что количество бактерий увеличивается за счет потребления субстрата и уменьшается вследствие уничтожения бактерий простейшими (Р), причем, из единицы количества бактерий образуется определенное количество простейших(W). Удельная скорость роста простейших достигает половины своего максимального значения (f) при концентрации бактерий, равной (L). Такая модель в значительно большей степени, чем рассмотренные ранее, отражает динамику роста и взаимоотношения основных групп микроорганизмов ила в процессе потребления органических веществ. Однако, эта модель не дает представлений о роли кислорода и его влияния на процесс. Модели механизма биохимической деструкции сложных органических молекул с участием внеклеточных ферментов до молекул мономеров, 169 способных проникать через K−Na каналы клетки внутрь клетки для последующих биохимических процессов, предложены в работах [80, 81]. В работе [82] показано, что многостадийный процесс внеклеточного расщепления сложных органических соединений протекает без участия кислорода. Однако, конечный продукт мономер, который затем проникает в клетку и является исходным субстратом для биохимической реакции в самой клетке (синтезе белка), зависит от концентрации кислорода. Авторы работа [83] показала, что в зависимости от концентрации растворенного кислорода формируется определенный симбиоз бактерий. В работе также показано, что наиболее деятельные группы микробного населения биоценоза при очистке сточных вод в количественном и видовом соотношении − это Pseudomonas и Bacterium. Исходя из вышеизложенного, в работе [84] считают, что внеклеточная деструкция органических веществ является лимитирующей стадией. Механизм процесса Используя фундаментальные представления, механизм процесса очистки сточных вод от органических веществ можно представить в виде следующих этапов трансформирования вещества и энергии: 170 − диффузия соединений субстрата к клеточной поверхности активного ила (к флокулам); − диффузия соединений субстрата к клеточной поверхности активного ила (к флокулам); − адсорбция субстрата на клеточной поверхности; − расщепление адсорбированного субстрата внеклеточными фермент − поглощение расщепленных мономеров клетками; − поглощение расщепленных мономеров клетками; − внутриклеточные процессы (рост и эндогенное дыхание); − высвобождение экстретируемых продуктов; − «выедание» первичной популяции организмов вторичными потребителями (простейшими) и диффузия (отвод) газообразных продуктов. Клеточная поверхность в аэротенке представлена в основном в виде флокул активного ила со средним размером 200 − 400 мкм. В работе [86], с использованием эволюционного метода сравнения времен диффузии Тр, и времени биохимической реакции показало, что диффузионные ограничения на стадиях подвода органических веществ к поверхности флокулы активного ила и внутри нее незначительны по сравнению с временем биохимической реакции. Длительность последней в 80 − 100 раз больше, чем время подвода реагентов, то есть процесс лимитируется скоростью биохимических реакции в клетке. Что касается кислорода, то он также сорбируется во флокулы. Область протекания реакции во флокуле по кислороду, согласно [86], можно отнести к диффузионно-кинетической. Это объясняется низкой концентрацией растворенного кислорода и высокой скоростью его потребления. Известно, что растворимость кислорода в воде при 302 К составляет 8,3 мг/дм3, концентрация растворенного кислорода − 2 г/м является оптимальной и отражает усредненные потребности микроорганизмов смешанных биоценозов. Внутреннее потребление кислорода, то есть процесс трансформации органического вещества и энергии в клетке можно представить в виде схемы: р р 1. Дыхание − окисление органического вещества: 1. Дыхание окисление органического вещества: 2 2 2 x y z x C H O O CO H O G     р 2 2 2 x y z x C H O O CO H O G     2 2 2 x y z x C H O O CO H O G     2. Синтез клеточного вещества: 3 2 x y z x C H O NH клеточное вещество H O G     3 2 x y z x C H O NH клеточное вещество H O G     3. Самоокисление: клеточное вещство + 2 2 2 3 x O CO H O NH G     Потребление кислорода внутри клетки осуществляется с помощью ферментов. Механизм процесса Полный состав белков − ферментов, необходимых для очистки сточных вод, неизвестен, однако чисто эмпирически установлено [88], что для процессов деструкции и окисления органических соединений важны ферменты, относящиеся к классам: оксидоредуктазы, трансферазы, гидролазы, лиазы. Например, первая группа ферментов отнимает ион водорода от окисляемого субстрата, а также способствует процессу: 2 2 4 2 O e O     . Другие классы ферментов способствуют разрыву связей в органических соединениях, например, негидролитическим путем, переносу атомных групп с одной молекулы на другую и т. п. Перенос молекул субстрата через цитоплазматическую мембрану субстрата через цитоплазматическую мембрану 171 осуществляется благодаря участию молекул переносчиков − специальных конферментов или пермеаз. Молекулы кислорода, согласно [88] беспрепятственно диффундируют через мембрану, и скорость диффузии зависит от скорости потребления кислорода внутри клетки. Если молекула субстрата имеет большие размеры, чем каналы в мембране, то внеклеточные ферменты аналогичных классов подвергают их деструкции в жидкой фазе, близкой к адсорбционной зоне флокулы ила [88]. Таким образом, можно утверждать, что на микроуровне процесс деструкции и окисления молекул субстрата лимитируется скоростью реакции биохимического окисления. Однако этот факт имеет место, если эффективны стадии 2, 3, а также равномерно поддерживается оптимальная концентрация растворенного кислорода (~ 2 г/м) во всем объеме аэротенка. Известно [89], что адсорбционная и перерабатывающая характеристика активного ила зависят от величины его удельной поверхности и ферментативной активности. Эти величины в свою очередь в значительной мере определяются величиной коэффициента зооглейности, который характеризует соотношение капсульных и безкапсульных штаммов Коэффициент (kz) зооглейности обнаруживает хорошо выраженную зависимость от специфических особенностей экологических группировок бактерий. Наиболее высоким kz обладают аблигатные аэробы (kz = 30), термотолеранты (kz = 40) и мезофилы (kz = 18). У микроаэрофильных и капнетических аэробов, окситолерантных анаэробов значение меньше 10, что, по мнению авторов, указывает на неблагоприятный технологический режим биологической очистки. В той же работе показано, что в случае использования сточных вод с условно постоянным биохимическим показателем (С), режим аэрации предопределяет биоценоз активного ила и при увеличении интенсивности аэрации возрастает процент аблигатных аэробов и уменьшается количество нитчатых бактерий. Это повышает коэффициент и интенсифицирует процесс очистки. Таким образом, можно утверждать, что на макроуровне процесс в аэротенке будет определяться следующими условиями: − скоростью растворения кислорода в сточных водах и равномерным распределением концентрации по всему объему. Эти условия фактически определяются гидродинамикой в аэротенке и величиной объемного коэффициента массопередачи кислорода. − скоростью растворения кислорода в сточных водах и равномерным распределением концентрации по всему объему. Эти условия фактически определяются гидродинамикой в аэротенке и величиной объемного коэффициента массопередачи кислорода. Механизм процесса При достижении определенных гидродинамических условий и скорости массопередачи обеспечивается формирование определенного симбиоза бактерий и соответственно получение флокул активного ила с требуемой адсорбционной способностью (удельной поверхностью) и ферментативной активностью, а также поддержание оптимальной концентрации растворенного кислорода в объеме обрабатываемых сточных вод. Для качественного подтверждения вышеизложенных положений было проведено обследование работы промышленных аэротенков на ряде водоочистных сооружений. При исследованиях определялось влияние интенсивности аэрации воздуха на видовой состав симбиоза активного ила, на удельную поверхность активного 172 ила и ферментативную активность. Определение вышеупомянутых показателей выполнялись методами, разработанными Институтом микробиологии и вирусологии интенсивность аэрации оценивалась качеством О2 (м3), подаваемого на 1м2 поверхности аэротенка в единицу времени (час.). Аэрация осуществлялась воздухом, пересчет на O2 (м3) осуществлялся по известным соотношениям. На обработку поступала смесь промышленных и хозяйственно бытовых сточных вод в соотношении − (10:1), биохимический показатель С − 0,32, количество органических загрязнений 60:80 мг/л, бихроматная окисляемость (ХПК) стоков составляла в среднем 750 мг О2/л, БПК − 86 мг О2/л, БПКпол − 245 мг О2/л, температура сточных вод была в пределах 291 − 298 К, рН среды было около 7,5. Процесс очистки проводился в двухступенчатом аэротенке, средняя скорость подачи потока воды в аэротенке составляла 1,8 м3/м2/час, гидравлическое время пребывания в аэротенке составляло 5 часов, время удержания (пребывания) активного ила составляло 10 суток. Аэрация жидкой фазы осуществлялась трубчатыми барботерами, уложенными по дну аэротенка. Интенсивность аэрации варьировалась в интервале 2 − 15 м3 на м3 стоков. В таблице 6.1 приведено влияние интенсивности аэрации на величину коэффициента kz активного ила. Таблица 6.1 – Влияние интенсивности аэрации на величину kz и R (усредненные показатели) № Интенсивность аэрации 1 3 5 7 9 Показатель 1 z k 4 12 18 21 10 2 R ru  3 10 20 22 9 ца 6.1 – Влияние интенсивности аэрации на величину kz и R ые показатели) r − удельная скорость биохимического окисления мг/г акт. ила; и − доля активного ила в иловой смеси г/л; и − доля активного ила в иловой смеси г/л; R − средняя скорость биохимического окисления; интенсивность аэрации м3/(м2∙ч). ( ) В таблице 6.2 приведена зависимость адсорбционной поверхности и ферментативной активности ила от интенсивности аэрации. Таблица 6.2 – Зависимость AП и Фа от интенсивности аэрации № Интенсивность аэрации, м3/(м2·ч) 1 5 7 9 Показатель 1 AП − удельная поверхность, м2/г 50 100 120 70 2 Фа − ферментативная активность, ат. ед. 40 90 100 80 Характер соотношения микробного населения биоценоза в проведенных сериях опытов (табл. 6.1 и 6.2) представлены ниже. Механизм процесса 3 2 В интервале интенсивности аэрации 3 − 7 м3/(м2·ч), достигалась почти оптимальная концентрация О, в воде − 1,72 г/м3, активный ил образован смесью 173 173 культур. В этом варианте преобладала наиболее деятельная группа Pseudomonas (Ps. desmoliticum, Рг. aeruginosa, Ps. zelinskii, Ps. dacunhae) клетки Pseudomonas составляли около 65 % всего населения биоценоза, 35 % микробного населения приходится на Bacterium, Bacillus. При этом капнетические и факультативные формы составляли − 20 % от общей численности бактерий, жгутиковые практически отсутствовали, а преобладали коловратки. р На выходе из аэротенка ХПК снизилось на 90 %, содержание органических примесей на 96 %, БПК5 на 98%, ВПК на 96%. На выходе из аэротенка ХПК снизилось на 90 %, содержание органических примесей на 96 %, БПК5 на 98%, ВПК на 96%. 3 2 р , 5 , При снижении интенсивности аэрации менее 3 м3/(м2ч), концентрация О2 в растворе снижалась до 0,8 г/м3, а плотность населения Pseudomonas уменьшилось на 40 − 50 %. На первое место по биогенности выдвигаются бактерии (Bact. aliphaticum, Bact. paraffinicum, Bact. filiforme). По сравнению с псевдомонадами бактерии накопляли биомассу значительно менее интенсивно, скорость окисления снизилась в 6 − 8 раз, ферментативная активность и удельная поверхность снизилась в 2 − 5 раза (см. табл. 6.1 и 6.2), капнептические и факультативно-анаэробные формы увеличились до 35 % от общей численности бактерий. При этом появились Amoeba limax, бесцветные жгутиковые. Повышение интенсивности аэрации более 9 м3/(м2ч) приводит к тому, что несмотря на концентрацию кислорода 4,2 г/м3 соотношение Pseudomonas и Bacterium с Bacillus становится примерно одинаковым, это, по видимому и приводит к снижению удельной поверхности, ферментативной активности и т. п. (табл. 6.1 и 6.2). Как следует из данных, приведенных в таблицах 1, 2, величина коэффициента как и предполагалось, зависит от видового состава и интенсивности аэрации. Наибольшее значение kz наблюдалось при интенсивности аэрации в интервале 5 − 7 м3/(м2ч) и соответствует вышерассмотренному видовому составу. Таким образом, приведенные экспериментальные данные свидетельствуют в пользу вышеприведенного механизма биохимической очистки сточных вод. Совершенно очевидно, что гидродинамическая обстановка в аэротенке и, по- видимому, значение объемного коэффициента массоотдачи по О2 определяют условия, при которых лимитирующей стадией биохимической очистки сточных вод является скорость биохимических реакций внутри клетки. С инженерной точки зрения, для обеспечения вышеупомянутых условий необходимо знать особенности гидродинамики в трехфазном реакторе псевдоожиженного слоя, содержащем частицы с низкой плотностью, закономерности массопередачи между газом и жидкостью в этих условиях и предельную величину объемного коэффициента массоотдачи. Кинетическая модель процесса 6.1), математическая модель будет описываться дифференциальными уравнениями материального баланса: d 2 0 0 0 1 01 2 02 0 03 0 1 0 0 1 04 2 2 0 0 1 01 2 dx k x x k x k x k x d dx k x x k x А d dx k x x k x d             . (6.9) 2 0 0 0 1 01 2 02 0 03 0 1 0 0 1 04 2 2 0 0 1 01 2 dx k x x k x k x k x d dx k x x k x А d dx k x x k x d             . (6.9) (6.9) Первое уравнение фактически описывает прирост биомассы в процессе очистки с учетом естественного отмирания основной и соответствующих групп микроорганизмов и скорости потребления органических загрязнений. Второе уравнение описывает изменение концентрации субстрата в процессе очистки. Полученная модель практически учитывает все недостатки известных моделей, описанных ранее. Кинетическая модель процесса Как видно из рассмотренного анализа при проведении процесса биохимической очистки сточных вод в аеротенках вводится ограничесние на минимально допустимое значение концентрации кислорода в рабочем объеме аеротенка. При температуре 298 − 308 К равновесная концентрация О2, составляет (8,3 − 7,5) мг/дм3. 174 Предпочтительная концентрация растворенного кислорода (О2) равна 2 г/м3, то есть в 3 − 4 раза меньше равновесной концентрации (Ср). Как видно из результатов обследования промышленных аэротенков при этой концентрации достигаются лучшие результаты. Таким образом, можно ввести ограничения на минимально допустимую концентрацию растворенного О2 (Сmin) в рабочем объеме аэротенка, при которой выполняются условия для эффективного протекания процесса. Растворимость кислорода вполне удовлетворительно описывается законом Генри: (6.8) e С H P   , (6.8) e С H P   , где С − растворимость газа; Р − парциальное давление газа; He − константа Генри. Тогда минимально допустимая концентрация растворенного определяется как 0,3 0,3 min p e p C H P С      Тогда минимально допустимая концентрация растворенного определяется как О2 да минимально допустимая концентрация растворенного 0,3 0,3 min p e p C H P С      0,3 0,3 min p e p C H P С      где Рр − равновесное давление. где Рр − равновесное давление. Структурная модель процесса биохимической очистки сточных вод при Сmin может быть представлена в следующем виде (рис. 6.1). А − поток сточных вод на входе; А1 − поток сточных вод на выходе; х0 − численность основной группы микроорганизмов; х1 − концентрация загрязняющих веществ на входе; х2 - концентрация промежуточных продуктов продуктов; х3 − концентрация загрязняющих веществ на выходе; k0 = k1 − коэффициент скорости реакции образования промежуточных продуктов питания основной группы k01 = k3+k2 − скорость естественной гибели основной группы микроорганизмов; k2 − скорость потребления загрязняющих веществ основной группой микроорганизмов; k02 = β – k3 − k4; β − коэффициент внутренней конкуренции основной группы; k3 − скорость гибели основной группы микроорганизмов; k4 − коэффициент скорости выхода из системы основной группы микроорганизмов; k03 = β; k04 = k3. − k4; β − коэффициент внутренней конкуренции основной группы; k3 − скорость гибели основной группы микроорганизмов; k4 − коэффициент скорости выхода из системы основной группы микроорганизмов; k03 = β; k04 = k3. Рисунок 6.1 Структурная модель процесса биохимической очистки в аэротенке (лимитирующая стадия биохимическая реакция при Сmin) 175 Согласно (рис. 6.1), математическая модель будет описываться дифференциальными уравнениями материального баланса: Согласно (рис. Кинетическая модель процесса 0 1 2 0; 0; 0 dx dx dx d d d             , если ввести В стационарном режиме 0 1 2 0; 0; 0 dx dx dx d d d             , если ввести б  безразмерные переменные в виде:     01 04 01 03 02 02 04 0 0 02 01 04 0 03 01 01 ; ; ; ; ; k k k k A k k k e m Q Q Q t k k k k k k k k k            , (6.10) 1; 0,1,2 k i   k уравнение можно представить в линейном виде: уравнение можно представить в линейном виде: уравнение можно представить в линейном виде:   0 1 2 dy l m y ly ly dt     , (6.11) 1 0 1 2 dy Qy Qy y dt       2 0 1 2 dy n y y y dt      0 1 2 dy l m y ly ly dt     , (6.11) 1 0 1 2 dy Qy Qy y dt       2 0 1 2 dy n y y y dt    (6.11) Анализ линеаризованной системы с учетом теории устойчивости систем (Ляпунова) показывает, что в диапазоне регулируемых параметров входного потока δ от δ = 0,5 до δ = 10,5, то есть в пределах, которые наблюдаются в промышленных условиях, стационарное состояние устойчиво. Аналогичные результаты получены и для параметра l. При решении характеристического уравнения системы получены: один корень действительный (λ0), два других корня (λ1, λ2) комплексные для всех корней численные значения меньше нуля. Таким образом, при Сmin система аэротенка работает устойчиво. Кинетическая модель процесса При условиях непрерывного процесса, ограничения скорости роста биомассы основной группы микроорганизмов по концентрации субстрата, а главное значительных колебаний концентрации растворенного по объему аэротенка необходимо учитывать объемный коэффициент массоотдачи по О2 В работе [90] показано, что в этом случае средняя скорость роста В работе [90] показано, что в этом случае средняя скорость роста 176 микроорганизмов μ может быть определена по уравнению: микроорганизмов μ может быть определена по уравнению: 1 m s c x C k S k C       , (6.12) 1 m s c x C k S k C       , (6.12) 1 m s c x C k S k C       , где μm− максимальная скорость роста микроорганизмов, ч"1; S и С − концентрации загрязнений в сточных водах и кислорода, г kC и kS − константы насыщения (константы моно) по кислороду и субстрату, г/л. kC и kS − константы насыщения (константы моно) по кислороду и субстрату, г/л. г/л. Можно составить математическое описание процесса при следующих допущениях: у − среда в аэротенке хорошо перемешивается, режим близок к идеальному смешению; − среда в аэротенке хорошо перемешивается, режим близок к идеальному смешению; ; − входной поток А и выходной поток равны А1; (А=А1); − процесс протекает при постоянных условиях (pH, давление, температура) и постоянстве экономических коэффициентов по субстрату и кислороду kC и kS. Согласно данных обследования, основные группы микроорганизмов Pseudomonas, Bacterium, Bacillus составляют 70 − 80 %. Исходя из этого, kS можно принять 70 %. Учитывая ограничения по кислороду, kS − можно принять 50 %. Согласно данных обследования, основные группы микроорганизмов Pseudomonas, Bacterium, Bacillus составляют 70 − 80 %. Исходя из этого, kS можно принять 70 %. Учитывая ограничения по кислороду, kS − можно принять 50 %. Кинетическая модель процесса Математическое описание процесса может быть представлено в виде системы трех обыкновенных нелинейных дифференциальных уравнений:     0 0 0 0 0 0 1 1 m s c m s s c La p m s s c dx Cx A S x d V k S k C Cx dS A S x S d V Y k S k C Cx dC S k C C d Y k S k C                            , (6.13) (6.13) где V − объем жидкости в аэротенке, л; где V − объем жидкости в аэротенке, л; kLa − объемный коэффициент массоотдачи по кислороду, ч−1. kLa − объемный коэффициент массоотдачи по кислороду, ч−1. kLa − объемный коэффициент массоотдачи по кислороду, ч−1. Приведем систему к безразмерному виду, введя безразмерные переменные: Приведем систему к безразмерному виду, введя безразмерные переменные:   0 0 0 ; ; ; ; ; ; ; ; p La p m c c s s c c m s m s s C k x C S k W W x y k k k y k x k y A y m n t k k y               . (6.14) огда модель будет иметь вид:   0 0 0 ; ; ; ; ; ; ; ; p La p m c c s s c c m s m s s C k x C S k W W x y k k k y k x k y A y m n t k k y               . (6.14) (6.14) s m  Тогда модель будет иметь вид: s m Тогда модель будет иметь вид: Тогда модель будет иметь вид: Тогда модель будет иметь вид: 177     0 1 1 1 1 1 1 p dx y W y mx x dt y W dy y W m y y x dt y W dW y W n nk W W x dt y W                   . Кинетическая модель процесса (6.15) (6.15) Решение системы, при 0; 0; 0 dx dy dW n dt dt dt    , показывает, что может быть два устойчивых стационарных состояния. Первое х1 = 0; у1 = 0; Wt = Wp соответ ствует режиму вымывания активного ила из рабочего объема аэротенка. Второе: Решение системы, при 0; 0; 0 dx dy dW n dt dt dt    , показывает, что может быть два устойчивых стационарных состояния. Первое х1 = 0; у1 = 0; Wt = Wp соответ ствует режиму вымывания активного ила из рабочего объема аэротенка. Кинетическая модель процесса Второе: 2 0 1 1 2 1 1 2 p m nk m x y W m m m                            (6 16)        2 0 2 0 0 1 1 2 1 1 2 4 1 1 1 1 1 p p p B m nk m x y W m m m m nk m nk y W y W m m m m m m m                                                           (6.16) 2 2 p m W W x nk   2 0 2 y y x              2 0 2 0 0 1 1 2 1 1 2 4 1 1 1 1 1 p p p B m nk m x y W m m m m nk m nk y W y W m m m m m m m                                                           (6.16) (6.16)        2 0 0 4 1 1 1 1 1 p p B m nk m nk y W y W m m m m m m m                                где    0 0 0 1 1 p p m y W y W        где    0 0 0 1 1 p p m y W y W        − скорость вымывания (минимальная) при й й Э где    0 0 0 1 1 p p m y W y W        − скорость вымывания (минимальная) при Э    0 0 0 p p y y   р ( ) р которой весь активный ил выносится из аэротенка. Кинетическая модель процесса Эта скорость определяет границу между режимами образования т < тB и вымывания биомассы m≥mB. При анализе решения было принято Wp = 10,2 ≤ у0 ≤ 10; пк − 0,07. Исходные концентрации биомассы, субстрата и кислорода в рабочем объеме приняты в диапазоне х = 0 − 10; у = 0 − 10; W = 2 − 10. Устойчивое динамическое состояние системы определяется тремя переменными: концентрацией биомассы активного ила «х», концентрацией загрязнений «у» и концентрацией кислорода W. Изменение состояний осуществляется с помощью скорости потока «m», концентрацией загрязнений во входном потоке у0 и интенсивности массопередачи кислорода на границе воздух − сточная вода − nk. Стационарные состояния x, у, W в аэротенке при вариации т, пk представлены на рисунке 6.2. которой весь активный ил выносится из аэротенка. Эта скорость определяет границу между режимами образования т < тB и вымывания биомассы m≥mB. При анализе решения было принято Wp = 10,2 ≤ у0 ≤ 10; пк − 0,07. Исходные концентрации биомассы, субстрата и кислорода в рабочем объеме приняты в диапазоне х = 0 − 10; у = 0 − 10; W = 2 − 10. Устойчивое динамическое состояние системы определяется тремя переменными: концентрацией биомассы активного ила «х», концентрацией загрязнений «у» и концентрацией кислорода W. Изменение состояний осуществляется с помощью скорости потока «m», концентрацией загрязнений во входном потоке у0 и интенсивности массопередачи кислорода на границе воздух − сточная вода − nk. Стационарные состояния x, у, W в аэротенке при вариации т, пk представлены на рисунке 6.2. Анализ данных приведенных на рисунке 6.2 показывает, что при пk1 в достаточно широком диапазоне т от 0,05 до 0,6 концентрация кислорода снижается ниже допустимого Wmin. Повышение пk до значений пk2, позволяет удержать концентрацию растворенного кислорода выше, чем Wmin также в широком диапазоне m. При дальнейшем повышении значении пk до nk3 минимум функции Ф(х, у, W) = 0 повышается и двигается в сторону больших значений скорости потока m, то есть наблюдается меньшее время пребывания очищаемой жидкости в аэротенке. 178 Рисунок 6.2 Зависимости x, y, w в объеме аэротенка от m и nk при 0 1 2 3 10; 10; 0,06; 0,45; 0,55 p y W nk nk nk      . Штриховая линия – нижний предел, ограничивающий допустимую концентрацию 2 min 0,3 p O W W   . Кинетическая модель процесса Рисунок 6.2 Зависимости x, y, w в объеме аэротенка от m и nk при 0 1 2 3 10; 10; 0,06; 0,45; 0,55 p y W nk nk nk      . Штриховая линия – нижний предел, ограничивающий допустимую концентрацию 2 min 0,3 p O W W   . По видимому, наблюдаемые нами более низкие показатели по очистке сточных вод при обследовании аэротенков за счет увеличения интенсивности аэрации выше 8 м3/(м2·час) обязаны этому явлению. Анализ данных, приведенных на рис. 6.2 позволяет также утверждать, что Wmin достигается при т, соответствующей максимальной продуктивности процесса по биомассе. Таким образом, анализ процесса в аэротенке по модели, описываемой системой уравнений, полностью подтверждает результаты, полученные при анализе модели, относительно устойчивости процесса, стационарности режима и продуктивности процесса. Pe1 − число Пекле по жидкости, Оценка величины kLa Система в аэротенке представляет собой трехфазный псевдоожиженный слой, содержащий частицы с низкой плотностью (активный ил). Взяв за основу известные исследования по массопередаче между газом и жидкостью для рассматриваемого случая [90] можно прийти к следующим выводам. В аэротенке стационарные профили растворенного кислорода можно рассчитывать по диффузионной модели. В этом случае уравнение материального баланса по жидкой фазе может быть записано в следующем виде:   1 2 ' 0 0 0 2 1 0 o e d C dC St C C P dz dz      , (6.17) (6.17) где Со − концентрация растворенного кислорода; где Со − концентрация растворенного кислорода; z − безразмерная продольная координата слоя; St − число Стентона; Pe1 − число Пекле по жидкости, 179 ' 0 C − равновесная концентрация кислорода на границе фаз газ − жидкость. Если принять, что мольная доля кислорода (в) газовой фазы − const, а профиль давлений в трехфазном слое линейный, тогда ' 0 C связана с высотой слоя соотношением:    ' 0 0 0 1 1 / T y P C y P z He He          , (6.18) (6.18) где Р − давление в псевдоожиженном слое; где Р − давление в псевдоожиженном слое; He − константа Генри; PT атмосферное давление; He − константа Генри; yo мольная доля кислорода в воздухе. yo мольная доля кислорода в воздухе. Переменная ν − определяется равенством: Переменная ν − определяется равенством:  Переменная ν − определяется равенством: (6.19)   / g g l l s s T Є Є Є gH P        , (6.19)   / g g l l s s Є Є Є gH        , , g l s Є − соответственно объемные доли газовой, жидкой и твердой фаз , , g l s − соответственно объемные доли газовой, жидкой и твердой фаз, , , g l s  − соответственно плотности фаз, , , g l s  − соответственно плотности фаз, соответственно плотности фаз, g − ускорение свободного падения, g − ускорение свободного падения, H − высота слоя. H − высота слоя. При решении вышеприведенной системы уравнений можно сделать следующие допущения: − характеристика слоя но его поперечному сечению постоянна. где L − координаты по высоте слоя H; 0 1 2 , , a a a − коэффициенты квадратного уравнения для l Є ; д рд ; 0 1 2 , , a a a − коэффициенты квадратного уравнения для l Є ; Z1 − коэффициент продольного перемешивания по жидкости; U1 − линейная скорость жидкости. 1 фф ц р д р д ; U1 − линейная скорость жидкости. U1 − линейная скорость жидкости. Решение уравнений с граничными условиями по Данквертсу можно получить с применением метода конечных разностей. Оценки можно получить с применением метода конечных разностей. Оценки параметров Z1 и kLa находятся в результате минимизации суммы квадратов разностей между измеренными и рассчитанными по моделям концентрациями кислорода. Оценка величины kLa Коэффициент продольного перемешивания по жидкости Z1 и kLa по жидкой пленке могут быть найдены в результате подгонки рассчитываемых по модели профилями концентраций с измеренными в аэротенке. − параметры Z1 и kLa не являются функциями от продольной координаты слоя, но учитывают продольное изменение перемешивания жидкости, что фиксируется экспериментально путем измерения элсктроповодности системы. Тогда с учетом продольного изменения удерживающей способности по жидкости и ее аппроксимации квадратным уравнением, материальный баланс по растворенному кислороду представляется следующим образом:         2 1 2 1 0 0 1 1 0 2 2 2 2 2 1 0 2 1 0 2 1 0 * 0 2 2 1 0 * 0 0 2 1 1 / La T H g g l l s s T L U a L a d C dC U z a C dL dL a L a L a a L a L a a L a L a k C a L a L a y P C He Є Є Є gdz P                                             , (6.20) , (6.20) где L − координаты по высоте слоя H; где L − координаты по высоте слоя H; 0 1 2 , , a a a − коэффициенты квадратного уравнения для l Є ; д рд ; 0 1 2 , , a a a − коэффициенты квадратного уравнения для l Є ; 0 1 2 , , a a a − коэффициенты квадратного уравнения для l Є ; 180 Z1 − коэффициент продольного перемешивания по жидкости; Выводы к разделу 6 1. В результате системного подхода к формализации феноменологических данных по биохимической очистке сточных вод от органических загрязнителей и результатов обследования работы промышленных аэротенков, предложена структурная схема процесса и механизм, разработаны математические модели макро уровня, позволяющие выявить стационарные состояния при управляющих переменных, в качестве которых являются скорости потока, концентрация загрязнений во входном потоке и интенсивности. 1. В результате системного подхода к формализации феноменологических данных по биохимической очистке сточных вод от органических загрязнителей и результатов обследования работы промышленных аэротенков, предложена структурная схема процесса и механизм, разработаны математические модели макро уровня, позволяющие выявить стационарные состояния при управляющих переменных, в качестве которых являются скорости потока, концентрация загрязнений во входном потоке и интенсивности. р р 2. Представлена модель и алгоритм расчета стационарных профилей растворенного кислорода в аеротенке, методы расчета и определение параметров Z1 и kLa. р р 2. Представлена модель и алгоритм расчета стационарных профилей растворенного кислорода в аеротенке, методы расчета и определение параметров Z1 и kLa. р р 3. Полученные результаты могут являться основанием для выбора конструкции аэротенков и прогнозирующих расчетов процесса очистки сточных вод от органических загрязнителей. 3. Полученные результаты могут являться основанием для выбора конструкции аэротенков и прогнозирующих расчетов процесса очистки сточных вод от органических загрязнителей. 181 181 СПИСОК ЛИТЕРАТУРЫ 1. Николаев, Л. А. (1984). Теоретическая химия. Москва: Высшая школа, 400. 1. Николаев, Л. А. (1984). Теоретическая химия. Москва: Высшая школа, 400. 2. Колмогоров, А. Н. (1988). Математика − наука и профессия. Москва: Наука, 288. 2. Колмогоров, А. Н. (1988). Математика − наука и профессия. Москва: Наука, 288. 3. 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ЭВОЛЮЦИЯ ХИМИЧЕСКИХ СИСТЕМ……………………………………….. 6 2 НЕКОТОРЫЕ НАУЧНЫЕ ОСНОВЫ ТЕХНОЛОГИЧЕСКИХ ПРОЦЕССОВ ХИМИЧЕСКОЙ И ПЕРЕРАБАТЫВАЮЩЕЙ ПРОМЫШЛЕННОСТИ…………………………………………………………… 22 3 МЕТОДОЛОГИЯ И ОРГАНИЗАЦИЯ НАУЧНЫХ ИССЛЕДОВАНИЙ В ИНЖЕНЕРНОЙ ХИМИИ………………………………………………………… 77 3.1 Определение объекта, цели и постановка задач научных исследований 79 4 ИНЖЕНЕРНОЕ ОФОРМЛЕНИЕ ХИМИЧЕСКИХ РЕАКТОРОВ…………. 83 4.1 Реакторы для проведения реакций в потоке гомогенной газовой фазы... 85 4.2 Результаты решений системы уравнений без учета закалки [а(т)]……... 93 4.3 Проведение некаталитических реакций в системе газ-твердое…………. 100 4.4 Реакторы для проведения каталитических реакций……………………... 127 5 АНАЛИЗ ЭКСПЛУАТАЦИОННОГО ФУНКЦИОНИРОВАНИЯ РЕАКТОРА ГЕТЕРОГЕННОГО КАТАЛИЗА В ПРОМЫШЛЕННЫХ УСЛОВИЯХ……………………………………………………………………….. 150 6 МЕХАНИЗМ И МАТЕМАТИЧЕСКАЯ МОДЕЛЬ ПРОЦЕССА БИОЛОГИЧЕСКОЙ ОЧИСТКИ СТОЧНЫХ ВОД ОТ ОРГАНИЧЕСКИХ СОЕДИНЕНИЙ В АЕРОТЕНКАХ………………………………………………. 167 СПИСОК ЛИТЕРАТУРЫ………………………………………………………… 182 189 Монография «ОСНОВЫ ТЕОРИИ ХИМИЧЕСКИХ ПРОЦЕССОВ И РЕАКТОРОВ» 101 «Екологія» и 133 «Галузеве машинобудування» Авторы: ПИТАК Инна Вячеславовна ШАПОРЕВ Валерий Павлович ПИТАК Олег Ярославович ГРУБНИК Алина Олеговна КОМАРИСТАЯ Богдана Николаевна Работу к изданию рекомендовала проф. Самойленко Н. Н. План 2017 р., поз. 111. Підписано до друку 27.11.2017р. Формат 60х80 1/16. Папір офсетний. Друк - ризографія. Гарнітура Times New Roman. Умов. друк. арк. 12. Наклад 300 прим. Зам №137-2017. Ціна договірна ____________________________________________________________________ Видавництво та друкарня "Технологічний Центр" Монография Авторы: Работу к изданию рекомендовала проф. Самойленко Н. Н. План 2017 р., поз. 111. Підписано до друку 27.11.2017р. Формат 60х80 1/16. Папір офсетний. Друк - ризографія. Гарнітура Times New Roman. Умов. друк. арк. 12. Наклад 300 прим. Зам №137-2017. Ціна договірна План 2017 р., поз. 111. Видавництво та друкарня "Технологічний Центр" Свідоцтво про внесення суб’єкта видавничої справи ДК №4452 від 10.12.2012 Адреса: 61145, м. Харків, вул. Шатилова дача, 4
https://openalex.org/W4310848559	https://ijpsat.org/index.php/ijpsat/article/download/4649/2857	English	null	Early Guessing of Performance Using Simulation as Part of Service Development	International Journal of Progressive Sciences and Technologies	2,022	cc-by	3,883	I I © I I © Vol. 34 No. 2 September 2022, pp. 410-416 Abstract— In Service-Oriented Architectures (SOA), the key problem is the quick and accurate evaluation of web service performance. Despite the fact that the integration of the simulation step into the development cycle of softwares/web services can allow to learn early the behavior of the performance of software/web service, it is still a challenge to use simulation as part of service development. This integration can be used to assess the performance of a family of web services by developing one of them. In this paper, we propose a methodology that shows how the simulation step can be integrated to the development cycle of a family of services using a model-based approach to describe the services and by choosing a reference web service to be developed and used to guess the performance of the remaining services in the family. Keywords— Model-based testing; Simulation; Performance; Web services; Zero-knownledge Early Guessing of Performance Using Simulation as Part of Service Development Information and Communication Technology department, Institut de Mathématiques et de Sciences Physiques (IMSP/UAC) Dangbo, Benin pelagie.houngue@imsp-uac.org Corresponding Author: Pélagie HOUNGUE II. OVERVIEW OF OUR ZERO-KNOWLEDGE SCENARIO Our zero-knowledge approach presented in [6] allows to integrate early the web service performance analysis to the development stage in order to have an estimate of the behavior of a given web service using only the information provided by the WSDL file, the SOAP message used to query the service and the STS-based model [9]. For this purpose, an approach is defined to assess the complexity of the web service in order to evaluate some factors used to evaluate the interval of service times. The first step of our approach built a profile table that associates parsing and construction times to the depth and the cardinality of the XML tree that encodes the input parameters of the web service (SOAP message). The second step is to determine the class of complexity of the service from the WSDL by defining an approach that makes easy the complexity evaluation. At the end of this step, a complexity factor called γ is computed for the web service to be simulated and a class of complexity is associated to it. In the last step the parsing and construction times (PT + CT) read from the profile table using the characteristics of the input SOAP message are used to estimate the interval in which the service times vary by applying the complexity factor γ and a factor α (0 < α ≤ 1) defining from a reference web service chosen for its class of complexity. In order to generate the bounds of the interval, α has two values denoted αmin and αmax to compute respectively the lower bound and the upper bound of the service times interval. After this estimation, a correction factor denoted FDI (Data Intensive Factor) is applied in order to take into account how the use of different types of resources can impact the service performance. With the interval of service times, a Monte Carlo [10] based approach is used to generate different random extractions of the partition for the different transitions of the STS-based model extended for simulation. One of the combinations is chosen randomly to annotate the STS-based model and the simulation script is generated to guess the behavior of the service. The results obtained demonstrate that this approach can provide an approximation of service behavior that will be similar to the behavior obtained by running a real service after the development [6], [7]. I. INTRODUCTION services as reference, by developing it and use it to perform the estimation of the performance for the others services members of the family. This work shows the integration of the performance analysis in the development process. The remaining of this paper is organized as follows. Section II presents the overview of our zero-knowledge scenario defined in our previous works. Section III gives the description of our concept and Section IV presents the integration of the simulation phase in the development cycle and describes the different steps to be followed to estimate the behavior of a family of services. Subsequently, Section V gives an overview of the related work and Section VI gives the conclusion. Finally, Section VII points out some future works. I. INTRODUCTION The development based on model allows to integrate early a performance analysis at the software development stage [1], [2], [3]. The evaluation of service performance is important [4], but in a Service-Oriented architecture there is some difficulties to assess the non-functional properties of the web service without any knowledge on the service’s behavior. Nowadays, early performance evaluation is a critical step in web service development [5] and it is important to have a prediction model for performance evaluation at this stage of the development cycle when no historical data are not yet available [6]. In [5], we propose a model-based approach that uses a Symbolic Transition System (STS) to describe the web services as finite state automata and evaluate their performance. This model was extended for simulation purposes by adding state transition probabilities and the delay distributions on the transitions. We evaluated the accuracy of this approach using two different scenarios, a full-knowledge and partial-knowledge scenarios which provide good results. Furthermore, in [6], we define a zero- knowledge scenario which defines a model that allows to estimate the performance of the service when no a priori knowledge is not available on its performance. This model uses the description of the service in the WSDL file, the SOAP message that encodes the input parameters of the service and the STS-based model to guess the behavior of the service. Moreover, in [7] we extends our previous works by using data obtained from our zero-knowledge approach, where neither the service code nor test- based the information on service execution times are available, to generate the SLA template and use it to negotiate the preliminary SLA and monitor it. In order to show the application of our methodology to estimate early the behavior of the service, we propose in this paper the different steps that need to be follow in order to use the simulation as part of the development [8]. This methodology will be applied to a family of services and will show how the performance of the entire family can be guessed by choosing one of the 410 Corresponding Author: Pélagie HOUNGUE Early Guessing of Performance Using Simulation as Part of Service Development services as reference, by developing it and use it to perform the estimation of the performance for the others services members of the family. This work shows the integration of the performance analysis in the development process. II. OVERVIEW OF OUR ZERO-KNOWLEDGE SCENARIO The following equations are used to compute the bounds of the service times interval and the steps to define them are detailed in [6]. Equations (1) and (2) allows to compute respectively the lower bound and the upper bound of the service times interval. γ is defined from the complexity and the values of αREF are defined from the reference web service using (3) and (4). 𝑆𝑇௠௜௡= ୔୘ାେ୘ [ఈೃಶ೘೔೙]ം∗𝐹஽ூ (1) 𝑆𝑇௠௜௡= ୔୘ାେ୘ [ఈೃಶ೘೔೙]ം∗𝐹஽ூ (1) 𝑆𝑇௠௔௫= ୔୘ାେ [ఈೃಶಷ೘ೌೣ]ം∗𝐹஽ூ (2) 𝛼ோாி೘೔೙= [୔୘ାେ୘]ೃಶಷ [ௌ்]ೃಶಷ೘೔೙ (3) 𝛼ோாி೘ೌೣ= [୔୘ାେ]ೃಶಷ [ௌ்]ೃಶಷ೘ೌೣ (4) Vol. 34 No. 2 September 2022 411 ISSN: 2509-0119 Early Guessing of Performance Using Simulation as Part of Service Development 3.1. Running Example or problem Jack is a developer and have to participate to a call that consists to develop a family of web services in the transportation domain. The winner will be the one who can make a proposition that can show a reliable behavior of the web services from the service time point of view. It can be noticed that the developers did not have enough to develop all the services in the set. Also, if they have time, they cannot develop all of the service without be sure to win the call, then the idea to develop all of the service for the call has to be avoided. Jack asks us if there are some strategies that can help him to win this call, since he has just much time to develop correctly one service. We say to him that there is a solution. He can develop one service as reference for the family and guess the behavior of the others from the performance measured for this service. In the following, we will give more detailed about the solution proposed to our friend Jack. III. THE CONCEPT OR SCENARIO The assessment of the service performance early during the development process can give good indicators to analyze the quality of the service under development. This approach can also help the developer when he has to develop a family of services. In this section, we present the story that justifies our concept and how a solution can be found using our approach and frameworks presented in our previous works [5], [6] and [7]. 3.2. Solution In order to estimate the behavior of a family of the web service, we propose to Jack a methodology that is based on the description of the web service as automaton using STS[11]. An STS-based model can be extended for simulation in order to assess the behavior of the service as shown in [5], [6], [7]. In this case, the developer has to: 1) Choose a reference web service for the family 1) Choose a reference web service for the family 2) Define the STS-based model of the reference service and describe the service using WSDL 3) Develop the reference web service 4) Measure the performance of the service developed 5) Save the performance in a database 6) Take the remaining web services and define for each of them the STS-based model, the WSDL file and the SOAP message that encodes the input parameters of the service 7) Apply our zero-knowledge scenario defined in [6] and presented in Section II using the first web service as reference to generate a simulation script for each service remaining in the family 7) Apply our zero-knowledge scenario defined in [6] and presented in Section II using the first web service as reference to generate a simulation script for each service remaining in the family 8) Run the simulation script to guess the behavior of the remaining services and save the outputs. This solution allows Jack to win the competition by providing a reliable estimation of the different services in the family. This solution allows Jack to win the competition by providing a reliable estimation of the different services in the family. The goal of this paper is to show how the simulation can be used as part of the development process and we present in Section IV the detailed methodology that makes this integration possible and solve this kind of situation with accurate precision. The goal of this paper is to show how the simulation can be used as part of the development process and we present in Section IV the detailed methodology that makes this integration possible and solve this kind of situation with accurate precision. IV. DEVELOPMENT CYCLE USING SIMULATION – OUR APPROACH This section gives a better definition of our methodology to simulation of the service performance as part of the development cycle. In our approach presented in [6], in order to guess the behavior of a service using simulation we need a reference web service choosing according to the class of the service to be simulate. In this case where we apply our approach on a family of the services, we choose a reference web service in the family and its performance will be used to guess the behavior of the service. Fig. 1 gives a complete view of the development of a class of service. In the following we describe the different steps shown on this figure. Fig. 1 gives a complete view of the development of a class of service. In the following we describe the different steps shown on this figure. In the first part of the process, the developer chooses the reference web service in the family (1). This service needs to be chosen as representative of the family according to the experience of the developer as example. After that the WSDL file describing the Vol. 34 No. 2 September 2022 412 ISSN: 2509-0119 Early Guessing of Performance Using Simulation as Part of Service Development service (2) and the STS-based model of the service (3) are defined and used (4 and 5) at the design stage to develop the service (6). The service developed is tested and the its performance is monitored (7) and save in a database prepared for a reference web service. These results will be used later in the performance estimation of the remaining services in the family. service (2) and the STS-based model of the service (3) are defined and used (4 and 5) at the design stage to develop the service (6). The service developed is tested and the its performance is monitored (7) and save in a database prepared for a reference web service. These results will be used later in the performance estimation of the remaining services in the family. The second part of the process consists to estimate the behavior of remaining services using the performance measured for the reference web service. The remaining family is taken (9) and the WSDL file, the STS-based model of the service and the input SOAP message (10, 11 and 12) are defined for each service. They will be used (13, 14 and 15) at the design stage to produce the STS-based model for simulation (16). This model is annotated with the transition probabilities and default distributions of delay needed to execute the task associated to the transition of the model. At the simulation stage (17), the delay distributions on the transition of the STS-model for simulation are updated. This information is computed using the profile table (8) and the performance measured for the reference service (19). The WSDL file of each service is used to compute the complexity of the service and the characteristics of the input SOAP message are used to read in the profile table the parsing and construction times, and use them to evaluate the bounds of the service times interval for each service. The interval of service times is divided randomly to annotate the STS-based models with the delay distributions (20). Fig. 1 gives a complete view of the development of a class of service. In the following we describe the different steps shown on this figure. In our case, by default a uniform distribution between two values is selected. After that the simulation script is produced for each service and the service can be simulated. The outputs are saved in a database for future use (21). The updated STS-based model obtained (20) is send back to the design step and make available for the development stage. At this step the behavior of the candidate web services can be guessed early using the simulation scripts produced. In order to verify the accuracy of our approach, the development can continue (23) and the services developed will be tested and monitored (24) and the results saved in a database (25). After the development, the simulation and the testing results are sent for comparison (26-27). The comparison will show the accuracy of the simulation results to guess the behavior of a family of services early using one of them as reference to simulate the others. We can note that the step (22) in the figure can loop in order to update the model to take into account new information from the reference service test. We can note that the step (22) in the figure can loop in order to update the model to take into account new information from the reference service test. Vol. 34 No. 2 September 2022 413 ISSN: 2509-0119 Early Guessing of Performance Using Simulation as Part of Service Development Fig. 1. Simulation in the development cycle V. RELATED WORKS hor of [8] gives an introduction of Software Development Life Cycle (SDLC), followed by the comp on among the various SDLC models. The authors of [12] show that testing is an important part of ment, while [13] define a new software development model, which prioritizes security aspects at the different ware life cycle and takes advantage of the benefits of the agile models. The authors of [14], [15] propose Fig. 1. Simulation in the development cycle V. RELATED WORKS VII. FUTURE WORKS In our future work, we will provide enough benchmarks on our approach to show the application of our methodology which integrate the simulation as part of development cycle on a real family of service development. We can improve our approach by making random selection of the reference web service to verify if the results will be better. With the evolution of machine learning and Artificial intelligence, it could be better to investigate new opportunities to combine simulations and machine learning to accelerate development processes and guess the behavior of the service at early stage. VI. CONCLUSION Simulation driven software development means making use of simulation at each stage of the service development process. In this work, we show how simulation can fit into software delivery life cycles and how it can help businesses reach their goals. Simulation most certainly has immense utility, but it should be used carefully following the situation when no better alternatives exist, and more accurate reference services can be provided to our model. The model proposed shows that there is a good perspective to have more information about performance of the future service in development. V. RELATED WORKS The author of [8] gives an introduction of Software Development Life Cycle (SDLC), followed by the comprehensive comparison among the various SDLC models. The authors of [12] show that testing is an important part of software development, while [13] define a new software development model, which prioritizes security aspects at the different phases of the software life cycle and takes advantage of the benefits of the agile models. The authors of [14], [15] propose simulation Vol. 34 No. 2 September 2022 414 ISSN: 2509-0119 Early Guessing of Performance Using Simulation as Part of Service Development modeling of UML software architectures by annotated UML diagrams with performance results. modeling of UML software architectures by annotated UML diagrams with performance results. The authors of [16], [17] propose to use simulation to evaluate the performance of the web services in order to have enough information on the behavior of the services and compose efficient web processes. The authors of [1] propose to extend the model-driven engineering with performance engineering in order to perform a performance evaluation process during the different development phases and. They extended a UML activity diagram with performance information and transform it in a simulation model for early performance prediction. They proposed to find the performance information used for the prediction from the developers experience and/or the collected performance data on existing systems or similar service. The authors of [18] show how to Increase the ROI of a Software Development Lifecycle by Managing the Risk using Monte Carlo and Discrete Event Simulation. As in [19] where challenges and opportunities of simulation-driven functional product development and operation are shown, and [20] show the way to assess the risk of software development with Agile methodologies using simulation, in this paper as in the previous [5], [6], [7], we try to show that we can guess the behavior of service at early stage of the development by proposed a methodology to integrate simulation in development cycle based on the performance of reference service. VIII. ACKNOWLEDGMENT We thank the African Center of Excellence in Mathematical Sciences and Applications (CEA-SMIA) of University of Abomey- Calavi for funding this work. We thank the African Center of Excellence in Mathematical Sciences and Applications (CEA-SMIA) of University of Abomey- Calavi for funding this work. REFERENCES [1] M. Fritzsche and J. Johannes, “Putting performance engineering into model-driven engineering: Model-driven performance engineering,” Model. Softw. Eng., pp. 164–175, 2008. [2] L. Cheung, L. Golubchik, and F. Shai, “A Study of Web Services Performance Prediction: A Client’s Perspective,” 2011. [3] D. C. Petriu, “Software model-based performance analysis,” John Wiley Sons, 2010. [4] M. P. Papazoglou, V. Andrikopoulos, and S. Benbernou, “Managing Evolving Services,” IEEE Softw., vol. 28, no. 3, pp. 49– 55, 2011. E. Damiani, and K. A. R. Sagbo, “Early Assessment of Service Performance Based on Simulation,” 2013. [5] C. A. Ardagna, E. Damiani, and K. A. R. Sagbo, “Early Assessment of Service Performance Based on [6] C. A. Ardagna, E. Damiani, K. A. R. Sagbo, and F. Frati, “Zero-knowledge evaluation of service performance based on simulation,” 2014. doi: 10.1109/HASE.2014.47. [7] K. A. R. Sagbo, Y. P. E. Houngue, and E. Damiani, “SLA Negotiation and Monitoring from Simulation Data,” 2017. doi: 10.1109/SITIS.2016.126. [8] V. Rastogi, “Software Development Life Cycle Models- Comparison , Consequences,” Int. J. Comput. Sci. Inf. Technol., 2015. 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Montalvo, “The application of a new secure software development life cycle (S-SDLC) with agile methodologies,” Electron., 2019, doi: 10.3390/electronics8111218. [14] S. Balsamo and M. Marzolla, “A simulation-based approach to software performance modeling,” ACM SIGSOFT Softw. Eng. Notes, 2003, doi: 10.1145/949952.940122. [15] S. Balsamo, A. Di Marco, P. Inverardi, and M. Simeoni, “Model-based performance prediction in software development: A survey,” Softw. Eng. IEEE Trans., vol. 30, no. 5, pp. 295–310, 2004. [16] S. Chandrasekaran, G. Silver, J. A. Miller, J. Cardoso, and A. P. Sheth, “XML-based modeling and simulation: web service technologies and their synergy with simulation,” in Proceedings of the 34th conference on Winter simulation: exploring new frontiers, 2002, pp. 606–615. [17] S. Chandrasekaran, J. A. Miller, G. S. Silver, B. Arpinar, and A. P. Sheth, “Performance Analysis and Simulation of Composite Web Services,” Electron. Mark., vol. 13, no. 2, pp. 120–132, 2003. [18] W. 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https://openalex.org/W3105439910	https://www.frontiersin.org/articles/10.3389/fmicb.2020.610179/pdf	English	null	Accessory Gvp Proteins Form a Complex During Gas Vesicle Formation of Haloarchaea	Frontiers in microbiology	2,020	cc-by	11,470	Accessory Gvp Proteins Form a Complex During Gas Vesicle Formation of Haloarchaea Kerstin Völkner, Alisa Jost and Felicitas Pfeifer* Microbiology and Archaea, Department of Biology, Technical University of Darmstadt, Darmstadt, Germany Halobacterium salinarum forms gas vesicles consisting of a protein wall surrounding a gas-ﬁlled space. The hydrophobic 8-kDa protein GvpA is the major constituent of the ribbed wall, stabilized by GvpC at the exterior surface. In addition, eight accessory Gvp proteins are involved, encoded by gvpFGHIJKLM that are co-transcribed in early stages of growth. Most of these proteins are essential, but their functions are not yet clear. Here we investigate whether GvpF through GvpM interact. Pull-down experiments performed in Haloferax volcanii with the cellulose-binding-domain as tag suggested many interactions, and most of these were supported by the split-GFP analyses. The latter study indicated that GvpL attracted all other accessory Gvp, and the related GvpF bound besides GvpL also GvpG, GvpH and GvpI. A strong interaction was found between GvpH and GvpI. GvpG showed afﬁnity to GvpF and GvpL, whereas GvpJ, GvpK and GvpM bound GvpL only. Using GvpA for similar analyses yielded GvpF as the only interaction partner. The contact site of GvpF was conﬁned to the N-terminal half of GvpA and subsequently mapped to certain amino acids. Taken together, our results support the idea that the accessory Gvp form a complex early in gas-vesicle assembly attracting GvpA via GvpF. Keywords: protein-protein interaction, split-GFP, protein network, cellulose binding domain, Haloferax volcanii, Halobacterium salinarum INTRODUCTION Specialty section: This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology Received: 28 September 2020 Accepted: 23 October 2020 Published: 12 November 2020 Citation: Völkner K, Jost A and Pfeifer F (2020) Accessory Gvp Proteins Form a Complex During Gas Vesicle Formation of Haloarchaea. Front. Microbiol. 11:610179. doi: 10.3389/fmicb.2020.610179 Specialty section: This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology Halophilic archaea (haloarchaea) thrive in hypersaline environments such as salt lakes or salterns containing up to 30% NaCl. They adapt to these salty conditions by maintaining a similarly high KCl concentration in the cytoplasm. The haloarchaea Halobacterium salinarum and Haloferax mediterranei produce gas vesicles allowing the cells to ﬂoat to the surface of the brine. These gas- ﬁlled vesicles consist of a wall exclusively formed by proteins. Major component is the hydrophobic, 8-kDa GvpA aggregating into a low-pitch helix seen by transmission electron microscopy as 4.6 nm wide ribs running perpendicular to the long axis of the gas vesicle (Walsby, 1994; Oﬀner et al., 1998; Pfeifer, 2012). The protein wall is stabilized on the exterior surface by the second structural protein, GvpC (Englert and Pfeifer, 1993). Gases are able to diﬀuse in and out, presumably via small holes in the wall. Water vapor might enter the hollow space, but is unable to precipitate because of the hydrophobic interior surface of the wall (Walsby, 1994). Electron microscopic studies of haloarchaeal cells indicate small bicones already ﬁlled with gas in early stages of growth; these bicones are enlarged to spindle- or cylinder-shaped structures. The ﬁnal diameter of the Received: 28 September 2020 Accepted: 23 October 2020 Published: 12 November 2020 ORIGINAL RESEARCH published: 12 November 2020 doi: 10.3389/fmicb.2020.610179 Edited by: Harold J. Schreier, University of Maryland, Baltimore County, United States Reviewed by: Jerry Eichler, Ben-Gurion University of the Negev, Israel Reviewed by: Jerry Eichler, Ben-Gurion University of the Negev, Israel Aharon Oren, Hebrew University of Jerusalem, Israel Ben-Gurion University of the Negev, Israel Aharon Oren, Hebrew University of Jerusalem, Israel Keywords: protein-protein interaction, split-GFP, protein network, cellulose binding domain, Haloferax volcanii, Halobacterium salinarum Correspondence: Felicitas Pfeifer pfeifer@bio.tu-darmstadt.de Citation: volcanii 1C transformants (the 1C construct contains except gvpC all gvp genes) leads to long and weak gas vesicles with altering diameters (Oﬀner et al., 1996), whereas the absence of GvpD and GvpE causes a low expression of the gvp gene cluster. In the case of GvpH and GvpI altered gas vesicle structures are observed. 1H transformants produce weaker gas vesicles of wild type shape, whereas extremely long and cylinder-shaped gas vesicles are found in 1I transformants. The lack of GvpN leads to small bicones that are not enlarged (Oﬀner et al., 2000). A deletion of any other gvp gene in the cluster leads to gas vesicle negative (Vac−) transformants (Oﬀner et al., 2000). Isolated gas vesicles cannot be disintegrated into their protein constituents and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); only GvpC is washed oﬀand separated on the gel (Englert and Pfeifer, 1993). Aggregates of the hydrophobic GvpA dissolve in 80% formic acid only, and dialysis to remove the formic acids leads to amorphous precipitates of GvpA (Belenky et al., 2004). The protein constituents of the gas vesicles were determined by MS/MS-based proteome analyses or immunological methods (Shukla and DasSarma, 2004; Chu et al., 2011). Except for GvpD, GvpE, and GvpK, all Gvp proteins are present in gas vesicle preparations suggesting that they are constituents of the wall or are attached to the structure during gas-vesicle assembly. In this report, we present a comprehensive study on the interactions of the accessory proteins GvpF through GvpM. Two diﬀerent methods were applied, i.e., pull-down experiments with the cellulose-binding domain, CBD, and the in vivo analysis by split-GFP. The cellulose binding domain derives from Clostridium thermocellum and is part of the CipB protein (Poole et al., 1992; Morag et al., 1995). Proteins tagged with CBD can be selected by cellulose in high salt solutions (Ortenberg and Mevarech, 2000; Irihimovitch and Eichler, 2003; Schlesner et al., 2012). The CBDGvp proteins and their putative Gvp binding partners were selected from lysates of Hfx. volcanii producing both bait and prey proteins. Each of the accessory Gvp proteins interacted with other Gvp, and CBDGvpM attracted all of them at once. In addition, each protein pair was tested by split- GFP in Hfx. volcanii transformants, and an interaction network was deduced. GvpL and GvpF had several interaction partners, whereas GvpH and GvpI bound each other and also interacted with GvpF and GvpL. Citation: Völkner K, Jost A and Pfeifer F (2020) Accessory Gvp Proteins Form a Complex During Gas Vesicle Formation of Haloarchaea. Front. Microbiol. 11:610179. doi: 10.3389/fmicb.2020.610179 November 2020 \| Volume 11 \| Article 610179 1 Frontiers in Microbiology \| www.frontiersin.org Accessory Gvp Form an Initiation Complex Völkner et al. of His-tagged Gvp to select putative binding partners and complicate the analysis. haloarchaeal gas vesicle is 200–250 nm; and the cylinder- shaped structures can grow as long as 2 µm (Walsby, 1994; Knitsch et al., 2017). To study putative interactions of Gvp proteins in high salt, we recently adapted the split-GFP method (Ghosh et al., 2000; Magliery et al., 2005) to high salt solutions and studied protein- protein interactions in Hfx. volcanii transformants (Winter et al., 2018). We used a derivative of the salt-adapted green ﬂuorescent protein smRS-GFP (Reuter and Maupin-Furlow, 2004) with higher ﬂuorescence, mGFP2 (Born and Pfeifer, 2019). The mGFP2 protein was split in the N- and C-terminal fragments (NGFP and CGFP) that were fused to the two proteins of interest. Both fragments do not assemble in trans, but will form a ﬂuorescent GFP when the two fusion partners interact. The ﬂuorescence of the transformants can be easily quantiﬁed (Winter et al., 2018). For each protein, four diﬀerent N/CGFP fusions (N- or CGFP fused N- or C-terminally) are produced and eight combinations tested per protein pair to exclude putative steric hindrances for the assembly of GFP. Using this method, the interaction of GvpL/GvpM was conﬁrmed, and the interaction site in GvpM conﬁned to the N-terminal 25-amino acid (aa) (Winter et al., 2018). GvpM also interacted with GvpF and GvpH, and both proteins bound to the C-terminal 25 aa of GvpM (Winter et al., 2018). Gas vesicles of haloarchaea are encoded by a gene cluster consisting of 14 gas vesicle protein (gvp) genes arranged as gvpACNO and gvpDEFGHIJKLM, and eight of these genes are essential to produce a gas-ﬁlled particle as determined in Haloferax volcanii transformants (Englert et al., 1992; Oﬀner et al., 2000). This moderately halophilic haloarchaeon is easy to transform, grows faster than Hbt. salinarum and lacks all of the gvp genes. The six non-essential Gvp proteins are the surface-attached GvpC, the two regulatory proteins GvpD and GvpE (Krüger et al., 1998; Zimmermann and Pfeifer, 2003; Hofacker et al., 2004), and the proteins GvpH, GvpI and GvpN (Oﬀner et al., 2000). The absence of GvpC in Hfx. Citation: In addition, the major gas vesicle structural protein GvpA was tested by split-GFP for interactions with these accessory proteins, and GvpF appeared to be the only binding partner. More detailed analyses with fragments of GvpA and variants of GvpA carrying single substitutions conﬁned the interaction site of GvpF in GvpA. The transcript encoding the accessory gas vesicle proteins GvpF through GvpM occurs in early exponential growth, implying that these proteins are required in early stages of gas- vesicle assembly (Oﬀner and Pfeifer, 1995). Putative interactions of GvpM have been studied by aﬃnity chromatography with His-tagged Gvp and Ni-NTA matrices, and the results suggest that GvpM is able to bind GvpH, GvpJ and GvpL, but not GvpG (Tavlaridou et al., 2014). For these analyses, HisGvp proteins were synthesized in Escherichia coli, puriﬁed under denaturing conditions and refolded in high salt solutions. Haloarchaea require 15–30% NaCl for growth, and adapt by maintaining a similarly high KCl concentration in the cytoplasm. The haloarchaeal proteins are usually adapted to these salt concentrations and often denature in low salt solutions. Thus, the puriﬁcation of these proteins from E. coli in low-salt solutions might have inﬂuenced the protein structure and interaction. Also, other histidine-rich haloarchaeal proteins bind non-speciﬁcally to the Ni-NTA or Ni-sepharose matrices used for the binding Frontiers in Microbiology \| www.frontiersin.org Western Analysis To verify the expression of gvp-gfp or gvp-cbd fusions, total protein was isolated from 50 mL cultures of the transformants in the late exponential growth phase. Cells were harvested by centrifugation (2,370 × g, 30 min, 4◦C) and re-suspended in 5 mL lysis buﬀer (2.5 M KCl, 50 mM MgCl2, 1 mM EDTA, 5% (v/v) glycerol, 50 mM Tris–HCl pH 8.0) + DNaseI (0.1 mg/mL). Cells were lysed by soniﬁcation on ice. The lysate was cleared from cell debris by centrifugation and dialyzed against 10 mM Tris–HCl pH 7.2 overnight at 20◦C to eliminate salts. 20 µg of total protein was separated by SDS-PAGE (Schägger and von Jagow, 1987) and transferred to a PVDF membrane (Roti Fluoro PVDF, Carl Roth) using a Semidry-Blotter (PerfectBlueTM, 30 min, 2 mA). The membrane was dried for one hour, reactivated with 100% (v/v) methanol and washed two times in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4) before blocking for an hour with Odyssey Blocking Buﬀer (Licor). The membrane was incubated with the respective antiserum raised against GvpF, GvpG, GvpH, GvpI, GvpJ, GvpK, GvpL, GvpM, or isolated gas vesicles (to detect GvpA). The membrane was washed four times in PBS + 0.1% (v/v) Tween20, incubated with the secondary antibodies labeled with IRDye 800 CW (Licor) for 1–2 h at 20◦C, and washed four times for 5 min in PBS + 0.1% (v/v) Tween20. Excessive Tween20 was removed by washing the membrane with PBS. The ﬂuorophore coupled to the secondary antibody is detectable at 800 nm with the Odyssey Fc Imager (Licor). The split-GFP shuttle vectors pJAS-NGFP-Nterm and -Cterm, as well as pWL-CGFP-Nterm and -Cterm have been described previously (Winter et al., 2018). They are based on the two compatible vector plasmids pJAS35 (Pfeifer et al., 1994) and pWLfdx (Scheuch and Pfeifer, 2007). The salt adapted mGFP2 is split between the residues 157 and 158, resulting in the NGFP and CGFP fragments. Nterm describes a fusion of the respective mgfp2 fragment to the 5′-terminus of the gene of interest, whereas Cterm stands for a fusion at the 3′-terminus. The respective mgfp2 fragments plus linker region are present in these vectors. The size of the linker encoded by the pJAS-derived shuttle vector is 14 aa [(GGSGSGS)2], whereas the linker derived from the pWLfdx vector is 16 aa long [(GGSG)4] (Winter et al., 2018). MATERIALS AND METHODS Strains and Cultivation Conditions The Escherichia coli strains One Shot Top10 (Invitrogen by Life Technologies) and GM1674 (dam−) (Palmer and Marinus, 1994) were grown in Luria-Bertani media at 37◦C overnight. For the selection of transformants, 100 µg/mL ampicillin was added. The haloarchaeon Haloferax volcanii WR340 was grown in salt media containing 3 M NaCl, 150 mM MgSO4, 50 mM KCl, November 2020 \| Volume 11 \| Article 610179 Frontiers in Microbiology \| www.frontiersin.org Frontiers in Microbiology \| www.frontiersin.org 2 Accessory Gvp Form an Initiation Complex Völkner et al. 0.05% (w/v) CaCl2, 25 mM Tris–HCl pH 7.2, 10 nM MnCl2, 0.5% (w/v) tryptone, 0.3% (w/v) yeast extract and 0.02% (w/v) histidine. For solid media, 1.8% (w/v) agar was added. To select Hfx. volcanii transformants, 6 µg/mL mevinolin (for selection of pWLfdx) and 0.2 µg/mL novobiocin (for selection of pJAS35) were added. Cultures on solid medium were incubated at 42◦C for 3–7 days in a humid atmosphere. Liquid cultures were grown for 3–5 days on a shaker at 180 rpm and 42◦C. Cultures forming GFP were initially incubated at 37◦C for 24 h before they were transferred to 30◦C overnight to enhance ﬂuorescence, always shaking at 180 rpm. pF-Lex, the gvpFGHIJKL reading frames of p-vac were ampliﬁed as XbaI–KpnI fragment and inserted in pJAS35 for expression under Pfdx control. f In all cases, the correct insertion was veriﬁed by DNA sequence analysis. To avoid a restriction barrier in Hfx. volcanii WR340, the plasmid DNA was demethylated by passage through E. coli GM1674 (dam−) (Palmer and Marinus, 1994). Hfx. volcanii was transformed simultaneously with the two vector plasmids as described previously (Pfeifer and Ghahraman, 1993). In each case, the presence of both plasmids was conﬁrmed by PCR, and the presence of the respective Gvp protein determined by Western analysis. Western Analysis The gvp reading frame under investigation was fused to ngfp or cgfp by inserting the respective fragment in these vector plasmids. The gvp reading frames were ampliﬁed by PCR using the p-vac region as template (containing the 14 gvp genes of Hbt. salinarum PHH1; Englert et al., 1992), and oligonucleotides including the desired restriction site for insertion (Supplementary Table 1). The NcoI-gvp-BlpI fragments ampliﬁed were inserted in pJAS- NGFP-Nterm or pJAS-NGFP-Cterm. For the insertion of gvp in pWL-CGFP-Nterm, BamHI and KpnI sites were used, and NcoI and BamHI for the insertion in pWL-CGFP-Cterm. In some cases, BspHI (gvpF, gvpI gvpJ, gvpL) or PciI (gvpG), was used instead of NcoI. Due to the presence of a KpnI site in gvpH, the ampliﬁed gvpH fragment was blunt-ended inserted in pWL-CGFP-Nterm. Afﬁnity Chromatography Using CBD-Tagged Proteins proteins (Supplementary Figure 2). Except for GvpJ and GvpM, the accessory Gvp were isolated in decent amounts (Table 1), and well visible in the Coomassie-stained gels (Supplementary Figure 2). GvpJ and GvpM were obtained in much lower amounts, presumably due to their hydrophobic nature and tendency to aggregate. Aggregates of GvpJ and GvpM might be present in the solid fraction of the cell extracts, and the addition of the detergents DDM (n-dodecyl-β-D-maltoside) or OGP (octyl- β-D-glucopyranoside) to the lysate and wash buﬀer slightly improved the presence of these proteins in the soluble fraction (Table 1). All of the accessory Gvp proteins including GvpJ and GvpM were detectable by Western analysis (Supplementary Figure 2). A smear of larger bands is always detectable with GvpJ, indicative of its ability to aggregate (Supplementary Figure 2). Thus, each of the CBDGvp proteins was selected and could be puriﬁed. To ensure that the untagged Gvp interacted neither with the cellulose matrix nor with CBD, transformants were produced harboring the plasmids pCBD (without gvp) and pXex (gvpX- pJAS35 expressing any gvp without cbd fusion). None of the Gvp proteins tested was detectable in the respective elution fraction demonstrating that none of them bound to cellulose or to CBD itself (data not shown). gg Each accessory Gvp was fused to the cellulose binding domain, CBD, at the N- or C-terminus (CBDX or XCBD) and lysates of the respective CBDX/Y transformants were tested in pulldown experiments (X, Y = any accessory Gvp). In the case of CBDM, also CBDM/pF-Lex transformants were tested. For each pull-down experiment, 400 mL cultures were grown at 37◦C, 180 rpm to the late exponential growth phase. The cells were harvested (12,210 × g, 20 min, 4◦C), and resuspended in 5 mL lysis buﬀer (2.5 M KCl, 50 mM MgCl2, 1 mM EDTA, 5% (v/v) glycerol, 50 mM Tris–HCl pH 8.0) plus DNaseI (0.1 mg/mL). The cells were lysed by ultrasound (Branson Soniﬁer 250, duty cycle: 55%, output control: 5, 2 min) and the suspension centrifuged for 20 min at 2,370 × g, 4◦C. The soluble protein fraction (7 mL) was incubated with 1 mL of a 10% (w/v) cellulose suspension (Avicel PH-101, Sigma Aldrich) for 30 min at room temperature on an overhead rotator. Quantitation of GFP Fluorescence The ﬂuorescence of Hfx. volcanii transformants was measured to determine the formation of ﬂuorescent GFP as a result of an interaction of the two fused proteins of interest. Transformants were grown as described to OD600nm 1.5–2. Two mL of the culture were harvested (2 min, 9,600 x g, 20◦C), the cells washed in 1 mL basal salts (3 M NaCl, 150 mM MgSO4, 50 mM KCl), and re-suspended in 500 µL basal salts. To investigate equal amounts of cells, the OD value was adjusted to OD600nm 1, and 300 µL of the culture transferred into a microtiter dish. For each Gvp protein combination, two biological and three technical replicates were investigated. The ﬂuorescence was measured (Phosphorimager FLA-5000 and the software Fujiﬁlm science lab image gauge ver. 4.24), and the ﬂuorescence intensity obtained in light absorbing units (LAU) per mm2. The relative ﬂuorescence, rf, was calculated as described (Winter et al., 2018). The p-value was determined by Student’s T-test. For pull-down assays of CBD-tagged proteins, the shuttle vector pCBD was used (Supplementary Figure 1). The cbd reading frame encodes the cellulose binding domain of the CipB protein from Clostridium thermocellum (Poole et al., 1992; Morag et al., 1995). The cbd reading frame was ampliﬁed from plasmid pWL-CBD-sec11b (Fine et al., 2006) and inserted in pWLfdx via NcoI and KpnI. Two additional cloning sites are present (BamHI, XbaI) allowing the insertion of the respective gvp reading frame upstream or downstream of cbd [NcoI and BamHI upstream of cbd are used to yield XCBD, and XbaI and KpnI downstream to yield CBDX (with X = GvpF, G, H, I, J, K, L, or M)]. The expression of the respective gene fusion is under control of the strong ferredoxin promoter, Pfdx (Pfeifer et al., 1994). The gvp sequences were ampliﬁed using p-vac as template. The oligonucleotides used are listed in Supplementary Table 1. For the construction of November 2020 \| Volume 11 \| Article 610179 Frontiers in Microbiology \| www.frontiersin.org 3 Accessory Gvp Form an Initiation Complex Völkner et al. Afﬁnity Chromatography Using CBD-Tagged Proteins The suspension was centrifuged (2,370 × g, 30 s), the supernatant removed and the resulting cellulose pellet with bound proteins resuspended in 600 µL washing buﬀer (2.5 M KCl, 50 mM Tris–HCl pH 8.0). The solution was transferred to an empty Mobicol column (Mobitec) and centrifuged (1,000 × g, 1 min), followed by six washing steps with 600 µl washing buﬀer. For protein elution, the cellulose was resuspended in 500 µL 100% ethylene glycol, incubated for 1 min at room temperature and centrifuged at 4,700 × g, 5 min. All fractions were dialyzed against 10 mM Tris–HCl, pH 7.2 overnight. For the analysis of proteins, 15 µL of each fraction were separated by SDS-PAGE (Schägger and von Jagow, 1987). The Gvp proteins were detected by Western analysis using the respective antisera. The CBD-tagged Gvp proteins were used as bait and tested for the selection of a putative Gvp interaction partner produced in the same cell. The ﬁrst protein pair tested was GvpL/GvpM, where an interaction has been demonstrated using His-tagged Gvp proteins. GvpL or GvpM carried CBD fused to the N- or C-terminus (CBDL, LCBD or CBDM, MCBD), and the combinations CBDL/M, LCBD/M, CBDM/L and MCBD/L were analyzed. Lysates of the CBDL/M and LCBD/M transformants were tested for the presence of GvpM, and monomers and dimers of this protein were identiﬁed by Western analysis (Supplementary Figure 3). Also, GvpL was present in the elution fractions of the CBDM/L and MCBD/L transformants indicating that GvpL bound GvpM (Supplementary Figure 3). These results RESULTS Two diﬀerent methods were applied to investigate protein- protein interactions. The ﬁrst approach was pull-down experiments using the cellulose-binding domain as tag, and the second approach was split-GFP to identify interaction partners in vivo. TABLE 1 \| Amount of CBDGvp recovered from a 400 ml culture by CBD. Protein Amount in µg CBDF 146 ± 44 CBDG 307 ± 41 CBDH 417 ± 86 CBDI 142 ± 18 CBDJ 23 ± 5 CBDK 214 ± 35 CBDL 484 ± 21 CBDM 13 ± 2 CBDJ (1.4 mM DDM) 67 ± 14 CBDJ (10.4 mM DDM) 132 ± 20 CBDJ (60 mm OGP) 90 ± 9 CBDM (1.4 mM DDM) 14 ± 0 CBDM (10.4 mM DDM) 33 ± 14 CBDM (60 mM OGP) 18 ± 5 The standard deviation was calculated from two biological and three technical replicates. TABLE 1 \| Amount of CBDGvp recovered from a 400 ml culture by CBD. TABLE 1 \| Amount of CBDGvp recovered from a 400 ml culture by CBD. TABLE 1 \| Amount of CBDGvp recovered from a 400 ml culture by CBD. Protein Amount in µg CBDF 146 ± 44 CBDG 307 ± 41 CBDH 417 ± 86 CBDI 142 ± 18 CBDJ 23 ± 5 CBDK 214 ± 35 CBDL 484 ± 21 CBDM 13 ± 2 CBDJ (1.4 mM DDM) 67 ± 14 CBDJ (10.4 mM DDM) 132 ± 20 CBDJ (60 mm OGP) 90 ± 9 CBDM (1.4 mM DDM) 14 ± 0 CBDM (10.4 mM DDM) 33 ± 14 CBDM (60 mM OGP) 18 ± 5 *The standard deviation was calculated from two biological and three technical replicates. Frontiers in Microbiology \| www.frontiersin.org Protein-Protein Interactions Investigated by Split-GFP Pairwise interactions of the accessory Gvp proteins were studied in Hfx. volcanii in vivo using the split-GFP method. Each gvp reading frame was ampliﬁed by PCR and inserted in the four vector plasmids to fuse the ngfp- or cgfp reading frame to the 5′ or 3′ terminus of each gvp (Winter et al., 2018). The N- and C-terminal GFP fragments NGFP and CGFP derive from the salt-adapted, green ﬂuorescent protein mGFP2 (Born and Pfeifer, 2019). A ﬂuorescent GFP is only formed when the two fusion partners interact and steric hindrance does not occur. The reading frame of each fusion protein is expressed under the Pfdx promoter control to yield similarly large amounts of these proteins. The four N/CGFP fusions were designated NX, or CX for the N-terminal fusion, and XC or XN for the C-terminal fusion with Gvp (with X = respective accessory Gvp; C = CGFP; N = NGFP). The eight combinations of a protein pair were tested in Hfx. volcanii transformants and the ﬂuorescence was measured in arbitrary light absorbing units per mm2. The highest relative ﬂuorescence (rf-values) calculated for each interaction pair are shown in Supplementary Figure 4, and the original data obtained in LAU/mm2 are presented in Supplementary Figure 5. A summary of these data is shown in Figure 3. The highest relative ﬂuorescence of all interactions was observed with the NG/CL transformant (rf 77.5) implying a strong interaction of GvpL with GvpG (Figure 3A). All other interaction partners of GvpL yielded rf-values below 20. Rf-values between 10 and 20 were observed for the interaction FC/NL, NI/LC, MC/LN, HN/LC, and JC/NL, and rf 7.4 was determined for KC/NL (Figures 3A,B and Supplementary Figure 5). These results implied that the 32-kDa GvpL interacted with all other accessory Gvp. GvpL is the largest of the accessory Gvp and might act as platform to bind all others. Also, GvpF interacted with several Gvp proteins (GvpL, GvpH, GvpI, and GvpG) (Figure 3B). The FC/LN transformant (rf 16.6) exhibited the highest relative ﬂuorescence, whereas the other three binding partners yielded lower rf-values (rf 5.0–6.3). In the case of GvpF it should be mentioned that the highest ﬂuorescence was always observed when N- or CGFP was fused to the C-terminus of GvpF, suggesting that a fusion to the N-terminus hinders the assembly of mGFP2. Pull-down Experiments Using the Cellulose Binding Domain Putative interactions of the accessory proteins GvpF through GvpM were investigated by pull-down experiments using the cellulose binding domain, CBD, allowing tagged proteins to bind cellulose at high salt concentrations (2–3 M KCl) (Ortenberg and Mevarech, 2000; Irihimovitch and Eichler, 2003; Schlesner et al., 2012). CBD was fused to the N- or C-terminus of each accessory Gvp using the vector plasmid pCBD (Supplementary Figure 1). The resulting CBDX or XCBD constructs (X = any accessory GvpF through GvpM) were used to transform Haloferax volcanii WR340. To test whether the CBD-fusion proteins can be indeed puriﬁed in 2 M KCl, lysates of the respective CBDX transformants were mixed with cellulose to bind the CBD-tagged Gvp, and the elution fractions were investigated by SDS-PAGE as well as Western analysis using antisera raised against the various Gvp Frontiers in Microbiology \| www.frontiersin.org November 2020 \| Volume 11 \| Article 610179 4 Accessory Gvp Form an Initiation Complex Völkner et al. conﬁrmed the GvpL/GvpM interaction already seen using the respective His-tagged Gvp proteins and a Ni-NTA matrix for aﬃnity chromatography (Tavlaridou et al., 2014). visible in addition to the expected 10-kDa GvpG protein, and GvpJ and GvpK also formed multimers (Figure 2). The monomer of the CBDM bait protein was also detected (Figure 2). Overall, CBDM was able to pull down GvpF through GvpL. It is possible that each of these Gvp proteins bound independently to CBDM, but it is also possible that some or all of them formed a complex that bound to GvpM. Similar pull-down experiments with CBD were performed with all other accessory Gvp proteins. The respective Hfx. volcanii transformants always carried two constructs, one for the bait protein (CBDX), and one for the prey GvpY (with X, Y = F, G, H, I, J, K, L, or M). Both reading frames encoding bait and prey were expressed under Pfdx promoter control to ensure a similarly high expression. Not all possible interactions were performed in both ways (CBDX+Y and CBDY+X), since protein interactions were already found with one protein pair. The results of these studies are presented in Figure 1 and summarized Table 2. The Western blots in Figure 1 are arranged according to the antiserum used to detect the prey protein. Using CBDX to select GvpM yielded monomeric GvpM with CBDH, and monomers as well as dimers with CBDF, CBDG, CBDJ, CBDK, and CBDL (Figure 1). Pull-down Experiments Using the Cellulose Binding Domain Dimers and multimers of GvpM were observed with CBDI (Figure 1). Thus, GvpM was pulled-down in all these cases. GvpL was selected by CBDF, but the results obtained with CBDH or CBDI were ambiguous (Figure 1). However, when CBDL was used as bait, GvpM, GvpK, GvpJ, or GvpG were selected again suggesting that all these accessory Gvp bound GvpL. Also, GvpK interacted with any of the accessory Gvp, since CBDF through CBDJ and CBDL pulled- down GvpK, and CBDK recovered GvpM. Monomers of GvpJ were selected by CBDX, but a smear including additional GvpJ multimers were found with CBDF, CBDH, CBDI, CBDL, or CBDM (Figure 1). GvpI was only used as bait, and CBDI pulled-down any of the accessory Gvp proteins. It is interesting to note that CBDI often induced the formation of larger oligomers, especially with GvpM, GvpK or GvpJ as prey. GvpH was selected by CBDF or CBDI, and CBDH pulled down GvpG, GvpJ, GvpK, GvpL or GvpM (Figure 1 and Table 2). GvpG monomers and dimers were selected by CBDF, whereas CBDH, CBDI and CBDL selected the dimer of GvpG (and additional multimers in the case of CBDI) (Figure 1). Since CBDG bound GvpM, GvpK and GvpJ, these results demonstrated that GvpG is able to interact with all accessory proteins. Similar results were obtained with GvpF, since GvpF was pulled down by CBDI, and CBDF pulled down GvpG, GvpH, and GvpJ through GvpM (Figure 1 and Table 2). Overall, the results of these pull-down experiments implied that any accessory Gvp had multiple interaction partners and suggested that these proteins might form a larger complex. Protein-Protein Interactions Investigated by Split-GFP The 23-kDa GvpF is smaller than GvpL, but both proteins exhibit sequence similarities and a similar 3D-structure when modeled using the crystal structure of the cyanobacterial GvpF as template (Xu et al., 2014; Winter et al., 2018). Frontiers in Microbiology \| www.frontiersin.org CBDM T CBDM To investigate whether GvpM was able to select all accessory Gvp at once, Hfx. volcanii transformants were produced carrying CBDM and in addition the plasmid pF-Lex expressing gvpFGHIJKL under the strong Pfdx promoter control. The interaction partners of CBDM were selected by pull-down experiments from the lysate of this transformant, and samples were tested by Western analyses using the diﬀerent antisera (Figure 2). All accessory Gvp were identiﬁed, implying that CBDM attracted all of them. In each case the respective monomeric Gvp was detected. In the case of GvpG, two 30–34 kDa bands were The second highest ﬂuorescence (rf 20) of all combinations was observed for the HC/NI transformant (Figure 3), implying that GvpH and GvpI attract each other. Both proteins also interacted with GvpF and GvpL (rf 6.3). Two partner proteins November 2020 \| Volume 11 \| Article 610179 Frontiers in Microbiology \| www.frontiersin.org 5 Accessory Gvp Form an Initiation Complex Völkner et al. FIGURE 1 \| Western analyses of the pull-down assays using CBDX. The bait CBDX and the prey protein Y (X, Y = any GvpF through GvpM protein) were synthesized in the same Hfx. volcanii cell. Both are marked on the top of the blots. Only the elution fraction of the cellulose matrix is shown. In each case, 15 µL of the elution fraction was separated by SDS-PAGE, the proteins transferred to a PVDF membrane and incubated with the respective antiserum indicated underneath (αF through αM). The putative interaction partners were visualized with the ﬂuorophore-labeled secondary antibody IRDye 800 CW (Licor). The blots are inverted to black-white and arranged according to the respective antiserum used. Arrows mark the expected Gvp monomer and dimer. Numbers on the left are size markers in kDa. FIGURE 1 \| Western analyses of the pull-down assays using CBDX. The bait CBDX and the prey protein Y (X, Y = any GvpF through GvpM protein) were synthesized in the same Hfx. volcanii cell. Both are marked on the top of the blots. Only the elution fraction of the cellulose matrix is shown. In each case, 15 µL of the elution fraction was separated by SDS-PAGE, the proteins transferred to a PVDF membrane and incubated with the respective antiserum indicated underneath (αF through αM). The putative interaction partners were visualized with the ﬂuorophore-labeled secondary antibody IRDye 800 CW (Licor). CBDM T The highest relative ﬂuorescence was obtained for the AC/FN transformant (rf 20), whereas all other transformants yielded rf- values < 1, suggesting very weak contacts between GvpA and the other Gvp proteins (Figure 4A). Except of NA/LC, the highest ﬂuorescence of a protein pair was always observed when N- or CGFP was fused to the C-terminus of GvpA. These results implied that GvpF is the only interaction partner of GvpA, were identiﬁed for GvpG (GvpL and GvpF), and GvpL appeared to be the only interaction partner of the three proteins GvpJ, GvpK and GvpM (Figure 3B). Overall, these results obtained by these split-GFP analyses demonstrated that all accessory Gvp proteins had at least one other Gvp protein as interaction partner. Since GvpL bound all of them it is possible that they form a larger complex. Compared to the results obtained by aﬃnity chromatography using CBDGvp, less interactions were observed with split-GFP, especially for the two hydrophobic proteins GvpJ (12 kDa) and GvpM (9.2 kDa), and for GvpK (12.6 kDa). CBDM T Interaction Partner(s) of GvpA To uncover interactions between the major structural gas vesicle protein GvpA and these accessory Gvp, each combination of A/X (X = GvpF through GvpM) was investigated by split-GFP. GvpA was fused at the N- or C-terminus to N- or CGFP and tested pairwise with the respective N/CGFP fusion variants of GvpF through GvpM. Eight diﬀerent combinations were investigated for each pair, and the highest rf-values calculated in each case are shown in Figure 4A. The original data obtained in these experiments is presented in Supplementary Figure 6. The highest relative ﬂuorescence was obtained for the AC/FN transformant (rf 20), whereas all other transformants yielded rf- values < 1, suggesting very weak contacts between GvpA and the other Gvp proteins (Figure 4A). Except of NA/LC, the highest ﬂuorescence of a protein pair was always observed when N- or CGFP was fused to the C-terminus of GvpA. These results implied that GvpF is the only interaction partner of GvpA, TABLE 2 \| Summary of the pull-down experiments using CBDGvp. A/X (X = GvpF through GvpM) was investigated by split-GFP. GvpA was fused at the N- or C-terminus to N- or CGFP and tested pairwise with the respective N/CGFP fusion variants of GvpF through GvpM. Eight diﬀerent combinations were investigated for each pair, and the highest rf-values calculated in each case are shown in Figure 4A. The original data obtained in these experiments is presented in Supplementary Figure 6. The highest relative ﬂuorescence was obtained for the AC/FN transformant (rf 20), whereas all other transformants yielded rf- values < 1, suggesting very weak contacts between GvpA and the other Gvp proteins (Figure 4A). Except of NA/LC, the highest ﬂuorescence of a protein pair was always observed when N- or CGFP was fused to the C-terminus of GvpA. These results implied that GvpF is the only interaction partner of GvpA, A/X (X = GvpF through GvpM) was investigated by split-GFP. GvpA was fused at the N- or C-terminus to N- or CGFP and tested pairwise with the respective N/CGFP fusion variants of GvpF through GvpM. Eight diﬀerent combinations were investigated for each pair, and the highest rf-values calculated in each case are shown in Figure 4A. The original data obtained in these experiments is presented in Supplementary Figure 6. CBDM T The blots are inverted to black-white and arranged according to the respective antiserum used. Arrows mark the expected Gvp monomer and dimer. Numbers on the left are size markers in kDa. TABLE 2 \| Summary of the pull-down experiments using CBDGvp. GvpF GvpG GvpH GvpI GvpJ GvpK GvpL GvpM CBDF GvpG GvpH + (GvpJ) (aggregate) GvpK (weak) GvpL GvpM CBDG + + + (GvpJ) (monomer+dimer) GvpK (weak) + GvpM CBDH + GvpG (dimer) + (GvpJ) (aggregate) GvpK (GvpL) GvpM (monomer) CBDI GvpF GvpG (dimer + multimer) GvpH + (GvpJ) (monomer+ aggregate) GvpK (Monomer+ multimer) (GvpL) GvpM (dimer + multimer) CBDJ + + + + GvpK + GvpM CBDK + + + + + + GvpM CBDL + GvpG (dimer) (−) (−) GvpJ (monomer) GvpK GvpM (monomer) CBDM + + + + GvpJ (aggregate) + + GvpX interaction detected. + interaction was shown in another combination. −no interaction identiﬁed. ( ) interaction not clear. TABLE 2 \| Summary of the pull-down experiments using CBDGvp. GvpF GvpG GvpH GvpI GvpJ GvpK GvpL GvpM CBDF GvpG GvpH + (GvpJ) (aggregate) GvpK (weak) GvpL GvpM CBDG + + + (GvpJ) (monomer+dimer) GvpK (weak) + GvpM CBDH + GvpG (dimer) + (GvpJ) (aggregate) GvpK (GvpL) GvpM (monomer) CBDI GvpF GvpG (dimer + multimer) GvpH + (GvpJ) (monomer+ aggregate) GvpK (Monomer+ multimer) (GvpL) GvpM (dimer + multimer) CBDJ + + + + GvpK + GvpM CBDK + + + + + + GvpM CBDL + GvpG (dimer) (−) (−) GvpJ (monomer) GvpK GvpM (monomer) CBDM + + + + GvpJ (aggregate) + + GvpX interaction detected. + interaction was shown in another combination. −no interaction identiﬁed. ( ) interaction not clear. were identiﬁed for GvpG (GvpL and GvpF), and GvpL appeared to be the only interaction partner of the three proteins GvpJ, GvpK and GvpM (Figure 3B). Overall, these results obtained by these split-GFP analyses demonstrated that all accessory Gvp proteins had at least one other Gvp protein as interaction partner. Since GvpL bound all of them it is possible that they form a larger complex. Compared to the results obtained by aﬃnity chromatography using CBDGvp, less interactions were observed with split-GFP, especially for the two hydrophobic proteins GvpJ (12 kDa) and GvpM (9.2 kDa), and for GvpK (12.6 kDa). Interaction Partner(s) of GvpA To uncover interactions between the major structural gas vesicle protein GvpA and these accessory Gvp, each combination of November 2020 \| Volume 11 \| Article 610179 Frontiers in Microbiology \| www.frontiersin.org 6 Accessory Gvp Form an Initiation Complex Völkner et al. FIGURE 2 \| Western analyses of proteins selected by CBDM in CBDM/pF-Lex transformants. In each case, 15 µL of the elution fraction was separated by SDS-PAGE, transferred to a PVDF membrane and incubated with the respective antiserum marked underneath (αF through αM). Reactions are visualized with the ﬂuorophore-labeled secondary antibody IRDye 800 CW (Licor). Arrows mark the expected Gvp monomer detected. The blots were inverted to black-white. Numbers on the left side are size markers in kDa. FIGURE 2 \| Western analyses of proteins selected by CBDM in CBDM/pF-Lex transformants. In each case, 15 µL of the elution fraction was separated by SDS-PAGE, transferred to a PVDF membrane and incubated with the respective antiserum marked underneath (αF through αM). Reactions are visualized with the ﬂuorophore-labeled secondary antibody IRDye 800 CW (Licor). Arrows mark the expected Gvp monomer detected. The blots were inverted to black-white. Numbers on the left side are size markers in kDa. FIGURE 3 \| Interactions of the accessory Gvp determined by split-GFP. (A) Highest relative ﬂuorescence (rf-values) determined for the protein-protein interactions calculated as described in the methods section. All experiments were performed with two biological and three technical replicates. (B) Summary of the different protein-protein interactions determined by split-GFP. The Gvp proteins tested are shown in the gray box on the left and their interaction partners are arranged according to the highest rf-value determined (given underneath). A ﬂuorescence exceeding rf > 5 was regarded as clear interaction. Rf-values < rf 4 are regarded as weak or no interaction. FIGURE 3 \| Interactions of the accessory Gvp determined by split-GFP. (A) Highest relative ﬂuorescence (rf-values) determined for the protein-protein interactions calculated as described in the methods section. All experiments were performed with two biological and three technical replicates. (B) Summary of the different protein-protein interactions determined by split-GFP. The Gvp proteins tested are shown in the gray box on the left and their interaction partners are arranged according to the highest rf-value determined (given underneath). A ﬂuorescence exceeding rf > 5 was regarded as clear interaction. Rf-values < rf 4 are regarded as weak or no interaction. Interaction Partner(s) of GvpA (B) Interaction of ﬁve different GvpA fragments with GvpF. (C) Sequence of the ﬁve GvpA fragments in relation to the aa sequence of GvpA shown on top. Numbers above the sequence depict the aa positions in GvpA. The helices α1 and α2 and the β-sheets β1 and β2 are in bold and marked on the bottom and also shaded in gray. as high as obtained for the interaction of GvpF with the entire GvpA (rf 20) (Figure 4B). Smaller protein fragments oﬀer less steric hindrance supporting the interaction (Ghosh et al., 2000; Winter et al., 2018). An increased ﬂuorescence was also found for the transformants harboring F/A1-34 and F/A1-43 (rf 33 – 35), whereas a very low relative ﬂuorescence (rf < 1) was obtained for the transformants harboring F/A20-47 or F/A44- 76. These results implied that the C-terminal portion of GvpA is not involved, and that the N-terminal portion contains the interaction site of GvpF. A27E, or G33V resulted in a low relative ﬂuorescence of the F/Amut transformants (Figure 5A and Supplementary Figure 6). Especially the charged aa in β1 and β2 (D24, R28, E40) and G20 of GvpA had a strong inﬂuence on the interaction with GvpF (Figure 5). All these aa are located in the N-terminal half of GvpA, whereas any of the single substitutions in the C-terminal half had no eﬀect on the interaction with GvpF, supporting the results described above. It is likely that these aa of GvpA are involved in the GvpF/GvpA interaction. In respect to the gas vesicle phenotype of the 1A+Amut transformants, most of these mutations result in a Vac−phenotype, except for D24A (cylindrical) and R28A (mini gas vesicles) (Knitsch et al., 2017; Figure 5B). It is possible that the Vac−phenotype is caused by the lack of an interaction between GvpF and GvpA, rather than (or in addition to) an inﬂuence of the mutation on the GvpA/GvpA contact in the gas vesicle wall. p To further conﬁne the interaction site of GvpF in GvpA, various substitution variants of GvpA were tested by split-GFP. Many of these point mutations in GvpA are known to inﬂuence the formation of gas vesicles in 1A+Amut transformants (Strunk et al., 2011; Knitsch et al., 2017). Interaction Partner(s) of GvpA These transformants carry two vector plasmids, the 1A construct (contains except gvpA all gvp genes of the p-vac region) and construct A (gvpA or mutant gvpA expressed in pMDS20 under the control of the native PA promoter). The diﬀerent GvpA variants were investigated by split-GFP analysis for their potential to interact with GvpF in Hfx. volcanii. The FN fusion protein was used as interaction partner in all these cases (Figure 5A). The strongest reductions in relative ﬂuorescence (rf < 6) compared to GvpA wild type (rf 15-18) were observed for the GvpA substitutions G20D, G20A, D24A, D24Y, R28A, R28D, or E40A, but also R15A, K19D, Interaction Partner(s) of GvpA FIGURE 3 \| Interactions of the accessory Gvp determined by split-GFP. (A) Highest relative ﬂuorescence (rf-values) determined for the protein-protein interactions calculated as described in the methods section. All experiments were performed with two biological and three technical replicates. (B) Summary of the different protein-protein interactions determined by split-GFP. The Gvp proteins tested are shown in the gray box on the left and their interaction partners are arranged according to the highest rf-value determined (given underneath). A ﬂuorescence exceeding rf > 5 was regarded as clear interaction. Rf-values < rf 4 are regarded as weak or no interaction. contains in addition β-sheet 1 (α1-β1), and A1-43 the α1-β1- β2 elements of GvpA (Figure 4C). Fragment A20-47 contains β1-β2, and A44-76 the α-helix 2 up to the C-terminus of GvpA (α2) (Figure 4C). Each of these GvpA fragments was fused at the N- or C-terminus to N- or CGFP and tested with the respective N/CGFP fusion of GvpF. Transformant FN/A1-22C yielded the highest ﬂuorescence (rf 41), i.e., more than twice and that the N-terminal region of GvpA might be involved in the interaction. To deﬁne the interaction site of GvpF in GvpA more precisely, ﬁve GvpA fragments harboring diﬀerent structural features (according to the model obtained by Strunk et al. (2011)) were studied by split-GFP. Fragment A1-22 encompasses the ﬁrst 22 amino acids of GvpA including α-helix 1 (α1), fragment A1-34 November 2020 \| Volume 11 \| Article 610179 Frontiers in Microbiology \| www.frontiersin.org Accessory Gvp Form an Initiation Complex Völkner et al. FIGURE 4 \| Split-GFP interaction studies with GvpA/GvpX. Only the highest relative ﬂuorescence determined for each combination is given. Two biological and three technical replicates were performed in each case. (A) Interaction study of GvpA with the eight accessory proteins GvpF through GvpM. (B) Interaction of ﬁve different GvpA fragments with GvpF. (C) Sequence of the ﬁve GvpA fragments in relation to the aa sequence of GvpA shown on top. Numbers above the sequence depict the aa positions in GvpA. The helices α1 and α2 and the β-sheets β1 and β2 are in bold and marked on the bottom and also shaded in gray. FIGURE 4 \| Split-GFP interaction studies with GvpA/GvpX. Only the highest relative ﬂuorescence determined for each combination is given. Two biological and three technical replicates were performed in each case. (A) Interaction study of GvpA with the eight accessory proteins GvpF through GvpM. DISCUSSION Gas-vesicle assembly involves twelve Gvp proteins, two of which, GvpA and GvpC, form the gas vesicle wall. A third protein, GvpN, is required for the enlargement of the structure, and the function of GvpO is not yet known (Pfeifer, 2012). The accessory proteins GvpF through GvpM are encoded by an November 2020 \| Volume 11 \| Article 610179 Frontiers in Microbiology \| www.frontiersin.org 8 Völkner et al. Accessory Gvp Form an Initiation Complex FIGURE 5 \| Split-GFP studies of GvpF and variants of GvpAmut. (A) Rf-values of the GvpF/GvpA (WT) and the various F/Amut transformants. The single substitutions in GvpA are indicated below. F/Amut transformants with relative ﬂuorescence <6 are labeled in blue. The ﬂuorescence was determined in LAU/mm2, and the relative ﬂuorescence in relation to the ﬂuorescence of Hfx. volcanii wild type calculated. Two biological and three technical replicates were performed in each case. (B). Sequence of GvpA and summary of the rf-values of the various GvpA mutants. The sequence of GvpA is presented on top and the structural features α1–β1–β2–α2 shaded in grey. The substitutions in GvpA are summarized underneath; each GvpA contains only a single substitution. Rf < 6 is regarded as weak interaction, whereas rf > 11 is similar to GvpA wild type. The Vac phenotype of the respective 1A+Amut transformants is indicated by color: red, Vac negative; green, cylinder shaped; yellow, mini gas vesicles; without color, wild type gas vesicles. FIGURE 5 \| Split-GFP studies of GvpF and variants of GvpAmut. (A) Rf-values of the GvpF/GvpA (WT) and the various F/Amut transformants. The single substitutions in GvpA are indicated below. F/Amut transformants with relative ﬂuorescence <6 are labeled in blue. The ﬂuorescence was determined in LAU/mm2, and the relative ﬂuorescence in relation to the ﬂuorescence of Hfx. volcanii wild type calculated. Two biological and three technical replicates were performed in each case. (B). Sequence of GvpA and summary of the rf-values of the various GvpA mutants. The sequence of GvpA is presented on top and the structural features α1–β1–β2–α2 shaded in grey. The substitutions in GvpA are summarized underneath; each GvpA contains only a single substitution. Rf < 6 is regarded as weak interaction, whereas rf > 11 is similar to GvpA wild type. The Vac phenotype of the respective 1A+Amut transformants is indicated by color: red, Vac negative; green, cylinder shaped; yellow, mini gas vesicles; without color, wild type gas vesicles. DISCUSSION operon and produced in minor amounts in early exponential growth. All eight accessory Gvp were investigated pairwise in respect to their interaction(s) and also with the major gas vesicle structural protein, GvpA. solutions. Only the prey protein was synthesized in Hfx. volcanii (Tavlaridou et al., 2014). The larger range of interactions observed with CBDX compared to HisX might be due to the correct folding of bait and prey under in vivo conditions. It should be noted that GvpJ and GvpM, but also GvpK oligomerized in the presence of CBDI, whereas other CBDX selected only monomers and/or dimers of these accessory Gvp. The very positively charged GvpI (pK 10.75) might be able to bind and connect the negatively charged Gvp (pK 4–5). GvpI contains 23 lysine and arginine residues in the N-terminal 53 aa, and the involvement of these aa in the oligomerization of the other proteins and also in the assembly of gas vesicles should be analyzed in further detail. Frontiers in Microbiology \| www.frontiersin.org Pull-Down Experiments Imply Complex Formation of the Accessory Gvp Only clear interactions (rf > 5) are incorporated. The respective rf-values are given at the connecting lines. Rf-values > 20 are indicated by thicker lines. of the accessory Gvp and GvpA as obtained by split-GFP. Only clear interactions (rf > 5) are incorporated. The respective rf-values . Rf-values > 20 are indicated by thicker lines. GURE 6 \| Interaction network of the accessory Gvp and GvpA as obtained by split-GFP. Only clear interactions (rf > 5) are incorpo e given at the connecting lines. Rf-values > 20 are indicated by thicker lines. FIGURE 6 \| Interaction network of the accessory Gvp and GvpA as obtained by split-GFP. Only clear interactions (rf > 5) are incorporated. The respective rf-values are given at the connecting lines. Rf-values > 20 are indicated by thicker lines. known so far whether the binding of the other Gvp occurs at the same time or sequentially. Vac+ or Vac−transformants were obtained, whereas variants in gas vesicle shape (as found with many 1A+Amut transformants) were not observed, implying that GvpM is involved in an early step in gas-vesicle assembly. The interaction partners of GvpF were GvpG, GvpH, GvpI, and GvpL as deduced from the split-GFP analyses (Figures 3, 6). GvpH (19.8 kDa) and GvpI (15.8 kDa) are interesting, since both are non-essential for gas vesicle formation and appear to interact with each other; a relatively high ﬂuorescence (rf 20) was observed for GvpH/GvpI. Both proteins interacted also with GvpL and GvpF. The binding to GvpL resulted in higher rf- values (rf 11.4, GvpH and rf 13.7, GvpI) compared to the rf-values obtained with GvpF (rf 6.3 in both cases) suggesting that the preferred interaction partner of GvpH and GvpI is GvpL. It is possible that GvpH and GvpI form a heterodimer that binds only temporary to the putative complex. GvpH inﬂuences the stability of the gas vesicles, whereas a lack of the positively charged GvpI results in extremely long gas vesicles (Oﬀner et al., 2000). Previous studies also suggest that the presence of GvpH prevents the aggregation of GvpM (Tavlaridou et al., 2014). As shown by the pull-down experiments performed in this report, CBDH selected GvpM as monomer and not as dimer, whereas all other accessory Gvp selected GvpM additionally as dimer (Figure 1). In contrast, GvpI induced the oligomerization of GvpJ, GvpK, and GvpM (Figure 1). Pull-Down Experiments Imply Complex Formation of the Accessory Gvp y p Earlier pull-down experiments with HisGvp proteins indicated non-speciﬁc binding of other histidine-rich proteins (such as PitA) to the Ni-NTA matrix, and also non-speciﬁc reactions of the Gvp antisera in Western analyses (Tavlaridou et al., 2014, and unpublished observations). In contrast, pull-down assays with CBDGvp on a cellulose matrix yielded a high speciﬁcity in the selection. In most cases, single protein bands were detected in the elution fraction, but also multimers were observed, especially with GvpJ, GvpK, and GvpM. None of the Gvp proteins bound to cellulose when the CBD tag was lacking, and the putative binding partner(s) of a given Gvp protein were selected in the respective CBDX/Y Hfx. volcanii transformants. Earlier studies with HisM implied an interaction of GvpM with GvpH, GvpJ, and GvpL, but not with GvpG (Tavlaridou et al., 2014). Testing the selection of these Gvp with CBDM veriﬁed these interactions, but GvpM also interacted with GvpG. A reason for this result could be that the pull-down experiments based on CBD-tagged proteins were performed in vivo with transformants synthesizing both bait and prey, whereas the earlier analyses with HisGvp were performed in vitro with proteins isolated from E. coli and refolded in high salt The results obtained by the pairwise CBDX/Y pull-down experiments suggested that all accessory Gvp proteins interact. In almost all cases, the prey proteins were detected by Western analyses. A quantitation of the binding eﬃciency is diﬃcult, since diﬀerent antisera were used. A quantitation of the interaction was easier using the split-GFP method and measuring the ﬂuorescence of the cells in vivo. The interaction network derived from the pull-down studies suggested that all Gvp proteins attract each other and possibly form a complex. Studies with CBDM+pF- Lex transformants demonstrated that GvpM indeed selected all the Gvp proteins at once (Figure 2). It is possible that any of these proteins interacted solely with GvpM, but it is likely that they bound as a complex to the bait. GvpM is required in early steps of gas-vesicle assembly as deduced from studies with 1M+Mmut transformants harboring construct 1M complemented by GvpM variants with single aa substitutions (Tavlaridou et al., 2014). Only November 2020 \| Volume 11 \| Article 610179 Frontiers in Microbiology \| www.frontiersin.org 9 Accessory Gvp Form an Initiation Complex Völkner et al. FIGURE 6 \| Interaction network of the accessory Gvp and GvpA as obtained by split-GFP. Pull-Down Experiments Imply Complex Formation of the Accessory Gvp To uncover the exact functions of GvpH and GvpI during gas vesicle formation, both proteins should be investigated in further detail. Frontiers in Microbiology \| www.frontiersin.org Gvp Interaction Network Deduced by Split-GFP The split-GFP method is easy to apply and the interactions can be semi-quantiﬁed. Each protein pair under investigation requires the analysis of eight combinations to exclude a possible steric hindrance in the assembly of GFP (Winter et al., 2018). Not all of the transformants tested were signiﬁcantly ﬂuorescent. The calculated relative ﬂuorescence (rf) varied from rf-values < 1, regarded as no protein-protein interaction, over rf 1–5 regarded as weak, to rf > 5 regarded as clear interaction. The data was used to establish a network of the accessory Gvp including all interactions exceeding rf 5 (Figure 6). The highest number of protein contacts was observed for GvpL that was shown to interact with all other accessory Gvp. The highest rf-value of all combinations tested was found with GvpL/GvpG (rf 77.45). The 10-kDa GvpG is relatively small, similar to GvpJ, GvpK, and GvpM (9.2–12.6 kDa). The latter three proteins yielded GvpL as sole interaction partner, whereas GvpG also contacted GvpF. It is possible that the hydrophobic nature of GvpM and GvpJ and their tendency to aggregate prevented the assembly of suﬃcient ﬂuorescent GFP in some cases. GvpL (32 kDa) and GvpF (23 kDa) are structurally related as determined by homology modeling using a crystal structure of GvpF derived from the cyanobacterium Microcystis aeruginosa as template (Xu et al., 2014; Winter et al., 2018). Since GvpL attracted all other accessory Gvp, this protein might play a central role in the formation of a protein complex. However, it is not Overall, the interaction studies by split-GFP yielded less interactions compared to the pull-down experiments, especially for the hydrophobic GvpJ and GvpM, but also for GvpK. These three proteins often form multimers as observed when investigated by SDS-PAGE. Studies on self-interactions of the Gvp proteins yielded only weak interactions for F/F (rf 1.5), or G/G (rf 3.5), whereas J/J, K/K or M/M transformants were not ﬂuorescent (Supplementary Figure 7). The formation of November 2020 \| Volume 11 \| Article 610179 Frontiers in Microbiology \| www.frontiersin.org Frontiers in Microbiology \| www.frontiersin.org 10 Accessory Gvp Form an Initiation Complex Völkner et al. FIGURE 7 \| 3D-model of GvpA and putative interaction site of GvpF. The GvpA in silico model is according to Strunk et al. (2011). The secondary structural features α1, β1, β2, and α2 are labeled. GvpF Is the Only Interaction Partner of p The analysis of the major gas vesicle structural protein, GvpA, by split-GFP uncovered GvpF as sole binding partner. This result implied that a GvpA/GvpF complex is formed, but since GvpF also attracts GvpG, GvpH, GvpI, and via GvpL all other accessory Gvp, it is also possible that a larger complex of the accessory Gvp binds GvpA via GvpF. The latter possibility is more likely, since the amount of GvpF is much smaller than the amount of the major gas vesicle protein GvpA. GvpF is only produced in early growth stages (together with GvpG through GvpM), and not in parallel to the massive production of GvpA later in growth. The PA promoter for the transcription of gvpA is 10-fold induced by GvpE, whereas the PF promoter for the transcription gvpFGHIJKLM is less active and not activated by GvpE as determined using GFP as reporter (Born and Pfeifer, 2019). Thus, the GvpF/GvpA binding cannot occur on a 1:1 basis throughout growth, and should happen in early stages of gas vesicle formation. DATA AVAILABILITY STATEMENT The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author. The contact site of GvpF in GvpA was conﬁned to the ﬁrst 40 aa of GvpA, since substitutions in the second half of this 76-aa protein had no inﬂuence on the GvpA/GvpF interaction. Several single substitutions of amino acids in the N-terminal half of GvpA inﬂuenced the GvpA/GvpF interaction, and all these positions are also important for gas vesicle formation (Knitsch et al., 2017). These aa are located in α1, β1 and β2 of GvpA, and the loop regions in between. Except for D24, the charged aa R15, K19, R28, E40 are located on the surface of GvpA (Figure 7; Strunk et al., Gvp Interaction Network Deduced by Split-GFP The amino acids marked and colored in blue are positions where a substitution resulted in a low relative ﬂuorescence of the F/Amut transformants. 2011). These aa are thought to form salt bridges to adjacent GvpA molecules in the gas vesicle wall (Sivertsen et al., 2010; Knitsch et al., 2017), but as shown here they also mediate the GvpA/GvpF contact (when GvpF is available). It is possible that GvpF contacts GvpA prior to the incorporation of GvpA in the gas vesicle wall, to keep this strongly hydrophobic protein in solution (especially by shielding β1-β2) and prevent an undesirable self-aggregation. It is likely that the accessory proteins including GvpF initiate gas vesicle formation by forming a nucleation complex that binds GvpA via GvpF and forces GvpA to start aggregating into the ribs of the gas vesicle wall; this hypothesis has been raised already in Shukla and DasSarma (2004). Frontiers in Microbiology \| www.frontiersin.org CONCLUSION A complex (or complexes) of the accessory Gvp proteins might initiate the formation of this gas-ﬁlled structure. The aggregation of GvpA to form the ribs of the gas vesicle wall most likely starts at the tips of the two conical end caps of each gas vesicle (Waaland and Branton, 1969), and the small bicones formed are enlarged by further addition of GvpA, and of GvpC at the exterior side. A larger protein complex is required at the tip that binds GvpA to initiate the formation of the gas vesicle wall. The hydrophobic interior surface of ribs formed by GvpA needs to be shielded, especially when the diameter of the conical structure is still small. A complex of the accessory Gvp in both tips could protect and ensure that the ribs become distant enough to expose the hydrophobic surface of GvpA at the interior side and form a hollow structure that rapidly ﬁlls with gas. The analysis of the cyanobacterial GvpF by immunogold labeling-based tomography suggests that GvpF is localized at the gas-facing surface of the gas vesicle wall (Xu et al., 2014), supporting this hypothesis. The accessory Gvp are presumably not only required in the tip, but also at the transition of the conical caps into the cylinder structure. In addition, helper proteins might be required at the site where the incorporation of GvpA occurs to enlarge the gas vesicle wall. Presumably this site is located in the center of the gas vesicle, and GvpN might be involved here. All these steps in gas-vesicle assembly are not yet understood, but the accessory Gvp proteins and their complex(es) certainly take part in these processes. Further studies will aim to identify the complex(es) formed by the accessory proteins and determine the location in the gas vesicles structure. FIGURE 7 \| 3D-model of GvpA and putative interaction site of GvpF. The GvpA in silico model is according to Strunk et al. (2011). The secondary structural features α1, β1, β2, and α2 are labeled. The amino acids marked and colored in blue are positions where a substitution resulted in a low relative ﬂuorescence of the F/Amut transformants. additional multimers might prevent the formation of ﬂuorescent GFP. These results implied that self-interaction of aggregating proteins is diﬃcult to detect by split-GFP. REFERENCES doi: 10.1016/S0723-2020(11)80327-9 Ghosh, I., Hamilton, A. D., and Regan, L. (2000). 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Transformation of halophilic archaea and investigation of gas vesicle synthesis. Syst. Appl. Microbiol. 16, 569–577. ACKNOWLEDGMENTS The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2020.610179/full#supplementary-material We thank Jerry Eichler (Ben-Gurion University, Israel) providing the vector encoding the cellulose binding domain, CBD. FUNDING The students Gayathiri Thavayogarajah, Regine Knitsch, and Sergio Leftheriotis (TU Darmstadt) are thanked to supply some of the vectors used in this study. Valuable discussions with Johannes Born and Arnulf Kletzin (TU Darmstadt) are gratefully acknowledged. This work was ﬁnancially supported by the German Research Foundation, DFG (PF 165/15–1). We also acknowledged support This work was ﬁnancially supported by the German Research Foundation, DFG (PF 165/15–1). 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. Frontiers in Microbiology \| www.frontiersin.org November 2020 \| Volume 11 \| Article 610179 REFERENCES No use, distribution or reproduction is permitted which does not comply with these terms. Xu, B. Y., Dai, Y. N., Zhou, K., Liu, Y. T., Sun, Q., Ren, Y. M., et al. (2014). Structure of the gas vesicle protein GvpF from the cyanobacterium Microcystis aeruginosa. Acta Crystallogr. D Biol. Crystallogr. 70, 3013–3022. doi: 10.1107/ S1399004714021312 November 2020 \| Volume 11 \| Article 610179 Frontiers in Microbiology \| www.frontiersin.org 13
https://openalex.org/W2960712532	https://bmcvetres.biomedcentral.com/track/pdf/10.1186/s12917-019-1982-x	English	null	Investigation of chronic and persistent classical swine fever infections under field conditions and their impact on vaccine efficacy	BMC veterinary research	2,019	cc-by	9,331	(2019) 15:247 (2019) 15:247 Coronado et al. BMC Veterinary Research (201 https://doi.org/10.1186/s12917-019-1982-x Open Access Investigation of chronic and persistent classical swine fever infections under field conditions and their impact on vaccine efficacy Liani Coronado1, Jose Alejandro Bohórquez2, Sara Muñoz-González2, Lester Josue Perez3, Rosa Rosell2,4, Osvaldo Fonseca1, Laiyen Delgado1, Carmen Laura Perera1, Maria Teresa Frías1 and Llilianne Ganges2* * Correspondence: llilianne.ganges@irta.cat * Correspondence: llilianne.ganges@irta.cat 2OIE Reference Laboratory for Classical Swine Fever, IRTA-CReSA, Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain Full list of author information is available at the end of the article * Correspondence: llilianne.ganges@irta.cat 2OIE Reference Laboratory for Classical Swine Fever, IRTA-CReSA, Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Recent studies have hypothesized that circulation of classical swine fever virus (CSFV) variants when the immunity induced by the vaccine is not sterilizing might favour viral persistence. Likewise, in addition to congenital viral persistence, CSFV has also been proven to generate postnatal viral persistence. Under experimental conditions, postnatal persistently infected pigs were unable to elicit a specific immune response to a CSFV live attenuated vaccine via the mechanism known as superinfection exclusion (SIE). Here, we study whether subclinical forms of classical swine fever (CSF) may be present in a conventional farm in an endemic country and evaluate vaccine efficacy under these types of infections in field conditions. Results: Six litters born from CSF-vaccinated gilts were randomly chosen from a commercial Cuban farm at 33 days of age (weaning). At this time, the piglets were vaccinated with a lapinized live attenuated CSFV C-strain vaccine. Virological and immunological analyses were performed before and after vaccination. The piglets were clinically healthy at weaning; however, 82% were viraemic, and the rectal swabs in most of the remaining 18% were positive. Only five piglets from one litter showed a specific antibody response. The tonsils and rectal swabs of five sows were CSFV positive, and only one of the sows showed an antibody response. After vaccination, 98% of the piglets were unable to clear the virus and to seroconvert, and some of the piglets showed polyarthritis and wasting after 36 days post vaccination. The CSFV E2 glycoprotein sequences recovered from one pig per litter were the same. The amino acid positions 72(R), 20(L) and 195(N) of E2 were identified in silico as positions associated with adaptive advantage. Conclusions: Circulation of chronic and persistent CSF infections was demonstrated in field conditions under a vaccination programme. Persistent infection was predominant. Here, we provide evidence that, in field conditions, subclinical infections are not detected by clinical diagnosis and, despite being infected with CSFV, the animals are vaccinated, rather than diagnosed and eliminated. These animals are refractory to vaccination, likely due to the SIE phenomenon. Improvement of vaccination strategies and diagnosis of subclinical forms of CSF is imperative for CSF eradication. Keywords: CSFV, Chronic infection, Persistent infection, Vaccination failures, Viral evolution Background [11, 15, 17, 18]. Long-term endemism of CSF is charac- terized by the circulation of moderate- and low- virulence strains [1, 11]. Therefore, laboratory support for accurate CSFV diagnosis is fundamental for efficient disease control. Unfortunately, not all endemic countries have the necessary resources to achieve this goal, and only clinical diagnosis is performed. Hence, the lack of laboratory diagnostics may also explain the complex epidemiological scenario in some endemic countries. g Classical swine fever (CSF) is one of the most devastat- ing diseases in the pig industry worldwide, affecting domestic pigs and wild boars. This disease is endemic in Asia, Central and South America, and some Eastern Euro- pean countries [1]. CSF virus (CSFV), the aetiological agent, belongs to the Pestivirus genus of the Flaviviridae family [2]. Three degrees of virulence (low, medium and high) have been described for CSFV strains, which have also been linked to different clinical manifestations of the disease [3]. Pigs infected with low-virulence strains may develop chronic CSF and can shed the virus continuously or intermittently for months, representing a constant source of reinfection [3]. Additionally, the role of low- virulence strains in “pregnant carrier sow syndrome”, which can lead to congenital infection of the foetus by trans-placental transmission, has been known for more than 50 years [4–7]. In addition to the persistent viral infection generated after trans-placental transmission, previous works have reported that CSFV is also able to generate persistent postnatal infection in both domestic pigs and wild boars [19, 20]. Subsequently, it was shown that pigs with per- sistent postnatal infection were unable to elicit a specific immune response to a CSFV live attenuated vaccine, with viral vaccine RNA undetectable in these pigs after vaccination via the superinfection exclusion (SIE) mech- anism [21, 22]. These studies were performed under ex- perimental conditions, with one of the few scientific reports showing the possible negative impact that persistently in- fected animals may have on vaccination programmes, which could complicate CSF control in endemic countries. Modified live attenuated vaccines based on the C- strain have played a relevant role in the implementation of eradication programmes for CSF [8–12]. These vaccines present several advantages that facilitate their use in developing countries, inducing high titres of neu- tralizing antibodies that provide protection against highly virulent CSFV strains [9, 13]. © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Coronado et al. BMC Veterinary Research (2019) 15:247 Coronado et al. BMC Veterinary Research (2019) 15:247 Page 2 of 13 Page 2 of 13 Background Moreover, this type of vaccine confers protection against CSFV even in the absence of neutralizing antibodies a few days post vac- cination (dpv) [8, 9, 13, 14]. In addition to horizontal protection, modified live attenuated vaccines based on the C-strain also induce protection in pregnant sows as early as 5 dpv, preventing viral trans-placental transmis- sion [8, 9]. Although the immune response induced by this type of vaccine does not allow differentiation of in- fected animals from vaccinated animals (the DIVA con- cept) [8, 9, 12], this vaccine continues to be used in endemic countries (a majority of which are developing countries). The reasons underlying the worldwide distri- bution of this vaccine are its high efficacy and safety as well as its relatively low cost of production [10]. Given this background, the present study aimed to determine whether subclinical forms of CSF, which may be misdiagnosed in the field, are present at the time of vaccination in a conventional farm from an endemic country. Likewise, this study aimed to evaluate vaccine efficacy for these types of infections in field conditions, an important aspect that is poorly understood to date. CSFV detection and antibody response in piglets at weaning g At the day of weaning (33 days after birth), the rectal swabs of all piglets in four of the six litters were CSFV RNA positive (litters B, C, D and F) and showed Ct values corresponding to high (Ct value less than 23) and moderate (Ct values between 23 and 28) viral RNA loads (Table 1). In the other two litters, only two out of nine animals were CSFV RNA positive with high RNA viral loads in rectal swabs in litter A, whereas in litter E, eight out of eleven were CSFV RNA positive with high and moderate RNA viral load in rectal swabs (Table 1). Like- wise, the results obtained for sera were similar to those obtained for rectal swabs. All serum samples from litters B, C, D and F were CSFV RNA positive, showing Ct values corresponding with high viral RNA loads (Ct value less than 23) in 38 out of 42 piglets (Table 1). Different profiles were observed in the two remaining litters. In litter E, five out of eleven piglets were CSFV RNA positive, with two animals exhibiting low viral RNA loads, one with a moderate RNA load and the other two with high viral RNA loads (Table 1). In litter A, the serum samples of four out of nine animals were positive (44.4%), three with Ct values corresponding to low RNA loads and one with a moderate viral RNA load. In general, the subclinical infection detected in the piglets from the present study at weaning included 82% animals that were viraemic. Among the remaining 18%, most of the piglets, although not viraemic, were positive in rectal swabs. CSFV detection in piglets after vaccination The presence of CSFV RNA in sera, rectal swabs and tonsils was monitored by qRT-PCR at different times post vaccination. After 21 dpv, 98% of the piglets from the six litters were unable to clear the virus, with a ma- jority of the piglets exhibiting high and moderate viral RNA loads in sera and rectal swabs (Tables 3 and 4). At 21 dpv, serum samples from the six litters that were positive by qRT-PCR with Ct values below 28 were veri- fied by a viral isolation test in PK-15 cells. CSFV detection and antibody response in piglets at weaning In terms of humoral response, only five out of eleven piglets from litter B showed a specific antibody response to E2 glycoprotein, as detected by ELISA, whereas the remaining piglets from the study were negative. Regard- ing the ELISA-positive samples, the antibody response levels measured as blocking percentage values were be- tween 42 and 47% (Table 1). These results were con- firmed by the NPLA, resulting in low neutralizing antibody titres of 1:25 in the five sera. y Notably, piglets from litters B, C and F showed the high- est viral RNA loads during the study, with strong viral RNA signals observed in sera, rectal swabs and tonsils (Tables 3, 4 and Fig. 1), and showed constant and similar Ct values during the study. In the remaining litters (A, D and E), the viral RNA load ranged from low to moderate and was undetected in some cases. In these litters, rectal swab samples allowed the detection of a greater number of positive animals than serum samples (Tables 3, 4 and Fig. 1). In particular, a majority of the pigs (seven out of nine) in litter A tested negative for viral RNA in serum samples at 21 dpv. After this time, at 33, 66 and 96 dpv, positive samples were detected intermittently, although with low viral RNA loads. Nevertheless, during the period from 21 to 96 dpv, the rectal swabs from all animals were positive at some point (Tables 3 and 4). Finally, in pigs from litter D, two different profiles were detected; three of these pigs developed some antibody response to the virus, as two animals were able to clear the virus in sera and rec- tal swabs from 33 to 96 dpv. In the remainder of the pigs, constant viraemia was observed during the study until 96 CSFV detection and antibody response in sows In the remaining litters, there were no obvious clinical signs; two cases of sudden death occurred in litters A and D, while cases of animals with polyarthritis were recorded in litters B and C. A mortality rate of 8% was obtained between 21 and 96 dpv, with a 14% mortality observed during the entire study. Additionally, the mor- tality for each litter was also determined (Table 2). Inter- estingly, in litters B, C and F, no anatomopathological lesions in internal organs were observed after necropsy. Finally, the three litters that remained until 96 dpv showed growth retardation from approximately 36 dpv until the end of the study (litters A, D and F), with clear signs of apathy, anorexia, diarrhoea, dyspnoea and con- junctivitis observed at that time in some animals. The largest accumulation of anatomopathological lesions of haemorrhages in tonsils and kidneys, enteritis and even development of interstitial pneumonia were observed in litters A and D, which remained in the study until 96 dpv; however, the mortality percentage in both litters was 22.2% (Table 2). bladder and stomach; marginal infarcts of the spleen; haemorrhages; and necrosis in the tonsils (Table 2). In the remaining litters, there were no obvious clinical signs; two cases of sudden death occurred in litters A and D, while cases of animals with polyarthritis were recorded in litters B and C. A mortality rate of 8% was obtained between 21 and 96 dpv, with a 14% mortality observed during the entire study. Additionally, the mor- tality for each litter was also determined (Table 2). Inter- estingly, in litters B, C and F, no anatomopathological lesions in internal organs were observed after necropsy. Finally, the three litters that remained until 96 dpv showed growth retardation from approximately 36 dpv until the end of the study (litters A, D and F), with clear signs of apathy, anorexia, diarrhoea, dyspnoea and con- junctivitis observed at that time in some animals. The largest accumulation of anatomopathological lesions of haemorrhages in tonsils and kidneys, enteritis and even development of interstitial pneumonia were observed in litters A and D, which remained in the study until 96 dpv; however, the mortality percentage in both litters was 22.2% (Table 2). CSFV detection and antibody response in sows CSFV detection and antibody response in sows Despite being vaccinated, the rectal swabs of five of the sows were CSFV RNA positive (from B to F), with Ct values corresponding to high (litter C) to moderate (litters B and D) and low (litters E and F) viral RNA loads. In contrast, the sow from litter A was negative. The results obtained from tonsil samples corresponded with those from rectal swabs, i.e., there were five positive sows (from B to F) and one negative sow (sow A). The sow from litter F showed a Ct value less than 28, and the others showed values greater than 29. Notably, although the rectal swabs of five sows were positive, the sera of only two of these sows were positive, showing low viral RNA loads (sows B and C had Ct values of 30 and 32, respectively). It has been previously discussed that CSF has not been controlled despite long-term vaccination programmes with live attenuated C-strain vaccines conducted in endemic areas [11, 15, 16]. Factors associated with vari- ability, quality, and stability of the vaccine batches pro- duced; loss of the cold chain; and lack of vaccination coverage, mainly in rural areas with poor accessibility and backyard farms, among others, have been used to explain this discrepancy [9, 11, 15, 16]. In terms of humoral response, notably, only the sow from litter E showed an antibody response (55.3% block- ing) in the ELISA test. With the exception of the animals from the six litters under study, all the animals from the farm were slaughtered once the samples collected at weaning were found to be CSFV RNA positive, and strict biosecurity measures were applied in the farm. Recent studies have demonstrated evolutionary cap- acity of CSFV in endemic conditions under inefficient vaccination programmes. In this regard, it has been hypothesized that circulation of new viral variants when the immunity induced by the vaccine is not sterilizing might favour viral persistence in the swine population Page 3 of 13 Page 3 of 13 Coronado et al. BMC Veterinary Research (2019) 15:247 Coronado et al. BMC Veterinary Research (2019) 15:247 bladder and stomach; marginal infarcts of the spleen; haemorrhages; and necrosis in the tonsils (Table 2). Table 1 Ct values from rectal swabs and sera obtained by CSFV-specific qRT-PCR and CSFV-specific antibodies at weaning Table 1 Ct values from rectal swabs and sera obtained by CSFV-specific qRT-PCR and CSFV-specific antibodies at weaning Table 1 Ct values from rectal swabs and sera obtained by CSFV-specific qRT-PCR and CSFV-specific antibodies at weaning The antibody test result against the E2 glycoprotein was considered positive when the blocking % was more than 40% (in bold). Values between 30 and 40% were considered doubtful (in black), and values less than 30% were considered negative (in grey) bThe antibody test result against the E2 glycoprotein was considered positive when the blocking % was more than 40% (in bold). Values between 30 and 40% were considered doubtful (in black), and values less than 30% were considered negative (in grey) bThe antibody test result against the E2 glycoprotein was considered positive when the blocking % was more than 40% (in bold). Va were considered doubtful (in black), and values less than 30% were considered negative (in grey) positive, although with low viral RNA loads. Finally, the tonsils of all the animals under study were positive at either 21 dpv or 96 dpv (Fig. 1). dpv with a high viral RNA load. Furthermore, the tonsils of the animals from this litter were also positive, with a low viral RNA load observed in the two animals that cleared the virus in sera. In contrast, animals that showed persistent viremia exhibited moderate and high viral RNA loads (Tables 3, 4 and Fig. 1). Additionally, despite the intermittent viral RNA detection during the study in litter A (Tables 3 and 4), the tonsils of all the animals were Antibody response profile detected after vaccination Notably, none of the animals in litters B, C and F showed an antibody response detected by ELISA at 21 dpv (litters B and C) or 96 dpv (litter F). Among the Clinical manifestations registered at weaning and after vaccination Notably, at weaning, all the animals were apparently clinically healthy (Table 2), exhibiting a weight gain simi- lar to the normal weight gain observed in Cuba in this type of commercial farm (data not shown); likewise, the absence of clinical symptoms was correlated with the absence of mortality at weaning. At 21 dpv, clinical signs were registered in only litter E, such as anorexia, conjunctivitis, weakness of hindquarters, dyspnoea, diar- rhoea, incoordination of movements and nervous disor- ders; three animals died suddenly, and the remaining animals were euthanized at 21 dpv. Lesions found after necropsy included petechial haemorrhages in the kidney, Coronado et al. BMC Veterinary Research (2019) 15:247 Page 4 of 13 Discussion In the present work, the coexistence of two forms of subclinical CSF was demonstrated: (i) chronic CSF and (ii) persistent CSF, likely transmitted congenitally from sows to their litters. Nevertheless, a postnatal persistent infection in some of these animals cannot be ruled out considering the ability of CSFV to generate this form of infection soon after birth [20]. Although both forms of infection can be considered as chronic CSF manifesta- tions, since both exceed 28 days of infection [1, 24], the knowledge and distinction of the pathogenesis of both types of infections is relevant for accurate diagnosis and control in endemic areas. One of the main differences Antibody response profile detected after vaccination Coronado et al. BMC Veterinary Research (2019) 15:247 Page 5 of 13 Table 2 CSFV-positive animals at weaning and clinical signs, mortality rates and pathological findings in the litters under study E2 gene of Pinar del Rio/2016 (LT985811), as well as all the available full-length E2 gene sequences from Cuban CSFV strains, were analysed as described in Methods sec- tion. By in silico analysis, the amino acid at position 72 (R) on the E2 glycoprotein was identified under positive selec- tion pressure for the strain LT985811 with a 0.01 level of significance (2LnL = 12.6 M2vsM1 and 2LnL = 36.8 M8vsM7). Likewise, sites 20 (L) and 195 (N) were also identified in silico as positions with adaptive advantage in the above-mentioned virus with a 0.01 significance level (θLTR = 28.85684). Other mutations without statistical significance were detected in the CSFV strain from the present study, namely, 3AxV and 7DxG in the external loop of the B/C domain, 91IxV in the A/D domain and 210DxN and 228AxV in the transmembrane domain (Additional file 1: Figure S1). remaining three litters, in litter A, five out of nine piglets were able to seroconvert at 21 dpv (Table 4 and Fig. 2). Furthermore, between 33 and 96 dpv, intermittent detec- tion of antibodies in animals from this litter was regis- tered. In terms of neutralizing antibody titres, only four pigs were capable of clearing the virus in sera, as determined by the NPLA at 96 dpv, with neutralizing antibody titres from 1:100 to 1:400 (Fig. 3). In the case of litter D, only three pigs showed CSFV- specific antibodies to E2 glycoprotein and neutralizing antibody titres at 21 dpv (Table 4 and Fig. 3). One of these pigs died (pig number 32) despite being the animal with the highest level of neutralizing antibodies gener- ated against infection. The other two antibody-positive pigs showed neutralizing antibody titres ranging from 1: 200 to 1:400 at 96 dpv (Fig. 3). Finally, in litter E, five pigs showed CSFV-specific anti- body responses, as measured by ELISA and the NPLA (Figs. 2 and 3). This group generated the highest neutral- izing antibody response at 21 dpv, with titres ranging from 1:400 to 1:800 (Fig. 3). ath or euthanasia, there is no sample. bThe antibody test result against the E2 glycoprotein was considered positive when the blockin bold). Values between 30 and 40% were considered inconclusive (in black), and values less than 30% were considered negative (in gre E2 sequence determination for each litter and sequence analysis The complete E2 sequences from tonsil samples of one animal from each litter studied were determined. Notably, the six consensus sequences obtained were identical and were deposited in the European Nucleotide Archive under the accession number LT985811. This viral strain was iso- lated and designated Pinar del Rio/2016. The full-length Coronado et al. BMC Veterinary Research (2019) 15:247 Page 6 of 13 Ct values in rectal swabs obtained by specific qRT-PCR at different times post vaccination Table 3 Ct values in rectal swabs obtained by specific qRT-PCR at different times post vaccination Coronado et al. BMC Veterinary Research (2019) 15:247 Page 7 of 13 Table 4 CSFV RNA detection in sera and CSFV-specific antibodies after vaccination aDeath or euthanasia, there is no sample. bThe antibody test result against the E2 glycoprotein was considered positive when the blocking % was more than 40% (in bold). Values between 30 and 40% were considered inconclusive (in black), and values less than 30% were considered negative (in grey) Table 4 CSFV RNA detection in sera and CSFV-specific antibodies after vaccination Coronado et al. BMC Veterinary Research (2019) 15:247 Page 8 of 13 Fig. 1 CSFV RNA detection in tonsil samples from piglets. CSFV viral RNA was detected using qRT-PCR [23] in litters either at 21 dpv (grey dots) or 96 dpv (white dots). Viral RNA load is expressed as the Ct value; the mean Ct value for each litter is also indicated (black triangle). Ct values equal to or less than 42 were considered to indicate positive results Fig. 1 CSFV RNA detection in tonsil samples from piglets. CSFV viral RNA was detected using qRT-PCR [23] in litters either at 21 dpv (grey dots) or 96 dpv (white dots). Viral RNA load is expressed as the Ct value; the mean Ct value for each litter is also indicated (black triangle). Ct values equal to or less than 42 were considered to indicate positive results Fig. 1 CSFV RNA detection in tonsil samples from piglets. CSFV viral RNA was detected using qRT-PCR [23] in litters either at 21 dpv (grey dots) or 96 dpv (white dots). Viral RNA load is expressed as the Ct value; the mean Ct value for each litter is also indicated (black triangle). E2 sequence determination for each litter and sequence analysis † indicates animals that died between 21 and 96 dpv Fig. 3 Neutralizing antibody response in piglets. Neutralizing antibody titres were detected by NPLA at 21 dpv (grey bars) and 96 dpv (white bars). Only the litters in which neutralizing antibody titres were detected are shown. * indicates animals that died before 21 dpv. † indicates animals that died between 21 and 96 dpv animals with persistent CSFV infection [20, 22]. This phenomenon explains the immunological anergy in pigs with persistent CSFV infection, blocking the immuno- logical mechanisms of the host to fight the infection [20, 26]. Notably, CSFV persistent form was the pre- dominant infection detected in the animals from the present study, including those from litters B, C and F and some piglets from litter D. animals showed permanent viremia until the end of the study (96 dpv; more than 4 months of infection), exhibit- ing the inability to seroconvert. Likewise, given that the animals in our study belonged to a commercial farm, dif- ferent aetiological agents could be associated with the clinical signs registered in some of these animals, which supports the importance of identification of concomitant infections that may be related, as described above; these infections will be identified in future studies. Although other porcine pathogens were not diagnosed in this study, previous work has shown that, in Cuba, CSFV is circulating in concomitance with other viral porcine pathogens, such as porcine circovirus 2 (PCV-2) and porcine parvovirus (PPV) [27]. However, consider- ing the high relevance, future studies will be carried out to understand the implications of other porcine patho- gens involved in coinfection with CSFV circulating in endemic scenarios. Unfortunately, currently, the re- sources have been used to detect the presence of sub- clinical forms of CSF, given the high impact of CSFV on the porcine immune system and given that CSF is con- sidered a notifiable disease by the World Organisation for Animal Health (OIE). Previous work has shown detection of a low RNA load of the lapinized C-strain vaccine by specific qRT-PCR [10] in serum and rectal swab samples soon after vaccin- ation in Pestivirus-free vaccinated pigs [21]. E2 sequence determination for each litter and sequence analysis Ct values equal to or less than 42 were considered to indicate positive results tonsils were considered to be chronically infected, such as piglets from litters A, D and E. between pigs with persistent CSFV infection and pigs suffering the chronic form is that, in chronic infec- tion, a specific immune response against CSFV can be generated, although inefficiently, and in some cases, the antibody response can mask the detection of the virus in sera [20, 25]. Considering this fact, even though antibodies were intermittently detected in some animals, the non-viraemic animals detected in this study that exhibited positive rectal swabs and In contrast, persistently infected animals have been previously defined as CSF-infected pigs that show per- manent viremia with high viral titres over long periods of time and are unable to generate a specific immune re- sponse to the virus, mainly in terms of the humoral im- mune response [20, 25]. Recently, a type I and II interferon response blockade has been demonstrated in Fig. 2 CSFV RNA detection in serum samples at weaning and 21 dpv versus antibody response after vaccination. CSFV RNA loads in sera expressed as Ct values (left Y axis) at weaning (white dots) and 21 dpv (white rhombuses). Ct values equal to or less than 42 were considered to indicate positive results (black line). Antibody response against the E2 glycoprotein detected by a commercial ELISA test (IDEXX) expressed as blocking % (right Y axis); samples with blocking % values more than 40 were considered positive (dotted line) Fig. 2 CSFV RNA detection in serum samples at weaning and 21 dpv versus antibody response after vaccination. CSFV RNA loads in sera expressed as Ct values (left Y axis) at weaning (white dots) and 21 dpv (white rhombuses). Ct values equal to or less than 42 were considered to indicate positive results (black line). Antibody response against the E2 glycoprotein detected by a commercial ELISA test (IDEXX) expressed as blocking % (right Y axis); samples with blocking % values more than 40 were considered positive (dotted line) Coronado et al. BMC Veterinary Research (2019) 15:247 Page 9 of 13 Fig. 3 Neutralizing antibody response in piglets. Neutralizing antibody titres were detected by NPLA at 21 dpv (grey bars) and 96 dpv (white bars). Only the litters in which neutralizing antibody titres were detected are shown. * indicates animals that died before 21 dpv. E2 sequence determination for each litter and sequence analysis Based on the levels of CSFV RNA loads detected in the rectal swabs and tonsils from vaccinated sows, as well as the inconsistent humoral response observed after vaccin- ation in these animals, we determined that five of these animals were asymptomatic CSFV carriers (sows from B to F). Only one vaccinated sow (sow E) was antibody positive at weaning, although at very low levels. The re- sults of the present study indicate that antibody response detection and CSFV diagnosis in serum samples from subclinically infected CSFV animals may be hindered by the coexistence of some antibodies and CSFV [20]. Therefore, in addition to detecting the virus in tonsils or tonsil scrapings, these results indicate the effectiveness of using samples such as rectal swabs for the detection of subclinical forms of CSF. Previous results showed that chronic forms of CSF can be fatal, with clinical manifestations similar to those described in the present study, as was the case for pig- lets from litter E [1, 24] (Table 2). This effect is probably due to the immunosuppression caused by persistent CSFV infections [20]. Previous work performed under laboratory conditions has indicated that pigs that exhibit CSFV permanent viremia for more than 30 days post in- fection may develop a similar clinical condition [20, 21]. Our data also show the clinical evolution over time of animals with persistent CSFV infection. In these ani- mals, signs of growth retardation between 21 and 36 dpv were observed. It was not until after 66 dpv (approxi- mately 3 months) that, in a sporadic manner, some of the animals developed severe symptoms, as previously described for the chronic form of CSF [24]. However, The failure in the response to vaccination in the sows was suggested by the lack of maternal immune response to protect piglets from either prenatal or postnatal infec- tions. Factors associated with poor handling and malpractice in vaccination have been widely discussed to explain the constant failures in vaccination in endemic areas [11, 15, 16]. Thus, these results prove how vaccine failure in the field may allow viral persistence in an en- demic scenario. Improvement in vaccine quality, as well as optimization of vaccination dose and schedule, in gilts Coronado et al. E2 sequence determination for each litter and sequence analysis BMC Veterinary Research (2019) 15:247 Page 10 of 13 Page 10 of 13 infections in animals are not detected by clinical diagno- sis and, despite being infected with CSFV, the animals are vaccinated, rather than diagnosed and eliminated. Animals with persistent and chronic CSFV infections are refractory to vaccination, likely due to the SIE phenomenon. Hence, CSFV eradication in some en- demic areas will require the elimination of persistently and chronically infected animals by relying heavily on laboratory tools for effective diagnosis. Likewise, CSF control programmes may include a vaccination strategy that confers sterilizing immunity to avoid the generation of subclinical infections in the field. Therefore, govern- ment support and financial aid will be key factors to ensure the institution of policies for effective control and eradication of this disease globally. and sows may aid the prevention of CSFV carrier gener- ation [28]. Nevertheless, the reason underlying the lack of effective maternal immunity in sows after vaccination must be studied in depth. Recently, SIE or homologous interference has been de- scribed as a mechanism that can interfere with CSFV vaccine efficacy in pigs with persistent postnatal CSFV infection under experimental conditions [21, 22]. Here, we show that this mechanism may also occur in animals suffering from subclinical CSF, either chronic or persist- ent, remaining unnoticed at the time of vaccination in field conditions. Pigs with persistent infection from the present study were unable to seroconvert at any time during more than 4 months under study. Likewise, vac- cination was also ineffective in litters suffering from a chronic form of CSF. Therefore, vaccination against sub- clinical forms of CSFV is counterproductive and gener- ates additional expenses for control programmes. Thus, along with improvement in the vaccination strategy, ef- forts must also be made in the diagnosis of these CSF disease forms. Experimental design This study was performed in a recently repopulated Cuban commercial pig farm located in the western region. Six randomly chosen sows and their respective litters (designated A to F) were included. The first-parity sows had been vaccinated with one dose of the lapinized CSFV C-strain vaccine (Labiofam strain) at weaning and again at 6 months before delivery. The experiment was initiated at weaning (33 days after birth), at which time, vaccination of piglets from the farm was carried out. At this time, piglets were identified individually (1 to 62), and the distribution among litters was as follows: litter A (piglets 1 to 9); B (10 to 20); C (21 to 30); D (31 to 39); E (40 to 50); F (51 to 62). After identification, sera and rectal swabs were collected from the litters and the corresponding sows. The sows were euthanized, and tonsil samples were collected. Subsequently, the piglets Cells and viruses The porcine kidney cell line PK-15 (ATCC CCL 33, U.S.A.) was cultured in pestivirus-free Dulbecco’s modi- fied Eagle medium (DMEM; Lonza, Switzerland) supple- mented with 5% foetal bovine serum (FBS) (Euroclone, Italy) at 37 °C in 5% CO2. The cell monolayer was incu- bated with 100 μl of serum. Following 1 h of adsorption, the serum was removed, and the cells were cultured in DMEM supplemented with 2% FBS. After 48 h of incu- bation, a peroxidase-linked assay (PLA) [31] was used for viral identification. With respect to the nature of the circulating viral strain, notably, the consensus sequence identified in the full-length E2 gene of the virus recovered from the six litters was the same regardless of the different CSF forms recorded. Therefore, our results corroborate, in a field infection, previous results showing CSFV stability [11, 29]. The CSFV Pinar del Rio strain isolated in 2010 (Pinar del Rio/2010) was phylogenetically the closest relative to the Pinar del Rio/2016 strain recovered in the present study. Previous studies have determined that the full-length E2 sequence is the most reliable sequence for phylogenetic analysis of CSFV [17, 30]. Position 72 (R) on the E2 protein, previously described in Pinar del Rio/ 2010 as a positive selection site for Cuban strains [15], was also identified in the Pinar del Rio/2016 isolate. This finding may suggest the relevance of this codon in the evolution of the CSFV 1.4 genotype in the Cuban endemic scenario. Likewise, codon 20 (L), previously described as a position associated with adaptive advan- tage in the CSFV Pinar del Rio/2010 strain [17], was also found in the strain from the present study. Moreover, codon 195 (N), present in only the CSFV Pinar del Rio/ 2016 strain, was also identified in silico as a position associated with adaptive advantage. Further studies will clarify the implications of these findings in the evolution and pathogenesis of CSFV. A lapinized live attenuated C-strain-based vaccine (Labiofam strain) belonging to subgenotype 1.1 (batch number 16001) and used in Cuba since 1965 for vaccin- ation against CSF [15, 32] was employed in this study. This vaccine batch had been previously tested in 33-day- old naïve pigs, and all the pigs showed a specific CSFV antibody response at 21 dpv, as measured by ELISA (IDEXX, Netherlands). Detection of CSFV RNA and virus isolation RNA was extracted from all of the samples using the RNeasy Mini Kit (Qiagen, Netherlands) according to the manufacturer’s instructions. The presence of CSFV RNA in sera, rectal swabs and tonsil samples was analysed by qRT-PCR [23]. Threshold cycle values (Ct) equal to or less than 42 were considered to indicate positive results. Samples in which fluorescence was undetectable were considered negative. Ct values below 23 were considered high, values from 23 to 28 were considered moderate, and values from 29 to 42 were considered low RNA viral loads [35]. Serum samples that were positive by qRT- PCR at 21 dpv were tested by virus isolation in PK15 cells, as described above. Additional file 1: Figure S1. Representation of the mutation pattern for Cuban CSFV strains on the 3D structure of the predicted model of the E2 protein of CSFV. The 3D structure and surface of the Cuban ancestral strain “Margarita/1958” [AJ704817], the previously described “Pinar del Rio/2010” [KX576461] strain [11] and the new strain “Pinar del Rio/ 2016”[LT985811] are presented. The in silico mutations associated with positive selection (red), associated with adaptation (blue) and not linked previously with any evolutionary advantage (yellow) are denoted. The antigenic region B/C (gold), antigenic region A/D (ruby red) and transmembrane domain (grey) are located on monomer B. (JPG 94 kb) Full-length E2 encoding sequence determination Full-length E2 encoding sequence determination The sequence of the full-length E2 region was deter- mined from tonsils obtained from one piglet of each litter as previously described [30]. The one-step RT-PCR protocol was performed using the commercially available One-Step RT-PCR Kit (Qiagen, Netherlands). The se- quencing reactions were conducted under the BigDye™ terminator-cycling conditions (Thermo Fisher Scientific, U.S.A.) using an ABI 3130 × L instrument. Forward and reverse sequences obtained from each amplicon were assembled using the Contig Express application in Vector NTI software, version 11 (Invitrogen, U.S.A.). All pigs were handled according to the rules described in the manual of good practices for porcine production from the Cuban Republic [33]. International standards for animal welfare were used following the regulations of the Animal Health Directory (DSA). The Ethics Com- mittee of the Cuban Ministry of Agriculture (MINAGRI) approved the study, and all efforts were made to minimize animal suffering. Animals were euthanized by bleeding by inserting an appropriately sized knife into major artery following stunning, according to the current protocol described in the OIE Terrestrial Animal Health Code [34]. Sequence analysis Multiple sequence alignment was conducted for all avail- able full-length E2 gene sequences from Cuban strains (CSFV/Margarita/1958 [JX028201, AJ704817], CSFV/ Santiago de Cuba/2011 [JX028203], CSFV/Holguin/ 2009[JX028202], CSFV/Pinar del Rio/2010 [JX028204, KX576461], and the new sequences obtained in the present study). Multiple sequence alignment was per- formed using the algorithm Clustal W (EMBL-EBI, United Kingdom) included in the program BioEdit Se- quence Alignment Editor [37]. Positive selection analysis from the complete E2 gene was carried out using several models (M0, M1, M2, M7 and M8) available in the CODEML module of the PAML 4.7 software package [38]. Finally, functional divergence among the Cuban se- quences was assessed by estimation of the coefficients of type-I functional divergence (θI) using DIVERGE 3.0 software [39]. The 3D structure and surface of the E2 protein was constructed as previously described [17] for the Cuban ancestral strain “Margarita/1958” [AJ704817] [11], the previously described “Pinar del Rio/2010” [KX576461] strain [11] and the new strain obtained in the present study. According to the rules for the CSF control programme in the country, in case of positive CSFV detection, only the animals under study were to be maintained following strict biosecurity measures, and the remaining animals from the farm were to be eliminated. Conclusion The presence of chronic and persistent CSF infections was demonstrated in an endemic scenario under vaccin- ation. The CSF persistent infection was the predominant form in the animals studied. Here, we provide one of the few reports showing that, in field conditions, subclinical Page 11 of 13 Coronado et al. BMC Veterinary Research (2019) 15:247 Page 11 of 13 Coronado et al. BMC Veterinary Research (2019) 15:247 were vaccinated using a pig dose, equivalent to 100 pro- tective doses (PDs), of a lapinized live attenuated C- strain-based vaccine (Labiofam strain) by intramuscular injection in the neck. At 21 dpv, sera and rectal swabs were obtained from the 62 piglets. The piglets from litters B, C and E were then euthanized, and tonsils were collected. Sera and rectal swabs were obtained from the animals from the remaining litters (A, D and F) at 36, 66 and 96 dpv (end of experiment), and tonsil samples were obtained. Additionally, clinical signs were registered by the veterinarian from the farm. were vaccinated using a pig dose, equivalent to 100 pro- tective doses (PDs), of a lapinized live attenuated C- strain-based vaccine (Labiofam strain) by intramuscular injection in the neck. At 21 dpv, sera and rectal swabs were obtained from the 62 piglets. The piglets from litters B, C and E were then euthanized, and tonsils were collected. Sera and rectal swabs were obtained from the animals from the remaining litters (A, D and F) at 36, 66 and 96 dpv (end of experiment), and tonsil samples were obtained. Additionally, clinical signs were registered by the veterinarian from the farm. sera from piglets were also evaluated at 21, 36, 66 and 96 dpv. Samples with blocking percentage values below 30% were considered negative, those with values be- tween 30 and 40% were considered doubtful and those with values ≥40% were considered positive. Acknowledgements The authors thank Dr. Félix Dominguez and all the field veterinarians who helped in the development of the present work. Funding 8. van Oirschot JT. Vaccinology of classical swine fever: from lab to field. Vet Microbiol. 2003;96:367–84. 8. van Oirschot JT. Vaccinology of classical swine fever: from lab to field. Vet Microbiol. 2003;96:367–84. This research was supported by Program I.1: Research grants for and about development from the MAEC–AECID programme of the Spanish government and by grants AGL2015–66907 and RTI2018-100887-B-100 from the Spanish government; the CERCA Programme, Generalitat de Catalunya, is also acknowl- edged for funding this research. The funding bodies did not play a role in the design of the study; collection, analysis, and interpretation of the data; or writ- ing of the manuscript. 9. Ganges L, Nuñez JI, Sobrino F, Borrego B, Fernández-Borges N, Frías- Lepoureau MT, et al. 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Transmission of classical swine fever virus depends on the clinical course of infection which is associated with high and low levels of virus excretion. Vet Microbiol. 2011; 147:262–73. https://doi.org/10.1016/j.vetmic.2010.06.032. CSF: Classical swine fever; CSFV: Classical swine fever virus; Ct: Threshold cycle; DIVA: Differentiating infected from vaccinated animals; DMEM: Dulbecco’s modified Eagle medium; dpv: Days post vaccination; DSA: Animal Health Directory; FBS: Foetal bovine serum; MINAGRI: Ministry of Agriculture; NPLA: Neutralization peroxidase-linked assay; PCV-2: Porcine circovirus 2; PD: Protective dose; PK-15 cells: Porcine kidney-15 cell line; PLA: Peroxidase-linked assay; PPV: Porcine parvovirus; qRT-PCR: Quantitative reverse transcription polymerase chain reaction; RNA: Ribonucleic acid; TCID50: 50% tissue culture infective dose 4. Aynaud JM, Corthier G, Vannier P, Tillon PJ. 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Classical swine fever and African swine fever. Brussels: Commission of the European Communities, Publication EUR 5904 EN; 1977. p. 214–30. Availability of data and materials 11. Coronado L, Liniger M, Muñoz-Gonzalez S, Postel A, Perez LJ, Perez- Simó M, et al. Novel poly-uridine insertion in the 3’UTR and E2 amino acid substitutions in a low virulent classical swine fever virus. Vet Microbiol. 2017; In press. The datasets used and/or analysed in the present study are available from the corresponding author on reasonable request. Received: 29 June 2018 Accepted: 26 June 2019 20. Muñoz-González S, Ruggli N, Rosell R, Pérez LJ, Frías-Leuporeau MT, Fraile L, et al. Postnatal persistent infection with classical swine fever virus and its immunological implications. PLoS One. 2015;10:e0125692. https://doi.org/1 0.1371/journal.pone.0125692. 20. Muñoz-González S, Ruggli N, Rosell R, Pérez LJ, Frías-Leuporeau MT, Fraile L, et al. Postnatal persistent infection with classical swine fever virus and its immunological implications. PLoS One. 2015;10:e0125692. https://doi.org/1 0.1371/journal.pone.0125692. Consent for publication 15. Pérez LJ, Díaz de Arce H, Perera CL, Rosell R, Frías MT, Percedo MI, et al. Positive selection pressure on the B/C domains of the E2-gene of classical swine fever virus in endemic areas under C-strain vaccination. Infect Genet Evol. 2012;12:1405–12. E2-specific and neutralizing antibody detection Piglet serum samples were tested with a neutralization PLA (NPLA) at weaning (33 days old, 0 dpv) and at 21 and 96 dpv [36]. The titres were expressed as the recip- rocal dilution of serum that neutralized 100 TCID50 of the Margarita strain in 50% of the culture replicates. Detection of E2-specific antibodies was performed using a commercial ELISA kit (IDEXX, Netherlands) following the manufacturer’s recommendations in serum samples collected at weaning from sows and piglets. In addition, Additional file 1: Figure S1. Representation of the mutation pattern for Cuban CSFV strains on the 3D structure of the predicted model of the E2 protein of CSFV. The 3D structure and surface of the Cuban ancestral strain “Margarita/1958” [AJ704817], the previously described “Pinar del Rio/2010” [KX576461] strain [11] and the new strain “Pinar del Rio/ 2016”[LT985811] are presented. The in silico mutations associated with positive selection (red), associated with adaptation (blue) and not linked previously with any evolutionary advantage (yellow) are denoted. The antigenic region B/C (gold), antigenic region A/D (ruby red) and transmembrane domain (grey) are located on monomer B. (JPG 94 kb) Page 12 of 13 Page 12 of 13 Page 12 of 13 Coronado et al. BMC Veterinary Research (2019) 15:247 Coronado et al. BMC Veterinary Research (2019) 15:247 Authors’ contributions 6. van Oirschot JT. Experimental production of congenital persistent swine fever infections. I Clinical, pathological and virological observations. Vet Microbiol. 1979;4:117–32. 6. van Oirschot JT. Experimental production of congenital persistent swine fever infections. I Clinical, pathological and virological observations. Vet Microbiol. 1979;4:117–32. LG and MTF conceived, designed and coordinated this study. LC, LJP, LD, CLP and MTF performed the experiments. LC, JAB, SM, LJP, CLP, OF, MTF, RR and LG analysed the data. LG, LJP, CLP contributed reagents/materials/analysis tools. LC, JAB, MTF and LG wrote the paper. SM, CLP, and LG critically read the manuscript. All authors have read and approved the final manuscript. 7. van Oirschot JT. Experimental production of congenital persistent swine fever infections: II Effect on functions of the immune system. Vet Microbiol. 1979;4:133-47. Competing interests The authors declare that they have no competing interests. 16. Ji W, Niu D-DD, Si H-LL, Ding N-ZZ, He C-QQ. Vaccination influences the evolution of classical swine fever virus. Infect Genet Evol. 2014;25:69–77. 16. Ji W, Niu D-DD, Si H-LL, Ding N-ZZ, He C-QQ. Vaccination influences the evolution of classical swine fever virus. Infect Genet Evol. 2014;25:69–77. Author details 1 17. Rios L, Coronado L, Naranjo-Feliciano D, Martínez-Pérez O, Perera CL, Hernandez-Alvarez L, et al. Deciphering the emergence, genetic diversity and evolution of classical swine fever virus. Sci Rep. 2017;7:17887. https:// doi.org/10.1038/s41598-017-18196-y. 17. Rios L, Coronado L, Naranjo-Feliciano D, Martínez-Pérez O, Perera CL, Hernandez-Alvarez L, et al. Deciphering the emergence, genetic diversity and evolution of classical swine fever virus. Sci Rep. 2017;7:17887. https:// doi.org/10.1038/s41598-017-18196-y. 1National Centre for Animal and Plant Health (CENSA), OIE Collaborating Centre for Disaster Risk Reduction in Animal Health, San José de las Lajas, Mayabeque, Cuba. 2OIE Reference Laboratory for Classical Swine Fever, IRTA-CReSA, Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain. 3University of Illinois, College of Veterinary Science, Department of Clinical Veterinary Medicine, Urbana, Illinois 61802, United States. 4Departament d’Agricultura Ramaderia i Pesca (DARP), Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain. 18. Yoo SJ, Kwon T, Kang K, Kim H, Kang SC, Richt JA, et al. Genetic evolution of classical swine fever virus under immune environments conditioned by genotype 1-based modified live virus vaccine. Transbound Emerg Dis. 2018: 1–11. https://doi.org/10.1111/tbed.12798. 19. Cabezón O, Colom-Cadena A, Muñoz-González S, Pérez-Simó M, Bohórquez JA, Rosell R, et al. Post-Natal persistent infection with classical swine fever virus in wild boar: a strategy for viral maintenance? Transbound Emerg Dis. 2015. https://doi.org/10.1111/tbed.12395. Received: 29 June 2018 Accepted: 26 June 2019 Ethics approval and consent to participate 12. Blome S, Moß C, Reimann I, König P, Beer M. Classical swine fever vaccines—state-of-the-art. Vet Microbiol. 2017;206:10–20. 12. Blome S, Moß C, Reimann I, König P, Beer M. Classical swine fever vaccines—state-of-the-art. Vet Microbiol. 2017;206:10–20. The animals in this study were from a recently repopulated Cuban commercial farm located in the western region. The authorities responsible for the farm provided written consent allowing the animals to be used in this study. International standards for animal welfare were used following the regulations of the Animal Health Directory (DSA). The Ethics Committee of the Cuban Ministry of Agriculture (MINAGRI) of the Cuban government approved the study, and all efforts were made to minimize animal suffering. 13. Graham SP, Haines FJ, Johns HL, Sosan O, Anna S, Rocca L, et al. Characterisation of vaccine-induced, broadly cross-reactive IFN-γ secreting T cell responses that correlate with rapid protection against classical swine fever virus. Vaccine. 2012;30:2742–8. 13. Graham SP, Haines FJ, Johns HL, Sosan O, Anna S, Rocca L, et al. Characterisation of vaccine-induced, broadly cross-reactive IFN-γ secreting T cell responses that correlate with rapid protection against classical swine fever virus. Vaccine. 2012;30:2742–8. 14. Suradhat S, Intrakamhaeng M, Damrongwatanapokin S. The correlation of virus-specific interferon-gamma production and protection against classical swine fever virus infection. Vet Immunol Immunopathol. 2001;83:177–89. https://doi.org/10.1016/S0165-2427(01)00389-0. 14. Suradhat S, Intrakamhaeng M, Damrongwatanapokin S. The correlation of virus-specific interferon-gamma production and protection against classical swine fever virus infection. Vet Immunol Immunopathol. 2001;83:177–89. https://doi.org/10.1016/S0165-2427(01)00389-0. 1. Blome S, Staubach C, Henke J, Carlson J, Beer M. Classical swine fever—an updated review. Viruses. 2017;9:1–24. https://doi.org/10.3390/v9040086. 2. Simmonds P, Becher P, Collett M, Gould EA, Heinz FX, Meyers G, et al. Family Flaviviridae. In: King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ, editors. Ninth report of the international Commitee on taxonomy of viruses. Ninth. San Diego: Elsevier Academic Press; 2012. p. 1004–20. References 1. Blome S, Staubach C, Henke J, Carlson J, Beer M. Classical swine fever—an updated review. Viruses. 2017;9:1–24. https://doi.org/10.3390/v9040086. 21. Muñoz-González S, Perez-Simó M, Muñoz M, Bohorquez JA, Rosell R, Summerfield A, et al. Efficacy of a live attenuated vaccine in classical swine fever virus postnatally persistently infected pigs. Vet Res. 2015;46:78. https:// doi.org/10.1186/s13567-015-0209-9. 21. Muñoz-González S, Perez-Simó M, Muñoz M, Bohorquez JA, Rosell R, Summerfield A, et al. Efficacy of a live attenuated vaccine in classical swine fever virus postnatally persistently infected pigs. Vet Res. 2015;46:78. https:// doi.org/10.1186/s13567-015-0209-9. 2. Simmonds P, Becher P, Collett M, Gould EA, Heinz FX, Meyers G, et al. Family Flaviviridae. In: King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ, editors. Ninth report of the international Commitee on taxonomy of viruses. Ninth. San Diego: Elsevier Academic Press; 2012. p. 1004–20. Page 13 of 13 Coronado et al. BMC Veterinary Research (2019) 15:247 Coronado et al. BMC Veterinary Research (2019) 15:247 22. Muñoz-González S, Pérez-Simó M, Colom-Cadena A, Cabezón O, Bohórquez JA, Rosell R, et al. 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https://openalex.org/W3089125418	https://www.mdpi.com/2073-4336/11/4/41/pdf?version=1600831562	English	null	Evolution of Cooperation in Social Dilemmas with Assortative Interactions	Games	2,020	cc-by	20,627	Received: 4 July 2020; Accepted: 16 September 2020; Published: 23 September 2020 Abstract: Cooperation in social dilemmas plays a pivotal role in the formation of systems at all levels of complexity, from replicating molecules to multi-cellular organisms to human and animal societies. Abstract: Cooperation in social dilemmas plays a pivotal role in the formation of systems at all levels of complexity, from replicating molecules to multi-cellular organisms to human and animal societies. In spite of its ubiquity, the origin and stability of cooperation pose an evolutionary conundrum, since cooperation, though beneﬁcial to others, is costly to the individual cooperator. Thus natural selection would be expected to favor selﬁsh behavior in which individuals reap the beneﬁts of cooperation without bearing the costs of cooperating themselves. Many proximate mechanisms have been proposed to account for the origin and maintenance of cooperation, including kin selection, direct reciprocity, indirect reciprocity, and evolution in structured populations. Despite the apparent diversity of these approaches they all share a uniﬁed underlying logic: namely, each mechanism results in assortative interactions in which individuals using the same strategy interact with a higher probability than they would at random. Here we study the evolution of cooperation in both discrete Abstract: Cooperation in social dilemmas plays a pivotal role in the formation of systems at all levels of complexity, from replicating molecules to multi-cellular organisms to human and animal societies. In spite of its ubiquity, the origin and stability of cooperation pose an evolutionary conundrum, since cooperation, though beneﬁcial to others, is costly to the individual cooperator. Thus natural selection would be expected to favor selﬁsh behavior in which individuals reap the beneﬁts of cooperation without bearing the costs of cooperating themselves. Many proximate mechanisms have been proposed to account for the origin and maintenance of cooperation, including kin selection, direct reciprocity, indirect reciprocity, and evolution in structured populations. Despite the apparent diversity of these approaches they all share a uniﬁed underlying logic: namely, each mechanism results in assortative interactions in which individuals using the same strategy interact with a higher probability than they would at random. Here we study the evolution of cooperation in both discrete strategy and continuous strategy social dilemmas with assortative interactions. Received: 4 July 2020; Accepted: 16 September 2020; Published: 23 September 2020 For the sake of tractability, assortativity is modeled by an individual interacting with another of the same type with probability r and interacting with a random individual in the population with probability 1 −r, where r is a parameter that characterizes the degree of assortativity in the system. For discrete strategy social dilemmas we use both a generalization of replicator dynamics and individual-based simulations to elucidate the donation, snowdrift, and sculling games with assortative interactions, and determine the analogs of Hamilton’s rule, which govern the evolution of cooperation in these games. For continuous strategy social dilemmas we employ both a generalization of deterministic adaptive dynamics and individual-based simulations to study the donation, snowdrift, and tragedy of the commons games, and determine the effect of assortativity on the emergence and stability of cooperation. Keywords: evolutionary game theory; replicator dynamics; adaptive dynamics; prisoners dilemma; hawk-dove game; coordination game; tragedy of the commons Swami Iyer 1 and Timothy Killingback 2,* 1 Department of Computer Science, University of Massachusetts, Boston, MA 02125, USA; swami.iyer@gmail.com 1 Department of Computer Science, University of Massachusetts, Boston, MA 02125, USA; swami.iyer@gmail.com 2 Department of Mathematics, University of Massachusetts, Boston, MA 02125, USA * Correspondence: timothy.killingback@umb.edu Evolution of Cooperation in Social Dilemmas with Assortative Interactions Swami Iyer 1 and Timothy Killingback 2,* www.mdpi.com/journal/games 1. Introduction The evolution and stability of cooperative behavior in social dilemmas is a key aspect of the formation of biological systems at multiple levels of complexity, ranging from replicating molecules, at the lower level, to multi-cellular organisms, at the mid level, to human societies, at the high level. Some examples of cooperation in social dilemmas include: formation of early replicating molecules to form larger replicating systems capable of enhanced information encoding [1,2]; integration of the originally autarchic prokaryote precursors of mitochondria and chloroplasts into eukaryotic cells [2]; differential production of replication enzymes in an RNA phage [3]; blood meal donation to roost mates by vampire bats [4]; predator inspection in ﬁsh [5]; allogrooming in social mammals [6]; Games 2020, 11, 41; doi:10.3390/g11040041 www.mdpi.com/journal/games 2 of 31 Games 2020, 11, 41 alarm calls in response to danger by mammals and birds [7]; contribution to a wide variety of public goods [8], including, social security, health and welfare programs; restraint in consuming common pool resources [9,10], such as responsible use of ﬁshing stocks, limiting the emission of pollution into the atmosphere, and sharing Internet bandwidth; correct implementation of the Transmission Control Protocol (TCP) so as to avoid congestion in Internet trafﬁc [11]; and ﬁle sharing over peer-to-peer networks [12]. Despite the widespread emergence of cooperation in social dilemmas it has proved to be fundamentally challenging to achieve a satisfactory understanding of the origin and maintenance of this phenomenon [2,13–18]. The difﬁculty in achieving such an understanding is a direct consequence of the nature of a social dilemma itself. A social dilemma may be deﬁned, in classical game theory, as a game which possesses at least one socially inefﬁcient Nash equilibrium [16,19], and in evolutionary game theory, as a game that possesses at least one socially inefﬁcient evolutionary attractor, such as an evolutionary stable strategy [20,21], a stable equilibrium point of the replicator dynamics [22,23], a convergent stable singular strategy of the adaptive dynamics [24–27], or an attracting state in stochastic evolutionary dynamics [28–33]. Since in a social dilemma adopting the strategy at the socially inefﬁcient equilibrium or attractor constitutes defection, while adopting the socially efﬁcient strategy is considered to be cooperation, the nature of the dilemma is that individuals employing strategies corresponding to the socially inefﬁcient attractor will be trapped there by the evolutionary dynamics, despite all individuals being better off if they adopted socially efﬁcient behavior. 1. Introduction As an aside it is interesting to note that many acts of cooperation (when the word cooperation is taken in the literal sense meaning the “process of working together to the same end”) may have a direct beneﬁt to the cooperating individual that exceeds the cost (see for example [34]). The emphasis on considering cooperation in social dilemmas in theoretical work is due to this case providing the most challenging theoretical problems, rather than cooperation in social dilemmas being necessarily more common than other forms of cooperation. A considerable number of different approaches to understanding the evolution of cooperation in social dilemmas have been studied [35]. These include: kin selection [13,36], direct reciprocity [14,37–39], indirect reciprocity [40,41], evolution in network structured populations [19,42–75], and evolution in group structured populations [76,77]. The underlying logic in all these approaches is that they all result in individuals assorting positively, that is, individuals of the same type interact with a greater probability than they would at random. The fundamental role of assortativity in promoting cooperative behavior was already clearly recognized by Hamilton in his work on inclusive fitness [78]. The central position occupied by assortativity in the evolution of cooperation through kin selection was made even more explicit by Grafen in his geometric interpretation of relatedness [79,80]. Grafen’s interpretation of relatedness in terms of assortativity is also discussed in [81]. Assortativity is clearly also a key feature of many other proximate mechanisms for promoting the evolution of cooperation. For example, in network structured populations [37,43–64,66–70,72,73] and in group structured populations [76,77], the formation of clusters of cooperators results in preferential interactions between cooperators [50]. The general role played by assortativity in the evolution of cooperation has also been considered recently in [82]. We should emphasize here that many of the most important challenges in understanding the evolution of cooperation center on identifying the different proximate mechanisms that give rise to the assortativity that results in cooperation. However, it is also important to study the effect of assortativity in its own right on the evolution of cooperation. The most signiﬁcant reason for studying assortativity in its own right is that if it can be shown that positive assortativity leads to some particular outcome in the evolution of cooperation then that provides a rationale for expecting that that outcome will occur for many different proximate mechanisms for obtaining cooperation. 1. Introduction In the present paper we give a detailed and systematic study of the effect of positive assortative interactions, modeled in the manner originally suggested by [79,80], on the evolution of cooperation in a wide variety of both discrete and continuous strategy social dilemmas. In such a system, an individual Games 2020, 11, 41 3 of 31 interacts with another of the same type with probability r and interacts with a random individual in the population with probability 1 −r (where r is a parameter that characterizes the degree of assortativity in the system). In this paper we take assortativity to be “positive” in the sense that a given individual has a higher probability of interacting with its own type than would be expected at random. The main reason why we focus here on this case is that most mechanisms through which assortativity naturally emerges result in positive assortativity. Perhaps the paradigm of such a situation is evolutionary dynamics in a spatially structured population. In such a population the spatially local nature of the interaction and reproduction processes typically results in the offspring of an individual being localized in the same region of space, and consequently individuals of the same type will often interact with a higher probability than expected from their frequencies in the total population. Thus, evolutionary dynamics in spatially structured populations often results in positive assortativity. It would be interesting to also consider the possibility of “negative” assortment, in which a given individual would interact with its own type with a lower probability than expected at random. To allow both positive and negative assortativity in a model would require that the degree of assortativity should be dependent on the strategy adopted by the individual, since an individual with a given strategy clearly cannot assort both positively and negatively. In the case of 2 × 2 games the generalization to also allow negative assortment would be relatively straight forward. However, for ﬁnite strategy games with more than two strategies or for continuous strategy games, the models would be considerably more complicated. For example, in the case of a continuous strategy game, it would be necessary to specify an “assortativity kernel” that would describe precisely the amount of positive or negative assortativity that any given strategy experiences. 1. Introduction For the continuous donation game, just as for the corresponding discrete game, in a well-mixed population cooperation will never evolve: that is, the investments made by individuals in the continuous game will evolve to zero for any cost and beneﬁt functions [50,99]. Various mechanisms have been proposed for the emergence and maintenance of cooperative investments in the game including: spatial [50] and network [53] structure, and reciprocal altruism [99]. For the continuous snowdrift game [26] and continuous tragedy of the commons game [27] in a well-mixed population a variety of different evolutionary outcomes are possible, depending on the nature of the cost and beneﬁt functions. For example, for some cost and beneﬁt functions the evolutionary end state of the population consists of all individuals making the same non-zero investment. However, for other cost and beneﬁt functions high and low investing individuals coexist—an outcome termed the “Tragedy of the Commune” [26]. In the Tragedy of the Commune there is a two-fold social dilemma—not only does evolutionary dynamics result in socially inefﬁcient behavior, but furthermore it forces an unequal outcome in which some individuals make large investments while others invest little or nothing. Here we consider the effects of positive assortativity on the evolution of cooperation in the continuous donation game, snowdrift game and tragedy of the commons game. We discuss the consequences of assortative interactions both for the level of cooperation that arises and for the emergence of dimorphic evolutionary end states. Related works consider the evolutionary dynamics of continuous strategy games with interaction structure [34,100,101] and the connection to inclusive ﬁtness theory [98]. The interesting work [101] is of particular relevance to our work here in that it considers the effect of relatedness in promoting cooperation in a multi-player version of the continuous snowdrift game. In this work relatedness is introduced in a more general manner to our deﬁnition of assortativity: the authors of [101] consider a probability distribution over the number of co-players that are identical-by-descent to a focal individual, and then quantify this assortment distribution based upon the mean and variance of the assortative interactions. The remainder of this article is organized as follows. In the Models section we formulate the three discrete games in terms of a single cost-to-beneﬁt ratio parameter ρ. 1. Introduction Games 2020, 11, 41 4 of 31 Cooperative behavior is often not discrete in nature. This is true of many of the examples of cooperation given above: for instance, when vampire bats share a blood meal with a roost mate [4]. Such situations can be described very naturally using social dilemmas formulated in terms of continuous strategy games. The strategies of individuals in the game represent the level of cooperation, or investment, that they make, and are described by continuous variables. The costs and beneﬁts associated with given investments are represented as continuous functions of the investments. Here we consider three continuous strategy games: the continuous donation game [50,53], in which a cooperative investment made by one individual (the donor or investor) towards another individual (the recipient) beneﬁts the recipient but is costly to the donor; the continuous snowdrift game [26], i hi h th i t t b ﬁt b th th d d th i i t b t i l tl t th d d th Cooperative behavior is often not discrete in nature. This is true of many of the examples of cooperation given above: for instance, when vampire bats share a blood meal with a roost mate [4]. Such situations can be described very naturally using social dilemmas formulated in terms of continuous strategy games. The strategies of individuals in the game represent the level of cooperation, or investment, that they make, and are described by continuous variables. The costs and beneﬁts associated with given investments are represented as continuous functions of the investments. Here we consider three continuous strategy games: the continuous donation game [50,53], in which a cooperative investment made by one individual (the donor or investor) towards another individual (the recipient) beneﬁts the recipient but is costly to the donor; the continuous snowdrift game [26], in which the investment beneﬁts both the donor and the recipient but is also costly to the donor; and the continuous tragedy of the commons game [27], in which the investment—in this context an investment typically represents the level of consumption of a limited common-pool resource, and cooperative behavior correspond to modest levels of consumption—beneﬁts the investor but is also costly to both the investor and the recipient. 1. Introduction Despite the possible complications that may arise, studying the effects of negative as well as positive assortment seems to be an interesting area for future research. It should also be noted that a different and important approach to elucidating the general role played by assortative interactions in promoting the evolution of cooperation focuses on the use of Price’s equation [83]. This approach was ﬁrst developed by Hamilton [78], and has been more recently studied in [84]. We consider here three discrete strategy social dilemmas: the donation game, the snowdrift game, and the sculling game. The donation game is the fundamental exemplar in the prisoner’s dilemma class of games, and provides the basic game theory model for altruism [13,17,18]. The snowdrift game is an exemplar of a social dilemma in the hawk-dove class of games [15,18]. While games of hawk-dove type have been extensively studied as models of conﬂicts and contests [20,21,85], the snowdrift game provides an interesting model for certain types of cooperative behavior that differ from pure altruism [15,17]. The sculling game, is an exemplar of a social dilemma in the coordination class of games. Games in this class have been widely used as models for conventions [28,29,86], but they have typically received little attention as models of cooperation, although interesting exceptions to this trend are [56,70,87,88]. The sculling game, as we deﬁne it [19], serves as a model for certain types of cooperative behavior not described by the donation or snowdrift games. We must emphasize here that there is a very substantial literature on the evolution of cooperation in the prisoner’s dilemma with assortative interactions, and we do not claim any great novelty for our results in this case. Equivalent results to those that we have obtained for the discrete donation game can therefore be found in the literature [13,79,80,89–98]. We have discussed the donation game essentially for completeness and to allow comparison with the more novel cases of the snowdrift game and the sculling game. Furthermore, the interesting work [98] considers some closely related issues. The minimum level of assortativity required to allow cooperation to be stably maintained in the prisoner’s dilemma, hawk-dove, and stag hunt games is described in terms of the payoff matrix entries in [98], and this leads to a different formulation of some of the results that we obtain here for the discrete strategy games. 2. Models 2.1. Discrete Games 2.1.1. Replicator Dynamics with Assortative Interactions In general, consider a 2-player game with m pure strategies σ1, . . . , σm and strategy space S = {σ1, . . . , σm}. Let P be a large population of individuals, each of which uses a strategy from S. We deﬁne assortative interactions as follows. An assortative interaction among individuals is an interaction that occurs preferentially among individuals of the same type, i.e., such an interaction occurs among individuals of the same type with a greater probability than would occur through random interactions. In the context of game theory, this means that individuals using the same strategy interact with a probability greater than that which would occur with random interactions. The most direct and convenient way to introduce assortative interactions in a population is to specify a parameter r ∈[0, 1], called the degree of assortativity or simply the assortativity, which is deﬁned as follows: with probability r, an individual interacts with another individual of its own type, and with probability 1 −r, the individual interacts with a randomly chosen individual from the population [79,80]. Thus, we say that the population P is assortatively-interacting or assortatively-mixed, with assortativity r, if an individual in P interacts with another individual in P with the same strategy with probability r and with a randomly picked individual in P with probability 1 −r. Let the payoff to strategy σi against σj be denoted by π(σi, σj). In biological evolution π(σi, σj) represents the change in the Darwinian ﬁtness (that is, the change in the expected number of offspring) of an individual using strategy σi in an interaction with an individual using σj. In social evolution through imitation dynamics π(σi, σj) measures the utility obtained by an individual using strategy σi in an interaction with an individual using σj. At time t, let N(t) be the total size of the population P, and let ni(t) be the number of individuals in P using strategy σi. The frequency of strategy σi is deﬁned to be pi(t) = ni(t) N(t). If p(t) = (p1(t), . . . , pm(t)) denotes the vector of frequencies, then the state of the population at time t is given by p(t) ∈∆m, where ∆m = {p(t) ∈Rm+ : ∑m i=1 pi(t) = 1} is the m-simplex. 1. Introduction We analyze the games using the framework of replicator dynamics generalized to include the degree of assortativity r, and derive mathematical relations (analogs of Hamilton’s rule) involving parameters ρ and r that must be satisﬁed for the evolution of complete cooperation. We also formulate and analyze the three continuous strategy games using the framework of adaptive dynamics generalized to allow for assortative interactions, and derive conditions that determine the effect of assortativity on the stability of cooperation. In the Results section we describe the results of studying the evolution of cooperation in these discrete and continuous strategy games with assortative interactions using an individual-based model, and we compare the results obtained from these simulations with those obtained analytically from the assortative generalizations of replicator dynamics and adaptive dynamics. Finally, in the 5 of 31 Games 2020, 11, 41 Games 2020, 11, 41 Discussion section we conclude the article with a discussion of the signiﬁcance of our results and with some suggestions for further inquiry. 2. Models We now consider how the frequencies of the different strategies σi change with time in an assortatively-interacting population P, with assortativity r, due to natural selection. The ﬁtness f r i of σi is the average payoff of σi, which, from the deﬁnition of assortativity, is given by f r i = rπ(σi, σi) + (1 −r) m ∑ j=1 pjπ(σi, σj). (1) (1) The dynamics of p(t) on ∆m in an assortatively-interacting population can be determined (in analogy with the non-assortative case [17,18,22,23,102]) as follows. The growth rate of the number of individuals ni using strategy σi is ˙ni = ni f r i . Therefore, ˙pi = d dt ni N = ˙niN −ni ∑j ˙nj N2 = pi f r i −pi ∑ j pj f r j = pi( f r i −¯f r), (2) = pi( f r i −¯f r), (2) (2) where ¯f r = ∑j pj f r j is the mean ﬁtness of the strategies in the population. This equation is the analog of the standard replicator equation for an assortatively-interacting population. We shall refer to this equation as the assortative replicator equation with assortativity r, or the r-replicator equation for where ¯f r = ∑j pj f r j is the mean ﬁtness of the strategies in the population. This equation is the analog of the standard replicator equation for an assortatively-interacting population. We shall refer to this equation as the assortative replicator equation with assortativity r, or the r-replicator equation for 6 of 31 Games 2020, 11, 41 short. The r-replicator equation is the natural generalization of the standard replicator equation when assortative interactions are included in the manner proposed by [79,80]. The r-replicator equation is equivalent to the standard (non-assortative) replicator equation under a transformation of the payoffs. If A = (aij), where aij = π(σi, σj), is the payoff matrix, then it is elementary to verify that the replicator equation with transformed payoff matrix ˆA = rB + (1 −r)A, where B = (bij) is the matrix such that bij = aii, is equivalent to the r-replicator equation. 2. Models Notwithstanding this equivalence, however, we feel that the best way to think about the replicator equation when there are assortative interactions is to consider the assortativity as directly affecting how the ﬁtness is deﬁned, as we have done here, since the change in the ﬁtness is clear and natural, whereas the necessary transformation in the payoffs is less clear. Grafen did not formulate the r-replicator equation in his work, since it was unnecessary for his aim of studying evolutionary stable strategies with assortative interactions. Equations similar to the r-replicator equation have been considered for assortativity that depends on the frequencies of the strategies by [91] and for different population structures (including those that include relatedness) by [92]. For r = 0, the r-replicator equation reduces to the standard replicator equation for a well-mixed population [17,18,22,23,102]: ˙pi = pi( fi −¯f ), (3) (3) re ¯f = ∑j pj fj is the mean ﬁtness of the population. where ¯f = ∑j pj fj is the mean ﬁtness of the population. where ¯f = ∑j pj fj is the mean ﬁtness of the population. where f = ∑j pj fj is the mean ﬁtness of the population. Consider now the case of a symmetric 2 × 2 game, with strategies denoted by C and D, and with payoff matrix π given by j j j Consider now the case of a symmetric 2 × 2 game, with strategies denoted by C and D, and with payoff matrix π given by j j j Consider now the case of a symmetric 2 × 2 game, with strategies denoted by C and D, and with payoff matrix π given by π = " C D C α β D γ δ # , (4) (4) where α, β, γ, δ ∈R. Let p denote the frequency of strategy C in the population, and thus 1 −p is the frequency of strategy D. where α, β, γ, δ ∈R. Let p denote the frequency of strategy C in the population, and thus 1 −p is the frequency of strategy D. Now consider a population of assortatively-interacting individuals who are playing this 2 × 2 game. Let r denote the degree of assortativity in the population. The ﬁtnesses f r C and f r D of the strategies C and D, with assortativity, are given by f r C = rπ(C, C) + (1 −r)[pπ(C, C) + (1 −p)π(C, D)] = rα + (1 −r)[pα + (1 −p)β] and (5) (5) f r D = rπ(D, D) + (1 −r)[pπ(D, C) + (1 −p)π(D, D)] = rδ + (1 −r)[pγ + (1 −p)δ], (6) (6) and the average ﬁtness of the population is ¯f r = p f r C + (1 −p) f r D. The evolutionary dynamics of the population, with assortativity, is thus given by the r-replicator equation ˙p = p( f r C −¯f r) = p(1 −p)( f r C −f r D) = p(1 −p){r(α −δ) + (1 −r)[p(α −γ) + (1 −p)(β −δ)]}. where ¯f = ∑j pj fj is the mean ﬁtness of the population. (7) (7) Games 2020, 11, 41 Games 2020, 11, 41 7 of 31 We note that for r = 0, this equation reduces to the standard replicator equation for a symmetric 2 × 2 game [17,18,22,23,102]): ˙p = p( fC −¯f ) = p(1 −p)( fC −fD) = p(1 −p)[p(α −β −γ + δ) + β −δ], (8) = p(1 −p)( fC −fD) = p(1 −p)[p(α −β −γ + δ) + β −δ], (8) (8) where fC = pπ(C, C) + (1 −p)π(C, D) and fD = pπ(D, C) + (1 −p)π(D, D) are the ﬁtnesses of the strategies C and D, respectively, and ¯f = p fC + (1 −p) fD is the mean ﬁtness of the population. Here, for simplicity, in the rest of this paper when we discuss discrete strategy games we shall restrict our attention to two-strategy games—however, our analysis of evolutionary dynamics in assortatively-mixed populations can be extended to games with any number of strategies, and this may represent an interesting topic for future study. 2.1.2. Donation Game The donation game is the fundamental exemplar in the prisoner’s dilemma class of games, and provides the basic game theory model for altruism [13,17,18]. We must emphasize here that there is signiﬁcant literature on the evolution of cooperation in the prisoner’s dilemma with assortative interactions, and we do not claim any particular originality for our results in this case. We discuss the donation game here essentially for completeness and to allow comparison with the more novel cases of the snowdrift game and the sculling game that follows. Equivalent results to those that we obtain here for the discrete donation game can therefore be found in the literature [13,79,80,89–98]. Consider the situation of two individuals, John and Bill, who donate blood to each other, as described in [19]. Suppose that the act of donating blood to someone incurs a cost c to the donor but confers a beneﬁt b to the recipient, where b, c ∈R+ and b > c. If we consider donation as the cooperative strategy C and non-donation as the defective strategy D, then the payoff matrix for the donation game is given by [19]: π = " C D C 1 −ρ −ρ D 1 0 # , (9) (9) where ρ ∈(0, 1) is the cost-to-beneﬁt ratio ρ = c b. It follows directly from the rank ordering of the elements of the payoff matrix that the game is in the prisoner’s dilemma class of symmetric 2 × 2 games. The standard replicator Equation (8) gives the evolutionary dynamics for the game without assortative interactions to be ˙p = −p(1 −p)ρ. The equilibrium points for the dynamics are ˆp = 0 and ˆp = 1, the former being asymptotically stable, while the latter is unstable. Thus, a well-mixed population of individuals playing the donation game, starting from an initial frequency p0 ∈(0, 1) of cooperators, will evolve towards the all-defector ( ˆp = 0) equilibrium state [19]. Consider now an assortatively-interacting population playing the donation game with the payoff matrix given by (9). Let the degree of assortativity be r. It follows directly from the r-replicator Equation (7) that the evolutionary dynamics with assortativity for the population is given by ˙p = p(1 −p)(r −ρ). (10) (10) Figure 1 shows the phase line diagrams and the bifurcation diagram for the game. The system has two equilibria, ˆp = 0 and ˆp = 1. 2.1.2. Donation Game (b) In the bifurcation diagram solid lines represent stable equilibrium points, dashed lines represent unstable equilibrium points, and arrows indicate the direction of evolutionary change. 2.1.2. Donation Game For r < ρ, the equilibrium point ˆp = 0 is asymptotically stable, while the equilibrium point ˆp = 1 is unstable. Thus, a population, starting from an initial frequency p0 ∈(0, 1) of cooperators, will evolve towards the stable equilibrium ˆp = 0, representing a state in which all individuals defect. However, a bifurcation occurs at rc = ρ, reversing the stability Figure 1 shows the phase line diagrams and the bifurcation diagram for the game. The system has two equilibria, ˆp = 0 and ˆp = 1. For r < ρ, the equilibrium point ˆp = 0 is asymptotically stable, while the equilibrium point ˆp = 1 is unstable. Thus, a population, starting from an initial frequency p0 ∈(0, 1) of cooperators, will evolve towards the stable equilibrium ˆp = 0, representing a state in which all individuals defect. However, a bifurcation occurs at rc = ρ, reversing the stability Games 2020, 11, 41 8 of 31 of the two equilibrium points. Hence, if r > ρ a population starting with any positive frequency of cooperators will evolve towards the stable equilibrium ˆp = 1, representing a state in which all individuals cooperate. A similar inequality is arrived at in [92] for the prisoner’s dilemma game that has constant gains from switching (that is, in which ﬁtness effects are additive), with the constant r representing the population structure and ρ the ratio of total costs to total beneﬁts (not including self). of the two equilibrium points. Hence, if r > ρ a population starting with any positive frequency of cooperators will evolve towards the stable equilibrium ˆp = 1, representing a state in which all individuals cooperate. A similar inequality is arrived at in [92] for the prisoner’s dilemma game that has constant gains from switching (that is, in which ﬁtness effects are additive), with the constant r representing the population structure and ρ the ratio of total costs to total beneﬁts (not including self). Thus, as is essentially well-known, assortative interactions provide a mechanism for promoting cooperative behavior in the donation game [13,79,80,89–98]. It should be noted that the condition for cooperation to evolve through assortativity in the donation game, r > ρ, is formally identical to Hamilton’s inequality, which governs the evolution of altruism through kin selection [13]. This correspondence is completely natural given Grafen’s geometric interpretation of relatedness in terms of assortativity [79,80]. 2.1.2. Donation Game p g p p ρ g Thus, as is essentially well-known, assortative interactions provide a mechanism for promoting cooperative behavior in the donation game [13,79,80,89–98]. It should be noted that the condition for cooperation to evolve through assortativity in the donation game, r > ρ, is formally identical to Hamilton’s inequality, which governs the evolution of altruism through kin selection [13]. This correspondence is completely natural given Grafen’s geometric interpretation of relatedness in terms of assortativity [79,80]. D C 0 1 (a) assortativity asymptotic frequency of cooperation 0 1 0 1 (b) Figure 1. Phase line diagrams and the bifurcation diagram for the donation game with assortative interactions. (a) In the phase line diagrams closed circles represent stable equilibrium points, open circles represent unstable equilibrium points, and the curved line connecting equilibrium points indicates the graph of the function on the right-hand side of the r-replicator equation. (b) In the bifurcation diagram solid lines represent stable equilibrium points, dashed lines represent unstable equilibrium points, and arrows indicate the direction of evolutionary change. assortativity asymptotic frequency of cooperation 0 1 0 1 (b) D C 0 1 (a) (b) (a) Figure 1. Phase line diagrams and the bifurcation diagram for the donation game with assortative Figure 1. Phase line diagrams and the bifurcation diagram for the donation game with assortative interactions. (a) In the phase line diagrams closed circles represent stable equilibrium points, open circles represent unstable equilibrium points, and the curved line connecting equilibrium points indicates the graph of the function on the right-hand side of the r-replicator equation. (b) In the bifurcation diagram solid lines represent stable equilibrium points, dashed lines represent unstable equilibrium points, and arrows indicate the direction of evolutionary change. Figure 1. Phase line diagrams and the bifurcation diagram for the donation game with assortative interactions. (a) In the phase line diagrams closed circles represent stable equilibrium points, open circles represent unstable equilibrium points, and the curved line connecting equilibrium points indicates the graph of the function on the right-hand side of the r-replicator equation. (b) In the bifurcation diagram solid lines represent stable equilibrium points, dashed lines represent unstable equilibrium points, and arrows indicate the direction of evolutionary change. open circles represent unstable equilibrium points, and the curved line connecting equilibrium points indicates the graph of the function on the right-hand side of the r-replicator equation. 2.1.3. Snowdrift Game The snowdrift game is an interesting exemplar of a social dilemma in the hawk-dove class of games, and provides a model for certain types of cooperative behavior that differ from pure altruism [15,17,18]. Consider the situation described in [19] of two individuals, John and Bill, who are stuck in a car on their way home because the road is blocked by a snowdrift. Let the beneﬁt of getting home be b and the cost of clearing the snow be c, where b, c ∈R+ and b > c. If we consider shoveling as the cooperative strategy C and non-shoveling as the defective strategy D, then the payoff matrix for the snowdrift game is given by [19]: π = " C D C 1 −ρ 2 1 −ρ D 1 0 # , (11) (11) where ρ ∈(0, 1) is the cost-to-beneﬁt ratio ρ = c b. It follows immediately from the rank ordering of the elements of the payoff matrix that the snowdrift game is in the hawk-dove class of symmetric 2 × 2 games. where ρ ∈(0, 1) is the cost-to-beneﬁt ratio ρ = c b. It follows immediately from the rank ordering of the elements of the payoff matrix that the snowdrift game is in the hawk-dove class of symmetric 2 × 2 games. Games 2020, 11, 41 9 of 31 In the absence of assortativity, the standard replicator Equation (8) gives the evolutionary dynamics for the game to be ˙p = p(1 −p)[p( ρ 2 −1) + 1 −ρ]. The equilibrium points for the dynamics are ˆp = 0 and ˆp = 1, and p⋆= 1−ρ 1−ρ 2 . The latter internal equilibrium is asymptotically stable, while the former two boundary equilibria are unstable. Thus, a population of individuals playing the snowdrift game, starting from an initial fraction p0 ∈(0, 1) of cooperators, will evolve towards the internal equilibrium state (p⋆= 1−ρ 1−ρ 2 ) in which cooperators and defectors coexist [19]. 1 2 Consider a population of assortatively-interacting individuals playing the snowdrift game with the payoff matrix given by (11). Let the degree of assortativity be r. The r-replicator Equation (7) gives the evolutionary dynamics with assortativity for the population to be ˙p = p(1 −p) h p(1 −r) ρ 2 −1 + rρ 2 −ρ + 1 i . 2.1.3. Snowdrift Game (12) (12) Figure 2 shows the phase line diagrams and the bifurcation diagram for the game. The system has three equilibria: ˆp = 0 and ˆp = 1 at the boundaries, and a possible internal equilibrium Figure 2 shows the phase line diagrams and the bifurcation diagram for the game. The system has three equilibria: ˆp = 0 and ˆp = 1 at the boundaries, and a possible internal equilibrium p⋆= rρ 2 + 1 −ρ (1 −r) 1 −ρ 2 . (13) (13) It is easy to verify that if r < rc = ρ 2 then the boundary equilibria are unstable, while there exists an internal equilibrium p⋆which is stable. Thus, if r < rc then a population starting with an initial frequency p0 ∈(0, 1) of cooperating individuals will evolve to a state of coexistence determined by the internal equilibrium, in which a fraction p⋆of the population will be cooperators and the remainder will be defectors. A bifurcation occurs at r = rc, in which the equilibrium p⋆passes through the boundary equilibrium ˆp = 1, resulting in a change of stability for the boundary equilibria. For r > rc there no longer exists any internal equilibrium, while the boundary equilibrium ˆp = 0 becomes unstable and the equilibrium ˆp = 1 becomes globally asymptotically stable. Therefore, for r > rc a population starting with any positive initial frequency p0 of cooperators will evolve to the completely cooperative state determined by the equilibrium ˆp = 1. For r < rc = ρ 2, in which case there is a stable internal equilibrium p⋆, it follows from ∂p⋆ ∂r = 1−ρ(1−ρ 4 ) [(1−r)(1−ρ 2 )]2 > 0, that increasing the assortativity r has the effect of increasing the frequency 2 1 of cooperators at equilibrium. Similarly, in this case, since ∂p⋆ ∂ρ = r−r2 2 −1 2 [(1−r)(1−ρ 2 )]2 < 0, increasing the cost-to-beneﬁt ratio ρ decreases the frequency of cooperators at equilibrium. Furthermore, for r > rc = ρ 2, the population is completely cooperative. Thus, for the snowdrift game, cooperative behavior is promoted in an assortatively-interacting population, and the condition r > ρ 2 governing the transition to complete cooperation is an analog of Hamilton’s inequality for the donation game. In related work, the minimal level of assortativity required for cooperation to be stably maintained in hawk-dove games is studied in [98]. 2.1.3. Snowdrift Game of cooperators at equilibrium. Similarly, in this case, since ∂p⋆ ∂ρ = r−r 2 −1 2 [(1−r)(1−ρ 2 )]2 < 0, increasing the cost-to-beneﬁt ratio ρ decreases the frequency of cooperators at equilibrium. Furthermore, for r > rc = ρ 2, the population is completely cooperative. Thus, for the snowdrift game, cooperative behavior is promoted in an assortatively-interacting population, and the condition r > ρ 2 governing the transition to complete cooperation is an analog of Hamilton’s inequality for the donation game. In related work, the minimal level of assortativity required for cooperation to be stably maintained in hawk-dove games is studied in [98]. 10 of 31 Games 2020, 11, 41 D C 0 1 (a) assortativity asymptotic frequency of cooperation 0 1 0 1 (b) Figure 2. Phase line diagrams and the bifurcation diagram for the snowdrift game with assortative interactions. (a) In the phase line diagrams closed circles represent stable equilibrium points, open circles represent unstable equilibrium points, and the curved line connecting equilibrium points indicates the graph of the function on the right-hand side of the r-replicator equation. (b) In the bifurcation diagram solid lines represent stable equilibrium points, dashed lines represent unstable equilibrium points, and arrows indicate the direction of evolutionary change. assortativity asymptotic frequency of cooperation 0 1 0 1 (b) D C 0 1 (a) (a) (b) Figure 2. Phase line diagrams and the bifurcation diagram for the snowdrift game with assortative interactions. (a) In the phase line diagrams closed circles represent stable equilibrium points, open circles represent unstable equilibrium points, and the curved line connecting equilibrium points indicates the graph of the function on the right-hand side of the r-replicator equation. (b) In the bifurcation diagram solid lines represent stable equilibrium points, dashed lines represent unstable equilibrium points, and arrows indicate the direction of evolutionary change. 2.1.4. Sculling Game Games 2020, 11, 41 11 of 31 Consider a population of assortatively-interacting individuals playing the sculling game with the payoff matrix (14). Let the degree of assortativity be r. The r-replicator Equation (7) gives that the evolutionary dynamics with assortativity for the population is given by ˙p = p(1 −p) h p(1 −r) + 3r 2 −ρ + 1 2 i . (15) (15) Figure 3 shows the phase line diagrams and the bifurcation diagram for the game. The system has three equilibria: ˆp = 0 and ˆp = 1 at the boundaries, and a possible internal equilibrium Figure 3 shows the phase line diagrams and the bifurcation diagram for the game. The system has three equilibria: ˆp = 0 and ˆp = 1 at the boundaries, and a possible internal equilibrium p⋆= −3r 2 + ρ −1 2 1 −r . (16) (16) It is straightforward to verify that if r < rc = 2ρ 3 −1 3 then the boundary equilibria ˆp = 0 and ˆp = 1 are stable, and there exists an unstable interior equilibrium p⋆. Thus, if r < rc = 2ρ 3 −1 3 then a population starting with an initial frequency p0 ̸= p⋆of cooperators will evolve to the all-defector state ˆp = 0 if p0 < p⋆and to the all-cooperator state ˆp = 1 if p0 > p⋆. It follows from this that, for r < rc, if r < ˆr = ρ−1 2 −p0 3 2 −p0 then such a population evolves to the all-defector equilibrium ˆp = 0, while if r > ˆr then the population evolves to the all-cooperator equilibrium ˆp = 1. For example, if a population starts with an initial frequency of cooperators p0 = 1 2, then ˆr = ρ −1, and the population will evolve to all-defection if r < ρ −1 and to all-cooperation if r > ρ −1. A bifurcation occurs at r = rc, in which the equilibrium p⋆passes through the boundary equilibrium ˆp = 0, resulting in a change of stability for the boundary equilibria. Consequently, for r > rc there does not exist any internal equilibrium, the boundary equilibrium ˆp = 0 is unstable, and the equilibrium ˆp = 1 is globally asymptotically stable. Thus, if r > rc then a population with any positive initial frequency p0 of cooperators will evolve to the completely cooperative state ˆp = 1. 2.1.4. Sculling Game Games in the coordination class have typically received little attention as models of cooperation, although for notable exceptions to this tendency see [56,70,87,88]. Here we consider the sculling game [19] as an exemplar of a social dilemma in the coordination class of games, with the aim of using it as a model for certain types of cooperative behavior not described by the donation or snowdrift games. Suppose individuals John and Bill are rowing in a double scull to get to their destination, as described in [19]. Let reaching the destination have a value of b 2 to both players, where b ∈R+, and let the cost of rowing be c ∈R+ to the rower. If we treat rowing as the cooperative strategy C and non-rowing as the defective strategy D, then the payoff matrix for the sculling game is given by [19]. π = " C D C 2 −ρ 1 2 −ρ D 1 2 0 # , (14) (14) where ρ ∈( 1 2, 3 2) is the cost-to-beneﬁt ratio ρ = c b. It follows directly from the rank ordering of the elements of the payoff matrix that the sculling game is in the coordination class of symmetric 2 × 2 games. Equation (8) gives the evolutionary dynamics for the game to be ˙p = p(1 −p)[p + 1 2 −ρ]. The equilibrium points for the dynamics are ˆp = 0, ˆp = 1, and p⋆= ρ −1 2. The former two boundary equilibria are asymptotically stable, while the latter internal equilibrium is unstable. Thus, a population of individuals playing the sculling game, starting from an initial fraction p0 ∈(0, 1)\{ρ −1 2} of cooperators, will evolve towards the all-defector ( ˆp = 0) state if p0 < ρ −1 2, and towards the all-cooperator ( ˆp = 1) state if p0 > ρ −1 2. We note that the equilibrium (C, C) is payoff-dominant over (D, D), for all ρ. Moreover, (C, C) is risk-dominant for ρ ∈( 1 2, 1), while (D, D) is risk-dominant for ρ ∈(1, 3 2). A coordination game in which one of the two equilibria is payoff dominant while the other is risk dominant is a stag hunt game [88]. Thus, for ρ ∈(1, 3 2) the sculling game is an exemplar of the stag hunt game [19]. 2.2. Continuous Games 2.2. Continuous Games 2.1.4. Sculling Game 2 For r < rc = 2ρ 3 −1 3, in which case there is an unstable internal equilibrium p⋆, it follows from ∂p⋆ ∂r = ρ−2 (1−r)2 < 0 that increasing the assortativity r has the effect of increasing the basin of attraction around ˆp = 1, thus decreasing the value of the initial frequency p0 of cooperators that is needed for the population to evolve to the all-cooperator state. Similarly, in this case, since ∂p⋆ ∂ρ = 1 1−r > 0, increasing the cost-to-beneﬁt ratio ρ has the effect of increasing the basin of attraction around ˆp = 0, thus increasing the value of the initial frequency p0 of cooperators that is needed for the population to evolve to the all-cooperator state. Furthermore, for r > rc = 2ρ 3 −1 3, the population becomes completely cooperative. Thus, for the sculling game, cooperative behavior is promoted in an assortatively-interacting population, and the condition r > 2ρ 3 −1 3 governing the transition to complete cooperation is an analog of Hamilton’s inequality for the donation game. In related work, the effect of assortativity on the maintenance of cooperation in stag hunt games is considered in [98]. 12 of 31 Games 2020, 11, 41 D C 0 1 (a) D C 0 1 (a) assortativity asymptotic frequency of cooperation 0 1 0 1 (b) Figure 3. Phase line diagrams and the bifurcation diagram for the sculling game with assortative interactions. (a) In the phase line diagrams closed circles represent stable equilibrium points, open circles represent unstable equilibrium points, and the curved line connecting equilibrium points indicates the graph of the function on the right-hand side of the r-replicator equation. (b) In the bifurcation diagram solid lines represent stable equilibrium points, dashed lines represent unstable equilibrium points, and arrows indicate the direction of evolutionary change. assortativity asymptotic frequency of cooperation 0 1 0 1 (b) D C 0 1 (a) (b) (a) Figure 3. Phase line diagrams and the bifurcation diagram for the sculling game with assortative interactions. (a) In the phase line diagrams closed circles represent stable equilibrium points, open circles represent unstable equilibrium points, and the curved line connecting equilibrium points indicates the graph of the function on the right-hand side of the r-replicator equation. 2.1.4. Sculling Game (b) In the bifurcation diagram solid lines represent stable equilibrium points, dashed lines represent unstable equilibrium points, and arrows indicate the direction of evolutionary change. 2.2.1. Adaptive Dynamics with Assortative Interactions (18) (18) We note that when r = 0, the above equation reduces to We note that when r = 0, the above equation reduces to We note that when r = 0, the above equation reduces to φ0 x(y) = π(y, x) −π(x, x), (19) (19) which is the standard equation for the invasion ﬁtness of a continuous strategy game in a well-mixed population [26]. The adaptive dynamics of a continuous strategy game in an assortatively-interacting population is determined by the invasion ﬁtness φr x(y) [17,23–26,104]. The evolution of the strategy x is governed by the selection gradient D(x) = ∂φrx ∂y y=x, and the adaptive dynamics of x is determined by the differential equation ˙x = mD(x), where m depends on the population size and on the mutational process at work [106]. For a constant population size, m simply scales the time variable, and thus we can set m = 1 without any loss of generality. Equilibrium points of the adaptive dynamics are called singular strategies and are solutions of D(x⋆) = 0. If no such solutions exist, then the strategy x monotonically increases or decreases under evolution, depending on the sign of D(x). If x⋆exists, it is convergent stable and, hence an attractor for the adaptive dynamics, if dD dx x=x⋆< 0. If this equality is reversed, x⋆is a repeller. Initially, the population will converge to a convergent stable singular point x⋆, but its subsequent evolutionary fate depends on whether x⋆is a maximum or minimum of the invasion ﬁtness φr x(y). If x⋆is a maximum, i.e., if ∂2φr x⋆ ∂y2 y=x⋆< 0, then x⋆is an evolutionarily stable strategy (ESS), representing an evolutionary end state in which all individuals adopt strategy x⋆. If, however, ∂2φr x⋆ ∂y2 y=x⋆> 0, then a population of x⋆-strategists can be invaded by mutant strategies on either side of x⋆. In this case the population undergoes evolutionary branching and splits into two distinct and diverging clusters of strategies. 2.2.1. Adaptive Dynamics with Assortative Interactions Let us now consider the evolutionary dynamics of continuous strategy social dilemmas with assortative interactions, which we model using continuous strategy games in assortatively-interacting populations. We quantify the probability that an individual interacts with another individual of its own type (i.e., in the context of games, the probability that an individual using a given strategy interacts with another individual using the same strategy) by the parameter r ∈[0, 1], the degree of assortativity, in exactly the same way as discussed above for the case of discrete games. Since the exact equivalent of replicator dynamics for continuous strategy games results in an inﬁnite dimensional dynamical system which is difﬁcult to study analytically, here we will adopt the standard approach of using deterministic, monomorphic, adaptive dynamics to study the evolutionary dynamics of the continuous strategy games [17,23–27,103–105]. We will study the evolutionary behavior of continuous games in assortatively-mixed populations analytically using a generalization of standard deterministic adaptive dynamics that accounts for the assortative interactions. Consider a large assortatively-mixed population of individuals, with assortativity r, playing a continuous game. We assume that the interactions are pairwise, with π(x, y) denoting the payoff to an x-strategist when interacting with a y-strategist. Let the population be initially monomorphic with all individuals using the same resident strategy x. Now consider a small fraction ξ of mutant individuals in the population playing strategy y. The ﬁtnesses f r x and f r y of the x and y strategies are given by f r x = rπ(x, x) + (1 −r)[(1 −ξ)π(x, x) + ξπ(x, y)] and f r y = rπ(y, y) + (1 −r)[(1 −ξ)π(y, x) + ξπ(y, y)]. (17) (17) Games 2020, 11, 41 13 of 31 The invasion ﬁtness φr x(y) of the mutant strategy y in the resident strategy x is the per capita growth rate of y when rare, and thus is given by φr x(y) = limξ→0 ˙ξ ξ . It follows from the r-replicator equation ˙ξ = ξ(1 −ξ)( f r y −f r x) that φr x(y) = lim ξ→0 ˙ξ ξ = rπ(y, y) + (1 −r)π(y, x) −π(x, x). (18) φr x(y) = lim ξ→0 ˙ξ ξ φr x(y) = lim ξ→0 ˙ξ ξ ξ = rπ(y, y) + (1 −r)π(y, x) −π(x, x). 2.2.2. Continuous Donation Game Consider the situation of two individuals, John and Bill, who donate blood to each other, as described in the discrete donation game formulated in [19]. Let us assume now that the amount of blood donated by each individual can vary. Interactions among pairs of individuals in which the cooperative investment (or donation) is continuously variable, and that made by one individual (the donor) beneﬁts the other individual (the recipient) but is costly to the donor, can be described using the continuous donation (CD) game (also referred to as the continuous prisoner’s dilemma game) [50,53,99]. The CD game involves the interaction between two individuals, making investments x and y, respectively, where x, y ∈R+. An investment x has the following effects: the payoff of the investor (donor) is reduced by C(x), where C is a function that speciﬁes the cost of making the investment, and the payoff of the beneﬁciary (recipient) is increased by B(x), where B is a function that speciﬁes the beneﬁt resulting from the investment. Therefore, the payoff to an x-investor interacting with a y-investor is given by the relation π(x, y) = B(y) −C(x). (20) (20) We shall assume here that there is an upper limit xm on the possible level of investment. We also assume that the cost and beneﬁt functions are smooth and monotonically increasing, Games 2020, 11, 41 14 of 31 satisfy C(0) = B(0) = 0, and B(x) > C(x), for x ∈(0, xc), where 0 < xc < xm. This latter assumption is a necessary condition for cooperation to evolve: if it does not hold, then if every individual invests x > 0, then each receives a lower payoff than if each had invested 0. In a well-mixed population (i.e., without assortativity) the adaptive dynamics of the investment x is given by ˙x = −C′(x), and thus since C′(x) > 0, for all x, it follows that x evolves to 0, the complete defection state. Hence, the CD game is a social dilemma: evolutionary dynamics results in all individuals making zero investment, with each individual consequently obtaining zero payoff, however, if each individual invested x ∈(0, xc), then all individuals would receive a payoff π(x, x) = B(x) −C(x) > 0. For an assortatively-mixed population of individuals with assortativity r, from Equations (18) and (20), we can write the invasion ﬁtness φr x(y) as φr x(y) = r[B(y) −B(x)] −C(y) + C(x). 2.2.2. Continuous Donation Game (21) φr x(y) = r[B(y) −B(x)] −C(y) + C(x). (21) The adaptive dynamics of the investment x is governed by The adaptive dynamics of the investment x is governed by ˙x = D(x) = ∂φr x(y) ∂y y=x = rB′(x) −C′(x). (22) (22) We note here that under quite general conditions the ﬁnal evolutionary outcome of the CD game in an assortatively-interacting population is completely determined by the analog of Hamilton’s inequality described above. Let us assume ﬁrst that there is no singular strategy x⋆∈(0, xm). In this case, if r > ρ = C′(0) B′(0) then it follows from the continuity of D(x) that D(x) > 0, for all x ∈(0, xm), and thus x will evolve to the maximally cooperative state x = xm. If, on the other hand, r < ρ = C′(0) B′(0) then x will evolve to the totally defective state x = 0. Let us now assume that there exists a unique singular strategy x⋆∈(0, xm), which is non-degenerate (i.e., D′(x⋆) ̸= 0). In this case, if r > ρ = C′(0) B′(0) then it follows from the continuity of D(x) that D′(x⋆) < 0, and thus x⋆is convergent stable, and as shown above x⋆is therefore also an ESS. Consequently, the population will evolve to a ﬁnal state in which all individuals are investing x⋆. If, however, r < ρ = C′(0) B′(0) then D′(x⋆) > 0, and hence x⋆is a repeller. In this latter case, if the initial state of the population x0 < x⋆then the population will evolve to the total defection state x = 0, while if x0 > x⋆then the population will evolve to the maximally cooperative state x = xm. Convex Cost and Concave Beneﬁt Functions Now we consider the case in which the cost function is convex (i.e., C′′(x) > 0) and the beneﬁt function is concave (B′′(x) < 0). There is good evidence that in many situations the beneﬁt function exhibits diminishing returns for sufﬁciently large levels of investment [108–110], and the cost is often well described by a convex quadratic function [111]. Thus, as an illustrative example we take the cost and beneﬁt functions to be quadratic functions of the investment x: C(x) = c1x2 and B(x) = −b2x2 + b1x, where c1, b1, b2, > 0. The evolution of cooperation is determined by the analog of Hamilton’s inequality given above: namely, cooperation will evolve if r > ρ = C′(0) B′(0). We now make the interesting observation that ρ = 0 (since C′(0) = 0 and B′(0) = b1 > 0) and thus the inequality r > ρ is satisﬁed for any r > 0. Hence, in this case a non-zero level of cooperation will evolve for any positive degree of assortativity. We obtain the singular strategy x⋆for the game as x⋆= rb1 2rb2 + 2c2 . (23) (23) We now choose xm = 1, and for simplicity take b1 = 2b2. Therefore, We now choose xm = 1, and for simplicity take b1 = 2b2. Therefore, x⋆= 1 1 + 2c1 rb1 , (24) (24) and x⋆∈[0, 1]. Since rB′′(x⋆) −C′′(x⋆) = −2b2r −2c1 < 0, the singular strategy x⋆is convergent stable, and hence also evolutionarily stable. As a result, an initially monomorphic population in which every individual invests any amount x0 ∈[0, 1], will evolve to a ﬁnal state in which all individuals cooperate by investing x⋆given by Equation (24). We note in particular that the completely defective initial state x0 = 0 will evolve to the cooperative state given by x⋆. Thus, in this case, cooperation will always evolve from the completely defective initial state x0 = 0 for any non-zero degree of assortativity. Furthermore, since ∂x⋆ ∂r = 2c1 r2b1(1+ 2c1 rb1 )2 > 0, the cooperative 1 investment x⋆made in the ﬁnal state increases with assortativity r. Therefore, assortative interactions provide a powerful mechanism for the origin and maintenance of cooperation in the social dilemma deﬁned by the CD game. Linear Cost and Beneﬁt Functions Suppose the cost and beneﬁt functions are linear functions of the investment x, i.e., suppose C(x) = cx and B(x) = bx, where b > c. Linear cost and beneﬁt functions are interesting because they arise as approximations to more general cost and beneﬁt functions. From Equation (22), ˙x = D(x) = rb −c. Since D(x) is constant, there are no singular strategies for the game, and the evolutionary fate of a mutant strategy is determined solely by the sign of D(x). An initially monomorphic population in which every individual invests an amount x0 ∈[0, xm], will evolve to the maximally cooperative state in which all individuals invest xm if r > ρ = C′(0) B′(0) = c b. If on the other hand the inequality is reversed, then the population will evolve to the state in which all individuals defect by making zero investment. Note that in this case the condition that governs the evolution of cooperation in the CD game is formally identical to the classical Hamilton’s rule [107]. Games 2020, 11, 41 15 of 31 2.2.3. Continuous Snowdrift Game Saturating beneﬁts are clearly realistic [108–110], and decelerating costs are reasonable when the initiation of cooperative acts is more costly than subsequent increases in cooperative investments. Suppose, as an illustrative example, that we take the cost and beneﬁt functions to be quadratic functions, i.e, suppose C(x) = −c2x2 + c1x and B(z) = −b2z2 + b1z, where c1, c2, b1, b2, > 0. We obtain the singular strategy x⋆for the CSD game as We obtain the singular strategy x⋆for the CSD game as x⋆= (1 + r)b1 −c1 4(1 + r)b2 −2c2 . (28) (28) The singular strategy is convergent stable if The singular strategy is convergent stable if b2 > c2 2(1 + r), (29) (29) and a repeller if the inequality is reversed. The singular strategy is an ESS if b2 > c2 1 + 3r, (30) b2 > c2 1 + 3r, (30) and an EBP if the inequality is reversed. Similar conditions for the convergence and evolutionary stability of a singular strategy are derived in [101] for a nonlinear public goods game with assortative interactions, in which relatedness is deﬁned as the expected value of a fraction of the group that is identical by descent to the focal individual. Thus, an initially monomorphic population in which every individual is investing x0 ∈[0, xm] will evolve to a ﬁnal state that crucially depends on the coefﬁcients c1, c2, b1, and b2 of the cost and beneﬁt functions, and on the assortativity r. If Equation (29) is satisﬁed, then the population initially evolves to a state in which all individuals are investing x⋆—the fate of the population thereafter depends on whether or not Equation (30) is satisﬁed. If it is satisﬁed, then x⋆is an ESS and the population remains at this end state permanently. Otherwise, x⋆is an EBP and the population splits into two distinct phenotypic clusters, which diverge evolutionarily from each other. If, however, Equation (30) is not satisﬁed, then x⋆is a repeller, and the fate of the population depends on the initial strategy x0—if x0 < x⋆, then the population evolves to the zero-investment state x = 0, while, if x0 > x⋆, then the population evolves to the maximum-investment state x = xm. 2.2.3. Continuous Snowdrift Game Consider again the case of two individuals, John and Bill, who are stuck in a car on their way home because the road is blocked by a snowdrift. Let us now assume that the amount of effort invested by each individual in shoveling to clear the snow can vary. Each individual beneﬁts from the total investment that they both make to clear the snow, however, each individual only bears the cost of their own investment. Such interactions among pairs of individuals in which the investment made by each individuals is beneﬁcial to both, but involves a cost only to the investor, can be described using the continuous snowdrift (CSD) game [26]. The CSD game concerns two individuals, making investments x, y ∈ R+, respectively. These investments have the following effects: the payoff of each individual is increased by B(x + y), where B(z) is a function that speciﬁes the beneﬁt to each individual resulting from the total amount of investment made by both participants, and the payoff to the investor, say the x-strategist here, is reduced by C(x), where C(x) is a function that speciﬁes the cost to an individual of making a given investment. Therefore, the payoff π(x, y) to an x-investor interacting with a y-investor is given by π(x, y) = B(x + y) −C(x). (25) (25) π(x, y) = B(x + y) −C(x). Games 2020, 11, 41 16 of 31 We shall again assume that there is an upper limit xm on the possible level of investment, that the cost and beneﬁt functions are smooth and monotonically increasing functions satisfying C(0) = B(0) = 0, and B(x) > C(x), for x ∈(0, xc), where 0 < xc < xm. For an assortatively-mixed population of individuals with assortativity r, from Equations (18) and (25), we can write the invasion ﬁtness φr x(y) as φr x(y) = (1 −r)B(x + y) + rB(2y) −C(y) −B(2x) + C(x). (26) (26) The adaptive dynamics of the investment x is governed by ˙x = D(x) = ∂φr x(y) ∂y y=x (27) = (1 + r)B′(2x) −C′(x). (27) Concave Cost and Beneﬁt Functions Concave Cost and Beneﬁt Functions We now consider the case in which the cost and beneﬁt functions are concave (C′′(x) < 0 and B′′(z) < 0). 2.2.4. Continuous Tragedy of the Commons Game Consider the case of two individuals, John and Bill, who jointly use a ﬁnite common-pool resource, such as a common ﬁshing ground or shared Internet bandwidth. Each of them beneﬁts from consuming the resource, but the costs incurred are shared among both. Such interactions between pairs of individuals sharing a common-pool resource in which consumption of the resource beneﬁts the consuming individual but is costly to both individuals, can be described using the continuous tragedy of the commons (CTOC) game [27]. The CTOC game involves two individuals, making investment x, y ∈R+, where in this context, the investment means the level of consumption of a limited common-pool resource, and thus cooperative behavior is identiﬁed with lower levels of consumption (i.e., lower levels of investment). The investments have the following effects: the payoff of the investor is increased by B(x), where B(x) is a function that speciﬁes the beneﬁt to an individual obtained from consuming a given amount of the resource, and the payoff of each individual is decreased by C(x + y), where C(z) is a function that speciﬁes the cost to both individuals resulting from a given total level of consumption. Therefore, the payoff π(x, y) to an x-investor interacting with a y-investor is given by π(x, y) = B(x) −C(x + y). (31) (31) Again we shall assume that there is an upper limit xm on the possible level of investment, that the cost and beneﬁt functions are smooth and monotonically increasing functions satisfying C(0) = B(0) = 0, and B(x) > C(x), for x ∈(0, xc), where 0 < xc < xm. These assumptions simply reﬂect the fact that the public resource is both ﬁnite and valuable to those consuming it. For an assortatively-mixed population of individuals with assortativity r, from Equations (18) and (31), we can write the invasion ﬁtness φr x(y) as φr x(y) = B(y) −rC(2y) −(1 −r)C(x + y) −B(x) + C(2x). (32) (32) The adaptive dynamics of the investment x is governed by The adaptive dynamics of the investment x is governed by ˙x = D(x) = ∂φr x(y) ∂y y=x = B′(x) −(1 + r)C′(2x). (33) (33) Convex Cost and Sigmoidal Beneﬁt Functions Convex Cost and Sigmoidal Beneﬁt Functions Convex Cost and Sigmoidal Beneﬁt Functions We now consider the case in which the cost function is convex and the benefit function is sigmoidal. 2.2.3. Continuous Snowdrift Game We note that ∂x⋆ ∂r = 2(2b2c1 −b1c2) > 0, if c1 b1 > c2 2b2 , and thus if the latter inequality holds then increasing assortativity results in higher levels of cooperation x⋆. For given cost and beneﬁt functions, both the location and the nature of the singular strategy x⋆varies as the degree of assortative interactions r changes. The varying nature 17 of 31 Games 2020, 11, 41 of the singular strategy x⋆with r in turn results in very different end-states for the evolutionary dynamics. These end-states are: an evolutionary repeller in which all individuals either invest nothing or invest the maximum possible amount, depending on the initial state of the population; an ESS, in which all individuals invest the same amount, given by the singular strategy x⋆; or an end-state in which evolutionary branching has occurred, leading to the coexistence of high and low investing individuals. This latter outcome represents a two-fold social dilemma—referred to as the “tragedy of the commune” [26]—not only is the total level of investment socially inefﬁcient, but in addition evolutionary dynamics forces an unequal division in the levels of investment. We observe from Equation (30) that increasing assortativity inhibits evolutionary branching in the CSD game. Thus, assortative interactions have a dual action on the evolutionary dynamics of the CSD game—increased assortativity ﬁrst leads to higher levels of cooperation and second reduces the potential for evolutionary branching, thereby reducing the likelihood of unequal levels of cooperation. and an EBP if the inequality is reversed. Thus, an initially monomorphic population in which every individual is investing x0 ∈[0, xm] will evolve to an end-state that depends crucially on the coefﬁcients c1, b1, and b3 of the cost and beneﬁt functions, and on the assortativity r. The population ﬁrst evolves to a state in which all individuals are investing x⋆, and the subsequent fate of the population depends on whether or not Equation (35) is satisﬁed. If it is satisﬁed, then x⋆is an ESS, and the population remains in this state permanently. Otherwise, x⋆is an EBP and the population splits into two distinct and diverging phenotypic clusters. We note that ∂x⋆ ∂r = 4b1( 4rb1 √ 16r2b2 1+12b1b3 −1) 6b3 < 0, if 4rb1 √ 16r2b2 1+12b1b3 < 1, and thus if the latter inequality ho then increasing assortativity results in higher levels of cooperation, i.e, lower values of x⋆. For given cost and beneﬁt functions, the location and the type of the singular strategy x⋆varies as the degree of assortative interactions r varies. Thus, as the degree of assortative interactions r changes, so does the form of the evolutionary dynamics. The end-state of the evolutionary dynamics can be either an ESS in which all individuals consume the same amount x⋆of the common resource, or it can be an end-state in which evolutionary branching has taken place, resulting in the coexistence of high and low consuming individuals. This latter outcome represents a second tragedy of the commons—not only is the resource over consumed to the detriment of all, but evolutionary dynamics forces an unequal division degree of consumption. We observe from Equation (35) that increasing assortativity inhibits evolutionary branching in the CTOC game. Thus, just as in the case of the CSD game, assortative interactions have a double action on the evolutionary dynamics of the CTOC game, with increased assortativity resulting both in higher levels of cooperation and in a reduced likelihood of unequal levels of consumption. 2.3. Individual-Based Model An individual-based model (IBM), or agent-based model [112], provides a natural alternative method of studying the evolutionary dynamics of a population with assortative interactions, which is complementary to the deterministic approaches discussed above. 2.2.4. Continuous Tragedy of the Commons Game Accelerating costs represent a realistic assumption and are often observed in nature [111]. 18 of 31 Games 2020, 11, 41 Benefits are also often accelerating initially and then saturate, resulting in sigmoidal benefit functions [21]. Suppose, as an illustrative example of this type of cost and benefit functions, we take the cost function to be a quadratic function and the benefit function to be a cubic function, i.e, suppose C(z) = c1z2 and B(x) = −b3x3 + b2x2 + b1x, where c1, b1, b2, b3 > 0. To simplify the analysis, we take b2 = 2b1 and c1 = b1. We obtain the singular strategy x⋆for the CTOC game as x⋆= q 16r2b2 1 + 12b1b3 −4rb1 6b3 . (34) (34) 6b3 The singular strategy is always convergent stable. Moreover, it is an ESS if The singular strategy is always convergent stable. Moreover, it is an ESS if b1 < q 16r2b2 1 + 12b1b3 2(1 −r) , (35) (35) and an EBP if the inequality is reversed. 2.3.1. Discrete Games We assume here that the individuals in the population play a symmetric 2 × 2 game with the payoff matrix given by Equation (4). We consider a population of n individuals, with an initial strategy proﬁle s0 = {s0 1, s0 2, . . . , s0 n} at generation 0, where s0 i ∈{C, D}, for 1 ≤i ≤n. Each generation of the evolutionary dynamics consists of an asynchronous interaction/update round, which involves sampling the population n times with replacement. Each interaction/update step at generation t is carried out as follows: in the interaction phase we pick uniformly at random two individuals i and j from the population. The two individuals 19 of 31 19 of 31 Games 2020, 11, 41 i and j interact (i.e., play the symmetric 2 × 2 game) with individuals k ̸= i and l ̸= j respectively. The interactions are assortative, i.e., with probability r, the individual k (respectively l) is of the same strategy type as i (respectively j), and with probability 1 −r, the individual k (respectively l) is chosen uniformly at random from the population. The change of individual i’s strategy from one generation to the next is determined by the payoff that player i receives from interacting with player k and the payoff that player j receives from interacting with player l. Precisely, if st−1 i , st−1 j , st−1 k , and st−1 l denote the strategies of i, j, k, and l, respectively, in generation t −1, then in generation t the payoff Pi received by individual i is π(st−1 i , st−1 k ) and the payoff Pj received by individual j is π(st−1 j , st−1 l ), where π is the payoff matrix for the game under consideration. In the update phase the probability that the focal individual i will inherit j’s strategy, pi←j, is determined using the Fermi update rule as [19] pi←j = 1 1 + e−β(Pj−Pi) , (36) (36) where the parameter β > 0 is the “selection strength” of the update rule. re the parameter β > 0 is the “selection strength” of the update rule. where the parameter β > 0 is the “selection strength” of the update rule. We note that the results of the individual-based simulations described in the next section are robust to changes in the update rule. 2.3.1. Discrete Games For example, in addition to employing the Fermi update rule given by Equation (36), we have also simulated the individual-based model using the replicator update rule, in which the probability pi←j that the focal individual i inherits individual j’s strategy is given by [19] pi←j = ( 0 if Pi ≥Pj Pj−Pi Pmax−Pmin otherwise, (37) (37) where Pmax = max(P1, P2, . . . , Pn), and Pmin = min(P1, P2, . . . , Pn). We ﬁnd that the evolutionary dynamics of the symmetric 2 × 2 games that we study is essentially identical irrespective of which of these update rules we employ. The results presented in the next section arise from simulations using the Fermi update rule (Equation (36)). 3.1.1. Donation Game Figure 4a,b, respectively, show how the analytically predicted and simulated values of p∞vary with the assortativity r ∈[0, 1] and the cost-to-beneﬁt ratio ρ ∈(0, 1). Figure 4c shows how p∞varies with r when ρ = 0.25 and Figure 4d shows how p∞varies with ρ when r = 0.25. The results are in excellent agreement with the analysis given above, showing that a population of individuals playing the donation game will evolve to the all-cooperator state (p∞= 1) if r > ρ and to the all-defector state (p∞= 0) if the inequality is reversed. 0.0 0.2 0.4 0.6 0.8 1.0 assortativity r 0.0 0.2 0.4 0.6 0.8 1.0 cost-to-benefit ratio ρ 0.0 0.2 0.4 0.6 0.8 1.0 asymptotic frequency of cooperation p∞ (a) 0.0 0.2 0.4 0.6 0.8 1.0 assortativity r 0.0 0.2 0.4 0.6 0.8 1.0 cost-to-benefit ratio ρ 0.0 0.2 0.4 0.6 0.8 asymptotic frequency of cooperation p∞ (b) 0.0 0.2 0.4 0.6 0.8 1.0 assortativity r 0.0 0.2 0.4 0.6 0.8 1.0 asymptotic frequency of cooperation p∞ simulated predicted (c) 0.0 0.2 0.4 0.6 0.8 cost-to-benefit ratio ρ 0.0 0.2 0.4 0.6 0.8 1.0 asymptotic frequency of cooperation p∞ simulated predicted (d) Figure 4. Variation of the long-term frequency p∞of cooperators with assortativity r ∈[0, 1] and cost-to-beneﬁt ratio ρ ∈(0, 1) for the donation game. (a) p∞(analytically predicted) versus r and ρ. (b) p∞(simulated) versus r and ρ. (c) p∞versus r when ρ = 0.25. (d) p∞versus ρ when r = 0.25. Parameters: n = 10,000, p0 = 0.5, and β = 1. 0.0 0.2 0.4 0.6 0.8 1.0 assortativity r 0.0 0.2 0.4 0.6 0.8 1.0 cost-to-benefit ratio ρ 0.0 0.2 0.4 0.6 0.8 asymptotic frequency of cooperation p∞ (b) 0.0 0.2 0.4 0.6 0.8 1.0 assortativity r 0.0 0.2 0.4 0.6 0.8 1.0 cost-to-benefit ratio ρ 0.0 0.2 0.4 0.6 0.8 1.0 asymptotic frequency of cooperation p∞ (a) (a) (b) 0.0 0.2 0.4 0.6 0.8 cost-to-benefit ratio ρ 0.0 0.2 0.4 0.6 0.8 1.0 asymptotic frequency of cooperation p∞ simulated predicted (d) 0.0 0.2 0.4 0.6 0.8 1.0 assortativity r 0.0 0.2 0.4 0.6 0.8 1.0 asymptotic frequency of cooperation p∞ simulated predicted (c) (d) (c) Figure 4. Variation of the long-term frequency p∞of cooperators with assortativity r ∈[0, 1] and cost-to-beneﬁt ratio ρ ∈(0, 1) for the donation game. (a) p∞(analytically predicted) versus r and ρ. (b) p∞(simulated) versus r and ρ. 2.3.2. Continuous Games An IBM also provides a natural way of studying the evolutionary dynamics of a population playing a pairwise continuous game with assortative interactions. We again consider a population of n individuals, now with an initial monomorphic strategy proﬁle x0 = {x0 1, x0 2, . . . , x0 n} in generation 0, where x0 1 = x0 2 = · · · = x0 n = x0 ∈(0, xm) and xm ∈R+. Thus in generation t = 0, every individual in the population uses the strategy x0. The interactions/update rounds are carried out in the same manner as in the case of discrete games, but with the following additional step: if during the update round at generation t, the focal individual i would have inherited the strategy xt−1 j of the individual j, then with probability µ it instead inherits a mutation of this strategy, picked from a normal distribution with mean xt−1 j and standard deviation σ. If the strategy space is a ﬁnite interval [a, b] then the mutations are taken from a truncated normal distribution on [a, b]. We note here that changing the precise way in which the random numbers are generated does not effect the outcome of the adaptive dynamics. For example, it was shown in [113] that including random numbers that are uniformly distributed in addition to having random numbers taken from a truncated normal distribution does not signiﬁcantly change the evolutionary dynamics of the system considered in that paper. The simulation results presented in the section below are based on the Fermi update rule given by Equation (36). We have also simulated the IBM using the replicator update rule (Equation (37)), and have found the results obtained using both rules to be qualitatively identical. 20 of 31 Games 2020, 11, 41 3.1. Discrete Games In this subsection we present the main results of simulations using the IBM introduced above for the donation, snowdrift, and sculling games. Additional results can be found in the Supplementary Materials document. 3.1.1. Donation Game (c) p∞versus r when ρ = 0.25. (d) p∞versus ρ when r = 0.25. Parameters: n = 10,000, p0 = 0.5, and β = 1. 3.1.2. Snowdrift Game Figure 5a,b, respectively, show how the analytically predicted and simulated values of p∞vary with the assortativity r ∈[0, 1] and the cost-to-beneﬁt ratio ρ ∈(0, 1). Figure 5c shows how p∞varies with r when ρ = 0.75 and Figure 5d shows how p∞varies with ρ when r = 0.25. The results agree very well with the analysis, which indicates that in a population of individuals playing the snowdrift game, increasing the assortativity r has the effect of increasing the fraction of cooperators at equilibrium, while increasing the cost-to-beneﬁt ratio ρ has the effect of decreasing the fraction of cooperators at Figure 5a,b, respectively, show how the analytically predicted and simulated values of p∞vary with the assortativity r ∈[0, 1] and the cost-to-beneﬁt ratio ρ ∈(0, 1). Figure 5c shows how p∞varies with r when ρ = 0.75 and Figure 5d shows how p∞varies with ρ when r = 0.25. The results agree very well with the analysis, which indicates that in a population of individuals playing the snowdrift game, increasing the assortativity r has the effect of increasing the fraction of cooperators at equilibrium, while increasing the cost-to-beneﬁt ratio ρ has the effect of decreasing the fraction of cooperators at 21 of 31 21 of 31 Games 2020, 11, 41 equilibrium. Thus, cooperation is promoted by increasing assortativity r, with a transition to complete cooperation occurring when r > ρ 2. 0.0 0.2 0.4 0.6 0.8 1.0 assortativity r 0.0 0.2 0.4 0.6 0.8 1.0 cost-to-benefit ratio ρ 0.2 0.4 0.6 0.8 1.0 asymptotic frequency of cooperation p∞ (a) 0.0 0.2 0.4 0.6 0.8 1.0 assortativity r 0.0 0.2 0.4 0.6 0.8 1.0 cost-to-benefit ratio ρ 0.0 0.2 0.4 0.6 0.8 asymptotic frequency of cooperation p∞ (b) 0.0 0.2 0.4 0.6 0.8 1.0 assortativity r 0.0 0.2 0.4 0.6 0.8 1.0 asymptotic frequency of cooperation p∞ simulated predicted (c) 0.0 0.2 0.4 0.6 0.8 1.0 cost-to-benefit ratio ρ 0.0 0.2 0.4 0.6 0.8 1.0 asymptotic frequency of cooperation p∞ simulated predicted (d) Figure 5. Variation of the long-term frequency p∞of cooperators with assortativity r ∈[0, 1] and cost-to-beneﬁt ratio ρ ∈(0, 1) for the snowdrift game. (a) p∞(analytically predicted) versus r and ρ. (b) p∞(simulated) versus r and ρ. (c) p∞versus r when ρ = 0.75. (d) p∞versus ρ when r = 0.25. Parameters: n = 10,000, p0 = 0.5, and β = 1. 3.1.2. Snowdrift Game 0.0 0.2 0.4 0.6 0.8 1.0 assortativity r 0.0 0.2 0.4 0.6 0.8 1.0 cost-to-benefit ratio ρ 0.0 0.2 0.4 0.6 0.8 asymptotic frequency of cooperation p∞ (b) 0.0 0.2 0.4 0.6 0.8 1.0 assortativity r 0.0 0.2 0.4 0.6 0.8 1.0 cost-to-benefit ratio ρ 0.2 0.4 0.6 0.8 1.0 asymptotic frequency of cooperation p∞ (a) (b) (a) 0.0 0.2 0.4 0.6 0.8 1.0 cost-to-benefit ratio ρ 0.0 0.2 0.4 0.6 0.8 1.0 asymptotic frequency of cooperation p∞ simulated predicted (d) 0.0 0.2 0.4 0.6 0.8 1.0 assortativity r 0.0 0.2 0.4 0.6 0.8 1.0 asymptotic frequency of cooperation p∞ simulated predicted (c) (d) (c) Figure 5. Variation of the long-term frequency p∞of cooperators with assortativity r ∈[0, 1] and cost-to-beneﬁt ratio ρ ∈(0, 1) for the snowdrift game. (a) p∞(analytically predicted) versus r and ρ. (b) p∞(simulated) versus r and ρ. (c) p∞versus r when ρ = 0.75. (d) p∞versus ρ when r = 0.25. Parameters: n = 10,000, p0 = 0.5, and β = 1. 3.1.3. Sculling Game Figure 6a,b respectively show how the predicted and simulated values of p∞vary with assortativity r ∈[0, 1] and cost-to-beneﬁt ratio ρ ∈( 1 2, 3 2). Figure 6c shows how p∞varies with r when ρ = 1.2 and Figure 6d shows how p∞varies with ρ when r = 0.25. The results are in very good agreement with the analysis, which indicates that in a population of individuals playing the sculling game, increasing the assortativity r has the effect of increasing the basin of attraction around ˆp = 1, thus decreasing the value of the initial fraction p0 of cooperators that is needed for the population to evolve to the all-cooperator state. Contrarily, increasing the cost-to-beneﬁt ratio ρ has the effect of increasing the basin of attraction around ˆp = 0, thus increasing the value of the initial fraction p0 of cooperators that is needed for the population to evolve to the all-cooperator state. Thus, cooperation is promoted by increasing assortativity r, with a transition to complete cooperation occurring when r > 2ρ 3 −1 3. 22 of 31 Games 2020, 11, 41 0.0 0.2 0.4 0.6 0.8 1.0 assortativity r 0.6 0.8 1.0 1.2 1.4 cost-to-benefit ratio ρ 0.0 0.2 0.4 0.6 0.8 1.0 asymptotic frequency of cooperation p∞ (a) 0.0 0.2 0.4 0.6 0.8 1.0 assortativity r 0.6 0.8 1.0 1.2 1.4 cost-to-benefit ratio ρ 0.0 0.2 0.4 0.6 0.8 asymptotic frequency of cooperation p∞ (b) 0.0 0.2 0.4 0.6 0.8 1.0 assortativity r 0.0 0.2 0.4 0.6 0.8 1.0 asymptotic frequency of cooperation p∞ simulated predicted (c) 0.6 0.8 1.0 1.2 1.4 cost-to-benefit ratio ρ 0.0 0.2 0.4 0.6 0.8 1.0 asymptotic frequency of cooperation p∞ simulated predicted (d) Figure 6. Variation of the long-term frequency p∞of cooperators with assortativity r ∈[0, 1] and cost-to-beneﬁt ratio ρ ∈( 1 2, 3 2) for the sculling game. (a) p∞(analytically predicted) versus r and ρ. (b) p∞(simulated) versus r and ρ. (c) p∞versus r when ρ = 1.2. (d) p∞versus ρ when r = 0.25. Parameters: n = 10,000, p0 = 0.5, and β = 1. 3.1.3. Sculling Game 0.0 0.2 0.4 0.6 0.8 1.0 assortativity r 0.6 0.8 1.0 1.2 1.4 cost-to-benefit ratio ρ 0.0 0.2 0.4 0.6 0.8 1.0 asymptotic frequency of cooperation p∞ (a) 0.0 0.2 0.4 0.6 0.8 1.0 assortativity r 0.6 0.8 1.0 1.2 1.4 cost-to-benefit ratio ρ 0.0 0.2 0.4 0.6 0.8 asymptotic frequency of cooperation p∞ (b) (b) 0.0 0.2 0.4 0.6 0.8 1.0 assortativity r 0.0 0.2 0.4 0.6 0.8 1.0 asymptotic frequency of cooperation p∞ simulated predicted (c) 0.6 0.8 1.0 1.2 1.4 cost-to-benefit ratio ρ 0.0 0.2 0.4 0.6 0.8 1.0 asymptotic frequency of cooperation p∞ simulated predicted (d) (d) (c) Figure 6. Variation of the long-term frequency p∞of cooperators with assortativity r ∈[0, 1] and cost-to-beneﬁt ratio ρ ∈( 1 2, 3 2) for the sculling game. (a) p∞(analytically predicted) versus r and ρ. (b) p∞(simulated) versus r and ρ. (c) p∞versus r when ρ = 1.2. (d) p∞versus ρ when r = 0.25. Parameters: n = 10,000, p0 = 0.5, and β = 1. 3.2. Continuous Games In this subsection we present the main results of simulations using the IBM introduced above for the continuous donation (CD), continuous snowdrift (CSD), and continuous tragedy of the commons (CTOC) games. Additional results can be found in the Supplementary Materials document. In this subsection we present the main results of simulations using the IBM introduced above for the continuous donation (CD), continuous snowdrift (CSD), and continuous tragedy of the commons (CTOC) games. Additional results can be found in the Supplementary Materials document. 3.2.1. Continuous Donation Game 3.2.1. Continuous Donation Game 3.2.1. Continuous Donation Game We ﬁrst consider the CD game with linear cost and beneﬁt functions C(x) = cx and B(x) = bx, where b > c. We denote the ratio c b by ρ and refer to it as the cost-to-beneﬁt ratio. The condition r > ρ that promotes cooperative investments, where r is the degree of assortativity, is similar to the one for the discrete donation game, and hence we study the CD game using a similar set of plots. We ﬁrst consider the CD game with linear cost and beneﬁt functions C(x) = cx and B(x) = bx, where b > c. We denote the ratio c b by ρ and refer to it as the cost-to-beneﬁt ratio. The condition r > ρ that promotes cooperative investments, where r is the degree of assortativity, is similar to the one for the discrete donation game, and hence we study the CD game using a similar set of plots. Figure 7a,b, respectively, show how the analytically predicted and simulated values of the long-term mean strategy ¯x∞(taken over the last 10% of the generations) varies with the assortativity r ∈[0, 1] and the cost-to-beneﬁt ratio ρ ∈(0, 1). Figure 7c shows how ¯x∞varies with r when ρ = 0.26 and Figure 7d shows how x∞varies with ρ when r = 0.26. The results are in excellent agreement with the analysis, which shows that in a population of individuals playing the CD game with linear cost and beneﬁt functions, individuals will asymptotically invest the maximum amount xm if r > ρ and make zero investment if the inequality is reversed. 23 of 31 Games 2020, 11, 41 0.0 0.2 0.4 0.6 0.8 1.0 assortativity r 0.0 0.2 0.4 0.6 0.8 1.0 cost-to-benefit ratio ρ 0.0 0.2 0.4 0.6 0.8 1.0 asymptotic strategy ¯x∞ (a) 0.0 0.2 0.4 0.6 0.8 1.0 assortativity r 0.0 0.2 0.4 0.6 0.8 1.0 cost-to-benefit ratio ρ 0.2 0.4 0.6 0.8 asymptotic strategy ¯x∞ (b) 0.0 0.2 0.4 0.6 0.8 1.0 assortativity r 0.0 0.2 0.4 0.6 0.8 1.0 asymptotic strategy ¯x∞ simulated predicted (c) 0.0 0.2 0.4 0.6 0.8 1.0 cost-to-benefit ratio ρ 0.0 0.2 0.4 0.6 0.8 1.0 asymptotic strategy ¯x∞ simulated predicted (d) Figure 7. 3.2.1. Continuous Donation Game Variation of the long-term mean strategy ¯x∞with assortativity r ∈[0, 1] and cost-to-beneﬁt ratio ρ ∈(0, 1) in the CD game with linear cost and beneﬁt functions: C(x) = cx and B(x) = bx with b > c. (a) ¯x∞(analytically predicted) versus r and ρ. (b) ¯x∞(simulated) versus r and ρ. (c) ¯x∞versus r when ρ = 0.26. (d) ¯x∞versus ρ when r = 0.26. Parameters: n =10,000, x0 = 0.1, xm = 1, µ = 0.01, σ = 0.005, and β = 1. 0.0 0.2 0.4 0.6 0.8 1.0 assortativity r 0.0 0.2 0.4 0.6 0.8 1.0 cost-to-benefit ratio ρ 0.0 0.2 0.4 0.6 0.8 1.0 asymptotic strategy ¯x∞ (a) 0.0 0.2 0.4 0.6 0.8 1.0 assortativity r 0.0 0.2 0.4 0.6 0.8 1.0 cost-to-benefit ratio ρ 0.2 0.4 0.6 0.8 asymptotic strategy ¯x∞ (b) (b) 0.0 0.2 0.4 0.6 0.8 1.0 assortativity r 0.0 0.2 0.4 0.6 0.8 1.0 asymptotic strategy ¯x∞ simulated predicted (c) 0.0 0.2 0.4 0.6 0.8 1.0 cost-to-benefit ratio ρ 0.0 0.2 0.4 0.6 0.8 1.0 asymptotic strategy ¯x∞ simulated predicted (d) (d) (c) Figure 7. Variation of the long-term mean strategy ¯x∞with assortativity r ∈[0, 1] and cost-to-beneﬁt ratio ρ ∈(0, 1) in the CD game with linear cost and beneﬁt functions: C(x) = cx and B(x) = bx with b > c. (a) ¯x∞(analytically predicted) versus r and ρ. (b) ¯x∞(simulated) versus r and ρ. (c) ¯x∞versus r when ρ = 0.26. (d) ¯x∞versus ρ when r = 0.26. Parameters: n =10,000, x0 = 0.1, xm = 1, µ = 0.01, σ = 0.005, and β = 1. We next consider the CD game with quadratic cost and beneﬁt functions C(x) = c1x2 and B(x) = −b2x2 + b1x, where c1, b1, b2 > 0. We let b1 = 2b2, in which case the singular strategy, given by Equation (24), is convergent stable and hence also evolutionarily stable. We next consider the CD game with quadratic cost and beneﬁt functions C(x) = c1x2 and B(x) = −b2x2 + b1x, where c1, b1, b2 > 0. We let b1 = 2b2, in which case the singular strategy, given by Equation (24), is convergent stable and hence also evolutionarily stable. 3.2.1. Continuous Donation Game q ( ), g y Figure 8 shows the variation of the distribution of the long-term values x∞(taken over the last 10% of the generations) of strategies with assortativity r; the dotted line indicates the singular strategy x⋆. The results are in very good agreement with the analysis, which indicates that in a population of individuals playing the CD game with quadratic cost and beneﬁt functions, individuals will in the long term invest an amount given by the evolutionarily stable singular strategy x⋆, which increases with assortativity r. Figure 8 shows the variation of the distribution of the long-term values x∞(taken over the last 10% of the generations) of strategies with assortativity r; the dotted line indicates the singular strategy x⋆. The results are in very good agreement with the analysis, which indicates that in a population of individuals playing the CD game with quadratic cost and beneﬁt functions, individuals will in the long term invest an amount given by the evolutionarily stable singular strategy x⋆, which increases with assortativity r. 24 of 31 Games 2020, 11, 41 0.0 0.2 0.4 0.6 0.8 1.0 assortativity r 0.0 0.2 0.4 0.6 0.8 1.0 asymptotic distribution of strategies x∞ singular strategy x 0.0 0.2 0.4 0.6 0.8 1.0 assortativity r 0.0 0.2 0.4 0.6 0.8 1.0 asymptotic distribution of strategies x∞ singular strategy x Figure 8. Variation of the distribution of long-term strategy values x∞with assortativity r in the CD game with quadratic cost and beneﬁt functions: C(x) = x2 and B(x) = −x2 + 2x. Parameters: x0 = 0.1, xm = 1, µ = 0.01, σ = 0.005, and β = 1. Arrows indicate the direction of evolutionary change. Figure 8. Variation of the distribution of long-term strategy values x∞with assortativity r in the CD game with quadratic cost and beneﬁt functions: C(x) = x2 and B(x) = −x2 + 2x. Parameters: x0 = 0.1, xm = 1, µ = 0.01, σ = 0.005, and β = 1. Arrows indicate the direction of evolutionary change. 3.2.2. Continuous Snowdrift Game 3.2.2. Continuous Snowdrift Game The next game we consider is the CSD game with quadratic cost and beneﬁt functions C(x) = −c2x2 + c1x2 and B(x) = −b2x2 + b1x, where c1, c2, b1, b1 > 0. The singular strategy for the game, given by Equation (28), is convergent stable if the inequality given by Equation (29) is satisﬁed and a repeller if the inequality is reversed. On the other hand, the singular strategy is an ESS if the inequality given by Equation (30) is satisﬁed and an EBP otherwise. Figure 9a,b show the variation of the distribution of the asymptotic strategy values x∞(taken over the last 10% of the generations) with assortativity r. The dotted line indicates the singular strategy x⋆. The value of r in (a) at which the singular strategy transitions from an EBP to an ESS is determined by equating the two sides of Equation (30) and solving for r. For the given choice of parameters, the transition point (indicated by the dashed vertical line) is r = 0.2. Similarly, the value of r in (b) where the singular strategy changes from a repeller where every individual makes zero asymptotic investment to one in which all individuals make the maximum asymptotic investment xm can be obtained from Equation (29). For the given choice of parameters, the transition point (indicated by the dashed vertical line) is r = 0.13. The results are in very good agreement with the analysis, which shows that in a population of individuals playing the CSD game with quadratic cost and beneﬁt functions, for suitable values of the coefﬁcients, the singular strategy can change from an EBP to an ESS as the assortativity increases. Similar results are presented in [101] for a nonlinear public goods game with assortative interactions, in which evolutionary attractors increase with relatedness (deﬁned as the expected value of a fraction of the group that is identical by descent to the focal individual) while evolutionary repellers decrease with relatedness. 25 of 31 Games 2020, 11, 41 0.0 0.1 0.2 0.3 0.4 0.5 0.6 assortativity r 0.0 0.2 0.4 0.6 0.8 1.0 asymptotic distribution of strategies x∞ singular strategy x (a) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 assortativity r 0.0 0.2 0.4 0.6 0.8 1.0 asymptotic distribution of strategies x∞ singular strategy x (b) Figure 9. 3.2.2. Continuous Snowdrift Game Variation of the distribution of asymptotic strategy values x∞with assortativity r, in a CSD game with quadratic cost function and quadratic beneﬁt function. (a) C(x) = −1.6x2 + 4.8x and B(x) = −x2 + 5x, and (b) C(x) = −1.5x2 + 4x and B(x) = −0.2x2 + 3x. Parameters: n = 10,000, x0 = 0.3, xm = 1, µ = 0.01, σ = 0.005, and β = 1. Arrows indicate the direction of evolutionary change. 3 2 3 Continuous Tragedy of the Commons Game 0.00 0.05 0.10 0.15 0.20 0.25 0.30 assortativity r 0.0 0.2 0.4 0.6 0.8 1.0 asymptotic distribution of strategies x∞ singular strategy x (b) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 assortativity r 0.0 0.2 0.4 0.6 0.8 1.0 asymptotic distribution of strategies x∞ singular strategy x (a) (b) (a) Figure 9. Variation of the distribution of asymptotic strategy values x∞with assortativity r, in a CSD game with quadratic cost function and quadratic beneﬁt function. (a) C(x) = −1.6x2 + 4.8x and B(x) = −x2 + 5x, and (b) C(x) = −1.5x2 + 4x and B(x) = −0.2x2 + 3x. Parameters: n = 10,000, x0 = 0.3, xm = 1, µ = 0.01, σ = 0.005, and β = 1. Arrows indicate the direction of evolutionary change. 3.2.3. Continuous Tragedy of the Commons Game 3.2.3. Continuous Tragedy of the Commons Game Finally, we consider the CTOC game with quadratic cost and cubic beneﬁt functions C(x) = c1x2 and B(x) = −b3x3 + b2x2 + b1x. If we let b2 = 2b1 and c1 = b1, the singular strategy for the game is given by Equation (34), and is an ESS if the inequality given by Equation (35) is satisﬁed and an EBP otherwise. Figure 10 shows the variation of the distribution of asymptotic strategy values x∞(taken over the last 10% of the generations) with assortativity r. The dotted line indicates the singular strategy x⋆. The value of r at which the singular strategy transitions from an EBP to an ESS is obtained by equating the two sides of Equation (35) and solving for r. For the given choice of parameters, the transition point (indicated by the dashed vertical line) is r = 0.3. 4. Discussion Comparing the condition found in [19] for complete cooperation to occur in the donation game on networks of mean degree k (namely, 1 ρ > k −1) with the condition obtained here for assortative interactions (r > ρ) suggests that the effective degree of assortativity resulting from network interactions in the framework studied in [19] is r = 1 k−1. Now taking this value of r and inserting it into the condition obtained here for the evolution of complete cooperation in the snowdrift game (r > ρ 2) then gives that the transition to complete cooperation in the snowdrift game on a network of mean degree k should be governed by the rule that complete cooperation prevails if 1 ρ > k−1 2 . This latter rule is consistent with the results found in [19]. Similarly, taking the value r = 1 k−1 in the condition derived here for the evolution of cooperation in the sculling game (r > ρ−1 2 −p0 3 2 −p0 , and taking the initial frequency of cooperators to be 1 p0 = 1 2 as was the case in [19]) gives the condition for cooperation to evolve in the sculling game on networks found in [19]: namely, that cooperation prevails if 1 ρ−1 > k −1. Thus we ﬁnd that despite the simple manner in which assortativity has been modeled here, we obtain results which are applicable to much more subtle systems in which assortativity emerges as a complex, self-organized, phenomenon. For the continuous strategy social dilemmas that we have considered—the continuous donation, continuous snowdrift and continuous tragedy of the commons games—we also ﬁnd that the level of cooperation increases with increasing assortativity. An additional interesting ﬁnding, and perhaps the most signiﬁcant result that we have obtained, is that the propensity for evolutionary branching to occur in the continuous snowdrift and continuous tragedy of the commons games is reduced as the degree of assortative interactions increases. Thus, assortativity plays a doubly beneﬁcial role in the evolution of cooperation in these games both by increasing the overall level of cooperation and by reducing the likelihood of unequal and unfair outcomes. We conjecture that this inhibiting effect of assortative interactions on evolutionary branching is a general phenomenon that holds for a broader class of evolutionary systems than merely the continuous strategy games that we have considered here. 4. Discussion In this work we have undertaken a detailed and systematic investigation of the evolution of cooperation in a wide variety of both discrete strategy and continuous strategy social dilemmas with assortative interactions modeled in the manner proposed by [79,80]. For the discrete strategy social dilemmas that we have studied—the donation, snowdrift and sculling games—we ﬁnd that in all cases the frequency of cooperation increases with increasing assortativity. In these social dilemmas the transition to complete cooperation is governed by Hamilton’s rule for the donation game, and natural analogs of it for the snowdrift and sculling games. Thus, the entire population evolves to cooperators in the donation game if r > ρ, in the snowdrift game if r > ρ 2, and in the sculling game if r > ρ−1 2 −p0 3 2 −p0 (where p0 is the initial frequency of cooperators). The results for the donation game are essentially well-known [13,79,80,89–98], while the results for the snowdrift and sculling games are more novel. Interesting related work is [98], which studies the effect of relatedness on the maintenance of cooperation in prisoner’s dilemma, hawk-dove, and stag hunt games. The deﬁnition of assortativity and of the games considered in [98] are formulated differently from those adopted here, and we hope that our results may be complementary to those of [98]. These results apply quite directly to games between relatives due to the fact that Grafen’s geometric interpretation of relatedness allows the degree of assortativity r to be identiﬁed with the degree of relatedness between the interacting individuals [79,80]. Thus, our results indicate that the emergence of complete cooperation in populations of related individuals playing the snowdrift and sculling games should be governed by the variants of Hamilton’s rule described above. g g g y Our results also elucidate the evolution of cooperation in discrete strategy social dilemmas on networks, since evolutionary dynamics in structured populations can result in assortative interactions between the individuals. For example, consider the results obtained for the evolution of cooperation in the donation, snowdrift and sculling games on networks in [19]. 3.2.2. Continuous Snowdrift Game The results are in excellent agreement with the analysis, which show that in a population of individuals playing the CTOC game with quadratic cost and cubic beneﬁt functions, for suitable values of the coefﬁcients, the singular strategy can transition from an EBP to an ESS with increasing assortativity. 0.0 0.2 0.4 0.6 0.8 1.0 assortativity r 0.0 0.5 1.0 1.5 2.0 2.5 3.0 asymptotic distribution of strategies x∞ singular strategy x Figure 10. Variation of the distribution of asymptotic strategy values x∞with assortativity r, in a continuous tragedy of the commons (CTOC) game with quadratic cost and cubic beneﬁt functions: C(x) = x2 and B(x) = −0.0834x3 + 2x2 + x. Parameters: n =10,000, x0 = 0.1, xm = 3, µ = 0.01, σ = 0.005, and β = 1. Arrows indicate the direction of evolutionary change. Figure 10. Variation of the distribution of asymptotic strategy values x∞with assortativity r, in a continuous tragedy of the commons (CTOC) game with quadratic cost and cubic beneﬁt functions: C(x) = x2 and B(x) = −0.0834x3 + 2x2 + x. Parameters: n =10,000, x0 = 0.1, xm = 3, µ = 0.01, σ = 0.005, and β = 1. Arrows indicate the direction of evolutionary change. Games 2020, 11, 41 26 of 31 26 of 31 4. Discussion The interesting work [101] has also explored similar topics to those studied in this paper in that it 27 of 31 Games 2020, 11, 41 considers the effect of relatedness on cooperation in a multi-player version of the continuous snowdrift game. In [101] relatedness is introduced in a different, and more general, manner to our deﬁnition of assortativity, through the consideration of a probability distribution over the number of co-players that are identical-by-descent to a focal individual. Thus, the results obtained in [101] for the evolutionary dynamics of the multi-player continuous snowdrift game include the results we have obtained for the two-player continuous snowdrift game as a special case (the precise connection between the two approaches is that if we take N = 2, Pr(1) = 1 −r and Pr(2) = r in Equation (3) for the invasion ﬁtness in [101] then we obtain an expression that is equivalent to our Equation (26) for the invasion ﬁtness). Of particular interest is the result found in [101] that increased relatedness reduces the possibility of evolutionary branching in the continuous multi-player snowdrift game. Thus, our result that the propensity for evolutionary branching to occur in the continuous snowdrift games decreases as the degree of assortative interactions increases is a special case of the ﬁndings of [101]. Since our formalism for studying the effect of assortativity on the evolution of cooperation in continuous strategy games is different from that of [101] we hope that these distinct methodologies may prove to be complementary. It would also be most interesting to apply the methods of [101] to study the evolutionary dynamics of the continuous donation and continuous tragedy of the commons games. Our results for evolutionary branching in the continuous snowdrift and continuous tragedy of the commons games with assortative interactions imply corresponding results for evolutionary branching in these games played between relatives. We expect, therefore, that evolutionary branching in these games will be inhibited as the degree of relatedness between the interacting individuals increases. Again, it is plausible to conjecture that this phenomenon is not restricted only to those games that we have considered here but holds more widely. Furthermore, we observe that our results on evolutionary branching in the continuous snowdrift and continuous tragedy of the commons games with assortative interactions may have interesting implications for the evolutionary dynamics of these games on networks. 4. Discussion Since one would expect the assortativity produced by interactions on a network to increase as the mean degree of the network decreases, it is therefore natural to conjecture that evolutionary branching in the continuous snowdrift and continuous tragedy of the commons games on networks will be inhibited as the mean degree of the network decreases. We have studied the evolutionary dynamics of the continuous snowdrift and continuous tragedy of the commons games on networks through simulations, and have found that evolutionary branching is inhibited on networks of low mean degree, exactly as predicted by our results (a detailed discussion of the effects of network structure on evolutionary branching in these games will be given elsewhere). Again, it is natural to conjecture that such an increase in the inhibition of evolutionary branching on networks of low mean degree may be a general phenomenon applicable to many evolutionary systems. Supplementary Materials: The following are available at http://www.mdpi.com/2073-4336/11/4/41/s1. Author Contributions: Conceptualization, T.K.; formal analysis, S.I. and T.K.; software, S.I.; visualization, S.I.; writing—original draft, S.I. and T.K.; writing—review and editing, S.I. and T.K. All authors have read and agreed to the published version of the manuscript. 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https://openalex.org/W4293660714	https://archimer.ifremer.fr/doc/00791/90257/95768.pdf	English	null	Rich and underreported: First integrated assessment of the diversity of mesopelagic fishes in the Southwestern Tropical Atlantic	Frontiers in marine science	2,022	cc-by	12,875	OPEN ACCESS OPEN ACCESS EDITED BY Lynne Jane Shannon, University of Cape Town, South Africa REVIEWED BY Cristina Lo´ pez-Pe´ rez, Spanish National Research Council (CSIC), Spain Tracey T. Sutton, Nova Southeastern University, United States CORRESPONDENCE Leandro Nole´ Eduardo leandronole@hotmail.com SPECIALTY SECTION This article was submitted to Marine Conservation and Sustainability, a section of the journal Frontiers in Marine Science RECEIVED 05 May 2022 ACCEPTED 25 July 2022 PUBLISHED 30 August 2022 EDITED BY Lynne Jane Shannon, University of Cape Town, South Africa REVIEWED BY Cristina Lo´ pez-Pe´ rez, Spanish National Research Council (CSIC), Spain Tracey T. Sutton, Nova Southeastern University, United States Leandro Nole´ Eduardo 1,2, Arnaud Bertrand 1,2,3, Fla´via Lucena-Fre´dou 1, Ba´rbara Teixeira Villarins 4, Ju´ lia Rodrigues Martins 4, Gabriel Vinı´cius Felix Afonso 5, Theodore Wells Pietsch 6, Thierry Fre´dou 1, Fabio Di Dario 7 and Michael Maia Mincarone 7 1Universidade Federal Rural de Pernambuco (UFRPE), Departamento de Pesca e Aquicultura, Recife, PE, Brazil, 2MARBEC, Univ. Montpellier, CNRS, Ifremer, IRD, Sète, France, 3Universidade Federal de Pernambuco (UFPE), Departamento de Oceanograﬁa, Recife, PE, Brazil, 4Programa de Po´ s- Graduac¸ão em Cieˆ ncias Ambientais e Conservac¸ão, Universidade Federal do Rio de Janeiro (UFRJ), Macae´ , RJ, Brazil, 5Programa de Po´ s-Graduac¸ão em Biologia Comparada, Faculdade de Filosoﬁa, Cieˆ ncias e Letras de Ribeirão Preto, Universidade de São Paulo (USP), Ribeirão Preto, SP, Brazil, 6School of Aquatic and Fishery Sciences and Burke Museum of Natural History and Culture, University of Washington, Seattle, WA, United States, 7Universidade Federal do Rio de Janeiro (UFRJ), Instituto de Biodiversidade e Sustentabilidade, Macae´ , RJ, Brazil CITATION Eduardo LN, Bertrand A, Lucena-Fre´ dou F, Villarins BT, Martins JR, Afonso GVF, Pietsch TW, Fre´ dou T, Di Dario F and Mincarone MM (2022) Rich and underreported: First integrated assessment of the diversity of mesopelagic ﬁshes in the Southwestern Tropical Atlantic. Front. Mar. Sci. 9:937154. doi: 10.3389/fmars.2022.937154 Mesopelagic ﬁshes play critical ecological roles by sequestering carbon, recycling nutrients, and acting as a key trophic link between primary consumers and higher trophic levels. They are also an important food source for harvestable economically valuable ﬁsh stocks and a key link between shallow and deep-sea ecosystems. Despite their relevance, mesopelagic ecosystems are increasingly threatened by direct and indirect human activities while representing some of the largest and least understood environments on Earth. TYPE Original Research PUBLISHED 30 August 2022 DOI 10.3389/fmars.2022.937154 TYPE Original Research PUBLISHED 30 August 2022 DOI 10.3389/fmars.2022.937154 TYPE Original Research PUBLISHED 30 August 2022 DOI 10.3389/fmars.2022.937154 COPYRIGHT © 2022 Eduardo, Bertrand, Lucena-Fre´ dou, Villarins, Martins, Afonso, Pietsch, Fre´ dou, Di Dario and Mincarone. 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. Introduction holds distinct biodiversity and includes several Marine Protected Areas and Ecologically or Biologically Signiﬁcant Marine Areas (EBSAs) that, by deﬁnition, are special places of fundamental importance for biodiversity and life cycles of marine species (CBD, 2014). Moreover, the SWTA includes different biogeographic provinces with contrasting thermodynamic features, current systems, and water-mass properties, leading to shifts in biodiversity and ecosystems (Bourlès et al., 1999; Pinheiro et al., 2018; Assunção et al., 2020; Costa da Silva et al., 2021; Dossa et al., 2021; Tosetto et al., 2021; Silva et al., 2022). Mesopelagic ﬁshes (200–1,000 m depth) are among the most abundant vertebrates in the biosphere (Gjøsaeter and Kawaguchi, 1980; Irigoien et al., 2014; Nelson et al., 2016). They often have a global distribution, vertical migratory behavior, and several adaptations to overcome challenges imposed by the deep-sea environment (Gjøsaeter and Kawaguchi, 1980; Sutton, 2013; Priede, 2017). Some of these adaptations include low metabolic rates, high tolerance to environmental changes, and complex visual and bioluminescence systems (Priede, 2017). Consequently, the mesopelagic zone holds one of the most diverse ﬁsh communities of the ocean, contributing to several ecosystem processes (Gjøsaeter and Kawaguchi, 1980; St. John et al., 2016). Mesopelagic ﬁshes play critical roles by sequestering carbon, recycling nutrients, and acting as key trophic links between primary consumers and higher trophic levels (e.g., larger ﬁshes, mammals, and seabirds) (e.g., Ariza et al., 2015; Cavan et al., 2019; Eduardo et al., 2020a; Eduardo et al., 2020b; Eduardo et al., 2021). They are also an important food source for harvestable ﬁsh stocks and a key link between shallow and deep- sea ecosystems (e.g., Cherel et al., 2010; Eduardo et al., 2020b; Eduardo et al., 2021). The ﬁrst collection of deep-sea ﬁshes in the SWTA was carried out by the HMS Challenger (1872–1876; Günther, 1887). Since then, mesopelagic ﬁshes have been sporadically collected by different vessels, such as the RV Akademik Kurchatov (1971– 1972; Parin et al., 1974), RV Walther Herwig (1966–1971; many authors), RV Marion Dufresne (1987; Séret and Andreata, 1992), RV Atlântico Sul (1996–1999; Figueiredo et al., 2002; Bernardes et al., 2005), RV Thalassa (1999–2000; Costa et al., 2007), RV Astro Garoupa (2003; Costa and Mincarone, 2010), RV Gyre (2008; Costa et al., 2015; Mincarone et al., 2017), and the RV Luke Thomas and RV Seward Johnson (2009, 2011; Lins Oliveira et al., 2015). OPEN ACCESS The composition, diversity, and other aspects of the most basic biological features of numerous mesopelagic groups of ﬁshes are still poorly known. Here, we provide the ﬁrst integrative study of the biodiversity of mesopelagic ﬁshes of the southwestern Tropical Atlantic (SWTA), based on two expeditions in northeastern Brazil in 2015 and 2017. A full list of mesopelagic ﬁshes of the region is provided, including rare species and new records for the Brazilian Exclusive Economic Zone and the indication of potentially new species in groups such as the Stomiiformes and Beryciformes. Key aspects of the diversity of mesopelagic ﬁshes of the region were also assessed, considering different depth strata and diel periods. At least 200 species, 130 genera, 56 families, and 22 orders of the Teleostei and one shark (Isistius brasiliensis, Dalatiidae, Squaliformes) were recorded, including potentially eight new species (4%) and 50 (25%) new records for Brazilian waters. Five families accounted for 52% of the diversity, 88% of specimens collected, and 66% of the total biomass: Stomiidae (38 spp., 8% of specimens, COPYRIGHT © 2022 Eduardo, Bertrand, Lucena-Fre´ dou, Villarins, Martins, Afonso, Pietsch, Fre´ dou, Di Dario and Mincarone. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Marine Science 01 frontiersin.org Eduardo et al. Eduardo et al. 10.3389/fmars.2022.937154 21% of biomass), Myctophidae (34 spp., 36%, 24%), Melamphaidae (11 spp., 2%, 7%), Sternoptychidae (9 spp., 26%, 10%), and Gonostomatidae (7 spp., 16%, 4%). During the day, richness and diversity were higher at lower mesopelagic depths (500–1000 m), with contributions of typically bathypelagic species likely associated with seamounts and oceanic islands. At night, richness and diversity increased at epipelagic depths, indicating the diel ascension of several species (e.g., myctophids and sternoptychids) that can endure temperature ranges of up to 25°C. Information on the geographic distribution of several rare species worldwide is also provided. KEYWORDS deep-sea, oceanic islands, seamounts, biodiversity, Brazil, Fernando de Noronha Ridge Introduction Although these expeditions substantially contributed to understanding the diversity and ecology of several groups, they were sparse and focused mostly on demersal species (Melo et al., 2020). Only a few studies focused on the mesopelagic communities of the SWTA, with most of them being restricted to the composition and taxonomy of speciﬁc groups (e.g., Lima et al., 2011; Mincarone et al., 2014). Consequently, an integrative overview of the mesopelagic ﬁsh community of the region is still lacking, leaving a “dark hole” in our understanding of their diversity, ecology, and function in marine ecosystems. Despite their importance, mesopelagic communities are increasingly threatened by climate change (Levin et al., 2019), plastic pollution (Ferreira et al., 2022; Justino et al., 2022), and exploitation of deep-sea resources (Hidalgo and Browman, 2019; Drazen et al., 2020). There is also a major lack of knowledge of the biology, ecology, distribution, and diversity of mesopelagic species, which are typically under-sampled and sparse in data (Glover et al., 2018; Hidalgo and Browman, 2019; Martin et al., 2020). The southwestern Tropical Atlantic (SWTA) encompasses oceanic islands, underwater canyons, and several seamounts (Travassos et al., 1999; Tchamabi et al., 2017). This region Tworecentexpeditionsfocusedonmesopelagicfaunaweremade aboard the RV Antea, as part of the project ABRACOS (Acoustics along the BRAzilian COaSt; Bertrand, 2015; Bertrand, 2017). For the Frontiers in Marine Science 02 frontiersin.org Eduardo et al. 10.3389/fmars.2022.937154 ﬁrst time, the mesopelagic zone of the SWTA was extensively surveyed, resulting in collections of thousands of deep-sea invertebrates and ﬁshes. Based on these collections, various studies have been published addressing the diversity and ecology of several ﬁsh groups, such as Argentiniformes (Mincarone et al., 2021a), Aulopiformes (Mincarone et al., 2022), Myctophiformes (Eduardo et al., 2021), Beryciformes (Afonso et al., 2021), Stomiiformes (Eduardo et al., 2020a; Eduardo et al., 2020b; Villarins et al., 2022), Ceratioidei (Mincarone et al., 2021a), Caristiidae (Mincarone et al., 2019), Howelidae (Eduardo et al., 2019), and Trichiuridae (Eduardo et al., 2018). However, most of the results of these cruises remains unpublished.Here,wepresentanintegrativestudyofthebiodiversity of mesopelagic ﬁshes from the SWTA. A full list of mesopelagic species collected during the ABRACOS expeditions, including a compilation of published new records and the indication of potentially new species, is provided. Key aspects of the mesopelagic ﬁsh diversity of the region were also addressed, considering different depth strata and diel periods. Data and specimen collection Data and specimens were collected during the Acoustics along the BRAzilian COaSt (ABRACOS) surveys, carried out from 29 August to 21 September 2015 (AB1) and from 9 April to 10 May 2017 (AB2), aboard the French RV Antea (Bertrand, 2015; Bertrand, 2017). Temperature proﬁles were collected using a CTDO SeaBird911+. Mesopelagic ﬁshes were collected day and night at 80 trawl stations by using mesopelagic (AB1; body mesh 30 mm, cod-end mesh 4 mm, size of the net mouth: 16.6 x 8.4 m; Bertrand, 2015) and micronekton (AB2; body mesh 40 mm, cod- end mesh 10 mm, size of the net mouth: 24 x 24 m; Bertrand, 2017) nets (Figure 1; Supplementary Material 1 and 2). Targeted depth ranged from 10 to 1,113 m and was deﬁned by the presence of acoustic scattered layers or patches detected by a Simrad EK60 (Kongsberg Simrad AS) split-beam scientiﬁc echosounder, operating at 38, 70, 120, and 200 kHz. Except for the layers 200– 300 and 700–800 at night, where no aggregation of organisms were observed through acoustics, all depth strata were sampled at least once (Supplementary Material 1). The net geometry was monitored using SCANMAR sensors, to give headline height, depth, and distance of wings and doors to ensure the net was ﬁshing correctly. Based on SCANMAR the estimated opening area of the micronekton trawl was 120 m2. For the mesopelagic trawl, however, the opening resembled an ellipse of 65 m2. As the trawl was not ﬁtted with an opening and closing mechanism, the collection of specimens during the lowering or hoisting of the net was reduced as much as possible by decreasing ship velocity and increasing winch speed. At the target depths, trawling activity lasted for about 30 minutes at 2–3 kt. Therefore, collection of specimens most likely occurred at target depths, which are indicated as capture depths in the species accounts. Other aspects of the biodiversity were assessed based on the sample-size-based rarefaction and extrapolation sampling curves, calculated for the species richness, Shannon diversity, and Simpson dominance, the three most widely used species diversity indexes (Magurran, 2004). For that, we used Hill’s numbers, which integrate species richness and relative abundance to propose a more intuitive and statistically rigorous alternative to calculate diversity measures (Chao et al., 2014). Statistical signiﬁcance was evaluated based on the conﬁdence interval overlapping of the curves. Study area The study area comprised the northeastern Brazilian coast, from Rio Grande do Norte to Alagoas states (5°–9°S), and the seamounts and oceanic islands of the Fernando de Noronha Ridge, including the Rocas Atoll (3°52′S, 33°49′W) and the Fernando de Noronha Archipelago (3°50′S, 32°25′W) (Figure 1). The main oceanographic physico-chemical features FIGURE 1 Study area with CTDO proﬁle (cross) and trawl samples (dots). Black and white symbols for ABRACOS 1 and ABRACOS 2, respectively. FIGURE 1 Study area with CTDO proﬁle (cross) and trawl samples (dots). Black and white symbols for ABRACOS 1 and ABRACOS 2, respectively 03 Frontiers in Marine Science frontiersin.org Eduardo et al. 10.3389/fmars.2022.937154 “Instituto de Biodiversidade e Sustentabilidade, Universidade Federal do Rio de Janeiro” (NUPEM/UFRJ). Taxonomic classiﬁcation follows Nelson et al. (2016), with exceptions noted in Villarins et al. (2022) for the Stomiiformes. “Instituto de Biodiversidade e Sustentabilidade, Universidade Federal do Rio de Janeiro” (NUPEM/UFRJ). Taxonomic classiﬁcation follows Nelson et al. (2016), with exceptions noted in Villarins et al. (2022) for the Stomiiformes. of the region were recently described by Assunção et al. (2020); Costa da Silva et al. (2021), and Dossa et al. (2021). Overall, the SWTA is considered oligotrophic. However, locally the banks and islands act as topographic obstacles to currents, driving subsurface enriched waters to the surface (Travassos et al., 1999; Tchamabi et al., 2017; Costa da Silva et al., 2021; Silva et al., 2022). This process increases primary production and enhances the mass and energy ﬂuxes throughout the food web (Travassos et al., 1999; Tchamabi et al., 2017). Richness estimators and biodiversity indexes We ﬁrst computed a randomised species accumulation curve to assess whether the ﬁsh community was exhaustively sampled with the gears employed (Gotelli and Colwell, 2001). This enables calculating a mean number of species for a given number of samples within a 95% conﬁdence interval. The Chao1 index, which extrapolates the total expected number of species in the area for a given sampling gear, was subsequently calculated (Magurran, 2004). Data and specimen collection Sample-size-based rarefaction and extrapolation sampling curves (Hsieh et al., 2016) were also constructed to test for differences in diversity indexes when considering depth strata (epipelagic 0–200 m; upper mesopelagic 200–500 m; lower mesopelagic 500–1000 m) and the diel period (day and night). As the sampling strategy employed in the AB2 expedition was much more efﬁcient in collecting mesopelagic ﬁshes (see Discussion), comparisons using diversity indexes were only made for this survey. Statistical analyses and the calculation of diversity indices were performed using the software R version 4.0.3 through the packages “iNext” (Hsieh et al., 2016) and “vegan” (Oksanen et al., 2017). Fish larvae and species traditionally classiﬁed as epipelagic were excluded from the species list, and they were not considered for the diversity assessments. Specimens identiﬁed at the genus level only (small- sized and/or damaged specimens), which might represent more than one species, were also excluded from the analyses (Supplementary Material 3). Specimens were sorted to the lowest taxonomic level and frozen or, in the case of rare species or taxonomic uncertainty, ﬁxed in 4% formalin and then preserved in a 70% alcohol solution (Eduardo et al., 2020a). In the laboratory, specimens were identiﬁed, measured (nearest 0.1 cm of standard length, SL), and weighed (nearest 0.01 g of total weight, TW). Excluding a few specimens of the Stomiidae, Sternoptychidae, and Myctophidae used for biological analyses (Eduardo et al., 2020a; Eduardo et al., 2020b; Eduardo et al., 2021), all specimens were deposited in the NPM – Fish Collection of the frontiersin.org Biodiversity Considering our two surveys, 7,119 specimens of mesopelagic ﬁshes, representing 200 species in 130 genera, 56 families, and 22 orders of the Teleostei and one shark (Isistius brasiliensis: Dalatiidae, Squaliformes), were collected and identiﬁed (Table 1). The species accumulation curve was steep, Frontiers in Marine Science 04 frontiersin.org frontiersin.org Eduardo et al. Eduardo et al. 10.3389/fmars.2022.937154 TABLE 1 Species recorded, survey (S) (1: ABRACOS 1; 2: ABRACOS 2), number of specimens (N), frequency of occurrence to overall samples (FO %), standard length (SL, mean and range), total wet weight (TW, mean and range), collection locality (PE, Pernambuco; PB, Paraı́ba; RN, Rio Grande do Norte; FNR, Fernando de Noronha Ridge), depth range (based on the target depth of each trawl), temperature range (T), and new records in the Brazilian Exclusive Economic Zone (EEZ). Biodiversity Species S N FO% SL (mm) TW (g) Locality Depth (m) T (°C) SQUALIFORMES Dalatiidae Isistius brasiliensis (Quoy & Gaimard, 1824) 1 1 1.2 172 (TL) 20.0 PB 100 24.4 NOTACANTHIFORMES Halosauridae Aldrovandia sp.* 2 1 1.2 167 3.4 FNR 900 4.3 ANGUILLIFORMES Eurypharyngidae Eurypharynx pelecanoides Vaillant, 1882 2 13 4.9 287 (99–524) 6.5 (1.0–33.9) FNR 780–900 4.3–4.7 Nemichthyidae Avocettina infans (Günther, 1878) 2 1 1.2 502 2.2 FNR 900 4.3 Labichthys carinatus Gill & Ryder, 1883 2 2 2.4 397 (227–568) 7.0 (0.5–13.5) FNR-PE 680–720 4.9–5.2 Nemichthys scolopaceus Richardson, 1848 1 7 3.7 290 (235–330) 2.9 (2.0–4.7) FNR 105–525 6.8–24.4 Serrivomeridae Serrivomer beanii Gill & Ryder, 1883 2 49 13.4 422 (60–592) 14.5 (0.5–65.4) FNR-PB-PE-RN 90–900 4.3–25.1 Serrivomer lanceolatoides (Schmidt, 1916) 2 1 1.2 413 4.6 FNR 900 4.3 Stemonidium hypomelas Gilbert, 1905 2 2 2.4 256 6.2 (3.4–9.0) FNR 800–900 4.3–4.7 ALEPOCEPHALIFORMES Platytroctidae Platytroctidae sp.* 2 1 1.2 55 0.8 FNR 610 5.6 Alepocephalidae Alepocephalidae sp.* 2 1 1.2 45 0.7 FNR 900 4.3 Photostylus pycnopterus Beebe, 19331 2 2 2.4 85 (75–95) 4.1 (2.7–5.5) FNR 800–900 4.3–4.7 ARGENTINIFORMES Opisthoproctidae Opisthoproctus soleatus Vaillant, 18882 2 1 1.2 49 1.0 FNR 385 9.2 Rhynchohyalus natalensis (Gilchrist & von Bonde, 1924)2 2 1 1.2 109 12.3 FNR 800 4.7 Winteria telescopa Brauer, 1901 2 31 9.8 95 (51–118) 6.7 (1.3–10.6) FNR-RN 440–900 4.3–8.5 Microstomatidae Xenophthalmichthys danae Regan, 19252 2 2 2.4 87 (60–114) 3.2 (2.1–4.3) FNR 385–505 7.0–9.2 Bathylagidae Dolicholagus longirostris (Maul, 1948) 2 8 7.3 79 (41–100) 3.3 (1.3–4.8) FNR 430–900 4.3–8.5 Melanolagus bericoides (Borodin, 1929) 2 9 3.7 148 (128–167) 17.8 (11.7–25.8) FNR 430–900 4.3–8.54 STOMIIFORMES Diplophidae Diplophos australis Ozawa, Oda & Ida, 1990 2 3 2.4 81 (71–99) 0.8 (0.5–1.3) FNR 780–800 4.6–4.7 Diplophos taenia Günther, 1873 1-2 25 12.2 71 (42–129) 1.9 (0.6–4.3) FNR-PB 25–800 4.7–28.8 Manducus maderensis (Johnson, 1890) 2 2 3.7 56 (42–65) 1.3 (0.7–1.4) FNR 90–615 5.6–25.1 Triplophos hemingi (McArdle, 1901)3 2 1 1.2 196 13.5 FNR 800 4.7 Gonostomatidae Cyclothone spp.* 1-2 874 28.0 33 (12–45) 1.4 (0.2–7.4) FNR-PB-PE-RN 350–1000 4.3–27.6 Gonostoma atlanticum Norman, 1930 1-2 67 18.3 51 (19–68) 1.8 (0.13–7.8) FNR-PB-PE-RN 100–900 4.3–24.6 Gonostoma denudatum Raﬁnesque, 18103 2 1 1.2 122 7.8 FNR 440 8.5 Margrethia obtusirostra Jespersen & Tåning, 1919 1 1 1.2 27 3.2 FNR 525 6.8 Sigmops bathyphilus (Vaillant, 1884) 2 1 1.2 155 17.3 FNR 800 4.7 Sigmops elongatus (Günther, 1878) 1-2 41 14.6 145 (45–250) 13.1 (0.5–26.8) FNR-PB-PE-RN 100–1000 4.3–24.6 S N FO% SL (mm) TW (g) Locality Depth (m) T (°C) S N FO% SL (mm) TW (g) Locality Depth (m) T (°C) Species Frontiers in Marine Science 05 frontiersin.org 10.3389/fmars.2022.937154 Eduardo et al. Species TABLE 1 Continued Species S N FO% SL (mm) TW (g) Locality Depth (m) T (°C) Zaphotias pedaliotus (Goode & Bean, 1896) 2 184 15.9 57 (37–81) 1.2 (0.5–4.7) FNR-PB 130–900 4.3–22.1 Sternoptychidae Argyropelecus aculeatus Valenciennes, 1850 2 51 12.2 56 (30–82) 6.1 (0.8–20.9) FNR-PB-PE-RN 100–900 4.3–24.6 Argyropelecus afﬁnis Garman, 1899 2 439 14.6 52 (27–82) 2.7 (0.5–6.9) FNR-PB-RN 30–800 4.6–28.7 Argyropelecus gigas Norman, 1930 2 9 2.4 86 (78–91) 14.2 (10.4–17.0) FNR-RN 610–700 5.2–5.6 Argyropelecus hemigymnus Cocco, 1829 1-2 80 22.0 24 (8–36) 2.4 (0.2–4.9) FNR-PE-RN 260–900 4.3–13.7 Argyropelecus sladeni Regan, 1908 2 27 11.0 57 (32–94) 4.1 (0.7–14.2) FNR 30–800 4.6–28.7 Sternoptyx diaphana Hermann, 1781 2 1091 20.7 24 (11–43) 2.0 (0.4–4.9) FNR-PB-PE-RN 65–900 4.3–26.5 Sternoptyx pseudobscura Baird, 1971 2 123 12.2 35 (13–59) 2.9 (0.5–9.9) FNR-PB-PE 520–900 4.3–6.3 Sternoptyx pseudodiaphana Borodulina, 1977 2 3 2.4 49 (42–59) 6.9 (5.2–9.9) FNR 800–900 4.4–4.7 Valenciennellus tripunctulatus (Esmark, 1871) 1-2 19 8.5 24 (23–32) 1.6 (1.0–2.3) FNR-PE 360–1000 4.3–10.9 Phosichthyidae Ichthyococcus polli Blache, 1964 1-2 14 9.8 52 (41–72) 2.5 (1.1–8.4) FNR-PB 385–900 4.3–9.2 Phosichthys argenteus Hutton, 1872 2 1 1.2 64 8.1 RN 630 5.6 Pollichthys mauli (Poll, 1953) 1 1 1.2 38 1.5 RN 75 25.7 Vinciguerria nimbaria (Jordan & Williams, 1895) 1-2 24 11.0 26 (17–49) 2.0 (0.4–6.5) FNR-PB-PE-RN 50–780 4.6–26.6 Stomiidae Aristostomias grimaldii Zugmayer, 19133 2 5 2.4 74 (65–86) 3.1 (1.8–5.0) FNR 700–800 4.7–5.26 Aristostomias tittmanni Welsh, 1923 2 3 3.7 43 (32–76) 2.5 (2.0–3.5) FNR-PB 30–800 4.6–28.7 Astronesthes atlantica Parin & Borodulina, 1996 1-2 3 3.7 38 (31–51) 1.2 (0.62–2.0) FNR 90–525 6.8–25.1 Astronesthes gemmifer Goode & Bean, 1896 2 1 1.2 146 21.6 FNR 430 8.5 Astronesthes gudrunae Parin & Borodulina, 20023 2 1 1.2 111 11.1 FNR 610 5.6 Astronesthes richardsoni (Poey, 1852) 2 7 6.1 71 (22–132) 5.7 (1.1–13.5) FNR 25–780 4.6–28.8 Astronesthes similus Parr, 1927 1-2 10 3.7 43 (36–75) 2.9 (0.5–4.9) FNR-PB 100–800 4.7–24.4 Bathophilus nigerrimus Giglioli, 18823 2 2 2.4 89 (84–95) 6.1 (5.1–7.2) FNR 90–610 5.6–25.1 Bathophilus pawneei Parr, 1927 2 4 3.7 66 (30–124) 3.2 (1.2–8.7) FNR 65–440 8.5–26.5 Borostomias elucens (Brauer, 1906)3 2 55 8.5 168 (46–299) 48.2 (0.5–218.9) FNR 610–900 4.3–5.6 Chauliodus sloani Bloch & Schneider, 1801 1-2 348 22.0 162 (55–270) 9.6 (0.3–53.9) FNR-PB-PE-RN 430–900 4.3–8.5 Eustomias bibulbosus Parr, 19273 2 1 1.2 87 0.6 PE 680 5.2 Eustomias braueri Zugmayer, 19113 2 2 1.2 69 (56–82) 1.6(0.6–2.6) PE 680 5.2 Eustomias brevibarbatus Parr, 1927 2 6 7.3 97 (85–128) 1.8 (0.5–4.6) FNR 90–900 4.3–25.1 Eustomias enbarbatus Welsh, 1923 2 2 2.4 54 (54–55) 2.1 (2.1–2.1) FNR-PE 680–780 4.6–5.2 Eustomias minimus Clarke, 19993 2 1 1.2 69 3.2 FNR 780 4.6 Eustomias schmidti Regan & Trewavas, 19303 2 1 1.2 68 4.9 FNR 780 4.6 Eustomias sp. Species 13 2 1 1.2 168 8.2 FNR 800 4.7 Eustomias sp. 23 2 1 1.2 120 2.3 FNR 430 8.5 Eustomias sp. 33 2 4 2.4 68 (49–78) 2.4 (1.6–2.8) FNR 90–720 4.9–25.1 Eustomias sp. 43 2 1 1.2 122 2.1 FNR 800 4.7 Eustomias sp. 53 2 3 1.2 54 (28–98) 0.5 (0.4–0.6) FNR 780 4.6 Grammatostomias dentatus Goode & Bean, 18963 1 1 1.2 114 3.5 PE 1000 4.3 Grammatostomias ovatus Prokoﬁev, 20143 1 1 1.2 67 1.5 PE 1000 4.3 Heterophotus ophistoma Regan & Trewavas, 1929 2 8 6.1 205 (96–253) 57.9 (0.7–107.6) FNR 430–900 4.3–8.5 Leptostomias gladiator (Zugmayer, 1911)3 2 1 1.2 83 0.9 FNR 780 4.6 Malacosteus niger Ayres, 1848 2 46 9.8 107 (633–181) 8.3 (1.4–34.4) FNR 610–900 4.3–5.6 Melanostomias bartonbeanis Parr, 19273 1-2 2 2.4 117 (50–185) 10.9 (2.3–19.6) FNR-PB 100–780 4.6–24.4 Melanostomias biseriatus Regan & Trewavas, 19303 2 2 2.4 103 (29–177) 11.1 (4.9–17.2) FNR-PE 610–680 5.6 Melanostomias tentaculatus (Regan & Trewavas, 1930) 1-2 5 4.9 162 (48–201) 15.7 (2.6–20.7) FNR-PB-PE 430–1000 4.3–8.5 Melanostomias sp.3 2 1 1.2 180 11.4 FNR 440 8.5 Species Frontiers in Marine Science 06 frontiersin.org Eduardo et al. Species 10.3389/fmars.2022.937154 TABLE 1 Continued Species S N FO% SL (mm) TW (g) Locality Depth (m) T (°C) Pachystomias microdon (Günther, 1878)3 2 9 8.5 137 (39–181) 23.2 (2.3–42.5) FNR 430–900 4.3–8.5 Photonectes achirus Regan & Trewavas, 19303 2 3 2.4 56 (33–79) 3.4 (1.2–3.4) PB-RN 100–800 4.7–24.6 Photostomias atrox (Alcock, 1890) 2 1 1.2 118 1.0 PE 680 5.2 Photostomias goodyeari Kenaley & Hartel, 20053 2 1 1.2 64 0.7 FNR 720 4.9 Stomias danae Ege, 1933 2 1 1.2 95 1.8 PB 800 4.7 Stomias longibarbatus (Brauer, 1902) 2 5 6.1 281 (173–390) 9.7 (1.4–25.7) FNR 260–800 4.7–13.7 Thysanactis dentex Regan & Trewavas, 1930 1-2 41 19.5 90 (43–150) 3.1 (0.5–10.6) FNR-RN 90–900 4.3–25.1 ATELEOPODIFORMES Ateleopodidae Ateleopodidae sp.* 2 1 1.2 122 0.6 FNR 800 4.7 AULOPIFORMES Anotopteridae Anotopterus pharao Zugmayer, 1911 1 1 1.2 27 1.0 RN 20 26.7 Giganturidae Gigantura chuni Brauer, 19011 2 3 2.4 111 (42–181) 19.4 (4.9–33.9) FNR 610–800 4.7–5.6 Gigantura indica Brauer, 1901 1-2 31 22.0 102 (16–190) 3.9 (0.6–11.7) FNR-PB-PE 50–900 4.3–27.6 Chlorophthalmidae Parasudis truculenta (Goode & Bean, 1896) 1 2 1.2 31 (30–33) 3.8 (3.2–4.5) FNR 105 24.4 Notosudidae Ahliesaurus berryi Bertelsen, Krefft & Marshall, 19761 2 1 1.2 198 17.8 FNR 800 4.7 Scopelosaurus smithii Bean, 1925 2 1 1.2 177 22.4 PE 680 4.5 Scopelarchidae Benthalbella infans Zugmayer, 19111 1 1 1.2 57 4.0 RN 560 5.9 Rosenblattichthys hubbsi Johnson, 19741 2 4 1.2 79 (40–100) 4.5 (0.5–6.9) PB 800–800 4.7–4.7 Scopelarchoides danae Johnson, 19741 2 1 1.2 80 2.3 FNR 780 4.6 Scopelarchus analis (Brauer, 1902) 1 2 2.4 103 (91–115) 7.9 (4.7–11.2) FNR 510–525 6.0–6.8 Scopelarchus guentheri Alcock, 1896 2 8 6.0 79 (38–113) 4.9 (0.5–12.2) FNR-PB-RN 385–900 4.3–9.2 Evermannellidae Odontostomops normalops (Parr, 1928) 2 4 3.7 134 (121–166) 11.9 (9.9–17.3) FNR 610–900 4.3–5.6 Paralepididae Lestidiops afﬁnis (Ege, 1930) 2 2 2.4 80 (58–102) 1.6 (1.6–1.7) FNR 110–430 8.5–24.1 Lestrolepis intermedia (Poey, 1868) 2 1 1.2 – 5.7 FNR 90 25.1 Stemonosudis gracilis (Ege, 1933) 2 1 1.2 217 3.9 FNR 100 24.6 Stemonosudis intermedia (Ege, 1933) 1-2 4 3.7 130 (71–205) 1.1 (0.5–2.26) FNR-PB 50–900 4.3–27.6 Stemonosudis siliquiventer Post, 1970 2 1 1.2 102 FNR 800 4.7 Alepisauridae Omosudis lowii Günther, 1887 2 10 7.3 82 (39–212) 7.2 (0.5–38.6) FNR 385–900 4.3–9.2 MYCTOPHIFORMES Neoscopelidae Scopelengys tristis Alcock, 18901 2 2 2.4 121 (98–145) 12.5 (5.34–19.71) FNR 780–800 4.6–4.71 Myctophidae Benthosema suborbitale (Gilbert, 1913) 1-2 20 8.5 24 (13–31) 1.7 (0.21–3.3) FNR-PB-RN 30–440 8.5–28.7 Bolinichthys distofax Johnson, 1975 2 85 11.0 62 (32–91) 6.4 (0.5–23.8) FNR-PB-PE-RN 430–900 4.3–8.5 Bolinichthys photothorax (Parr, 1928) 1-2 55 13.4 53 (22–67) 5.8 (0.51–27.8) FNR-PB 510–900 4.3–6.0 Bolinichthys supralateralis (Parr, 1928) 2 4 3.7 75 (50–92) 10.2 (6.6–16.3) FNR 720–900 4.3–4.9 Ceratoscopelus warmingii (Lütken, 1892) 1-2 41 20.7 50 (18–74) 2.6 (0.5–6.2) FNR-RN 30–900 4.3–28.7 Dasyscopelus asper (Richardson, 1845) 1-2 53 13.4 58 (14–75) 3.8 (0.9–7.1) FNR-PE-RN 25–900 4.3–28.8 Dasyscopelus obtusirostre (Tåning, 1928) 1-2 17 9.8 66 (25–84) 5.0 (0.6–7.8) FNR-PB-PE-RN 30–800 4.7–28.7 ( d) Species (Ramsay, 1881) 2 5 6.1 36 (18–55) 3.1 (0.1–7.0) FNR-PE-RN 100–510 6.0–24.4 Zu cristatus (Bonelli, 1819)1 1-2 9 11.0 57 (10–89) 14.7 (0.1–93.1) FNR-RN 20–720 4.9–26.7 STYLEPHORIFORMES Stylephoridae Stylephorus chordatus Shaw, 17911 1-2 64 18.3 176 (59–279) 3.7 (0.5–11.0) FNR-PB-RN 25–900 4.3–28.8 GADIFORMES Melanonidae Melanonus zugmayeri Norman, 1930 2 21 11.0 115 (64–265) 11.6 (1.0–11.9) FNR 95–900 4.3–24.7 Macrouridae Bathygadus sp.* 2 2 1.2 76 (72–81) 1.3 (1.0–1.5) FNR 900 4.3 Macrouroides inﬂaticeps Smith & Radcliffe, 1912 2 2 2.4 197 (179–215) 91.4 (67.4–115.4) FNR 800–900 4.3–4.7 Bregmacerotidae Bregmaceros cf. Species 07 Frontiers in Marine Science frontiersin.org Eduardo et al. Species 10.3389/fmars.2022.937154 TABLE 1 Continued Species S N FO% SL (mm) TW (g) Locality Depth (m) T (°C) Dasyscopelus selenops (Tåning, 1928) 2 2 3.7 45 (27–59) 2.5 (2.2–2.8) FNR-PE 430–900 4.3–8.5 Diaphus bertelseni Nafpaktitis, 1966 2 2 2.4 84 (74–94) 8.0 (6.8–9.3) FNR-RN 100–385 9.2–24.7 Diaphus brachycephalus Tåning, 1928 1-2 470 29.3 38 (09–58) 1.5 (0.5–17) FNR-PE-RN 30–1000 4.3–28.7 Diaphus dumerilii (Bleeker, 1856) 1-2 114 24.4 45 (26–59) 2.4 (0.5–11) FNR-PB-PE-RN 65–900 4.3–26.5 Diaphus fragilis Tåning, 1928 1-2 147 24.4 49 (14–86) 2.6 (0.4–11.7) FNR-PB-PE-RN 65–900 4.3–26.5 Diaphus garmani Gilbert, 1906 1-2 137 11.0 41 (25–51) 2.6 (0.5–9.9) FNR-PE-RN 65–900 4.3–26.5 Diaphus lucidus (Goode & Bean, 1896) 2 43 11.0 76 (31–96) 5.3 (1.3–9.7) FNR-PB-PE-RN 25–800 4.7–28.8 Diaphus mollis Tåning, 1928 1-2 52 20.7 48 (15–59) 1.9 (0.2–4.0) FNR-RN 105–900 4.3–24.4 Diaphus perspicillatus (Ogilby, 1898) 1-2 279 20.7 49 (18–69) 2.1 (0.5–4.9) FNR-PB-PE-RN 65–900 4.3–26.5 Diaphus problematicus Parr, 1928 1-2 4 3.7 69 (52–77) 4.1 (1.7–5.8) FNR 430–720 4.9–8.5 Diaphus splendidus (Brauer, 1904) 1-2 241 18.3 53 (20–85) 2.3 (0.5–6.6) FNR-PB-PE-RN 100–900 4.3–24.6 Diogenichthys atlanticus (Tåning, 1928) 1 9 3.7 18 (15–23) 0.5 (0.2–1.0) FNR 60–525 6.0–26.6 Electrona risso (Cocco, 1829) 2 76 17.1 66 (50–81) 7.4 (3.2–12.4) FNR-PB-RN 385–900 4.3–9.2 Hygophum hygomii (Lütken, 1892) 2 2 1.2 53 (52–54) 2.2 (1.9–2.4) FNR 800 4.7 Hygophum macrochir (Günther, 1864) 1-2 28 8.5 50 (34–60) 1.9 (0.5–8.0) FNR-PB 30–800 4.6–28.7 Hygophum reinhardtii (Lütken, 1892) 1-2 5 3.7 51 (24–76) 2.5 (1.2–6.8) FNR 30–150 20.0–28.7 Hygophum taaningi Becker, 1965 1-2 108 12.2 51 (26–66) 1.9 (0.9–3.1) FNR-RN 90–900 4.3–25.1 Lampadena luminosa (Garman, 1899) 1-2 29 4.9 28 (19–51) 2.2 (0.5–5.4) FNR-PB-RN 100–900 4.3–24.6 Lampanyctus alatus Goode & Bean, 1896 2 2 1.2 37 (37–38) 3.5 (2.9–4.2) FNR 430 8.5 Lampanyctus lineatus (Tåning, 1928) 1-2 5 4.9 137 (26–178) 19.0 (0.63–29.46) FNR-PB 50–900 4.3–26.5 Lampanyctus festivus Tåning, 1928 2 4 1.2 87 (56–120) 6.8 (1.3–13.7) FNR 900 4.3 Lampanyctus nobilis Tåning, 1928 1-2 285 29.3 19 (57–120) 2.6 (0.4–14) FNR-PB-PE-RN 25–900 4.3–28.8 Lampanyctus tenuiformis (Brauer, 1906) 2 26 9.8 111 (44–149) 16.0 (0.7–46.4) FNR-PE-RN 25–900 4.3–28.8 Lepidophanes guentheri (Goode & Bean, 1896) 1-2 219 29.3 48 (22–62) 3.6 (0.5–9.9) FNR-PB-PE-RN 25–1000 4.3–28.8 Myctophum nitidulum Garman, 1899 1-2 12 11.0 59 (38–70) 3.6 (1.8–5.1) FNR-PB-RN 30–800 4.7–28.7 Notoscopelus resplendens (Richardson, 1845) 2 2 2.4 75 (67–84) 3.1 (2.7–3.5) FNR 430–780 4.6–8.54 Taaningichthys bathyphilus (Tåning, 1928) 2 10 4.9 62 (54–71) 1.7 (1.1–2.8) FNR 720–900 4.3–4.98 LAMPRIFORMES Lophotidae Eumecichthys ﬁski (Günther, 1890) 2 1 1.2 1880 2190.0 FNR 780 4.6 Trachipteridae Desmodema polystictum (Ogilby, 1898) 2 1 1.2 74 1.0 FNR 800 4.7 Trachipterus sp. Species atlanticus Goode & Bean, 1886 1-2 20 7.3 65 (32–85) 1.9 (0.5–4.4) FNR-RN 90–800 4.7–25.1 TRACHICHTHYIFORMES Anoplogastridae Anoplogaster cornuta (Valenciennes, 1833) 2 4 3.7 100 (85–107) 31.2 (18.3–43.4) FNR-RN 610–800 4.7–5.6 S N FO% SL (mm) TW (g) Locality Depth (m) T (°C) 2 2 3.7 45 (27–59) 2.5 (2.2–2.8) FNR-PE 430–900 4.3–8.5 2 2 2.4 84 (74–94) 8.0 (6.8–9.3) FNR-RN 100–385 9.2–24.7 1-2 470 29.3 38 (09–58) 1.5 (0.5–17) FNR-PE-RN 30–1000 4.3–28.7 1-2 114 24.4 45 (26–59) 2.4 (0.5–11) FNR-PB-PE-RN 65–900 4.3–26.5 1-2 147 24.4 49 (14–86) 2.6 (0.4–11.7) FNR-PB-PE-RN 65–900 4.3–26.5 1-2 137 11.0 41 (25–51) 2.6 (0.5–9.9) FNR-PE-RN 65–900 4.3–26.5 2 43 11.0 76 (31–96) 5.3 (1.3–9.7) FNR-PB-PE-RN 25–800 4.7–28.8 1-2 52 20.7 48 (15–59) 1.9 (0.2–4.0) FNR-RN 105–900 4.3–24.4 1-2 279 20.7 49 (18–69) 2.1 (0.5–4.9) FNR-PB-PE-RN 65–900 4.3–26.5 1-2 4 3.7 69 (52–77) 4.1 (1.7–5.8) FNR 430–720 4.9–8.5 1-2 241 18.3 53 (20–85) 2.3 (0.5–6.6) FNR-PB-PE-RN 100–900 4.3–24.6 1 9 3.7 18 (15–23) 0.5 (0.2–1.0) FNR 60–525 6.0–26.6 2 76 17.1 66 (50–81) 7.4 (3.2–12.4) FNR-PB-RN 385–900 4.3–9.2 2 2 1.2 53 (52–54) 2.2 (1.9–2.4) FNR 800 4.7 1-2 28 8.5 50 (34–60) 1.9 (0.5–8.0) FNR-PB 30–800 4.6–28.7 1-2 5 3.7 51 (24–76) 2.5 (1.2–6.8) FNR 30–150 20.0–28.7 1-2 108 12.2 51 (26–66) 1.9 (0.9–3.1) FNR-RN 90–900 4.3–25.1 1-2 29 4.9 28 (19–51) 2.2 (0.5–5.4) FNR-PB-RN 100–900 4.3–24.6 2 2 1.2 37 (37–38) 3.5 (2.9–4.2) FNR 430 8.5 1-2 5 4.9 137 (26–178) 19.0 (0.63–29.46) FNR-PB 50–900 4.3–26.5 2 4 1.2 87 (56–120) 6.8 (1.3–13.7) FNR 900 4.3 1-2 285 29.3 19 (57–120) 2.6 (0.4–14) FNR-PB-PE-RN 25–900 4.3–28.8 2 26 9.8 111 (44–149) 16.0 (0.7–46.4) FNR-PE-RN 25–900 4.3–28.8 1-2 219 29.3 48 (22–62) 3.6 (0.5–9.9) FNR-PB-PE-RN 25–1000 4.3–28.8 1-2 12 11.0 59 (38–70) 3.6 (1.8–5.1) FNR-PB-RN 30–800 4.7–28.7 2 2 2.4 75 (67–84) 3.1 (2.7–3.5) FNR 430–780 4.6–8.54 2 10 4.9 62 (54–71) 1.7 (1.1–2.8) FNR 720–900 4.3–4.98 2 1 1.2 1880 2190.0 FNR 780 4.6 2 1 1.2 74 1.0 FNR 800 4.7 2 5 6.1 36 (18–55) 3.1 (0.1–7.0) FNR-PE-RN 100–510 6.0–24.4 1-2 9 11.0 57 (10–89) 14.7 (0.1–93.1) FNR-RN 20–720 4.9–26.7 1-2 64 18.3 176 (59–279) 3.7 (0.5–11.0) FNR-PB-RN 25–900 4.3–28.8 2 21 11.0 115 (64–265) 11.6 (1.0–11.9) FNR 95–900 4.3–24.7 2 2 1.2 76 (72–81) 1.3 (1.0–1.5) FNR 900 4.3 2 2 2.4 197 (179–215) 91.4 (67.4–115.4) FNR 800–900 4.3–4.7 1-2 20 7.3 65 (32–85) 1.9 (0.5–4.4) FNR-RN 90–800 4.7–25.1 2 4 3.7 100 (85–107) 31.2 (18.3–43.4) FNR-RN 610–800 4.7–5.6 (Continued) Species Species 10.3389/fmars.2022.937154 TABLE 1 Continued Species S N FO% SL (mm) TW (g) Locality Depth (m) T (°C) Diretmidae Diretmoides pauciradiatus (Woods, 1973) 1-2 23 8.5 26 (4–62) 3.4 (0.5–8.5) FNR 85–900 4.3–25.4 Diretmus argenteus Johnson, 1864 2 116 13.4 53 (14–75) 8.1 (0.6–67.4) FNR 430–900 4.3–8.5 Trachichthyidae Aulotrachichthys argyrophanus (Woods, 1961) 2 6 3.7 28 (24–34) 1.2 (0.7–1.5) FNR 230–780 4.6–12.4 BERYCIFORMES Rondeletiidae Rondeletia loricata Abe & Hotta, 1963 1-2 3 3.7 55 (32–78) 4.8 (1.2–10.4) FNR 525–900 4.3–6.8 Cetomimidae Cetomimus sp.4 2 2 2.4 64 (39–65) 1.8 (1.5–2.1) FNR-PE 680–780 4.6–5.2 Cetostoma regani Zugmayer, 1914 1-2 5 4.9 98 (81–114) 5.8 (1.6–18.4) FNR 525–900 4.3–6.8 Ditropichthys storeri (Goode & Bean, 1895)4 2 1 1.2 49 1.5 FNR 610 5.6 Gyrinomimus bruuni Rofen, 19594 2 2 1.2 63 (60–66) 8.6 (1.2–16.1) FNR 900 4.3–4.3 Melamphaidae Melamphaes eulepis Ebeling, 19624 2 10 4.9 43 (35–47) 20.3 (10.8–24.6) FNR 430–900 4.3–8.5 Melamphaes leprus Ebeling, 19624 2 1 1.2 80 14.0 FNR 430 8.5 Melamphaes longivelis Parr, 19334 2 2 2.4 75 (74–75) 46.8 (8.0–85.7) FNR 630–780 4.6–5.6 Melamphaes polylepis Ebeling, 1962 2 37 9.8 53 (36–70) 34.1 (2.0–60.0) FNR-PE 610–900 4.3–5.6 Melamphaes typhlops (Lowe, 1843) 2 7 7.3 54 (37–71) 31.6 (1.0–60.8) FNR-PE 430–900 4.3–8.5 Melamphaes sp.* 4 2 1 1.2 62 43.9 FNR 900 4.3 Poromitra megalops (Lütken, 1878) 1-2 27 9.8 46 (25–59) 1.8 (0.5–4.2) FNR-RN 525–900 4.3–6.8 Poromitra sp.** 1-2 28 11.0 85 (48–121) 10.6 (1.3–37.3) FNR-PE-RN 45–1000 4.3–8.5 Scopeloberyx opercularis Zugmayer, 1911 2 1 1.2 32 3.9 FNR 780 4.6 Scopeloberyx opisthopterus (Parr, 1933) 2 4 3.7 29 (25–32) 2.7 (1.9–3.6) FNR 720–900 4.7–4.9 Scopelogadus mizolepis (Günther, 1878) 1-2 19 9.8 54 (37–70) 9.8 (0.8–39.1) FNR 430–900 4.3–8.5 OPHIDIIFORMES Bythitidae Bythitidae sp.* 2 2 2.4 87 (86–89) 2.7 (2.6–2.8) FNR-PE 680–900 4.3–5.26 KURTIFORMES Apogonidae Paroncheilus afﬁnis (Poey, 1875) 1 1 1.2 28 4.2 RN 75 25,6 PERCIFORMES Howellidae Bathysphyraenops simplex Parr, 19335 1 3 3.7 65 (41–78) 7.5 (5.0–9.0) FNR 525–900 4.3–6.8 Howella atlantica Post & Quéro, 1991 2 25 8.5 58 (52–69) 4.0 (2.6–6.5) FNR-PE 680–900 4.3–5.2 Bramidae Brama brama (Bonaterre, 1788) 2 1 1.2 28 1.0 FNR 900 4.3 Brama caribbea Mead, 1972 1-2 64 15.9 25 (12–55) 2.0 (0.4–9.8) FNR-PE-RN 58–900 4.3–26.6 Taractichthys longipinnis (Lowe, 1843) 2 1 1.2 32 1.3 PE 240 14.8 Caristiidae Paracaristius nudarcus Stevenson & Kenaley, 20116 2 1 1.2 175 181 FNR 430 8.5 Platyberyx andriashevi (Kukuev, Parin & Trunov, 2012)6 2 3 2.4 68 (24–149) 31.2 (1.1–87.8) FNR 230–800 4.7–12.4 Platyberyx paucus Stevenson & Kenaley, 20136 2 3 3.7 95 (92–98) 33.4 (31.1–36.7) FNR-RN 630–800 4.7–5.6 Platyberyx pietschi Stevenson & Kenaley, 20136 2 1 1.2 74 9.2 RN 630 5.6 SCOMBROLABRACIFORMES Scombrolabracidae Scombrolabrax heterolepis Roule, 1921 2 1 1.2 76 6.1 FNR 900 4.3 SCOMBRIFORMES (Continued) TABLE 1 Continued S N FO% SL (mm) TW (g) Locality Depth (m) T (°C) 1-2 23 8.5 26 (4–62) 3.4 (0.5–8.5) FNR 85–900 4.3–25.4 2 116 13.4 53 (14–75) 8.1 (0.6–67.4) FNR 430–900 4.3–8.5 2 6 3.7 28 (24–34) 1.2 (0.7–1.5) FNR 230–780 4.6–12.4 1-2 3 3.7 55 (32–78) 4.8 (1.2–10.4) FNR 525–900 4.3–6.8 2 2 2.4 64 (39–65) 1.8 (1.5–2.1) FNR-PE 680–780 4.6–5.2 1-2 5 4.9 98 (81–114) 5.8 (1.6–18.4) FNR 525–900 4.3–6.8 2 1 1.2 49 1.5 FNR 610 5.6 2 2 1.2 63 (60–66) 8.6 (1.2–16.1) FNR 900 4.3–4.3 2 10 4.9 43 (35–47) 20.3 (10.8–24.6) FNR 430–900 4.3–8.5 2 1 1.2 80 14.0 FNR 430 8.5 2 2 2.4 75 (74–75) 46.8 (8.0–85.7) FNR 630–780 4.6–5.6 2 37 9.8 53 (36–70) 34.1 (2.0–60.0) FNR-PE 610–900 4.3–5.6 2 7 7.3 54 (37–71) 31.6 (1.0–60.8) FNR-PE 430–900 4.3–8.5 2 1 1.2 62 43.9 FNR 900 4.3 1-2 27 9.8 46 (25–59) 1.8 (0.5–4.2) FNR-RN 525–900 4.3–6.8 1-2 28 11.0 85 (48–121) 10.6 (1.3–37.3) FNR-PE-RN 45–1000 4.3–8.5 2 1 1.2 32 3.9 FNR 780 4.6 2 4 3.7 29 (25–32) 2.7 (1.9–3.6) FNR 720–900 4.7–4.9 1-2 19 9.8 54 (37–70) 9.8 (0.8–39.1) FNR 430–900 4.3–8.5 2 2 2.4 87 (86–89) 2.7 (2.6–2.8) FNR-PE 680–900 4.3–5.26 1 1 1.2 28 4.2 RN 75 25,6 1 3 3.7 65 (41–78) 7.5 (5.0–9.0) FNR 525–900 4.3–6.8 2 25 8.5 58 (52–69) 4.0 (2.6–6.5) FNR-PE 680–900 4.3–5.2 2 1 1.2 28 1.0 FNR 900 4.3 1-2 64 15.9 25 (12–55) 2.0 (0.4–9.8) FNR-PE-RN 58–900 4.3–26.6 2 1 1.2 32 1.3 PE 240 14.8 2 1 1.2 175 181 FNR 430 8.5 6 2 3 2.4 68 (24–149) 31.2 (1.1–87.8) FNR 230–800 4.7–12.4 2 3 3.7 95 (92–98) 33.4 (31.1–36.7) FNR-RN 630–800 4.7–5.6 2 1 1.2 74 9.2 RN 630 5.6 2 1 1.2 76 6.1 FNR 900 4.3 (Continued) Species Frontiers in Marine Science 08 frontiersin.org Eduardo et al. Species *Potential new species. 1Mincarone et al. (2022), 2Mincarone et al. (2021a), 3Villarins et al. (2022), 4Afonso et al. (2021), 5Eduardo et al. (2019), 6Mincarone et al. (2019) 7Eduardo et al (2018b) 8Mincarone et al (2021b) TABLE 1 Continued S N FO% SL (mm) TW (g) Locality Depth (m) T (°C) 1-2 3 3.7 68 (44–112) 1.1 (0.9–1.3) FNR 70–900 4.3–25.8 1 1 1.2 36 4.1 FNR 110 24.0 2 4 3.7 107 (85–145) 1.7 (0.7–2.8) FNR 90–800 4.7–25.1 1 15 1.2 154 (112–191) 20.2 (15.0–34.0) FNR 150 20.6 2 10 7.3 91 (75–129) 13.2 (6.7–30.3) FNR 65–720 4.9–26.5 1-2 5 3.7 86 (14–133) 38.9 (8.9–70.2) FNR 25–570 6.3–28.8 2 1 1.2 720 550 FNR 610 5.7 2 2 1.2 82 (70–95) 2.9 (2.2–3.7) FNR 900 4.3 2 1 1.2 90 7.9 FNR 800 4.7 2 5 4.9 127 (69–170) 11.8 (1.1–29.3) FNR 720–800 4.6–4.9 2 2 2.4 44 (31–57) 3.3 (2.2–4.3) FNR-PE 240–800 4.7–14.8 2 2 2.4 71 (67–75) 2.3 (2.0–2.7) FNR 430–900 4.3–8.5 2 27 4.9 167 (29–234) 144.0 (0.8–290.3) FNR 90–800 4.7–25.1 2 1 1.2 29 1.9 FNR 440 8.5 2 1 1.2 38 2.7 FNR 800 4.7 2 1 1.2 76 8.1 FNR 800 4.7 1-2 13 12.2 30(9–50) 1.6(0.5–5.3) FNR-RN 35–900 4.6–27.4 1-2 5 4.9 16 (14–19) 1.6 (0.7–3.3) FNR 58–900 4.3–26.6 2 1 1.2 32 0.3 FNR 900 4.3 2 2 2.4 72 (55–90) 60.3 (12.0–108.7) FNR-PE 680–900 4.3–5.2 2 4 4.9 41 (32–50) 3.6 (2.5–6.2) FNR-PE 505–800 4.7–7.0 2 1 1.2 35 0.7 FNR 900–900 4,3 2 2 2.4 39 (30–48) 3.0 (1.1–4.9) FNR 505–900 4.3–7.0 2 2 2.4 59 (19–98) 32.6 (0.4–64.8) FNR-PE 680–720 4.9–5.2 1 1 1.2 6 0.2 FNR 68 24.5 9818 2 1 1.2 170 45.1 FNR 900 4.7 2 2 2.4 78 (42–113) 6.7 (4.0–9.4) FNR-RN 720–780 4.6–4.9 2022), 2Mincarone et al. (2021a), 3Villarins et al. (2022), 4Afonso et al. (2021), 5Eduardo et al. (2019), 6Mincarone et al. S N FO% SL (mm) TW (g) Locality Depth (m) T (°C) Species Frontiers in Marine Science 09 frontiersin.org Eduardo et al. 10.3389/fmars.2022.937154 TABLE 1 Continued Species S N FO% SL (mm) TW (g) Locality Depth (m) T (°C) Gempylidae Gempylus serpens Cuvier, 1829 1-2 3 3.7 68 (44–112) 1.1 (0.9–1.3) FNR 70–900 4.3–25.8 Lepidocybium ﬂavobrunneum (Smith, 1843) 1 1 1.2 36 4.1 FNR 110 24.0 Nesiarchus nasutus Johnson, 1862 2 4 3.7 107 (85–145) 1.7 (0.7–2.8) FNR 90–800 4.7–25.1 Promethichthys prometheus (Cuvier, 1832) 1 15 1.2 154 (112–191) 20.2 (15.0–34.0) FNR 150 20.6 Nomeidae Cubiceps pauciradiatus Günther, 1872 2 10 7.3 91 (75–129) 13.2 (6.7–30.3) FNR 65–720 4.9–26.5 Psenes cyanophrys Valenciennes, 1833 1-2 5 3.7 86 (14–133) 38.9 (8.9–70.2) FNR 25–570 6.3–28.8 Trichiuridae Aphanopus intermedius Parin, 19837 2 1 1.2 720 550 FNR 610 5.7 TRACHINIFORMES Chiasmodontidae Chiasmodon braueri Weber, 1913 2 2 1.2 82 (70–95) 2.9 (2.2–3.7) FNR 900 4.3 Chiasmodon niger Johnson, 1864 2 1 1.2 90 7.9 FNR 800 4.7 Kali kerberti (Weber, 1913) 2 5 4.9 127 (69–170) 11.8 (1.1–29.3) FNR 720–800 4.6–4.9 Pseudoscopelus cordilluminatus Melo, 20101 2 2 2.4 44 (31–57) 3.3 (2.2–4.3) FNR-PE 240–800 4.7–14.8 Pseudoscopelus scutatus Krefft, 1971 2 2 2.4 71 (67–75) 2.3 (2.0–2.7) FNR 430–900 4.3–8.5 SCORPAENIFORMES Setarchidae Ectreposebastes imus Garman, 1899 2 27 4.9 167 (29–234) 144.0 (0.8–290.3) FNR 90–800 4.7–25.1 CAPROIFORMES Caproidae Antigonia capros Lowe, 1843 2 1 1.2 29 1.9 FNR 440 8.5 Antigonia combatia Berry & Rathjen, 1959 2 1 1.2 38 2.7 FNR 800 4.7 LOPHIIFORMES Ceratiidae Ceratias uranoscopus Murray, 1877 2 1 1.2 76 8.1 FNR 800 4.7 Himantolophidae Himantolophus spp. 1-2 13 12.2 30(9–50) 1.6(0.5–5.3) FNR-RN 35–900 4.6–27.4 Melanocetidae Melanocetus johnsonii Günther, 1864 1-2 5 4.9 16 (14–19) 1.6 (0.7–3.3) FNR 58–900 4.3–26.6 Thaumatichthyidae Thaumatichthys sp.8 2 1 1.2 32 0.3 FNR 900 4.3 Oneirodidae Chaenophryne draco Beebe, 1932 2 2 2.4 72 (55–90) 60.3 (12.0–108.7) FNR-PE 680–900 4.3–5.2 Chaenophryne ramifera Regan & Trewavas, 19328 2 4 4.9 41 (32–50) 3.6 (2.5–6.2) FNR-PE 505–800 4.7–7.0 Dolopichthys sp.8 2 1 1.2 35 0.7 FNR 900–900 4,3 Oneirodes anisacanthus (Regan, 1925)8 2 2 2.4 39 (30–48) 3.0 (1.1–4.9) FNR 505–900 4.3–7.0 Oneirodes carlsbergi (Regan & Trewavas, 1932)8 2 2 2.4 59 (19–98) 32.6 (0.4–64.8) FNR-PE 680–720 4.9–5.2 Caulophrynidae Caulophryne sp.8 1 1 1.2 6 0.2 FNR 68 24.5 Gigantactinidae Gigantactis watermani Bertelsen, Pietsch & Lavenberg, 19818 2 1 1.2 170 45.1 FNR 900 4.7 Rhynchactis sp.8 2 2 2.4 78 (42–113) 6.7 (4.0–9.4) FNR-RN 720–780 4.6–4.9 Specimen(s) damaged. Classiﬁcation follows Nelson et al. (2016), with exceptions noted in Villarins et al. (2022) for the Stomiiformes. Superscript numbers indicate species recorded for the ﬁrst time in the Brazilian Exclusive Economic Zone and their respective references. Species 10 Frontiers in Marine Science frontiersin.org 10.3389/fmars.2022.937154 Eduardo et al. indicating that more species would be recorded with additional sampling using the same gears (Figure 2). Indeed, richness estimators indicated that about 100 (50%) additional mesopelagic species of ﬁshes are expected to occur in the area (Figure 2). Additionally, 759 specimens representing about 40 ﬁsh taxa were sampled. However, they could not be identiﬁed to species level given their small size and/or poor condition. As it was not possible to determine whether these specimens belong to species not listed in Table 1, they were placed in a separate list to ensure a more robust assessment of species diversity (Supplementary Material 3). 2% of the total number of specimens collected. At the alpha taxonomic level, the following taxa represented almost 50% of all specimens collected: Sternoptyx diaphana (14%), Cyclothone spp. (11%; see Discussion), Diaphus brachycephalus (6%), Argyropelecus afﬁnis (6%), Chauliodus sloani (5%), Lampanyctus nobilis (4%), and Diaphus perspicillatus (4%). About 126 species were represented by ﬁve specimens or less, of which 62 were represented by a single specimen. Considering biomass, the most representative families were the Myctophidae (24%), Stomiidae (21%), Setarchidae (Scorpaeniformes, 11%), Sternoptychidae (10%), and Melamphaidae (7%) (Figure 3). These families together accounted for 73% of the biomass of all ﬁshes collected. The remaining families individually accounted for less than 4% of the total weight. At the speciﬁc level, the following species represented 42% of the biomass: Ectreposebastes imus (11%), Chauliodus sloani (9%), Borostomias elucens (6%), Eumecichthys ﬁski (6%, a single specimen), Sternoptyx diaphana (4%), Melamphaes polylepis (3%), and Argyropelecus afﬁnis (3%). Ranges of standard length (SL) and wet weight for all species collected on the two surveys are provided in Table 1. Overall, a wide size range was sampled, from 4 mm (Diretmoides pauciradiatus) to 1,880 mm SL (Eumecichthys ﬁski, Lophotidae). However, 90% of the specimens measured between 30 and 200 mm SL (Supplementary Material 4) The ﬁve orders with the highest number of species were the Stomiiformes (at least 62 species, four families), Myctophiformes (35 spp., two families), Aulopiformes (18 spp., seven families), Beryciformes (16 spp., three families), and Lophiiformes (12 spp., seven families), accounting for 70% of the total number of species recorded on the two surveys. Thirteen orders included less than ﬁve species each. Species Considering families, the most representative were the Stomiidae (38 spp.), Myctophidae (34 spp.), Melamphaidae (11 spp.), Sternoptychidae (10 spp.), and Gonostomatidae (7 spp.) (Figure 3). Half of the families (28) were represented by a single species. Distribution, vertical migration, biodiversity indexes, and size Based on the two campaigns, 60 species (29%) were recorded in a wide longitudinal distribution (Table 1). In contrast, 133 species (64%) were collected only in a few localities, with 116 being restricted to the Fernando de Noronha Ridge area, which aggregates most specimens collected (Table 1). Considering depth and period, the highest diversity, abundance, and biomass were found between depths of 700 and 1,000 m during the day (Figure 4). At night, the highest number of species was recorded at lower mesopelagic depths (500–1,000 m). However, much larger values of number of species, abundance, and biomass were detected in shallow waters (0– In terms of abundance, the most representative families when considering the two surveys were the Myctophidae (Myctophiformes; 36%), Sternoptychidae (Stomiiformes; 26%), Gonostomatidae (Stomiiformes; 16%), Stomiidae (Stomiiformes; 8%), and Melamphaidae (Beryciformes; 2%) (Figure 3). These families together accounted for 88% of all specimens collected. The remaining families represented individually no more than FIGURE 2 Species accumulation (S) and Chao1 estimator for ABRACOS 1 and 2 together. Dashed lines represent the conﬁdence interval of 95%. RE 2 cies accumulation (S) and Chao1 estimator for ABRACOS 1 and 2 together. Dashed lines represent the conﬁdence interval of 95%. FIGURE 2 Species accumulation (S) and Chao1 estimator for ABRACOS 1 and 2 together. Dashed lines represent the conﬁdence interval of 95%. 11 11 Frontiers in Marine Science frontiersin.org frontiersin.org Eduardo et al. 10.3389/fmars.2022.937154 FIGURE 3 Main mesopelagic ﬁsh families collected on the surveys ABRACOS 1 and ABRACOS 2 when considering diversity, abundance, and biomass. Fish images represent only examples of species included in the group. FIGURE 3 Main mesopelagic ﬁsh families collected on the surveys ABRACOS 1 and ABRACOS 2 when considering diversity, abundance, and biomass. Fish images represent only examples of species included in the group. biogeographic classiﬁcation of the mesopelagic zone (Sutton et al., 2017), the Tropical and western Equatorial Atlantic, which is the larger area encompassing the SWTA, was not considered a region particularly diverse in terms of mesopelagic ﬁshes. However, the mesopelagic species richness revealed by our two campaigns is higher than those reported for other parts of the world, such as the Mediterranean (25 spp.; Olivar et al., 2012), central Equatorial Paciﬁc (113 spp.; Barnett, 1984), southwestern Indian Ocean (121 spp.; Cherel et al., 2020), eastern Equatorial Atlantic (132 spp.; Olivar et al., 2017), and South China Sea (169 spp.; Wang et al., 2019). Distribution, vertical migration, biodiversity indexes, and size The species richness of mesopelagic ﬁshes in the SWTA is actually more similar to that reported for the North Paciﬁc (228 spp.; Barnett, 1984) and the Gulf of Mexico (approximately 300 spp.; Sutton et al., 2020), which are considered as comprising some of the most speciose deep-sea ichthyofaunas of the world (Sutton et al., 2017). Major factors driving deep-sea biodiversity, such as climate, seabed structure, water masses, and phylogenetic history, are likely responsible for the variation in species richness of different parts of the world. However, an asymmetry in collecting effort is certainly affecting the values recorded so far. In the Gulf of Mexico, a much higher sampling effort has been deployed to assess the deep-sea diversity compared with most regions of the world, with several expeditions conducted only in the last decade (Sutton et al., 2020). That situation is in striking contrast with the SWTA, where just a handful of expeditions to assess deep-sea diversity have been conducted in the last centuries. 200 m), likely reﬂecting the ascent in the water column of several species at night. At least 50 species seem to have a wide range of depth distribution and tolerance to variations in water temperature (up to 800 m and 25°C; e.g., species of Sternoptychidae and Myctophidae). However, 66 species seem to be restricted to deeper (> 600 m) and colder waters (< 6° C) regardless of the time period (e.g., Lophiiformes and Beryciformes; Table 1). Signiﬁcant differences in biodiversity indexes (calculated only for ABS2, see methodology) were found when considering diel periods and depth. Higher values of richness and diversity were found in lower mesopelagic waters and during the daytime. However, dominance values were signiﬁcantly higher in epipelagic waters and also during daytime (Figure 5). Detailed values for the calculated indexes are given in the Supplementary Material 5. Frontiers in Marine Science frontiersin.org Diversity and distribution Based on our two campaigns, at least 201 species of mesopelagic ﬁshes occur in the SWTA. Results also indicate that about 100 additional species could have been collected if sampling efforts were increased. The taxonomically diverse pool of mesopelagic species recorded in our surveys also reveals a vast array of diversity not only in terms of the number of species but also in terms of size, anatomy, and behaviour. In a recent global The relatively high number of mesopelagic species of ﬁshes recorded in our two campaigns is likely related to the diversity of Frontiers in Marine Science 12 frontiersin.org frontiersin.org Eduardo et al. 10.3389/fmars.2022.937154 FIGURE 4 Sample-size-based rarefaction (solid line segment) and extrapolation (dotted line segments) sampling curves for species richness, diversity, and dominance of mesopelagic ﬁsh data at different depth categories and diel periods. Curves include the conﬁdence intervals of 95% (shaded areas). For this analysis, only species recorded in the ABRACOS 2 survey were considered. FIGURE 4 Sample-size-based rarefaction (solid line segment) and extrapolation (dotted line segments) sampling curves for species richness, diversity, and dominance of mesopelagic ﬁsh data at different depth categories and diel periods. Curves include the conﬁdence intervals of 95% (shaded areas). For this analysis, only species recorded in the ABRACOS 2 survey were considered. present in the SWTA (Bourlès et al., 1999; Assunção et al., 2020; Costa da Silva et al., 2021; Dossa et al., 2021; Tosetto et al., 2021). This results in a high complexity of habitats and oceanographic conditions that likely contribute to higher levels of species diversity (Levin et al., 2001). habitats and the high variability of oceanographic processes of the SWTA. Despite being located in an oligotrophic portion of the ocean, this region is also characterized by the presence of underwater canyons, oceanic islands, and several seamounts that interact with local currents and enhance marine productivity (Travassos et al., 1999; Tchamabi et al., 2017; Costa da Silva et al., 2021). As an example, small uplifting processes have been reported along the shelf-break and oceanic islands of the region (Travassos et al., 1999; MMA, 2006; Tchamabi et al., 2017; Silva et al., 2022), a situation that has been directly associated with the occurrence of hotspots of ﬁsh biodiversity (Hazin, 1993; Eduardo et al., 2018; Eduardo et al., 2020a). frontiersin.org Notable records In terms of taxonomic composition, ﬁve families of the Teleostei accounted for 52% of the species richness, 88% of the specimens, and 66% of the total biomass collected on the two surveys: the Stomiidae (38 spp., 8% of the specimens, 21% of the biomass), Myctophidae (34 spp., 36%, 24%), Melamphaidae (11 spp., 2%, 7%), Sternoptychidae (9 spp., 26%, 10%), and Gonostomatidae (7 spp., 16%, 4%). These families, therefore, seem to be the most represented in the mesopelagic ﬁsh fauna of the SWTA. The dominance of these families in mesopelagic waters has also been reported in other parts of the world (e.g., Gjøsaeter and Kawaguchi, 1980; Olivar et al., 2017; Wang et al., 2019; Cherel et al., 2020). A strong pattern of dominance was also observed within these families, with few species accounting for 50% of the total number of specimens: Sternoptyx diaphana (14%), Cyclothone spp. (11%), Argyropelecus afﬁnis (6%), Diaphus brachycephalus (6%), Chauliodus sloani (5%), Diaphus perspicillatus (4%), and Lampanyctus nobilis (4%). The pattern of dominance at the species level detected in the SWTA was, however, distinct from other parts of the world. In the Eastern Tropical Atlantic, for instance, the lanternﬁshes B. suborbitale, C. warmingii, and H. macrochir were dominant (Olivar et al., 2017), whereas these same species were considered rare in our study. The viperﬁsh C. sloani is usually globally recorded in low abundances (e.g., Olivar et al., 2017; Among the 201 species of mesopelagic ﬁshes recorded during the ABRACOS expeditions, 50 (25%) represent new records for Brazilian waters, all of which have been dealt with in a series of recent papers (Table 1). In addition to these new records, eight species (ﬁve Eustomias, one Melanostomias, one Melamphaes, and one Poromitra) are potentially new and will be formally described later. Several species recorded here are also rare worldwide, and their occurrence in the SWTA adds new information on their global distribution. For instance, three specimens of Platyberyx paucus and one of Platyberyx pietschi were collected during the AB2. Before these records, only four specimens of P. paucus were known, from the central North Paciﬁc and western Central Atlantic. Platyberyx pietschi, in turn, was known from just two specimens collected in the western Central Atlantic, one specimen collected in the central Paciﬁc, and another from the western South Paciﬁc (Stevenson and Kenaley, 2013; Mincarone et al., 2019). Diversity and distribution Distinct biogeographic provinces, with different thermodynamic features, current systems and water mass properties, are also This results in a high complexity of habitats and oceanographic conditions that likely contribute to higher levels of species diversity (Levin et al., 2001). The highest levels of richness and diversity (considering only specimens collected during AB2, see methodology) were found at lower mesopelagic depths (500–1,000 m), with several species collected only at these depths (e.g., species of the Beryciformes and Lophiiformes). Interestingly, many of these species are considered bathypelagic and/or benthopelagic (Priede, 2017; Melo et al., 2020). The collection of those species in mesopelagic waters is likely related to the presence of seamounts and oceanic islands. In addition to being related to Frontiers in Marine Science 13 frontiersin.org frontiersin.org Eduardo et al. 10.3389/fmars.2022.937154 FIGURE 5 Number of species and average values of abudance (individuals.hour-1) and biomass (kg.hour-1x102) of mesopelagic species of ﬁshes collected on the survey ABRACOS 2. Depth strata not sampled. FIGURE 5 Number of species and average values of abudance (individuals.hour-1) and biomass (kg.hour-1x102) of mesopelagic species of ﬁshes collected on the survey ABRACOS 2. Depth strata not sampled. FIGURE 5 Number of species and average values of abudance (individuals.hour-1) and biomass (kg.hour-1x102) of mesopelagic species of ﬁshes collected on the survey ABRACOS 2. Depth strata not sampled. 14 Frontiers in Marine Science frontiersin.org frontiersin.org Eduardo et al. 10.3389/fmars.2022.937154 an increase in habitat complexity, seamounts may increase the occurrence of pelagic and benthic predators that actively seek these areas to hunt for prey trapped by ﬂow-topographic processes (Cascão et al., 2019). For instance, in the Azorean seamounts plateau, the micronekton community is dominated by non- or weakly migratory benthopelagic ﬁshes (Cascão et al., 2019). In summary, our results also seem to indicate that seamounts play a signiﬁcant role in the biodiversity structuring and ecology of mesopelagic ﬁshes in the SWTA. Wang et al., 2019; Cherel et al., 2020), whereas the species is among the most relevant mesopelagic species in the SWTA considering the abundance and total weight (Eduardo et al., 2020c). Diversity and distribution These differences in the pattern of dominance at the species level in different parts of the world are likely associated with different sampling strategies employed and differences in oceanographic and biogeographic features (e.g., seabed structure, water masses, and hydrographic fronts), which are major factors driving the structure and composition of mesopelagic assemblages (Hulley and Krefft, 1985; Olivar et al., 2017; Cascão et al., 2019). Cyclotone is another seemingly abundant genus of mesopelagic ﬁsh in the SWTA (Olivar et al., 2017). Eight species of the genus were reported for the SWTA: C. acclinidens, C. alba, C. braueri, C. microdon, C. obscura, C. pallida, C. parapallida, and C. pseudopallida (Villarins et al., 2022) The sampling gears employed in the study, however, seemed to be only partially adequate to collect specimens of the genus. In several trawls we observed onboard that a substantial number of specimens of Cyclothone escaped back into the sea during the hoisting of the net. Additionally, given their poor condition of preservation, specimens of the genus could not be identiﬁed at species level. Therefore, the abundance of species of Cyclothone presented here is underestimated. The two surveys conducted during this study resulted in different patterns of species richness. For example, 17 species were exclusively recorded in AB1 (mesopelagic trawl), whereas 136 species were recorded only in AB2 (micronekton trawl). The two campaigns were conducted in different seasons. However, since the study area is located in a tropical region, few oceanographic differences were noted in the mesopelagic zone (for further info refer to Assunção et al., 2020; Costa da Silva et al., 2021; Dossa et al., 2021). Therefore, the signiﬁcant disparity in species richness between the two expeditions is clearly related to differences in sampling strategies. The use of multiple sampling gears is vital to maximizing the representation of ﬁsh diversity (Magurran, 2004), especially in the deep-sea. However, the sampling strategy used in AB2, which included the use of larger gear, with greater mesh sizes, deeper hauls, and broader sampling area, resulted in the collection of a higher number of specimens of different species in a broader size range (Supplementary Material 2 and 3). Role of international cooperation for the decade of deep ocean science their support. For providing literature we thank Alexei Orlov (P.P. Shirshov Institute of Oceanology), Artem Prokoﬁev (A.N. Severtsov Institute of Ecology and Evolution), Jørgen Nielsen (Natural History Museum of Denmark), Ron Fricke (State Museum of Natural History Stuttgart), Sergei Evseenko (in memorian, P.P. Shirshov Institute of Oceanology), and Thomas Munroe (National Systematics Laboratory, NOAA). This study is part of the PhD thesis conducted by the ﬁrst author, who is especially grateful to members of the examination committee, Anne Lebourges-Dhaussy, Emmanuel Paradis, Heino Fock, Juan Carlos Molinero, Luiz A. Rocha, Paulo Travassos, Rosângela Lessa, and Yves Cherel, for their critical reviews and constructive comments. CAPES (Coordination for the Improvement of Higher Education Personnel) provided a student scholarship to Leandro Eduardo, who is also supported by FUNBIO and Humanize under the grant “Programa Bolsas Funbio - Conservando o Futuro 2018” (011/2019). We thank CNPq (Brazilian National Council for Scientiﬁc and Technological Development) for providing research grants to Flávia Lucena- Frédou, Thierry Frédou, and Michael Mincarone (grants 308554/ 2019-1, 307422/2020-8, and 314644/2020-2, respectively). Gabriel Afonso was supported by PIBIC/CNPq, Bárbara Villarins was supported by PIBIC/CNPq and PROTAX/CNPq (443302/2020), and Júlia Martins was supported by CAPES, FUNBIO, and PROTAX/CNPq (443302/2020). This study is a contribution to the LMI TAPIOCA, program CAPES/COFECUB (88881.142689/ 2017-01), EU H2020 TRIATLAS project (grant agreement 817578). The NPM Fish Collection is supported by the Project MULTIPESCA (FUNBIO) under the grant ‘Pesquisa Marinha e Pesqueira’ and FAPERJ (grant E-26/210.290/2021). The high number of new records made during the ABRACOS expeditions reﬂects not only the high diversity of the SWTA, but also the overall lack of scientiﬁc information on deep-sea diversity in the region, as noted previously (e.g., Reis et al., 2016; Mincarone et al., 2022). The United Nations Decade of Ocean Science roadmap recognizes the deep-sea as a frontier of science and discovery (Ryabinin et al., 2019). There is an unequal capacity to conduct science among nations, with developing economies facing substantial barriers to participating in deep-sea research. Consequently, the least-studied parts of the deep-sea are located off the least economically developed countries (Howell et al., 2020). These biases are highlighted by the fact that a French research institution ﬁnanced the surveys described here, and that those expeditions are among the very few that have addressed the mesopelagic ichthyofauna of Brazil. To achieve sustainability, we need a well-known and predictable ocean. Notable records Other species considered rare worldwide that were collected in the ABRACOS expeditions are Aulotrachichthys argyrophanus, Rhynchohyalus natalensis, Eumecichthys ﬁski, Macrouroides inﬂaticeps, Pseudoscopelus cordilluminatus, Melamphaes leprus, and Gigantactis watermani (Pimentel et al., 2020; Afonso et al., 2021; Mincarone et al., 2021b; Mincarone et al., 2022). frontiersin.org Frontiers in Marine Science 15 10.3389/fmars.2022.937154 Eduardo et al. Author contributions All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. All co-authors declare no competing interests and agree with the submission of this manuscript. All authors signiﬁcantly contributed to the development of the article. Role of international cooperation for the decade of deep ocean science Only by thinking globally and strengthening international cooperation we will develop an ocean research that corrects asymmetry in funding and knowledge among countries, meeting the crucial need for a more encompassing deep-sea knowledge aimed at the conservation and sustainable use of its unique habitats. Ethics 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. All collecting methods and specimen handling procedures were approved and carried out in accordance with relevant guidelines and regulations of the Brazilian Ministry of Environment (SISBIO; authorization number: 47270–5). 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https://openalex.org/W4319080204	https://eprints.gla.ac.uk/288740/1/288740.pdf	English	null	Exploring the Effects of EEG-Based Alpha Neurofeedback on Working Memory Capacity in Healthy Participants	Bioengineering	2,023	cc-by	18,826	Article Exploring the Effects of EEG-Based Alpha Neurofeedback on Working Memory Capacity in Healthy Participants Rab Nawaz 1,2, Guilherme Wood 3,4, Humaira Nisar 1,5,* and Vooi Voon Yap 1,6 1 Department of Electronic Engineering, Faculty of Engineering and Green Technology, Universiti Tunku Abdul Rahman, Kampar 31900, Malaysia 2 Biomedical Engineering Research Division, University of Glasgow, Glasgow G12 8QQ, UK 3 Department of Psychology, University of Graz, Universitaetsplatz 2/III, 8010 Graz, Austria 4 BioTechMed-Graz, 8010 Graz, Austria 5 Centre for Healthcare Science and Technology, Universiti Tunku Abdul Rahman, Sungai Long 31900, Malaysia 6 Department of Computer Science, Aberystwyth University, Penglais SY23 3FL, UK * Correspondence: humaira@utar.edu.my 1 Department of Electronic Engineering, Faculty of Engineering and Green Technology, Universiti Tunku Abdul Rahman, Kampar 31900, Malaysia 2 Biomedical Engineering Research Division, University of Glasgow, Glasgow G12 8QQ, UK 3 Department of Psychology, University of Graz, Universitaetsplatz 2/III, 8010 Graz, Austria 4 BioTechMed-Graz, 8010 Graz, Austria 5 Centre for Healthcare Science and Technology, Universiti Tunku Abdul Rahman, Sungai Long 31900, Malaysia 6 Department of Computer Science, Aberystwyth University, Penglais SY23 3FL, UK * Correspondence: humaira@utar.edu.my 1 Department of Electronic Engineering, Faculty of Engineering and Green Technology, Universiti Tunku Abdul Rahman, Kampar 31900, Malaysia 2 Biomedical Engineering Research Division, University of Glasgow, Glasgow G12 8QQ, UK 3 Department of Psychology, University of Graz, Universitaetsplatz 2/III, 8010 Graz, Austria 4 BioTechMed-Graz, 8010 Graz, Austria 5 Centre for Healthcare Science and Technology, Universiti Tunku Abdul Rahman, Sungai Long 31900, Malaysia 6 Department of Computer Science, Aberystwyth University, Penglais SY23 3FL, UK * Correspondence: humaira@utar.edu.my 1 Department of Electronic Engineering, Faculty of Engineering and Green Technology, Universiti Tunku Abdul Rahman, Kampar 31900, Malaysia 2 Biomedical Engineering Research Division, University of Glasgow, Glasgow G12 8QQ, UK 3 Department of Psychology, University of Graz, Universitaetsplatz 2/III, 8010 Graz, Austria 4 BioTechMed-Graz, 8010 Graz, Austria 5 Centre for Healthcare Science and Technology, Universiti Tunku Abdul Rahman, Sungai Long 31900, Malaysia 6 Department of Computer Science, Aberystwyth University, Penglais SY23 3FL, UK Abstract: Neurofeedback, an operant conditioning neuromodulation technique, uses information from brain activities in real-time via brain–computer interface (BCI) technology. This technique has been utilized to enhance the cognitive abilities, including working memory performance, of human beings. The aims of this study are to investigate how alpha neurofeedback can improve working memory performance in healthy participants and to explore the underlying neural mechanisms in a working memory task before and after neurofeedback. Citation: Nawaz, R.; Wood, G.; Nisar, H.; Yap, V.V. Exploring the Effects of EEG-Based Alpha Neurofeedback on Working Memory Capacity in Healthy Participants. Bioengineering 2023, 10, 200. https://doi.org/ 10.3390/bioengineering10020200 Keywords: EEG; neurofeedback training; working memory capacity; functional connectivity; N-back Article Exploring the Effects of EEG-Based Alpha Neurofeedback on Working Memory Capacity in Healthy Participants Thirty-six participants divided into the NFT group and the control group participated in this study. This study was not blinded, and both the participants and the researcher were aware of their group assignments. Increasing power in the alpha EEG band was used as a neurofeedback in the eyes-open condition only in the NFT group. The data were collected before and after neurofeedback while they were performing the N-back memory task (N = 1 and N = 2). Both groups showed improvement in their working memory performance. There was an enhancement in the power of their frontal alpha and beta activities with increased working memory load (i.e., 2-back). The experimental group showed improvements in their functional connections between different brain regions at the theta level. This effect was absent in the control group. Furthermore, brain hemispheric lateralization was found during the N-back task, and there were more intra-hemisphere connections than inter-hemisphere connections of the brain. These results suggest that healthy participants can beneﬁt from neurofeedback and from having their brain networks changed after the training. bioengineering bioengineering bioengineering bioengineering 1. Introduction Received: 26 December 2022 Revised: 20 January 2023 Accepted: 31 January 2023 Published: 3 February 2023 Neurofeedback training (NFT) is an operant conditioning procedure that can modulate brain electrical activity such that one can learn to control one’s own activity. In late 1950s and early 1960s, Joe Kamiya was the ﬁrst to demonstrate the ability of brainwaves through feedback and is, therefore, known as the father of neurofeedback. Barry Sterman discovered the clinical potential of NFT in the early 1970s and evaluated, for the ﬁrst time, the associa- tion between brain activity and operant conditioning procedure by training cats to increase their sensorimotor rhythm (SMR) [1], which is usually considered a part of the alpha EEG rhythm [2]. During NFT, through a real-time interface with a computer, information about one’s brain activity is received as feedback and the desired modulation of brain activity leads to cognitive function, mood, motor function, and behavioural improvements [2,3]. 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/). Characterized by individual differences, an alpha band NFT design is one of the most effective protocols [4] and its functional role in neurofeedback (NF) protocols of cognitive performance has been increasingly discussed in the literature [3,5]. Over the https://www.mdpi.com/journal/bioengineering Bioengineering 2023, 10, 200. https://doi.org/10.3390/bioengineering10020200 Bioengineering 2023, 10, 200 2 of 22 past two decades, research has reported a relationship between alpha activity and memory functions [6,7] and has shown that alpha and theta activities are related to memory per- formance [8]. However, alpha reactivity and event-related changes in alpha power also show that, during actual task demands, the cognitive performance (in particular memory performance) increases with the suppression of alpha activity [8]. Memory processing is also associated with pre-stimulus alpha EEG activity [9]. Similarly, alpha NFT has been shown to produce signiﬁcant improvements in working memory [10–12]. These studies indicated that improvements in short-term memory are positively correlated with an in- crease in the relative amplitude in the individual upper alpha band during training when the EEG signal is recorded from the Cz channel [11]. In another study, subjects with mild cognitive impairment were trained to increase the power of their individual upper alpha band of the electroencephalogram (EEG) signal over the central parietal region. An increase in peak alpha frequency was observed throughout the period of training, and the memory performance also improved signiﬁcantly following the training [13]. Another NFT study provided promising results that demonstrate the trainability of frontoparietal alpha rhythm and its functional correlations with working memory and episodic memory [14]. These ﬁndings support the importance of alpha modulation in cognitive enhancements [5]. Although during NFT, a speciﬁc EEG activity over a speciﬁc brain region is used for training, this produces concomitant changes in brain regions and activities other than the trained one [12,15,16]. This indicates the interdependency of different oscillations of brain signals. The conventional taxonomy of brain oscillations is arbitrary, and brain signals are naturally non-isolated in-term of frequency ranges, which leads to this interdepen- dency [17]. Besides the brain region and the frequency band, which is used during the training, investigating the effects of NFT in other brain regions and frequency bands can demonstrate the global underlying neural changes in the brain. The goal of the current study was, therefore, to analyse the data in the alpha band (8–13 Hz) (i.e., trained activity), as well as in the theta (4–8 Hz) and beta (13–30 Hz) bands (i.e., non-trained activities). ( ) ( ) ( ) Cognitive activity recruits functional connectivity (FC) between regions distributed over the whole brain instead of a single brain region [18,19]. Along with the studies reporting the increase in spectral power, NFT modulates large-scale neural networks [20], and there is evidence of changes in the brain FC after NFT [17]. Such FC changes are observed in a task-based EEG activity after NFT [20]. The FC changes after NFT have been extensively studied in patients with neurological diseases, such as traumatic brain injuries [21] and autism [22]. However, the exploration of changes in FC after NFT in task-based EEG in healthy participants is lacking. The processing of information in the left and right hemispheres of the human brain has unique properties that lead to functional asymmetry during memory tasks [23,24]. This asymmetric behaviour of the brain is known as hemispheric lateralization [25]. Different studies have identiﬁed a decrease in hemispheric lateralization associated with the cogni- tive demands during controlled inhibition [26], conﬂict resolution [27], and memory [28]. Asymmetric brain activation can also lead to differential FC in the two hemispheres. In general, the rationale behind this study is to explore the effect of alpha NFT on cognitive abilities. Speciﬁcally, in this paper, we want to see its effect on the working memory ca- pacity in healthy participants by exploring the changes in the FC after NFT in an N-back memory task. Therefore, our ﬁrst hypothesis is that the brain network in the left and right hemispheres are involved in different degrees during the N-back (1- and 2-back) task. Additionally, our second hypothesis is that FC in both hemispheres will be enhanced after NFT. For this reason, we explored the hemispheric lateralization of FC using an N-back working memory task in two different memory loads (1- and 2-back) [29] and evaluated the changes in FC before and after NFT, with the aim to assess our hypothesis. The n-back task is a continuous performance task to measure working memory capacity in a way com- plementary to other established working memory tasks [19], so that it provides evidence on facets of working memory not typically addressed by other well-established working memory tasks. 3 of 22 Bioengineering 2023, 10, 200 2.1. Participants Initially, 50 healthy participants (18 females and 32 males) were recruited via email advertisement over the campus. The participants were equally divided into two groups, the NFT group and the control group, and the assignments in terms of number of females in each group, were roughly equal. They were university students. The mean age of the NFT group was 23.32 ± 4.66 years, and that of the control group was 26 ± 4.84 years, with no signiﬁcant difference in the group ages. Due to unavoidable circumstances (e.g., their scheduled session clashed with their classes), some of the participants could not participate in the study until the end, and therefore, they were excluded from the analysis. Additionally, the data of some the participants were also excluded from the analysis due to the excessive artifacts in their N-back EEG recordings. The ﬁnal sample size for the analysis in the current study included the data from 36 participants (25.45 ± 5.36 years old; 12 females and 24 males). Before conducting the experiments, the researcher conducted a one-to-one interview with each participant and verbally asked about their health (e.g., if they currently have any health issues or had any psychological problem in the past). All participants were healthy and had no psychological problems. Participants were randomly assigned to either the NFT group (17 participants) or the control group (19 participants). They voluntarily participated in the study, and no remuneration was provided. All experimental procedures were evaluated and approved by the scientiﬁc and ethical review committee of the Institute of Post-graduate Studies and Research (IPSR), Universiti Tunku Abdul Rahman, Malaysia (ref. number U/SERC/90/2018) and were in accordance with the Declaration of Helsinki. Before the start of the experiments, each participant was briefed by the researcher in a one-on-one meeting about the purpose and procedures of the study. A written informed consent form was signed by each participant. 2.2. Neurofeedback Training An Acer swift, 8 GB RAM, Windows 10 OS, 13.5-inch native display computer was used for data acquisition and processing. The participants viewed the vertical alpha power bar, which was updated in real time, on a 17-inch LCD monitor connected to the main computer. The participants were seated in a chair in front of the 17-inch display monitor at a comfortable distance during the NFT session. The participants had to look at the vertical bar, so they were asked to keep their eyes open and to minimize the blinking. The NFT group participants received training for a total of 600 min, which was di- vided into 20 sessions, where each session had a duration of 30 min (20 × 30 min = 600 min). Each 30 min session was further divided into two sub-sessions, each of 15 min du- ration. A short break of 1–2 min was provided between the two sub-sessions. Each partic- ipant received a minimum of one and a maximum of three NFT sessions per week. The participants in the NFT group took around 10 weeks from the beginning of the first NFT session to complete 20 sessions. Therefore, there was approximately 10 weeks in the gap between the pre-NF and post-NF N-back task. The time slot for the training was kept con- sistent across all the sessions for the participants to minimize the influence of circadian rhythm [38]. The present study is not a clinical study and should not be interpreted as such. The study’s aims were to provide new evidence on working memory mechanisms, not on the efficacy of alpha NFT in enhancing working memory capacity. Thus, the par- ticipants in the control group did not perform any training. They performed the N-back task at the start (which counted as the pre-NF session) and at the end (which counted as the post-NF session). To compare the NFT effects in an unbiased manner in terms of The NFT group participants received training for a total of 600 min, which was divided into 20 sessions, where each session had a duration of 30 min (20 × 30 min = 600 min). Each 30 min session was further divided into two sub-sessions, each of 15 min duration. A short break of 1–2 min was provided between the two sub-sessions. Each participant received a minimum of one and a maximum of three NFT sessions per week. 2.2. Neurofeedback Training Figure 1B shows Bioengineering 2023, 10, 200 4 of 22 to the ar was the connections between OpenVibe, Unity 3D, and LSL and the ﬂow of the signal from the EEG device to the ﬁnal output of the feedback bar. y p g y ( ) work protocol. Figure 1B shows the connections between OpenVibe, Unity 3D, and LSL and the flow of the signal from the EEG device to the final output of the feedback bar. Figure 1. Representation of the NFT training system. (A) A participant is sitting in front of the feed- back screen, and the visual feedback is shown as a red vertical bar on the screen. (B) The EEG data are recorded for the offline analysis, and the signal processing module in OpenVibe is connected with the Unity3D via LSL protocol. Figure 1. Representation of the NFT training system. (A) A participant is sitting in front of the feedback screen, and the visual feedback is shown as a red vertical bar on the screen. (B) The EEG data are recorded for the ofﬂine analysis, and the signal processing module in OpenVibe is connected with the Unity3D via LSL protocol. Figure 1. Representation of the NFT training system. (A) A participant is sitting in front of the feed- back screen, and the visual feedback is shown as a red vertical bar on the screen. (B) The EEG data are recorded for the offline analysis, and the signal processing module in OpenVibe is connected with the Unity3D via LSL protocol. Figure 1. Representation of the NFT training system. (A) A participant is sitting in front of the feedback screen, and the visual feedback is shown as a red vertical bar on the screen. (B) The EEG data are recorded for the ofﬂine analysis, and the signal processing module in OpenVibe is connected with the Unity3D via LSL protocol. An Acer swift, 8 GB RAM, Windows 10 OS, 13.5-inch native display computer was used for data acquisition and processing. The participants viewed the vertical alpha power bar, which was updated in real time, on a 17-inch LCD monitor connected to the main computer. The participants were seated in a chair in front of the 17-inch display monitor at a comfortable distance during the NFT session. The participants had to look at the vertical bar, so they were asked to keep their eyes open and to minimize the blinking. 2.2. Neurofeedback Training The NFT was performed using Openvibe [30], which is an open source BCI software toolbox. A customized NFT protocol was designed. An Emotiv Epoc+ device was used for EEG signal acquisition; it consists of 14 active EEG sensors, which were placed on the scalp at the AF3, F7, F3, FC5, T7, P7, O1, O2, P8, T8, FC6, F4, F8, and AF4 locations according to 10–20 international standards. Despite the fact that the placement of the sensors from this device are not customizable, it has been used in many studies and has proved to be an effective wireless and low-cost device for NFT research [31]. The frontal lobe of the brain plays an important role in the higher level of cognitive functions and is responsible for immediate and sustained attention, time management, so- cial skills, emotions, empathy, working memory, and executive planning [32]. Furthermore, the alpha band is the most prevalent rhythm in adult EEG and related to psychological states and cognitive processing [8]. The frontal alpha activity caused by thalamic and anterior cingulate cortex activity addresses attention and working memory [33]. Therefore, for neurofeedback, the Alpha EEG rhythm (8–13 Hz) from the frontal sensors F7, F3, F4, and F8 were processed in real time via the signal processing module designed in OpenVibe [34]. The Butterworth ﬁlter with 0.5 dB pass band ripples was used to extract the alpha band from the raw EEG signals through the “Modiﬁable Temporal Box” in OpenVibe. Welch’s method with a Hamming window was used to compute the absolute value of the alpha band power using a 5 seconds window [35]. Human psychophysics indicates an impairment in the visual perception and performance with unsuitable feedback latency [36]. Therefore, considering these factors and the visual ergonomics of healthy people, the feedback was updated every second, meaning that there was 80% overlap with the previous segment. Power was computed for each sensor separately and, then, averaged over the sensors to use as feedback. The feedback was displayed to the participants in the form of a vertical bar on a monitor as feedback. This vertical bar was designed in Unity3D [37], which is also an open source software toolbox. OpenVibe and Unity3D communicate with each other with the help of a Lab Streaming Layer (LSL) network protocol. 2.3. Experimental Protocol 3. Experimental Protocol The current study emp 2.3. Experimental Protocol 3. Experimental Protocol The current study emp The current study employed an N-back memory task to assess working memory capacity with two memory load factors, 1-back (low memory load) and 2-back (high memory load). The structure of the N-back test is shown in Figure 2. Twelve English letters—B, D, H, J, K, M, N, P, R, T, V, and X—in the 1-back condition and thirteen letters—B, C, F, H, J, K, M, N, P, Q, S, V, and X—in the 2-back condition in capital font were presented to the participant on the screen as the test stimuli. For each stimulus, they were required to press a key (1 or 2) on the keyboard to respond to the stimulus. If the stimulus letter was a target, the participant needed to press 1, and if the stimulus letter was a non-target, the participant needed to press 2. The test consisted of two conditions, 1-back and 2-back, each containing 36 stimuli; 12 of these were targets, and the other 24 were non-targets. In the 1-back condition, if the letter was the same as the one presented just before, it was the target letter; otherwise, it was a non-target. Similarly, in the 2-back condition, a letter was a target if it was the same as the letter presented two before. Each stimulus appeared for a maximum of 1500 ms on the screen. The participant was required to respond to the stimulus within this time. Between the two consecutive stimuli, an inter stimulus interval (ISI) of 500 ms was presented in the form of a ﬁxation (i.e., a plus sign). y p y y g y acity with two memory load factors, 1-back (low memory load) and 2-back (high memory load). The structure of the N-back test is shown in Figure 2. Twelve English let- ers—B, D, H, J, K, M, N, P, R, T, V, and X—in the 1-back condition and thirteen letters— , C, F, H, J, K, M, N, P, Q, S, V, and X—in the 2-back condition in capital font were pre- ented to the participant on the screen as the test stimuli. For each stimulus, they were equired to press a key (1 or 2) on the keyboard to respond to the stimulus. 2.3. Experimental Protocol 3. Experimental Protocol The current study emp If the stimulus tter was a target, the participant needed to press 1, and if the stimulus letter was a non- arget, the participant needed to press 2. The test consisted of two conditions, 1-back and -back, each containing 36 stimuli; 12 of these were targets, and the other 24 were non- argets. In the 1-back condition, if the letter was the same as the one presented just before, was the target letter; otherwise, it was a non-target. Similarly, in the 2-back condition, a tter was a target if it was the same as the letter presented two before. Each stimulus ppeared for a maximum of 1500 ms on the screen. The participant was required to re- pond to the stimulus within this time. Between the two consecutive stimuli, an inter stim- lus interval (ISI) of 500 ms was presented in the form of a fixation (i.e., a plus sign). igure 2. Structure of the N-back task. Figure 2. Structure of the N-back task. igure 2. Structure of the N-back task. Before starting the test, the researcher orally gave the instructions to the participant. The task initiated with the 1-back condition, and then, the 2-back condition followed. The equence of test presentation was the same for all participants. Twenty practice trials pre- eded 36 test trials for each condition. When the practice trials were completed, the par- icipant was informed via onscreen instructions that “practice trials are finished and now ou will be presented with actual test trials”. The stimuli were presented on an extended display of 17-inch LCD via E-Prime version 3.0 (Psychology Software Tools, Pittsburgh, PA, USA) stimulus presentation software. Figure 2. Structure of the N-back task. Before starting the test, the researcher orally gave the instructions to the participant. The task initiated with the 1-back condition, and then, the 2-back condition followed. The sequence of test presentation was the same for all participants. Twenty practice trials preceded 36 test trials for each condition. When the practice trials were completed, the participant was informed via onscreen instructions that “practice trials are ﬁnished and now you will be presented with actual test trials”. The stimuli were presented on an extended display of 17-inch LCD via E-Prime version 3.0 (Psychology Software Tools, Pittsburgh, PA, USA) stimulus presentation software. gure 2. Structure of the N-back task. Figure 2. Structure of the N-back task. 2.2. Neurofeedback Training The participants in the NFT group took around 10 weeks from the beginning of the ﬁrst NFT session to complete 20 sessions. Therefore, there was approximately 10 weeks in the gap between the pre-NF and post-NF N-back task. The time slot for the training was kept consistent across all the sessions for the participants to minimize the inﬂuence of circadian rhythm [38]. The present study is not a clinical study and should not be interpreted as such. The study’s aims were to provide new evidence on working memory mechanisms, not on the efﬁcacy of alpha NFT in enhancing working memory capacity. Thus, the participants in the control group did not perform any training. They performed the N-back task at the start (which counted as the pre-NF session) and at the end (which counted as the post-NF session). To compare the NFT effects in an unbiased manner in terms of timing, the participants in the control group were invited for a second attempt at the task approximately 10 weeks after the ﬁrst attempt (i.e., pre-NF session). Therefore, the gap between the pre-NF and post-NF N-back tasks were approximately equal for both groups. pp y q g p Before starting the NFT session, the researcher instructed the participants to increase the height of the red vertical bar showing alpha power in real time (i.e., feedback bar) by adopting their own strategy. The complete design of the NFT protocol can be seen in Figure 1. 5 of 22 Fig- Bioengineering 2023, 10, 200 2.3. Experimental Protocol 3. Experimental Protocol The current study emp Before starting the test, the researcher orally gave the instructions to the participant. he task initiated with the 1-back condition, and then, the 2-back condition followed. The equence of test presentation was the same for all participants. Twenty practice trials pre- eded 36 test trials for each condition. When the practice trials were completed, the par- cipant was informed via onscreen instructions that “practice trials are finished and now ou will be presented with actual test trials”. The stimuli were presented on an extended isplay of 17-inch LCD via E-Prime version 3.0 (Psychology Software Tools, Pittsburgh, A USA) stimulus presentation software Before starting the test, the researcher orally gave the instructions to the participant. The task initiated with the 1-back condition, and then, the 2-back condition followed. The sequence of test presentation was the same for all participants. Twenty practice trials preceded 36 test trials for each condition. When the practice trials were completed, the participant was informed via onscreen instructions that “practice trials are ﬁnished and now you will be presented with actual test trials”. The stimuli were presented on an extended display of 17-inch LCD via E-Prime version 3.0 (Psychology Software Tools, Pittsburgh, PA, USA) stimulus presentation software. 2.4.1. EEG Recordings and Processing 2.4.1. EEG Recordings and Processing The continuous EEG data during the task performance were recorded using an Emotiv Epoc+ EEG device containing 14 active EEG sensors located at AF3, F7, F3, FC5, T7, P7, O1, O2, P8, T8, FC6, F4, F8, and AF4 and two reference electrodes placed on the mastoid bones behind the ears. The sampling rate of the device was 128 samples/seconds. We combined the AF3, F7, F3, F4, F8, and AF4 sensors into the frontal lobes; FC5 and FC6 into the fronto-central lobes; P7 and P8 into the parietal lobes; and O1 and O2 into the occipital lobes to perform analyses on each lobe separately. The recorded EEG signal was ﬁltered ofﬂine using a 0.1 Hz high-pass FIR ﬁlter to remove the slow drift and a 40 Hz low-pass FIR ﬁlter via the pop_eegﬁltnew function in Bioengineering 2023, 10, 200 6 of 22 the EEGLAB toolbox [39] with a Hamming window and default options of the transition bandwidth. The narrowband EEG data were then passed through multiple data cleaning stages to remove the artifacts from the data. Artifact Subspace Reconstruction (ASR) is a widely used method for cleaning EEG data and uses the variance-based algorithm presented in [40]. This algorithm creates a statistical model of the clean EEG portion in the data and applies principal component analysis (PCA) to new incoming raw signal and transforms it into the principal component (PC) space. If any of the PCs have larger variances than the variance of the calibration data, it is rejected, and the signal is reconstructed and projected back into the original channel data [41]. This method is implanted with the pop_cleanrawdata function in the EEGLAB. We cleaned the data with ASR and, then, segmented it into short epochs from −500 to 1000 ms around the stimulus onset time (i.e., 0 ms), resulting in a 1500 ms epoch length. The EEG segments of correct-only trials were included in further processing and the ﬁnal analysis [42]. Each EEG segment was inspected visually, and every segment containing artifacts was rejected. Secondly, the independent component analysis (ICA) method using the infomax algorithm [43] was applied to each segment of the data. ICA is a signal processing tool that separates linearly mixed sources. In EEG recordings, these sources are the brain signals (i.e., true EEG) plus some common physiological artifacts, such as an eye artifact or EMG. 2.4.1. EEG Recordings and Processing These artifacts are not the true EEG signals and are referred to as non-brain activity. An eye artifact has an almost entirely frontal distribution over the scalp, and ICA has shown great success in identifying these artifacts in EEG data. Using ICA, the data were transformed into the component time-series space. A thorough visual inspection of each independent component (IC) was undertaken within EEGLAB by simultaneously looking at the time-series of that IC, its time–frequency plot, and its topographical map. The ICs that had mostly a frontal distribution, sudden peaks at the frontal regions (i.e., Fp1 and Fp2 electrodes), and characteristics that looked like non-brain activity were identiﬁed as eye artifacts and removed from the data. The signal was reconstructed with the remaining ICs. To avoid the chances of losing excess real EEG data, the maximum number of ICs to reject per recording was restricted to 3 out of 14, meaning that the number of ICs rejected per recording was between 1 and 3. The average number of trials per subject included in the ﬁnal analysis for the NFT group in 1-back were 30.3 and 28.8 and in 2-back were 26.5 and 27.8 in the pre-NF and post-NF sessions, respectively. Similarly, in the control group, the average trials in 1-back were 31.5 and 32.1 and those in 2-back were 26.1 and 29.6 in the pre-NF and post-NF sessions, respectively. The Laplacian ﬁlter is a spatial high-pass ﬁlter that effectively attenuates the low spatial frequencies that can potentially cause volume conduction artifacts in the sensor- level EEG signals. Thus, applying the Laplacian ﬁlter is appropriate for EEG data before the connectivity analysis [44]. Filtering the data using a Laplacian ﬁlter enhances the signal-to-noise ratio and has proved effective in the EEG data, e.g., in brain–computer interfaces [45], it reasonably suppresses the volume conduction artifact in the data and makes it appropriate for the connectivity analysis [46]. Therefore, we also applied the Laplacian ﬁlter as a pre-processing step to attenuate the volume conduction artifacts before the functional connectivity (FC) analysis. We implemented the Laplacian ﬁlter using the spherical splines approach, and the ﬁlter has an order of 2, as described in [47]. 2.4.3. Power Computation The time–frequency decomposition of EEG trials resulted in a complex output. The post-stimulus instantaneous power for each trial was computed by taking the square of the real and imaginary parts of the output [17]. Each trial was baseline-corrected using a pre-stimulus sub-window from −300 ms to −100 ms (where 0 ms is stimulus onset). For power-law compensation, a baseline normalization was performed, and the values were converted to the decibel scale (i.e., decibel transformation) [49] using Equation (3). Power (in dB) = 10 power baseline (3) (3) where the baseline activity was taken between −300 ms and −100 ms. Finally, the trials were averaged to compute the mean power. 2.4.2. Time–Frequency Decomposition The EEG signal is highly non-stationary in nature [35]. To overcome the non-stationarity characteristics of the EEG data, the signal is decomposed into short time windows, which are considered roughly stationary via time–frequency decomposition [48]. Complex Morlet wavelet (CMW) convolution is one of the time–frequency decomposition methods that results in optimal trade-off between the time precision and spectral resolution, which is the main factor in a time–frequency analysis [48]. Therefore, we also used the CMW Bioengineering 2023, 10, 200 7 of 22 7 of 22 convolution method for time–frequency decomposition. CMW is deﬁned by multiplying a complex sine wave with the Gaussian wave, as given in Equation (1); convolution method for time–frequency decomposition. CMW is deﬁned by multiplying a complex sine wave with the Gaussian wave, as given in Equation (1); cmw = ei2π f te(−t2/2σ2) (1) (1) where t is the time and f is the frequency. In the current analysis, the frequency is deﬁned from 1 to 30 Hz using 40 logarithmic steps. σ is the Full-Width at Half-Maximum (FWHM) parameter and is given by Equation (2); FWHM = n 2π f (2) (2) In Equation (2), n represents the number of wavelet cycles, which is ﬁxed from 3 to 8 in 40 logarithmic steps in the current study. In Equation (2), n represents the number of wavelet cycles, which is ﬁxed from 3 to 8 in 40 logarithmic steps in the current study. 2.4.4. Functional Connectivity Measurement To identify individual rhythmic components that compose the measured data, specif- ically to study rhythmic neuronal interactions, frequency domain representation of the signal is often considered convenient [50]. Subsequently, frequency-domain connectivity metrics can be estimated to evaluate the neuronal interactions. These matrices include the quantiﬁcation of consistency across observations of the phase difference between the oscil- latory components in the signals, for example, phase locking value (PLV). The distribution of the phase differences computed via the PLV method could be indicative of functionally meaningful synchronization between neural populations. Therefore, we preferred to use this method in the current analysis. PLV is a phase synchronization-based measure for FC analysis in BCI research [51] to measure the FC between different EEG electrodes. PLV represents the average phase difference between two electrodes, ranges between 0 (no synchronization) and 1 (full synchronization), and is given by Equation (4). PLV(t) = 1 N N ∑ n=1 exp( j(∆ϕn(t)) ) (4) (4) where ∆ϕn(t) = ϕx(t) −ϕy(t) represents the phase difference between electrode x and elec- trode y, t is time, and N is the length of the time-series. The broadband signal was ﬁltered in the desired EEG band, and the analytic signal was computed. Fast Fourier transform (FFT) and Hilbert transform are two prominent methods to compute the analytic signal. We chose to use Hilbert transform because it is very useful in analysing nonstationary signals such as EEG [52]. Then, the time series of the phase angles from the analytic signal were obtained for each EEG electrode. Finally, PLV was computed for each epoch using Equation (4) and, then, averaged across epochs. The PLV values were computed for each epoch; therefore, the window length of the PLV calculation was equal to the length of the Bioengineering 2023, 10, 200 8 of 22 epoch (i.e., 1500 ms). The brain networks were visualized using BrainNet Viewer [53], a MATLAB toolbox for brain connectivity analysis. epoch (i.e., 1500 ms). The brain networks were visualized using BrainNet Viewer [53], a MATLAB toolbox for brain connectivity analysis. 2.5. EEG Data Collected during the NFT Sessions The pre-processing for the EEG-data collected during the NFT sessions is described in detail in our publication [34] and is presented here for completeness. The pre-processing was partly similar to the pre-processing of task-based EEG data. First, continuous EEG data were ﬁltered via a FIR ﬁlter between 0.5 and 40 Hz using a Hamming window with the help of the pop_eegﬁltnew function in EEGLAB. After that, ASR was used to reconstruct the artifact portion of the data with clean data [40]. This method removes the non-stationary high-variance signal and, then, reconstructs the missing data using a spatial mixing matrix. Then, the data were segmented into 2 s short epochs, and each epoch was inspected for the artifact using the pop_autorej function in EEGLAB. An epoch was selected as a bad epoch if any of the data points exceeded ﬁve standard deviations of the amplitude and was rejected from the data. This algorithm was iteratively implemented via a custom-written MATLAB script. If the number of epochs selected for rejection was greater than 5% of the data, the procedure was repeated with a more liberal threshold (increased by 0.5 SD) [54]. Absolute alpha power was computed using Welch’s averaged, modiﬁed periodogram method (512 DFT points and 75% overlap between consecutive windows) and a Hann tapering window. The power was computed for each channel separately, and then, the mean power was computed across all 14 EEG channels. 2.6. Statistical Analysis 2.6. Statistical Analysis In the current study, we have two groups (NFT and control) and two sessions (Pre-NF and Post-NF). Therefore, a 2(Group: NFT vs. Control) × 2(Session: Pre-NF vs. Post-NF) two-way repeated measure analysis of variance (ANOVA) was performed. In the ANOVA model, the between-subject factor was Group and the within-subject factor was Session. The same ANOVA model was applied separately for the 1-back and 2-back conditions and were repeated for behavioural results (i.e., RT and error rates), band powers, and FC. A paired t-test with Bonferroni correction was performed for pairwise comparisons where needed [55]. All statistics were performed using the Pingouin statistical package implemented in Python [56]. p y The EEG data collected during the NFT sessions were analysed to investigate whether the within-session alpha power changed in response to the training. For this purpose, we compared the mean alpha power within the 1st half (i.e., ﬁrst 15 min) and 2nd half (i.e., last 15 min) of the NFT session to the baseline using separate one-way repeated measure ANOVA. The EEG data collected before starting the experiments were used as the baseline in this case. Additionally, we explored the within-session changes by investigating each half of the NFT session. We divided each half of the NFT session into three ﬁve-minute sub-blocks (B1, B2, and B3). Then, we applied a 2 (half session: 1st half and 2nd half) × 3(sub-blocks: B1, B2 and B3) 2-way ANOVA to investigate the alpha power differences between the 1st half and 2nd half and among B1, B2, and B3. 3.1. Behavioural Results (Response Time and Error Rate) The behavioural results are shown in Figure 3. In the response times (RT), there was an expected signiﬁcant main effect of Session in the 1-back condition, F(1, 34) = 5.25, p = 0.028, η2 = 0.133. RT signiﬁcantly reduced from 549.2 (ms) to 544.69 (ms) in the Post-NF Session for the NFT group and from 604.27 (ms) to 533.89 (ms) in the control group. Similarly, in the 2-back condition, there was a signiﬁcant main effect of Session, F(1, 34) = 5.26, p = 0.000016, η2 = 0.426, as well as an interaction effect between Group and Session, F(1, 34) = 4.48, p = 0.041, η2 = 0.116. RT in the Post-NF Session signiﬁcantly reduced from 740.23 (ms) to 684.80 (ms) in the NFT group and from 759.25 (ms) to 619.40 (ms) in the control group. 9 of 22 40 Bioengineering 2023, 10, 200 gure 3. Behavioural results: (A) response time and (B) error rates. (* p < 0.05, p < 0.005, * p < 005 **** p < 0 00005) The diamond shape in the box plots represents the outliers in the data Figure 3. Behavioural results: (A) response time and (B) error rates. (* p < 0.05, p < 0.005, * p < 0.0005, **** p < 0.00005). The diamond shape in the box plots represents the outliers in the data. ure 3. Behavioural results: (A) response time and (B) error rates. (* p < 0.05, p < 0.005, * p < 05 **** 0 00005) Th di d h i th b l t t th tli i th d t Figure 3. Behavioural results: (A) response time and (B) error rates. (* p < 0.05, p < 0.005, * p < 0.0005, **** p < 0.00005). The diamond shape in the box plots represents the outliers in the data. p p p p In the 1-back condition, there was a significant main effect of Session on the error es, 𝐹(1,34) = 5.98, 𝑝= 0.019, 𝜂2 = 0.149. The error rates in the Post-NF Session de- ased to 2.2% from 4.9% in the NFT group and to 4.9% from 7.3% in the control group. milarly, the main effect of the Group was also significant, 𝐹(1,34) = 8.21, 𝑝= 07, 𝜂2 = 0.194. However, there was no interaction effect between Group and Session, 1,34) = 0.01, 𝑝= 0.892, 𝜂2 = 0.0005. 3.1. Behavioural Results (Response Time and Error Rate) In the 2-back condition, the main effects of Session d Group were significant 𝐹(1,34) = 15.70, 𝑝= 0.0003, 𝜂2 = 0.315 and 𝐹(1,34) = 34, 𝑝= 0.044, 𝜂2 = 0.113, respectively. The error rates in the Post-NF session decreased 9.34% from 13.5% in the NFT group and to 12.42% from 20.17% in the control group. wever, the interaction effect between Group and Session was not significant 𝐹(1,34) = In the 1-back condition, there was a signiﬁcant main effect of Session on the error rates, F(1, 34) = 5.98, p = 0.019, η2 = 0.149. The error rates in the Post-NF Session decreased to 2.2% from 4.9% in the NFT group and to 4.9% from 7.3% in the control group. Similarly, the main effect of the Group was also signiﬁcant, F(1, 34) = 8.21, p = 0.007, η2 = 0.194. However, there was no interaction effect between Group and Session, F(1, 34) = 0.01, p = 0.892, η2 = 0.0005. In the 2-back condition, the main effects of Session and Group were signiﬁcant F(1, 34) = 15.70, p = 0.0003, η2 = 0.315 and F(1, 34) = 4.34, p = 0.044, η2 = 0.113, respectively. The error rates in the Post-NF session decreased to 9.34% from 13.5% in the NFT group and to 12.42% from 20.17% in the control group. However, the interaction effect between Group and Session was not signiﬁcant F(1, 34) = 1.26, p = 0.26, η2 = 0.036. 6, 𝑝= 0.26, 𝜂= 0.036. 3.2. Within-Session Alpha Power Within-Session Alpha Power The within-session mean alpha power for the 1st half and 2nd half of the NFT session hown in Figure 4. One-way repeated measure ANOVA revealed that the within-ses- n alpha power was significantly greater than the baseline alpha power (p = 0.00207). e post hoc test revealed that the alpha power in both halves of the session was signifi- ntly greater than that of the baseline, with p = 0.0255 and p = 0.046 for the first half and The within-session mean alpha power for the 1st half and 2nd half of the NFT session is shown in Figure 4. One-way repeated measure ANOVA revealed that the within-session alpha power was signiﬁcantly greater than the baseline alpha power (p = 0.00207). The post hoc test revealed that the alpha power in both halves of the session was signiﬁcantly greater than that of the baseline, with p = 0.0255 and p = 0.046 for the ﬁrst half and second half, respectively. However, the alpha power in the ﬁrst half and second half was not signiﬁcantly different from each other (p = 0.25956). g y p Overall, the alpha power within each half of the session signiﬁcantly increased from the baseline. However, this increase reduced from the beginning (B1) to the end (B3) of the session, as shown in Figure 4. From the 2-way ANOVA, we found that this decrease (from B1 to B3) within each half of the session was signiﬁcant (p = 0.00076). The post hoc test revealed that this signiﬁcant main effect was observed between B1 and B3 (p = 0.0060) and between B2 and B3 (p = 0.0183). However, the difference between B1 and B2 was not signiﬁcant (p = 0.22516). 10 of 22 Bioengineering 2023, 10, 200 10 of 22 Figure 4. Absolute alpha power at baseline and within-session (1st half = first 15 min, 15 min) and within-session (first half and second half) sub-blocks (B1, B2, and B3, each (* p < 0.05, p < 0.005, * p < 0.0005). The diamond shape in the box plots represents h d Figure 4. Absolute alpha power at baseline and within-session (1st half = ﬁrst 15 min, 2nd half = last 15 min) and within-session (ﬁrst half and second half) sub-blocks (B1, B2, and B3, each ﬁve minutes) (* p < 0.05, p < 0.005, * p < 0.0005). 6, 𝑝= 0.26, 𝜂= 0.036. 3.2. Within-Session Alpha Power The diamond shape in the box plots represents the outliers in the data. Figure 4. Absolute alpha power at baseline and within-session (1st half = first 15 min 15 min) and within-session (first half and second half) sub-blocks (B1, B2, and B3, eac (* p < 0.05, p < 0.005, * p < 0.0005). The diamond shape in the box plots represents Figure 4. Absolute alpha power at baseline and within-session (1st half = ﬁrst 15 min, 2nd half = last 15 min) and within-session (ﬁrst half and second half) sub-blocks (B1, B2, and B3, each ﬁve minutes) (* p < 0.05, p < 0.005, * p < 0.0005). The diamond shape in the box plots represents the outliers in the data. the data. 3.3. EEG Post-Stimulus Power 3.3.1. Frontal Lobe Post-stimulus power in frontal lobe ( p < 0.005): 1-back post-stimulus power in (A1) Figure 5. Post-stimulus power in frontal lobe ( p < 0.005): 1-back post-stimulus power in (A1) NFT group and (B1) control group; 2-back post-stimulus power in (A2) NFT group and (B2) control group. Post-stimulus power in different EEG bands within each group in (C1) 1-back and (C2) 2- back conditions. The colour bar of the TF plots represents the value of EEG power in dB scale. The diamond shape in the box plots represents the outliers in the data. Figure 5. Post-stimulus power in frontal lobe ( p < 0.005): 1-back post-stimulus power in (A1) NFT group and (B1) control group; 2-back post-stimulus power in (A2) NFT group and (B2) control group. Post-stimulus power in different EEG bands within each group in (C1) 1-back and (C2) 2-back conditions. The colour bar of the TF plots represents the value of EEG power in dB scale. The diamond shape in the box plots represents the outliers in the data. e ( p < 0.005): 1-back post-stimulus power in (A1) NF stimulus power in (A2) NFT group and (B2) contr be ( p < 0.005): 1-back post-stimulus power in (A1 group and (B1) control group; 2-back post-stimulus power in (A2) NFT group and (B2) control group. Post-stimulus power in different EEG bands within each group in (C1) 1-back and (C2) 2- back conditions. The colour bar of the TF plots represents the value of EEG power in dB scale. The diamond shape in the box plots represents the outliers in the data. NFT group and (B1) control group; 2-back post-stimulus power in (A2) NFT group and (B2) control group. Post-stimulus power in different EEG bands within each group in (C1) 1-back and (C2) 2-back conditions. The colour bar of the TF plots represents the value of EEG power in dB scale. The diamond shape in the box plots represents the outliers in the data. 3.3.1. Frontal Lobe p p g y the baseline. However, this increase reduced from the beginning (B1) to th the session, as shown in Figure 4. From the 2-way ANOVA, we found that (from B1 to B3) within each half of the session was significant (p = 0.00076). test revealed that this significant main effect was observed between B1 and B and between B2 and B3 (p = 0.0183). However, the difference between B1 an significant (p = 0.22516). 3.3. EEG Post-Stimulus Power In 1-back condition, there was no signiﬁcant main effect of Group or Session in frontal theta, alpha, and beta activities. Similarly, in the 2-back condition, no signiﬁcant main effect of Group or Session was found in frontal beta activity. The main effect of Group in frontal alpha activity and the main effect of Session in theta activity were also non- signiﬁcant. However, a signiﬁcant main effect of Group in frontal theta, F(1, 34) = 9.01, p = 0.0049, η2 = 0.20, and a marginally signiﬁcant effect of Session in frontal alpha, F(1, 34) = 3.91, p = 0.055, η2 = 0.10, were observed. The post-stimulus power for the frontal lobe is shown in Figure 5. 11 of 22 Bioengineering 2023, 10, 200 Figure 5. Post-stimulus power in frontal lobe ( p < 0.005): 1-back post-stimulus power in (A1) NFT group and (B1) control group; 2-back post-stimulus power in (A2) NFT group and (B2) control group. Post-stimulus power in different EEG bands within each group in (C1) 1-back and (C2) 2- back conditions. The colour bar of the TF plots represents the value of EEG power in dB scale. The diamond shape in the box plots represents the outliers in the data. Figure 5. Post-stimulus power in frontal lobe ( p < 0.005): 1-back post-stimulus power in (A1) NFT group and (B1) control group; 2-back post-stimulus power in (A2) NFT group and (B2) control group. Post-stimulus power in different EEG bands within each group in (C1) 1-back and (C2) 2-back conditions. The colour bar of the TF plots represents the value of EEG power in dB scale. The diamond shape in the box plots represents the outliers in the data. Figure 5. Post-stimulus power in frontal lobe ( p < 0.005): 1-back post-stimulus power in (A1) NFT group and (B1) control group; 2 back post stimulus power in (A2) NFT group and (B2) control Figure 5. 3.3.2. Fronto-Central Lobe I th 1 b k diti 3.3.2. Fronto-Central Lobe In the 1-back condition, the main effect of both factors, Group and Session, were non- significant in alpha and beta activities. Similarly, the main effect of Session was also non- significant in theta activity. However, the main effect of Group was significant in theta activity, 𝐹(1,34) = 5.42, 𝑝= 0.025, 𝜂2 = 0.137. In the 2-back condition, there was no sig- nificant main effect of Group or Session in beta activity. On the other hand, in theta activ- ity, the main effect of Group, 𝐹(1,34) = 9.79, 𝑝= 0.0035, 𝜂2 = 0.223, as well as of Session, 𝐹(1,34) = 7.22, 𝑝= 0.011, 𝜂2 = 0.175, were significant. Similarly, in alpha activity, the main effect of both factors, Group, 𝐹(1,34) = 4.55, 𝑝= 0.040, 𝜂2 = 0.118, and Session, 𝐹(1,34) = 8.18, 𝑝= 0.0071, 𝜂2 = 0.194, were significant. The fronto-central lobe post- stimulus power results are shown in Figure 6. In the 1-back condition, the main effect of both factors, Group and Session, were non-signiﬁcant in alpha and beta activities. Similarly, the main effect of Session was also non-signiﬁcant in theta activity. However, the main effect of Group was signiﬁcant in theta activity, F(1, 34) = 5.42, p = 0.025, η2 = 0.137. In the 2-back condition, there was no signiﬁcant main effect of Group or Session in beta activity. On the other hand, in theta activity, the main effect of Group, F(1, 34) = 9.79, p = 0.0035, η2 = 0.223, as well as of Session, F(1, 34) = 7.22, p = 0.011, η2 = 0.175, were signiﬁcant. Similarly, in alpha activity, the main effect of both factors, Group, F(1, 34) = 4.55, p = 0.040, η2 = 0.118, and Session, F(1, 34) = 8.18, p = 0.0071, η2 = 0.194, were signiﬁcant. The fronto-central lobe post-stimulus power results are shown in Figure 6. 12 of 22 Bioengineering 2023, 10, 200 Figure 6. Post-stimulus power fronto-central lobe (* p < 0.05, ** p < 0.005): 1-back post-stimulus power in (A1) NFT group and (B1) control group; 2-back post-stimulus power in (A2) NFT group and (B2) control group. Post-stimulus power in different EEG bands within each group in (C1) 1- back and (C2) 2-back conditions. The colour bar of the TF plots represents the value of EEG power in dB scale. The diamond shape in the box plots represents the outliers in the data. 3.3.3. Parietal Lobe I th 1 b k 3.3.3. Parietal Lobe In the 1-back condition, the main effect of Group was significant in theta, 𝐹(1,34) = 8.19, 𝑝= 0.0071, 𝜂2 = 0.194 , and alpha activities, 𝐹(1,34) = 10.34, 𝑝= 0.0028, 𝜂2 = 0.233, and non-significant in beta activity. The main effect of Session was non-significant in theta and alpha activities. However, there was a significant effect of Session in beta activity, 𝐹(1,34) = 4.80, 𝑝= 0.0352, 𝜂2 = 0.123. In the 2-back condition, no significant main effects for Group or Session, were observed in theta, alpha, or beta activities. How- ever, the main effect of Group was significant in theta activity, 𝐹(1,34) = 8.71, 𝑝= 0.0056, 𝜂2 = 0.204. The post-stimulus power results for the parietal lobe are shown in Fig- ure 7 In the 1-back condition, the main effect of Group was significant in theta, F(1,34) = 8.19, p = 0.0071, η2 = 0.194, and alpha activities, F(1, 34) = 10.34, p = 0.0028, η2 = 0.233, and non-signiﬁcant in beta activity. The main effect of Session was non-signiﬁcant in theta and alpha activities. However, there was a signiﬁcant effect of Session in beta activity, F(1, 34) = 4.80, p = 0.0352, η2 = 0.123. In the 2-back condition, no signiﬁcant main effects for Group or Session, were observed in theta, alpha, or beta activities. However, the main effect of Group was signiﬁcant in theta activity, F(1, 34) = 8.71, p = 0.0056, η2 = 0.204. The post-stimulus power results for the parietal lobe are shown in Figure 7. In the 1-back condition, the main effect of Group was significant in theta, 𝐹(1,34) = 8.19, 𝑝= 0.0071, 𝜂2 = 0.194 , and alpha activities, 𝐹(1,34) = 10.34, 𝑝= 0.0028, 𝜂2 = 0.233, and non-significant in beta activity. The main effect of Session was non-significant in theta and alpha activities. However, there was a significant effect of Session in beta activity, 𝐹(1,34) = 4.80, 𝑝= 0.0352, 𝜂2 = 0.123. In the 2-back condition, no significant main effects for Group or Session, were observed in theta, alpha, or beta activities. How- ever, the main effect of Group was significant in theta activity, 𝐹(1,34) = 8.71, 𝑝= 0.0056, 𝜂2 = 0.204. 3.3.2. Fronto-Central Lobe I th 1 b k diti 3.3.2. Fronto-Central Lobe 3 3 3 Parietal Lobe Figure 6. Post-stimulus power fronto-central lobe (* p < 0.05, ** p < 0.005): 1-back post-stimulus power in (A1) NFT group and (B1) control group; 2-back post-stimulus power in (A2) NFT group and (B2) control group. Post-stimulus power in different EEG bands within each group in (C1) 1-back and (C2) 2-back conditions. The colour bar of the TF plots represents the value of EEG power in dB scale. The diamond shape in the box plots represents the outliers in the data. Figure 6. Post-stimulus power fronto-central lobe (* p < 0.05, ** p < 0.005): 1-back post-stimulus power in (A1) NFT group and (B1) control group; 2-back post-stimulus power in (A2) NFT group and (B2) control group. Post-stimulus power in different EEG bands within each group in (C1) 1- back and (C2) 2-back conditions. The colour bar of the TF plots represents the value of EEG power in dB scale. The diamond shape in the box plots represents the outliers in the data. Figure 6. Post-stimulus power fronto-central lobe (* p < 0.05, ** p < 0.005): 1-back post-stimulus power in (A1) NFT group and (B1) control group; 2-back post-stimulus power in (A2) NFT group and (B2) control group. Post-stimulus power in different EEG bands within each group in (C1) 1-back and (C2) 2-back conditions. The colour bar of the TF plots represents the value of EEG power in dB scale. The diamond shape in the box plots represents the outliers in the data. 3.3.3. Parietal Lobe I th 1 b k 3.3.3. Parietal Lobe Post-stimulus power parietal lobe (* p < 0.05, ** p < 0.005): 1-back post-stimulus power in (A1) NFT group and (B1) control group; 2-back post-stimulus power in (A2) NFT group and (B2) control group. Post-stimulus power in different EEG bands within each group in (C1) 1-back and (C2) 2-back conditions. The colour bar of the TF plots represents the value of EEG power in dB scale. The diamond shape in the box plots represents the outliers in the data. Figure 7. Post-stimulus power parietal lobe (* p < 0.05, ** p < 0.005): 1-back post-stimulus power in (A1) NFT group and (B1) control group; 2 back post stimulus power in (A2) NFT group and (B2) Figure 7. Post-stimulus power parietal lobe (* p < 0.05, ** p < 0.005): 1-back post-stimulus power (A1) NFT group and (B1) control group; 2-back post-stimulus power in (A2) NFT group and (B2) control group. Post-stimulus power in different EEG bands within each group in (C1) 1-back and (C2) 2-back conditions. The colour bar of the TF plots represents the value of EEG power in dB scale. The diamond shape in the box plots represents the outliers in the data. g p p ( p p ) p p in (A1) NFT group and (B1) control group; 2-back post-stimulus power in (A2) NFT group and (B2) control group. Post-stimulus power in different EEG bands within each group in (C1) 1-back and (C2) 2-back conditions. The colour bar of the TF plots represents the value of EEG power in dB scale. The diamond shape in the box plots represents the outliers in the data. 3.3.3. Parietal Lobe I th 1 b k 3.3.3. Parietal Lobe The post-stimulus power results for the parietal lobe are shown in Fig- ure 7 In the 1-back condition, the main effect of Group was significant in theta, F(1,34) = 8.19, p = 0.0071, η2 = 0.194, and alpha activities, F(1, 34) = 10.34, p = 0.0028, η2 = 0.233, and non-signiﬁcant in beta activity. The main effect of Session was non-signiﬁcant in theta and alpha activities. However, there was a signiﬁcant effect of Session in beta activity, F(1, 34) = 4.80, p = 0.0352, η2 = 0.123. In the 2-back condition, no signiﬁcant main effects for Group or Session, were observed in theta, alpha, or beta activities. However, the main effect of Group was signiﬁcant in theta activity, F(1, 34) = 8.71, p = 0.0056, η2 = 0.204. The post-stimulus power results for the parietal lobe are shown in Figure 7. 13 of 22 Bioengineering 2023, 10, 200 Figure 7. Post-stimulus power parietal lobe (* p < 0.05, ** p < 0.005): 1-back post-stimulus power in (A1) NFT group and (B1) control group; 2-back post-stimulus power in (A2) NFT group and (B2) control group. Post-stimulus power in different EEG bands within each group in (C1) 1-back and (C2) 2-back conditions. The colour bar of the TF plots represents the value of EEG power in dB scale. The diamond shape in the box plots represents the outliers in the data. Figure 7. Post-stimulus power parietal lobe (* p < 0.05, ** p < 0.005): 1-back post-stimulus power in (A1) NFT group and (B1) control group; 2-back post-stimulus power in (A2) NFT group and (B2) control group. Post-stimulus power in different EEG bands within each group in (C1) 1-back and (C2) 2-back conditions. The colour bar of the TF plots represents the value of EEG power in dB scale. The diamond shape in the box plots represents the outliers in the data. Figure 7. Post-stimulus power parietal lobe (* p < 0.05, ** p < 0.005): 1-back post-stimulus power in (A1) NFT group and (B1) control group; 2-back post-stimulus power in (A2) NFT group and (B2) control group. Post-stimulus power in different EEG bands within each group in (C1) 1-back and (C2) 2-back conditions. The colour bar of the TF plots represents the value of EEG power in dB scale. The diamond shape in the box plots represents the outliers in the data. Figure 7. 3.3.4. Occipital Lobe I th 1 b k 3.3.4. Occipital Lobe In the 1-back condition, the interaction effect of Group and Session was significant in theta activity, 𝐹(1,34) = 4.55, 𝑝= 0.0402, 𝜂2 = 0.118. The main effect of Group was sig- nificant in theta, 𝐹(1,34) = 12.89, 𝑝= 0.0010, 𝜂2 = 0.275; alpha, 𝐹(1,34) = 13.58, 𝑝= 0.00079, 𝜂2 = 0.285; and beta activities, 𝐹(1,34) = 6.71, 𝑝= 0.0139, 𝜂2 = 0.165, whereas the main effect of Session was non-significant. In the 2-back condition, the main effect of Group was significant only in theta activity, 𝐹(1,34) = 9.08, 𝑝= 0.0048, 𝜂2 = 0.210. The main effect of Session was significant in theta, 𝐹(1,34) = 5.56, 𝑝= 0.0241, 𝜂2 = 0.140, and beta activities, 𝐹(1,34) = 4.20, 𝑝= 0.0480, 𝜂2 = 0.110. No significant interaction ef- fect was observed in the 2-back condition. The post-stimulus power results for occipital lobe are shown in Figure 8. In the 1-back condition, the interaction effect of Group and Session was signiﬁcant in theta activity, F(1, 34) = 4.55, p = 0.0402, η2 = 0.118. The main effect of Group was signiﬁcant in theta, F(1, 34) = 12.89, p = 0.0010, η2 = 0.275; alpha, F(1, 34) = 13.58, p = 0.00079, η2 = 0.285; and beta activities, F(1, 34) = 6.71, p = 0.0139, η2 = 0.165, whereas the main effect of Session was non-signiﬁcant. In the 2-back condition, the main effect of Group was signiﬁcant only in theta activity, F(1, 34) = 9.08, p = 0.0048, η2 = 0.210. The main effect of Session was signiﬁcant in theta, F(1, 34) = 5.56, p = 0.0241, η2 = 0.140, and beta activities, F(1, 34) = 4.20, p = 0.0480, η2 = 0.110. No signiﬁcant interaction effect was observed in the 2-back condition. The post-stimulus power results for occipital lobe are shown in Figure 8. 14 of 22 Bioengineering 2023, 10, 200 Figure 8. Post-stimulus power occipital lobe (* p < 0.05, p < 0.005, * p < 0.0005): 1-back post- stimulus power in (A1) NFT group and (B1) control group; 2-back post-stimulus power in (A2) NFT group and (B2) control group. Post-stimulus power in different EEG bands within each group in (C1) 1-back and (C2) 2-back conditions. The colour bar of the TF plots represents the value of EEG power in dB scale. The diamond shape in the box plots represents the outliers in the data. 3.4.1. Theta Band Th i 3.4.1. Theta Band The connectivity results for theta band are shown in Figure 9. There was no signifi- cant main effect of Group or Session on the FC in the frontal lobe. However, the interaction effect between Group and Session was significant in the 1-back condition, 𝐹(1,34) = 9.538, 𝑝= 0.0040, 𝜂2 = 0.219, as well as in the 2-back condition, 𝐹(1,34) = 6.68, 𝑝= 0.0142, 𝜂2 = 0.164. A post hoc test with Bonferroni correction revealed that the FC in the frontal lobe of NFT group in both experimental conditions, 1-back and 2-back, signifi- cantly increased in the Post-NF session when compared with the Pre-NF session (p < 0.05). The connectivity results for theta band are shown in Figure 9. There was no signiﬁcant main effect of Group or Session on the FC in the frontal lobe. However, the interaction effect between Group and Session was signiﬁcant in the 1-back condition, F(1, 34) = 9.538, p = 0.0040, η2 = 0.219, as well as in the 2-back condition, F(1, 34) = 6.68, p = 0.0142, η2 = 0.164. A post hoc test with Bonferroni correction revealed that the FC in the frontal lobe of NFT group in both experimental conditions, 1-back and 2-back, signiﬁcantly increased in the Post-NF session when compared with the Pre-NF session (p < 0.05). p (p ) The main effect of Group was also non-signiﬁcant in the fronto-central lobe. How- ever, the main effect of Session (1-back: F(1, 34) = 5.85, p = 0.0210, η2 = 0.146, 2- back: F(1, 34) = 4.96, p = 0.0326, η2 = 0.127) and the interaction effect between Group and Session (1-back: F(1, 34) = 6.25, p = 0.0174, η2 = 0.155, 2-back: F(1, 34) = 5.17, p = 0.0293, η2 = 0.132) were signiﬁcant. The post hoc test again revealed a signiﬁcant increase in FC of the NFT group in both conditions, 1-back and 2-back, in the Post-NF session when compared to the Pre-NF session (p < 0.05). Similarly, in the parietal lobe, the main effect of Group was non-signiﬁcant and the main effect of Session, F(1, 34) = 4.29, p = 0.0460, η2 = 0.112, as well as the interaction effect, F(1, 34) = 9.83, p = 0.0035, η2 = 0.224, were signiﬁcant in the 1-back condition. 3.3.4. Occipital Lobe I th 1 b k 3.3.4. Occipital Lobe 3 4 EEG F ti l C ti it g p p ( p , p , p ) p stimulus power in (A1) NFT group and (B1) control group; 2-back post-stimulus power in (A2) NFT group and (B2) control group. Post-stimulus power in different EEG bands within each group in (C1) 1-back and (C2) 2-back conditions. The colour bar of the TF plots represents the value of EEG power in dB scale. The diamond shape in the box plots represents the outliers in the data. 3.3.4. Occipital Lobe I th 1 b k 3.3.4. Occipital Lobe 3 4 EEG F ti l C ti it Figure 8. Post-stimulus power occipital lobe (* p < 0.05, p < 0.005, * p < 0.0005): 1-back post- stimulus power in (A1) NFT group and (B1) control group; 2-back post-stimulus power in (A2) NFT group and (B2) control group. Post-stimulus power in different EEG bands within each group in (C1) 1-back and (C2) 2-back conditions. The colour bar of the TF plots represents the value of EEG power in dB scale. The diamond shape in the box plots represents the outliers in the data. Figure 8. Post-stimulus power occipital lobe (* p < 0.05, p < 0.005, * p < 0.0005): 1-back post- stimulus power in (A1) NFT group and (B1) control group; 2-back post-stimulus power in (A2) NFT group and (B2) control group. Post-stimulus power in different EEG bands within each group in (C1) 1-back and (C2) 2-back conditions. The colour bar of the TF plots represents the value of EEG power in dB scale. The diamond shape in the box plots represents the outliers in the data. Figure 8. Post-stimulus power occipital lobe (* p < 0.05, p < 0.005, * p < 0.0005): 1-back post- stimulus power in (A1) NFT group and (B1) control group; 2-back post-stimulus power in (A2) NFT group and (B2) control group. Post-stimulus power in different EEG bands within each group in (C1) 1-back and (C2) 2-back conditions. The colour bar of the TF plots represents the value of EEG power in dB scale. The diamond shape in the box plots represents the outliers in the data. Figure 8. Post-stimulus power occipital lobe (* p < 0.05, p < 0.005, * p < 0.0005): 1-back post- stimulus power in (A1) NFT group and (B1) control group; 2 back post stimulus power in (A2) NFT Figure 8. Post-stimulus power occipital lobe (* p < 0.05, p < 0.005, * p < 0.0005): 1-back post- stimulus power in (A1) NFT group and (B1) control group; 2-back post-stimulus power in (A2) NFT group and (B2) control group. Post-stimulus power in different EEG bands within each group in (C1) 1-back and (C2) 2-back conditions. The colour bar of the TF plots represents the value of EEG power in dB scale. The diamond shape in the box plots represents the outliers in the data. 3.4.1. Theta Band Th i 3.4.1. Theta Band In the 2-back condition, the main effect of Session was non-signiﬁcant, but the interaction effect was signiﬁcant, F(1, 34) = 8.47, p = 0.0063, η2 = 0.199. The post hoc test revealed a signiﬁcant increase in Post-NF FC in the NFT group only (p < 0.05). Finally, in the occipital lobe, the main effect of Group and Session was non-signiﬁcant in both conditions, 1-back and 2-back. However, the interaction effect between Group and Session was signiﬁcant in the 1-back condition, F(1, 34) = 11.31, p = 0.0019, η2 = 0.249, Bioengineering 2023, 10, 200 15 of 22 15 of 22 as well as in the 2-back condition, F(1, 34) = 10.22, p = 0.0030, η2 = 0.231. Similarly, to other brain regions, the post hoc test revealed a signiﬁcant increase in Post-NF FC in the NFT group only (p < 0.05). REVIEW 15 of 22 as well as in the 2-back condition, F(1, 34) = 10.22, p = 0.0030, η2 = 0.231. Similarly, to other brain regions, the post hoc test revealed a signiﬁcant increase in Post-NF FC in the NFT group only (p < 0.05). REVIEW 15 of 22 Figure 9. Theta band functional connectivity (* p < 0.05, ** p < 0.005). Connectivity matrices shown in (A1) for NFT group and (B1) for control group and brain connections in (C1) for NFT group and (D1) for control group in 1-back condition. Similarly, connectivity matrices shown in (A2) for NFT group and (B2) for control group and brain connections in (C2) for NFT group and (D2) for control group in 2-back condition. The box plots in (E1,E2) show the phase synchronization index in differ- ent brain lobes in 1-back and 2-back conditions, respectively. The colour bar of the connectivity matrices and brain connections represent the strength of synchronization between the EEG chan- nels. It ranges from 0 (no synchronization) to 1 (full synchronization). The diamond shape in the box plots represents the outliers in the data. Th i ff f G l i ifi i h f l l b H Figure 9. Theta band functional connectivity (* p < 0.05, ** p < 0.005). Connectivity matrices shown in (A1) for NFT group and (B1) for control group and brain connections in (C1) for NFT group and (D1) for control group in 1-back condition. 3.4.1. Theta Band Th i 3.4.1. Theta Band Connectivity matrices shown in Figure 9. Theta band functional connectivity (* p < 0.05, ** p < 0.005). Connectivity matrices shown in (A1) for NFT group and (B1) for control group and brain connections in (C1) for NFT group and (D1) for control group in 1-back condition. Similarly, connectivity matrices shown in (A2) for NFT group and (B2) for control group and brain connections in (C2) for NFT group and (D2) for control group in 2-back condition. The box plots in (E1,E2) show the phase synchronization index in differ- ent brain lobes in 1-back and 2-back conditions, respectively. The colour bar of the connectivity matrices and brain connections represent the strength of synchronization between the EEG chan- nels. It ranges from 0 (no synchronization) to 1 (full synchronization). The diamond shape in the box plots represents the outliers in the data. Figure 9. Theta band functional connectivity (* p < 0.05, ** p < 0.005). Connectivity matrices shown in (A1) for NFT group and (B1) for control group and brain connections in (C1) for NFT group and (D1) for control group in 1-back condition. Similarly, connectivity matrices shown in (A2) for NFT group and (B2) for control group and brain connections in (C2) for NFT group and (D2) for control group in 2-back condition. The box plots in (E1,E2) show the phase synchronization index in different brain lobes in 1-back and 2-back conditions, respectively. The colour bar of the connectivity matrices and brain connections represent the strength of synchronization between the EEG channels. It ranges from 0 (no synchronization) to 1 (full synchronization). The diamond shape in the box plots represents the outliers in the data. 3.4.1. Theta Band Th i 3.4.1. Theta Band Similarly, connectivity matrices shown in (A2) for NFT group and (B2) for control group and brain connections in (C2) for NFT group and (D2) for control group in 2-back condition. The box plots in (E1,E2) show the phase synchronization index in different brain lobes in 1-back and 2-back conditions, respectively. The colour bar of the connectivity matrices and brain connections represent the strength of synchronization between the EEG channels. It ranges from 0 (no synchronization) to 1 (full synchronization). The diamond shape in the box plots represents the outliers in the data. Figure 9. Theta band functional connectivity (* p < 0.05, ** p < 0.005). Connectivity matrices shown in (A1) for NFT group and (B1) for control group and brain connections in (C1) for NFT group and (D1) for control group in 1-back condition. Similarly, connectivity matrices shown in (A2) for NFT group and (B2) for control group and brain connections in (C2) for NFT group and (D2) for control group in 2-back condition. The box plots in (E1,E2) show the phase synchronization index in differ- ent brain lobes in 1-back and 2-back conditions, respectively. The colour bar of the connectivity matrices and brain connections represent the strength of synchronization between the EEG chan- nels. It ranges from 0 (no synchronization) to 1 (full synchronization). The diamond shape in the box plots represents the outliers in the data. Figure 9. Theta band functional connectivity (* p < 0.05, ** p < 0.005). Connectivity matrices shown in (A1) for NFT group and (B1) for control group and brain connections in (C1) for NFT group and (D1) for control group in 1-back condition. Similarly, connectivity matrices shown in (A2) for NFT group and (B2) for control group and brain connections in (C2) for NFT group and (D2) for control group in 2-back condition. The box plots in (E1,E2) show the phase synchronization index in different brain lobes in 1-back and 2-back conditions, respectively. The colour bar of the connectivity matrices and brain connections represent the strength of synchronization between the EEG channels. It ranges from 0 (no synchronization) to 1 (full synchronization). The diamond shape in the box plots represents the outliers in the data. Figure 9. Theta band functional connectivity (* p < 0.05, ** p < 0.005). Connectivity matrices shown Figure 9. Theta band functional connectivity (* p < 0.05, ** p < 0.005). The main effec ever the main effe 3.4.2. Alpha Band ever, the main effect of Session (1 back: 𝐹(1,34) 5.85, 𝑝 0.0210, 𝜂 0.146, 2 back: 𝐹(1,34) = 4.96, 𝑝= 0.0326, 𝜂2 = 0.127) and the interaction effect between Group and Ses- sion (1-back: 𝐹(1,34) = 6.25, 𝑝= 0.0174, 𝜂2 = 0.155 , 2-back: 𝐹(1,34) = 5.17, 𝑝= 0.0293, 𝜂2 = 0.132) were significant. The post hoc test again revealed a significant increase in FC of the NFT group in both conditions, 1-back and 2-back, in the Post-NF session when compared to the Pre-NF session (p < 0.05). Similarly, in the parietal lobe, the main effect of Group was non-significant and the The results of the alpha band connectivity are shown in Figure 10. There was no main effect of Group or Session or an interaction effect between Group and Session in the 1-back condition in the frontal or fronto-central lobes. Similarly, in the 2-back condition, the main effect of Session and the interaction effect were non-signiﬁcant in the fronto-central lobe. However, the main effect of Group in the 2-back condition in the fronto-central lobe was signiﬁcant, F(1, 34) = 4.58, p = 0.0395, η2 = 0.118. 16 of 22 16 of 22 Bioengineering 2023, 10, 200 Figure 10. Alpha band functional connectivity (* p < 0.05). Connectivity matrices shown in (A1) for NFT group and (B1) for control group and brain connections in (C1) for NFT group and (D1) for control group in 1-back condition. Similarly, connectivity matrices shown in (A2) for NFT group and (B2) for control group and brain connections in (C2) for NFT group and (D2) for control group in 2-back condition. The box plots in (E1,E2) show the phase synchronization index in different brain lobes in 1-back and 2-back conditions, respectively. The colour bars of the connectivity matrices and brain connections represent the strength of synchronization between the EEG channels. It ranges from 0 (no synchronization) to 1 (full synchronization). The diamond shape in the box plots repre- sents the outliers in the data. The main effect of Group and Session and the interaction effect were also non signif Figure 10. Alpha band functional connectivity (* p < 0.05). Connectivity matrices shown in (A1) for NFT group and (B1) for control group and brain connections in (C1) for NFT group and (D1) for control group in 1-back condition. The main effec ever the main effe 3.4.2. Alpha Band Connectivity matrices shown in (A1) for NFT group and (B1) for control group and brain connections in (C1) for NFT group and (D1) for Figure 10. Alpha band functional connectivity (* p < 0.05). Connectivity matrices shown in (A1) for p g icant in the parietal lobe in both conditions, 1-back and 2-back. Similarly, in the occipital lobe, the interaction effect and main effect of Session were non-significant in the 1-back condition. However, the main effect of Group was significant, 𝐹(1,34) = 4.19, 𝑝= 0.0483, 𝜂2 = 0.109, in the occipital lobe in the 1-back condition. No main or interaction effect of Group and Session was observed in the occipital lobe in the 2-back condition. The main effect of Group and Session and the interaction effect were also non- signiﬁcant in the parietal lobe in both conditions, 1-back and 2-back. Similarly, in the occipital lobe, the interaction effect and main effect of Session were non-signiﬁcant in the 1-back condition. However, the main effect of Group was signiﬁcant, F(1, 34) = 4.19, p = 0.0483, η2 = 0.109, in the occipital lobe in the 1-back condition. No main or interaction effect of Group and Session was observed in the occipital lobe in the 2-back condition. The main effec ever the main effe 3.4.2. Alpha Band Similarly, connectivity matrices shown in (A2) for NFT group and (B2) for control group and brain connections in (C2) for NFT group and (D2) for control group in 2-back condition. The box plots in (E1,E2) show the phase synchronization index in different brain lobes in 1-back and 2-back conditions, respectively. The colour bars of the connectivity matrices and brain connections represent the strength of synchronization between the EEG channels. It ranges from 0 (no synchronization) to 1 (full synchronization). The diamond shape in the box plots represents the outliers in the data. Figure 10. Alpha band functional connectivity (* p < 0.05). Connectivity matrices shown in (A1) for NFT group and (B1) for control group and brain connections in (C1) for NFT group and (D1) for control group in 1-back condition. Similarly, connectivity matrices shown in (A2) for NFT group and (B2) for control group and brain connections in (C2) for NFT group and (D2) for control group in 2-back condition. The box plots in (E1,E2) show the phase synchronization index in different brain lobes in 1-back and 2-back conditions, respectively. The colour bars of the connectivity matrices and brain connections represent the strength of synchronization between the EEG channels. It ranges from 0 (no synchronization) to 1 (full synchronization). The diamond shape in the box plots repre- sents the outliers in the data. The main effect of Group and Session and the interaction effect were also non signif Figure 10. Alpha band functional connectivity (* p < 0.05). Connectivity matrices shown in (A1) for NFT group and (B1) for control group and brain connections in (C1) for NFT group and (D1) for control group in 1-back condition. Similarly, connectivity matrices shown in (A2) for NFT group and (B2) for control group and brain connections in (C2) for NFT group and (D2) for control group in 2-back condition. The box plots in (E1,E2) show the phase synchronization index in different brain lobes in 1-back and 2-back conditions, respectively. The colour bars of the connectivity matrices and brain connections represent the strength of synchronization between the EEG channels. It ranges from 0 (no synchronization) to 1 (full synchronization). The diamond shape in the box plots represents the outliers in the data. Figure 10. Alpha band functional connectivity (* p < 0.05). 3.4.3. Beta Band The beta ban 3.4.3. Beta Band The beta band connectivity results are shown in Figure 11. There was no main or interaction effect of Group and Session in the frontal lobe in either condition, 1-back or 2- back. In the fronto-central lobe, the main effect of Group was significant in the 1-back condition, 𝐹(1,34) = 7.138, 𝑝= 0.0114, 𝜂2 = 0.173, as well as in the 2-back condition, 𝐹(1,34) = 5.65, 𝑝= 0.0231, 𝜂2 = 0.142 . However, the main effect of Session and the The beta band connectivity results are shown in Figure 11. There was no main or interaction effect of Group and Session in the frontal lobe in either condition, 1-back or 2-back. In the fronto-central lobe, the main effect of Group was signiﬁcant in the 1-back condition, F(1, 34) = 7.138, p = 0.0114, η2 = 0.173, as well as in the 2-back condition, F(1, 34) = 5.65, p = 0.0231, η2 = 0.142. However, the main effect of Session and the interaction effect were non-signiﬁcant in the fronto-central lobe in both conditions, 1-back and 2-back. In the parietal and occipital lobes, no main or interaction effects were observed in either condition. 17 of 22 served 17 of 22 served Bioengineering 2023, 10, 200 Figure 11. Beta band functional connectivity (* p < 0.05). Connectivity matrices shown in (A1) for NFT group and (B1) for control group and brain connections in (C1) for NFT group and (D1) for control group in 1-back condition. Similarly, connectivity matrices shown in (A2) for NFT group and (B2) for control group and brain connections in (C2) for NFT group and (D2) for control group in 2-back condition. The box plots in (E1) and (E2) show the phase synchronization index in different brain lobes in 1-back and 2-back conditions, respectively. The coloured bars of the connectivity ma- trices and brain connections represent the strength of synchronization between the EEG channels. It ranges from 0 (no synchronization) to 1 (full synchronization). The diamond shape in the box plots represents the outliers in the data. 4 Discussion Figure 11. Beta band functional connectivity (* p < 0.05). Connectivity matrices shown in (A1) for NFT group and (B1) for control group and brain connections in (C1) for NFT group and (D1) for control group in 1-back condition. The rationa 4. Discussion y g p the alpha as well as theta and beta bands. The real-time feedback activity was recorded at the frontal lobe, and the offline analyses were extended to the frontal, fronto-central, pa- rietal, and occipital brain regions. Additionally, based on our hypothesis, we visually ob- served the hemispheric lateralization of FC using the N-back task and found an enhance- ment in the brain FC in the theta band after NFT. Regarding the effect of NFT on the brain alpha, theta, and beta activities in the N-back task, we found different results in the post- stimulus power of both groups. At the frontal region before and after NFT the post-stimulus alpha power presented The rationale behind the current study was to investigate the effects of alpha NFT on the alpha as well as theta and beta bands. The real-time feedback activity was recorded at the frontal lobe, and the ofﬂine analyses were extended to the frontal, fronto-central, parietal, and occipital brain regions. Additionally, based on our hypothesis, we visually observed the hemispheric lateralization of FC using the N-back task and found an enhancement in the brain FC in the theta band after NFT. Regarding the effect of NFT on the brain alpha, theta, and beta activities in the N-back task, we found different results in the post-stimulus power of both groups. At the frontal region, before and after NFT, the post stimulus alpha power presented similar levels in the low-memory condition (i.e., 1-back) and increased power in the high- memory condition (2-back). The increased activation in the 2-back condition for both groups in the post-NF session is suggestive of the involvement of the frontal region in the more-demanding high-memory condition [57]. From the pre- to post-NF sessions in the frontal lobe, a similar change in both groups was observed, which indicated a non-specific activation that cannot be attributed to NFT effects. Previous research stated that a more circumscribed cortical activation is related to better working memory performance [58]. At the frontal region, before and after NFT, the post-stimulus alpha power presented similar levels in the low-memory condition (i.e., 1-back) and increased power in the high- memory condition (2-back). The increased activation in the 2-back condition for both groups in the post-NF session is suggestive of the involvement of the frontal region in the more-demanding high-memory condition [57]. 3.4.3. Beta Band The beta ban 3.4.3. Beta Band Similarly, connectivity matrices shown in (A2) for NFT group and (B2) for control group and brain connections in (C2) for NFT group and (D2) for control group in 2-back condition. The box plots in (E1) and (E2) show the phase synchronization index in different brain lobes in 1-back and 2-back conditions, respectively. The coloured bars of the connectivity matrices and brain connections represent the strength of synchronization between the EEG channels. It ranges from 0 (no synchronization) to 1 (full synchronization). The diamond shape in the box plots represents the outliers in the data. Figure 11. Beta band functional connectivity (* p < 0.05). Connectivity matrices shown in (A1) for NFT group and (B1) for control group and brain connections in (C1) for NFT group and (D1) for Figure 11. Beta band functional connectivity (* p < 0.05). Connectivity matrices shown in (A1) for Figure 11. Beta band functional connectivity (* p < 0.05). Connectivity matrices shown in (A1) for NFT group and (B1) for control group and brain connections in (C1) for NFT group and (D1) for control group in 1-back condition. Similarly, connectivity matrices shown in (A2) for NFT group and (B2) for control group and brain connections in (C2) for NFT group and (D2) for control group in 2-back condition. The box plots in (E1) and (E2) show the phase synchronization index in different brain lobes in 1-back and 2-back conditions, respectively. The coloured bars of the connectivity ma- trices and brain connections represent the strength of synchronization between the EEG channels. It ranges from 0 (no synchronization) to 1 (full synchronization). The diamond shape in the box plots represents the outliers in the data. 4 Discussion Figure 11. Beta band functional connectivity (* p < 0.05). Connectivity matrices shown in (A1) for NFT group and (B1) for control group and brain connections in (C1) for NFT group and (D1) for control group in 1-back condition. Similarly, connectivity matrices shown in (A2) for NFT group and (B2) for control group and brain connections in (C2) for NFT group and (D2) for control group in 2-back condition. The box plots in (E1) and (E2) show the phase synchronization index in different brain lobes in 1-back and 2-back conditions, respectively. The coloured bars of the connectivity matrices and brain connections represent the strength of synchronization between the EEG channels. It ranges from 0 (no synchronization) to 1 (full synchronization). 3.4.3. Beta Band The beta ban 3.4.3. Beta Band The diamond shape in the box plots represents the outliers in the data. The rationa 4. Discussion In the current study, the enhanced FC in the theta band suggests the presence of stronger interregional synchronization in the NFT group when compared with the control group: an important ingredient for the co-activation of neural structures, which is involved in different sub-processes of working memory function [67]. These results suggest that upper alpha training elicited reorgani- zation processes in the brain, which are not only predominant in the right hemisphere but also visible in a different frequency band, the theta band. Research has indicated that working memory deﬁcits are associated with lower connectivity of the dorsolateral prefrontal cortex (DLPFC) with other regions, and the higher global brain connectivity in our results within the NFT group predicts better working memory performance as well as general ﬂuid intelligence [68]. Interactions between the alpha and theta bands have also been reported [69]. Both alpha and theta rhythms have been postulated to mediate the interaction between distal and widely distributed connections. Since the alpha rhythm is distributed across the whole scalp, it is highly suitable to engender neural plasticity and, consequently, adaptive changes in the brain activity that can be seen foremost in the theta frequency. The increase in theta band power has been associated with increased encoding success in memory tasks [8]. Theta oscillations have been related to control processes and regional integration during working memory [67]. These results indicate that FC strength in the theta band was sensitive to working memory capacity and was aided by neurofeedback. A generally lower FC is associated with a lower performance in several cognitive domains and is mostly observed empirically in patients, e.g., those with schizophrenia [70]. The results from the present study particularly highlight a segregation of theta FC between the left and right hemispheres when assessing working memory capacity. Furthermore, neurofeedback seems to play a major role in manipulating the theta network of working memory capacity, which is in line with our hypotheses. Previous research has demonstrated that FC in the resting condition is inversely related to cognitive reserve [71], but there is also evidence of a higher FC in the theta band, which suggests higher cognitive reserve scores for active engagement and attention during a task [72]. The relevance of theta activity for attention in humans has been reported in [73], and increased global connectivity over the brain in theta band has been reported to enhance cognitive performance. The rationa 4. Discussion From the pre- to post-NF sessions in the frontal lobe, a similar change in both groups was observed, which indicated a non-speciﬁc activation that cannot be attributed to NFT effects. Previous research stated that a more circumscribed cortical activation is related to better working memory performance [58]. A load-dependent change in theta and alpha band at the fronto-central, parietal, and occipital positions was observed with increased theta and alpha activities in the high-load condition (i.e., 2-back task). Regarding alpha activity, a similar effect was observed, where an increase in alpha activity with increasing memory load was observed [59]. Similarly, research Bioengineering 2023, 10, 200 18 of 22 18 of 22 studies have shown an increase in theta EEG power with the increase in working memory load [60–62]. The power changes observed in different EEG bands in the current study contribute to an understanding of the neural mechanism of working memory. Along with the increase in training band (i.e., alpha), enhanced activity in the non-training band (i.e., theta) was also observed. This increase was not really a result of the NFT but it represents a strong coordination between alpha and theta activities during a memory task per se. g p g y p A visual observation of FC across the theta, alpha, and beta bands revealed hemi- spheric lateralization. Our statistical analysis conﬁrmed that only the theta band FC was correlated with working memory and was increased after training. Previous research associated the theta band in a human being with working memory [63], and a signiﬁcantly lower FC in the theta band was reported in memory impairments [64]. The integration of a brain process to confront the increase in memory demands during a working memory task was indicated only in the increase in theta FC [65]. A higher FC in the more efﬁcient group within the theta band was observed [66], indicating that their working memory function seems to be more efﬁcient in contrast with others. Sauseng et al. proposed a ‘process level’ mechanism for theta synchronization during the encoding phase of working memory, which integrates processes such as attention across brain regions [67]. These brain processes are very crucial in the formation of a memory trace. The rationa 4. Discussion Finally, we conclude that the increased FC in the theta band in the NFT group after training in the current study reﬂects the positive effect of training on continuous cognitive processing (attention and executive control) during task performance [74]. As an indicator of higher cognitive reserve scores [75], the theta FC patterns in our results reﬂect the more efﬁcient functional networks of attention and executive function during memory processing after the training [74]. 19 of 22 Bioengineering 2023, 10, 200 19 of 22 The following limitations of the present study need to be addressed in future studies: A gender-balanced design is necessary to understand the extent and generality of lateral- ization effects. Since brain lateralization is not comparable in male and female individuals, a sample with a balance of genders should help to provide a more precise generalization of the study results [76]. Moreover, the addition of a second control group receiving the placebo NFT would extend the present results to the realm of clinical usability. 5. Conclusions In our results, we found that alpha neurofeedback training might improve the working memory capacity of healthy participants, as evidenced by the enhanced functional brain connections in the theta band. From the visual inspection of the plots of the brain networks, we observed that the functional connectivity of working memory within each hemisphere of the brain is stronger in comparison with the connectivity between the two hemispheres. Author Contributions: R.N., G.W. and H.N. contributed to the conception and design of the study. R.N. collected, processed, and analysed the data. R.N. performed the statistical analysis and wrote the ﬁrst draft of the manuscript. H.N. and V.V.Y. secured the funding for data collection from the Universiti Tunku Abdul Rahman Research Fund, Malaysia. G.W. provided support in securing the funding from the Austrian Agency for International Cooperation in Education and Research (OeAD). All authors have read and agreed to the published version of the manuscript. Funding: This research work is supported by the Universiti Tunku Abdul Rahman Research Fund (UTARRF) (grant No. IPSR/RMC/UTARRF/2021-C2/H03), Malaysia; the Excellent Research Centre Award Fund, Centre for Healthcare Science and Technology, UTAR, Malaysia; the Austrian Agency for International Cooperation in Education and Research (OeAD) (Reference No. ICM-2020-00116); and the Institute of Psychology, University of Graz, Austria. Institutional Review Board Statement: The study was conducted in accordance with the Decla- ration of Helsinki, and approved by the Scientiﬁc and Ethical Review Committee of Institute of Post-graduate Studies and Research (IPSR), Universiti Tunku Abdul Rahman, Malaysia (Reference No. U/SERC/90/2018). 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[CrossRef] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
https://openalex.org/W2781762021	https://humanfactors.jmir.org/2018/1/e1/PDF	English	null	Second Version of Google Glass as a Wearable Socio-Affective Aid: Positive School Desirability, High Usability, and Theoretical Framework in a Sample of Children with Autism	JMIR human factors	2,018	cc-by	10,528	Original Paper Original Paper Second Version of Google Glass as a Wearable Socio-Affective Aid: Positive School Desirability, High Usability, and Theoretical Framework in a Sample of Children with Autism ed T Sahin1,2, PhD; Neha U Keshav1, PhD; Joseph P Salisbury1, PhD; Arshya Vahabzadeh1,3, MD 1Brain Power, Cambridge, MA, United States 2Department of Psychology, Harvard University, Cambridge, MA, United States 3Massachusetts General Hospital, Boston, MA, United States Corresponding Author: Ned T Sahin, PhD Brain Power 1 Broadway Cambridge, MA, United States Phone: 1 617 514 7300 Email: sahin@post.harvard.edu Corresponding Author: Ned T Sahin, PhD Brain Power 1 Broadway Cambridge, MA, United States Phone: 1 617 514 7300 Email: sahin@post.harvard.edu Abstract Background: Computerized smartglasses are being developed as an assistive technology for daily activities in children and adults with autism spectrum disorder (ASD). While smartglasses may be able to help with educational and behavioral needs, their usability and acceptability in children with ASD is largely unknown. There have been reports of negative social perceptions surrounding smartglasses use in mainstream populations, a concern given that assistive technologies may already carry their own stigma. Children with ASD may also have a range of additional behavioral, developmental, and social challenges when asked to use this emerging technology in school and home settings. Objective: The usability and acceptability of Glass Enterprise Edition (Glass), the successor to Google Glass smartglasses, were explored in children with ASD and their caregivers. Methods: Eight children with ASD and their caregivers were recruited to attend a demonstration session with Glass smartglasses the week they were publicly released. The children had a wide range of ability, including limited speech to speaking, and represented a full range of school ages (6 to 17 years). Children and caregivers were interviewed about their experience of using the smartglasses and whether they would use them at school and home. Results: All 8 children succeeded in using Glass and did not feel stressed (8/8, 100%) or experience any overwhelming sensory or emotional issues during the session (8/8, 100%). All 8 children (8/8, 100%) endorsed that they would be willing to wear and use the device in both home and school settings. Caregivers felt the experience was fun for the children (8/8, 100%), and most caregivers felt the experience was better than they had expected (6/8, 75%). Conclusions: A wide age and ability range of children with ASD used Glass immediately after it was released and found it to be usable and acceptable. Despite concerns about potential stigma or social acceptability, all of the children were prepared to use the technology in both home and school settings. Encouragingly, most caregivers noted a very positive response. There were no behavioral, developmental, or social- or stigma-related concerns during or after the session. Smartglasses may be a useful future technology for children with ASD and are readily accepted for use by children with ASD and their caregivers. (JMIR Hum Factors 2018;5(1):e1) doi: 10.2196/humanfactors.8785 JMIR Hum Factors 2018 \| vol. 5 \| iss. 1 \| e1 \| p. 1 (page number not for citation purposes) JMIR HUMAN FACTORS JMIR HUMAN FACTORS Sahin et al Modern Assistive-Reality Smartglasses The emergence of a new crop of smartglasses is encouraging, especially because the initial public reaction to the widely recognized original Google Glass resulted in some negative social reactions. Modern smartglasses vary in terms of physical dimensions, functionality, and intended user group. For the purposes of this report, we decided to investigate the acceptability and usability of the most recently released lightweight smartglasses, Glass Enterprise Edition (Glass). Glass was released by X (a subsidiary of Alphabet Inc, formerly known as Google X) in July 2017. Glass is an assistive-reality technology, and it is the successor to Google Glass, one of the most recognizable smartglasses in the world [19]. Glass, like its predecessor, is a head-mounted, wearable computer that has demonstrated utility in a variety of situations where operating a computer hands-free and while heads-up is of particular advantage. Glass has been creatively developed as a technology that can deliver social and cognitive skills coaching to children and adults with ASD [8]. To our knowledge, we have reported on the first studies of ASD-related software on the original Google Glass (Explorer Edition) [8,9,15,16], and here we present the first appearance of Glass (Enterprise Edition) in the literature. Smartglasses can deliver a large range of experiences, including augmented and virtual reality [10]. They are also sensor-rich and can collect a wide range of quantitative user data [9,11,12]. These data can be monitored and analyzed on a real-time basis, allowing for the smartglasses to dynamically change the user experience to optimize learning—effectively placing the user and the smartglasses in a closed feedback loop [8,13,14]. Given the proximity of smartglasses to the sensory organs contained in the human head, this type of computing may enable a higher level of human-computer interaction than other devices [13]. Smartglasses are already being developed as a social and behavioral communication aid for people with ASD [8,15,16]. It would seem that the Enterprise Edition (which has updates to the form factor, usability, central processor, display, audio system, and other features) would represent a substantial advantage for assistive technology apps and algorithms for ASD. However, it remains unknown whether people with ASD would actually desire to wear the new device. Assistive apps for people with ASD on the original Google Glass have been shown to be tolerable [20], safe [15], and to reduce hyperactivity in an ASD sample [8,16]. KEYWORDS autism; technology; digital health; augmented reality; virtual reality; smartglasses; usability; schools; education; classroom; IDEA; IEP; special education JMIR Hum Factors 2018 \| vol. 5 \| iss. 1 \| e1 \| p. 1 (page number not for citation purposes) http://humanfactors.jmir.org/2018/1/e1/ XSL•FO RenderX JMIR HUMAN FACTORS Sahin et al potentially encouraging children to remain heads-up while using them. This heads-up posture when using smartglasses can allow for better user engagement with people and the social world (Figure 1 A, right). Background Autism spectrum disorder (ASD) is a childhood-onset developmental disorder, with an estimated 3.5 million people being diagnosable with ASD in the United States alone [1]. Innovative assistive technologies may help to address the unmet educational and therapeutic resource demands of the ASD community [2]. While there are many different types of assistive technology, the portability, capability, and ubiquity of smartphone and tablet devices has led to considerable growth in assistive apps for these devices [3,4]. More recent technological advances have resulted in lightweight smartglasses: face-worn computers with a visual display and in-built sensors [5-7] that can also deliver assistive apps [8,9]. JMIR Hum Factors 2018 \| vol. 5 \| iss. 1 \| e1 \| p. 2 (page number not for citation purposes) JMIR HUMAN FACTORS Sahin et al Figure 1. Head-worn computers encourage users to be heads-up and allow them to be hands-free in contrast to screen-based technologies such as phones and tablets. (A) Demonstrative example of a person using a tablet while her sibling uses Glass Enterprise Edition, days after it was released. Both siblings have autism spectrum disorder. Tablet use encourages a heads-down stance, suboptimal posture, and visual disconnection from the social world. (B) The Glass Enterprise Edition device from multiple views. Modern Assistive-Reality Smartglasses However, small changes in devices can greatly affect the desire of potential users to wear them. There are a number of important differentiating factors to consider when smartglasses are compared to handheld devices. Handheld devices such as tablets and smartphones require one or both hands to hold the device and encourage a heads-down posture (Figure 1 A, left) [17]. Evidence suggests that smartphone use may decrease user awareness of their social and physical environment. This is a particular concern in people with ASD, given that they already often face challenges engaging with the social world around them [18]. In contrast, head-worn computers pose an advantage in allowing and http://humanfactors.jmir.org/2018/1/e1/ XSL•FO RenderX http://humanfactors.jmir.org/2018/1/e1/ Understanding the Needs of People With Autism Spectrum Disorder Given that the initial entry of Google Glass and other smartglasses raised privacy concerns and some negative public reaction, the announcement of a major new release of head-worn computing [19] signaled a potentially major advance for assistive technology targeting populations who traditionally face significant social challenges [17]. Google Glass was ahead of its time and may have been held back by perceptions around desirability and social acceptability of wearing this new category of device in public [21,22]. It is therefore reassuring to developers that head-worn computer platforms have received public backing from one of the largest companies in the world [19], in this case the inventor of the product [23]. As with any assistive technology, it is important to investigate and understand the attitudes of children and young adults with ASD, especially because children with special needs are often forced to use devices and systems they do not actually like or want to be associated with [24,25]. This is ultimately less effective because aversion leads to lower adherence. Poor adherence and problems with maintaining lasting engagement are some of the largest issues facing educational devices and apps as well as well-being and lifestyle tools [26,27]. JMIR Hum Factors 2018 \| vol. 5 \| iss. 1 \| e1 \| p. 3 (page number not for citation purposes) JMIR Hum Factors 2018 \| vol. 5 \| iss. 1 \| e1 \| p. 3 (page number not for citation purposes) XSL•FO RenderX JMIR HUMAN FACTORS Sahin et al the wearer’s face that is visible to others, especially the central information-rich parts of the face such as the eye regions [43]. Many people with ASD use assistive technology to help them with communication skills, social and emotional skills, and adaptive/daily activities and living skills [28]. Assistive technologies elicit a range of responses from individuals and their peers, and they can be considered cool [25], weird, desirable, or a source of stigma [29,30]. Users of assistive technologies can often express a preference for the type of assistive technology that they want to use [31,32], even at a young age [33]. Additionally, the social acceptability of an assistive technology may be one of the most important elements in determining if that technology gets used by people with developmental disabilities [30,34]. Understanding the Needs of People With Autism Spectrum Disorder These individuals have often had to use technologies that have been selected for them and their families while having little input to the potential negative image, stigma, or embarrassment of using such technologies [30]. Understanding and implementing user preference of assistive technologies empowers self-determination in these individuals [31]. The preferences and views of the family and caregivers of these individuals are also important as they impact the acceptance and effective use of such technologies in the household [28,35]. These issues are pertinent to smartglasses in light of past reports of negative public perception (eg, around privacy concerns [22]). The relative effect of this obscuring of the facial region may be dependent on the size of the individual’s face relative to the smartglasses, which may correlate with the age of the individual given that biologic age determines an individual’s head size [44]. It may also depend on the ability of the receiver to successfully compensate for partly missing facial data and to make inferences about a sender (a common application of this in ASD research is the “Reading the Eyes in the Mind” test [45]). Since people with and without ASD find it more difficult to read the facial emotional expressions of people with ASD [41], it is conceivable that further obscuring the amount of visible facial information could make the interaction even more arduous. This point may be particularly relevant to interactions between people with ASD and their unaffected family members. ASD is a highly hereditable condition with a complex genetic basis [46], and many unaffected relatives of children with ASD have been found to have subclinical autistic traits [47]. The parents of children with ASD may demonstrate subtle deficits in social communication and face processing [48,49]. Given these reports and considerations, the physical presence of smartglasses may affect social communication, and it may be sensible to attempt to minimize such facial obscuration to enhance social communication between people with ASD and their family members. There have only been a handful of reports on the use of smartglasses in people with ASD [8,15,16], and the attitudes toward and acceptability of such devices to people with ASD remains unclear. The use of smartglasses in people with ASD also requires discussion of their potential impact on social communication from a cognitive neuroscience standpoint and their prospective influence on child development from ecological, psychosocial, and cognitive child development theories. Understanding the Needs of People With Autism Spectrum Disorder The presence of face-worn smartglasses may also influence social relationships, of the adults or children who wear them, as they alter a user’s facial appearance. Unlike many other assistive technologies, they are not easy to hide. Wearing smartglasses may not only alter how the user perceives the world but may alter how the world perceives the user. Facial appearance plays a key role in determining how people interact with one another [50], including whom they help, hire, or want to date [51]. Human faces may also be judged based on their symmetry, a marker of attractiveness and an indicator of optimal developmental outcome despite environmental stressors [52]. Greater facial symmetry has been linked to increased perceived trustworthiness and a decreased risk of being bullied [53]. Facial symmetry may be perceived as demonstrating genetic quality and therefore suitability of an individual as a mate [52], while facial asymmetry may be a predictor of long-term psychological, emotional, and physiological distress [54]. Users of smartglasses that are asymmetrical, such as those that are monocular, could be perceived as being less attractive and trustworthy due to the aforementioned principle of evolutionary psychology. By extension, “asymmetric” smartglasses users may also be at greater risk of bullying [53]. On the other hand, smartglasses that are asymmetrical may obscure less of the wearer’s face from the view of others. As discussed earlier, maximizing how much of the face is visible may help facilitate social communication. Even nontechnological face-worn glasses are associated with impaired interpersonal relationships: for example, wearing prescription glasses or having a history of using eye patches has been associated with a 35% increase in the likelihood of receiving physical or verbal bullying [55]. http://humanfactors.jmir.org/2018/1/e1/ JMIR Hum Factors 2018 \| vol. 5 \| iss. 1 \| e1 \| p. 4 (page number not for citation purposes) Smartglasses in the Context of Child Development rtglasses in the Context of Child Developmen The perceptual impact of smartglasses and their ability to augment a child’s cognitive and emotional functioning may have a central and influential role in childhood development if we consider Bronfenbrenner and Ceci’s bioecological model [56] and Bronfenbrenner’s earlier ecological systems theory [57]. According to the bioecological model, children are active participants in their environments and they have unique bidirectional interactions with each of their contextually separate environments, including home and school. This model places increased emphasis on the cognitive, emotional, and physical attributes of the child in his or her development and in how the child and environments interact with one another. As outlined in Bronfenbrenner’s ecological systems theory [57], the school environment, like the home environment, is one of the most intimate and influential environments affecting childhood development, as it lies in the child’s microsystem. When we consider that smartglasses may enhance the cognitive and emotional functioning of children within their microsystem, we can see that they may have a highly influential role in child development. Even within the microsystem, the contextual differences between the most intimate of environments may affect a child’s view toward using assistive technology. Research has shown that children have different attitudes and levels of enthusiasm toward using assistive technology depending on whether they are asked to use it at home or at school [32]. Learning happens continuously in childhood, and the use of smartglasses technology may provide a digital means of enabling learning to occur, as in Vygotsky’s zone of proximal development (ZPD) [61]. Vygotsky originally described his ZPD as being “the distance between the actual development level as determined by independent problem solving and the level of potential development as determined through problem solving under adult guidance or in collaboration with more capable peers” [62]. These smartglasses designed as assistive technologies may allow children to undertake and learn tasks that they would have found impossible or very difficult to do independently. A child with ASD normally has a number of challenges in being in the ZPD, such as becoming overwhelmed with new experiences, struggling with transitions in environment or activities, and coping with sensory stimuli [18]. Sensor-rich smartglasses may be of particular utility here in that they can be used, with the right software, to monitor the behavioral and physiologic functioning of a child. Smartglasses in the Context of Child Development For instance, they can be transformed by software to be able to detect when children are under- or overstimulated and to accordingly adapt the learning experience in real time to keep a child engaged and in the ZPD [8]. Furthermore, use of smartglasses by future school-age children and adolescents should prompt a discussion of Erikson’s 4th and 5th psychosocial stages [58]. Erikson identified a range of psychosocial developmental stages from birth through death. School-age children experience Erikson’s 4th psychosocial stage, described as a psychosocial crisis of industry versus inferiority. A child in this stage is often expected to learn and demonstrate new skills, productively complete tasks, and meet the expectations of parents and teachers. During this stage, a child becomes aware of his or her abilities and the abilities of his or her peers. A child who cannot master these expected skills risks a sense of inferiority and failure. The potential impact of smartglasses on this developmental stage is not known. They may aid children in successfully mastering this psychosocial stage by allowing them to be productive and giving them a sense of achievement. There is also a risk that children may feel inferior if they feel that without the smartglasses they are incompetent or if they feel ridiculed for wearing such devices. Each child may face a unique situation based on his or her own personal attributes and the support received from key people such as teachers, parents, and peers. This highlights the importance of ensuring that these key people are familiar with smartglasses technology and understand its capabilities and functionality. Potential Impact of Smartglasses on Social Communication The human face, a complex and dynamic system, is our most powerful means of social communication [36]. To successfully transmit social information to another person, the sender must have the mental and physical means of generating a facial and bodily representation of the social information that she or he wishes to send, while the receiver must be in a position to see and decode the facial and bodily representations into social information. The social communication deficits seen in ASD may impede the ability to send and receive social information. People with ASD are reported to have deficits in facial perception [37,38], emotion recognition [39], eye gaze [40], and production of facial expressions [41]. It is important to consider the possibility that social communication may be further impacted by the physical presence of smartglasses on a sender’s face. Smartglasses may impede social communication if, for example, the sender demonstrates a hesitancy in producing natural head movements or expressing large magnitude facial emotional expressions due to concern that the smartglasses may fall off the face or be damaged. Smartglasses may also impair social interaction if the user feels the assistive device is socially undesirable [42] or a source of stigma [30]. In these situations, users may not use the device or may alter their facial and bodily actions to minimize attention to themselves. Furthermore, the physical form factor of smartglasses may obscure a portion of JMIR Hum Factors 2018 \| vol. 5 \| iss. 1 \| e1 \| p. 4 (page number not for citation purposes) XSL•FO RenderX JMIR HUMAN FACTORS Sahin et al of belonging. Many questions remain unanswered about how smartglasses may impact people with ASD during this stage, especially given the many social challenges people with ASD encounter during this transition from childhood to adulthood [60]. How will ASD and these technologies define the individual? Will these technologies help individuals to find their purpose or hinder them? The impact of such technology may depend on smartglasses’ physical attributes, their impact on social relationships, or individual person characteristics (as discussed above within the scope of the bioecological model [56]). JMIR Hum Factors 2018 \| vol. 5 \| iss. 1 \| e1 \| p. 5 (page number not for citation purposes) http://humanfactors.jmir.org/2018/1/e1/ Victimization, Socialization, and the School Environment The questions were adapted or simplified based on the child’s speaking ability and were repeated if needed. Study staff interacted with the child and caregiver and spent time ensuring the questions were understood, considered, and accurately answered. the population, given the risk of stigma and social isolation [30]. This is especially true for a device that is worn on the face. Participants Individuals who had a known history of epilepsy or seizure disorder were not asked to take part in this study. Individuals who had any uncontrolled or severe medical or mental health condition that would make participation in the study predictably hazardous were also not invited to participate. Eight children with clinically diagnosed ASD and their caregivers were entered into this study. The participants represented a wide range of school-aged children, ages 6.7 to 17.2 years (mean 11.7 [SD 3.3] years), including 7 males and 1 female. Participants were recruited from a user research database created from Web-based research interest forms. Written consent for study participation was obtained from the legal guardians, and children from age 7 to 17 years provided written assent. In this report, every participant was accompanied by a parent or guardian caregiver to the session, and participants and caregivers could exit the session at any time and for any reason. It was explained that the main aim of the study was to understand the acceptability and usability of modern smartglasses technology in children with ASD. Study Outline We gave 8 children with ASD an opportunity to try the Glass smartglasses in a controlled, recorded environment and to explore its features, usability, and visual characteristics. We observed and recorded the interaction of the children with the device. We also conducted a postsession semistructured interview with the children and their caregivers, who accompanied the child and observed the whole session. Our sample represented a broad age range and severity spectrum of ASD. Participants were then asked whether they would consider wearing and using the device for 1 hour each day in their school and separately asked the same question about using the device at home. The caregiver was also interviewed in order to rate whether they felt the experience was fun for the participant and whether they felt the experience with the smartglasses went better than they had expected. Results All 8 children, who represented the full range of school ages (6 to 17 years), successfully wore, interacted with, and explored one or more Glass smartglasses (Figure 2). The smartglasses were loaded with a suite of assisted-reality apps for social-emotional learning and self-coaching related to brain-based challenges and needs, as discussed elsewhere [8]. Participants explored the devices at their leisure, putting them on and taking them off and exploring the style, size, weight, shape, and features such as foldability, and spoke out loud in some cases (children with greater speaking ability) about their observations and questions. All children successfully transitioned to the interview room, where they responded to questions by the experimenter, accompanied and assisted by their caregivers as needed. There were no negative effects reported or observed. Caregivers rated the participant level of overall ASD functioning according to a subjective 7-point scale (1=lowest-functioning/severe to 7=highest-functioning/mild). Caregivers also rated speaking ability on a similar scale (1=nonspeaking to 7=fully conversational). Participants represented a large range of overall ASD functioning (range 4 to 7 out of 7; mean 5.6 [SD 1.1]) and speaking ability (range 4 to 7 out of 7; mean 5.5 [SD 1.3]). All participants noted that they did not feel stressed (8/8, 100%, Table 1) or have an overwhelming sensory or emotional experience when using the smartglasses (8/8, 100%). The participants all reported that they would be agreeable to using the smartglasses in both home (8/8, 100%) and school settings (8/8, 100%). Caregivers reported no concerns with the children using the smartglasses, and all caregivers reported that their child appeared to have fun using the device (8/8, 100%). The majority of caregivers felt the interaction of the child with the smartglasses went better than they had expected (6/8, 75%; Table 2). Of the remaining 2, 1 parent said that the experience Institutional Review Board Statement The use of the Brain Power Autism System running on multiple head-worn computing devices by children and adults with autism was approved by Asentral Inc Institutional Review Board, an affiliate of the Commonwealth of Massachusetts Department of Public Health. The study was performed in accordance with relevant guidelines and regulations. http://humanfactors.jmir.org/2018/1/e1/ Victimization, Socialization, and the School Environment School-age children with ASD are at risk of being stigmatized [63] and being victims of bullying [64] for multiple reasons. They have different developmental trajectories that may put them at greater risk of victimization than their neurotypically developing peers, especially when they have challenges in social skills and communication [64]. They may struggle to recognize social cues and develop relationships with their peers, impeding their ability to be better integrated by the community [65-67]. Bullying may be particularly problematic at school, where academic and social factors may be a source of considerable stress, anxiety, and mental health concerns in children [68-70]. A school represents not only an academic establishment but a complicated and highly social environment. Children in schools often balance interpersonal relationships with peers and staff, complex social hierarchies, and school rules that can dictate the most basic elements of children’s day (whom to play with, where to sit, and when to talk to others [65-67,71]). Some reports have suggested that children with ASD have inherently low motivation or desire to join social groups, but recent evidence indicates this is not the case and many have a strong desire for acceptance [72-74]. Therefore, it is important to consider the acceptability and design of any assistive device in Following this stage is Erikson’s 5th psychosocial stage that occurs in adolescence, described as a psychosocial crisis between identity versus role confusion [58]. Adolescence is a time of tremendous biological and psychological change [59], and during this stage individuals seek to define their role in the world, seeking to address the existential question, who am I and what can I be? Individuals will try to find like-minded social groups, focus on relationships with peers, and pursue a sense XSL•FO RenderX JMIR HUMAN FACTORS Sahin et al Following the testing, participants and caregivers went into a separate room where they were questioned about their experience as part of a semistructured interview. The participants were asked to compare their experience of Glass with previously tested assistive devices and gamified apps related to ASD. As previously noted, the participants were recruited from a research database for technology-related studies in ASD, and all had seen and tried the original Google Glass. Participants were asked if they became stressed when using the device and if the session was an overwhelming sensory or emotional experience for them. JMIR Hum Factors 2018 \| vol. 5 \| iss. 1 \| e1 \| p. 6 (page number not for citation purposes) JMIR HUMAN FACTORS Sahin et al Data Collection Procedure Participants and their caregivers were orientated to the testing room where they had an opportunity to learn about the Glass smartglasses and to physically wear and use them. They were provided with any assistance they required to properly place the smartglasses on their heads and align it with their eyes, although little assistance was needed. They were able to use any of the apps on the smartglasses. Testing sessions were recorded via video and photographs. All participants and/or caregivers gave written consent for their images and video to be used in current and future research analyses. XSL•FO RenderX JMIR HUMAN FACTORS JMIR Hum Factors 2018 \| vol. 5 \| iss. 1 \| e1 \| p. 7 (page number not for citation purposes) was not tabulated as a yes but as an undetermined. Children with ASD and their caregivers may be particularly discerning about factors that could impact the use and social acceptance of such technologies in educational settings such as schools and in the home environment. The results demonstrate that Glass was acceptable and desirable sway a child’s enthusiasm toward the device. Many children in this study mentioned favoring Glass because of its unobtrusive, sleek design; having a device that is less noticeable and designed to be “cool” may help with its social acceptance and may not carry the stigma of assistive technology with it. The desirability of Glass in this case was predicated on a prediction of social acceptability (colloquially, the “cool factor”) in a social situation. Many factors may be included in a participant’s prediction of the cool factor of a device. Such factors may include unobtrusiveness, lightness, futuristic look, comfort, ease of storing, ease of transport, durability, ruggedness, styling, ability to give others experiences they could not otherwise have (conferring to the child an ability to control a social situation in a positive way), ability to initiate a conversation with decreased anxiety over selecting the topic of the conversation (ice-breaker), and more. Sahin et al JMIR HUMAN FACTORS Limitations The unanimous willingness of participants to wear the smartglasses in school is also important. The school setting is a place of high risk relative to social integration and stigma that could result from an undesirable or socially inappropriate device or behavior. This is one reason we chose the question of acceptability of the device at school as a high-bar test for how desirable and acceptable this new device may be. However, a limitation of this work is that we asked for the opinion of the target users, and such an opinion is necessarily based on a prediction. It may be hard to predict how a device or behavior will actually be received in the complex and changing social hierarchy of a school environment. Additionally, children with ASD may have extra challenges in predicting the emotional reactions and behaviors of their classmates, especially if they are in an integrated school environment with neurotypical or typically developing children their same chronological age. For all these reasons, further research is needed to test the acceptability within school environments. These results are important for a number of reasons. Children with ASD are frequently not involved in providing design or usability feedback to interventions and technologies developed for them. Involving children when choosing an assistive device is crucial to ensure that the device is socially appropriate for the environment, which will likely lead to greater compliance in wearing the device. It also appears that these children are accepting of new technologies, even on relatively uncommon and highly visible platforms such as head-mounted computers. The children who participated in this study were more open to using Glass in a public environment than many adults have been [22]. With this in mind, it will be equally as important to ensure caregivers and peers in the child’s microsystem are accepting of the assistive technology [57], as their opinions will likely Principal Findings Smartglasses are an emerging technology that could hold much promise as an assistive technology for children and young adults with ASD. It is important to seek the opinions of children with ASD and their caregivers when considering the use of a new assistive device. This is especially true of smartglasses given their high level of visibility, prior reports of negative social perceptions, and the potential interplay of such devices with social communication and child development. Children with ASD and their caregivers may be particularly discerning about factors that could impact the use and social acceptance of such technologies in educational settings such as schools and in the home environment. The results demonstrate that Glass was acceptable and desirable by all participants, who spanned the full range of school ages (6 to 17 years). It was encouraging to find that all 8 school-aged children with ASD felt that using these smartglasses was not a stressful experience and denied being overwhelmed in a sensory or emotional way. Additionally, it was also promising to see that all of the children expressed a willingness to use these devices in both school and home settings. Caregivers noted that children had fun using the device, and most caregivers felt their expectations of how the children would interact with the smartglasses were surpassed. http://humanfactors.jmir.org/2018/1/e1/ JMIR Hum Factors 2018 \| vol. 5 \| iss. 1 \| e1 \| p. 8 (page number not for citation purposes) was not tabulated as a yes but as an undetermined. had proceeded “as expected” and another answered the question conversationally but without a direct response, so the response was not tabulated as a yes but as an undetermined. had proceeded “as expected” and another answered the question conversationally but without a direct response, so the response Figure 2. Children on the autism spectrum using and exploring the Glass Enterprise Edition device during a testing session at Brain Power. Each of the 8 participants, who represent the entire range of school ages, range from mild to moderate autism severity, and demonstrate a wide breadth of speaking ability (from moderate impairments in speech to being fully conversational), rated Glass Enterprise Edition as desirable to wear on their heads and use daily in the often-complex social environment of school and at home. often complex social environment of school and at home. http://humanfactors.jmir.org/2018/1/e1/ http://humanfactors.jmir.org/2018/1/e1/ JMIR Hum Factors 2018 \| vol. 5 \| iss. 1 \| e1 \| p. 7 (page number not for citation purposes) XSL•FO RenderX JMIR HUMAN FACTORS Sahin et al Table 1. Participant responses following use of smartglasses. Neutral or undetermined response n (%) No n (%) Yes n (%) Question 0 (0) 0 (0) 8 (100) Would you wear the smartglasses for 1 hour each day at school? 0 (0) 0 (0) 8 (100) Would you wear the smartglasses for 1 hour a day at home? 0 (0) 8 (100) 0 (0) Did you feel stressed while wearing the smartglasses? 0 (0) 8 (100) 0 (0) Did you feel overwhelmed (emotionally/sensory)? Table 2. Caregiver responses following use of smartglasses. Neutral or undetermined response n (%) No n (%) Yes n (%) Question 0 (0) 0 (0) 8 (100) Was it fun for your child to use Glass? 2 (25) 0 (0) 6 (75) Did the experience go better than you anticipated? Discussion Principal Findings Smartglasses are an emerging technology that could hold much promise as an assistive technology for children and young adults with ASD. It is important to seek the opinions of children with ASD and their caregivers when considering the use of a new assistive device. This is especially true of smartglasses given their high level of visibility, prior reports of negative social perceptions, and the potential interplay of such devices with social communication and child development. References 1. Buescher AVS, Cidav Z, Knapp M, Mandell DS. Costs of autism spectrum disorders in the United Kingdom and the United States. JAMA Pediatr 2014 Aug;168(8):721-728. [doi: 10.1001/jamapediatrics.2014.210] [Medline: 24911948] 1. Buescher AVS, Cidav Z, Knapp M, Mandell DS. Costs of autism spectrum disorders in the United Kingdom and the United States. JAMA Pediatr 2014 Aug;168(8):721-728. [doi: 10.1001/jamapediatrics.2014.210] [Medline: 24911948] 2. Odom SL, Thompson JL, Hedges S, Boyd BA, Dykstra JR, Duda MA, et al. Technology-aided interventions and instruction for adolescents with autism spectrum disorder J Autism Dev Disord 2015 Dec;45(12):3805 3819 [doi: 1. Buescher AVS, Cidav Z, Knapp M, Mandell DS. 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Comput Hum Behav 2015 Aug;49:635-647. [doi: 10.1016/j.chb.2015.03.003] g j 6. Rauschnabel P, Brem A, Ro Y. Augmented reality smart glasses: definition, conceptual insights, and managerial importance [unpublished working paper]. 2015. URL: https://www.researchgate.net/publication/ 6. Conflicts of Interest This report was supported by Brain Power, a neurotechnology company developing a range of artificially intelligent wearable technologies. Brain Power has engineering and technical partnerships with major technology companies and also receives funding support from federal and congressional sources. Acknowledgments The authors thank Google, Inc. for a generous grant and also the Glass team at X (formerly Google X) for technical guidance on Glass software development. This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs through the Autism Research Program under Award No. W81XWH 17-1-0449. Early work to transform smartglasses into biomedical sensors was supported in part by the United States Army Medical Research and Materiel Command under Contract No. W81XWH-14-C-0007 (awarded to TIAX, LLC). Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the Department of Defense. The authors would also like to acknowledge Krystal Huey for her help with study coordination. Conclusions These results suggest that a smartglasses platform may be an acceptable base for assistive software apps that could promote self-sufficiency. For instance, they may have a desirable new platform for gamified, social-emotional self-coaching apps XSL•FO RenderX XSL•FO RenderX XSL•FO RenderX JMIR HUMAN FACTORS Sahin et al populations. Further research is clearly needed to address these and more limitations or open questions of this work. This report represents part of a larger, ongoing research initiative. based in neuroscience and artificial intelligence that have been deployed on other head-worn computer platforms [8]. The results are promising at a broader level for those who wish to use or develop apps that harness the unique features of this family of devices, such as their ability to allow the user to be heads-up, hands-free, and able to perceive and engage with the world around while receiving additional assistance. The results suggest that the newest entrant into the still-emerging family of devices may be well received, at least by some discerning This paper represents the first published work, to our knowledge, using Glass (Enterprise Edition). It also represents the first published use of Glass as an assistive or assessment device for people with different abilities or intellectual disabilities or challenges. 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Abbreviations ASD: autism spectrum disorder Glass: Glass Enterprise Edition ZPD: zone of proximal development ASD: autism spectrum disorder Glass: Glass Enterprise Edition ZPD: zone of proximal development Edited by G Eysenbach; submitted 18.08.17; peer-reviewed by S Shamsuddin, S Clough, I Montagni; comments to au revised version received 08.10.17; accepted 29.10.17; published 04.01.18 Please cite as: Sahin NT, Keshav NU, Salisbury JP, Vahabzadeh A Second Version of Google Glass as a Wearable Socio-Affective Aid: Positive School Desirability, High Usability, and Theoretical Framework in a Sample of Children with Autism JMIR Hum Factors 2018;5(1):e1 URL: http://humanfactors.jmir.org/2018/1/e1/ doi: 10.2196/humanfactors.8785 PMID: 29301738 Please cite as: Sahin NT, Keshav NU, Salisbury JP, Vahabzadeh A Second Version of Google Glass as a Wearable Socio-Affective Aid: Positive School Desirability, High Usability, and Theoretical Framework in a Sample of Children with Autism JMIR Hum Factors 2018;5(1):e1 URL: http://humanfactors.jmir.org/2018/1/e1/ doi: 10.2196/humanfactors.8785 PMID: 29301738 ©Ned T Sahin, Neha U Keshav, Joseph P Salisbury, Arshya Vahabzadeh. Originally published in JMIR Human Factors (http://humanfactors.jmir.org), 04.01.2018. 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 JMIR Human Factors, is properly cited. The complete bibliographic information, a link to the original publication on http://humanfactors.jmir.org, as well as this copyright and license information must be included. JMIR Hum Factors 2018 \| vol. 5 \| iss. 1 \| e1 \| p. 12 (page number not for citation purposes) http://humanfactors.jmir.org/2018/1/e1/ XSL•FO RenderX
https://openalex.org/W3022878380	https://scindeks-clanci.ceon.rs/data/pdf/1451-2092/2020/1451-20922002383X.pdf	Latin	null	Investigation and optimization of machining parameters influence on surface roughness in turning AISI 4340 steel	FME Transactions	2,020	cc-by	5,485	1. INTRODUCTION technique during dry turning and found that feed rate has the most significant effect on surface roughness. Tamizharasan and Senthilkumar [8] analyzed the effect of various cutting tool geometries over surface roughness and MRR using Taguchi’s technique and by ANOVA. Nalbant et al. [9] optimized the cutting parameters in turning AISI 1030 steel based on the surface roughness produced and found that insert radius and feed rate are the most contributing factors. Ramesh et al. [10] predicted the effect of cutting parameters on surface roughness during turning aerospace titanium alloy (gr5) using response surface methodology model and found that feed rate is the most influencing factor. Kopac et al. [11] determined optimal conditions to achieve desired surface roughness in turning C15 E4 steel by varying the cutting speed, tool and workpiece material, depth of cut and no. of cuts and using coated inserts. Turning is a process of producing axisymmetric surface by removing unwanted material from the work-piece to produce a desired shape, where the tool moves in a perpendicular plane and the workpiece is hold on a spindle and rotated [1]. The interesting parameters that were associated with the process of turning were cutting speed, feed and depth of cut [2]. The cutting speed can be defined as the relative surface speed of the workpiece with respect to the tool, which is responsible for material removal. The relative motion of the tool with respect to the job in perpendicular direction to cutting speed for the purpose of reaching unmachined surface is called feed [3]. The penetration of the cutting tool into the job, that is beneath the job surface is depth of cut, which is the radial distance in turning, from the unmachined surface of the job to the tool tip [4]. Turning is the finishing operation performed on com- ponents to produce final shape of the component with adequate dimension and tolerances [5], for which optimization of machining parameters is performed by most of the researchers. Asilturk and Neseli [12] determined optimum mac- hining parameters for better surface roughness during dry turning of AISI 304 steel using coated carbide insert by response surface methodology and predicted it using the developed mathematical model and found that feed rate is the most contributing factor. Bernardos and Vos- niakos [13] have presented various methodologies that are to be followed in predicting the surface roughness and how to reduce it. © Faculty of Mechanical Engineering, Belgrade. All rights reserved Received: January 2019, Accepted: January 2020 Correspondence to: Mr Anantha Raman Lakshmipathi Department of Mechanical Engineering, S.A Engineering College, Chennai, India. E-mail: raman3366@gmail.com doi:10.5937/fme2002383B Jayakumar Shivasankar UG Scholar Department of Mechanical Engineering Manakula Vinayagar Institute of Technology Kalitheerthalkuppam, Puducherry India UG Scholar Department of Mechanical Engineering Manakula Vinayagar Institute of Technology Kalitheerthalkuppam, Puducherry India UG Scholar Department of Mechanical Engineering Manakula Vinayagar Institute of Technology Kalitheerthalkuppam, Puducherry India Keywords: Machining parameter; Surface roughness; Taguchi’s Technique; ANOVA. Balakrishnan Deepanraj Associate Professor Department of Mechanical Engineering Jyothi Engineering College Thrisur, Kerala India Associate Professor Department of Mechanical Engineering Jyothi Engineering College Thrisur, Kerala India Natarajan Senthilkumar Professor Department of Mechanical Engineering Adhiparasakthi Engineering College Melmaruvathur, Tamil Nadu India Professor Department of Mechanical Engineering Adhiparasakthi Engineering College Melmaruvathur, Tamil Nadu India Lakshmipathi Anantha Raman This paper focuses on the experimental investigation of machining parameters such as cutting speed, feed rate and depth of cut influence over surface roughness parameters (Ra, Ry and Rt) during turning AISI 4340 steel. Further, in order to achieve smaller surface roughness parameter values, the machining parameters are optimized using Taguchi’s technique Signal-to-Noise ratio (S/N ratio). Analysis of Variance (ANOVA) is performed to determine the most contributing factor that influences the surface roughness parameters. It is observed that the feed rate is the most significant factor contributing by 70.50%, depth of cut by 18.54% and cutting speed by 9.15%. From the optimum condition obtained, a confirmation experiment is performed and the results obtained shows that the surface roughness parameter values are reduced by 31.63% than the designed experimental values. Assistant Professor Department of Mechanical Engineering S.A Engineering College Avadi, Chennai, Tamil Nadu India Figure 1. Surface roughness parameters [6] Akhyar et al. [17] optimized the cutting parameters during turning Ti-6%Al-4%V with coated and uncoated cemented carbide tool and found that cutting speed and tool grade have a significant effect on surface rough- ness. Mahdavinejad and Bidgoli [18] used an adaptive neural fuzzy intelligent system to predict the roughness in turning process and found that surface quality dec- reases with depth of cut. Ramesh et al. [19] optimized the turning conditions based on surface roughness while turning Duplex stainless steel 2205 using CVD trian- gular carbide insert and developed a regression equation to predict it. Aruna and Dhanalakshmi [20] optimized surface roughness, when turning Inconel 718 with cer- met inserts based on response surface methodology and developed a second order quadratic models to predict it and concluded that cutting speed has the strongest effect on surface roughness. Saini et al. [21] developed an accurate predictive model using response surface met- hodology for surface roughness and tool wear and pre- dicted it for various cutting conditions and found that decrease in feed rate and increase in cutting speed re- sults in increased surface quality with higher tool wear. Bhushan et al. [22] investigated the influence of machi- ning parameters on surface roughness during machining 7075 Al alloy and 10% SiC particulate MMC and found that for optimum surface roughness, cutting speed sho- uld be within 180 to 220 m/min, feed rate between 0.1 to 0.3 mm/rev and depth of cut within 0.5 to 1.5 mm. 1. INTRODUCTION Sahin and Motorcu [14] used response surface methodology to develop a mathe- matical formula to predict surface roughness and found that feed rate is the most significant parameter con- tributing towards surface roughness using ANOVA. Palanikumar and Karthikeyan [15] determined the factors that influence surface roughness during turning Al/SiC particulate composite using carbide tool insert and found that feed rate is the most significant factor, responsible for surface roughness. Verma et al. [16] used Taguchi’s technique to optimize machining para- meters in turning ASTM A242 Type-1 steel over sur- Surface roughness parameters such as Ra – Average roughness value, Rt – Maximum height of roughness profile and Ry – Average maximum height of profile are of importance in dimensional stability of a machined component subjected to assembly. Fig. 1 shows the surface roughness profile of various parameters [6]. Asilturk and Akkus [7] minimized surface roughness by optimizing the turning parameter using Taguchi’s FME Transactions (2020) 48, 383-390 383 FME Transactions (2020) 48, 383-390 383 face roughness and found that cutting speed is the most significant parameter responsible for surface roughness. face roughness and found that cutting speed is the most significant parameter responsible for surface roughness. From the literature review, it was identified that, most of the research works were centered towards optimization of average surface roughness (Ra) and not on the other surface roughness parameters such as Ry, Rt, etc, which is targeted through the experimental investigation. In this work the dimensional accuracy and surface integrity of low alloy steel used in shafts is analyzed for better fit and tolerances through analysis and optimization using Taguchi’s approach for various surface roughness parameters, which is the novelty of the present study. Apart from this, the influence of machining parameters on the surface roughness conditions were evaluated and the optimized conditions were identified. Figure 1. Surface roughness parameters [6] FME Transactions 2. WORKPIECE AND CUTTING INSERT MATERIAL For analysis, the workpiece material selected is AISI 4340 steel, which is highly ductile, resistance to shock, wear resistance and having high tensile strength used in construction of aircrafts and heavy vehicle crankshaft, gear shaft, camshaft and propeller shaft etc. The ele- mental composition of the workpiece is shown in Table 1. Table 1. Chemical composition of AISI 4340 steel Table 1. Chemical composition of AISI 4340 steel Sl. No Elements Present Alloying % 1 Carbon 0.372 2 Silicon 0.278 3 Manganese 0.570 4 Phosphorus 0.030 5 Sulphur 0.026 6 Chromium 1.106 7 Molybdenum 0.320 8 Nickel 1.467 9 Aluminum 0.023 10 Copper 0.14 11 Niobium 0.064 12 Vanadium 0.033 13 Iron 95.571 The chemical composition of the sample is in agre- ement with the specified value as per B.S. 970 grade EN- 24. The Hardness is 217 BHN. The SEM image of the workpiece material is shown in Fig. 2. The microstructure shows fine pearlite grains in a matrix of ferrite, with fine dispersion of the chromium carbides. The size of the grains shows that the material is in mild annealed con- dition and the hardness measured is in agreement with it. p Singh and Rao [23] determined the effects of cutting conditions and tool geometry on the surface roughness in the finish hard turning of AISI 52100 bearing steel using mixed ceramic inserts made up of aluminium oxide and titanium carbonitride and found that feed is the dominant factor determining the surface finish fol- lowed by nose radius and cutting velocity. Chavoshi and Tajdari [24] studied the influence of hardness (H) and spindle speed (N) on surface roughness (Ra) in hard turning operation of AISI 4140 using CBN cutting tool has been studied and predicted it using ANN and regre- ssion models. Senthilkumar and Tamizharasan [25] pre- dicted flank wear and surface roughness using artificial neural network model, based on the experiments per- formed using Taguchi’s DoE for different tool geo- metries and machining parameters during turning practically used automobile axles. Cakir et al. [26] examined the effects of cutting parameters onto the surface roughness during turning AISI P20 steel with CNMG 120408 carbide inserts having completely the same geometry and substrate but different coating layers through mathematical models. Figure 2. SEM image of AISI 4340 steel Figure 2. SEM image of AISI 4340 steel 384 ▪ VOL. 3.1 Taguchi’s Technique In optimization, Taguchi’s tool is a powerful technique, which applies a special design of Orthogonal Array (OA), used to examine the output characteristics through lesser number of experiments [27-31]. For evaluating the outputs, experimental results determined from the ortho- gonal array are transformed into Signal-to-Noise (S/N) ratios [32-33]. Considering 3 parameters varied through 3 levels in this research, Taguchi’s Design of Experiments (DoE) is applied. The control parameters and their levels chosen [34] are shown in Table 3. Smaller-the-better (Minimize): Smaller-the-better (Minimize): 2 1 1 / 10log n i i S N y n = ⎛ ⎞ = − ⎜ ⎟ ⎜ ⎟ ⎝ ⎠ ∑ (1) (1) Larger-the-better (Maximize): 2 1 1 1 / 10log n i i S N n y = ⎛ ⎞ = − ⎜ ⎟ ⎜ ⎟ ⎝ ⎠ ∑ (2) (2) Figure 3. SEM image of cutting insert Nominal-the-best: 2 / 10log y y S N s ⎛ ⎞ ⎜ ⎟ = ⎜ ⎟ ⎝ ⎠ (3) 3. METHODOLOGY USED (3) 3.2 Analysis of Variance A statistical tool for evaluating the difference between the available set of scores are Analysis of variance (ANOVA), which is applied to data set for dividing the total variance measured into sources [39]. Depending upon the type of analysis, it may be important to deter- mine which factors have a significant effect on the res- ponse, and how much of the variability in the response variable is attributable to each factor [40-41]. For deter- mining the significant factor, which contributes more, ANOVA is done [42]. The contribution of each para- meter is determined by means of ANOVA. In parametric optimization, least number of expe- riments identified is calculated as, Minimum expe- riments = [(L-1) x P] + 1 = [(3-1) x 3] + 1 = 7 ≈ L9 [35- 37]. The various combinations of feed rate, cutting speed and depth of cut based on which the experiments were to be conducted is presented in Table 4. 2. WORKPIECE AND CUTTING INSERT MATERIAL 48, No 2, 2020 FME Transactions FME Transactions The cutting tool insert used in this study is uncoated cemented carbide insert, whose ISO designation is CNMG 120404, whose hardness is 1433 BHN. The cutting tool insert chemical composition is given in Table. 2. will be robust against the noise parameters [38]. For Smaller-the-better analysis, the obtained outputs are usually an undesired output and for Larger-the-better category, output responses are usually a desired one and for Nominal-the-best category, the quality characte- ristics are usually a nominal output. Table 2. Chemical composition of cutting insert Sl. No Elements/compound Alloying % 1 Tungsten carbide 96.4 2 Titanium carbide 0.5 3 Tantalum carbide 0.8 4 Cobalt 2.19 Table 2. Chemical composition of cutting insert Table 3. Control Parameters and their Levels Parameter / Level Symbol Level I Level 2 Level 3 Cutting Speed (m/min) A 136 122 108 Feed Rate (mm/rev) B 0.203 0.330 0.432 Depth of Cut (mm) C 0.1 0.2 0.3 Table 3. Control Parameters and their Levels The SEM photomicrograph of the tungsten carbide cutting tool insert is shown in Fig. 3. The SEM image of the cutting insert shows tungsten carbide (WC) particles which are predominant with titanium carbide (TiC). The structure is variable composition of solid solution phases of WC and TiC. Table 4. Inner Array of Taguchi L9 Orthogonal Array Table 4. Inner Array of Taguchi L9 Orthogonal Array Sl. No Cutting Speed (m/min) Feed Rate (mm/rev) Depth of Cut (mm) 1 136 0.203 0.1 2 136 0.330 0.2 3 136 0.432 0.3 4 122 0.203 0.2 5 122 0.330 0.3 6 122 0.432 0.1 7 108 0.203 0.3 8 108 0.330 0.1 9 108 0.432 0.2 Figure 3. SEM image of cutting insert Smaller-the-better (Minimize): 3.3 Experimental Setup The experiments are conducted on a CNC Turning centre, Lokesh make 2 axes CNC TL-20 lathe. After performing the machining process, the surface rough- ness values of the machined surfaces are recorded by For analyzing the output responses, three categories were available, (i.e.) Smaller-the-better, Larger-the-bet- ter and Nominal-the-better to determine the S/N ratio in Taguchi’s technique. For larger S/N ratio, the process VOL. 48, No 20, 2020 ▪ 385 FME Transactions Figure 5. Area graph of surface roughness parameters using Surfcorder SE3500. Its specification is, measuring range of Z: 600 μm X: 100 mm, measuring magnifi- cation of Z: 50-500,000 X: 1-5,000, measuring speed of 0.05-2 mm/s, Z traverse range of 250 mm. Fig. 4 shows the machining operation performed, along with cutting insert and insert holder. Specification of cutting insert holder used is WIDAX D 9K, CCLNL 25 25 M12. Figure 4. Experimental setup with cutting insert and tool holder Figure 5. Area graph of surface roughness parameters Initially, Ry value decreases by 12.5%, when cutting speed is increased from 108 m/min to 122 m/min and then it increases by 2.3% when cutting speed is increa- sed to 136 m/min. when feed rate is increased, Ry value increases initially by 24.9% and then by 36%. When depth of cut increases from 0.1 to 0.2, Ry decreases by 21.6% and then by 17.2% when depth of cut is 0.3 mm. 17.6% decrease in Rt value is obtained when cutting speed is increased from 108 m/min to 122 m/min and 5.2% decrease when cutting speed is increased from 122 m/min to 136 m/min. When feed rate is increased from 0.203 mm/rev to 0.33 mm/rev and then to 0.432 mm/ rev, an increase in Rt value by 24.9% and 38% is observed. A decrease in Rt value by 19.5% and then by 26.6% is observed when depth of cut is increased from 0.1 mm to 0.3 mm. Figure 4. Experimental setup with cutting insert and tool holder For this analysis, the surface roughness values have to be lower for a given set of input parameters. Hence, the Smaller-the-better condition is selected as given in Equ. 1. From the combined S/N ratio determined, average values are calculated for each level of cutting speed, feed rate and depth of cut, which is given in Table 6. From the response table, main effects plot is drawn as shown in Fig. 6. 3.3 Experimental Setup Based on this, the optimum machining parameters determined are cutting speed of 122 m/min, feed rate of 0.203 mm/rev and depth of cut of 0.3 mm (A2B1C3). 4. RESULTS AND DISCUSSION Based on the L9 orthogonal array designed using Taguchi’s DoE, experiments are conducted. In this study, 9 different workpieces are taken and for each experiment a separate workpiece is used. After perfor- ming the operation, the surface roughness is measured with a Surfcorder. The various surface roughness values measured are given in the Table 5, along with the combined S/N ratio [43]. Table 6. Surface roughness values measured Table 6. Surface roughness values measured Level / Parameter Cutting Speed Feed Rate Depth of Cut 1 -21.91 -19.27 -22.75 2 -20.63 -21.80 -20.89 3 -21.97 -23.45 -20.88 Max – Min 1.34 4.18 1.87 Fig. 5 shows the area graph for surface roughness values determined. It is observed that all the surface roughness values Ra, Ry and Rt increases or decreases in the same trend [44]. When the cutting speed is increased from 108 m/min to 122 m/min, Ra value decreases by 13.3% and when increased to 136 m/min, it further decreases by 35.5%. When feed rate is increased from 0.203 mm/rev, the Ra value increases by 50.9% and then by 65.7%. When depth of cut is increased from 0.1 mm to 0.2 mm, Ra value decreases by 16.3% and when it is further increased to 0.3 mm, Ra increases by 0.8%. FME Transactions Figure 6. Main effects plot for combined S/N ratio Table 5. Surface roughness values measured Table 5. Surface roughness values measured Sl. No Ra Ry Rt Combined S/N Ratio 1 2.46 13.39 14.89 -21.3253 2 2.34 13.38 14.87 -21.3099 3 3.71 16.75 18.58 -23.2875 4 1.43 8.39 10.35 -17.7709 5 2.44 12.73 13.39 -20.6358 6 3.37 16.67 19.50 -23.4863 7 2.18 10.01 10.87 -18.7140 8 2.57 16.23 19.83 -23.4455 9 3.45 16.29 20.16 -23.5774 Figure 6. Main effects plot for combined S/N ratio 386 ▪ VOL. 48, No 2, 2020 FME Transactions FME Transactions observed that a higher level of interaction effect exists in between the cutting speed and depth of cut. 3.4 Interaction Effect of Machining Parameters Interaction plot is drawn to study the combining influ- ence of chosen input parameters [45]. Fig. 7 shows the interaction plot for Ra surface roughness value. No interaction effect is visualized between cutting speed and feed rate. A higher correlation effect is observed between cutting speed and depth of cut. For a cutting speed of 108 m/min and for a depth of cut of 0.3 mm, lower Ra value is obtained. In between feed rate and depth of cut, except for a feed rate of 0.203 mm/rev, no interaction effect is seen. Figure 9. Interaction plot for Rt Figure 7. Interaction plot for Ra Figure 9. Interaction plot for Rt Figure 9. Interaction plot for Rt 3.5 Confirmation Experiment Based on the optimum machining parameters deter- mined, experiment is conducted and the surface roughness values are measured. The surface roughness parameter values obtained are, Ra-1.58 µm, Ry-9.57 µm and Rt-12.06 µm. The output results obtained shows that the optimum machining parameters produces a lower surface roughness values than the experiments conducted based on orthogonal array by 31.63%. Fig. 10 shows the surface roughness profile obtained for the validation experiment based on the optimum condition reached. It shows the P-profile and F-profile of the surface roughness values obtained. Figure 7. Interaction plot for Ra Fig. 8 shows the interaction effect for Ry values. A lower interaction effect is observed between cutting speed and feed rate for a cutting speed of 108 m/min than the other cutting speeds. Higher interaction effect is seen between cutting speed and depth of cut, and for a cutting speed of 122 m/min, the effect is higher. No significant interaction effect is seen between feed rate and depth of cut and for a feed rate of 0.203 mm/rev, a lower interaction effect is seen. Figure 10. Surface roughness profile of validated experiment ANOVA is performed to determine the most contributing machining parameter using Minitab-18 software and the results obtained are shown in Table. 7. From the ANOVA table, it is observed that the feed rate is the most significant factor that contributes 70.50% of surface roughness in the machined surface. Depth of cut contributes up to 18.54% and the least contributing factor is cutting speed, contributing by 9.15%. Figure 8. Interaction plot for Ry Fig. 9 shows the interaction plot for Rt values of surface roughness. A smaller interaction is seen between cutting speed and feed rate. For a cutting speed of 108 m/min, Rt value is more corresponding to the feed rate of 0.330 mm/rev. A higher interaction effect is observed between cutting speed and depth of cut and for a cutting speed of 122 m/min and for a depth of cut of 0.2 mm, the surface roughness value is lower. No interaction effect is seen between feed rate and depth of cut except for a feed rate of 0.432 mm/rev. From all the interaction plots, it is Figure 8. Interaction plot for Ry Figure 8. Interaction plot for Ry Figure 8. Interaction plot for Ry Fig. 9 shows the interaction plot for Rt values of surface roughness. 3.5 Confirmation Experiment A smaller interaction is seen between cutting speed and feed rate. For a cutting speed of 108 m/min, Rt value is more corresponding to the feed rate of 0.330 mm/rev. Figure 10. Surface roughness profile of validated experiment ANOVA is performed to determine the most contributing machining parameter using Minitab-18 software and the results obtained are shown in Table. 7. From the ANOVA table, it is observed that the feed rate is the most significant factor that contributes 70.50% of surface roughness in the machined surface. Depth of cut contributes up to 18.54% and the least contributing factor is cutting speed, contributing by 9.15%. A higher interaction effect is observed between cutting speed and depth of cut and for a cutting speed of 122 m/min and for a depth of cut of 0.2 mm, the surface roughness value is lower. No interaction effect is seen between feed rate and depth of cut except for a feed rate of 0.432 mm/rev. From all the interaction plots, it is VOL. 48, No 20, 2020 ▪ 387 5. CONCLUSIONS [8] Tamizharasan, T. and Senthilkumar, N.: Analysis of Surface Roughness and Material Removal Rate in Turning using Taguchi’s Technique, In Proceedings of IEEE International Conference on Advances in Engineering, Science and Management, Nagapattinam, India, pp. 231-236, 2012. In this work, Taguchi’s technique is used to optimize the machining parameters such as cutting speed, feed rate and depth of cut. From the analysis, some of the outcomes are, • The optimum condition obtained is cutting speed of 122 m/min, feed rate of 0.203 mm/rev and depth of cut of 0.2 mm. [9] Nalbant, M., Gokkaya, H. and Sur, G.: Application of Taguchi method in the optimization of cutting parameters for surface roughness in turning, Materials & Design, Vol. 28, pp. 1379-1385, 2007. • The most significant parameter responsible for surface roughness values is feed rate, contributing by 74.20%. The least contributing factors are depth of cut and cutting speed, contributing by 14.31% and 9.35% respectively. [10] Ramesh, S., Karunamoorthy, L., Palanikumar, K.: Measurement and analysis of surface roughness in turning of aerospace titanium alloy (gr5), Measu- rement, Vol. 45, pp. 1266-1276, 2012. • It is observed that the interaction effect between cutting speed and depth of cut is higher than the interaction between cutting speed and feed rate and between feed rate and depth of cut. [11] Kopac, J., Bahor, M., Sokovic, M.: Optimal machi- ning parameters for achieving the desired surface roughness in fine turning of cold pre-formed steel workpieces, International Journal of Machine Tools & Manufacture, Vol. 42, pp. 707-716, 2002. • Confirmation experiment shows a reduction in various surface roughness values by 31.63% when compared to the experimental results. The obtained surface roughness parameter values are Ra-1.58 μm, Ry-9.57 μm and Rt-12.06 μm. [12] Asilturk, I., Neseli, S.: Multi response optimisation of CNC turning parameters via Taguchi method- based response surface analysis, Measurement, Vol. 45, pp. 785-794, 2012. FME Transactions FME Transactions Table 7. ANOVA for Combined S/N ratio Table 7. ANOVA for Combined S/N ratio Source DoF Seq. SS Adj. MS F P % Contribution Cutting speed 2 3.4500 1.725 5.03 0.166 9.15% Feed rate 2 26.5944 13.2972 38.78 0.025 70.50% Depth of cut 2 6.9924 3.4962 10.20 0.089 18.54% Residual error 2 0.6858 0.3429 - - 1.82% Total 8 37.7226 - - - 100% REFERENCES [13] Benardos, P. G. and Vosniakos, G. C.: Predicting surface roughness in machining: a review, International Journal of Machine Tools & Manufacture, Vol. 43, pp. 833-844, 2003. [1] Groover, M. P.: Fundamentals of Modern Manu- facturing - Materials, Processes and Systems, John Wiley & Sons, Inc., USA, 2010. [14] Sahin, Y., Motorcu, A. 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[41] Gajalakshmi, K., Senthilkumar, N. and Prabu, B.: Multi-response Optimization of Dry Sliding Wear Parameters of AA6026 Using Hybrid Grey Relational Analysis Coupled with Response Surface Method, Measurement and Control, Vol. 52, No. 5-6, pp. 540-553, 2019. [29] Senthilkumar, N. and Tamizharasan, T.: Impact of Interface Temperature over Flank Wear in Hard Turning using Carbide Inserts, Procedia Engineering, Vol. 38, pp. 613-621, 2012. [42] Senthilkumar, N., Sudha, J. and Muthukumar, V.: A grey-fuzzy approach for optimizing machining parameters and the approach angle in turning AISI 1045 steel, Advances in Production Engineering & Management, Vol. 10, No. 4, pp. 195-208, 2015. [30] Ross, P. J.: Taguchi Techniques for Quality Engineering, Tata McGraw Hill publishing company Ltd., New Delhi, 2005. [31] Marichamy, S., Ravichandran, M., Stalin, B. and Babu, B. S.: Optimization of Abrasive Water Jet Machining Parameters for α-β brass using Taguchi [43] Senthilkumar, N. and Tamizharasan, T.: Effect of Tool geometry in Turning AISI 1045 steel: VOL. 48, No 20, 2020 ▪ 389 FME Transactions Experimental Investigation and FEM analysis, Arabian Journal for Science and Engineering, Vol. 39, No. 6, pp. 4963-4975, 2014. 390 ▪ VOL. 48, No 2, 2020 Б. Депанраџ, Л.А. Раман, Н. Сентилкумар, Ј. Шивансанкар Рад се бави експерименталним истраживањем утицаја параметара обраде као што је брзина резања, брзина помоћног кретања и дубина резања на параметре храпавости површине приликом обраде на стругу AISI 4340 челика. [44] Suhail, A. H. et al.: Surface Roughness Identification Using the Grey Relational Analysis with Multiple Performance Characteristics in Turning Operations, Arabian Journal for Science and Engineering, Vol. 37, No. 4, pp.1111-1117, 2012. Да би се добиле мање вредности параметара храпа- вости површине, параметри обраде су оптимизирани применом Тагучијеве технике односа сигнал-бука. АНОВА је коришћена да би се утврдило који фактор највише утиче на параметре храпавости. Утврђено је да фактор брзина помоћног кретања има највећи утицај (70,50%), дубина резања (18,54%) и брзина резања (9,15%). [45] Senthilkumar, N., Selvakumar, V. and Tamizharasan, T.: Optimization and Performance Analysis of Uncoated and Coated Carbide Inserts during Hard Turning AISI D2 Steel Using Hybrid GRA-PCA Technique, Applied Mechanics and Materials, Vol. 852, pp 151-159, 2016. На основу добијеног стања извршен је експеримент чији резултати показују да су вредности параметара храпавости површине редуковане за 31,63% у поређењу са вредностима пројектованим експери- ментом. ИСТРАЖИВАЊЕ И ОПТИМИЗАЦИЈА УТИЦАЈА ПАРАМЕТАРА ОБРАДЕ НА ХРАПАВОСТ ПОВРШИНЕ КОД ОБРАДЕ АISI 4340 ЧЕЛИКА 390 ▪ VOL. 48, No 2, 2020 FME Transactions FME Transactions
https://openalex.org/W4388699133	https://www.mdpi.com/1422-0067/24/22/16376/pdf?version=1700056247	English	null	Comparison of the Effects of Recombinant and Native Prolactin on the Proliferation and Apoptosis of Goose Granulosa Cells	International journal of molecular sciences	2,023	cc-by	11,915	Article Comparison of the Effects of Recombinant and Native Prolactin on the Proliferation and Apoptosis of Goose Granulosa Cells Donghang Deng 1,2,3,†, Wen Li 2,3,†, Xiaopeng Li 1,2,3, Xin Yuan 2,3 , Liang Li 1,2,3, Jiwen Wang 1,2,3, Chunchun Han 1,2,3,* and Shenqiang Hu 1,2,3,* 1 State Key Laboratory of Swine and Poultry Breeding Industry, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130, China; dengdh94@163.com (D.D.); 1 State Key Laboratory of Swine and Poultry Breeding Industry, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130, China; dengdh94@163.com (D.D.); lixp0715@163.com (X.L.); cnliliang@foxmail.com (L.L.); wjw2886166@163.com (J.W.) p ( ) g ( ) j (J ) 2 Key Laboratory of Livestock and Poultry Multi-omics, Ministry of Agriculture and Rural Affairs, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130, China; 15520739895@163.com (W.L.); nihaoyuanxin88@outlook.com (X.Y.) y 3 Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, China g y g * Correspondence: 13839@sicau.edu.cn (C.H.); shenqiang.hu@sicau.edu.cn (S.H.) * Correspondence: 13839@sicau.edu.cn (C.H.); shenqi † These authors contributed equally to this work. Abstract: In poultry, prolactin (PRL) plays a key role in the regulation of incubation behavior, hormone secretion, and reproductive activities. However, previous in vitro studies have focused on the actions of PRL in ovarian follicles of poultry, relying on the use of exogenous or recombinant PRL, and the true role of PRL in regulating ovarian granulosa cell (GC) functions in poultry awaits a further investigation using endogenous native PRL. Therefore, in this study, we ﬁrst isolated and puriﬁed recombinant goose PRL protein (rPRL) and native goose PRL protein (nPRL) using Ni-afﬁnity chromatography and rabbit anti-rPRL antibodies-ﬁlled immunoafﬁnity chromatography, respectively. Then, we analyzed and compared the effects of rPRL and nPRL at different concentrations (0, 3, 30, or 300 ng/mL) on the proliferation and apoptosis of both GCs isolated from goose ovarian pre-hierarchical follicles (phGCs) and from hierarchical follicles (hGCs). Our results show that rPRL at lower concentrations increased the viability and proliferation of both phGCs and hGCs, while it exerted anti-apoptotic effects in phGCs by upregulating the expression of Bcl-2. On the other hand, nPRL increased the apoptosis of phGCs in a concentration-dependent manner by upregulating the expressions of caspase-3 and Fas and downregulating the expressions of Bcl-2 and Becn-1. Citation: Deng, D.; Li, W.; Li, X.; Yuan, X.; Li, L.; Wang, J.; Han, C.; Hu, S. Comparison of the Effects of Recombinant and Native Prolactin on the Proliferation and Apoptosis of Goose Granulosa Cells. Int. J. Mol. Sci. 2023, 24, 16376. https:// doi.org/10.3390/ijms242216376 Academic Editor: Honoo Satake Keywords: goose; prolactin; protein puriﬁcation; follicular development; proliferation; apoptosis International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences Article Comparison of the Effects of Recombinant and Native Prolactin on the Proliferation and Apoptosis of Goose Granulosa Cells In conclusion, this study not only obtained a highly pure nPRL for the ﬁrst time, but also suggested a dual role of PRL in regulating the proliferation and apoptosis of goose GCs, depending on its concentration and the stage of follicle development. The data presented here can be helpful in purifying native proteins of poultry and enabling a better understanding of the roles of PRL during the ovarian follicle development in poultry. Citation: Deng, D.; Li, W.; Li, X.; Yuan, X.; Li, L.; Wang, J.; Han, C.; Hu, S. Comparison of the Effects of Recombinant and Native Prolactin on the Proliferation and Apoptosis of Goose Granulosa Cells. Int. J. Mol. Sci. 2023, 24, 16376. https:// doi.org/10.3390/ijms242216376 The orderly and progressive growth and development of ovarian follicles is the basis of egg production in poultry. These follicles, at different stages of development within the ovary of a goose during the egg-laying period, are generally categorized into the pre- hierarchical (<10 mm in diameter) and hierarchical (also called preovulatory, i.e., F6–F1) follicles [1]. Each follicle is mainly composed of three types of cells, including oocytes, granulosa cells (GCs), and theca cells. Among them, the GCs form the layers surrounding the oocytes, and are mainly responsible for synthesizing and secreting hormones and cytokines and providing nutrients for oocyte maturation and follicular development [2]. In addition, previous studies have indicated that the proliferation, differentiation, apoptosis, 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/). https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2023, 24, 16376. https://doi.org/10.3390/ijms242216376 Int. J. Mol. Sci. 2023, 24, 16376 2 of 15 and autophagy of GCs play important roles in regulating ovarian follicular development, selection, and atresia [3,4]. and autophagy of GCs play important roles in regulating ovarian follicular development, selection, and atresia [3,4]. [ ] Prolactin (PRL), known as a polypeptide hormone and mainly synthesized and se- creted by lactotroph cells in the anterior pituitary gland, plays a crucial role in regulating female reproductive functions such as incubation behavior, hormone secretion, and fol- licular development in poultry [5,6]. It is widely accepted that the seasonal changes in breeding activity of both wild and domestic birds are attributed to the alternating secre- tions of luteinizing hormone (LH) and PRL, with plasma PRL levels reaching their peak, while LH levels decline sharply during the broodiness period [7]. Many studies have also demonstrated that the elevated level of plasma PRL is the main factor that induces an incubation behavior in poultry [8]. The inﬂuence of PRL on avian seasonal reproductive activities depends on its plasma levels, which are strongly regulated by a photoperiod [9]. It has been reported that, following immunization with recombinant PRL, a signiﬁcant reduction in incubation behavior and altered egg production performance are observed in both turkeys and chickens [10,11]. Similarly, the number of large white follicles in a hen ovary was observed to be higher in PRL-immunized groups than in BSA-immunized ones, suggesting that PRL plays an important role in regulating the ovarian follicular develop- ment of poultry [11,12]. Abundant evidence supports the indirect and direct actions of PRL on the hypothalamic–hypophyseal–ovarian axis that regulates the incubation behavior and ovarian activities of poultry [6,9,13]. Furthermore, several recent in vitro studies using exogenous or recombinant PRL have demonstrated that PRL can act directly on both basal and gonadotrophin-mediated follicular cell steroidogeneses, depending on its concentra- tion, the follicular size, and the stage of the ovulation cycle in chickens [14,15]. Additionally, lower concentrations of recombinant PRL promoted in vitro oocyte maturation and early embryonic development in mice [16], while the mice lacking PRL or PRL receptors exhibited complete infertility and abnormal ovarian follicular development [17,18]. Taken together, these results indicate that PRL has a dual role in either promoting or inhibiting ovarian follicular development, which is supposed to depend on its concentrations and the poultry species involved. p However, considering that previous studies on the actions and mechanisms of PRL in avian ovarian follicular cells rely on the use of exogenous or recombinant PRL rather than endogenous native PRL, deciphering its true roles awaits a further investigation. Moreover, compared to chickens and ducks, domestic geese generally show a stronger reproductive seasonality and, consequently, a lower egg production performance, which is assumed to be associated with the species/breed-speciﬁc pituitary secretions of PRL in response to photoperiodic changes [19–24]. Therefore, it is important to reveal the true role of PRL in regulating goose ovarian cell functions. In the present study, we ﬁrstly generated and puriﬁed the polyclonal antibodies against the recombinant goose PRL (rPRL). Subsequently, we constructed an immunoafﬁnity chromatography (IAC) column to isolate and purify the endogenous native PRL (nPRL) from geese pituitary glands. Finally, we compared the effects of rPRL and nPRL at different concentrations on the proliferation and apoptosis of the GCs isolated from goose ovarian pre-hierarchical follicles (phGCs) and hierarchical follicles (hGCs). These data will not only provide a better understanding of the role of PRL during avian ovarian follicular development, but also be helpful for controlling reproductive seasonality in practical goose production. 2.2. Puriﬁcation and Identiﬁcation of Goose Recombinant PRL Protein and Its Antibody 2.2. Puriﬁcation and Identiﬁcation of Goose Recombinant PRL Protein and Its Antibody The prokaryotic expression products of the pET-28a-PRL were obtained fro The prokaryotic expression products of the pET-28a-PRL were obtained from the soluble fraction in E. coli BL21 (DE3) using diﬀerent optimization conditions (Supplementary Figure S1). The obtained supernatants were puriﬁed using Ni-aﬃnity chromatography, and the results are shown in Figure 2A. The rPRL protein was collected after being washed with diﬀerent concentrations of eluent buﬀer. The band of the recombinant protein appeared close to 25 kDa, which corresponds to the estimated molecular mass of the goose mature PRL. Then, the rPRL protein was characterized by quadrupole time-of-ﬂight tandem mass spectrometry (Q-TOF-MS) and subsequent sequencing, and was veriﬁed to be the goose recombinant PRL protein (Figure 2B,C). These results demonstrate that the rPRL was successfully obtained in this study. In addition, the rabbit-anti-rPRL antibody, with a molecular mass of approximately 50 kDa, was puriﬁed using self-made rPRL protein-coupled aﬃnity chromatography, as shown The prokaryotic expression products of the pET-28a-PRL were obtained from the solu- ble fraction in E. coli BL21 (DE3) using different optimization conditions (Supplementary Figure S1). The obtained supernatants were puriﬁed using Ni-afﬁnity chromatography, and the results are shown in Figure 2A. The rPRL protein was collected after being washed with different concentrations of eluent buffer. The band of the recombinant protein appeared close to 25 kDa, which corresponds to the estimated molecular mass of the goose mature PRL. Then, the rPRL protein was characterized by quadrupole time-of-ﬂight tandem mass spectrometry (Q-TOF-MS) and subsequent sequencing, and was veriﬁed to be the goose recombinant PRL protein (Figure 2B,C). These results demonstrate that the rPRL was suc- cessfully obtained in this study. In addition, the rabbit-anti-rPRL antibody, with a molecular mass of approximately 50 kDa, was puriﬁed using self-made rPRL protein-coupled afﬁnity chromatography, as shown in Figure 2D. soluble fraction in E. coli BL21 (DE3) using diﬀerent optimization conditions (Supplementary Figure S1). The obtained supernatants were puriﬁed using Ni-aﬃnity chromatography, and the results are shown in Figure 2A. The rPRL protein was collected after being washed with diﬀerent concentrations of eluent buﬀer. The band of the recombinant protein appeared close to 25 kDa, which corresponds to the estimated molecular mass of the goose mature PRL. Then, the rPRL protein was characterized by quadrupole time-of-ﬂight tandem mass spectrometry (Q-TOF-MS) and subsequent sequencing, and was veriﬁed to be the goose recombinant PRL protein (Figure 2B,C). These results demonstrate that the rPRL was successfully obtained in this study. 2.1. Ampliﬁcation of PRL Gene and Construction of Recombinant pET-28a-PRL 2.1. Ampliﬁcation of PRL Gene and Construction of Recombinant pET-28a-PRL The genomic DNA extracted from the pituitary gland of Sichuan White Goose served as the template for PCR ampliﬁcation. The cDNA encoding the mature peptide of goose PRL, with an approximate length of 600 bp, was successfully obtained using RT-PCR, and was further validated with sequencing (Figure 1A). The recombinant plasmid pET-28a-PRL was successfully synthesized by T4 ligase after cleavage of PRL and pET-28a fragments Int. J. Mol. Sci. 2023, 24, 16376 3 of 15 using bi t g binant 3 of 15 using bi t g binant using restriction endonucleases Nco I and Xho I, and was validated by restriction enzymes digestion, as showed in Figure 1B. and pET-28a fragments using restriction endonucleases Nco I and Xho I, and was validated by restriction enzymes digestion, as showed in Figure 1B. p g g validated by restriction enzymes digestion, as showed in Figure 1B. using restriction endonucleases Nco I and Xho I, and was validated by restriction enzymes digestion, as showed in Figure 1B. and pET-28a fragments using restriction endonucleases Nco I and Xho I, and was validated by restriction enzymes digestion, as showed in Figure 1B. validated by restriction enzymes digestion, as showed in Figure 1B. Figure 1. Ampliﬁcation of PRL gene and construction of the recombinant goose pET-28a-PRL. (A) The PCR products of the cDNAs encoding goose mature PRL were visualized on an agarose gel. Lanes 1–3, the fragments of the PCR products of PRL. (B) Double enzyme digestion veriﬁcation of the recombinant plasmid pET-28a-PRL plasmid. Lane 1: recombinant plasmid pET-28a-PRL. Lane 2, the double digestion products of recombinant plasmid pET-28a-PRL by digestion with the Nco I and Xho I restriction enzymes. M, DNA marker. Figure 1. Ampliﬁcation of PRL gene and construction of the recombinant goose pET-28a-PRL. (A) The PCR products of the cDNAs encoding goose mature PRL were visualized on an agarose gel. Lanes 1–3, the fragments of the PCR products of PRL. (B) Double enzyme digestion veriﬁcation of the recombinant plasmid pET-28a-PRL plasmid. Lane 1: recombinant plasmid pET-28a-PRL. Lane 2, the double digestion products of recombinant plasmid pET-28a-PRL by digestion with the Nco I and Xho I restriction enzymes. M, DNA marker. Figure 1. Ampliﬁcation of PRL gene and construction of the recombinant goose pET-28a-PRL. (A) The PCR products of the cDNAs encoding goose mature PRL were visualized on an agarose gel. 2.1. Ampliﬁcation of PRL Gene and Construction of Recombinant pET-28a-PRL Lanes 1–3, the fragments of the PCR products of PRL. (B) Double enzyme digestion veriﬁcation of the recombinant plasmid pET-28a-PRL plasmid. Lane 1: recombinant plasmid pET-28a-PRL. Lane 2, the double digestion products of recombinant plasmid pET-28a-PRL by digestion with the Nco I and Xho I restriction enzymes. M, DNA marker. 2.2. Puriﬁcation and Identiﬁcation of Goose Recombinant PRL Protein and Its Antibody Figure 1. Ampliﬁcation of PRL gene construction of the recombinant goose pET-28a-P Figure 1. Ampliﬁcation of PRL gene and construction of the recombinant goose pET-28a-PRL. (A) The PCR products of the cDNAs encoding goose mature PRL were visualized on an agarose gel. Lanes 1–3, the fragments of the PCR products of PRL. (B) Double enzyme digestion veriﬁcation of the recombinant plasmid pET-28a-PRL plasmid. Lane 1: recombinant plasmid pET-28a-PRL. Lane 2, the double digestion products of recombinant plasmid pET-28a-PRL by digestion with the Nco I and Xho I restriction enzymes. M, DNA marker. Figure 1. Ampliﬁcation of PRL gene and construction of the recombinant goose pET-28a-PRL. (A) The PCR products of the cDNAs encoding goose mature PRL were visualized on an agarose gel. Lanes 1–3, the fragments of the PCR products of PRL. (B) Double enzyme digestion veriﬁcation of the recombinant plasmid pET-28a-PRL plasmid. Lane 1: recombinant plasmid pET-28a-PRL. Lane 2, the double digestion products of recombinant plasmid pET-28a-PRL by digestion with the Nco I and Xho I restriction enzymes. M, DNA marker. The PCR products of the cDNAs encoding goose mature PRL were visualized on an agarose gel. Lanes 1–3, the fragments of the PCR products of PRL. (B) Double enzyme digestion veriﬁcation of the recombinant plasmid pET-28a-PRL plasmid. Lane 1: recombinant plasmid pET-28a-PRL. Lane 2, the double digestion products of recombinant plasmid pET-28a-PRL by digestion with the Nco I and Xho I restriction enzymes. M, DNA marker. 2.2. Puriﬁcation and Identiﬁcation of Goose Recombinant PRL Protein and Its Antibody 2.2. Puriﬁcation and Identiﬁcation of Goose Recombinant PRL Protein and Its Antibody 2.2. Puriﬁcation and Identiﬁcation of Goose Recombinant PRL Protein and Its Antibody The prokaryotic expression products of the pET-28a-PRL were obtained fro 2.2. Puriﬁcation and Identiﬁcation of Goose Recombinant PRL Protein and Its Antibody 2.2. Puriﬁcation and Identiﬁcation of Goose Recombinant PRL Protein and Its Antibody The prokaryotic expression products of the pET-28a-PRL were obtained fro In addition, the rabbit-anti-rPRL antibody, with a molecular mass of approximately 50 kDa, was puriﬁed using self-made rPRL protein-coupled aﬃnity chromatography, as shown in Figure 2D. in Figure 2D. Figure 2. Puriﬁcation and identiﬁcation of goose recombinant PRL protein (rPRL) and the rabbit- anti-rPRL antibodies. (A) The SDS-PAGE results showing the rPRL solutions puriﬁed by Ni afﬁnity chromatography. M, protein marker; Lane 1, the supernatant after lysis of pET-28a-PRL-BL21; Lane 2, n Figure 2D. in Figure 2D. Figure 2. Puriﬁcation and identiﬁcation of goose recombinant PRL protein (rPRL) and the rabbit- anti-rPRL antibodies. (A) The SDS-PAGE results showing the rPRL solutions puriﬁed by Ni afﬁnity chromatography. M, protein marker; Lane 1, the supernatant after lysis of pET-28a-PRL-BL21; Lane 2, Int. J. Mol. Sci. 2023, 24, 16376 4 of 15 rrow ref d b Q the sample of the supernatant after ﬂowing through the Ni column; Lane 3–8, the sample after cleaning the Ni column with eluent buffer. The red arrow refers to the band of rPRL. (B) Peptide score distribution of the recombinant protein analyzed by Q-TOF-MS. (C) Sequence coverage of the peptides characterized by Q-TOF-MS. These matched peptides are shown in bold green when compared with the goose mature PRL. (D) SDS-PAGE results of the rabbit-anti-rPRL sera before and after being puriﬁed using the rPRL protein-coupled afﬁnity chromatography. Lane 1, the diluted rabbit-anti-PRL sera; Lanes 2–6, the solutions puriﬁed from the diluted rabbit-anti-rPRL sera using the Tris-HCl elution buffer. The red arrow refers to the band of the rabbit-anti-rPRL antibody. shown in bold green when compared with the goose mature PRL. (D) SDS-PAGE resu rabbit-anti-rPRL sera before and after being puriﬁed using the rPRL protein-couple chromatography. Lane 1, the diluted rabbit-anti-PRL sera; Lanes 2–6, the solutions puri the diluted rabbit-anti-rPRL sera using the Tris-HCl elution buﬀer. The red arrow ref band of the rabbit-anti-rPRL antibody. 2.3. Puriﬁcation and Identiﬁcation of Goose Native PRL Protein The total protein extracted from the pituitary glands of Sichuan White G 2.3. Puriﬁcation and Identiﬁcation of Goose Native PRL Protein lysed with tissue protein lysis buﬀer, followed The total protein extracted from the pituitary glands of Sichuan White Geese was lysed with tissue protein lysis buffer, followed by incubation on ice with an immunoafﬁnity chromatography column constructed using NHS-Activated beads and rabbit-anti-rPRL anti- bodies. These antibodies can speciﬁcally bind to the nPRL, allowing for a collection of native PRL proteins through the utilization of a low-pH eluent buffer (Figure 3A). Subsequently, the immunoreactivity of nPRL was tested using Western blot, with a rabbit-anti-rPRL antibody being the primary antibody (Figure 3B), resulting in two immunoreactive bands that appeared at approximately 23 kDa and 26 kDa (corresponding to the non-glycosylated and glycosylated PRL), respectively. These results demonstrate that the goose native PRL protein was successfully obtained in this study. immunoaﬃnity chromatography column constructed using NHS-Activated be rabbit-anti-rPRL antibodies. These antibodies can speciﬁcally bind to the nPRL, for a collection of native PRL proteins through the utilization of a low-pH elue (Figure 3A). Subsequently, the immunoreactivity of nPRL was tested using West with a rabbit-anti-rPRL antibody being the primary antibody (Figure 3B), res two immunoreactive bands that appeared at approximately 23 kDa and (corresponding to the non-glycosylated and glycosylated PRL), respectively results demonstrate that the goose native PRL protein was successfully obtaine study. Figure 3. Puriﬁcation and identiﬁcation of the native PRL protein from Sichuan White G The steps of puriﬁcation. (B) The Western blot results of the goose native PRL protein puriﬁed using a rabbit anti-rPRL antibodies-ﬁlled aﬃnity chromatograph column. Lane goose native PRL protein solutions that were eluted using 0.1 M Glycine at a pH of 3. Figure 3. Puriﬁcation and identiﬁcation of the native PRL protein from Sichuan White Goose. (A) The steps of puriﬁcation. (B) The Western blot results of the goose native PRL protein that was puriﬁed using a rabbit anti-rPRL antibodies-ﬁlled afﬁnity chromatograph column. Lanes 1–3, the goose native PRL protein solutions that were eluted using 0.1 M Glycine at a pH of 3. Figure 3. Puriﬁcation and identiﬁcation of the native PRL protein from Sichuan White G The steps of puriﬁcation. (B) The Western blot results of the goose native PRL protein puriﬁed using a rabbit anti-rPRL antibodies-ﬁlled aﬃnity chromatograph column. Lane goose native PRL protein solutions that were eluted using 0.1 M Glycine at a pH of 3. Figure 3. Puriﬁcation and identiﬁcation of the native PRL protein from Sichuan White Goose. (A) The steps of puriﬁcation. 2.3. Puriﬁcation and Identiﬁcation of Goose Native PRL Protein lysed with tissue protein lysis buﬀer, followed (B) The Western blot results of the goose native PRL protein that was puriﬁed using a rabbit anti-rPRL antibodies-ﬁlled afﬁnity chromatograph column. Lanes 1–3, the goose native PRL protein solutions that were eluted using 0.1 M Glycine at a pH of 3. Figure 3. Puriﬁcation and identiﬁcation of the native PRL protein from Sichuan White G The steps of puriﬁcation. (B) The Western blot results of the goose native PRL protein puriﬁed using a rabbit anti-rPRL antibodies-ﬁlled aﬃnity chromatograph column. Lane goose native PRL protein solutions that were eluted using 0.1 M Glycine at a pH of 3. Figure 3. Puriﬁcation and identiﬁcation of the native PRL protein from Sichuan White Goose. (A) The steps of puriﬁcation. (B) The Western blot results of the goose native PRL protein that was puriﬁed using a rabbit anti-rPRL antibodies-ﬁlled afﬁnity chromatograph column. Lanes 1–3, the goose native PRL protein solutions that were eluted using 0.1 M Glycine at a pH of 3. Int. J. Mol. Sci. 2023, 24, 16376 5 of 15 5 of 15 2.4. Effects of rPRL and nPRL on Goose Pre-Hierarchical and Hierarchical Granulosa Cell Viability hGCs, both cell types were treated with diﬀerent concentrations of eit (0 3 30 or 300 ng/mL) for 24 h The results show that both rPR To investigate the effects of nPRL and rPRL on the viability of goose phGCs and hGCs, both cell types were treated with different concentrations of either nPRL or rPRL (0, 3, 30, or 300 ng/mL) for 24 h. The results show that both rPRL and nPRL at a concentration of 3 or 30 ng/mL signiﬁcantly upregulated the viability of phGCs (p < 0.05), while 300 ng/mL of nPRL and rPRL showed no signiﬁcant effects (Figure 4A). In hGCs, the nPRL exerted a stimulatory effect on cell viability in a concentration-dependent manner, whereas the trend for rPRL in hGCs was consistent with that in phGCs (Figure 4B). (0, 3, 30, or 300 ng/mL) for 24 h. The results show that both rPRL concentration of 3 or 30 ng/mL signiﬁcantly upregulated the viability of while 300 ng/mL of nPRL and rPRL showed no signiﬁcant eﬀects (Figu the nPRL exerted a stimulatory eﬀect on cell viability in a concentr manner, whereas the trend for rPRL in hGCs was consistent with that i 4B). Figure 4. 2.3. Puriﬁcation and Identiﬁcation of Goose Native PRL Protein lysed with tissue protein lysis buﬀer, followed “*” indicates an extremely signiﬁcant di to the control group (p < 0.001), “” indicates an extremely signiﬁcant diﬀerenc control group (p < 0 01) “” indicates a signiﬁcant diﬀerence compared to the Figure 4. Comparison of the effects of rPRL and nPRL, at different concentrations, on the viability of goose phGCs (A) and hGCs (B). The viability of GCs was determined using the CCK-8 assay. Data are presented as the mean ± S.D. “” indicates an extremely signiﬁcant difference compared to the control group (p < 0.001), “” indicates an extremely signiﬁcant difference compared to the control group (p < 0.01), “” indicates a signiﬁcant difference compared to the control group (p < 0.05). of goose phGCs (A) and hGCs (B). The viability of GCs was determined usin Data are presented as the mean ± S.D. “” indicates an extremely signiﬁcant to the control group (p < 0.001), “” indicates an extremely signiﬁcant diﬀeren control group (p < 0 01) “” indicates a signiﬁcant diﬀerence compared to th goose phGCs (A) and hGCs (B). The viability of GCs was determined using the CCK-8 assay. Data are presented as the mean ± S.D. “” indicates an extremely signiﬁcant difference compared to the control group (p < 0.001), “” indicates an extremely signiﬁcant difference compared to the control group (p < 0.01), “” indicates a signiﬁcant difference compared to the control group (p < 0.05). control group (p < 0.01), indicates a signiﬁcant diﬀerence compar 0.05). 2.5. Effects of rPRL and nPRL on Goose Pre-Hierarchical and Hierarchical Granulosa Cell Proliferation control group (p 0.01), indicates a signiﬁcant diﬀerence compar 0.05). 2.5. Effects of rPRL and nPRL on Goose Pre-Hierarchical and Hierarchical Granulosa Cell Proliferation 2.5. Eﬀects of rPRL and nPRL on Goose Pre-Hierarchical and Hierarchical Gra Cell Proliferation To further investigate the eﬀects of nPRL and rPRL on the prolife and hGCs, we conducted the EdU assay. As shown in Figure 5, our r t t t ith 30 / L f PRL i iﬁ tl t d ( 0 05) h To further investigate the effects of nPRL and rPRL on the proliferation of phGCs and hGCs, we conducted the EdU assay. As shown in Figure 5, our results show that treatment with 30 ng/mL of nPRL signiﬁcantly promoted (p < 0.05), while nPRL at 3 or 300 ng/mL did not signiﬁcantly affect (p > 0.05), the proliferation of phGCs. 2.3. Puriﬁcation and Identiﬁcation of Goose Native PRL Protein lysed with tissue protein lysis buﬀer, followed Comparison of the eﬀects of rPRL and nPRL, at diﬀerent concentratio of goose phGCs (A) and hGCs (B). The viability of GCs was determined using Data are presented as the mean ± S.D. “” indicates an extremely signiﬁcant di to the control group (p < 0.001), “” indicates an extremely signiﬁcant diﬀerenc control group (p < 0.01), “” indicates a signiﬁcant diﬀerence compared to the 0.05). Figure 4. Comparison of the effects of rPRL and nPRL, at different concentrations, on the viability of goose phGCs (A) and hGCs (B). The viability of GCs was determined using the CCK-8 assay. Data are presented as the mean ± S.D. “” indicates an extremely signiﬁcant difference compared to the control group (p < 0.001), “” indicates an extremely signiﬁcant difference compared to the control group (p < 0.01), “” indicates a signiﬁcant difference compared to the control group (p < 0.05). 2.5. Effects of rPRL and nPRL on Goose Pre-Hierarchical and Hierarchical Granulosa Cell Proliferation Figure 4. Comparison of the eﬀects of rPRL and nPRL, at diﬀerent concentratio Figure 4. Comparison of the effects of rPRL and nPRL, at different concentrations, on the viability of Figure 4. Comparison of the eﬀects of rPRL and nPRL, at diﬀerent concentratio of goose phGCs (A) and hGCs (B). The viability of GCs was determined using Data are presented as the mean ± S.D. “” indicates an extremely signiﬁcant di to the control group (p < 0.001), “” indicates an extremely signiﬁcant diﬀerenc control group (p < 0 01) “” indicates a signiﬁcant diﬀerence compared to the Figure 4. Comparison of the effects of rPRL and nPRL, at different concentrations, on the viability of goose phGCs (A) and hGCs (B). The viability of GCs was determined using the CCK-8 assay. Data are presented as the mean ± S.D. “” indicates an extremely signiﬁcant difference compared to the control group (p < 0.001), “” indicates an extremely signiﬁcant difference compared to the control group (p < 0.01), “” indicates a signiﬁcant difference compared to the control group (p < 0.05). Figure 4. Comparison of the eﬀects of rPRL and nPRL, at diﬀerent concentratio of goose phGCs (A) and hGCs (B). The viability of GCs was determined using Data are presented as the mean ± S.D. 2.3. Puriﬁcation and Identiﬁcation of Goose Native PRL Protein lysed with tissue protein lysis buﬀer, followed However, in hGCs, compared to the control group, neither rPRL nor nPRL treatments signiﬁcantly changed the cell proliferation ratio (p > 0.05). 6 of 156 Int. J. Mol. Sci. 2023, 24, 16376 Mol. Sci. 2023, 24, x FOR PEER Figure 5. Comparison of the eﬀects of rPRL and nPRL, at diﬀerent concentrations, on proliferation of goose phGCs (A,C) and hGCs (B,D). The cell proliferation was tested using EdU assay. Data are presented as the mean ± SD. “” indicated a signiﬁcant diﬀerence compa to the control group (p < 0.05). Figure 5. Comparison of the effects of rPRL and nPRL, at different concentrations, on the proliferation of goose phGCs (A,C) and hGCs (B,D). The cell proliferation was tested using the EdU assay. Data are presented as the mean ± SD. “” indicated a signiﬁcant difference compared to the control group (p < 0.05). 2 6 Eff f PRL d PRL G P Hi hi l d Hi hi l G l gure 5. Comparison of the eﬀects of rPRL and nPRL, at diﬀerent concentrations, on oliferation of goose phGCs (A,C) and hGCs (B,D). The cell proliferation was tested using dU assay. Data are presented as the mean ± SD. “” indicated a signiﬁcant diﬀerence comp the control group (p < 0.05). Figure 5. Comparison of the effects of rPRL and nPRL, at different concentrations, on the proliferation of goose phGCs (A,C) and hGCs (B,D). The cell proliferation was tested using the EdU assay. Data are presented as the mean ± SD. “” indicated a signiﬁcant difference compared to the control group (p < 0.05). 6. Eﬀects of rPRL and nPRL on Goose Pre-Hierarchical and Hierarchical Granul ll A t i 2.6. Effects of rPRL and nPRL on Goose Pre-Hierarchical and Hierarchical Granulosa Cell Apoptosis 6. Eﬀects of rPRL and nPRL on Goose Pre-Hierarchical and Hierarchical Granu ll A t i 2.6. Effects of rPRL and nPRL on Goose Pre-Hierarchical and Hierarchical Granulosa Cell Apoptosis Cell Apoptosis We further examined the eﬀects of rPRL and nPRL on the apoptosis of goose ph nd hGCs using a ﬂow cytometer. As shown in Figure 6, compared to the control gro ng/mL of nPRL had no signiﬁcant eﬀect on the apoptotic rates of both phGCs GCs. However, as the concentration of nPRL increased, it signiﬁcantly increased poptotic rates of both phGCs (300 ng/mL) and hGCs (30 and 300 ng/mL). In cont ower concentrations of rPRL (3 and 30 ng/mL) signiﬁcantly reduced the apoptotic ra hGCs (p < 0.05), while 300 ng/mL of rPRL showed no signiﬁcant eﬀect. In hGCs, We further examined the effects of rPRL and nPRL on the apoptosis of goose phGCs and hGCs using a ﬂow cytometer. As shown in Figure 6, compared to the control group, 3 ng/mL of nPRL had no signiﬁcant effect on the apoptotic rates of both phGCs and hGCs. However, as the concentration of nPRL increased, it signiﬁcantly increased the apoptotic rates of both phGCs (300 ng/mL) and hGCs (30 and 300 ng/mL). In contrast, lower concentrations of rPRL (3 and 30 ng/mL) signiﬁcantly reduced the apoptotic rate of phGCs (p < 0.05), while 300 ng/mL of rPRL showed no signiﬁcant effect. In hGCs, the apoptotic rate was not signiﬁcantly changed by rPRL at any concentration (p > 0.05). 7 of 157 o 7 of 157 o Int. J. Mol. Sci. 2023, 24, 16376 Int. J. Mol. Sci. 2023, 24, x FOR Figure 6. Comparison of the eﬀects of rPRL and nPRL, at diﬀerent concentrations, o apoptosis of goose phGCs (A,C) and hGCs (B,D). The cells stained with annexin-V-APC considered as apoptotic. The Q4 quadrant (Annexin V−/PI−), Q3 quadrant (Annexin V+/PI− Q2 quadrant (Annexin V+/PI+) represent the percentages of healthy cells, cells during apoptosis, and cells during late apoptosis, respectively. The apoptotic rate was deﬁned a percentage of apoptotic cells: the cells during early and late apoptosis/total cells × 100%. Da presented as the mean ± S.D. “” indicates an extremely signiﬁcant diﬀerence compared control group (p < 0.01), while “” indicates a signiﬁcant diﬀerence compared to the control g (p < 0.05). Figure 6. Comparison of the effects of rPRL and nPRL, at different concentrations, on the apoptosis of goose phGCs (A,C) and hGCs (B,D). The cells stained with annexin-V-APC were considered as apoptotic. 6. Eﬀects of rPRL and nPRL on Goose Pre-Hierarchical and Hierarchical Granu ll A t i 2.6. Effects of rPRL and nPRL on Goose Pre-Hierarchical and Hierarchical Granulosa Cell Apoptosis The Q4 quadrant (Annexin V−/PI−), Q3 quadrant (Annexin V+/PI−), and Q2 quadrant (Annexin V+/PI+) represent the percentages of healthy cells, cells during early apoptosis, and cells during late apoptosis, respectively. The apoptotic rate was deﬁned as the percentage of apoptotic cells the cells during early and late apoptosis/total cells × 100%. Data are presented as the mean ± S.D “*” indicates an extremely signiﬁcant difference compared to the control group (p < 0.01), while “” indicates a signiﬁcant difference compared to the control group (p < 0.05). 2 7 Effects of rPRL and nPRL on the Expression of Several Key Genes Involved in Cell Apoptosis Figure 6. Comparison of the eﬀects of rPRL and nPRL, at diﬀerent concentrations, on apoptosis of goose phGCs (A,C) and hGCs (B,D). The cells stained with annexin-V-APC w considered as apoptotic. The Q4 quadrant (Annexin V−/PI−), Q3 quadrant (Annexin V+/PI−), Q2 quadrant (Annexin V+/PI+) represent the percentages of healthy cells, cells during e apoptosis, and cells during late apoptosis, respectively. The apoptotic rate was deﬁned as percentage of apoptotic cells: the cells during early and late apoptosis/total cells × 100%. Data presented as the mean ± S.D. “*” indicates an extremely signiﬁcant diﬀerence compared to control group (p < 0.01), while “” indicates a signiﬁcant diﬀerence compared to the control gr (p < 0 05) Figure 6. Comparison of the effects of rPRL and nPRL, at different concentrations, on the apoptosis of goose phGCs (A,C) and hGCs (B,D). The cells stained with annexin-V-APC were considered as apoptotic. The Q4 quadrant (Annexin V−/PI−), Q3 quadrant (Annexin V+/PI−), and Q2 quadrant (Annexin V+/PI+) represent the percentages of healthy cells, cells during early apoptosis, and cells during late apoptosis, respectively. The apoptotic rate was deﬁned as the percentage of apoptotic cells: the cells during early and late apoptosis/total cells × 100%. Data are presented as the mean ± S.D. “*” indicates an extremely signiﬁcant difference compared to the control group (p < 0.01), while “” indicates a signiﬁcant difference compared to the control group (p < 0.05). p 2.7. Eﬀects of rPRL and nPRL on the Expression of Several Key Genes Involved in Cell Apop 2.7. Effects of rPRL and nPRL on the Expression of Several Key Genes Involved in Cell Apoptosis in Goose Pre-Hierarchical and Hierarchical Granulosa Cells 2.7. Eﬀects of rPRL and nPRL on the Expression of Several Key Genes Involved in Cell Apo 2.7. 3. Discussion 3. Discussion Prolactin is widely recognized as one of the main factors causing incubati behavior, implying a negative role in the regulation of poultry ovarian follicul development and egg production performance [25]. However, several recent studies chickens have reported that PRL may play a stimulatory role in ovarian follic development by inﬂuencing the synthesis and secretion of follicular cell stero hormones, depending on its concentration and the stage of follicle development [14,1 Considering that almost all previous in vitro studies on the roles of poultry PRL us either exogenous PRL puriﬁed from other species [26] or recombinant PRL [2 deciphering its exact roles awaits a further investigation using endogenous native PR Thus, herein we ﬁrstly isolated and puriﬁed the nPRL, and subsequently compared t Prolactin is widely recognized as one of the main factors causing incubation behav- ior, implying a negative role in the regulation of poultry ovarian follicular development and egg production performance [25]. However, several recent studies in chickens have reported that PRL may play a stimulatory role in ovarian follicle development by inﬂu- encing the synthesis and secretion of follicular cell steroid hormones, depending on its concentration and the stage of follicle development [14,15]. Considering that almost all previous in vitro studies on the roles of poultry PRL used either exogenous PRL puriﬁed from other species [26] or recombinant PRL [27], deciphering its exact roles awaits a further investigation using endogenous native PRL. Thus, herein we ﬁrstly isolated and puriﬁed the nPRL, and subsequently compared the effects of rPRL and nPRL on the proliferation and apoptosis of goose phGCs and hGCs. eﬀects of rPRL and nPRL on the proliferation and apoptosis of goose phGCs and hGCs The prokaryotic expression vector pET-28a-PRL was constructed to express th rPRL protein in E. coli. The rPRL was puriﬁed using Ni-aﬃnity chromatography, and w then used to immunize rabbits for preparing the rabbit-anti-rPRL polyclonal antibodie Compared to our previous research [28], in this study, we replaced the prokaryo expression vector pET-32a with pET-28a to optimize the recombinant plasm construction eﬃciency and to reduce the molecular mass of the rPRL protein from 37 kD to 25 kDa, which may be closer to that of the nPRL protein. 6. Eﬀects of rPRL and nPRL on Goose Pre-Hierarchical and Hierarchical Granu ll A t i 2.6. Effects of rPRL and nPRL on Goose Pre-Hierarchical and Hierarchical Granulosa Cell Apoptosis Effects of rPRL and nPRL on the Expression of Several Key Genes Involved in Cell Apoptosis in Goose Pre-Hierarchical and Hierarchical Granulosa Cells in Goose Pre-Hierarchical and Hierarchical Granulosa Cells To further evaluate the eﬀects of rPRL and nPRL on the apoptosis of goose phG and hGCs, the mRNA expression levels of Bcl2, Caspase3, Fas, and Beclin1 were detec by qPCR. As depicted in Figure 7, treatment with rPRL at doses of 3 or 30 ng/ To further evaluate the effects of rPRL and nPRL on the apoptosis of goose phGCs and hGCs, the mRNA expression levels of Bcl2, Caspase3, Fas, and Beclin1 were detected by qPCR. As depicted in Figure 7, treatment with rPRL at doses of 3 or 30 ng/mL signiﬁcantly upregulated the mRNA expression of the anti-apoptotic marker gene Bcl2 (p < 0.05). On Int. J. Mol. Sci. 2023, 24, 16376 8 of 15 8 of 15 the other hand, nPRL inhibited the expression of Bcl2. Meanwhile, nPRL promoted the expression of Caspase3 and Fas in phGCs in a dose-dependent manner. Both rPRL and nPRL at medium to high concentrations signiﬁcantly reduced the mRNA levels of Beclin1 in phGCs and hGCs (p < 0.05). However, neither rPRL nor nPRL signiﬁcantly changed the expressions of Bcl2, Caspase3, and Fas in hGCs. Both rPRL and nPRL at medium to high concentrations signiﬁcantly reduced the mRN levels of Beclin1 in phGCs and hGCs (p < 0.05). However, neither rPRL nor nPR signiﬁcantly changed the expressions of Bcl2, Caspase3, and Fas in hGCs. Figure 7. Eﬀects of rPRL and nPRL on the expressions of several key genes involved in ce apoptosis in goose phGCs (A) and hGCs (B). Data are presented as the mean ± S.D. Diﬀere lowercase letters indicate signiﬁcant diﬀerences among diﬀerent treatment groups at p < 0.05. Figure 7. Effects of rPRL and nPRL on the expressions of several key genes involved in cell apoptosis in goose phGCs (A) and hGCs (B). Data are presented as the mean ± S.D. Different lowercase letters indicate signiﬁcant differences among different treatment groups at p < 0.05. Figure 7. Eﬀects of rPRL and nPRL on the expressions of several key genes involved in c apoptosis in goose phGCs (A) and hGCs (B). Data are presented as the mean ± S.D. Diﬀere lowercase letters indicate signiﬁcant diﬀerences among diﬀerent treatment groups at p < 0.05. Figure 7. 6. Eﬀects of rPRL and nPRL on Goose Pre-Hierarchical and Hierarchical Granu ll A t i 2.6. Effects of rPRL and nPRL on Goose Pre-Hierarchical and Hierarchical Granulosa Cell Apoptosis Effects of rPRL and nPRL on the expressions of several key genes involved in cell apoptosis in goose phGCs (A) and hGCs (B). Data are presented as the mean ± S.D. Different lowercase letters indicate signiﬁcant differences among different treatment groups at p < 0.05. 3. Discussion 3. Discussion In support of this, it has been previously suggested that the folded chicken PRL showed high bioac- tivities in a homologous system [31]. Moreover, there is evidence that post-translational modiﬁcations of chicken PRL signiﬁcantly affected both its biological activities during the reproductive cycle [32] and ovarian follicular cell steroidogenesis [15]. Regarding the effects of PRL on avian ovarian follicular cell functions, it has been previously shown that PRL regulated both the basal and gonadotropin-stimulated estrogen and progesterone production in chicken ovarian follicular cells, depending on the concentration, the degree of glycosylation, the type of gonadotropin, and the stage of follicle development [14,15]. Besides this, it is noticeable that this study represents the ﬁrst to reveal the effects of PRL on the viability, proliferation, and apoptosis of avian GCs. Consistent with our results, a low concentration of PRL signiﬁcantly increased the viability and antioxidant capac- ity, while high PRL concentrations induced the oxidative stress and apoptosis of ovine ovarian GCs [33,34]. A recent study reported that plasma PRL levels, ranging from 25 to 100 ng/mL, could be beneﬁcial for metabolic ﬁtness, which is crucial for maintaining metabolic homeostasis [35]. The circulating PRL, at either extremely low or high levels, disrupted the metabolic homeostasis in diabetes and induced obesity-derived metabolic diseases [36,37]. It has also been reported that moderate levels of PRL played a positive role in glucose-stimulated insulin secretion, while higher PRL levels impaired the insulin- secretory capacity in diabetic mice [38]. Similar to our results, PRL exerted a biphasic effect on the growth of human endometrial cells, playing a positive role at low concentrations and a negative role at high concentrations [39]. Thus, it was proposed that a FIT-PRL concentration ranging from 3 to 30 ng/mL could promote the viability and proliferation, while high PRL concentrations induce the apoptosis, of goose GCs, depending on the stage of follicle development. p It is well known that apoptosis is a form of programmed cell death, characterized by a series of morphological and biochemical changes, which is attributed to accurately regulated molecular events or signaling cascades. The Bcl-2 family plays an important role in the regulation of the mitochondria-mediated pathway of apoptosis. Among them, the anti-apoptotic protein Bcl2 is located on the surface of the mitochondrion and inhibits the progression of apoptosis by regulating the permeability of the mitochondrial outer membrane [40]. 3. Discussion 3. Discussion Then, the nPRL protein w puriﬁed from the pituitary glands of Sichuan White Geese using an immune-aﬃni column Thus this study represents the ﬁrst to obtain a highly pure nPRL and The prokaryotic expression vector pET-28a-PRL was constructed to express the rPRL protein in E. coli. The rPRL was puriﬁed using Ni-afﬁnity chromatography, and was then used to immunize rabbits for preparing the rabbit-anti-rPRL polyclonal antibodies. Com- pared to our previous research [28], in this study, we replaced the prokaryotic expression vector pET-32a with pET-28a to optimize the recombinant plasmid construction efﬁciency and to reduce the molecular mass of the rPRL protein from 37 kDa to 25 kDa, which may be closer to that of the nPRL protein. Then, the nPRL protein was puriﬁed from the pitu- itary glands of Sichuan White Geese using an immune-afﬁnity column. Thus, this study represents the ﬁrst to obtain a highly pure nPRL and to investigate its effects on follicular cell functions. column. Thus, this study represents the ﬁrst to obtain a highly pure nPRL and investigate its eﬀects on follicular cell functions. Previous studies have suggested that PRL can act directly on GCs to regulate avia ovarian follicular development [29,30]. In this study, both goose phGCs and hGCs we t eated ith ou u iﬁed PRL a d PRL at diﬀe e t o e t atio e e ti ely Ou Previous studies have suggested that PRL can act directly on GCs to regulate avian ovarian follicular development [29,30]. In this study, both goose phGCs and hGCs were treated with our puriﬁed rPRL and nPRL at different concentrations, respectively. Our results show that both rPRL and nPRL, at a low concentration, increased the viability of Int. J. Mol. Sci. 2023, 24, 16376 9 of 15 phGCs and hGCs, but only nPRL at moderate-to-high concentrations promoted the viability and proliferation of phGCs. Meanwhile, we also found that nPRL at moderate-to-high concentrations exerted pro-apoptotic effects on both phGCs and hGCs, while lower concen- trations of rPRL decreased the apoptotic rate of phGCs. These data collectively demonstrate the differences in the bioactivity of rPRL and nPRL in goose ovarian GCs, and such dif- ferences could be due to the fact that the rPRL is an unfolded and post-translationally unmodiﬁed protein, while the nPRL is folded and post-translationally modiﬁed. 4.1. Ethics Statement All experimental procedures involving the manipulation of animals in this study were conducted in concordance with the “Guidelines for Experimental Animals” of the Ministry of Science and Technology (Beijing, China). This study has been reviewed and approved by the Sichuan Agricultural University Animal Ethical and Welfare Committee (Approval No.: 20190035). 4.2. Birds and Tissue Collection All the experimental geese were provided by the Waterfowl Breeding Experimental Farm of Sichuan Agricultural University (Ya’an Campus, Sichuan, China). A total of 50 female Sichuan White Geese were used for tissue collection. Thirty Sichuan White Geese were euthanized by carbon dioxide, and the pituitary glands were immediately isolated. One of the pituitary glands was used for amplifying the cDNA encoding the mature peptide of PRL, and the others were used for the puriﬁcation of nPRL protein. Twenty Sichuan White Geese, aged between 35 and 45 weeks and selected based on their egg-laying records to ensure regular sequences of at least 2–3 eggs, were used for the isolation of phGCs and hGCs. 4.3. Construction of the Goose Recombinant pET28a-PRL Genomic DNA was extracted from the pituitary gland of a Sichuan White Goose using the FastPure Cell/Tissue DNA Isolation Mini Kit (Vazyme, Nanjing, China). According to the reported nucleotide sequence of the Sichuan White Goose PRL gene (GenBank accession number: GQ202542.1), the cDNA encoding the goose PRL mature peptide was obtained by polymerase chain reaction (PCR) ampliﬁcation using our designed primers F (5′-CATGCCATGGGCTTGCCTATCTGCCCCAATGGATCTG-3′) and R (5′-CCCTCGAGGC AATTGCTATCATGTATTAGGCGGC-3′). The PCR was performed using a T100TM thermal cycler (Bio-Rad, Hercules, CA, USA) and the reaction conditions are as follows: 95 ◦C for 5 min; 30 cycles of 95 ◦C for 30 s, 65 ◦C for 30 s, and 72 ◦C for 45 s; 72 ◦C for 7 min. The PCR product was identiﬁed by 1.5% agarose gel electrophoresis and recovered by gel extraction kit (Omega Bio-tek, Norcross, GA, USA). g The puriﬁed PCR products were transformed into competent E. coli DH5á cells. Posi- tive clones were screened by RT-PCR and sent to Tsingke Biotechnology Co., Ltd. (Chengdu, China) for sequencing analysis. Subsequently, both the PRL fragment and the pET-28a plasmid (Yeasen, Shanghai, China) were digested with NcoI and XhoI restriction enzymes, followed by ligation using the T4 DNA ligase at 16 ◦C for 12 h. Finally, the recombinant plasmid pET28a-PRL was introduced into E. coli BL21 (DE3) for protein expression. 3. Discussion 3. Discussion As a member of the tumor necrosis factor receptor superfamily, the Fas-mediated oligomerization and conformational changes via ligand binding can result in downstream caspase activation and cell apoptosis [41]. Moreover, caspases are the conserved executioners of apoptosis, and can be activated by the upstream apoptotic signals emanating from external and internal stimuli [42]. In the present study, our results show that low concentrations of rPRL signiﬁcantly upregulated the levels of Bcl-2 to inhibit the apoptosis of phGCs, which is consistent with the observations made using ﬂow cytometry. However, the expression of Bcl-2 was downregulated after treatment with nPRL, and higher concentrations of nPRL increased the apoptotic rate of hGCs by upregulating the expressions of Fas and Caspase3. Similar to what was found in our study, with increasing concentrations of ovine PRL, the viability of ovine ovarian GCs increased ﬁrst and then decreased. Moreover, a low concentration (below 20 ng/mL) of PRL decreased the apoptosis of GCs, while 500 ng/mL of PRL increased the apoptosis and inhibited the steroid hormone secretion of GCs [43]. These results suggest that the effects of PRL on the proliferation and apoptosis of either mammalian or avian GCs depend on its concentration. Becn1, known as an autophagy-related protein, plays a role in preventing the assembly of the pre-autophagosomal structure and inhibiting autophagy by binding to Bcl-2 [44]. It has Int. J. Mol. Sci. 2023, 24, 16376 10 of 15 been reported that autophagy is involved in regulating the apoptosis of GCs to accelerate follicular atresia [45]. In this study, the expression levels of Becn1 and Bcl-2 in phGCs and hGCs were signiﬁcantly decreased after treatment with high concentrations of either rPRL or nPRL. There is evidence that Becn1 plays important roles in the regulation of the life span of human corpus luteum and ovarian androgen-secreting cells by maintaining autophagy at levels that promote cell survival rather than cell death [46]. Additionally, the siRNA-mediated knockdown of Becn1 showed that the inhibition of autophagy resulted in a decreased expression of genes involved in the differentiation of GCs [47]. These results imply that high PRL concentrations may promote the apoptosis of goose GCs, at least by part, by disrupting the autophagy process. 4.6. Preparation of the Immunoafﬁnity Chromatography (IAC) Column and Puriﬁcation of the Goose Native PRL (nPRL) Protein 4.6. Preparation of the Immunoafﬁnity Chromatography (IAC) Column and Puriﬁcation of the Goose Native PRL (nPRL) Protein To purify the nPRL protein, the rabbit-anti-rPRL antibody was used for speciﬁc cou- pling with nPRL. In detail, the rabbit-anti-rPRL antibody was ﬁrstly coupled to the NHS- activated beads 4FF (Smart-Lifesciences, Changzhou, China) in a coupling buffer (0.2 M NaHCO3, 0.5 M NaCl, pH 8.0) at 4 ◦C for 1 h. Subsequently, the liquid in the column was discarded, and the column was sealed with a sealing solution (0.1 M Tris-base, pH 8.5) at 28 ◦C for 1 h. After being washed with PBS, the IAC column was successfully prepared. Then, the goose pituitary glands were lysed in a lysis buffer (Beyotime Biotech, Nantong, China) and centrifuged at 10,000× g at 4 ◦C for 10 min, and the resulting supernatant was loaded onto the self-made IAC column and incubated at 4 ◦C for 1 h. After being washed with PBS, the nPRL protein was ﬁnally eluted using an elution buffer (0.1 M Glycine, pH 3.0) at a ﬂow rate of about 1 mL/min. The protein concentration measurement, SDS-PAGE, and Western blot analyses were performed to characterize the puriﬁed nPRL. 4.5. Preparation and Puriﬁcation of Polyclonal Antibodies against the rPRL Protein 4.5. Preparation and Puriﬁcation of Polyclonal Antibodies against the rPRL Protein The rabbits were immunized with our puriﬁed rPRL protein four times with a two- week interval between each immunization. Subsequently, the antiserum was collected from these rabbits. To purify the anti-rPRL antibodies from the antiserum, an empty column was ﬁlled using the CNBR-activated Sepharose 4B ﬁller. Thereafter, 1 mg of rPRL protein together with the coupling solution (0.1 M NaHCO3, 0.5 M NaCl, pH 8.3) was introduced into the column at 4 ◦C for 12 h. Next, the sealing solution (Tris-HCl, pH 8.0) was added to fully seal any unbound protein sites for 6 h. The afﬁnity chromatography column was prepared and cleaned with the appropriate phosphate-buffered saline (PBS), consisting of 50 mM NaH2PO4 and15 mM NaCl. The diluted serum was loaded onto the self-made columns and incubated at 4 ◦C for 12 h. After being washed three times with PBS, the antibodies were ﬁnally eluted using an elution buffer (0.15 mM NaCl, pH 2.5) at a ﬂow rate of about 1 mL/min, and immediately frozen at −80 ◦C. 4.4. Prokaryotic Expression and Puriﬁcation of the Goose Recombinant PRL (rPRL) Protein The E. coli BL21 (DE3) strain carrying the Pet28a-PRL plasmid was cultured in Luria– Bertani medium, supplemented with 50 g/L kanamycin at 37 ◦C, until reaching an OD600 of 0.4–0.6. Subsequently, the conditions for protein expression, including the concentration of isopropyl-β-D-thiogalactopyranoside (IPTG), induction temperature, and induction Int. J. Mol. Sci. 2023, 24, 16376 11 of 15 11 of 15 time, were assessed and optimized. Finally, protein expression was induced at 37 ◦C using a concentration of 0.5 mmol/L IPTG for a duration of 6 h. The culture medium was centrifuged at 10,000× g for 10 min to collect the sediment, which was broken by sonication for 30 min followed by centrifugation to remove the supernatants. The rPRL protein was ﬁnally puriﬁed using Ni afﬁnity chromatography and analyzed by both sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and quadrupole time-of-ﬂight tandem mass spectrometry (Q-TOF-MS). 4.8. Cell Viability Assay The viability of GCs was evaluated using the CCK-8 assay. Brieﬂy, the GCs were ﬁrstly seeded into 96-well plates and cultured for 24 h. Then, the cells were treated with nPRL and rPRL at different concentrations (0, 3, 30, and 300 ng/mL) for another 24 h. After treatment, the CCK-8 solution (APExBIO, Houston, TX, USA) (10 µL/well) was added to each well and incubated at 37 ◦C for 4 h. Finally, the absorbance was measured at 450 nm using spectrophotometry. 4.7. Cell Culture and Treatment The ovarian follicles were obtained from the abdominal cavity of Sichuan White Geese during the egg-laying period, and the GC layers were subsequently isolated from both the pre-hierarchical (6–10 mm in diameter) and hierarchical follicles (F3-F1). After being washed with PBS, the isolated GC layers were cut into small pieces and subjected to di- gestion with 0.1% collagenase II. The resulting cell suspensions were then mixed with DMEM/F12 supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO, USA) to halt the digestion process. After passing through a 200-mesh sieve and centrifugation at 1200 g/min for 10 min, the supernatant was discarded while the cells were resus- pended in the aforementioned medium. The number of GCs in each follicular category was determined using a hemocytometer. Then, the cells were diluted to approximately 6 × 105 cells /mL in media, seeded into different culture plates, and incubated at 37 ◦C under a humidiﬁed atmosphere containing 95% air and 5% CO2 until reaching a conﬂuence of >70%. Following an initial medium change after 6 h of incubation, non-adherent cells were aspirated, while the remaining were used for subsequent treatments. After incubating for 24 h, both phGCs and hGCs were treated with nPRL and rPRL at the concentrations of 0, 3, 30, or 300 ng/mL for another 24 h, and were ﬁnally used for cell viability, proliferation, Int. J. Mol. Sci. 2023, 24, 16376 12 of 15 12 of 15 and apoptosis, as well as gene expression analysis. The concentrations of rPRL and nPRL were selected based on both the observed ﬂuctuations in circulating PRL levels of female geese throughout the reproductive cycle [22,23] and the doses used in previous in vitro studies [14,15,31]. 4.10. Annexin V-FITC/PI Double Staining in the Detection of Apoptosis by Flow Cytometry 4.10. Annexin V-FITC/PI Double Staining in the Detection of Apoptosis by Flow Cytometry The GCs were ﬁrstly seeded onto 24-well plates and incubated for 24 h. Subsequently, the GCs were treated with rPRL and nPRL at different concentrations for an additional 24 h. To assess the apoptosis rate induced by PRL, the cells were harvested and double-stained using the Annexin V-FITC/PI apoptosis detection kit (Vazyme Biotech, Nanjing, China) according to the manufacturer’s instructions. The apoptotic rate was detected using a BD Accuri C6 Flow cytometer and analyzed using FlowJo software (v10.8). A total of 10,000 cells per sample were analyzed, and the apoptotic rate was determined by summing up the cell proportions of early and late apoptotic cells. 4.9. EdU and Hoechst 33,342 Staining Assay The GCs were ﬁrstly seeded onto 96-well plates and incubated for 24 h. After treatment with nPRL and rPRL at different concentrations for 24 h, the GCs were exposed to 50 µM of 5-ethynyl-2′-deoxyuridine (RiboBio, Nantong, China) at 37 ◦C for 4 h according to the manufacturer’s instructions. Then, the GCs were ﬁxed with a solution containing 4% paraformaldehyde for 30 min and permeabilized with a solution containing 0.5% TritonX- 100 for 10 min. After being washed with PBS, the GCs were incubated with a reaction cocktail consisting of Apollo dye (1×) in a volume of 100 µL for 30 min. Finally, the DNA content within cells was stained using Hoechst33342 dye for 30 min and visualized under a ﬂuorescence microscope. 5. Conclusions In conclusion, the present study obtained a highly pure nPRL for the ﬁrst time. More- over, our results have demonstrated that the rPRL at lower concentrations increased the viability and proliferation of both phGCs and hGCs, while exerting anti-apoptotic effects only in phGCs. By comparison, the nPRL showed more pronounced stimulatory effects on the viability and proliferation of both GC types, but increased their apoptosis at higher concentrations. These results suggest a dual role of PRL in regulating the proliferation and apoptosis of goose GCs, depending on its concentration and the stage of follicle de- velopment. These data presented here provide a better understanding of the roles of PRL in regulating avian ovarian follicular development, and can be helpful for purify- ing native proteins of poultry and for controlling reproductive seasonality in practical goose production. Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/ijms242216376/s1. Author Contributions: Conceptualization, S.H., C.H. and D.D.; methodology, S.H. and C.H.; formal analysis, D.D. and W.L.; investigation, D.D., W.L., X.Y., X.L. and L.L.; writing—original draft prepa- ration, D.D. and W.L.; writing—review and editing, S.H., C.H. and J.W. All authors have read and agreed to the published version of the manuscript. Funding: This study was ﬁnancially supported by the National Natural Science Foundation of China (31802064 and 32202676), the Sichuan Science and Technology Program (2023NSFSC0227), the Key Technology Support Program of Sichuan Province (2021YFYZ0014), and the International Cooperation Project of Sichuan Province (2022YFH0035). Institutional Review Board Statement: This study has been reviewed and approved by the Sichuan Agricultural University Animal Ethical and Welfare Committee (Approval No.: 20190035). Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: All data supporting our reported results have been given in the Results section of this article. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 4.12. Statistical Analysis The SPSS 22.0 and GraphPad Prism 8 software were used for data analysis. The results are presented as the mean ± S.D. Statistical signiﬁcance was determined using one-way ANOVA followed by the Duncan’s multiple range test. Differences between groups were considered statistically signiﬁcant at p < 0.05 and extremely signiﬁcant at p < 0.01. 4.11. Quantitative Real-Time PCR The extracted RNA was reverse transcribed into cDNA using the Prime Script RT reagent kit (Takara, Dalian, China) according to the manufacture’s guidelines. The qRT-PCR reaction solution was prepared in a total volume of 12.5 µL containing 1 µL cDNA, 6.25 mL of 2× SYBR Premix Ex Taq (Vazyme Biotech, Nanjing, China), 4.25 µL of ddH2O, and 0.5 µL of each speciﬁc primer pair (10 µM). The reactions were conducted under the following conditions: pre-denaturation at 95 ◦C for 10 s, followed by 40 cycles of denaturation at 95 ◦C for 5 s and annealing/extension at the corresponding temperature of each primer pair for 30 s. An 80-cycle melting curve was performed, with the temperature ranging from 60 ◦C to 95 ◦C and increasing by 0.5 ◦C every 10 s. Each sample were ampliﬁed in triplicate, and the relative mRNA expression levels of target genes were normalized to the reference genes GAPDH and β-Actin using the comparative Cq method (∆∆Cq) [48]. The primer pairs used for qRT-PCR are listed in Table 1. Int. J. Mol. Sci. 2023, 24, 16376 13 of 15 13 of 15 Table 1. Primer pairs for real-time quantitative PCR. Gene Forward Primer (5′–3′) Reversed Primer (5′–3′) Tm (◦C) Product Length (bp) Bcl2 CCTTCGTGGAGTTGTATGGCA CCACCAGAACCAAACTCAGGATA 60 100 Caspase3 CTGGTATTGAGGCAGACAGTGG CAGCACCCTACACAGAGACTGAA 60 158 Fas CACTCCCACAAGTCAAG AGTAGGGTTCCATAGGC 60 163 Becn1 CGCTGTGCCAGATGTGGAAGG CAGAAGGAATACTGCGAGTTCAAGA 60 151 GAPDH GCTGATGCTCCCATGTTCGTGAT GTGGTGCAAGAGGCATTGCTGAC 60 86 β-Actin CAACGAGCGGTTCAGGTGT TGGAGTTGAAGGTGGTCTCG 60 92 Table 1. Primer pairs for real-time quantitative PCR. 1. Deng, Y.; Gan, X.; Chen, D.; Huang, H.L.; Yuan, J.S.; Qiu, J.M.; Hu, S.Q.; Hu, J.W.; Wang, J.W. Comparison of growth characteristics of in vitro cultured granulosa cells from geese follicles at different developmental stages. Biosci. Rep. 2018, 38, BSR20171361. [CrossRef] [PubMed] p 3. Johnson, A.L.; Woods, D.C. Dynamics of avian ovarian follicle development: Cellular mechanisms of granulosa cell differentiation. Gen. Comp. Endocrinol. 2009, 163, 12–17. [CrossRef] [C oss e ] [ e ] 2. 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[CrossRef] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
https://openalex.org/W4393148851	https://link.springer.com/content/pdf/10.1007/s11469-024-01282-4.pdf	English	null	The Effect of Mindfulness-based Psychoeducation on Negative Automatic Thoughts and Medication Adherence in Individuals with Cannabis Use Disorder: a Randomized Controlled Trial	International journal of mental health and addiction	2,024	cc-by	7,284	International Journal of Mental Health and Addiction https://doi.org/10.1007/s11469-024-01282-4 International Journal of Mental Health and Addiction https://doi.org/10.1007/s11469-024-01282-4 ORIGINAL ARTICLE The Effect of Mindfulness‑based Psychoeducation on Negative Automatic Thoughts and Medication Adherence in Individuals with Cannabis Use Disorder: a Randomized Controlled Trial Funda Kavak Budak1 · Aysel Akbeniz2 · Fatma Melike Erkan1 · Abdurrezzak Gültekin3 · Hatice Birgül Cumurcu4 Accepted: 13 March 2024 © The Author(s) 2024 Accepted: 13 March 2024 © The Author(s) 2024 Extended author information available on the last page of the article Abstract on cannabis users reported a negative correlation between mindfulness and negative automatic thoughts (Budak et al., 2021). Miller and Fleming reported that substance addicts had a low level of mindfulness (Miller & Ve Fleming, 2008). Also, negative automatic thoughts negatively affect the medication adherence of individuals (Artıran & Şeker, 2020). Medication adherence generally refers to as the degree of the patient to follow the rec- ommended treatment regimen (Yel & Karadakovan, 2022). Medication non-adherence is an important issue in substance use disorders. In a study on individuals with substance use disorders, it was found that 47.7% of patients suffered from problems with medication adherence during the treatment (Blum et al., 2018). Even though pharmacological therapies are promising in the treatment of cannabis use disorder, no pharmacological approach with proven efficacy is available. Therefore, psychosocial-based interventions such as cognitive behavioural therapy, motivational inter- view techniques, and deprivation-based emergency management are used alongside phar- macological treatment (Connor et al., 2021; Patel, 2022). One of these psychosocial meth- ods is mindfulness-based intervention (Bayır & Aylaz, 2021; Kargın & Hiçdurmaz, 2020). The concept of mindfulness is a mental process based on the observation of emotions, thoughts, and bodily sensations by focusing attention on the present moment without any judgement (Çetin & Aylaz, 2018). Mindfulness-based interventions are used in psychother- apy sessions, psychoeducational sessions, and some meditation practices (Groves, 2016). Mindfulness-based interventions help individuals to notice and regulate underlying mala- daptive thoughts, emotional reactions, and automatic and impulsive behaviours in mental health disorders and strengthen individuals to cope with distress by increasing their toler- ance for it (Davis et al., 2019; Zümbül, 2021). The results of studies on cannabis use have indicated that mindfulness reduces cannabis abuse and associated adversities (Barrington et al., 2019; Lin et al., 2021; Schneegans et al., 2021). The study by Barrington et al. indi- cated that individuals with a high level of mindfulness reported lower levels of cannabis abuse (Barrington et al., 2019). Mindfulness-based psychoeducation would produce effec- tive results in the treatment of cannabis use disorder, as in other substance use disorders (Sripada, 2022), by reducing negative automatic thoughts. The literature includes studies in which mindfulness-based interventions have been applied with individuals diagnosed with substance use disorders (Amaro & Black, 2017; Black & Amaro, 2019; Roos et al., 2017). Abstract The aim of this study is to evaluate the effect of mindfulness-based psychoeducation on negative automatic thoughts and medication adherence in individuals with cannabis use disorder. The randomized controlled study was conducted between September 2021 and December 2022 in the psychiatry clinic of a university hospital in Turkey. The sample size of the study consisted of 60 individuals (30 experimental groups, 30 control groups). Descriptive Characteristics Form, Negative Automatic Thoughts Questionnaire, and Morisky Medication Adherence Scale were used to collect data. In the study, while eight sessions of mindfulness-based psychoeducation were given to the experimental group, no intervention was applied to the control group. Experimental group posttest negative auto- matic thoughts and medication adherence scale total mean scores were found to be statisti- cally significant (p < 0.05). It was determined that the posttest negative automatic thoughts scale total score mean in the experimental group decreased compared to the control group, and the medication adherence scale total score mean increased compared to the control group. It was determined that mindfulness-based psychoeducation given to individuals with cannabis use disorder was effective in negative automatic thoughts and adherence to treatment. Keywords Mindfulness · Cannabis · Substance · Patient · Negative automatic thought · Psychiatric nursing · Psychoeducation · Adherence to treatment Cannabis use disorder is the most common illegal substance use disorder in the general population (Pinto et al., 2019). The World Drug Report 2021 by the World Health Organi- sation states that approximately 4% of the world population abused cannabis in 2019 (WHO- World Health Organization, 2021). While one study reported that individuals with more negative automatic thoughts are more likely to abuse cannabis (Roos & Witkiewitz, 2017), another study revealed that individuals with cannabis use disorder had higher levels of negative automatic thoughts (Budak et al., 2021). Extended author information available on the last page of the article 56789) 3 23451 International Journal of Mental Health and Addiction The cognitive approach indicates that negative automatic thoughts affect the emo- tions and behaviours of individuals due to their dysfunctional and detrimental effects on the assessment and interpretation of the environment (Artıran & Şeker, 2020). While one study showed that individuals with substance use disorders had more negative automatic thoughts (Gülen, 2019), another study revealed a significant positive correlation between negative automatic thoughts and the potential for substance use disorder (Karami, 2018). A study conducted by Budak et al. Abstract However, no studies have been found that espe- cially examined the effect of mindfulness-based psychoeducation on negative automatic thoughts and medication adherence in individuals with cannabis use disorder. The intro- duction of approaches, effectiveness of which is proven in the treatment of cannabis use disorder, leading to serious problems, would contribute to the improvement of the physical and mental health of patients. Psychiatric nurses assume major responsibilities in the treat- ment of substance use disorders such as cannabis, as it requires a multidisciplinary team approach (Kargın & Hiçdurmaz, 2018). Scientific publications that address these concepts related to the group should be available so that psychiatric nurses can deliver the appropri- ate psychosocial care. Therefore, this study is believed to serve as a guideline for psychiat- ric nurses. Study Design The study design was randomized controlled trial. 1 3 1 3 International Journal of Mental Health and Addiction This study aims to evaluate the effect of mindfulness-based psychoeducation on negative automatic thoughts and medication adherence in individuals with cannabis use disorder. Place and Time of the Study The study was conducted in the psychiatry clinic of Turgut Özal Medical Centre, İnönü University, in Turkey, between September 2021 and December 2022. The psychiatry clinic has a capacity of 52 beds. The cases of anxiety disorder, mood disorder, psychosis, related disorders, and substance use disorder are treated in the clinic. A total of 45 healthcare professionals on staff, including 12 nurses, 7 faculty members, 13 assistants, and 13 other staff members, work in this clinic. There is a library, garden, table tennis board, sports garden, and hobby room for patients in the Psychiatry Clinic of Turgut Özal Medical Centre. In clinics, patients are subjected to regular psychiatric examinations and participate in interviews and group therapies with a psychologist. At the same time, psychiatric nurses working in the clinic write their daily observations about the patients in observation reports and report them to the physicians. Therefore, if the patient has a psy- chiatric problem in addition to addiction, it is stated in the clinical reports and treatment. Hypotheses H1: Mindfulness-based psychoeducation reduces negative automatic thoughts in indi- viduals with cannabis use disorder. H2: Mindfulness-based psychoeducation increases medication adherence in individuals with cannabis use disorder. Descriptive Characteristics Form This form was prepared by the researcher upon the literature review and consists of a total of ten questions including the socio-demographic characteristics of the patients. Randomization Randomization list for assigning patients to groups MedCalc version 18.11.3 made using. Guided by the clinician, who meet the inclusion criteria, agree to participate in the study, and routinely patients receiving treatment (similar drug therapy) are listed in the order of referral. Equal distribution according to socio-demographic characteristics (age, gender, marital status, education level, working status, people living with, status of having chil- dren, cannabis starting age, getting treatment to quit cannabis) was taken into account in the randomization of the experimental and control groups. The randomization number obtained from MedCalc of the number received by the patient. The last part of the list indi- cates that the patient is in the experimental or control group has determined. Patients were not told which group they were in. Exclusion Criteria • Being under 18 years of age • Having a comorbid psychiatric diagnosis (psychotic disorder, personality disorder, depres- sion, etc.) • Being in their period of substance deprivation • Having communication problems • Having previously or currently participate in any mindfulness programme • Having previously or currently participate in any mindfulness programm Inclusion Criteria • Meeting the criteria for substance abuse according to DSM-V • Being currently abusing cannabis Population and Sample Two patients in the control group changed the city they lived in, and three patients were assigned to another city for work reasons. The research was completed with 60 patients, 30 in the experimental group, and 30 in the control group (Fig. 1). Population and Sample The population of the study consisted of individuals diagnosed with substance use disorders in the psychiatry clinic of Turgut Özal Medical Centre. Based on hospital records, there were 200 individuals with substance use disorders who were treated in the psychiatry clinic in 2021–2022. One hundred thirty-five out of 200 individuals with substance use disor- ders used only cannabis, and 27 out of 200 individuals with substance use disorders have no diagnosis of an additional psychiatric disorder. It was obtained from the records of the patients that they only used cannabis and did not have an additional diagnosis of mental ill- ness. The sample size was calculated for 108 individuals. The sample size determined by power analysis was calculated as at least 30 individuals for each group and 60 individuals in total (30 in experimental groups, 30 in control groups), assuming that the mean score of being affected by cannabis use, which was 37.83 (standard deviation 31) in the group who abused cannabis, would decrease by 5 points, at an error level of 5%, a two-way significance 1 1 3 3 International Journal of Mental Health and Addiction level, a confidence interval of 95%, and an ability to represent the population of 80% (Kavak Budak et al., 2021). Considering that there would be losses in the research, 70 patients (35 experimental, 35 control) were interviewed. Three patients from the experimental group stated that they gave up participating in the training after the preliminary tests were carried out, and two patients reported that they could not spare time for the training hours and days. Two patients in the control group changed the city they lived in, and three patients were assigned to another city for work reasons. The research was completed with 60 patients, 30 in the experimental group, and 30 in the control group (Fig. 1). level, a confidence interval of 95%, and an ability to represent the population of 80% (Kavak Budak et al., 2021). Considering that there would be losses in the research, 70 patients (35 experimental, 35 control) were interviewed. Three patients from the experimental group stated that they gave up participating in the training after the preliminary tests were carried out, and two patients reported that they could not spare time for the training hours and days. Negative Automatic Thoughts Questionnaire Hollon and Kendall developed the questionnaire in 1980 (Hollon & Kendal, 1984). Şahin and Şahin conducted the second validity and reliability study in 1992 (Şahin 1 3 1 International Journal of Mental Health and Addiction Outpatient Clinical Application (n=200) Excluded from the Study (n= 92) • Using substances other than cannabis (n=65) • Those with comorbid psychiatric illness (n=27) Included as a result of Power Analysis (n=70) Control Group (n=35) Analyzed (n=30) No intervention was made. Analyzed (n=30) 8-Session Mindfulness-Based Psychoeducation was given. Experimental Group (n=35) Determination of Groups Record Analysis 5 patients were excluded from the study since they did not participate in the training at different times. 5 patients were excluded from the study since they could not be reached while collecting the post-test data. Fig 1 Research consort flow chart Outpatient Clinical Application (n=200) • Those with comorbid psychiatric illness (n=27) & Şahin, 1992). It requires a scoring on a 5-point Likert scale. All items are scored straight. Total score ranges between 30 and 150. A score of 67 and above indicates a high level of negative automatic thoughts. The Cronbach’s alpha internal consistency coefficient of the questionnaire was determined as 0.93 (Şahin & Şahin, 1992). In this study, the Cronbach’s alpha coefficient of the questionnaire was found to be 0.94. Included as a result of Power Analysis (n=70) Control Group (n=35) Analyzed (n=30) No intervention was made. Analyzed (n=30) 8-Session Mindfulness-Based Psychoeducation was given. Experimental Group (n=35) Determination of Groups Analysis 5 patients were excluded from the study since they did not participate in the training at different times. 5 patients were excluded from the study since they could not be reached while collecting the post-test data. Fig. 1 Research consort flow chart Determination of Groups Experimental Group (n=35) Control Group (n=35) No intervention was made. 8-Session Mindfulness-Based Psychoeducation was given. 5 patients were excluded from the study since they did not participate in the training at different times. 5 patients were excluded from the study since they could not be reached while collecting the post-test data. Analyzed (n=30) Analyzed (n=30) Fig. 1 Research consort flow chart Fig. 1 Research consort flow chart & Şahin, 1992). It requires a scoring on a 5-point Likert scale. All items are scored straight. Total score ranges between 30 and 150. A score of 67 and above indicates a high level of negative automatic thoughts. Negative Automatic Thoughts Questionnaire The Cronbach’s alpha internal consistency coefficient of the questionnaire was determined as 0.93 (Şahin & Şahin, 1992). In this study, the Cronbach’s alpha coefficient of the questionnaire was found to be 0.94. & Şahin, 1992). It requires a scoring on a 5-point Likert scale. All items are scored straight. Total score ranges between 30 and 150. A score of 67 and above indicates a high level of negative automatic thoughts. The Cronbach’s alpha internal consistency coefficient of the questionnaire was determined as 0.93 (Şahin & Şahin, 1992). In this study, the Cronbach’s alpha coefficient of the questionnaire was found to be 0.94. 3 International Journal of Mental Health and Addiction Data Collection The data were collected in a private room in the clinic by the researcher using face-to- face interview method. Pretest forms (Descriptive Characteristics Form, Negative Auto- matic Thoughts Questionnaire and Morisky Medication Adherence Scale) and posttest forms (Negative Automatic Thoughts Questionnaire and Morisky Medication Adher- ence Scale) were applied in the experimental and control groups. The researcher dis- tributed the forms to the patients, and the patients filled out the forms themselves. The researcher collected the completed forms. It took approximately 15 min to complete the data collection tools. Nursing Intervention The researcher who applied the mindfulness training attended the training on “Mindful- ness and Its Use in Therapeutic Process” provided by Assoc. Prof. Dr. Zümra Atalay on 29 April 2018 and received its certificate. i The individuals in the experimental group were informed about mindfulness-based psychoeducation, and psychoeducation days were set. The individuals in the experimen- tal group were divided into three groups of ten people. The psychoeducation programme was provided twice a week (eight sessions) as group education over 4 weeks. Each session lasted for approximately 40 min. No intervention was applied to the control group. Patients coming to the clinic were first included in the experimental group. After reaching the num- ber in the experimental group, the control group was reached. In both groups, 15 days were waited for the posttest after the pretest. The individuals in the control and experimental groups continued their medication, education and routine practices, in the clinic during this period. Morisky Medication Adherence Scale Morisky, Green, and Levine developed the scale in 1986 (Morisky et al., 1986). Yılmaz conducted the Turkish validity and reliability study of the scale in 2004 (Yılmaz, 2004). The Cronbach’s alpha coefficient of the scale ranges between 0.64 and 0.96. The scale consists of four questions to measure medication adherence. The questions are answered as “yes/no”. If the answers to all questions are “no”, medication adherence is rated as high (4 points); if the answer to one or two questions is “yes”, medication adherence is rated as moderate (between 2 and 3 points); and if the answer to three or four questions is “yes”, medication adherence is rated as low (between 0 and 1 point) (Yılmaz, 2004). In this study, the Cronbach’s alpha coefficient of the scale was found to be 0.92. Mindfulness‑based Psychoeducation Sessions Session 1: The technique of meeting with mindfulness allowed the group members to introduce themselves to one another during the session. According to this technique, each person paired up with the patient next to him/her within the given time frame and explained themselves only to the person with whom they are paired. When the time was up, everyone took turns introducing the person they were paired up with to the 1 3 International Journal of Mental Health and Addiction group, and the introduction was completed in this way. The group members were asked to share their experiences with the practice. group, and the introduction was completed in this way. The group members were asked to share their experiences with the practice. Session 2: During this session, they were informed about addiction, substance use dis- order, and cannabis abuse and tried to make them recognise their bodies with mindful- ness exercises. Session 3: This session involved a raisin practice to explore mindfulness. Breathing and body meditation practices were realised. They were told to notice the sounds that they hear from inside and outside with their senses during the practice. This allowed them to accept what was going on in their inner world and their surroundings as what they were. The patients were told to be aware of themselves while performing their daily activities, and mindfulness breathing exercises were assigned as homework. Session 4: This session was based on exploring their bodies, and the patients did body meditation. The body meditation focused on the body. The session tried to raise mind- fulness and alleviate stress in daily life with a habit-breaker: spending some time in nature. Session 5: The causes of anger in patients and methods of coping with anger were dis- cussed. They were informed about anger and coping strategies. The meditation on fac- ing difficulties was practised. This meditation enabled the patient to develop the ability to face the difficulties occasionally encountered in daily life. A letter to the self-habit breaker made the patient aware of his/her own feelings and thoughts. Session 6: The patients were made to do mindfulness breathing exercises and breathing meditation. This meditation was intended to teach coping with difficult circumstances and new coping strategies. The habit breaker “going for a walk” was used to raise mind- fulness and reduce stress in daily life. Mindfulness‑based Psychoeducation Sessions Session 7: The patients were told about the causes of stress and coping strategies. It aimed to alleviate the patient’s stress by increasing their joy in life. The patients did the treasure of pleasure meditation. They were enabled to use their whole consciousness to focus on extremely simple pleasures such as the warmth of the hands or the taste of the favourite food, thus trying to increase the joy of life.f Session 8: The effectiveness of the programme was assessed. What they learned were reinforced. Concerning the whole training, the group members were asked to evaluate themselves and the programme (Table 1). Statistical Analysis The SPSS 25.0 for Windows software (SPSS, Chicago, IL, USA) was used for statistically analysis of the data. Before the analysis, the Kolmogorov–Smirnov test was run to assess the compatibility of the scales for normal distribution. Percentage distribution, mean, Pear- son chi-square, independent samples t-test, and paired t-test were applied to assess the nor- mally distributed data. Ethical Considerations Approval from the Scientific Research and Publication Ethics Committee of Inonu Univer- sity (IRB NO: 2018/10–15) and then legal permission from the related institutions were obtained to conduct the study. The participants were informed about the aim of the study, 3 3 International Journal of Mental Health and Addiction Table 1 Mindfulness-based psychoeducation programme Table 1 Mindfulness-based psychoeducation programme Session 1: Preparation session Acquaintance Explaining group rules Introducing the programme Session 2: Recognition of the disease Cannabis and its effects Explaining the concept of addiction, Brain and addiction Explaining cannabis and its effects, Session 3: Mindfulness • Mindfulness • Mindfulness breathing exercise • Raisin meditation • Mindfulness in daily activities Session 4: Don’t forget the body Body scanning meditation Being aware of daily activities Habit breaker Session 5: Coping with anger and anger Understanding the nature of anger Anger and emotion, thought, and behavior relationship Ways to control anger Meditation to face difficulties Habit breaker Session 6: Applying mindfulness to daily life • Mindfulness breathing exercise • Breathing meditation • Habit breaker Session 7: Methods of coping with stress What is stress? What are the effects of stress? How can we deal with stress? Actions to deal with cannabis-related thoughts Mindfulness breathing exercise Pleasure treasure meditation Session 8: Evaluation Reinforcement of the learned information Evaluation of the programme Receiving feedbacks Session 8: Evaluation Reinforcement of the learned information Evaluation of the programme Receiving feedbacks and then their questions were responded. Afterwards, their verbal and written consents were obtained. After the training given to the experimental group was completed, patients in the control group were also given 8 weeks of training. and then their questions were responded. Afterwards, their verbal and written consents were obtained. After the training given to the experimental group was completed, patients in the control group were also given 8 weeks of training. Findings It can be asserted that the total mean score of the individuals in the control and experimen- tal groups in the Negative Automatic Thoughts Questionnaire in the pretest was high when the total mean score of the scale was taken into consideration (30–150). Upon intra-group and inter-group comparison of the control and experimental groups in terms of total mean score of the Negative Automatic Thoughts Questionnaire, no statistically significant dif- ference was found in the Negative Automatic Thoughts Questionnaire total mean scores of the control group in the pretest and posttest, whereas a statistically significant difference was found between the Negative Automatic Thoughts Questionnaire total mean scores of the experimental group in the pretest and posttest (p < 0.05). There was a decrease in the total mean score of the Negative Automatic Thoughts Questionnaire in the posttest in the experimental group compared to the control group (Tables 2 and 3). In the experimental group, negative automatic thoughts decreased by approximately 31 points in the posttest compared to the pretest, while in the control group, negative automatic thoughts increased by approximately 1 point in the posttest compared to the pretest. When the Morisky Medication Adherence Scale total mean scores of the control and experimental groups in the pretest were taken into consideration (0–1 [low], 2–3 [moderate], and 4 [high]), it was determined that their medication adherence was at a moderate level. Findings Upon intra-group and inter-group comparison of the control and exper- imental groups in terms of total mean score of the Morisky Medication Adherence 1 3 International Journal of Mental Health and Addiction International Journal of Mental Health and Addiction Table 2 Descriptive characteristics of the control and experimental groups Group Control group (n:30) Experi- mental group (n:30) Test and value Descriptive characteristics n % n % Age 18–28 15 50 16 53.33 X2 = 0.310 p = 0.605 29–39 10 33.33 11 36.67 40–50 5 16.67 3 10 Gender Male 30 100 30 100 X2 = 5.339 p = 0.588 Female 0 0 0 0 Marital status Married 12 40 11 36.67 X2 = 1.398 p = 0.134 Single 18 60 19 63.33 Education level Literate 2 6.66 3 10 Primary education 8 26.67 7 23.33 Secondary 12 40 11 36.67 X2 = 3.605 p = 0.269 University 8 26.67 9 30 Working status Yes 4 13.33 5 16.67 X2 = 0.990 p = 0.863 No 26 86.67 25 83.33 People living with Family 26 86.67 27 90 Alone 4 13.33 3 10 X2 = 5.157 p = 0.461 Status of having children Yes 4 13.33 4 13.33 X2 = 0.409 p = 0.210 No 26 86.67 26 86.67 Cannabis starting age 18–28 27 90 28 93.33 X2 = 4.339 p = 0.688 29–39 3 10 2 6.67 Getting treatment to quit cannabis Yes 15 50 15 50 X2 = 1.339 p = 0.785 No 15 50 15 50 Group Control group (n:30) Experi- mental group (n:30) Test and value Descriptive characteristics n % n % Age 18–28 15 50 16 53.33 X2 = 0.310 p = 0.605 29–39 10 33.33 11 36.67 40–50 5 16.67 3 10 Gender Male 30 100 30 100 X2 = 5.339 p = 0.588 Female 0 0 0 0 Marital status Married 12 40 11 36.67 X2 = 1.398 p = 0.134 Single 18 60 19 63.33 Education level Literate 2 6.66 3 10 Primary education 8 26.67 7 23.33 Secondary 12 40 11 36.67 X2 = 3.605 p = 0.269 University 8 26.67 9 30 Working status Yes 4 13.33 5 16.67 X2 = 0.990 p = 0.863 No 26 86.67 25 83.33 People living with Family 26 86.67 27 90 Alone 4 13.33 3 10 X2 = 5.157 p = 0.461 Status of having children Yes 4 13.33 4 13.33 X2 = 0.409 p = 0.210 No 26 86.67 26 86.67 Cannabis starting age 18–28 27 90 28 93.33 X2 = 4.339 p = 0.688 29–39 3 10 2 6.67 Getting treatment to quit cannabis Yes 15 50 15 50 X2 = 1.339 p = 0.785 No 15 50 15 50 Scale, no statistically significant difference was found in the Morisky Medication Adherence Scale total mean scores of the control group in the pretest and posttest, whereas a statistically significant difference was found in the Morisky Medication Adherence Scale total mean score of the experimental group in the pretest and post- test (p < 0.05). Findings There was a decrease in the Morisky Medication Adherence Scale total mean score in the posttest in the experimental group compared to the control group (Table 4). 1 3 International Journal of Mental Health and Addiction Table 3 Comparison of the pretest and posttest negative automatic thoughts scale total score mean of the individuals in the experimental and control groups (n = 60) Independent samples t test. *Paired t test, p < 0.05 is significant Negative automatic thoughts Control group experimental group Groups Pretest Posttest Test and value (X ± S.S.) (X ± S.S.) Experimental group (n = 30) 110.23 ± 5.88 79.35 ± 4.76 t = − 1.480 p = 0.001 Control group (n = 30) 111.56 ± 5.62 112.38 ± 6.06 t = 1.983 p = 0.246 *Test and value t = 0.131 p = 0.132 t = − 1.309 p = 0.001 Table 3 Comparison of the pretest and posttest negative automatic thoughts scale total score mean of the individuals in the experimental and control groups (n = 60) Table 4 Comparison of the pretest and posttest Morisky Medication Adherence Scale total score mean of the individuals in the experimental and control groups (n = 60) Independent samples t test, *Paired t test, p < 0.05 is significant Morisky medication adherence scale Control group Experimental group Groups Pretest Posttest Test and value (X ± S.S.) (X ± S.S.) Experimental group (n = 30) 2.46 ± 1.08 3.97 ± 1.86 t = 1.140 p = 0.001 Control group (n = 30) 2.56 ± 1.13 2.51 ± 1.06 t = 2.347 p = 0.001 *Test and value t = 1.073 p = 0.865 t = − 2.106 p = 0.001 Table 4 Comparison of the pretest and posttest Morisky Medication Adherence Scale total score mean the individuals in the experimental and control groups (n = 60) Independent samples t test, *Paired t test, p < 0.05 is significant 1 3 Discussion The training showed that individuals may enjoy a temporary state of pleasure when abusing substances, but may regret it afterwards. Explanation of the cognitive, emotional, and physi- cal damages of cannabis abuse to the abusers during the training may have strengthened the willpower of the abusers due to the ability of the abusers to think and recognise themselves better through the training. From this point of view, the training may have affected the nega- tive automatic thoughts of individuals with cannabis use disorder. Before the training, it was found that the medication adherence of the individuals in the control and experimental groups was at a moderate level. One of the most important chal- lenges in substance use disorders is medication adherence. Considerations such as low belief in treatment for substance use disorders and less willpower to quit the substance may make it difficult to adhere to medication. A study on individuals with substance use disorders found that 47.7% of patients suffered from problems with medication adherence during the treat- ment (Blum et al., 2018). Çelikay Söyler et al. found that there were problems in medication adherence in a study conducted with individuals with cannabis use disorder (Çelikay Söyler et al., 2022). This finding of the present study is compatible with the literature. i When the Morisky Medication Adherence Scale total mean score of the control and experimental groups after the training was compared, a statistically significant difference was determined (p < 0.05). After the training, there was no change in the medication adher- ence of the control group in the posttest, whereas there was an increase in the medica- tion adherence of the experimental group. Bayır and Aylaz determined in their study on substance use disorders that mindfulness-based education was effective in medication adherence in substance use disorders (Bayır & Aylaz, 2021). In their study on women with substance use disorders, Amaro and Black found that mindfulness-based therapy increased medication adherence (Amaro & Black, 2017). The increase in the medication adherence of the individuals in the experimental group after mindfulness-based psychoeducation con- firms one of the hypotheses of the study, “Mindfulness-based psychoeducation increases medication adherence in individuals with cannabis use disorder”. Discussion The findings of the study, which were conducted to determine the effect of mindfulness- based psychoeducation on negative automatic thoughts and medication adherence in indi- viduals with cannabis use disorder, were discussed based on the literature. Individuals with substance use disorders may exhibit high levels of negative emotions and thoughts. Swendsen et al. determined that alcohol addicts had high levels of negative emotions and thoughts (Swendsen et al., 2002). Cannabis abuse, one of the substance use disorders, may impair the quality of life of individuals. Cannabis abuse may lead to nega- tive effects on the cognitive, emotional, and thinking abilities of individuals. These nega- tive effects may bring about automatic negative thoughts of individuals. The study by Roos Witkiewitz reported that individuals who held more negative automatic thoughts exhibited higher levels of cannabis abuse (Roos & Witkiewitz, 2017). The study by Budak et al. also reported that individuals with cannabis use disorder had high levels of negative automatic thoughts (Budak et al., 2021). It can be asserted that the individuals in the control and experimental groups had high level of the negative automatic thoughts before the training. 1 3 International Journal of Mental Health and Addiction When the total mean scores of the control and experimental groups in the negative auto- matic thoughts questionnaire after the training was compared, a statistically significant dif- ference was determined between them (p < 0.05). In a study conducted by Öğütçü Zeman et al. with individuals with a history of substance abuse, they found a significant correla- tion between cannabis abuse and negative automatic thoughts (Öğütçü Zeman et al., 2019). In their study, Barrington et al. revealed that individuals with a high level of mindfulness reported lower levels of cannabis abuse (Barrington et al., 2019). Mindfulness-based psy- choeducation has been reported to be effective in treating cannabis use disorder by reduc- ing negative automatic thoughts (Sripada, 2022). After the training, there was no change in the negative automatic thoughts of the control group in the posttest, whereas the nega- tive automatic thoughts of the experimental group decreased. The reduction in the negative automatic thoughts of the individuals in the experimental group after the mindfulness-based psychoeducation confirms one of the hypotheses of the study, “Mindfulness-based psych- oeducation reduces negative automatic thoughts in individuals with cannabis use disorder”. Conclusion The results of the study revealed that mindfulness-based psychoeducation given to indi- viduals with cannabis use disorder was effective in negative automatic thoughts and adher- ence to treatment. It would be an appropriate intervention to organize mindfulness-based psychoeducation programs in psychiatric clinics and alcohol and substance abuse centres in addition to routine treatment. Psychiatric nurses do a one-to-one patient follow-up in the field and actively exercise their basic roles such as teaching, counselling, rehabilitation, case management, and caregiving. The nurses who work in psychiatric clinics should be certified in mindfulness-based psychoeducation training and should be able to practice it on all patients with cannabis use disorder, and this programme must try with other sub- stance use disorders . Discussion Mindfulness-based inter- ventions help individuals to notice and regulate underlying maladaptive thoughts, emo- tional reactions, and automatic and impulsive behaviours of mental health disorders and strengthen individuals to cope with distress by increasing their tolerance for it (Davis et al., 2019; Zümbül, 2021). The results of studies on cannabis abuse have indicated that mindful- ness reduces cannabis abuse and associated adversities (Barrington et al., 2019; Lin et al., 2021; Schneegans et al., 2021). Individuals who abuse cannabis might have raised their mindfulness of medication adherence due to the training they received about medication adherence. This may be considered to have improved their medication adherence. 1 3 International Journal of Mental Health and Addiction Limitations The limitation of the study is that it was conducted in a single centre. Another limitation is that there was no female participant. Acknowledgements The authors would like to thank all patients who gave their time to participate in this research. Funding Open access funding provided by the Scientific and Technological Research Council of Türkiye (TÜBİTAK). This research did not receive any specific grant from funding agencies in the public, commer- cial, or not-for-profit sectors. Data Availability The data used to support the findings of this study are available from the corresponding author on request. Declarations Ethics Approval The present study was approved by the local research ethics committee of Inonu University (and was carried out following the Code of Ethics of the World Medical Association (Declaration of Helsinki) for medical research involving humans (World Medical Association, 2013). Informed Consent Before participating, verbal informed consent was obtained from the patients. In the informed consent, ethical and privacy issues were covered. Conflict of Interest The authors declare no competing interests. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Com- mons licence, and indicate if changes were made. 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Funda Kavak Budak1 · Aysel Akbeniz2 · Fatma Melike Erkan1 · Abdurrezzak Gültekin3 · Hatice Birgül Cumurcu4 Funda Kavak Budak1 · Aysel Akbeniz2 · Fatma Melike Erkan1 · Abdurrezzak Gültekin3 · Hatice Birgül Cumurcu4 Funda Kavak Budak funda.kavak@inonu.edu.tr Aysel Akbeniz aysel_akbeniz@hotmail.com Fatma Melike Erkan fatma.erkan@inonu.edu.tr Abdurrezzak Gültekin abdibey1989@hotmail.com Hatice Birgül Cumurcu hatice.cumurcu@inonu.edu.tr 1 Department of Psychiatric Nursing * Funda Kavak Budak funda.kavak@inonu.edu.tr Aysel Akbeniz aysel_akbeniz@hotmail.com Fatma Melike Erkan fatma.erkan@inonu.edu.tr Abdurrezzak Gültekin abdibey1989@hotmail.com Hatice Birgül Cumurcu hatice.cumurcu@inonu.edu.tr 1 Department of Psychiatric Nursing, Inonu University, Malatya Post Code: 44100, Turkey 2 Department of Nursing, Faculty of Health Sciences, Tarsus University, Tarsus, Mersin Post Code: 33400, Turkey 3 Department of Nursing, Kahramanmaraş Sütçü Imam University, Afsin Health College Afş Kahramanmaraş, Turkey 4 Department of Psychiatry, Inonu University, Malatya Post Code: 44100, Turkey * Funda Kavak Budak funda.kavak@inonu.edu.tr Authors and Affiliations Funda Kavak Budak1 · Aysel Akbeniz2 · Fatma Melike Erkan1 · Abdurrezzak Gültekin3 · Hatice Birgül Cumurcu4 * Funda Kavak Budak funda.kavak@inonu.edu.tr * Funda Kavak Budak funda.kavak@inonu.edu.tr Aysel Akbeniz aysel_akbeniz@hotmail.com Fatma Melike Erkan fatma.erkan@inonu.edu.tr Abdurrezzak Gültekin abdibey1989@hotmail.com Abdurrezzak Gültekin abdibey1989@hotmail.com Hatice Birgül Cumurcu hatice.cumurcu@inonu.edu.tr 1 Department of Psychiatric Nursing, Inonu University, Malatya Post Code: 44100, Turkey 2 Department of Nursing, Faculty of Health Sciences, Tarsus University, Tarsus, Mersin Post Code: 33400, Turkey 3 Department of Nursing, Kahramanmaraş Sütçü Imam University, Afsin Health College Afşin, Kahramanmaraş, Turkey 4 Department of Psychiatry, Inonu University, Malatya Post Code: 44100, Turkey 1 3
https://openalex.org/W3081524466	https://www.nature.com/articles/s41598-020-71317-y.pdf	English	null	Serum BDNF levels correlate with regional cortical thickness in minor depression: a pilot study	Scientific reports	2,020	cc-by	9,235	www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports OPEN Serum BDNF levels correlate with regional cortical thickness in minor depression: a pilot study M. Polyakova1,2,3,9, F. Beyer1,10, K. Mueller1, C. Sander2,3, V. Witte1,3, L. Lampe1,3, F. Rodrigues3,4, S. Riedel‑Heller3,4, J. Kratzsch3,5, K. T. Hoffmann6, A. Villringer1,3,7, P. Schoenknecht2,3,8 & M. L. Schroeter1,3,9 Serum BDNF levels correlate with regional cortical thickness in minor depression: a pilot study M. Polyakova1,2,3,9, F. Beyer1,10, K. Mueller1, C. Sander2,3, V. Witte1,3, L. Lampe1,3, F. Rodrigues3,4, S. Riedel‑Heller3,4, J. Kratzsch3,5, K. T. Hoffmann6, A. Villringer1,3,7, P. Schoenknecht2,3,8 & M. L. Schroeter1,3,9 Serum brain-derived neurotrophic factor (BDNF) reflects state changes in mood disorders. But its relation to brain changes in depression has rarely been investigated in humans. We assessed the association between serum BDNF, cortical thickness, or gray matter volume in 20 subjects with a minor depressive episode and 40 matched healthy subjects. Serum BDNF positively correlated with cortical thickness and volume in multiple brain regions in the minor depression group: the bilateral medial orbitofrontal cortex and rostral anterior cingulate cortex, left insula, and cingulum, right superior frontal gyrus, and other regions—regions typically affected by major depression. Interestingly, these correlations were driven by subjects with first episode depression. There was no significant association between these imaging parameters and serum BDNF in the healthy control group. Interaction analyses supported this finding. Our findings point to a specific association between serum BDNF and magnetic resonance imaging parameters in first-episode minor depression in a region- and condition-dependent manner. A positive correlation between serum BDNF and structural gray matter estimates was most consistently observed for cortical thickness. We discuss why cortical thickness should be preferred to volumetric estimates for such analyses in future studies. Results of our pilot study have to be proven in future larger-scale studies yielding higher statistical power. Minor depression is a subclinical depressive state characterized by depressed mood or lack of interest, combined with one to three other depressive symptoms disturbing a patient over two weeks. In later life minor depres- sion becomes more prevalent than major depressive disorder (MDD)1. Patients suffering from this have an increased risk of developing MDD2 or attempting suicide3. The pathophysiology of minor depression remains largely unexplored4. Its clinical proximity to MDD makes minor depression a good clinical model for examining the earliest pathophysiological changes in depression. Here one has to differentiate between minor depressive episode and minor depressive disorder. For the diagnosis of minor depressive disorder, in contrast to episode, an exclusion of depression history is crucial5.h p y The neurotrophic hypothesis of depression is highly discussed today. It postulates that mood disorders are related to decreased synthesis of brain-derived neurotrophic factor (BDNF) in the brain resulting in impaired synaptogenesis and neuronal activity6. Treatment with antidepressants, on the other hand, increases BDNF secre- tion in the brain7 and in serum8, whereas the latter is associated with recovery from depression9. Methods Subjects. Subjects. Twenty subjects satisfying DSM-IV criteria5 for minor depressive episode were selected from the database of the population-based LIFE-Adult study18. In accordance with Structured Psychiatric Interview for DSM-IV Disorders (SKID), every subject had one to four depressive symptoms for at least two weeks, with depressed mood or loss of interest being one of them. Forty healthy volunteers from the same study were free from depressive symptoms or cognitive impairment and were matched at a 1:2 ratio by sex and age to the sub- jects with minor depression. The study was carried out in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the University of Leipzig. All participants gave written informed consent. pp y y p g p p g Mild and major neurocognitive disorders2–4 (formerly known as mild cognitive impairment and dementia) were excluded according to DSM-5 diagnostic criteria for mild Neurocognitive Disorder (NCD). These criteria require: (A) presence of subjective cognitive disturbance; (B) objective cognitive decline 1–2 standard deviations (SD) below sex- and age-adjusted norms in at least one of five cognitive domains; (C) preserved activities of daily living according to the Activities of Daily Living scale (ADL); (D) absence of delirium and major psychiatric illness (E). ( ) Cognitive testing was performed using the German version of the Consortium to Establish a Registry for Alzheimer’s Disease (CERAD)-plus test battery and a Stroop test. Specific tests or subtests were assigned to each DSM-5 cognitive domain. With Trail Making Test (TMT)-A and Stroop neutral we evaluated attention, with TMT-B/A and Stroop incongruent/neutral executive function. The word list subtest from the CERAD-plus test battery was used for assessment of learning and memory, figure drawing test was used for the visuo-construction/ perception domains. Participants’ scores were compared to normative values adjusted for sex, age, and educa- tion, obtained from the Basel memory clinic (www.memoryclinic.ch). A mean deviation from the norms was calculated for each cognitive domain if this domain was assessed with more than one test.” BDNF measurement. Blood samples were withdrawn from subjects by venipuncture, between 7:25 and 10:45 in the morning, after an overnight fasting. Serum was prepared using the standard operating procedures. In brief, samples were left for 45 min for clotting, followed by a centrifugation step (10 min, 2,750 g, 15 °C). Samples were then filled in straws (CryoBioSytems IMV, France) by an automatic aliquoting system (DIVA, CryoBioSytems IMV, France). www.nature.com/scientificreports/ attempted to relate sBDNF to brain imaging parameters. Some region-of-interest-based analyses revealed a posi- tive correlation between sBDNF and the volume of the hippocampus in healthy subjects10,11. Others found no correlation of hippocampal and amygdala volumes with sBDNF, neither in healthy subject12 nor in subjects with mood disorders13 or schizophrenia12. One study did not find any relation of cortical thickness across the brain to sBDNF in healthy subjects and subjects with recurrent MDD14, and another reported a negative correlation in patients with schizophrenia12. attempted to relate sBDNF to brain imaging parameters. Some region-of-interest-based analyses revealed a posi- tive correlation between sBDNF and the volume of the hippocampus in healthy subjects10,11. Others found no correlation of hippocampal and amygdala volumes with sBDNF, neither in healthy subject12 nor in subjects with mood disorders13 or schizophrenia12. One study did not find any relation of cortical thickness across the brain to sBDNF in healthy subjects and subjects with recurrent MDD14, and another reported a negative correlation in patients with schizophrenia12. Histologically, parameters such as gray matter volume and cortical thickness measured by magnetic resonance imaging (MRI) in vivo represent distinct brain features15. Gray matter volume is, mathematically, a product of thickness and area, where area has more weight16,17. In ontogenesis, cortical surface area is defined by the number of neuronal columns and cortical thickness is defined by the number of neurons within the columns. Moreover, these brain features are related to distinct sets of genes17. In neuroimaging studies, the histological underpinnings of imaging parameters are rarely taken into account. g g p y In this perspective, studies on the correlation between sBDNF and MRI parameters lack a systematic approach, investigating different diseases using different analysis methods, with potentially improper parameters. Since cortical thickness and volume are distinct measures of the brain16, we performed a systematic whole-brain structural MRI study correlating sBDNF levels to these imaging parameters estimated with FreeSurfer. Due to the neurotrophic effects of BDNF we generally hypothesized a positive correlation between sBDNF and corti- cal estimates, modified due to the reduction of sBDNF and regional gray matter volume/cortical thickness in depressive disorders. Differences between subjects with or without a history of depression were assessed in an explorative analysis. Serum BDNF levels correlate with regional cortical thickness in minor depression: a pilot study M. Polyakova1,2,3,9, F. Beyer1,10, K. Mueller1, C. Sander2,3, V. Witte1,3, L. Lampe1,3, F. Rodrigues3,4, S. Riedel‑Heller3,4, J. Kratzsch3,5, K. T. Hoffmann6, A. Villringer1,3,7, P. Schoenknecht2,3,8 & M. L. Schroeter1,3,9 y p In this study, we investigated whether serum (s)BDNF levels are related to changes in human gray matter parameters in subjects with minor depression and in healthy controls. To our knowledge, very few studies have 1Max Planck Institute for Human Cognitive and Brain Sciences, Leipzig, Germany. 2Clinic for Psychiatry and Psychotherapy, University of Leipzig, Leipzig, Germany. 3LIFE—Leipzig Research Center for Civilization Diseases, University of Leipzig, Leipzig, Germany. 4Institute of Social Medicine, Occupational Health and Public Health (ISAP), Leipzig University, Leipzig, Germany. 5Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, Leipzig University, Leipzig, Germany. 6Department of Neuroradiology, Leipzig University, Leipzig, Germany. 7Berlin School of Mind and Brain and the Mind‑Brain Institute, Humboldt-University of Berlin, Berlin, Germany. 8Department of Psychiatry and Psychotherapy, University Affiliated Hospital Arnsdorf, Technical University of Dresden, Dresden, Germany. 9Clinic for Cognitive Neurology, University of Leipzig, Leipzig, Germany. 10CRC Obesity Mechanisms, Subproject A1, University of Leipzig, Leipzig, Germany. email: polyakova@cbs.mpg.de \| https://doi.org/10.1038/s41598-020-71317-y Scientific Reports \| (2020) 10:14524 www.nature.com/scientificreports/ Results i i Participants’ characteristics. Subjects with minor depressive episode were not significantly different from control subjects in terms of age, sex, body mass index (BMI), and amount of white matter hyperintensities as rated using the Fazekas scale. Levels of sBDNF were also comparable, i.e. not significantly different, between both groups (see Table 1). Cortical thickness. Cortical thickness, and gray matter volume, were not statistically different between both groups (Supplementary Table 1 and 2), whereas sBDNF correlated with imaging parameters. At p < 0.05, we observed a positive correlation between sBDNF and cortical thickness only in the minor depression group as illustrated in Fig. 1 and Table 2. On the uncorrected level, sBDNF positively correlated with cortical thickness in the left medial orbitofrontal, the rostral and caudal anterior cingulate cortex, posterior and isthmus cingulate cortex, and the insula and precuneus. In the right hemisphere we observed positive correlations between sBDNF and cortical thickness in the medial orbitofrontal, superior frontal, rostral anterior cingulate cortex, superior parietal cortex, temporal pole and transverse temporal, as well as with the supramarginal, postcentral and peri- calcarine gyrus (Fig. 1). No regions remained significant after the FDR correction for multiple comparisons pFDR < 0.05 (see Table 2). h l h b h h l d d b ( bl ) h In healthy subjects, contrary to our hypothesis, correlations tended to be negative (Table 2). On the uncor- rected level (p < 0.05), we observed significant negative correlations between sBDNF and cortical thickness of the bilateral cuneus, right lingual gyrus, and insula. Positive correlations were observed only for the left caudal anterior cingulate cortex and right entorhinal region. Interestingly, a negative correlation between sBDNF and thickness in the right cuneus was significant at the pFDR < 0.05 threshold.fi i Between-group interaction effects were significant for correlations between sBDNF and cortical thickness in the bilateral cuneus and insula, left medial orbitofrontal cortex, precuneus, isthmus and posterior cingulate cortex, as well as the right pericalcarine and lingual gyrus, pars opercularis and superior parietal lobule. In all these cases, we observed positive correlations in subjects with minor depression and near-zero or negative cor- relations in healthy participants (Table 2). Cortical volume. Correlations between sBDNF and volumetric data are illustrated in Fig. 2 and Table 3. The regional correlation pattern was similar between the volumetric and cortical thickness data (see Figs. 1 and 2). Methods Subjects. To minimize freeze–thaw cycles, samples were sorted in a cryogenic work bench (temperatures below − 100 °C) and automatically stored in tanks with a coolable top frame in the gas phase of liquid nitrogen (Askion, Germany) and stored for analysis18. Serum BDNF was assessed using an ELISA kit manufactured by R&D Systems (Wiesbaden, Germany) as previously described4. Neuroimaging—measurement of gray matter volume & thickness. T1-weighted images were acquired with a 3-T Magnetom Verio Scanner (Siemens Healthcare, Erlangen, Germany) using three-dimen- sional magnetization-prepared rapid gradient-echo imaging (3D MP-RAGE) protocol with the following parameters: inversion time 900 ms; repetition time 2,300 ms; echo time 2.98 ms; flip angle 9°; field of view 256 × 240 × 176 mm; voxel size 1 × 1 × 1 mm. To analyze gray matter volume and cortical thickness, T1-weighted images were preprocessed using FreeSurfer version 5.3.0 (https://surfer.nmr.mgh.harvard.edu/) 19.t MR images were preprocessed using the standard pipeline recon-all. After normalization and skull-stripping of the T1-weighted images, cortical tissue boundaries were reconstructed and transformed to a subject-specific surface mesh. The distance between pial and gray/white matter surfaces at each vertex location of the mesh was calculated in order to obtain cortical thickness measurements20. Based on Desikan-Killiany’s cortical parcellation, regional cortical thickness and gray matter volume was extracted separately for the several brain regions in each hemisphere and averaged for the analysis. All images were visually checked for misplaced tissue boundaries and manually corrected if necessary. Scientific Reports \| (2020) 10:14524 \| https://doi.org/10.1038/s41598-020-71317-y www.nature.com/scientificreports/ Table 1. Participants’ characteristics. Chi-square test for sex, independent sample t test for age, body mass index (BMI), serum brain derived neurotrophic factor (sBDNF), Mann–Whitney U test for the Fazekas score. Subjects with minor depression Healthy subjects p-value N (with history of depression) 20 (12) 40 – Sex (male/female) 5/15 10/30 1.0 Age (years) 70.3 (4.3) 69.6 (4.3) 0.57 Fazekas score (0/1/2) 6/12/2 12/23/5 0.90 BMI (kg/m2) 28.3 (5.2) 28.4 (5.2) 0.91 sBDNF (µg/l) 26.0 (5.1) 25.7 (7.1) 0.84 Subjects with minor depression Healthy subjects p-value N (with history of depression) 20 (12) 40 – Sex (male/female) 5/15 10/30 1.0 Age (years) 70.3 (4.3) 69.6 (4.3) 0.57 Fazekas score (0/1/2) 6/12/2 12/23/5 0.90 BMI (kg/m2) 28.3 (5.2) 28.4 (5.2) 0.91 sBDNF (µg/l) 26.0 (5.1) 25.7 (7.1) 0.84 Table 1. Participants’ characteristics. Methods Subjects. Chi-square test for sex, independent sample t test for age, body mass index (BMI), serum brain derived neurotrophic factor (sBDNF), Mann–Whitney U test for the Fazekas score. Statistics. The statistical analysis was performed in SPSS Version 24 (IBM Corp., Armonk, NY, USA). After the visual assessment of data distributions, gray matter volume, normalized to total intracranial volume (TIV), and cortical thickness estimates were correlated with sBDNF levels by calculating Pearson’s correlation coeffi- cients separately for each group. First, we used the uncorrected p value < 0.05 (one-tailed, directed hypothesis). We subsequently corrected for multiple comparisons using the false discovery rate (FDR) approach as suggested by Benjamini–Hochberg21 with a threshold of 0.05. The family of tests included all segmented brain regions and mean thickness (68 regions left/right tests for the left/right analysis). We report uncorrected p values along with the calculated FDR p value21. These are labelled accordingly throughout the tables in bold. Interaction effects were tested between the significant correlations in minor depression and healthy control groups by using Fisher’s z-test. Subgroup analysis was performed post hoc according to the same procedures as the main analysis. Figures were prepared by MP in Blender 2.78 software (https://www.blender.org/) using the Desikan-Killiani template by Prof. Anderson Winkler (https://brainder.org/research/brain-for-blender/). Results i i In subjects with minor depression at p < 0.05, sBDNF correlated positively with bilateral medial orbitofrontal and pericalcarine cortical volume. Additionally, in the left hemisphere, we observed positive correlations between sBDNF and volumes of the left rostral, caudal, and anterior cingulate, as well as the posterior cingulate cortex, precuneus, fusiform, entorhinal, and postcentral gyrus. In the right hemisphere, sBDNF positively correlated with volumes of the isthmus cingulate, lateral orbitofrontal, precentral cortex, pars orbitalis of the inferior fron- tal gyrus, superior parietal and superior temporal gyrus, as well as with the temporal pole and supramarginal gyrus. gy In healthy subjects, negative correlations at p < 0.05 were found between sBDNF and volumes of the right superior parietal cortex, right cuneus, lingual and fusiform, as well as with the left postcentral, and lingual gyrus. None of these correlations remained significant after FDR correction. Scientific Reports \| (2020) 10:14524 \| https://doi.org/10.1038/s41598-020-71317-y www.nature.com/scientificreports/ Figure 1. Correlation of serum BDNF with cortical thickness and normalized cortical volume in subjects with minor depression and healthy controls. (A) Correlation of sBDNF with cortical thickness in subjects with minor depression; (B) Correlation of sBDNF with cortical thickness in healthy controls; (C) Correlation of sBDNF with cortical volume normalized to total intracranial volume in subjects with minor depression; (D) Correlation of sBDNF with cortical volume normalized to total intracranial volume in healthy controls; BDNF Brain- Derived Neurotrophic Factor, ACC anterior cingulate cortex, OFC orbitofrontal cortex, PCC posterior cingulate cortex. Figures were prepared in Blender 2.78 software (https://www.blender.org/) using the Desikan-Killiani template by Anderson Winkler (https://brainder.org/research/brain-for-blender/). Figure 1. Correlation of serum BDNF with cortical thickness and normalized cortical volume in subjects with minor depression and healthy controls. (A) Correlation of sBDNF with cortical thickness in subjects with minor depression; (B) Correlation of sBDNF with cortical thickness in healthy controls; (C) Correlation of sBDNF with cortical volume normalized to total intracranial volume in subjects with minor depression; (D) Correlation of sBDNF with cortical volume normalized to total intracranial volume in healthy controls; BDNF Brain- Derived Neurotrophic Factor, ACC anterior cingulate cortex, OFC orbitofrontal cortex, PCC posterior cingulate cortex. Figures were prepared in Blender 2.78 software (https://www.blender.org/) using the Desikan-Killiani template by Anderson Winkler (https://brainder.org/research/brain-for-blender/). Results i i Interaction effects were significant for correlations of sBDNF with volumes of the left posterior and rostral anterior cingulate cortex, precuneus, postcentral, lingual gyrus, as well as for correlations with right medial orbitofrontal, middle temporal, lingual, superior parietal, superior temporal and supramarginal volumes. Simi- lar to cortical thickness, positive correlations characterized the minor depression group, and negative ones the healthy control group. Subgroup analysis. Finally, we performed a post hoc subgroup analysis to investigate potential differences between persons with and without a history of depression (n = 8 vs n = 12). The results are depicted in Supple- mentary Tables 3–6 and Fig. 2. Interestingly, cortical thickness was larger in subjects without history of depres- sion (Supplementary Table 3). Further analysis showed that correlation between cortical thickness and sBDNF in the minor depression group was driven by subjects without a history of depression. Correlation between sBDNF and right medial orbitofrontal cortical thickness in this subgroup remained significant after FDR correction. Interaction effects between both subgroups were significant for the left lateral orbitofrontal gyrus, right medial orbitofrontal gyrus, right pars triangularis of the inferior frontal gyrus, the rostral anterior cingulate cortex, and superior frontal gyrus. In all regions, correlations in subjects with first-episode minor depression were significantly higher than in subjects with recurrent depression. j p Gray matter volume correlated both positively and negatively with sBDNF in both subgroups. However, none of these correlations remained significant after FDR correction. Interaction effects were significant for correla- tion between sBDNF and left middle temporal, right pericalcarine, and right posterior cingulate volumes. In all these cases, negative correlations were observed in subjects with first-episode minor depression and positive correlations in subjects with recurrent episode. Discussion To our knowledge, this is the first structural MRI study investigating the correlation between sBDNF and gray matter parameters in minor depression. At the uncorrected level (p < 0.05) positive correlation was detected in multiple depression-related regions in subjects with minor depressive episode, but not in the control group. The respective interaction effects were significant. The post hoc analysis revealed that correlations with cortical thick- ness were driven by subjects with first-episode minor depression, while volumetric data showed mixed effects. Discussion Scientific Reports \| (2020) 10:14524 \| https://doi.org/10.1038/s41598-020-71317-y www.nature.com/scientificreports/ Region of interest Subjects with minor depression Healthy controls Interaction analysis Pearson’s correlation p-value pFDR 0.05 Pearson’s correlation p-value pFDR 0.05 Fisher’s z p-value Left hemisphere Bank of the superior temporal sulcus − 0.21 0.18 0.03 − 0.03 0.44 0.04 – – Caudal anterior cingulate 0.39 0.05 0.01 0.40 0.01 0.001 − 0.04 0.48 Caudal middle frontal 0.16 0.25 0.04 − 0.03 0.44 0.04 – – Cuneus 0.24 0.16 0.03 − 0.35 0.01 0.004 2.08 0.02 Entorhinal cortex 0.28 0.12 0.03 0.17 0.14 0.02 – – Frontal pole 0.11 0.33 0.04 0.14 0.19 0.02 – – Fusiform gyrus 0.34 0.07 0.02 0.19 0.13 0.01 – – Inferior parietal gyrus 0.03 0.45 0.05 0.25 0.06 0.01 – – Inferior temporal gyrus 0.35 0.06 0.01 0.23 0.08 0.01 – – Insula 0.40 0.04 0.01 − 0.14 0.20 0.02 1.90 0.03 Isthmus cingulate 0.49 0.01 0.004 − 0.04 0.40 0.03 1.96 0.03 Lateral occipital sulcus − 0.04 0.44 0.05 0.01 0.49 0.05 – – Lateral orbitofron- tal cortex 0.24 0.15 0.03 0.03 0.44 0.04 – – Lingual gyrus 0.18 0.22 0.04 − 0.23 0.07 0.01 – – Medial orbitofron- tal cortex 0.49 0.01 0.003 0.19 0.12 0.01 1.16 0.12 Middle temporal gyrus 0.12 0.31 0.04 0.23 0.08 0.01 – – Paracentral gyrus 0.16 0.25 0.04 − 0.02 0.46 0.04 – – Parahippocampal gyrus − 0.04 0.44 0.05 0.13 0.21 0.02 – – Pars opercularis 0.31 0.09 0.02 0.02 0.45 0.04 – – Pars orbitalis − 0.04 0.44 0.05 − 0.001 0.50 0.05 – – Pars triangularis 0.04 0.44 0.05 − 0.24 0.07 0.01 – – Pericalcarine cortex 0.35 0.07 0.02 − 0.27 0.05 0.01 2.17 0.15 Postcentral gyrus 0.13 0.29 0.04 − 0.20 0.11 0.01 – – Posterior cingu- late cortex 0.63 0.002 0.001 0.18 0.14 0.01 1.90 0.03 Precentral gyrus 0.10 0.34 0.04 0.07 0.34 0.03 – – Precuneus 0.46 0.02 0.01 − 0.03 0.43 0.04 1.69 0.05 Rostral anterior cingulate cortex 0.42 0.03 0.01 0.04 0.41 0.04 1.39 0.82 Rostral middle frontal cortex 0.32 0.08 0.02 0.14 0.19 0.02 – – Superior frontal gyrus 0.32 0.09 0.02 0.03 0.44 0.04 – – Superior parietal gyrus − 0.16 0.25 0.04 − 0.02 0.44 0.04 – – Superior temporal gyrus 0.25 0.15 0.03 0.09 0.30 0.03 – – Supramarginal gyrus 0.17 0.24 0.04 − 0.04 0.41 0.04 – – Temporal pole 0.18 0.22 0.03 0.24 0.07 0.01 – – Transverse tempo- ral gyrus 0.25 0.15 0.03 0.14 0.20 0.02 – – Right hemisphere Banks of the superior temporal sulcus 0.32 0.09 0.02 − 0.01 0.48 0.05 – – Caudal anterior cingulate 0.15 0.26 0.04 0.09 0.29 0.03 – – Caudal middle frontal 0.49 0.02 0.01 0.08 0.31 0.03 1.54 0.62 Continued https://doi.org/10.1038/s41598-020-71317-y Scientific Reports \| (2020) 10:14524 \| www.nature.com/scientificreports/ Table 2. Discussion Correlation between cortical thickness and serum BDNF in subjects with minor depression and healthy controls. BDNF brain derived neurotrophic factor, 1-tailed p-values are reported, FDR p value is Region of interest Subjects with minor depression Healthy controls Interaction analysis Pearson’s correlation p-value pFDR 0.05 Pearson’s correlation p-value pFDR 0.05 Fisher’s z p-value Cuneus 0.26 0.13 0.03 − 0.50 0.001 0.001 2.79 < 0.001 Entorhinal cortex 0.29 0.11 0.03 0.36 0.01 0.003 − 0.26 0.40 Frontal pole − 0.03 0.45 0.05 0.17 0.14 0.02 – – Fusiform gyrus 0.30 0.10 0.02 − 0.17 0.15 0.02 – – Inferior parietal gyrus 0.29 0.11 0.03 0.19 0.12 0.01 – – Inferior temporal gyrus 0.30 0.10 0.02 0.20 0.11 0.01 – – Insula 0.32 0.08 0.02 − 0.31 0.03 0.004 2.24 0.01 Isthmus cingulate 0.10 0.34 0.04 0.06 0.37 0.03 – – Lateral occipital sulcus 0.34 0.07 0.02 0.02 0.46 0.04 – – Lateral orbitofron- tal cortex 0.13 0.29 0.04 0.10 0.26 0.03 – – Lingual gyrus 0.33 0.08 0.02 − 0.38 0.01 0.002 2.53 0.01 Medial orbitofron- tal cortex 0.57 0.005 0.001 − 0.13 0.21 0.02 2.65 < 0.001 Middle temporal gyrus 0.34 0.07 0.02 0.14 0.19 0.02 – – Paracentral gyrus 0.17 0.24 0.04 0.05 0.38 0.03 – – Parahippocampal gyrus 0.06 0.40 0.05 0.16 0.17 0.02 – – Pars opercularis 0.48 0.02 0.01 0.03 0.42 0.04 1.68 0.05 Pars orbitalis 0.34 0.07 0.02 0.03 0.44 0.04 – – Pars triangularis 0.30 0.10 0.03 0.07 0.34 0.03 – – Pericalcarine cortex 0.52 0.01 0.00 − 0.11 0.25 0.02 2.35 0.01 Postcentral gyrus 0.23 0.17 0.03 − 0.08 0.32 0.03 – – Posterior cingu- late cortex 0.12 0.30 0.04 0.11 0.26 0.03 – – Precentral gyrus 0.41 0.04 0.01 0.01 0.47 0.05 1.44 0.07 Precuneus 0.19 0.21 0.03 − 0.12 0.24 0.02 – – Rostral anterior cingulate cortex 0.41 0.04 0.01 − 0.08 0.31 0.03 1.78 0.38 Rostral middle frontal cortex 0.22 0.18 0.03 − 0.01 0.47 0.05 – – Superior frontal gyrus 0.38 0.05 0.01 0.01 0.48 0.05 1.35 0.09 Superior parietal gyrus 0.41 0.04 0.01 − 0.07 0.34 0.03 1.70 0.04 Superior temporal gyrus 0.36 0.06 0.01 − 0.01 0.47 0.04 – – Supramarginal gyrus 0.49 0.01 0.004 0.01 0.47 0.05 1.78 0.38 Temporal pole 0.44 0.03 0.01 0.24 0.07 0.01 0.77 0.22 Transverse tempo- ral gyrus 0.48 0.02 0.01 0.08 0.31 0.03 1.49 0.07 Table 2. Discussion (A) Correlation of sBDNF with cortical thickness in subjects without history of depression; (B) Correlation of sBDNF with cortical thickness in subjects with history of depression; (C) Correlation of sBDNF with cortical volume normalized to total intracranial volume in subjects without history of depression; (D) Correlation of sBDNF with cortical volume normalized to total intracranial volume in subjects with history of depression; BDNF Brain-Derived Neurotrophic Factor, ACC anterior cingulate cortex, OFC orbitofrontal cortex, PCC posterior cingulate cortex. Figures were prepared in Blender 2.78 (https://www.blender.org/) using the Desikan-Killiani template (https://brainder.org/research/brain -for-blender/). individual variability, volumetric measures are more influenced by the area estimates16. Moreover, the FreeSurfer algorithm has shown a tendency to misestimate cortical volume22.i Histologically, cortical area is defined by the number of neuronal columns, while cortical thickness by the number of neurons and their connections within the column23. The change of clinical state from euthymic to depressed is unlikely to alter the number of neuronal columns, and, therefore, cortical area and volume. Further- more, sBDNF is a dynamic measure24,25. In light of the neurotrophic hypothesis, a number of neuronal connec- tions is thought to decrease due to deficiency of neurotrophic factors in depression6. Therefore, we suggest that cortical thickness is much more useful for clinical studies compared to cortical volume to examine state changes in depression. Accordingly, we will further discuss results for this parameter only. Correlation between serum BDNF and regional cortical thickness seems to be relevant in early minor depressive states. In this study, sBDNF correlated positively with cortical thickness of numerous brain regions in minor depression. Though none of these correlations remained significant after the rigorous FDR correction, the total number of correlations was substantially higher than the expected at 5% false-positive rate (3.4 significant results are expected out of 68). Moreover, note that correlation coefficients reached relatively high values, explaining a high amount of variability in the data. sBDNF correlated positively with the thickness of the bilateral medial orbitofrontal cortex and rostral anterior cingulate, left cingulate cortex, insula, and right superior frontal gyrus. Discussion Correlation between cortical thickness and serum BDNF in subjects with minor depression and healthy controls. BDNF brain derived neurotrophic factor, 1-tailed p-values are reported, FDR p value is derived using the Benjamini–Hochberg procedure, Fisher’s z-test for interaction analysis was performed only for significant correlations. Regions significantly correlating with sBDNF at p < 0.05 are marked as bold. Table 2. Correlation between cortical thickness and serum BDNF in subjects with minor depression and healthy controls. BDNF brain derived neurotrophic factor, 1-tailed p-values are reported, FDR p value is derived using the Benjamini–Hochberg procedure, Fisher’s z-test for interaction analysis was performed only for significant correlations. Regions significantly correlating with sBDNF at p < 0.05 are marked as bold. Though most of these correlations remained non-significant after the FDR correction, they should inform future studies about the effect direction, effect size, and required sample size. Imaging phenotype matters—cortical thickness should be preferred to cortical volume in depression. Following a recent publication from the field of imaging genetics17, it is reasonable to argue that thickness and volume estimates are not interchangeable also in clinical investigations. In the FreeSurfer estima- tions gray matter volume is a product of cortical area by cortical thickness16,17. Since cortical area has larger inter- https://doi.org/10.1038/s41598-020-71317-y Scientific Reports \| (2020) 10:14524 \| www.nature.com/scientificreports/ Figure 2. Subgroup analysis: Correlation of serum BDNF with cortical thickness and normalized cortical volume in subjects with or without history of depression. (A) Correlation of sBDNF with cortical thickness in subjects without history of depression; (B) Correlation of sBDNF with cortical thickness in subjects with history of depression; (C) Correlation of sBDNF with cortical volume normalized to total intracranial volume in subjects without history of depression; (D) Correlation of sBDNF with cortical volume normalized to total intracranial volume in subjects with history of depression; BDNF Brain-Derived Neurotrophic Factor, ACC anterior cingulate cortex, OFC orbitofrontal cortex, PCC posterior cingulate cortex. Figures were prepared in Blender 2.78 (https://www.blender.org/) using the Desikan-Killiani template (https://brainder.org/research/brain -for-blender/). Figure 2. Subgroup analysis: Correlation of serum BDNF with cortical thickness and normalized cortical volume in subjects with or without history of depression. Discussion These regions are typically activated in functional MRI paradigms that assess emotion regulation in healthy subjects26,27 and in major depression22,27,28, and show changes in structure and glucose metabolism in MDD as revealed by systematic and quantitative meta-analyses29 and histopathological studies with glial and later neuronal alterations30–32.i g Whether this correlation is specific to minor depression as compared to major depression remains to be investigated. Some considerations may be drawn from other studies of cortical thickness and sBDNF. Cortical thinning was robustly detected in patients with first episode major depression in a large scale study of ENIGMA consortium33, as well as smaller studies34–36. In minor depression we did not observe these effects37. Serum BDNF has been unchanged in first episode major depression38 and in minor depression4. One study has reported a positive correlation between sBDNF and hippocampal volume in first episode major depression in a region-of- interest analysis39. Scientific Reports \| (2020) 10:14524 \| Discussion Correlation between normalized gray matter volume and serum BDNF in subjects with minor depression and healthy controls. BDNF brain derived neurotrophic factor, 1-tailed p values are reported, FDR p value is derived using the Benjamini–Hochberg procedure, Fisher’s z-test for interaction analysis was performed only for significant correlations. Regions significantly correlating with sBDNF at p = 0.05 are marked as bold. Table 3. Correlation between normalized gray matter volume and serum BDNF in subjects with minor depression and healthy controls. BDNF brain derived neurotrophic factor, 1-tailed p values are reported, FDR p value is derived using the Benjamini–Hochberg procedure, Fisher’s z-test for interaction analysis was performed only for significant correlations. Regions significantly correlating with sBDNF at p = 0.05 are marked as bold. An earlier study, investigating the relation of sBDNF to cortical thickness in patients with recurrent major depression, did not show such a correlation14. These patients had a recurrent severe (major) depressive disorder, which likely exhausted BDNF resources. Our previous meta-analysis investigating the effects of electro-convulsive therapy on BDNF in such patients showed no response of sBDNF to therapy40. In patients with less severe depres- sive disorder sBDNF responds much better to anti-depressive treatment9. In line with this argument, our minor depression subtype analyses revealed that the correlation between sBDNF and cortical thickness was driven by subjects without a history of depression. In summary, a significant positive correlation between sBDNF and corti- cal thickness might be only relevant in early depressive states and might indicate a compensatory mechanism, because it was neither detected in healthy controls nor in minor depressive states with a history of depression. With respect to healthy controls, we replicated previous findings14. p y p pi g Regions correlating with sBDNF in our study substantially overlapped with regional cortical thinning in MDD shown in a recent very powerful meta-analysis33. Here, the bilateral medial orbitofrontal cortex, fusiform gyrus, insula, rostral anterior and posterior cingulate cortex and, unilaterally, the left middle temporal gyrus, right inferior temporal gyrus, and right caudal anterior cingulate cortex were significantly thinner in the MDD group than in healthy controls. The obtained effect sizes for cortical thinning were relatively small in this meta- analysis (Cohen’s d − 0.13 to 0.49)33. Discussion Scientific Reports \| (2020) 10:14524 \| https://doi.org/10.1038/s41598-020-71317-y www.nature.com/scientificreports/ Region of Interest Subjects with minor depression Healthy subjects Interaction analysis Pearson’s correlation p-value pFDR 0.05 Pearson’s correlation p-value pFDR 0.05 Fisher’s z p-value Left hemisphere Banks of the superior temporal sulcus − 0.05 0.42 0.05 0.13 0.21 0.02 – – Caudal anterior cingulate cortex 0.46 0.02 0.01 0.20 0.11 0.01 1.00 0.16 Caudal middle frontal 0.15 0.26 0.04 − 0.07 0.33 0.03 – – Cuneus 0.25 0.14 0.03 − 0.14 0.19 0.02 – – Entorhinal cortex 0.40 0.04 0.01 0.31 0.02 0.003 0.34 0.37 Frontal pole 0.21 0.19 0.03 0.24 0.06 0.01 – – Fusiform gyrus 0.47 0.02 0.01 0.09 0.29 0.03 1.44 0.07 Inferior parietal gyrus 0.12 0.30 0.04 0.06 0.35 0.04 – – Inferior temporal gyrus 0.17 0.23 0.04 0.16 0.16 0.02 – – Insula 0.35 0.07 0.02 0.33 0.02 0.001 0.08 0.47 Isthmus cingulate cortex 0.21 0.19 0.03 0.08 0.32 0.03 – – Lateral occipital sulcus − 0.11 0.33 0.05 0.07 0.33 0.04 – – Lateral orbitofrontal cortex 0.30 0.10 0.02 0.16 0.16 0.01 – – Lingual gyrus 0.19 0.21 0.03 − 0.25 0.06 0.01 – – Medial orbitofrontal cortex 0.63 0.001 0.001 0.32 0.02 0.001 1.39 0.08 Middle temporal gyrus 0.13 0.29 0.04 0.12 0.24 0.03 – – Paracentral gyrus − 0.23 0.16 0.03 − 0.09 0.29 0.03 – – Parahippocampal gyrus 0.11 0.32 0.05 0.21 0.10 0.01 – – Pars opercularis 0.27 0.12 0.02 − 0.07 0.33 0.04 – – Pars orbitalis 0.15 0.27 0.04 0.13 0.20 0.02 – – Pars triangularis 0.14 0.27 0.04 − 0.15 0.17 0.02 – – Pericalcarine cortex 0.40 0.04 0.01 − 0.14 0.20 0.02 1.9 0.03 Postcentral gyrus 0.45 0.02 0.01 − 0.26 0.05 0.01 2.6 0.005 Posterior cingulate cortex 0.58 0.004 0.002 − 0.02 0.46 0.05 2.3 0.01 Precentral gyrus − 0.01 0.48 0.05 0.02 0.44 0.05 – – Precuneus 0.57 0.004 0.003 − 0.11 0.25 0.03 2.57 0.005 Rostral anterior cingulate cortex 0.59 0.003 0.001 0.13 0.21 0.02 1.84 0.03 Rostral middle frontal cortex 0.26 0.13 0.03 0.11 0.24 0.03 – – Superior frontal gyrus 0.09 0.36 0.05 0.05 0.37 0.04 – – Superior parietal gyrus 0.17 0.23 0.04 − 0.06 0.35 0.04 – – Superior temporal gyrus 0.29 0.10 0.02 0.09 0.28 0.03 – – Supramarginal gyrus 0.28 0.11 0.02 − 0.12 0.23 0.03 – – Temporal pole 0.18 0.22 0.03 0.24 0.07 0.01 – – Transverse temporal gyrus 0.24 0.15 0.03 0.08 0.32 0.03 – – Right hemisphere Banks of the superior temporal sulcus − 0.16 0.25 0.04 0.19 0.12 0.01 – – Caudal anterior cingulate − 0.19 0.21 0.03 0.07 0.33 0.04 – – Caudal middle frontal cortex 0.28 0.12 0.02 − 0.02 0.46 0.05 – – Cuneus 0.09 0.35 0.05 − 0.27 0.05 0.004 1.24 0.11 Entorhinal cortex − 0.16 0.25 0.04 0.16 0.16 0.01 – – Frontal pole 0.24 0.15 0.03 − 0.03 0.42 0.05 – – Fusiform gyrus 0.16 0.25 0.04 − 0.23 0.08 0.01 – – Inferior parietal gyrus 0.14 0.28 0.04 0.06 0.35 0.04 – – Inferior temporal gyrus 0.34 0.07 0.02 0.23 0.08 0.01 – – Insula 0.30 0.10 0.02 0.14 0.19 0.02 – – Isthmus cingulate cortex 0.42 0.03 0.01 − 0.02 0.45 0.05 1.6 0.05 Lateral occipital gyrus 0.37 0.05 0.01 0.05 0.37 0.04 1.15 0.12 Lateral orbitofrontal sulcus 0.38 0.05 0.01 0.12 0.23 0.03 0.95 0.17 Continued Scientific Reports \| (2020) 10:14524 \| https://doi.org/10.1038/s41598-020-71317-y www.nature.com/scientificreports/ Region of Interest Subjects with minor depression Healthy subjects Interaction analysis Pearson’s correlation p-value pFDR 0.05 Pearson’s correlation p-value pFDR 0.05 Fisher’s z p-value Lingual gyrus 0.34 0.07 0.02 − 0.24 0.07 0.01 – – Medial orbitofrontal cortex 0.50 0.01 0.004 − 0.05 0.38 0.04 2.03 0.02 Middle temporal gyrus 0.22 0.18 0.03 − 0.07 0.34 0.04 – – Paracentral gyrus − 0.22 0.17 0.03 0.12 0.23 0.03 – – Parahippocampal gyrus − 0.14 0.28 0.04 0.17 0.15 0.01 – – Pars opercularis 0.31 0.09 0.02 0.01 0.47 0.05 – – Pars orbitalis 0.39 0.04 0.01 0.11 0.25 0.03 1.03 0.15 Pars triangularis 0.26 0.14 0.03 0.15 0.19 0.02 – – Pericalcarine cortex 0.40 0.04 0.01 − 0.04 0.40 0.04 1.59 0.06 Postcentral gyrus − 0.003 0.50 0.05 − 0.14 0.20 0.02 – – Posterior cingulate cortex 0.34 0.07 0.02 0.06 0.35 0.04 – – Precentral gyrus 0.48 0.02 0.01 0.07 0.34 0.04 1.55 0.06 Precuneus 0.36 0.06 0.02 − 0.06 0.36 0.04 – – Rostral anterior cingulate cortex − 0.21 0.19 0.03 0.13 0.21 0.02 – – Rostral middle frontal cortex 0.16 0.25 0.04 − 0.14 0.19 0.02 – – Superior frontal gyrus 0.17 0.23 0.04 0.19 0.11 0.01 – – Superior parietal gyrus 0.39 0.04 0.01 − 0.27 0.05 0.004 2.34 0.01 Superior temporal gyrus 0.42 0.03 0.01 − 0.09 0.29 0.03 – – Supramarginal gyrus 0.55 0.01 0.004 − 0.15 0.18 0.02 2.6 0.004 Temporal pole 0.49 0.02 0.01 0.34 0.02 0.001 0.6 0.27 Transverse temporal gyrus 0.35 0.07 0.02 0.01 0.48 0.05 – – Table 3. Discussion This suggests that large sample sizes are required for such studies.i h gg g We have shown here for the first time that in minor depression the correlation of sBDNF with cortical thick- ness is significantly different from controls. The subtype analysis suggested that this correlation was mainly driven by subjects with first episode depression. These data provide insight into the early mechanisms of depression with a focus on neuroendocrine mechanisms, possibly indicating an early compensatory mechanism, similar to other diseases41,42. Furthermore, it also shows that no universal positive correlation between brain measures and BDNF exists. Similarly, animal studies have shown that correlations between brain BDNF and sBDNF is very much region- and strain-specific40.l y g pi Whilst cortical thickness is a relatively straightforward measure, biological processes, reflected by sBDNF, are less understood. It has been long supposed that sBDNF reflects cortical and hippocampal secretion of BDNF43,44. However, a recent study has shown that sBDNF is instead derived from megakaryocytes45 and not from the brain. Therefore, mechanisms linking brain and serum BDNF are yet to be further examined. h g y Both cortical thickness46 and sBDNF9,47 are reduced in MDD. In our previous reports comparing subjects with minor depression, we found neither sBDNF differences4 nor differences in cortical measures37. The evidence we provide here is correlational and by no means causative. However, we might have observed an early sign of Scientific Reports \| (2020) 10:14524 \| https://doi.org/10.1038/s41598-020-71317-y www.nature.com/scientificreports/ neurotrophic function in early subclinical depression, not yet visible on the biomarker or whole-brain level. This observation should be confirmed by future studies. neurotrophic function in early subclinical depression, not yet visible on the biomarker or whole-brain level. This observation should be confirmed by future studies. Limitations Our study has a number of limitations. Firstly, due to the unexpectedly low prevalence of minor depression in our sample, which originated from a large population-based study with approximately 2,500 participants, our sample size was relatively small and we had to include subjects with and without a history of depression. Because our study is the first one in minor depression, an a priori power analysis was not feasible. A previous study inves- tigating the correlation between sBDNF and hippocampal volumes in early major depression used a comparable sample size n = 2539, suggesting we had enough statistical power. To increase power, we matched our sample on a 1:2 basis to healthy controls. Secondly, we did not have precise information on the duration of minor depressive state burden, which might be an additional parameter of interest for further analyses. Although the ELISA kits used for sBDNF quantification were not optimal according to a recent publication48, these kits were purchased prior to this publication. We used a whole-brain approach guaranteeing data-driven statistics in both cortical thickness and volume. Although only a minority of results survived correction for multiple comparisons using the FDR procedure, we underlined validity of our findings by interaction analyses demonstrating specificity compared to healthy subjects. Future studies are necessary to prove our pilot findings in larger and preferably multi-centric cohorts. Finally, we did not use the voxel-wise estimation, because we wanted to make our data comparable to the recent meta-analysis by the ENIGMA consortium. Summary y In this study, we observed a positive correlation between serum BDNF measurements and structural gray mat- ter estimates in minor depression. The correlation between sBDNF and imaging parameters was region- and condition-dependent. These findings require verification in larger samples considering a-priori power estima- tions and controlling for the duration of depression burden. Furthermore, our analysis suggests that cortical thickness is a more suitable structural parameter for biomarker studies than gray matter volume, at least in studies of depression. Received: 6 March 2020; Accepted: 10 August 2020 References erences olyakova, M. et al. Prevalence of minor depression in elderly persons with and without mild cognitive impairment: A systematic eview. J. Affect. Disord. 152, 28–38 (2014). 1. Polyakova, M. et al. Prevalence of minor depression in elderly persons with and without mild cognitive impairment: A system review. J. Affect. Disord. 152, 28–38 (2014). ff 2. 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Schizophr. Res. 129(2–3), 201–204 (2011).f 12. Zugman, A. et al. Serum brain-derived neurotrophic factor and cortical thickness are differently related in patients with schizo- phrenia and controls. Psychiatry Research-Neuroimaging 234(1), 84–89 (2015).f d controls. www.nature.com/scientificreports/ A. Decreased peripheral brain-derived neurotrophic factor levels are a biomarker of disease activity in major psychiatric disorders: A comparative meta-analysis. Mol. Psychiatry 2, 2 (2013). 45. Chacon-Fernandez, P. et al. Brain-derived neurotrophic factor in megakaryocytes. J. Biol. Chem. 291(19), 9872–9881 (2016). levels are a biomarker of disease activity in major psychiatric disorders: A comparative meta-analysis. Mol. Psychiatry 2, 2 (2013) 5. Chacon-Fernandez, P. et al. Brain-derived neurotrophic factor in megakaryocytes. J. Biol. Chem. 291(19), 9872–9881 (2016). y j p y p y y y ( ) 45. Chacon-Fernandez, P. et al. Brain-derived neurotrophic factor in megakaryocytes. J. Biol. Chem. 291(19), 9872–9881 (2016). g y y 6. Schmaal, L. et al. Cortical abnormalities in adults and adolescents with major depression based on brain scans from 20 cohorts worldwide in the ENIGMA Major Depressive Disorder Working Group. Mol. Psychiatry 2, 2 (2016). j p g p y y 7. Molendijk, M. L. et al. Serum levels of brain-derived neurotrophic factor in major depressive disorder: State-trait issues, clinica features and pharmacological treatment. Mol. Psychiatry 16(11), 1088–1095 (2011). j p g p y y 47. Molendijk, M. L. et al. Serum levels of brain-derived neurotrophic factor in major depressive disorder: State-trait issues, clinical features and pharmacological treatment. Mol. Psychiatry 16(11), 1088–1095 (2011). g y y 8. Polacchini, A. et al. A method for reproducible measurements of serum BDNF: Comparison of the performance of six commercia assays. Sci. Rep. 5, 2 (2015). 48. Polacchini, A. et al. A method for reproducible measurements of serum BDNF: Comparison of the performance of six commercial assays. Sci. Rep. 5, 2 (2015). Author contributions M.P., M.L.S., K.M., P.S. designed the study, M.P. analyzed the data, M.P. and M.L.S. wrote the manuscript; C..S, F.R., S.R.H., J.K., A.V., V.W. contributed to data collection and laboratory measurements, F.B. and M.P. con- tributed to data preprocessing, L.L., K.T.H. and white matter lesions assessment, all of the authors edited and reviewed the final version of the manuscript Funding Open access funding provided by Projekt DEAL. Acknowledgements We thank all participants and the team at the LIFE study center, who made this study possible. This study was supported by LIFE—Leipzig Research Center for Civilization Diseases, Universität Leipzig. LIFE is funded by the European Union, the European Regional Development Fund (ERDF), and by the Free State of Saxony within the framework of the excellence initiative (project numbers 713-241202, 713-241202, 14505/2470, 14575/2470). MP acknowledges support from the International Max Planck Research School on Neuroscience of Communi- cation: Function, Structure, and Plasticity (IMPRS NeuroCom) by the Max Planck Society. MLS was supported by the German Research Foundation (DFG; SCHR 774/5-1). MP, LL, and MLS were supported by the German Consortium for Frontotemporal Lobar Degeneration, which is funded by the German Federal Ministry of Edu- cation and Research, by the Parkinson’s Disease Foundation (Grant No. PDF-IRG-1307), and the Michael J. Fox Foundation (Grant No. MJFF-11362). We also thank Prof. Anderson Winkler for sharing brain templates for blender under the Creative Commons Attribution-ShareAlike 3.0 Unported License at his blog https://brainder. org/research/brain-for-blender/. 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Psychiatry 22(6), 900–909 (2017).i 34. Han, K. M. et al. Cortical thickness, cortical and subcortical volume, and white matter integrity in patients with their first episode of major depression. J. Affect. Disord. 155, 42–48 (2014).i j p ff 35. Zhao, K. et al. Funding O Funding Open access funding provided by Projekt DEAL. Scientific Reports \| (2020) 10:14524 \| https://doi.org/10.1038/s41598-020-71317-y www.nature.com/scientificreports/ Competing interests The authors declare no competing interests. Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-71317-y. Correspondence and requests for materials should be addressed to M.P. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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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 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 Author(s) 2020 Scientific Reports \| (2020) 10:14524 \| https://doi.org/10.1038/s41598-020-71317-y
https://openalex.org/W4206588072	https://repository.kulib.kyoto-u.ac.jp/dspace/bitstream/2433/267448/1/TASLP.2021.3138719.pdf	English	null	Word-Region Alignment-Guided Multimodal Neural Machine Translation	IEEE/ACM transactions on audio, speech, and language processing	2,022	cc-by	13,860	Yuting Zhao , Mamoru Komachi, Tomoyuki Kajiwara, and Chenhui Chu Yuting Zhao , Mamoru Komachi, Tomoyuki Kajiwara, and Chenhui Chu many studies have focused on incorporating images to improve translations [3]–[5]. Abstract—We propose word-region alignment-guided multi- modal neural machine translation (MNMT), a novel model for MNMT that links the semantic correlation between textual and visual modalities using word-region alignment (WRA). Existing studies on MNMT have mainly focused on the effect of integrat- ing visual and textual modalities. However, they do not leverage the semantic relevance between the two modalities. We advance the semantic correlation between textual and visual modalities in MNMT by incorporating WRA as a bridge. This proposal has been implemented on two mainstream architectures of neural machine translation (NMT): the recurrent neural network (RNN) and the transformer. Experiments on two public benchmarks, English–German and English–French translation tasks using the Multi30k dataset and English–Japanese translation tasks using the Flickr30kEnt-JP dataset prove that our model has a signiﬁ- cant improvement with respect to the competitive baselines across different evaluation metrics and outperforms most of the existing MNMT models. For example, 1.0 BLEU scores are improved for the English–German task and 1.1 BLEU scores are improved for the English–French task on the Multi30k test2016 set; and 0.7 BLEU scores are improved for the English–Japanese task on the Flickr30kEnt-JP test set. Further analysis demonstrates that our model can achieve better translation performance by integrating WRA, leading to better visual information use. The study of the potential for improving translation qual- ity using images was pioneered by [6]. Subsequent studies have contextualized textual representations using global vi- sual features extracted from an entire image to initialize tex- tual encoder/decoder recurrent neural network (RNN) hidden states [7]–[10]. However, the effect of the image cannot be fully exerted because the single global visual features of an entire image are complex. To effectively utilize an image, some researchers incorpo- rate spatially grid-based local visual features extracted from a convolutional neural network (CNN) into MNMT [11]–[15]. As theseequallysizedgrid-basedlocalvisualfeaturesdonotconvey speciﬁc semantics, the role of visual features is dispensable in translation. As reported in [16], MNMT models disregard visual features because the quality of the image features or the manner in which they are integrated into the model is not satisfactory. y g y Consequently, current studies have incorporated richer local visual features such as object-level regional features [17], [18]. These studies mainly relied on object detection to automati- cally extract objects in an image. 244 244 IEEE/ACM TRANSACTIONS ON AUDIO, SPEECH, AND LANGUAGE PROCESSING, VOL. 30, 2022 Yuting Zhao , Mamoru Komachi, Tomoyuki Kajiwara, and Chenhui Chu Although regional features containing semantic information can assist in generating trans- lations [19], the lack of guidance to focus on image regions that are semantically relevant to the source words during translation can lead to incorrect translations. Therefore, the latest study [20] relates multimodal inputs in a graphical manner. Their method is aimed at reﬁning multimodal representation learning using a graph-based multimodal fusion encoder. Thus far, fully exploit- ing the semantic relevance between multimodal inputs remains challenging. Index Terms—Multi30k, multimodal machine translation, semantic correlation, vision and language, word-region alignment. Manuscript received June 29, 2021; revised October 24, 2021; accepted December 11, 2021. Date of publication December 28, 2021; date of current version January 8, 2022. This work was supported by the Grant-in-Aid for YoungScientists#19K20343,JSPS.Theassociateeditorcoordinatingthereview of this manuscript and approving it for publication was Prof. Mohit Bansal. (Corresponding author: Yuting Zhao.) Yuting Zhao and Mamoru Komachi are with Tokyo Metropolitan Univer- sity, Hino 191-0065, Japan (e-mail: zhao-yuting@ed.tmu.ac.jp; komachi@ tmu.ac.jp). der a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ Tomoyuki Kajiwara is with Ehime University, Matsuyama 790-8577, Japan (e-mail: kajiwara@cs.ehime-u.ac.jp). Chenhui Chu is with Kyoto University, Kyoto 606-8501, Japan (e-mail: chu@i.kyoto-u.ac.jp). This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommon Manuscript received June 29, 2021; revised October 24, 2021; accepted December 11, 2021. Date of publication December 28, 2021; date of current version January 8, 2022. This work was supported by the Grant-in-Aid for YoungScientists#19K20343,JSPS.Theassociateeditorcoordinatingthereview of this manuscript and approving it for publication was Prof. Mohit Bansal. (Corresponding author: Yuting Zhao.) Yuting Zhao and Mamoru Komachi are with Tokyo Metropolitan Univer- sity, Hino 191-0065, Japan (e-mail: zhao-yuting@ed.tmu.ac.jp; komachi@ tmu.ac.jp). Tomoyuki Kajiwara is with Ehime University, Matsuyama 790-8577, Japan (e-mail: kajiwara@cs.ehime-u.ac.jp). Chenhui Chu is with Kyoto University, Kyoto 606-8501, Japan (e-mail: chu@i.kyoto-u.ac.jp). Digital Object Identiﬁer 10.1109/TASLP.2021.3138719 Digital Object Identiﬁer 10.1109/TASLP.2021.3138719 ZHAO et al.: WORD-REGION ALIGNMENT FOR MULTIMODAL NEURAL MACHINE TRANSLATION By advancing the correlation between textual and visual modal- ities by integrating WRA, the textual and visual context can provide semantically relevant information to generate accurate translations.Thisstudyhasthepotentialtobeextendedtosubtitle translation in news, movies, TV shows, etc., where the alignment between text and visual information is crucial. Consequently, in recent studies, images are represented us- ing multiple object-level regional features to solve the afore- mentioned limitations by attempting the following integration strategies: 1) generating a single representation from multiple object-level features to initialize the source textual sequence [30] or reﬁne the target language textual context [31]; and 2) learning to focus on speciﬁc regional features using an additional image attention mechanism [19]. Although regional features aid object localization or semantic information presentation, the manner in which they are integrated into the model still needs to be improved. Overall, the main contributions of this study are four-fold: Overall, the main contributions of this study are four-fold: r We proposed WRA, an intermediate component as an addi- tional input to bridge multimodal inputs based on semantic relevance. r We proposed a novel integration strategy W2R of the MNMT model that leverages the WRA to guide the model to translate certain source words into target words while attending to semantically relevant image regions. r We implemented our proposal on both RNN-based and transformer-based architectures, and evaluated it on English–German and English–French tasks using the Multi30k dataset [21] and on the English–Japanese trans- lation tasks using the Flickr30kEnt-JP dataset [22]. Ex- tensive experiments validated the consistent efﬁcacy of the proposed method and revealed that it signiﬁcantly improved baselines based on different evaluation metrics and outperformed most of the existing methods. Based on [16], if the textual modality is sufﬁcient to accomplish the translation task, the visual modality should be integrated to play a complementary role rather than a redundant role. Toward this end, an emerging trend of exploiting correlations between modalities has been considered promising. ZHAO et al.: WORD-REGION ALIGNMENT FOR MULTIMODAL NEURAL MACHINE TRANSLATION ZHAO et al.: WORD-REGION ALIGNMENT FOR MULTIMODAL NEURAL MACHINE TRANSLATION 245 Fig.1. ExampleofWRA-guidedMNMT.TheWRAbuildssemanticrelevance between the vision and language. Speciﬁcally, each region-level visual feature is annotated using a visual concept that is used to create a relationship with every source word. When generating the “rouge,” similar attention weights (“att” in the ﬁgure) are assigned to both the corresponding source word “red” and image region “red shirt.” and/or decoder RNN hidden states with them [9]; 3) interacting with them elementwise using textual annotations or target word embeddings [10]; and 4) inﬂuencing the textual encoder by learning the visual representation alongside them [7]. Although these models were designed to enrich the textual context using sufﬁcient visual information to improve translation, it is difﬁcult tosummarizeallthesemanticinformationofanentireimageinto a single global visual feature. To address this issue, subsequent researchers represented visual information using a set of convolutional local features that are equally sized grid local features. These features were used to preserve spatial correspondence with the image. The following integration methods were investigated: 1) computing a multimodal context using a multimodal attention mechanism that simultaneously focuses on an image and its source descrip- tion [12], [24]; 2) conjecturing a learnable masking operation over the convolutional feature maps to help the attention mech- anism ﬁlter out local features that are irrelevant to translation and focus on the most important part of the visual inputs [15]; 3) focusing on textual and visual features independently using different attention strategies [13], [14], [25]; and 4) attending to local features by setting an additional attention sublayer after self-attention [26]–[28]. However, in the aforementioned approaches, the attention mechanism cannot easily distinguish equally sized local features. As proved in [29], attending to spe- ciﬁc regions of the image is crucial to improving the translation. Fig.1. ExampleofWRA-guidedMNMT.TheWRAbuildssemanticrelevance between the vision and language. Speciﬁcally, each region-level visual feature is annotated using a visual concept that is used to create a relationship with every source word. When generating the “rouge,” similar attention weights (“att” in the ﬁgure) are assigned to both the corresponding source word “red” and image region “red shirt.” withsemanticallyrelevantregionalvisualfeatures.Additionally, two modality-dependent attention mechanisms are utilized to generate textual and visual contexts for decoding target words. ZHAO et al.: WORD-REGION ALIGNMENT FOR MULTIMODAL NEURAL MACHINE TRANSLATION Some strategies have been developed: 1) jointly learning a shared vision-language embedding space and a translator [32]; 2) modeling the interaction between visual and textual features using a latent variable model alongside a translation model [33]; 3) training multi-head co-attention to capture the interaction between visual and textual features in multiple subspaces [18]; 4) learning a universal visual representation by retrieving associ- ated images for words in a source sentence [34]; 5) utilizing man- ually annotated datasets to train supervised visual attention [35]; and 6) integrating multimodal graph neural networks [20] and dynamic context-guided capsule networks [36] into the MNMT. Although these researchers successfully prove the effectiveness of relating textual and visual information for MNMT, there are lingering concerns. First, jointly learning visual and textual r We conducted detailed analyses to prove the effectiveness of the proposed method and demonstrate that our method can lead to better visual information use. I. INTRODUCTION M l i (N M ULTIMODAL neural machine translation (MNMT) ex- tends the conventional text-to-text neural machine trans- lation (NMT) [1], [2] by exploiting an auxiliary source modality, speciﬁcally images, to translate source sentences paired with images into a target language. The main motivation behind this is that the translation is expected to be more accurate than tex- tual translation because there are numerous situations in which textual context alone is insufﬁcient for correct translation such as for ambiguous words and grammatical gender. Therefore, An overview of our proposal is shown in Fig. 1. This study proposes a novel facility called word-region alignment (WRA) that explicitly correlates source words with image regions as an additional input in the proposed MNMT model. Unlike existing MNMT models, we design the WRA as an intermediate component to bridge multimodal inputs. Speciﬁcally, as visual concepts can summarize the semantics of image regions, we utilize these visual concepts as a medium to pre-process the semantic relevance between source words and image regions. In terms of architecture, we propose a novel integration strategy word-to-region (W2R) that leverages the WRA, facilitating the interaction between semantically relevant textual and visual features. Under the integration strategy W2R, the pre-processed WRA is leveraged as a bridge to link textual and visual inputs, acting as an auxiliary cue to guide textual features to interact Digital Object Identiﬁer 10.1109/TASLP.2021.3138719 Digital Object Identiﬁer 10.1109/TASLP.2021.3138719 III. METHODOLOGY In this section, we describe our methodology as follows: 1) We introduce the proposed WRA in Section III-A, according to Fig. 2; 2) The details of the integration of WRA into the RNN- based MNMT model shown in Fig. 3 and the transformer-based MNMT model shown in Fig. 4 are presented in Sections III-B and III-C, respectively. Fig. 3. WRA-guided RNN-based MNMT model. attribute class followed by an object class (see Fig. 2). We extract m image regions along with visual concepts for each image that are used to annotate the semantics of the corresponding regions. Then, we convert the source words and visual concepts into subword units. II. RELATED WORK Subsequently, it was be used to create a relationship with each source word based on semantic similarity. The WRA represents the semantic correlation between each regional visual feature and all words in a sentence. Fig. 2. WRA generation. Each region-level visual feature was annotated using a visual concept consisting of an attribute class, followed by an object class. Subsequently, it was be used to create a relationship with each source word based on semantic similarity. The WRA represents the semantic correlation between each regional visual feature and all words in a sentence. Fig. 3. WRA-guided RNN-based MNMT model. Overall, this study differs from previous works in the fol- lowing two techniques: First, this study is the ﬁrst to propose an integration facility called WRA, utilizing visual concepts as an intermediary to pre-process the semantic relevance between source words and image regions. Second, it proposes a novel integration strategy called W2R, advancing the interaction be- tween textual and visual modalities by leveraging WRA as a bridge without disturbing the textual context. 1[Online]. Available: https://github.com/facebookresearch/fastText II. RELATED WORK Early MNMT models integrated visual information using a single global visual feature extracted by a CNN. They used the global visual feature to contextualize textual representations in the following ways: 1) appending them at the head/tail to the original textual sequence [23]; 2) initializing the textual encoder IEEE/ACM TRANSACTIONS ON AUDIO, SPEECH, AND LANGUAGE PROCESSING, VOL. 30, 2022 246 Fig. 2. WRA generation. Each region-level visual feature was annotated using a visual concept consisting of an attribute class, followed by an object class. Subsequently, it was be used to create a relationship with each source word based on semantic similarity. The WRA represents the semantic correlation between each regional visual feature and all words in a sentence. representations with latent space requires large-scale training data that MNMT lacks. Second, in multimodal tasks, different modalities do not usually have equal importance. It is suggested that texts are obviously more important than images [37]. Likewise, the impact of textual predominance has been revealed by [38]. Therefore, effectively correlating multimodal inputs is a lingering challenge for MNMT. Meanwhile, some methods have been developed for correlat- ing modalities in other multimodal tasks that focus on image- to-text one-way operation: 1) Correlating textual and visual modalities by a multimodal embedding space [39], [40]. Rather than learning a joint space, a pre-processed facility WRA using visual concepts as an intermediary to build semantic relevance between words and regions is proposed in this paper; 2) Aligning textual and visual features by different attention mechanisms, such as a mutual attention mechanism [41], a stack of co- attention layers [42], and self-attention [43] or cross-modality attention [44]. In contrast, in our study, double attention is collocated for learning alignment between source words and target words and between image regions and target words; and 3) Jointly training visual and textual attention mechanisms [45] or jointly learning word-tag-region triple embeddings [46] to ﬁnd sharedsemanticsbetweenimagesandsentences.Differently,our work pre-processes the semantic relevance between images and sentences using WRA rather than joint training. Moreover, rep- resentations of visual concepts do not need to be jointly learned. Instead, the visual concepts act as an intermediary to indicate the explicit semantic information of the corresponding image regions to help words and regions build semantic relevance. Fig. 2. WRA generation. Each region-level visual feature was annotated using a visual concept consisting of an attribute class, followed by an object class. B. WRA-Guided RNN-Based MNMT Model By concatenating the forward and backward annotations, the textual representation is denoted as H = (h1, h2, h3, . . . , hn) ∈Rdh. −→ hn) ∈Rds and updates the backward with annotations (←− h1, ←− h2, ←− h3, · · · , ←− hn) ∈Rds. By concatenating the forward and backward annotations, the textual representation is denoted as H = (h1, h2, h3, . . . , hn) ∈Rdh. → hn) ∈Rds and updates the backward with annotations (← h1, ←− h2, ←− h3, · · · , ←− hn) ∈Rds. By concatenating the forward and backward annotations, the textual representation is denoted as H = (h1, h2, h3, . . . , hn) ∈Rdh. Here, gi,j represents the similarity score between the i-th region and the j-th word. Here, gi,j represents the similarity score between the i-th region and the j-th word. 2) Visual Encoder: The visual encoder is an object- detection-based approach [47] to regional feature extraction. Given an input image, the visual encoder employs the faster R-CNN [49] in conjunction with ResNet-101 [50] as its back- bone, which is pre-trained on the Visual Genome [51] dataset to extract m regional visual features from each image. Each regional feature is represented as a vector a and the visual representation is denoted by A = (a1, a2, a3, . . . , am) ∈Rdr. Finally, we deﬁne the semantic relevance between the i-th region and the whole source sentence using a similarity vec- tor gi ∈Rdn. Then, the soft WRA is represented as Gsa = {g1, g2, g3, . . . , gm} ∈Rdn. p g p y R-CNN [49] in conjunction with ResNet-101 [50] as its back- bone, which is pre-trained on the Visual Genome [51] dataset to extract m regional visual features from each image. Each regional feature is represented as a vector a and the visual representation is denoted by A = (a1, a2, a3, . . . , am) ∈Rdr. 2) Hard WRA: The hard WRA is generated as a maximum similarity matrix along the source word axis based on the soft WRA. A. WRA Generation We identify two types of explicit WRA: As shown in Fig. 2, we propose a WRA containing explicit semantic interactions between the source words and image re- gions. The WRA is pre-processed; it acts as an auxiliary input to guide interactions between the textual and visual information inside the entire model. 1) Soft WRA: The soft WRA is generated as a cosine simi- larity matrix that presents the semantic similarity score between source words and image regions. Tocalculatecosinesimilarityscoresbetweenthesourcewords and image regions, ﬁrst, we utilize fastText1 to learn subword representations of the source words and visual concepts. We use For regions, we use the object detection method in [47] to extract object-level regions for each image. Speciﬁcally, each image region is not only denoted by a bounding box in the image but also detected along with a visual concept consisting of an ZHAO et al.: WORD-REGION ALIGNMENT FOR MULTIMODAL NEURAL MACHINE TRANSLATION 247 Fig. 4. WRA-guided transformer-based MNMT model. Fig. 4. WRA-guided transformer-based MNMT model. Finally, the hard WRA can be represented using m one-hot vectors by Gha = {g′ 1, g′ 2, g′ 3, . . . , g′ m} ∈Rdn. apre-trainedmodel2 containingtwomillionwordvectorstrained on subword information on Common Crawl (600B tokens). The subword embeddings of source words can be generated directly, whereas the subword embeddings of visual concepts should take an average of all the constituent subwords because they are phrases. Then, these embeddings provide a mapping function from a subword to a 300-dim vector, where semantically similar subwords are embedded close to each other. 2[Online]. Available: https://fasttext.cc/docs/en/english-vectors.html B. WRA-Guided RNN-Based MNMT Model As illustrated in Fig. 3, based on text-to-text RNN archi- tecture [2], our WRA-guided RNN-based MNMT model com- prises four parts: textual encoder, visual encoder, word-to-region (W2R), and decoder. The most unique stage is the W2R, where the soft/hard WRA are integrated to guide interactions between textual and visual representations. As shown in Fig. 2, the source words are represented by W = {w1, w2, w3, . . . , wn} ∈Rd300, and visual concepts are represented by C = {c1, c2, c3, . . . , cm} ∈Rd300, where n de- notes the source length and m denotes the region amount. 1) Textual Encoder: The textual encoder is a bi-directional RNN with gated recurrent unit (GRU) [48]. Given a source sentence X = (x1, x2, x3, · · · , xn), the encoder updates the forward hidden states with annotations (−→ h1, −→ h2, −→ h3, · · · , → ← 1) Textual Encoder: The textual encoder is a bi-directional RNN with gated recurrent unit (GRU) [48]. Given a source sentence X = (x1, x2, x3, · · · , xn), the encoder updates the forward hidden states with annotations (−→ h1, −→ h2, −→ h3, · · · , −→ hn) ∈Rds and updates the backward with annotations (←− h1, ←− h2, ←− h3, · · · , ←− hn) ∈Rds. By concatenating the forward and backward annotations, the textual representation is denoted as H = (h1, h2, h3, . . . , hn) ∈Rdh. 1) Textual Encoder: The textual encoder is a bi-directional RNN with gated recurrent unit (GRU) [48]. Given a source sentence X = (x1, x2, x3, · · · , xn), the encoder updates the forward hidden states with annotations (−→ h1, −→ h2, −→ h3, · · · , −→ hn) ∈Rds and updates the backward with annotations (←− h1, ←− h2, ←− h3, · · · , ←− hn) ∈Rds. By concatenating the forward and backward annotations, the textual representation is denoted as H = (h1, h2, h3, . . . , hn) ∈Rdh. g g We deﬁne the soft WRA by computing the similarity score between the source words and visual concepts as follows: gi,j = cT i · wj ∥ci∥· ∥wj∥, i ∈[1, m], j ∈[1, n] −→ hn) ∈Rds and updates the backward with annotations (←− h1, ←− h2, ←− h3, · · · , ←− hn) ∈Rds. IEEE/ACM TRANSACTIONS ON AUDIO, SPEECH, AND LANGUAGE PROCESSING, VOL. 30, 2022 248 r Under the guidance of the soft WRA: where V txt, U txt, W txt are the training parameters; etxt t,i is the attention energy; αtxt t,i is the attention weight matrix. h′ i = T 1 n ⊙(gi · H) , Likewise, visual attention focuses on every multimodal rep- resentation ri in R by assigning an attention weight. Then, the multimodal context vector ct is generated as follows: r Under the guidance of the hard WRA: r Under the guidance of the hard WRA: h′ i = T(g′ i · H) eimg t,j = (V img)Ttanh(U imgs′ t + W imgri) αimg t,j = softmax(eimg t,j ) ct = m j=1 αimg t,j ri, eimg t,j = (V img)Ttanh(U imgs′ t + W imgri) where gi is from the Gsa; g′ i is from the Gha; H is the textual representation;nisthesourcelength;andalineartransformation function is deﬁned by T : Rdh →Rdr. where gi is from the Gsa; g′ i is from the Gha; H is the textual representation;nisthesourcelength;andalineartransformation function is deﬁned by T : Rdh →Rdr. αimg t,j = softmax(eimg t,j ) ct = m j=1 αimg t,j ri, ii) In the second stage, the WRA-guided textual repre- sentations H′ = (h′1, h′2, h′3, . . . , h′m) ∈Rdr are combined with the visual representations A = (a1, a2, a3, . . . , am) ∈Rdr throughconcatenation,toenricheachimageregionusingseman- tically relevant textual features. Through this transformation, the interaction between the independently represented textual and visual features are effectively facilitated. where V img, U img, W img are the training parameters; αimg t,j is a weight matrix; eimg t,j is the attention energy. ,j Based on the textual context vector zt and previous hidden state proposal s′t, a hidden state proposal s′′t is computed in the second GRU (fgru2). Similarly, based on the multimodal context vector ct and the s′′t, the ﬁnal hidden state st is generated in the third GRU (fgru3). Because the calculation of fgru2 and fgru3 are similar to the function of fgru1, we organize them as follows: ri = CONCAT(h′ i, ai) = h′i ai (1) (1) Thus, the visual representations are semantically enhanced by combining WRA-guided textual features and advanced to mul- timodal representations, denoted as R = (r1, r2, r3, . . . , rm) ∈ Rdr. IEEE/ACM TRANSACTIONS ON AUDIO, SPEECH, AND LANGUAGE PROCESSING, VOL. 30, 2022 st = fgru3([ct, yt−1], s′′ t) s′′ t = fgru2(zt, s′ t) Weensurethatbothrepresentationshavetheirownprojections to compute the candidate probabilities by obtaining textual and visual GRU blocks [53] as follows: 4) Doubly Attentive Decoder: The doubly attentive decoder inspired by [13] functions in the textual and multimodal contexts based on two independent attention mechanisms, then predicts the probability of a target word. It is a deepGRU [52] with three stacked GRUs derived from the convoluted gated recurrent units (cGRUs).3 bv t = fght(W v b st) bt t = fght(W t bs′′ t) To generate the target word yt at time step t, a hidden state proposal s′t is computed in the ﬁrst GRU (fgru1) using the previous target word yt−1 and hidden state st−1 as follows: yt ∼pt = softmax(W t projbt t + W v projbv t ). yt ∼pt = softmax(W t projbt t + W v projbv t ). where W v b , W t b, W t proj, W v proj are training parameters, and fght is a gated hyperbolic tangent activation [54] substituted for the tanh function. s′ t = fgru1(yt−1, st−1) s′ t = (1 −ˆξt) ⊙˙st + ˆξt ⊙st−1 ˙st = tanh(WEY [yt−1] + ˆγt ⊙(Ust−1)) ˆγt = σ(WγEY [yt−1] + Uγst−1) ˆξt = σ(WξEY [yt−1] + Uξst−1) B. WRA-Guided RNN-Based MNMT Model We make a hard choice to pair each region with only one word in the whole sentence by aligning the most semantically relevant words to each region based on similarity score gi,j: 3) Word-to-Region (W2R): After generating textual and vi- sual representations independently, the WRA is integrated as an additional input to bridge them, acting as an auxiliary cue to guide source words to interact with regional features. g′ i,j = 1, if arg max j∈[1,n] (gi) = j, 0, otherwise The formulation, which entails two stages, is illustrated in Fig. 3. Here, g′ i,j represents the replacement of the maximum sim- ilarity score between the i-th region and the j-th word with 1, and the replacement of others with 0. i) In the ﬁrst stage, intermediate textual representations H′ = (h′1, h′2, h′3, . . . , h′m) ∈Rdr are associated with each image region under the guidance of the WRA. Subsequently, two deﬁnitions are used to calculate the WRA-guided textual feature h′i, with respect to the i-th region. i) In the ﬁrst stage, intermediate textual representations H′ = (h′1, h′2, h′3, . . . , h′m) ∈Rdr are associated with each image region under the guidance of the WRA. Subsequently, two deﬁnitions are used to calculate the WRA-guided textual feature h′i, with respect to the i-th region. 3[Online]. Available: https://github.com/nyu-dl/dl4mt-tutorial/blob/master/ docs/cgru.pdf 4[Online]. Available: https://github.com/moses-smt/mosesdecoder 5[Online]. Available: https://github.com/rsennrich/subword-nmt 6[Online]. Available: https://github.com/nlab-mpg/Flickr30kEnt-JP 7[Online]. Available: http://bryanplummer.com/Flickr30kEntities/ 8[Online]. Available: http://shannon.cs.illinois.edu/DenotationGraph/ 9[Online]. Available: https://nlab-mpg.github.io/wat2021-mmt-jp/ 10[Online]. Available: https://taku910.github.io/mecab/ 11[Online]. Available: http://www.phontron.com/kytea/index.html 12Weusedaconsistentwordsegmentationmethodwiththepreviouswork[35] during testing. As their work used different data settings with the WAT 2021 ofﬁcial dataset, we did not compare our results with theirs in this work. A. Datasets 2) Flickr30kEnt-JP: We conducted experiments on the En- glish →Japanese (En →Jp) task using the Flickr30kEnt-JP dataset [22], an extension of Flickr30k [56] and Flickr30k enti- ties [57] with newly included manual Japanese translations. For training and validation, we used the Flickr30kEnt-JP dataset6 for Japanese sentences, the Flickr30k entities dataset7 for English sentences, and the Flickr30k dataset8 for images. They are sharing the same splits of training and validation data made in Flickr30k entities. For the test data, we used WAT 2021 ofﬁcial test set,9 whose corresponding images were in the Flickr30k dataset. The conventional textual cross-attention used in the trans- former, called the multi-head scaled dot-product attention, is simultaneously calculated on a set of queries, keys, and values with M heads in parallel. Then, the generation of each head is packed together into a matrix Q, Kt, Vt. The output matrix is computed as follows: MultiHead(Q, Kt, Vt) = Concat(head1 t, . . . , headM t )W O where headi∈[1,M] t = Attention(QW Q i , KtW K i , VtW V i ) = softmax(QW Q i (KtW K i )T √dm )VtW V i MultiHead(Q, Kt, Vt) = Concat(head1 t, . . . , headM t )W O where headi∈[1,M] t = Attention(QW Q i , KtW K i , VtW V i ) QW Q(K W K)T MultiHead(Q, Kt, Vt) = Concat(head1 t, . . . , headM t )W O MultiHead(Q, Kt, Vt) = Concat(head1 t, . . . , headM t )W O Note that the Japanese training data size was originally 148,915 sentences; however, ﬁve sentences were missing. Therefore, we used 148,910 sentences for training. In summary, we used 148,910 pairs for training, 5 k pairs for validation, and 1 k monolingual English sentences for testing. where headi∈[1,M] t = Attention(QW Q i , KtW K i , VtW V i ) where headi∈[1,M] t = Attention(QW Q i , KtW K i , VtW V i ) where headi∈[1,M] t = Attention(QW Q i , KtW K i , VtW V i ) = softmax(QW Q i (KtW K i )T √dm )VtW V i For English sentences, we applied lowercase, punctuation normalization, and the tokenizer in the Moses Toolkit. Then, we converted space-separated tokens into subword units using the BPE model with 10 k merge operations. A. Datasets 1) Multi30k: WeexperimentedonEnglish→German(En→ De) and English →French (En →Fr) tasks using the Multi30k dataset [21]. The dataset contained 29 k training images and 1,014 validation images. For testing, we used three public test sets to evaluate our models: the Test2016 and Test2017 sets, each containing 1 k images, and the ambiguous MSCOCO set [4] containing 461 images. Each image was paired with image descriptions expressed by both the original English sentences and the German and French translations. 3) Word-to-Region (W2R): Based on the two stages of WRA integration method introduced in Section III-B3, we simi- larly combine semantically relevant textual features into vi- sual features guided by soft/hard WRA. Consequently, the visual representations are combined with the WRA-guided textual representations and converted into semantically en- hanced multimodal representations, which are denoted as R = (r1, r2, r3, . . . , rm) ∈Rdr. We lowercased and tokenized the English, German, and French descriptions and English visual concepts using the script in the Moses Toolkit.4 We converted space-separated tokens into subword units using a byte pair encoding (BPE) model.5 With 10 k merge operations, the resulting vocabulary sizes of each language pair were 5,202→7,065 tokens for En →De and 5,833→6,575 tokens for En →Fr. ( ) 4) Doubly Attentive Decoder: The decoder also comprises a stack of N identical layers. In addition to the two sublayers, similar to the encoder, a decoder with double cross-attention mechanisms over the sources inspired by [27] is implemented. The proposed model attends to textual representations and multimodal representations simultaneously to generate textual and multimodal contexts. Each of them is attended to us- ing the same set of queries, that is, the output of the self- attention sublayer. A residual connection link is used between the queries and summed context vectors from the parallel double cross-attentions. 4) Doubly Attentive Decoder: The decoder also comprises a stack of N identical layers. In addition to the two sublayers, similar to the encoder, a decoder with double cross-attention mechanisms over the sources inspired by [27] is implemented. The proposed model attends to textual representations and multimodal representations simultaneously to generate textual and multimodal contexts. Each of them is attended to us- ing the same set of queries, that is, the output of the self- attention sublayer. A residual connection link is used between the queries and summed context vectors from the parallel double cross-attentions. A. Datasets For Japanese sentences, we used MeCab10 for word segmentation during training with the IPA dictionary and used KyTea11 for word segmentation during testing12 with the Full SVM model. where W ∗ i and W O are learnable parameter matrices. where W ∗ i and W O are learnable parameter matrices. Similarly, the visual cross-attention mechanism performs the multi-head scaled dot-product attention on multimodal repre- sentations with M heads in parallel, as follows: MultiHead(Q, Kr, Vr) = Concat(head1 r, . . . , headM r )W o where headi∈[1,M] r = Attention(QW q i , KrW k i , VrW v i ) = softmax(QW q i (KrW k i )T √dm )VrW v i MultiHead(Q, Kr, Vr) = Concat(head1 r, . . . , headM r )W o where headi∈[1,M] r = Attention(QW q i , KrW k i , VrW v i ) = softmax(QW q i (KrW k i )T √dm )VrW v i IV. EXPERIMENTS 2) Visual Encoder: The internal structure of the visual encoder is the same as that introduced in Section III- B2. Similarly, the visual representation is denoted by A = (a1, a2, a3, . . . , am) ∈Rdr. 2) Visual Encoder: The internal structure of the visual encoder is the same as that introduced in Section III- B2. Similarly, the visual representation is denoted by A = (a1, a2, a3, . . . , am) ∈Rdr. 2) Visual Encoder: The internal structure of the visual encoder is the same as that introduced in Section III- B2. Similarly, the visual representation is denoted by A = (a1, a2, a3, . . . , am) ∈Rdr. C. WRA-Guided Transformer-Based MNMT Model As illustrated in Fig. 4, based on text-to-text transformer architecture [55], our proposed model also comprises four parts: textual encoder, visual encoder, word-to-region (W2R), and decoder. The W2R is the core stage to leverage WRA. where Wξ, Uξ, Wγ, Uγ, W, and U are the training parameters and EY is the target word embedding. 1) Textual Encoder: In the transformer [55], a source sen- tence is encoded by a textual encoder with multiple layers. The encoder is composed of a stack of N identical layers, each of which included two sublayers. The ﬁrst and second sublayers are the multi-head attention and position-wise fully connected feed-forward network (FFN). Residual connection and layer normalization are used between sublayers. Formally, the output of each sublayer is deﬁned as LayerNorm(x + Sublayer(x)), where Sublayer() is the function implemented by the sublayer itself. To encourage these residual connections, all the sublayers and embedding layers produce outputs of dimension dm. Each source word is encoded as a vector h and the textual represen- tation is denoted by H = (h1, h2, . . . , hn) ∈Rdm. Textual attention focuses on every textual representation hi in H by assigning an attention weight, following which the textual context vector zt is generated as follows: etxt t,i = (V txt)Ttanh(U txts′ t + W txthi) αtxt t,i = softmax(etxt t,i ) zt = n i=1 αtxt t,i hi, 3[Online]. Available: https://github.com/nyu-dl/dl4mt-tutorial/blob/master/ docs/cgru.pdf ZHAO et al.: WORD-REGION ALIGNMENT FOR MULTIMODAL NEURAL MACHINE TRANSLATION 249 IV. EXPERIMENTS A. Results on the En →De Task For training En →Jp task, we stopped training when the BLEU score did not improve over 20 evaluations on the valida- tion set, and one validation evaluation was performed after every epoch. 1) Results Within Our RNN-Based Models: As shown in Table I, compared with the text-to-text NMT, the MNMTR con- sistently improved the translation performance, beneﬁting from integrating regional features. Nevertheless, the improvements were less signiﬁcant across different metrics on all the test sets. From this point, we observed that even if high-quality regional features are fused, the role of the visual feature is limited by the integration method and is not fully realized. In contrast, the proposed MNMTW2R(sa) and MNMTW2R(ha) models yielded signiﬁcantly improved translation results, compared to the NMT baseline, and consistently obtained a larger margin than the MNMTR. 1) Results Within Our RNN-Based Models: As shown in Table I, compared with the text-to-text NMT, the MNMTR con- sistently improved the translation performance, beneﬁting from integrating regional features. Nevertheless, the improvements were less signiﬁcant across different metrics on all the test sets. 2) Settings of the Transformer-Based Models: We set the encoder and decoder to contain N = 6 layers. The dimensions of all the input and output layers were set to dm = 512. The textual representation dimension was dm = 512 and the dimensions of the inner feed-forward neural network layer were dﬀ= 2, 048. The number of all the multi-head modules in the encoder and decoder layers was set to M = 8. For training En →De and En →Fr tasks, the sentence- minibatch size was set to 64, the label smoothing value was set at 0.1, and the attention dropout and residual dropout were 0.3. We used the Adam optimizer with β1 = 0.9, β2 = 0.98. The learning rate was tuned to 0.05, with a warm-up step of 4,000 for the NMT model; 0.03 with a warm-up step of 4,000 for the MNMTR, MNMTW2R(sa), and MNMTW2R(ha) models. We The key difference between the MNMTW2R and MNMTR was the integration of WRA. It was veriﬁed that integrating the WRA enabled better use of the visual features; therefore, the performance was better than that of the model without the WRA. We think that the signiﬁcant improvements could be attributed to two aspects of the proposed model: r The WRA bridges vision and language well. IEEE/ACM TRANSACTIONS ON AUDIO, SPEECH, AND LANGUAGE PROCESSING, VOL. 30, 2022 IEEE/ACM TRANSACTIONS ON AUDIO, SPEECH, AND LANGUAGE PROCESSING, VOL. 30, 2022 250 stopped training when the METEOR score did not improve over 10 evaluations on the validation set and one validation evaluation was performed every 1,000 iterations. the average score over three runs. We conducted a statistical signiﬁcance test with bootstrap resampling [60] for the merger of three test translations using the script in Moses Toolkit. We reported a statistically signiﬁcant improvement in BLEU if the p-value is < 0.05.13 For training En →Jp task, different settings were used. The sentence-minibatch size was set at 48, the Adam optimizer was tuned using β1 = 0.9, β2 = 0.998. Training was stopped when the BLEU score did not improve over 20 evaluations on the validation set and one validation evaluation was performed after each epoch. C. Setup In our experiments, we split two branches based on the architecture of the models: RNN-based models and transformer- based models. Each branch includes the following types of models for comparison. V. RESULTS A. Results on the En →De Task D. Further Experimental Comparison r NMT: the text-to-text NMT model, wherein only the tex- tual sentences were used. To empirically verify the merit of our proposed model, we also presented the performance of the following state-of-the-art MNMT models for comparison, namely: r MNMTR: the doubly attentive MNMT model [19] using regional visual features, without integrating WRA to pro- cess W2R strategy. r VAG-NMT [32]: Jointly optimizes a translation model and learns a shared vision-language space. r MNMTW2R(sa): the proposed MNMT model incoporating soft WRA to guide W2R stage. g g p r VMMTF [33]: An MNMT model that incorporates image context learnt by a latent variable model. r MNMTW2R(ha): the proposed MNMT model incorporat- ing hard WRA to guide W2R stage. r Del+Obj [31]: A transformer-based deliberation model enriched using object-level features. All the models were implemented with Nmtpytorch [61].14 r Trans+VR [34]: A transformer model with universal visual representation by a topic-image lookup table. In the visual encoder for all the MNMT models, the number of regional features was set to m = 36, and the dimensions of the regional feature vectors were set to dr = 2, 048. r VAR-{S2S, TF} (hard) [18]: Jointly trains the source-to- target and target-to-source translation models through hard visual agreement regularization. 1) Settings of the RNN-Based Models: We set the dimensions of the encoder and decoder hidden states at ds = 256, the textual representation dimension at dh = 512, word embedding at 128- dim, batch size at 46, textual dropout at 0.3, visual dropout at 0.5, model dropout at 0.5, and both blocks bt t and bv t at 0.5. We used the Adam optimizer [62] with a learning rate of 0.0004 for all the models. r MNMT+SVA [35]: A transformer-based MNMT model with the supervised visual attention mechanism. r GMFE-NMT [20]: A graph-based multimodal fusion en- coder to conduct graph encoding for NMT. r MTF [37]: A transformer-based NMT model with multi- modal self-attention to integrate text and image features. For training En →De and En →Fr tasks, we consistently stopped training when the METEOR score did not improve over 10 evaluations on the validation set, and one validation evaluation was performed every 1,000 iterations. 13We did not report on METEOR due to the statistical signiﬁcance test for METEOR is not implemented in the Moses script. 14[Online]. Available: https://github.com/lium-lst/nmtpytorch B. Evaluation We evaluated the quality of the translation according to the token level BLEU [58] and METEOR [59] metrics and reported where W ∗ i and W o are learnable parameter matrices. Therefore, the summarized output from the two cross- attentions is fed into the residual connection and layer normal- ization. Then, the output is fed into the FFN sublayer, where the dimensions of the input and output are dm and dﬀ. Finally, the decoder is capped using a linear layer and a softmax layer to predict the probability of a target word. 13We did not report on METEOR due to the statistical signiﬁcance test for METEOR is not implemented in the Moses script. 14[Online]. Available: https://github.com/lium-lst/nmtpytorch TABLE I TABLE I TABLE I BLEU AND METEOR SCORES ON MULTI30K EN →DE TASK. THE RESULTS ARE SIGNIFICANTLY BETTER THAN THOSE OF NMT (†) AND MNMTR (‡) WITH p-VALUE < 0.05. THE GAP IN AN INCREASE (↑) OR DECREASE (↓) IN RELATION TO THE NMT BASELINE IS SHOWN IN PARENTHESES. THE BEST PERFORMANCE IN OUR MODELS AND EXISTING MNMT MODELS APPEAR IN BOLD. ALL OUR RESULTS ARE THE AVERAGE SCORES OVER THREE RUNS TABLE I BLEU AND METEOR SCORES ON MULTI30K EN →DE TASK. THE RESULTS ARE SIGNIFICANTLY BETTER THAN THOSE OF NMT (†) AND MNMTR (‡) WITH p-VALUE < 0.05. THE GAP IN AN INCREASE (↑) OR DECREASE (↓) IN RELATION TO THE NMT BASELINE IS SHOWN IN PARENTHESES. THE BEST PERFORMANCE IN OUR MODELS AND EXISTING MNMT MODELS APPEAR IN BOLD. ALL OUR RESULTS ARE THE AVERAGE SCORES OVER THREE RUNS TABLE I BLEU AND METEOR SCORES ON MULTI30K EN →DE TASK. THE RESULTS ARE SIGNIFICANTLY BETTER THAN THOSE OF NMT (†) AND MNMTR (‡) WITH p-VALUE < 0.05. THE GAP IN AN INCREASE (↑) OR DECREASE (↓) IN RELATION TO THE NMT BASELINE IS SHOWN IN PARENTHESES. THE BEST PERFORMANCE IN OUR MODELS AND EXISTING MNMT MODELS APPEAR IN BOLD. ALL OUR RESULTS ARE THE AVERAGE SCORES OVER THREE RUNS TABLE I BLEU AND METEOR SCORES ON MULTI30K EN →DE TASK. THE RESULTS ARE SIGNIFICANTLY BETTER THAN THOSE OF p-VALUE < 0.05. THE GAP IN AN INCREASE (↑) OR DECREASE (↓) IN RELATION TO THE NMT BASELINE IS SHOWN IN PAREN IN OUR MODELS AND EXISTING MNMT MODELS APPEAR IN BOLD. ALL OUR RESULTS ARE THE AVERAGE SCO In general, both the MNMTW2R(sa) and MNMTW2R(ha) models performed well, and there was almost no gap in trans- lation results between the integration of soft WRA and hard WRA. In more detail, the integration of soft WRA could help visual attention focus on regional visual features by considering all textual features according to the semantic relevance of each image region. On the other hand, the integration of hard WRA could assist visual attention to focus on regional visual features by indicating the most semantically relevant textual features for each of them. Therefore, the integration of either soft WRA or hard WRA could help the visual attention pay attention to regional visual features that were semantically related to the textual features, leading to better visual information use. the text-to-text NMT model. TABLE I The signiﬁcant improvements show that the proposed MNMTW2R(sa) and MNMTW2R(ha) models overcome both problems. Speciﬁcally, in our proposed models, we maintained the textual context while enriching the visual features with WRA-guided textual features to generate a multi- modal context such that the multimodal context can play a more effective role than the pure visual context. 3) Comparison of Proposed Model and Existing Ones: We conducted early stopping on the METEOR metric; therefore, we mainly compared our METEOR results with existing models. As shown in Table I, the METEOR scores of the transformer- based MNMTW2R(sa) and MNMTW2R(ha) models surpassed all the state-of-the-art MNMT results. The best performance was yielded by the proposed transformer-based MNMTW2R(sa) model. Moreover, the METEOR scores of the RNN-based MNMTW2R(sa) and MNMTW2R(ha) models were comparable to the state-of-the-art MNMT results. This demonstrates that our proposed models are competitive among all the existing MNMT models. 2) Results Within Our Transformer-Based Models: As shown in Table I, based on the transformer architecture, the MNMTR model cannot outperform the state-of-the-art text-to- text NMT model. This may be attributed to two factors: r When the primary modality (text) is sufﬁcient to accom- plish the translation task, the visual context cannot play a supplementary role; however, it may interfere with the effect of the textual context. A. Results on the En →De Task r Integrating WRA-guided textual features with visual fea- tures can promote visual features utilization. ZHAO et al.: WORD-REGION ALIGNMENT FOR MULTIMODAL NEURAL MACHINE TRANSLATION 251 TABLE II TABLE II BLEU AND METEOR SCORES ON MULTI30K EN →FR TASK. THE RESULTS ARE SIGNIFICANTLY BETTER THAN THOSE OF NMT (†) AND MNMTR (‡) WITH p-VALUE OF < 0.05. THE INCREASE (↑) OR DECREASE (↓) IN RELATION TO THE NMT BASELINE IS SHOWN IN PARENTHESES. THE BEST PERFORMANCE IN OUR MODELS AND EXISTING MNMT MODELS APPEAR IN BOLD. ALL OUR RESULTS ARE THE AVERAGE SCORES OVER THREE RUNS TABLE II BLEU AND METEOR SCORES ON MULTI30K EN →FR TASK. THE RESULTS ARE SIGNIFICANTLY BETTER THAN THOSE OF NMT (†) AND MNMTR (‡) WITH p-VALUE OF < 0.05. THE INCREASE (↑) OR DECREASE (↓) IN RELATION TO THE NMT BASELINE IS SHOWN IN PARENTHESES. THE BEST PERFORMANCE IN OUR MODELS AND EXISTING MNMT MODELS APPEAR IN BOLD. ALL OUR RESULTS ARE THE AVERAGE SCORES OVER THREE RUNS TABLE III BLEU AND METEOR SCORES ON FLICKR30KENT-JP EN →JP TASK. THE RESULTS ARE SIGNIFICANTLY BETTER THAN THOSE OF NMT (†) AND MNMTR (‡) WITH p-VALUE < 0.05. THE INCREASE (↑) OR DECREASE (↓) IN RELATION TO THE NMT BASELINE IS SHOWN IN PARENTHESES. THE BEST PERFORMANCE ARE SHOWN IN BOLD. ALL OUR RESULTS ARE THE AVERAGE SCORES OVER THREE RUNS TABLE III B. Results on the En →Fr Task r When encoding source words, the transformer considers the association between words and the entire sentence. However, there was no relationship among the regional features. Therefore, the visual context may not be as useful as textual context. As shown in Table II, the results of the En →Fr task showed a consistent trend with the results of the En →De task. Thus, the generality of the proposed framework was established. First, based on either the RNN or transformer architecture, the proposed MNMTW2R(sa) and MNMTW2R(ha) models con- sistently surpassed the NMT model by a signiﬁcant margin. In contrast, the proposed MNMTW2R(sa) and MNMTW2R(ha) models consistently improved the translation performance over IEEE/ACM TRANSACTIONS ON AUDIO, SPEECH, AND LANGUAGE PROCESSING, VOL. 30, 2022 IEEE/ACM TRANSACTIONS ON AUDIO, SPEECH, AND LANGUAGE PROCESSING, VOL. 30, 2022 252 A. Ablation Study To further verify the effectiveness of the different components in our proposed model, we also showed the performance of the following ablated versions. All the ablated versions were implementedonbothRNN-basedandtransformer-basedmodels with soft/hard WRA. Tables V and VI show their corresponding results. All the reported results are the average score over three runs. Therefore, results in Table III proved the effectiveness of integratingtheWRAandvalidatedthegeneralityoftheproposed model for different language pairs. The En →Jp results for previous works were unavailable because the WAT 2021 ofﬁcial dataset was newly created. VI. ANALYSES VI. ANALYSES of WRA, the proposed models are more effective in terms of improving translation performance than the MNMTR model, which can encourage visual features to play a positive role. C. Results on the En →Jp Task In contrast, the MNMTR without the WRA failed to achieve a uniﬁed improvement over NMT and the improvement was less signiﬁcant. This validates the effectiveness of our postula- tion that translation performance can be effectively improved through the integration of WRA. As shown in Table III, based on either the RNN or transformer architecture, the proposed MNMTW2R(sa) and MNMTW2R(ha) models consistently outperformed the NMT baselines on both BLEU and METEOR scores. In contrast, the MNMTR model without the integration of WRA failed to overcome its transformer-based NMT, and the improvement over its RNN-based NMT was less signiﬁcant than the proposed MNMTW2R(sa) and MNMTW2R(ha) models. This result may be attributed to the fact that, when textual modality is sufﬁcient to perform well, inadequate use of visual context may help less in translation performance and may even interfere with the effect of text context in transformer-based NMT. Second, the results of the proposed transformer-based MNMTW2R(sa) and MNMTW2R(ha) models surpassed all the state-of-the-art MNMT results based on both the BLEU and ME- TEOR metrics, except for the out-of-domain MSCOCO test set. The best performance was achieved by the transformer-based MNMTW2R(sa) model, which is consistent with the result of the En →De task. It is worthy of note that the previous researchers mainly compared the experimental results on the En →De task such that the existing results on the En →Fr task were fewer than those of the En →De task. In detail, the only difference between the proposed MNMTW2R(sa)/MNMTW2R(ha) model and the MNMTR base- line is the integration of WRA. Beneﬁtting from the integration ZHAO et al.: WORD-REGION ALIGNMENT FOR MULTIMODAL NEURAL MACHINE TRANSLATION 253 TABLE IV RIBES SCORES ON EN →DE, EN →FR, AND EN →JP TASKS. THE BEST PERFORMANCE IS SHOWN IN BOLD. ALL RESULTS ARE THE AVERAGE SCORES OVER THREE RUNS. THE RIBES SCORES REPORTED FOR EN →DE AND EN →FR TASKS ARE EVALUATED ON THE MULTI30K TEST2016 SET, AND EN →JP TASK IS EVALUATED ON THE WAT 2021 OFFICIAL TESTSET TABLE IV RIBES SCORES ON EN →DE, EN →FR, AND EN →JP TASKS. THE BEST PERFORMANCE IS SHOWN IN BOLD. ALL RESULTS ARE THE AVERAGE SCORES OVER THREE RUNS. THE RIBES SCORES REPORTED FOR EN →DE AND EN →FR TASKS ARE EVALUATED ON THE MULTI30K TEST2016 SET, AND EN →JP TASK IS EVALUATED ON THE WAT 2021 OFFICIAL TESTSET TABLE IV RIBES SCORES ON EN →DE, EN →FR, AND EN →JP TASKS. THE BEST PERFORMANCE IS SHOWN IN BOLD. ALL RESULTS ARE THE AVERAGE SCORES OVER THREE RUNS. THE RIBES SCORES REPORTED FOR EN →DE AND EN →FR TASKS ARE EVALUATED ON THE MULTI30K TEST2016 SET, AND EN →JP TASK IS EVALUATED ON THE WAT 2021 OFFICIAL TESTSET TABLE V ABLATION STUDY ON DIFFERENT INTEGRATION STRATEGIES OF WRA. BLEU AND METEOR SCORES ON EN →DE AND EN →FR TASKS USING THE MULTI30K DATASET. THE BEST PERFORMANCE IS SHOWN IN BOLD. ALL RESULTS ARE THE AVERAGE SCORES OVER THREE RUNS TABLE V IEEE/ACM TRANSACTIONS ON AUDIO, SPEECH, AND LANGUAGE PROCESSING, VOL. 30, 2022 IEEE/ACM TRANSACTIONS ON AUDIO, SPEECH, AND LANGUAGE PROCESSING, VOL. 30, 2022 254 TABLE VI LATION STUDY ON DIFFERENT INTERMODAL FUSION OPERATIONS. BLEU AND METEOR SCORES ON EN →DE AND EN →FR TASKS USING THE MULTI3 DATASET. THE BEST PERFORMANCE IS SHOWN IN BOLD. ALL RESULTS ARE THE AVERAGE SCORES OVER THREE RUNS TABLE VI ABLATION STUDY ON DIFFERENT INTERMODAL FUSION OPERATIONS. BLEU AND METEOR SCORES ON EN →DE AND EN →FR TASKS USING THE MULTI30K DATASET. THE BEST PERFORMANCE IS SHOWN IN BOLD. ALL RESULTS ARE THE AVERAGE SCORES OVER THREE RUNS TABLE VI ABLATION STUDY ON DIFFERENT INTERMODAL FUSION OPERATIONS. BLEU AND METEOR SCORES ON EN →DE AN DATASET. THE BEST PERFORMANCE IS SHOWN IN BOLD. ALL RESULTS ARE THE AVERAGE SCOR corresponding textual features to enrich the textual context and realize a multimodal context. From Table VI, it can be observed that the CONCAT operation was the most effective fusion operation to generate the multi- modal context in our proposal. The results were slightly worse when the fusion was realized using the SUM and MULTIPLY operators. This difference could be attributed to the fact that concatenation could make use of a linear layer that learned how to integrate the modality-speciﬁc activations into the multimodal context vector, as demonstrated in [12]. W2R and R2W (R ⇌W): We implemented W2R together with R2W to achieve a bi-directional integration strategy called R ⇌ W. In R ⇌W, both the visual context enriched by WRA-guided textualfeaturesandthetextualcontextenrichedbyWRA-guided visual features became multimodal contexts. As shown in Table V, the proposed MNMTW2R(sa) and MNMTW2R(ha) models achieved the best performance among all the integration strategies. When the WRA was integrated with the R2W and R ⇌W strategies, the results were worse than the W2R integration strategy because it might disturb the textual context. It was suggested that maintaining the textual context while enriching the visual context into a multimodal context by WRA-guided textual features was the most appropriate integra- tion strategy for WRA. D. Additional Results for Different Language Pairs 1) Different Integration Strategies of WRA: In the proposed MNMTW2R(sa) and MNMTW2R(ha) models, we integrate WRA to guide textual features into corresponding visual features to generate multimodal context during the W2R stage. In the ablation study, we conduct extensive experiments on different integration strategies of WRA to conﬁrm the effectiveness of the integration method of WRA. We further proved the superiority of our proposed models by measuring the RIBES [63] for different language pairs, which is an evaluation metric that is more sensitive to reordering. As shown in Table IV , the proposed MNMTW2R(sa) and MNMTW2R(ha) models achieved better RIBES scores than the NMT and MNMTR baselines based on either RNN or trans- former architecture across different language pairs. For instance, 0.5 and 0.4 RIBES scores were improved for En →De and En → Fr tasks, respectively; and 0.2 RIBES scores were improved for the En →Jp task. The results in Table IV further proved that the proposedmodelscouldachievesuperiortranslationperformance for different language pairs. Region-to-Word (R2W): Unlike the MNMTW2R(sa) and MNMTW2R(ha) models, we implemented a region-to-word (R2W) stage instead of the W2R stage introduced in Sec- tions III-B3 and III-C3. In R2W, we maintained the pure visual context and integrated the WRA-guided visual features with B. Human Evaluation The human evaluation results of the En →De, En →Fr, and En →Jp tasks are summarized in Figs. 5 and 6. We compared the translation quality of the RNN-based models and transformer- based models on all test sets among the MNMTW2R(sa), MNMTR, and NMT, or among the MNMTW2R(ha), MNMTR, and NMT. We randomly selected 50 samples for the evaluation of each test set and list the number of times that raters preferred one translation over another or judged them to be of the same quality (Tie). Moreover, compared with the R2W strategy, the R ⇌W strategywasslightlybetter.Weconjecturedthatwheninterfering with the textual context in the text-to-text task, enriching the visual context using WRA-guided textual features, instead of the pure visual context, enabled better visual information use in the image-to-text task. These results validated the effectiveness of the proposed WRA integration strategy. We employed two bilingual raters of German and English to evaluate the En →De translation quality, two bilingual raters of French and English to evaluate the En →Fr translation quality, and two bilingual raters of Japanese and English to evaluate the En →Jp translation quality. The raters were informed to focus more on the semantic meaning (adequacy) than on grammatical correctness (ﬂuency) when indicating reference translations. They were given the option of a tie if they could not make a decision. For the evaluation result, we counted the number of times the raters ranked one translation over others or considered that the translations were tied. We measured the agreement using the kappa coefﬁcient (K) with 20% of the samples of each language pair overlapped and evaluated twice. The agreement p p g gy 2) Different Intermodal Fusion Operations: We explored the impact of different intermodal fusion operations during the generation of a multimodal context on the overall performance. In the proposed W2R stage, we combined the WRA-guided textual features and visual features to generate a multimodal context with CONCAT as the fusion operator, which is deﬁned in 1). Instead of CONCAT, we investigated the SUM and MUL- TIPLY operations to fuse modalities for generating multimodal contexts. p p g gy 2) Different Intermodal Fusion Operations: We explored the impact of different intermodal fusion operations during the generation of a multimodal context on the overall performance. MNMTW MNMTW2R(ha) on different language pairs, respectively. For the Test2016 set of the En →De/En →Fr task and the ofﬁcial test set of the En →Jp task, we generated textual and visual representations as follows: 1) Text: the hidden representations for textual features generated by the textual encoder. 2) Im- age_(independent): representations of regional visual features generated by the visual encoder, independent of textual features. 3) Image_(MNMTW2R(sa/ha)): the learned representations for regional visual features generated by the W2R integration strat- egy using soft/hard WRA, which are enriched with semantically relevant textual features guided by WRA. We took the average of word/region representations to obtain the representations for each sentence and image and visualized them using the T-SNE toolkit. Fig. 6 Human evaluation results on the En →Jp task. The number of times the MNMTW2R(sa)/MNMTW2R(ha) ranked ﬁrst on test set is shown in red. for bilingual raters of German and English was K = 0.62, the agreement for bilingual raters of French and English was K = 0.69, and the agreement for bilingual raters of Japanese and English was K = 0.65. In the interpretation of the Kappa coefﬁcient, 0.61–0.8 is substantial. As shown in Fig. 7, the representations learned by the proposed RNN-based/Transformer-based MNMTW2R(sa) and MNMTW2R(ha) models are visualized, respectively. The representation distribution of different proposed mod- els conveys the same commonality that the distribution of image_(MNMTW2R(sa/ha)) is always in the middle of the text and image_(independent). It can be observed that the distri- bution of image_(MNMTW2R(sa/ha)) is always closer to the text than the image_(independent). Furthermore, although the image_(MNMTW2R(sa/ha)) is close to the text, the distribution of the text is not disturbed by the image_(MNMTW2R(sa/ha)). In general, we observed that the proposed MNMTW2R(sa) and MNMTW2R(ha) models based on both the RNN and transformer ranked top consistently across different language pairs. It further veriﬁed that not only under the automatic evaluation metrics but also under the human judgments, the proposed MNMTW2R(sa) and MNMTW2R(ha) models could achieve better translation performance. In more detail, when analyzing the same test set, the number of times that the MNMTW2R(sa) ranked top was close to that when the MNMTW2R(ha) ranked top. The integration of soft WRA could enrich regional features with an average of all textual features according to different semantic relevance. In contrast, the integration of hard WRA could explicitly enhance regional features with their most semantically relevant textual features. MNMTW When translating a source word, both can help the visual attention mechanism pay attention to regional features enriched by their relevant textual features. Consequently, the integration of either soft or hard WRA could lead to a better interaction between textual and visual features to make effective visual information utilization. y g ( / ) Visualizations in Fig. 7 further prove the contributions of each key component of the proposed method as follows: First, the utilization of WRA is a key component that causes the distribution of the image_(independent) and image_(MNMTW2R(sa/ha)) to be different. It can be found that, after enriching the independent visual features with related tex- tual features guided by WRA, the proposed method can bring the visual features closer to the textual features. This demonstrates that the intermediate facility WRA plays a crucial role as a bridge, connecting independent textual and visual features that are far from each other. B. Human Evaluation In the proposed W2R stage, we combined the WRA-guided textual features and visual features to generate a multimodal context with CONCAT as the fusion operator, which is deﬁned in 1). Instead of CONCAT, we investigated the SUM and MUL- TIPLY operations to fuse modalities for generating multimodal contexts. In the proposed W2R stage, we combined the WRA-guided textual features and visual features to generate a multimodal context with CONCAT as the fusion operator, which is deﬁned in 1). Instead of CONCAT, we investigated the SUM and MUL- TIPLY operations to fuse modalities for generating multimodal contexts. ZHAO et al.: WORD-REGION ALIGNMENT FOR MULTIMODAL NEURAL MACHINE TRANSLATION 255 Fig. 5 Human evaluation results on the En →De and En →Fr tasks. The number of times the MNMTW2R(sa)/MNMTW2R(ha) ranked ﬁrst on each test set is shown in red. Fig. 5 Human evaluation results on the En →De and En →Fr tasks. The number of times the MNMTW2R(sa)/MNMTW2R(ha) ranked ﬁrst on each test set is shown in red. the En →De and En →Fr tasks. The number of times the MNMTW2R(sa)/MNMTW2R(ha) ranked ﬁrst on each test set is 5 Human evaluation results on the En →De and En →Fr tasks. The number of times the MNMTW2R(sa)/MNMTW2R(h wn in red. Fig. 6 Human evaluation results on the En →Jp task. The number of times the MNMTW2R(sa)/MNMTW2R(ha) ranked ﬁrst on test set is shown in red. C. Visualization Second, the W2R integration strategy is another key compo- nent that brings the image_(MNMTW2R(sa/ha)) close to the text without disturbing the distribution of the text. By concatenating visual features with WRA-guided textual features in the W2R strategy, the proposed method can encourage visual features to In Fig. 7, we visualized the learned textual and visual rep- resentations to further analyze the proposed method. In detail, we visualized the textual and visual representations, which are learned by RNN-based/Transformer-based MNMTW2R(sa) and IEEE/ACM TRANSACTIONS ON AUDIO, SPEECH, AND LANGUAGE PROCESSING, VOL. 30, 2022 256 Fig. 7 Representation visualization for textual features, independent visual features, and enriched visual features with soft/hard WRA-guided textual features Representations are learned by RNN-based/Transformer-based MNMTW2R(sa) and MNMTW2R(ha) on the En →De, En →Fr and En →Jp tasks, respectively Text (blue): the textual representations generated by textual encoder. Image_(independent) (orange): the visual representations generated by visual encoder befor conducting W2R, which are independent of textual representations. Image_(MNMTW2R(sa/ha)) (green): the enriched visual representations generated by W2R which have been related with textual features by leveraging soft/hard WRA as a bridge. Fig. 7 Representation visualization for textual features, independent visual features, and enriched visual features with soft/hard WRA-guided textual features. Representations are learned by RNN-based/Transformer-based MNMTW2R(sa) and MNMTW2R(ha) on the En →De, En →Fr and En →Jp tasks, respectively. Text (blue): the textual representations generated by textual encoder. Image_(independent) (orange): the visual representations generated by visual encoder before conducting W2R, which are independent of textual representations. Image_(MNMTW2R(sa/ha)) (green): the enriched visual representations generated by W2R, which have been related with textual features by leveraging soft/hard WRA as a bridge. noun of “arrière-cour.” However, the NMT and MNMTR with- out the WRA models mistranslated it as “cour,” which means “yard” in English. Through visualization, we observed that the regional visual feature utilized by the MNMTW2R(ha) model provided more helpful information for generating more accurate translations than the MNMTR model. This showed that the pro- posed model can fully utilize visual information to complement textual information to learn more accurate translations. interact with semantically relevant textual features to help them be closer without disturbing the textual features. Therefore, it can be demonstrated that the W2R strategy plays a crucial role in advancing interactions between textual and visual modalities without disturbing the textual features. D. Case Study In Fig. 8, we showed two cases to analyze the translation qual- ity. The left case shows the results from the RNN-based models and the right case shows the results from the transformer-based models. We performed the visualization as following: 1) We visualized the source-target alignment of textual attention; and 2) We visualized the region-target alignment of visual attention at a time step that generated a certain target word while attending to the most weighted region. The region was denoted by a bounding box along with the target word. In the left example, the MNMTW2R(ha) correctly translated “backyard” to a compound In the right example, the MNMTW2R(sa) correctly translated the source phrase “tank tops” to the target word “débardeurs,” whereastheNMTandMNMTR withouttheWRAmodelsfailed. From the visualization, we observed that the textual attention weights for the source tokens “tank” and “tops” to the target word “débardeurs” were the highest. At the same time, the region with the highest visual attention weight provided semantically relevant information about “tank tops” to help generate the target word “débardeurs.” This showed that in the proposed model, the textual and visual context provided semantically ZHAO et al.: WORD-REGION ALIGNMENT FOR MULTIMODAL NEURAL MACHINE TRANSLATION 257 Fig. 8 Improved examples for case study. The improved translation is highlighted in green. Fig. 8 Improved examples for case study. The improved translation is highlighted in green. Fig. 9 Negative cases for error analysis. The translation errors are highlighted in red. The translation errors are highlighted in red. is. The translation errors are highlighted in red. to be redundant, which does not have a positive effect on the translation performance. relevant information interactively to generate more accurate translation. Additionally, there is another shortcoming of the WRA worth considering. Our method proposes to use visual concepts as an intermediary to pre-process the semantic relevance between image regions and source words into WRA. However, as we use the object detection method to extract visual concepts for object-level regions in images, the classiﬁed visual concepts are all in English. Therefore, this condition limits the source language to English in our proposed model. We think that this limitation can be solved. However, it is essential to use additional methods, such as cross-lingual word embedding. These cases revealed that our proposal can lead to better visual information use and improved translation accuracy. A. Shortcomings In Fig. 9, we showed two negative cases to analyze the shortcomings of the proposed method. In the upper case, the MNMTW2R(sa)/MNMTW2R(ha) mis- translated “moderately populated” that consisted of an adverb and a verb. After checking the WRA, we found no visual concepts related to “moderately” or “populated” included in the WRA. In the lower case, the MNMTW2R(sa)/MNMTW2R(ha) mistranslated the verb “breaking.” Similarly, we found no re- lated visual concepts for “breaking” included in the WRA. VIII. CONCLUSION [13] I. Calixto, Q. Liu, and N. Campbell, “Doubly-attentive decoder for multi- modal neural machine translation,” in Proc. 55th Annu. Meeting Assoc. Comput. Linguistics, 2017, pp. 1913–1924. In this study, we proposed WRA to link textual and visual features based on semantic relevance. To facilitate the semantic correlation between textual and visual contexts, we proposed a novel integration strategy W2R. The W2R integration method guided by WRA effectively maintained the textual context while transforming the visual context into a multimodal one by en- riching it using WRA-guided textual features. Extensive exper- imental results showed that the proposed model signiﬁcantly outperformed the competitive baselines on the En →De, En →Fr, and En →Jp language pairs consistently. Moreover, the performance of the proposed model surpassed most of the existing MNMT methods. Further analysis demonstrated that the proposed model’s superior translation performance was at- tributable to better visual information utilization. [14] J. Delbrouck and S. Dupont, “An empirical study on the effectiveness of images in multimodal neural machine translation,” in Proc. Conf. Empirical Methods Natural Lang. Process., 2017, pp. 910–919. [15] O. Caglayan et al., “LIUM-CVC submissions for WMT18 multimodal translation task,” in Proc. 3rd Conf. Mach. Transl., 2018, pp. 597–602. [16] O. Caglayan, P. Madhyastha, L. Specia, and L. Barrault, “Probing the need for visual context in multimodal machine translation,” in Proc. Conf. North Amer. Chapter Assoc. Comput. Linguistics, Hum. Lang. Technol., 2019, pp. 4159–4170. [17] H. Tan and M. Bansal, “LXMERT: Learning cross-modality encoder representations from transformers,” in Proc. Conf. Empirical Methods Natural Lang. Process. 9th Int. Joint Conf. Natural Lang. Process., 2019, pp. 5099–5110. pp [18] P. Yang, B. Chen, P. Zhang, and X. Sun, “Visual agreement regularized training for multi-modal machine translation,” in Proc. AAAI Conf. Artif. Intell., 2020, pp. 9418–9425. [19] Y. Zhao, M. Komachi, T. Kajiwara, and C. Chu, “Double attention-based multimodal neural machine translation with semantic image regions,” in Proc. 22nd Annu. Conf. Eur. Assoc. Mach. Transl., 2020, pp. 105–114. In the future, we plan to explore multimodal integration methods that can elevate the visual context from redundant to complementary. In this study, we have found that the use of visual features for non-visual words, such as verbs and adverbs, in the proposed model tends to be redundant. VIII. CONCLUSION To lead the visual context to act as a complementary role rather than a redundant role, the MNMT model needs to be able to understand which attributes of words require images and which attributes of words do not require images. Therefore, the word components in the sentence structure should be considered to improve MNMT performance in the next research step. [20] Y. Yin et al., “A novel graph-based multi-modal fusion encoder for neural machine translation,” in Proc. 58th Annu. Meeting Assoc. Comput. Linguistics, 2020, pp. 3025–3035. [21] D. Elliott, S. Frank, K. Sima’an, and L. Specia, “Multi30K: Multilingual english-german image descriptions,” in Proc. 5th Workshop Vis. Lang., 2016, pp. 70–74. [22] H. Nakayama, A. Tamura, and T. Ninomiya, “A visually-grounded parallel corpus with phrase-to-region linking,” in Proc. 12th Lang. Resour. Eval. Conf., 2020, pp. 4204–4210. [23] P. Huang, F. Liu, S. Shiang, J. Oh, and C. Dyer, “Attention-based multi- modal neural machine translation,” in Proc. 1st Conf. Mach. Transl., 2016, pp. 639–645. pp [24] O. Caglayan et al., “Does multimodality help human and machine for translation and image captioning?,” in Proc. 1st Conf. Mach. Transl., 2016, pp. 627–633. IEEE/ACM TRANSACTIONS ON AUDIO, SPEECH, AND LANGUAGE PROCESSING, VOL. 30, 2022 258 IEEE/ACM TRANSACTIONS ON AUDIO, SPEECH, AND LANGUAGE PROCESSING, VOL. 30, 2022 We think that, as the sentence structure and word order between English and German/French almost correspond, the improvement of word translation accuracy is signiﬁcant for the overall improvement of translation performance. However, because the basic sentence structure and word order between En- glish (strongly head-initial) and Japanese (strongly head-ﬁnal) are opposite, they lack the innate word order correspondence like English and German/French, the improvement of word accuracy is not enough to bring about a signiﬁcant improvement in the overall translation performance. Therefore, it is difﬁcult for the proposed method to improve the En →Jp task as much as it does for En →De and En →Fr tasks. We suggest that special care of word order is essential for improving MNMT translation performance between distant language pairs such as Japanese and English, and wrong word order may lead to misunderstanding or incomprehensibility. [4] D. Elliott, S. Frank, L. Barrault, F. Bougares, and L. Specia, “Findings of the second shared task on multimodal machine translation and mul- tilingual image description,” in Proc. 2nd Conf. Mach. Transl., 2017, pp. 215–233. [5] L. Barrault, F. Bougares, L. Specia, C. Lala, D. Elliott, and S. Frank, “Findings of the third shared task on multimodal machine translation,” in Proc. 3rd Conf. Mach. Transl., 2018, pp. 304–323. f pp [6] D. Elliott, S. Frank, and E. Hasler, “Multi-language image description with neural sequence models,” CoRR, vol. abs/1510.04709, 2015. Á [7] D. Elliott and Á. Kádár, “Imagination improves multimodal translation,” in Proc. 8th Int. Joint Conf. Natural Lang. Process., 2017, pp. 130–141. Proc. 8th Int. Joint Conf. Natural Lang. 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https://openalex.org/W3091080084	https://diposit.ub.edu/dspace/bitstream/2445/176292/1/706939.pdf	English	null	Dietary Strategies for Metabolic Syndrome: A Comprehensive Review	Nutrients	2,020	cc-by	13,646	Dietary Strategies for Metabolic Syndrome: A Comprehensive Review Sara Castro-Barquero 1,2,3 , Ana María Ruiz-León 2,3, Maria Sierra-Pérez 1, Ramon Estruch 1,2,3 and Rosa Casas 1,2,3,* Sara Castro-Barquero 1,2,3 , Ana María Ruiz-León 2,3, Maria Sierra-Pérez 1, Ramon Estruch and Rosa Casas 1,2,3,* 1 Department of Medicine, Faculty of Medicine and Life Sciences, University of Barcelona, 08036 Barcelona, Spain; sara.castro@ub.edu (S.C.-B.); mariasipe4@gmail.com (M.S.-P.); restruch@clinic.cat (R.E.) 1 Department of Medicine, Faculty of Medicine and Life Sciences, University of Barcelona, 08036 Barcelona, Spain; sara.castro@ub.edu (S.C.-B.); mariasipe4@gmail.com (M.S.-P.); restruch@clinic.cat (R.E.) 2 Institut d’Investigacions Biomèdiques August Pi I Sunyer (IDIBAPS), 08036 Barcelona, Spain; amruiz@clinic.cat 2 Institut d’Investigacions Biomèdiques August Pi I Sunyer (IDIBAPS), 08036 Barcelona, Spain; amruiz@clinic.cat 3 Centro de Investigación Biomédica en Red, Fisiopatología de la Obesidad y la Nutrición (CIBEROBN), Instituto de Salud Carlos III, 28029 Madrid, Spain * Correspondence: rcasas1@clinic.cat; Tel.: +34-932275400; Fax: +34-932272907 Received: 7 September 2020; Accepted: 27 September 2020; Published: 29 September 2020 Abstract: Metabolic syndrome is a cluster of metabolic risk factors, characterized by abdominal obesity, dyslipidemia, low levels of high-density lipoprotein cholesterol (HDL-c), hypertension, and insulin resistance. Lifestyle modiﬁcations, especially dietary habits, are the main therapeutic strategy for the treatment and management of metabolic syndrome, but the most eﬀective dietary pattern for its management has not been established. Speciﬁc dietary modiﬁcations, such as improving the quality of the foods or changing macronutrient distribution, showed beneﬁcial eﬀects on metabolic syndrome conditions and individual parameters. On comparing low-fat and restricted diets, the scientiﬁc evidence supports the use of the Mediterranean Dietary Approaches to Stop Hypertension (DASH) diet intervention as the new paradigm for metabolic syndrome prevention and treatment. The nutritional distribution and quality of these healthy diets allows health professionals to provide easy-to-follow dietary advice without the need for restricted diets. Nonetheless, energy-restricted dietary patterns and improvements in physical activity are crucial to improve the metabolic disturbances observed in metabolic syndrome patients. Keywords: metabolic syndrome; dietary pattern; Mediterranean diet; plant-based diet; DASH diet; low-carbohydrate diet; high-protein diet; low-fat diet nutrients nutrients 1. Introduction Following unhealthy dietary patterns and sedentary lifestyles has led to a notable increase in the prevalence of overweight and obesity worldwide. Non-communicable chronic diseases (NCDs) related to unhealthy dietary patterns and weight gain have expanded in parallel, being the major cause of morbidity and mortality both in developed and underdeveloped countries [1]. Among NCDs, cardiovascular diseases (CVD) and type 2 diabetes mellitus (T2DM) are public health priorities, not only for their high prevalence and outcomes but also for the huge economic burden imposed on the health system [2,3]. Metabolic syndrome (MetS) is a clinical condition characterized by a clustering of metabolic risk factors, which is deﬁned by the simultaneous occurrence of at least three of the following components: central obesity, dyslipidemia, impaired glucose metabolism, elevated blood pressure (BP), and low levels of high-density lipoprotein cholesterol (HDL-c), according to the consensual deﬁnition of the International Diabetes Federation, the American Heart Association, and the National Heart, Lung and Nutrients 2020, 12, 2983; doi:10.3390/nu12102983 www.mdpi.com/journal/nutrients www.mdpi.com/journal/nutrients 2 of 21 Nutrients 2020, 12, 2983 Blood Institute [4]. In developed countries, the prevalence of MetS has risen up to 20–25% in the adult population, and its incidence continues to increase over time [5–8]. In Spain, the prevalence of MetS is currently reaching epidemic proportions, aﬀecting approximately 22.7% of the population, taking into account that its incidence increases with age [5]. In addition, MetS increases the risk of T2DM onset and major cardiovascular events by two-fold and ﬁve-fold, respectively, and other chronic disease such as cancer, neurodegenerative diseases, non-alcoholic fatty liver disease, the risk of reproductive, lipid and circulatory disorders, atherosclerosis, and all cause-mortality are also increased [8–13]. Blood Institute [4]. In developed countries, the prevalence of MetS has risen up to 20–25% in the adult population, and its incidence continues to increase over time [5–8]. In Spain, the prevalence of MetS is currently reaching epidemic proportions, aﬀecting approximately 22.7% of the population, taking into account that its incidence increases with age [5]. In addition, MetS increases the risk of T2DM onset and major cardiovascular events by two-fold and ﬁve-fold, respectively, and other chronic disease such as cancer, neurodegenerative diseases, non-alcoholic fatty liver disease, the risk of reproductive, lipid and circulatory disorders, atherosclerosis, and all cause-mortality are also increased [8–13]. Recent evidence has demonstrated the association between the incidence and prevention of MetS and modiﬁable lifestyle factors, especially dietary habits. Steckhan et al. 1. Introduction analyzed the positive eﬀects of diﬀerent dietary approaches on MetS inﬂammatory markers [14]. Regarding the prevention of MetS, Godos et al. also conducted a meta-analysis to demonstrate the preventive role of the promotion of healthy dietary patterns to reduce the prevalence of MetS [15]. Furthermore, some sub-studies from the PREDIMED-Plus cohort showed associations between some dietary components of the traditional Mediterranean Diet (MedDiet) and improvement in MetS components [16–20]. The aim of the present review was to analyze the potential beneﬁts of diﬀerent dietary approaches on MetS status and their use as eﬃcient strategies to prevent and treat MetS and its comorbidities. Dietary Patterns A single-nutrient dietary intervention has several limitations, and dietary advice must be focused on the overall dietary pattern as part of MetS treatment. Recent evidence supports the implementation of healthy food-based dietary interventions instead of calorie or isolated nutrient restriction [1,21] diets. The health beneﬁts, regarding MetS, of dietary macronutrient patterns and diﬀerent dietary approaches are summarized in Table 1. 3 of 21 Nutrients 2020, 12, 2983 Table 1. Dietary strategies and potential health beneﬁts for Metabolic Syndrome (MetS). Dietary Pattern Nutritional Distribution Improvements in MetS Criteria Ref. Mediterranean diet I 35–45% kcal/d from total fat (mainly MUFA 1, EVOO and nuts being the principal source) I 35–45% kcal/d from CH I 15–18% kcal/d from protein Reduction of CVD incidence and outcomes [22–29] Decreased BP (systolic and diastolic) [15,26] Inverse association with mortality [24,30] Improvements in dyslipemia [26] Decreased incidence of T2DM [12,22,23,29,31] DASH diet I Total fats 27% kcal/d I Saturated fats 6% kcal/d I Dietary cholesterol I CH 55% kcal/d I Proteins 18% kcal/d Reduction of BP (systolic and diastolic) [32,33] Reduction in BMI and waist circumference [34,35] Improvement in cardiometabolic proﬁle [36–39] Reduction in T2DM incidence [40] Plant-based diets I Reduction or restriction of animal-derived foods I High intake of plant-source foods I Fat proﬁle rich in UFAs Reduction of BP (systolic and diastolic) [41,42] Decreased body weight and risk of obesity [43–45] Reduction of the risk of CVD [46] Decreased all-cause mortality [43,47,48] Decreased risk of T2DM [43,47,48] Low CH diets and very low CH diets (ketogenic diets) I <50% kcal/d from carbohydrates and <10% kcal/d from CH in ketogenic diets I High protein (20–30% kcal/d) I High fat intake (30–70% kcal/d) Weight-loss and weight-loss maintenance [49–52] Reduction of DBP [52] Reduction of LDL-c and triglycerides levels [49–51] Increase of HDL-c levels [49–51] Improvements in insulin resistance [53,54] Reduction of HbA1c levels [49,51] Low-fat diet I <30% kcal/d from total fat (<10% of saturated fat) I 15–17% kcal/d from protein I 50–60% kcal/d from CH Reduction of BP (systolic and diastolic) [33,55] Short-term improvement of cholesterol proﬁle [33,55] Short-term weight loss [55] Reduced risk of all-cause mortality [56] High protein diet I High protein (20–30% kcal/d) or 1.34–1.50 g/Kg body weight/d from protein I Low CH (40–50% kcal/d) Reduction of triglycerides levels [57,58] le 1. Dietary strategies and potential health beneﬁts for Metabolic Syndrome (MetS). 4 of 21 Nutrients 2020, 12, 2983 Table 1. Cont. 2. Mediterranean Diet The MedDiet refers to the dietary pattern, culture and culinary techniques adhering to countries and populations living in the Mediterranean Sea basin [64]. This dietary pattern has stimulated a great deal of scientiﬁc evidence, demonstrating the potential health beneﬁts associated with adherence, and the primary and secondary prevention of many health outcomes, such as CVD, T2DM, and MetS [10,22]. Recent scientiﬁc evidence concluded that the MedDiet not only has beneﬁcial eﬀects on health but also has beneﬁcial eﬀects on sustainability and culture [22,65]. Additionally, the MedDiet has been recognized by UNESCO as an Intangible Cultural Heritage of Humanity [66] and the 2015–2020 American Dietary guidelines referred to the MedDiet as an example of a healthy dietary pattern [21]. The MedDiet is a plant-based diet characterized by a high intake of vegetables including leafy green vegetables, fruits, whole-grain cereals, pulses, legumes, nuts, and extra virgin (cold pressed) olive oil (EVOO) as the main source of fat. Moreover, classical recipes are seasoned with sauces such as sofrito, whose main ingredients are olive oil, tomato, garlic, onion or leek, rich in phenolic compounds and carotenoids, such as naringenin, hydroxy-tyrosyl, lycopene and β-carotene [67]. Moderate alcohol intake of fermented alcoholic beverages such as red wine, mainly during meals, is also characteristic of the MedDiet, which also comprises a low to moderate intake of ﬁsh and poultry, and low consumption of red meat, butter, sweets, pastries and soft drinks [12,23,68] The traditional MedDiet is a high fat and low-carbohydrate (CH) dietary pattern, which provides a 35–45% of total daily energy intake from fat, about 15% from protein, and 40–45% energy from CH [12,68]. However, the proﬁle of this fat is mainly one of monounsaturated (MU) and polyunsaturated (PU) fatty acids (FA) and the main food sources of total fat intake are EVOO and nuts. EVOO is one of the key foods of the MedDiet and is the main contributor of monounsaturated fatty acids (MUFAs) in MedDiet countries. Oleic acid is the major component of EVOO and many studies have linked MUFA intake to improvements in insulin resistance, one of the main risk factors for MetS, and in blood lipid proﬁle, and a reduction in both systolic and diastolic BP levels [12,24,69]. Table 1. Cont. Table 1. Cont. Dietary Pattern Nutritional Distribution Improvements in MetS Criteria Ref. Other dietary patterns and strategies Nordic diet I High content of whole-grain high-ﬁbre products I Low in meat and processed foods Reduction of BP (systolic and diastolic) [59] Increase of HDL-c levels [59] Intermittent fasting I Fasting for a long period of time Weight loss [60–62] Improvements in insulin resistance [60–62] Improvements in dyslipidaemia [60–62] Reduction of BP (systolic and diastolic) [60–62] Decreased risk of T2DM [63] Decreased risk of CVD [63] 1 Monounsaturated fatty acids, MUFA; extra virgin olive oil, EVOO; carbohydrates, CH; cardiovascular disease, CVD; blood pressure, BP; type 2 diabetes mellitus, T2DM; Dietary Approaches to Stop Hypertension, DASH; unsaturated fatty acids, UFAs; body mass index, BMI; diastolic blood pressure, DBP; low-density lipoprotein cholesterol, LDL-c; high-density lipoprotein cholesterol, HDL-c, glycated hemoglobin, HbA1c; monounsaturated fatty acids, MUFA. 5 of 21 Nutrients 2020, 12, 2983 2. Mediterranean Diet EVOO is also rich in polyphenols, which present anti-inﬂammatory and antioxidant eﬀects and contribute to improving the lipid proﬁle and endothelial function [70] Besides the beneﬁcial eﬀects of unsaturated fats, the whole dietary pattern characterized by the high intake of fruits and vegetables together with moderate red wine consumption provides wide nutritional components, such as antioxidant vitamins (vitamin C, E and β-carotene), phytochemicals (such as polyphenols), folates and minerals, which may exert beneﬁcial eﬀects [31,70]. Considering the eﬀects of the MedDiet on MetS, Di Daniele et al. conducted a review addressing the impact of MedDiet adherence on MetS criteria, obesity and adipose tissue dysfunction [10]. The authors reported that prescription of the MedDiet can be used as a possible therapy for MetS, as it prevents the excess of adiposity and obesity-related inﬂammatory response. Franquesa et al. concluded that there is a strong evidence for the eﬀect of the MedDiet on obesity and on MetS prevention in healthy or high-CVD risk individuals, as well as on the risk of mortality in overweight or obese individuals [22]. As previously cited, a meta-analysis of 12 cross-sectional and prospective cohorts showed that higher adherence to the MedDiet was associated with a 19% lower risk of developing MetS (relative risk (RR): 0.81 (95% conﬁdence interval (CI) 0.71 to 0.92)), and individual components, such as waist circumference and BP, were also improved (RR: 0.82 (95% CI 0.70 to 0.96); RR: 0.87 (95% CI 0.77 to 0.97), respectively) [15]. Several prospective studies observed the same protective eﬀects in Mediterranean and non-Mediterranean countries [25–27]. The CARDIA (Coronary Artery Risk Development in Young Adults) study is a prospective study including 4713 individuals which evaluated the evolution of CVD risk factors in black and white populations in the United States [28]. They observed a lower incidence of MetS in individuals with a higher adherence to the MedDiet (Hazard ratio (HR): 0.67 (95% CI 0.49 to 0.90)) compared to those with lower adherence, showing a linear trend according to the ﬁve score categories (p for trend = 0.005) [25]. Kesse-Guyot et al. conducted a prospective 6-year follow-up with 3232 subjects in the SU.VI.MAX study to evaluate the association between diﬀerent MedDiet adherence scores and the incidence of MetS. 3. DASH Diet In 1997, the Dietary Approaches to Stop Hypertension (DASH) diet became a promising strategy for the treatment of high BP [74], and subsequent randomized clinical trials (RCTs) have supported this evidence [32]. This eating pattern promotes vegetables, fruits, whole grains, low- or free-fat dairy products, legumes and nuts intake, while restricting the intake of red and processed meat and sugar-sweetened beverages [74,75]. The DASH diet is characterized by a low-fat content (27% of daily calorie intake from fat), especially saturated fats (6% of energy) and dietary cholesterol (150 mg/d approximately), and reduced sodium content (from 1500 to 2300 mg/day), but it is rich in ﬁber (>30 g/day), potassium, magnesium and calcium compared to other dietary patterns [55,76]. The DASH diet has proven to be a useful strategy for the treatment of hypertension [32,33,55,77], and several epidemiological studies have associated higher adherence to the DASH diet with a better cardiometabolic proﬁle [34,36–39,78–80]. In a meta-analysis of several cohort studies, Schwingshackl et al. reported that higher adherence to the DASH diet was associated with a signiﬁcant reduction in the risk of all-cause mortality (RR: 0.78 (95% CI 0.77 to 0.80), the incidence of or mortality by CVD and cancer (RR: 0.78 (95% CI 0.76 to 0.80); RR: 0.84 (95% CI 0.82 to 0.87), respectively) and the incidence of T2DM (RR: 0.82 (95% CI 0.78 to 0.85)) [40]. Regarding the use of DASH diet as an approach for the treatment of hypertension, a recent meta-analysis of 30 RCT with 5545 hypertensive and non-hypertensive participants concluded that the DASH diet together with lifestyle interventions signiﬁcantly decreased systolic and diastolic BP measurements compared with a control diet (mean diﬀerences: −3.2 mm Hg (95% CI −4.2 to −2.3) and −2.5 mm Hg (95% CI −3.5 to −1.5), respectively) [32]. This eﬀect was more pronounced when sodium intake was lower than 2400 mg/d, in subjects under the age of 50, and in participants with hypertension but without antihypertensive medication [32]. Moreover, on comparing the antihypertensive eﬀects of the DASH diet with 13 other eating patterns (including low-fat diet, Nordic diet, MedDiet, Paleolithic diet and low-sodium diet), the DASH diet was the most eﬀective, especially in comparison with low-fat diets [33]. In contrast, Ge et al. 2. Mediterranean Diet They found that participants with higher adherence had a 53% lower 6 of 21 Nutrients 2020, 12, 2983 risk compared to the lowest tertile of the MedDiet score (odds ratio (OR): 0.47 (95% CI 0.32 to 0.69) and 0.50 (95% CI 0.32 to 0.77 for each MedDiet score) [26]. In addition, MedDiet adherence scores were associated with improvements in some individual criteria for MetS, such as waist-circumference, BP, triglycerides and HDL-c levels [26]. Moreover, lower MetS prevalence was observed in Korean adults with medium to high MedDiet adherence (OR: 0.73 (95% CI 0.56 to 0.96) and 0.64 (95% CI 0.46 to 0.89), respectively) [30]. MedDiet adherence has been inversely associated with the incidence of CVD and mortality, as well as cancer and degenerative diseases [23,71]. In the case of CVD, the MedDiet is associated with clinically meaningful reductions in the risk of developing the main CVD outcomes, including coronary heart disease and stroke [72]. In a prospective cohort study with 25,994 healthy women from the US Women’s Health Study, Ahmad et al. observed an inverse association between the highest MedDiet adherence score and the incidence of CVD compared to the lowest score (HR: 0.72 (95% CI 0.61 to 0.86), p for trend < 0.001) [73]. Among the health eﬀects observed, MedDiet interventions have shown improvements in body composition by reducing total and segmental fat, which might have an eﬀect on metabolic proﬁle [10]. Furthermore, the MedDiet has contributed to a decrease in the incidence of T2DM and CVD, while lessening severity and associated complications in individuals who have already been diagnosed [12,22,23,29,31]. Due to the health beneﬁts associated with this easy-to follow dietary pattern, the MedDiet should be considered as one of the ﬁrst treatment strategies for the prevention and management of MetS. 3. DASH Diet identiﬁed the Paleolithic and Atkins diets as the most eﬀective dietary patterns for both systolic and diastolic BP management after six months of intervention compared to usual dietary advice, although this eﬀect was not observed after one year of intervention [55]. The DASH diet intervention has also shown potential eﬀects against excess body weight and abdominal obesity [35]. Middle-term dietary interventions have shown a signiﬁcant reduction in The DASH diet intervention has also shown potential eﬀects against excess body weight and abdominal obesity [35]. Middle-term dietary interventions have shown a signiﬁcant reduction in 7 of 21 Nutrients 2020, 12, 2983 body mass index (BMI) (weighted mean diﬀerence: −0.42 kg/m2 (95% CI −0.64 to −0.20)) and waist circumference (−1.05 cm (95% CI −1.61 to −0.49)) [35]. Nevertheless, in overweight or obese individuals, DASH dietary approaches showed signiﬁcant weight loss compared to other dietary patterns (−3.63 kg (95% Credible Interval −2.52 to −4.76)) whereas this weight loss was lower after one year of intervention (−3.08 kg (95% Credible Interval −0.48 to −5.66)) [55]. g The results are not consistent in the case of blood lipoproteins [55,77]. Ge et al. did not observe signiﬁcant diﬀerences in HDL-c or low-density lipoprotein-cholesterol (LDL-c) levels after a DASH dietary intervention versus usual diet [55], whereas in a meta-analysis of 1917 participants with some CVD risk factors, Siervo et al. observed a reduction in total cholesterol and LDL-c levels after the DASH intervention (mean diﬀerences: −0.20 mmol/L (95% CI −0.31 to −0.10) and −0.10 mmol/L (95% CI −0.20 to 0.01), respectively), but reported no signiﬁcant diﬀerences in HDL-c and triglyceride levels [77]. Similar results were obtained in a recently published controlled trial in 80 T2DM patients after 12 weeks following the DASH diet compared to an antidiabetic diet based on American Diabetes Association guidelines [81]. Both dietary interventions signiﬁcantly reduced triglycerides, total cholesterol and very-low-density lipoproteins. Epidemiological evidence suggests an association between higher adherence to the DASH diet and a better cardiometabolic proﬁle and lower risk of CVD [36–39]. A cross-sectional study of 1493 adults showed that higher adherence to the DASH diet was associated with 48% less risk of developing MetS, whereas BMI, waist circumference, pro-inﬂammatory markers and adiposity measures were signiﬁcantly lower compared to individuals with lower adherence [34]. Interestingly, Ashari et al. 3. DASH Diet observed that higher adherence to the DASH diet was associated with a 64% lower risk of MetS in 425 healthy children and adolescents from 6–18 years of age [82]. In addition, the authors also observed inverse associations among adherence to the DASH diet and BP, fasting plasma glucose levels and abdominal obesity [82]. In this sense, adaptation of the DASH diet to type 1 diabetes glucose requirements (a reduction in CH of around 10% and 15% increase in fat content) resulted in better glucose control and improved the quality of the whole diet, showing a higher intake of fruits, vegetables, ﬁber and protein compared to the usual intake [83]. The health beneﬁts associated with the DASH diet are probably due to its nutritional quality and distribution. The DASH diet is rich in vegetables and fruits, which translate into high potassium, magnesium and ﬁber intake, and these nutrients have shown to have a role in BP control, glucose metabolism and insulin response [84]. Furthermore, vegetables and fruits are the main food source of antioxidants and polyphenols, which have been linked to better glucose and insulin blood levels [84]. Moreover, it is limited in sodium and fat, mainly saturated fatty acids (SFA), which are closely related to CVD [84]. Nonetheless, Pickering et al. suggested that the potential health eﬀects of the DASH diet are dependent on eating pattern adherence, with subjects with lower adherence to the DASH diet showing greater beneﬁt from DASH dietary interventions in BP control than those with higher adherence before the dietary intervention [85]. Nonetheless, the commitment and implication of the patient are critical in all life-style interventions based on dietary modiﬁcations [86,87]. 4. Plant-Based Diets Plant-based diets include a wide variety of dietary patterns, which are characterized by a reduction or restriction in animal-derived food intake and the promotion of plant-source food intake, such as fruits, vegetables, nuts, legumes, and grains. Among plant-based diets, strict vegetarian diets, also known as vegan diets, are deﬁned by the exclusion of all animal-derived products, including dairy products, eggs and honey; lacto-vegetarian diets restrict animal food intake except for dairy products; lacto-ovo-vegetarian diets exclude meat, seafood and poultry but include eggs and dairy products; and pesco-vegetarians or pescatarians are similar to lacto-ovo-vegetarian but include ﬁsh [88]. Despite the fact that plant-based diets are deﬁned by the exclusion of some or all animal products, recent evidence deﬁnes plant-based diets as dietary patterns that promote a reduction in animal-source food intake along with an increase in plant-based food intake, such as the MedDiet [41,88–90]. Nutrients 2020, 12, 2983 8 of 21 Plant-based diets have consistently been associated with beneﬁcial cardiometabolic eﬀects, speciﬁcally with a lower risk of developing MetS and all of its components [91]. Moreover, these dietary patterns are associated with decreased all-cause mortality and a decreased risk of obesity, T2DM and CVD [43,47,48]. Some studies have found a lower risk of mortality from ischemic heart disease in vegetarians compared with non-vegetarians [43]. Additionally, recent systematic reviews and meta-analyses found signiﬁcant associations between adherence to the MedDiet and DASH diets and a 38% and 20% lower risk of CVD, respectively, while a 28% reduction in the risk of coronary heart disease was observed following a vegetarian diet [46]. Regarding BP, a meta-analysis of seven RCTs reported a mean reduction of 4.8 mmHg in systolic BP (95% CI −3.1 to −6.6; p < 0.001) and a 2.2 mmHg reduction in diastolic BP (95% CI −1.0 to −3.5; p < 0.001) in participants following a vegetarian diet compared to an omnivorous diet [42]. These results were conﬁrmed by the same authors in a meta-analysis of 32 observational studies including 604 participants, in which an association was observed between vegetarian diets and reductions in systolic and diastolic BP (−6.9 mmHg (95% CI −9.1 to −4.7; p < 0.01) and −4.7 mmHg (95% CI −6.3 to −3.1; p < 0.01), respectively) [41,42]. p p y The eﬀects of plant-based diets on blood lipid concentrations are controversial. Wang et al. 4. Plant-Based Diets conducted a meta-analysis of 11 RCTs to evaluate the eﬀects of vegetarian diet on triglycerides, LDL-c, HDL-c and non-HDL-c levels [92]. Total cholesterol levels, LDL-c and HDL-c, were signiﬁcantly reduced after following a vegetarian diet compared to an omnivorous control diet (0.36 mmol/L (95% CI 0.55 to 0.17; p < 0.001), 0.34 mmol/L (95% CI 0.57 to 0.11; p < 0.001) and 0.10 mmol/L (95% CI 0.14 to 0.06; p < 0.001), respectively). No signiﬁcant eﬀects were observed for triglyceride levels. This study also described a signiﬁcant weight-loss in participants who followed the vegetarian compared to the omnivorous diet (−2.88 kg (95% CI −3.56 to −2.20; p < 0.001)). Similar results were observed in another meta-analysis of 12 RCTs involving 1151 individuals, in which subjects randomized to the vegetarian diet intervention group showed signiﬁcant weight loss compared to the non-vegetarian group (mean diﬀerence −2.02 kg (95% CI −2.80 to −1.23; p < 0.001)) [44]. Other studies assessing plant-based dietary patterns, such as the MedDiet, have also described positive eﬀects on body weight and waist circumference [45]. The health beneﬁts observed are mainly explained by the nutritional quality of plant-based diets as they promote the intake of a wide variety of plant-based foods while cutting down the intake of animal-derived products, such as red and processed meat, which have been associated with a higher risk of developing T2DM, CVD and certain types of cancer [93]. However, it is important to consider that the term “plant-based” does not necessary mean “healthy”, as there is evidence supporting adverse health eﬀects of the excessive intake of some plant-derived foods, such as reﬁned grains, snacks, pastries or sugar-sweetened beverages [41,88,94]. A healthy plant-based diet promotes the intake of whole grains, fruits, vegetables, legumes, and non-hydrogenated vegetable oils, such as EVOO. Thus, plant-based diets have low-energy density and high ﬁber content, which may contribute to CVD prevention, weight loss and long-term body weight maintenance [41,44,88]. Moreover, the proﬁle of fat is mainly MUFA and polyunsaturated fatty acids (PUFA), while SFA intake is lower compared to other dietary patterns. Replacing SFA by MUFA and PUFA has been linked with anti-inﬂammatory eﬀects and improvements in insulin sensitivity [88]. Among plant-derived foods, the antioxidant eﬀect exerted by several nutrients and bioactive compounds such as vitamin C and E, β-carotenes and polyphenols has been linked to the prevention of CVD and MetS [41,88,95]. 5. Low-Carbohydrate Diet Low-CH dietary patterns are characterized by a reduction of total CH intake (<50% of daily calorie intake from CH). This type of diet implies a restriction in the intake of several ultra-processed foods, reﬁned grains, starches and foods rich in simple or added sugars [1]. The association between CH intake and the prevalence and management of the MetS is discrepant [97]. In a meta-analysis of 18 studies with 69,554 MetS patients, Lui et al. concluded that the risk of developing MetS was increased in individuals with higher CH intake (2.5% increase in the risk of MetS per 5% energy from CH intake (95% CI 0.4 to 4.8)) [97]. Moreover, some eﬀects on lipid proﬁle were observed in individuals with high CH intake, such as elevated BP, triglycerides and LDL-c and reduced HDL-c levels [98,99]. The mechanisms underlying the health beneﬁts observed in low-CH diets are the avoidance of the rapid absorption associated with some types of CH, such as glucose and reﬁned grains, which leads to an increase in insulin resistance and insulin demand [53,54]. Therefore, in the case of T2DM, recent clinical guidelines do not recommend a speciﬁc CH distribution or restriction, and dietary individualization must be prioritized in the treatment and management of this condition [49]. Bazzano et al. conducted a RCT to analyze the eﬀect of a low-CH diet (<40% of total energy intake from CH) compared with a low-fat diet (<30% of total energy from fat, <7% SFA) without energy restriction or physical activity advice in obese adults (BMI 30 to 45 kg/m2) [50]. After 1 year of intervention, subjects on the low-CH diet without energy restriction showed greater weight loss (−3.5 kg (95% CI −5.6 to −1.4 kg)), speciﬁcally in fat mass (−1.5% (CI −2.6% to −0.4%)). Moreover, some cardiovascular risk factors were improved in the low-CH group, such as triglycerides, HDL-c and total cholesterol to HDL-c ratio [50]. Regarding the management of T2DM, low-CH compared to low-fat dietary interventions (<30% of total energy from fat) showed higher reductions of body weight, glycosylated hemoglobin (HbA1c), triglycerides and BP levels and increased HDL-c concentrations and, consequently, a modiﬁcation in glucose-lowering medications was observed [49,51]. Recent evidence has shown an association between dietary CH intake and the risk of mortality. The Prospective Urban Rural Epidemiology (PURE) study is a cohort study of 135,335 individuals aged 35–70 years from 18 countries from ﬁve continents [98]. 4. Plant-Based Diets Finally, the replacement of some animal-derived foods implies intake restriction of the harmful components mainly present in red and processed meat, such as excessive sodium, heme iron, nitrates and nitrites, which have been linked to CVD outcomes [41,88,96]. In conclusion, recent evidence has demonstrated the protective eﬀect of plant-based diets against MetS, CVD and their individual risk factors. However, healthy plant-derived food choices are crucial to ensure these beneﬁcial eﬀects. Thus, dietary guidelines should consider healthy plant-based dietary patterns as a potential dietary strategy for the prevention and treatment of MetS. 9 of 21 Nutrients 2020, 12, 2983 5. Low-Carbohydrate Diet The aim of this study was to assess the association of dietary fat and CH intake and total mortality and CVD, diﬀerentiating this intake according to the proﬁle of FA and CH. The ﬁndings of this study suggest the need for an update in dietary guidelines, with emphasis on fat restriction to promote low-fat and CH dietary patterns (around 50–55% of daily energy intake from CH) rich in PUFA and whole-grain CH. Other studies have also observed an association between reﬁned CH intake and a higher risk of cardiovascular events, such as stroke or myocardial infarction [100,101]. However, there is insuﬃcient scientiﬁc evidence on low-CH diets (<50–55% of total energy intake) and metabolic improvements have not been demonstrated in order to support or recommend very-low CH diets [98]. Seidelmann et al. observed that with high (>70% of total energy intake) and low (<40%) CH diets the total mortality increased, with 50–55% showing the lowest risk of mortality, representing a U-shaped association [102]. The replacement of CH with other nutrients has shown diﬀerent eﬀects on total mortality, which was increased in low-CH diets rich in animal-derived fat and/or protein. By deﬁnition, low-CH diets promote the restriction of foods rich in CH, such as vegetables, fruits, whole-grain cereals, legumes, etc. Consequently, low-CH diets, compared to diets with 50–55% of energy from CH, showed lower amounts of bioactive compounds such as ﬁber, PUFAs, polyphenols, vitamins and minerals [103]. Therefore, the use of very low-CH diets as a dietary approach for MetS should promote plant-based fat and/or protein food sources [102]. Among low-CH diets, it has been postulated that very low CH ketogenic diets have a therapeutic role in several NCDs, including overweight and obesity, CVD and MetS [104]. Although there is no standardized deﬁnition of the ketogenic diet, it is characterized by a reduction in CH to less than 10% of daily energy intake, which means around 30 to 50 g of CH per day, and a relative increase of fat intake (fat to CH and protein intake ratio of 3:1 to 4:1) [105]. This restrictive dietary pattern has shown protective eﬀects for obesity and CVD by reducing body weight and improving the lipid proﬁle [104,106–108]. The meta-analysis of Bueno et al. 6. Low-Fat Diet By deﬁnition, the fat content of the low-fat diet comprises less than 30% of total energy, of which <10% are SFA, with a moderate PUFA content and limited trans FA [111,112]. In proportion, CH intake is higher and protein intake is moderate (around 15–17% of total energy intake). Low-fat diets usually include foods and products with reduced total fat content, such as low-fat dairy products instead of whole-fat products and derivatives. Low fat diets in weight-loss oriented dietary interventions showed a reduction in the risk of premature mortality in obese adults [56]. In this sense, a meta-analysis of 34 RCTs observed an 18% lower risk of all-cause mortality in weight-loss oriented dietary interventions in obese adults (95% CI 0.71 to 0.95), while no signiﬁcant eﬀects were observed in CVD mortality or incidence [56]. Recently, a network meta-analysis described the eﬀectiveness of the low-fat diet for body weight reduction compared to the usual dietary advice and dietary patterns after short-term intervention (6 months), with this eﬀect being attenuated after one year [55]. Clinical trials evaluating the eﬀect of a low-fat diet on the prevalence of MetS have shown conﬂicting results [113–116]. Dietary interventions based on low-fat intake (around 20% of total energy intake from fat) slightly reduced MetS components, but no signiﬁcant eﬀects were observed for CVD or the incidence of coronary heart disease in postmenopausal women compared to the usual diet [113,114]. Nevertheless, following a low-fat diet was not associated with a lower prevalence of MetS in older subjects at high CVD risk [115]. In this sense, Veum et al. compared the eﬀect of a low-fat, high-CH diet (around 30% of total energy intake from fat) vs. a very high-fat, low-CH diet (around 73% total energy intake from fat) on MetS components [117]. No signiﬁcant diﬀerences were observed in MetS components, body weight and body composition in the medium-term [117]. Similar ﬁndings were described by Gardner et al. in the DIETFITS trial, in which both low-fat and low-CH dietary approaches showed signiﬁcant weight loss with no diﬀerences between the two interventions [118]. Regarding BP and blood lipoproteins, a low-fat diet showed beneﬁcial eﬀects on systolic and diastolic BP management, and improved HDL-c and LDL-c levels in the short term compared to usual diet, but these eﬀects were reduced in long-term interventions [33,55]. However, in a meta-analysis of RCTs including 17,230 hypertensive and pre-hypertensive participants, Schwingshackl et al. 5. Low-Carbohydrate Diet observed greater weight loss (weighted mean 10 of 21 Nutrients 2020, 12, 2983 diﬀerence −0.91 kg (95% CI −1.65 to −0.17 kg)), and reduced triglyceride (−0.18 mmol/L (95% CI −0.27 to −0.08)) and diastolic BP levels (−1.43 mmHg (CI −2.49 to −0.37)), while HDL-c levels increased (0.09 mmol/L (95% CI 0.06 to 0.12)) after following a ketogenic diet compared to a low-fat diet [52]. The mechanisms of action underlying these protective eﬀects are as follows: the absence of dietary CH intake leads to a decrease in insulin secretion, which is translated into an inhibition of lipogenesis and fat accumulation and an increase in lipolysis; a satiety eﬀect of protein intake and its eﬀect on appetite control hormones, such as leptin and ghrelin; and the modulation of insulin secretion and ketone body production which might lead to metabolic improvements, especially in insulin signaling [104,109]. Moreover, CH restriction and the glycogen depletion characteristic of this type of diet lead to the use of ketone bodies as the main source of energy. Nevertheless, energy restriction is necessary to maintain ketone body production. Thus, recent evidence suggests that body weight and CVD beneﬁts observed with ketogenic dietary interventions are due to energy restriction, in spite of the macronutrient distribution of the diet [104,110]. However, health care professionals should consider the diﬃculties in following a ketogenic diet and the absence of healthy foods such as vegetables, fruits and whole-grain cereals, the intake of which is associated with a lower risk of developing chronic diseases such as CVD, T2DM and some types of cancer. 7. High-Protein Diet Recent evidence suggests that a high-protein dietary pattern leads to greater weight-loss and CVD improvements than standard protein diets (0.8 g protein/kg body weight). High-protein diets are characterized by a 20–30% of daily energy intake from protein, which means around 1.34 to 1.5 g protein/kg body weight [57]. Currently, the use of high-protein dietary interventions has been postulated for the treatment of obesity, MetS and glycemic control [58,93]. The eﬀect of high-protein dietary strategies for weight management is controversial. A meta-analysis of 18 studies on the eﬀect of a high-protein diet in T2DM patients showed that a high-protein diet did not signiﬁcantly decrease body weight compared to a regular protein diet [135]. Moreover, no signiﬁcant eﬀects where observed for glycemic control parameters, such as fasting glucose, insulin and HbA1c, blood lipid proﬁle or BP levels. Nevertheless, a signiﬁcant reduction of triglyceride levels was observed in participants who followed a high-protein diet. In the case of MetS, high-protein diets with CH restriction have shown eﬀective weight-loss in obese adults with MetS [57,58]. Campos-Nonato et al. performed a RCT in 118 adults with MetS to evaluate the eﬀect of a hypocaloric high-protein diet compared to a hypocaloric standard protein diet (500 kcal/day less than the metabolic rate and 1.34 g protein/kg body weight or 0.8 g protein/kg body weight, respectively) [57]. Weight-loss after 6 months of the dietary interventions was signiﬁcantly higher in participants who followed a high-protein diet (−7.0 kg ± 3.7; p-value = 0.046) compared to the standard protein diet (−5.1 kg ± 3.6; p-value = 0.157) [57]. MetS criteria, including fasting blood glucose, insulin, homeostatic model assessment for insulin resistance (HOMA-IR) index, and triglyceride, and cholesterol levels, improved in both intervention arms, but non-signiﬁcant diﬀerences were observed in the comparison between groups. The Optimal Macronutrient Intake Trial to Prevent Heart Disease (OmniHeart) study was a randomized, controlled, three-period, crossover nutritional study with 164 participants with overweight or obesity and prehypertension or stage 1 hypertension free of T2DM [136]. 6. Low-Fat Diet suggested that the MedDiet and the DASH diet are more eﬀective in long-term BP management compared to low-fat diets [33]. Likewise, low-CH diets showed greater eﬀects on the control of glycated hemoglobin and blood lipid levels than low-fat diets in a short to medium term intervention [52,112,119,120]. In the case of glucose metabolism and insulin control, some RCTs have not identiﬁed signiﬁcant eﬀects of low-fat dietary interventions versus other dietary approaches, while higher triglyceride levels were observed, mostly when simple CH proportion is increased [121–125]. In the case of T2DM management, Nutrients 2020, 12, 2983 11 of 21 Basterra-Gortari et al. found that a low-fat diet did not have an eﬀect on glucose-lowering medication management while the MedDiet supplemented with EVOO could delay its requirement in older people at high CVD risk [126]. MetS is associated with a pro-inﬂammatory state, and it has been proposed that a low-fat dietary intervention inducing weight loss slightly reduces inﬂammatory biomarkers such as high-sensitive c-reactive protein (CRP), interleukin−6 (IL) and tumor necrosis factor alpha (TNF-α) levels [16,127–129]. These results are inconclusive, and the eﬀects observed depend on weight loss and diet composition, particularly dietary ﬁber, fruits and vegetables [16,128]. Additionally, some studies observed that low-fat dietary interventions could improve the microbiome dysbiosis linked to MetS by increasing α-diversity [130,131]. However, limited results are available and more evidence regarding long-term response is needed [132]. Furthermore, nutrigenetic interactions have been described between dietary fat content and metabolic response [133]. Based on the evidence available, current dietary guidelines, such as the 2015–2020 American and European Dietary Guidelines, should avoid stating upper limits of total fat intake, mainly from healthy unsaturated FA. Moreover, this recommendation should include not exceeding 10% of total energy intake from SFA and the replacement of SFA by MUFA and PUFA [22,134]. 8. Other Dietary Patterns and Strategies Other dietary alterations have been shown to improve the MetS condition, such as the Nordic Diet, which is characterized by a high content of whole-grain high-ﬁber products (such as rye, barley, oat, rice, vegetables, fruits and nuts), with rapeseed oil as the main source of dietary fat and a high intake of ﬁsh and shellﬁsh [142,143]. Similar to the DASH and the MedDiet, the Nordic diet is considered to be a healthy dietary pattern in that it promotes the intake of vegetables, fruits, ﬁsh, poultry, nuts, and is low in sodium, red meat and processed foods. A recent meta-analysis of 5 RCTs including 513 participants demonstrated the eﬀectiveness of the Nordic diet in improving some MetS criteria, mainly systolic and diastolic BP (weighted mean diﬀerences −3.97 mmHg (95% CI −6.40 to −1.54; p < 0.001); −2.08 mmHg (95% CI −3.43 to −0.72; p = 0.003), respectively) [59]. Moreover, improvements in LDL-c (0.30 mmol/l (95% CI −0.54 to −0.06; p = 0.013)), but not in HDL-c and TG levels, were observed compared to control diets [59]. Further studies are needed to evaluate the beneﬁcial eﬀects of the Nordic diet on MetS management and prevention. Among dietary strategies, intermittent fasting has shown beneﬁts for CVD, T2DM, metabolic disturbances and cancer, mainly because of the daily caloric restriction involved [63]. The main cardiometabolic eﬀects observed after an intermittent-fasting intervention are weight loss and improvements in insulin resistance, dyslipidemia, BP levels and inﬂammation [60–62]. Despite the evidence and potential health beneﬁts of intermittent fasting, the applicability of this dietary strategy is complex and trained health care providers are needed to avoid side eﬀects. Furthermore, De la Iglesia et al. postulated other potential dietary approaches for the prevention and treatment of MetS, such as diets rich in omega−3 FA, low glycemic index, high antioxidant capacity or high meal frequency dietary interventions [144]. Thus, dietary intervention based on energy restriction, independently of the distribution of macronutrients, might inﬂuence BP and CVD. Accordingly, most scientiﬁc evidence highlights the relevance of dietary quality rather than quantity, especially in the management and prevention of MetS [8,145–147]. Moreover, the eﬀectiveness of every dietary intervention is associated with the previous metabolic state (e.g., presence of insulin resistance, T2DM, altered fasting glucose levels, etc.) [148,149]. 8. Other Dietary Patterns and Strategies While multifaceted lifestyle interventions focus on weight-loss and the promotion of physical activity, adherence is the key factor in achieving the beneﬁcial eﬀects observed in each dietary pattern, with intervention adherence being decisive in the results observed independently of the type of diet [150–152]. 7. High-Protein Diet This study aimed to evaluate insulin sensitivity with the quantitative insulin sensitivity check index among three dietary interventions: a high-CH diet (58% of daily kcal from CH; 15% from protein and 27% from fat); a protein diet (replacement of 10% of total CH to protein, 25% of daily kcal intake from protein, mainly from plant-based protein sources); and an unsaturated diet (replacement of 10% of total CH to unsaturated fat, 37% of daily kcal intake from fat, mainly from seeds and oils such as olive, canola and saﬄower oils and nuts). The protein and high-CH dietary patterns did not aﬀect insulin sensitivity, while the unsaturated diet showed improvements in insulin sensitivity, suggesting that the replacement of CH by unsaturated fat, such as in the MedDiet patterns, are alternative dietary approaches to improve insulin sensitivity. The mechanism underlying the potential health beneﬁts of a high-protein diet is that protein induces satiety, which is translated into Nutrients 2020, 12, 2983 12 of 21 reduced energy intake in the next meals [137,138]. Furthermore, high protein intake avoids muscle mass loss during energy-restrictive dietary interventions for weight loss [139]. Among protein food sources, meat and meat derived products have been associated with a higher risk of developing T2DM, CVD and MetS [12,13,140]. Dietary guidelines recommend prioritizing plant-based protein food sources such as soy, legumes, beans, nuts and seeds instead of meat and processed meat [21]. Plant-based protein food sources are rich in ﬁbre, phenolic compounds and PUFA, while cholesterol, trans or SFA are in lower proportions [141]. In a recent meta-analysis of 36 RCTs, red meat consumption had no eﬀect on the blood lipid proﬁle or BP, while after analyses stratiﬁed by the type of comparison diet, the substitution of red meat with plant-based protein foods showed a reduction in total cholesterol and LDL-c levels [93]. Thus, strong evidence promotes the intake of plant-based protein food sources, and this should also be recommended to promote environmental sustainability. 9. Conclusions The protective eﬀects of healthy dietary patterns on MetS seem to be due to the sum of small dietary changes rather than the restriction of any single nutrient. On comparing low-fat diets and very-restricted diets, the scientiﬁc evidence supports the use of the MedDiet intervention as the new paradigm for MetS prevention and treatment. The nutritional distribution and quality of the MedDiet allows health professionals to provide easy-to-follow dietary advice without the need for a restricted 13 of 21 Nutrients 2020, 12, 2983 diet. Nonetheless, RCTs on the eﬀects of a low-CH MedDiet style diet, promoting the intake of whole grain and plant-based protein food sources in patients with MetS, are needed to demonstrate the eﬃcacy of this dietary pattern. diet. Nonetheless, RCTs on the eﬀects of a low-CH MedDiet style diet, promoting the intake of whole grain and plant-based protein food sources in patients with MetS, are needed to demonstrate the eﬃcacy of this dietary pattern. Author Contributions: Conceptualization, R.C. and S.C.-B.; writing—original draft preparation, S.C.-B.; A.M.R.-L.; M.S.-P.; writing—review and editing, R.E. and R.C.; supervision, R.C. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. 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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W4206822618	https://www.mdpi.com/2075-4418/12/1/138/pdf?version=1641535478	English	null	Beyond the In-Practice CBC: The Research CBC Parameters-Driven Machine Learning Predictive Modeling for Early Differentiation among Leukemias	Diagnostics	2,022	cc-by	9,177	Rana Zeeshan Haider 1,2,* ,Ikram Uddin Ujjan 3, Najeed Ahmed Khan 4, Eloisa Urrechaga 5 and Tahir Sultan Shamsi 2,† Rana Zeeshan Haider 1,2,* ,Ikram Uddin Ujjan 3, Najeed Ahmed Khan 4, Eloisa Urrechaga 5 and Tahir Sultan Shamsi 2,† 1 Baqai Institute of Hematology, Baqai Medical University, Karachi 75340, Pakistan q gy q y 2 National Institute of Blood Disease (NIBD), Karachi 75300, Pakistan 4 Department of Computer Science, NED University of Engineering and Technology, Karachi 75270, Pakistan najeed@neduet.edu.pk 4 Department of Computer Science, NED University of Engineering and Technology, Karachi 75270, Pakistan; najeed@neduet.edu.pk j 5 Core Laboratory, Galdakao-Usansolo Hospital, 48960 Galdakao, Spain; eloisamaria.urrechagaigartua@osakidetza.eus 5 Core Laboratory, Galdakao-Usansolo Hospital, 48960 Galdakao, Spain; * Correspondence: zeeshan3335@yahoo.com; Tel.: +92-343-507-1271 † The author passed away. Abstract: A targeted and timely treatment can be a beneﬁcial tool for patients with hematological emergencies (particularly acute leukemias). The key challenges in the early diagnosis of leukemias and related hematological disorders are their symptom-sharing nature and prolonged turnaround time as well as the expertise needed in reporting conﬁrmatory tests. The present study made use of the potential morphological and immature fraction-related parameters (research items or cell population data) generated during complete blood cell count (CBC), through artiﬁcial intelligence (AI)/machine learning (ML) predictive modeling for early (at the pre-microscopic level) differentia- tion of various types of leukemias: acute from chronic as well as myeloid from lymphoid. The routine CBC parameters along with research CBC items from a hematology analyzer in the diagnosis of 1577 study subjects with hematological neoplasms were collected. The statistical and data visualiza- tion tools, including heat-map and principal component analysis (PCA,) helped in the evaluation of the predictive capacity of research CBC items. Next, research CBC parameter-driven artiﬁcial neural network (ANN) predictive modeling was developed to use the hidden trend (disease’s signature) by increasing the auguring accuracy of these potential morphometric parameters in differentiation of leukemias. The classical statistics for routine and research CBC parameters showed that as a whole, all study items are signiﬁcantly deviated among various types of leukemias (study groups). The CPD parameter-driven heat-map gave clustering (separation) of myeloid from lymphoid leukemias, followed by the segregation (nodding) of the acute from the chronic class of that particular lineage. Furthermore, acute promyelocytic leukemia (APML) was also well individuated from other types of acute myeloid leukemia (AML). The PCA plot guided by research CBC items at notable variance vindicated the aforementioned ﬁndings of the CPD-driven heat-map. diagnostics diagnostics diagnostics Rana Zeeshan Haider 1,2,* ,Ikram Uddin Ujjan 3, Najeed Ahmed Khan 4, Eloisa Urrechaga 5 and Tahir Sultan Shamsi 2,† Through training of ANN predictive modeling, the CPD parameters successfully differentiate the chronic myeloid leukemia (CML), AML, APML, acute lymphoid leukemia (ALL), chronic lymphoid leukemia (CLL), and other related hematological neoplasms with AUC values of 0.937, 0.905, 0.805, 0.829, 0.870, and 0.789, respectively, at an agreeably signiﬁcant (10.6%) false prediction rate. Overall practical results of using our ANN model were found quite satisfactory with values of 83.1% and 89.4.7% for training and testing datasets, respectively. We proposed that research CBC parameters could potentially be used for early differentiation of leukemias in the hematology–oncology unit. The CPD-driven ANN modeling is a novel practice that substantially strengthens the predictive potential of CPD items, allowing the clinicians to be conﬁdent about the typical trend of the “disease ﬁngerprint” shown by these automated potential morphometric items. Citation: Haider, R.Z.; Ujjan, I.U.; Khan, N.A.; Urrechaga, E.; Shamsi, T.S. Beyond the In-Practice CBC: The Research CBC Parameters-Driven Machine Learning Predictive Modeling for Early Differentiation among Leukemias. Diagnostics 2022, 12, 138. https://doi.org/10.3390/ diagnostics12010138 1. Introduction Artiﬁcial intelligence (AI) has seen remarkable development and increased value during the past two decades along with its successful introduction for solving complex data-related problems [1]. In fact, in recent years, a signiﬁcant increase in this trend has been noted for the use of AI in clinical aims for all three conventional medical tasks: diagnosis, therapy, and prognosis, but comparatively more for diagnosis [2,3]. A well-known and commonly used process or tool thorough which AI can achieve its learning objectives is Machine Learning (ML). For clinicians, a major question is whether ML will prove itself as an applied clinical tool in medical diagnostics. Through a brief literature survey, we can easily ﬁnd various studies regarding successful applications of the ML approach in specialized diagnostic ﬁelds [4–8]. However, for complex ﬁelds of clinical diagnostics such as hematology, limited examples of successful applications of ML have been reported. as hematology, limited examples of successful applications of ML have been reported. For medical diagnoses where a physician needs to determine which disease most likely explains the patient’s signs and symptoms, personal medical skills, knowledge, and experience are interlinked and together play a key role [9]. Laboratory tests assist in the conﬁrmation, exclusion, classiﬁcation, and/or monitoring of diseases along with further guidance for treatment [10]. Sadly, the typical practice of clinicians and diagnostic staff is aimed exclusively at targeting ‘out of reference range’ parameters (values) in the entire test report, which can limit the extended and even actual diagnostic potential of laboratory tests. This approach has a direct link with the underestimation and overlooking of the actual power of diagnostic test results [11]. Heavy workloads and a lack of convenient solutions for assistance in the prompt screening of the entire details generated along with routine laboratory testing may be behind this current clinical/diagnostic practice. The diagnostic workup for hematological diseases, especially neoplasms like leukemias, are ordinarily led by a basic blood test: complete blood cell count (CBC), and even a well- trained technologist/hematologist can pass over the trends, relations, and deviations among the increasing values of classical and additional (extended) data/parameters generated by modern hematology analyzers. The particularly advanced hematologic analyzers are now able to count and recognize the morphological characteristics of the types/subtypes of blood cell subpopulations by using the principles of electrical impedance, radiofrequency conductivity, light scattering, and/or Cytochemistry [12]. Citation: Haider, R.Z.; Ujjan, I.U.; Khan, N.A.; Urrechaga, E.; Shamsi, T.S. Beyond the In-Practice CBC: The Research CBC Parameters-Driven Machine Learning Predictive Modeling for Early Differentiation among Leukemias. Diagnostics 2022, 12, 138. https://doi.org/10.3390/ diagnostics12010138 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. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/diagnostics Diagnostics 2022, 12, 138. https://doi.org/10.3390/diagnostics12010138 Diagnostics 2022, 12, 138 2 of 11 Keywords: leukemia; complete blood cell count; cell population data; CBC research parameters; machine learning; artiﬁcial neural network 1. Introduction The above-mentioned analyzers also incorporate innovative computer algorithms and advanced hardware technology that help them in the collection and generation of the cell’s morphologic data, which are called Research CBC parameters or cell population data (CPD), a potential automated quantitative morphologic item. The Research CBC parameters are generated by multiple channels corresponding to the size, complexity (cytoplasmic granularity), and DNA/RNA content [12]. The presence of immature/abnormal white blood cells (WBCs) in peripheral blood deviate the values of CPD parameters, thus able to offer some sort of ‘disease signature’. The degree of modiﬁcation in the values of CPD parameters is linked with the number and immaturity or abnormality of that individual type of immature/abnormal white blood cell (WBCs). The clinical utility of these research CBC parameters, particularly for sepsis, infection, and hematological disorders, are well reported [13–27]. Morphological assessments through these CPD items might be preeminently objective, quantitative, and automated and can minimize the risk of subjective interpretation [22,28]. Compared to other hematological disorders, leukemias have highly bizarre clinical, morphological, and biological characteristics. Starting treatment without waiting for a deﬁnitive diagnosis or delaying for other concerns is an effective practice in treating leukemias [29,30]. In routine practice in a hematology clinic, ML tools can effectively facilitate the clinical personals by smartly handling hundreds of attributes (items) like Diagnostics 2022, 12, 138 3 of 11 3 of 11 CPD parameters, and they have the potential for sensible detection and utilization of various patterns (disease’s ﬁngerprints) among routine and research CBC parameters for various diseases. The particular disease signatures can make this specialization of diagnostics especially tempting for AI/ML applications. Apart from the morphology- based classiﬁcation systems and their limitations, we worked to propose a novel prediction model based on the ML framework that would utilize the deviation trends among the values of CBC data, especially CPD parameters, as ‘ﬁngerprints’ for leukemias and related disorders. This CBC data-driven ML (artiﬁcial neural network (ANN)) tool will offer efﬁcient screening for backing early expulsion and directing presentation of “patients’ ﬂow-from” in hematology–oncology departments. 2. Material and Methods 2.1. Study Population The study population consisted of newly diagnosed acute leukemia cases that were presented to the academic research center (National Institute of Blood Diseases and Bone Marrow Transplantation, Karachi, Pakistan) from February 2014 to December 2020. A total of 1577 cases having >1000 WBCs and at least a 2% immature/abnormal WBCs cell count in peripheral blood with a complete diagnostic work-up are included in this study. The diagnosis and subtypes of leukemias and related hematological disorders were conﬁrmed through bone marrow examination, immunophenotyping, cytogenetic and/or molecular tests based on initial workups, along with clinical and demographic information according to the WHO classiﬁcation of tumors of the hematopoietic and lymphoid tissues (2008). In the ﬁnal reports from diagnostic work-ups, all cases were allocated to one of the six principle disease groups and thus 354, 96, 213, 272, 153, and 489 cases were of acute myeloid leukemia (AML), acute promyelocytic leukemia (APML) PML-RARA, chronic myeloid leukemia (CML), acute lymphoid leukemia (ALL), chronic lymphoid leukemia (CLL), and ‘others (’Non-Hodgkin’s lymphoma, Plasma cell dyscrasia, and etc.), respectively. The collection of patient data and samples (blood and bone marrow) has been carried out in accordance with the Declaration of Helsinki, under the terms of all relevant local legislation. The responsible ethical committee of the National Institute of Blood Disease (NIBD) reviewed and approved the study in accordance with the ‘medical research involving human subjects act’ on permit number: NIBD/RD-167/14-201 dated 16th December 2013. Each study subject gave informed consent. 2.2. Sample Preparation and Methods Overall, 1577 peripheral whole blood samples were collected in K3EDTA blood tubes (Becton Dickinson, Franklin Lakes, NJ, USA). The analysis of all samples was performed with the Sysmex XN-Module (Kobe, Japan) by strictly following the manufacturer’s instructions, and the quality of the data was validated by regular analysis of internal quality control material (XN-CHECK levels 1, 2 and 3; Streck Laboratories Inc., Omaha, NE, USA). A peripheral blood smear was also prepared for all samples followed by May–Grunwald—Giemsa staining. Peripheral blood morphological examination was performed by optical microscopy (OM), in accordance with the recommendations of the International Council for Standardization in Hematology (ICSH) [31]. Briefly, differential count analysis by OM was carried out by two skilled hematologists (the opinion of a third hematologist was called upon where results were found to have >5% disagreement) on 200 cells at 100× magnification, as recommended by the CLSI document H20-A2 [32] and by the ICSH guidelines [31]. 2.4. AI Based Approach For AI applications, artiﬁcial neural network (ANN) from ML tools was selected for predictive modeling. The primary reason for selecting ANN is its superiority over other AI tools for ﬁnding patterns that are far too complex or numerous for a human programmer to extract and teach the machine to recognize. To conduct ANN predictive modeling with the aim of prediction/differentiation and classiﬁcation, and to determine the nature of our dataset, ﬁrst we tried the two most ﬁtted modeling tools: Radial basis function network (RBFN) and Multiple perceptron (MLP) networking. In the training and testing stages, RBFN out-performed MLP with ‘lower percent incorrect prediction’ rates, so we continued with RBFN. It is a computational non-linear data modeling having three (input, hidden, and output) layers and a feed-forward, supervised learning network that can smartly classify the cases through the input layer (variables—in our case, CBC parameters) similarity measurement with respect to examples from a training (data) set. Each hidden layer stores a ‘prototype’ that is an individual example of many more present in the training set. For the classiﬁcation of a new case, each variable computes the Euclidean distance among a new input and its prototype. An input layer/factor (which provides information from the outside world to the network), a hidden layer also called the radial basis function layer (which has no direct connection with the outside world and performs computations and transfers information from input nodes to output nodes), and an output layer/dependent variables (which is responsible for computations and transferring information from the network to the outside world). The hidden layer transforms the input vectors into radial basis functions. RBFN algorithm. To create an RBFN predictive model, SPSS syntax-programming language was used that allowed operators for any possible modiﬁcation, and in this way, we tried various dataset partitions of 50, 60, 70 and 50, 40, 30 for training and testing, respectively, for our network. In this regard, cases were randomly assigned based on relative numbers of cases without using any portioning variable to assign cases. 2.3. Classical Statistical Data Analysis Data was analyzed using SPSS version 23.0 (New York, NY, USA) and visualized through Clustvis (Institute of Computer Science, University of Tartu, J. Liivi-Tartu, Estonia), which is a web tool for visualizing the clustering of multivariate data (inspired by the PREDECT project and mostly based on BoxPlotR codes). The calculation of mean, standard Diagnostics 2022, 12, 138 4 of 11 deviation (SD), and signiﬁcance (P-) values among study groups were also carried out through SPSS. To delve into and obtain visualization of the subtle patterns of the Research CBC parameters among study groups, heat map (a supervised data visualization tool) and principal component analyses (PCA) were conducted. Heat map and PCA plots were generated through “Clustvis” https://biit.cs.ut.ee/clustvis/ (accessed on 1 December 2021), which is a web tool for visualizing the clustering of multivariate data. Aimed clustering (nodding) of study parameters, the function ‘correlation’ for the “clustering distance”, ‘average’ for the “clustering methods” and ‘tightest cluster ﬁrst’ considering the “tree ordering of columns” were used. For the color grading scheme of the heat map, the command of function ‘diverging: RdBu (Red to Blue)’ at ‘minimum-2 to maximum-2′ was used. The diverging: RdBu contains diverging palette options, more suitable for data with both negative and positive values, which is the same as in our data. 3. Results MO-X; m E W Baseline characteristics (routine CBC along with research items) of the analytic cohort in consonance with study groups are presented in Table 1 as mean, standard deviation (SD), and signiﬁcance. Exceptionally, NRBC (%) was noted as insigniﬁcant while all other study parameters showed signiﬁcant difference by classical statistical analysis. Furthermore, correlation-based clustering of study groups on the heat map (Figure 1) underpinned the subtle trends of CBC research items for the types of leukemias. ter, NE-WX; neutrophil side scatter distribution width, NE-WY; neutrophil side fluo tribution width, NE-WZ; neutrophil forward scatter distribution width, LY-WX; lym scatter distribution width, LY-WY; lymphocyte side fluorescence distribution width, trophil forward scatter distribution width, MO-WX; monocyte side scatter distributio MO-WY; monocyte side fluorescence distribution width, MO-WZ; monocyte forwar tribution width. Figure 1. The heat map: color grading and clustering trends of CBC Research parame Scheme 1. color-grading the parameters (rows) allows a quick view of hot and cold sp the dataset but also clusters (rearranges) the study groups (columns) with identical p nodding (branching) them. As a whole, hot spots were noted for CML followed by A Figure 1. The heat map: color grading and clustering trends of CBC Research parameters among study groups. For heat map color grading ‘diverging Red to Blue’ scheme (for higher to lower values, respectively) was used. The clustering of study groups (columns) is presented on function ‘correlation’. Figure 1. The heat map: color grading and clustering trends of CBC Research parame Scheme 1. color-grading the parameters (rows) allows a quick view of hot and cold s the dataset but also clusters (rearranges) the study groups (columns) with identical p nodding (branching) them. As a whole, hot spots were noted for CML followed by A Figure 1. The heat map: color grading and clustering trends of CBC Research parameters among study groups. For heat map color grading ‘diverging Red to Blue’ scheme (for higher to lower values, respectively) was used. The clustering of study groups (columns) is presented on function ‘correlation’. APML while CLL followed by ALL presented greater numbers of cold spots. Notabl ers’ showed a mixed pattern (both cold and hot spots). The nodding trends help us to find how closely patterned to each oth parameters are. 2.4. AI Based Approach In the architecture of our predictive network, we used options to automatically compute ranges in ﬁnding the best number of units within this range, and for numbers of units in the hidden layer, the normalized radial basis function was selected for the activation function of the hidden layer, and we tabbed an automatic computation of the amount of overlap to allow for overlapping among hidden units. Methodologically, the RBFN model was developed in two steps: ﬁrst, by using clustering methods, radial basis functions were determined and the width and center of each of the radial basis functions were calculated. In the second step, the network determined the synaptic weights given the radial basis functions. Both classiﬁcation and prediction through the output layer were laid by sum-of-squares error functions with the identity activation function. y We estimated the importance of set of the selected CBC attributes with our normalized radial basis function algorithm and evaluated our ML network results for classiﬁcation (diagnostic) problems by observing the classiﬁcation accuracy (true positive rate) generated Diagnostics 2022, 12, 138 MO WZ 5 of 11 cell hem 5 of 11 cell hem after training the network against relevant data set. To avoid false negative and false positive results that may put the patient’s life at risk by causing delays in treatment as well as potentially resulting in unnecessary health care costs, we added a normal (healthy) population in the training set of our data. Furthermore, to visualize the network behavior, an ROC curve, predicted-by-observed chart, cumulative gains chart, and lift chart were generated. neutrophil, LYMPH# (10 /μL); absolute lymphocyte count, MONO# (10 /μL); absolu count, EO# (103/μL); absolute eosinophil count, BASO# (103/μL); absolute basophil co (%); percent neutrophil count, LYMP (%); percent lymphocyte count, MONO (%); pe cyte count, EO (%); percent eosinophil count, BASO (%); percent basophil count, IG# solute immature granulocyte count, IG (%); percent immature granulocyte count. NE phil side scatter, NE-SFL; neutrophil side fluorescence, NE-FSC; neutrophil forward lymphocyte side scatter LY-Y; lymphocyte side fluorescence LY-Z; lymphocyte forw 3. Results MO-X; m E W The step/level of any particular node where it groups to oth scribes its degree of clustering (correlation in our case) The first step of n In the heat map illustration (Figure 1), color-grading the parameters (rows) allows a quick view of hot and cold spots within the dataset but also clusters (rearranges) the study groups (columns) with identical patterns by nodding (branching) them. As a whole, hot spots were noted for CML followed by AML and APML while CLL followed by ALL presented greater numbers of cold spots. Notably, group ‘others’ showed a mixed pattern (both cold and hot spots). scribes its degree of clustering (correlation, in our case). The first step of observed between CML and all other study groups. At the second step (level The nodding trends help us to ﬁnd how closely patterned to each other our study parameters are. The step/level of any particular node where it groups to other node/s describes its degree of clustering (correlation, in our case). The ﬁrst step of nodding was observed between CML and all other study groups. At the second step (level of nodding), AML and APML were separated from ALL, CLL, and group ‘others’. Through the third level, nodding group ‘others’ were distinct from ALL and CLL. Various levels of clustering at our Research CBC parameter driven heat map suggested the predictive potential of our study parameters, particularly for CML from other leukemias, as well as for differentiation Diagnostics 2022, 12, 138 6 of 11 of myeloid from lymphoid leukemias. The lower the level of nodding, the closer the values of the study (lower predictive potential). of myeloid from lymphoid leukemias. The lower the level of nodding, the closer the values of the study (lower predictive potential). Table 1. Baseline characteristics (classical CBC and research CBC (CPD) of analytic cohorts, according to types of leukemias and related hematological disorders). Table 1. Baseline characteristics (classical CBC and research CBC (CPD) of analytic cohorts, according to types of leukemias and related hematological disorders). Study Parameters Study Groups Sig. 3. Results MO-X; m E W AML APML CML ALL CLL Others Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Automated Classical CBC Parameters Hb 8.19 ± 2.10 8.56 ± 1.61 9.38 ± 1.87 8.17 ± 2.59 10.64 ± 2.47 9.69 ± 3.24 <0.005 RBC (1012/L) 2.78 ± 0.82 2.93 ± 0.61 3.49 ± 0.8 3.02 ± 1.29 3.92 ± 0.99 3.68 ± 1.56 <0.005 PCV 25.09 ± 8.09 25.6 ± 5.29 28.86 ± 6.24 24.73 ± 7.64 34.39 ± 7.51 30.27 ± 10.53 <0.005 MCV 90.34 ± 10.47 88.18 ± 8.06 83.45 ± 10.14 83.94 ± 9.12 88.93 ± 9.01 85.47 ± 10.93 <0.005 MCH 29.39 ± 3.53 29.01 ± 3.05 27.1 ± 3.9 27.36 ± 2.91 27.41 ± 3.29 27.15 ± 4.18 <0.005 MCHC 32.35 ± 1.93 32.95 ± 2.34 32.15 ± 2.25 32.64 ± 1.93 30.81 ± 2.44 31.65 ± 1.92 <0.005 WBC (109/L) 39.66 ± 66.75 26.8 ± 47.65 192.39 ± 142.46 70.91 ± 107.47 95.81 ± 123.45 16.99 ± 36.84 <0.005 PLT (103/µL) 60.88 ± 83.18 53.73 ± 85.94 438.42 ± 292.94 53.74 ± 62.92 187.03 ± 105.8 304.35 ± 406.31 <0.005 NEUT# (103/µL) 9.59 ± 29.59 10.35 ± 18.81 161.65 ± 125.42 3.25 ± 4.22 5.83 ± 4.49 8.39 ± 13.65 <0.005 LYMPH# (103/µL) 9.07 ± 12.93 4.77 ± 11.01 9.62 ± 5.23 47.76 ± 77.4 82.74 ± 115.79 6.19 ± 31.27 <0.005 MONO# (103/µL) 21.29 ± 42.93 12.05 ± 25.11 8.25 ± 8.81 20.09 ± 42.26 6.8 ± 17 1.91 ± 5.26 <0.005 EO# (103/µL) 0.18 ± 0.99 0.07 ± 0.15 5.1 ± 5.24 0.13 ± 0.29 0.3 ± 0.42 0.36 ± 1.35 <0.005 BASO# (103/µL) 0.07 ± 0.21 0.06 ± 0.13 5.32 ± 5.22 0.15 ± 0.39 0.15 ± 0.23 0.08 ± 0.15 <0.005 NEUT (%) 22.16 ± 19.91 35.91 ± 19.93 81.92 ± 11.61 13.12 ± 17.24 12 ± 12.26 54.22 ± 23.9 <0.005 LYMPH (%) 37.9 ± 22.28 37.91 ± 26.65 7.19 ± 5.86 64.36 ± 22.07 80.56 ± 17.37 31.97 ± 22.22 <0.005 MONO (%) 39.09 ± 23.61 25.37 ± 21.72 4.88 ± 4.61 21.07 ± 18.04 6.48 ± 10.98 11.35 ± 11.68 <0.005 EO (%) 0.67 ± 2.07 0.71 ± 1.5 3.16 ± 5.5 0.5 ± 0.96 0.73 ± 1.5 1.96 ± 2.45 <0.005 BASO (%) 0.18 ± 0.36 0.1 ± 0.16 2.85 ± 2.04 0.22 ± 0.3 0.23 ± 0.33 0.49 ± 0.75 <0.005 IG# (103/µL) 1.86 ± 4.83 1.53 ± 3.76 65.04 ± 57.27 0.73 ± 1.51 0.45 ± 1.62 1.18 ± 3.61 <0.005 IG (%) 4.38 ± 6.33 5.08 ± 7.77 30.31 ± 9.61 1.76 ± 2.93 0.54 ± 1.33 4.07 ± 6.64 <0.005 NRBC# (103/µL) 0.35 ± 1.07 0.11 ± 0.22 2.16 ± 3.42 0.51 ± 1.56 0.05 ± 0.27 0.47 ± 4.46 <0.005 NRBC (%) 1.61 ± 3.55 0.91 ± 1.35 1.15 ± 1.38 1.4 ± 3.87 0.28 ± 1.51 1.56 ± 8.43 0.549 PDW (fL) 8.76 ± 7.24 6.19 ± 7.35 11.09 ± 6.35 7.03 ± 6.72 11.92 ± 4.6 8.31 ± 6.58 <0.005 MPV (fL) 7.23 ± 5.46 5.05 ± 5.67 8.83 ± 4.6 5.89 ± 5.35 9.93 ± 3.39 6.97 ± 5.25 <0.005 PCT (%) 0.05 ± 0.09 0.04 ± 0.09 0.41 ± 0.35 0.04 ± 0.07 0.19 ± 0.12 0.28 ± 0.42 <0.005 Retic count 1.93 ± 11.84 1.15 ± 1.51 3.13 ± 2.21 0.55 ± 1.07 0.2 ± 0.54 1.88 ± 1.63 0.015 Automated Research CBC (CPD) Parameters NE–SSC(ch) 140.81 ± 14.05 143.08 ± 10.87 149.05 ± 6.53 149.64 ± 9.26 150.16 ± 7.89 147.15 ± 10.04 <0.005 NE–SFL(ch) 51.43 ± 17.33 65.85 ± 22.71 45.86 ± 5.12 50.71 ± 8.3 45.81 ± 8.45 45.66 ± 7.27 <0.005 NE–FSC(ch) 72.29 ± 11.15 72.59 ± 11.81 84.04 ± 5.57 80.89 ± 7.03 82.23 ± 5.73 78.92 ± 7.69 <0.005 LY–X(ch) 87.33 ± 10.39 84.5 ± 10.35 81.63 ± 8.89 84.75 ± 7.25 79.58 ± 4.46 81.45 ± 4.49 <0.005 LY–Y(ch) 68.65 ± 12.29 65.54 ± 9.37 42.89 ± 19.68 68.91 ± 16.15 59.04 ± 8.9 65.11 ± 6.06 <0.005 LY–Z(ch) 56.68 ± 3.74 57.32 ± 3.02 52.44 ± 3.49 58.2 ± 3.79 57.78 ± 2.94 56.66 ± 2.39 <0.005 MO–X(ch) 118.05 ± 8.27 120.75 ± 9.83 126.3 ± 6.91 110.2 ± 7.39 109.97 ± 6.14 119.14 ± 5.74 <0.005 MO–Y(ch) 114.65 ± 23.51 115.35 ± 25.35 112.09 ± 24.26 108.43 ± 23.79 101.6 ± 9.56 105.47 ± 17.45 <0.005 MO–Z(ch) 62.66 ± 4.97 65.49 ± 7.92 60.28 ± 2.89 65.29 ± 6.54 64.9 ± 3.53 62.82 ± 4.76 <0.005 NE–WX 435.71 ± 127.01 419.16 ± 119.61 501.29 ± 76.69 386.73 ± 108.58 323.69 ± 61.47 368.47 ± 88.09 <0.005 NE–WY 1388.88 ± 755.01 1262.53 ± 829.7 2467.69 ± 693.2 1226.47 ± 616.41 740.42 ± 279.96 897.1 ± 471.45 <0.005 NE–WZ 825.5 ± 257.67 801.79 ± 213.15 847.02 ± 109.49 721.08 ± 203.64 650.14 ± 154.81 691.02 ± 156.02 <0.005 LY–WX 533.66 ± 118.75 550.86 ± 136.81 695.52 ± 168.56 535.53 ± 119.29 530.33 ± 115.78 536.78 ± 109.45 <0.005 LY–WY 1069.66 ± 267.76 994.91 ± 184.93 1929.71 ± 1070.73 1060.03 ± 231.82 960.37 ± 169.92 1007.77 ± 220.04 <0.005 LY–WZ 568.06 ± 115.83 586.67 ± 142.48 801.74 ± 165.36 578.5 ± 138.35 460.95 ± 102.18 527.32 ± 122.95 <0.005 MO–WX 340.51 ± 75.02 301.81 ± 104.41 357.22 ± 65.23 319.04 ± 90.03 285.66 ± 66.46 291.38 ± 73.36 <0.005 MO–WY 873.84 ± 282.05 701.67 ± 446.57 1146.88 ± 346.87 878.07 ± 317.66 832.36 ± 218.58 736.74 ± 258.89 <0.005 MO–WZ 616.05 ± 112.94 601.16 ± 204.8 767.94 ± 100.79 681.88 ± 226.76 636 ± 255.98 597.25 ± 156.62 <0.005 Hb; hemoglobin, RBC; red blood cell, PCV; packed cell volume, MCH; mean cell hemoglobin, MCHC; mean cell hemoglobin, WBC; white blood cell, PLT; platelet, NEUT# (103/µL); absolute neutrophil, LYMPH# (103/µL); absolute lymphocyte count, MONO# (103/µL); absolute monocyte count, EO# (103/µL); absolute eosinophil count, BASO# (103/µL); absolute basophil count, NEUT (%); percent neutrophil count, LYMP (%); percent lymphocyte count, MONO (%); percent monocyte count, EO (%); percent eosinophil count, BASO (%); percent basophil count, IG# (103/µL); absolute immature granulocyte count, IG (%); percent immature granulocyte count. 3. Results MO-X; m E W PC1 and PC2 correspond to a linear merger of our Research CBC parameters aimed at the identiﬁcation of directions/principal com- Diagnostics 2022, 12, 138 7 of 11 near m incipal 7 of 11 near m incipal ponents along which the variation in the data is maximal. For the most part, the PCA plot vindicates the impression from heat map that CML from other study groups is highly distinct while myeloid vs. lymphoid leukemias are decidedly apparent from each other. Barely a minor degree of overlaps was noted for AML with APML, and ALL with group ‘others’ and CLL. vindicates the impression from heat map that CML from other study groups is h distinct while myeloid vs. lymphoid leukemias are decidedly apparent from each o Barely a minor degree of overlaps was noted for AML with APML, and ALL with g others’ and CLL. Figure 2. Principal Component Analysis (PCA) plot demonstrating Research CBC parameters driven relatedness among various types of leukemias (our study groups). Figure 2. Principal Component Analysis (PCA) plot demonstrating Research CBC parameters driven relatedness among various types of leukemias (our study groups). igure 2. Principal Component Analysis (PCA) plot demonstrating Research CBC parameter driven relatedness among various types of leukemias (our study groups). Figure 2. Principal Component Analysis (PCA) plot demonstrating Research CBC parameters driven relatedness among various types of leukemias (our study groups). The model summary of our CBC research-driven RBFN predictive framework in ure 3 presents various positive signs. The sum of squared error remained 46.59 and or training and testing, respectively. The smaller value of the squared error in testing raining indicates the most-fit number of hidden units/layers to minimize error func Furthermore, the percentage of incorrect predictions was noticeably lower at 16.9 for ng and 10.6 in consideration of the testing dataset. Additionally, practical results of u he network as shown in the classification table likewise decidedly remained deno The model’s performance-related scales in terms of predictive-pseudo probability, s ivity and specificity (AUC), gain, and lift charts were found to be promisingly convin or the predictive ability of the network. The ROC curve gives more powerful and m cleaner visual presentation of the specificity and sensitivity in a single plot than the s of tables. 3. Results MO-X; m E W The ROC chart presents all six curves of AML, APML, CML, ALL, CLL group ‘others’ with area values of 0.810, 0.789, 0.937, 0.829, 0.905, and 0.805, respect n the predicted-by-observed chart, the ‘observed response’ and ‘predicted categ were aligned with the x-axis and y-axis, respectively. The prediction is considered ect’ when the boxplot is found near the level of ‘0.5′ for the y-axis. In our case, all box are noted near the 0.5 mark. In the present analysis, a cumulative gains chart was The model summary of our CBC research-driven RBFN predictive framework in Figure 3 presents various positive signs. The sum of squared error remained 46.59 and 22.18 for training and testing, respectively. The smaller value of the squared error in testing over training indicates the most-ﬁt number of hidden units/layers to minimize error func- tions. Furthermore, the percentage of incorrect predictions was noticeably lower at 16.9 for training and 10.6 in consideration of the testing dataset. Additionally, practical results of using the network as shown in the classiﬁcation table likewise decidedly remained de- noting. The model’s performance-related scales in terms of predictive-pseudo probability, sensitivity and speciﬁcity (AUC), gain, and lift charts were found to be promisingly con- vincing for the predictive ability of the network. The ROC curve gives more powerful and much cleaner visual presentation of the speciﬁcity and sensitivity in a single plot than the series of tables. The ROC chart presents all six curves of AML, APML, CML, ALL, CLL, and group ‘others’ with area values of 0.810, 0.789, 0.937, 0.829, 0.905, and 0.805, respectively. In the predicted-by-observed chart, the ‘observed response’ and ‘predicted categories’ were aligned with the x-axis and y-axis, respectively. The prediction is considered ‘Correct’ when the boxplot is found near the level of ‘0.5′ for the y-axis. In our case, all boxplots are noted near the 0.5 mark. In the present analysis, a cumulative gains chart was also presented wherein ‘target’ (total number of cases) and ‘gained’ (in reference to the target of the overall ﬁgure of cases for a particular class) were shown. In addition to points along both axes, a ‘baseline’ curve is indicated in the shape of the diagonal line and curve laid above the baseline and is accepted as a greater gain. The lift chart provides a different view of the cumulative gains chart. 3. Results MO-X; m E W NE-SSC; neutrophil side scatter, NE-SFL; neutrophil side ﬂuorescence, NE-FSC; neutrophil forward scatter, LY-X; lymphocyte side scatter, LY-Y; lymphocyte side ﬂuorescence, LY-Z; lymphocyte forward scatter, MO-X; monocyte side scatter, MO-Y; monocyte side ﬂuorescence, MO-Z; monocyte forward scatter, NE-WX; neutrophil side scatter distribution width, NE-WY; neutrophil side ﬂuorescence distribution width, NE-WZ; neutrophil forward scatter distribution width, LY-WX; lymphocyte side scatter distribution width, LY-WY; lymphocyte side ﬂuorescence distribution width, LY-WZ; neutrophil forward scatter distribution width, MO-WX; monocyte side scatter distribution width, MO-WY; monocyte side ﬂuorescence distribution width, MO-WZ; monocyte forward scatter distribution width. The Principal Component Analysis (PCA) plot (Figure 2) was used to graphically visualize our multivariate data and to extract the key information as a set of new attributes called principle components (PC1 and PC2) PC1 and PC2 correspond to a linear merger Table 1. Baseline characteristics (classical CBC and research CBC (CPD) of analytic cohorts, according to types of leukemias and related hematological disorders). Hb; hemoglobin, RBC; red blood cell, PCV; packed cell volume, MCH; mean cell hemoglobin, MCHC; mean cell hemoglobin, WBC; white blood cell, PLT; platelet, NEUT# (103/µL); absolute neutrophil, LYMPH# (103/µL); absolute lymphocyte count, MONO# (103/µL); absolute monocyte count, EO# (103/µL); absolute eosinophil count, BASO# (103/µL); absolute basophil count, NEUT (%); percent neutrophil count, LYMP (%); percent lymphocyte count, MONO (%); percent monocyte count, EO (%); percent eosinophil count, BASO (%); percent basophil count, IG# (103/µL); absolute immature granulocyte count, IG (%); percent immature granulocyte count. NE-SSC; neutrophil side scatter, NE-SFL; neutrophil side ﬂuorescence, NE-FSC; neutrophil forward scatter, LY-X; lymphocyte side scatter, LY-Y; lymphocyte side ﬂuorescence, LY-Z; lymphocyte forward scatter, MO-X; monocyte side scatter, MO-Y; monocyte side ﬂuorescence, MO-Z; monocyte forward scatter, NE-WX; neutrophil side scatter distribution width, NE-WY; neutrophil side ﬂuorescence distribution width, NE-WZ; neutrophil forward scatter distribution width, LY-WX; lymphocyte side scatter distribution width, LY-WY; lymphocyte side ﬂuorescence distribution width, LY-WZ; neutrophil forward scatter distribution width, MO-WX; monocyte side scatter distribution width, MO-WY; monocyte side ﬂuorescence distribution width, MO-WZ; monocyte forward scatter distribution width. The Principal Component Analysis (PCA) plot (Figure 2) was used to graphically visualize our multivariate data and to extract the key information as a set of new attributes called principle components (PC1 and PC2). 4. Discussion The Research CBC parameters (CPD items) generated by modern hematology ana- lyzers are promising automated quantitative morphological parameters (by quantifying the changes in morphological characteristics of blood cells) to introduce as signatory CBC- based diagnostic items for hematological neoplasms, especially leukemias. The potential clinical utility of CPD items for early exclusion of hematological disorders is well reported in various studies [21,22,25]. However, none of these studies applies AI tools and only a few studies report the utility of these CPD parameters by aiding statistical equations and simple models/classiﬁers. As an illustration, a study by Yang et al. evaluated CPD (white cell scattering) items for differentiation of acute leukemia lineage by using the Coulter DxH800 analyzer. In this study, the authors derive a 21-CBC items-based model (generated by the KYL program, an Excel macro program) and reported very high (100%) speciﬁcity and sensitivity for differentiation of acute promyelocytic leukemia (APL) cases, while for ALL, comparably less signiﬁcant speciﬁcity and sensitivity was achieved [33]. In another study by Virk et al., the clinical utility of the CPD parameters, scatter grams, and ﬂags by generating statistical equations for screening of AML cases was published [34]. Here, an exception is found in studies conducted by Shabbir et al. [35] and our work (Haider et al.) [36], wherein ML tools were challenged for discernment between just two study groups (hematologic vs. non-hematologic malignancies and acute promyelocytic leukemia vs. other hematologic malignancies, respectively). g g p y) The ﬁndings from our study’s dataset present a promise for this early pre-microscopic prediction of leukemias and related common hematological neoplasms, since the Research CBC parameters are not only able to detect the presence of leukemia but can assist in prediction of the lineage and type of leukemias. In addition, we showed that an AI approach, using an ML algorithm trained on the Research CBC parameters along with routine items-based results (dataset), is not only able to differentiate the lineage of leukemia (either myeloid or lymphoid) but can also remain predictive for the type (acute or chronic or other) of leukemia and related disorders. The result showed that our RFBN model performed the classiﬁcation with high accuracy and successfully differentiated the study groups with a signiﬁcant (10.6 percent) in-correction rate. 3. Results MO-X; m E W The values along the y-axis represent the ratio of the cumulative gains for each curve (category/ subgroups) against the baseline curve. In this way, the lift at 10% for category AML is 25%/10% = 2.5. Both cumulative and lift charts are based on combined testing and training samples. Diagnostics 2022, 12, 138 8 of 11 based on combined testing and training samples. Diagnostics 2022, 12, x FOR PEER REVIEW 9 of 12 Figure 3. The model summary, classification table, predicted-by-observed chart, ROC curve, cumu- lative gains and lift chart for the Research CBC parameters driven Radial Basis Function (RBF) pre- dictive model. 4. Discussion The Research CBC parameters (CPD items) generated by modern hematology ana AUC AML 0.810 APML 0.789 CML 0.937 ALL 0.829 CLL 0.905 Others 0.805 0 0 0 0 0 0 0 0 0 0 Figure 3. The model summary, classiﬁcation table, predicted-by-observed chart, ROC curv mulative gains and lift chart for the Research CBC parameters driven Radial Basis Function predictive model. Diagnostics 2022, 12, x FOR PEER REVIEW 9 of 12 gnostics 2022, 12, x FOR PEER REVIEW gnostics 2022, 12, x FOR PEER REVIEW AUC AML 0.810 APML 0.789 CML 0.937 ALL 0.829 CLL 0.905 Others 0.805 0 0 0 0 0 0 0 0 0 0 Figure 3. The model summary, classification table, predicted-by-observed chart, ROC curve, cumu- lative gains and lift chart for the Research CBC parameters driven Radial Basis Function (RBF) pre- dictive model. 4. Discussion AUC AML 0.810 APML 0.789 CML 0.937 ALL 0.829 CLL 0.905 Others 0.805 0 0 0 0 0 0 0 0 0 0 Figure 3. The model summary, classiﬁcation table, predicted-by-observed chart, ROC curv mulative gains and lift chart for the Research CBC parameters driven Radial Basis Function predictive model. AUC AML 0.810 APML 0.789 CML 0.937 ALL 0.829 CLL 0.905 Others 0.805 0 0 0 0 0 0 0 0 0 0 Figure 3. The model summary, classification table, predicted-by-observed chart, ROC curve, cumu- lative gains and lift chart for the Research CBC parameters driven Radial Basis Function (RBF) pre- dictive model. 4 Di i Figure 3. The model summary, classiﬁcation table, predicted-by-observed chart, ROC curve, cu- mulative gains and lift chart for the Research CBC parameters driven Radial Basis Function (RBF) predictive model. Diagnostics 2022, 12, 138 9 of 11 9 of 11 4. Discussion This limitation can be addressed by validation/re-validation, ideally through external independent datasets at multiple centers. 5. Conclusions In the present study, a particular disease’s signature raised by the research CBC parameters is illustrated for early differentiation among various types of leukemias and related disorders. The CPD-driven ANN modeling can be a novel practice that substantially strengthens the disease signatory characteristics from these research CBC items in different types of leukemias. The suggested approach can reduce the frequency of extra and irrational diagnostic test ordering that is not only time-consuming but is also an extra burden on patients and laboratory staff. Here, the application of CPD-driven predictive modeling as an assistant predictive tool in the decision support system of hematology laboratory/clinics is suggested. Author Contributions: Conceptualization, R.Z.H.; Data curation, R.Z.H.; Methodology, R.Z.H., N.A.K. and E.U.; Resources, I.U.U. and N.A.K.; Software, N.A.K.; Supervision; T.S.S.; Visualization, R.Z.H.; Writing—original draft, R.Z.H.; Writing—review & editing, I.U.U., N.A.K., E.U. and T.S.S. All authors have read and agreed to the published version of the manuscript. Funding: The authors declare that they did not receive funding. Institutional Review Board Statement: The National Institute of Blood Disease Research Ethics Committee approved our study (Permit number: NIBD/RD-167/14-2014). All patients provided written informed consent prior to enrollment in the study. Informed Consent Statement: Written informed consent has been obtained. Acknowledgments: The authors are grateful to lab members of clinical hematology laboratory-NIBD for assistance in patient’s diagnosis. Acknowledgments: The authors are grateful to lab members of clinical hematology laboratory-NIBD for assistance in patient’s diagnosis. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 4. Discussion The utility of our suggested Research CBC parameters-driven predictive model for leukemias and related disorders was established through its practical results of 83.1% and 89.4% for training and testing datasets, respectively (Figure 1). The higher accuracy from RFBN in our case can be justiﬁed as RFBN increases the feature vector (hidden layers). When the dimension of hidden layers is increased, the linear reparability of hidden layers increases. The importance of the proposed CBC data-driven ML predictive modeling increases for clinics and/or diagnostic setups not specializing in hematology by serving as a screening tool to aid them in diagnostic procedures and the proper and early referral of patients with hematological emergencies such as acute leukemias. Furthermore, our proposed model can minimize irrational ordering of laboratory tests (usually used for additional conﬁrmations) in order to research the correct diagnosis. AI applications in laboratory diagnostics facilitates early (decisions) diagnosis through a limited proﬁle of laboratory tests [10]. The suggested approach has the potential to be replicated to differentiate other hematological disorders. The results of this study are appealing and serve to legitimize the in-practice clinical approach. Clinicians have acknowledged that results of laboratory investigations, examined in isolation, have deﬁnite diagnostic importance in clinical decision-making, typically in environments where clinical knowledge and expertise play a critical role [11]. This belief is logical considering the deﬁciency of the reported work on the Research CBC parameters such as CPD items and AI tools in hematology (laboratory) diagnostics. Accordingly, the use of the AI approach in the clinical interpretation of laboratory tests can be more diagnostically valuable as a speedy, directed, and more persuasive practice in lieu of trailing the probable diagnosis through an increasing number of irrational and expensive tests. The utility of AI/ML predictive models can be effectively enhanced provided that information regarding clinical (patient’s) history and physical examination of patients Diagnostics 2022, 12, 138 10 of 11 10 of 11 become an integral part by adding more valuable attributes to such models. Here, it is necessary to mention that the scope of our study is limited in its boarder clinical utility. This limitation can be addressed by validation/re-validation, ideally through external independent datasets at multiple centers. become an integral part by adding more valuable attributes to such models. Here, it is necessary to mention that the scope of our study is limited in its boarder clinical utility. 13. Lee, S.-E.; Lim, J.; Kim, Y.; Min, W.-S.; Han, K. Leukocyte Cell Population Analysis from the Coulter Automatic Blood Cell Analyzer DxH800 to Monitor the Effect of G-CSF. J. Clin. Lab. Anal. 2012, 26, 194–199. [CrossRef] [PubMed] 12. Ward, P.C. The CBC at the turn of the millennium: An overview. Clin. Chem. 2000, 46, 1215–1220. [Cro References 1. Jordan, M.I.; Mitchell, T.M. Machine learning: Trends, perspectives, and prospects. Science 2015, 349, 255–260. [CrossRef] 2 S l i P P il R S S h W B A iﬁi l I lli i M di l Di i A I t M d 1988 108 80 87 [C R f 1. 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https://openalex.org/W4295269419	https://www.frontiersin.org/articles/10.3389/fgene.2022.923078/pdf	English	null	Discovery and Validation of Grain Shape Loci in U.S. Rice Germplasm Through Haplotype Characterization	Frontiers in genetics	2,022	cc-by	11,308	ORIGINAL RESEARCH published: 12 September 2022 doi: 10.3389/fgene.2022.923078 Reviewed by: Reviewed by: Yingpeng Han, Northeast Agricultural University, China Reviewed by: Yingpeng Han, Northeast Agricultural University, China Vanisri Satturu, Professor Jayashankar Telangana State Agricultural University, India China Vanisri Satturu, Professor Jayashankar Telangana State Agricultural University, India Correspondence: Brijesh Angira bangira@agcenter.lsu.edu Adam N. Famoso AFamoso@agcenter.lsu.edu †Present address: Tommaso Cerioli, Corteva Agriscience, Cremona, Lombardy, Italy †Present address: Tommaso Cerioli, Corteva Agriscience, Cremona, Lombardy, Italy Specialty section: This article was submitted to Plant Genomics, a section of the journal Frontiers in Genetics Received: 18 April 2022 Accepted: 10 June 2022 Published: 12 September 2022 Citation: Angira B, Cerioli T and Famoso AN (2022) Discovery and Validation of Grain Shape Loci in U.S. Rice Germplasm Through Haplotype Characterization. Front. Genet. 13:923078. doi: 10.3389/fgene.2022.923078 Specialty section: This article was submitted to Plant Genomics, a section of the journal Frontiers in Genetics Discovery and Validation of Grain Shape Loci in U.S. Rice Germplasm Through Haplotype Characterization Brijesh Angira 1, Tommaso Cerioli 2† and Adam N. Famoso 1* 1H. Rouse Caffey Rice Research Station, Louisiana State University Agricultural Center, Baton Rouge, LA, United States, 2School of Plant, Environmental and Soil Science, Louisiana State University, Baton Rouge, LA, United States Rice grain shape is a major determinant of rice market value and the end-use. We mapped quantitative trait loci (QTL) for grain shape traits in a bi-parental recombinant inbred line population (Trenasse/Jupiter) and discovered two major grain length QTLs—qGL3.1 and qGL7.1. Previously, a major grain shape gene GS3 was reported in the qGL3.1 region and grain length gene GL7 was reported to be encompassing qGL7.1 locus. The re-sequencing SNP data on the International Rice Research Institute (IRRI) 3K Rice Genome Project (RGP) panel were obtained from the IRRI SNP-Seek database for both genes and haplotype diversity was characterized for each gene in this diverse panel. United States rice germplasm was not well represented in the IRRI 3K RGP database. Therefore, a minimum SNP set was identiﬁed for each gene that could differentiate all the characterized haplotypes. These haplotypes in the 3K RGP panel were screened across 323 elite U.S. genotypes using the minimum SNP set. The screening of haplotypes and phenotype association conﬁrmed the role of GS3 under qGL3.1. However, screening of the GL7 haplotypes in the U.S. germplasm panel showed that GL7 did not play a role in qGL7.1, and in addition, GL7.1 did not segregate in the Trenasse/ Jupiter RIL population. This concluded that qGL7.1 is a novel QTL discovered on chr7 for grain shape in the Trenasse/Jupiter RIL population. A high-throughput KASP-based SNP marker for each locus (GS3 and qGL7.1) was identiﬁed and validated in elite U.S. rice germplasm to be used in an applied rice breeding program. Keywords: rice, grain shape, grain length, QTL, haplotypes, high-throughput KASP marker, SNP INTRODUCTION Rice is a major source of calories for human diets, and the direct consumption of the grain makes grain quality a major component for rice industries. Grain shape is a major grain quality component of rice and directly inﬂuences market value. Consumers make decisions based on the cooking quality and presumed taste of rice depending on its grain length, grain width, aroma, and whiteness. Regions, their cultures, and rice cuisines also drive the preference for different types of rice. Rice is classiﬁed by grain types based on the length and width of the grains. However, there is no global standard for rice classiﬁcation based on shape and size. In the United States, medium grain is predominantly grown in California, accounting for 70% of the U.S. medium-grain rice acres. Long grain is predominantly grown in the southern United States, accounting for 99% of the U.S. long-grain rice acres (USDA and National Agricultural Statistics Service, 2021). Received: 18 April 2022 Accepted: 10 June 2022 Published: 12 September 2022 Edited by: Jyothi Badri, Indian Institute of Rice Research (ICAR), India (ICAR), India Reviewed by: Yingpeng Han, Northeast Agricultural University, China Vanisri Satturu, Professor Jayashankar Telangana State Agricultural University, India *Correspondence: Brijesh Angira bangira@agcenter.lsu.edu Adam N. Famoso AFamoso@agcenter.lsu.edu †Present address: Tommaso Cerioli, Corteva Agriscience, Cremona, Lombardy, Italy United States Germplasm Panel United States Germplasm Panel A United States rice germplasm panel (U.S. germplasm panel) was compiled to characterize grain shape phenotypes. Additionally, this panel was used to discover and validate grain shape loci and trait markers. The panel consists of 323 lines and represents the genetic diversity present in modern and elite southern U.S. rice germplasm. The panel consists of key historical varieties and germplasm, advanced breeding lines, and modern released varieties from the rice breeding programs of Louisiana, Arkansas, Texas, Mississippi, California, and Missouri. Grain shape traits are controlled by variation in naturally occurring genes that are often ﬁrst identiﬁed as quantitative trait loci (QTLs). Some grain shape genes in rice are also responsible for grain weight and yield. Chen et al. (2022) reported KRN2/ OsKRN2 genes in rice and maize and their study showed that a complete loss of function allele of these genes increased grain numbers and yield without a negative impact on other agronomic traits. Many major genes and QTLs have been reported for grain shape traits in rice (Sun et al., 2018). The ﬁrst grain size QTL to be cloned and characterized was GS3 (Fan et al., 2006; Takano-Kai et al., 2009). GS3 encodes a heterotrimeric G protein that interacts with other G-protein subunits as part of a signaling pathway that regulates grain size in rice (Botella, 2012; Sun et al., 2018). The wild-type GS3 protein produces medium grain, a loss-of-function allele results in long grain, while truncated forms lacking the C-terminus produce very short grain (Mao et al., 2010; Takano- Kai et al., 2013). Other grain length QTLs have been identiﬁed on chromosome (chr) three using different mapping populations. These include GW3.1 (Li et al., 2004), GL3.1/GL3 (Qi et al., 2012), qGL3 (Zhang et al., 2012), and TGW3/GL3.3 (Hu et al., 2018; Xia et al., 2018). Several grain length QTLs have also been reported on chr7, including qGL7 (Bai et al., 2010), qGL7.2 (Shao et al., 2010), GLW7/OsSPL13 (Si et al., 2016), SLG7 (Zhou et al., 2015), and GL7/GW7 (Wang S. et al., 2015). Copy number variation at the GL7 locus contributes to grain size diversity in O. sativa (Wang Y. et al., 2015). Citation: Angira B, Cerioli T and Famoso AN (2022) Discovery and Validation of Grain Shape Loci in U.S. Rice Germplasm Through Haplotype Characterization. Front. Genet. 13:923078. doi: 10.3389/fgene.2022.923078 September 2022 \| Volume 13 \| Article 923078 1 Frontiers in Genetics \| www.frontiersin.org Grain Shape Traits Haplotype Characterization Angira et al. Two primary subspecies or varietal groups—Indica and Japonica—represent two genetically distinct genetic pools of O. sativa (Oka, 1988; Wang and Tanksley, 1989; Sun et al., 2002; Childs, 2014). The varietal group Indica is adapted and predominantly grown in tropical latitudes; whereas, Japonica is grown in both temperate and tropical climates (Mackill and Lei, 1997). Each varietal group can be further divided into genetic subpopulations. The varietal group Japonica comprises temperate japonica, tropical japonica, and aromatic subpopulations, while Indica comprises indica and aus subpopulations (Second, 1985; Glaszmann, 1987; Garris et al., 2005; Caicedo et al., 2007; McCouch et al., 2016). Signiﬁcant variation for grain length and shape exists between and within each group, and certain subpopulations are associated with speciﬁc grain classes. For example, the temperate japonica subpopulation is associated with short and round grains, aus with short and slender grains, indica with long, slender grains, and aromatic with both very short and extra-long, fragrant rice (Juliano and Villareal, 1993). Almost all cultivated varieties in the United States belong to the Japonica varietal group. United States long-grain rice is tropical japonica, while U.S. medium-grain rice is a mix of temperate and tropical japonica (Lu et al., 2005). validate trait SNPs for applied breeding purposes. High- throughput SNP markers were identiﬁed and validated for semi-dwarf gene sd1 (Angira et al., 2019), Cercospora disease locus CRSP2.1 (Addison et al., 2021), and an aroma gene BADH2 (Addison et al., 2020) of rice using haplotype characterization approach in the recent studies. Similar haplotype analysis approaches were reported in other crops (Maldonado et al., 2019; Brinton et al., 2020; Bruce et al., 2020; Marsh et al., 2022) and proven to be vital in understanding the trait genetic architecture in a particular germplasm. We developed KASP (Kompetitive allele speciﬁc polymerase) SNP markers using the haplotype characterization approach. KASP is an allele- speciﬁc oligo extension and ﬂuorescence-based cost-effective genotyping technology (Kumpatla et al., 2012; Semagn et al., 2014). In this study, we identiﬁed and validated SNPs for the identiﬁed loci and developed the SNPs as KASP assays to be used in an applied breeding program. “Trenasse” x ‘Jupiter’ Recombinant Inbred Line Population p A bi-parental recombinant inbred line (RIL) population consisting of 286 RILs was developed from a cross between the varieties Trenasse (T) and Jupiter (J) and is referred to as the ‘TJ RIL’ population. Trenasse is a typical U.S. long-grain variety (Linscombe et al., 2006), and Jupiter is a typical U.S. medium-grain variety (Sha et al., 2006). The TJ RIL population was developed using single seed decent breeding method and derived at the F8 generation. Jupiter is widely grown medium grain variety in the southern United State and Trenasse, released in 2006 and still exists in the pedigree of modern long grain varieties. The reported average yield of Trenasse and Jupiter was 8089 kg ha−1 across 21 statewide trials during 2001–2003 (Linscombe et al., 2006) and 8734 kg ha−1 across 37 statewide trials during 2002–2004 (Sha et al., 2006), respectively. Numerous Grain width and grain weight loci are also found on different chromosomes, including GW2 (Song et al., 2007), GIF1 (Wang et al., 2008), GW5/qGW5/SW5, (Shomura et al., 2008; Weng et al., 2008), GS5 (Li et al., 2011), GW6a (Song et al., 2015), TGW6 (Ishimaru et al., 2013), OsSPL16/GW7 (Wang S. et al., 2015), and GW8/OsSPL16 (Wang et al., 2012). Frontiers in Genetics \| www.frontiersin.org Quantitative Trait Loci Mapping for Grain Dimension Traits Quantitative trait loci mapping was conducted using the TJ RIL population. The population was genotyped using KASP SNP assays at the HRCRRS. Linkage map construction and QTL mapping of grain shape traits were executed using composite interval Mapping implemented in QTL IciMapping software V4.1 (Meng et al., 2015). The Kosambi mapping function was used for linkage map development, and the signiﬁcant logarithm of the odds (LOD) threshold was calculated using 1000 permutations and Type I error rate. Additive QTLs were detected using a 1.0 cM stepwise regression with a 0.001 probability threshold. Phenotyping Methods The TJ RIL population (n = 286) was planted at the H. Rouse Caffey Rice Research Station (HRCRRS) near Crowley, LA in 2017 and 2018 and phenotyped for grain traits–grain length and grain width. The U.S. germplasm panel was planted at the HRCRRS in 2019 and phenotyped for grain traits. The panel was phenotyped only for 1 year because the grain dimension traits are highly heritable and the environment does not have a signiﬁcant effect on the traits (Fan et al., 2006; Gao et al., 2016). Both the TJ RILs and the U.S. germplasm panels were planted with a Hege seed drill, and each row was 1.8 m in length. Standard agronomic practices were followed according to the Louisiana Rice Production handbook (LSU Agcenter, 2014). The SeedCount SC6000R - Reﬂectance Image Analysis System (nextinstruments.net) was used to phenotype grain traits: length, width, and length/width ratio (LWR). The instrument uses software and ﬂatbed scanner technology to rapidly and accurately analyze the physical characteristics of a grain sample. The instrument reliably eliminates broken and double grains in a scanning plate. Similar haplotype characterization steps were followed for the GL7 grain shape gene. Single nucleotide polymorphism data for the gene GL7 (LOC_Os07g41200), located at 24,664,168–24,669,324 bp (IRGSP v.1) on chr7, consisted of 82 SNPs identiﬁed in the 3,024 genotypes available in the SNP- Seek portal. Heterozygous SNP calls were considered as missing data. Single nucleotide polymorphisms having >0.2 missing data across 3,024 germplasm samples were omitted. Next, samples having >0.2 missing data across the remaining SNPs were removed from further consideration. In the ﬁnal ﬁltering step, SNPs having minor allele frequency <0.001 were omitted. Following these ﬁltering steps, 2,993 samples remained, and they had genotyping data at 71 SNPs which were then assigned to haplotype groups. Haplotypes present in less than 35 (<0.012) samples were classiﬁed as rare haplotypes. Samples that could not be classiﬁed into haplotype groups due to missing data were assigned to the Unclassiﬁed group. Nine haplotype groups accounted for 96% of the 2,993 samples. Similar haplotype characterization methods in the 3K RGP panel were previously reported by Angira et al. (2019) and Addison et al. (2021). Public Database For this study, we used the International Rice Informatics Consortium SNP-Seek (IRRI SNP-Seek) database available at snp-seek.irri.org (Alexandrov et al., 2014; Mansueto et al., 2016, 2017). The SNP-Seek portal hosts multiple genomic Haplotype characterization approach is a powerful tool that help in understanding a particular germplasm and identify and September 2022 \| Volume 13 \| Article 923078 Frontiers in Genetics \| www.frontiersin.org 2 Grain Shape Traits Haplotype Characterization Angira et al. datasets and formats including the sequencing data of the 3,000 rice genomes project (3K RGP) (Li et al., 2014) and the High- Density Rice Array (HDRA) dataset (McCouch et al., 2016). The HDRA dataset contains 700,000 SNPs (single nucleotide polymorphism) genotyped on 1,568 diverse rice accessions, including 92 U.S. rice varieties (McCouch et al., 2016). Of the ﬁve SNP sets available for panel—All (32M), All biallelic (29M), Base (18M), Filtered (4.8M), and Core (404k) (Mansueto et al., 2017)—this study utilized the 18M SNP set (3KBase) as well as the HDRA dataset to characterize haplotypes in the U.S. germplasm diversity panel. subpopulation groups. However, modern U.S. germplasm is not well represented in this dataset, which contains only 32 U.S. varieties. Therefore, the haplotypes identiﬁed in the 3K RGP diversity panel have to be surveyed across the U.S. rice germplasm panel. g SNP data for the GS3 gene (LOC_OS03G0407400), located at 16,729,501–16,735,109 base pair (bp) (IRGSP v.1) on chr3, consisted of 169 SNPs identiﬁed in the 3,024 genotypes available in the SNP-Seek portal. Heterozygous SNP calls were considered as missing data. Single nucleotide polymorphisms having missing data >0.2 across the 3,024 germplasm samples were omitted from the analysis. Next, samples having >0.2 missing SNP calls were removed from further analysis, and then SNPs with minor allele frequency <0.001 were omitted. After these ﬁltering steps, 2,426 samples remained, and these had genotyping data at 96 SNPs, which were organized into haplotype groups. A haplotype is deﬁned as having identical alleles at all 96 SNP loci. A haplotype number was assigned to haplotypes found in more than 30 samples (>0.012); haplotypes found in less than 30 samples were considered rare haplotypes. This SNP haplotype characterization identiﬁed eight GS3 haplotypes, which accounted for 92% of the 2,426 samples. Haplotype Characterization of Candidate Genes Across the 3K RGP Panel Upon assigning haplotypes for the GS3 and GL7 genes in the 3K RGP panel, a set of minimum SNPs was developed that accurately differentiated all haplotypes. The minimum SNP sets consisted of seven and eight SNPs for GS3 and GL7 genes, respectively. Flanking sequences (100 bp each side) for each SNP were extracted using the Ricebase database (Edwards et al., 2016), and these SNPs were developed into KASP assays for screening across the U.S. germplasm panel. Kompetitive allele speciﬁc polymerase chain reaction genotyping was conducted on the Single nucleotide polymorphism data for the GS3 and GL7 genes were extracted from the IRRI SNP-Seek database using the 3K RGP dataset and 3kbase dataset ﬁlters (iric.irri.org; snp-seek.irri. org) (Alexandrov et al., 2014; Mansueto et al., 2017). The dataset consisted of 3,024 genotypes representing the global diversity of rice germplasm including aus, indica, indica admix, japonica admix, tropical japonica, temperate japonica, and aromatic September 2022 \| Volume 13 \| Article 923078 Frontiers in Genetics \| www.frontiersin.org 3 Grain Shape Traits Haplotype Characterization Angira et al. U.S. germplasm panel at the HRCRRS following protocols developed for the LGC SNPline system (biosearchtech.com/ products/instruments-and-consumables/genotyping- instruments/snpline-genotyping-automation, accessed 22 October 2021). The minimum SNP set markers were screened across the U.S. germplasm panel for both genes to document the frequency of the haplotype groups characterized using the 3K RGP panel. 1547 cM was constructed. Two major QTLs were detected for grain length (qGL3.1 and qGL7.1), grain width (qGW3.1 and qGW7.1), and LWR (qGLWR3.1 and qGLWR7.1). The long-grain parent, Trenasse, contributed the alleles for increased grain length and LWR at both QTLs. The medium-grain parent, Jupiter, contributed the alleles for increased grain width at both grain width QTLs. All QTLs were detected within and across both years. Major QTLs for grain length, qGL3.1 and qGL7.1, explained about 53% and 22% of phenotypic variation, respectively (Table 1). U.S. germplasm panel at the HRCRRS following protocols developed for the LGC SNPline system (biosearchtech.com/ products/instruments-and-consumables/genotyping- g y g instruments/snpline-genotyping-automation, accessed 22 October 2021). The minimum SNP set markers were screened across the U.S. germplasm panel for both genes to document the frequency of the haplotype groups characterized using the 3K RGP panel. Haplotype Characterization of the qGL7.1 QTL Region in HDRA Dataset The QTLs detected for the three traits co-localized to the same two regions on chr3 and chr7, with both regions encompassing previously identiﬁed grain shape genes in rice. The grain dimension QTLs detected on chr3 were mapped between 16.73 and 24.00 Mb, a region that harbors the major grain size gene GS3 (Fan et al., 2006, 2009; Takano-Kai et al., 2009). The QTLs detected on chr7 were mapped between 24.85 and 25.32 Mb, which is 176 kb from the closest ﬂanking marker of the GL7 locus (Wang Y. et al., 2015). Grain dimension traits (grain length, grain width, and LWR) were highly correlated and the QTLs for both the traits were co-localized. Thus, we focused on only grain length trait in this study. However, we explored all the reported grain shape traits in the QTLs. To investigate whether the genes GS3 and GL7 underlie the QTLs detected in this study, qGL3.1 and qGL7.1, respectively, the haplotypes of each gene were characterized using the global rice diversity 3K RGP panel and evaluated across the U.S. germplasm panel that had been phenotyped for grain dimension traits. g To explore haplotype variation across the more extensive qGL7.1 QTL region, SNP data were extracted from the HDRA dataset using the SNP-Seek portal. The dataset consisted of 1,568 germplasm samples, and 763 SNPs were detected in the 475 Kb qGL7.1 QTL region. To explore haplotype variation only in modern U.S. germplasm, we focused on the 34 U.S. genotypes in the HDRA dataset that were released after 1980. Single nucleotide polymorphisms having >0.15 missing data across the 34 U.S. samples, samples having >0.2 missing data across the SNPs, SNPs with minor allele frequency <0.02, and monomorphic SNPs were omitted from the further analysis. After these ﬁltering steps, genotyping data for 358 SNPs on 34 U.S. lines remained for analysis. Haplotypes at a frequency of ≥0.05 were characterized in this dataset and a minimum SNP set was developed that could accurately classify all of the samples into the correct haplotype groups. This minimum SNP set was developed into KASP assays and screened across the U.S. germplasm panel. Interaction of GS3 and qGL7.1 in the TJ RIL Population, Segregating Populations, and Advanced Breeding Lines The IRRI SNP-Seek database contains re-sequencing data for 3,024 diverse rice samples (3K RGP panel). However, the U.S. rice germplasm is not well represented in this panel. To leverage the 3K RGP re-sequencing data for use evaluating our speciﬁc germplasm of interest, we ﬁrst characterized the haplotype diversity of the GS3 and GL7 genes across the 3K RGP panel and identiﬁed the minimum set of SNPs necessary to distinguish the haplotypes of each gene. These minimum sets of SNPs for both genes were subsequently used to develop KASP SNP assays to evaluate the TJ RIL mapping population and the U.S. rice germplasm diversity panel. g The TJ RIL population was used to examine the interaction between the GS3 and qGL7.1 genes. The RILs were classiﬁed into four classes—Class 1: GS3-Long/qGL7.1-Long; Class 2: GS3- Long/qGL7.1-Med; Class 3: GS3-Med/qGL7.1-Long; and Class 4: GS3-Med/qGL7.1-Med, based on the alleles at both loci. Grain length phenotypes were correlated with these classes to understand the effect of the combination of alleles at both loci. We also used 78 advanced breeding lines that were developed by crossing long- and medium-grain varieties. These varieties were phenotyped for grain length and grain width, in addition to yield, for 2 years. Haplotype Characterization of the GS3 Gene Across the 3K RGP Panel A total of 169 SNPs were identiﬁed in the GS3 gene (LOC_OS03G0407400) based on the re-sequencing data for the 3K RGP panel. After ﬁltering SNPs and genotypes based on missing data (>0.2) and minor allele frequency (<0.001), a dataset was extracted consisting of genotypes for 2,426 samples evaluated at 96 SNP loci (Figure 1A, Supplementary Table S1). Based on these 96 SNPs, eight haplotype groups were identiﬁed with a frequency of >0.012. These eight haplotypes described the variation in GS3 found in 2,222 (92%) of the samples. Haplotypes in 204 samples (8%) could either not be classiﬁed Haplotype Characterization of the GL7 Gene Across 3K RGP Panel A total of 78 SNPs were identiﬁed in the GL7 gene (LOC_ Os07g41200) based on the re-sequencing data for the 3K RGP panel. After ﬁltering SNPs and genotypes based on missing data (>0.2) and minor allele frequency (<0.001), a dataset consisting of genotypes for 2,993 samples evaluated at 71 SNPs was extracted. Based on these 71 SNPs, nine haplotypes were identiﬁed with a frequency >0.012. These nine haplotypes described the variation in GL7 found in 2,873 (96%) samples (Figure 1B, Supplementary Table S2). Haplotypes in 120 samples (4%) could either not be classiﬁed due to missing data or were rare (<1.5%), so these samples were omitted from further analysis. Based on the haplotype assignments from the 71 SNPs, a subset of eight SNPs (minimum SNP set) was identiﬁed that could accurately differentiate the nine haplotype groups. This minimum SNP set was used to represent haplotype variation in the GL7 gene (Figure 1B). For this study, we were most interested in haplotypes that could be used to differentiate Japonica samples, as U.S. rice germplasm is predominantly of Japonica ancestry. GS3-Hap1 was the most common SNP haplotype across the 3K RGP panel, found in 1,047 samples, of which about 58% were of Indica ancestry (n = 601), 40% were of Japonica ancestry (n = 424), and 2% were admixtures (n = 22) (Figure 1A, Supplementary Table S1). Within the tropical japonica subpopulation, GS3-Hap1 was the predominant haplotype, accounting for 76% (n = 332) of tropical japonica genotypes. GS3-Hap1 was distinguished from all other haplotypes by the previously reported casual C-A mutation allele in the second exon of GS3 at 16,733,441 bp (GS3-SNP1), containing the long-grain “T” allele (Fan et al., 2009; Takano- Kai et al., 2009). GS3-Hap3 was present in 329 genotypes, of which 73% belonged to the Japonica varietal group (n = 240), 21% were Indica (n = 68), and 6% were admixed (n = 21). GS3- Hap3 was the third most common haplotype across the 3K RGP panel and the predominant haplotype within the temperate japonica subpopulation, accounting for 52% (n = 139) of all temperate japonica lines (Figure 1A, Supplementary Table S1). Based on the association of GS3 haplotypes to speciﬁc subpopulations, we anticipated that the GS3-Hap1 allele would be predominant in U.S. long-grain germplasm and that GS3-Hap3 would be predominant in U.S. medium-grain germplasm. Quantitative Trait Loci Mapping for Grain Dimension Traits A total of 286 RILs, were phentotyped for grain length and grain width for 2 years and genotyped with 139 Kompetitive Allele-Speciﬁc PCR (KASP) single nucleotide polymorphism (SNP) markers. ICIMapping software was used to map the QTLs. LOD, logarithm of the odds; PVE, percent variation explained; Add = additive effect; Pos = position; chr = chromosome. aDonor indicates the parent that contributed the allele for the increased value of the trait in the Jupiter/Trenasse RIL, population. samples of Indica ancestry and were rare or not present in Japonica samples. due to missing data or were rare; these samples were omitted from further analysis. Based on the haplotype assignments from the 96 SNPs, a subset of seven SNPs (minimum SNP set) was identiﬁed that could accurately differentiate the eight haplotype groups and correctly assign samples to each group (Figure 1A). This minimum SNP set was used to represent the haplotype diversity in the GS3 gene. Frontiers in Genetics \| www.frontiersin.org Quantitative Trait Loci Mapping for Grain Dimension Traits Quantitative trait loci (QTL) mapping was conducted for grain dimension traits using 286 RILs derived from a cross between Trenasse (long-grain) and Jupiter (medium-grain) based on phenotype data collected over 2 years. The population was genotyped with 139 KASP SNPs, and a genetic linkage map of September 2022 \| Volume 13 \| Article 923078 Frontiers in Genetics \| www.frontiersin.org 4 Grain Shape Traits Haplotype Characterization Angira et al. TABLE 1 \| Grain dimension Quantitative trait loci (QTL) detected in the Trenasse/Jupiter Recombinant Inbred Line population. Trait QTL name chr Pos (cM) QTL interval start QTL interval stop LOD PVE Add Donora Marker name Pos (cM) IRGSP V.1 Marker name Pos (cM) IRGSP V.1 Length qGL3.1 3 89 LSU-963 88.58 16,733,441 LSU-92 111.08 23,999,431 74.91 53.34 −0.36 Trenasse Length qGL5.1 5 63 LSU-48 46.58 16,473,206 LSU-329 66.57 19,504,928 5.03 2.67 0.08 Jupiter Length qGL7.1 7 124 LSU-323 124.00 24,845,026 LSU-159 125.84 25,319,809 43.00 22.36 −0.23 Trenasse Width qGW3.1 3 49 LSU-1253 48.04 9,828,999 LSU-309 53.52 10,500,404 6.21 4.11 0.03 Jupiter Width qGW3.2 3 89 LSU-963 88.58 16,733,441 LSU-92 111.08 23,999,431 18.60 13.11 0.06 Jupiter Width qGW3.3 3 177 LSU-10 171.96 32,674,916 LSU-240 178.10 34,454,081 6.10 4.14 0.03 Jupiter Width qGW7.1 7 124 LSU-323 124.00 24,845,026 LSU-159 125.84 25,319,809 39.92 32.70 0.09 Jupiter Width qGW8.1 8 200 LSU-445 199.62 18,966,446 LSU-481 214.61 21,589,285 9.29 6.01 −0.04 Trenasse Width qGW12.1 12 81 LSU-1266 70.13 24,395,807 LSU-265 81.52 26,133,920 5.99 3.80 0.03 Jupiter LWR qLWR3.1 3 49 LSU-1253 48.04 9,828,999 LSU-309 53.52 10,500,404 5.99 2.28 −0.05 Trenasse LWR qLWR3.2 3 89 LSU-963 88.58 16,733,441 LSU-92 111.08 23,999,431 63.29 38.24 −0.19 Trenasse LWR qLWR5.1 5 14 LSU-587 12.11 3,341,107 LSU-340 28.11 6,607,675 6.42 2.63 0.05 Jupiter LWR qLWR7.1 7 124 LSU-323 124.00 24,845,026 LSU-159 125.84 25,319,809 58.98 33.12 −0.18 Trenasse LWR qLWR8.1 8 203 LSU-445 199.62 18,966,446 LSU-481 214.61 21,589,285 7.13 3.07 0.06 Jupiter A total of 286 RILs, were phentotyped for grain length and grain width for 2 years and genotyped with 139 Kompetitive Allele-Speciﬁc PCR (KASP) single nucleotide polymorphism (SNP) markers. ICIMapping software was used to map the QTLs. LOD, logarithm of the odds; PVE, percent variation explained; Add = additive effect; Pos = position; chr = chromosome. aDonor indicates the parent that contributed the allele for the increased value of the trait in the Jupiter/Trenasse RIL, population. Haplotype Characterization of the GL7 Gene Across 3K RGP Panel Other common haplotypes observed in the 3K RGP panel were predominant in GL7-Hap1 was the most common SNP haplotype in the 3K RGP panel and was found in 1,257 samples, of which 97% were of Indica ancestry and about 3% were admixtures. GL7-Hap2 was present in 595 samples, of which 90% belonged to the Japonica varietal group, 7% were Indica, and the remaining 3% were admixed (Figure 1B, Supplementary Table S2). GL7-Hap2 was the second most common haplotype group in the panel and the most common haplotype in Japonica samples. GL7-Hap2 was present in 93% of tropical japonica samples in the 3K RGP panel. GL7-Hap3 was present in 337 samples, of which 52% belonged to the temperate japonica subpopulation. GL7-Hap3 was the predominant haplotype within temperate japonica, September 2022 \| Volume 13 \| Article 923078 Frontiers in Genetics \| www.frontiersin.org 5 Grain Shape Traits Haplotype Characterization Angira et al. IGURE 1 \| SNP data was obtained from SNP-Seek online portal of the International Rice Research Institute for GS3 and GL7 genes and haplotypes were haracterized based on speciﬁed criteria with each gene. (A) Haplotype characterization of the GS3 gene in IRRI SNP-Seek 3K RGP genotype data. (B) Haploty haracterization of the GL7 gene in IRRI SNP-Seek 3K RGP genotype data. FIGURE 1 \| SNP data was obtained from SNP-Seek online portal of the International Rice Research Institute for GS3 and GL7 genes and haplotypes were characterized based on speciﬁed criteria with each gene. (A) Haplotype characterization of the GS3 gene in IRRI SNP-Seek 3K RGP genotype data. (B) Haplotype characterization of the GL7 gene in IRRI SNP-Seek 3K RGP genotype data. in the TJ RIL population. The minimum SNP sets that differentiated GS3 (7 SNPs) and GL7 (8 SNPs) haplotypes were converted into KASP assays and used to screen the U.S. germplasm panel. accounting for 61% of all temperate japonica samples. Based on these observations, we anticipated that the GL7-Hap2 would be the most common haplotype within U.S. long-grain germplasm and GL7-Hap3 would predominate within U.S. medium-grain germplasm. Of the eight GS3 haplotypes observed in the 3K RGP panel, only two haplotypes were observed within the U.S. rice germplasm panel (Figure 2A, Supplementary Table S3). GS3- Hap1 was present in 281 genotypes, all of which were previously classiﬁed as long-grain germplasm. GS3-Hap3 was present in 42 genotypes, all were previously classiﬁed as medium or short grain germplasm. Haplotype Characterization of the GL7 Gene Across 3K RGP Panel A comparison of grain length in samples carrying GS3-Hap1 or GS3-Hap3 was highly signiﬁcant (p < 0.0001). The Haplotype Characterization of GS3 and GL7 in U.S. Germplasm The haplotypes of GS3 and GL7 observed in the 3K RGP panel were investigated in the U.S. germplasm panel to determine whether they underlie the qGS3.1 and qGL7.1 QTLs detected September 2022 \| Volume 13 \| Article 923078 Frontiers in Genetics \| www.frontiersin.org Grain Shape Traits Haplotype Characterization Angira et al. FIGURE 2 \| (A) Haplotype characterization of GS3 in U.S. rice germplasm. Haplotypes that were characterized in the IRRI 3K RGP panel were screened in U.S. rice germplasm panel based on the minimum SNP set identiﬁed to differentiate the eight haplotype groups in IRRI 3K RGP dataset. (B) Phenotypic association of haplotype/ SNP alleles and grain length across U.S. rice germplasm. Different alphabet letters on each class represent the statistically signiﬁcant difference p < 0.001 within each graph. FIGURE 2 \| (A) Haplotype characterization of GS3 in U.S. rice germplasm. Haplotypes that were characterized in the IRRI 3K RGP panel were screened in U.S. rice germplasm panel based on the minimum SNP set identiﬁed to differentiate the eight haplotype groups in IRRI 3K RGP dataset. (B) Phenotypic association of haplotype/ SNP alleles and grain length across U.S. rice germplasm. Different alphabet letters on each class represent the statistically signiﬁcant difference p < 0.001 within each graph. as did all the individual RILs, demonstrating that GL7 is not the gene underlying the QTL qGL7.1 detected in this study. average grain length of the entire panel was 6.98 mm, with a standard deviation of 0.47 mm, and a range of 5.37–7.95 mm. The GS3-Hap1 group had an average grain length of 7.13 mm, while the GS3-Hap3 group had an average length of 5.94 mm. GS3- Hap1 and GS3-Hap3 explained ~75% of grain length variation in the U.S. germplasm panel and accurately differentiated long- and medium-grain varieties (Figures 2A,B). Thus, we concluded that the GS3 gene underlies the qGS3.1 QTL and that it is a major gene underlying grain length variation in U.S. germplasm, accurately distinguishing grain market classes. The SNP GS3-SNP1, which represents the known casual C-A point mutation in the GS3 gene, accurately differentiates long- and medium-grain varieties and is an ideal functional SNP for use as a trait marker. Frontiers in Genetics \| www.frontiersin.org Haplotype Characterization of the qGL7.1 QTL Region Using the HDRA Dataset rice germplasm panel. qGL7.1-SNP1 was identiﬁed in the HDRA dataset that was obtained from SNP-Seek online portal within the qGL7.1 QTL region. QTL qGL7.1 was detected on Chromosome seven in Trenasse/Jupiter RIL population. qGL7.1-SNP1 differentiated long-grain haplotypes from medium-grain haplotypes within U.S. lines of HDRA dataset. Allele A is associated with the medium-grain genotypes and allele G is associated with long-grain genotypes. Different alphabet letters on each class represent the statistically signiﬁcant difference p < 0.001 within each graph. The SNP qGL7.1-SNP1 (25,211,630 bp) was able to differentiate qGL7.1-Hap1 (predominantly found in long-grain varieties) from qGL7.1-Hap3 (predominantly found in medium-grain varieties), but it does not differentiate qGL7.1-Hap3 from qGL7.1-Hap4. If used together, GL7.1-SNP1 and qGL7.1-SNP3 can differentiate all four haplotypes found in the U.S. germplasm, but a single SNP capable of differentiating long-grain qGL7.1-Hap1 from medium-grain qGL7.1-Hap3 would be sufﬁcient for applied breeding programs focusing on the southern United States. When used alone, SNP qGL7.1-SNP1 explained 43% (Figure 3) of the phenotypic variation for grain length. We recommend that it be used as a diagnostic marker to identify the long-grain allele at qGL7.1 in applied breeding applications in the United States. was identiﬁed that accurately differentiated the four haplotypes. The four haplotypes were perfectly associated with the previously deﬁned grain classes. qGL7.1-Hap1 (n = 13; 38%) and qGL7.1- Hap2 (n = 2; 6%) were present in samples classiﬁed as long grain, where qGL7.1-Hap3 (n = 7; 21%) and qGL7.1-Hap4 (n = 2; 0.1%) were present in samples classiﬁed as medium/short grain (Supplementary Figure S1A; Supplementary Table S4). The three SNPs that differentiated the qGL7.1 haplotypes were developed into KASP assays and surveyed across the entire U.S. germplasm panel. Three of the four haplotypes (qGL7.1-Hap1, qGL7.1-Hap3, and qGL7.1-Hap4) were detected in modern U.S. germplasm. The most frequent haplotype was qGL7.1-Hap1 (n = 271; 83.9%), found in 266 long-grain and ﬁve medium-grain varieties. Haplotype qGL7.1-Hap3 (n = 43; 13.3%) was observed in 14 long-grain, 27 medium-grain, and two short-grain samples, and was the predominant haplotype in medium grains. qGL7.1- Hap4 (n = 5; 1.5%) was observed in two long-grain, one medium- grain, one short-grain samples, and was rare in southern U.S. rice germplasm. Four samples (1.2%) had unclassiﬁed haplotypes due to heterozygous SNP calls (Supplementary Figure S1B; Supplementary Table S5). Together, qGL7.1 haplotypes explained 46% of the variation for grain length across the U.S. germplasm panel. Haplotype Characterization of the qGL7.1 QTL Region Using the HDRA Dataset qGL7.1-Hap1 was associated with long-grain length (7.1 mm) that was signiﬁcantly longer than either qGL7.1- Hap3 (6.3 mm) or qGL7.1-Hap4 (5.6 mm). Haplotype Characterization of the qGL7.1 QTL Region Using the HDRA Dataset QTL Region Using the HDRA Dataset Upon concluding that the candidate gene GL7 did not underlie the QTL qGL7.1, haplotype analysis was conducted across the qGL7.1 QTL region. The QTL was mapped to a region of 474,783 bp (1.84 cM) on chr7, which was too large to conduct haplotype analysis based on re-sequencing data. We therefore utilized the HDRA dataset (McCouch et al., 2016) and identiﬁed 763 SNPs across the QTL region based on genotypes from 1,568 samples. To prioritize the identiﬁcation and characterization of the haplotypes most likely to be present in modern U.S. rice germplasm, we focused on the 34 U.S. varieties in the HDRA dataset that were released after 1980. Single nucleotide polymorphisms having >0.15 missing data across 34 U.S. varieties, samples with >0.2 missing data across the SNPs, SNPs with minor allele frequency <0.02, and monomorphic SNPs were excluded from the analysis. Upon ﬁltering of SNPs, 358 SNPs remained for haplotype characterization across 34 U.S. lines. Based on these 358 SNPs, four haplotype groups were identiﬁed at a frequency of ≥0.05. A minimum set of three SNPs Of the nine GL7 haplotypes observed in the 3K RGP panel, only two haplotypes (GL7-Hap2 and GL7-Hap3) were present in U.S. rice germplasm (Supplementary Table S3). GL7-Hap2 was present in 98% of the U.S. panel, including 100% of long-grain varieties and 86% of medium/short-grain varieties. Only six U.S. varieties (S-201, S-102, M-206, Acadia, Pirogue, and Vista) contained the GL7-Hap3 allele, all of which were either California medium-grain varieties and/or historical varieties. No modern Southern U.S. medium-grain variety contained GL7-Hap3. The parents of the TJ RIL population contained GL7-Hap2 at the GL7 gene, September 2022 \| Volume 13 \| Article 923078 Frontiers in Genetics \| www.frontiersin.org 7 Grain Shape Traits Haplotype Characterization Angira et al. FIGURE 3 \| Grain length variation explained by qGL7.1-SNP1 (LSU-1480) across a U.S. rice germplasm panel. qGL7.1-SNP1 was identiﬁed in the HDRA dataset that was obtained from SNP-Seek online portal within the qGL7.1 QTL region. QTL qGL7.1 was detected on Chromosome seven in Trenasse/Jupiter RIL population. qGL7.1-SNP1 differentiated long-grain haplotypes from medium-grain haplotypes within U.S. lines of HDRA dataset. Allele A is associated with the medium-grain genotypes and allele G is associated with long-grain genotypes. Different alphabet letters on each class represent the statistically signiﬁcant difference p < 0.001 within each graph. FIGURE 3 \| Grain length variation explained by qGL7.1-SNP1 (LSU-1480) across a U.S. Frontiers in Genetics \| www.frontiersin.org Interaction of GS3 and qGL7.1 Within a RIL Population, Segregating Populations, and Advanced Breeding Lines The allelic states of GS3 and qGL7.1 were highly correlated across the U.S. germplasm panel, with 96% of long-grain varieties containing the long-grain allele at both loci and 95% of medium-grain varieties containing the medium-grain allele at both loci. To understand the interaction and effect of each locus in a controlled genetic background, the TJ RILs were grouped into four classes based on alleles at GS3-SNP1 and qGL7.1-SNP1, and these classes were compared for grain length. The classes were deﬁned as Class1: GS3-Long/qGL7.1-Long; Class2: GS3-Long/ To identify a single SNP that could reliably distinguish long- and medium-grain varieties for routine use in southern U.S. breeding programs, the three haplotype SNPs were compared. September 2022 \| Volume 13 \| Article 923078 Frontiers in Genetics \| www.frontiersin.org 8 Angira et al. Grain Shape Traits Haplotype Characterization FIGURE 4 \| Genotypic classes of Trenasse/Jupiter RILs based on the alleles at GS3 and qGL7.1 in the RIL population. Four genotypic classes were identiﬁed based on the allele state at GS3 and qGL7.1. Classes denoted by a different letter indicate signiﬁcant differences between classes (p < 0.001). FIGURE 4 \| Genotypic classes of Trenasse/Jupiter RILs based on the alleles at GS3 and qGL7.1 in the RIL population. Four genotypic classes were identiﬁed based on the allele state at GS3 and qGL7.1. Classes denoted by a different letter indicate signiﬁcant differences between classes (p < 0.001). qGL7.1-Med; Class3: GS3-Med/qGL7.1-Long; Class4: GS3-Med/ qGL7.1-Med. Signiﬁcant differences in grain length were observed among classes (p < 0.001). Class1 (7.2 mm) had the longest grain length and Class4 had the shortest (6.0 mm). Class2 exhibited a longer grain length than Class3, demonstrating that GS3 has a larger effect than qGL7.1 in this population (Figure 4). Selecting long-grain alleles at both loci would help develop a longer grain rice variety using U.S. germplasm. qGL7.1 had a longer grain length (7.1 mm) compared to the class containing the homozygous medium-grain allele at GS3 and homozygous long-grain (6.7 mm) allele at qGL7.1 (Supplementary Figure S2). Thus, GS3 again appears to have a larger effect than qGL7.1 in these additional populations, consistent with observations in the TJ RIL population. Among the lines homozygous for the long-grain GS3 allele, the allelic state (homozygous long, homozygous medium, heterozygous) at qGL7.1 did not signiﬁcantly contribute to grain length. September 2022 \| Volume 13 \| Article 923078 Frontiers in Genetics \| www.frontiersin.org Interaction of GS3 and qGL7.1 Within a RIL Population, Segregating Populations, and Advanced Breeding Lines However, among the lines that were homozygous for the medium-grain allele at the GS3 gene, signiﬁcantly different grain lengths were observed for the qGL7.1 allele classes (Supplementary Figure S2). To further validate the effect of GS3 and qGL7.1 in different genetic backgrounds, four F3 populations (CL272/Mermentau, CL272/Presidio, CRPS/Titan, and Mermentau/Titan) that segregated for both GS3 and qGL7.1 loci were examined. The populations were divided into nine genotypic classes based on the allele conﬁgurations [homozygous (homo) long (L) allele, heterozygous (het), or homozygous medium (M) allele] at both loci. The genotypic classes were deﬁned as GS3-homo L/ qGL7.1-homo L; GS3-homo L/qGL7.1-homo M; GS3-homo L/ qGL7.1-het; GS3-homo M/qGL7.1-homo L; GS3-homo M/ qGL7.1-homo M; GS3-homo M/qGL7.1-het; GS3- het/qGL7.1- homo L; GS3-het/qGL7.1-homo M; and GS3-het/qGL7.1-het). The average grain length of the genotypic class that was homozygous long at both loci was 7.2 mm, compared to 6.4 mm for the genotypic class that was homozygous medium at both loci. The genotypic class that was heterozygous at both loci was 6.6 mm. The genotypic class containing the homozygous long-grain allele at GS3 and homozygous medium-grain allele at In addition to the RIL population and segregating populations, we also investigated the effect of these loci on grain length in advanced breeding lines that were developed by crossing long- and medium-grain varieties. Seventy eight breeding lines were genotyped at both loci and phenotyped for grain length across 2 years. These lines were classiﬁed into four classes based on allele state at GS3 and qGL7.1. Lines that had the long-grain allele at both loci had an average grain length of 6.9 mm, compared to a grain length of 6.1 mm for lines with the medium-grain allele at both loci. Lines with a long-grain allele at GS3 and medium-grain allele at qGL7.1 had an average grain length of 6.5 mm, compared to 6.3 mm for lines that had the medium-grain allele at GS3 and the long-grain allele at qGL7.1. These results conﬁrm that both loci have a signiﬁcant effect across multiple populations and September 2022 \| Volume 13 \| Article 923078 Frontiers in Genetics \| www.frontiersin.org 9 Grain Shape Traits Haplotype Characterization Angira et al. genetic backgrounds and that GS3 has a larger effect on grain length than qGL7.1. the class that had long-grain allele but was signiﬁcantly longer from the class that had medium-grain allele at qGL7.1 (Supplementary Figure S2). DISCUSSION Many genes conferring grain shape have been reported in previous studies. Here, a novel QTL, qGL7.1, for grain shape was mapped to a 475 kb region and explained 22% of the phenotypic variation in the TJ RIL population. We explored candidate genes in this region using data available on SNP-Seek platform (Mansueto et al., 2016, 2017). This region harbors 80 genes including 25 expressed proteins, 13 retrotransposons, seven DC1 domain-containing proteins, three C1-like domain- containing proteins, three transposon proteins, two sulfotransferase domain-containing proteins, and two stress responsive A/B barrel domain-containing proteins (Supplementary Table S6). The most promising candidate gene is LOC_Os07g41590, located between 24,931,739–24,933,197 bp. It is a gibberellin (GA) receptor gene, and GAs are reported to be crucial in many developmental processes, including stem elongation, leaf expansion, trichome development, seed germination, pollen maturation, and ﬂowering initiation (Achard and Genschik, 2009). Vaughan et al. (2011) documented the role of GA in determining wheat grain size. They reported that an increase in GA production resulted in up to a 15% increase in grain weight. Therefore, LOC_Os07g41590 may be a grain shape candidate gene underlying the qGL7.1 QTL. We also explored mutants in the qGL7.1 region and identiﬁed the Giant Embryo (GE) gene (Koh et al., 1996; Nagasawa et al., 2013) located at 25.373,984–25,375,652 bp and the BUI1 (BENT UPPERMOST INTERNODE1) gene (Yang et al., 2011) at 25,373,984–25,375,652 bp. GE controls cell size in the embryo and cell death in the endosperm. Endogenous GE expression is upregulated in mutants, and GE overexpression causes a small embryo and enlarged endosperm. Thus, GE might be another candidate gene underlying grain length in the qGL7.1 region. Further investigation is needed to identify other candidate genes in this region that may play a role in grain shape. Interaction of GS3 and qGL7.1 Within a RIL Population, Segregating Populations, and Advanced Breeding Lines These results do not provide sufﬁcient evidence to conclude dominance/recessive nature of qGL7.1. Further studies using true F1 plants are required to conclude the gene action of qGL7.1. Validation and Technical Performance of the Identiﬁed Grain Length Trait SNPs Haplotype characterization and phenotypic association identiﬁed a single candidate SNP/locus for use as a trait marker for the major grain size QTLs, qGS3.1 and qGL7.1. For a trait marker to have broad utility in an applied breeding program, it should be informative across the target breeding germplasm and provide high-quality, cost-effective, and reproducible genotype data. GS3- SNP1 and qGL7.1-SNP1 were validated across the U.S. germplasm panel and shown to be effective at differentiating long- and medium-grain cultivars based on alleles at GS3 and qGL7.1. Thus, these SNPs have potential utility for use in U.S. rice breeding programs. However, for a SNP marker to be effectively deployed in an applied breeding program, it is also essential that the marker provide reproducible results and that it is amenable to high-throughput screening and scoring. To ensure that each of the selected SNPs provides reliable genotyping results for high- throughput molecular breeding activities, the technical performance of the KASP assay was tested. Each assay was tested with three replicates across the U.S. germplasm panel, and a plate of 96 samples from the F1 population that included all three genotypic classes was used to evaluate SNP clustering in the presence of heterozygous plants. Each assay provided >99% data return, consistent allele calls across the replicates, and exhibited clear and distinct clusters that could be accurately assessed using auto-scoring (Supplementary Figure S3). Frontiers in Genetics \| www.frontiersin.org SUPPLEMENTARY MATERIAL The results of this study conﬁrmed that GS3 is a major grain length gene, and qGL7.1 is a novel locus on chr7 that signiﬁcantly affects grain shape in the U.S. germplasm. We identiﬁed and validated GS3-SNP1 and qGL7.1-SNP1 markers which accurately identify long- and medium-grain alleles at GS3 and qGL7.1. Alleles at GS3 and qGL7.1 do not segregate within the long- or the medium-grain class, but they do segregate when long- and medium-grain varieties are crossed. Selecting for long-grain alleles at both loci will help increase grain length, and selecting for medium-grain alleles at both loci will facilitate the development of shorter grain varieties. Further studies are needed to investigate the gene action of qGL7.1 and to discover the genes in the locus. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fgene.2022.923078/ full#supplementary-material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fgene.2022.923078/ full#supplementary-material Supplementary Figure 1 \| (A) Haplotype characterization within QTL qGL7.1 region using HDRA SNP-Seek genotype dataset to study the diversity in the QTL region. The haplotype characterization was conducted using only US varieties that were release after 1980. (B) Haplotype characterization of the QTL qGL7.1 across US germplasm using minimum SNP set developed from HDRA data within the QTL region. Supplementary Figure 2 \| Four segregating F3 populations - CL272/Mermentau, CL272/Presidio, CRPS/Titan, and Mermentau/Titan, that segregated for both GS3 and qGL7.1 genes were used to study the effect of the genes on grain length. Each population had about 70 panicles. A total of 276 panicles were genotyped for GS3 and qGL7.1, and all the seeds of each panicle were phenotyped for grain length. First letter in class name is allele at GS3 gene, and the second letter followed by forward slash is allele state at qGL7.1 gene. L stands for long-grain allele, M stands for medium-grain allele, and H stands for heterozygote state at the gene. (A) Average grain length of the deﬁned classes. Different alphabet letters on each class represent the statistically signiﬁcant difference p < 0.001 within each graph. Investigation Into the Gene Action and Inheritance of GS3 and qGL7.1 q The four F3 populations (CL272/Mermentau, CL272/Presidio, CRPS/Titan, and Mermentau/Titan) segregating for both GS3 and qGL7.1 loci were used to investigate the gene action and inheritance of the two loci. Grain length of the lines that were heterozygous at GS3 and homozygous for the long allele at qGL7.1 had grain lengths similar to the lines that were homozygous for the medium-grain allele at GS3 and for the long-grain allele at qGL7.1. Similarly, lines that were heterozygous at GS3 and homozygous for the medium-grain allele at qGL7.1 had grain lengths similar to lines that were homozygous for the medium alleles at GS3 and qGL7.1. However, lines that were homozygous for the long alleles at GS3 and qGL7.1 were signiﬁcantly longer than lines that were heterozygous at the GS3 gene and homozygous for the long allele at qGL7.1 (Supplementary Figure S2). These results demonstrate that the long-grain allele of GS3 is recessive, consistent with previous reports. The effects of GS3 and qGL7.1 on grain length were studied in a wide range of materials including a U.S. germplasm panel (n = 323), a RIL (TJ) population (n = 286), four F2:F3 breeding populations, and 78 advanced breeding lines, developed by crossing long and medium-grain parents segregating at both GS3 and qGL7.1 loci. In all these materials, both loci have a signiﬁcant effect on grain length, with GS3 having a larger effect than qGL7.1. Lines carrying the long-grain allele at both loci had the longest grains, and lines carrying medium-grain alleles at both loci exhibited the shortest grains, with other combinations of alleles conferring intermediate grain lengths. These results across a wide range of plant material clearly showed the beneﬁt of using allele-speciﬁc markers at both loci to select for grain length in U.S. rice germplasm. Compared to GS3, the gene action of the qGL7.1 locus was less clear. Among the F2:F3 lines that were homozygous for the long- grain GS3 allele, no signiﬁcant differences in grain length were observed among the three qGL7.1 genetic classes (homozygous long, heterozygous, and homozygous medium). However, among F2:F3 lines homozygous for the medium-grain alleles at GS3, the heterozygous class at qGL7.1 was not signiﬁcantly different from September 2022 \| Volume 13 \| Article 923078 10 Grain Shape Traits Haplotype Characterization Angira et al. AUTHOR CONTRIBUTIONS United States long-grain and medium-grain rice varieties differ in their cooking qualities in addition to their grain length. Long-grain rice has an intermediate amylose content of 21–24% and an intermediate gel temperature of 71–74°C as indicated by intermediate alkali spreading value of three–5 (Webb, 1985; Gravois and Webb, 1997; Linscombe, 2017). In contrast, short-to medium-grain rice has a lower amylose content of 15–19% and a low gel temperature of 65–68°C as indicated by alkali spreading value of 6–7 (Webb, 1985; Gravois and Webb, 1997; Linscombe, 2017). Due to these differences in both cooking quality and grain length, it is challenging to cross long- and medium-grain varieties and to select a long- or medium-grain quality line from the segregating population. In this study, we reported high- throughput markers for a major grain length locus GS3 and a novel QTL qGL7.1. Within each grain class (long or medium), these loci do not segregate, and the reported markers may not play any role. However, when crosses are made between long- and medium-grain parents, validated markers GS3-SNP1 and qGL7.1-SNP1 can play an important role in selecting either long-grain or medium- grain lines. Major waxy alleles are characterized and reported in previous studies (Yang et al., 2019; Shao et al., 2020). Markers are also available for amylose and gel temperature traits. These cooking quality SNP markers along with the validated GS3-SNP1 and qGL7.1-SNP1 markers for grain length will facilitate breeders’ efforts to select the desired grain class and cooking quality when crossing long- and medium-grain varieties. BA mapped QTLs for grain shape traits using KASP markers, analyzed the data, executed haplotype characterization in IRRI 3K RGP dataset and the U.S. germplasm, identiﬁed and validated markers for use in applied breeding programs, and lead manuscript writing. AF assisted in designing the project, troubleshooting research problems, ﬁeld planting and design, and contributed in writing the manuscript. TC developed advanced breeding lines crossing long- and medium- grain varieties. These advanced breeding lines were used to validate the effect of the novel locus and to validate the markers. ACKNOWLEDGMENTS The authors thank Valerie Dartez for contribution in manuscript preparation and proofreading. The authors thank Valerie Dartez for contribution in manuscript preparation and proofreading. FUNDING This research study was supported by Louisiana Rice Research Board (grant GR- 59400008264, PI: AF and grant GR-00006303 and GR-00008230, PI: BA), Horizon Ag. (grant GR-00004555, PI: AF), National Science Foundation (grant number (FAIN) 1826836, PI: A. Pereira), Hatch project LAB94468 (PI: BA), and Hatch project LAB94342 (PI: AF). This research study was supported by Louisiana Rice Research Board (grant GR- 59400008264, PI: AF and grant GR-00006303 and GR-00008230, PI: BA), Horizon Ag. (grant GR-00004555, PI: AF), National Science Foundation (grant number (FAIN) 1826836, PI: A. Pereira), Hatch project LAB94468 (PI: BA), and Hatch project LAB94342 (PI: AF). 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https://openalex.org/W4287777551	https://zenodo.org/records/5517498/files/Paper_BeersLaw_final_manuscript.pdf	English	null	Optimal surface estimation and thresholding of confocal microscope images of biofilms using Beer's Law	Zenodo (CERN European Organization for Nuclear Research)	2,020	cc-by	12,528	Optimal surface estimation and thresholding of confocal 1 microscope images of biofilms 2 using Beer’s Law 3 A. E. Parkera,b,∗, J. A. Christenc, L. Lorenza, H.Smitha,d ∗Corresponding author Email address: parker@math.montana.edu (A. E. Parker) URL: www.math.montana.edu/parker (A. E. Parker) Preprint submitted to Elsevier May 8, 2020 Optimal surface estimation and thresholding of confocal 1 microscope images of biofilms 2 using Beer’s Law 3 A. E. Parkera,b,∗, J. A. Christenc, L. Lorenza, H.Smitha,d 4 aCenter for Biofilm Engineering, Montana State University, Bozeman, MT, USA 5 bDepartment of Mathematical Sciences, Montana State University, Bozeman, MT, USA 6 cCentro de Investigación en Matemáticas, Jalisco S/N, Valenciana, Guanajuato, GTO, 7 36023, MEXICO 8 dDepartment of Microbiology and Immunology, Montana State University, Bozeman, 9 MT, USA 10 Optimal surface estimation and thresholding of confocal 1 microscope images of biofilms 2 using Beer’s Law 3 A. E. Parkera,b,∗, J. A. Christenc, L. Lorenza, H.Smitha,d 4 aCenter for Biofilm Engineering, Montana State University, Bozeman, MT, USA 5 bDepartment of Mathematical Sciences, Montana State University, Bozeman, MT, USA 6 cCentro de Investigación en Matemáticas, Jalisco S/N, Valenciana, Guanajuato, GTO, 7 36023, MEXICO 8 dDepartment of Microbiology and Immunology, Montana State University, Bozeman, 9 MT, USA 10 ∗Corresponding author P i t b itt d t El Abstract 11 Beer’s Law explains how light attenuates into thick specimens, including thick biofilms. We use a Bayesian optimality criterion, the maximum of the posterior probability distribution, and computationally efficiently fit Beer’s Law to the 3D intensity data collected from thick living biofilms by a confocal scanning laser microscope. Using this approach the top surface of the biofilm and an optimal image threshold can be estimated. Biofilm characteristics, such as bio-volumes, can be calculated from this surface. Results from the Bayesian approach are compared to other approaches including the method of maximum likelihood or simply counting bright pixels. Uncertainty quan- tification (i.e., error bars) can be provided for the parameters of interest. This approach is applied to confocal images of stained biofilms of a common lab strain of Pseudomonas aeruginosa, stained biofilms of Janthinobacterium isolated from the Antarctic, and biofilms of Staph aureus that have been genetically modified to fluoresce green. May 8, 2020 May 8, 2020 Keywords: Attenuation, thresholding, Maximum likelihood, Beer-Lambert 12 Law, Bayesian, confocal microscope image analysis 13 1. Introduction 14 Confocal Scanning Laser Microscopy (CSLM) has become an essential 15 instrument in biofilm research because of the ability to generate 3D visu- 16 alizations of hydrated biofilms noninvasively and in real-time [e.g., 25, 11]. 17 Fluorophores are introduced into the microbes by either staining or geneti- 18 cally modifying the species to express a fluorescent protein. CSLM utilizes 19 the same lens to excite and to detect emitted light from these fluorophores 20 [20]. The pinhole system enables the removal of out-of-focus light necessary 21 for the generation of high resolution 3D images (108 pixels or more) at spatial 22 resolutions less than 1µm (see Figure 1). 23 When imaging thick biofilms, the laser’s intensity attenuates markedly 24 as it passes through the top layers of the biofilm so that the bacteria deep 25 in the biofilm do not fluoresce at all [23]. Hence, CSLM image resolution is 26 restricted by the depth of sample penetration which has made the quantita- 27 tive assessment of CSLM data challenging. With conventional CSLM, light 28 begins to scatter severely between 60-80µm [4]. The penetration depth can 29 be greater for more advanced CSLM, e.g. multiphoton confocal microscopes, 30 but attenuation still occurs for thick enough specimens. This phenomenon 31 is predicted by Beer’s Law (or the Beer-Lambert Law), a physics model that 32 describes how light transmission decays as the microscope’s laser is focused 33 deeper into a specimen [6, 26]. Our investigations confirm that this model 34 also well explains, quantitatively, the phenomenon in biofilms. 35 2 2 A B C Figure 1: CSLM images of thick biofilms (> 100µm) visualized with Imaris. These images are analyzed in this paper. A: Using a 25x objective, a 620µm × 620µm field of view over 1024 × 1024 × 163 pixels of a 192µm thick stained Pseudomonas aeruginosa biofilm. B: Using a 25x objective, a 620µm × 620µm field of view over 512×512×17 pixels of a 112µm thick biofilm of GFP Staph. aureus. The hollowed out appearance of the biofilm in the side-panels is due to the attenuation of light because of Beer’s Law. C: When using a 63x objective, a 246µm × 246µm field of view over 1024 × 1024 × 286 pixels of a 141µm thick stained Janthinobacterium biofilm isolated from Antarctica. 1. Introduction 14 3 A B C A B A B A B C Figure 1: CSLM images of thick biofilms (> 100µm) visualized with Imaris. These images are analyzed in this paper. A: Using a 25x objective, a 620µm × 620µm field of view over 1024 × 1024 × 163 pixels of a 192µm thick stained Pseudomonas aeruginosa biofilm. B: Using a 25x objective, a 620µm × 620µm field of view over 512×512×17 pixels of a 112µm thick biofilm of GFP Staph. aureus. The hollowed out appearance of the biofilm in the side-panels is due to the attenuation of light because of Beer’s Law. C: When using a 63x objective, a 246µm × 246µm field of view over 1024 × 1024 × 286 pixels of a 141µm thick stained Janthinobacterium biofilm isolated from Antarctica. 3 We present a novel approach for the analysis of CSLM data that fits 36 Beer’s Law to the intensities in these 3D images. Our approach considers 37 a Beer’s threshold, that identifies which bright pixels (with fluorescence in- 38 tensities above Beer’s threshold) are associated with the top surface of the 39 biofilm. This is in contrast to a conventional image threshold that specifies 40 which bright pixels (with intensities above the threshold) are associated with 41 biomass regardless of the spatial location. Our approach considers a range of 42 possible Beer’s threshold values provided by the user (e.g., the microscopist 43 or data analyst). For each possible Beer’s threshold, a linear regression model 44 that describes Beer’s Law is fit to all of the intensity data below the surface 45 by least squares. Reported are two Beer’s thresholds that are optimal com- 46 pared to any other candidate in the range of possible thresholds: (1) the 47 maximum likelihood estimate (MLE) that minimizes the mean squared er- 48 ror (or, equivalently, maximizes the R2 as for any regression model); and 49 (2) the maximum of the Bayesian posterior distribution (MAP) that max- 50 imizes the probability of the threshold given the data. Importantly, from 51 the biofilm’s surface, our approach can quantify other biofilm characteris- 52 tics, such as bio-volume, from a CSLM image. Methods that require the user 53 to subjectively choose a single threshold can result in quantitative outcomes, 54 like bio-volumes, that are biased [e.g., see 18]. 1. Introduction 14 55 There are a number of automatic threshold detection schemes already 56 available in image analysis software such as COMSTAT (implemented in 57 MATLAB) [14], COMSTAT2 (implemented in ImageJ) [27], Imaris [16], and 58 daime [8]. Otsu’s method for threshold selection, used by the COMSTAT im- 59 plementations in MATLAB and ImageJ, maximizes the intra-class correlation 60 4 [19], where each class is composed of pixels with intensities either above or 61 below the threshold. Imaris implements a method due to [7] that iteratively 62 reduces the threshold until the correlation of the image using only pixels with 63 intensities below the threshold reaches 0 [16, p. 241]. The Objective Thresh- 64 old Selection (OTS) method [28], based on Robust Automated Threshold 65 Selection (RATS-L), is implemented by the software daime [8] and uses an 66 edge matrix to estimate the threshold, then keeps modifying the threshold 67 until bio-volume, defined as the number of bright pixels above the threshold, 68 stabilizes. A variant of OTS, the bio-volume elasticity method [18], uses a 69 heuristic approach to choose a threshold based on the rate of decrease of the 70 bio-volume as the threshold is increased. Due to Beer’s Law, quantifying 71 bio-volume simply by counting bright pixels is unbiased only if the biofilm 72 being imaged is thin (e.g., less than 100µm). For thick biofilms (Figure 1), 73 counting bright pixels would underestimate the bio-volume [22]. We compare 74 our approach to Otsu’s (via COMSTAT2 in ImageJ), the method due to [7] 75 (via Imaris) and RATS-L (via daime). 76 Uncertainty quantification via a Bayesian probability interval (also called 77 a credible interval), or a confidence interval, can be provided by our approach 78 for all parameters of interest. For example, our approach provides the uncer- 79 tainty in the Beer’s threshold, the location of the top surface of the biofilm, 80 and the bio-volume from a single image. In contrast, uncertainty quan- 81 tification for characteristics from single images are not provided by Costes’ 82 approach (in Imaris) or by Otsu’s (COMSTAT2 in ImageJ). 1. Introduction 14 OTS determines 83 a confidence interval for the bio-volume by generating a threshold for each 84 column in the image and then considering the distribution of threshold values 85 5 5 To illustrate that our technique can be applied successfully to many dif- 87 ferent types of microbiological samples, we analyze CSLM images from three 88 individual organisms: (1) a lab strain of P. aeruginosa (ATCC #15442), 89 likely the most studied biofilm forming bacterium ever; (2) a genetically 90 modified green fluorescing Staphylococcus aureus; and (3) an environmen- 91 tal Janthinobacterium isolate (Figure 1). To demonstrate the accuracy of 92 our method, results are compared to other available image analysis software 93 packages when possible. 94 2. Methods 95 2.1. Biofilm growth and image acquisition 96 2.1. Biofilm growth and image acquisition 96 Pseudomonas aeruginosa biofilms (ATCC 15442, https://www.atcc.org/prod 97 were grown in the high shear environment in the CDC Biofilm Reactor using 98 ASTM method E2542 [13]. A biofilm sample was then stained with 200µL 99 of a diluted 1:1 mixture of Syto 9 and Propidium Iodide (LIVE/DEAD Ba- 100 cLight Bacterial Viability Kit stain (Invitrogen, catalog #L7012)) and then 101 imaged. 102 Green-fluorescing protein (GFP) Staphylococcus aureus AH2547 were kindly 103 provided by Dr. Alexander Horswill [21]. These GFP S. aureus biofilms were 104 grown in a separate CDC Biofilm Reactor. The growth medium was supple- 105 mented with 10µg/mL of chloramphenicol to retain the GFP plasmid. A 106 biofilm sample was imaged over time using the Treatment Imaging Flow Cell 107 (BioSurface Technologies Corporation, catalog #FC 30). The time series was 108 analyzed by [22]; here we analyze a single image from that series. 109 2.1. Biofilm growth and image acquisition 96 2.1. Biofilm growth and image acquisition 96 Pseudomonas aeruginosa biofilms (ATCC 15442, https://www.atcc.org/products/all/15442.asp 97 were grown in the high shear environment in the CDC Biofilm Reactor using 98 ASTM method E2542 [13]. A biofilm sample was then stained with 200µL 99 of a diluted 1:1 mixture of Syto 9 and Propidium Iodide (LIVE/DEAD Ba- 100 cLight Bacterial Viability Kit stain (Invitrogen, catalog #L7012)) and then 101 imaged. 102 Pseudomonas aeruginosa biofilms (ATCC 15442, https://www.atcc.org/products/all/15442.aspx 97 were grown in the high shear environment in the CDC Biofilm Reactor using 98 ASTM method E2542 [13]. A biofilm sample was then stained with 200µL 99 of a diluted 1:1 mixture of Syto 9 and Propidium Iodide (LIVE/DEAD Ba- 100 cLight Bacterial Viability Kit stain (Invitrogen, catalog #L7012)) and then 101 imaged. 102 Green-fluorescing protein (GFP) Staphylococcus aureus AH2547 were kindly 103 provided by Dr. Alexander Horswill [21]. These GFP S. aureus biofilms were 104 grown in a separate CDC Biofilm Reactor. The growth medium was supple- 105 mented with 10µg/mL of chloramphenicol to retain the GFP plasmid. A 106 biofilm sample was imaged over time using the Treatment Imaging Flow Cell 107 (BioSurface Technologies Corporation, catalog #FC 30). The time series was 108 analyzed by [22]; here we analyze a single image from that series. 109 6 Biofilm formation was also investigated for Antarctic isolate Janthinobac- 110 terium sp. strain CG23_2, a psychrotolerant, aerobic, violacein-pigmented, 111 rod-shaped, Gram-negative, catalase-positive organism [24]. Janthinobac- 112 terium biofilms were grown in the CDC Biofilm Reactor following the same 113 procedure as above [13] with the following modifications. The reactor was in- 114 oculated with a second-generation mid exponential phase bacterial inoculum 115 grown in a minimal R2A medium (BD Difco, purchased from Fisher Scien- 116 tific, catalog number DF186-07-3) at 15 degrees C. Bacteria were grown in 117 full strength R2A media for 48 hours to allow for initial colonization. After 118 48 hours the pump was turned on with a flow rate of 1.67 mL/min, the inflow 119 media was a 1 to 10 dilution of R2A to induce biofilm production. Through- 120 out the reactor run, biofilms were subjected to a constant shear of 80 rpm. 121 Following seven days of growth coupons were removed from the rods and 122 stained with a 1:1 mixture of Syto 9 and Propidium Iodide (LIVE/DEAD 123 BacLight Bacterial Viability Kit stain (Invitrogen, catalog #L7012)). 2.1. Biofilm growth and image acquisition 96 124 The fully intact and hydrated biofilm samples described above were im- 125 aged on an upright Leica TCS-SP5 CSLM using using either a 25X/0.95NA 126 2.5mmWD water objective or a 63X/0.9NA 2.2mmWD water objective. Flu- 127 orophore excitation lasers and emission bandwidths were as follows: Syto9 128 (live) 488 nm excitation, 500–550 nm emission collection; Propidium iodide 129 (dead), 561 nm excitation, 580–700 nm emission collection. 130 Gray-scale (8-bit) images were generated using Imaris [16]. Only the 131 “LIVE” (green) channel was analyzed for each of the 3 bugs. The analysis 132 methodology presented here can be updated for the simultaneous analysis of 133 multiple florescence channels. We investigate this possibility elsewhere. 134 7 7 2.2. Model description 135 g The CSLM images in Figure 1 were collected over different lattice sizes 137 nx × ny × nz, chosen by the microscopist. In our context, a lattice is an 138 ordered array of points describing the arrangement of pixels that form a 3D 139 CSLM image. The x- and y-dimensions of the lattice are associated with the 140 horizontal dimensions parallel to the surface where the biofilm is attached. 141 The z-dimension is associated with the vertical dimension orthogonal to the 142 surface. A CSLM constructs 3D images by a z-stack; that is, by first col- 143 lecting a single nx × ny planar image called a z-slice at each vertical location 144 or distance (z) into the biofilm and then stacking the z-slices together. The 145 number of x and y pixels are usually the same, nx = ny. Each z-slice is 146 generated by a raster laser scanner that causes microbes and/or other mate- 147 rial in the biofilm that have been stained or genetically modified to fluoresce 148 when excited by the laser. 149 At each spatial location corresponding to a pixel in the image, the CSLM 150 records the intensity of the biofilm’s fluorescence as an 8-bit integer (i.e., an 151 integer value between 0 and 255). In our examples, the horizontal (xy) field 152 of view for each planar z-slice is captured by either a 25x or 63x objective 153 lens resulting in either a 620µm × 620µm or 246µm × 246µm field of view 154 respectively. Because microsopists desire high spatial resolutions to achieve 155 appealing visualizations, they typically choose high dimensional pixelations, 156 nx = ny = 1024 (Figure 1A and 1C). 2.1. Biofilm growth and image acquisition 96 However, when generating a movie of 157 images [e.g., see 22], the microscopist may back off on the spatial resolution, 158 say nx = ny = 512 (Figure 1B) to increase the temporal resolution of the time 159 8 8 lapse image frames in the movie. In our examples, the vertical dimension (z) 160 ranges from 100µm to 200µm. The microscopist sets the number (nz) of 161 z-slices over this range depending on the thickness of the biofilm. In the 162 analyses that follow, each z-slice is identified with an integer value between 163 z = 1 (where, ideally, the biofilm is attached to a substratum) and z = nz 164 (the z-slice at the very top of the image). In a similar way each pixel in 165 the horizontal x and y dimensions can also be identified with an integer so 166 that each pixel in the 3D image can be identified by the coordinates (x, y, z) 167 where 1 ≤x ≤nx, 1 ≤y ≤ny and 1 ≤z ≤nz. 168 2.2.2. Beer’s Law 169 2.2.2. Beer’s Law 169 When there is uniform attenuation of light throughout the depth of the 170 biofilm, then Beer’s Law takes the simple form 171 I = I0e−ψcℓ (1) (1) I I0e (1) [26] where 172 [26] where 172 [26] where • I0 is the intensity of light received (from the laser) by a single z-slice 173 in the CSLM 174 • I is the intensity of light transmitted by a single z-slice in the CSLM 175 • ψ is the attenuation cross section of the fluorophore in the z-slice 176 • ψ is the attenuation cross section of the fluorophore in the z-slice 176 • c is the three-dimensional volumetric number density of the fluorophore 177 in the z-slice. This is an important feature of the model for biofilms 178 because it is known that the bacteria and the matrix that glues them to- 179 gether are spatially heterogeneous, and so we expect c to vary through- 180 out the biofilm. 181 9 • ℓis the path length of the beam of light through the z-slice 182 We fit this physical model to the intensities in each 3D image. 183 • ℓis the path length of the beam of light through the z-slice 182 We fit this physical model to the intensities in each 3D image. 2.1. Biofilm growth and image acquisition 96 183 To fit Beer’s Law to data it is crucial to know where the top surface of 185 the biofilm is. A key feature in the modeling process is that an edge detector 186 is applied to the 3D light intensity image data to estimate the location of the 187 surface of the biofilm. The simple edge detection scheme that we use was 188 described previously in [22] and is summarized as follows. First the data is 189 thresholded so that pixels with an intensity value less than some candidate 190 Beer’s threshold θ are set to 0, and other pixels are set to 1. The value θ is 191 called a Beer’s threshold because, unlike conventional thresholding, it is only 192 used to find the biofilm’s top surface. The biofilm’s surface, zsurf is estimated 193 in each column of the image (at each (x, y) location) by applying the edge 194 detector to the thresholded image data, starting at the top (z = nz) and 195 moving toward the bottom of the image (z = 1) until an edge is detected (at 196 z = zsurf). Thresholding is a common feature to finding edges in any kind of 197 image. Any edge detector can be used, but we use a simple computationally 198 inexpensive scheme that identifies the set of z values for which there are two 199 adjacent z-slices with non-zero pixels in the thresholded data. The biofilm’s 200 surface or thickness, zsurf, is then set to the largest value of z in this identified 201 set. At each planar spatial location (x, y), the top surface of the biofilm is 202 denoted by zsurf(x, y\|θ), that emphasizes the dependence on the candidate 203 Beer’s threshold θ. 2.1. Biofilm growth and image acquisition 96 204i Hence, there is a set of candidate biofilm surfaces found by the edge 205 Hence, there is a set of candidate biofilm surfaces found by the edge 205 Hence, there is a set of candidate biofilm surfaces found by the edge 10 A B 0 20 40 60 80 100 120 140 160 180 z-pixel 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 log intensity CSLM intensities noise above biofilm biofilm surface fluorescing biofilm severe attenuation 0 20 40 60 80 100 120 140 160 180 z-pixel 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 log intensity CSLM intensities noise above biofilm biofilm surface fluorescing biofilm severe attenuation Figure 2: Visualization of the quadratic and linear regression model for Beer’s Law fit to a single column from the CSLM image of the P. aeruginosa biofilm in Figure 1A. A: Fit of the model to the column of intensity data (I) at (x = 300, y = 100) when the parabola models a single region of high fluorescence (i.e., β3 < 0). B: Fit of the model to the column of intensity data (I) at (x = 300, y = 200) when the parabola models two regions of high fluorescence (i.e., β3 > 0). A B 0 20 40 60 80 100 120 140 160 180 z-pixel 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 log intensity CSLM intensities noise above biofilm biofilm surface fluorescing biofilm severe attenuation 0 20 40 60 80 100 120 140 160 180 z-pixel 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 log intensity CSLM intensities noise above biofilm biofilm surface fluorescing biofilm severe attenuation Figure 2: Visualization of the quadratic and linear regression model for Beer’s Law Figure 2: Visualization of the quadratic and linear regression model for Beer’s Law fit to a single column from the CSLM image of the P. aeruginosa biofilm in Figure 1A. A: Fit of the model to the column of intensity data (I) at (x = 300, y = 100) when the parabola models a single region of high fluorescence (i.e., β3 < 0). B: Fit of the model to the column of intensity data (I) at (x = 300, y = 200) when the parabola models two regions of high fluorescence (i.e., β3 > 0). 2.1. Biofilm growth and image acquisition 96 detector, one for each candidate threshold θ in the range supplied by the 206 user (for integer valued θ ∈[θmin, θmax]): zsurf\|θ = θmin; zsurf\|θ = (θmin + 1); 207 ...; zsurf\|θ = θmax. 208 detector, one for each candidate threshold θ in the range supplied by the 206 user (for integer valued θ ∈[θmin, θmax]): zsurf\|θ = θmin; zsurf\|θ = (θmin + 1); 207 ...; zsurf\|θ = θmax. 208 detector, one for each candidate threshold θ in the range supplied by the 206 user (for integer valued θ ∈[θmin, θmax]): zsurf\|θ = θmin; zsurf\|θ = (θmin + 1); 207 ...; zsurf\|θ = θmax. 208 Given a candidate Beer’s threshold (θ) and an estimate of where the 209 surface of the biofilm is (zsurf), then a linear regression model is fit to the 210 log-transformed intensity data: 211 ln(I(x, y, z\|, θ)) =    β0(x, y\|θ) + ε(x, y, z\|θ) for z > zsurf(x, y\|θ) f(β(x, y\|θ)) + ε(x, y, z\|θ) for z ≤zsurf(x, y\|θ) .(2) Note that the regression equation is different for pixels above the biof 212 Note that the regression equation is different for pixels above the biofilm 212 Note that the regression equation is different for pixels above the biofilm 212 11 versus pixels below the biofilm’s surface. The pixel at (x, y, z) for which the 213 z-coordinate is z > zsurf(x, y) is above the biofilm; otherwise the pixel is 214 inside the biofilm. 215 The regression equation in (2) specifies a set of regression coefficients for 216 each column in the 3D CSLM image (at location (x, y)) for each value of 217 the threshold θ, so we write β := β(x, y\|θ). We will drop the dependence 218 on (x, y) for brevity in the equations below. The ε(x, y, z\|θ) term is the 219 measurement error of the log-transformed intensities at a (x, y, z) location 220 that is described further in section 2.3.1. The regression function f in (2) 221 describes the intensities generated by the biofilm excited by the laser: 222 f(β1, β2, β3, β4, β5) =    β1 + β2z + β3z2 for I(x, y, z) ≥θ β4 + β5z for I(x, y, z) < θ . (3) (3) The terms in the linear regression model in (2) and (3) are interpreted as 223 follows (cf. 2.1. Biofilm growth and image acquisition 96 (1)): 224 The terms in the linear regression model in (2) and (3) are interpreted as 223 follows (cf. (1)): 224 The terms in the linear regression model in (2) and (3) are interpreted as 223 follows (cf. (1)): 224 • The surface of the biofilm is at zsurf (see the vertical pink line in Figure 225 2); 226 • The surface of the biofilm is at zsurf (see the vertical pink line in Figure 225 2); 226 • β0(x, y) is the noise level of the intensities above the biofilm (see the 227 horizontal black dashed line in Figure 2); 228 • β1(x, y), β2(x, y) and β3(x, y) are the regression parameters for the 229 parabola that models the log intensities of the brightly fluorescing 230 biofilm just below the surface (see the green dashed-dotted parabola in 231 Figure 2) 232 • the parameters β4(x, y) and β5(x, y)) model the exponential decay of 233 • β0(x, y) is the noise level of the intensities above the biofilm (see the 227 horizontal black dashed line in Figure 2); 228 • β1(x, y), β2(x, y) and β3(x, y) are the regression parameters for the 229 parabola that models the log intensities of the brightly fluorescing 230 biofilm just below the surface (see the green dashed-dotted parabola in 231 Figure 2) 232 • the parameters β4(x, y) and β5(x, y)) model the exponential decay of 233 12 the light intensities deep in the biofilm as prescribed by Beer’s Law 234 (see the solid blue line in Figure 2) 235 the light intensities deep in the biofilm as prescribed by Beer’s Law 234 (see the solid blue line in Figure 2) 235 In summary, according to Beer’s Law, the intensities of the 3D image of 236 the biofilm depends on an estimate of the location of the biofilm’s surface; 237 and the location of the biofilm’s surface in turn depends on identifying a 238 Beer’s threshold θ of the data. In contrast to other methodologies, the Beer’s 239 threshold does not indicate that we drop any pixels from consideration in 240 our analysis. 2.1. Biofilm growth and image acquisition 96 A correlated structure 267 may be incorporated into the likelihood, i.e., ε ∼N(0, σ2Ω(γ)), where 268 the parameter(s) γ can be estimated as in spatial statistics. Or, as in 269 [15, 22], one may assume non-correlated errors in the likelihood as we 270 do while the spatial correlation is imposed via an informative (normal) 271 prior on βθ or zsurf, e.g. with a scaled Laplacian as the prior precision 272 matrix. The results presented here using non-correlated errors in the 273 likelihood appear satisfactory. Imposing a correlation structure will 274 require far more computations, as discussed more in the Discussion in 275 section 4. 276 Given a threshold θ, it is well known that the maximum likelihood esti- 277 mator (MLE) for the 6nxny regression parameters is 278 • The data d = ln(I) is a vector of length nxnynz that includes all inten- 256 sities over all (x, y, z) spatial locations. 257 • The data d = ln(I) is a vector of length nxnynz that includes all inten- 256 sities over all (x, y, z) spatial locations. 257 • βθ is a vector of length 6nxny because there are 6 regression coefficients 258 (β0, β1, β2, β3, β4, β5) per column of image data. The θ subscript in βθ 259 is a reminder that a set of 6nxny regression coefficients is found for 260 each θ. 261 • βθ is a vector of length 6nxny because there are 6 regression coefficients 258 (β0, β1, β2, β3, β4, β5) per column of image data. The θ subscript in βθ 259 is a reminder that a set of 6nxny regression coefficients is found for 260 each θ. 261 • Fθ is the model matrix (with dimensions nxnynz ×6nxny ) so that Fθβθ 262 represents the simple model in (2) and (3), conditioned on the Beer’s 263 threshold θ. 264 • ε is the vector (of length nxnynz) of measurement errors, ε ∼N(0, σ2I). 265 That is, the measurement error is assumed to be independent and iden- 266 tically normally distributed with variance σ2. A correlated structure 267 may be incorporated into the likelihood, i.e., ε ∼N(0, σ2Ω(γ)), where 268 the parameter(s) γ can be estimated as in spatial statistics. 2.1. Biofilm growth and image acquisition 96 Instead, the threshold only aids in fitting the correct regression 241 model to all of the intensities in the 3D image by identifying: (1) the top 242 surface of the biofilm via an edge detector; (2) which pixels are “close to the 243 surface” to be modeled as one or two bright parabolic regions (see Figure 2); 244 and (3) which pixels are “deep in the biofilm” to be modeled by exponential 245 decay as predicted by Beer’s Law. 246 2.3.1. Finding optimal values for Beer’s Law parameters for a single Beer’s 248 threshold 249 2.3.1. Finding optimal values for Beer’s Law parameters for a single Beer’s 248 threshold 249 Given CSLM image data, the inverse problem is to estimate the param- 250 eters for the model (i.e., equations (2) and (3)) that describe the data. The 251 parameters of interest are the Beer’s threshold θ and the regression coeffi- 252 cients {βi}5 i=0. To formulate the inverse problem, we consolidate the notation 253 from equations (2) and (3): 254 Given CSLM image data, the inverse problem is to estimate the param- 250 eters for the model (i.e., equations (2) and (3)) that describe the data. The 251 parameters of interest are the Beer’s threshold θ and the regression coeffi- 252 cients {βi}5 i=0. To formulate the inverse problem, we consolidate the notation 253 from equations (2) and (3): 254 d = Fθβθ + ε d = Fθβθ + ε where 255 13 • The data d = ln(I) is a vector of length nxnynz that includes all inten- 256 sities over all (x, y, z) spatial locations. 257 • βθ is a vector of length 6nxny because there are 6 regression coefficients 258 (β0, β1, β2, β3, β4, β5) per column of image data. The θ subscript in βθ 259 is a reminder that a set of 6nxny regression coefficients is found for 260 each θ. 261 • Fθ is the model matrix (with dimensions nxnynz ×6nxny ) so that Fθβθ 262 represents the simple model in (2) and (3), conditioned on the Beer’s 263 threshold θ. 264 • ε is the vector (of length nxnynz) of measurement errors, ε ∼N(0, σ2I). 265 That is, the measurement error is assumed to be independent and iden- 266 tically normally distributed with variance σ2. 2.1. Biofilm growth and image acquisition 96 Or, as in 269 [15, 22], one may assume non-correlated errors in the likelihood as we 270 do while the spatial correlation is imposed via an informative (normal) 271 prior on βθ or zsurf, e.g. with a scaled Laplacian as the prior precision 272 matrix. The results presented here using non-correlated errors in the 273 likelihood appear satisfactory. Imposing a correlation structure will 274 require far more computations, as discussed more in the Discussion in 275 section 4. 276 • ε is the vector (of length nxnynz) of measurement errors, ε ∼N(0, σ2I). 265 That is, the measurement error is assumed to be independent and iden- 266 tically normally distributed with variance σ2. A correlated structure 267 may be incorporated into the likelihood, i.e., ε ∼N(0, σ2Ω(γ)), where 268 the parameter(s) γ can be estimated as in spatial statistics. Or, as in 269 [15, 22], one may assume non-correlated errors in the likelihood as we 270 do while the spatial correlation is imposed via an informative (normal) 271 prior on βθ or zsurf, e.g. with a scaled Laplacian as the prior precision 272 matrix. The results presented here using non-correlated errors in the 273 likelihood appear satisfactory. Imposing a correlation structure will 274 require far more computations, as discussed more in the Discussion in 275 section 4. 276 Given a threshold θ, it is well known that the maximum likelihood esti- 277 mator (MLE) for the 6nxny regression parameters is 278 ˆβMLE\|θ = (F T θ Fθ)−1F T θ d (4) ˆβMLE\|θ = (F T θ Fθ)−1F T θ d (4) ˆβMLE\|θ = (F T θ Fθ)−1F T θ d (4) 14 [e.g., see 17]. The standard deviations SD(ˆβMLE\|θ) are equal to the square 279 root of the diagonal of the variance-covariance matrix 280 [e.g., see 17]. The standard deviations SD(ˆβMLE\|θ) are equal to the square 279 root of the diagonal of the variance-covariance matrix 280 V ar(ˆβMLE\|θ) = MSEθ × (F T θ Fθ)−1; (5) (5) the mean squared error is the mean squared error is MSEθ = 1 nxnynz −6nxny nxnynz ∑ i=1 (di −ˆdi)2; and the predicted log intensities are ˆd = Fθ × ( ˆβMLE\|θ ) (here × refers to 281 matrix-vector multiplication) [17]. The equation for MSE uses the fact that 282 the degrees of freedom for error is DFE = nxnynz −6nxny as is typical for 283 regression. 2.1. Biofilm growth and image acquisition 96 284 The Bayesian MAP estimator for the regression coefficients maximizes the posterior probability distribution (π(β\|θ, d)) of the regression coefficients given a threshold θ and the data [2]. When uniform priors are chosen for the regression coefficients {βi} as we do here, then ˆβMAP\|θ = ˆβMLE\|θ [3, 5, 15]. Moreover, in this case π(β\|θ, d) is a t-distribution with mean 285 equal to ˆβMAP\|θ and variance given by (5) (see section 2.3.3). Using these 286 results, uncertainty quantification of the regression coefficients, including how 287 to construct probability intervals, is provided in section 2.3.3. 288 2.3.2. Finding the optimal Beer’s threshold 289 We use a brute force technique to find the Beer’s threshold ˆθMAP, over the range of possible integer threshold values supplied by the user, that max- imizes the Bayesian marginal posterior distribution, ˆθMAP = argmaxθ∈[θmin,θmax]π(θ\|d) ˆθMAP = argmaxθ∈[θmin,θmax]π(θ\|d) 15 (see section 2.3.3 for the definition of π(θ\|d)). 290 The MLE for the Beer’s threshold minimizes the mean squared error, The MLE for the Beer’s threshold minimizes the mean squared error, ˆθMLE = argminθ∈[θmin,θmax]MSEθ. ˆθMLE = argminθ∈[θmin,θmax]MSEθ. We use a brute force approach to find the MLE by first finding the MLE for 291 the regression parameters via (4) for each integer threshold value in the user 292 provided range, and then calculating MSEθ. 293 We use a brute force approach to find the MLE by first finding the MLE for 291 the regression parameters via (4) for each integer threshold value in the user 292 provided range, and then calculating MSEθ. 293 The optimal MAP ˆθMAP and MLE ˆθMLE for the Beer’s threshold are 294 each associated with a biofilm surface (section 2.2.3). The biofilm surface 295 zsurf\|θ = ˆθMAP maximizes the probability of the threshold given the model 296 for Beer’s Law and the data. The biofilm surface zsurf\|θ = ˆθMLE minimizes 297 the mean squared error of the fit of Beer’s Law to the data. The optimal 298 MAP and MLE threshold and surfaces may be different as we investigate in 299 the examples. 300 2.3.3. Uncertainty Quantification of threshold and regression parameters 301 2.3.3. Uncertainty Quantification of threshold and regression parameters 301 Uncertainties are quantified for the parameters (θ and β) by the Bayesian 302 posterior probability distribution. One advantage of the Bayesian approach 303 is that it allows any prior information that the researcher has available about 304 the parameters to be incorporated into the model by probability statements 305 (via a probability distribution π(·)). The prior probability distributions for 306 the parameters in (2) and (3) are: 307 2.3.3. Uncertainty Quantification of threshold and regression parameters 301 Uncertainties are quantified for the parameters (θ and β) by the Bayesian 302 posterior probability distribution. One advantage of the Bayesian approach 303 is that it allows any prior information that the researcher has available about 304 the parameters to be incorporated into the model by probability statements 305 (via a probability distribution π(·)). 2.3.2. Finding the optimal Beer’s threshold 289 The prior probability distributions for 306 the parameters in (2) and (3) are: 307 π(β0, β1, β2, β3, β4, β5) = UNIF(−∞, ∞) (6) π(θ) = UNIF(all integers from θmin to θmax) (7) π(σ2) ∝ 1/σ2. (8) π(β0, β1, β2, β3, β4, β5) = UNIF(−∞, ∞) (6) π(θ) = UNIF(all integers from θmin to θmax) (7) π(σ2) ∝ 1/σ2. (8) π(β0, β1, β2, β3, β4, β5) = UNIF(−∞, ∞) (6) (6) (8) 16 The prior for the Beer’s Law parameters (βi) is given by a flat uniform distri- 308 bution over the real numbers in (6). Such a flat uniform prior is sometimes 309 described as “non-informative” because it does not prefer any values for the 310 βi over any other. If we wanted to constrain some parameter βi to be pos- 311 itive, then we might impose a prior π(βi) = UNIF(0, ∞). Or if there was 312 some preferred value for βi, say β∗ i , then we might impose a normal prior 313 centered at β∗ i , that is, π(βi) = N(β∗ i , σ2 β). 314 The prior for the Beer’s Law parameters (βi) is given by a flat uniform distri- 308 bution over the real numbers in (6). Such a flat uniform prior is sometimes 309 described as “non-informative” because it does not prefer any values for the 310 βi over any other. If we wanted to constrain some parameter βi to be pos- 311 itive, then we might impose a prior π(βi) = UNIF(0, ∞). Or if there was 312 some preferred value for βi, say β∗ i , then we might impose a normal prior 313 centered at β∗ i , that is, π(βi) = N(β∗ i , σ2 β). 314 The prior for the Beer’s Law parameters (βi) is given by a flat uniform distri- 308 bution over the real numbers in (6). Such a flat uniform prior is sometimes 309 described as “non-informative” because it does not prefer any values for the 310 βi over any other. If we wanted to constrain some parameter βi to be pos- 311 itive, then we might impose a prior π(βi) = UNIF(0, ∞). Or if there was 312 some preferred value for βi, say β∗ i , then we might impose a normal prior 313 centered at β∗ i , that is, π(βi) = N(β∗ i , σ2 β). 2.3.2. Finding the optimal Beer’s threshold 289 Equation (11) shows that π(β\|d) 336 is a mixture of t distributions with mixture weights π(θi\|d). 337 Uncertainty quantification for θ and β, via probability statements and 338 interval estimates, can be constructed using (10) and (11) respectively. An 339 immediate consequence is that probability intervals for {βi} cannot be con- 340 structed using the standard t(DFE) distribution unless π(θ\|d) has most of 341 its probability mass at ˆθMAP as in the examples we present here (see Figure 342 4). 343 2.3.2. Finding the optimal Beer’s threshold 289 314 The prior for the Beer’s threshold in (7) is informed by the range, [θmin, θmax], 315 provided by the user. The uniform prior over the integers in this range en- 316 forces the constraint that the Beer’s threshold is constrained to be between 317 θmin and θmax but with no preference for any of the values in this range over 318 any other. 319 The prior specification (8) for the variance of the measurement error 320 prefers smaller values. 321 The prior specification (8) for the variance of the measurement error 320 prefers smaller values. 321 The prior specification (8) for the variance of the measurement error 320 prefers smaller values. 321 The results in the rest this section presume the use of the priors (6)-(8). 322 Using non-uniform priors for either θ or β would encapsulate more specific 323 knowledge of the value of the threshold or of the regression parameters de- 324 scribing Beer’s Law and could, for example, result in ˆβMAP\|θ ̸= ˆβMLE\|θ (c.f. 325 section 2.3.1) and change the form of the posterior distributions in equations 326 (9) and (10) below (see section 5). 327 The posterior probability distribution of interest that incorporates the 328 information from the prior distributions with the likelihood of the data is: 329 π(β, θ\|d) ∝ π(β\|θ, d) × π(θ\|d) ∝ t(ˆβMLE\|θ, V ar(ˆβMLE\|θ), df = DFE) × π(θ\|d) (9) ∝ t(ˆβMLE\|θ, V ar(ˆβMLE\|θ), df = DFE) × π(θ\|d) (9) ∝ t(ˆβMLE\|θ, V ar(ˆβMLE\|θ), df = DFE) × π(θ\|d) (9) (9) 17 17 where 330 π(θ\|d) = c √ (MSEθ/2)nxny(6−nz) det(F T θ Fθ) (10) π(θ\|d) = c √ (MSEθ/2)nxny(6−nz) det(F T θ Fθ) (10) (10) and c is a normalization constant (see section 5 and Figure 4). 331 A very convenient consequence of (9) is that the marginal posterior for 332 the regression coefficients, that encapsulates uncertainty in the thresholding 333 value is 334 π(β\|d) = ∑ i t(ˆβMLE\|θi, V ar(ˆβMLE\|θi)) × π(θi\|d) (11) (11) where the mean and covariance of each multivariate t is calculated via (4) 335 and (5), and π(θi\|d) is calculated via (10). Equation (11) shows that π(β\|d) 336 is a mixture of t distributions with mixture weights π(θi\|d). 337 where the mean and covariance of each multivariate t is calculated via (4) 335 and (5), and π(θi\|d) is calculated via (10). 2.3.4. Optimally Estimating Bio-volumes 344 For a fixed threshold, the volume V is estimated by integrating under the biofilm’s surface for a fixed threshold, ˆVθ = ∑ x,y zsurf(x, y\|θ)∆xyz ˆVθ = ∑ x,y zsurf(x, y\|θ)∆xyz where ∆xyz gives the voxel (“3D pixel”) size in cubic microns (µm3). Over 345 all thresholds, the biofilm’s volume can be estimated from the mean and 346 18 standard deviation (SD) of the marginal posterior for the volume (see section 347 5) 348 standard deviation (SD) of the marginal posterior for the volume (see section 347 5) 348 Mean(V \|d) = ∑ i ˆVθi × π(θi\|d); SD(V \|d) = √∑ i (nxnyˆσ2 surf\|θi) × π(θi\|d)2 where π(θi\|d) is calculated via (10); and ˆσ2 surf\|θi is calculated via (14) in the 349 Appendix. 350 where π(θi\|d) is calculated via (10); and ˆσ2 surf\|θi is calculated via (14) in the 349 Appendix. 350 3. Results 351 3.1. Image Analysis 352 3.1. Image Analysis 352 We use our optimal Bayesian (MAP) and likelihood (MLE) approaches 353 to analyze the biofilm images in Figure 1. 354 Figure 2 illustrates the fit of Beer’s Law to two different columns of the 355 P. aeruginosa image data (see the description of the figure in the context 356 of the model in the text after equation (3)). This plot makes clear that 357 the model is fit to the intensity data for every pixel in the image; no data 358 were excluded prior to the model fit. Figure 3A shows the surface of the P. 359 aeruginosa biofilm over all of the 1024 columns in a single x-slice and Figure 360 3B shows the biofilm’s surface over the entire 620µm × 620µm field of view. 361 Figure 4A shows the marginal posterior (in (10)) for the Beer’s threshold 362 parameter (maximized by ˆθMAP = 7) alongside the likelihood (maximized by 363 ˆθMLE = 7). In this example, the MAP and MLE are the same. Because the 364 log-transformed posterior at the MAP is orders of magnitude larger than at 365 other values, the Beer’s threshold value is 7 with probability close to 1. 366 19 A B 100 200 300 400 500 600 700 800 900 1000 y-pixel 20 40 60 80 100 120 140 160 z-pixel 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 Figure 3: Visualizations of the surface of the 192µm thick biofilm of P. aeruginosa from Figure 1A based on the optimal fit of Beer’s Law to the intensity data. A: An estimate of the biofilm’s surface in a single x-slice (x = 300). Pixels are colored according to the value of the log-intensity. B: An estimate of the surface over the entire field of view. The vertical dimension is colored according to the biofilm’s thickness. A 100 200 300 400 500 600 700 800 900 1000 y-pixel 20 40 60 80 100 120 140 160 z-pixel 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 B A B Figure 3: Visualizations of the surface of the 192µm thick biofilm of P. aeruginosa from Figure 1A based on the optimal fit of Beer’s Law to the intensity data. A: An estimate of the biofilm’s surface in a single x-slice (x = 300). Pixels are colored according to the value of the log-intensity. 3. Results 351 B: An estimate of the surface over the entire field of view. The vertical dimension is colored according to the biofilm’s thickness. 20 A B 4 6 8 10 12 14 θ=threshold -0.3 -0.25 -0.2 -0.15 -0.1 -0.05 0 log probability 1/c, c > 108 π(θ \| d) π(d \| θ,β, σ2) 4 6 8 10 12 14 16 18 20 θ=threshold -0.5 -0.45 -0.4 -0.35 -0.3 -0.25 -0.2 -0.15 -0.1 -0.05 0 0.05 log probability 1/c, c > 106 π(θ \| d) π(d \| θ,β, σ2) Figure 4: The log marginal posterior, π(θ\|d), for the Beer’s threshold (θ) compared to the likelihood π(d\|θ, β, σ2) given CSLM data (d). A. For P. aeruginosa, the MLE and the MAP are clearly the same at θ = 7. B. For S. aureus, the MLE is at θ = 6, whereas the MAP is at θ = 8. A B A 4 6 8 10 12 14 θ=threshold -0.3 -0.25 -0.2 -0.15 -0.1 -0.05 0 log probability 1/c, c > 108 π(θ \| d) π(d \| θ,β, σ2) B 4 6 8 10 12 14 16 18 20 θ=threshold -0.5 -0.45 -0.4 -0.35 -0.3 -0.25 -0.2 -0.15 -0.1 -0.05 0 0.05 log probability 1/c, c > 106 π(θ \| d) π(d \| θ,β, σ2) Figure 4: The log marginal posterior, π(θ\|d), for the Beer’s threshold (θ) compared to the likelihood π(d\|θ, β, σ2) given CSLM data (d). A. For P. aeruginosa, the MLE and the MAP are clearly the same at θ = 7. B. For S. aureus, the MLE is at θ = 6, whereas the MAP is at θ = 8. Figure 4: The log marginal posterior, π(θ\|d), for the Beer’s threshold (θ) compared to the likelihood π(d\|θ, β, σ2) given CSLM data (d). A. For P. aeruginosa, the MLE and the MAP are clearly the same at θ = 7. B. For S. aureus, the MLE is at θ = 6, whereas the MAP is at θ = 8. Figure 4B shows different results for the MAP and the MLE for the Beer’s 367 threshold for the S. aureus image data. With probability close to 1, the Beer’s 368 threshold is equal to the MAP at ˆθMAP = 8, whereas with probability close 369 to 0 it is equal to the MLE at ˆθMLE = 6. The associated MLE and MAP 370 estimates for the surface of the S. Figure 6 shows results when our Bayesian image analysis approach is ap- 378 3. Results 351 aureus biofilm are presented in Figure 371 5. The differences in the two surface estimates are subtle. Upon closer 372 inspection (compare the black rectangular region in both panes), the MAP 373 estimate in Figure 5B illustrates a smoother fit to the data whereas the MLE 374 surface in Figure 5A is more noisy (i.e., it does not fit the data as well). 375 Inspection of residual plots (similar to Figure 3A, not shown) confirm this 376 conclusion. 377 Figure 6 shows results when our Bayesian image analysis approach is ap- 378 21 A B Figure 5: Visualizations of two different surface estimates of the 112µm thick biofilm of Staph. aureus based on two optimal fits of Beer’s Law to the intensity data. The vertical dimension is colored according to the biofilm’s thickness. The black rectangle in each pane highlights a region of the biofilm’s surface where the MLE is much noisier than the MAP. A: The biofilm’s surface estimated via the MLE at ˆθMLE = 6. B: The surface estimated via the Bayesian MAP for the Beer’s threshold at ˆθMAP = 8. A B A B Figure 5: Visualizations of two different surface estimates of the 112µm thick biofilm of Staph. aureus based on two optimal fits of Beer’s Law to the intensity data. The vertical dimension is colored according to the biofilm’s thickness. The black rectangle in each pane highlights a region of the biofilm’s surface where the MLE is much noisier than the MAP. A: The biofilm’s surface estimated via the MLE at ˆθMLE = 6. B: The surface estimated via the Bayesian MAP for the Beer’s threshold at ˆθMAP = 8. 22 A B 100 200 300 400 500 600 700 800 900 1000 y-pixel 50 100 150 200 250 z-pixel 0 1 2 3 4 5 Figure 6: Visualizations of the surface of the 141µm thick biofilm of Janthinobac- terium from Figure 1C based on the optimal fit of Beer’s Law to the intensity data. A: The estimate of the biofilm’s surface in an x slice (x = 325). Pixels are colored according the value of the log-intensity. B: The surface of the biofilm using the Bayesian MAP for the Beer’s threshold, ˆθMAP = 26. The vertical dimension is colored according to the biofilm’s thickness. 3. Results 351 B A 100 200 300 400 500 600 700 800 900 1000 y-pixel 50 100 150 200 250 z-pixel 0 1 2 3 4 5 A B 00 500 60 y-pixel y-pixel Figure 6: Visualizations of the surface of the 141µm thick biofilm of Janthinobac- terium from Figure 1C based on the optimal fit of Beer’s Law to the intensity data. A: The estimate of the biofilm’s surface in an x slice (x = 325). Pixels are colored according the value of the log-intensity. B: The surface of the biofilm using the Bayesian MAP for the Beer’s threshold, ˆθMAP = 26. The vertical dimension is colored according to the biofilm’s thickness. 23 plied to CSLM data collected from an environmental isolate from the Antarc- 379 tic, Janthinobacterium. The x-slice in Figure 6A shows a biofilm architecture 380 that is much different than the stained P. aeruginosa biofilm or the GFP S. 381 aureus biofilm. Still, the x-slice suggests that our approach provides a rea- 382 sonable estimate of the biofilm’s surface. Figure 6B shows an estimate of 383 the surface over all of the x-slices. The MLE is ˆθMLE = 24 and the MAP 384 is ˆθMAP = 26 (the posterior and likelihood curves similar to Figure 4 not 385 shown). However, unlike the differing results for S. aureus (Figure 5), there 386 is little qualitative difference in the MLE and MAP estimates of the surface 387 for this example. 388 3.2. Assessing model fit 389 3.2. Assessing model fit 389 Figures 2, 3A and 6A function like residual plots that can be generated 390 after any regression analysis. These plots allow the data analyst to assess 391 the fit of the model to the data. Unfortunately the other image analyses 392 discussed here (COMSTAT in MATLAB or ImageJ, Imaris and daime) do 393 not provide residual diagnostics. 394 For example, there are two artifacts in the estimated surfaces for P. aerug- 395 inosa that would be missed without assessing the fit of the model to the data. 396 First, sometimes the technician may inadvertently collect image data into the 397 substratum where the biofilm is attached, as occurs for the bottom-most z- 398 slices in Figure 3A. This can easily be remedied by excluding these z-slices 399 from the analysis. Second, there are some surface model misfits, identified in 400 Figure 3A by the high frequency in the black curve at the substratum where 401 there is little biofilm. One approach to dealing with such misfit is to smooth 402 the estimate of the biofilm’s surface. This possibility is addressed further in 403 24 section 4. 404 section 4. 404 3.3. Comparisons with Imaris, COMSTAT2 and daime 405 In addition to the Bayesian and MLE approaches that we present, the 406 analysis of each of the three biofilm images in Figure 1 was attempted using 407 three other methods: Imaris, COMSTAT2 (in ImageJ) [27], and daime [8]. 408 Table 1 compares the thresholds and volumes estimated by the different 409 methods. 410 In addition to the Bayesian and MLE approaches that we present, the 406 analysis of each of the three biofilm images in Figure 1 was attempted using 407 three other methods: Imaris, COMSTAT2 (in ImageJ) [27], and daime [8]. 408 Table 1 compares the thresholds and volumes estimated by the different 409 methods. 410 Unfortunately, neither COMSTAT2 nor daime were able to analyze all 3 411 images. Only the P. aeruginosa image was able to be analyzed with COM- 412 STAT2, as the file sizes of the other two images were too large for the program 413 to handle. Even though the pixelation was less for the Staph data, the image 414 included here is part of a larger file containing a movie of the biofilm over 415 time [22]. 3.2. Assessing model fit 389 Only the Staph GFP image could be analyzed with the daime 416 software, as the other two images contained a z-stack that was too thick for 417 the program to handle. Our Bayesian and likelihood approaches were able to 418 process all 3 images because, by design, they are computationally efficient. 419 For example, it took 40s to consider a single threshold for the Pseudomonas 420 data, about 1s to consider a single threshold for Staph, and 2m 45s to consider 421 a single threshold for Janth using a machine with an Intel (R) Core i7-6900K 422 CPU at 3.2 GHz. Therefore, our approach, Imaris and COMSTAT2 (in Im- 423 ageJ) can only be compared for the P. aeruginosa data; and our approach, 424 Imaris and daime can only be compared for the Staph data. 425 Based on the information provided in Table 1, the COMSTAT2 output 426 for the volume of the P. aeruginosa biofilm is similar to the Bayesian MAP 427 (although the thresholds are different). This is because COMSTAT2 cal- 428 Based on the information provided in Table 1, the COMSTAT2 output 426 for the volume of the P. aeruginosa biofilm is similar to the Bayesian MAP 427 (although the thresholds are different). This is because COMSTAT2 cal- 428 25 Biofilm Image from Figure 1 Method Software Characteristic P. aeruginosa S. aureus Janth. Beer’s MAP MATLAB Threshold 7 8 26 Bio-volume 2.55 × 107 1.21 × 107 6.51 × 106 Beer’s MLE MATLAB Threshold 7 6 24 Bio-volume 2.55 × 107 1.38 × 107 6.55 × 106 Costes’ Imaris Threshold 23 58 50 Bio-volume 2.8 × 106 3.6 × 106 5.4 × 105 Otsu’s COMSTAT2 Threshold 3 NC NC Bio-olume 2.32 × 107 NC NC RATS-L daime Threshold NC NA NC Bio-volume NC 6.66 × 105 NC Table 1: Comparison of image analysis results for 5 different approaches applied to P. aeruginosa, S. aureus, and Janthinobacterium biofilms. Reported thresholds are intensity values ranging from 0 to 255; Biofilm bio-volumes reported as µm3. Approaches presented herein are listed as Beer’s MAP and Beer’s MLE. COMSTAT2 was implemented in ImageJ. NA indicates that daime did not provide the thresholding value. NC indicates quantities that could not be calculated due to restrictions on the file type and/or file size. Table 1: Comparison of image analysis results for 5 different approaches applied to P. aeruginosa, S. aureus, and Janthinobacterium biofilms. 3.2. Assessing model fit 389 Reported thresholds are intensity values ranging from 0 to 255; Biofilm bio-volumes reported as µm3. Approaches presented herein are listed as Beer’s MAP and Beer’s MLE. COMSTAT2 was implemented in ImageJ. NA indicates that daime did not provide the thresholding value. NC indicates quantities that could not be calculated due to restrictions on the file type and/or file size. 26 culates a “connected volume” which, similar to our approach, includes all 429 pixels under the top of the biofilm in the volume calculation. The Imaris 430 and daime analyses underestimate the volume compared to our Bayesian 431 and MLE approaches. This is not surprising since Imaris [16, p. 235-8] and 432 daime [8] calculate the volume by considering only bright pixels (above the 433 image threshold). Unfortunately, it is challenging to determine specifically 434 what the other software programs are doing in the absence of diagnostics of 435 model fit as in Figures 2, 3 and 6. 436 A last crucial point is that the Bayesian MAP is the only approach to 437 provide uncertainty quantification for the threshold. The MAP estimate 438 for the Beer’s threshold for each microbial species is preferred over the other 439 possible threshold values with probability close to 1 (Figure 4). Furthermore, 440 the MAP and MLE approaches are the only two that provide uncertainty 441 quantification for the bio-volume for each of the 3 species. The Bayesian 442 MAP for the bio-volumes in Table 1 are associated with SD(V \|d) of 1.64 × 443 104, 2.35 × 104 and 1.61 × 103 (see section 2.3.4). The MLE bio-volumes 444 in Table 1 had SDs of 1.64 × 104, 2.42 × 104 and 1.57 × 103. From these, 445 because most of the probability mass for π(θ\|d) is at ˆθMAP (Figure 4), then 446 probability or confidence intervals can be calculated using the t(df = nxny − 447 1) distribution by ˆV ± tdfSD. 448 4. Discussion 449 We analyze spatial intensity data generated by CSLM. This technology 450 is advancing. In addition to the intensity data that we focus on here, state- 451 of-the-art Raman CSLMs can also provide high dimensional spectroscopy 452 27 information for chemical species at every point in the high dimensional spatial 453 lattice. Our regression apporach is flexible enough to allow the user to add 454 covariates and/or factors to the regression equations to investigate spatial 455 relationships between the biofilm species and chemical species. Instead of 456 regression models, one could use ODE or PDE models of these relationships 457 (e.g., to model reaction and diffusion of substrates into the biofilm), but at 458 an increase in computational expense. 459 Previous Bayesian statistical analyses of CSLM images of thin layers of 460 human cells considered less severe attenuation effects [1]. [26] looked at much 461 thicker specimens of starfish oocytes and empirically assessed the attenuation 462 effects due to Beer’s Law using different CSLM objectives lenses (e.g., 25x 463 and 63x) and media (e.g., oil or water). [22] used an edge detector as we do 464 here to find a Bayesian estimate of the surface of the biofilm, but they used 465 a fixed threshold. We expand upon those results by considering a range of 466 user-supplied thresholds and selecting the one that optimizes the fit of Beer’s 467 Law to the data. 468 Our parabolic regression model for Beer’s Law explicitly accounts for one 469 or two bright regions just below the biofilm’s surface (Figure 2). It is not 470 clear what is occurring in these highly fluorescing regions of the image, but 471 three plausible yet speculative explanations are: higher cell densities; higher 472 levels of cell activity (especially when imaging GFP organisms); or a spatial 473 architecture of the biofilm that allows the fluorescent signal to be readily 474 collected. 475 The use of the edge detector is crucial. When developing our approach 476 we attempted fitting quadratic and quartic polynomial regression models 477 28 directly to each column of the data (i.e., without identification of the surface). 478 Unfortunately, these approaches were adversely influenced by either the noisy 479 low intensity pixels above the biofilm or the attenuating low intensity pixels 480 in the depth of the biofilm. 4. Discussion 449 Our approach minimizes this limitation by using 481 an edge detector to find the biofilm’s top surface then fitting a regression 482 model that depends on the surface. Furthermore, our approach provides the 483 optimal Beer’s threshold that gives the “best” fit model. The “best” Beer’s 484 thresholds we report are the Bayesian maximum of the posterior (MAP) and 485 the maximum likelihood estimate (MLE). In our Staph example, the MAP 486 appeared to better fit the data, whereas for the Janth example there was 487 little difference. In all of the examples, the MAP for the Beer’s threshold 488 was preferred with probability close to 1 over any other threshold value. 489 In contrast to the other automatic threshold methods that we discuss, the 490 threshold we find is based on the optimal fit of Beer’s Law to the data, not 491 on a heuristic that requires tweaking the threshold until some biofilm char- 492 acteristic like bio-volume “stabilizes.” Furthermore, our approach is compu- 493 tationally efficient, taking between 1 second and 3 minutes to assess a single 494 threshold on a single image in the examples presented, whereas some of the 495 other methods presented could not process the large CSLM image data sets 496 at all (Table 1). 497 A major benefit of our approach is that we are able to assess the fit of 498 the model using diagnostic plots, analogous to checking residuals for any 499 regression analysis [17]. Because such assessments are not available from 500 other software packages, it is not clear to the user what other packages are 501 doing, or how to assess what they are doing. 502 29 One approach to dealing with data that have detrimental influence on the 503 fit of the regression model (e.g., identified by Figure 3) is to smooth the sur- 504 face. Smoothing is a consequence of imposing a spatial correlation structure 505 on the surface or on the intensities [15]. While implementing smoothing is 506 easy (e.g., by convolution), we recommend: (1) Using a Bayesian statistical 507 process to choose the amount of smoothing (i.e., depending on posterior prob- 508 abilities) as opposed to a data analyst choosing; (2) generating some measure 509 of uncertainty (e.g., probability intervals) for the smoothing parameter; and 510 (3) incorporating the uncertainty in the smoothing into the uncertainty for 511 all downstream quantities of interest. Data Availability 534 The data and MATLAB computer code are available on request from the 535 authors. 536 The data and MATLAB computer code are available on request from the 535 authors. 536 5. Appendix Acknowledgements 530 We thank the Industrial Associates of the CBE for partially support- 531 ing this work. JAC is partially funded by CONACYT CB-2016-01-284451, 532 RDECOMM and ONRG grants. 533 4. Discussion 449 Not surprisingly, implementing these 512 steps is computationally expensive. Because of the expense, unfortunately, 513 it is common to let a data analyst choose the level of smoothing and to ig- 514 nore the uncertainty in the smoothing term in biofilm CSLM image analysis 515 [e.g., see 18]. Our approach is flexible enough to allow these steps to be 516 implemented although at increased computational cost [22]. We investigate 517 simultaneous threshold estimation with computationally efficient approaches 518 to choosing the smoothing parameter (e.g., following [9] or [10]) elsewhere. 519 One drawback of our approach is that it does not model holes or overhang- 520 ing features within the biofilm. That is, our computationally efficient model 521 may be too simplistic for biofilms that have many holes and/or overhang- 522 ing features. Independent analyses (i.e., cryosectioning, optical coherence 523 tomography or inverted CSLM) suggest that in many cases, biofilms contain 524 viable bacteria all the way through [22]. The diagnostic graphical tools dis- 525 played in Figures 3, 5 and 6 allow the user to assess the appropriateness of 526 the model presented here We are currently developing a more computation One drawback of our approach is that it does not model holes or overhang- 520 ing features within the biofilm. That is, our computationally efficient model 521 may be too simplistic for biofilms that have many holes and/or overhang- 522 ing features. Independent analyses (i.e., cryosectioning, optical coherence 523 tomography or inverted CSLM) suggest that in many cases, biofilms contain 524 viable bacteria all the way through [22]. The diagnostic graphical tools dis- 525 played in Figures 3, 5 and 6 allow the user to assess the appropriateness of 526 the model presented here. We are currently developing a more computation- 527 30 ally expensive technique that implements a more complex statistical model 528 that can deal with these types of features. 529 ally expensive technique that implements a more complex statistical model 528 that can deal with these types of features. 529 5. Appendix 537 By Bayes Rule, the full posterior probability distribution for all of the parameters in our problem is π(β, σ2, θ\|d) = π(d\|β, σ2, θ)π(β\|σ2, θ)π(σ2\|θ)π(θ) π(d) . The first three factors in the numerator on the right hand side are propor- 538 tional to π(β, σ2\|θ, d): 539 The first three factors in the numerator on the right hand side are propor- 538 tional to π(β, σ2\|θ, d): 539 π(β, σ2\|θ, d) = π(d\|β, σ2, θ)π(β\|σ2, θ)π(σ2\|θ) π(d\|θ) . (12) (12) The specification for the model d = Fθβθ + ε in section 2.3.1 implies that the likelihood π(d\|β, σ2, θ) is normal. The specification of the priors in section The specification for the model d = Fθβθ + ε in section 2.3.1 implies that the likelihood π(d\|β, σ2, θ) is normal. The specification of the priors in section 31 2.3.3 implies that π(β, σ2\|θ, d) is a normal-gamma distribution [3, 15]. It follows that integrating out σ2 from (12) gives the multivariate t, 2.3.3 implies that π(β, σ2\|θ, d) is a normal-gamma distribution [3, 15]. It follows that integrating out σ2 from (12) gives the multivariate t, π(β\|θ, d) = t(ˆβMLE\|θ, V ar(ˆβMLE\|θ)) π(β\|θ, d) = t(ˆβMLE\|θ, V ar(ˆβMLE\|θ)) [12, 3]. This substantiates the claim that the first factor in equation (9) is a 540 multivariate t. We derive the second factor π(θ\|d) in (9) and (10) next. 541 Rearranging equation (12) we get that [12, 3]. This substantiates the claim that the first factor in equation (9) is a 540 multivariate t. We derive the second factor π(θ\|d) in (9) and (10) next. 541 Rearranging equation (12) we get that Rearranging equation (12) we get that π(d\|θ) = π(d\|β, σ2, θ)π(β\|σ2, θ)π(σ2\|θ) π(β, σ2\|θ, d) π(d\|θ) = (normal likelihood) × priors (normalgamma) . After cancellation of like terms [3] we get: 542 After cancellation of like terms [3] we get: 542 π(d\|θ) ∝ ( (dTd −ˆβT MLEF T θ Fθ ˆβMLE)/2 )−DFE/2 det(F T θ Fθ)1/2 where DFE = nxnynz −6nxny. Similar calculations to derive π(d\|θ) were 543 performed by [10] although they assumed normal instead of uniform priors 544 on the {βi}. If we let ˆd = Fθ ˆβMLE, then the numerator can be simplified by 545 noting that DFE · MSEθ = (d −ˆd)T(d −ˆd) = dTd −2dT ˆd + ˆdT ˆd. 5. Appendix 537 Because 546 dT ˆd = √ dTd · √ ˆdT ˆd cos(∠(d, ˆd)) and cos(∠(d, ˆd)) = √ ˆdT ˆd/ √ dTd it follows 547 that DFE · MSEθ = dTd −ˆdT ˆd. So 548 π(d\|θ) ∝ √ (DFE · MSEθ/2)−DFE det(F T θ Fθ) . (13) (13) 32 The determinant calculation det(F T θ Fθ) needed to evaluate (13) is straight- 549 forward even for our high dimensional problem because Fθ is composed of 550 nxny blocks (one for each column), each of size nz × 6. Hence det(F T θ Fθ) is 551 the product of the determinant of nxny blocks, each of size 6 × 6. 552 By Bayes Rule, the marginal posterior probability distribution for the Beer’s threshold is (d\|θ) (θ) π(θ\|d) = π(d\|θ)π(θ) ∑ θ′ π(d\|θ′)π(θ′) where π(d\|θ) is calculated via (13). This equation shows how to incorporate prior information for θ for any prior π(θ). Under the assumption of a flat prior over the possible integer values for θ (as in section 2.3.3), then π(θ\|d) = π(d\|θ) ∑ θ′ π(d\|θ′) where π(d\|θ) is calculated via (13). This substantiates equation (10). 553 To estimate bio-volume (V ) based on the surface, we consider the follow- ing model of the biofilm’s surface To estimate bio-volume (V ) based on the surface, we consider the follow- ing model of the biofilm’s surface zsurf(x, y\|θ) = τ(x, y\|θ) + ϵ where τ is the true thickness of the biofilm and ϵ ∼N(0, σ2 surfI). Because zsurf depends on the Beer’s threshold θ (through the edge detector and the regression equations (2) and (3) describing Beer’s Law), then, similar to the calculations leading to (11), π(V \|d) = ∑ i t( ˆVMAP\|θi, nxnyˆσ2 surf\|θi, df = nxny −1)) × π(θi\|d) where the bio-volume for a single Beer’s threshold θi is estimated by ˆVMAP\|θi = 554 ∑ x,y zsurf(x, y\|θi)∆xyz; 555 ∑ x,y zsurf(x, y\|θi)∆xyz; 555 ˆσ2 surf\|θi = (zsurf − ˆVMAP\|θi nxny )T(zsurf − ˆVMAP\|θi nxny )/(nxny −1); (14) (14) 33 and π(θi\|d) is calculated by (10). This substantiates the equations for Mean(V \|d) 556 and SD(V \|d) in section 2.3.4. 557 and π(θi\|d) is calculated by (10). This substantiates the equations for Mean(V \|d) 556 and SD(V \|d) in section 2.3.4. 557 References 558 [1] Al-Awadhi, F., Hurn, M., and Jennison, C. (2011). Three-dimensional 559 Bayesian analysis and confocal microscopy. Journal of Applied Statistics, 560 38(1):29–46. 561 [2] Bernardo, J. and Smith, A. (1994). Bayesian Theory. 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https://openalex.org/W1991417373	https://bmcpublichealth.biomedcentral.com/counter/pdf/10.1186/1471-2458-12-1051	English	null	Regulation of antibiotic sales in Mexico: an analysis of printed media coverage and stakeholder participation	BMC public health	2,012	cc-by	9,071	CORRESPONDENCE Open Access * Correspondence: veronika.wirtz@insp.mx Center for Health Systems Research, National Institute of Public Health, Av. Universidad 655, Cuernavaca, Mor C.P. 62100, Mexico © 2012 Dreser 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. Regulation of antibiotic sales in Mexico: an analysis of printed media coverage and stakeholder participation Anahí Dreser, Edna Vázquez-Vélez, Sandra Treviño and Veronika J Wirtz* Anahí Dreser, Edna Vázquez-Vélez, Sandra Treviño and Veronika J Wirtz* Dreser et al. BMC Public Health 2012, 12:1051 http://www.biomedcentral.com/1471-2458/12/1051 Dreser et al. BMC Public Health 2012, 12:1051 http://www.biomedcentral.com/1471-2458/12/1051 Open Access Abstract Background: Restricting antibiotics sales to those with medical prescriptions only is a central strategy for promoting appropriate use and containing antibiotic resistance; however, many low and middle income countries have not enforced policies that prevent widespread self-medication with antibiotics. In 2010, the Mexican government announced the enforcement of antibiotic sales regulations, a policy that gained media prominence. This study analyzes media coverage of issues, stakeholder representation, and positions taken during policy agenda setting, drafting, and implementation to shed light on policy making to promote appropriate antibiotic utilization. Methods: We carried out a quantitative content analysis of 322 newspaper articles published between January 2009 and December 2010 in 18 national and regional newspapers. Additionally, we conducted a qualitative content analysis to understand the positions adopted and strategies developed by nine key stakeholders. Framing theory guided the analysis. Results: The Ministry of Health dominated media coverage, justifying the enforcement policy by focusing on risks of self-medication, and to a lesser degree dangers of increasing antibiotic resistance. Pharmacy associations appeared to be the leading opponents, arguing that the policy created logistical difficulties and corruption, and had negative economic impact for pharmacies and their clients. The associations developed strategies against the regulation such as attempting to delay implementation and installing physicians’ consultation offices within pharmacies. While medical associations and academic institutions called for a comprehensive strategy to combat antibiotic resistance, improve prescription quality, and create public awareness, these issues had little impact on media coverage. Consumer groups and legislators received very little media coverage. Conclusions: The narrowly-focused and polarized media coverage ─centred on problems of self- medication and economic impact ─was a missed opportunity to publicly discuss and to develop a comprehensive national strategy on antibiotic use in Mexico. It highlights the need for discussing and developing interventions within the framework of a pharmaceutical policy. Keywords: Antibiotics, Mexico, Over-the-counter sales, Antibiotic resistance, Media analysis, Pharmaceutical policy Dreser et al. BMC Public Health 2012, 12:1051 http://www.biomedcentral.com/1471-2458/12/1051 Dreser et al. BMC Public Health 2012, 12:1051 http://www.biomedcentral.com/1471-2458/12/1051 Dreser et al. BMC Public Health 2012, 12:1051 http://www.biomedcentral.com/1471-2458/12/1051 Page 2 of 11 status for antibiotic sales. It created an unprecedented public debate that gained prominence and space in the media. The announcement followed a declaration by the Minister of Health in May 2009 −in the midst of the H1N1 influenza outbreak −associating high mortality amongst influenza patients with self-medication, particu- larly with antibiotics [12]. Antibiotic policies in Mexico It has been recognized that the media not only have the potential to diffuse health information in society and to influence health behavior and attitudes [13,14], but also have the potential to influence public perceptions of health policy issues, the political elite’s policy considera- tions, and, eventually, the final policy product [15,16]. The media’s primary role in public policy making has been related to the process of agenda setting, through the ability to raise and shape issues and public opinion which, in turn, can impel governments to act [17]. The way in which issues are discussed in the media influ- ences how problems are defined and, consequently, which policy alternatives are considered to address them. One manner in which the media can shape public opin- ion of health policy issues is by framing or emphasizing them in particular ways [18]. Although there is no consistency in how “framing” is referred to in the litera- ture, a frame can be considered an emphasis on particu- lar aspects of a topic [19]. Entman [20] defines framing as follows: “[to] frame is to select some aspects of a per- ceived reality and make them more salient in a commu- nicating text in such a way as to promote a particular problem definition, causal interpretation, moral evalu- ation, and/or treatment recommendation.” Policy issues reported in the media depend on which facts are reported and which frames are chosen to address them. Mexico is a middle-income country characterized by a fragmented health care system; expenditure in medicines occurs largely as out-of-pocket [7]. Until recently, anti- biotics were among medicines most commonly sold in private pharmacies (approximately 40% without pre- scription) [8], and their use for many years exceeded that of other Latin American countries [9]. Inappropriate antibiotic prescriptions and lack of enforcement of prescription-only sales of antibiotics are both well- documented problems that Mexico shared with other Latin American countries [8,10]. Inappropriate antibiotic use and resistance have been very low on the govern- ment’s policy agenda, partly due to the lack of clear problem indicators, ignorance of strategies promoted internationally, and perception by policy makers that ac- cess to and supply of medicines should receive priority [6]. Background Aiming to address the global public health threat caused by antimicrobial resistance, the World Health Assembly in 1998 urged member countries to promote the appro- priate use of antimicrobials by improving prescribing, dispensing, surveillance and legislation [1]. In the same year, the Pan American Conference on Antimicrobial Resistance in the Americas recommended that countries improve antibiotic use, including restriction on antibiot- ic sales to prescription-only and education on the proper use of antibiotics for health professionals and com- munity members [2]. In 2001, the World Health Organization (WHO) launched its Global Strategy for Containment of Antimicrobial Resistance. Recognizing that isolated interventions have little impact on improv- ing antibiotic use, the Global Strategy proposed a range of interventions, organized under the umbrella of na- tional health and medicine policies [3]. These recom- mendations were updated in 2011 [4]. Only a few, largely high-income, countries have devel- oped national strategies to promote appropriate anti- biotic use; many countries have had difficulty translating international recommendations into national action plans. In part, difficulties have resulted from multiple stakeholder coordination, rigid legal frameworks, and potential conflicts between guaranteeing access to medi- cines and assuring rational use [5,6]. Abstract The policy, enacted as a Min- isterial Agreement, additionally required all antibiotic prescriptions to be retained and registered in pharma- cies, and imposed high penalties for non-compliance, in- cluding rescinding business licenses. Soon after the announcement, the policy was opened to public discus- sion in a governmental website. Although several actors from the private sector declared opposition to the policy, it was finally approved with only minor changes, one of them delaying implementation for some months. The enforcement policy applied to 2,000 medicines – roughly one fifth of all medicines available commercially in the country. Drafting and implementing actions to enforce regulations on antibiotic sales were accompanied by press round-ups and press bulletins organized by diverse stakeholders, opinion columns in newspapers, discussion forums on the internet, and special programmes on radio and television. This paper analyzes media coverage of that policy process. Antibiotic policies in Mexico A White Paper by the Mexican Ministry of Health (MOH) proposing a National Pharmaceutical Policy in 2005 argued that regulating medicines sales to prescription-only by physicians – as stipulated by law since 1984 – had not been enforced as a measure to pro- mote access to medicines because universal health care coverage has not been achieved [11]. In March 2010, the Mexican National Health Council announced a new policy enforcing the prescription-only Dreser et al. BMC Public Health 2012, 12:1051 http://www.biomedcentral.com/1471-2458/12/1051 Page 3 of 11 Table 1 Newspapers searched and number of articles included in the analysis Newspaper Estimated number of copies sold per day Number of articles included in the analysis El Universal 180, 000 45 Reforma 175, 000 37 El Sol de México 60,500 32 Milenio diario 40,000 30 Excélsior 90,000 24 La Prensa 150, 000 24 Ovaciones 82,082 21 La Jornada 38,759 19 El financiero 72,000 14 El economista 37,000 13 Diario de México 14,500 13 El Gráfico 300,000 11 Uno más uno 67,672 10 La Crónica de Hoy 70,000 9 Impacto 65,000 6 Rumbo de México 225,245 6 La Razón 61,075 4 Metro 143,000 4 Total number of articles 322 Table 1 Newspapers searched and number of articles included in the analysis The choice of frame reflects journalistic conventions; the interaction among journalists and elite constituents, so- cial movements, and scientific bodies; as well as a frame’s resonance with broader political values [19,21,22]. De Vreese [19] proposed distinguishing be- tween “issue specific” frames and “generic” frames which are not related to a specific topic or a specific cultural or historical context. Actors use diverse frames to draw at- tention to issues of concern implying that certain types of solutions are called for [23]. Elite stakeholders have significant resources for shaping journalistic frames to serve their specific interests [21], which include reinfor- cing the status quo. However, the media can also give voice to less powerful stakeholders in order to reflect dissension or give a new direction to a policy debate. As Entman [20] explains, frames reveal the “imprint of power” when reflecting the identity of actors and inter- ests competing to dominate news texts. Because framing emphasizes one aspect of a problem at the expense of others, it can restrict public debate on policies in the range of issues and policy alternatives considered [15,23]. Data analysis In the identified newspaper articles, we conducted: 1) a quantitative content analysis to determine the frequency of topics and stakeholders covered; and 2) a qualitative content analysis to gain insight into stakeholder posi- tions and actions in relation to the enforcement policy. We were interested in the content of the articles rather than in the presentation of the stories (such as headlines or graphical images). All articles were coded for the ana- lysis. To develop the codebook, each author coded 20 articles identifying emerging themes through inductive reasoning [25]. Those themes were then structured and sorted into subject categories and each of the codes defined. The reliability and validity of the codification was checked by using investigator triangulation with 20 randomly selected newspaper articles. Discrepancies were discussed and adjustment made to the codebook Antibiotic policies in Mexico The objective of this study was to analyze media coverage of the process that led to the enforcement policy on antibiotic sales regulations in Mexico focusing, firstly, on 1) Which themes gained more or less promin- ence; 2) Which stakeholder groups dominated the public debate; 3) What were their positions and actions vis-a- vis the enforcement policy. The results are discussed in relation to the processes of agenda setting, policy draft- ing, and policy implementation in order to highlight po- tential media and stakeholder effects on the overall development process. Secondly, with this study we aimed to shed light on the process of policy making for medicines in Mexico that could be relevant for other low and middle income countries (LMICs) wishing to develop policies directed at improving the use of antibiotics. December 2010 on the website and included in our study only those that covered issues related to antibiotic use or antibiotics sales regulation. The time period cov- ers three stages of the policy process: 1) policy agenda setting (01 January 2009 to 24 March 2010); 2) policy drafting (25 March 2010 to 24 August 2010); and 3) pol- icy implementation (25 August 2010 to 31 December 2010). Results In total, 322 newspaper articles were included in the analysis; of them 18 were published during the agenda setting period, 185 during policy drafting, and 119 dur- ing implementation. Despite official statements regard- ing the problem of self-medication with antibiotics and the need to control antibiotic sales (following the influ- enza epidemic in 2009), these topics had little media coverage in the agenda setting period. During the study period (see Figure 1) we observed two peaks of media coverage on the topics of antibiotic use and antibiotic regulation. The first followed the public declaration by a member of the National Health Council, on 24 March In order to understand the position and actions of sta- keholders in relation to the new policy during each stage of the policy making process, a qualitative content ana- lysis was performed of all the notes in which a Table 2 Summary of the codebook Thematic category Description of the category and subcategories included Self-medication (SM) Purchase and use of prescription-only-medicines, including antibiotics, without medical prescription. Subcategories: i) SM to treat viral infections (influenza, other respiratory or gastro-intestinal infections); ii) causes and scope of SM; iii) solutions to the problem of SM, including the new policy to enforce sales regulation. Antibiotic resistance (AR) Antimicrobial or antibiotic resistance. Subcategories: i) causes and scope, including relation to self-medication and prescription ii) solutions, ii) using AR as an argument to support the new policy to enforce regulation on antibiotic sales. Prescription (PM) Medical PM of antibiotics. Subcategories: i) unjustified PM of antibiotics (for instance, for influenza and other respiratory diseases) ii) perception of low quality of PM; iii) need to train physicians and improve their PM of antibiotics Economic impact (EI) Direct and indirect EI resulting from the enforcement policy. Subcategories: i) EI in the private sector such as pharmacies and the pharmaceutical industry; ii) EI in the population. Corruption (CO) Corruption in the pharmaceutical sector derived from the enforcement policy. Subcategories: i) Black market of counterfeit medicines; ii) Black market of prescriptions; iii) CO in the process of pharmacy supervision; iv) physicians involved in CO. Regulation (RE) Regulation and policies related to the sales of antibiotics in Mexico. Results Subcategories: i) Legislative framework and lack of enforcement; ii) processes of drafting and implementing the enforcement policy; iii) need to develop an impact evaluation of the enforcement policy; iv) objective of the enforcement policy; v) need to disseminate information about the enforcement policy. Health System (HS) Functioning of the Mexican health system in relation to antibiotic use or the enforcement policy. Subcategories: i) problems of the HS; ii) impact of the enforcement policy on the health services provided in the HS; iii) access to health services for the population without health insurance. Rational use of medicines (RUM) Rational use of medicines in the national and international context. Subcategories: i) International guidelines on RUM; ii) recommendations to achieve RUM in Mexico; iii) causes and consequences of inappropriate use of antibiotics, including adverse reactions (but excluding references related solely to self-medication). Pharmacies (PH) Functioning of pharmacies in relation to antibiotic use or the enforcement policy. Subcategories: i) Operation of PH, ii) quality of services provided by pharmacy staff, and training of pharmacy staff; iii) position of pharmacy associations towards the enforcement policy; iv) demand of the pharmacy associations owners towards the government. Purchase and use of prescription-only-medicines, including antibiotics, without medical prescription. Subcategories: i) SM to treat viral infections (influenza, other respiratory or gastro-intestinal infections); ii) causes and scope of SM; iii) solutions to the problem of SM, including the new policy to enforce sales regulation. Antimicrobial or antibiotic resistance. Subcategories: i) causes and scope, including relation to self-medication and prescription ii) solutions, ii) using AR as an argument to support the new policy to enforce regulation on antibiotic sales. Medical PM of antibiotics. Subcategories: i) unjustified PM of antibiotics (for instance, for influenza and other respiratory diseases) ii) perception of low quality of PM; iii) need to train physicians and improve their PM of antibiotics Direct and indirect EI resulting from the enforcement policy. Subcategories: i) EI in the private sector such as pharmacies and the pharmaceutical industry; ii) EI in the population. Corruption in the pharmaceutical sector derived from the enforcement policy. Subcategories: i) Black market of counterfeit medicines; ii) Black market of prescriptions; iii) CO in the process of pharmacy supervision; iv) physicians nvolved in CO. Regulation and policies related to the sales of antibiotics in Mexico. Data sources and collection Secondly, stakeholder appearance was counted as the number of times a stakeholder was men- tioned in a newspaper article: the same association or in- stitution was counted only once per article even if it was mentioned several times in the same article. We counted separately different actors in the same stakeholder group mentioned in the same articles. Data sources and collection We conducted a systematic review of printed newspaper articles in Mexico using a media service that publishes daily on its website [24] all Mexican newspaper articles focussed on pharmaceutical issues. The service searches text electronically in 11 national newspapers in addition to seven in the capital district and other important cities. These newspapers represent the primary print media in terms of copies sold and political relevance in Mexico (see Table 1). The key words used for the electronic search include, among others, “medicines”, “antibiotics”, “Ministry of Health”, “pharmaceutical industry”, and “pharmacies”. We manually screened all of the news- paper articles published between January 2009 and Dreser et al. BMC Public Health 2012, 12:1051 http://www.biomedcentral.com/1471-2458/12/1051 Page 4 of 11 stakeholder code appeared. Firstly, we concentrated the analysis on the voice of stakeholders, particularly quotes, rather than the voice of journalists (except for editorials and opinion columns for which we considered journalists as another stakeholder group). Here, we looked for statements about the policy and its objec- tives, and for arguments against or supporting the new policy. Secondly, we looked, both in the voice of stake- holders and journalists, for references to specific actions developed by the actors in relation to the new policy development. During the analysis, we searched for simi- larities and differences between actors within the same stakeholder group, and between different stakeholder groups. (Table 2). All other newspaper articles were coded by one author using Atlas ti 5.2 software. In addition to the codebook, each article was coded according to stake- holders covered by the media: 1) government (execu- tive); 2) congress (legislative); 3) pharmacies and outlet associations; 4) pharmaceutical industry; 5) medical associations; 6) civil society groups; 7) academic institu- tions; 8) journalists (in editorials and opinion columns); and 9) private sector other than the pharmaceutical in- dustry. Finally, each time a stakeholder code was attached to a newspaper article, it was linked to the cor- responding subject category so that we could link the stakeholder with the topics covered. For the quantitative content analysis, we first calcu- lated the frequency of thematic categories for each of the three time periods separately. If a thematic code was mentioned several times in the same article, we counted it only once. Topics covered by the news media Overall, the most prominent themes found during the policy development process were regulation (29%), self- medication (13%), and economic impact (12%). Rational use of medicines, medical prescription, and health sys- tems were least frequently mentioned. Figure 2 shows the prevalence of each theme category by stage during the development process. Policy implementation: During this period, the theme “regulation” also dominated media coverage, but in rela- tion to information and impact of the policy. Increasing media coverage included the categories “economic im- pact” on the retail sector, “corruption,” and “pharmacy”. The category “health system” also gained relevance, with most of the notes covering the impact of the enforce- ment on the health system and the inability of health system capacity to provide health care to the population. In contrast, discussions about the rational use of medi- cines became less prominent. Agenda setting: "Self-medication" was the most fre- quent thematic category, particularly the risks of in- appropriate treatment of respiratory diseases, including H1N1 influenza, with antibiotics. The second most fre- quently mentioned thematic category was “regulation,” with most of the discussion focusing on the lack of en- forcement for antibiotic sales. The third category, “anti- biotic resistance,” was covered mainly as a consequence of self-medication. The need to promote the rational use of antibiotics – including improving medical prescribing – was also addressed during this stage. Results Subcategories: i) Legislative framework and lack of enforcement; ii) processes of drafting and implementing the enforcement policy; iii) need to develop an impact evaluation of the enforcement policy; iv) objective of the enforcement policy; v) need to disseminate information about the enforcement policy. Functioning of the Mexican health system in relation to antibiotic use or the enforcement policy. Subcategories: i) problems of the HS; ii) impact of the enforcement policy on the health services provided in the HS; iii) access to health services for the population without health insurance. Rational use of medicines in the national and international context. Subcategories: i) International guidelines on RUM; ii) recommendations to achieve RUM in Mexico; iii) causes and consequences of inappropriate use of antibiotics, including adverse reactions (but excluding references related solely to self-medication). Functioning of pharmacies in relation to antibiotic use or the enforcement policy. Subcategories: i) Operation of PH, ii) quality of services provided by pharmacy staff, and training of pharmacy staff; iii) position of pharmacy associations towards the enforcement policy; iv) demand of the pharmacy associations owners towards the government. Dreser et al. BMC Public Health 2012, 12:1051 http://www.biomedcentral.com/1471-2458/12/1051 Page 5 of 11 notes were coded in this category) and the objective of the enforcement. “Self-medication” and “antibiotic resist- ance” were frequently mentioned in relation to each other; both were used as arguments to support the new policy. The thematic category “pharmacy” was men- tioned more frequently in relation to the role of pharma- cies in implementing the policy, and to the demands made by the pharmacies to the government. The “eco- nomic impact” of the policy emerged as a theme: almost two-thirds of the notes mentioned the economic impact on the retail sector, while less than half referred to the economic impact on the population. “Corruption” was mentioned in relation to the risks of prescription falsifi- cation and counterfeit antibiotics subsequent to the policy. 2010, announcing the Ministerial Agreement to enforce regulation of antibiotic sales. The second peak coincided with the start of the enforcement policy (i.e. the Agree- ment) implementation in August 2010. The newspaper sections in which the articles appeared were: front page or front section 36%; business or financial 11%; editorials and columns 9%; health or society sections 8%; politics 6%; others 30%. Stakeholders covered by the media Policy drafting: “Regulation” was the main thematic category during this phase, and included sub-categories such as the process of policy development (a third of the Figure 3 shows the frequency with which stakeholders were mentioned at each stage of the policy making process. Overall, the executive government dominated media 0 20 40 60 80 100 120 140 Number of articles Months MoH underlines in press conference the relation between novel influenza deaths and self-medication with antibiotics MoH declares that a measure to control the sales of antibiotic will be introduced Official announcement of a Ministerial Agreement to enforce regulation on antibiotic sales Publication of the Ministerial Agreement Implementation begins Figure 1 Monthly coverage on antibiotic use and regulation in Mexican printed media, and main policy milestones 2009-2010. Publication of the Ministerial Agreement Implementation begins Official announcement of a Ministerial Agreement to enforce regulation on antibiotic sales MoH underlines in press conference the relation between novel influenza deaths and self-medication with antibiotics MoH declares that a measure to control the sales of antibiotic will be introduced Months Dreser et al. BMC Public Health 2012, 12:1051 http://www.biomedcentral.com/1471-2458/12/1051 Page 6 of 11 0% 5% 10% 15% 20% 25% 30% 35% 40% 45% Regulation Self-medication Economic impact Pharmacies Corruption Antibiotic resistance Health System Rational use Prescription Percentage Categories Agenda-setting Policy drafting Policy implementation 28.6% 12.7% 12.2% 11.3% 10.4% 9.8% 5.9% 4.8% 4.2% N=950 references to categories in newspaper notes Figure 2 Theme categories covered by the printed media by stage of the policy process. particularly consulting agencies) appeared later on in the policy process. coverage (35% of the notes), particularly the ministry of health (MOH) and the medicines regulatory authority (“COFEPRIS”), and to a much lesser extent other minis- tries. Second ranked were pharmacies and outlet asso- ciations (23%), particularly the association of independent pharmacies, “ANAFARMEX”. Editorials and opinion columns represented 9% of the notes. While academic institutions (9%), medical associations (6%), and the pharmaceutical industry (4%) had greater salience during the first two stages, their presence decreased in the last phase. In contrast, the coverage of legislators (6%), civil society groups (5%), and other private enterprises (3%, Stakeholder positions and strategies Ministry of health The MOH, as the main supporter of the policy, focused initially on the dangers of self-medication, especially in relation to influenza, as the main justification for the policy. Among the first declarations published were: “No more self-medication damaging the Mexicans” [26]; “. . . the use of antibiotics in cases of influenza H1N1 has been 0% 5% 10% 15% 20% 25% 30% 35% 40% 45% Government Pharmacies & outlets associations Academic institutions Journalists Medical associations Congress Civil society groups Pharmaceutical industry Other private sector Percentage Stakeholders Agenda-setting Policy drafting Policy implementation 34.9% 23.2% 8.7% 8.7% 6.4% 6.0% 4.6% 4.1% 3.3% N=482 references to stakeholders in newspaper notes Figure 3 Printed media coverage of stakeholders by policy stage. Figure 3 Printed media coverage of stakeholders by policy stage. Page 7 of 11 Dreser et al. BMC Public Health 2012, 12:1051 http://www.biomedcentral.com/1471-2458/12/1051 fatal [. . .]” [27]. Later, the problem of antimicrobial resistance was also mentioned, linking it only to self- medication; as the Health Minister explained: “The regulation is not a caprice. In Mexico, the population is generating a resistance to antibiotics which arises from self-medication” [28]. provision of loans to modernize small pharmacies, as well as the publication of a comprehensive database of all registered physicians in Mexico to detect false pre- scriptions. Another association (UNEFARM) organized a protest at the regulatory agency building against the regulation and sent a petition to the President. In con- trast, declarations by large pharmacy chains were very scarce. When faced with arguments by other actors against the policy and the demand to delay its implementation, the MOH defended the enforcement arguing that access to medical care was guaranteed with “Seguro Popular,” a recently-introduced form of health insurance. Even the Mexican President was quoted supporting the policy [29]. Later, the MOH recognized the arguments regard- ing counterfeit prescriptions and the lack of an informa- tion campaign, declaring that they would be addressed. Arguments that could have been used to gain support for the policy, such as protecting against adverse drug reactions and unnecessary expenses, were scarcely addressed. During the implementation period, ANAFARMEX demanded that the government permit sales of analge- sics and cold and cough preparations only in pharmacies (and not in other outlets) as a way of compensating for economic loss. Stakeholder positions and strategies Ministry of health A later strategy developed by large phar- macy chains buffered the impact of the regulation by offering a “discount” on antibiotics, and creating phys- ician offices attached to pharmacies to offer cheap or free medical consultation. Independent pharmacy asso- ciations pointed out that these “express clinics” gener- ated unfair competition for smaller pharmacies. Six months after the implementation, four pharmacy chains plus some independent pharmacies had included con- sultation offices within drugstores; one large pharmacy chain declared that each of its 920 branches had physi- cians who offered free “medical counseling” for over 30,000 consumers each day [31]. When, because of policy implementation, physician consultation offices attached to pharmacies began to emerge, the position of the MOH was portrayed as vague, and only some statements cautioned the popula- tion about the quality of these consultations. In contrast, the MOH stressed the “positive” impact of the policy, de- claring that a 35% reduction in antibiotic sales had been achieved [30]. Pharmacies and outlet associations According to the media, these stakeholders were pre- dominantly in favor of the policy, highlighting antimicro- bial resistance and adverse drug reactions as serious public health problems. At the same time, they stressed the need for an integrated action plan to improve med- ical prescribing, increase public awareness regarding the prudent use of antibiotics, and professionalize pharma- cies. Some medical associations mentioned concerns about limited access to antibiotic prescriptions in poor rural communities and the quality of care offered in clinics attached to pharmacies. The position of these groups – as portrayed by the media – was predominantly against the enforcement policy. Even though initial statements were generally favourable (recognizing the problems of self-medication and antibiotic resistance), later these groups appeared as leading opponents. Main arguments conveyed were eco- nomic losses and logistical difficulties for the pharmacies (now forced to pursue additional tasks when selling anti- biotics) as well as the negative health and economic effects particularly on poor populations with scarce ac- cess to healthcare. Other arguments were that the regu- lation would foster corruption, namely a black market in antibiotics and counterfeit prescriptions, as well as false inspectors blackmailing pharmacies. During the policy drafting period, the action taken by these groups included the publication of open letters to the MOH and dissemination of a policy brief suggesting priority actions. Excerpts of the policy brief were later incorporated into the declarations of the MOH and in the official publication of the Ministerial Agreement. Early strategies used against the policy included creat- ing alliances (among some independent pharmacy asso- ciations, medicine distributors and outlet associations) demanding the government delay implementation in order to develop first a detailed impact assessment. Later they demanded a public education campaign (to change the “culture of self-medication”) and the provision of in- formation systems for pharmacies to facilitate the new dispensing procedures. The association of independent pharmacies ANAFARMEX asked the government to stop the “witch hunt” against them and demanded the Patients and civil society groups There were only a few declarations by patients and civil society organizations. Although the media reflected the views of citizens (in letters to the editors, interviews to the population, and on-line discussion forums), the declarations of only one association (“El poder del con- sumidor”) were reflected in the media. This association underlined the possible negative effect of the enforce- ment policy for the poorest populations and stressed the need to improve access to health care, provide the public with more information and monitor user fees for private medical services. Besides the interests and resources of stakeholders, the predominant themes reflected in news coverage of anti- biotic use and antibiotic regulation can also be explained by journalistic conventions. These conventions tend to favour certain generic frames such as “episodic framing” [19] in which social issues and public affairs are por- trayed as limited to events only, and not discussed in a broader context (e.g. coverage centred on the policy enact- ment, rather than in the problem of antibiotic use). Other common generic frames found in our study are “conflict” (winners and losers, corruption) and “economic Congress Congress ─the Mexican legislative body─declarations were scarcely mentioned in the media during the initial phase and reflected divided positions by political parties. While legislators of the ruling political party were sup- portive of the policy, members of opposing parties stressed the adverse health and economic impact on the population (now “forced to pay a doctor”) given the Dreser et al. BMC Public Health 2012, 12:1051 http://www.biomedcentral.com/1471-2458/12/1051 Page 8 of 11 medicines stock outs) on the other. Some columns ques- tioned the real reasons for the regulation. Interestingly, many editorials were also critical about the lack of trans- parency and clear leadership in the process of policy making. Some editorials also questioned the feasibility of implementing the regulation in a country inundated by medicines promotion, where traditionally the law is not enforced, and there are too few inspectors to supervise the pharmaceutical sector. insufficient availability and quality of public health ser- vices. Some legislators in the Congress tried to promote an agreement to revoke the policy, arguing that it was “a restrictive measure that did not correspond with a health policy for a country facing a political, economic and so- cial crisis” [32]. Strategies of the Congress to intervene included the proposal of a law reform to introduce higher punishments for those who use false prescrip- tions, and demands to the executive branch of the gov- ernment to develop information campaigns and an independent evaluation of the policy impact. Pharmaceutical industry and other private firms During the early policy stages, media coverage addressed the declarations by some national pharmaceutical com- panies regarding administrative uncertainty around the policy change, as well as demanding the government de- velop an impact assessment and delay implementation. The generic medicines industry underlined the import- ance of prescribing antibiotics by generic name. Even though the theme of regulation dominated media coverage, the focus was primarily on the problems of developing the policy, and little, except for self- medication, about its objectives or its relation to public health issues. The other dominant themes in media debates mirrored the voice of the two major stake- holders involved. On one hand, the MOH defended the policy by citing the dangers of self-medication with anti- biotics; on the other, pharmacy associations opposed it, citing issues of economic impact and corruption. These divergent frames were scarcely addressed by the media on the common ground of a wider public debate on ra- tional use of medicines and pharmaceutical policy. The emphasis on the problem of self-medication probably minimized public concerns about new physician offices attached to pharmacies and contributed to the belief that, with the policy, problems in antibiotic use had been solved. This represented a missed opportunity to discuss in-depth pharmaceutical policies and the development of a national strategy on antibiotic use. Additionally, media coverage also represented a missed opportunity to sensitize the public about the problem of antibiotic re- sistance and the need to use antibiotics prudently, which coincides with other studies which stress the scarcity of key mobilising information on health directed to the public [34]. The statements by transnational pharmaceutical com- panies and consulting agencies were more frequent dur- ing the implementation period and focused on the high value of the antibiotic market in Mexico, the resultant “reconfiguration of the market,” the economic losses that the pharmaceutical sector would face and the need to re- invent the pharmacies. Deloitte, a private consulting agency, suggested that among the new business models to avoid economic drawbacks for pharmacies were the “in-situ physicians” [33]. They also warned that other regulations to control sales of other medicines could be expected. Except for one company, these groups omitted the issue of antimicrobial resistance. Discussion The results highlight a number of important points about media coverage and stakeholder participation in relation to antibiotic use and the process of regulating antibiotic sales in Mexico that could be relevant for other LMICs aiming to develop policies directed to im- proving the use of medicines. Journalists This coincides with other authors pointing out that, although Mexico has attained a media establishment relatively independent of govern- ment control, it has also been increasingly beholden to commercial interests [38,39]. However, even when these association groups had a strong voice, their effect appears to be limited, suggesting that the influence of political factors may outweigh media framing effects on shaping policies, as other studies have pointed up [15]. Our results coincide largely with the findings of an analysis of printed media coverage of tobacco policies in Mexico [41] which reported the overwhelming presence of governmental actors, and limited inclusion of aca- demic centers and civil society organizations. However, because the tobacco study included coverage of the de- velopment of two laws, the presence of actors from the legislative branch was significantly larger. Interestingly, our results on actor participation contrast sharply with a similar study on medicines policy developed in Canada [15] where civil society was the dominant voice, demon- strating the strategic use of the media by these groups. Although Mexico is undergoing a democratic transition, the experience of media advocacy by academic and civil society groups is still developing. Large pharmacy chains had a minor presence in media debates, as did the transnational pharmaceutical com- panies because their interests might not be greatly affected by the policy. Some large chains buffered the impact of the policy by opening new physician offices, which might have favored the prescription of branded medicines. Private enterprises largely framed the policy debate as a business issue, rather than a health issue, which resulted in a focus on maximizing profits rather than improving medicines use. This highlights the inher- ent complexities of pharmaceutical policies competing with both economic and public health interests. Some lessons can be drawn from our study which are relevant for other low and middle-income countries aim- ing to develop policies directed at improving antibiotic use. First, active opposition by some stakeholders to the enforcement of regulations on antibiotic sales should be expected, particularly arguing the economic impact on the population. Actions seeking to counteract the policy goals include opening physician offices next to pharma- cies. Second, even if the voices of governmental and business-related groups are favored in media coverage, the policy views of other groups can also achieve visibil- ity in the media which could contribute to a more informed policy development process. Journalists During the formulation period, several editorials and columns discussed the appropriateness of the regulation in recognizing the conflict between the problems of self- medication and antimicrobial resistance on one side, and the problem of low access to and low quality of public medical services (including long waiting times and Dreser et al. BMC Public Health 2012, 12:1051 http://www.biomedcentral.com/1471-2458/12/1051 Page 9 of 11 consequences”. Coverage might also be explained by a regional trend characteristic of Latin America of health news reporting leaning towards frames of political con- flict [35]. demands of interest groups (such as developing a public information campaign), reflect a political context charac- terized by vertical and closed decision-making. In addition, remarks in the media and editorials speculating about the true reasons for the enforcement suggest a lack of transparency and lack of trust in the authorities. The present study also sheds light on the actors and processes involved in health and pharmaceutical policy making in Mexico. The executive government, mainly the MOH and the medicines regulatory authority domi- nated media coverage. This agrees with studies portray- ing the process of public policy making in Mexico as state-centered, with low levels of pluralistic debate [36]. The scant involvement of the legislature and the fact that coverage reflected contradictory views of individual legislators instead of that of an ad-hoc expert commis- sion also coincides with other studies highlighting the traditionally weak role of the legislature and its advisory commissions in decision-making [36,37]. Traditionally, civil society has had a very marginal role in decision-making in Mexico [36,37] with the voice of these actors barely present in media coverage. The ab- sence of opinions by pharmacist associations can be explained by the small number of pharmacists working in private sector pharmacies. The large majority of phar- macy staff lack university degrees and undergo very little professional training in pharmaceutical sciences or related areas [40]. Hence, pharmacy associations with a business focus rather than professional associations em- phasizing professional credentials dominated the media debate. The associations of small independent pharmacies mostly affected by the regulation were successful in or- ganizing press round-ups to push their views in media debates and make specific demands to the government. These groups also took advantage of this momentum to advance their own agenda, such as the threat of compe- tition by pharmacy chains. Journalists Third, this study showed that even if governmental action focuses on a single problem and a single intervention (enforcement of sales regulations to avoid self-medication), other related issues will soon arise in public debate. These issues include access to medicines (a very sensitive issue for the population), the quality of medical services, the tracking of prescriptions and verification of their authen- ticity, and the adverse impact on commercial interests. Academic institutions and medical associations also took advantage of the policy momentum to push their own agendas, especially during the agenda setting and the early drafting periods. They used the media to stress the need to develop a comprehensive strategy on anti- biotic use, a theme that had little resonance in the media, and had little impact on the final policy. The scarcity of newspaper coverage describing dia- logue or interaction between interest groups and the MOH, and the lack of governmental response to the Page 10 of 11 Dreser et al. BMC Public Health 2012, 12:1051 http://www.biomedcentral.com/1471-2458/12/1051 Page 10 of 11 of a pharmaceutical policy, and to mobilize political support. These concerns highlight the need to discuss and de- velop interventions within the common framework of a pharmaceutical policy, and to engage stakeholders during the policy process. Finally, the scarcity of in- depth reporting on issues of antibiotic use and regula- tion in Mexico and Latin America has also been related to the limited availability of specialized journalism and independent information sources [39]. To address these limitations and to generate political priorities on antimicrobial use and resistance, the South American Infectious Disease Initiative [42] has worked with the news media in three countries, resulting in an improve- ment on the quantity and quality of coverage of these issues [39], an experience that might be worth replicat- ing in other countries. Our study presents some limitations. First, our analysis used a unique coding scheme (“issue-specific” instead of “generic” frames) which limits our ability to generalize and compare the results [19]. However, whenever pos- sible, we discussed our coding scheme in relation to gen- eric frames. Other limitations concern excluding an analysis of electronic media (radio, television and inter- net) which are important in Mexico and other countries in the region [39] as well as the fact that our analysis was limited to the period of only five months after policy implementation. Journalists Relevant discussions and stakeholder actions that might have appeared later in the policy process or in other media outlets were excluded. Finally, as others pointed out, we can describe the representa- tion of issues and stakeholder participation in the media, but we can only infer their potential effects on the pub- lic and on the policy process [15]. To provide a more comprehensive picture of stakeholder participation in the policy process it is necessary to use alternative data sources. How does the Mexican case compare with other rele- vant experiences? The cases of India and Brazil, both middle-income countries that undertook antibiotic pol- icies contemporaneously to Mexico are worth discussing. During 2010, international attention turned to the spread of the New Delhi metallo-beta-lactamase 1 (NDM-1) gene, believed to have originated in India, which causes several types of bacteria to become resist- ant. The naming of the gene and a description of its spread from that country to Europe in an influential international medical journal was followed by an intense public debate in India [43]. Controversy triggered action and, in 2011, the Indian government published the National Policy for Containment of Antimicrobial Resistance. The policy included, among many other interventions, the banning of over-the-counter antibiotic sales. However, later the government decided to put this regulation on hold indefinitely, arguing concerns over access to medicines, particularly in rural areas [44]. In the case of Brazil it was the spread of the multi- resistant bacterium CRKP (Carbapenem-Resistant Klebsiella pneumoniae), also followed by the media, which gave place to the resolution RDC 44/2010 of the Brazilian regulatory agency, regulating antibiotic sales to medical prescription only, to be retained at pharmacies. The resolution, supported by medical groups, faced the opposition of pharmacy associations, citing scarce access to medical services for the poorest populations, as well as the risk of triggering a parallel antibiotic market [45]. Taken together, the cases of these three countries underline the relevance of focusing events (influenza outbreak, NDM-1 and CRKP spread) to place issues of antibiotic use and resistance on the governmental agenda. However, these cases also show that an appar- ently technical issue (regulating antibiotic sales) can be very politically sensitive – because economic interests are affected and because of the relation to wider com- plex societal issues – underlying again the need to dis- cuss antibiotic policies within the common framework Authors’ contributions d d f AD and VJW conceived of the study and wrote the manuscript. EVV analyzed the data and drafted a first layout of the manuscript under supervision of AD and VJW. ST participated in the study design and data analysis. AD revised the entire manuscript which was approved by all other co-authors. Conclusions Aspects of greater salience on media coverage were the issue of self medication used to justify the policy to en- force antibiotic sales regulations, and the negative aspects (economic impact and corruption) which largely represented the views of the two main stakeholders in the debate. Neither of these perspectives was conducive to public discussion on the wider problems of antibiotic misuse and resistance. Because framing emphasizes some aspects of the issues at the expense of others, the extensive coverage of the conflicts around developing and implementing the enforcement policy left little room to discuss wider societal issues, with the exception of discussions concerning access to medicines and health services. The absence of debate on the relationship be- tween the policy and promoting rational use of medi- cines, improving the quality of services provided in pharmacies, and addressing the global public health threat posed by antimicrobial resistance meant a missed opportunity for discussing and developing a comprehen- sive national strategy on antibiotic use in Mexico. Competing interests The authors declare that they have no competing interests. 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https://openalex.org/W4312350887	https://periodicals.karazin.ua/politology/article/download/18660/16983	Ukrainian	null	ARTIFICIAL INTELLIGENCE AS FACTOR OF GEOPOLITICAL POWER	Vìsnik Harkìvsʹkogo nacìonalʹnogo unìversitetu ìmenì V.N. Karazìna. Serìâ Pitannâ polìtologìï	2,021	cc-by	4,547	In cites: Vinnykova, Nataliya. 2021. Artificial Intelligence аs Factor оf Geopolitical Power. The journal of V. N. Karazin Kharkiv National University. Series «Issues of Political Science» 40: 21-28. https://doi.org/10.26565/2220-8089-2021-40-03 ISSN 2220-8089 Вісник Харківського національного університету імені В. Н. Каразіна, серія «Питання політології», 2021, випуск 40 ISSN 2220-8089 Вісник Харківського національного університету імені В. Н. Каразіна, серія «Питання політології», 2021, випуск 40 DOI: 10.26565/2220-8089-2021-40-03 УДК 327.2 Наталія Анатоліївна Вінникова професор, д.політ.н., Харківський національний університет імені В.Н. Каразіна, майдан Свободи, 4, Харків, 61022, vinnykova@karazin.ua, https://orcid.org/0000-0001-5941-7562 Наталія Анатоліївна Вінникова професор, д.політ.н., Харківський національний університет імені В.Н. Каразіна, майдан Свободи, 4, Харків, 61022, vinnykova@karazin.ua, https://orcid.org/0000-0001-5941-7562 © Вінникова Н.А., 2021. Як цитувати: Вінникова, Н.A. 2021. Штучний інтелект як новий чинник геополітичного впливу. Вісник Харківського національного університету імені В.Н. Каразіна, серія «Питання політології» 40: 21-28. https://doi.org/10.26565/2220-8089-2021-40-03 ISSN 2220-8089 Вісник Харківського національного університету імені В. Н. Каразіна, серія «Питання політології», 2021, випуск 40 більша частина зосереджена на сфері наукових досліджень, охорони здоров’я, бізнесу, кібербезпеки, освіти (OECD AI 2021). Технології штучного інтелекту є привабливими для багатьох країн в аспекті використання у воєнних цілях. Швидкість поширення технологій має значення, адже країна, яка першою запускає технологію, має перевагу над своїми конкурентами. більша частина зосереджена на сфері наукових досліджень, охорони здоров’я, бізнесу, кібербезпеки, освіти (OECD AI 2021). Технології штучного інтелекту є привабливими для багатьох країн в аспекті використання у воєнних цілях. Швидкість поширення технологій має значення, адже країна, яка першою запускає технологію, має перевагу над своїми конкурентами. Вплив технологій на політику, економіку, оборонно-військову сферу залежить від того, як уряди та організації здійснюють імплементацію та використання нових можливостей. У контексті чинної Четвер- тої технологічної революції на порядок денний виходить питання регулювання технологій штучного інтелекту (ШІ). Нор- мотворча та інвестиційна активність, яку демонструють держави і міжнародні орга- нізації у розвитку та впровадженні штуч- ного інтелекту свідчать про перетворення ШІ з чинника інноваційного розвитку на важіль геополітичного впливу. Протягом останніх років держави, одна за одною, затверджують стратегії розвитку ШІ. Наочним доказом визнання значущості технологій ШІ у внутрішній і зовніній політиці є міжнародне рейтингування дер- жав за критеріями готовності урядів до розвитку та впровадження ШІ (AI Readi- ness Index 2020; The Global AI Index 2021). Д і ШІ і і У міжнародному рейтингу готовності урядів до впровадження технологій штуч- ного інтелекту топові місця посідають країни євроатлантичного регіону. У першій десятці зі 172 держав представлені США, Велика Британія, Фінляндія, Німеч- чина, Швеція, Данія, Нідерланди, Норвегія (AI Readiness Index 2020). Водночас країни Південної Азії і також демонструють високі показники в цій сфері. Так, Китай, Сінгапур і Корея в обох глобальних індексах з розвитку ШІ входять до першої десятки, займаючи 2, 6 і 7 позиції відповідно (AI Readiness Index 2020; The Global AI Index 2021). Дослідження впливу ШІ на міжнародні відносини лише починають розвиватися. У цьому ракурсі оцінюється роль технологій ШІ у військовій могутності держав (Horowitz 2018; Shaw 2017) і та їхні позиції у цифровому вимірі міжнародних відносин (Granados & De la Peña 2021; Kai-Fu Lee 2018, Kissinger, Schmidt & Huttenlocher 2021). Натомість праць, які б містили аналіз ролі технологій штучного інтелекту в структуруванні міжнародної системи, бракує. ) На тлі стратегічних інтенцій держав з розвитку технологій штучного інтелекту визнаними лідерами у новій світовій гонці озброєнь за впровадження ШІ, на думку науковців, є Сполучені Штати Америки та Китай (Granados & De la Peña 2021:11; Miailhe 2018:111). ШТУЧНИЙ ІНТЕЛЕКТ ЯК ЧИННИК ГЕОПОЛІТИЧНОГО ВПЛИВУ Висвітлено роль технологій штучного інтелекту (ШІ) у формуванні цифрового світового ладу. Проаналізовано загальносвітові тенденції політико-нормативного забезпечення розвитку та впровадження ШІ, розкрито особливості державних стратегій у цій галузі. Доведено, що наразі найпотужнішими акторами міжнародної системи у використанні ШІ є Сполучені Штати Америки та Китай. Завдяки інвестиціям, нормативно-інституційному забезпеченню та впровадженню ШІ технологій у сферу зовнішньої політики ці дві держави формують дуополію цифрового виміру світового порядку. Значущим фактором переваги США та КНР у просуванні штучного інтелекту є наявність транснаціональних техногігантів, як-от Google і Tencent та ін. Зазначено, що європейські країни, насамперед ті, що входять до складу ЄС, об’єднує етико-спрямований підхід до виробництва та впровадження технологій ШІ. Утім вони поступаються США та КНР в інвестиційному й інфраструктурному забезпеченні розвитку технологій штучного інтелекту. р у у у З’ясовано, що викликом для держав, які претендують на технологічне домінування в міжнародній системі, є доступ до даних. У цьому аспекті перевага на боці Китаю. Останній має не лише повномасштабний доступ до персональних даних своїх громадян, а й інтенсивно розвиває інструменти збору даних в інших державах, зокрема шляхом реалізації стратегії «Цифрового шовкового шляху». у Розкрито причиново-наслідкові зв’язки між впровадженням нових технологій і реконфігурацією геополітичного балансу сил в історичній ретроспективі. Обґрунтовано, що особливістю поточного етапу технологічного розвитку є те, що першість у впровадженні ШІ-технологій забезпечує перевагу лише в короткостроковій перспективі. На відміну від попередніх технологічних революцій, чинна характеризуються швидкістю адаптації та всебічним впровадженням ШІ і, таким чином, надає можливості загальносистемного впливу будь-якому актору міжнародних відносин, державного чи приватного сектора, що може різко змінити рівновагу сил на міжнародній арені. Ключові слова: технології штучного інтелекту, геополітичний баланс сил, Сполучені Штати Америки, Китай, світовий порядок. Як цитувати: Вінникова, Н.A. 2021. Штучний інтелект як новий чинник геополітичного впливу. Вісник Харківського національного університету імені В.Н. Каразіна, серія «Питання політології» 40: 21-28. https://doi.org/10.26565/2220-8089-2021-40-03 © Вінникова Н.А., 2021. 21 ISSN 2220-8089 Вісник Харківського національного університету імені В. Н. Каразіна, серія «Питання політології», 2021, випуск 40 стільникові мережі 5G, центри зберігання даних та глобальні супутникові навігаційні системи) (State Council 2015). Ця стратегія зосереджена не лише на міжнародних інфраструктурних проєктах Інтернет- зв’язку, але й стимулює місцеві цифрові фірми доповнювати інші галузі, як-от фінансові, у своїх закордонних підприєм- ствах. Зокрема «Цифровий шовковий шлях» передбачає створення конкуренто- спроможних платформ і додатків для надання Інтернет-послуг, таких як хмарні обчислення та аналіз великих даних, китайським і світовим компаніям (State Council 2015). Сполученим Штатам першість у розвитку ШІ (AI Readiness Index 2020; The Global AI Index 2021). Закон США «Про національну ініціативу в галузі штучного інтелекту» передбачає скоординовану програму для всього федерального уряду з метою забезпечення постійного лідерства США в дослідженнях і розробках штучного інтелекту, використанні штучного інтелекту в державному та приватному секторах, а також підготовки робочої сили до інтеграції ШІ в усі сектори економіки та суспільства (National AI Initiative Act 2020). Створення Національного офісу ініціа- тиви зі штучного інтелекту в рамках Управління науково-технічної політики Білого дому та фінансування, яке закладає федеральний уряд США в галузь ШІ свідчать про її пріоритетність. Витрати федеральних цивільних і оборонних контрактів на штучний інтелект і машинне навчання за прогнозами зростуть до 4,3 млрд доларів у 2023 фінансовому році (Bloomberg Government 2021). В оборон- ному секторі витрати на розвиток штуч- ного інтелекту оцінюються в 1,4 млрд доларів у 2020 році; 2 млрд доларів у 2021 році; 2,4 млрд доларів у 2022 році та 2,8 млрд доларів у 2023 році (Bloomberg Government 2021). Хоча в міжнародних рейтингах з розвитку ШІ Китай поступається США (AI Readiness Index 2020; The Global AI Index 2021), на стадії впровадження йому про- рокують лідерство. Адже КНР має суттєві переваги в забезпеченні чотирьох чинни- ків, що визначатимуть еру впровадження ШІ: наявність даних, фахівців у галузі штучного інтелекту, підприємців, які хочуть розвивати бізнес на основі ШІ та масштабну державну підтримку (Kai-Fu Leе 2018). ) У доступності даних, необхідних для машинного навчання та живлення ШІ, Китай має конкурентну перевагу з-понад 939,8 мільйонами користувачів Інтернету та 1,61 мільярда користувачів мобільного зв’язку (Kemp 2021a), порівняно з 298,8 мільйонами та 353,8 мільйонами користу- вачів Інтернету та мобільного зв’язку в Сполучених Штатах (Kemp 2021b). Інший претендент на світове лідерство в галузі технологій штучного інтелекту, Китай, концентрує ресурси на освіті для забезпечення громадян відповідними навичками користування, виробництва та впровадження ШІ. Амбітний «План май- бутнього покоління для штучного інте- лекту» передбачає поступальне набуття Китаєм до 2030 року статусу світового центру інновацій у сфері штучного інте- лекту (State Council, 2017: 6). ISSN 2220-8089 Вісник Харківського національного університету імені В. Н. Каразіна, серія «Питання політології», 2021, випуск 40 Хоча США та Китай є найбільшими економіками світу, з потужними кадровими ресурсами і транснаціональними техногігантами, як-от Google, Apple, Amazon, Facebook та Alibaba, Tencent, WeChat, Baidu, інтенсив- ному розвитку та впровадженню ШІ сприяє, насамперед, державна підтримка. Дві події артикулювали увагу урядів цих країн до можливостей і загроз, які несе з собою штучний інтелект. Зовнішній вплив на перебіг президентської електоральної кампанії у 2016 році, здійснений за допо- могою технологій штучного інтелектту, виявив вразливість інформаційного прос- тору Сполучених Штатів. Перемога про- грами Google Alpha Go над чемпіоном світу в грі Го того ж року стала спусковим механізмом для мобілізації урядом КНР ресурсів з метою набуття абсолютної світової першості в галузі розвитку та впровадження штучного інтелекту. Отже, метою нашого дослідження є визначення ролі технологій штучного інтелекту в контексті формування цифро- вого світового порядку. Дослідження складається з кількох блоків. По-перше, необхідно виявити найпотужніших акторів міжнародної системи у просуванні ШІ технологій. По-друге, розкрити особивос- ті підходів цих акторів до впровадження ШІ. Третім кроком дослідження стане проєктування перспектив розвитку цифрового світового ладу. Протягом останніх років держави, одна за одною, затверджують стратегії розвитку ШІ. Якщо на попередніх етапах розвитку ШІ основними рушіями висту- пали дослідницькі установи та приватні компанії IT-сектору, то успіх у чинній стадії впровадження штучного інтелекту залежить від державної підтримки. З-понад 700 державних програм з розвитку ШІ Широкий спектр державних заходів, зокрема великі інвестиції та інституціона- лізація цього напряму забезпечують 22 ISSN 2220-8089 Вісник Харківського національного університету імені В. Н. Каразіна, серія «Питання політології», 2021, випуск 40 ISSN 2220-8089 Вісник Харківського національного університету імені В. Н. Каразіна, серія «Питання політології», 2021, випуск 40 важко запобігти купівлі європейських компаній американськими та китайськими гігантами, як це було з британською компанією Deep Mind, піонером у галузі штучного інтелекту, купленої Google у 2014 р., або придбання Kuka, потужного концерну німецької робототехніки, китайським гігантом електропобутової техніки Midea у 2016 р. (Miailhe 2018:114). США, і 4,8 млрд доларів – азійським. Однак азійські фірми залучили цю суму майже в стільки ж угод (321), як і в США (324), що свідчить про те, що середній розмір угоди там був меншим. За вказаний період у США укладено 24 мегараундові угоди, в Азії – 13 угод (CB Insights 2021). Інвестиції в європейські ШІ-компанії склали 1,6 млрд доларів, канадійські – 0,4 млрд доларів. ШІ-стартапи країни Цент- ральної та Південної Америки отримали 0,5 млрд доларів інвестицій. У венчурний бізнес з розвитку ШІ в інших регіонах світу інвестовано 0,1 млрд (CB Insights 2021). Серед найбільш проблемних аспектів розвитку ШІ-технологій у державах-чле- нах та в ЄС у цілому можна виділити такі: у у  реактивність політичних заходів щодо стимулювання впровадження ШІ; у у  реактивність політичних заходів щодо стимулювання впровадження ШІ;  відсутність глобальних техногігантів;  відсутність глобальних техногігантів;  ) Нарощування Сполученими Штатами Америки та КНР цифрових потужностей у галузі ШІ-технологій зумовлює виклики для інших країн у питанні власного стату- су та ролі в цифровому вимірі світового порядку. Зокрема в ЄС визнається проб- лема наростання конкуренції у сфері роз- витку ШІ. Наразі в Європейському Союзі напрацьовано понад 60 програм і планів з розвитку ШІ (OECD. AI powered by EC/ OECD 2021). Водночас кожна держава- член ЄС має власну стратегію вироб- ництва та впровадження технологій ШІ (National Strategies on Artificial Intelligence 2021). 23 держави-члени ЄС входять до топ-50 міжнародного рейтингу з найкра- щою готовністю до впровадження ШІ (AI Readiness Index 2020).  необхідність узгодження національних стратегій держав-членів у межах загально- європейського нормативно-інституційного фрейму;  відсутність єдиної транс’європейської структури, відповідальної за формування політики з розвитку та впровадження технологій штучного інтелекту; у у  відсутність чіткого бачення своєї ролі у цифровому світі, що формується. Таким чином, попри прагнення багатьох країн не відставати в розвитку ШІ, формування дуополії між США та Китаєм у цифровому вимірі світового ладу вбачається неминучим. у Технологічні революції спричинили значні зміни в балансі сил, міжнародній конкуренції та міжнародних конфліктах. Перша промислова революція принесла прибуток, який стимулював розширення Британської імперії і забезпечила глобаль- не лідерство Великобританії на десятиліт- тя. ISSN 2220-8089 Вісник Харківського національного університету імені В. Н. Каразіна, серія «Питання політології», 2021, випуск 40 Уряд Китаю має на меті розвинути індустрію штучного інтелекту вартістю в 1 трлн юанів із суміж- ними галузями на суму 10 трлн юанів (State Council, 2017:6). Крім того, уряд також співпрацює з національними техно- логічними компаніями для розвитку науко- во-дослідного та промислового лідерства в окремих галузях штучного інтелекту. Наявність сприятливої екосистеми, що передбачає розвинуту нормативно-регуля- торну базу, фінансову та інституційну підтримку на державному рівні, дозволяє залучати більшу кількість компаній у галузі виробництва і впровадження штуч- ного інтелекту. Станом на 2021 рік 11382 ШІ-стартапів знаходилися в США (Tracxn 2021а), що є найбільшим показником у світі. У КНР функціонувало 1730 компаній з ШІ-технологій (Tracxn 2021b). Напрями інвестування в ШІ підтверд- жують першість позицій США та КНР з розвитку ШІ, а також окреслюють лінії двох цифрових блоків на світовій арені. Із 17,9 мільярдів доларів, зібраних ШІ- стартапами по всьому світі в третьому кварталі 2021 року, 10,4 млрд доларів надійшли компаніям, які знаходяться З 2015 року урядом КНР запущена ініціатива «Цифровий шовковий шлях» для будівництва інфраструктури, що з’єд- нує Азію, Африку та Європу (наземні та підводні кабелі передачі даних, 23 ISSN 2220-8089 Вісник Харківського національного університету імені В. Н. Каразіна, серія «Питання політології», 2021, випуск 40 точніший аналіз і можливість вироблення ефективніших рішень, що забезпечує конкурентну перевагу. Оскільки дані використовуються різними джерелами одночасно, проблема полягає в тому, щоб отримати доступ до даних, а не володіти ними. Вимоги до функціонування ШІ надають перевагу країнам з широким спектром інструментів доступу до даних. І, як вже зазначалося, такою державою наразі є КНР. На відміну від США, і тим більше від країн Європи, Китай менше опікується питаннями захисту приватності та персональних даних. Натомість плат- форми, на кшталт WeChat, та системи повсюдного відеоспостереження збирають безпрецедентно великі обсяги даних про соціальне та особисте життя громадян у КНР. Також це забезпечує досвід для впровадження технологій збору даних в інших країнах. У такий спосіб Китай формує потужний важіль впливу на внут- рішню і зовнішню політику інших держав. точніший аналіз і можливість вироблення ефективніших рішень, що забезпечує конкурентну перевагу. Оскільки дані використовуються різними джерелами одночасно, проблема полягає в тому, щоб отримати доступ до даних, а не володіти ними. Вимоги до функціонування ШІ надають перевагу країнам з широким спектром інструментів доступу до даних. І, як вже зазначалося, такою державою наразі є КНР. На відміну від США, і тим більше від країн Європи, Китай менше опікується питаннями захисту приватності та персональних даних. Натомість плат- форми, на кшталт WeChat, та системи повсюдного відеоспостереження збирають безпрецедентно великі обсяги даних про соціальне та особисте життя громадян у КНР. Також це забезпечує досвід для впровадження технологій збору даних в інших країнах. У такий спосіб Китай формує потужний важіль впливу на внут- рішню і зовнішню політику інших держав. однополярного світового ладу на чолі зі Сполученими Штатами Америки. Тому не дивно, що Сполучені Штати лідирують у світі – в економічному плані з такими компаніями, як Google, і у військовому плані, зі зброєю цифрової епохи. Спільним для цих промислових рево- люцій є зміна характеру війни та ключових інструментів влади. Технології можуть безпосередньо впливати на здатність країн вести війни та вигравати. За допомогою технологій можна змінювати баланс сил, впливаючи на економічну могутність країни. Зокрема Китай спроможний вико- ристовувати штучний інтелект, щоб зба- лансувати військову перевагу США шля- хом ведення війни розвідок, яка ґрунтує- ться на створенні альтернативної логістики та машинному навчанні, а також розвитку алгоритмів ведення бойових дій. Техно- логії штучного інтелекту можуть змен- шити витрати на ведення воєнних операцій та знизити ризики для військових. ISSN 2220-8089 Вісник Харківського національного університету імені В. Н. Каразіна, серія «Питання політології», 2021, випуск 40 Технологічна революція кінця ХІХ-го – початку ХХІ-го століття призвела до поновлення міжнародної конкуренції та формування багатополярного міжнарод- ного середовища. Велика Британія, Фран- ція, Німеччина, Сполучені Штати та Японія змагалися за контроль над такими природними ресурсами, як нафта та вугілля для розвитку хімічної і автомо- більної промисловості (Horowitz 2018:51). Жорстка конкуренція зумовила ескалацію міжнародної напруженості, що зрештою вибухнула у Першій світовій війні ) Утім, експерти стримано оцінюють просування ЄС у розвитку ШІ, насамперед у контексті зовнішньої політики, вказуючи на наочний контраст у пріоритетах відпо- відних стратегій США та Європейського Союзу (Franke 2019). На відміну від Спо- лучених Штатів та держав, які концент- рують ресурси на сферах дослідження та оборони, Євросоюз підкреслює важливість штучного інтелекту в таких секторах, як охорона здоров’я, транспорт та комунальні послуги. Крім того, дотримуючись мульти- латералізму в своїй політиці з розвитку штучного інтелекту, Євросоюз може стати вразливим для геополітичних конкурентів, які роблять ставку на інновації та технона- ціоналізм, і ризикує бути поглинутим зовнішніми хвилями цифрової експансії. Третя промислова революція з масш- табним впровадженням комп’ютерних технологій, мікропроцесорів, Інтернету, глобальних виробничих ланцюгів та електроніки породила хвилю інновацій на початку 1990-х років, у період формування Європа поступається Китаю та Сполу- ченим Штатам у техніко-індустріальних ресурсах. Європейський цифровий ринок не достатньо інтегрований. За таких умов 24 ISSN 2220-8089 Вісник Харківського національного університету імені В. Н. Каразіна, серія «Питання політології», 2021, випуск 40 ISSN 2220-8089 Вісник Харківського національного університету імені В. Н. Каразіна, серія «Питання політології», 2021, випуск 40 https://flia.org/wp-content/uploads/2017/07/A- New-Generation-of-Artificial-Intelligence- Development-Plan-1.pdf 13. State Council. 2015. Guiding Opinions of the State Council on Actively Propelling the Internet Plus Action Plan, 1 July 2015 no. 40. Lexis China. URL: https://flia.org/wp-content/uploads/2017/07/A- New-Generation-of-Artificial-Intelligence- Development-Plan-1.pdf 13. State Council. 2015. Guiding Opinions of the State Council on Actively Propelling the Internet Plus Action Plan, 1 July 2015 no. 40. Lexis China. URL: ISSN 2220-8089 Вісник Харківського національного університету імені В. Н. Каразіна, серія «Питання політології», 2021, випуск 40 р у р Країни, що повільно реагують на означені технологічні виклики, ризикують опинитися в стані «цифрової вассалізації» або взагалі перетворитися на «цифрову колонію» в одній із цифрових імперій нового світового ладу. Однак протягом наступних років розроблення нових під- ходів до штучного інтелекту іншими акторами міжнародних відносин може змінити геополітичний баланс сил. На відміну від попередніх історичних етапів, що характеризувалися розподілом геогра- фічних зон впливу, чинна технологічна революція нівелює зонування як таке. Штучний інтелект є частиною комплексної міжнародної системи, а не є ізольованим технологічним процесом, керованим гру- пою компаній. Водночас саме приватні компанії є головними розробниками ШІ. Фінансово-економічні можливості транс- національних корпорацій, підсилені техно- логіями ШІ, виводять їх на новий рівень могутності, релевантний дипломатичним і військовим ресурсам найпотужніших дер- жав. Отже, питання, що потребує подаль- шого дослідження, пов’язане з роллю, яку відіграватимуть техногіганти в геополі- тичній боротьбі держав за встановлення цифрової гегемонії. Комерційні аспекти впровадження штучного інтелекту зумовлюють багато- сторонність конкуренції за участі країн і компаній з усього світу. Досягнення комерційно керованих технологій штуч- ного інтелекту спрямовані на побудову нових і зміну характеру наявних галузей та забезпечення того, щоб провідні транснаціональні корпорації базувалися у власній країні. Отже, існують стимули майже для всіх типів політичних режимів для розроблення технологій ШІ. На відміну від ядерної технології, яка була елітною технологією з особливими цілями, ШІ має загальне призначення. Технології штучного інтелекту можна інтерпретувати як вимір влади, оскільки ШІ впливає на військово-оборонну, бізнесову сфери, галузі охорони здоров’я, освіти, науки, урядування та дипломатії. Технології ШІ є наскрізним компонентом комплексної структури міжнародних відносин. Таким чином, як продемонстрував наш аналіз, у розвитку та впровадженні техно- логій штучного інтелекту лідирують дві країни, США та Китай, які фактично є хабами цифрового виміру світового ладу. Ключовим фактором для впроваджен- ня ШІ є дані. Великі дані – це «паливо» для ШІ. Машинне навчання вдосконалює- ться шляхом отримання більшої кількості та більш різноманітних даних. Це означає Стаття надійшла до редакції 15.10.2021. Стаття надійшла до редакції 15.10.2021. Стаття рекомендована до друку 1.11.2021. 25 REFERENCES 1. AI Readiness Index. 2020. Oxford Insights. URL: https://www.oxfordinsights.com/government-ai- readiness-index-2020 2. The Global AI Index. 2021. The Tortoise. URL: https://www.tortoisemedia.com/intelligence /global-ai/ https://hk.lexiscn.com/law/guiding-opinions-of- the-state-council-on-actively-propelling-the- internet-plus-action-plan.html https://hk.lexiscn.com/law/guiding-opinions-of- the-state-council-on-actively-propelling-the- internet-plus-action-plan.html 14. Kemp, Simon. 2021a. Digital 2021: China. Report. Datareportal. 9 February 2021. URL: https://datareportal.com/reports/digital-2021-china 15. Kemp, Simon. 2021b. Digital 2021: the United States of America. Report. Datareportal. 9 February 2021. URL: https://datareportal.com/reports/digital-2021- united-states-of-america?rq=USA 16. Tracxn. 2021а. Artificial Intelligence Startups in United States. 5 November 2021. URL: https://tracxn.com/explore/Artificial- Intelligence Startups in United States g 3. Horowitz, Michael. C. 2018. “Artificial Intelligence, International Competition, and the Balance of Power”, Texas National Security Review 1(3) : 37-57 14. Kemp, Simon. 2021a. Digital 2021: China. Report. Datareportal. 9 February 2021. URL: https://datareportal.com/reports/digital-2021-china 15. Kemp, Simon. 2021b. Digital 2021: the United States of America. Report. Datareportal. 9 February 2021. URL: ( ) https://doi.org/10.15781/T2639KP49 4. Shaw, Ian. G. 2017. “Robot Wars: US Empire and geopolitics in the robotic age”, Security Dialogue, 48(5): 451-470 https://doi.org/10.1177/0967010617713157 https://datareportal.com/reports/digital-2021- united-states-of-america?rq=USA q 16. Tracxn. 2021а. Artificial Intelligence Startups in United States. 5 November 2021. URL: https://tracxn.com/explore/Artificial- Intelligence-Startups-in-United-States 17. Tracxn. 2021b. Artificial Intelligence Startups in China. 8 November 2021. URL: https://tracxn.com/explore/Artificial-Intelligence- Startups-in-China 18. CB Insights. 2021. State Of AI Q3’21 Report. 11 November 2021. URL: https://www.cbinsights.com/research/report/ai- trends-q3-2021/ 16. Tracxn. 2021а. Artificial Intelligence Startups in United States. 5 November 2021. URL: https://tracxn.com/explore/Artificial- 5. Granados, Oscar M. and De la Peña, Nicolas. 2021. “Artificial Intelligence and International System Structure”, Revista Brasileira de Política Internacional 64(1): e003. https://doi.org/10.1590/0034-7329202100103 p p Intelligence-Startups-in-United-States 17. Tracxn. 2021b. Artificial Intelligence f g Startups in China. 8 November 2021. URL: https://tracxn.com/explore/Artificial-Intelligence- Startups-in-China 6. Kai-Fu, Lee. 2018. AI Superpowers: China, Silicon Valley, and the New World Order. Boston, Mass: Houghton Mifflin. p 18. CB Insights. 2021. State Of AI Q3’21 Report. 11 November 2021. URL: https://www.cbinsights.com/research/report/ai- trends-q3-2021/ 7. Kissinger, Henry, Schmidt, Eric, Huttenlocher, Daniel. 2021. The Age of AI: and Our Human Future. New York: Little, Brown and Company; First Edition. q 19. OECD.AI powered by EC/OECD. 2021. Database of national AI policies. OECD.AI. URL: https://oecd.ai/en/dashboards/countries/European Union. p y 8. OECD.AI. 2021. Database of national AI policies. OECD.AI. URL: https://oecd.ai/en/ 8. OECD.AI. 2021. Database of national AI policies. OECD.AI. URL: https://oecd.ai/en/ 9. Miailhe, Nicolas. 2018. “The geopolitics of artificial intelligence: The return of empires”, Politique étrangère I: 105-117. DOI: https://doi.org/10.3917/pe.183.0105 10. National AI Initiative Act of 2020. 2020. H.R.6216 – 116th Congress 03 December 2020. URL: 9. Miailhe, Nicolas. 2018. ARTIFICIAL INTELLIGENCE AS FACTOR OF GEOPOLITICAL POWER Каразина площадь Свободы, 4, Харьков, 61022, vinnykova@karazin.ua, http://orcid.org/0000-0001-5941-7562 ISSN 2220-8089 Вісник Харківського національного університету імені В. Н. Каразіна, серія «Питання політології», 2021, випуск 40 Nataliya Vinnykova Professor, Doctor of Political Sciences V.N. Karazin Kharkiv National University Svoboda Sq., Kharkiv, 61022, Ukraine, Nataliya Vinnykova Professor, Doctor of Political Sciences V.N. Karazin Kharkiv National University Svoboda Sq., Kharkiv, 61022, Ukraine, Nataliya Vinnykova Professor, Doctor of Political Sciences V.N. Karazin Kharkiv National University Svoboda Sq., Kharkiv, 61022, Ukraine, vinnykova@karazin.ua, http://orcid.org/0000-0001-5941-7562 vinnykova@karazin.ua, http://orcid.org/0000-0001-5941-7562 vinnykova@karazin.ua, http://orcid.org/0000-0001-5941-7562 ARTIFICIAL INTELLIGENCE AS FACTOR OF GEOPOLITICAL POWER The article provides insight into how developments in artificial intelligence (AI) affect the balance of power in the digital world order. This study reveals the global trends in the political and regulatory support for the development and implementation of AI, as well as peculiarities of government strategies in this area.The United States of America and China have been proven to be the most powerful actors in the international system in using AI. Thanks to investments, regulatory and institutional support and the introduction of AI technologies in the foreign policy’s domain, these two states form a duopoly of the digital world order. A significant advantage of the United States and China in promoting artificial intelligence is the presence of transnational tech giants such as Google and Tencent, etc. It is noted that European countries, primarily those that are part of the EU, are united by an ethical-oriented approach to the production and implementation of AI technologies. However, they are inferior to the United States and China in investment and infrastructure support for the development of artificial intelligence technologies. Our findings indicate that access to data is a challenge for states claiming technological dominance in the international system. In this aspect, the advantage is on the side of China. The latter not only has full-scale access to the personal data of its citizens, but is also actively developing tools for collecting data in other states, in particular by implementing the «Digital Silk Road» strategy. The paper highlights the correlations between the introduction of new technologies and the reconfiguration of the geopolitical balance of forces in the historical retrospective. Our analysis confirms that a feature of the current stage of technological development is that the primacy in the implementation of AI technologies provides an advantage only in the short term. Unlike previous technological revolutions, the current one is characterized by the rapidity of adaptation and the comprehensive implementation of AI, and thus provides an opportunity for any actor in international relations, public or private to make systemic impact, which can dramatically change the geopolitical balance of power on the international stage. g g p f p g Keywords: artificial intelligence technologies, geopolitical balance of power, USA, China, world order. Наталья Анатольевна Винникова профессор, д.полит.н. Харьковский национальный университет имени В.Н. Каразина площадь Свободы, 4, Харьков, 61022, vinnykova@karazin.ua, http://orcid.org/0000-0001-5941-7562 Наталья Анатольевна Винникова профессор, д.полит.н. Харьковский национальный университет имени В.Н. REFERENCES “The geopolitics of artificial intelligence: The return of empires”, Politique étrangère I: 105-117. DOI: https://doi.org/10.3917/pe.183.0105 20. National Strategies on Artificial Intelligence. 2021. AI Watch. European Commission. Brussels. URL: https://knowledge4policy.ec.europa.eu/ai- watch/national-strategies-artificial-intelligence _en. 21 Franke Ulrike 2019 Harnessing artificial 20. National Strategies on Artificial Intelligence. 2021. AI Watch. European Commission. Brussels. 20. National Strategies on Artificial Intelligence. 2021. AI Watch. European Commission. Brussels. URL: https://knowledge4policy.ec.europa.eu/ai- watch/national-strategies-artificial-intelligence _en. 21. Franke, Ulrike. 2019. Harnessing artificial intelligence. Policy Brief. European Council on Foreign Relations. URL: https://ecfr.eu/publication/harnessing_artificial_in telligence/ URL: https://knowledge4policy.ec.europa.eu/ai- watch/national-strategies-artificial-intelligence _en. URL: https://knowledge4policy.ec.europa.eu/ai- watch/national-strategies-artificial-intelligence _en. 10. National AI Initiative Act of 2020. 2020. H.R.6216 – 116th Congress 03 December 2020. URL: 21. Franke, Ulrike. 2019. Harnessing artificial intelligence. Policy Brief. European Council on Foreign Relations. URL: https://www.congress.gov/116/bills/hr6216/BILL S-116hr6216ih.pdf p 11. Bloomberg Government. 2021. Artificial Intelligence & Machine Learning. BGOV Market Profile. Bloomberg Government. URL: https://about.bgov.com/reports/market-profile- artificial-intelligence-and-machine-learning/ 12. State Council. 2017. Next Generation Artificial Intelligence Development Plan. The Foundation for Law and International Affairs. No. 35. 8 July 2017. URL: 11. Bloomberg Government. 2021. Artificial Intelligence & Machine Learning. BGOV Market Profile. Bloomberg Government. URL: https://about.bgov.com/reports/market-profile- artificial-intelligence-and-machine-learning/ https://ecfr.eu/publication/harnessing_artificial_in telligence/ https://ecfr.eu/publication/harnessing_artificial_in telligence/ The article was received by the editors 15.10.2021 The article is recommended for printing 01.11.2021 g g 12. State Council. 2017. Next Generation Artificial Intelligence Development Plan. The Foundation for Law and International Affairs. No. 35. 8 July 2017. URL: The article was received by the editors 15.10.2021 The article is recommended for printing 01.11.2021 26 ИСКУССТВЕННЫЙ ИНТЕЛЛЕКТ КАК ФАКТОР ГЕОПОЛИТИЧЕСКОГО ВЛИЯНИЯ Отражена роль технологий искусственного интеллекта (ИИ) в формировании цифрового мирового строя. Проанализированы общемировые тенденции политико- нормативного обеспечения развития и внедрения ИИ, раскрыты особенности государственных стратегий в этой области. 27 у р р Доказано, что наиболее мощными акторами международной системы в использовании ИИ являются Соединенные Штаты Америки и Китай. Благодаря инвестициям, нормативно- 27 ISSN 2220-8089 Вісник Харківського національного університету імені В. Н. Каразіна, серія «Питання політології», 2021, випуск 40 институциональному обеспечению и внедрению ИИ технологий в сферу внешней политики, эти два государства формируют дуополию цифрового измерения мирового порядка. Значимым фактором преимущества США и КНР в продвижении искусственного интеллекта является наличие транснациональных техногигантов, таких как Google и Tencent и т.д. Отмечено, что европейские страны, прежде всего входящие в состав ЕС, объединяет этико-ориентированный подход к производству и внедрению технологий ИИ. Впрочем, они уступают США и КНР в инвестиционном и инфраструктурном обеспечении развития технологий искусственного интеллекта. Выяснено, что вызовом для государств, претендующих на технологическое доминирование в международной системе, является доступ к данным. В этом аспекте преимущество на стороне Китая. Последний имеет не только полномасштабный доступ к персональным данным своих граждан, но и активно развивает инструменты сбора данных в других государствах, в частности путем реализации стратегии «Цифрового шелкового пути». Раскрыты причинно-следственные связи между внедрением новых технологий и реконфигурацией геополитического баланса сил в исторической ретроспективе. Обосновано, что особенностью текущего этапа технологического развития является то, что первенство во внедрении ИИ-технологий обеспечивает преимущество только в краткосрочной перспективе. В отличие от предыдущих технологических революций, нынешняя характеризуется быстротой адаптации и всесторонним внедрением ИИ, и таким образом предоставляет возможности общесистемного воздействия любому актору международных отношений, государственного или частного сектора, что может резко изменить геополитический баланс сил на международной арене. у р р Ключевые слова: технологии искусственного интеллекта, геополитический баланс сил, США, Китай, мировой порядок. 28 28
https://openalex.org/W2981756253	https://europepmc.org/articles/pmc6899025?pdf=render	English	null	Star-PAP regulates tumor protein D52 through modulating miR-449a/34a in breast cancer	Biology open	2,019	cc-by	7,161	ABSTRACT Tumor protein D52 (TPD52) is an oncogene amplified and overexpressed in various cancers. Tumor-suppressive microRNA- 449a and microRNA-34a (miR-449a/34a) were recently reported to inhibit breast cancer cell migration and invasion via targeting TPD52. However, the upstream events are not clearly defined. Star-PAP is a non-canonical poly (A) polymerase which could regulate the expression of many miRNAs and mRNAs, but its biological functions are not well elucidated. The present study aimed to explore the regulative roles of Star-PAP in miR-449a/34a and TPD52 expression in breast cancer. We observed a negative correlation between the expression of TPD52 and Star-PAP in breast cancer. Overexpression of Star-PAP inhibited TPD52 expression, while endogenous Star-PAP knockdown led to increased TPD52. Furthermore, RNA immunoprecipitation assay suggested that Star-PAP could not bind to TPD52, independent of the 3′-end processing. RNA pull-down assay showed that Star-PAP could bind to 3′region of miR-449a. In line with these results, blunted cell proliferation or cell apoptosis caused by Star-PAP was rescued by overexpression of TPD52 or downregulation of miR-449a/34a. Our findings identified that Star-PAP regulates TPD52 by modulating miR- 449a/34a, which may be an important molecular mechanism underlying the tumorigenesis of breast cancer and provide a rational therapeutic target for breast cancer treatment. In our further investigation of the regulatory mechanism of Star-PAP in tumor progression, we noticed that it was reported that Star-PAP knockdown dramatically increased Tumor protein D52 (TPD52) expression (Mellman et al., 2008). Inspired by that, TPD52 is a well-known oncogene overexpressed in breast cancer (Roslan et al., 2014; Byrne et al., 2014), we sought to determine whether regulation between Star-PAP and TPD52 was also involved in breast cancer development besides BIK. TPD52 is one member of the TPD52-like protein family, which was originally found upregulated in human breast carcinoma and further identified overexpressed in a variety of malignancies (Byrne et al., 2014; Chen et al., 2013; Tennstedt et al., 2014). Overexpressed TPD52 has been identified as a potential driver gene that is highly associated with regeneration and poor prognosis of breast cancer (Aure et al., 2013; Shehata et al., 2008a). Roslan et al. found that TPD52 was increased in breast cancer, especially in HER2 positive human breast cancer and cell lines (Roslan et al., 2014). As a kind of single-stranded non- coding RNA, microRNAs (miRNAs) play a profound role in regulating gene expression in eukaryotes. © 2019. Published by The Company of Biologists Ltd \| Biology Open (2019) 8, bio045914. doi:10.1242/bio.045914 RESEARCH ARTICLE 1State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201, P.R. China. 2University of Chinese Academy of Sciences, Beijing, 100049, P.R. China. 3Institute of Materia Medica, School of Pharmacy, North Sichuan Medical College, Nanchong, Sichuan, 637100, P.R. China. Star-PAP regulates tumor protein D52 through modulating miR-449a/34a in breast cancer Aizhu Duan1,2, Lingmei Kong1, Tao An1, Hongyu Zhou1, Chunlei Yu3,* and Yan Li1,* Star-PAP is a non-canonical poly (A) polymerase encoding by TUT1, and plays a critical role in the 3′-end processing and expression of a specific set of mRNAs and microRNAs. This process is very critical to stability and post-transcription of RNAs in human pathophysiology, including tumor progression (Li et al., 2013; Mellman et al., 2008; Knouf et al., 2013; Singh et al., 2009). Several mRNAs involved in oxidative stress response, DNA damage, apoptosis and EMT, such as HO-1, BIK and NME1 have been reported to be regulated by Star-PAP (Mellman et al., 2008; Li et al., 2017, 2012; Li and Anderson, 2014; A and Laishram, 2018). Our recent study found that Star-PAP is downregulated in breast cancer cells, and the overexpression of Star-PAP induced cell apoptosis and inhibited proliferation partly through upregulating BIK expression, suggesting that Star-PAP functions as a tumor suppressor (Yu et al., 2017). However, the other action mechanisms of Star-PAP in breast cancer still remain unknown. 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 that the original work is properly attributed. A.D., 0000-0001-5265-9219; Y.L., 0000-0002-5721-7062 Authors for correspondence (liyan@mail.kib.ac.cn, yu_chunlei@foxmail.com) ABSTRACT Recent studies showed that tumor-suppressive microRNA-449a and microRNA-34a (miR-449a/34a) inhibit breast cancer cell migration and invasion via targeting TPD52 (Li et al., 2016; Zhang et al., 2017). However, the upstream events that regulate these two microRNAs are not clearly defined. INTRODUCTION Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death worldwide (Bray et al., 2018). Although optimization of therapy with better surgery, cytotoxic agents and endocrine drugs have highly improved the prognosis, the knowledge about the underlying mechanisms of breast cancer pathogenesis is still limited (Shiovitz and Korde, 2015; Hart et al., 2015). Therefore, a better understanding of the molecular mechanisms is imperative for discovering the potential therapeutic targets for prevention and treatment of breast cancer. Due to the important roles of Star-PAP in regulating the expression of a global miRNAs and mRNAs, the present study aimed to explore the effects of Star-PAP on miR-449a/34a and TPD52 expression in breast cancer. Our studies identified that Star-PAP regulated TPD52 through modulating miR-449/34a, which plays a critical role in inhibiting the cell proliferation and inducing the apoptosis of breast cancer cells. Our findings provided a rational therapeutic target for breast cancer treatment. Biology Open Authors for correspondence (liyan@mail.kib.ac.cn, yu_chunlei A.D., 0000-0001-5265-9219; Y.L., 0000-0002-5721-7062 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 that the original work is properly attributed. Received 4 July 2019; Accepted 20 October 2019 1 Biology Open (2019) 8, bio045914. doi:10.1242/bio.045914 RESEARCH ARTICLE play an important role in regulating TPD52 expression in breast cancer cells. RESULTS Star-PAP and TPD52 are inversely expressed in human breast cancer To explore the correlation between Star-PAP and TPD52 in human breast cancer cells, we first examined the expression of Star-PAP and TPD52 in a panel of breast cancer cell lines and two mammary epithelial cell lines. Compared with the mammary epithelial cells, the breast cancer cell lines expressed lower levels of Star-PAP mRNA and higher levels of TPD52 mRNA, as quantified by qPCR (Fig. 1A,B). The results were further confirmed by the RT-PCR and western blot assays (Fig. 1C,D). Moreover, the correlation analysis of the clinical data (264 breast cancer patients) from R2 database showed that there was a negative correlation between Star-PAP and TPD52 mRNA (r=−0.2036, Y=−0.1398X+6.684, P=0.0009, Fig. 1E). These data suggested that Star-PAP might Star-PAP regulated TPD52 expression in an indirect manner To elucidate the regulatory effect of Star-PAP on TPD52, siRNA targeting Star-PAP was employed to knockdown the endogenous Star-PAP in MDA-MB-468 and SK-BR-3 cells. As the result, downregulation of Star-PAP led to increased expression of TPD52 in both cell lines (Fig. 2A,B). In contrast, when Star-PAP was overexpressed, the expression of TPD52 mRNA and protein levels were both markedly reduced (Fig. 2C,D). The above-mentioned results suggested that Star-PAP is an important regulator of TPD52. Given the role of Star-PAP in processing the 3′-end of some specific mRNAs, we employed RNA immunoprecipitation (RIP) assay to investigate whether Star-PAP directly bind to TPD52 mRNA. Fig. 1. The expressions of Star-PAP and TPD52 in breast cancer cells. (A) Expression levels of Star-PAP mRNA in breast cancer cell lines were detected by qPCR and normalized to MCF-12A. (B) TPD52 mRNA levels were detected by qPCR and normalized to MCF-12A. (C) Star-PAP and TPD52 mRNA were examined by RT-PCR and the products were analyzed in agarose gel. (D) Protein levels of Star-PAP and TPD52 were examined by western blot. (E) The negative correlation between Star-PAP and TPD52 mRNA in 264 breast cancer patients from the R2 database. Biology Open Fig. 1. The expressions of Star-PAP and TPD52 in breast cancer cells. (A) Expression levels of Star-PAP mRNA in breast cancer cell lines were detected by qPCR and normalized to MCF-12A. (B) TPD52 mRNA levels were detected by qPCR and normalized to MCF-12A. (C) Star-PAP and TPD52 mRNA were examined by RT-PCR and the products were analyzed in agarose gel. (D) Protein levels of Star-PAP and TPD52 were examined by western blot. Star-PAP regulated TPD52 by modulating miR-449a/34a Star-PAP regulated TPD52 by modulating miR-449a/34a A previous study showed that the suppression of Star-PAP resulted in an approximately 40% decrease in the global miRNAs levels, as measured by real-time PCR-based miRNA profiling (Knouf et al., 2013). In this work, we found that when Star-PAP overexpressed in the three cell lines, the levels of both miR-449a and miR-34a were significantly upregulated (P<0.01 versus control) (Fig. 4A). Moreover, the expression levels of miR-449a/34a were both significantly downregulated following Star-PAP knockdown (Fig. 4B). To further clarify the underlying mechanisms, RNA pull-down assay was performed and the result showed that Star-PAP could selectively bind to 3′region of miR-449a (Fig. 4C). Accordingly, we suggested that Star-PAP may regulate TPD52 through modulating miR-449a/34a. Subsequently, the dual luciferase reporter assay was conducted, and the results showed that the co-transfection of Star-PAP with wild-type TPD52 reduced the relative luciferase activity (Fig. 4D). Whereas, co-transfection of Star-PAP with TPD52 3′-UTR mutation had no effects on the luciferase activity (Fig. 4D). Moreover, the decreased levels of TPD52 mRNA and protein caused by Star-PAP were rescued or aggravated by the co-transfection with miR-449a inhibitor or miR- 449a mimic, respectively (Fig. 4E,F). Taken together, the results indicated that Star-PAP suppressed the expression of TPD52 by a miR-449a/34a-mediated mechanism. RESULTS Star-PAP and TPD52 are inversely expressed in human breast cancer (E) The negative correlation between Star-PAP and TPD52 mRNA in 264 breast cancer patients from the R2 database. Fig. 1. The expressions of Star-PAP and TPD52 in breast cancer cells. (A) Expression levels of Star-PAP mRNA in breast cancer cell lines were detected by qPCR and normalized to MCF-12A. (B) TPD52 mRNA levels were detected by qPCR and normalized to MCF-12A. (C) Star-PAP and TPD52 mRNA were examined by RT-PCR and the products were analyzed in agarose gel. (D) Protein levels of Star-PAP and TPD52 were examined by western blot. (E) The negative correlation between Star-PAP and TPD52 mRNA in 264 breast cancer patients from the R2 database. 2 Biology Open (2019) 8, bio045914. doi:10.1242/bio.045914 RESEARCH ARTICLE Fig. 2. Star-PAP regulated TPD52 expression in breast cancer cells. (A,B) Indicated cells were transfected with Star-PAP siRNAs for 48 h, the TPD52 mRNA level (A) and protein level (B) were detected by qPCR and western blot. (C,D) Cells were transfected with plasmids inserting with Star-PAP for 24 h, then TPD52 mRNA (C) and protein level (D) were detected by qPCR and western blot. (E) TPD52 mRNA was detected by RT-PCR after RIP of Star-PAP in SK-BR-3 cells. GAPDH (non-Star-PAP target mRNA) and HO-1 (Star-PAP target mRNA) were used as the negative and positive control, respectively. Data are means±s.d. (n=3) with three independent repeats, P<0.05, P<0.01, *P<0.001. Fig. 2. Star-PAP regulated TPD52 expression in breast cancer cells. (A,B) Indicated cells were transfected with Star-PAP siRNAs for 48 h, the TPD52 mRNA level (A) and protein level (B) were detected by qPCR and western blot. (C,D) Cells were transfected with plasmids inserting with Star-PAP for 24 h, then TPD52 mRNA (C) and protein level (D) were detected by qPCR and western blot. (E) TPD52 mRNA was detected by RT-PCR after RIP of Star-PAP in SK-BR-3 cells. GAPDH (non-Star-PAP target mRNA) and HO-1 (Star-PAP target mRNA) were used as the negative and positive control, respectively. Data are means±s.d. (n=3) with three independent repeats, P<0.05, P<0.01, P<0.001. suggested that TPD52 was directly modulated by miR-449a and miR-34a in human breast cancer cell lines. Unexpectedly, the data showed that Star-PAP could bind to its direct target gene HO-1 but could not bind to TPD52, suggesting that Star-PAP may regulate TPD52 expression in an indirect manner (Fig. 2E). TPD52 was modulated by miR-449a and miR-34a To clarify the regulatory mechanism of TPD52 in breast cancer, Targetscan (http://www.targetscan.org/ V7.0) was used to predict the regulator(s) of TPD52. Bioinformatics prediction showed that there was a putative sequence region in miR-449a and miR-34a to bind 3′-untranslated region (UTR) of TPD52 (Fig. 3A). To confirm the binding, we constructed firefly luciferase reporters containing the wild type or 3′-UTR-mutant of TPD52. The dual luciferase reporter assays were performed in MDA-MB-468, MCF-7 and SK-BR-3 cell lines. In all the tested cell lines, when the wild-type TPD52 was co-transfected with miR-449a/34a mimics could significantly inhibit the relative luciferase activity, while miR-449a/34a inhibitors increased luciferase activity (Fig. 3B). Whereas, mutation of the perfectly complementary sites in the 3′-UTR of TPD52 abolished the suppressive or upregulated effects of miR-449a/34a mimics and inhibitors, respectively (Fig. 3B). These results indicated that miR-449a/ 34a could suppress the expression of TPD52 by interacting with its 3′-UTR complementary sequences. Moreover, in the same cell lines, the transfection of miR-449a/34a mimics or inhibitors could decrease or increase the expression levels of TPD52 mRNA and protein, respectively (Fig. 3C,D). Taken together, these data Biology Open 3 RESEARCH ARTICLE Biology Open (2019) 8, bio045914. doi:10.1242/bio.045914 Fig. 3. MiR-449a/34a inhibited TPD52 expression. (A) The predicted miR-449a/34a binding sequences and the mutant sequence at the 3′-UTR of TPD52 mRNA. (B) Cells were co-transfected with either 50 nM miR-449a/34a mimics or inhibitors and 80 ng plasmid carrying either WT or Mut 3′-UTR of TPD52. The relative firefly luciferase activity normalized with Renilla luciferase was measured 48 h after transfection. (C,D) Cells were transfected with miR-449a/34a mimics or inhibitors for 48 h, and the expressions of TPD52 were detected by qPCR (C), and the protein levels were detected by western blot (D). Data are means±s.d. (n=3) with three independent repeats, P<0.05, P<0.01, P<0.001. gy p ( ) Biology Open Fig. 3. MiR-449a/34a inhibited TPD52 expression. (A) The predicted miR-449a/34a binding sequences and the mutant sequence at the 3′-UTR of TPD52 mRNA. (B) Cells were co-transfected with either 50 nM miR-449a/34a mimics or inhibitors and 80 ng plasmid carrying either WT or Mut 3′-UTR of TPD52. The relative firefly luciferase activity normalized with Renilla luciferase was measured 48 h after transfection. (C,D) Cells were transfected with miR-449a/34a mimics or inhibitors for 48 h, and the expressions of TPD52 were detected by qPCR (C), and the protein levels were detected by western blot (D). Data are means±s.d. TPD52 was modulated by miR-449a and miR-34a Star-PAP regulated miR-449a/34a expressions. (A) Overexpression of Star-PAP for 24 h. The expression of miR-449a/34a was detected (B) Decreased levels of miR-449a/34a following Star-PAP knockdown by siRNAs for 48 h. (C) Cells were co-transfected with either pFlagcmv2-Sta Fig. 4. Star-PAP regulated miR-449a/34a expressions. (A) Overexpression of Star-PAP for 24 h. The expression of miR-449a/34a was detected by qPCR. (B) Decreased levels of miR-449a/34a following Star-PAP knockdown by siRNAs for 48 h. (C) Cells were co-transfected with either pFlagcmv2-Star-PAP and 80 ng plasmid carrying either WT or Mut 3′-UTR of TPD52. The relative firefly luciferase activity normalized with Renilla luciferase was measured 24 h after transfection (D) RNA pull down assay was performed by 5′-biotin-miR-449a and biotin-Scramble 293 FT cells were transfected with pFlagcmv2-Star-PAP Fig. 4. Star-PAP regulated miR-449a/34a expressions. (A) Overexpression of Star-PAP for 24 h. The expression of miR-449a/34a was detected by qPCR. (B) Decreased levels of miR-449a/34a following Star-PAP knockdown by siRNAs for 48 h. (C) Cells were co-transfected with either pFlagcmv2-Star-PAP and 80 ng plasmid carrying either WT or Mut 3′-UTR of TPD52. The relative firefly luciferase activity normalized with Renilla luciferase was measured 24 h after transfection. (D) RNA pull down assay was performed by 5′-biotin-miR-449a and biotin-Scramble. 293 FT cells were transfected with pFlagcmv2-Star-PAP for 24 h. Cells were lysed in RIPA buffer, then incubated with biotin-miR-449a or biotin-scramble for 4 h, before that they were pre-incubated with miR-449a or Scramble for 1 h, followed by adding avidin beads, and finally examined by western blot. (E) Cells were co-transfected with Star-PAP and miR-449a mimic or inhibitor, the TPD52 mRNA level was detected by qPCR. (F) The same treatment as with E, and the protein levels were examined. (G) Quantification of TPD52 protein level in F by NIH ImageJ software. Data are means±s.d. (n=3) with three independent repeats, P<0.05, P<0.01, P<0.001. TPD52 was modulated by miR-449a and miR-34a (n=3) with three independent repeats, P<0.05, P<0.01, P<0.001. 4 Biology Open (2019) 8, bio045914. doi:10.1242/bio.045914 RESEARCH ARTICLE Star-PAP suppressed breast cancer cell growth and prognosis the expression pattern of TPD52 was firstly analyze Fig. 4. Star-PAP regulated miR-449a/34a expressions. (A) Overexpression of Star-PAP for 24 h. The expression of miR-449a/34a was detected by qP (B) Decreased levels of miR-449a/34a following Star-PAP knockdown by siRNAs for 48 h. (C) Cells were co-transfected with either pFlagcmv2-Star-PAP 80 ng plasmid carrying either WT or Mut 3′-UTR of TPD52. The relative firefly luciferase activity normalized with Renilla luciferase was measured 24 h af transfection. (D) RNA pull down assay was performed by 5′-biotin-miR-449a and biotin-Scramble. 293 FT cells were transfected with pFlagcmv2-Star-PA for 24 h. Cells were lysed in RIPA buffer, then incubated with biotin-miR-449a or biotin-scramble for 4 h, before that they were pre-incubated with miR-44 or Scramble for 1 h, followed by adding avidin beads, and finally examined by western blot. (E) Cells were co-transfected with Star-PAP and miR-449a m or inhibitor, the TPD52 mRNA level was detected by qPCR. (F) The same treatment as with E, and the protein levels were examined. (G) Quantification TPD52 protein level in F by NIH ImageJ software. Data are means±s.d. (n=3) with three independent repeats, P<0.05, P<0.01, P<0.001. gy p ( ) Fig. 4. Star-PAP regulated miR-449a/34a expressions. (A) Overexpression of Star-PAP for 24 h. The expression of miR-449a/34a was detected by q (B) Decreased levels of miR-449a/34a following Star-PAP knockdown by siRNAs for 48 h. (C) Cells were co-transfected with either pFlagcmv2-Star-PA 80 ng plasmid carrying either WT or Mut 3′-UTR of TPD52. The relative firefly luciferase activity normalized with Renilla luciferase was measured 24 h a transfection. (D) RNA pull down assay was performed by 5′-biotin-miR-449a and biotin-Scramble. 293 FT cells were transfected with pFlagcmv2-Star-P for 24 h. Cells were lysed in RIPA buffer, then incubated with biotin-miR-449a or biotin-scramble for 4 h, before that they were pre-incubated with miR-44 or Scramble for 1 h, followed by adding avidin beads, and finally examined by western blot. (E) Cells were co-transfected with Star-PAP and miR-449a m or inhibitor, the TPD52 mRNA level was detected by qPCR. (F) The same treatment as with E, and the protein levels were examined. (G) Quantification TPD52 protein level in F by NIH ImageJ software. Data are means±s.d. (n=3) with three independent repeats, P<0.05, P<0.01, P<0.001. Fig. 4. Star-PAP suppressed breast cancer cell growth and promoted apoptosis through inhibiting TPD52 expression doi:10.1242/bio.045914 Modification of miRNA sequences by the 3′ addition of nucleotides is a conserved physiological and common post- transcriptional event that shows selectivity for specific miRNAs and is also observed across species ranging from Caenorhabditis elegans to human (Wyman et al., 2011). The modifications resulting predominantly from adenylation and uridylation could influence miRNA stability and efficiency of target repression (Wyman et al., 2011). As a poly (A) and poly (U) polymerase, Star-PAP regulates both 3′A and 3′U additions in a variety of substrates, including miRNAs and mRNAs (Mellman et al., 2008; Trippe et al., 1998; Wyman et al., 2011). For example, the suppression of Star-PAP could significantly reduce miR-200a with a 3′U and miR-31 with 3′A in HCT-116 cells (Wyman et al., 2011). A previous study suggested that Star-PAP suppression resulted in a global decrease in miRNA levels of approximately 40%, as measured by real-time PCR-based miRNA profiling (Knouf et al., 2013). Herein, we evaluated the effects of Star-PAP on miR-449a and miR-34a in breast cancer cells, and the results showed that both miR-449a and miR-34a were upregulated following Star-PAP overexpression, while they were reduced by Star-PAP knockdown (Fig. 4A,B). It has been found that Star-PAP modifies miRNAs 3′ nucleotide additions and as opposed to the canonical poly (A) polymerases (PAPs), Star-PAP directly binds its target and plays a structural role to help assemble the cleavage and polyadenylation complex (Wyman et al., 2011; Laishram and Anderson, 2010). Consistently, we found that Star-PAP directly binds to the 3′ region of miR-449a, as identified by RNA pull-down assay (Fig. 4C). Our results proved that Star-PAP is an important regulator of miR-449a and miR-34a in human breast cells. between TPD52 expression and the prognosis across the entire spectrum of breast cancer patients, and the results showed that high levels of TPD52 was associated with relatively decreased relapse- free survival (Fig. 5B). In addition, the elevated TPD52 expression also indicated a lower relapse-free survival in the four major breast cancer subtypes (basal-like, HER2-enriched, luminal A and luminal B) (Fig. S2). We also found that TPD52 expression was significantly higher both in NSCLC and ovarian cancer. Consistently, the poor prognosis was also observed in TPD52- high NSCLC or ovarian cancer patients (Fig. S3). Collectively, these data indicated that the upregulation of TPD52 was positively correlated with the poor prognosis in various types of cancer. Star-PAP suppressed breast cancer cell growth and promoted apoptosis through inhibiting TPD52 expression prognosis, the expression pattern of TPD52 was firstly analyzed in Oncomine database. The breast cancer patients (n=1992) expressed significantly higher levels of TPD52 than the normal donors (n=144) (Fig. 5A). Then KM-plotter, an online cancer survival analysis database, was further exploited to evaluate the relationship As a potential driver gene, TPD52 is highly associated with regeneration in breast cancer (Aure et al., 2013). In order to investigate the correlation between TPD52 levels and clinical 5 Biology Open (2019) 8, bio045914. doi:10.1242/bio.045914 RESEARCH ARTICLE Fig. 5. Star-PAP suppressed MDA-MB-468 proliferation and induced apoptosis through inhibiting TPD52 expression. (A) Expression levels of TPD52 in 1992 breast cancer patients and 144 healthy donors. Dataset accession number and P-value were shown. (B) KM-plotter analysis of relapse-free survival of breast cancer patients stratified by the expression levels of TPD52. Number of patients, log-rank P-value and hazard ratio (HR) were shown. (C) Cells transfected with Star-PAP, siTPD52 or co-transfected with Star-PAP and TPD52 were seeded in 4000/well, and cell viability was measured by MTS. (D) The same treatment with C for 48 h, and the apoptotic cells were analyzed by Flow cytometry. (E) Cells transfected with Star-PAP and miR-449a mimic or inhibitor were seeded in 4000/well, and cell viability was measured by MTS. (F) The same treatment as C for 48 h, and cell apoptosis was analyzed. Data are means±s.d. (n=3) with three independent repeats, P<0.05, P<0.01, P<0.001. 6 Biology Open Fig. 5. Star-PAP suppressed MDA-MB-468 proliferation and induced apoptosis through inhibiting TPD52 expression. (A) Expression levels of TPD52 in 1992 breast cancer patients and 144 healthy donors. Dataset accession number and P-value were shown. (B) KM-plotter analysis of relapse-free survival of breast cancer patients stratified by the expression levels of TPD52. Number of patients, log-rank P-value and hazard ratio (HR) were shown. (C) Cells transfected with Star-PAP, siTPD52 or co-transfected with Star-PAP and TPD52 were seeded in 4000/well, and cell viability was measured by MTS. (D) The same treatment with C for 48 h, and the apoptotic cells were analyzed by Flow cytometry. (E) Cells transfected with Star-PAP and miR-449a mimic or inhibitor were seeded in 4000/well, and cell viability was measured by MTS. (F) The same treatment as C for 48 h, and cell apoptosis was analyzed. Data are means±s.d. (n=3) with three independent repeats, P<0.05, P<0.01, P<0.001. 6 RESEARCH ARTICLE Biology Open (2019) 8, bio045914. Star-PAP suppressed breast cancer cell growth and promoted apoptosis through inhibiting TPD52 expression Considering the regulatory relationship between Star-PAP and TPD52, we hypothesized that the tumor suppressing effect of Star- PAP in breast cancer cells might be mediated by modulating TPD52 expression. Indeed, Star-PAP overexpression suppressed cell proliferation and promoted apoptosis, which was similar to the effects of TPD52 downregulation (Roslan et al., 2014; Han et al., 2015; Chen et al., 2016). Whereas the inhibitory effect of Star-PAP overexpression was reversed by co-transfection with TPD52 (Fig. 5C,D). Furthermore, to confirm the roles of miR-449a/34a in the Star-PAP-TPD52 pathway, Star-PAP was co-transfected with miR-449a mimic or inhibitor. The miR-449a mimic exhibited synergistic effects with Star-PAP on cell proliferation suppression and apoptosis promotion. In contrast, the effects of Star-PAP were weakened by the co-transfection of miR-449a inhibitor (Fig. 5E,F). Similar results were obtained when the cells were co-transfected with miR-34a (Fig. S1). These data verified that Star-PAP regulated TPD52 via modulating miR-449a/34a, as a molecular mechanism underlying the function of Star-PAP as a tumor suppressor gene. mRNA maturation involves the addition of a poly (A) tail (∼250 adeno sine residue) at the 3′-end by PAPs in the nucleus (Zhao et al., 1999; Edmonds, 2002; Laishram, 2014; Moore and Proudfoot, 2009; Proudfoot and O’Sullivan, 2002), which is essential for mRNA stability, translation and exporting the transcript from the nucleus to the cytoplasm (Zhao et al., 1999; Laishram, 2014; Proudfoot, 2004). Previous studies have shown that mRNA maturation of several apoptosis-related genes, such as BIK, was regulated by Star-PAP. Our previous work showed that Star-PAP controls BIK expression and induced cell apoptosis through the mitochondrial pathway (Yu et al., 2017). However, investigation of the biological functions of mRNA 3′-end processing by Star-PAP is still at a nascent stage. Given that TPD52 is closely associated with tumor progression, our study here identified a clearly regulatory relationship that Star-PAP regulates the expression of TPD52 via modulating miR-449a/34a, rather than the direct 3′-end processing way in breast cancer cells (Fig. 2E). Furthermore, the proposed Star- PAP-miR-449a/34a-TPD52 axis is involved in proliferation and apoptosis of breast cancer cells (Fig. 5; Fig. S1). Our findings thus provide a potential therapeutic target for breast cancer treatment. DISCUSSION For the first time, we illuminated the regulatory relationship among Star-PAP, miR-449a/34a and TPD52. We found that Star- PAP indirectly regulated TPD52 through modulating miR-449a/ 34a. TPD52 is an oncogene overexpressed in various cancers, including breast, prostate, lung, gastric, ovarian and pediatric cancers (Byrne et al., 2014; Shehata et al., 2008b; Chen et al., 1997; Cheng et al., 2012). Knockdown of TPD52 induced G1 arrest or disrupted the mitochondrial membrane potential and activated caspase 9 and caspase 3 (Chen et al., 2016; Ummanni et al., 2008). In the present study, we found that TPD52 is generally upregulated in breast cancer cell lines, especially in MDA- MB-468, MCF-7 and SK-BR-3 cells (Fig. 1B). Consistently, we also found that TPD52 was significantly upregulated in breast cancer samples (Fig. 5A). Recently, miRNAs were reported to suppress the expression of TPD52 via its 3′-UTR at transcriptional level (Byrne et al., 1995). MiR-449a/34a have been reported to inhibit breast cancer cell migration and invasion through targeting TPD52 (Li et al., 2016; Zhang et al., 2017). Since miR-449a and miR-34a share the same seed sequence, they are expected to regulate the overlapping target genes (Bou Kheir et al., 2011). Herein, we found that miR-449a and miR-34a could inhibit cell proliferation of breast cancer cells through suppressing TPD52. The expression level of TPD52 was decreased by transfecting with miR-449a/34a mimics but increased with miR-449a/34a inhibitors (Fig. 3B–D). MiR-34a and miR-449a played key roles in tumorigenesis, specifically, miR-34a has been described as a master regulator of tumor suppression (Bader, 2012). Although the bioactivity of these miRNAs has been well studied, their upstream regulatory events are not clearly defined. Author contributions Author contributions Methodology: L.K.; Data curation: A.D.; Writing - original draft: A.D.; Writing - review & editing: T.A., H.Z., C.Y.; Supervision: Y.L.; Project administration: C.Y., Y.L.; Funding acquisition: C.Y., Y.L., L.K., T.A. GGGACTGGGTCTGTTC-3′; HO-1-F: 5′-CCACCAAGTTCAAGCAGC- TCTA-3′, HO-1-R: 5′-GCTCCTGCAACTCCTCAAAGAG-3′. Cell apoptosis analysis p p y Cell apoptosis was analyzed by the Annexin V-FITC/Propidium Iodide (PI) Apoptosis kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s protocol. MDA-MB-468 cells were collected after transfection for 48 h, washed twice with cold PBS, and then suspended in a binding buffer containing Annexin V and PI. After incubation for 15 min at room temperature in the dark, the samples were immediately analyzed by using the FACSCalibur flow cytometer (BD Biosciences) and analyzed using FlowJo (FlowJo LLC, Ashland, OR, USA). p p y Cell apoptosis was analyzed by the Annexin V-FITC/Propidium Iodide (PI) Apoptosis kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s protocol. MDA-MB-468 cells were collected after transfection for 48 h, washed twice with cold PBS, and then suspended in a binding buffer containing Annexin V and PI. After incubation for 15 min at room temperature in the dark, the samples were immediately analyzed by using the FACSCalibur flow cytometer (BD Biosciences) and analyzed using FlowJo (FlowJo LLC, Ashland, OR, USA). Cell apoptosis was analyzed by the Annexin V-FITC/Propidium Iodide (PI) Apoptosis kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s protocol. MDA-MB-468 cells were collected after transfection for 48 h, washed twice with cold PBS, and then suspended in Cell apoptosis was analyzed by the Annexin V-FITC/Propidium Iodide (PI) Apoptosis kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s protocol. MDA-MB-468 cells were collected after transfection for 48 h, washed twice with cold PBS, and then suspended in a binding buffer containing Annexin V and PI. After incubation for 15 min at room temperature in the dark, the samples were immediately analyzed by using the FACSCalibur flow cytometer (BD Biosciences) and analyzed using FlowJo (FlowJo LLC, Ashland, OR, USA). using the FACSCalibur flow cytometer (BD Biosciences) and analyzed using FlowJo (FlowJo LLC, Ashland, OR, USA). Cell lines and cell culture All breast cancer cell lines except SUM-159PT were obtained from ATCC (Manassas, VA, USA) and authenticated by short tandem repeat profiling. MDA-MB-231, MDA-MB-436, MDA-MB-468, BT-20 and MCF-7 were cultured in DMEM supplemented with 10% FBS and 1% penicillin/ streptomycin (P/S) (HyClone, Logan, UT, USA). HCC1937 and SK-BR-3 were cultured in RPMI 1640 with 10% FBS and 1% P/S. MCF-10A and MCF-12A were cultured in DMEM/F12 supplemented with 5% horse serum, 20 ng/ml EGF, 100 ng/ml cholera toxin, 10 μg/ml insulin, 500 ng/ml hydrocortisone. SUM-159PT was obtained from Asterand (Hertfordshire, UK) and cultured in Ham’s F12 medium supplemented with 5% FBS, 1 μg/ml hydrocortisone and 5 μg/ml insulin. HS578t was cultured in Biology Open 7 RESEARCH ARTICLE Biology Open (2019) 8, bio045914. doi:10.1242/bio.045914 DMEM with 10% FBS, 10 μg/ml insulin, and 30 ng/ml EGF. All cell lines were mycoplasma free and passaged no more than ten passages after resuscitation. DMEM with 10% FBS, 10 μg/ml insulin, and 30 ng/ml EGF. All cell lines were mycoplasma free and passaged no more than ten passages after resuscitation. control (GenePharma) in 4°C for 4 h, followed by adding avidin beads (Thermo Fisher Scientific) overnight. Finally, the beads were washed and boiled for western blot detection. Statistical analysis Data were presented as mean±standard deviation (s.d.) and analyzed for significance using GraphPad Prism 7 software (La Jolla, CA, USA). Two- tailed Student’s t-test was used to determine the statistical significance. P<0.05, P<0.01, *P<0.001 were considered to be statistically significant. p q y GAPDH-F: 5′-ACGACCACTTTGTCAAGCTCA-3′, GAPDH-R: 5′- TCTCTCTTCCTCTTGTGCTCT-3′; Star-PAP-F: 5′-GAGTTCTTCCCT- GGCTGTGT-3′, Star-PAP-R: 5′- AGCGATGGAGATTCTGGAGC-3′; TPD52-F: 5′- ACTACCAGTCCCCGTTTGATT-3′, TPD52-R: 5′-CTCA- GGGACTGGGTCTGTTC-3′; HO-1-F: 5′-CCACCAAGTTCAAGCAGC- TCTA-3′, HO-1-R: 5′-GCTCCTGCAACTCCTCAAAGAG-3′. Funding RIP was performed as reported with antibody against Star-PAP (Yu et al., 2017). A total of 107 cells were used for the preparation of mRNP lysate. Finally, RNA was purified with TRIzol (Thermo Fisher Scientific) and reverse transcribed by RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). This research was funded by the National Natural Science Foundation of China [81773783, 81603161 and 81803587]. The West Light Foundation of the Chinese Academy of Sciences (L.K. and T.A.) and the project of the Applied Basic Research Foundation of Yunnan Province [2018FB147]. The project support for L.K. and T.A. from the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences is also acknowledged. qPCR and RT-PCR assay To investigate the mRNA levels of TPD52, the public data sets EGAS00000000083, GSE19188, GSE12470 were analyzed in Oncomine (www.oncomine.org) according to the instructions. The online survival analysis software KM-plotter was employed for cancer survival analysis as previously described (Yu et al., 2017). The correlation analysis of TPD52 and Star-PAP was analyzed in R2 database. Total RNA was prepared with TRIzol (Thermo Fisher Scientific) according to the manufacturer’s protocol. Reverse transcription was performed using RevertAid H Minus First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific). For qPCR, SYBR Select Master Mix (Roche) was used with ABI 7500 Real-Time PCR System (Thermo Fisher Scientific). For RT-PCR, PrimeSTAR HS DNA Polymerase (Takara, Kusatsu, Japan) was used, and the products were analyzed in agarose gel. For detection of mature miR- 449a/34a, total RNA was subjected to reverse TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems), and TaqMan MicroRNA Assay Kit (Applied Biosystems) was used for qPCR analysis. U6 or GAPDH was measured as an internal control for miRNA or mRNA. Relative changes in gene and miRNA expression were determined using the 2−ΔΔCt method. The primers for qPCR and RT-PCR analysis of mRNA were: References A, P. S. and Laishram, R. S. (2018). Nuclear phosphatidylinositol-phosphate type I kinase alpha-coupled star-PAP Polyadenylation regulates cell invasion. Mol. Cell. Biol. 38, e0057. doi:10.1128/MCB.00457-17 Biology Open Aure, M. R., Steinfeld, I., Baumbusch, L. O., Liestol, K., Lipson, D., Nyberg, S., Naume, B., Sahlberg, K. K., Kristensen, V. N., Borresen-Dale, A. L. et al. (2013). 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Western blotting was performed with Primary antibodies for Star-PAP and TPD52 (Abcam, Cambridge, UK), β-actin and GAPDH (Sigma-Aldrich, St Louis, MO, USA) were used according to the manufacturer’s instructions. Supplementary information Supplementary information available online at http://bio.biologists.org/lookup/doi/10.1242/bio.045914.supplemental ttp://bio.biologists.org/lookup/doi/10.1242/bio.045914.supplemen Plasmids, siRNAs and luciferase reporter assay y Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) was used for both siRNA and plasmid transfection according to the manufacturer’s instructions. Star-PAP cDNA (GeneCopoeia, Rockville, MD, USA) was cloned into pFlag-CMV2 and pCDNA6-myc-his vector for transient overexpression. Two reported siStar-PAPs were used for knockdown, and the sequences were as follows: siStar-PAP-1: 5′-AACU- ACGAGCTGCGAGAAA-3′; siStar-PAP-2:5′-GUGUGUUUGUCAGU- GGCUU-3′; and siTPD52: 5′-GGAAGAGCUAAGAGAATT-3′. 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This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, 1. Introduction The study of component and process reliability is the basis of many efficiency evaluations in Operations Management discipline. For example, in the calculation of the Overall Equipment Effectiveness (OEE) introduced by Nakajima [1], it is necessary to estimate a crucial parameter called availability. This is strictly related to reliability. Still as an example, consider how, in the study of service level, it is important to know the availability of machines, which again depends on their reliability and maintainability. Reliability is defined as the probability that a component (or an entire system) will perform its function for a specified period of time, when operating in its design environment. The elements necessary for the definition of reliability are, therefore, an unambiguous criterion for judging whether something is working or not and the exact definition of environmental conditions and usage. Then, reliability can be defined as the time dependent probability of correct operation if we assume that a component is used for its intended function in its design environment and if we clearly define what we mean with "failure". For this definition, any discussion on the reliability basics starts with the coverage of the key concepts of probability. A broader definition of reliability is that "reliability is the science to predict, analyze, prevent and mitigate failures over time." It is a science, with its theoretical basis and principles. It also has sub-disciplines, all related - in some way - to the study and knowledge of faults. Reliability is closely related to mathematics, and especially to statistics, physics, chemistry, mechanics and electronics. In the end, given that the human element is almost always part of the systems, it often has to do with psychology and psychiatry. In addition to the prediction of system durability, reliability also tries to give answers to other questions. Indeed, we can try to derive from reliability also the availability performance of a Operations Management 82 system. In fact, availability depends on the time between two consecutive failures and on how long it takes to restore the system. Reliability study can be also used to understand how faults can be avoided. You can try to prevent potential failures, acting on the design, materials and maintenance. 1. Introduction Reliability involves almost all aspects related to the possession of a property: cost management, customer satisfaction, the proper management of resources, passing through the ability to sell products or services, safety and quality of the product. This chapter presents a discussion of reliability theory, supported by practical examples of interest in operations management. Basic elements of probability theory, as the sample space, random events and Bayes' theorem should be revised for a deeper understanding. 2. Reliability basics The period of regular operation of an equipment ends when any chemical-physical phenom‐ enon, said fault, occurred in one or more of its parts, determines a variation of its nominal performances. This makes the behavior of the device unacceptable. The equipment passes from the state of operation to that of non-functioning. In Table 1 faults are classified according to their origin. For each failure mode an extended description is given. Failure cause Description Stress, shock, fatigue Function of the temporal and spatial distribution of the load conditions and of the response of the material. The structural characteristics of the component play an important role, and should be assessed in the broadest form as possible, incorporating also possible design errors, embodiments, material defects, etc.. Temperature Operational variable that depends mainly on the specific characteristics of the material (thermal inertia), as well as the spatial and temporal distribution of heat sources. Wear State of physical degradation of the component; it manifests itself as a result of aging phenomena that accompany the normal activities (friction between the materials, exposure to harmful agents, etc..) Corrosion Phenomenon that depends on the characteristics of the environment in which the component is operating. These conditions can lead to material degradation or chemical and physical processes that make the component no longer suitable. Table 1. Main causes of failure. The table shows the main cases of failure with a detailed description To study reliability you need to transform reality into a model, which allows the analysis by applying laws and analyzing its behavior [2]. Reliability models can be divided into static and dynamic ones. Static models assume that a failure does not result in the occurrence of other Reliability and Maintainability in Operations Management http://dx.doi.org/10.5772/54161 83 faults. Dynamic reliability, instead, assumes that some failures, so-called primary failures, promote the emergence of secondary and tertiary faults, with a cascading effect. In this text we will only deal with static models of reliability. faults. Dynamic reliability, instead, assumes that some failures, so-called primary failures, promote the emergence of secondary and tertiary faults, with a cascading effect. In this text we will only deal with static models of reliability. In the traditional paradigm of static reliability, individual components have a binary status: either working or failed. Systems, in turn, are composed by an integer number n of compo‐ nents, all mutually independent. 2. Reliability basics Depending on how the components are configured in creating the system and according to the operation or failure of individual components, the system either works or does not work. Let’s consider a generic X system consisting of nelements. The static reliability modeling implies that the operating status of the i - th component is represented by the state function Xi defined as: Xi ={ 1 if the i - th component works 0 if the i - th component fails (1) Xi ={ 1 if the i - th component works 0 if the i - th component fails (1) (1) The state of operation of the system is modeled by the state function Φ(X ) Φ(X )={ 1 if the system works 0 if the system fails (2) Φ(X )={ 1 if the system works 0 if the system fails (2) (2) The most common configuration of the components is the series system. A series system works if and only if all components work. Therefore, the status of a series system is given by the state function: Φ(X )=∏ i=1 n Xi = min i∈{1,2,…,n} Xi (3) (3) where the symbol ∏ indicates the product of the arguments. where the symbol ∏ indicates the product of the arguments. where the symbol ∏ indicates the product of the arguments. System configurations are often represented graphically with Reliability Block Diagrams (RBDs) where each component is represented by a block and the connections between them express the configuration of the system. The operation of the system depends on the ability to cross the diagram from left to right only by passing through the elements in operation. Figure 1 contains the RBD of a four components series system. Figure 1. Reliability block diagram for a four components (1,2,3,4) series system. Figure 1. Reliability block diagram for a four components (1,2,3,4) series system. Operations Management 84 The second most common configuration of the components is the parallel system. A parallel system works if and only if at least one component is working. A parallel system does not work if and only if all components do not work. So, if Φ - (X ) is the function that represents the state of not functioning of the system and X - i indicates the non-functioning of the i - th element, you can write: The second most common configuration of the components is the parallel system. 2. Reliability basics A parallel system works if and only if at least one component is working. A parallel system does not - work if and only if all components do not work. So, if Φ(X ) is the function that represents the t t f t f ti i f th t d X - i di t th f ti i f th i th l t state of not functioning of the system and X i indicates the non-functioning of the i - th element, you can write: Φ - (X )=∏ i=1 n X - i (4) (4) Accordingly, the state of a parallel system is given by the state function: Accordingly, the state of a parallel system is given by the state function: Φ(X )=1 - ∏ i=1 n (1 - X i)=∐ i=1 n Xi = max i∈{1,2,…,n} Xi (5) (5) (5) where the symbol ∐ indicates the complement of the product of the complements of the arguments. Figure 2 contains a RBD for a system of four components arranged in parallel. where the symbol ∐ indicates the complement of the product of the complements of the arguments. Figure 2 contains a RBD for a system of four components arranged in parallel. Figure 2. Parallel system. The image represents the RBD of a system of four elements (1,2,3,4) arranged in a reliability parallel configuration. Figure 2. Parallel system. The image represents the RBD of a system of four elements (1,2,3,4) arranged in a reliability parallel configuration. Another common configuration of the components is the series-parallel systems. In these systems, components are configured using combinations in series and parallel configurations. An example of such a system is shown in Figure 3. State functions for series-parallel systems are obtained by decomposition of the system. With this approach, the system is broken down into subsystems or configurations that are in series or in parallel. The state functions of the subsystems are then combined appropriately, de‐ pending on how they are configured. A schematic example is shown in Figure 4. Reliability and Maintainability in Operations Management http://dx.doi.org/10.5772/54161 85 Figure 3. Series-parallel system. The picture shows the RBD of a system due to the series-parallel model of 9 elementa‐ ry units. Figure 3. Series-parallel system. The picture shows the RBD of a system due to the series-parallel model of 9 elementa‐ ry units. Figure 4. 2. Reliability basics Calculation of the state function of a series-parallel. Referring to the configuration of Figure 3, the state function of the system is calculated by first making the state functions of the parallel of{1,2}, of {3,4, 5} and of {6,7, 8 , 9}. Then we evaluate the state function of the series of the three groups just obtained. Figure 4. Calculation of the state function of a series-parallel. Referring to the configuration of Figure 3, the state function of the system is calculated by first making the state functions of the parallel of{1,2}, of {3,4, 5} and of {6,7, 8 , 9}. Then we evaluate the state function of the series of the three groups just obtained. Figure 4. Calculation of the state function of a series-parallel. Referring to the configuration of Figure 3, the state function of the system is calculated by first making the state functions of the parallel of{1,2}, of {3,4, 5} and of {6,7, 8 , 9}. Then we evaluate the state function of the series of the three groups just obtained. 86 Operations Management A particular component configuration, widely recognized and used, is the parallel kout ofn. A system kout of nworks if and only if at least kof the ncomponents works. Note that a series A particular component configuration, widely recognized and used, is the parallel kout ofn. A system kout of nworks if and only if at least kof the ncomponents works. Note that a series system can be seen as a system nout of nand a parallel system is a system 1 out ofn. The state function of a system kout of nis given by the following algebraic system: A system kout of nworks if and only if at least kof the ncomponents works. Note that a series system can be seen as a system nout of nand a parallel system is a system 1 out ofn. The state function of a system kout of nis given by the following algebraic system: Φ(X )={ 1 if ∑ i=1 n Xi ≥k 0 otherwise (6) Φ(X )={ 1 if ∑ i=1 n Xi ≥k 0 otherwise (6) (6) The RBD for a system kout of nhas an appearance identical to the RBD schema of a parallel system of ncomponents with the addition of a label "k out of n". 2. Reliability basics For other more complex system configurations, such as the bridge configuration (see Figure 5), we may use more intricate techniques such as the minimal path set and the minimal cut set, to construct the system state function. A Minimal Path Set - MPS is a subset of the components of the system such that the operation of all the components in the subset implies the operation of the system. The set is minimal because the removal of any element from the subset eliminates this property. An example is shown in Figure 5. Figure 5. Minimal Path Set. The system on the left contains the minimal path set indicated by the arrows and shown in the right part. Each of them represents a minimal subset of the components of the system such that the operation of all the components in the subset implies the operation of the system. Figure 5. Minimal Path Set. The system on the left contains the minimal path set indicated by the arrows and shown in the right part. Each of them represents a minimal subset of the components of the system such that the operation of all the components in the subset implies the operation of the system. A Minimal Cut Set - MCS is a subset of the components of the system such that the failure of all components in the subset does not imply the operation of the system. Still, the set is called min‐ imal because the removal of any component from the subset clears this property (see Figure 6). MCS and MPS can be used to build equivalent configurations of more complex systems, not referable to the simple series-parallel model. The first equivalent configuration is based on the consideration that the operation of all the components, in at least a MPS, entails the operation Figure 6. Minimal Cut Set. The system of the left contains the minimal cut set, indicated by the dashed lines, shown in the right part. Each of them represents a minimum subset of the components of the system such that the failure of all components in the subset does not imply the operation of the system. Reliability and Maintainability in Operations Management http://dx.doi.org/10.5772/54161 Reliability and Maintainability in Operations Management http://dx.doi.org/10.5772/54161 87 Reliability and Maintainability in Operations Management http://dx.doi.org/10.5772/54161 87 Figure 6. Minimal Cut Set. 2. Reliability basics The system of the left contains the minimal cut set, indicated by the dashed lines, shown in the right part. Each of them represents a minimum subset of the components of the system such that the failure of all components in the subset does not imply the operation of the system. of the system. This configuration is, therefore, constructed with the creation of a series subsystem for each path using only the minimum components of that set. Then, these subsys‐ tems are connected in parallel. An example of an equivalent system is shown in Figure 7. 1 2 1 3 5 4 3 2 4 5 4 1 5 2 3 1 2 1 3 5 4 3 2 4 5 Figure 7. Equivalent configurations with MPS. You build a series subsystem for each MPS. Then such subsystems are connected in parallel. 4 1 5 2 3 Figure 7. Equivalent configurations with MPS. You build a series subsystem for each MPS. Then such subsystems are connected in parallel. The second equivalent configuration, is based on the logical principle that the failure of all the components of any MCS implies the fault of the system. This configuration is built with the creation of a parallel subsystem for each MCS using only the components of that group. Then, these subsystems are connected in series (see Figure 8). After examining the components and the status of the system, the next step in the static modeling of reliability is that of considering the probability of operation of the component and of the system. Operations Management 88 Figure 8. Equivalent configurations with MCS. You build a subsystem in parallel for each MCS. Then the subsystems are connected in series. Figure 8. Equivalent configurations with MCS. You build a subsystem in parallel for each MCS. Then the subsystems are connected in series. The reliability Ri of the i - th component is defined by: Ri = P(Xi =1) (7) (7) while the reliability of the systemR is defined as in equation 8: while the reliability of the systemR is defined as in equation 8: R = P(Φ(X )=1) (8) (8) The methodology used to calculate the reliability of the system depends on the configuration of the system itself. 2. Reliability basics For a series system, the reliability of the system is given by the product of the individual reliability (law of Lusser, defined by German engineer Robert Lusser in the 50s): R =∏ i=1 n Ri since R = P(⋂ i=1 n (Xi =1))=∏ i=1 n P(Xi =1)=∏ i=1 n Ri (9) For an example, see Figure 9. Figure 9. serial system consisting of 4 elements with reliability equal to 0.98, 0.99, 0.995 and 0.975. The reliability of the whole system is given by their product: R = 0.98 · 0.99 · 0.995 · 0.975 = 0.941 R =∏ i=1 n Ri since R = P(⋂ i=1 n (Xi =1))=∏ i=1 n P(Xi =1)=∏ i=1 n Ri (9) (9) For an example, see Figure 9. Figure 9. serial system consisting of 4 elements with reliability equal to 0.98, 0.99, 0.995 and 0.975. The reliability of the whole system is given by their product: R = 0.98 · 0.99 · 0.995 · 0.975 = 0.941 Figure 9. serial system consisting of 4 elements with reliability equal to 0.98, 0.99, 0.995 and 0.975. The reliability of the whole system is given by their product: R = 0.98 · 0.99 · 0.995 · 0.975 = 0.941 For a parallel system, reliability is: Reliability and Maintainability in Operations Management http://dx.doi.org/10.5772/54161 8 Reliability and Maintainability in Operations Management http://dx.doi.org/10.5772/54161 89 89 R =1 - ∏ i=1 n (1 - Ri)=∐ i=1 n Ri (10) (10) In fact, from the definition of system reliability and by the properties of event probabilities, it follows: In fact, from the definition of system reliability and by the properties of event probabilities, it follows: R = P(⋃ i=1 n (Xi =1))=1 - P(⋂ i=1 n (Xi =0))=1 - ∏ i=1 n P(Xi =0)= =1 - ∏ i=1 n 1 - P(Xi =1) =1 - ∏ i=1 n (1 - Ri)=∐ i=1 n Ri (11) (11) In many parallel systems, components are identical. In this case, the reliability of a parallel system with n elements is given by: In many parallel systems, components are identical. In this case, the reliability of a parallel system with n elements is given by: R =1 - (1 - Ri)n (12) Figure 10. A parallel system consisting of 4 elements with the same reliability of 0.85. The system reliability s given by their co-product: 1 - (1 - 0.85)4=0.9995. 2. Reliability basics R =1 - (1 - Ri)n (12) R =1 - (1 - Ri)n (12) Figure 10. A parallel system consisting of 4 elements with the same reliability of 0.85. The system reliability s given by their co-product: 1 - (1 - 0.85)4=0.9995. For a series-parallel system, system reliability is determined using the same approach of decomposition used to construct the state function for such systems. Consider, for instance, the system drawn in Figure 11, consisting of 9 elements with reliability R1= R2=0.9; R3= R4= R5=0.8 and R6= R7= R8= R9=0.7. Let’s calculate the overall reliability of the system. system. system. Operations Management 90 Figure 11. The system consists of three groups of blocks arranged in series. Each block is, in turn, formed by elements in parallel. First we must calculate R1,2=1 - (1 - 0.8)2=0.99. So it is possible to estimated R3,4,5=1 - (1 - 0.8)3=0.992. Then we must calculate the reliability of the last parallel block R6,7,8,9=1 - (1 - 0.7)4=0.9919. Finally, we proceed to the series of the three blocks: R = R1,2∙R3,4,5∙R6,7,8,9=0.974. Figure 11. The system consists of three groups of blocks arranged in series. Each block is, in turn, formed by elements in parallel. First we must calculate R1,2=1 - (1 - 0.8)2=0.99. So it is possible to estimated R3,4,5=1 - (1 - 0.8)3=0.992. Then we must calculate the reliability of the last parallel block R6,7,8,9=1 - (1 - 0.7)4=0.9919. Finally, we proceed to the series of the three blocks: R = R1,2∙R3,4,5∙R6,7,8,9=0.974. To calculate the overall reliability, for all other types of systems which can’t be brought back to a series-parallel scheme, it must be adopted a more intensive calculation approach [3] that is normally done with the aid of special software. Reliability functions of the system can also be used to calculate measures of reliability importance. Reliability functions of the system can also be used to calculate measures of reliability importance. These measurements are used to assess which components of a system offer the greatest opportunity to improve the overall reliability. The most widely recognized definition of reliability importance I ' i of the components is the reliability marginal gain, in terms of overall system rise of functionality, obtained by a marginal increase of the component reliability: I ' i = ∂R ∂Ri (13) (13) For other system configurations, an alternative approach facilitates the calculation of reliability importance of the components. 2. Reliability basics Let R(1i)be the reliability of the system modified so that Ri =1 and R(0i)be the reliability of the system modified withRi =0, always keeping unchanged the other components. In this context, the reliability importance Ii is given by: Ii = R(1i) - R(0i) (14) (14) In a series system, this formulation is equivalent to writing: es system, this formulation is equivalent to writing: Reliability and Maintainability in Operations Management http://dx.doi.org/10.5772/54161 Reliability and Maintainability in Operations Management http://dx.doi.org/10.5772/54161 Reliability and Maintainability in Operations Management http://dx.doi.org/10.5772/54161 91 Ii = ∏ j=1 j≠i n R j (15) (15) Thus, the most important component (in terms of reliability) in a series system is the less reliable. For example, consider three elements of reliability R1=0.9, R2=0.8 e R3=0.7. It is therefore: I1=0.8∙0.7=0.56, I2=0.9∙0.7=0.63 and I3=0.9 · 0.8=0.72 which is the higher value. If the system is arranged in parallel, the reliability importance becomes as follows: Ii = ∏ j=1 j≠i n (1 - R j) (16) (16) It follows that the most important component in a parallel system is the more reliable. With the same data as the previous example, this time having a parallel arrangement, we can verify Eq. 16 for the first item: I1= R(11) - R(01)= 1 - (1 - 1) · (1 - 0.8)∙(1 - 0.7) - 1 - (1 - 0) · (1 - 0.8)∙(1 - 0.7) =1 - 0 - 1 + (1 - 0.8)∙(1 - 0.7)=(1 - 0.8)∙(1 - 0.7). For the calculation of the reliability importance of components belonging to complex systems, which are not attributable to the series-parallel simple scheme, reliability of different systems must be counted. For this reason the calculation is often done using automated algorithms. 3. Fleet reliability Suppose you have studied the reliability of a component, and found that it is 80% for a mission duration of 3 hours. Knowing that we have 5 identical items simultaneously active, we might be interested in knowing what the overall reliability of the group would be. In other words, we want to know what is the probability of having a certain number of items functioning at the end of the 3 hours of mission. This issue is best known as fleet reliability. Consider a set of midentical and independent systems in a same instant, each having a reliabilityR. The group may represent a set of systems in use, independent and identical, or could represent a set of devices under test, independent and identical. A discrete random variable of great interest reliability isN , the number of functioning items. Under the assump‐ tions specified, N is a binomial random variable, which expresses the probability of a Bernoulli process. The corresponding probabilistic model is, therefore, the one that describes the extraction of balls from an urn filled with a known number of red and green balls. Suppose that the percentage Rof green balls is coincident with the reliability after 3 hours. After each extraction from the urn, the ball is put back in the container. Extraction is repeated mtimes, and we look for the probability of finding ngreen. The sequence of random variables thus obtained is a Bernoulli process of which each extraction is a test. Since the probability of obtaining N successes in mextractions from an urn, with restitution of the ball, follows the binomial distribution B(m, R)B, the probability mass function of N is the well-known: Operations Management 2 92 P(N =n)= m ! n ! (m - n) ! R n(1 - R)m-n (17) (17) The expected value of N is given by: E(N )=μN =m∙Rand the standard deviation is: σN = m∙R ∙(1 - R). σN = m∙R ∙(1 - R). Let’s consider, for example, a corporate fleet consisting of 100 independent and identical systems. All systems have the same mission, independent from the other missions. Each system has a reliability of mission equal to 90%. We want to calculate the average number of missions completed and also what is the probability that at least 95% of systems would complete their mission. 4. Time dependent reliability models When reliability is expressed as a function of time, the continuous random variable, not negative, of interest is T , the instant of failure of the device. Let f (t) be the probability density function of T , and let F(t) be the cumulative distribution function of T . F(t) is also known as failure function or unreliability function [4]. In the context of reliability, two additional functions are often used: the reliability and the hazard function. Let’s define Reliability R(t)as the survival function: R(t)= P(T ≥t)=1 - F(t) (19) (19) The Mean Time To Failure - MTTF is defined as the expected value of the failure time: MTTF = E(T )=∫0 ∞t ∙f (t)∙dt (20) (20) Integrating by parts, we can prove the equivalent expression: Integrating by parts, we can prove the equivalent expression: Integrating by parts, we can prove the equivalent expression: MTTF = E(T )=∫0 ∞R(t)∙dt (21) (21) 3. Fleet reliability This involves analyzing the distribution of the binomial random variable character‐ ized by R = 0.90andm = 100. The expected value is given by E(N )=μN =100∙0.9=90. The probability that at least 95% of the systems complete their mission can be calculated as the sum of the probabilities that complete their mission 95, 96, 97, 98, 99 and 100 elements of the fleet: P(N ≥n)= ∑ n=95 100 m ! n ! (m - n) ! R n(1 - R)m-n =0,058 (18) (18) 5. Hazard function Another very important function is the hazard function, denoted by λ(t), defined as the trend of the instantaneous failure rate at time t of an element that has survived up to that time t. The Another very important function is the hazard function, denoted by λ(t), defined as the trend of the instantaneous failure rate at time t of an element that has survived up to that time t. The Reliability and Maintainability in Operations Management http://dx.doi.org/10.5772/54161 93 failure rate is the ratio between the instantaneous probability of failure in a neighborhood of t- conditioned to the fact that the element is healthy in t- and the amplitude of the same neighborhood. The hazard function λ(t) [5] coincides with the intensity function z(t) of a Poisson process. The hazard function is given by: λ(t)= lim Δt→0 P(t ≤T < t + ∆t \| T ≥t) ∆t (22) (22) Thanks to Bayes' theorem, it can be shown that the relationship between the hazard function, density of probability of failure and reliability is the following: Thanks to Bayes' theorem, it can be shown that the relationship between the hazard function, density of probability of failure and reliability is the following: λ(t)= f (t) R(t) (23) λ(t)= f (t) R(t) (23) (23) Thanks to the previous equation, with some simple mathematical manipulations, we obtain the following relation: Thanks to the previous equation, with some simple mathematical manipulations, we obtain the following relation: R(t)=e -∫ 0 t λ(u)∙du (24) R(t)=e -∫ 0 t λ(u)∙du (24) (24) In fact, since ln R(0) =ln 1 =0, we have: In fact, since ln R(0) =ln 1 =0, we have: In fact, since ln R(0) =ln 1 =0, we have: R(t)= f (t) λ(t) = 1 λ(t) ∙ dF (t) dt = - 1 λ(t) ∙ dR(t) dt → 1 R(t) dR(t)= - λ(t)dt →ln R(t) - ln R(0) = - ∫0 tλ(u)du (25) (25) From equation 24 derive the other two fundamental relations: From equation 24 derive the other two fundamental relations: F(t)=1 - e -∫ 0 t λ(u)∙du f (t)=λ(t)∙e -∫ 0 t λ(u)∙du (26) (26) The most popular conceptual model of the hazard function is the bathtub curve. According to this model, the failure rate of the device is relatively high and descending in the first part of the device life, due to the potential manufacturing defects, called early failures. 5. Hazard function They manifest themselves in the first phase of operation of the system and their causes are often linked to structural deficiencies, design or installation defects. In terms of reliability, a system that manifests infantile failures improves over the course of time. Later, at the end of the life of the device, the failure rate increases due to wear phenomena. They are caused by alterations of the component for material and structural aging. The beginning of the period of wear is identified by an increase in the frequency of failures which continues as time goes by. The wear-out failures occur around the average age of operating; the only way to avoid this type of failure is to replace the population in advance. Operations Management 94 Between the period of early failures and of wear-out, the failure rate is about constant: failures are due to random events and are called random failures. They occur in non-nominal operating conditions, which put a strain on the components, resulting in the inevitable changes and the consequent loss of operational capabilities. This type of failure occurs during the useful life of the system and corresponds to unpredictable situations. The central period with constant failure rate is called useful life. The juxtaposition of the three periods in a graph which represents the trend of the failure rate of the system, gives rise to a curve whose characteristic shape recalls the section of a bathtub, as shown in Figure 12. Between the period of early failures and of wear-out, the failure rate is about constant: failures are due to random events and are called random failures. They occur in non-nominal operating conditions, which put a strain on the components, resulting in the inevitable changes and the consequent loss of operational capabilities. This type of failure occurs during the useful life of the system and corresponds to unpredictable situations. The central period with constant failure rate is called useful life. The juxtaposition of the three periods in a graph which represents the trend of the failure rate of the system, gives rise to a curve whose characteristic shape recalls the section of a bathtub, as shown in Figure 12. Figure 12. Bathtub curve. The hazard function shape allows us to identify three areas: the initial period of the early failures, the middle time of the useful life and the final area of wear-out. Figure 12. Bathtub curve. 5. Hazard function The function is defined as follows: Reliability and Maintainability in Operations Management http://dx.doi.org/10.5772/54161 95 R(t + t0\|t0)= P(T >t + t0\|T >t0) R(t + t0\|t0)= P(T >t + t0\|T >t0) (27) (27) Applying Bayes' theorem we have: Applying Bayes' theorem we have: Applying Bayes' theorem we have: P(T >t + t0\|T >t0)= P(T > t0 \| T > t + t0) ∙P(T > t + t0) P(T > t0) (28) (28) And, given that P(T >t0\|T >t + t0)=1, we obtain the final expression, which determines the residual reliability: P(T >t0\|T >t + t0)=1, we obtain the final expression, which determines the ty: And, given that P(T >t0\|T >t + t0)=1, we obtain the final expression, which determines the residual reliability: R(t + t0\|t0)= R(t + t0) R(t0) (29) (29) The residual Mean Time To Failure – residual MTTF measures the expected value of the residual life of a device that has already survived a time t0: The residual Mean Time To Failure – residual MTTF measures the expected value of the residual life of a device that has already survived a time t0: (30) MTTF(t0)= E(T - t0\|T >t0)=∫0 ∞R(t + t0\|t0)∙dt (30) For an IFR device, the residual reliability and the residual MTTF, decrease progressively as the device accumulates hours of operation. This behavior explains the use of preventive actions to avoid failures. For a DFR device, both the residual reliability and the residual MTTF increase while the device accumulates hours of operation. This behavior motivates the use of an intense running (burn-in) to avoid errors in the field. The Mean Time To Failure –MTTF, measures the expected value of the life of a device and coincides with the residual time to failure, where t0=0. 5. Hazard function The hazard function shape allows us to identify three areas: the initial period of the early failures, the middle time of the useful life and the final area of wear-out. The most common mathematical classifications of the hazard curve are the so called Constant Failure Rate - CFR, Increasing Failure Rate - IFR and Decreasing Failure Rate - DFR. The most common mathematical classifications of the hazard curve are the so called Constant Failure Rate - CFR, Increasing Failure Rate - IFR and Decreasing Failure Rate - DFR. The CFR model is based on the assumption that the failure rate does not change over time. Mathematically, this model is the most simple and is based on the principle that the faults are purely random events. The IFR model is based on the assumption that the failure rate grows up over time. The model assumes that faults become more likely over time because of wear, as is frequently found in mechanical components. The DFR model is based on the assumption that the failure rate decreases over time. This model assumes that failures become less likely as time goes by, as it occurs in some electronic components. Since the failure rate may change over time, one can define a reliability parameter that behaves as if there was a kind of counter that accumulates hours of operation. The residual reliabili‐ ty function R(t + t0\|t0), in fact, measures the reliability of a given device which has already survived a determined time t0. 5. Hazard function In this case we have the following relationship: MTTF =MTTF(0)= E(T \|T >0)=∫0 ∞R(t)∙dt (31) MTTF =MTTF(0)= E(T \|T >0)=∫0 ∞R(t)∙dt (31) (31) aracteristic life of a device is the time tC corresponding to a reliability R(tC) equal to The characteristic life of a device is the time tC corresponding to a reliability R(tC) equal to 1 e, that is the time for which the area under the hazard function is unitary: The characteristic life of a device is the time tC corresponding to a reliability R(tC) equal to 1 e, that is the time for which the area under the hazard function is unitary: 1 e, that is the time for which the area under the hazard function is unitary: 1 e, that is the time for which the area under the hazard function is unitary: 1 e, that is the time for which the area under the hazard function is unitary: R(tC)=e -1=0,368 →R(tC)= ∫ 0 tC λ(u)∙du =1 (32) (32) Let us consider a CFR device with a constant failure rate λ. The time-to-failure is an exponential random variable. In fact, the probability density function of a failure, is typical of an expo‐ nential distribution: Let us consider a CFR device with a constant failure rate λ. The time-to-failure is an exponential random variable. In fact, the probability density function of a failure, is typical of an expo‐ nential distribution: Operations Management 96 Operations Management 6 f (t)=λ(t)∙e -∫ 0 t λ(u)∙du =λe-λ∙t (33) f (t)=λ(t)∙e -∫ 0 t λ(u)∙du =λe-λ∙t (33) (33) The corresponding cumulative distribution function F(t)is: The corresponding cumulative distribution function F(t)is: F(t)= ∫ -∞ t f (z)dz = ∫ -∞ t λe-λ∙zdz =1 - e -λ∙t (34) The reliability function R(t)is the survival function: R(t)=1 - F(t)=e -λ∙t (35) (34) The reliability function R(t)is the survival function: The reliability function R(t)is the survival function: R(t)=1 - F(t)=e -λ∙t (35) (35) For CFR items, the residual reliability and the residual MTTF both remain constant when the device accumulates hours of operation. In fact, from the definition of residual reliability, ∀t0∈0, ∞, we have: For CFR items, the residual reliability and the residual MTTF both remain constant when the device accumulates hours of operation. 5. Hazard function In fact, from the definition of residual reliability, ∀t0∈0, ∞, we have: R(t + t0\|t0)= R(t + t0) R(t0) = e -λ∙(t +t0) e -λ∙t0 =e -λ∙(t+t0)+λ∙t0=e -λ∙t = R(t) (36) (36) Similarly, for the residual MTTF, is true the invariance in time: the residual MTTF, is true the invariance in time: Similarly, for the residual MTTF, is true the invariance in time: MTTF(t0)=∫0 ∞R(t + t0\|t0)∙dt =∫0 ∞R(t)∙dt ∀t0∈0, ∞ (37) (37) This behavior implies that the actions of prevention and running are useless for CFR devices. Figure 13 shows the trend of the function f (t)=λ ∙e -λ∙t and of the cumulative distribution function F(t)=1 - e -λ∙t for a constant failure rate λ =1. In this case, since λ =1, the probability density function and the reliability function, overlap: f (t)= R(t)=e -t. This behavior implies that the actions of prevention and running are useless for CFR devices. Figure 13 shows the trend of the function f (t)=λ ∙e -λ∙t and of the cumulative distribution function F(t)=1 - e -λ∙t for a constant failure rate λ =1. In this case, since λ =1, the probability density function and the reliability function, overlap: f (t)= R(t)=e -t. The probability of having a fault, not yet occurred at time t, in the next dt, can be written as follows: P(t <T <t + dt \|T >t) (38) P(t <T <t + dt \|T >t) (38) (38) Recalling the Bayes' theorem, in which we consider the probability of an hypothesis H, being known the evidence E: Recalling the Bayes' theorem, in which we consider the probability of an hypothesis H, being known the evidence E: P(H \| E)= P(E \| H ) ∙P(H ) P(E) (39) (39) we can replace the evidence E with the fact that the fault has not yet taken place, from which we obtain P(E)→P(T >t). We also exchange the hypothesis H with the occurrence of the fault in the neighborhood of t, obtaining P(H )→ P(t <T <t + dt). So we get: we can replace the evidence E with the fact that the fault has not yet taken place, from which we obtain P(E)→P(T >t). We also exchange the hypothesis H with the occurrence of the fault in the neighborhood of t, obtaining P(H )→ P(t <T <t + dt). So we get: Reliability and Maintainability in Operations Management http://dx.doi.org/10.5772/54161 97 Figure 13. 5. Hazard function Probability density function and cumulative distribution of an exponential function. In the figure is seen the trend of f (t)=λ ∙e -λ∙t and of f (t)=λ ∙e -λ∙t with λ =1. Figure 13. Probability density function and cumulative distribution of an exponential function. In the figure is seen Figure 13. Probability density function and cumulative distribution of an exponential function. In the figure is seen the trend of f (t)=λ ∙e -λ∙t and of f (t)=λ ∙e -λ∙t with λ =1. P(t <T <t + dt \|T >t)= P(T > t \| t < T < t + dt) ∙P(t < T < t + dt) P(T > t) (40) (40) Since P(T >t \|t <T <t + dt)=1, being a certainty, it follows: Since P(T >t \|t <T <t + dt)=1, being a certainty, it follows: Since P(T >t \|t <T <t + dt)=1, being a certainty, it follows: P(t <T <t + dt \|T >t)= P(t < T < t + dt) P(T > t) = f (t)dt e -λ∙t = λe -λ∙tdt e -λ∙t =λ ∙dt (41) (41) As can be seen, this probability does not depend on t, i.e. it is not function of the life time already elapsed. It is as if the component does not have a memory of its own history and it is for this reason that the exponential distribution is called memoryless. The use of the constant failure rate model, facilitates the calculation of the characteristic life of a device. In fact for a CFR item, tCis the reciprocal of the failure rate. In fact: R(tC)=e -λ∙tC =e -1 →tC = 1 λ (42) (42) Therefore, the characteristic life, in addition to be calculated as the time value tC for which the reliability is 0.368, can more easily be evaluated as the reciprocal of the failure rate. Therefore, the characteristic life, in addition to be calculated as the time value tC for which the reliability is 0.368, can more easily be evaluated as the reciprocal of the failure rate. The definition of MTTF, in the CFR model, can be integrated by parts and give: MTTF =∫0 ∞R(t)∙dt =∫0 ∞e -λ∙t ∙dt =- 1 λ e -λ∙t\| ∞ 0 = - 0 λ + 1 λ = 1 λ (43) (43) In the CFR model, then, the MTTF and the characteristic life coincide and are equal to 1 λ. 5. Hazard function Operations Management 98 Let us consider, for example, a component with constant failure rate equal to λ =0.0002failures per hour. We want to calculate the MTTF of the component and its reliability after 10000 hours of operation. We’ll calculate, then, what is the probability that the component survives other 10000 hours. Assuming, finally, that it has worked without failure for the first 6000 hours, we’ll calculate the expected value of the remaining life of the component. From equation 43 we have: MTTF = 1 λ = 1 0.0002 failures h =5000 h (44) For the law of the reliability R(t)=e -λ∙t, you get the reliability at 10000 hours: From equation 43 we have: MTTF = 1 λ = 1 0.0002 failures h =5000 h (44) For the law of the reliability R(t)=e -λ∙t, you get the reliability at 10000 hours: From equation 43 we have: ave: MTTF = 1 λ = 1 0.0002 failures h =5000 h (44) (44) For the law of the reliability R(t)=e -λ∙t, you get the reliability at 10000 hours: R(10000)=e -0.0002∙10000=0.135 (45) R(10000)=e -0.0002∙10000=0.135 (45) The probability that the component survives other 10000 hours, is calculated with the residual reliability. Knowing that this, in the model CFR, is independent from time, we have: R(t + t0\|t0)= R(t)→R(20000\|10000)= R(10000)=0.135 (46) (46) Suppose now that it has worked without failure for 6000 hours. The expected value of the residual life of the component is calculated using the residual MTTF, that is invariant. In fact: MTTF(t0)=∫0 ∞R(t + t0\|t0)∙dt →MTTF(6000)=∫0 ∞R(t + 6000\|6000)∙dt =∫0 ∞R(t)∙dt =MTTF =5000 h (47) (47) 6. CFR in series Let us consider n different elements, each with its own constant failure rate λi and reliability Ri =e -λi∙t, arranged in series and let us evaluate the overall reliability RS. From equation 9 we have: RS =∏ i=1 n Ri =∏ i=1 n e -λi∙t =e -∑ i=1 n λi∙t (48) (48) Since the reliability of the overall system will take the form of the type RS =e -λs∙t, we can conclude that: Since the reliability of the overall system will take the form of the type RS =e -λs∙t, we can conclude that: RS =e -∑ i=1 n λi∙t =e -λs∙t →λs = ∑ i=1 n λi (49) (49) Reliability and Maintainability in Operations Management http://dx.doi.org/10.5772/54161 99 In a system of CFR elements arranged in series, then, the failure rate of the system is equal to the sum of failure rates of the components. The MTTF can thus be calculated using the simple relation: In a system of CFR elements arranged in series, then, the failure rate of the system is equal to the sum of failure rates of the components. The MTTF can thus be calculated using the simple relation: MTTF = 1 λs = 1 ∑ i=1 n λi (50) (50) For example, let me show the following example. A system consists of a pump and a filter, used to separate two parts of a mixture: the concentrate and the squeezing. Knowing that the failure rate of the pump is constant and is λP =1,5∙10-4 failures per hour and that the failure rate of the filter is also CFR and is λF =3∙10-5, let’s try to assess the failure rate of the system, the MTTF and the reliability after one year of continuous operation. To begin, we compare the physical arrangement with the reliability one, as represented in the following figure: Figure 14. physical and reliability modeling of a pump and a filter producing orange juice. Figure 14. physical and reliability modeling of a pump and a filter producing orange juice. Figure 14. physical and reliability modeling of a pump and a filter producing orange juice. 6. CFR in series As can be seen, it is a simple series, for which we can write: λs = ∑ i=1 n λi =λP + λF =1.8∙10-4 failures h (51) As can be seen, it is a simple series, for which we can write: λs = ∑ i=1 n λi =λP + λF =1.8∙10-4 failures h (51) As can be seen, it is a simple series, for which we can write: simple series, for which we can write: λs = ∑ i=1 n λi =λP + λF =1.8∙10-4 failures h (51) (51) MTTF is the reciprocal of the failure rate and can be written: MTTF is the reciprocal of the failure rate and can be written: MTTF = 1 λs = 1 1.8 ∙10-4 =5,555 h (52) (52) As a year of continuous operation is 24 · 365=8,760 hours, the reliability after one year is: As a year of continuous operation is 24 · 365=8,760 hours, the reliability after one year is: RS =e -λs∙t =e -1.8∙10-4·8760=0.2066 As a year of continuous operation is 24 · 365=8,760 hours, the reliability after one year RS =e -λs∙t =e -1.8∙10-4·8760=0.2066 -λs∙t =e -1.8∙10-4·8760=0.2066 RS =e -λs∙t =e -1.8∙10-4·8760=0.2066 RS =e -λs∙t =e -1.8∙10-4·8760=0.2066 100 Operations Management 7. CFR in parallel It is a parallel arrangement, for which the following equation is applicable It is a parallel arrangement, for which the following equation is applicable: It is a parallel arrangement, for which the following equation is applicable: Reliability and Maintainability in Operations Management http://dx.doi.org/10.5772/54161 101 101 λP = 2 3 λ = 2 3 9∙10-6=6∙10-6 guasti h (56) (56) The MTTF is the reciprocal of the failure rate and is: The MTTF is the reciprocal of the failure rate and is: MTTF = 1 λp = 1 6 ∙10-6 =166,666 h (57) (57) As a year of continuous operation is 24 · 365=8,760 hours, the reliability after one year is: As a year of continuous operation is 24 · 365=8,760 hours, the reliability after one year is: RP =e -λp∙t =e -6∙10-6·8,760=0.9488 (58) (58) It is interesting to calculate the reliability of a system of identical elements arranged in a parallel configuration k out of n. The system is partially redundant since a group of k elements is able to withstand the load of the system. The reliability is: It is interesting to calculate the reliability of a system of identical elements arranged in a parallel configuration k out of n. The system is partially redundant since a group of k elements is able to withstand the load of the system. The reliability is: Rk out of n = P(k ≤j ≤n)= ∑ j=k n ( n j )R j ∙(1 - R)n- j (59) (59) Let us consider, for example, three electric generators, arranged in parallel and with failure rate λ =9 · 10-6. In order for the system to be active, it is sufficient that only two items are in operation. Let’s get the reliability after one year of operation. Let us consider, for example, three electric generators, arranged in parallel and with failure rate λ =9 · 10-6. In order for the system to be active, it is sufficient that only two items are in operation. Let’s get the reliability after one year of operation. We’ll have: n =3, k =2. So, after a year of operation (t =8760 h ), reliability can be calculated as follows: R2 out of 3= ∑ j=2 3 ( 3 j )R j ∙(1 - R)3- j =( 3 2 )e-2λt(1 - e-λt)3-2 + ( 3 3 )e-λt(1 - e-λt)3-3= = 3 ! 2 ! (3 - 2) ! 7. CFR in parallel If two components arranged in parallel are similar and have constant failure rate λ, the reliability of the system RP can be calculated with equation 10, wherein RC is the reliability of the component RC =e -λt: RP =1 - ∏ i=1 2 (1 - Ri)=1 - (1 - R1)2=2RC - RC 2=2e-λ∙t - e-2λ∙t (53) (53) The calculation of the MTTF leads to MTTF = 3 2λ . In fact we have: The calculation of the MTTF leads to MTTF = 3 2λ . In fact we have: MTTF =∫0 ∞R(t)∙dt =∫0 ∞2e-λ∙t - e-2λ∙t ∙dt = - 2 λ e -λt + 1 2λ e -2λt\| ∞ 0 = 2 λ (0 - 1) + 1 2λ (0 - 1)= 3 2λ (54) (54) Therefore, the MTTF increases compared to the single component CFR. The failure rate of the parallel system λP, reciprocal of the MTTF, is: Therefore, the MTTF increases compared to the single component CFR. The failure rate of the parallel system λP, reciprocal of the MTTF, is: λP = 1 MTTF = 2 3 λ (55) (55) As you can see, the failure rate is not halved, but was reduced by one third. As you can see, the failure rate is not halved, but was reduced by one third. For example, let us consider a safety system which consists of two batteries and each one is able to compensate for the lack of electric power of the grid. The two generators are equal and have a constant failure rate λB =9∙10-6 failures per hour. We’d like to calculate the failure rate of the system, the MTTF and reliability after one year of continuous operation. As in the previous case, we start with a reliability block diagram of the problem, as visible in Figure 15. Figure 15. Physical and reliability modeling of an energy supply system. It is a parallel arrangement, for which the following equation is applicable: Figure 15. Physical and reliability modeling of an energy supply system. Thus, in parallel with stand-by, the MTTF is doubled.= Thus, in parallel with stand-by, the MTTF is doubled.= 7. CFR in parallel e -2λt ∙(1 - e -λt)3-2 + 3 ! 3 ! (3 - 3) ! e -λt ∙(1 - e -λt)3-3= =3∙e -2λt ∙(1 - e -λt)3-2 + 1∙e -λt ∙(1 - e -λt)3-3=0.963 A particular arrangement of components is that of the so-called parallel with stand-by: the second component comes into operation only when the first fails. Otherwise, it is idle. Figure 16. RBD diagram of a parallel system with stand-by. When component 1 fails, the switch S activates compo‐ nent 2. For simplicity, it is assumed that S is not affected by faults. Figure 16. RBD diagram of a parallel system with stand-by. When component 1 fails, the switch S activates compo‐ nent 2. For simplicity, it is assumed that S is not affected by faults. 102 Operations Management If the components are similar, then λ1=λ2. It’s possible to demonstrate that for the stand-by parallel system we have: If the components are similar, then λ1=λ2. It’s possible to demonstrate that for the stand-by parallel system we have: MTTF = 2 λ (60) MTTF = 2 λ (60) 8. Repairable systems The CBM approach consists of the following steps: • group the data from the sensors; Reliability and Maintainability in Operations Management http://dx.doi.org/10.5772/54161 103 • diagnose the condition; • estimate the Remaining Useful Life – RUL; • decide whether to maintain or to continue to operate normally • decide whether to maintain or to continue to operate normally. CBM schedule is modeled with algorithms aiming at high effectiveness, in terms of cost minimization, being subject to constraints such as, for example, the maximum time for the maintenance action, the periods of high production rate, the timing of supply of the pieces parts, the maximization of the availability and so on. In support of the prognosis, it is now widespread the use of diagrams that do understand, even graphically, when the sensor outputs reach alarm levels. They also set out the alert thresholds that identify ranges of values for which maintenance action must arise [9]. Starting from a state of degradation, detected by a measurement at the time tk, we calculate the likelihood that the system will still be functioning within the next instant of inspection tk+1. The choice to act with a preventive maintenance is based on the comparison of the expected value of the cost of unavailability, with the costs associated with the repair. Therefore, you create two scenarios: Starting from a state of degradation, detected by a measurement at the time tk, we calculate the likelihood that the system will still be functioning within the next instant of inspection tk+1. The choice to act with a preventive maintenance is based on the comparison of the expected value of the cost of unavailability, with the costs associated with the repair. Therefore, you create two scenarios: • continue to operate: if we are in the area of not alarming values. It is also possible that being in the area of preventive maintenance, we opt for a postponement of maintenance because it has already been established replacement intervention within a short interval of time • stop the task: if we are in the area of values above the threshold established for preventive maintenance of condition. The modeling of repairable systems is commonly used to evaluate the performance of one or more repairable systems and of the related maintenance policies. The information can also be used in the initial phase of design of the systems themselves. 8. Repairable systems The modeling of repairable systems is commonly used to evaluate the performance of one or more repairable systems and of the related maintenance policies. The information can also be used in the initial phase of design of the systems themselves. In the traditional paradigm of modeling, a repairable system can only be in one of two states: working (up) or inoperative (down). Note that a system may not be functioning not only for a fault, but also for preventive or corrective maintenance. 8. Repairable systems The devices for which it is possible to perform some operations that allow to reactivate the functionality, deserve special attention. A repairable system [6] is a system that, after the failure, can be restored to a functional condition from any action of maintenance, including replacement of the entire system. Maintenance actions performed on a repairable system can be classified into two groups: Corrective Maintenance - CM and Preventive Maintenance - PM. Corrective maintenance is performed in response to system errors and might correspond to a specific activity of both repair of replacement. Preventive maintenance actions, however, are not performed in response to the failure of the system to repair, but are intended to delay or prevent system failures. Note that the preventive activities are not necessarily cheaper or faster than the corrective actions. As corrective actions, preventive activities may correspond to both repair and replacement activities. Finally, note that the actions of operational maintenance (servicing) such as, for example, put gas in a vehicle, are not considered PM [7]. Preventative maintenance can be divided into two subcategories: scheduled and on-condi‐ tion. Scheduled maintenance (hard-time maintenance) consists of routine maintenance operations, scheduled on the basis of precise measures of elapsed operating time. Condition-Based Maintenance - CBM [8] (also known as predictive maintenance) is one of the most widely used tools for monitoring of industrial plants and for the management of maintenance policies. The main aim of this approach is to optimize maintenance by reduc‐ ing costs and increasing availability. In CBM it is necessary to identify, if it exists, a measur‐ able parameter, which expresses, with accuracy, the conditions of degradation of the system. What is needed, therefore, is a physical system of sensors and transducers capable of moni‐ toring the parameter and, thereby, the reliability performance of the plant. The choice of the monitored parameter is crucial, as is its time evolution that lets you know when mainte‐ nance action must be undertaken, whether corrective or preventive. To adopt a CBM policy requires investment in instrumentation and prediction and control systems: you must run a thorough feasibility study to see if the cost of implementing the apparatus are truly sustainable in the system by reducing maintenance costs. 9. Availability Availability may be generically be defined as the percentage of time that a repairable system is in an operating condition. However, in the literature, there are four specific measures of repairable system availability. We consider only the limit availability, defined with the limit of the probability A(t) that the system is working at time t, when t tends to infinity. A=lim t→∞A(t) (61) A=lim t→∞A(t) (61) The limit availability just seen is also called intrinsic availability, to distinguish it from the technical availability, which also includes the logistics cycle times incidental to maintenance The limit availability just seen is also called intrinsic availability, to distinguish it from the technical availability, which also includes the logistics cycle times incidental to maintenance Operations Management 104 actions (such as waiting for the maintenance, waiting for spare parts, testing...), and from the operational availability that encompasses all other factors that contribute to the unavailability of the system such as time of organization and preparation for action in complex and specific business context [10]. The models of the impact of preventive and corrective maintenance on the age of the compo‐ nent, distinguish in perfect, minimal and imperfect maintenance. Perfect maintenance (perfect repair) returns the system as good as new after maintenance. The minimal repair, restores the system to a working condition, but does not reduce the actual age of the system, leaving it as bad as old. The imperfect maintenance refers to maintenance actions that have an intermediate impact between the perfect maintenance and minimal repair. The average duration of maintenance activity is the expected value of the probability distri‐ bution of repair time and is called Mean Time To Repair - MTTR and is closely connected with the concept of maintainability. This consists in the probability of a system, in assigned operating conditions, to be reported in a state in which it can perform the required function. Figure 17 shows the state functions of two repairable systems with increasing failure rate, maintained with perfect and minimal repair. Figure 17. perfect maintenance vs minimal repair. In figure are represented the state functions of two systems both with IFR. Y (t) is equal to 1 when the system wotks, otherwise it’s 0. The left system is subject to a policy of perfect repair and shows homogeneous durations of the periods of operation. 9. Availability The right system adopts the minimal repair for which the durations of the periods of operation are reducing as time goes by. Figure 17. perfect maintenance vs minimal repair. In figure are represented the state functions of two systems both with IFR. Y (t) is equal to 1 when the system wotks, otherwise it’s 0. The left system is subject to a policy of perfect repair and shows homogeneous durations of the periods of operation. The right system adopts the minimal repair for which the durations of the periods of operation are reducing as time goes by. 11. The substitution model for CFR Let us consider the special case of the general substitution model where Ti is an exponential random variable with constant failure rate λ. Let also Di be an exponential random variable with constant repair rate μ. Since the reparable system has a constant failure rate (CFR), we know that aging and the impact of corrective maintenance are irrelevant on reliability performance. For this system it can be shown that the limit availability is: A= μ λ + μ (63) (63) Let us analyze, for example, a repairable system, subject to a replacement policy, with failure and repair times distributed according to negative exponential distribution. MTTF=1000 hours and MTTR=10 hours. Let’s calculate the limit availability of the system. The formulation of the limit availability in this system is given by eq. 63, so we have: A= μ λ + μ = 1 10 1 1000 + 1 10 = 0,1 0.101 =0.990 (64) (64) This means that the system is available for 99% of the time. This means that the system is available for 99% of the time. This means that the system is available for 99% of the time. 10. The general substitution model The general substitution model, states that the failure time of a repairable system is an unspecified random variable. The duration of corrective maintenance (perfect) is also a random variable. In this model it is assumed that preventive maintenance is not performed. Reliability and Maintainability in Operations Management http://dx.doi.org/10.5772/54161 105 Let’s denote by Ti the duration of the i - th interval of operation of the repairable system. For the assumption of perfect maintenance (as good as new), {T1, T2, …, Ti, …, Tn} is a sequence of independent and identically distributed random variables. Let’s denote by Ti the duration of the i - th interval of operation of the repairable system. For the assumption of perfect maintenance (as good as new), {T1, T2, …, Ti, …, Tn} is a sequence of independent and identically distributed random variables. Let us now designate with Di the duration of the i - th corrective maintenance action and assume that these random variables are independent and identically distributed. Therefore, each cycle (whether it is an operating cycle or a corrective maintenance action) has an identical probabilistic behavior, and the completion of a maintenance action coincides with time when system state returns operating Regardless of the probability distributions governing Ti and Di, the fundamental result of the general pattern of substitution is as follows: A= E(T i) E(T i) + E(Di) = MTTF MTTF + MTTR = MTTF MTBF (62) A= E(T i) E(T i) + E(Di) = MTTF MTTF + MTTR = MTTF MTBF (62) on model for CFR (62) 12. General model of minimal repair After examining the substitution model, we now want to consider a second model for repairable system: the general model of minimal repair. According to this model, the time of Operations Management 106 system failure is a random variable. Corrective maintenance is instantaneous, the repair is minimal, and not any preventive activity is performed. system failure is a random variable. Corrective maintenance is instantaneous, the repair is minimal, and not any preventive activity is performed. The times of arrival of faults, in a repairable system corresponding to the general model of minimal repair, correspond to a process of random experiments, each of which is regulated by the same negative exponential distribution. As known, having neglected the repair time, the number of faults detected by time t, {N (t), t ≥0}, is a non-homogeneous Poisson process, described by the Poisson distribution. 13. Minimal repair with CFR 13. Minimal repair with CFR A well-known special case of the general model of minimal repair, is obtained if the failure time T is a random variable with exponential distribution, with failure rate λ. In this case, the general model of minimal repair is simplified because the number E N (t) of faults that occur within the time t: {N (t), t ≥0} is described by a homogeneous Poisson process with intensity z(t)=λ, and is: E N (t) =μN (t)=Z(t)=∫ 0 t z(u)∙du =∫ 0 t λ ∙du =λt (65) (65) If, for example, we consider λ =0.1 faults/hour, we obtain the following values at time 100, 1000 and 10000: E N (100) =0,1∙100=10; E N (1000) =0,1∙1000=100; E N (10000) =0,1∙10000=1000. It If, for example, we consider λ =0.1 faults/hour, we obtain the following values at time 100, 1000 and 10000: E N (100) =0,1∙100=10; E N (1000) =0,1∙1000=100; E N (10000) =0,1∙10000=1000. It should be noted, as well, a linear trend of the expected number of failures given the width of the interval taken. and 10000: E N (100) =0,1∙100=10; E N (1000) =0,1∙1000=100; E N (10000) =0,1∙10000=1000. It should be noted, as well, a linear trend of the expected number of failures given the width of the interval taken. Finally, we can obtain the probability mass function of N (t), being a Poisson distribution: P N (t)=n = Z (t)n n ! e -Z (t)= (λt)n n ! e -λt (66) P N (t)=n = Z (t)n n ! e -Z (t)= (λt)n n ! e -λt (6 (66) Also, the probability mass function of N (t + s) - N (s), that is the number of faults in a range of amplitude t shifted forward of s, is identical: P N (t + s) - N (s)=n = (λt)n n ! e -λt (67) (67) Since the two values are equal, the conclusion is that in the homogeneous Poisson process (CFR), the number of faults in a given interval depends only on the range amplitude. Since the two values are equal, the conclusion is that in the homogeneous Poisson process (CFR), the number of faults in a given interval depends only on the range amplitude. The behavior of a Poisson mass probability distribution, with rate equal to 5 faults each year, representing the probability of having n ∈N faults within a year, is shown in Figure 18. 13. Minimal repair with CFR Reliability and Maintainability in Operations Management http://dx.doi.org/10.5772/54161 107 Figure 18. Poisson distribution. In the diagram you can see the probability of having N faults within a year, having a homogeneous Poisson process with a rate of 5 faults each year. Figure 18. Poisson distribution. In the diagram you can see the probability of having N faults within a year, having a homogeneous Poisson process with a rate of 5 faults each year. Since in the model of minimal repair with CFR, repair time is supposed to be zero (MTTR = 0), the following relation applies: MTBF =MTTF + MTTR =MTTF = 1 λ (68) (68) Suppose that a system, subjected to a repair model of minimal repair, shows failures according to a homogeneous Poisson process with failure rate λ = 0.0025 failures per hour. We’d like to estimate the average number of failures that the system will have during 5000 hours. Then, determine the probability of having not more than 15 faults in a operation period of 5000 hours. The estimate of the average number of failures in 5000 hours, can be carried out with the expected value function: E N (t) =λ ∙t → E N (5000) =0.0025 failures h ∙5000 h =12.5 failures (69) (69) The probability of having not more than 15 faults in a period of 5000 hours of operation, is calculated with the sum of the probability mass function evaluated between 0 and 15: The probability of having not more than 15 faults in a period of 5000 hours of operation, is calculated with the sum of the probability mass function evaluated between 0 and 15: P N (5000)≤15 = ∑ n=0 15 (λt)n n ! e -λ∙t = ∑ n=0 15 12.5n n ! e -12.5=0.806 (70) (70) 108 Operations Management 14. Minimal repair: Power law A second special case of the general model of minimal repair, is obtained if the failure time T is a random variable with a Weibull distribution, with shape parameter β and scale parameter α. In this case the sequence of failure times is described by a Non-Homogeneous Poisson Process - NHPP with intensity z(t) equal to the probability density function of the Weibull distribution: z(t)= β α β t β-1 (71) (71) Since the cumulative intensity of the process is defined by: Since the cumulative intensity of the process is defined by: Z(t)=∫ 0 t z(u)∙du (72) (72) the cumulative function is: the cumulative function is: Z(t)=∫ 0 t β α β u β-1∙du = β α β ∙ u β β \|0 t = t β α β =( t α )β (73) (73) As it can be seen, the average number of faults occurring within the time t ≥0 of this not homogeneous poissonian process E N (t) =Z(t), follows the so-called power law. As it can be seen, the average number of faults occurring within the time t ≥0 of this not homogeneous poissonian process E N (t) =Z(t), follows the so-called power law. If β >1, it means that the intensity function z(t) increases and, being this latter the expression of the average number of failures, it means that faults tend to occur more frequently over time. Conversely, if β <1, faults decrease over time. In fact, if we take α =10 hours (λ =0.1 failures/h) and β =2, we have: E N (100) =(0.1∙100)2=100=102; E N (1000) =(0.1∙1000)2=10000=1002; t, if we take α =10 hours (λ =0.1 failures/h) and β =2, we have: 0) =(0.1∙100)2=100=102; E N (1000) =(0.1∙1000)2=10000=1002; E N (10000) =(0.1∙10000)2=1000000=10002. We can observe a trend no longer linear but increasing according to a power law of a multiple of the time width considered. The probability mass function of N (t) thus becomes: unction of N (t) thus becomes: P N (t)=n = Z (t)n n ! e -Z (t)= ( t α )β∙n n ! e -( t α )β (74) P N (t)=n = Z (t)n n ! e -Z (t)= ( t α )β∙n n ! e -( t α )β (74) (74) For example, let us consider a system that fails, according to a power law, having β =2.2 and α =1500 hours. 14. Minimal repair: Power law What is the average number of faults occurring during the first 1000 hours of operation? What is the probability of having two or more failures during the first 1000 hours of operation? Which is the average number of faults in the second 1000 hours of operation? For example, let us consider a system that fails, according to a power law, having β =2.2 and α =1500 hours. What is the average number of faults occurring during the first 1000 hours of operation? What is the probability of having two or more failures during the first 1000 hours of operation? Which is the average number of faults in the second 1000 hours of operation? The average number of failures that occur during the first 1000 hours of operation, is calculated with the expected value of the distribution: The average number of failures that occur during the first 1000 hours of operation, is calculated with the expected value of the distribution: Reliability and Maintainability in Operations Management http://dx.doi.org/10.5772/54161 1 Reliability and Maintainability in Operations Management http://dx.doi.org/10.5772/54161 109 Reliability and Maintainability in Operations Management http://dx.doi.org/10.5772/54161 109 E N (t) =μN (t)=Z(t)=( t α )β →E N (1000) =( 1000 1500 )2.2=0.41 (75) (75) The probability of two or more failures during the first 1000 hours of operation can be calculated as complementary to the probability of having zero or one failure: P N (1000)≥2 =1 - P N (1000)<2 =1 - ∑ n=0 1 ( t α )β∙n n ! e -( t α )β =1 - 0.410 0 ! e -0.41 - 0.411 1 ! e -0.41=1 - 0.663 - 0.272=0.064 (76) (76) The average number of faults in the succeeding 1000 hours of operation is calculated using the equation: The average number of faults in the succeeding 1000 hours of operation is calculated using the equation: E N (t + s) - N (s) =Z(t + s) - Z(s) (77) E N (t + s) - N (s) =Z(t + s) - Z(s) (77) that, in this case, is: that, in this case, is: E N (2000) - N (1000) =Z(2000) - Z(1000)=1.47 (78) (78) 15. Conclusion After seeing the main definitions of reliability and maintenance, let's finally see how we can use reliability knowledge also to carry out an economic optimization of replacement activities. Consider a process that follows the power law with β >1. As time goes by, faults begin to take place more frequently and, at some point, it will be convenient to replace the system. Let us define with τthe time when the replacement (here assumed instantaneous) takes place. We can build a cost model to determine the optimal preventive maintenance time τ * which optimizes reliability costs. Let’s denote by C f the cost of a failure and with Cr the cost of replacing the repairable system. Let’s denote by C f the cost of a failure and with Cr the cost of replacing the repairable system. If the repairable system is replaced every τ time units, in that time we will have the replacement costs Cr and so many costs of failure C f as how many are the expected number of faults in the time range (0;τ . The latter quantity coincides with the expected value of the number of faults E N (τ) . The average cost per unit of time c(τ), in the long term, can then be calculated using the following relationship: c(τ)= C f ∙E N (τ) + Cr τ (79) c(τ)= C f ∙E N (τ) + Cr τ (79) Then follows: Then follows: Operations Management c(τ)= C f ∙Z (τ) + Cr τ (80) (80) Differentiating c(τ) with respect to τ and placing the differential equal to zero, we can find the relative minimum of costs, that is, the optimal time τ * of preventive maintenance. Manipulat‐ ing algebraically we obtain the following final result: Differentiating c(τ) with respect to τ and placing the differential equal to zero, we can find the relative minimum of costs, that is, the optimal time τ * of preventive maintenance. Manipulat‐ ing algebraically we obtain the following final result: τ =α ∙ Cr C f (β - 1) 1 β (81) τ =α ∙ Cr C f (β - 1) 1 β (81) (81) Consider, for example, a system that fails according to a Weibull distribution with β =2.2 and α =1500 hours. 15. Conclusion Knowing that the system is subject to replacement instantaneous and that the cost of a fault C f =2500 € and the cost of replacing Cr =18000 €, we want to evaluate the optimal interval of replacement. The application of eq. 81 provides the answer to the question: The application of eq. 81 provides the answer to the question: τ *=α ∙ Cr C f (β - 1) 1 β =1500∙ 18000 2500(2.2 - 1) 1 2.2 =1500 · 2.257=3387 h (82) (82) Author details Filippo De Carlo Address all correspondence to: filippo.decarlo@unifi.it Address all correspondence to: filippo.decarlo@unifi.it Industrial Engineering Department, University of Florence, Florence, Italy RBD: Reliability Block Diagram CBM: Condition-Based Maintenance CFR: Constant Failure Rate CM: Corrective Maintenance DFR: Decreasing Failure Rate IFR: Increasing Failure Rate MCS: Minimal Cut Set MPS: Minimal Path Set MTTF: Mean Time To Failure MTTR: Mean Time To Repair NHPP: Non-Homogeneous Poisson Process PM: Preventive Maintenance Reliability and Maintainability in Operations Management http://dx.doi.org/10.5772/54161 111 References [1] Nakajima S. Introduction to TPM: Total Productive Maintenance. Productivity Press, Inc., 1988, 1988:129. ] Barlow RE. Engineering Reliability. SIAM; 2003. [3] De Carlo F. Impianti industriali: conoscere e progettare i sistemi produttivi. New York: Mario Tucci; 2012. ] O’Connor P, Kleyner A. Practical Reliability Engineering. John Wiley & Sons; 2011. ] Meyer P. Understanding Measurement: Reliability. Oxford University Press; 2010. [6] Ascher H, Feingold H. Repairable systems reliability: modeling, inference, misconcep‐ tions and their causes. M. Dekker; 1984. [7] De Carlo F, Borgia O, Adriani PG, Paoli M. New maintenance opportunities in legacy plants. 34th ESReDA Seminar, San Sebastian, Spain: 2008. [8] Gertler J. Fault detection and diagnosis in engineering systems. Marcel Dekker; 1998. [9] Borgia O, De Carlo F, Tucci M. From diagnosis to prognosis: A maintenance experience for an electric locomotive. Safety, Reliability and Risk Analysis: Theory, Methods and Applications - Proceedings of the Joint ESREL and SRA-Europe Conference, vol. 1, 2009, pp. 211–8. [10] Racioppi G, Monaci G, Michelassi C, Saccardi D, Borgia O, De Carlo F. Availability assessment for a gas plant. Petroleum Technology Quarterly 2008;13:33–7.
https://openalex.org/W4254007466	https://aladabj.uobaghdad.edu.iq/index.php/aladabjournal/article/download/722/568	Arabic	null	تقنيات القناع	Al-ādāb	2,020	cc-by	7,676	جملة اآل ادا اعدداد 115 2016 م1437 هـ تقني ات القناع أقنعة مسرح(النو) أ منوذجا .م زيد سالن سليواى جاهعة بغداد/ هركس دراسات الورأة ث: تقني ات القناع أقنعة مسرح(النو) أ منوذجا .م زيد سالن سليواى جاهعة بغداد/ هركس دراسات الورأة ث: جملة اآل ادا اعدداد 115 جملة اآل ادا اعدداد 115 :ثانٍا: اهمٍت انبحث رزِخـ اٛٔ٤خ اُجؾش ك٢ اُزؼش٣ق ثبُو٘بع اُز١ ع٤ٌٔ٘٘ب ٖٓ اعزوقبءٚأٗٞاػ ٚٝأف٘بك ٝأُٞاد اُز٢ . ٙف٘غ ٜٓ٘ب ٝاٗزٔبءٙ ٝسٓٞصٙ ٝرؼبث٤ش جملة اآل ادا اعدداد 115 جملة اآل ادا اعدداد 115 :اُخؾت ٝرزٞصع ػِ٠ صالصخ اٗٔبه ٖٓ اُؾخق٤بد ٝٛ٢ٍاألثيب ٝاألؽجبػ )ُٞ٘ٝأُٜشعٕٞ، ٝ (ا . ك٢ اُ٤بثبٕ اُز١ اػزٔذ اُزو٘غ ًوبػذح اعبع٤خ ٝاداح عٔبُ٤خ رغزؼَٔ ٖٓ اعَ اؿشاك ك٘٤خ ٖٝٗؾً ٜٔزٔ٤ٖ ثٜزا اُ٘ٞع ٖٓ اُلٖ ٝالع٤ٔب ك٢ اُغبٗت االًبد٣ٔ٢، كوذ ؽـِ٘ب اُو٘بع ٝثذد ُ٘ب ك٢ ؽبٗٚ اُؼذ٣ذ ٖٓ االعئِخ اُز٢ ٗيشؽٜب ٖٝٓ اٜٛٔب: ً٤ل٤خ اُؼَٔ ػِ٠ اُو٘بع؟ ٓب ٛ٢ اُزو٘٤بد اُز٢ ٜٚٔ٣لشمٜب؟ ٝٓب ٛ٢ ٓ٤ٌبٗ٤ضٓبرٚ؟ كخ٤شٗب إ ٣ٌٕٞ ٓٞمٞع دساعز٘ب ٛزٙ ؽز٠ ٣زغ٘٠ ُ٘ب ك . ٚٝاعز٤ؼبثٚ ٝاُؼَٔ ػِ٤ مهخص انبحث: عغَ اُو٘بع ؽنٞسٙ ك٢ عَ اُؾنبساد ٝاُضوبكبد أُزؼبهجخ ٝالع٤ٔب خالٍ االؽ زلبالد اُيوغ٤خ ٝاُذ٣٘٤خ، ًٔب ثذا ٛزا اُؾنٞس ك٢ اُؾ٤بح اُ٤ٞٓ٤خ صْ اٗزوَ اُ٠ أُغشػ ٖٓ ُ٤قجؼ رؼج٤شا عٔبُ٤ب ؽ٤ش رٌٞ٣ٖ أُٔضَ ٝعٔبُ٤خٟأخش رٞظق ُ٘زبط . اُؼشٝك أُغشؽ٤خ ى٤ِخ كزشاد ربس٣خ أُغشػ ٚإ اخز٤بسٗب ُٔٞمٞع (رو٘٤خ اُو٘بع) ًزٞعٚ عٔبُ٢ ٣لشك ثبُنشٝسح اخز٤بساد ٝرو٘٤بد رزؼِن ث ٝرخزِق ػٖ اُغٔبُ٤بدٟاألخش ػِ٠ ٓغزٟٞ رٔض٤َ أُٔضَ ًبُؾشًخ ٝاُقٞد ٝاإلُوبء ٝاإل٣ٔبءح ٖٓ ٝثو٤خ ػ٘بفش اُؼشك أص٣بء ٝد٣ٌٞس ٝؿ٤شٛب . ٖٝٓ ٛ٘ب عبءد دساعز٘ب ُزجؾش ك٢ رو٘٤بد اُو٘بع ، ُٝززخز ٖٓ ٓغشػ( ُٞ٘ا) . اُ٤بثبٗ٢ اٗٔٞرعب ُٜب انفصم االول ًاالطار انمنهج اوال: مشكهت انبحث وانحاجت إنٍه: ثذأ ظٜٞس اُو٘بع ػ٘ذ االؿش٣ن، ٝٓغشػ األعشاس ٝأُؼغضاد ثأٝسثب خالٍ اُوشٗ٤ٖ اُخبٓظ ػؾش . ٝاُغبدط ػؾش، ًٝزُي ك٢ اٌُٞٓ٤ذ٣ب د١ السر٢ ثب٣يبُ٤ب ػِ٠ ٝعٚ اُخقٞؿ )ٝٓغ ثذا٣خ اُوشٕ اُؼؾش٣ٖ اظٜش (ًٞسدٕ ًش٣ظ اٛزٔبٓب ثبُو٘بع ثٞفلٚ (اُشاط أُضبُ٢) اُز١ . ٟ٣ض٣َ اُيبثغ اُؾخق٢ ػٖ أُٔضَ، ٝ٣ضش١ االؽزـبٍ ثبٌٓبٗبد اُغغذ االخش )صْ (ثشٝرُٞذ ثشؽذ اُز١ اػزٔذ اُزو٘غ ٖٓ اعَ رؾو٤ن أُغخ ًبعشاء عٔبُ٢ ُ٤ؾون اٛذاكب ع٤بع٤خ، ًٝزُي (ٓب)٣شُٛٞذ )اُز١ ٣شٟ اُو٘بع رؼج٤شا ػٖ عٞٛش اٌُبئٖ ٝ (ث٤زش ثشٝى . ْٛثبًزؾبكٚ اُِؾظخ اُز٢ رقيذّ خالُٜب رار٤خ أُٔضَ ثؾذٝدٛب االٗغبٗ٤خ .... ٝؿ٤ش آب ك٢ أُغبسػ اُؾشه٤خ كوذ ٗؾأ (اٌُبربًبُ٢) ثبُٜ٘ذ ك٢ ٚاُوشٕ اُغبثغ ػؾش ؽ٤ش ثِؾ ك٤ٚ ُؼت ٝع ً أُٔضَ اػِ٠ دسعبد ٚٔبُٚ ثٞفل– ٚا١ اُٞع– اداح ُزُٞ٤ذ اه٘ؼخ ٓزؼذدح، ٝظٜش (اُزٞث٤٘ؾ) ك٢ (ثبُ٢)، ٝ٣ؼذ ٖٓ اثشص االٗٞاع أُغشؽ٤خ أُو٘ؼخ، ٝهذ اػزٔذٝا ُق٘بػخ ٛزٙ االه٘ؼخ ػِ٠ ٓبدح 572 جملة اآل ادا اعدداد 115 ب - ػنذ:االنهت انمصزٌت اؽزٜشد ٓقش اُوذ٣ٔخ ثبُؼذ٣ذ ٖٓ االُٜخ، ًٝزُي ثٔغٔٞػخ ٖٓ اُؾ٤ٞاٗبد االُٜخ اٝ أُوذعخ ." ًٔب اؽزٜش ٙد ٓقش ثز٘ٞع اُٜزٜب ٝاخزالكبرٜب، ؽ٤ش ػشكذ ًَ ٓذ٣٘خ ثبُٜبٜٛب اٝ اُٜزٜب، ًَٝ ٛز "االُٜخ ًبٗذ ُٜب ٓؼبدالد ٓغ اُؾ٤ٞاٗبد أُؤُٜخvii . جملة اآل ادا اعدداد 115 جملة اآل ادا اعدداد 115 2016 م1437 هـ "صوت فـ٤ش رغزؼَٔ ُزوجَ اُقٞسح أُ٘جضوخ ٖٓ اعٜضح اُزِلبصiv ّ، آب ك٢ ػِْ دساعخ افٍٞ اٌُال ( كبٕ ُلظMasco )  ٣شاد اُزؼش٣ق ثبُو٘بع ٗٝاُغبؽشح ٓؼب، ٝٛزا ٣وٞد ب اُ٠ أُؼزوذاد ٝاُؼبداد ٍٝالع٤ٔب اُش٣ل٤خ ٜٓ٘ب ًٝزُي ػبُْ االىلب . ًٝزُي ًبٕ اال٣يبُ٤ٕٞ ٣غزخذٓٞٙ االسعزوشاى٤ٕٞ ك٢ . اُـشة ك٢ ٓؾبٛذ مبؽٌخ ُؾخق٤بد ٓو٘ؼخ، ًٝزُي ك٢ اُؾلالد اُزٌ٘ش٣خ ٝؿ٤شٛب ِٚٔٛزا ثؼل ٖٓ اُزؼبس٣ق اُؼبٓخ ُِو٘بع ثٔب اؽز ثجؼل ٖٓ اُٞمؼ٤بد ٝاُؾبالد، اال إ ثؾض٘ب . ع٤شرٌض اعبعب ػِ٠ اُو٘بع ك٢ أُغشػ :ثانٍا: جذور انمناع أ - ًف:انمٍثٍىنىجٍا االغزٌمٍت انزومانٍت رٔ٤ض اُ٤ٞٗبٗ٤٤ٕٞ ثبالؽزلبالد اُذ٣٘٤خ أُو٘ؼخ اُز٢ أٗزغذ أُغشػ، ٝثبُزبُ٢ كبٕ " اُو٘بع اُيوغ٢ ك٢ اُ٤ٞٗبٕ اُوذ٣ٔخ ُ٤ظ إال "عضءا ٖٓ ٓغبُخ اػْ ٝاؽَٔ ٝٛ٢ رغغ٤ذ االُٜخv َ، كبُؾٌَ اُز١ ٣ٔض االُٜخ ك٢ اُؼقش اٌُالع٤ٌ٢ ٛٞ اُقٞسح االٗغبٗ٤خ، رُي إ رٔضبٍ اُيوظ ٣غغذ االُٚ ػجش اُخقبئـ االًضش اثٜبسا ك٢ اُغغذ االٗغبٗ٢ ًبُغٔبٍ ٝاُؾجبة ٝاُوٞح ٝؿ٤شٛب، ػِ٠ اٗٚ ُجؼل اُوٟٞ االُٜ٤خ فالد ثبُو٘بع ثؾ . ٤ش ٣قِؼ اُو٘بع ُِزؼج٤ش ػٖ ٓظبٛش اخشٟ ُِلٞم اُيج٤ؼ٢ ُِٝؾذ٣ش ػٖ االه٘ؼخ االُٜ٤ خ ٝ )ػ٘ذ اُ٤ٞٗبٕ هذ٣ٔب ع٘زيشم اُ٠ (هٞسهٞ) ٝ (أسر٤ٔظ (د٣ٞٗ٤غٞط)، كبٓب ه٘بع (هٞسهٞ) ك٤ٔضط ث٤ٖ االٗغبٕ ٝاُؾ٤ٞإ ػجش ؽٌَ ٓضدٝط (ؽخق٤خ اٗضٞ٣خ ُْ راد ٝعٚ ٝؽؾ٢)، آب (أسر٤ٔظ) كٜ٢ اُٜخ رغغذ ػجش ه٘بع، ٌُٜ٘ب رخقـ مٖٔ ىوغٜب ٌٓبٗب ٛبٓب ُاله٘ؼخ ُٝٔظبٛش اُزو٘غ، ار ٣زْ اُزؼج٤ش ػٖ أُٞاهق أُخزِلخ ٝأُز٘بهنخ ًبُزؾلظ االٗضٞ١ )ٝاالكزشاط اُؾ٤ٞاٗ٢ ٝاُؼلخ ٝاُلغٞس ُ٘وذ ٓخزِق اُو٤ْ االعزٔبػ٤خ ٝاُزٔشد ػِ٤ٜب، آب (د٣ٞٗ٤غٞط ك٤ؼذ االُٚ أُو٘غ ثبٓز٤بص ػ٘ذ ا ُٚ٤ٞٗبٗ٤٤ٖ اُوذآ٠، ٖٝٓ خقبئق األعبع٤خ اُز٢ رؤًذٛب اُٞصبئن االسً٤ُٞٞع٤خ ٝاألدث٤خ ٖاُٞع٤ٜخ ٓٔضِخ ثبُخقٞؿ ك٢ عبرث٤خ اُؼ٤٘٤ )ٞؽأٗٚ ؽإٔ (هٞسهvi . :ثانثا أهذاف :انبحث . ٚاُٞهٞف ػِ٠ ٌٓٞٗبد اُؼَٔ ػِ٠ اُو٘بع ٝرو٘٤بر اُٞهٞف ػِ٠ ٌٓٞٗبد اُؼَٔ ػِ٠ اُو٘بع ٝر :رابؼا: حذود انبحث . ٣زؾذد اُجؾش اُؾبُ٢ ثذساعخ رو٘٤خ اُو٘بع ك٢ ٓغشػ اُ٘ٞ اُ٤بثبٗ٢ ًانفصم انثان اإلطار اننظزي . ٣زؾذد اُجؾش اُؾبُ٢ ثذساعخ رو٘٤خ اُو٘بع ك٢ ٓغشػ اُ٘ٞ اُ٤بثبٗ٢ ًانفصم انثان اإلطار اننظزي انمبحث األول:- :انمناع " :٣وٍٞ اثٖ ٓ٘ظٞس اُو٘بع اٝعغ ٖٓ أُو٘ؼخ ٝهذ رو٘ؼذ ثٚ ٝه٘ؼذ سأعٜب ٝه٘ؼزٜب أُجغزٜب اُو٘بع "ٚكزو٘ؼذ ثi ٞٛٝ ، ٝرشٟ ُغ٤٘٤ل٤بف اُالس ثبٕ اُو٘بع ٛٞ " اُظٜٞس ثٞعٚ ؿ٤ش ؽو٤و٢ ٝٓخزِق "ا٣نب اُزٌ٘ش ثويؼخ ٖٓ اُؤبػii، ٝك٢ ٓغبٍ اُلٖ ا ُزؾٌ٤ِ٢ ك٤ؤًذ ث٤زش كب٣ظ ثبٕ اُو٘بع ٛٞ " اػبدح "ٕاٗزبط اُٞعٚ ٝٗؾذ اُشاط ٣زْ ثٞاعيخ اُزضٝ٣ن ٝاُض٣٘خ ٖٓ رُي رؾخ٤ـ ٝعٚ اٗغبٕ اٝ ؽ٤ٞاiii ًٔب ٣ٌٖٔ اُؾذ٣ش ػٖ اُٞاه٢ ُٞعٚ أُجبسص٣ٖ ثبُغ٤ق، ٝٛٞ ػجبسح ػٖ ه٘بع ٓق٘ٞع ُؾٔب٣خ اُٞعٚ . ٝٓب ٣٘يجن ػِ٠ أُجبسص٣ٖ ثبُغ٤ق ،٣٘غؾت ػِ٠ ٓشث٢ اُ٘ؾَ ػ٘ذ اهزشاثْٜ ٖٓ أُ٘ؾالد ٝٛٞ ا٣نب " ٝام ٖٓ اُلٞالر ك٢ ؽٌَ هيغ فـ٤شح ٣غزؼِٔٚ اُجؾبسح، ًٝزُي هيؼخ ٖٓ اُؾذ٣ذ راد 572 جملة اآل ادا اعدداد 115 جملة اآل ادا اعدداد 115 جملة اآل ادا اعدداد 115 - " إ ُِوٞح أُوذعخ ٓغ اُو٘بع ػالهخ ٝى٤ذح رزٔضَ ك٢ إػيبء ٌٓبٗخ هٞ٣خ ُذٟ االُٜخ "االؿش٣و٤خ اُز٢ ُٜب ٗلظ اُو٤ٔخ ٝاُزوذ٣شix . إ ؽبَٓ اُو٘بع ٣ٔضَ دائٔب اُز١ ٣ؾِٔٚ ٝ٣قجؼ اُو٘بع را عذٟٝ ػ٘ذٓب ٣ؤد١ أُٔضَ دٝسٙ ثٌَ دهخ ٝٛٞ دٝس ٣زٔبؽ٠ ٓؼٚ، ٝ٣ٌٕٞ اُو٘بع ٗٞػب ٖٓ اُو٤بكخ اٝ ُجبعب اٝ ٓالٓؼ رذٍ ػِ٠ اُؾخق٤خ اُز٢ ،ٝهغ روِ٤ذٛب كبُٔٔضَ ارا ال ٣وز٘غ ثو٘بػٚ اُٞؽ٤ذ ٝاٗٔب ٣غؼ٠ اُ٠ روِ٤ذ االخش، ٝٛزا اُزوِ٤ذ ٣٘ذسط ٚمٖٔ اُخ٤بٍ ٝ٣ذخَ ك٢ ٓغبٍ ٓب كٞم اُيج٤ؼخ . ٝثٜزا كبٕ أُزوجَ اُز١ ٣زٞعٚ اُ٤ٚ أُٔضَ ػِ٤ إ ٣ؾؼش ٝ٣زبصش ثٔب ٣وذّ اُ٤ٚ، ٝػِ٤ٚ اُزبهِْ ٓغ اُؾبالد أُخزِلخ اُز٢ ٣ؼب٣ؾٜب ؽز٠ ٣قجؼ ًٚٗٝب ك٢ ؽبُخ اُؾخـ أُوِذ ٝثبُزبُ٢ ثٔب ٛٞ كٞم اُيج٤ؼ٢، ٝٛزٙ اُؾبُخ رؾَٔ ًَ اُذ٣بٗبد ؽز٠ اُز٢ ْٜال رؤٖٓ ثٞعٞد االسٝاػ، ٝٛ٘بى ٓؼزوذاد رؤٖٓ ثٌَ ٓب ٣وّٞ ثٚ اُغؾشح اُز٣ٖ ٣غؼٕٞ اُ٠ ؿب٣بر . ثبعزؼٔبٍ اُو٘بع :ثانثا: انىاع انمناع ثؼذ ْإ ًبٕ اُو٘بع اُز١ ٣غزؼَٔ ك٢ االؽزلبالد اُذ٣٘٤خ ثٔؼظ اُؾنبساد ٝ ٙعزٝس اُزبس٣خ٤خ ، كوذ َاٗزغذ ٛزٙ االؽزلبالد أُغشػ ُذٟ اُ٤ٞٗبٗ٤٤ٖ، ٝثبُزبُ٢ اُو٘بع أُغشؽ٢ اُز١ اعزؼِٔٚ أُٔض ٙ(ص٤غجظ) ػ٘ذٓب ًبٕ ٣يِ٢ ٝعٜٚ ٝروبع٤ٔٚ ثجؼل أُغبؽ٤ن الداء ادٝاس ٓخزِلخ، ٝرٔضِذ ٛز ا"ُٔغبؽ٤ن ك٢ " ثوب٣ب اُخٔشح ٝ اٝسام اُؾغشx ًٔب اْٜٗ ًبٗٞا ٣غزؼِٕٔٞ " دّ اُؾ٤ٞاٗبد اُز٢ "ٚ٣وذٜٓٞٗب هشثبٗب ُالُٜخ ُيالء اُٞعxi . ٝسؿْ ػذ ٛزا اُو٘بع رٌ٘شا اًضش ٓ٘ٚ ه٘بػب، كوذ اسرأ٣٘ب إ ٗذسط أٗٞاع اُو٘بع ٓؼزٔذ٣ٖ ك٢ رُي ػِ٠ . عزٝس اُو٘بع  انمناع ًانطمس:ػنذ االغزٌك ادٟ اُو٘بع اُيوغ٢ دٝسا ٜٓٔب ك٢ االؽزلبالد اُذ٣٘٤خ اُيو غ٤خ ػ٘ذ االؿش٣ن، كبسرجو ثبالُٜخ، ُٝزُي كبٕ " ٓغبُخ اُو٘بع اُيوغ٢ ػ٘ذ االؿش٣ن اُوذ٣ٔخ ُ٤غذ عٟٞ عضءا ٖٓ ٓغبُخ اػْ ٛ٢ رغغ٤ذ ٍاالُٜخ، كبُؾٌَ أُوٖ٘ ُِزٔض٤َ اال ٛٞ اُقٞسح االٗغبٗ٤خ، كزٔضب اُيوظ ٣غغذٙ اُٚ ػجش اًضش ٟٞاُخقبئـ اثٜبسا ك٢ عغذ االٗغبٕ ًبُغٔبٍ ٝاُؾجبة . ٝثبُزٞاصٕ ٓغ رُي كبٕ ُجؼل اُو "االُٜ٤خ فالد خيٞف٤خ ثبُو٘بع ٣قِؼ ُِزؼج٤ش سٓض٣ب ػٖ ٓظبٛش اخشٟ ُِلٞم ىج٤ؼ٢xii . ث - :انمناع وخزق انطبٍؼت إن اإلنسان بطبعه يسعى دائما الى اختراق حىاسه الخمس الى ما فىق الطبيعت، وبالعىدة الى أصل ( كلمتmasco) التي تعني (ساح رة) كما اسلفنا، فان القناع ظهر تعبير ا رمزي ا لبعض الحاالث غير ( الطبيعيت، فقذ حذدJean Pierre vernant :) مشكالث ثالث مرتبطت بما هى فىق الطبيعت - إ اُوٞح اُظبٛشح ٖٓ خالٍ اُو٘بع ُ٤ظ ُذ٣ٜب ا١ ؽٌَ اخش ُِزؼج٤ش ػذٟ اُو٘بعviii . -إ االُٚ اُز١ ُ٤ظ ُٚ ه٘بع، . ِٚٓعذ٣ش ثبالؽزشاّ ػجش ىوٞعٚ ثزوذ٣ش اُو٘بع ٝؽب - إ اُوٞح اُظبٛشح ٖٓ خالٍ اُو٘بع ُ٤ظ ُذ٣ٜب ا١ ؽٌَ اخش ُِزؼج٤ش ػذٟ اُو٘بعviii . - إ اُوٞح اُظبٛشح ٖٓ خالٍ اُو٘بع ُ٤ظ ُذ٣ٜب ا١ ؽٌَ اخش ُِزؼج٤ش ػذٟ اُو٘بعviii . - إ االُٚ اُز١ ُ٤ظ ُٚ ه٘بع، . ِٚٓعذ٣ش ثبالؽزشاّ ػجش ىوٞعٚ ثزوذ٣ش اُو٘بع ٝؽب - إ االُٚ اُز١ ُ٤ظ ُٚ ه٘بع، . ِٚٓعذ٣ش ثبالؽزشاّ ػجش ىوٞعٚ ثزوذ٣ش اُو٘بع ٝؽب 577 جملة اآل ادا اعدداد 115 جملة اآل ادا اعدداد 115 جملة اآل ادا اعدداد 115 ًكبالؽزلبالد اُز٢ رظٜش ك٤ٜب االه٘ؼخ رغزٜذف اُزز َ٤ش ثبالؽذاس االعيٞس٣خ اُز٢ ٝهؼذ ك٢ االف "ٝادد اُ٠ ر٘ظ٤ْ اُؼبُْ ك٢ ؽٌِٚ اُؾبُ٢xiii . ٚٝرخزِق اؽٌبٍ االه٘ؼخ ٝاٗٞاػٜب ك٢ أُغزٔغ االكش٣و٢ ٝرزؼذد خقٞف٤برٚ ٝٓؼبٗ٤ٚ، كٔالٓؼ اُٞع ،ك٢ اُلخ االكش٣و٢ ُٜب دالالد ٝامؾخ ٝػذ٣ذ االؽٌبٍ افجؾذ ٓزلن ػِ٤ٜب ػِ٠ ٓغزٟٞ اُذالالد ك نال ػٖ عزٝسٙ اُذ٣٘٤خ، ار " إ ُاله٘ؼخ اُيوغ٤خ ك٢ هجبئَ اكش٣و٤ب ٝأُؾ٤يبد ًٝزُي ك٢ ثالد "االعٌ٤ٔٞ ٝاُٜ٘ٞد ك٢ آش٣ٌب ٝظ٤لخ ٓوذعخxiv . ًكبالؽزلبالد اُز٢ رظٜش ك٤ٜب االه٘ؼخ رغزٜذف اُزز َ٤ش ثبالؽذاس االعيٞس٣خ اُز٢ ٝهؼذ ك٢ االف "ٝادد اُ٠ ر٘ظ٤ْ اُؼبُْ ك٢ ؽٌِٚ اُؾبُ٢xiii . ٚٝرخزِق اؽٌبٍ االه٘ؼخ ٝاٗٞاػٜب ك٢ أُغزٔغ االكش٣و٢ ٝرزؼذد خقٞف٤برٚ ٝٓؼبٗ٤ٚ، كٔالٓؼ اُٞع ًكبالؽزلبالد اُز٢ رظٜش ك٤ٜب االه٘ؼخ رغزٜذف اُزز َ٤ش ثبالؽذاس االعيٞس٣خ اُز٢ ٝهؼذ ك٢ االف "ٝادد اُ٠ ر٘ظ٤ْ اُؼبُْ ك٢ ؽٌِٚ اُؾبُ٢xiii . ٚٝرخزِق اؽٌبٍ االه٘ؼخ ٝاٗٞاػٜب ك٢ أُغزٔغ االكش٣و٢ ٝرزؼذد خقٞف٤برٚ ٝٓؼبٗ٤ٚ، كٔالٓؼ اُٞع ،ك٢ اُلخ االكش٣و٢ ُٜب دالالد ٝامؾخ ٝػذ٣ذ االؽٌبٍ افجؾذ ٓزلن ػِ٤ٜب ػِ٠ ٓغزٟٞ اُذالالد ك نال ػٖ عزٝسٙ اُذ٣٘٤خ، ار " إ ُاله٘ؼخ اُيوغ٤خ ك٢ هجبئَ اكش٣و٤ب ٝأُؾ٤يبد ًٝزُي ك٢ ثالد "االعٌ٤ٔٞ ٝاُٜ٘ٞد ك٢ آش٣ٌب ٝظ٤لخ ٓوذعخxiv .  انمناع ًانطمس:ػنذ انٍابانٍٍن اخزِق اُ٤بثبٗ٤٤ٕٞ ػٖ ؿ٤شْٛ ك٢ اُؼذ٣ذ ٖٓ أُغالد، ٝٛزا االخزالف عؼَ ْٜٓ٘ ؽؼجب ٓزٔ٤ضا ٖٓ ؽنبس٣ب ؽ٤ش اُؼبداد ٝاُزوبُ٤ذ ٝاُيوٞط، ٝثبُزبُ٢ اُضوبكخ اُز٢ ٣ؾٌَ اُو٘بع اُيوغ٢ ٖٓ خالُٜب ػ٘قشا اعبع٤ب ٖٓ ٓٔبسعبرٜب عٞاء ك٢ اُؾلالد اُذ٣٘٤خ اٝ اُؼشٝك أُغشؽ٤خ، ٝك٢ ٛزا "االىبس كبٕ " أُؼ٘٠ اُيوغ٢ ُِو٘بع ٣زٞاعذ ك٢ أُغشؽ٤خ االؽزلبُ٤خxv . ب - :ًانمناع انمسزح اٗيِن اُو٘بع ٓغ اٗيالم أُغشػ اُ٤ٞٗبٗ٢ ك٢ ػٜذ (ص٤غجظ)، كٌبٗذ اُزشاع٤ذ٣ب روذّ ثٞاعيخ صالس ٖٓ ٌٖٔٓٔضِ٤ٖ، ٝػ٘ذ ظٜٞس اُو٘بع ٝاعزؼٔبُٚ افجؼ أُٔضَ هبدسا ػِ٠ اداء اًضش ٓب ٣ األدٝاس . ٚٝهذ ػشكذ ٓؼظْ اُؾؼٞة ٝاُضوبكبد اُو٘بع ك٢ ىوٞعٜب ٝاؽزلبالرٜب ٝرج٘بٙ أُغشػ ٝاعزؼِٔز ثؼل أُذ اسط ثأٗٞاع ٝأف٘بف ٓخزِلخ ُٝـب٣بد ٓؼ٤٘خ ، ٜٝٓ٘ب .  انمناع ًانطمس:ًاالفزٌم  انمناع ًانطمس:ًاالفزٌم اسرجو اُو٘بع اُيوغ٢ ك٢ أُغزٔؼبد االكش٣و٤خ ثبُؼبد ٌَاد ٝاُزوبُ٤ذ ٝاُؾ٤بح اُ٤ٞٓ٤خ اُخبفخ ث ْٓغزٔغ ٖٓ ٛزٙ أُغزٔؼبد ػِ٠ اخزالكٜب ٝر٘ٞػٜب ٝرؼذدٛب، االٓش اُز١ عؼَ اُو٘بع ٣٘زؾش ثٔؼظ ،ىوٞعٚ ٖٓ ثِذ اُ٠ اخش ٓضَ "اُيوٞط اُضساػ٤خ ٝأُبرٔ٤خ اٝ اُذ٣٘٤خ ُِٔغزٔؼبد اُجؾش٣خ اسرجو اُو٘بع اُيوغ٢ ك٢ أُغزٔؼبد االكش٣و٤خ ثبُؼبد ٌَاد ٝاُزوبُ٤ذ ٝاُؾ٤بح اُ٤ٞٓ٤خ اُخبفخ ث ْٓغزٔغ ٖٓ ٛزٙ أُغزٔؼبد ػِ٠ اخزالكٜب ٝر٘ٞػٜب ٝرؼذدٛب، االٓش اُز١ عؼَ اُو٘بع ٣٘زؾش ثٔؼظ ،ىوٞعٚ ٖٓ ثِذ اُ٠ اخش ٓضَ "اُيوٞط اُضساػ٤خ ٝأُبرٔ٤خ اٝ اُذ٣٘٤خ ُِٔغزٔؼبد اُجؾش٣خ 572 جملة اآل ادا اعدداد 115  انمناع فً انمسزح:انٍىنانً انمذٌم ٍعجن ٓ٘ب اُوٍٞ ثبٕ اٍٝ ٖٓ اعزؼَٔ اُو٘بع ٛٞ (ص٤غجظ) ًٔب رؤًذ ٓبسر٤ٖ داك٤ذ " ٣ؼٞد اعزؼٔب "اُو٘بع ٝأُالثظ اُزشاع٤ذ٣خ اُ٠ ػٜذ ص٤غجظxvi ، ًّٝبٗذ اُٝ٠ أُغشؽ٤بد االؿش٣و٤خ ًبٗذ روذ ثٔٔضَ ٝاؽذ صْ ام٤ق أُٔضَ اُضبٗ٢ ٝاُضبُش ك٢ ػٜذ (عٞكًِٞ٤ظ)، ٝثٜزا اؽزبط أُٔضِٕٞ اُ٠ ،اه٘ؼخ الداء ؽخق٤بد ٓزؼذدح، ٝرْ ريٞ٣غ اه٘ؼخ اُيوٞط (د٣ٞٗ٤غٞط ٝ أسر٤ٔ٤ظ) اُ٠ اُخؾجخ . )ٝٛزا ٓب ًبٕ ٣وّٞ ثٚ (ص٤غجظ "ًٝبٗذ ٛزٙ االه٘ؼخ ٓق٘ٞػخ ٖٓ اُؤبػ ٝاُغجظ ٝرـي٢ ًبَٓ اُش ْاط، ٣زخِِٜب صوت ُِؼ٤٘٤ٖ ٝاُل ٝرؾَٔ ؽؼشا ُٝؾ٠، ًٝبٗذ روبع٤ْ اُٞعٚ ٓوجُٞخ ٝٓقٞؿخ ٝٓيِ٤خ، ُٜٝزٙ االه٘ؼخ ٝأُالثظ ٚٝظ٤لخ سٓض٣خ ٝرٔضَ ٗٞػ٤خ ٖٓ اُؾخق٤بد ٖٝٓ ؿب٣زٜب ا٣نب اُزبص٤ش ٝرؾش٣ي أُؾبػش ٝاُزشك٤ "ٖػِ٠ أُزلشع٤xvii 572 جملة اآل ادا اعدداد 115 جملة اآل ادا اعدداد 115 ٖٝٓ ٝظبئق اُو٘بع ك٢ أُغشػ اُ٤ٞٗبٗ٢ ٕاٗٚ ًب َ٣غزؼَٔ ُزنخ٤ْ اُؾخق٤خ اُز٢ ٣وّٞ ثٜب أُٔض ٖٝرلخ٤ٜٔب، كنال ػ . ِٕٞدٝسٙ ك٢ اُزلش٣ن ث٤ٖ اُؾخق٤بد اُز٢ ًبٕ ٣ؤد٣ٜب أُٔض  ًانمناع فًانكىمٍذٌا دي الرت:  ًانمناع فًانكىمٍذٌا دي الرت: ًبٗذ رغٔ٠ ك٢ كشٗغب اٌُٞٓ٤ذ٣ب اال٣يبُ٤خ ًٝٞٓ٤ذ٣ب األه٘ؼخ ، ٝك٢ اُوشٕ اُضبٖٓ ػؾش أىِن ػِ٤ٜب ٖاعْ اٌُٞٓ٤ذ٣ب د١ السر٢ ٝرؼ٘٢ اُلٖ ٝأُٜبسح اُزو٘٤خ ٝاُغبٗت االؽزشاك٢ ُِٔٔضِ٤xviii ، اسرٌضد ٛزٙ اٌُٞٓ٤ذ٣ب اُز٢ افجؾذ رٞعٜب عٔبُ٤ب خبفب ك٢ اُوشٕ اُغبدط ػؾش " ،ٍػِ٠ االسرغب ٝ٣لشك ُؼت أُٔضَ اال٣يبُ٢ ثبُو٘بع رؾٌٔب ك٢ اُغغذ ًٔب ك٢ اُٞعٚ ٝٛزا ٣زيِت ٓ٘ٚ ؽشك٤خ ٖٜٝٓبسح ًج٤شر٤ ٚ. إ ٗقق اُو٘بع اُز١ ٣ـي٢ اُ٘قق االػِ٠ ٖٓ اُٞعٚ ٣زشى روبع٤ْ اُٞع ثبسصح ُِؼ٤بٕ ػٌظ االه٘ؼخ االؿش٣و٤خxix . انمبحث انثانً: مسزح(اننى): اوال: نمحت ػن مسزح(اننى): ( ُٞ٘ا:) ٓبعبح ؽؼش٣خ ٣بثبٗ٤خ ٖٓ ث٤ٖ صالصخ اف٘بف ٓغشؽ٤خ ًالع٤ٌ٤خ ك٢ أُغشػ اُ٤بثبٗ٢ ٝ٣ؼذ ( ُٞ٘ا) ٝاهذٜٓب، " ٝاُ٘ٞ هق٤ذ ؿ٘بئ٢ ٓ٤ٔ٢ ٓقبؽت ثبٝسًغزشا رٌٕٞ ػٔٞٓب ٓغضاح ػجش سهقخ ا "سهقبد ُ٤غذ ُٜب ػالهخ ثبُٔٞمٞعxx ّ. ٝروذ ػشٝك اُ٘ٞ ك٢ أُ٘بعجبد اُذ٣٘٤خ ٝاُيوغ٤خ اُز٢ ر٘ظْ ٓيِغ ًَ ع٘خ عذ٣ذح، ٝروذّ خٔظ ٓغشؽ٤بد ك٢ ٣ّٞ ٝاؽذ، ٝرغٔغ ٓب ث٤ٖ اٌُٞٓ٤ذ٣خ. ُٞ٘ٝا ٝأُالؽظ ُزبس٣خ أُغشػ ٝاُلٖ ثقلخ ػبٓخ ٗغز٘زظ ؽلبظٚ ػِ٠ اُؾٌَ اُزوِ٤ذ١ ٝ٣زؾبؽ٠ اُزـ٤٤ش ٝاُزجذ٣َ ٝٛٞ ٓب ٣ؤًذٙ كٞث٤ٕٞ ثبٝسص ثوُٞٚ " رٔزبص اُ٤بثبٕ ػٖ اع٤ب ًِٜب اُز٢ روذط اُؼشف ثقلخ ٓ ػبٓخ ٝرزؾبؽ٠ اُزـ٤٤ش ٝاُزجذ٣َ، ثبٜٗب اُذُٝخ اُٞؽ٤ذح اُز٢ ُْ ٣يشأ ػِ٠ ُْٝ ٍٞغشؽٜب ثزبرب ا١ اك ٣غش ػِ٤ٚ ا١ اؽ٤بء اٝ رغذ٣ذ كؼبٍ.. ُْٝ ٣ضٍ ًَ ٖٓ سهقٜب ٝٓغشؽٜب عِ٤ٔب ًٔب ًبٕ ٓيبثوب "ُٙٔلبٛ٤ٔٚ االف٤ِخ ٓؾزلظب ثٌَ روبُ٤ذxxi . ٝرز٘بٍٝ أُغشؽ٤بد اُخٔظ اُز٢ ٣وذٜٓب اُ٘ٞ ؽٍٞ اُؾخق٤بد االُٜ٤خ ٝػشٝك األؽجبػ اُز٢ ٖرزؾذس ػٖ أُؾبسث٤ ٝاألػذاء ، ٝأُٞام٤غ أُأخٞرح ٖٓ اُزبس٣خ اٌُالع٤ٌ٢ اُوذ٣ْ، ٝٓغشؽ٤بد اُ٘غبء أُغ٘ٞٗبد، ٝاُز٢ رشٝ١ اهبف٤ـ ػٖ ؽ٤بح رزبُْ الٜٓبد كوذٕ اث٘بءٖٛ، ٝٓغشؽ٤بد اُٞؽٞػ ٝاُؾ٤بى٤ٖ . آب خقٞف٤بد ٓغشػ اُ٘ٞ كٜ٢ اُزبً٤ذ ػِ٠ اُؾخق٤بد أُؾٞس٣خ ٝاعزؼٔبٍ االه٘ؼخ ٝاالػزٔبد ػِ٠ اُشهـ ًٞع٤ ِخ ٜٓٔخ ُِزؼج٤شxxii . جملة اآل ادا اعدداد 115 جملة اآل ادا اعدداد 115 2016 م1437 هـ 2016 م1437 هـ :ثانٍا:ػناصز اننى وتمنٍاته أ - :انفضاء ْ٣ؼذ كنبء اُ٘ٞ ٖٓ اُلنبءاد أُزٔ٤ضح، ٝٛٞ كنبء اػذ اعبعب ُٜزا اُؾٌَ أُغشؽ٢، كـ " رقٔ٤ ٓغشػ اُ٘ٞ ٓزٔ٤ض ثذسعخ ال ٣قِؼ اال ُٔغشؽ٤بد اُ٘ٞ ٝؽذٛب، كال ٣ٌٖٔ إ ٣ؼشك ك٤ٚ ٗٔو "ُٞ٘ٓغشؽ٢ خالف اxxiii ، ٝ٣زٌٕٞ اُلنبء " ػجبسح ػٖ سثبػ٢ ُالمالع ػبس ٝٓلزٞػ ٖٓ صالصخ ٖعٞاٗت ث٤ أػٔذح هبئٔخ ٝالفوخ ثبُغذسإ ٝٓزٌٞٗخ ٖٓ اُق٘ٞثش ٝٛ٢ اُز٢ رؾذد صٝا٣ب "اُلنبءxxiv . ٝخؾجخ ٓغشػ اُ٘ٞ ٓشرلؼخ ٝرـي٠ ثغوق سؿْ ٝعٞدٙ داخَ كنبء، ٝ٣ؾ٤و ثٚ ػَ ًبَٓ االسم٤خ ؽق٠ اث٤ل، ؿشعذ خالُٚ ؽغ٤شا د فـ٤شح ٖٓ اُق٘ٞثش ؽٍٞ االػٔذح، ٝرؾذ اُخؾجخ رٞعذ . ٗٞع ٖٓ اُغشاس اُنخٔخ ف٘ؼذ ٖٓ اُخضف ٝٛ٢ اُز٢ رنخْ االفٞاد خالٍ اُشهـ ،ٝٗظشا ُشؽبثخ خؾجخ أُغشػ كبٕ اُغٜٔٞس ٣غزي٤غ إ ٣غِظ ك٢ صالس عٜبد ؽٍٞ اُخؾجخ َٔٝػِ٠ أُٔضَ ك٢ ٛزٙ اُؾبُخ إ ٣ؼي٢ اٛٔ٤خ هقٟٞ ُزٔٞهؼٚ ٝ٣غزؼ ٖٓ ٟاُغبس٣خ اُ٤غش اُخؾجخ ُِزٞهق ٝرُي الٕ اُو٘بع ٣ؾذ ٖٓ ؽوِٚ اُجقش١xxv . :ثانٍا:ػناصز اننى وتمنٍاته أ - :انفضاء 522 522 جملة اآل ادا اعدداد 115 جملة اآل ادا اعدداد 115 جملة اآل ادا اعدداد 115 ٝاُشهـ ٣شرجو ثبُـ٘بء اسرجبىب ٝصغوب اُ٠ ؽذ ػذٙ آشا ال ه٤ٔخ ُٚ ٝال اؽبع ٤ظ ٝال ٓؾبس٣غ ٝال ربص٤ش ، ٝرقْٔ اُشهقبد ثؼ٘ب٣خ كبئوخ كوذ " رؼ٘٢ اُخيٞح اُٞاؽذح سؽِخ ًبِٓخ ٝسكغ اُ٤ذ هذ ٣ؼ٘٢ "اُجٌبء ٝادٗ٠ اُزلبرخ ٖٓ اُشاط هذ رؼ٘٢ ٗٞػب ٖٓ اُشكل اٝ االٌٗبسxxviii . ٝك٢ اُ٤بثبٕ ٣ؼٞد اُشهـ اُ٠ االؽزلبالد اُز٢ رؾٞ١ اُضوبكخ اُؾؼج٤خ رِي اُز٢ ٍرزٔظٜش ٖٓ خال اُشهقبد أُو٘ؼخ ك٢ اؽزلبالد اُوشٟ، ٝاُضوبكخ االسعزوشاى٤خ ك٢ اُوقٞس ؽز٠ اُوشٕ اُضبٗ٢ ػؾش ًٝزُي اُ٘بثؼخ ٖٓ االس عزوشاى٤خ ؽز٠ اُوشٕ اُغبثغ ػؾش، ك٤ب ظَ اُو٘بع ٓوذعب ُِشاهق٤ٖ رغغ٤ذا . ُُٞ٘الُٜخ ٝاُؾ٤بى٤ٖ، ُ٤زؾٍٞ اُ٠ هيؼخ ك٘٤خ ٝثبُزبُ٢ اه٘ؼخ ا ما ا سفز ػنه االطار اننظزي من مؤشزاث 1 - رؼٞد عزٝس اُو٘بع ك٢ ٓؼظْ اُؾن بساد اُ٠ االؽزلبالد اُذ٣٘٤خ، ٓؤد٣ب دٝسا ىوغ٤ب ٝٓ٤زبك٤ض٣و٤ب ٜٓٔب، ٝهذ ظٜش اعزؼٔبُٚ ُزٔض٤َ االُٜخ اٝ اعزؾنبس االسٝاػ اٝ اُيوٞط . اُضساػ٤خ، ًٝزُي اُززً٤ش ثبالؽذاس االعيٞس٣خ 2 - ٖٓ اخزِق اعزؼٔبٍ اُو٘بع اُ٤ٞٗبٕ اُ٠ االكبسهخ ٝاُ٤بثبٗ٤٤ٖ، ؽ٤ش اعزؼِٔزٚ ٛزٙ اُؾؼٞة ،ثيشم ٓخزِلخ، اال اٜٗب رغزٔغ ك٢ اػزجبس اُو٘بع رؼج٤شا سٓض٣ب ػٖ ٓظبٛش كٞم اُيج٤ؼ٢ ٝثبُزبُ٢ ػِْ عؾش١ ٣ؤد١ ك٤ٚ اُو٘بع دٝس اُٞع٤و ث٤ٖ ٛزا اُؼبُْ أُوذط ٝاُٞاهغ. 2 - ٖٓ اخزِق اعزؼٔبٍ اُو٘بع اُ٤ٞٗبٕ اُ٠ االكبسهخ ٝاُ٤بثبٗ٤٤ٖ، ؽ٤ش اعزؼِٔزٚ ٛزٙ اُؾؼٞة ،ثيشم ٓخزِلخ، اال اٜٗب رغزٔغ ك٢ اػزجبس اُو٘بع رؼج٤شا سٓض٣ب ػٖ ٓظبٛش كٞم اُيج٤ؼ٢ ٝثبُزبُ٢ ػِْ عؾش١ ٣ؤد١ ك٤ٚ اُو٘بع دٝس اُٞع٤و ث٤ٖ ٛزا اُؼبُْ أُوذط ٝاُٞاهغ. 3 - إ ؽبَٓ اُو٘بع ٣ٔضَ دائٔب اُز١ ٣ؾِٔٚ ٝ٣قجؼ ٙاُو٘بع را عذٟٝ ػ٘ذٓب ٣ؤد١ أُٔضَ دٝس ثٌَ دهخ ٝٛٞ دٝس ٣زٔبؽ٠ ٓؼٚ، ٝ٣ٌٕٞ اُو٘بع ٗٞػب ٖٓ اُو٤بكخ اٝ ُجبعب اٝ ٓالٓؼ رذٍ ػِ٠ اُؾخق٤خ اُز٢ ٝهغ روِ٤ذٛب . اا 2 - ٖٓ اخزِق اعزؼٔبٍ اُو٘بع اُ٤ٞٗبٕ اُ٠ االكبسهخ ٝاُ٤بثبٗ٤٤ٖ، ؽ٤ش اعزؼِٔزٚ ٛزٙ اُؾؼٞة ،ثيشم ٓخزِلخ، اال اٜٗب رغزٔغ ك٢ اػزجبس اُو٘بع رؼج٤شا سٓض٣ب ػٖ ٓظبٛش كٞم اُيج٤ؼ٢ ٝثبُزبُ٢ ػِْ عؾش١ ٣ؤد١ ك٤ٚ اُو٘بع دٝس اُٞع٤و ث٤ٖ ٛزا اُؼبُْ أُوذط ٝاُٞاهغ. 3 - إ ؽبَٓ اُو٘بع ٣ٔضَ دائٔب اُز١ ٣ؾِٔٚ ٝ٣قجؼ ٙاُو٘بع را عذٟٝ ػ٘ذٓب ٣ؤد١ أُٔضَ دٝس ثٌَ دهخ ٝٛٞ دٝس ٣زٔبؽ٠ ٓؼٚ، ٝ٣ٌٕٞ اُو٘بع ٗٞػب ٖٓ اُو٤بكخ اٝ ُجبعب اٝ ٓالٓؼ رذٍ ػِ٠ اُؾخق٤خ اُز٢ ٝهغ روِ٤ذٛب . ٢ ع اغ ٢ ٝثبُزبُ٢ ػِْ عؾش١ ٣ؤد١ ك٤ٚ اُو٘بع دٝس اُٞع٤و ث٤ٖ ٛزا اُؼبُْ أُوذط ٝاُٞاهغ. 3 - إ ؽبَٓ اُو٘بع ٣ٔضَ دائٔب اُز١ ٣ؾِٔٚ ٝ٣قجؼ ٙاُو٘بع را عذٟٝ ػ٘ذٓب ٣ؤد١ أُٔضَ دٝس ثٌَ دهخ ٝٛٞ دٝس ٣زٔبؽ٠ ٓؼٚ، ٝ٣ٌٕٞ اُو٘بع ٗٞػب ٖٓ اُو٤بكخ اٝ ُجبعب اٝ ٓالٓؼ رذٍ ػِ٠ اُؾخق٤خ اُز٢ ٝهغ روِ٤ذٛب . 3 - إ ؽبَٓ اُو٘بع ٣ٔضَ دائٔب اُز١ ٣ؾِٔٚ ٝ٣قجؼ ٙاُو٘بع را عذٟٝ ػ٘ذٓب ٣ؤد١ أُٔضَ دٝس ثٌَ دهخ ٝٛٞ دٝس ٣زٔبؽ٠ ٓؼٚ، ٝ٣ٌٕٞ اُو٘بع ٗٞػب ٖٓ اُو٤بكخ اٝ ُجبعب اٝ ٓالٓؼ رذٍ ػِ٠ اُؾخق٤خ اُز٢ ٝهغ روِ٤ذٛب . 4 - رؤًذ خقٞف٤بد ٓغشػ اُ٘ٞ ػِ٠ اُؾخق٤بد أُؾٞس٣خ ٝاعزؼٔبٍ االه٘ؼخ ٝاالػزٔبد ػِ٠ أُٞع٤و٠ ٝاُشهـ ٝاُـ٘بء ًٞع٤ِخ . ٜٓٔخ ُِزؼج٤ش 4 - رؤًذ خقٞف٤بد ٓغشػ اُ٘ٞ ػِ٠ اُؾخق٤بد أُؾٞس٣خ ٝاعزؼٔبٍ االه٘ؼخ ٝاالػزٔبد ػِ٠ أُٞع٤و٠ ٝاُشهـ ٝاُـ٘بء ًٞع٤ِخ . ب - :انغناء وانمىسٍمى وانزلص ٓب ٣ؼشف ػٖ اُ٘ٞ ٛٞ اػزٔبدٙ اٌُج٤ش ػِ٠ اُقٞد ثٔب ك٢ رُي أُٞع٤و٠ ٝاُـ٘بء ٝأُؤصشاد . اُقٞر٤خ اُزقٞ٣ش٣خ - :أُٞع٤و٠ ٣غِظ أُٞع٤و٤ٕٞ ك٢ ٓؤخشح اُخؾجخ، ٝاُغٞهخ ك٢ اُغبٗت اال٣ٖٔ ٓ٘ٚ، ٝرزٌٕٞ اُغٞهخ ٖٓ صٔبٗ٤خ اُ٠ اص٘٢ ػؾش ؽخقب، آب االالد أُٞع٤و٤خ كٜ٢ اُيجٍٞ ٝأُضٓبس، ك٢ ؽ٤ٖ رٌٕٞ ُِٔٞع٤و٠ . ٝظ٤لخ ك٢ خِن اُغٞ ٝاالػالٕ ػٖ ثذا٣خ اُؼشك ٣غِظ أُٞع٤و٤ٕٞ ك٢ ٓؤخشح اُخؾجخ، ٝاُغٞهخ ك٢ اُغبٗت اال٣ٖٔ ٓ٘ٚ، ٝرزٌٕٞ اُغٞهخ ٖٓ صٔبٗ٤خ اُ٠ اص٘٢ ػؾش ؽخقب، آب االالد أُٞع٤و٤خ كٜ٢ اُيجٍٞ ٝأُضٓبس، ك٢ ؽ٤ٖ رٌٕٞ ُِٔٞع٤و٠ . ٝظ٤لخ ك٢ خِن اُغٞ ٝاالػالٕ ػٖ ثذا٣خ اُؼشك - :اُـ٘بء ٌَ٣ٌٕٞ اُـ٘بء ك٢ ػشك اُ٘ٞ كشد٣ب اٝ عٔبػ٤ب، ٝ٣ؼٞد ؿ٘بء اُغٞهخ ك٢ اُجذا٣خ اُ٠ ٓؾ رو٘٢ ْٜكشمٚ ه٘بع اُ٘ٞ اُوذ٣ْ " اُز١ ًبٕ ٣ؾذ ثٞاعيخ اُلْ ُٝزُي ال٣غزي٤غ أُٔضِٕٞ ٗين ٗقٞف ْٜ٘كٌبٗذ اُغٞهخ رزٌِْ ػٞمب ػ"، آب اُ٤ّٞ كوذ افجؾذ اُغٞهخ روّٞ ثذٝس اُشاٝ١xxvi ، ٝاُغجت اُٞؽ٤ذ ُٞعٞد اُـ٘بء ك٢ اُ٘ٞ ُغزٝسٙ ٝٓ٘يِوبرٚ أُغزٔذح ٖٓ ٓغٔٞػخ كٕ٘ٞ اُؼشك ثٔب ك٢ ر ُي االالػ٤ت اُجِٜٞاٗ٤خxxvii . ٣ٌٕٞ اُـ٘بء ك٢ ٓغشػ اُ٘ٞ ثجذا٣خ اُؼشك ٝٝعيٚ ٜٝٗب٣زٚ، ًٝزُي ُِٔ٢ء اُلشاؿبد ا١ ػ٘ذٓب ،٣ٌٕٞ أُٔضَ ٌٜٓ٘ٔب ك٢ رـ٤٤ش ه٘بػٚ اٝ ٓالثغٚ، كنال ػٖ ٓقبؽجزٚ ُِشهقبد ٝاُؾذس 522 جملة اآل ادا اعدداد 115 ما ا سفز ػنه االطار اننظزي من مؤشزاث ٜٓٔخ ُِزؼج٤ش 5 - ٖٓ ٣ؼٞد اُشهـ ك٢ اُ٤بثبٕ اُ٠ االؽزلبالد اُز٢ رؾٞ١ اُضوبكخ اُؾؼج٤خ رِي اُز٢ رزٔظٜش خالٍ اُشهقبد أُو٘ؼخ ك٢ اؽزلبالد اُوشٟ، ٝاُضوبكخ االسعزوشاى٤خ ك٢ اُوقٞس، اُ٠ عبٗت ثوبء اُو٘بع ٓوذعب ُِشاهق٤ٖ رغغ٤ذا ُالُٜخ ٝاُؾ٤بى٤ٖ، ُ٤زؾٍٞ اُ٠ هيؼخ ك٘٤خ ٝثب. ُُٞ٘زبُ٢ اه٘ؼخ ا 5 - ٖٓ ٣ؼٞد اُشهـ ك٢ اُ٤بثبٕ اُ٠ االؽزلبالد اُز٢ رؾٞ١ اُضوبكخ اُؾؼج٤خ رِي اُز٢ رزٔظٜش خالٍ اُشهقبد أُو٘ؼخ ك٢ اؽزلبالد اُوشٟ، ٝاُضوبكخ االسعزوشاى٤خ ك٢ اُوقٞس، اُ٠ عبٗت ثوبء اُو٘بع ٓوذعب ُِشاهق٤ٖ رغغ٤ذا ُالُٜخ ٝاُؾ٤بى٤ٖ، ُ٤زؾٍٞ اُ٠ هيؼخ ك٘٤خ ٝثب. ُُٞ٘زبُ٢ اه٘ؼخ ا 525 جملة اآل ادا اعدداد 115 جملة اآل ادا اعدداد 115 2016 م1437 هـ انفصم انثانث اجزاءاث انبحث انفصم انثانث اجزاءاث انبحث انفصم انثانث اجزاءاث انبحث :اوال: مجتمغ انبحث ؽَٔ ٓغزٔغ اُجؾش ك٢ اُٞهٞف ػِ٠ رو٘٤بد اُو٘بع ك٢ ٓغشػ( ُٞ٘ا) . اُ٤بثبٗ٢ :ثانٍا: ػٍنت انبحث رْ اٗزوبء ػ٤٘خ اُجؾش ػِ٠ ٝكن اُيش٣وخ اُوقذ٣خ، ٝرُي ثبخز٤بسٙ ٓغشػ( ُٞ٘ا) اُ٤بثبٗ٢ اٗٔٞرعب . ُِزؾِ٤َ، ُزٞكش اُذساعبد ٝأُشاعغ ك٢ ٓٞمٞع اُجؾش :ثانثا أداة :انبحث . أُؤؽشاد اُز٢ اعلش ػٜ٘ب االىبس اُ٘ظش١ . أُؤؽشاد اُز٢ اعلش ػٜ٘ب االىبس اُ٘ظش١ أوال:: تمنٍت انمناع 1 - :انمناع فً انمسزح انمؼاصز 1 - :انمناع فً انمسزح انمؼاصز ٕٞربصش أُخشع ك٢ أُغشػ أُؼبفش ثزو٘٤بد اُو٘بع، ٝال ع٤ٔب ك٢ اُزٞعٜبد اُغٔبُ٤خ اٝ ك٢ اداء ٕأُٔضَ ، ٌُٖٝ اخزِلذ ىشم اعزؼٔبُٚ ٝدٝاػ٤ٚ ٖٓ ٓغشؽ٢ اُ٠ اخش، كوذ اعزؼِٔٚ (ًٞسد ًش٣ظ) اُو٘بع المبكخ اُزؼبث٤ش اُز٢ ٣ش٣ذ ُٜب إ رجشص ػِ٠ ٝعٚ أُٔضَ ثؼ٤ذا ػٖ اٗلؼبالرٚ اُزار٤خ ٝاهقبء الٌَٓبٗبرٚ اُغغذ٣خ، كوذ ًبٕ (ًش٣ظ)، ٣ؼذ اُو٘بع اداح الصاُخ اُيبثغ اُؾخق٢ ػٖ أُٔض ، ،ٝ٣شٟ إ أُٔضَ ر٘ؼٌظ ػِ٤ٚ اٌُض٤ش ٖٓ االٗلؼبالد اُزار٤خ ؿ٤ش أُشؿٞثخ ٝ٣ؾذد اؽزـبالد اُغغذ ُزا كبُو٘بع ثبُ٘غجخ اُ٤ٚ ٛٞ اُشاط أُضبُ٢ ُِٔغشػxxix )، ٝ٣شٟ (عبٕ ُٞى ٞٛ " إ ؽَٔ اُو٘بع "ٚاٝال رـ٤٤ش اُغغذ ثزـ٤٤ش اُٞعxxx )ٖ، ٝرشٟ (اس٣بٕ ٓ٘ٞؽٌ٤ ٌَ" إ اُو٘بع ٗظبّ اعبع٢ الٗٚ ؽ "ٖٖٓ االؽٌبٍ، ًَٝ ؽٌَ ٣خنغ ُ٘ظبّ ٓؼ٤xxxi . ٛزٙ ثؼل ٖٓ ٗٔبرط اُزغبسة أُغشؽ٤خ اُز٢ اػزٔذد اُو٘بع، ًٝبٗذ ؿب٣زٜب اثشاص اٛٔ٤زٚ ك٢ أُغزٔؼبد ٝاُؾنبساد ٝربس ٣خ )ٞٗ( ٍأُغشػ كنال ػٖ اٛٔ٤زٚ ك٢ أُغبسػ اُؾشه٤خ ٝال ع٤ٔب ا .)ٝٛٞ (اٗٔٞرط ثؾض٘ب 528 جملة اآل ادا اعدداد 115 جملة اآل ادا اعدداد 115 2016 م1437 هـ 2 - :ارتباط انمناع بانممذس عجن ٝإ اؽشٗب اُ٠ اسرجبه ثبُٔوذط ٝأُ٤زبك٤ض٣و٢، ٝهذ ظٜش اعزؼٔبُٚ ُزٔض٤َ االُٜخ اٝ اعزؾنبس . االسٝاػ اٝ اُيوٞط اُضساػ٤خ، ًٝزُي اُززً٤ش ثبالؽذاس االعيٞس٣خ ٞٛ ٝٛٞ " ػِٔ٤خ رغغ٤ذ ُٔب ٝال ٓشئ٢ خ٤بُ٢ ػجش ٝع٤و ٓبد١ ٝاهؼ٢ ؽ٤ش رجذ ٛ٘ب ًٝبٜٗب ػِٔ٤خ ٝعبىخ اهشة اُ٠ "اُغؾشxxxii ، ٝ٣ؼذ اُو٘بع رو٘٤خ ٝاداح ُزؾو٤ن ًَ ٓب رًش ُٔب رٔ٤ض ثٚ ٖٓ خشم ُِؼبُْ اُٞاهؼ٢ اُ٠ . اُؼبُْ اُخ٤بُ٢ 1 - انمناع وأداء :انممثم إ اُٞعبئَ اُز٢ رٞكشد ُذٟ أُٔضَ ُِزؼج٤ش ٛ٢ اُغغذ ٝاُقٞد ٝٛ٢ رُٞذ ك٢ ر٘بؿٜٔب ٝر٘بعوٜب ٚٗخيبثب ٓؼ٤٘ب، ُِٝو٘بع ػالهخ ٓجبؽشح ثٜز٣ٖ اُؼ٘قش٣ٖ، كبُو٘بع ٣غٔؼ ُِٔٔضَ ثزغبٝص ً٤ب اُؾخق٢ ٝاُزؾِ٤ن اُ٠ اكبم اُخ٤بٍ اُغؾش٣خ اُز٢ ٣وٞدٙ اُ٤ٜب اُؾٌَ اُال ٓؼزبد اُز١ رٌٕٞ ػِ٤ ٚ ٚؽشًبر ٚرِي اُز٢ ٣لشمٜب ػِ٤ٚ ؽٌَ اُو٘بع اُز١ ٣ِضّ ؽبِٓٚ ثبُزو٤ذ ثخيٞىٚ اُز٢ ر٘زظ ؽشًز "ٝفٞرٚ ٝىبهزٚ ٝهٞرٚ ٝثبُزبُ٢ كبُو٘بع " ؽبُخ ٖٓ ؽبالد كوذإ اُشٝػ اُجذائ٤خ ثؾٌَ ٓؤهذxxxiii . ٝٛزا ٓب ٣غؼَ ٓ٘ٚ ٝع٤ِخ ٖٓ ٝعبئَ اُزؾشس ٝاُو٘بع ٣ٌؾق عغذ أُٔضَ ٝ٣ذكؼٚ ال٣غبد ٝعبئ َ رؼج٤ش اخشٟ ؿ٤ش اُٞعٚ الًزؾبف ُـخ عذ٣ذح ال ػالهخ ُٜب ثـ(اُجبٗزٞٓب٣ْ) ٝال ثبُزؼج٤ش اُغغٔبٗ٢ أُذسٝط، ٝرُي ثٜذف اُؾقٍٞ ػِ٠ ؽو٤وخ ك٘٤خ . ٗو٤خ ٣لشمٜب اُو٘بع ثؼ٤ذا ػٖ االمؾبى ْإ اُو٘بع ٣لشك ثبُنشٝسح رؾٞ٣َ االؽبع٤ظ اُذاخِ٤خ ٝا٣ٔبءاد اُٞعٚ اُ٠ ؽشًبد عغذ٣خ رزشع ٛزٙ االؽبع٤ظ، ك٤ٔب رزٌٕٞ ػِٔ٤خ رشً٤ت اُؾخق٤خ اُز٢ ٣ؾِٜٔب اُو٘بع ثزؾٞ٣َ ٓالٓؼ اُو٘بع َٔٝخيٞىٚ اُؼش٣نخ اُ٠ اُغغذ ُِؾقٍٞ ػِ٠ رشً٤جخ عغذ٣خ ُِؾخق٤خ رزٔبؽ٠ ٓغ اُو٘بع، ٝاُؼ ٚػِ٠ اُغغذ ػٖ ىش٣ن اُو٘بع ٣٘ل٢ ًَ أُؼي٤بد اُذخ٤ِخ ػ٘ٚ ا١ اُو٘بع ٝٓالٓؾٚ ٝخيٞى " ٚٝرنبس٣غ سؿجخ ك٢ ربع٤ظ عغذ ؽش ىِ٤ن ال رؼٞهٚ اُزٔ٤ضاد ٝال ٣ؼزشف ثبُؾٞاعض اُذ٣٘٤خ ٝاالهزقبد٣خ ٝاالعزٔبػ٤خ، ٝٓؾووب ُزارٚ ُٝٔزِو٤ٚ ٗٞػب ٖٓ اُ٘ؾٞح اُجبى٘٤خ اُز٢ رزخِن ٖٓ عشاء "أُؼغضح اُؾشً٤خ ُِغغذ االٗغبٗ٢ ُِٔٔضَ ٝهذسارٚ اُال ٓز٘بٛ٤خ ٝؿ٤ش أُؾذٝدحxxxiv . ثانٍا: انمناع فً م:سزح اننى 1 - :شكم انمناع واستؼمانه 1 - :شكم انمناع واستؼمانه 1 - :شكم انمناع واستؼمانه ٚإ ه٘بع اُ٘ٞ ٓق٘ٞع ٖٓ اُخؾت ٝال ٣خل٢ ًبَٓ اُٞعٚ ثوذس ٓب ٣جشص رهٖ أُٔضَ ٝخذ٣ ٌٕٞ، ٝ٣ . ثبُزبُ٢ هِ٤َ االٗؾذاس ٗؾٞ االعلَ، ًَٝ ه٘بع ٣زٔبؽ٠ ٓغ سٝػ اُؾخق٤خ ٝىج٤ؼزٜب ٝٝمغ اُو٘بع ك٢ اُ٘ٞ ُٚ ٓشؽِٚ ٝىوٞعٚ اُخبفخ، ار إ ٣جذا ثٞمغ اُؾؼش أُغزؼبس ػِ٠ عٔغٔخ اح ذٛب اُ ضَ ا ب وق اُو٘بع ن ص ٖ اُؾ ا يخ ؽ و ذا صوٚ اُ ضَ 1 - :شكم انمناع واستؼمانه ٚإ ه٘بع اُ٘ٞ ٓق٘ٞع ٖٓ اُخؾت ٝال ٣خل٢ ًبَٓ اُٞعٚ ثوذس ٓب ٣جشص رهٖ أُٔضَ ٝخذ٣ ٌٕٞ، ٝ٣ . ثبُزبُ٢ هِ٤َ االٗؾذاس ٗؾٞ االعلَ، ًَٝ ه٘بع ٣زٔبؽ٠ ٓغ سٝػ اُؾخق٤خ ٝىج٤ؼزٜب ٝٝمغ اُو٘بع ك٢ اُ٘ٞ ُٚ ٓشؽِٚ ٝىوٞعٚ اُخبفخ، ار إ ٣جذا ثٞمغ اُؾؼش أُغزؼبس ػِ٠ عٔغٔخ أُٔضَ ٝ٣ٞصوٚ ع٤ذا ثٞاعيخ ؽش٣و ٖٓ اُؾؼش، صْ ٣نغ اُو٘بع ٝ٣وق ثؼذٛب أُٔضَ آبّ ٓشاح ُٔؾبٛذح رؾشًٚ " ٝك٢ ؿشكخ أُشاح ٣غِظ أُٔضَ ٓذح ىٞ٣ِخ ُِزبَٓ ك٢ ؽخق٤زٚ اُ٠ إ ٣ؾؼش "ٚثبُوٞح اُذاخِ٤خ رغزو٤ظ ك٤xxxv . ٚإ ه٘بع اُ٘ٞ ٓق٘ٞع ٖٓ اُخؾت ٝال ٣خل٢ ًبَٓ اُٞعٚ ثوذس ٓب ٣جشص رهٖ أُٔضَ ٝخذ٣ ٌٕٞ، ٝ٣ . ثبُزبُ٢ هِ٤َ االٗؾذاس ٗؾٞ االعلَ، ًَٝ ه٘بع ٣زٔبؽ٠ ٓغ سٝػ اُؾخق٤خ ٝىج٤ؼزٜب ٝٝمغ اُو٘بع ك٢ اُ٘ٞ ُٚ ٓشؽِٚ ٝىوٞعٚ اُخبفخ، ار إ ٣جذا ثٞمغ اُؾؼش أُغزؼبس ػِ٠ عٔغٔخ أُٔضَ ٝ٣ٞصوٚ ع٤ذا ثٞاعيخ ؽش٣و ٖٓ اُؾؼش، صْ ٣نغ اُو٘بع ٝ٣وق ثؼذٛب أُٔضَ آبّ ٓشاح ُٔؾبٛذح رؾشًٚ " ٝك٢ ؿشكخ أُشاح ٣غِظ أُٔضَ ٓذح ىٞ٣ِخ ُِزبَٓ ك٢ ؽخق٤زٚ اُ٠ إ ٣ؾؼش "ٚثبُوٞح اُذاخِ٤خ رغزو٤ظ ك٤xxxv . ُٝاعزؼٔبٍ اُو٘بع ك٢ ا ٘ٞ سٓض ُِؼٞدح اُ٠ االفَ أُوذط، ٝاػبدح سثو اُقِخ ثبالُٜخ ٝثبُيج٤ؼخ ٝرؾو٤ن أُقبُؾخ ٓغ إٌُٞ، ٝأُالؽظ ك٢ عَ اه٘ؼخ اٜٗب رِخـ رٞعٜ٤ٖ سئ٤غ٤٤ٖ ُِلٖ اُ٤بثبٗ٢ ،٣زٔضالٕ ك٢ اُؾبػش٣خ اٝ اُزقٞ٣ش أُغخ٢ (اٌُبسًز٤ش) ٝاُغٔبٍ أُضبُ٢ ٝاُٞاهؼ٤خ اُٜضُ٤خ ٝ٣غبػذ اُو٘بع ػِ٠ ٙرغغ٤ذٛب ثٞاعيخ رٌض٤ق اُيبهخ " ٝثزؼذ٣َ أُٔضَ ُغًِٞٚ اُغغذ١ ٝرؾٞ٣ش اُ٠ ٓغٔٞػخ ٓزشاثيخ ٖٓ االؽبساد ٝاال٣ٔبءاد، كؼِ٠ أُٔضَ رٌض٤ق اال٣ؾبء ػٖ ىش٣ن ثؼش "َٓاُؾ٤بح ك٢ اُو٘بع اُز١ ٣ٌٕٞ ك٢ اُؼبدح رِخ٤قب ُو٤ٔخ اٝ عِٞى اٗغبٗ٢ ًبxxxvi . 2 - :تكىٌن انممثم فً مسزح اننى ربعظ ٖٓغشػ اُ٘ٞ ػِ٠ ٓجذأ اُزٔش٣ش اُز١ ٓش ٖٓ ع٤َ اُ٠ اخش اػزٔبدا ػِ٠ اػشاف ٝهٞاٗ٤ عًِٞ٤خ رؾٌَ ٓالٓؼ أُغزٔغ اُ٤بثبٗ٢ اُز١ ربعغذ ػِ٤ٚ اُيوٞط اُذ٣٘٤خ ٝآزذد اُ٠ االؽزلبالد اُذ٣٘٤خ ٝاُيوغ٤خ ٝرُي ثبالػزٔبد ػِ٠ ٓٔضِ٤ٖ ٓؾٌ٘٤ٖ ٝٓزٔشع٤ٖ رٞاسصٞا ٛزا اُؾٌَ ٖٓ أُغشػ ع٤ال ثؼذ ع٤َ ٝخنؼٞا ُزٌٞ٣ٖ فبسّ ٣زٌٕٞ ٖٓ عجغ ٓشاؽَ ػٔش٣خ، ٝخالٍ ٛزٙ اُغ٘ٞاد ٖٓ ٌٖٔاُزٌٞ٣٘٤خ ُٖ ٣ؤد١ ٓٔضَ اُ٘ٞ ًَ االدٝاس، ثَ ع٤وغ رشً٤ضٙ ػِ٠ دٝس ٝاؽذ ؽز٠ ٣ز ٞٛ ،اُؾقٍٞ ػِ٠ رو٘٤بد فٞر٤خ ٝعغذ٣خ رغؼِٚ ٣ؾخـ اُذٝس اُز١ ٣وّٞ ثٚ ػجش ٝع٤و ٓبد١ ُاُو٘بع " ثؾنٞس اُزٌ٘٤ي ا ٙجذٗ٢ ُِٔٔضَ اُز١ ال ٣ؼجش ػٖ ؽ٢ء ك٢ رِي اُِؾظخ ٌُٝ٘ٚ ٣غِت اٗزجب "أُزلشط ثٞاعيخ اُ٘ٔو اُلش٣ذ ٖٓ اُوذسح ػِ٠ اُزؼج٤ش اُغِج٢ ثزوذ٣ٔٚ ُ٘ٔو ٗلغ٢ اعزٔبػ٢xxxvii . جملة اآل ادا اعدداد 115 جملة اآل ادا اعدداد 115 2016 م1437 هـ 1 - :شكم انمناع واستؼمانه ثانٍا: انمناع فً م:سزح اننى ٣ؼذ اُو٘بع ػ٘قشا اعبع٤ب ك٢ ٓغشػ اُ٘ٞ، ثٞفق إ ػشك اُ٘ٞ ٣ؼزٔذ ػِ٠ ٓٔضَ ٝاؽذ ٝعبػذ ُٚ، ٖٝٓ اعَ رُي كبٕ اُو٘بع ٛٞ أُؾذد اُشئ٤غ٢ ُِخيٞه أُؤعغخ ُِؾخق٤خ، ٝثبُزبُ٢ رشً٤جزٜب ٕاُغغٔبٗ٤خ ٝؽشًزٜب ٝؽز٠ إ ًبٕ أُٔضَ ٣ظٜش ثٞعٚ ػبس ٝثذٕٝ ه٘بع، كبٕ ػِ٤ٚ ا ٚٗ٣ؤد١ ًٝب . ٣ؾَٔ اُو٘بع 522 جملة اآل ادا اعدداد 115 3 - األداء باأللنؼت: اعزؼٔبٍ اُو٘بع ٣لشك ثبُنشٝسح ٓالثظ ٝرشً٤جخ عغٔبٗ٤خ ٝثبُزبُ٢ ؽشًخ ٝرؾشًب ٝا٣ٔبءح رٔ٤ض ؽخق٤خ ػٖ اخشٟ، ٝال ع٤ٔب إ ؿبُجٜب ك٢ اُ٘ٞ ٣ؤد٣ٜب ٓٔضَ ٝاؽذ ، ار " إ رو٘٤بد اُزٔض٤َ ك٢ ،ٓغشػ اُ٘ٞ رٜذف اُ٠ االدٛبػ ٝرؾٞ٣ش اُغغذ ثقلزٜب رو٘٤بد خبفخ رٜذف اُ٠ ٓ٘ؼ أُؼِٞٓبد ك"ٜ٢ رنغ اُغغذ ك٢ اُؾٌَ أُيِٞةxxxviii . ،ٝ٣زْ اُؼَٔ ػِ٠ اُؾخق٤خ ك٢ ٓغشػ اُ٘ٞ ثبُجذء ػِ٠ ا٣غبد اُزشً٤جخ اُغغذ٣خ ٝاُغغٔبٗ٤خ . ٝاٗيالهب ٖٓ ٛزٙ اُزشً٤جخ ٣زْ اُؼَٔ ػِ٠ اُؾشًخ ٝٗٞػ٤زٜب ٝٓغزٞ٣بد اسرٌبصٛب 522 جملة اآل ادا اعدداد 115 جملة اآل ادا اعدداد 115 2016 م1437 هـ 4 - :انتضخٍم 4 - :انتضخٍم جملة اآل ادا اعدداد 115 جملة اآل ادا اعدداد 115 2016 م1437 هـ 2016 م1437 هـ 2 - ُِٔو٘بع ُـخ خبفخ ٣ ٌٖ رذٝ٣ٜ٘ب ثغجت اُؼالهخ اُـش٣جخ ث٤ٖ أُ٘ؾٞد ٝاُغغذ اُجؾش١ . ِٚٓرخنغ اُ٠ عِٔخ ٖٓ اُوٞاػذ ٝاُزو٘٤بد ُزؾو٤ن اُزٌبَٓ ث٤ٖ اُو٘بع ٝؽب 2 - ُِٔو٘بع ُـخ خبفخ ٣ ٌٖ رذٝ٣ٜ٘ب ثغجت اُؼالهخ اُـش٣جخ ث٤ٖ أُ٘ؾٞد ٝاُغغذ اُجؾش١ . ِٚٓرخنغ اُ٠ عِٔخ ٖٓ اُوٞاػذ ٝاُزو٘٤بد ُزؾو٤ن اُزٌبَٓ ث٤ٖ اُو٘بع ٝؽب 3 - ٣ؼذ اُو٘بع ك٢ أُغشػ ُ٤ظ اًغغٞاسا ثغ٤يب ثوذس ٓب ٛٞ ٓؾذد ُٔغَٔ ػ٘بفش اُؼشك ًبُ٘ـ ٝاال٣وبع ٝربص٤ش اُلنبء ٝاُؼالهخ ٓغ اُغٜٔٞس، ُٝثز ي كبٕ اُو٘بع ُ٤ظ ػِٔب . فؾ٤ؾب ٌُٝ٘ٚ كٖ فؾ٤ؼ 3 - ٣ؼذ اُو٘بع ك٢ أُغشػ ُ٤ظ اًغغٞاسا ثغ٤يب ثوذس ٓب ٛٞ ٓؾذد ُٔغَٔ ػ٘بفش اُؼشك ًبُ٘ـ ٝاال٣وبع ٝربص٤ش اُلنبء ٝاُؼالهخ ٓغ اُغٜٔٞس، ُٝثز ي كبٕ اُو٘بع ُ٤ظ ػِٔب . فؾ٤ؾب ٌُٝ٘ٚ كٖ فؾ٤ؼ 4 - ُِو٘بع ٛٞ٣خ ك٘٤خ صوبك٤خ ٝاعزٔبػ٤خ ٝع٤بع٤خ رٔزذ اُ٠ اُؼالط اُ٘لغ٢ ٝاالعزٔبػ٢ ٝاُغغذ١ ٚٝاُزٛ٘٢ ػ٘ذٓب ٣ؼغض اُِلظ ػٖ ىشد االالّ ٝاالٝعبع الٕ ُٚ ػالهخ ثبالٗغبٕ ك٢ ٓغزٞ٣بر . أُزؼذدح 4 - ُِو٘بع ٛٞ٣خ ك٘٤خ صوبك٤خ ٝاعزٔبػ٤خ ٝع٤بع٤خ رٔزذ اُ٠ اُؼالط اُ٘لغ٢ ٝاالعزٔبػ٢ ٝاُغغذ١ ٚٝاُزٛ٘٢ ػ٘ذٓب ٣ؼغض اُِلظ ػٖ ىشد االالّ ٝاالٝعبع الٕ ُٚ ػالهخ ثبالٗغبٕ ك٢ ٓغزٞ٣بر . أُزؼذدح 5 - ٣جؾش اُو٘بع ػٖ اُزاد ٖٓ عٜخ أُش٣ل ٝك٢ ٗلظ اُٞهذ ٝع٤ِخ ُِزؾشس الٗٚ ٣ٔزِي ٖٓ ٖػالهخ ٓغ االٗغبٕ ك٢ ٓغزٞ٣بد ٜٓ٘ب اُغغذ١ ٝاُ٘لغ٢ ٝاُشٝؽ٢، ًٔب ٣ٌٖٔ أُؼبُغ٤ اًزؾبف خجب٣ب ؽخق٤خ أُش٣ل ػجش اُؾلشاد اُز٢ ٣٘زغٜب، ٓضِٔب ٣غٔؼ ثزؾشس عغذ١ . ٝرٞافَ ٗلغ٢ ٝٝمٞػ رٛ٘٢ 5 - ٣جؾش اُو٘بع ػٖ اُزاد ٖٓ عٜخ أُش٣ل ٝك٢ ٗلظ اُٞهذ ٝع٤ِخ ُِزؾشس الٗٚ ٣ٔزِي ٖٓ ٖػالهخ ٓغ االٗغبٕ ك٢ ٓغزٞ٣بد ٜٓ٘ب اُغغذ١ ٝاُ٘لغ٢ ٝاُشٝؽ٢، ًٔب ٣ٌٖٔ أُؼبُغ٤ اًزؾبف خجب٣ب ؽخق٤خ أُش٣ل ػجش اُؾلشاد اُز٢ ٣٘زغٜب، ٓضِٔب ٣غٔؼ ثزؾشس عغذ١ . ٝرٞافَ ٗلغ٢ ٝٝمٞػ رٛ٘٢ :االستنتاجاث 1 - ،٣ؼذ اُو٘بع عضءا ٖٓ أُغشػ ٝ٣خنغ ثبُنشٝسح اُ٠ اُوبػذح، ٝٛزا ٓب ٣غؼَ ٓب ٝفَ اُ٤ٚ ٖٓ هٞاػذ ٝرو٘٤بد ٓشرجيخ ثبعزؼٔبٍ اُو٘بع ؿ٤ش صبثزخ ثوذس ٓب ٛ٢ هبثِخ ُالمبكخ اٝ اُؾزف اٝ اُزـ٤٤ش ؽغت اُـب٣خ ٖٓ اعزؼٔبُٚ ٝثبُزبُ٢ اُخيبة اُز١ . ٚ٣ؤعغ 1 - ،٣ؼذ اُو٘بع عضءا ٖٓ أُغشػ ٝ٣خنغ ثبُنشٝسح اُ٠ اُوبػذح، ٝٛزا ٓب ٣غؼَ ٓب ٝفَ اُ٤ٚ ٖٓ هٞاػذ ٝرو٘٤بد ٓشرجيخ ثبعزؼٔبٍ اُو٘بع ؿ٤ش صبثزخ ثوذس ٓب ٛ٢ هبثِخ ُالمبكخ اٝ اُؾزف اٝ اُزـ٤٤ش ؽغت اُـب٣خ ٖٓ اعزؼٔبُٚ ٝثبُزبُ٢ اُخيبة اُز١ . ٚ٣ؤعغ 2 - رأصش ثؼل أُخشع٤ٖ ٝاٌُزبة اُؼبُٔ٤٤ٖ ثزو٘٤بد اُو٘بع، ار ا عزؼبٗٞا ثٚ، ٓغغال َٔؽنٞسٙ ك٢ اُؼشٝك أُغشؽ٤خ، الٕ اُزقٞس اُغٔبُ٢ ُؼشك ٓغشؽ٢ ٣ؾز ٖٓ اُؼذ٣ذ ٖٓ االٌٓبٗبد ٝاالخز٤بساد اُز١ ٣شعغ اُ٠ اُؼشك أُغشؽ٢ اٝ اُٜذف ٝساءٙ ٝاُـشك اُز١ اػذ ٖٓ اعِٚ، ٝرؾذد ٛزٙ االٌٓبٗ٤بد ثزؾذد اُٞمؼ٤بد . ٝاُغٔبُ٤بد أُشاد االؽزـبٍ ػِ٤ٜب 2 - رأصش ثؼل أُخشع٤ٖ ٝاٌُزبة اُؼبُٔ٤٤ٖ ثزو٘٤بد اُو٘بع، ار ا عزؼبٗٞا ثٚ، ٓغغال َٔؽنٞسٙ ك٢ اُؼشٝك أُغشؽ٤خ، الٕ اُزقٞس اُغٔبُ٢ ُؼشك ٓغشؽ٢ ٣ؾز ٖٓ اُؼذ٣ذ ٖٓ االٌٓبٗبد ٝاالخز٤بساد اُز١ ٣شعغ اُ٠ اُؼشك أُغشؽ٢ اٝ اُٜذف ٝساءٙ ٝاُـشك اُز١ اػذ ٖٓ اعِٚ، ٝرؾذد ٛزٙ االٌٓبٗ٤بد ثزؾذد اُٞمؼ٤بد . ٝاُغٔبُ٤بد أُشاد االؽزـبٍ ػِ٤ٜب 527 4 - :انتضخٍم ٣ؼذ اُزنخ٤ْ ك٢ أُغشػ ػ٘قشا ٖٓ اُؼ٘بفش اُز٢ ٣ؼَٔ ػِ٤ٜب ٙأُجذػٕٞ الثشاص ٓب ٣ٌٖٔ اثشاص ك٢ اُؾخق٤خ اٝ ُزج٤بٕ ٓب ٣ش٣ذٕٝ اُٞفٍٞ اُ٤ٚ، ٝاؽ٤بٗب ٗغذ اُؾخق٤خ رارٜب روزن٢ رنخ٤ٜٔب ػِ٠ ٓغزٟٞ اُؾشًخ ٝاُ٘ظش ٝأُالثظ ٝاُزشً٤جخ اُغغذ٣خ، ٝك٢ أُغشػ اُ٤بثبٗ٢ ؽ٤ش اُلنبء اُز١ ٣زغْ ثبُلشاؽ ٝاالرغبع ٝٝعٞد (عشاس) رؾذ كنبء خؾجخ أُغ شػ ُزنخ٤ْ االفٞاد ٝٝهغ االهذاّ ٝفٞال اُ٠ أُالثظ اُز٢ رزٔ٤ض ثٌضشح هيؼٜب ٝكنلنزٜب ٝمخبٓزٜب ًٝضشح اُٞاٜٗب ٝىُٜٞب ، ٝ٣وغ ًَ ٛزا ٓشٝسا ثبُؾشًخ اُز٢ رزٔ٤ض ثبُخلخ ٝاُذهخ ٝاُنخبٓخ اُز٢ رؼٞد اعبعب اُ٠ اعزؼٔبٍ اُو٘بع، ار إ " ٓب ٣غِت االٗزجبٙ ك٢ اعزؼٔبٍ اُو٘بع ٛٞ اد ٚاء أُٔضَ ؽ٤ش ارغٔذ ؽشًبر ثبُشفبٗخ ٝمخبٓخ ارو٘ذ اُ٠ اثؼذ اُؾذٝد ٝهذ ًبٕ فٞرٚ ٣ؼْ هبػخ ًبِٓخ ِٓ٤ئخ ثبُغٜٔٞس، ؽز٠ "ك٢ ٓغبسػ اُٜٞاء اُيِن كبٕ االُوبء ٣ٌٕٞ ثي٤ئب ٝٝامؾبxxxix . ٝاُ٠ عبٗت إ اُزنخ٤ْ رو٘٤خ ٓشرجيخ ثبُو٘بع، اال اٜٗب رلشك ا٣نب ىج٤ؼخ اُؾخق٤خ ٝال ع٤ٔب ك٢ ٝٓغشػ اُ٘ٞ اُز١ رزٔ٤ض ٓغَٔ ؽخق٤برٚ ثبُخ٤بٍ ُٝ٤غذ ؽخق٤بد ػبد٣خ ٓضَ االُٚ ٝاُؾ٤يبٕ ا اُٞؽؼ ٝاسٝاػ أُؾبسث٤ٖ، ٝأُشاح ثٞفلٜب ؽخقب ؿش٣جب العزجؼبدٛب ػٖ ٓنٔبس اُؾ٤بح اُؼِٔ٤خ خبسط أُ٘ضٍ . ٝرغغ٤ذ ٛزٙ اُؾخق٤بد ٣زيِت " رؼذ٣َ اُغِٞى اُغغذ١ ٝرؾٞ٣شٙ اُ٠ ٓغٔٞػخ ٖٓ اال ؽبساد ٝاال٣ٔبءاد ثٔغبػذح اُو٘بع اُز١ ٣ؼَٔ ػَٔ ؽبَٓ ٓشئ٢ ُِوٟٞ اُال ٓشئ٤خ اُز٢ "رغٌٖ اُيج٤ؼخ، ٝرُي ثزٌض٤ق اال٣ؾبء ػٖ ىش٣ن ثؼش اُؾ٤بح ك٢ اُو٘بعxl . ٝاالداء ثبُو٘بع ٣غزٞعت اعبعب رؾٞ٣َ عٔ٤غ االؽبع٤ظ ٝاالكٌبس اُ٠ ؽشًبد عغذ٣خ ٝامؾخ ُُِٞؼ٤بٕ، ٝرُي ك٢ ؿ٤بة رؼبث٤ش ا عٚ، ٝإ ًبٕ اُٞعٚ ػبس٣ب ثذٕٝ ه٘بع كؼِ٠ أُٔضَ ا ٕ ٣ؤد١ ثٞعٚ ٓؾب٣ذ ا١ دٕٝ رؼبث٤ش. َإ االداء ثٞاعيخ اُو٘بع ٣غؼ أُٔضَ ٣٘زوَ اُ٠ ػبُْ ًِٚ ؿشاثخ ٝ٣لٞم ًَ ٓب ٛٞ ٓبُٞف ٝٓؼزبد ك٢ ػبُٔ٘ب اُ٤ٞٓ٢ " ك٤زـ٤ش اُٞعٚ ثزـ٤ش اُغغذ ٝٗذخَ ك٢ اداء اًضش ٖٓ االداء اُ٤ٞٓ٢، ٝثزُي ٣ٌٖٔ ػذ اُزنخ٤ْ رو٘٤خ ٓشرجيخ ثبعزؼٔبٍ اُو٘بع ًٞع٤ِخ ُِزؼج٤ش رخشط ػٖ أُؼزبد ٝاُ٤ٞٓ٢ اُ٠ "ػبُْ عؾش١xli ٣ؼزوذ ٖٓ خالُٚ اُ٤بثبٗ٤ٕٞ إ اُو٘بع رغٌ٘ٚ اسٝاػ ؽش٣شح اٝ خ٤شح ٝػجش رو٘٤بد . ٓزؼِوخ ثبعزؼٔبٍ اُو٘بع انفصم انزابغ اننتائج ومنالشتها 1 - ٓش اُو٘بع ثزؾٞالد ٓخزِلخ ٝٓز٘ٞػخ اثزذاء ٖٓ روبع٤ٔٚ ٝروغ٤ٔبرٚ ٖٓ اُيوغ٢ اُ٠ ٚأُغشؽ٢، ٝٛ٘بى ػالهخ ث٤ٖ اُ٘ٞ اُ٤بثبٗ٢ ٝأُٔضَ ًبٗغبٕ ٣ؼَٔ اُو٘بع ػِ٠ ر٘ٔ٤خ ؽٞاع َٝهذسارٚ، ًٔب ٣ؼ٤ذ ُغغذٙ اُزٞاصٕ ثبُز٘بؿْ ٝاُشؽبهخ ٝأُشٝٗخ ٝاُزٞاكن ث٤ٖ ٗلظ أُٔض . ٚٝٓشاًض اُغبرث٤خ ُذ٣ جملة اآل ادا اعدداد 115 :اوال: انمزاجغ بانهغت انؼزبٍت :زبٍ ز جغ ب و ا: 1 - ثبٝسصٕٞ، كٞث٤ ، أُغشػ ك٢ اُؾشم، دساعخ ك٢ اُشهـ أُغشؽ٢ ك٢ اع٤ب، رشعٔخ اؽذ ،سمب ٓؾٔذ، ٓشًض اُؾبسهخ ُالثذاع اُلٌش١، ٌٓزجخ أُغشػ1995 . 2 -ص٣بٓ٢، ٓٞرًٞ٤ٞ ٝاخشٕٝ، ٓغشػ اُ٘ٞ اُ٤بثبٗ٢ ٓغِٔٚ ٖٓ أُغشػ اُؼبُٔ٢، رشعٔخ عَٔ٤ 1 - ثبٝسصٕٞ، كٞث٤ ، أُغشػ ك٢ اُؾشم، دساعخ ك٢ اُشهـ أُغشؽ٢ ك٢ اع٤ب، رشعٔخ اؽ ،سمب ٓؾٔذ، ٓشًض اُؾبسهخ ُالثذاع اُلٌش١، ٌٓزجخ أُغشػ1995 . ػ ١ عا 2 - ص٣بٓ٢، ٓٞرًٞ٤ٞ ٝاخشٕٝ، ٓغشػ اُ٘ٞ اُ٤بثبٗ٢ ٓغِٔٚ ٖٓ أُغشػ اُؼبُٔ٢، رشعٔخ ع َٔ٤ اُنؾبى، ع303 ٝ 309 ٕ، ٓب٣ظ ٝؽض٣شا1993 ، اٌُٞ٣ذ، أُغِظ اُٞى٘٢ ُِضوبكخ . ٝاُلٕ٘ٞ ٝاالداة 2 - ص٣بٓ٢، ٓٞرًٞ٤ٞ ٝاخشٕٝ، ٓغشػ اُ٘ٞ اُ٤بثبٗ٢ ٓغِٔٚ ٖٓ أُغشػ اُؼبُٔ٢، رشعٔخ ع َٔ٤ اُنؾبى، ع303 ٝ 309 ٕ، ٓب٣ظ ٝؽض٣شا1993 ، اٌُٞ٣ذ، أُغِظ اُٞى٘٢ ُِضوبكخ . ٝاُلٕ٘ٞ ٝاالداة 3 - عبٓؼٕ، ٜٓشا ، اُغغذ أُ٘قٞؿ ُٚ ٝاُغغذ اُؾش ك٢ ٗظش٣بد االخشاط اُؾذ٣ضخ، ط1 ، ٓغِخ أُغشػ، ع33 ، ٓبسط1996 . ٕ، داس اُغ٤َ ُِ٘ؾش ٝاُزٞص٣غ ٝاُيجبػخ ث٤شٝد ُج٘ب 3 - عبٓؼٕ، ٜٓشا ، اُغغذ أُ٘قٞؿ ُٚ ٝاُغغذ اُؾش ك٢ ٗظش٣بد االخشاط اُؾذ٣ضخ، ط1 ، ٓغِخ أُغشػ، ع33 ، ٓبسط1996 . ٕ، داس اُغ٤َ ُِ٘ؾش ٝاُزٞص٣غ ٝاُيجبػخ ث٤شٝد ُج٘ب 4 - ،فبُؼ، د. عؼذ ، رو٘٤بد أُٔضَ ك٢ أُغشػ اُؾشه٢، ٓغِخ أُغشػ، ػذد56 ، عٞ٣ِ٤خ 1993 . 4 - ،فبُؼ، د. عؼذ ، رو٘٤بد أُٔضَ ك٢ أُغشػ اُؾشه٢، ٓغِخ أُغشػ، ػذد56 ، عٞ٣ِ٤خ 1993 . 5 - ك٤بك، د. ُ٤ِ٠ ِٓ٤ؾخ، ٓؼغْ اُيالة، داس اٌُزت اُؼِٔ٤خ، ه1 ،ٕ، ث٤شد ُج٘ب2005 . 5 - ك٤بك، د. ُ٤ِ٠ ِٓ٤ؾخ، ٓؼغْ اُيالة، داس اٌُزت اُؼِٔ٤خ، ه1 ،ٕ، ث٤شد ُج٘ب2005 . - ٓ٘ظٞس، اثٖ، ُغبٕ اُؼشة، داس ث٤شٝد، ط11 - 12 ، 1990 . 7 - ُ٣ٞعل٢، د. ؽغٖ، أُغشػ ٝاالٗزشٝثُٞٞع٤ب، داس اُضوبكخ، ٓؤعغخ ا ٘ؾش ٝاُزٞص٣غ، اُذاس ،اُج٤نبء2002 . جملة اآل ادا اعدداد 115 2016 م1437 هـ :ثانٍا: بانهغت انفزنسٍت 1- Allard, Genevieve, Le masque Serie que-saige? Imprimerie de presses universitaires de France Paris octobre 1998. 1- Allard, Genevieve, Le masque Serie que-saige? Imprimerie de presses universitaires de France Paris octobre 1998. 2- Ariane, Mnoouchkhine, Le masque une discipline de base au theatre du soleil. Le masque du rite au theatre Op. Cit . 2- Ariane, Mnoouchkhine, Le masque une discipline de base au theatre du soleil. Le masque du rite au theatre Op. Cit . 3- David, Martine, Le theatre, Edition Beline 75006 Paris 1995 . 3- David, Martine, Le theatre, Edition Beline 75006 Paris 1995 . 4- Dieterlen, Germaine, Societes traditionnelles d Afrique ocidentale . Le masque du rite au theatre CNRS Edition 75005 Paris 1995. 4- Dieterlen, Germaine, Societes traditionnelles d Afrique ocidentale . Le masque du rite au theatre CNRS Edition 75005 Paris 1995. 5- Hubert Marie Claude, le theatre. Editions les essentions. Milan OULOUSE Mars 1996 . 6- LE Cop Jacques, Role du masque dans la formation de l acteur IBID . 7- Le Larousse de poche 2002, Edition mis a jour 2001. 7- Le Larousse de poche 2002, Edition mis a jour 2001. 8- Pavis, Patrice, Dictionnaire de theatre, Armmand Colin, Paris 2002. 527 جملة اآل ادا اعدداد 115 جملة اآل ادا اعدداد 115 جملة اآل ادا اعدداد 115 2016 م1437 هـ 9- Pignard, Robert, histoire du theatre, serie: que sais-je? Presses universitaire de France, Paris 1945. 10- Sieffert, rene, Connaissance de l orient. Collection UNESCO d ceuvres representatives. Edition Gallimard 1960 . 10- Sieffert, rene, Connaissance de l orient. Collection UNESCO d ceuvres representatives. Edition Gallimard 1960 . 11- Zeami, La tradition secrete du No suivi de: Uve Journee NO, Traduction et commentaire de ReneK, Sieffert, La tradition secrete du NO> Edition Gallinard 1960 . 522 522 جملة اآل ادا اعدداد 115 :ثالثا: الوواقع االلكترونية http:Theatre NO, Article de Wikipidia, L encyclopedie libre. 522 جملة اآل ادا اعدداد 115 جملة اآل ادا اعدداد 115 2016 م1437 هـ 2016 م1437 هـ : انهىامش  جوردن كريج، (1872-1966)، مخرج انكليزي وسينوغراف ومنظر مسرحين ويعد من اهم التجريبيين الاصلاحيين لحركة المسرح العالمي، وهو مبدع نظرية (خشبة المسرح التشكيلية الجديدة) وخلق مفهوم لعناصر العرض المسرحية(الباحث) .  بروتولد برشت، (1898-1956)، مخرج ومنظر مسرحي الماني (الباحث) .  مايرخولد، (1874-1940)، مخرج ومصمم الماني، اثر منهجه (البيوميكانيكا) في عمل الممثل في المسرح العالمي عموما، له تجارب ومفاهيم في الاخراج والفضاء المسرحي ونظريته حول الجسد (الباحث) .  بيتر بروك، ولد عام 1925في لندن وهو من ابويين روسيين، يعد واحدا من ابرز المخرجين المسرحيين في العالم (الباحث) . i -ابن منظور، لسان العرب، دار بيروت، ج 11-12، 1990، ص 2003. i -ابن منظور، لسان العرب، دار بيروت، ج 11-12، 1990، ص 2003. ii - Genevieve Allard, Le masque Serie que-saige? Imprimerie de presses universitaires de France Paris octobre 1998. P 3 . ii - Genevieve Allard, Le masque Serie que-saige? Imprimerie de presses universitaires de France Paris octobre 1998. P 3 . iii - Patrice Pavis, Dictionnaire de theatre, Armmand Colin, Paris 2002 . P 198 . iv - I Bid, P 4 . iii - Patrice Pavis, Dictionnaire de theatre, Armmand Colin, Paris 2002 . P 198 . iv - I Bid, P 4 .  سيعتمد الباحث المصطلحات الفرسية، فضلا عن المصادر والمراجع في اللغة نفسها، لسهولة حصول Iعليها و توفرها من جهة، ولتمكنه من ايجاد المترجم المتخصص بها من جهة اخرى .  سيعتمد الباحث المصطلحات الفرسية، فضلا عن المصادر والمراجع في اللغة نفسها، لسهولة حصول Iعليها و توفرها من جهة، ولتمكنه من ايجاد المترجم المتخصص بها من جهة اخرى .  سيعتمد الباحث المصطلحات الفرسية، فضلا عن المصادر والمراجع في اللغة نفسها، لسهولة حصول Iعليها و توفرها من جهة، ولتمكنه من ايجاد المترجم المتخصص بها من جهة اخرى . v - Frantisi-Ducroux (Francoise) et Vernant (Jean Pierre), Divinites au masque dans la Grece ancienn, la masque du rite au theatre . CNRS Editions Paris 1995, P 19 . v - Frantisi-Ducroux (Francoise) et Vernant (Jean Pierre), Divinites au masque dans la Grece ancienn, la masque du rite au theatre . CNRS Editions Paris 1995, P 19 . vi -ينظر: د. حسن يوسفي، المسرح والانتروبولوجيا، دار الثقافة، مؤسسة النشر والتوزيع، الدار البيضاء، 2002، ص ص 14-13. - Frantisi-Ducroux (Francoise) et Vernant (Jean Pierre), Divinites au masque dans la rece ancienn, la masque du rite au theatre . CNRS Editions Paris 1995, P 19 . : انهىامش v - Frantisi-Ducroux (Francoise) et Vernant (Jean Pierre), Divinites au masque dans la Grece ancienn, la masque du rite au theatre . CNRS Editions Paris 1995, P 19 . vi -ينظر: د. حسن يوسفي، المسرح والانتروبولوجيا، دار الثقافة، مؤسسة النشر والتوزيع، الدار البيضاء، 2002، ص ص 14-13. Grece ancienn, la masque du rite au theatre . CNRS Editions Paris 1995, P 19 . vi -ينظر: د. حسن يوسفي، المسرح والانتروبولوجيا، دار الثقافة، مؤسسة النشر والتوزيع، الدار البيضاء، 2002، ص ص 14-13. vi -ينظر: د. حسن يوسفي، المسرح والانتروبولوجيا، دار الثقافة، مؤسسة النشر والتوزيع، الدار البيضاء، 2002، ص ص 14-13. vii - Genevieve Allard, Op. Cit. p.p. 11-12 . iii - Genevieve Allard, Op. Cit. p.p. 11-12 . viii - Le Larousse de poche 2002, Edition mis a jour 2001. P 892 . i viii - Le Larousse de poche 2002, Edition mis a jour 2001. P 892 . i ix - Genevieve Allard, Op. Cit, P 14 . x - Martine David, Le theatre, Edition Beline 75006 Paris 1995 . P 237 . i xi - Patrice Pavis, Op. Cit. P 196 . xi - Patrice Pavis, Op. Cit. P 196 . xii -د. حسن يوسفي، المسرح والانتروبولوجيا، مرجع سابق، ص 13. xii -د. حسن يوسفي، المسرح والانتروبولوجيا، مرجع سابق، ص 13. xii -د. حسن يوسفي، المسرح والانتروبولوجيا، مرجع سابق، ص 13. xii -د. حسن يوسفي، المسرح والانتروبولوجيا، مرجع سابق، ص 13. xiii - Germaine Dieterlen, Societes traditionnelles d Afrique ocidentale . Le masque du rite au theatre CNRS Edition 75005 Paris 1995, P 27 . xiv i id O Ci 269 xiii - Germaine Dieterlen, Societes traditionnelles d Afrique ocidentale . Le masque du rite au theatre CNRS Edition 75005 Paris 1995, P 27 . i xiii - Germaine Dieterlen, Societes traditionnelles d Afrique ocidentale . Le masque du rite au theatre CNRS Edition 75005 Paris 1995, P 27 . xiv - Martine David, Op. Cit P 269 . x xvi - Martine David, Op. Cit P 273 . ii xvi - Martine David, Op. Cit P 273 . ii xvii - Martine David, Op. Cit P 273-274 . xvii - Martine David, Op. Cit P 273-274 . iii xviii - Patrice Pavis, Op. Cit. P 59 . i xviii - Patrice Pavis, Op. Cit. P 59 . i xix - Marie Claude Hubert, le theatre. Editions les essentions. Milan OULOUSE Mars 1996, P 20 . xix - Marie Claude Hubert, le theatre. Editions les essentions. Milan OULOUSE Mars 1996, P 20 . 522 جملة اآل ادا اعدداد 115 جملة اآل ادا اعدداد 115 2016 م1437 هـ 2016 م1437 هـ xx - Zeami, La tradition secrete du No suivi de: Uve Journee NO, Traduction et commentaire de rene sieffert. Connaissance de l orient. Collection UNESCO d ceuvres representatives. Edition Gallimard 1960, P 15 . xx - Zeami, La tradition secrete du No suivi de: Uve Journee NO, Traduction et commentaire de rene sieffert. Connaissance de l orient. Collection UNESCO d ceuvres representatives. Edition Gallimard 1960, P 15 . xxi -فوبيون باورز، المسرح في الشرق، دراسة في الرقص المسرحي في اسيا، ترجمة احد رضا محمد، مركز الشارقة للابداع الفكري، مكتبة المسرح، 1995، ص 502. xxi -فوبيون باورز، المسرح في الشرق، دراسة في الرقص المسرحي في اسيا، ترجمة احد رضا محمد، مركز الشارقة للابداع الفكري، مكتبة المسرح، 1995، ص 502. i - Le No en quelques lignes, Op. Cit, P 1 . iii xxii - Le No en quelques lignes, Op. Cit, P 1 . iii xxii - Le No en quelques lignes, Op. Cit, P 1 . xxiii xxiv - Robert Pignard, histoire du theatre, serie: que sais-je? Presses universitaire de France, Paris 1945, P 38 . xxiv - Robert Pignard, histoire du theatre, serie: que sais-je? xii -د. حسن يوسفي، المسرح والانتروبولوجيا، مرجع سابق، ص 13. Presses universitaire de France, Paris 1945, P 38 . xxv - http:Theatre NO, Article de Wikipidia, L encyclopedie libre. xxvi - Wikipidia IBID. P 4 . xxv - http:Theatre NO, Article de Wikipidia, L encyclopedie libre. xxvi - Wikipidia IBID. P 4 . xxvii -د. ليلى مليحة فياض، معجم الطلاب، دار الكتب العلمية، ط1، بيرت لبنان، 2005، ص 8. xxviii -د. سعد صالح، تقنيات الممثل في المسرح الشرقي، مجلة المسرح، عدد 56، جويلية 1993، ص 84. xxix -ينظر: د. يوسفي حسن، مرجع سابق، ص 22. -(جون لوك 16321704) فيلسوف تجريبي ومفكر سياسي إنكليزي .(الباحث) -(جون لوك 16321704) فيلسوف تجريبي ومفكر سياسي إنكليزي .(الباحث) Role du masque dans la formation de l acteur, Le masque du rite au 65 -(جون لوك 16321704) فيلسوف تجريبي ومفكر سياسي إنكليزي .(الباحث) xxx - Lecoq Jacques, Role du masque dans la formation de l acteur, Le masque du rite au theatre, Op. Cit. P 265 . xxx - Lecoq Jacques, Role du masque dans la formation de l acteur, Le masque du rite au theatre, Op. Cit. P 265 . xxx - Lecoq Jacques, Role du masque dans la formation de l acteur, Le masque du rite au theatre, Op. Cit. P 265 .  (آريان منوستكين1940) وُلدت في باريس من أبوين روسيين وتخرجت من جامعة (السوربون) أسست فرقة فرقة (مسرح الشمس) عام1964والتي ضمت مجموعة من الهواة وعلى شكل جمعية تعاونية عمالية تحولت فيما بعد إلى فرقة مسرحية. xxxi - Mnoouchkhine Ariane, Le masque une discipline de base au theatre du soleil. Le masque du rite au theatre Op. Cit, P 233 . xxxi - Mnoouchkhine Ariane, Le masque une discipline de base au theatre du soleil. Le masque du rite au theatre Op. Cit, P 233 . xxxii -موتوكيو زيامي واخرون، مسرح النو الياباني، ترجمة جميل الضحاك، مجلة المسرح العالمي، ع 308-309، المجلس الوطني للثقافة والفنون والاداب، الكويت، مايس، 1998، ص 15. xxxiii -المرجع السابق، ص 16. xxxiv -مهران سامح، الجسد المنصوص له والجسد الحر في نظريات الاخراج الحديثة، ج1، مجلة المسرح، ع 88، مارس 1996، دار الجيل للنشر والتوزيع والطباعة بيروت لبنان، ص 69. xxxv -د. يوسفي حسن، مرجع سابق، ص 34. xxxvi -د. سعد صالح، مرجع سابق، ص 87. xxxvii -موتوكيو زيامي واخرون، مرجع سابق، ص 15. xxxviii -المرجع السابق، ص 17. xii -د. حسن يوسفي، المسرح والانتروبولوجيا، مرجع سابق، ص 13. xxxix Sieffert ReneK La tradition secrete du NO> Edition Gallinard 1960 P 31 xxxii -موتوكيو زيامي واخرون، مسرح النو الياباني، ترجمة جميل الضحاك، مجلة المسرح العالمي، ع 308-309، المجلس الوطني للثقافة والفنون والاداب، الكويت، مايس، 1998، ص 15. xxxiii -المرجع السابق، ص 16. xxxiv -مهران سامح، الجسد المنصوص له والجسد الحر في نظريات الاخراج الحديثة، ج1، مجلة المسرح، ع 88، مارس 1996، دار الجيل للنشر والتوزيع والطباعة بيروت لبنان، ص 69. xxxv -د. يوسفي حسن، مرجع سابق، ص 34. xxxvi -د. سعد صالح، مرجع سابق، ص 87. xxxvii -موتوكيو زيامي واخرون، مرجع سابق، ص 15. xxxviii -المرجع السابق، ص 17. xxxix Si ff t R K L t diti t d NO> Editi G lli d 1960 P 31 xxxii -موتوكيو زيامي واخرون، مسرح النو الياباني، ترجمة جميل الضحاك، مجلة المسرح العالمي، ع 308-309، المجلس الوطني للثقافة والفنون والاداب، الكويت، مايس، 1998، ص 15. xxxiii -المرجع السابق، ص 16. -المرجع السابق، ص 16. xxxiv -مهران سامح، الجسد المنصوص له والجسد الحر في نظريات الاخراج الحديثة، ج1، مجلة المسرح، ع 88، مارس 1996، دار الجيل للنشر والتوزيع والطباعة بيروت لبنان، ص 69. xxxv -د. يوسفي حسن، مرجع سابق، ص 34. xxxvi -د. سعد صالح، مرجع سابق، ص 87. xxxvii -موتوكيو زيامي واخرون، مرجع سابق، ص 15. xxxviii -المرجع السابق، ص 17. xxxiv -مهران سامح، الجسد المنصوص له والجسد الحر في نظريات الاخراج الحديثة، ج1، مجلة المسرح، ع 88، مارس 1996، دار الجيل للنشر والتوزيع والطباعة بيروت لبنان، ص 69. xxxv -د. يوسفي حسن، مرجع سابق، ص 34. xxxvii -موتوكيو زيامي واخرون، مرجع سابق، ص 15. xxxvii -موتوكيو زيامي واخرون، مرجع سابق، ص 15. xxxviii xxxix - Sieffert ReneK La tradition secrete du NO> Edition Gallinard 1960, P 31 . xxxix - Sieffert ReneK La tradition secrete du NO> Edition Gallinard 1960, P 31 . xl -سعد صالح، مرجع سابق، ص 78. xli - LE Cop Jacques, Role du masque dans la formation de l acteur IBID, P 265 . 522 جملة اآل ادا اعدداد 115 2016 م1437 هـ Mask Technology Masks Theater (Nau) a model Research by Zaid Salim Suliman Baghdad University / Center for Women's Studies Research Summary: Mask his presence record in almost all civilizations and cultures successive particularly during liturgical celebrations and religious, as this presence in everyday life seemed then go to the theater to become an expression of aesthetically pleasing in terms of formation Representative and other aesthetic of the product employs theatrical performances throughout the periods of the history of the theater. Our selection of the subject (the mask technique) necessarily imposes an orientation aesthetic choices and techniques related to it and different from other aesthetics on the level of representation of actor such as animation, sound, speech and gesture and the rest of the display elements of fashion and decoration and others. Hence our study to look at the mask techniques, and taken from the scene (Nau) Japanese have a model. 525
https://openalex.org/W2105212323	https://www.zora.uzh.ch/id/eprint/27028/11/Jefimovs.pdf	English	null	Advanced X-ray diffractive optics	Journal of physics. Conference series	2,009	cc-by	2,280	Zurich Open Repository and Archive University of Zurich University Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Originally published at: Vila-Comamala, J; Jefimovs, K; Pilvi, T; Ritala, M; Sarkar, S S; Solak, H H; Guzenko, V A; Stampanoni, M; Marone, F; Raabe, J; Tzvetkov, G; Fink, R H; Grolimund, D; Borca, C N; Kaulich, B; David, C (2009). Advanced X-ray diffractive optics. Journal of Physics: Conference Series, 186:012078. DOI: https://doi.org/10.1088/1742-6596/186/1/012078 Advanced X-ray diffractive optics Vila-Comamala, J ; Jefimovs, K ; Pilvi, T ; Ritala, M ; Sarkar, S S ; Solak, H H ; Guzenko, V A ; Stampanoni, M ; Marone, F ; Raabe, J ; Tzvetkov, G ; Fink, R H ; Grolimund, D ; Borca, C N ; Kaulich, B ; David, C DOI: https://doi.org/10.1088/1742-6596/186/1/012078 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-27028 Journal Article Published Version The following work is licensed under a Creative Commons: Attribution 4.0 International (CC BY 4.0) License. Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-27028 Journal Article Published Version The following work is licensed under a Creative Commons: Attribution 4.0 ollowing work is licensed under a Creative Commons: Attribution 4.0 International (CC BY 4.0) License. Originally published at: Vila-Comamala, J; Jefimovs, K; Pilvi, T; Ritala, M; Sarkar, S S; Solak, H H; Guzenko, V A; Stampanoni, M; Marone, F; Raabe, J; Tzvetkov, G; Fink, R H; Grolimund, D; Borca, C N; Kaulich, B; David, C (2009). Advanced X-ray diffractive optics. Journal of Physics: Conference Series, 186:012078. DOI: https://doi.org/10.1088/1742-6596/186/1/012078 IOP Publishing doi:10.1088/1742-6596/186/1/012078 9th International Conference on X-Ray Microscopy Journal of Physics: Conference Series 186 (2009) 012078 J Vila-Comamala1, K Jeﬁmovs1,2, T Pilvi3, M Ritala3, S S Sarkar1, H H Solak1, V A Guzenko1, M Stampanoni1,4, F Marone1, J Raabe1, G Tzvetkov1, R H Fink5, D Grolimund1, C N Borca1, B Kaulich6 and C David1 1Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland 2 1Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland 3Department of Chemistry, FIN-00014 University of Helsinki, Finland 4Institute for Biomedical Engineering, CH-8092 University and ETH Z¨urich, Switzerland 5 5Friedrich-Alexander Universit¨at, D-91058 Erlangen, Germany 6 5Friedrich-Alexander Universit¨at, D-91058 Erlangen, Germany E-mail: joan.vila@psi.ch Abstract. X-ray microscopy greatly beneﬁts from the advances in x-ray optics. At the Paul Scherrer Institut, developments in x-ray diﬀractive optics include the manufacture and optimization of Fresnel zone plates (FZPs) and diﬀractive optical elements for both soft and hard x-ray regimes. In particular, we demonstrate here a novel method for the production of ultra-high resolution FZPs. This technique is based on the deposition of a zone plate material (iridium) onto the sidewalls of a prepatterned template structure (silicon) by atomic layer deposition. This approach overcomes the limitations due to electron-beam writing of dense patterns in FZP fabrication and provides a clear route to push the resolution into sub-10 nm regime. A FZP fabricated by this method was used to resolve test structures with 12 nm lines and spaces at the scanning transmission x-ray microscope of the PolLux beamline of the Swiss Light Source at 1.2 keV photon energy. Advanced X-Ray Diﬀractive Optics J Vila-Comamala1, K Jeﬁmovs1,2, T Pilvi3, M Ritala3, S S Sarkar1, H H Solak1, V A Guzenko1, M Stampanoni1,4, F Marone1, J Raabe1, G Tzvetkov1, R H Fink5, D Grolimund1, C N Borca1, B Kaulich6 and C David1 J Vila-Comamala1, K Jeﬁmovs1,2, T Pilvi3, M Ritala3, S S Sarkar1, H H Solak1, V A Guzenko1, M Stampanoni1,4, F Marone1, J Raabe1, G Tzvetkov1, R H Fink5, D Grolimund1, C N Borca1, B Kaulich6 and C David1 2. Zone-doubling technique for high resolution FZPs Up to date, x-ray FZPs are mostly prepared by electron-beam lithography tools. Even though these systems are capable of writing patterns with spot sizes and position accuracies in the sub- 10 nm range, the ﬁnal size of the patterned structure is mainly depending on the range of the secondary electrons created in the sensitive resist layer. This eﬀect is particularly detrimental when writing dense patters of lines such as gratings or FZPs, and it explains the stagnation at 25-20 nm in terms of spatial resolution of FZP-based x-ray microscopy. Here we report on a simple nanofabrication method that overcomes the diﬃculty of high density patterning. Since this approach only requires a single lithography step and no need for alignment, this method is very reproducible. Moreover, it allows the fabrication of structures with extremely high aspect ratios. It is based on a deposition of thin layer of a high refractive index material onto the sidewalls of template made of a low-index material. This leads to a doubling of the eﬀective line density of the deposited material comparing to that of the template, which in turn improves the spatial resolution of the FZP by a factor of two. The fabrication steps are schematically shown in Figure 1. The process starts with the electron-beam exposure of the FZP pattern on a high resolution negative tone resist. Then, the pattern is transferred to an intermediate chromium layer which is used as mask during the reactive ion etching process to create the template structure on a previously prepared silicon membrane. The ﬁnal step in this fabrication technique requires a highly conformal deposition of the high refractive index material with accurate control of the thickness. Because of its unique self-limiting growth mechanism, atomic layer deposition [9] is the best choice for this purpose and was employed in this work. A cross section of the 15 nm wide outermost zones of a zone-doubled FZP is shown in Figure 2a. The aspect ratio of these structures is around 12. 1) Substrate preparation and e-beam lithography (0.25 duty cycle at the outer regions) 2) Cr mask dry etching in a Cl :CO plasma 2 2 3) Silicon RIE in a CHF :SF :O plasma 3 6 2 4) Iridium coating by atomic layer deposition Silicon Single Crystal Substrate Cr layer e-beam resist Ir layer Figure 1. 1. Introduction New developments in x-ray optics are improving the performance of x-ray microscopes, which in turn beneﬁt a lot the investigation of both inorganic and biological materials. At the Paul Scherrer Institut, research activities in x-ray diﬀractive optics include the manufacture of Fresnel zone plates (FZPs) and diﬀractive optical elements for both soft and hard x-rays by several methods: Au-electroplated FZPs useful in the multi-keV range have been produced [1, 2] and a new type of diﬀractive optical elements to be used as condenser in full-ﬁeld transmission x-ray microscope has been prepared [3, 4]. For soft x-rays, silicon-etched FZPs that can withstand high radiation heat loads have been manufactured [5] and a new technique to produce the FZP patterns using extreme ultraviolet holographic lithography is under development [6]. In particular, we want to introduce here a novel technique for the fabrication of FZPs with extremely narrow outermost zone widths. In a FZP-based microscope, there exists an intimate relationship between the spatial resolution and the outermost zone width of the FZP, the two of them being essentially comparable. Due to manufacturing limitations, the spatial resolution of x-ray microscopes is currently stagnating at 25-20 nm, and higher resolution has only been reported in very few exceptional cases [7, 8]. The method we present can overcome one of the c⃝2009 IOP Publishing Ltd 1 IOP Publishing doi:10.1088/1742-6596/186/1/012078 9th International Conference on X-Ray Microscopy Journal of Physics: Conference Series 186 (2009) 012078 IOP Publishing doi:10.1088/1742-6596/186/1/012078 IOP Publishing doi:10.1088/1742-6596/186/1/012078 main limitations during the manufacture of FZPs and it provides a clear route to push the spatial resolution of x-ray microscopes to the sub-10 nm regime. main limitations during the manufacture of FZPs and it provides a clear route to push the spatial resolution of x-ray microscopes to the sub-10 nm regime. 3. High resolution scanning transmission x-ray microscopy f f f To demonstrate the feasibility of the proposed fabrication technique a zone-doubled FZP with an outermost zone width of 15 nm and a diameter of 100 µm was produced and tested at the scanning transmission x-ray microscope (STXM) of the PolLux beamline of the Swiss Light Source at 1.2 keV photon energy. During the experiments, a GaAs/AlGaAs heterostructure containing several line widths ranging from 40 nm down to 9 nm was used as a test sample. Figure 2b shows the STXM image of the sample where the smallest visible features consist of 3 lines of 12 nm width. Figure 2. a) Cross section of zone-doubled FZP with an outermost zone width of 15 nm. b) STXM image of GaAs/AlGaAs heterostructure with several line widths. Smallest visible features consist of 3 lines of 12 nm width. Figure 2. a) Cross section of zone-doubled FZP with an outermost zone width of 15 nm. b) STXM image of GaAs/AlGaAs heterostructure with several line widths. Smallest visible features consist of 3 lines of 12 nm width. In conclusion, we have developed a novel technique that frees us from the present limitation of electron-beam lithography in the production of FZPs. It improves the spatial resolution by a factor of 2 and provides a clear route to push the resolution of FZP-based x-ray microscopy into the sub-10 nm regime. [1] Jeﬁmovs K, Bunk O, Pfeiﬀer F, Grolimund D and van der Veen J F 2007 Microelectron. Eng. 84 1467 [2] Borca C N, Grolimund D, Willimann M, Meyer B, Jeﬁmovs K, Vila-Comamala J and David C 2008 These proceedings [3] Jeﬁmovs K, Vila-Comamala J, Stampanoni M, Kaulich B and David C 2008 J. Synchrotron Rad. 15 106 [4] Stampanoni M, Marone F, Mikuljan G, Jeﬁmovs K, Trtik P, Vila-Comamala J, David C and Abela R 2008 These proceedings [5] Vila-Comamala J, Jeﬁmovs K, Raabe J, Kaulich B and David C 2008 Microelectron. Eng. 85 1241 [6] Sarkar S S, Sahoo P K, H H Solak C D and van der Veen J F 2008 J. Vac. Sci. Tech. B (accepted) [7] Chao W, Harteneck B D, Liddle J A, Anderson E H and Attwood D T 2005 Nature 435 1210 [8] Jeﬁmovs K, Vila-Comamala J, Pilvi T, Raabe J, Ritala M and David C 2007 Phys. Rev. Lett. 99 264801 [9] Leskel¨a M and Ritala M 2003 Angew. Chem., Int. Ed. 42 5548 IOP Publishing doi:10.1088/1742-6596/186/1/012078 In addition, theoretical calculations [8] show that the proposed focusing zone-doubled structures can provide diﬀraction eﬃciencies slightly below but comparable to those of ordinary FZPs (∼10—20 %). For instance, 9.1% diﬀraction eﬃciency is expected for a zone-doubled FZP made of silicon and iridium, considering a diameter of 100 µm and an outermost zone width of 20 nm at 1.0 keV. Further calculations also show that the zone-doubled structures are more eﬃcient at higher energies, for which the absorption of the silicon template is highly reduced. [9] Leskel¨a M and Ritala M 2003 Angew. Chem., Int. Ed. 42 5548 [ ] [8] Jeﬁmovs K, Vila-Comamala J, Pilvi T, Raabe J, Ritala M and David C 2007 Phys. Rev. Lett. 99 26 [9] Leskel¨a M and Ritala M 2003 Angew. Chem., Int. Ed. 42 5548 2. Zone-doubling technique for high resolution FZPs Zone-doubled FZP manufacturing steps: 1) electron-beam lithography, 2) pattern transfer to chromium layer by reactive ion etching, 3) pattern transfer to silicon by reactive ion etching and 4) iridium coating by atomic layer deposition. e-beam resist 2) Cr mask dry etching in a Cl :CO plasma 2 2 g p y (0.25 duty cycle at the outer regions) 4) Iridium coating by atomic layer deposition Figure 1. Zone-doubled FZP manufacturing steps: 1) electron-beam lithography, 2) pattern transfer to chromium layer by reactive ion etching, 3) pattern transfer to silicon by reactive ion etching and 4) iridium coating by atomic layer deposition. 2 IOP Publishing doi:10.1088/1742-6596/186/1/012078 9th International Conference on X-Ray Microscopy Journal of Physics: Conference Series 186 (2009) 012078 [ ] , , , g [2] Borca C N, Grolimund D, Willimann M, Meyer B, Jeﬁmovs K, Vila-Comamala J and David C 2008 These proceedings [3] Jeﬁmovs K, Vila-Comamala J, Stampanoni M, Kaulich B and David C 2008 J. Synchrotron Rad. 15 106 [4] Stampanoni M, Marone F, Mikuljan G, Jeﬁmovs K, Trtik P, Vila-Comamala J, David C and Abela R 2008 These proceedings [ ] , , ( p ) [7] Chao W, Harteneck B D, Liddle J A, Anderson E H and Attwood D T 2005 Nature 435 1210 [5] Vila-Comamala J, Jeﬁmovs K, Raabe J, Kaulich B and David C 2008 Microelectron. Eng. 85 1241 [6] Sarkar S S, Sahoo P K, H H Solak C D and van der Veen J F 2008 J. Vac. Sci. Tech. B (accepted) Jeﬁmovs K, Bunk O, Pfeiﬀer F, Grolimund D and van der Veen J F 2007 Microelectron. Eng. 84 1467 [2] Borca C N, Grolimund D, Willimann M, Meyer B, Jeﬁmovs K, Vila-Comamala J and David C 2008 These proceedings [3] Jeﬁmovs K, Vila-Comamala J, Stampanoni M, Kaulich B and David C 2008 J. Synchrotron Rad. 15 106 [4] Stampanoni M, Marone F, Mikuljan G, Jeﬁmovs K, Trtik P, Vila-Comamala J, David C and Abela R 2008 These proceedings [5] Vila-Comamala J, Jeﬁmovs K, Raabe J, Kaulich B and David C 2008 Microelectron. Eng. 85 1241 [5] Vila-Comamala J, Jeﬁmovs K, Raabe J, Kaulich B and David C 2008 Microelectron. Eng. 85 1241 [6] Sarkar S S, Sahoo P K, H H Solak C D and van der Veen J F 2008 J. Vac. Sci. Tech. B (accepted) [7] Chao W Harteneck B D Liddle J A Anderson E H and Attwood D T 2005 Nature 435 1210 Vila-Comamala J, Jeﬁmovs K, Raabe J, Kaulich B and David C 2008 Microelectron. Eng. 85 1241 Sarkar S S, Sahoo P K, H H Solak C D and van der Veen J F 2008 J. Vac. Sci. Tech. B (accepted) References 3
https://openalex.org/W4200156722	https://www.degruyter.com/document/doi/10.1515/opphil-2020-0163/pdf	English	null	Rewatching, Film, and New Television	Open Philosophy	2,021	cc-by	11,756	Research Article https://doi.org/10.1515/opphil-2020-0163 received January 31, 2021; accepted June 29, 2021 https://doi.org/10.1515/opphil-2020-0163 received January 31, 2021; accepted June 29, 2021 https://doi.org/10.1515/opphil-2020-0163 received January 31, 2021; accepted June 29, 2021 Abstract: Those of us who are captivated by new television (the sort of serialized television that began largely in the early 1990s), often ﬁnd ourselves rewatching episodes or whole series. Why? What is the philosophical signiﬁcance of the phenomenon of rewatching? In what follows, I engage with the ontology of television series in order to think about these questions around rewatching. I conclude by reﬂecting on what the entire discussion might suggest about the medium of new television, about ourselves, and also about our world and the possibilities of art in it. Keywords: Stanley Cavell, aesthetics, television, art, phenomenology, worldhood, world, meaning, herme- neutics, genre Open Philosophy 2022; 5: 17–30 * Corresponding author: Martin Shuster, Department of Philosophy, Center for Geographies of Justice, Goucher College, Baltimore, United States, e-mail: martin.shuster@goucher.edu * Corresponding author: Martin Shuster, Department of Philosophy, Center for Geographies of Justice, Goucher College, Baltimore, United States, e-mail: martin.shuster@goucher.edu Open Access. © 2022 Martin Shuster, published by De Gruyter. This work is licensed under the Creative Commons Attribution , 4 Deeper discussions of this point can be found in Shuster, “The Ordinariness and Absence of the World.” Open Access. © 2022 Martin Shuster, published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License. 1 Cavell, The Senses of Walden, 150. 2 Ibid. Emphasis added. 3 See Shuster, New Television. 4 Deeper discussions of this point can be found in Shuster, “The Ordinariness and Absence of the World.” 2 World Central to Cavell’s understanding of ﬁlm ontology is the question of having and screening a world, and that question hinges on understanding that the screen is a barrier. Here’s how Cavell puts it: “A screen is a barrier. What does the silver screen screen? It screens me from the world it holds – that is, it makes me invisible. And it screens that world from me – that is, screens its existence from me. That the projected world does not exist (now) is its only diﬀerence from reality. (There is no feature, or set of features, in which it diﬀers. Existence is not a predicate.)”⁵ There are two points that I want to stress in thinking about this quote. First, note that Cavell is here linking ﬁlm and philosophy rather closely.⁶ To anyone versed in Western philosophy, Cavell’s phrasing will call to mind Kant’s discussion of the category of existence in the Critique of Pure Reason,⁷ where he notes that existence is not a predicate. As Kant points out, an existing hundred thalers (dollars) is not any diﬀerent than an imaginary hundred thalers: a dollar is a dollar regardless of whether it exists, even though it may make a big diﬀerence to my practical life whether it does at this moment exist – reside – in my wallet. Second, note the importance of the category of world. The invocation of this category connects Cavell to an entire philosophical (speciﬁcally: phenomenological) tradition (more on this shortly), exempliﬁed for Cavell above all in the work of Thoreau and Heidegger.⁸ What does it mean to have a world? Or to be in one? Or, relevant to understanding our relationship to ﬁlm and it being screened before us, to be absent from a world? Cavell speaks “of ﬁlm as satisfying the wish for the magical reproduction of the world by enabling us to view it unseen.”⁹ When talking about this phenomenon of “viewing the world unseen,” Cavell frames his discussion around conceptions and tradi- tions of modern philosophy. Here’s what he writes: “What we wish to see in this way is the world itself – that is to say, everything. 5 Cavell, The World Viewed, 24. Emphasis added. 6 For more on what’s at stake with such a link, see Wartenberg, “On the Possibility of Cinematic Philosophy.” See also the essays in Herzogenrath, Film as Philosophy. 7 Kant, Critique of Pure Reason, A596–B624. 8 Cavell, “Night and Day.” For more on Cavell’s indebtedness to phenomenology around this topic, see the discussion in Shuster, “The Ordinariness and Absence of the World.” See also Techio, “The World Viewed and the World Lived.” 9 Cavell, The World Viewed, 101. 10 Ibid., 102. 11 This is a suggestion that even critics of Cavell’s ultimate conclusions about/from ﬁlm agree with. For example, see, for example, notably Foa Dienstag, Cinema, Democracy and Perfectionism, 68–9. 18 important task for philosophy (“the world in a piece of wax” is surely meant to call to mind Descartes’s famous invocation of a ball of wax in the second meditation). In what follows, I engage with this shared ontology only in a limited fashion in order to think about what it means to be drawn – and importantly redrawn – to what’s on the screen. What does this suggest about new television, about ourselves, and about our world? Much of my argument will hinge on exploring the sort of pleasure involved in watching and rewatching. , 11 This is a suggestion that even critics of Cavell’s ultimate conclusions about/from ﬁlm agree with. For example, see, for example, notably Foa Dienstag, Cinema, Democracy and Perfectionism, 68–9. 5 Cavell, The World Viewed, 24. Emphasis added. 1 Introduction: Watching and rewatching Those of us who are captivated by new television (the sort of serialized television that began largely in the early 1990s), often ﬁnd ourselves rewatching episodes or whole series. Why? What is the (philosophical) signiﬁcance of the phenomenon of rewatching, if any? As an opening, let me recall how at a certain point in Senses of Walden, Stanley Cavell claims that “without the mode of perception inspired … by the everyday, the near, the low, the familiar, one is bound to be blind to the poetry of ﬁlm, to the sublimity of it.”¹ Cavell continues noting that without such a mode we would also be cut oﬀfrom “some of the best poetry of philosophy – not now its mythological ﬂights, nor its beauty or purity of argumentation, but now its power of exempliﬁcation, the world in a piece of wax.”² There is a lot in these quotes, and it may not be immediately obvious why these remarks about ﬁlm (or poetry) might be useful when thinking about new television let alone rewatching. My thought, which I can only gesture to here, but for which I have argued elsewhere,³ is that the ontology of ﬁlm – that is, how we ought to understand the aesthetic objects that appear on the screen – is consonant with the ontology of television series. This is so because both fundamentally rely on screening a world to us. In this regard, I ﬁnd Cavell’s work on the ontology of ﬁlm fruitful and useful.⁴ Notice in the above quotations that Cavell is already raising several issues that are equally important for television series: that what’s screened in ﬁlm or new television might be seen as poetic or sublime, somehow bound up with who we most fundamentally are in our most ordinary lives; that somehow such aesthetic objects are themselves intimately related to the practice or pursuit of philosophy; and that these two points must somehow be understood and contextua- lized amidst a broader discussion of having a world, being in a world, and relating to that world as an 18  Martin Shuster Martin Shuster 2 World Nothing less than that is what modern philosophy has told us (whether for Kant’s reasons, or for Locke’s, or Hume’s) is metaphysically beyond our reach or (as Hegel or Marx or Kierkegaard or Nietzsche might rather put it) beyond our reach metaphysically.”¹⁰ Cavell’s suggestion is that the aesthetic signiﬁcance of ﬁlm ought to be linked somehow to the project of modern philosophy. Both might be seen as underwritten by a shared reliance on our visual sensibilities in the context of our existence as creatures with a world (whether a world of objects or concepts or whatever else, i.e., the world of, say, what appears as much as the world of what interests us).¹¹ This way of framing the connection appears very much to be indebted to the way that Heidegger himself approached the problems of modern philosophy. Cavell stresses this link to Heidegger’s understanding of modern 19 Rewatching, Film, and New Television philosophy when Cavell notes that: “Our condition has become one in which our natural mode of percep- tion is to view, feeling unseen. We do not so much look at the world as look out at it, from behind the self.”¹² Compare this to Heidegger’s claims in “The Age of the World Picture,”¹³ where Heidegger claims that, “the fact that the world becomes picture at all is what distinguishes the essence of the modern age.”¹⁴ In modernity, the world itself comes to be understood as a sort of image, where our relationship to that world is fundamentally – chieﬂy and almost exclusively – representational. In Heidegger’s words, “world picture, when understood essentially, does not mean a picture of the world but the world conceived and grasped as picture.”¹⁵ Such a view leads to a particular picture of ourselves as embodied within the world: our relationship to the world is such that the world is out there, and our mode of access to it is mediated by means of our capacity for representing it. Due to a particular conception of our sensibility as fundamentally representational, there also emerges a particular epistemological paradigm, namely one that admits a distinct sort of skepticism.¹⁶  12 Cavell, The World Viewed, 102. 13 Cavell mentions this essay in the World Viewed, but it is unclear whether it directly inﬂuences Cavell in The World Viewed. 14 Heidegger, “The Age of the World Picture,” in The Question Concerning Technology and Other Essays, 130. 15 Ibid., 129. 16 On this point, see Rorty, “The World Well Lost.” 17 According to Heidegger, he pursues a sharp break from Husserl’s approach to phenomenology and to worldhood. I think this is debatable. For more on this point, especially around the topic of world and worldhood, see Overgaard, Husserl and Heidegger on Being in the World. 18 Husserl, Ideas Pertaining to a Pure Phenomenology and to a Phenomenological Philosophy, 52. 19 Ibid. 20 Ibid. 21 Heidegger, Being and Time, 12. 12 Cavell, The World Viewed, 102. 13 Cavell mentions this essay in the World Viewed, but it is unclear whether it directly inﬂuences Cavell in The World Viewed. 14 Heidegger, “The Age of the World Picture,” in The Question Concerning Technology and Other Essays, 130. 15 Ibid 129 21 Heidegger, Being and Time, 12. 12 Cavell, The World Viewed, 102. 3 Having a world and skepticism A commentator on Heidegger proﬁtably terms 20  Martin Shuster 20 this an “absorbed coping.”²² On such a view, “self and world belong together … self and world are not two beings, like subject and object … but self and world are the basic determination … in the unity of the structure of being-in-the-world.”²³ Only when this absorbed coping falters, for example if something doesn’t make sense or if our engage- ment with the world is halted, by, say, a problem or an unexpected occurrence or discovery or an object or whatever else, then do we switch gears into a representational stance where we fundamentally relate to something outside of us, as a subject to an object. Only when our practical engagement with the world comes to a halt do we undertake a way of being in the world where we are situated opposite objects as opposed to amidst them. When this happens, we are more likely to engage in various epistemological modes of inquiry: we question rather than use. For both Husserl and Heidegger – and importantly, for Cavell – the world is thereby not simply the relationship between a subject and an object (let alone a collection of objects), but rather the horizon by which we have any objects altogether; that is, any particular object only becomes what it is for us in a broader contextual horizon, and, importantly, that horizon can itself aﬀect what we see (or don’t). Objects are never solitary (nor are we), instead always ﬁtting into a region of a broader world, occupying a broader web of signiﬁcance.²⁴ Cavell’s point is therefore that the world of the screen – in this phenomenological sense – is screened to us and therefore screened oﬀfrom us. The world of the screen will never be our world, except as an object in our world (i.e., a screen … and if that’s our experience of a ﬁlm or television show, then our aesthetic relationship to what’s on the screen has either broken down, or never existed). Because the world is screened to us, we are forever – ontologically – sealed oﬀfrom it. Given this discussion, one signiﬁcance of ﬁlm that emerges is that it oﬀers a response to the modern representational paradigm and concomitant predicament that Heidegger diagnoses. 3 Having a world and skepticism To see how this is all the case, let’s note that ﬁlm exhibits a world by screening that world to us. Such a screened world can be considered – just like any other world, including our own – from the perspective of and in relation to a particular epistemological paradigm. How do we understand the nature and existence of the world that we are perceiving before us? This question, importantly, can be posed equally well of the world in which I am writing this, as much as of the world that the screen is exhibiting before me (a world that I perceive but do not access in the same way – admittedly a lot hinges on what it means to access the world, which is roughly the topic of this section). Although Cavell’s reference point for conceiving of a world is Heidegger, it is useful – with an eye to what I’ll suggest shortly – tointroduce Heidegger’sthought with referenceto the workof his teacher, Edmund Husserl.¹⁷ In the ﬁrst book of Ideas Pertaining to a Pure Phenomenology and to a Phenomenological Philosophy, Husserl introduces the notion of world with the thought of a horizon. Husserl homes in on how no matter what we perceive, it is always already somehow situated for us amidst a broader horizon. Such a horizon “makes up a constant halo around the ﬁeld of actual perception.”¹⁸ He goes on, noting that such a world is never “exhausted,” and instead “is ‘on hand’ for me in the manner peculiar to consciousness at every waking moment.”¹⁹ This is so much the case that “in the ﬁxed order of its being, it reaches into the unlimited,” i.e., this is always the case in perception, and “my indeterminate surroundings are inﬁnite, the misty and never fully determinable horizon is necessarily there.”²⁰ In Being and Time, Heidegger highlights how the aforementioned modern view – where we understand our relationship to our world in fundamentally representational terms – is in fact derivative of a more fundamental way of being in the world. According to Heidegger, we are not “next” to a world, rather we are always in one.²¹ The overlap with Husserl’s claims, about us having an inﬁnite horizon in which we locate ourselves and what we perceive, should be apparent. 3 Having a world and skepticism Because the world is screened to us, in the case of ﬁlm and new television, the responsibility for the world is “out of our hands.”²⁵ In other words, unlike in our world, we can have no eﬀect on the world screened to us; we are a mere ghost in that world (almost always, even less than that, because the denizens of that world cannot even acknowledge that we are haunting them).²⁶  22 See Dreyfus, Being-in-the-World, 69ﬀ. 23 Heidegger, The Basic Problems of Phenomenology, 297. 24 In Cavell’s work, this is not a merely ontological or phenomenological point, as if these approaches could be divorced from ethical and political conclusions. On this point, see especially Panagia, “Why Film Matters to Political Theory;” and Panagia, “Blankets, Screens, and Projections: Or, the Claim of Film.” 25 Cavell, The World Viewed, 102. 26 I say “almost always,” because there are interesting cases – in shows like Fleabag, House of Cards, House of Lies – where characters do address us directly and thereby appear aware of us. I explore the signiﬁcance of this phenomenon in Shuster, “Fleabag, Modernism, and New Television.” 27 Cavell, The World Viewed, 73. 28 Ibid., 185. 29 Ibid., 24. 23 Heidegger, The Basic Problems of Phenomenology, 297. 22 See Dreyfus, Being-in-the-World, 69ﬀ. , , 26 I say “almost always,” because there are interesting cases – in shows like Fleabag, House of Cards, House of Lies – where characters do address us directly and thereby appear aware of us. I explore the signiﬁcance of this phenomenon in Shuster, “Fleabag, Modernism, and New Television.” 25 Cavell, The World Viewed, 102. 24 In Cavell’s work, this is not a merely ontological or phenomenological point, as if these approaches could be divorced from ethical and political conclusions. On this point, see especially Panagia, “Why Film Matters to Political Theory;” and Panagia, “Blankets, Screens, and Projections: Or, the Claim of Film.” 21 Rewatching, Film, and New Television These thoughts prompt Cavell to call ﬁlm a “moving image of skepticism,” thereby invoking here the epistemological paradigm highlighted above. Let me pause here in order to ﬂag the precise way in which skepticism is being invoked. Commonly, skepticism might be tied to a particular scheme of representation, where a particular paradigm conception of our senses takes hold, then, as Heidegger notes, our relationship to the world becomes one lodged around representation. Richard Rorty captures this point well when he notes that, “any theory which views knowledge as accuracy of representation, and which holds that certainty can only be rationally had about representations, will make skepticism inevitable.”³⁰ On such a view we can always ask whether our representations “match up” to what’s “out there” in the world ³¹ can always ask whether our representations “match up” to what’s “out there” in the world.³¹ Cavell’s view of skepticism, however, diﬀers from this more common view. When Cavell stresses that ﬁlm is a “moving image of skepticism,” he follows up his comment with an explanation that stresses, “not only is there a reasonable possibility, [but rather] it is a fact that here our normal senses are satisﬁed of reality while reality does not exist – even, alarmingly, because it does not exist, because viewing it is all it takes.”³² In other words, the world we see on screen does not exist as part of our world in the phenomenological sense – it is a (phenomenological) world of its own whose deﬁning property in distinction to our world is that it is a diﬀerent world, one that is inaccessible to us in that phenomenological way. Our relationship to it can only ever be one of viewing it, not of being in it, in the way that we are in our world. Cavell follows up these comments with a crucial claim, he notes that even in light of all of this, “to deny, on skeptical grounds, just this satisfaction – to deny that it is ever reality which ﬁlm projects and screens – is a farce of skepticism.”³³ Cavell is thereby locating ﬁlm in a very distinct register: it is not the case that ﬁlm screens to us something that is not real. It is real, but it is a reality from which we are barred.  30 Rorty, Philosophy and the Mirror of Nature, 113. 31 There is a lot more than might be said here, especially about how this paradigm diﬀers from pre-modern ones, where instead of something coming in to our sensory apparatus, something was being sent out. Central to this entire paradigm shift is the Muslim polymath, Ibn al-Haytham (Alhazen). See Lindberg, “Alhazen’s Theory of Vision and Its Reception in the West;” and El-Bizri, “A Philosophical Perspective on Alhazen’s Optics.” 32 Cavell, The World Viewed, 189–90. First emphasis added. 33 Ibid., 190. 34 Ibid., 189. 35 Ibid., 22. 36 Ibid. 37 There is a lot more that might be said here in the context of the relationship between these claims in The World Viewed and in Cavell’s, The Claim of Reason (which while published after The World Viewed was largely written before). I pursue this in more detail in Shuster, New Television, 22–30. 4 Skepticism and ﬁlm, and new television This ontological split between us and the world screened to us in ﬁlm and new television, forms the basis for Cavell’s suggestion that ﬁlm is fundamentally an “automatic world projection.”²⁷ Cavell highlights how, as in Husserl’s conception, an entire horizon is presented to us before any particular objects are; thus he notes that with ﬁlm, reality is present “before its appearances are known.”²⁸ Film presents us with a world in this deep phenomenological sense, ﬁlled with a horizon that far outstrips what we see on screen. The crucial feature of such a screened world, however, is that we are forever absent from it. We can never enter it, we may be said to haunt it. This is what Cavell highlights when he stresses that the screen is “not a support, not like a canvas; there is nothing to support, that way,” rather the “screen is a barrier,” where “it screens me from the world it holds – that is, makes me invisible.”²⁹ The same is true for our experience of new television. 24 In Cavell’s work, this is not a merely ontological or phenomenological point, as if these approaches could be divorced from ethical and political conclusions. On this point, see especially Panagia, “Why Film Matters to Political Theory;” and Panagia, “Blankets, Screens, and Projections: Or, the Claim of Film.” 26 I say “almost always,” because there are interesting cases – in shows like Fleabag, House of Cards, House of Lies – where characters do address us directly and thereby appear aware of us. I explore the signiﬁcance of this phenomenon in Shuster, “Fleabag, Modernism, and New Television.” 27 Cavell The World Viewed 73 30 Rorty, Philosophy and the Mirror of Nature, 113. 31 There is a lot more than might be said here, especially about how this paradigm diﬀers from pre-modern ones, where instead of something coming in to our sensory apparatus, something was being sent out. Central to this entire paradigm shift is the Muslim polymath, Ibn al-Haytham (Alhazen). See Lindberg, “Alhazen’s Theory of Vision and Its Reception in the West;” and El-Bizri, “A Philosophical Perspective on Alhazen’s Optics.” 32 Cavell, The World Viewed, 189–90. First emphasis added. 36 b d. 37 There is a lot more that might be said here in the context of the relationship between these claims in The World Viewed and in Cavell’s, The Claim of Reason (which while published after The World Viewed was largely written before). I pursue this in more detail in Shuster, New Television, 22–30. 22  Martin Shuster 22 kind of world rather than simply in each – that is exactly what guarantees their signiﬁcance and thereby their ultimate reality to us as modern subjects. The question, as much for our world as the world of the screen, then, is how we relate to each. In The Claim of Reason, Cavell poses a series of questions about our relationship to the world we’re in, but they apply as much to the world of the screen. He asks: “Shall we say that we have faith that the things of our world exist? But how is that faith achieved, how expressed, how maintained, how deepened, how threatened, how lost?”³⁸ In the context of ﬁlm, Cavell summarizes the issues at stake as follows: I described the artistic signiﬁcance of the motion picture … as its apparent and unpredictable solution of the problem of reality at a stroke, by its miraculous neutralizing of the need to connect with reality through representing it, by its stroke of acquiring this connection through successive projections of reality itself. And I went on to say that this had also not solved the problem of reality but brought it to some ultimate head, since the connection is established by putting us in the condition of “viewing unseen,” which establishes the connection only at the price of establishing our absolute distance and isolation. And this is exactly the price of skepticism.³⁹ The ontology of the screen, in turn, demands that the director undertake particular strategies – genre conventions, types, various automatisms, as Cavell calls them – in order to compel conviction in the worlds on the screen.⁴⁰ As with other modernist arts, it may be the case that these automatisms fail, that the work of art in question simply fails. The ontology of the screen, in turn, demands that the director undertake particular strategies – genre conventions, types, various automatisms, as Cavell calls them – in order to compel conviction in the worlds on the screen.⁴⁰ As with other modernist arts, it may be the case that these automatisms fail, that the work of art in question simply fails. As earlier, while I cannot present this claim in its entirety here (although I have laid out the case for it elsewhere),⁴¹ the modality of new television – instantiated across a range of genres from police procedurals to court dramas to period pieces to sci-ﬁoperas to military epics to whatever else – is deﬁned by two features: (1) the exhibition of a world where all human institutions have been emptied of normative authority, and (2) a concomitant exception with regards to (the institution of) the family. Think, just for example, with regard to this suggestion, of shows as diverse as Breaking Bad, where even the powerful normativity of science is brought into question as Walter White pursues his bloody crime saga “for” his family, to The Sopranos, where modern America is presented merely as one giant criminal enterprise with family at the heart of it, to 24, where there simply is no norm that will not be sacriﬁced to family (imagined either as one’s immediate family or one’s extended family, i.e., the nation), to even The Wire, which presents the modern American city as entirely absent of any normative authority, oﬀering the family as the single glimmer of something possibly better. Furthermore, these two aspects of the mode of new television are often presented using traditional televisual genres (e.g., police procedural or sitcom) while eschewing the regular predictable natures inherent to such forms (more on this shortly). 40 I discuss this in more detail in Shuster, New Television, 30–47. 38 Cavell, The Claim of Reason, 243. 39 Cavell, The World Viewed, 195. 42 A theory of moods is developed prior to Heidegger in Husserl’s work, but is found across several sources, including unpublished ones. For more on this, see Lee, “Edmund Husserl’s Phenomenology of Mood;” and Ramírez, Feeling and Value, Willing and Action. Why would alleging that the world screened to us in ﬁlm (or new television) is not real make a farce of skepticism? Cavell’s answer to this question once again trades on the phenomenological tradition elaborated above. He notes that while such a claim “seems to remember that skepticism concludes against our conviction in the existence of the external world,” it nonetheless “seems to forget that skepticism begins in an eﬀort to justify that conviction … to yield here to the familiar wish to speak of ﬁlm as providing in general an ‘illusion of reality’ would serve to disguise this latent anxiety – as does the conclusion of philosophical skepticism itself.”³⁴ Cavell’s point is that once such an epistemological, representational paradigm is on the scene, then any response to it, whether one that denies our knowledge in the face of it or one that aﬃrms it, is already a skeptical one; all such responses take our relationship to the world to be a problem that somehow involves knowledge. As Cavell puts it, “at some point the unhinging of our consciousness from the world interposed our subjectivity between us and our presentness to the world,”³⁵ i.e., the pos- sibility for our being in the world – our absorbed coping – to break down emerged. In such case, “our subjectivity became what is present to us, individuality became isolation,”³⁶ i.e., our relationship to the world became a problem.³⁷ Given that our relationship to the world of the screen is fundamentally diﬀerentiated from our relation- ship to our world not by whether one or the other world is “real,” but rather by how seriously it can present a world to us, and in which world we are in, we need to assess the signiﬁcance of the world of the screen in quite particular terms. To the extent that the world on the screen and the world in which we are in are both real, our relationship to each is thereby a problem for us. It is that fact – that we exist in relation to each 36 Ibid. 37 There is a lot more that might be said here in the context of the relationship between these claims in The World Viewed and in Cavell’s, The Claim of Reason (which while published after The World Viewed was largely written before). I pursue this in more detail in Shuster, New Television, 22–30. objects), rather we are attuned in such a way so that there is a certain sort of equipoise between what the world reveals about itself and what it reveals about us at any moment.⁴³ Think of this as a phenomeno- logical feature of being in a world. It is important to note furthermore that, in this tradition, mood is not merely some subjective state. The world suggests a particular mood; for example, something (in the world) frightens me. This is not to say, of course, that there is no subjective component; it is I who become frightened, but not for no reason (this is not to say, however, that there may not be bad reasons, as in, for example, a cop who is frightened of any non-white body because of explicit or implicit racism). Moods are, again in Heidegger’s terminology, disclosive (again, as Heidegger notes, for example, fear reveals something threatening).⁴⁴ This phenomenological understanding of mood oﬀers a means to think about the relationship between this world and the world of the screen. To begin to unpack that idea, let me introduce a remark of Wittgenstein’s about pleasure. Late in the Philosophical Investigations, Wittgenstein states: “See it not as obvious, but as a strange fact (merkwürdig Faktum), that pictures and ﬁctitious narratives give us pleasure (Vergnügen), occupy our minds (Geist).”⁴⁵ What is beyond my scope here is the aesthetic basis of that pleasure – there are a slew of options here, from Kantian inspired accounts that focus on the pleasures involved with the absence of ﬁxed conceptual categories for judgment (i.e., in the context of judgments of beauty) to accounts that prioritize the pleasures inherent to rhythm, aﬀect, or whatever else. It seems to me less important at this juncture to settle the why or how of the pleasure than to admit that there is some pleasure involved with both watching and rewatching; or, again, let’s just agree to acknowledge the importance of mood in this context. With that in mind, let me admit something: I think that I have now seen The Wire in its entirety more than ﬁve times all the way through. Several of those times involved rewatching it as I was preparing a book, but other times, it just struck me as something I wanted to do.  43 See Heidegger, Being and Time, 29–30. 44 See ibid., 30. 45 Wittgenstein, Philosophical Investigations, 524. Translation modiﬁed. Some political conclusions are teased from this remark in the context of Cavell’s work on ﬁlm in Panagia, “Why Film Matters to Political Theory,” 103ﬀ. 46 For an exploration of this mood in Heidegger, especially as an “authentic” possibility of human beings, see Smith, “On Heidegger’s Theory of Moods.” 47 We might parse this as a sort of voyeurism. See Cavell, The World Viewed, 45; and see also Laugier and Cerisuelo, Stanley Cavell, 34. 5 Rewatching, having a world, and (new) television It is with this last point about predictability that I want ﬁnally to raise the question of rewatching, especially around new television. To do that, I want to return to the earlier points about a world and especially about having a horizon as a central feature of having a world. Any such horizon, as Heidegger and other philosophers in the phenomenological tradition ultimately came to argue, is inﬂected by particular moods.⁴² In Heidegger’s terminology, being in the world involves an attunement (Beﬁndlichkeit): as we have already seen, we are not a mere subject relating to an object (or 23 Rewatching, Film, and New Television hooked an epistemological ﬁngernail into the world of the screen. We haunt it, but now in the way that on certain conceptions of ultimate reality, God might be said to haunt the world (think, for example, of the way that, say, Boethius envisions God’s providence about human aﬀairs, as a view from a place to which we are oblivious and from which we are absent, and vice versa).⁴⁸ Let me start this paragraph now with a spoiler warning about The Wire, Breaking Bad, The Sopranos, and The Shield (just to name a few random series). Note that the very idea of a spoiler warning itself suggests something about the epistemological issues and pleasures involved in watching and rewatching. What happens when I watch The Wire or Breaking Bad when I know that Omar or Walter White is killed, respectively? How about when I suspect that Tony Soprano is also killed in the notorious ﬁnal black screen of The Sopranos? Or when I know that Vic Mackey will get his due punishment and lose his family at the end of The Shield? What relationship, if any, exists between such epistemological frames and the joy we might normally get from watching these television series? One way to tackle these questions is to note that they have something in common with questions already in circulation around ﬁlm and new television, namely the question of genres. For example, genre conventions also already suggest a kind of epistemological ﬁngernail into the world. When we watch a (traditional) western or a romance (or remarriage) comedy or police procedural or courtroom drama, we experience “a narrative whose outcome is clearly signaled from the outset.”⁴⁹ There are two diﬀerent ways to register the signiﬁcance of this fact. On one hand, we might claim that television genres and the episodes that actualize them are deﬁned by a sort of “reductive pressure,”⁵⁰ where every facet of an episode is already delineated by the parameters of the show in procedural terms: the characters do not change in meaningful ways, and every episode is a sort of hard reset where the characters move, perhaps through new situations, but in highly predictable ways.  48 See Book IV of Boethius, The Consolation of Philosophy. 49 Stevens, “Romantic Comedy and the Virtues of Predictability,” 33. 50 VanArendonk, “Theorizing the Television Episode,” 69ﬀ. 51 The story can, of course, be tempered with some “intermediary species.” For such an account, see Sepinwall, The Revolution Was Televised. 52 Erich Fromm puts this point about masochism well when he notes that “the masochistic character worships the past” (Der masochistische Charakter vergottet die Vergangenheit). For more on this point, see Fromm, “Studien Über Autorität Und Familie,” 119. 53 Stevens, “Romantic Comedy and the Virtues of Predictability,” 44. 48 See Book IV of Boethius, The Consolation of Philosoph 49 Stevens, “Romantic Comedy and the Virtues of Predictability,” 33. Notably, choosing to rewatch a television series is a diﬀerent prospect than choosing to rewatch a ﬁlm; most saliently, the time commitments are quite distinct (I am interested here in rewatching an entire series, like one might a ﬁlm, rather than rewatching a particular episode or episodes, like one might rewatch the scenes of a ﬁlm). There is, it might be said, more of a commitment involved here, a temporal seriousness to it that far exceeds mere entertain- ment, or at least suggests something diﬀerent. Furthermore, there is also the issue, in common with ﬁlm, that I now know what will happen, thereby seemingly undercutting, at least when it comes to rewatching, one avenue of explanation for the sort of attraction(s) that new television series might oﬀer (say, unpredict- ability or the discovery or unmasking of something). Finally, as with the object of fright, something about the (aesthetic) object in question invites watching it again; it is appropriate here to speak of a mood that might compel rewatching. Given Wittgenstein’s suggestion above about pleasure, say, provisionally, that the mood involved is joy.⁴⁶ Note that with reference to the ontology of what’s being screened, everything stays the same: whether we’re watching or rewatching, we are still always screened oﬀfrom the world of the screen. What we are seeing, however, is inﬂected diﬀerently. It is the case that our experience of rewatching is diﬀerent to watching. We know what will happen. That feature – that certainty – both entirely accords with the skepti- cism that follows from the ontological facts and also rubs against it. Cavell’s image of skepticism as a state of “being sealed oﬀfrom the world” – an image that’s harnessed so proﬁtably in the analysis of the signiﬁcance of the ontology of the screen as a barrier⁴⁷ – no longer feels entirely as apt, or at least requires some sort of contextualization in the context of rewatching. Even as we are ontologically sealed oﬀ, we have 24  Martin Shuster 24 51 The story can, of course, be tempered with some “intermediary species.” For such an account, see Sepinwall, Th Was Televised. 0 VanArendonk, “Theorizing the Television Episode,” 69ﬀ. that “there are no secrets about what will happen,” reveals new television’s eschewing of such traditional genre traditions as a fetishization of epistemology, no diﬀerent than “the longstanding association” between truth and the feminine, with “philosophers seeking to strip her [truth] nude.”⁵⁴ On such a view, pre-new television, predictable television shows “attest that we do largely know how things will go, even if we ﬁnd enjoyment in pretending that we do not. We do know, really, that social and political institutions which precede our own introduction into the world allow relatively few prospects of profound change. And, of course, we all know that each of our stories will end in death.”⁵⁵ The joy that emerges from the constancy of genre – of predictability – ultimately rests on the fact that this predictability “feels like freedom,” ulti- mately “from epistemological and narrative structures,” and also thereby “from the gendered connotations they entail,” and – it might be said given the above reference to our ﬁnitude – from our very mortal coils. I see no way to settle for one view over the other. There are pleasures to be found in predictability and unpredictability, and also dangers. I suggest drawing inspiration here from another remark by Wittgenstein, who once wrote “don’t get involved in partial problems, but always take ﬂight to where there is a free view over the whole single problem, even if this view is still not a clear one.”⁵⁶ Such a higher view in this context would involve trying to integrate these positions in some dialectical fashion. One way to proceed might be to understand all such genre conventions, and the associated judgments and categories and moods associated with them, as a sort of sundial to the present moment, revealing broader social and political conditions, conditions which may in fact reveal contradictory impulses, indeed, contradictions.⁵⁷ Given these dialectical possibilities and my earlier invocation of phenomenology, and given the phi- losophical stakes involved in thinking about (new) television, it strikes me as worthwhile to register, in this context, the phenomenological impulses and underpinnings of two now classic texts in Anglophone philosophy – John McDowell’s Mind and World,⁵⁸ and Rorty’s aforementioned, Philosophy and the Mirror of Nature. I am led here because of two traits these texts share.  54 Ibid., 43. 55 Ibid., 45–46. 56 Wittgenstein, Notebooks, 1914–1916, 23. 57 Such an account of, say, older television can be gleaned from Ngai, Our Aesthetic Categories. See especially, for example, the discussion of I Love Lucy at 182ﬀ. For new television, I would cite my own Shuster, New Television. 58 McDowell, Mind and World. 59 For more on their relationship, see Scheibler, Gadamer; Coltman, The Language of Hermeneutics; and Kusch, Language as Calculus Vs. Language as Universal Medium. 60 Cavell, The World Viewed, 23. 61 Ibid., 82. This is a point that is oftentimes overlooked in discussions of The World Viewed and Cavell’s book. It is elegantly and importantly presented in Morgan, “Modernist Investigations.” Often this is exactly how people periodize television prior to the advent of new television.⁵¹ Notably, we rewatch such television also, but we do so perhaps allegedly to sulk in our powerlessness, to revel in it: to retreat from the world in order to ﬁnd comfort in abdication and repetition; in short, we allegedly rewatch to pass the time. In psychoanalytic terms, we might say that this conception of rewatching is masochistic, in the sense that it worships the past, taking what’s shown as immutable and exactly drawing pleasure from that in order to assuage the resentment we otherwise repress due to our overall state of powerlessness (this might be the same mindlessness that also allegedly endlessly compels us to scroll through social media).⁵² On such a view, what’s remarkable about so-called “new television” might be the fact that it eschews many of these conventions, aims to break out of such reductive pressure by producing highly evolving characters, distinct genre combinations, or episodic and storytelling paradigms that fundamentally push against the sort of epis- temic certainty that such reductive pressure seems to guarantee or oﬀer. New television seeks to oﬀer, say, a pleasure that is not masochistic. On the other hand, there is no reason to attribute mindlessness nor masochism to any such reductive pressure, and, indeed, the very idea of it being “reductive” may already be sneaking in a value judgment that is simply inappropriate. For example, it may be the case that even shows as allegedly as “simple” as, say, All in the Family or The Love Boat (just to name two) are in fact shows that virtuously avoid a certain kind of epistemological paradigm, an avoidance whereby a particular “masculinist history of unveiling, and its critical derision” of predictability is seen as problematic, involved fundamentally with “unmasking as an epistemological strategy” that references “both misogyny and moral censorship.”⁵³ On such a view, the fact 53 Stevens, “Romantic Comedy and the Virtues of Predictability,” 44. 25 Rewatching, Film, and New Television 56 Wittgenstein, Notebooks, 1914–1916, 23. g 57 Such an account of, say, older television can be gleaned from Ngai, Our Aesthetic Categories. See especially, for example, the discussion of I Love Lucy at 182ﬀ. For new television, I would cite my own Shuster, New Television. 58 McDowell Mind and World 61 Ibid., 82. This is a point that is oftentimes overlooked in discussions of The World Viewed and Cavell’s book. It is elegantly and importantly presented in Morgan, “Modernist Investigations.” 59 For more on their relationship, see Scheibler, Gadamer; Coltman, The Language of Hermeneutics; and Kusch, L Calculus Vs. Language as Universal Medium. e on their relationship, see Scheibler, Gadamer; Coltman, The Language of Hermeneutics; and Kusch, Language as Language as Universal Medium. The World Viewed, 23. 54 Ibid., 43. 55 Ibid., 45–46. To get a grip on the dialectical picture that’s emerging here – spread out seemingly across several modalities, from questions of predictability to questions of optimism or pessimism – let me cite a remark of the great ﬁlm critic, Victor Perkins, who noted that, “what we see in the world is so much more a product of our will and whim than what we see on the screen.”⁶³ Perkins continues, teasing out a signiﬁcance of this point: “since it is not possible to aﬀect the course of events [on the screen], it is not necessary either. If we are without power we are also without responsibility. Our exclusion from the world so vividly represented frees us from the need to consider what we see in terms of an active response.”⁶⁴ This is so much the case that “we can observe the progress of a ﬁre with that much more attention when it can be neither our business to put it out nor our concern to escape.”⁶⁵ Keeping Perkins’s remarks in mind, imagine then, that the ontology of the screen makes available a sense of our own temporality. In watching, because we are barred from that world, we cannot but realize that we are a creature with a past and a future; it remains open to us with any watching to judge which element – past or future – to prioritize. In stressing the possibility of what’s screened to us as being of the past or of the future, there is an implicit prioritization of the present: only we can decide what sort of world is being screened to us, and in doing so we register a judgment about both our world and the world of the screen, not to mention the possibilities of each, and indeed, of art altogether (this is one way to understand the forcefulness and the ultimate aesthetic success of Black Mirror: it urgently raises the question of whether what’s being shown to us is already here, i.e., a world past, or whether it is a world that might yet still be, i.e., a world of the future). First, each of them revolves around how we need to understand our relationship to the world, indeed how we ought to understand the very notion of a world. Second, and more importantly, both books strikingly conclude with an invocation of the work of Hans-Georg Gadamer, and especially the idea of a worldly horizon. Gadamer, a student both of Husserl and Heidegger,⁵⁹ should be viewed as exactly involved in the sort of phenomenological approach invoked above. McDowell marshals Gadamer and the notion of a horizon as a repository for tradition, while Rorty marshals the notion of a horizon as the means by which we change and constitute ourselves. At a very high altitude, it might be said that McDowell focuses on the ways in which our worldly horizon is inherently conservative, the way in which it sets the parameters by which the world and the objects in it are given to us, while Rorty focuses on the way in which our worldly horizon perpetually harbors progressive potential, allowing for radical change in our world simply by means of how that horizon is constituted. These two views are not unrelated to the question of the import of predictability rehearsed above. Recall especially the suggestion that predictability within televisual genres oﬀers a possibility for acknowledging the impossibility of truly radical change – to which we might say: is it truly impossible? This seems to me neither a simple nor an idle question. Note furthermore in this context how Cavell frequently stresses that the world on screen is a world that is past. As he puts it, it is a world I “see, but to which I am nevertheless not present … is a world past.”⁶⁰ Yet, notably, Cavell also speaks of ﬁlm as screening “a world of an immediate future,” i.e., not “a world just past nor a world of make-believe.”⁶¹ In this way, “the temporality 26  Martin Shuster 26 of cinema [but also of new television] is radically open – at least once we factor in the experience of the viewer’s engagement with the ﬁlm. This is why some ﬁlms evoke the ‘Once upon a time’ while other ﬁlms ask us to imagine ‘What if one day.’”⁶² It is of course an empirical fact which judgment applies when, and what (dis)pleasures it brings (or fails to). This sense of the importance of temporality is only potentially heightened when we rewatch because the entire machinery of judgment is so potentially heighted, as when we rewatch to get at a prior judgment, to conﬁrm or disconﬁrm or, at the very least, to test it (more on this shortly). Our fundamental relationship to the world – any world – is one of acknowledgment.⁶⁶ A screened work of art demands that we acknowledge, whether explicitly or implicitly the separateness of our world and the world of the screen. In doing so, however, we are oﬀered the possibility of achieving conviction in our experience, conviction in the world of the screen, and therefore, ultimately, conviction in and about our world (and our place, and the place of art, in it). The psychoanalytic economy of viewing screened works of art is such that even as it exhibits and prioritizes our powerlessness as humans, it also – at least poten- tially – makes the case for the powers of (televisual) art.  62 Ibid., 232. Morgan is quoting Cavell, The World Viewed, 83. 63 Perkins, Film as Film, 71. 64 Ibid. 65 Ibid. 66 “I do not propose the idea of acknowledging as an alternative to knowing, but rather as an interpretation of it, as I take the word acknowledge, containing knowledge, itself to suggest (or perhaps it suggests that knowing is an interpretation of acknowl- edging.” See Cavell, “The Philosopher in American Life,” 39. 6 Conclusion One striking fact about rewatching is that it naturally pushes any genre toward predictability. Rewatching reorients even “epistemophilic narratives” (narratives that prioritize unmasking something that wasn’t known prior) toward the predictability of traditional genre conventions (like older forms of television). In the context of new television this takes on a particular tenor. In a prior work, I have argued that, at its best, the genre of new television – with its prioritization of an exhibition of the total loss of normative 27 Rewatching, Film, and New Television authority and its recourse to the family – oﬀers its greatest potential and most interesting aesthetic accom- plishments when it acknowledges these features through an understanding of the family, not as the site of biological necessity or rigid gender or identity categories, but rather as the site of the raw potential involved in being human, of what, following Hannah Arendt, can be termed human natality: the fact that we can each potentially introduce something new into the world (think of 24 as an example of the former and of, say, Weeds as an example of the latter).⁶⁷ On such an understanding of new television, I take it that one possibility for rewatching signals, as I suggested above, a desire to test one’s judgment about a particular piece of new television. Given where the world is now and what the show achieved (achieves?) as a work of art (given that is, the mood of the present moment), will this show continue to compel conviction? Note that this is a question as much about me as about the world as about the show (the aesthetic object). With Cavell, it might be said that what’s screened in this context “promises us happiness exactly not because we are rich or beautiful or perfectly expressive, but because we can tolerate individuality, separateness, and inexpressiveness. 6 Conclusion In particular, because we can maintain a connection with reality.”⁶⁸ In other words, aesthetic experience – our conviction that this piece of new television matters to us as art – is exactly a way of maintaining connection to reality, to the world, both of the show and its creator(s).⁶⁹ In this way, I think it is fundamentally mistaken, as one commentator claims, that ﬁlm and television restrict our vision in terms of “reciprocity,” locating us as having “the vision of the animal, which sees everything except another perspective.”⁷⁰ There are, of course, other reasons for rewatching a series, many – like instances of new television themselves – are regressive (avoidance rather than acknowledgment, resignation rather than potentiality, passing the time rather than inspiration). But this is just another way to say that there are many kinds of joys. My point has been that it is possible to locate one kind of joy in (re)watching new television, one that is “not predicated on a freezing of the world or a desire for mastery over it, but rather on the hope to see it anew and, in light of that re-seeing, to perhaps ﬁnd diﬀerent ways to be and act in the future.”⁷¹ I have written “rewatching” as “(re)watching” just now exactly to acknowledge that rewatching in this context means testing again an original judgment for the potentiality of a particular work of art, of a particular piece of new television. In this way, given the way in which new television oftentimes unfolds in new, genre bending, “complex” ways,⁷² there are deep analogies between watching and rewatching in the context of new television, where both often revolve around testing one’s intuitions about a particular genre, seeing whether one’s initial (or subsequent) judgments about a particular piece of new television will turn out to be correct.⁷³ To the extent that we are all, in Robert Warshow’s words, “self-made,” forming “ourselves in terms of the particular choices we make from among the confusing multitude of stimuli that present themselves to us,”⁷⁴ rewatching oﬀers another site for such formation. It must be stressed that, as with all art, there is no reason to see it merely as imitative, but rather it may in fact be seen as constitutive of our world. 74 Warshow, The Immediate Experience, xxxvii 72 Mittell, Complex Tv. 72 Mittell, Complex Tv. 73 On this point, see the discussion of True Detective in Nannicelli, Appreciating the Art of Television, 133ﬀ. 74 Warshow, The Immediate Experience, xxxvii.  67 This overall point (with reference to these shows and others) is argued for in Shuster, New Television, 170–74. 68 Cavell, The World Viewed, 215. 67 This overall point (with reference to these shows and others) is argued for in Shuster, New Television, 170–74. 68 Cavell, The World Viewed, 215. 69 This need not be unpacked ultimately as a commitment to auteur theory, at least not in any traditional sense. The point will stand even if we acknowledge that shows – like ﬁlms I might add – are assembled in complex ways. On the former in the context of new television, see Nochimson, Television Rewired. 70 See Foa Dienstag, Cinema, Democracy and Perfectionism, 70. I am also not sure that this is true of animals, see Crary, Inside Ethics. 71 Rennebohm, “The ‘Cinema Remarks’,” 75–6. As should be obvious, I am quite sympathetic to Rennebohm’s account and intend for this account to be complementary. 72 Mittell, Complex Tv. 73 On this point, see the discussion of True Detective in Nannicelli, Appreciating the Art of Television, 133ﬀ. 74 Warshow, The Immediate Experience, xxxvii. 72 Mittell, Complex Tv. 73 On this point, see the discussion of True Detective in Nannicelli, Appreciating the Art of Television, 133ﬀ. 74 W h Th I di E i ii p pp g f 74 Warshow, The Immediate Experience, xxxvii. Ethics. 71 Rennebohm, “The ‘Cinema Remarks’,” 75–6. As should be obvious, I am quite sympathetic to Rennebohm’s account and intend for this account to be complementary.  Martin Shuster world a deﬁcient mode of being, instead of looking at things the other way round, instead of seeing in the imaginary – that is, human – mode of existence, a deﬁcient or secondary mode of being?⁷⁵ I once mentioned this to some colleagues only to be met with the mocking response that: “Everything is water” (mockingly hearkening back to Thales and the prehistory of philosophy, when seemingly any idea could be asserted). Of course, the suggestion that Castoriadis is letting his philosophical fancy run amuck is not entirely without substance. There are indeed problematic ways of unpacking what he is suggesting. I once mentioned this to some colleagues only to be met with the mocking response that: “Everything is water” (mockingly hearkening back to Thales and the prehistory of philosophy, when seemingly any idea could be asserted). Of course, the suggestion that Castoriadis is letting his philosophical fancy run amuck is not entirely without substance. There are indeed problematic ways of unpacking what he is suggesting. At the same time, in conclusion, I think, for example, of the way in which Michaela Coel’s brilliant show, I May Destroy You, intervened in the present #MeToo moment, oﬀering at one time an understanding of how gender and race come to be intertwined in the present moment of late capitalism, while also proposing a potential way forward, one that eschews the typical binary that society presents (i.e., eschews both retribution and revenge, and passivity and subjugation). In the concluding scenes of I May Destroy You (spoiler warning!), the protagonist, Arabella, has ﬁnally remembered the trauma of her rape in its entirety (the premise of the show is that the trauma unfolds non-linearly, episodically, hesitantly). In the prior episode she ﬁnally remembers the identity of her attacker. What occurs in this ﬁnal episode is an exhibition of three distinct desires and fantasies: one of vengeance (where she kills her attacker), one of a kind of rapprochement (where she and her attacker encounter each other, engage in dialogue, revealing him also as a victim of earlier violence), and some mixture of the two (where they encounter one another sexually again, this time with her penetrating him). The three are built on and around each other, often bleeding into each other, so that the viewer is unsure which is prioritized, or, indeed, whether it may even make sense to speak in terms of prioritization here. Its stylistic approach and composition force us to confront our own desires in response to I May Destroy You. The entire show then concludes with a sort of coda, almost an add- on, where Arabella appears to be at peace, involved in having produced a piece of art (a book) that also works through this question, i.e., through her trauma. It is meant, I believe, to be a meditation on the show, the trauma that underwrites it (the show’s creator, Michaela Coel, has noted that she herself was sexually assaulted), and the place of art in each of these. It is meant also to signal our place in this drama. There are no easy answers around any of these topics, but I May Destroy You invites rewatching exactly to try to get at the best way forward given the intimate relationship between late capitalism and misogyny on the one hand,⁷⁶ and between that and the role of art (if any) in our world. And, in this way, with Castoriadis, it seems to me entirely appropriate to highlight how our current (binary or simplistic) practices are entirely deﬁcient when compared to the possibilities that this work of art proposes, and thereby the possibilities it oﬀers. Or at least I’d like to watch it again and see Acknowledgment: The topic of this paper was inspired by one strand of comments that Robert Pippin made on my television book during a MLA panel; I am grateful to him for those comments and also to the other respondents – Yi-Ping Ong, James South, Stefanie Dunning – for related comments. I shared a very early draft of this paper at the conference “The Thought of the Movies: En hommage à Stanley Cavell” held at the Université Paris 1 – Panthéton-Sorbonne in June of 2019. Thank you to Sandra Laugier for the invitation and to her and others for comments at that conference. Thank you also to Ada Jaarsma for reading a draft of this paper and for her comments, and also to the comments of the anonymous reviewers. Conﬂict of interest: Author states no conﬂict of interest. Boethius, Anicius Manlius Severinus. The Consolation of Philosophy, translated by V. E. Watts. London: Penguin, 1969. Castoriadis, Cornelius. World in Fragments: Writings on Politics, Society, Psychoanalysis, and the Imagination, translated by David Ames Curtis. Palo Alto: Stanford University Press, 1997. 75 Castoriadis, World in Fragments, 5. 76 Penny, Meat Market. 76 Penny, Meat Market. 6 Conclusion Let me conclude with a thought by Cornelius Castoriadis, who wrote: No philosopher ever starts by saying: ‘Let Mozart’s Requiem be a paradigm of being, let us start from that.’ Why would we not start by positing a dream, a poem, a symphony as paradigmatic of the fullness of being and by seeing in the physical  67 This overall point (with reference to these shows and others) is argued for in Shuster, New Television, 170–74. 68 Cavell, The World Viewed, 215. 70 See Foa Dienstag, Cinema, Democracy and Perfectionism, 70. I am also not sure that this is true of animals, see Crary, Inside Ethics. 71 Rennebohm, “The ‘Cinema Remarks’,” 75–6. 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https://openalex.org/W4283695018	https://zenodo.org/records/6769671/files/GSCARR-2022-0108.pdf	English	null	Combination of ketamine, corticosteroids and sevoflurane inhibits the risk of bronchospasm in intubated children under general anesthesia	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	1,219	2. Case Study A 6-year-old child weighing 30 kg with a recent cold, nasal congestion and hearing of both amphibians and with a known history of frequent asthma attacks underwent emergency appendectomy. It was given intravenously before the introduction of anesthesia solu medrol 60 mg, Dexaton 3 mg, onda 3 mg. Introduction to anesthesia was by intravenous administration of Fentanyl 60 mcg, Propofol 120 mg, Esmeron 30 mg. After intubation, intrabronchial aspiration was performed, intrabronchial Flixotide 50 mcg και Aerolin 100 mcg was given and mechanically ventilated with 50% N2O and 2% sevoflurane. Ketamine 6 mg was given intravenously, plus 30 mcg Fentanyl, Apotel 350 mg, and morphine 1.5 mg. The monitoring included ECG, NBP, SpO2 and the ventilation model in Drager machine was Volune Control. Towards the end of the operation and with pure inhaled oxygen he was put in a Pressure Control model and the awakening was done smoothly after intravenous Bridium 0.6 mg. 1. Introduction Asthma is a chronic respiratory disease that affects people of all ages and is characterized by episodic and reversible attacks of wheezing, chest tightness, shortness of breath, and coughing. According to the ATS/ERS guideline, severe asthma is defined as asthma which requires treatment with high dose inhaled corticosteroids (ICS) plus a second controller (and/or systemic corticosteroid) to prevent it from becoming “uncontrolled” or remains “uncontrolled“despite this therapy [1-7]. Abstract Asthma in children is associated with significant morbidity. Children with severe asthma are at increased risk for adverse outcomes including medication-related side effects, life-threatening exacerbations, and impaired quality of life. In the study, an asthmatic child with a recent cold, received general anesthesia for emergency surgery centered on sevoflurane, corticosteroids and ketamine. The purpose of this study is to demonstrate the beneficial effects of the combination of sevoflurane, ketamine and corticosteroids in asthmatic children and to prevent complications when they are given general anesthesia for emergency surgery. Keywords: Corticosteroids; Sevoflurane; Bronchospasm; Ketamine; Anesthesia; Astma Maria I Dalamagka * Department of Anesthesia, General Hospital of Larisa, Greece. GSC Advanced Research and Reviews, 2022, 11(01), 165–166 GSC Advanced Research and Reviews, 2022, 11(01), 165–166 ublication history: Received on 09 March 2022; revised on 17 April 2022; accepted on 17 April 2022 Publication history: Received on 09 March 2022; revised on 17 April 2022; accepted on 17 April 2 Article DOI: https://doi.org/10.30574/gscarr.2022.11.1.0108 Corresponding author: Maria I Dalamagka Anesthesia Department, General Hospital of Larisa, Greece. Combination of ketamine, corticosteroids and sevoflurane inhibits the risk of bronchospasm in intubated children under general anesthesia Maria I Dalamagka * Copyright © 2022 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons  Corresponding author: Maria I Dalamagka Acknowledgments General Hospital of Larisa. Statement of informed consent Written informed consent was obtained from the patient for publication of this case report. A copy of the written consent is available. 5. Conclusion In conclusion, the combination of Ketamine, corticosteroids and sevoflurane has been shown to inhibit possible complications such as bronchospasm when asthmatic children implied in general anesthesia. 3. Management and Outcome The common denominator underlined in all forms of asthma is bronchial hyperresponsiveness to various stimuli. Inhaled glucocorticoids have long been used as a first-line treatment for persistent pediatric asthma, as they are the or(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. GSC Advanced Research and Reviews, 2022, 11(01), 165–166 most effective intervention for the treatment of asthma. Thus, Solu medrol (kg x 2), Dexaton (up to 0.1x kg) and Flixotide 50 mcg and Aerolin 100 mcg were administered intravenously. Ketamine also causes bronchodilation and was administered at its appropriate titrated dose (0.2 x kg), as anesthesia was maintained with sevoflurane which does not irritate the respiratory system. 4. Discussion Corticosteroids have inhibitory properties in many effects on many stem cells and inflammatory cells, which are activated in asthma. Inhaled steroids reduce the number and activation of inflammatory cells in the epithelium and submucosa by clogging the damaged epithelium and, potentially, inhibiting the production of proinflammatory cytokines, and reducing the survival time of the epithelium. This action of corticosteroids in combination with the ketamine which has a bronchodilator effect and the property of sevoflurane as it does not irritate the airway, they eliminate bronchospasm and prevent possible laryngospasm. [7] J Corren, JR Parnes, L Wang, M Mo, SL Roseti, JM Griffiths, et al. Tezepelumab in adults with uncontrolled asthma N Engl J Med. 2017; 377: 936-946. [6] H Farne, DJ Jackson, SL Johnston Are emerging PGD2 antagonists a promising therapy class for treating asthma? Expert Opin Emerg Drugs. 2016; 21: 359-364. References [1] KF Chung, SE Wenzel, JL Brozek, A Bush, M Castro, PJ Sterk, et al. International ERS/ATS guidelines on definition, evaluation and treatment of severe asthma Eur Respir J. 2014; 43; 343-373. [1] KF Chung, SE Wenzel, JL Brozek, A Bush, M Castro, PJ Sterk, et al. International ERS/ATS guidelines on definition, evaluation and treatment of severe asthma Eur Respir J. 2014; 43; 343-373. [2] HS Zahran, CM Bailey, SA Damon, PL Garbe, PN Breysse. Vital signs: asthma in children – United States, 2001- 2016 MMWR Morb Mortal Wkly Rep. 2018; 67: 149-155. [2] HS Zahran, CM Bailey, SA Damon, PL Garbe, PN Breysse. Vital signs: asthma in children – United States, 2001- 2016 MMWR Morb Mortal Wkly Rep. 2018; 67: 149-155. [3] WG Teague, BR Phillips, JV Fahy, SE Wenzel, AM Fitzpatrick, WC Moore. et al.Baseline features of the severe asthma Research program (SARP III) cohort: differences with age J Allergy Clin Immunol Pract. 2018; 6: 545-554. [3] WG Teague, BR Phillips, JV Fahy, SE Wenzel, AM Fitzpatrick, WC Moore. et al.Baseline features of the severe asthma Research program (SARP III) cohort: differences with age J Allergy Clin Immunol Pract. 2018; 6: 545-554. [4] F Rabe, P Nair, G Brusselle, JF Maspero, M Castro, L.Sher et al. Efficacy and safety of dupilumab in glucocorticoid- dependent severe asthma N Engl J Med. 2018; 378: 2475-2485. [4] F Rabe, P Nair, G Brusselle, JF Maspero, M Castro, L.Sher et al. Efficacy and safety of dupilumab in glucocorticoid- dependent severe asthma N Engl J Med. 2018; 378: 2475-2485. [5] S Gonem, R Berair, A Singapuri, R Hartley, MFM Laurencin, G Bacher, et al. Fevipiprant, a prostaglandin D2 receptor 2 antagonist, in patients with persistent eosinophilic asthma: a single-centre, randomised, double-blind, parallel-group, placebo-controlled trial Lancet Respir Med. 2016; 4: 699-707. [5] S Gonem, R Berair, A Singapuri, R Hartley, MFM Laurencin, G Bacher, et al. Fevipiprant, a prostaglandin D2 receptor 2 antagonist, in patients with persistent eosinophilic asthma: a single-centre, randomised, double-blind, parallel-group, placebo-controlled trial Lancet Respir Med. 2016; 4: 699-707. [6] H Farne, DJ Jackson, SL Johnston Are emerging PGD2 antagonists a promising therapy class for treating asthma? Expert Opin Emerg Drugs. 2016; 21: 359-364. [6] H Farne, DJ Jackson, SL Johnston Are emerging PGD2 antagonists a promising therapy class for treating asthma? Expert Opin Emerg Drugs. 2016; 21: 359-364. References [7] J Corren, JR Parnes, L Wang, M Mo, SL Roseti, JM Griffiths, et al. Tezepelumab in adults with uncontrolled asthma N Engl J Med. 2017; 377: 936-946. [7] J Corren, JR Parnes, L Wang, M Mo, SL Roseti, JM Griffiths, et al. Tezepelumab in adults with uncontrolled asthma N Engl J Med. 2017; 377: 936-946. 166
https://openalex.org/W3093001044	https://www.mdpi.com/1420-3049/25/20/4709/pdf?version=1602678894	English	null	LPS Induces GM-CSF Production by Breast Cancer MDA-MB-231 Cells via Long-Chain Acyl-CoA Synthetase 1	Molecules/Molecules online/Molecules annual	2,020	cc-by	8,173	Received: 4 September 2020; Accepted: 12 October 2020; Published: 14 October 2020 Abstract: Granulocyte–macrophage colony-stimulating factor (GM-CSF) is a monomeric glycoprotein that has been implicated in the tumor growth and progression of diﬀerent types of cancer. GM-CSF is produced by various non-immune cells including MDA-MB-231 in response to various stimuli. However, the role of lipopolysaccharide (LPS) in the regulation of GM-CSF in MDA-MB-231 breast cancer cells so far remains unclear. Herein, we asked whether LPS could induce GM-CSF production in MDA-MB-231 cells, and if so, which signaling pathway was involved. MDA-MB-231 cells were treated with LPS or tumor necrosis factor alpha (TNF-α; positive control), and GM-CSF expression levels were determined by qRT-PCR, ELISA, and confocal microscopy. Phosphorylation of the mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-kB) signaling proteins were evaluated by ﬂow cytometry. Our results show that LPS induces GM-CSF expression at both mRNA and protein levels in MDA-MBA-231 cells. Inhibition of acyl-CoA synthetase 1 (ACSL1) activity in the cells with triacsin C signiﬁcantly reduces the secretion of GM-CSF. Furthermore, the inhibition of ACSL1 activity signiﬁcantly blocks the LPS-mediated phosphorylation of p38 MAPK, MEK1/2, extracellular signal-regulated kinase (ERK)1/2, c-Jun NH2-terminal kinase (JNK), and nuclear factor-κB (NF-kB) in the cells. These ﬁndings provide the ﬁrst evidence that LPS induces ACSL1-dependent GM-CSF gene expression in MDA-MB-231 breast cancer cells, which requires the activation of p38 MAPK, MEK1/2, ERK1/2, JNK, and NF-kB. Keywords: LPS; ACSL1; GM-CSF; MDA-MB-231; MAPK LPS Induces GM-CSF Production by Breast Cancer MDA-MB-231 Cells via Long-Chain Acyl-CoA Synthetase 1 Fatema Al-Rashed 1,† , Reeby Thomas 1,†, Areej Al-Roub 1,†, Fahd Al-Mulla 2 and Rasheed Ahmad 1,* 1 Immunology & Microbiology Department, Dasman Diabetes Institute, Kuwait City 15462, Kuwait; fatema.alrashed@dasmaninstitute.org (F.A.-R.); reeby.thomas@dasmaninstitute.org (R.T.); areej.abualroub@dasmaninstitute.org (A.A.-R.) 1 Immunology & Microbiology Department, Dasman Diabetes Institute, Kuwait City 15462, Kuwait; fatema.alrashed@dasmaninstitute.org (F.A.-R.); reeby.thomas@dasmaninstitute.org (R.T.); areej.abualroub@dasmaninstitute.org (A.A.-R.) 1 Immunology & Microbiology Department, Dasman Diabetes Institute, Kuwait City 15462, Kuwait; fatema.alrashed@dasmaninstitute.org (F.A.-R.); reeby.thomas@dasmaninstitute.org (R.T.); areej.abualroub@dasmaninstitute.org (A.A.-R.) j g 2 Genetics and Bioinformatics, Dasman Diabetes Institute, Kuwait City 15462, Kuwait; fahd.almulla@dasmaninstitute.org * Correspondence: rasheed.ahmad@dasmaninstitute.org † These authors contributed equally to this work. molecules molecules molecules 1. Introduction Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a monomeric glycoprotein/ cytokine that is involved in the immune modulation and hematopoiesis [1]. GM-CSF is produced by activated monocytes, macrophages, T cells, B cells, ﬁbroblasts, mast cells, vascular endothelial cells, and a wide variety of cancer cell types, including MDA-MB-231 [2,3] in response to various stimuli. The excessive production of GM-CSF is involved in chronic inﬂammatory disorders by maintaining the existence of target cells and promoting the renewal of macrophages [4]. In this regard, a pathogenic role of increased GM-CSF has been well established in autoimmune diseases that are associated with cellular immune responses such as multiple sclerosis (MS) and rheumatoid arthritis (RA) [5,6]. Many studies show that GM-CSF is involved in promoting tumor growth and progression. GM-CSF regulates cancer Molecules 2020, 25, 4709; doi:10.3390/molecules25204709 www.mdpi.com/journal/molecules 2 of 12 Molecules 2020, 25, 4709 cell proliferation and migration in various types of cancer. Constitutive GM-CSF protein expression and secretion along with its association with tumor growth and poor prognosis have been noted in multiple cancer models [7–9]. Elevated GM-CSF in serum underlies obesity-associated breast cancer metastasis. Obesity increases the risk for the promotion and progression of various cancers. One important mechanism leading this association is the obesity-induced chronic low-grade inﬂammation. Overnutrition and high-fat and high-energy diets have been shown to facilitate the absorption of bacterial lipopolysaccharide (LPS) from intestinal bacteria in the stings of obesity [10,11]. It has been reported that increased inﬂammatory mediators contribute to the aggressive breast cancer phenotype in obesity, which increases the risk of mortality in breast cancer patients [12]. The inflammatory state of obesity enhanced GM-CSF production in obese cancer patients, and it led to promote tumor growth and progression. GM-CSF regulates cell proliferation and migration in various types of cancer, including breast cancer, and it is upregulated by various stimuli. It remains unclear how GM-CSF production is upregulated in obese breast cancer patients. Since LPS and GM-CSF are elevated in a state of obesity, we asked whether LPS could trigger GM-CSF production in MDA-MB-231 cells. In addressing our study’s hypothesis that LPS could activate the GM-CSF gene expression in MDA-MB-231 cells, herein we report that LPS induces GM-CSF expression in MDA-MB-231 cells, and it involves the activation of acyl-CoA synthetase 1 (ACSL1), p38 mitogen-activated protein kinase (MAPK), MEK1/2, extracellular signal-regulated kinase (ERK1/ERK2), c-Jun NH2-terminal kinase (JNK) and nuclear factor-κB (NF-kB). 2. Results 2.1. LPS Induces GM-CSF Gene Expression in Human MDA-MB-231 Cells 2.1. LPS Induces GM-CSF Gene Expression in Human MDA-MB-231 Cells GM-CSF supports angiogenesis in primary breast tumor and further triggering cancer invasion and metastatic spread [3]. Elevated GM-CSF in serum underlies obesity-associated breast cancer metastasis. Since LPS and GM-CSF levels are higher in obesity settings, we wanted to investigate whether LPS induced GM-CSF production in MDA-MB-231 cells. This was accomplished by exposing MDA-MB-231 cells to vehicle or LPS or TNF-α (as a positive control) for 24 h. Our data show that LPS treatment of the MDA-MB-231 cells induced high GM-CSF mRNA expression level compared to the cells treated with vehicle (Figure 1A). We also measured the GM-CSF protein level in the supernatant of the MDA-MB-231 cells incubated with vehicle or LPS or TNF-α. Similar to mRNA, GM-CSF secreted protein levels were signiﬁcantly higher in MDA-MB-231 cells supernatant when stimulated with LPS (Figure 1B). Confocal microscopy along with the ﬂuorescence intensity of MDA-MB-231 cells also showed that there was a signiﬁcant increase in the expression of GM-CSF in the cells treated with LPS or TNF-α (positive control) (Figure 1C,D). 2.2. LPS-Induced GM-CSF Production Is Downregulated by the Inhibition of ACSL1 GM-CSF production requires ACSL1 when MDA-MB-231 cells are stimulated with TNFα. To see whether the inhibition of ACSL1 activity would inﬂuence the expression of GM-CSF, MDA-MB-231 cells were treated with triacsin C prior to LPS treatment. ACSL1 inhibition with triacsin signiﬁcantly suppressed the LPS-mediated production of GM-CSF by MDA-MB-231 cells (Figure 2A,B). We also conﬁrmed these ﬁndings with confocal microscopy (Figure 2C). Confocal microscopy data revealed that the ﬂuorescence intensity of GM-CSF staining content was reduced in MDA-MB-231 cells pretreated with triacsin C following exposure to LPS (Figure 2D). 3 of 123 o Molecules 2020, 25, 4709 ecules 2020 25 x Molecules 2020, 25, 4709 3 of 12 les 2020, 25, x 3 igure 1. Effect of lipopolysaccharide (LPS) on granulocyte–macrophage colony-stimulating factor GM-CSF) production in human MDA-MB-231 cells. MDA-MB-231 cells were cultured in six-well plates at a concentration of 1 × 106 cells/well. Cells were treated with vehicle, LPS (10 ng/mL), and TNF-α (10 ng/mL; positive control) separately. After 24 h incubation, cells and supernatants were ollected. (A) Total cellular RNA was isolated, and GM-CSF mRNA expression was determined by eal-time RT-PCR. (B) Secreted GM-CSF protein in culture media was determined by ELISA. (C) MDA-MB-231 cells were treated with vehicle, LPS, or TNF-α for 24 h and then were stained with GM- CSF (red) and 4′,6-diamidino-2-phenylindole (DAPI) (blue). White arrows indicate typical stained ells. (D) GM-CSF fluorescence intensity was determined for 10 random images. The results obtained rom three independent experiments are shown. All data are expressed as mean ± SEM (n ≥ 3). ** p < Figure 1. Eﬀect of lipopolysaccharide (LPS) on granulocyte–macrophage colony-stimulating factor (GM-CSF) production in human MDA-MB-231 cells. MDA-MB-231 cells were cultured in six-well plates at a concentration of 1 × 106 cells/well. Cells were treated with vehicle, LPS (10 ng/mL), and TNF-α (10 ng/mL; positive control) separately. After 24 h incubation, cells and supernatants were collected. (A) Total cellular RNA was isolated, and GM-CSF mRNA expression was determined by real-time RT-PCR. (B) Secreted GM-CSF protein in culture media was determined by ELISA. (C) MDA-MB-231 cells were treated with vehicle, LPS, or TNF-α for 24 h and then were stained with GM-CSF (red) and 4′,6-diamidino-2-phenylindole (DAPI) (blue). White arrows indicate typical stained cells. (D) GM-CSF ﬂuorescence intensity was determined for 10 random images. The results obtained from three independent experiments are shown. All data are expressed as mean ± SEM (n ≥3). 2.2. LPS-Induced GM-CSF Production Is Downregulated by the Inhibition of ACSL1 (C) Representative figures for confocal microscopy. MDA-MB-231 cells were stained with GM-CSF (green) and DAPI (blue). White arrows indicate typical stained cells. White arrows indicate typical stained cells. (D) GM-CSF fluorescence intensity was determined for 10 random images. All data are expressed as mean ± SEM (n ≥ 3). * p < 0.05, p < 0.02, ** p < 0.001 versus vehicle. Figure 2. Eﬀect of acyl-CoA synthetase 1 (ACSL1) inhibition on LPS mediated GM-CSF production in MDA-MB-231 cells. MDA-MB-231 cells were pretreated with a long chain acyl-CoA synthetase (ACSL1) inhibitor (triacsin C: (1 µM) or vehicle for 1 h and then incubated with LPS for 24 h. (A) Total cellular RNA was isolated, and GM-CSF mRNA expression was determined by real-time RT-PCR. (B) Secreted GM-CSF protein in culture media was determined by ELISA. (C) Representative ﬁgures for confocal microscopy. MDA-MB-231 cells were stained with GM-CSF (green) and DAPI (blue). White arrows indicate typical stained cells. White arrows indicate typical stained cells. (D) GM-CSF ﬂuorescence intensity was determined for 10 random images. All data are expressed as mean ± SEM (n ≥3). * p < 0.05, p < 0.02, ** p < 0.001 versus vehicle. Figure 2. Effect of acyl-CoA synthetase 1 (ACSL1) inhibition on LPS mediated GM-CSF production in MDA-MB-231 cells. MDA-MB-231 cells were pretreated with a long chain acyl-CoA synthetase (ACSL1) inhibitor (triacsin C: (1 μM) or vehicle for 1 h and then incubated with LPS for 24 h. (A) Total cellular RNA was isolated, and GM-CSF mRNA expression was determined by real-time RT-PCR. (B) Secreted GM-CSF protein in culture media was determined by ELISA. (C) Representative figures for confocal microscopy. MDA-MB-231 cells were stained with GM-CSF (green) and DAPI (blue). White arrows indicate typical stained cells. White arrows indicate typical stained cells. (D) GM-CSF fluorescence intensity was determined for 10 random images. All data are expressed as mean ± SEM (n ≥ 3). * p < 0.05, p < 0.02, p < 0.001 versus vehicle. Figure 2. Eﬀect of acyl-CoA synthetase 1 (ACSL1) inhibition on LPS mediated GM-CSF production in MDA-MB-231 cells. MDA-MB-231 cells were pretreated with a long chain acyl-CoA synthetase (ACSL1) inhibitor (triacsin C: (1 µM) or vehicle for 1 h and then incubated with LPS for 24 h. (A) Total cellular RNA was isolated, and GM-CSF mRNA expression was determined by real-time RT-PCR. 2.2. LPS-Induced GM-CSF Production Is Downregulated by the Inhibition of ACSL1 p < 0.01, * p < 0.001, p < 0.0001 versus vehicle. ure 1. Effect of lipopolysaccharide (LPS) on granulocyte–macrophage colony-stimulating fac M-CSF) production in human MDA-MB-231 cells. MDA-MB-231 cells were cultured in six-w tes at a concentration of 1 × 106 cells/well. Cells were treated with vehicle, LPS (10 ng/mL), a F-α (10 ng/mL; positive control) separately. After 24 h incubation, cells and supernatants we lected. (A) Total cellular RNA was isolated, and GM-CSF mRNA expression was determined l-time RT-PCR. (B) Secreted GM-CSF protein in culture media was determined by ELISA. ( DA-MB-231 cells were treated with vehicle, LPS, or TNF-α for 24 h and then were stained with G F (red) and 4′,6-diamidino-2-phenylindole (DAPI) (blue). White arrows indicate typical stain s. (D) GM-CSF fluorescence intensity was determined for 10 random images. The results obtain m three independent experiments are shown All data are expressed as mean ± SEM (n ≥3) Figure 1. Eﬀect of lipopolysaccharide (LPS) on granulocyte–macrophage colony-stimulating factor (GM-CSF) production in human MDA-MB-231 cells. MDA-MB-231 cells were cultured in six-well plates at a concentration of 1 × 106 cells/well. Cells were treated with vehicle, LPS (10 ng/mL), and TNF-α (10 ng/mL; positive control) separately. After 24 h incubation, cells and supernatants were collected. (A) Total cellular RNA was isolated, and GM-CSF mRNA expression was determined by real-time RT-PCR. (B) Secreted GM-CSF protein in culture media was determined by ELISA. (C) MDA-MB-231 cells were treated with vehicle, LPS, or TNF-α for 24 h and then were stained with GM-CSF (red) and 4′,6-diamidino-2-phenylindole (DAPI) (blue). White arrows indicate typical stained cells. (D) GM-CSF ﬂuorescence intensity was determined for 10 random images. The results obtained from three independent experiments are shown. All data are expressed as mean ± SEM (n ≥3). p < 0.01, * p < 0.001, **** p < 0.0001 versus vehicle. 4 of 12 4 of 12 Molecules 2020, 25, 4709 M l l 2020 25 Figure 2. Effect of acyl-CoA synthetase 1 (ACSL1) inhibition on LPS mediated GM-CSF production in MDA-MB-231 cells. MDA-MB-231 cells were pretreated with a long chain acyl-CoA synthetase (ACSL1) inhibitor (triacsin C: (1 μM) or vehicle for 1 h and then incubated with LPS for 24 h. (A) Total cellular RNA was isolated, and GM-CSF mRNA expression was determined by real-time RT-PCR. (B) Secreted GM-CSF protein in culture media was determined by ELISA. 2.2. LPS-Induced GM-CSF Production Is Downregulated by the Inhibition of ACSL1 (B) Secreted GM-CSF protein in culture media was determined by ELISA. (C) Representative ﬁgures for confocal microscopy. MDA-MB-231 cells were stained with GM-CSF (green) and DAPI (blue). White arrows indicate typical stained cells. White arrows indicate typical stained cells. (D) GM-CSF ﬂuorescence intensity was determined for 10 random images. All data are expressed as mean ± SEM (n ≥3). * p < 0.05, p < 0.02, p < 0.001 versus vehicle. 2 3 ACSL1 Is Involved in LPS Activated MAPK and NF-kB Signaling Pathways 2.3. ACSL1 Is Involved in LPS Activated MAPK and NF-kB Signaling Pathways g g y LPS activate a network of signaling pathways that influence the expression of several genes. It is known that LPS stimulates the activation of mitogen-activated protein kinases (MAPKs) (p38, MEK1/2, ERK1/2, JNK) and NF-kB signaling pathways [13,14]. In agreement with these reports, our flow cytometry data show that LPS treatment of MDA-MB-231 induces the phosphorylation of p38 MAPK, MEK1/2, ERK1/2, JNK (Figure 3A–D) and NF-kB; Figure 4A–D). Next, we explore whether ACSL1 is involved in the LPS-mediated phosphorylation of p38 MAPK, ERK1/2, JNK, and NF-kB. MDA-MB-231 cells were treated with LPS in the presence or absence of triacsin C and the phosphorylation of key signaling molecules was determined by flow cytometry. Our flow cytometry results showed that ACSL1 inhibition with triacsin C significantly attenuated the LPS-induced phosphorylation of p38 MAPK, ERK1/2, JNK (Figure 3A–D), and NF-kB (Figure 4A–C). Our Western LPS activate a network of signaling pathways that inﬂuence the expression of several genes. It is known that LPS stimulates the activation of mitogen-activated protein kinases (MAPKs) (p38, MEK1/2, ERK1/2, JNK) and NF-kB signaling pathways [13,14]. In agreement with these reports, our ﬂow cytometry data show that LPS treatment of MDA-MB-231 induces the phosphorylation of p38 MAPK, MEK1/2, ERK1/2, JNK (Figure 3A–D) and NF-kB; Figure 4A–D). Next, we explore whether ACSL1 is involved in the LPS-mediated phosphorylation of p38 MAPK, ERK1/2, JNK, and NF-kB. MDA-MB-231 cells were treated with LPS in the presence or absence of triacsin C and the phosphorylation of key signaling molecules was determined by ﬂow cytometry. Our ﬂow cytometry results showed that ACSL1 inhibition with triacsin C signiﬁcantly attenuated the LPS-induced phosphorylation of p38 MAPK, ERK1/2, JNK (Figure 3A–D), and NF-kB (Figure 4A–C). Our Western blot protein data for 5 of 12 5 f 12 Molecules 2020, 25, 4709 phospho NF-KB and total NF-kB clearly exhibited a similar treatment response (Figure 4D,E) to that seen that by ﬂow cytometry. Overall, our results are signifying that p38 MAPK, ERK1/2, and NF-kB molecules were downstream of LPS/ACSL1 signaling. blot protein data for phospho NF-KB and total NF-kB clearly exhibited a similar treatment response (Figure 4D,E) to that seen that by flow cytometry. Overall, our results are signifying that p38 MAPK, ERK1/2, and NF-kB molecules were downstream of LPS/ACSL1 signaling. molecules were downstream of LPS/ACSL1 signaling. ERK1/2, and NF kB molecules were downstream of LPS/ACSL1 signaling. Figure 3. 2 3 ACSL1 Is Involved in LPS Activated MAPK and NF-kB Signaling Pathways 2.3. ACSL1 Is Involved in LPS Activated MAPK and NF-kB Signaling Pathways Mitogen-activated protein kinase (MAPK) signaling molecules activation in LPS-treated MDA-MBA-231 was inhibited by triacsin C. MDA-MBA-231 cells were pretreated with ACSL1 inhibitor (Triacsin C: (1 μM) or vehicle for 1 hr and then incubated with LPS for 15 min. There is no difference between vehicle and Triacsin C cells. Therefore, for the histogram, we used only vehicle- treated cells. (A–D) The phosphorylation of p38, extracellular signal-regulated kinase (ERK1/2) and c-Jun NH2-terminal kinase (JNK) was determined by flow cytometry presented in the form of staining intensity and representative histograms. The data are representative of three independent Figure 3. Mitogen-activated protein kinase (MAPK) signaling molecules activation in LPS-treated MDA-MBA-231 was inhibited by triacsin C. MDA-MBA-231 cells were pretreated with ACSL1 inhibitor (Triacsin C: (1 µM) or vehicle for 1 hr and then incubated with LPS for 15 min. There is no diﬀerence between vehicle and Triacsin C cells. Therefore, for the histogram, we used only vehicle-treated cells. (A–D) The phosphorylation of p38, extracellular signal-regulated kinase (ERK1/2) and c-Jun NH2-terminal kinase (JNK) was determined by ﬂow cytometry presented in the form of staining intensity and representative histograms. The data are representative of three independent experiments. All data are expressed as mean ± SEM (n ≥3). p < 0.01, * p < 0.001, p < 0.0001 versus LPS. Figure 3. Mitogen-activated protein kinase (MAPK) signaling molecules activation in LPS-treated MDA-MBA-231 was inhibited by triacsin C. MDA-MBA-231 cells were pretreated with ACSL1 inhibitor (Triacsin C: (1 μM) or vehicle for 1 hr and then incubated with LPS for 15 min. There is no difference between vehicle and Triacsin C cells. Therefore, for the histogram, we used only vehicle- treated cells. (A–D) The phosphorylation of p38, extracellular signal-regulated kinase (ERK1/2) and c-Jun NH2-terminal kinase (JNK) was determined by flow cytometry presented in the form of staining intensity and representative histograms. The data are representative of three independent Figure 3. Mitogen-activated protein kinase (MAPK) signaling molecules activation in LPS-treated MDA-MBA-231 was inhibited by triacsin C. MDA-MBA-231 cells were pretreated with ACSL1 inhibitor (Triacsin C: (1 µM) or vehicle for 1 hr and then incubated with LPS for 15 min. There is no diﬀerence between vehicle and Triacsin C cells. Therefore, for the histogram, we used only vehicle-treated cells. 2 3 ACSL1 Is Involved in LPS Activated MAPK and NF-kB Signaling Pathways 2.3. ACSL1 Is Involved in LPS Activated MAPK and NF-kB Signaling Pathways (E) Phosphorylation intensity of NF-κB was quantiﬁed using Image Lab software (version 6.0.1, Bio-Rad, Hercules, CA, USA) and are presented in arbitrary units, which indicated that the Western blot protein data for phospho NF-KB and NF-kB clearly exhibited a similar treatment response to that seen that by ﬂow cytometry. All data are expressed as mean ± SEM (n ≥3). p < 0.01, * p < 0.001 versus LPS. Figure 4. Nuclear factor-κB (NF-κB) activation in LPS-treated monocytes was inhibited by triacsin C. MDA-MB-231 cells were pretreated with ACSL1 inhibitor (Triacsin C: (1 μM) or vehicle for 1 h and then incubated with LPS for 15 min. NF-κB phosphorylation was determined by flow cytometry. (A) R t ti fl t t d t l t f NF B fl t t l i hibit f k B Figure 4. Nuclear factor-κB (NF-κB) activation in LPS-treated monocytes was inhibited by triacsin C. MDA-MB-231 cells were pretreated with ACSL1 inhibitor (Triacsin C: (1 µM) or vehicle for 1 h and then incubated with LPS for 15 min. NF-κB phosphorylation was determined by ﬂow cytometry. (A) Representative ﬂow cytometry dot plots of p-NF-κB ﬂuorescence versus total inhibitor of kappa B t i l h (IkB ) ll (B) St i i i t it f ﬂ t t l i f NF kB h h l ti Figure 4. Nuclear factor-κB (NF-κB) activation in LPS-treated monocytes was inhibited by triacsin C. MDA-MB-231 cells were pretreated with ACSL1 inhibitor (Triacsin C: (1 μM) or vehicle for 1 h and then incubated with LPS for 15 min. NF-κB phosphorylation was determined by flow cytometry. (A) Representative flow cytometry dot plots of p-NF-κB fluorescence versus total inhibitor of kappa B protein alpha (IkBα) cells. (B) Staining intensity of flow cytometry analysis of NF-kB phosphorylation. (C) Representative histograms of one experiment. (D) MDA-MB-231 were pretreated with ACSL1 inhibitor (Triacsin C: (1 μM) or vehicle for 1 hr and then incubated with LPS for 15 min. Cell lysates were prepared as described in Materials and Methods. Samples were run on denaturing gels. Phosphorylated NF-κB is depicted in the upper panel, and total respective proteins are shown in the lower panel. (E) Phosphorylation intensity of NF-κB was quantified using Image Lab software (version 6.0.1, Bio-Rad, Hercules, CA, USA) and are presented in arbitrary units, which indicated that Figure 4. 2 3 ACSL1 Is Involved in LPS Activated MAPK and NF-kB Signaling Pathways 2.3. ACSL1 Is Involved in LPS Activated MAPK and NF-kB Signaling Pathways (A–D) The phosphorylation of p38, extracellular signal-regulated kinase (ERK1/2) and c-Jun NH2-terminal kinase (JNK) was determined by ﬂow cytometry presented in the form of staining intensity and representative histograms. The data are representative of three independent experiments. All data are expressed as mean ± SEM (n ≥3). p < 0.01, * p < 0.001, p < 0.0001 versus LPS. 6 of 12 01 Molecules 2020, 25, 4709 experiments. All versus LPS olecules 2020, 25, 4709 6 of experiments. All data are expressed as mean ± SEM (n ≥ 3). p < 0.01, p < 0.001, p < 0.0001 versus LPS. Figure 4. Nuclear factor-κB (NF-κB) activation in LPS-treated monocytes was inhibited by triacsin C. MDA-MB-231 cells were pretreated with ACSL1 inhibitor (Triacsin C: (1 μM) or vehicle for 1 h and then incubated with LPS for 15 min. NF-κB phosphorylation was determined by flow cytometry. (A) Representative flow cytometry dot plots of p-NF-κB fluorescence versus total inhibitor of kappa B protein alpha (IkBα) cells. (B) Staining intensity of flow cytometry analysis of NF-kB phosphorylation. (C) Representative histograms of one experiment. (D) MDA-MB-231 were pretreated with ACSL1 inhibitor (Triacsin C: (1 μM) or vehicle for 1 hr and then incubated with LPS for 15 min. Cell lysates were prepared as described in Materials and Methods. Samples were run on denaturing gels. Phosphorylated NF-κB is depicted in the upper panel, and total respective proteins are shown in the lower panel. (E) Phosphorylation intensity of NF-κB was quantified using Image Lab software (version 6.0.1, Bio-Rad, Hercules, CA, USA) and are presented in arbitrary units, which indicated that Figure 4. Nuclear factor-κB (NF-κB) activation in LPS-treated monocytes was inhibited by triacsin C. MDA-MB-231 cells were pretreated with ACSL1 inhibitor (Triacsin C: (1 µM) or vehicle for 1 h and then incubated with LPS for 15 min. NF-κB phosphorylation was determined by ﬂow cytometry. (A) Representative ﬂow cytometry dot plots of p-NF-κB ﬂuorescence versus total inhibitor of kappa B protein alpha (IkBα) cells. (B) Staining intensity of ﬂow cytometry analysis of NF-kB phosphorylation. (C) Representative histograms of one experiment. (D) MDA-MB-231 were pretreated with ACSL1 inhibitor (Triacsin C: (1 µM) or vehicle for 1 hr and then incubated with LPS for 15 min. Cell lysates were prepared as described in Materials and Methods. Samples were run on denaturing gels. 2 3 ACSL1 Is Involved in LPS Activated MAPK and NF-kB Signaling Pathways 2.3. ACSL1 Is Involved in LPS Activated MAPK and NF-kB Signaling Pathways Phosphorylated NF-κB is depicted in the upper panel, and total respective proteins are shown in the lower panel. (E) Phosphorylation intensity of NF-κB was quantiﬁed using Image Lab software (version 6.0.1, Bio-Rad, Hercules, CA, USA) and are presented in arbitrary units, which indicated that the Western blot protein data for phospho NF-KB and NF-kB clearly exhibited a similar treatment response to that seen that by Figure 4. Nuclear factor-κB (NF-κB) activation in LPS-treated monocytes was inhibited by triacsin C. MDA-MB-231 cells were pretreated with ACSL1 inhibitor (Triacsin C: (1 μM) or vehicle for 1 h and then incubated with LPS for 15 min. NF-κB phosphorylation was determined by flow cytometry. (A) Representative flow cytometry dot plots of p-NF-κB fluorescence versus total inhibitor of kappa B protein alpha (IkBα) cells. (B) Staining intensity of flow cytometry analysis of NF-kB phosphorylation. (C) Representative histograms of one experiment. (D) MDA-MB-231 were pretreated with ACSL1 inhibitor (Triacsin C: (1 μM) or vehicle for 1 hr and then incubated with LPS for 15 min. Cell lysates were prepared as described in Materials and Methods. Samples were run on denaturing gels. Phosphorylated NF-κB is depicted in the upper panel, and total respective proteins are shown in the lower panel. (E) Phosphorylation intensity of NF-κB was quantified using Image Lab software (version 6.0.1, Bio-Rad, Hercules, CA, USA) and are presented in arbitrary units, which indicated that Figure 4. Nuclear factor-κB (NF-κB) activation in LPS-treated monocytes was inhibited by triacsin C. MDA-MB-231 cells were pretreated with ACSL1 inhibitor (Triacsin C: (1 µM) or vehicle for 1 h and then incubated with LPS for 15 min. NF-κB phosphorylation was determined by ﬂow cytometry. (A) Representative ﬂow cytometry dot plots of p-NF-κB ﬂuorescence versus total inhibitor of kappa B protein alpha (IkBα) cells. (B) Staining intensity of ﬂow cytometry analysis of NF-kB phosphorylation. (C) Representative histograms of one experiment. (D) MDA-MB-231 were pretreated with ACSL1 inhibitor (Triacsin C: (1 µM) or vehicle for 1 hr and then incubated with LPS for 15 min. Cell lysates were prepared as described in Materials and Methods. Samples were run on denaturing gels. Phosphorylated NF-κB is depicted in the upper panel, and total respective proteins are shown in the lower panel. factor. 3. Discussion . Discussion In this study, we report for the first time that LPS induced GM-CSF production by MDA-MB 31 cancer cells. LPS regulates the production of IL-6, IL-8, and GM-CSF in monocytic cells [15], whic upported our findings. In addition, a growing body of evidence suggests that GM-CSF is produce nd secreted by a wide variety of non-immune cell types, including fibroblasts, keratinocyte ndothelial cells, and MDA-MB-231 cells in response to appropriate stimuli [3,16]. LPS activate nflammatory responses in monocytes/macrophages via ACSL1 [17]. Our data showed that th nhibition of ACSL1 significantly blocked the LPS-induced production of GM-CSF, which t d b th t d th t ACSL1 d fi i t h di l d d i fl t In this study, we report for the ﬁrst time that LPS induced GM-CSF production by MDA-MB-231 cancer cells. LPS regulates the production of IL-6, IL-8, and GM-CSF in monocytic cells [15], which supported our ﬁndings. In addition, a growing body of evidence suggests that GM-CSF is produced and secreted by a wide variety of non-immune cell types, including ﬁbroblasts, keratinocytes, endothelial cells, and MDA-MB-231 cells in response to appropriate stimuli [3,16]. LPS activates inﬂammatory responses in monocytes/macrophages via ACSL1 [17]. Our data showed that the inhibition of ACSL1 signiﬁcantly blocked the LPS-induced production of GM-CSF, which is supported by the study that ACSL1-deﬁcient mouse macrophages display reduced inﬂammatory responses after long-term stimulation with LPS [18]. upported by the study that ACSL1-deficient mouse macrophages display reduced inflammator esponses after long-term stimulation with LPS [18]. Regarding molecular mechanisms underlying the LPS-induced production of GM-CSF, It i nown that LPS induces the activation and phosphorylation of MAP kinase signaling pathways (p38 RK1/2, JNK) [13,14] that regulate the expression of several genes. In agreement with previou eports, our flow cytometry data show that LPS treatment of MDA-MB-231 induces th hosphorylation of p38 MAPK, ERK1/2, and JNK. It has been reported that ACSL1 function in TNF α induced the activation of MAPK and NF-κB signaling proteins. 2 3 ACSL1 Is Involved in LPS Activated MAPK and NF-kB Signaling Pathways 2.3. ACSL1 Is Involved in LPS Activated MAPK and NF-kB Signaling Pathways Nuclear factor-κB (NF-κB) activation in LPS-treated monocytes was inhibited by triacsin C. MDA-MB-231 cells were pretreated with ACSL1 inhibitor (Triacsin C: (1 µM) or vehicle for 1 h and then incubated with LPS for 15 min. NF-κB phosphorylation was determined by ﬂow cytometry. (A) Representative ﬂow cytometry dot plots of p-NF-κB ﬂuorescence versus total inhibitor of kappa B protein alpha (IkBα) cells. (B) Staining intensity of ﬂow cytometry analysis of NF-kB phosphorylation. (C) Representative histograms of one experiment. (D) MDA-MB-231 were pretreated with ACSL1 inhibitor (Triacsin C: (1 µM) or vehicle for 1 hr and then incubated with LPS for 15 min. Cell lysates were prepared as described in Materials and Methods. Samples were run on denaturing gels. Phosphorylated NF-κB is depicted in the upper panel, and total respective proteins are shown in the lower panel. (E) Phosphorylation intensity of NF-κB was quantiﬁed using Image Lab software (version 6.0.1, Bio-Rad, Hercules, CA, USA) and are presented in arbitrary units, which indicated that the Western blot protein data for phospho NF-KB and NF-kB clearly exhibited a similar treatment response to that seen that by ﬂow cytometry. All data are expressed as mean ± SEM (n ≥3). p < 0.01, *** p < 0.001 versus LPS. 7 of 12 0.01, Molecules 2020, 25, 4709 response to that se The thematic illustration presented below summarizes the aforementioned ﬁndings of this study (Figure 5). The thematic illustration presented below summarizes the aforementioned findings of this stud Figure 5). The thematic illustration presented below summarizes the aforementioned ﬁndings of this study (Figure 5). The thematic illustration presented below summarizes the aforementioned findings of this study Figure 5). Figure 5. A novel role of ACSL1 in LPS induced GM-CSF production in MDA-MB-231 breast cancer cells. LPS: Lipopolysaccharide; ACSL1: long-chain acyl-CoA synthetase 1; p38 MAPK: p38 mitogen- activated protein kinase ERK: extracellular signal-regulated kinase; NF-κB: nuclear factor kappa- light-chain-enhancer of activated B cells; GM-CSF: granulocyte-macrophage colony-stimulating factor. Figure 5. A novel role of ACSL1 in LPS induced GM-CSF production in MDA-MB-231 breast cancer cells. LPS: Lipopolysaccharide; ACSL1: long-chain acyl-CoA synthetase 1; p38 MAPK: p38 mitogen- activated protein kinase ERK: extracellular signal-regulated kinase; NF-κB: nuclear factor kappa-light- chain-enhancer of activated B cells; GM-CSF: granulocyte-macrophage colony-stimulating factor. Di i Figure 5. A novel role of ACSL1 in LPS induced GM-CSF production in MDA-MB-231 breast cancer ells. 2 3 ACSL1 Is Involved in LPS Activated MAPK and NF-kB Signaling Pathways 2.3. ACSL1 Is Involved in LPS Activated MAPK and NF-kB Signaling Pathways LPS: Lipopolysaccharide; ACSL1: long-chain acyl-CoA synthetase 1; p38 MAPK: p38 mitogen activated protein kinase ERK: extracellular signal-regulated kinase; NF-κB: nuclear factor kappa ight-chain-enhancer of activated B cells; GM-CSF: granulocyte-macrophage colony-stimulating a to Figure 5. A novel role of ACSL1 in LPS induced GM-CSF production in MDA-MB-231 breast cancer cells. LPS: Lipopolysaccharide; ACSL1: long-chain acyl-CoA synthetase 1; p38 MAPK: p38 mitogen- activated protein kinase ERK: extracellular signal-regulated kinase; NF-κB: nuclear factor kappa-light- chain-enhancer of activated B cells; GM-CSF: granulocyte-macrophage colony-stimulating factor. factor. 3. Discussion It is well documented that TNF- timulates MAPK and NF-κB signaling pathways involved in the regulation of several inflammator ytokines that contribute to the pathogenesis of different inflammatory conditions including state o besity [19,20], suggesting the role of ACSL1 and its association with MAPKs/NF-kB signalin Regarding molecular mechanisms underlying the LPS-induced production of GM-CSF, It is known that LPS induces the activation and phosphorylation of MAP kinase signaling pathways (p38, ERK1/2, JNK) [13,14] that regulate the expression of several genes. In agreement with previous reports, our ﬂow cytometry data show that LPS treatment of MDA-MB-231 induces the phosphorylation of p38 MAPK, ERK1/2, and JNK. It has been reported that ACSL1 function in TNF-α induced the activation of MAPK and NF-κB signaling proteins. It is well documented that TNF-α stimulates MAPK and NF-κB signaling pathways involved in the regulation of several inﬂammatory cytokines that contribute to the pathogenesis of diﬀerent inﬂammatory conditions including state of obesity [19,20], suggesting the role of ACSL1 and its association with MAPKs/NF-kB signaling pathways in the development of chronic low-grade inﬂammation. LPS also plays a key role in the activation of MAPKs and NF-kB signaling pathways and interestingly, our results for the ﬁrst time show that the inhibition of ACSL1 8 of 12 Molecules 2020, 25, 4709 reduced the LPS-induced activation of MAPK and NF-kB signaling proteins in a similar pattern, as we previously demonstrated that the disruption of the activity of ACSL1 in MDA-MB-231 suppressed the phosphorylation of p38 MAPK, ERK1/2, and JNK ([3]). Thus, ACSL1 appears to play a central role in MDA-MB-231 for the LPS-mediated activation of MAPK signaling pathways in parallel to the production of GM-CSF. It is well documented that LPS stimulates MAPK signaling pathways involved in the regulation of several inﬂammatory cytokines that contribute to the pathogenesis of diﬀerent inﬂammatory conditions. Meja et al. reported that LPS-induced GM-CSF secretion by human monocytic cells was partially blocked by p38 MAPK and ERK1/2 [21]. The transcription factor NF-κB induces the expression of various pro-inﬂammatory genes, including those encoding cytokines and chemokines. LPS induced NF-kB activation and cytokine (IL-6 and IL-8) production in human myeloid and non-myeloid cells [22]. The dysregulation of NF-κB activation contributes to the pathogenic processes of various inﬂammatory diseases [23]. 4.1. Cell Culture Human MDA-MB-231 cells were purchased from the American Type Culture Collection (ATCC), grown in Dulbecco’s Modiﬁed Eagle Medium (DMEM) culture medium (Gibco, Life Technologies, Grand Island, NE, USA) supplemented with 10% fetal bovine serum (Gibco, Life Technologies, Grand Island, NE, USA), 2 mM glutamine (Gibco, Invitrogen, Grand Island, NE, USA), 1 mM sodium pyruvate, 10 mM N-2-hydroxyethylpiperazine-N-ethanesulfonic acid (HEPES), 100 ug/mL normocin, 50 U/mL penicillin, and 50 µg/mL streptomycin (P/S; (Gibco, Invitrogen, Grand Island, NE, USA), and incubated at 37 ◦C (with humidity) in 5% CO2. factor. 3. Discussion Our results showed that the inhibition of ACSL1 activity reduces the phosphorylation of NF-kB in response to LPS, indicating a very interesting role of ACSL in LPS-mediated activation of major transcription factor NF-kB, which was supported by the ﬁndings indicating that ACSL inhibition attenuates NF-κB activity resulting from TNF-α stimulation [3]. These results indicate that ACSL1 acts upstream of the NF-κB pathway in a similar fashion as noted in case of GM-CSF production by TNF-α [3]. We speculate that LPS interacts with MDA-MB-231 cells and activates a complex process, which includes the participation of several molecules including MAPK signaling molecules (p38, ERK1/2, JNK, C-Jun) interacting with each other along with having extensive cross-talk to other inﬂammatory pathways (including NF-kB) in the orchestration of inﬂammatory responses that regulate GM-CSF. In summary, our ﬁndings show that LPS induces the production of GM-CSF by MDA-MB-231 breast cancer cells. Furthermore, our study demonstrates a distinct role of ACSL1 in the regulation of LPS-mediated GM-CSF production in MDA-MB-231 cells. Interestingly, ACSL1 acts upstream of p38, ERK1/2, JNK, and NF-kB signaling molecules. 4.2. Cell Stimulation MDA-MB-231 cells were plated in 12-well plates (Costar, Corning Incorporated, New York, NY, USA) at 1 × 106 cells/well concentration unless indicated otherwise. Cells were treated with vehicle or TNF-α (2 ng/mL; 210-TA, R&D Systems, Minneapolis, MN, USA) or lipopolysaccharide (LPS) (10ng/mL; L4391, Sigma Aldrich, Merck KGaA, Darmstadt, Germany) for 24 h at 37 ◦C. After incubation, cells were harvested for RNA isolation, and conditioned media were collected for measuring secreted GM-CSF. Then, cells were stimulated with TNF-α (2 ng/mL; 210-TA, R&D Systems, Minneapolis, MN, USA) or lipopolysaccharide (LPS) (10 ng/mL; L4391, Sigma Aldrich, Merck KGaA, Darmstadt, Germany) overnight at 37 ◦C unless otherwise speciﬁed. 4.6. Immunocytoﬂuorescence MDA-MB-231 cells (106/mL) were seeded on coverslips and cultured in 6-well plates at 37 ◦C. After incubation, the slides were ﬁxed in 4% formaldehyde and washed three times in cold PBS. Then, cells were permeabilized using in 0.1% Triton X-100, followed by three washes in cold PBS. The cells were blocked in 1% bovine serum albumin for 1 hr. The slides were incubated overnight at room temperature with a primary antibody of rabbit polyclonal anti GM-CSF antibody (GTX51383 Genetex, Irvine, CA, USA) in 1:200 dilution. Then, the cells were washed in PBS containing 0.05% Tween three times and again incubated with the secondary antibody conjugated with Alexa Fluor 647 or Alexa Fluor 488 (abcam® ab150079, abcam® ab150077) 1:200 dilution for 1 h. After washing the slide several times in PBS, the cells were counterstained and mounted with a coverslip using mountant containing DAPI (Vectashield, Vectorlab, H1500). The confocal images of the MDA-MB-231 cells were collected on an inverted Zeiss LSM710 AxioObsever microscope (Carl Zeiss, Gottingen, Germany) using Plan-Apochromat 40X/1.40 oil DIC M27 objective lens. Excitation was via a 647 nm HeNe solid-state laser and the 405 nm line of an argon ion laser. After laser excitation of the samples, optimized emission detection bandwidths were conﬁgured by Zeiss Zen 2010 control software; subsequently, the confocal images were captured, and ﬂuorescence was measured using Zeiss Zen 2012 software. 4.3. Real-Time Quantitative RT-PCR Total RNA was extracted from MDA-MB-231 cells using a RNeasy Mini Kit (Qiagen, Valencia CA, USA) per the manufacturer’s instructions [24]. The cDNA was synthesized using 1 µg of total 9 of 12 9 of 12 Molecules 2020, 25, 4709 RNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster city, CA, USA). Real-time PCR was performed on a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using TaqMan® Gene Expression Master Mix (Applied Biosystems). Each reaction contained 500 ng of cDNA that was ampliﬁed with Inventoried TaqMan Gene Expression Assay products (CSF2: Hs00929873; ACSL1: Hs00960561; GAPDH: Hs03929097_g1). The threshold cycle (Ct) values were normalized to the housekeeping gene GAPDH, and the amounts of target mRNA relative to control were calculated with the ∆∆Ct method [25,26]. Relative mRNA expression was expressed as fold expression over average of control gene expression. The expression level in control treatment was assumed to be 1. Values are presented as mean ± SEM. Results were analyzed statistically; p < 0.05 was considered signiﬁcant [27,28]. 4.4. Intracellular Staining and Flow Cytometry Human MDA-MB-231 cells were pretreated with ACSL1 inhibitor (Triacsin C: (1 µM) or vehicle for 1 h and then incubated with LPS for 15 min. The samples were ﬁxed and permeabilized with a BD Cytoﬁx/Cytoperm™ﬁxation/permeabilization kit (Cat no. 554714, BD Biosciences, San Jose, CA, USA) according to the manufacturer’s protocol at 4 ◦C for 15 min. Then, the cells were washed 2 times in cold phosphate-buﬀered saline (PBS), and the permeabilized cells were incubated with 5 µL of phosphorylated antibodies against p38 MAPK Alexa Fluor® 488 (Cat no. 612594), MEK1 PE (Cat no. 560099), ERK1/2 PE-Cy7 (Cat no. 560116), JNK Alexa Fluor® 647 (Cat no. 562481), IκBα Alexa Fluor® 647 (Cat no. 560817), and NFκB PE (Cat no. 558423) antibodies for 20 min in 4 ◦C. All antibodies were purchased from Biosciences, San Jose, CA, USA. The cells were washed twice in FACS buﬀer (PBS containing 5% FBS and 2mM EDTA) and resuspended in FACS buﬀer and analyzed using BD FACSDiva Software (V.8.0.2, BD Biosciences San Jose, CA, USA). 4.5. GM-CSF Determination Secreted GM-CSF protein in supernatants of MDA-MB-231 cells stimulated with TNF-α was quantiﬁed using sandwich ELISA following the manufacturer′s instructions (DY215-05, R&D systems, Minneapolis, MN, USA). 5. Statistical Analysis GraphPad Prism software (La Jolla, CA, USA) was used for statistical analysis. Data were presented as mean ± standard error of the mean (SEM). For comparison between means of the groups, an unpaired Student t-test and one-way ANOVA were used followed by Tukey′s test. p value < 0.05 was considered significant. Ns: no significance, * p < 0.05, p < 0.01, * p < 0.001 and **** p < 0.0001). Author Contributions: Conceptualization, F.A.-R., R.T. and R.A.; methodology, F.A.-R.; R.T.; A.A.-R.; formal analysis, F.A.-R.; R.T.; A.A.-R.; data curation, F.A.-R; R.T.; A.A.-R.; writing—original draft preparation, F.A.-R.; R.T.; A.A.-R., R.A.; writing—review and editing, R.A.; F.A.-M.; supervision, R.A.; funding acquisition, R.A. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, F.A.-R., R.T. and R.A.; methodology, F.A.-R.; R.T.; A.A.-R.; formal analysis, F.A.-R.; R.T.; A.A.-R.; data curation, F.A.-R; R.T.; A.A.-R.; writing—original draft preparation, F.A.-R.; R.T.; A.A.-R., R.A.; writing—review and editing, R.A.; F.A.-M.; supervision, R.A.; funding acquisition, R.A. All authors have read and agreed to the published version of the manuscript. Funding: This work was ﬁnancially supported by Kuwait Foundation for the Advancement of Sciences (KFAS) (Grants #: RA AH 2016-007). Funding: This work was ﬁnancially supported by Kuwait Foundation for the Advancement of Sciences (KFAS) (Grants #: RA AH 2016-007). Acknowledgments: BioRender.com was used for Figure 5. Conﬂicts of Interest: The authors declare there are no conﬂicts of interest involved. 4.7. Western Blotting MDA-MB-231cellsweretreatedwithLPSandincubatedfor30minwithlysisbuffer(10XLysisBuffer, Cell Signaling, Danvers, MA, USA). The protein lysates were prepared and resolved by 12% SDS-PAGE, 10 of 12 10 of 12 Molecules 2020, 25, 4709 as described earlier [26]. Cellular proteins were transferred to an Immuno-Blot polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA, USA) by electroblotting. Then, the membranes were blocked with 5% non-fat milk in PBS for 1 h, which was followed by incubation with primary antibodies against p-NF-κB (cat# 3033) and NF-κB (cat# 3034) in 1:1000 dilution at 4 ◦C overnight. All primary antibodies were purchased from Cell Signaling (Cell Signaling Technology Inc., Danvers, MA, USA). Then, the blots were washed three times with Tris-buffered saline with tween (TBS-T) and incubated for 2 h with HRP-conjugated secondary antibody (Promega, Madison, WI, USA). 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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional aﬃliations. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2108042993	https://zenodo.org/records/2406720/files/article.pdf	German	null	Oberflächenspannungen auf dem Ölgebiete II	Angewandte Chemie	1,920	public-domain	1,625	K o h 1 e n s t o f f. Die Wertigkeit des Kohlcnstoffs gegen Wasserstoff sowohl als auch gegen oxydierende Elenicnte ka.nn 1, 2, 3 und 4 bet,ragen. Nebenvalenzen koiinen auftreten. Die Summe von wirksamen Haupt- und Nebenvalenzen uberschreitet bei Wasserstoffverbin dungen nie und erreicht meistens die Zahl 4, die in diesen Vcrbin- dungen auch als Koordiiiationszahl gelten muB. Die Theorie cler organischen Verbindungen macht wedkr einen Unterschied zwischen (+)- und (-)-Hauptvalenzen, noch zwiechen Haupt- und Nebenvalenzen, noch zwischen der Koordiiiationszahl und der Summe der Valenzzahlen und stellt den Satz auf : Die Wertigkeit des Kohlen- stoffs betragt 4. g Mir varen die entgegenpehaltcnen Zahlen anniihernd aus der Arbeit v. D a l l w i t z - W e g e n e r bek-nnt; sie beziehen sich, wie aus jener Arbeit hervorgcht und durch die mir unterbreiteten Zitrte RUS <em Protokoll cies gmannten Autors best!tigt wird, nur auf die Schmierergiebigkcit des Oles, d. h. nur auf r'en aus den Kapillaritiitseig~~schaften b e r e c h n e t e n voraussichtlichen V e r b r a u c h des Oles im Betriebe, nicht aber suf die bekanntcn anderen Eigenschaften der Schn ierole (innere Reibung, Bestlndig- keit usw.), denen v. D a 11 w i t z - W e g e n e r neben seintn kapillari- metrischen Feststellungen der Schmierei giebigbcit eincn selb- standigen P1 tz in der Gesamtbenrteilung eines Schmierols einraumt, und auch nicht auf Betriebserfahrungen. Das apodiktische sum- niarische Urteil ,,Knochenol ist ein sehlechtes Schmierol" erschien mir daher nicht geniigend gestiitzt und widersprach auch allen uns Chemik rn seit Jahrzchnten gelaufigen Erfahrugen, r.ach denen bekanntlich gerade feines saurefrcies Knoch< nt 1 fur die subtileren Schmierzwecke, z. B. Uhren, Chronometer, Torpedos, Schreibmaechinen, Nahma=chinen usw. ein bewahrtes Schmierol ist. Nach den aus dem D a 1 I w i t z schen Protokoll gegebenen Erkliirun- gen ist aber der eusdruck ,,schlechtes Schmi, rol" selbst in bezug auf die Kapillareigenschaft,en und die Schmierergic bigkcit bereits sehr abr eschwacht, da hier nur noch von < inem ,,guten Mittelol" cie Rede ist. Weiterhin ist aber zu Ee enken, daB die geringere Schmierergiebigkeit des Oles, soweit Mitteilungen hieruber vorliegen, seii ens v. l) Vgl. Angew. Chem. 33, I, 267 [1920]. 2, Petroleum 16, 259ff. und Z85ff. [1920]. 3, p ist die sog. L e n a r dzahl und stellt die Oberfliichen- fliehkraft p = OL ' cos 0 dar, in der 0 den Rapdwinkel bedeutet, den ein Tropfen des Oles gegen die nietallene Unterlage bildet. 4, Siehe friiheres Zitat. B o r. Gegen Wasserst,off ist das Bor ein-, zwei- und dreiwertig. Die Nebenvalenzen ermoglichen weitgehende Aneinanderlagerung von Boranen, so daB die einfachen Borwasserstoffe BH, BH, und BH, fiir sich nicht bestancndig sind, sondcrn sich vereinigen zu Verbindunpen wie D i b o r a n BH,. BH,, T e t r a b o r a n BH,. BH,. BH,. BH, usw. Durchgangig bemerkt man die Koordinationszahl 4, wahrend v i e r w e r t i g e s Bor noch nicht gefunden worden ist. g g Grgen oxydierende Elemente wirkt das Bor nur dreiwertig. Das Bor(3)-oxyd wird durch Wasser leicht in M e t a b o r s a u r e verwandelt, die erst in der Gliihhitze Wasser abspaltet uncl Bortrioxyd zuriickbildet. Die Metaborsaure betatigt Neben- valt.11 zcn, indem sie Wasser anlagert und dabei 0 r t. h o b or s a u r c [ B':' :;I, 1 bildet, oder indem sie mit Bortrioxyd zu komplexen Borsaurcn zusammentritt. Das B o r f 1 u or i d , BE,, vereinigt sich mit Fluorwasserstoff zu Fluorborsaure B/O [ 801 [ B':' :;I, 1 bildet, oder indem sie mit Bortrioxyd zu komplexen B i D B f 1 i d BE i i i h mit Fluorwasserstoff zu Fluorborsaure K o h 1 e n s t o f f. D a 11 w i t z - W e g e n e r nur aus den Kapillai k nstanten berechnet, nicht aber wie in verschiedenen anderen Fallen, auch durch praktische Versuche bestatigt erscheii t, und daB der seitens D a 11 w i t z - W e g en e r ermittelte Wert von a bedeutend holier liegt, 91s die von G r u n m a c h und B e i n4) fi;.: oleinreiche, dem dem Knochenol physikaliscb gleichzustellende~ Ole (Oliveno1 und Mandclol) und von mir fur Olein und Knochenol g fundene Werte von a (3.33-3,40 und 3,18-3,29). Uberhaupt liegen a'le W e d fur OL axch bei anderen Schmierolen, die v. D a I 1 w i t z - W e g e n e r und nicin Mitarbeiter Dr. M e y e r h e i m nach der vcn erEterem benutzten Methode von P r o k t e r - H a J 1 ermittelt haben, .wesent- lich hoher als die von mir und den oten genarnten anderen Autoren festgestellten Werte. Schon aus diesem .Grunde niuseen natiirlich die Schlufifolgerungen aus xroglicherweiee zu hohen Werten mit entsprechendem Vorb halt aufgenommen werden. Auf diese Unterschiede wird an anderer Stelle naher einzugehen sein, g Die ungehemmte Entwicklung der organischen Cheniie seit der Aufstellung dieses Satzes beweist, daB dieser auf die iiberwiegende Mehrzahl der Kohlenstoffverbindungen anwendbar und wegen seiner Einfachheit als Grundlage einer Theorie brauchbar ist. Da aber fiir kein anderes Element ein Lhnlicher Satz formuliert werden kann, so muBte die Verallgemeinerung der Theroic der organischen Ver- bindungen scheitern. Die Lehre von den Nebenvalenzen ist aber allgemeiner Anwendung fahig. Leider inussen wir es uns versagen, an dieser Stelle die Kohlenstoffverbindungen zu analysieren und zu zeigen, daB man folgerichtig den Kohlenstoff in seincn Verbindungen null- bis vierwertig annehmen muB. Oberflachenspannungen auf dem Olgebiete 11, Von D. HOLDE. (Eingeg.~am %?./lo. 1920.) Gegen SchluB der van mir Fublizierten auszugsweieen Bearbeitung meines in Hannqyer gehakenen Vcrtrags uber ,,Obei flachenspan- nungen alif den1 Olgebiete" wies ich darauf hinl), daB v. D a 11 - w i t z - W e ge n e r in seiner letzten Publikation iiber Kapillaritats- priifungen bei SchmierClen2), das bichcr als eines der besten Schmier- ole geltende Knochenol als ein ,,s c h 1 e c h t e s S.c h m i e r o 1" bezeichnet hatte, ohne daB ersichtlich ware, auf welche Eigen- schaften sich dieses befrc mderde Urteil griinde. Von seiten cines Mitarbeit rs we: de ich darauf aufme7 ksam genacht, daB v. D a 11 - w i t z - W e g e n e r in der oben zitiertcn Arbeit seine Ansicht auf die von ihm erinittelte hohe Oberflachenspannung OL = 4,11 -4,15, die kapillare Steighohe 19,G7 bei C , 4 irm Spaltbreite und den daraus berechneten Raddwinkel 32" 50' griinde. In einem Laboratoriumsprotokoll sol1 nach diesen Feststellungen, die ic h in der gensnnten Art-eit annahernd bestatigt iand, v. D a 1 1 w i t z - W e g e n e r Kncchenol ,,beziiglich der Schmierergiebigkcit unter die g u t e n M i t t e l o l e (also nicht unter die s c h l e c h t e n S c h m i e r o 1 e ) einrangieren, es sei jedenfa Is kein ,,hervorragendes OP. ,,Fiir Knochenol berechne sich c ie scg. Repul zahl A = a-P3) zu 0,658, was wieder ein , , g u t e s Mitteliil" beziiglich der Schmierergiebigkeit ankiinS igt, wahrcnd Minc ralole gev ohnlich kleinere, d. h. bessera Wertc von -4 geben." Phosphor (5)- Meta- Pyro- Ortho- oxyd phosphorsaure phosphorsaure phosphorsaure Phosphor (5)- oxyd Phosphor (5)- oxyd Pyro- phosphorsaure Meta- phosphorsaure Meta- phosphorsaure Der Unterschied zwischen dem von Hauptbindungen gehaltenen, die beiden Phosphoratome vereinigenden Sauerstoff a t o m in dem Pentoxycl und deni durch Nebenbindungen gehaltenen Wasscr- m o 1 e k ti 1 in der Pyrophosphorsaure ist einleuchtend. 290 290 [ Zeitschrift fur aogewandte Chemie Holde: Oberflachenspannungen auf dem Olgebiete 11. P h o s p h o r . sein, denn Wasserstoff und oxydierende Elemente vereinigen sich zu indifferenten Verbindungen, wie Wasser oder Chlorwasserstoff, die nur in Nebenbindung angelagert werden. Die Wertigkeit eines Elementes in einer Verbindung wird nur durch Oxydatjon oder Reduktion, nicht durch Betatigung von Nebenbindungen ver- andert. [A. 203.J p Die Kebenvalenzen sind in den Wasserstoffverbindungen des Phosphors schwach ausgepragt, aber doch in den Phosphonium- salzen, die dcn Ammoniumsalzen entsprechen, und in der Bildung polymerer Formen er kennbar. leitet sich Die u n t c r p h o s p h o r i g e S a u r e PRO [ :OH2] I vom einwertigen, die p h o s p h o r i g e S a u r e P=O H, und [PFaH]H2 vom rlreiwertigen und die P h o s p h o r s u r e voni funfww tigen Phosphor ab. Die genetischen Beziehungen der Phos- phorsauren zueinander geht aus folgenden Fornieln hervor : [ ] [ ::I l) Vgl. Angew. Chem. 33, I, 267 [1920]. Z 11 s a in ni e n f a s s u n g. Aus den angcfuhrten Beispielen, deren Zahl noch beliebig ver- mehrt werden konnte, geht hervor, daB wir die Wert,ipkeit eines Elenientes in einer Verbindung eindeutig ermitteln konnen, wenn wir die eben dargelegten Voraussetzungen machen, die wir hier kurz wiederholen werden. g [A. ZOOS.] Unter Wertigkeit eines Elementes verstehen wir nicht allgemcin die Hochstzahl der Hauptbindungen, die es betatigen kann, sondern die Zahl der Hauptveibindungen, die in einer bestimmten Ver- bindung tatsachlich wirksam sind. Wir unterscheiden die Wertigktit gegen Waeserstoff, oder ( +)-Wertigkeit, von der Wertigkeit gegen oxydierende Elemerite, oder (-)-Wert,igkeit. ( +)- und (-)-Haupt- bindungen konnen nicht gleichzeitig an einem Elemente wirksani
https://openalex.org/W3128378071	https://www.research.ed.ac.uk/files/202049588/20210326_Brechin_molecules_26_00757.pdf	English	null	[CrIII8NiII6]n+ Heterometallic Coordination Cubes	Molecules/Molecules online/Molecules annual	2,021	cc-by	8,550	General rights C i h f h General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. [CrIII 8NiII 6]n+ Heterometallic Coordination Cubes Citation for published version: O'Connor, HM, Sanz, S, Scott, AJ, Pitak, MB, Klooster, WT, Coles, SJ, Chilton, NF, McInnes, EJL, Lusby, PJ, Weihe, H, Piligkos, S & Brechin, EK 2021, '[CrIII 8NiII 6]n+ Heterometallic Coordination Cubes', Molecules (Basel, Switzerland), vol. 26, no. 3, 757. https://doi.org/10.3390/molecules26030757 Citation for published version: O'Connor, HM, Sanz, S, Scott, AJ, Pitak, MB, Klooster, WT, Coles, SJ, Chilton, NF, McInnes, EJL, Lusby, PJ, Weihe, H, Piligkos, S & Brechin, EK 2021, '[CrIII 8NiII 6]n+ Heterometallic Coordination Cubes', Molecules (Basel, Switzerland), vol. 26, no. 3, 757. https://doi.org/10.3390/molecules26030757 Published In: Molecules (Basel, Switzerland) Edinburgh Research Explorer Citation: O’Connor, H.M.; Sanz, S.; Scott, A.J.; Pitak, M.B.; Klooster, W.T.; Coles, S.J.; Chilton, N.F.; McInnes, E.J.L.; Lusby, P.J.; Weihe, H.; et al. [CrIII8NiII6]n+ Heterometallic Coordination Cubes. Molecules 2021, 26, 757. https://doi.org/10.3390/ molecules26030757 Citation: O’Connor, H.M.; Sanz, S.; Scott, A.J.; Pitak, M.B.; Klooster, W.T.; Coles, S.J.; Chilton, N.F.; McInnes, E.J.L.; Lusby, P.J.; Weihe, H.; et al. [CrIII8NiII6]n+ Heterometallic Coordination Cubes. Molecules 2021, 26, 757. https://doi.org/10.3390/ molecules26030757 Keywords: molecular magnetism; supramolecular chemistry; heterometallic clusters; magnetometry; EPR spectroscopy Academic Editors: William T. A. Harrison, R. Alan Aitken and Paul Waddell Received: 19 January 2021 Accepted: 30 January 2021 Published: 2 February 2021 molecules molecules Article II Article [CrIII8NiII6]n+ Heterometallic Coordination Cubes Alan Aitken and Paul Waddell Received: 19 January 2021 Accepted: 30 January 2021 Published: 2 February 2021 Take down policy Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 molecules Article [CrIII8NiII6]n+ Heterometallic Coordination Cubes Helen M. O’Connor 1 , Sergio Sanz 1 , Aaron J. Scott 1, Mateusz B. Pitak 2, Wim T. Klooster 2, Simon J. Coles 2 , Nicholas F. Chilton 3, Eric J. L. McInnes 3, Paul J. Lusby 1, Høgni Weihe 4, Stergios Piligkos 4,* and Euan K. Brechin 1,* 1 EaStCHEM School of Chemistry, The University of Edinburgh, David Brewster Road, Edinburgh EH3 5JF, UK; oconnoh7@tcd.ie (H.M.O.); s.calvo@fz-juelich.de (S.S.); Aaron.Scott@ed.ac.uk (A.J.S.); Paul.Lusby@ed.ac.uk (P.J.L.) 1 EaStCHEM School of Chemistry, The University of Edinburgh, David Brewster Road, Edinburgh EH3 5JF, UK; oconnoh7@tcd.ie (H.M.O.); s.calvo@fz-juelich.de (S.S.); Aaron.Scott@ed.ac.uk (A.J.S.); 2 UK National Crystallography Service, Chemistry, Highﬁeld Campus, University of Southampton, Southampton SO17 1BJ, UK; Mateusz.Pitak@matthey.com (M.B.P.); W.T.Klooster@soton.ac.uk (W.T.K.); S.J.Coles@soton.ac.uk (S.J.C.) 2 UK National Crystallography Service, Chemistry, Highﬁeld Campus, University of Southampton, Southampton SO17 1BJ, UK; Mateusz.Pitak@matthey.com (M.B.P.); W.T.Klooster@soton.ac.uk (W.T.K.); S.J.Coles@soton.ac.uk (S.J.C.) 3 Department of Chemistry, The University of Manchester, Oxford Road, Manchester M13 9PL, UK; nicholas.chilton@manchester.ac.uk (N.F.C.); eric.mcinnes@manchester.ac.uk (E.J.L.M.) 4 Department of Chemistry, University of Copenhagen, Universitetsparken 5, Copenhagen DK-2100, Denmark; weihe@chem.ku.dk * Correspondence: piligkos@kiku.dk (S.P.); ebrechin@ed.ac.uk (E.K.B.) Abstract: Three new heterometallic [CrIII8NiII6] coordination cubes of formulae [CrIII8NiII6L24(H2O)12] (NO3)12 (1), [CrIII8NiII6L24(MeCN)7(H2O)5](ClO4)12 (2), and [CrIII8NiII6L24Cl12] (3) (where HL = 1-(4-pyridyl)butane-1,3-dione), were synthesised using the paramagnetic metalloligand [CrIIIL3] and the corresponding NiII salt. The magnetic skeleton of each capsule describes a face-centred cube in which the eight CrIII and six NiII ions occupy the eight vertices and six faces of the structure, respectively. Direct current magnetic susceptibility measurements on (1) reveal weak ferromagnetic interactions between the CrIII and NiII ions, with JCr-Ni = + 0.045 cm−1. EPR spectra are consistent with weak exchange, being dominated by the zero-ﬁeld splitting of the CrIII ions. Excluding wheel- like structures, examples of large heterometallic clusters containing both CrIII and NiII ions are rather rare, and we demonstrate that the use of metalloligands with predictable bonding modes allows for a modular approach to building families of related polymetallic complexes. Compounds (1)–(3) join the previously published, structurally related family of [MIII8MII6] cubes, where MIII = Cr, Fe and MII = Cu, Co, Mn, Pd. Citation: O’Connor, H.M.; Sanz, S.; Scott, A.J.; Pitak, M.B.; Klooster, W.T.; Coles, S.J.; Chilton, N.F.; McInnes, E.J.L.; Lusby, P.J.; Weihe, H.; et al. [CrIII8NiII6]n+ Heterometallic Coordination Cubes. Molecules 2021, 26, 757. https://doi.org/10.3390/ molecules26030757 Academic Editors: William T. A. Harrison, R. 1. Introduction Heterometallic coordination complexes have seen application in areas as diverse as metalloprotein chemistry [1,2], catalysis [3], porous materials [4,5], and magnetism [6]. The latter includes three-dimensional (3D) networks [7], two-dimensional (2D) sheets [8], one-dimensional (1D) chains [9], and zero-dimensional (0D) (molecular) polygons and polyhedra [10,11], investigating controllable exchange interactions [12], enhanced mag- netocaloric effects [13], spin frustration [14], slow relaxation of the magnetisation [15,16], and quantum coherence timescales [17]. A search of the Cambridge Structural Database (CSD) reveals that heterometallic wheels of varying size and metal ratios dominate the chemistry of polymetallic clusters containing both CrIII and NiII ions with a nuclearity of four or more. Examples include [Cr7Ni], [Cr9Ni], [Cr8Ni2], [Cr7Ni2], [Cr6Ni2], [Cr2Ni5], [CrNi6] wheels and discs (centred/Anderson wheels), and [Cr14Ni2] and [Cr28Ni4] ‘linked rings’ [18–23]. Surprisingly, the search reveals only two other unique structural motifs, a rather unusual [Cr3Ni2] linear complex [24], and an ‘S-shaped’ [Cr12Ni3] chain [25]. We have previously reported a metalloligand approach that enabled us to synthesise high- nuclearity heterometallic coordination capsules of paramagnetic transition metal ions in a 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. 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/molecules Molecules 2021, 26, 757. https://doi.org/10.3390/molecules26030757 Molecules 2021, 26, 757 2 of 9 ne-1,3 modular and predictable fashion [26–29]. This metalloligand, based on the tritopic [MIIIL3] moiety shown in Figure 1 (HL = 1-(4-pyridyl)butane-1,3-dione), features a tris(acac) co- ordinated octahedral transition metal ion, in which the ligand is functionalised with a p-pyridyl donor group. In the fac-isomer of this metalloligand, the three N-donor groups are orientated in such a way that combination with a square-planar metal ion leads to the en- tropically favoured self-assembly of a cubic structure [30]. Herein, we report the syntheses, structures, and magnetic properties of three novel tetradecanuclear [CrIII8NiII6]n+ cubes, namely [CrIII8NiII6L24(H2O)12](NO3)12 (1), [CrIII8NiII6L24(MeCN)7(H2O)5](ClO4)12 (2), and [CrIII8NiII6L24Cl12] (3), which join the growing family of [MIII8MII6] cubes constructed from [MIIIL3] and a variety of MII salts (MIII = Cr, Fe; MII = Cu, Co, Mn, Pd) [26–29]. ised with a p pyridyl donor group. 2. Material 2.1. Synthesis 2. Materials and Methods 2.1. Synthesis 1-(4-pyridyl)butane-1,3-dione (HL) and the metalloligand [CrIIIL3 i l bli h d d [26 31 32] All ti i d 1-(4-pyridyl)butane-1,3-dione (HL) and the metalloligand [CrIIIL3] were prepared by previously published procedures [26,31,32]. All reactions were carried out under aerobic conditions. Solvents and reagents were used as received from commercial suppliers. Cau- tion: perchlorate salts of metal complexes with organic ligands are potentially explosive. previously published procedures [26,31,32]. All reactions were carrie conditions. Solvents and reagents were used as received from commer tion: perchlorate salts of metal complexes with organic ligands are po Synthesis of [CrIII8NiII6L24(H2O)12](NO3)12 (1). To a solution of mmol) in 10 mL of dichloromethane, a solution of Ni(NO3)2∙6H2O (30 Synthesis of [CrIII8NiII6L24(H2O)12](NO3)12 (1). To a solution of [CrIIIL3] (54 mg, 0.1 mmol) in 10 mL of dichloromethane, a solution of Ni(NO3)2·6H2O (30 mg, 0.1 mmol) was added in 10 mL of methanol. The solution was stirred for 18 h before being ﬁltered and allowed to stand. Dark orange X-ray quality crystals were obtained from the diffusion of diethyl ether into the mother liquor. Yield of (1) = 69%. Elemental analysis (%) calculated (found): C 46.16 (46.01) H 3.87 (3.78) N 8.97 (8.63). o ) i 0 o i o o e a e, a o u io o Ni(NO ) O ( 0 added in 10 mL of methanol. The solution was stirred for 18 h befor allowed to stand. Dark orange X-ray quality crystals were obtained fr diethyl ether into the mother liquor. Yield of (1) = 69%. Elemental ana (found): C 46.16 (46.01) H 3.87 (3.78) N 8.97 (8.63). S th i f [C III NiII L (M CN) (H O) ](ClO ) (2) T l t ( ) ( ) ( ) ( ) Synthesis of [CrIII8NiII6L24(MeCN)7(H2O)5](ClO4)12 (2). To a solution of [CrIIIL3] (108 mg, 0.2 mmol) in 10 mL of dichloromethane, a solution of Ni(ClO4)2·6H2O (73 mg, 0.2 mmol) was added in 10 mL of acetonitrile. The solution was stirred for 18 h before being ﬁltered and allowed to stand. Brown X-ray quality crystals were obtained after 5 days from the diffusion of pentane into the mother liquor. Yield of (2) = 81%. Elemental analysis (%) calculated (found): C 44.34 (44.06) H 3.61 (3.59) N 6.97 (7.11). Synthesis of [CrIII8NiII6L24(MeCN)7(H2O)5](ClO4)12 (2). 1. Introduction In the fac isomer of this metallol donor groups are orientated in such a way that combination with a s ion leads to the entropically favoured self-assembly of a cubic structu report the syntheses, structures, and magnetic properties of three nov [CrIII8NiII6]n+ cubes, namely [CrIII8NiII6L24(H2O)12 [CrIII8NiII6L24(MeCN)7(H2O)5](ClO4)12 (2), and [CrIII8NiII6L24Cl12] (3), whi family of [MIII8MII6] cubes constructed from [MIIIL3] and a variety of M MII = Cu, Co, Mn, Pd) [26–29]. Figure 1. Molecular structure of [MIIIL3], where HL = 1-(4-pyridyl)butane-1,3- MIII = green, O = red, N = blue, C = grey. H-atoms have been omitted for clarit Figure 1. Molecular structure of [MIIIL3], where HL = 1-(4-pyridyl)butane-1,3-dione. Colour code: MIII = green, O = red, N = blue, C = grey. H-atoms have been omitted for clarity. Figure 1. Molecular structure of [MIIIL3], where HL = 1-(4-pyridyl)butane-1,3-d MIII = green, O = red, N = blue, C = grey. H-atoms have been omitted for clarity Figure 1. Molecular structure of [MIIIL3], where HL = 1-(4-pyridyl)butane-1,3-dione. Colour code: MIII = green, O = red, N = blue, C = grey. H-atoms have been omitted for clarity. Figure 1. Molecular structure of [MIIIL3], where HL = 1-(4-pyridyl)butane-1,3- MIII = green O = red N = blue C = grey H-atoms have been omitted for clarit Figure 1. Molecular structure of [MIIIL3], where HL = 1-(4-pyridyl)butane-1,3-dione. Colour code: MIII = green, O = red, N = blue, C = grey. H-atoms have been omitted for clarity. 2.2. Crystallographic Details ( ) ( ) Crystal Data for [CrIII8NiII6L24Cl12] (3). C216H192Cl12Cr8N24Ni6O48, Mr = 5085.58, triclinic, a = 28.171(16) Å, b = 30.225(16) Å, c = 32.40(2) Å, α = 72.27(6)◦, β = 72.08(6)◦, γ = 64.04(6)◦, V = 22,417(27) Å3, Z = 2, P-1, Dc = 0.753 g cm−3, µ = 0.543 mm−1, T = 100.0(1) K, 134,344 reﬂections measured, 66,105 unique (Rint = 0.1446), which were used in all calcula- tions, wR2 (all data) = 0.5544, and R1 [I > 2(I)] = 0.1938. CCDC 1977312. 2. Material 2.1. Synthesis To a solut mg, 0.2 mmol) in 10 mL of dichloromethane, a solution of Ni(ClO4 mmol) was added in 10 mL of acetonitrile. The solution was stirred fo filtered and allowed to stand. Brown X-ray quality crystals were ob from the diffusion of pentane into the mother liquor. Yield of (2) = 81% ( ) ( ) ( ) ( ) Synthesis of [CrIII8NiII6L24Cl12] (3). To a solution of [CrIIIL3] (108 mg, 0.2 mmol) in 10 mL of dichloromethane, a solution of NiCl2 (20 mg, 0.15 mmol) was added in 10 mL of tetrahydrofuran. The solution was stirred for 18 h before being ﬁltered and allowed to stand. Brown X-ray quality crystals were obtained after room temperature evaporation of the mother liquor for 5 days. Yield of (3) = 58%. Elemental analysis (%) calculated (found): C 51.01 (50.79) H 3.81 (3.71) N 6.61 (6.68). Molecules 2021, 26, 757 3 of 9 2.2. Crystallographic Details C216H216Cr8N24Ni6O60, Mr = 4876.38, monoclinic, a = 25.754(3) Å, b = 41.336(5) Å, c = 43.217(5) Å, α = 90◦, β = 90.6450(10)◦, γ = 90◦, V = 46,004(9) Å3, Z = 4, P21/n, Dc = 0.704 g cm−3, µ = 9.18 mm−1, T = 100.15(10) K, 370,995 reﬂections measured, 81,102 unique (Rint = 0.1902), which were used in all calculations, wR2 (all data) = 0.3687, and R1 [I > 2(I)]= 0.1242. CCDC 1977309. p Crystal Data for [CrIII8NiII6L24(H2O)12](NO3)12 (1). C216H216Cr8N24Ni6O60, Mr = 4876.38, monoclinic, a = 25.754(3) Å, b = 41.336(5) Å, c = 43.217(5) Å, α = 90◦, β = 90.6450(10)◦, γ = 90◦, V = 46,004(9) Å3, Z = 4, P21/n, Dc = 0.704 g cm−3, µ = 9.18 mm−1, T = 100.15(10) K, 370,995 reﬂections measured, 81,102 unique (Rint = 0.1902), which were used in all calculations, wR2 (all data) = 0.3687, and R1 [I > 2(I)]= 0.1242. CCDC 1977309. Crystal Data for [CrIII8NiII6L24(MeCN)7(H2O)5](ClO4)12 (2). C230H218Cr8N31Ni6O54, Mr = 5048.60, monoclinic, a = 25.788(6) Å, b = 41.606(9) Å, c = 45.869(11) Å, α = 90◦, β = 90.785(2)◦, γ = 90◦, V = 49,210(20) Å3, Z = 4, P21/n, Dc = 0.681 g cm−3, µ = 0.412 mm−1, T = 100.15(10) K, 391,278 reﬂections measured, 85,150 unique (Rint = 0.2371), which were used in all calculations, wR2 (all data) = 0.4444, and R1 [I > 2(I)] = 0.1521. CCDC 1977311. Crystal Data for [CrIII8NiII6L24Cl12] (3). C216H192Cl12Cr8N24Ni6O48, Mr = 5085.58, triclinic, a = 28.171(16) Å, b = 30.225(16) Å, c = 32.40(2) Å, α = 72.27(6)◦, β = 72.08(6)◦, γ = 64.04(6)◦, V = 22,417(27) Å3, Z = 2, P-1, Dc = 0.753 g cm−3, µ = 0.543 mm−1, T = 100.0(1) K, 134,344 reﬂections measured, 66,105 unique (Rint = 0.1446), which were used in all calcula- tions, wR2 (all data) = 0.5544, and R1 [I > 2(I)] = 0.1938. CCDC 1977312. Crystal Data for [CrIII8NiII6L24(MeCN)7(H2O)5](ClO4)12 (2). C230H218Cr8N31Ni6O54, Mr = 5048.60, monoclinic, a = 25.788(6) Å, b = 41.606(9) Å, c = 45.869(11) Å, α = 90◦, β = 90.785(2)◦, γ = 90◦, V = 49,210(20) Å3, Z = 4, P21/n, Dc = 0.681 g cm−3, µ = 0.412 mm−1, T = 100.15(10) K, 391,278 reﬂections measured, 85,150 unique (Rint = 0.2371), which were used in all calculations, wR2 (all data) = 0.4444, and R1 [I > 2(I)] = 0.1521. CCDC 1977311. 2.2. Crystallographic Details 2.2. Crystallographic Details Single-crystal X-ray diffraction data were collected for (1)–(3) at T = 100 K on a Rigaku AFC12 goniometer equipped with an enhanced sensitivity (HG) Saturn 724+ detector mounted at the window of an FR-E+ Superbright MoKα rotating anode generator with HF Varimax optics (70 µm focus) [33]. The CrysalisPro software package was used for instru- ment control, unit cell determination, and data reduction [34]. Due to very weak scattering power, single-crystal X-ray diffraction data for (1) and (2) were collected at T = 30.15 K us- ing a synchrotron source (λ = 0.6889 Å) on the I19 beam line at Diamond Light Source on an undulator insertion device with a combination of double crystal monochromator, vertical and horizontal focussing mirrors, and a series of beam slits. The same software as above was used for data reﬁnement. Crystals of all samples were sensitive to solvent loss, which resulted in crystal delamination and poor-quality X-ray diffraction data. To slow down crystal degradation, crystals of (1)–(3) were “cold-mounted” on MiTeGen MicromountsTM at T = 203 K using Sigma-Aldrich Fomblin Y® LVAC (3300 mol. wt.), with the X-Temp 2 crystal cooling system attached to the microscope [35]. This procedure protected crystal quality and permitted collection of usable X-ray data. Unit cell parameters in all cases were reﬁned against all data. Crystal structures were solved using Intristic Phasing as implemented in SHELXT [36]. All non-hydrogen atoms were reﬁned with anisotropic displacement parameters, and all hydrogen atoms were added at calculated positions and reﬁned using a riding model with isotropic displacement parameters based on the equiva- lent isotropic displacement parameter (Ueq) of the parent atom. All three crystal structures contain large accessible voids and channels that are ﬁlled with diffuse electron density belonging to uncoordinated solvent, whose electron contribution was accounted for by the PLATON/SQUEEZE routine ((1) and (2)) [37], or by the SMTBX solvent masking routine, as implemented in OLEX2 software (3). To maintain reasonable molecular geometry, DFIX restraints were used in all three complexes. p Crystal Data for [CrIII8NiII6L24(H2O)12](NO3)12 (1). 2.3. Magnetic and Spectroscopic Measurements Direct current (dc) susceptibility and magnetisation data were measured on powdered, polycrystalline samples of (1) using a Quantum Design SQUID MPMS-XL magnetometer, operating between 1.8 and 300 K for dc applied magnetic ﬁelds ranging from 0 to 5 T. X-band EPR spectra were collected on powdered microcrystalline samples of (1) using a Bruker EMX spectrometer at the EPSRC UK National EPR Facility at The University of Manchester. 3.1. Structural Description H-atoms have been omitted for clarity. ure 2. From left to right, molecular structures of (1), (2), and (3). Colour code: CrIII = green, NiII = yellow, O = red ue, Cl = light green, C = grey. H-atoms have been omitted for clarity. ue, Cl = light green, C = grey. H-atoms have been omitted for clarity. The CrIII ions are all octahedral, possessing {CrO6} coordination spheres with CrII The CrIII ions are all octahedral, possessing {CrO6} coordination spheres with CrIII-O distances between 1.9 and 2.2 Å, and cis/trans angles in the range 82.7–97˚/171.3–179.4˚, respectively. The equatorial positions of the octahedral NiII ions are occupied by four pyridyl donors from four distinct [CrIIIL3] subunits, with NiII–N distances in the range 1.9– 3.0 Å. For (1) and (2), the axial positions are occupied by twelve water and twelve acetoni- trile/water molecules (NiII-O ≈2.1 Å; NiII-N ≈2.1 Å), respectively. The cubes are therefore cationic (12+). The charge balancing nitrate or perchlorate anions for (1) and (2) respec- tively, are located both within the central cavity of the cube and in the void spaces between cubes. In contrast to (1) and (2), complex (3) is neutral, with the axial positions of the NiII ions occupied by chloride anions (NiII-Cl ≈2 7 Å) The CrIII ions are all octahedral, possessing {CrO6} coordination spheres with CrIII-O distances between 1.9 and 2.2 Å, and cis/trans angles in the range 82.7–97 ions are all octahedral, possessing {CrO6} coordination spheres with CrIII-O ween 1.9 and 2.2 Å, and cis/trans angles in the range 82.7–97˚/171.3–179.4˚, The equatorial positions of the octahedral NiII ions are occupied by four rs from four distinct [CrIIIL3] subunits, with NiII–N distances in the range 1.9– and (2), the axial positions are occupied by twelve water and twelve acetoni- olecules (NiII-O ≈2.1 Å; NiII-N ≈2.1 Å), respectively. The cubes are therefore . The charge balancing nitrate or perchlorate anions for (1) and (2) respec- ated both within the central cavity of the cube and in the void spaces between trast to (1) and (2), complex (3) is neutral, with the axial positions of the NiII d by chloride anions (NiII-Cl ≈2.7 Å). /171.3–179.4 The CrIII ions are all octahedral, possessing {CrO6} coordination spheres with CrIII-O distances between 1.9 and 2.2 Å, and cis/trans angles in the range 82.7–97˚/171.3–179.4˚, respectively. 3.1. Structural Description The equatorial positions of the octahedral NiII ions are occupied by four pyridyl donors from four distinct [CrIIIL3] subunits, with NiII–N distances in the range 1.9– 3.0 Å. For (1) and (2), the axial positions are occupied by twelve water and twelve acetoni- trile/water molecules (NiII-O ≈2.1 Å; NiII-N ≈2.1 Å), respectively. The cubes are therefore cationic (12+). The charge balancing nitrate or perchlorate anions for (1) and (2) respec- tively, are located both within the central cavity of the cube and in the void spaces between cubes. In contrast to (1) and (2), complex (3) is neutral, with the axial positions of the NiII ions occupied by chloride anions (NiII-Cl ≈2.7 Å). , respectively. The equatorial positions of the octahedral NiII ions are occupied by four pyridyl donors from four distinct [CrIIIL3] subunits, with NiII–N distances in the range 1.9–3.0 Å. For (1) and (2), the axial positions are occupied by twelve water and twelve acetonitrile/water molecules (NiII-O ≈2.1 Å; NiII-N ≈2.1 Å), respectively. The cubes are therefore cationic (12+). The charge balancing nitrate or perchlorate anions for (1) and (2) respectively, are located both within the central cavity of the cube and in the void spaces between cubes. In contrast to (1) and (2), complex (3) is neutral, with the axial positions of the NiII ions occupied by chloride anions (NiII-Cl ≈2.7 Å). distances between 1.9 and 2.2 Å, and cis/trans angles in the range 82.7–97˚/171.3–179.4˚, respectively. The equatorial positions of the octahedral NiII ions are occupied by four pyridyl donors from four distinct [CrIIIL3] subunits, with NiII–N distances in the range 1.9– 3.0 Å. For (1) and (2), the axial positions are occupied by twelve water and twelve acetoni- trile/water molecules (NiII-O ≈2.1 Å; NiII-N ≈2.1 Å), respectively. The cubes are therefore cationic (12+). The charge balancing nitrate or perchlorate anions for (1) and (2) respec- tively, are located both within the central cavity of the cube and in the void spaces between cubes. In contrast to (1) and (2), complex (3) is neutral, with the axial positions of the NiII ions occupied by chloride anions (NiII-Cl ≈2.7 Å). There are several close intermolecular contacts (Figure 3) between the cages in the ions occupied by chloride anions (Ni Cl ≈2.7 Å). There are several close intermolecular contacts (Figure 3) between the cages in the extended structures of (1)–(3). 3.1. Structural Description In (1), the closest inter-cluster contact is between the aro- matic protons of the pyridyl group and the O-atom (2.3 Å) of a neighbouring L- ligand. In (2) and (3), the closest contact is between the protons of the metalloligand methyl group, and the O-atom of a neighbouring L− ligand (2.3 Å) and the protons of a neighbouring methyl group (2.3 Å), respectively. Several other close inter-cluster contacts between neighbouring cubes exist, for example: Ar-H∙∙∙O ≈2.5 Å and C-H∙∙∙O ≈2.7 Å for (1), H2C- H∙∙∙O ≈2 5 Å and H2O∙∙∙H-CH2 ≈2 7 Å for (2) and Ar-H∙∙∙Cl ≈2 7 Å and C-H∙∙∙Cl ≈2 8 Å for d by chloride anions (Ni Cl 2.7 Å). e several close intermolecular contacts (Figure 3) between the cages in the uctures of (1)–(3). In (1), the closest inter-cluster contact is between the aro- s of the pyridyl group and the O-atom (2.3 Å) of a neighbouring L- ligand. In he closest contact is between the protons of the metalloligand methyl group, om of a neighbouring L- ligand (2.3 Å) and the protons of a neighbouring p (2.3 Å), respectively. Several other close inter-cluster contacts between g cubes exist, for example: Ar-H∙∙∙O ≈2.5 Å and C-H∙∙∙O ≈2.7 Å for (1), H2C- and H2O···H-CH2 ≈2.7 Å for (2), and Ar-H∙∙∙Cl ≈2.7 Å and C-H∙∙∙Cl ≈2.8 Å for ions occupied by chloride anions (Ni Cl 2.7 Å). There are several close intermolecular contacts (Figure 3) between the cages in the extended structures of (1)–(3). In (1), the closest inter-cluster contact is between the aro- matic protons of the pyridyl group and the O-atom (2.3 Å) of a neighbouring L- ligand. In (2) and (3), the closest contact is between the protons of the metalloligand methyl group, and the O-atom of a neighbouring L- ligand (2.3 Å) and the protons of a neighbouring methyl group (2.3 Å), respectively. Several other close inter-cluster contacts between neighbouring cubes exist, for example: Ar-H∙∙∙O ≈2.5 Å and C-H∙∙∙O ≈2.7 Å for (1), H2C- H∙∙∙O ≈2.5 Å and H2O···H-CH2 ≈2.7 Å for (2), and Ar-H∙∙∙Cl ≈2.7 Å and C-H∙∙∙Cl ≈2.8 Å for p y There are several close intermolecular contacts (Figure 3) between the cages in the extended structures of (1)–(3). In (1), the closest inter-cluster contact is between the aromatic protons of the pyridyl group and the O-atom (2.3 Å) of a neighbouring L−ligand. 3.1. Structural Description The inter- ume of the cube is approximately 1400 Å3. REVIEW 4 of 8 like metallic skeleton, with the eight CrIII ions located at the corners and the six NiII ions located on the faces, approximately 1.4–2.3 Å above the Cr⋯Cr⋯Cr⋯Cr plane. The inter- nal cavity volume of the cube is approximately 1400 Å3. like metallic skeleton, with the eight CrIII ions located at the corners and the six NiII ions located on the faces, approximately 1.4–2.3 Å above the Cr⋯Cr⋯Cr⋯Cr plane. The inter- nal cavity volume of the cube is approximately 1400 Å3. ure 2. From left to right, molecular structures of (1), (2), and (3). Colour code: CrIII = green, NiII = yellow, O = red, lue, Cl = light green, C = grey. H-atoms have been omitted for clarity. Figure 2. From left to right, molecular structures of (1), (2), and (3). Colour code: CrIII = green, NiII = yellow, O = red, N = blue, Cl = light green, C = grey. H-atoms have been omitted for clarity. ft to right, molecular structures of (1), (2), and (3). Colour code: CrIII = green, NiII = yellow, O = red, N green, C = grey. H-atoms have been omitted for clarity. ure 2. From left to right, molecular structures of (1), (2), and (3). Colour code: CrIII = green, NiII = yellow, O = red, ue, Cl = light green, C = grey. H-atoms have been omitted for clarity. ure 2. From left to right, molecular structures of (1), (2), and (3). Colour code: CrIII = green, NiII = yellow, O = red, ue, Cl = light green, C = grey. H-atoms have been omitted for clarity. The CrIII ions are all octahedral, possessing {CrO6} coordination spheres with CrIII- ure 2. From left to right, molecular structures of (1), (2), and (3). Colour code: CrIII = green, NiII = yellow, O = red lue, Cl = light green, C = grey. H-atoms have been omitted for clarity. Figure 2. From left to right, molecular structures of (1), (2), and (3). Colour code: CrIII = green, NiII = yellow, O = red, N = blue, Cl = light green, C = grey. H-atoms have been omitted for clarity. ft to right, molecular structures of (1), (2), and (3). Colour code: CrIII = green, NiII = yellow, O = red, N green, C = grey. 3.1. Structural Description The heterometallic cubes [CrIII8NiII6L24(H2O)12](NO3)12 (1), [CrIII8NiII6L24(MeCN)7 (H2O)5](ClO4)12 (2), and [CrIII8NiII6L24Cl12] (3) were formed from the reaction of [CrIIIL3] Molecules 2021, 26, 757 4 of 9 with the corresponding NiII salt in CH2Cl2/MeOH, CH2Cl2/MeCN, and CH2Cl2/THF, respectively. All three structures (Figure 2) reveal a similar [CrIII8NiII6] cube-like metallic skeleton, with the eight CrIII ions located at the corners and the six NiII ions located on the faces, approximately 1.4–2.3 Å above the Cr· · · Cr· · · Cr· · · Cr plane. The internal cavity volume of the cube is approximately 1400 Å3. R REVIEW 4 of 8 like metallic skeleton, with the eight CrIII ions located at the corners and the six NiII ions located on the faces, approximately 1.4–2.3 Å above the Cr⋯Cr⋯Cr⋯Cr plane. The inter- nal cavity volume of the cube is approximately 1400 Å3. 4 of 8 skeleton, with the eight CrIII ions located at the corners and the six NiII ions he faces, approximately 1.4–2.3 Å above the Cr⋯Cr⋯Cr⋯Cr plane. The inter- lume of the cube is approximately 1400 Å3. R REVIEW 4 of 8 like metallic skeleton, with the eight CrIII ions located at the corners and the six NiII ions located on the faces, approximately 1.4–2.3 Å above the Cr⋯Cr⋯Cr⋯Cr plane. The inter- nal cavity volume of the cube is approximately 1400 Å3. like metallic skeleton, with the eight CrIII ions located at the corners and the six NiII ions located on the faces, approximately 1.4–2.3 Å above the Cr⋯Cr⋯Cr⋯Cr plane. The inter- nal cavity volume of the cube is approximately 1400 Å3. with the corresponding NiII salt in CH2Cl2/MeOH, CH2Cl2/MeCN, and CH2Cl2/THF, respectively. All three structures (Figure 2) reveal a similar [CrIII8NiII6] cube-like metallic skeleton, with the eight CrIII ions located at the corners and the six NiII ions located on the faces, approximately 1.4–2.3 Å above the Cr· · · Cr· · · Cr· · · Cr plane. The internal cavity volume of the cube is approximately 1400 Å3. R REVIEW 4 of 8 like metallic skeleton, with the eight CrIII ions located at the corners and the six NiII ions located on the faces, approximately 1.4–2.3 Å above the Cr⋯Cr⋯Cr⋯Cr plane. The inter- nal cavity volume of the cube is approximately 1400 Å3. 4 of 8 skeleton, with the eight CrIII ions located at the corners and the six NiII ions e faces, approximately 1.4–2.3 Å above the Cr⋯Cr⋯Cr⋯Cr plane. 3.2. Magnetic Properties susceptibility, χM field B of 0 1 T Plot of χMT (open circles) versus T for complex (1), with the sum of the Curie constants the uncorrelated ions and the best-ﬁt data represented by the dashed and solid lines respectively Figure 4. Plot of χMT (open circles) versus T for complex (1), with the su the uncorrelated ions and the best fit data represented by the dashed an Figure 4. Plot of χMT (open circles) versus T for complex (1), with the sum of the Curie constants of the uncorrelated ions and the best-ﬁt data represented by the dashed and solid lines, respectively. p y Upon cooling, the value of χMT remains essentially constant K, where it begins to increase, reaching a maximum of 21.8 cm3 m this temperature, χMT falls rapidly to a minimum value of 18.5 The behaviour is suggestive of weak ferromagnetic exchange b ions, with the decrease in χMT below 6 K attributed to intermol exchange interactions, and/or zero-field splitting (zfs) effects prim NiII ions. Quantitative analysis of the susceptibility data via stan tion techniques is non-trivial due to the large nuclearity of the c enormous dimensions of the spin-Hamiltonian matrices. Even th trices used in approaches based on Irreducible Tensor Operator mensions than what is realistic for exact numerical matrix diagon Upon cooling, the value of χMT remains essentially constant to approximately T = 75 K, where it begins to increase, reaching a maximum of 21.8 cm3 mol−1 K at T = 6 K. Below this temperature, χMT falls rapidly to a minimum value of 18.5 cm3 mol−1 K at T = 2.0 K. The behaviour is suggestive of weak ferromagnetic exchange between the CrIII and NiII ions, with the decrease in χMT below 6 K attributed to intermolecular antiferromagnetic exchange interactions, and/or zero-ﬁeld splitting (zfs) effects primarily associated with the NiII ions. Quantitative analysis of the susceptibility data via standard matrix diagonaliza- tion techniques is non-trivial due to the large nuclearity of the cluster and the associated enormous dimensions of the spin-Hamiltonian matrices. Even the total spin (S) block matrices used in approaches based on Irreducible Tensor Operator algebra are of larger dimensions than what is realistic for exact numerical matrix diagonalization. Previously, we reported the use of computational techniques, known in theoretical nuclear physics as statistical spectroscopy [38], to analyse the structurally similar [MIII8MII6]n+ (MIII = Cr, Fe; MII = Co, Cu, Ni; n = 0–12) cubes [26–28]. 3.1. Structural Description In (2) and (3), the closest contact is between the protons of the metalloligand methyl group, and the O-atom of a neighbouring L−ligand (2.3 Å) and the protons of a neighbouring methyl group (2.3 Å), respectively. Several other close inter-cluster contacts between neighbouring cubes exist, for example: Ar-H···O ≈2.5 Å and C-H···O ≈2.7 Å for (1), H2C-H···O ≈2.5 Å and H2O···H-CH2 ≈2.7 Å for (2), and Ar-H···Cl ≈2.7 Å and C-H···Cl ≈2.8 Å for (3). extended structures of (1)–(3). In (1), the closest inter-cluster contact is between the aro- matic protons of the pyridyl group and the O-atom (2.3 Å) of a neighbouring L- ligand. In (2) and (3), the closest contact is between the protons of the metalloligand methyl group, and the O-atom of a neighbouring L− ligand (2.3 Å) and the protons of a neighbouring methyl group (2.3 Å), respectively. Several other close inter-cluster contacts between neighbouring cubes exist, for example: Ar-H∙∙∙O ≈2.5 Å and C-H∙∙∙O ≈2.7 Å for (1), H2C- H∙∙∙O ≈2.5 Å and H2O∙∙∙H-CH2 ≈2.7 Å for (2), and Ar-H∙∙∙Cl ≈2.7 Å and C-H∙∙∙Cl ≈2.8 Å for (3). (3). (3). (3). Figure 3. Packing diagrams of (1)–(3) viewed down the a-, a-, and c-axis, respectively. Colour code as in Figure 1. Figure 3. Packing diagrams of (1)–(3) viewed down the a-, a-, and c-axis, respectively. Colour code as in Figure 1. (3 gure 3. Packing diagrams of (1)–(3) viewed down the a-, a-, and c-axis, respectively. Colour code as in Figu igure 3. Packing diagrams of (1)–(3) viewed down the a-, a-, and c-axis, respectively. Colour code as in Figu Molecules 2021, 26, 757 5 of 9 or th 3.2. Magnetic Properties susceptibility, χM field B of 0 1 T 3.2. Magnetic Properties susceptibility, χM field B of 0 1 T As complexes (1)–(3) are structurally analogous, and for the sake of brevity, we discuss only the behaviour of a representative example, complex (1). The dc molar magnetic susceptibility, χM, of a polycrystalline sample of (1) was measured in an applied magnetic ﬁeld, B, of 0.1 T, over the 2–300 K temperature, T, range. The experimental results are shown in Figure 4 in the form of the χMT product versus temperature, where χM = M/B, and M is the magnetization of the sample. Due to the loss of lattice solvent during the evacuation of the sample chamber of the SQUID magnetometer, leading to an uncertainty in the molar mass of the measured sample, the T = 300 K χMT product of (1) was scaled to 21.00 cm3 mol−1 K, the expected value from the sum of Curie constants for a [CrIII8NiII6] unit, with gCr = gNi = 2.0, where gCr and gNi are the g-factors of CrIII and NiII, respectively. Note that this rescaled value has a maximum deviation of 15% from the unscaled data. field, B, of 0.1 T, over the 2 300 K temperature, T, range. The shown in Figure 4 in the form of the χMT product versus tempe and M is the magnetization of the sample. Due to the loss of l evacuation of the sample chamber of the SQUID magnetometer, in the molar mass of the measured sample, the T = 300 K χMT pro 21.00 cm3 mol–1 K, the expected value from the sum of Curie c unit, with gCr = gNi = 2.0, where gCr and gNi are the g-factors of C Note that this rescaled value has a maximum deviation of 15% fr Figure 4. Plot of χMT (open circles) versus T for complex (1), with the su the uncorrelated ions and the best-fit data represented by the dashed an Figure 4. Plot of χMT (open circles) versus T for complex (1), with the sum of the Curie constants of the uncorrelated ions and the best-ﬁt data represented by the dashed and solid lines, respectively. Figure 4. Plot of χMT (open circles) versus T for complex (1), with the Figure 4. 3.2. Magnetic Properties susceptibility, χM field B of 0 1 T We now extend this methodology to quantify the exchange interactions present in (1). Due to the fact that the inﬂuence of the zfs of the NiII ions will mainly affect the measured properties at low temperatures, the use of the Molecules 2021, 26, 757 6 of 9 isotropic spin-Hamiltonian (1) is sufﬁcient to model the exchange interactions between CrIII and NiII ions in the T = 300–6 K region: ˆHiso = −2JCr−Ni ∑ allCr−Nipairs ˆSCr · ˆSNi + µBBg∑ i ˆSZ i (1) (1) with i running over all constitutive metal centres, g is the isotropic g-factor, ˆS is a spin- operator, JCr-M is the isotropic exchange parameter between CrIII and MII centres, and µB is the Bohr magneton. We assume common g-factors for both CrIII and NiII (gCr = gNi = 2.0) since the 300 K χMT product of (1) was scaled to the sum of its Curie constants, as ex- plained above. We neglect any JCr-Cr and JNi-Ni terms as these centres are not connected as ﬁrst neighbours. Using Hamiltonian (1), JCr-Ni was determined to be +0.045 cm−1. Variable-temperature and variable-ﬁeld (VTVB) magnetization studies of (1) collected in the T = 2–10 K and B = 0.5–5 T temperature and ﬁeld ranges (Figure 5) are consistent with this picture. M reaches a value of 32.8 µB at B = 5 T and T = 2 K, approaching the saturation value of 36 µB, consistent with relatively small exchange-induced splittings that lead to the mS = 18 projection of the S = 18 total spin state, being the ground state at the highest measured magnetic ﬁeld. The weak ferromagnetic exchange between the d3 CrIII ions and the d8 NiII ions is as one would expect, mediated via the 1-(4-pyridyl)butane-1,3-dione ligand [26–28]. first neighbours. Using Hamiltonian (1), JCr-Ni was determined to temperature and variable-field (VTVB) magnetization studies of 10 K and B = 0.5–5 T temperature and field ranges (Figure 5) are ture. M reaches a value of 32.8 μB at B = 5 T and T = 2 K, approach of 36 μB, consistent with relatively small exchange-induced splitt 18 projection of the S = 18 total spin state, being the ground state magnetic field. The weak ferromagnetic exchange between the d ions is as one would expect, mediated via the 1-(4-pyridyl)buta 28]. Figure 5. 3.2. Magnetic Properties susceptibility, χM field B of 0 1 T VTVB magnetisation data for (1) in the temperature and field r 0 5–5 T Figure 5. VTVB magnetisation data for (1) in the temperature and ﬁeld ranges T = 2–10 K and B = 0.5–5 T. Figure 5. VTVB magnetisation data for (1) in the temperature and field r 0 5–5 T Figure 5. VTVB magnetisation data for (1) in the temperature and ﬁeld ranges T = 2–10 K and B = 0.5–5 T. 0.5 5 T. 3.3. EPR Spectroscopy 0.5 5 T. 3.3. EPR Spectroscopy 0 5 5 3.3. EPR Spectroscopy 3.3. EPR Spectroscopy X-band EPR spectra of a powdered sample of (1) at 5 and 1 feature at ca. 2 kG (Figure 6). This is similar to spectra from the is and related [CrIII8MII6] and [CrIII2MII3] species [26,29], and arises fr with a near-axial zero-field splitting of \|DCr\| ca. 0.5–0.6 cm−1. Th a weak exchange interaction \|JCr-Ni\| with respect to \|DCr\|, and h X-band EPR spectra of a powdered sample of (1) at 5 and 10 K are dominated by a feature at ca. 2 kG (Figure 6). This is similar to spectra from the isolated [CrIIIL3] complex, and related [CrIII8MII6] and [CrIII2MII3] species [26,29], and arises from the CrIII (S = 3/2) ions with a near-axial zero-ﬁeld splitting of \|DCr\| ca. 0.5–0.6 cm−1. This is only consistent with a weak exchange interaction \|JCr-Ni\| with respect to \|DCr\|, and hence consistent with the magnetic data. There are no clear features arising from the NiII (S = 1) ions, which implies that \|DNi\| must be much larger than the microwave energy. We also observed this for a related [FeIII8MII6] cube, which only showed EPR features due to FeIII [28]. This is consistent with \|DNi\| values of 5–10 cm−1 determined from magnetization studies Molecules 2021, 26, 757 7 of 9 o FeIII zatio of isolated [NiII(pyridine)4X2] complexes [39], and with high-ﬁeld EPR studies of NiII complexes with mixed N,O-donor sets [40]. ixed N,O-donor sets [40]. of isolated [NiII(pyridine)4X2] complexes [39], and with high-ﬁeld EPR studies of NiII complexes with mixed N,O-donor sets [40]. ixed N,O-donor sets [40]. i u e 6 X ba d ( a 9 4 GH ) EPR e t u of a o de ed a Figure 6. X-band (ca. 9.4 GHz) EPR spectrum of a powdered sample of (1) at 5 K. Figure 6. X-band (ca. 9.4 GHz) EPR spectrum of a powdered sample of (1) at 5 K. gure 6. X-band 4. Conclusions We have shown that the modular self-assembly of [MIIIL3] metalloligands with simple MII salts can be exploited to construct large heterometallic coordination compounds of CrIII and NiII. Compounds (1)–(3) join a growing family of [MIII8MII6] cubes, where MIII = Cr and Fe and MII = Cu, Co, Mn, Pd, and Ni. The ability to build families of isostructural com- plexes containing different combinations of paramagnetic (and diamagnetic) metal centres aids the qualitative and quantitative understanding of magnetic properties and the under- lying structural parameters that govern behaviour. Examples of large, heterometallic cages in which the 3d metal ions can be exchanged with other 3d metal ions are extremely rare. g y Magnetic susceptibility and magnetization data show the presence of weak ferromag- netic exchange between the CrIII and NiII ions, with JCr-Ni = +0.045 cm−1. EPR spectroscopy is consistent with the exchange interactions being much weaker than the zero-ﬁeld split- tings of both the CrIII and NiII ions. Author Contributions: E.K.B., P.J.L., H.M.O. and S.S. designed the study. H.M.O., S.S. and A.J.S. performed the synthetic work. M.B.P., W.T.K. and S.J.C. performed the crystallographic work. H.W. and S.P. performed the magnetometry measurements and associated analysis. N.F.C. and E.J.L.M. performed the EPR spectroscopic experiments and associated analysis. H.M.O. and E.K.B. wrote the paper with input from all authors. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the EPSRC (UK), grant numbers EP/N01331X/1 and EP/P025986/1, and by the VILLUM FONDEN (Denmark), grant 13376. We also thank the EP- SRC for funding the UK National EPR Facility. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding authors. Conﬂicts of Interest: The authors declare that there is no conﬂict of interest. 8 of 9 Molecules 2021, 26, 757 References 1. Barton, B.E.; Whaley, C.M.; Rauchfuss, T.B.; Gray, D.L. Nickel−Iron Dithiolato Hydrides Relevant to the [NiFe]-Hydrogenase Active Site. J. Am. Chem. Soc. 2009, 131, 6942–6943. [CrossRef] 2. 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https://openalex.org/W2949716378	https://uwspace.uwaterloo.ca/bitstream/10012/13117/1/171511.full.pdf	English	null	Modelling science trustworthiness under publish or perish pressure	Royal Society open science	2,018	cc-by	14,643	Research 1School of Mathematics and Physics, Queen’s University Belfast, BT7 1NN, UK 2Department of Oncology, University of Oxford, Old Road Campus, Oxford OX3 7DQ, UK 3Department of Applied Mathematics, University of Waterloo, 200 University Avenue W, Waterloo, Ontario, Canada N2L 3G1 4Meta-Research Innovation Center at Stanford (METRICS), 5Department of Medicine, 6Department of Health Research and Policy, 7Department of Biomedical Data Science, and 8Department of Statistics, Stanford University, SPRC, MSOB X306, 1265 Welch Road, Stanford, CA 94305, USA 1School of Mathematics and Physics, Queen’s University Belfast, BT7 1NN, UK 2Department of Oncology, University of Oxford, Old Road Campus, Oxford OX3 7DQ, UK 3Department of Applied Mathematics, University of Waterloo, 200 University Avenue W, Waterloo, Ontario, Canada N2L 3G1 4Meta-Research Innovation Center at Stanford (METRICS), 5Department of Medicine, 6Department of Health Research and Policy, 7Department of Biomedical Data Science, and 8Department of Statistics, Stanford University, SPRC, MSOB X306, 1265 Welch Road, Stanford, CA 94305, USA 1School of Mathematics and Physics, Queen’s University Belfast, BT7 1NN, UK 2Department of Oncology, University of Oxford, Old Road Campus, Oxford OX3 7DQ, UK 3Department of Applied Mathematics, University of Waterloo, 200 University Avenue W, Waterloo, Ontario, Canada N2L 3G1 4Meta-Research Innovation Center at Stanford (METRICS), 5Department of Medicine, 6Department of Health Research and Policy, 7Department of Biomedical Data Science, and 8Department of Statistics, Stanford University, SPRC, MSOB X306, 1265 Welch Road, Stanford, CA 94305, USA Received: 29 September 2017 Accepted: 1 December 2017 DRG, 0000-0003-3140-3278; CTB, 0000-0001-6214-6601 Scientiﬁc publication is immensely important to the scientiﬁc endeavour. There is, however, concern that rewarding scientists chieﬂy on publication creates a perverse incentive, allowing careless and fraudulent conduct to thrive, compounded by the predisposition of top-tier journals towards novel, positive ﬁndings rather than investigations conﬁrming null hypothesis. This potentially compounds a reproducibility crisis in several ﬁelds, and risks undermining science and public trust in scientiﬁc ﬁndings. To date, there has been comparatively little modelling on factors that inﬂuence science trustworthiness, despite the importance of quantifying the problem. We present a simple phenomenological model with cohorts of diligent, careless and unethical scientists, with funding allocated by published outputs. This analysis suggests that trustworthiness of published science in a given ﬁeld is inﬂuenced by false positive rate, and pressures for positive results. We ﬁnd decreasing available funding has negative consequences for resulting trustworthiness, and examine strategies to combat propagation of irreproducible science. Scientiﬁc publication is immensely important to the scientiﬁc endeavour. Keywords: research ethics, research fraud, science trustworthiness, public trust in science, publish or perish Author for correspondence: David Robert Grimes e-mail: d.r.grimes@qub.ac.uk on April 18, 2018 http://rsos.royalsocietypublishing.org/ Downloaded from on April 18, 2018 http://rsos.royalsocietypublishing.org/ Downloaded from David Robert Grimes1,2, Chris T. Bauch3 and John P. A. Ioannidis4,5,6,7,8 David Robert Grimes1,2, Chris T. Bauch3 and John P. A. Ioannidis4,5,6,7,8 Research There is, however, concern that rewarding scientists chieﬂy on publication creates a perverse incentive, allowing careless and fraudulent conduct to thrive, compounded by the predisposition of top-tier journals towards novel, positive ﬁndings rather than investigations conﬁrming null hypothesis. This potentially compounds a reproducibility crisis in several ﬁelds, and risks undermining science and public trust in scientiﬁc ﬁndings. To date, there has been comparatively little modelling on factors that inﬂuence science trustworthiness, despite the importance of quantifying the problem. We present a simple phenomenological model with cohorts of diligent, careless and unethical scientists, with funding allocated by published outputs. This analysis suggests that trustworthiness of published science in a given ﬁeld is inﬂuenced by false positive rate, and pressures for positive results. We ﬁnd decreasing available funding has negative consequences for resulting trustworthiness, and examine strategies to combat propagation of irreproducible science. Subject Areas: j mathematical modelling/computer modelling and simulation/statistics rsos.royalsocietypublishing.org Research Cite this article: Grimes DR, Bauch CT, Ioannidis JPA. 2018 Modelling science trustworthiness under publish or perish pressure. R.Soc.opensci. 5: 171511. http://dx.doi.org/10.1098/rsos.171511 Research Cite this article: Grimes DR, Bauch CT, Ioannidis JPA. 2018 Modelling science trustworthiness under publish or perish pressure. R.Soc.opensci. 5: 171511. http://dx.doi.org/10.1098/rsos.171511 2 rsos.royalsocietypublishing.org R.Soc. opensci. 5: 171511 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . pp p y Despite their vital importance in conveying accurate science, top-tier journals possess a limited number of publication slots and are thus overwhelmingly weighted towards publishing only novel or signiﬁcant results. Despite the fact that null results and replications are important scientiﬁc contributions, the reality is that journals do not much care for these ﬁndings. Researchers are not rewarded for submitting these ﬁndings nor for correcting the scientiﬁc record, as high-proﬁle examples attest [15,16]. This pressure to produce positive results may function as a perverse incentive. Edwards & Roy [17] argue that such incentives encourage a cascade of questionable ﬁndings and false positives. Heightened pressure on academics has created an environment where ‘Work must be rushed out to minimize the danger of being scooped’ [18]. The range of questionable behaviour itself is wide [19]. Classic ‘fraud’ (falsiﬁcation, fabrication and plagiarism (FFP) [20]) may be far less important than more subtle questionable research practices, which might include selective reporting of (dependent) variables, failure to disclose experimental conditions and unreported data exclusions [21]. hing.org R.Soc. opensci. 5: 171511 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . So how common are such practices? A study of National Institute of Health (NIH)-funded early and mid-career scientists (n = 3247) found that within the previous 3 years, 0.3% admitted to falsiﬁcation of data, 6% to a failure to present conﬂicting evidence and a worrying 15.5% to changing of study design, methodology or results in response to funder pressure [22]. An overview by Fanelli [23] has shown that questionable research practices are as common as 75%, while fraud per se occurs only in 1–3% of scientists. These ﬁndings are alarming, yet quantiﬁcation of these perverse incentives is vital if we are to understand the potential extent of the underlying problem, and formulate strategies to address it. This is an underdeveloped area, but one which is slowly growing—recent works by Smaldino & McElreath [24,25] have employed elegant dynamic models to demonstrate that even when there is no attempts at fraud or untoward research practices, selection based solely on published output tends to produce poorer methods and higher false discovery rates, a phenomenon they term ‘the natural selection of bad science’. Suboptimal science and fraud can take myriad forms which renders it difﬁcult to detect [26]. 1. Introduction In academia, the phrase ‘publish or perish’ is more than a pithy witticism—it reﬂects the reality that researchers are under immense pressure to continuously produce outputs, with career advancement dependent upon them [1,2]. Academic publications are deemed a proxy for scientiﬁc productivity and ability, and with an increasing number of scientists competing for funding, the previous decades have seen an explosion in the 2018 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. on April 18, 2018 http://rsos.royalsocietypublishing.org/ Downloaded from on April 18, 2018 http://rsos.royalsocietypublishing.org/ Downloaded from rate of scientiﬁc publishing [3]. Yet while output has increased dramatically, increasing publication volume does not imply that the average trustworthiness of publications has improved. As science pivots on replicable ﬁndings, for the purposes of this work we deﬁne a trustworthy ﬁnding as one that can be replicated independently, corresponding to a true positive or negative. A previous paper by Ioannidis [4] has outlined the reasons why many published research ﬁndings are false, and the dubious use of p-values for signiﬁcance in research ﬁndings has of late been widely discussed [5–9]. Across much of experimental science from psychology [10] to biomedical science [11–13] and cancer research [14], there is concern over an apparent reproducibility crisis. 2 pT + pF + n = 1. In certain ﬁelds, the false positive rate may be high, and so diligent researchers take measures to falsify positive results and test their results multiple times. Even when research groups are very diligent, they may occasionally happen upon a false or misleading result which is hard to eliminate and due to experimental or theoretical difﬁculty rather than carelessness. For the diligent cohort, this will be as low as can reasonably be achieved and so we state they submit a small fraction, ϵ, of their false positives for publication. Researchers exist on a spectrum, but for simplicity we may broadly sub-divide this spectrum into three distinct classes. (i) Diligent cohort. This group take pains to replicate experiments and do not dishonestly manipulate results. Their false positive submission fraction is ϵ, thus as low as reasonably possible. They account for a fraction fD of the initial total, and parameters relating to them have subscript D. (i) Diligent cohort. This group take pains to replicate experiments and do not dishonestly manipulate results. Their false positive submission fraction is ϵ, thus as low as reasonably possible. They account for a fraction fD of the initial total, and parameters relating to them have subscript D. (ii) Careless cohort. This group do not falsify results, but are much less careful at eliminating spurious positive results. They may also have questionable practices that lead them to false positives. As a result, they have a false positive submission rate of cϵ where c > 1. They account for a fraction fC of the initial total, and parameters relating to them have subscript C. (iii) Unethical cohort. This group appear broadly similar to the diligent group, but with one crucial difference in that they may occasionally manipulate data or knowingly submit dubious results at a rate of δ beyond global discovery rate. For convenience, instead of deﬁning a higher value of DR in this group to account for the higher ‘discovery’ rate, we retain the same parameter value of DR for the unethical cohort but allow pT + pF + n + δ > 1, so that their realized ‘discovery’ rate is higher than the other groups. They account for a fraction fU of the initial total, and parameters relating to them have subscript U. 2 rsos.royalsocietypublishing.org R.Soc. opensci. 5: 171511 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . For the purposes of this article, we deﬁne fraud as an explicit ‘intention to deceive’ [27]. A more recent investigation [23] put the weighted mean percentage of scientists committing research fraud as high as 1.97%, with over a third admitting to questionable research practices. The same investigation found that about 14.12% of scientists reported observing fraudulent research behaviour in colleagues. Another study [28] found that 5% of responding authors claimed to be personally aware of fabricated or misrepresented data in a trial they had participated in. A study of bio-statisticians [29] found that over half of respondents reported being aware of research misconduct. A 2012 [30] analysis found that FFP offenses rather than honest error accounted for 67.4% of retracted publications, with the rate of retraction due to fraud increasing 10-fold since 1975. An important question is whether scientists who are unethical (fraudulent) or sloppy (careless) may thrive and even outperform diligent scientists in a system driven by publish or perish pressure. As it is impossible to identify all unethical and careless scientists, one can perform mathematical modelling of science under different assumptions and ﬁnd out how these scientists fare and what the implications are for the overall trustworthiness of science. To better understand the impact of publish or perish on scientiﬁc research, and to garner insight into what practices drive the trustworthiness of published science is of paramount importance if we are to counteract any detrimental impacts of such practices. In this work, we present a simple but instructive model of scientiﬁc publishing trustworthiness under the assumption that researchers are rewarded for their published output, taking account of ﬁeld-speciﬁc differences and the proportion of resources allocated with funding cycle. The factors that inﬂuence resultant trustworthiness in simulation are quantiﬁed and discussed, as well as implications for improving the trustworthiness of scientiﬁc publishing. It is important to note that the motivations of scientists and ecosystem of scientiﬁc publishing are inherently complex, and we do not expect the model discussed to be deterministically predictive— rather, the model is presented as a tool for allowing us to formulate publication dynamics in a population of scientists in a formal and precise way, subject to certain caveats. on April 18, 2018 http://rsos.royalsocietypublishing.org/ Downloaded from 2.1. Basic model and assumptions rsos.royalsocietypublishing.org R.Soc. opensci. 5: 171511 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . To construct a simple model of publication rewards, we deﬁne the total amount of available funding for research as R(t). Per unit of funding in a given ﬁeld, there is a global discovery rate of DR, which includes a proportion pT of positive/signiﬁcant results, a proportion pF of false positives, and a proportion n of null results. Null results in principle can include both true negatives and false negatives, but given the bias towards positive results we will not discriminate between these two in this investigation. The relative proportion of positives and nulls will be inherently ﬁeld-speciﬁc—certain disciplines will be more prone to false positives, while others tend to yield less ambiguous results. As the quantities are proportions, we have that rg R.Soc. opensci. 5: 171511 pT + pF + n = 1. (2.1) (2.1) 2. Model outline 3 on April 18, 2018 http://rsos.royalsocietypublishing.org/ Downloaded from (2.8) 2 2 Variable funding resources ⎛ ⎜⎝ x(t + 1) y(t + 1) z(t + 1) ⎞ ⎟⎠= ⎛ ⎜⎜⎜⎜⎜⎜⎜⎜⎝ LD(t) A(t) x(t) LC(t) A(t) y(t) LU(t) A(t) z(t) ⎞ ⎟⎟⎟⎟⎟⎟⎟⎟⎠ . (2.8) 2.2. Variable funding resources ⎛ ⎜⎝ x(t + 1) y(t + 1) z(t + 1) ⎞ ⎟⎠= ⎛ ⎜⎜⎜⎜⎜⎜⎜⎜⎝ LD(t) A(t) x(t) LC(t) A(t) y(t) LU(t) A(t) z(t) ⎞ ⎟⎟⎟⎟⎟⎟⎟⎟⎠ . (2.8) (2.8) on April 18, 2018 http://rsos.royalsocietypublishing.org/ Downloaded from results may not ever see the light of publication, the so-called ‘ﬁle drawer’ problem. We assume that each cohort submit only a fraction of their null results in the proportions βD, βC, βU such that 4 ⎛ ⎜⎝ SD− SC− SU− ⎞ ⎟⎠= DRn ⎛ ⎜⎝ βD βC βU ⎞ ⎟⎠. (2.4) (2.4) Equations (2.1)–(2.4) comprise the researcher-speciﬁc parameters, and we must further quantify the journal-speciﬁc elements also. Competition for space in ﬁeld-speciﬁc top-tier journals is ﬁerce, and we denote the combined carrying-capacity of these ﬁeld-speciﬁc top-tier journals as J(t). These journals exhibit a clear bias towards positive results, with a positive-publication weighing fraction of published articles, B, describing signiﬁcant results. Thus, presuming that more submissions are obtained than can be published, we can quantify the probability that a positive result (νP(t)) or a negative result (νN(t)) is published. These probabilities are given by g R.Soc. opensci. 5: 171511 νP(t) νN(t) = ⎛ ⎜⎜⎜⎝ JB x(t)SD+ + y(t)SC+ + z(t)SU+ J(1 −B) x(t)SD−+ y(t)SC−+ z(t)SU− ⎞ ⎟⎟⎟⎠. (2.5) From this, we can then yield an expression for the publication rate per unit of funding for the diligent, careless and unethical cohorts, which are, respectively, ⎛ ⎜⎝ LD(t) LC(t) LU(t) ⎞ ⎟⎠= νP(t) ⎛ ⎜⎝ SD+ SC+ SU+ ⎞ ⎟⎠+ νN(t) ⎛ ⎜⎝ SD− SC− SU− ⎞ ⎟⎠. (2.6) (2.6) The average rate of publications per unit of funding per unit time in top-tier journals for a given ﬁeld is thus The average rate of publications per unit of funding per unit time in top-tier journals for a given ﬁeld is thus A(t) = J x(t) + y(t) + z(t). (2.7) (2.7) If researchers are rewarded with funding based solely on their published output, we can quantify the impact of this with time by employing a recursive series solution at discrete time steps, corresponding to funding cycles. If funding is allocated to each cohort based upon their output at the beginning of the previous funding cycling, and we assume total funding remains constant (dR/dt = 0) then the funding available for each cohort at each successive time step is ⎛ ⎜⎝ x(t + 1) y(t + 1) z(t + 1) ⎞ ⎟⎠= ⎛ ⎜⎜⎜⎜⎜⎜⎜⎜⎝ LD(t) A(t) x(t) LC(t) A(t) y(t) LU(t) A(t) z(t) ⎞ ⎟⎟⎟⎟⎟⎟⎟⎟⎠ . pT + pF + n = 1. The funding held by the diligent cohort at a given time is x(t), with y(t) held by the careless cohort and z(t) by the unethical cohort, so that x(t) + y(t) + z(t) = R(t). (2.2) (2.2) With these assumptions, we can model the theoretical impact of a paradigm where researchers are rewarded with funding and success in direct relation to their publication output. As outlined in the Introduction, there is huge pressure on scientists to submit positive or ‘novel’ ﬁndings, while ﬁndings conﬁrming the null hypothesis are frequently side-lined. Under such a selection pressure, all researchers will aim to submit their signiﬁcant positive results for publication. The respective rates of submission per unit funding for the diligent, careless and unethical cohorts are accordingly ⎛ ⎜⎝ SD+ SC+ SU+ ⎞ ⎟⎠= DR ⎛ ⎜⎝ pT + ϵpF pT + cϵpF pT + ϵpF + δ ⎞ ⎟⎠ (2.3) (2.3) The rate at which null results are submitted is less clear—in general, there is a signiﬁcant bias in publication towards signiﬁcant results. As a consequence, negative ﬁndings are often shunned by high- impact journals, and scientists are disinclined to submit them, meaning that potentially important null on April 18, 2018 http://rsos.royalsocietypublishing.org/ Downloaded from on April 18, 2018 http://rsos.royalsocietypublishing.org/ Downloaded from 2.5. Counteracting publication bias It is also possible to envision a situation where journals do not give any preference to positive results over null results. In this case, we would expect researchers to submit all their results so that βD = βC = βU = 1. In this case, νP and νN are replaced by a single function of time ν, given by ν(t) = J x(t)(SD+ + SD−) + y(t)(SC+ + SC−) + z(t)(SU+ + SU−). (2.12) (2.12) 2.6. Trustworthiness of published science Finally, we deﬁne a metric for the trustworthiness of published science, deﬁned as the proportion of reproducible results, T(t). This is given by T(t) = 1 −νpDR(x(ϵpF) + y(cϵpF) + z(ϵpF + δ)) J , (2.13) (2.13) where the time arguments of x,y,z and νp have been excluded for clarity. where the time arguments of x,y,z and νp have been excluded for clarity. where the time arguments of x,y,z and νp have been excluded for clarity. 2.2. Variable funding resources We also consider the fact that the total amount of funding may not remain constant, so we may model the impact of changing funding scenarios. For simplicity, we assume it changes at some constant rate G, which can be negative (for diminishing funding, the likes of which might occur with a decrease in NIH or EU funding budgets), zero (for constant funding, as in equation (2.8)) or positive (increasing funding). New funding is allocated at random in proportions reﬂecting the typical make-up of new researchers, and accordingly the reﬁned equations are ⎛ ⎜⎝ x(t + 1) y(t + 1) z(t + 1) ⎞ ⎟⎠= ⎛ ⎜⎜⎜⎜⎜⎜⎜⎜⎝ LD(t) A(t) x(t) + fDG LC(t) A(t) y(t) + fCG LU(t) A(t) z(t) + fUG ⎞ ⎟⎟⎟⎟⎟⎟⎟⎟⎠ . (2.9) (2.9) on April 18, 2018 http://rsos.royalsocietypublishing.org/ Downloaded from on April 18, 2018 http://rsos.royalsocietypublishing.org/ Downloaded from 2.3. Research fraud detection 5 For unethical researchers, we can look at a slightly more complicated scenario where dubious publications have a probability of detection leading to denial of funding, η. We further assume this penalization only applies to dubious results which were published rather than just submitted. If this consideration is taken into account, then we modify the last part of equation (2.9) to reﬂect this so that z(t + 1) = LU(t) A(t) −DRηδνp(t) z(t) + fUG. (2.10) 2.4. Rewarding diligence The diligent cohort have intrinsically lower submission rates than other groups, and consequently are more likely to suffer under a publish or perish regime, despite the importance of their reproducible work. To counteract this, it has been suggested that rewarding diligence might counteract this trend [31,32]. We might envision an ideal situation where scientiﬁc works are audited for reproducibility by independent bodies, with groups who keep their reproducibility high and error rates below a certain unavoidable threshold (given by DRνPϵ) garnering a reward of RW. This in practice could only be achieved by the diligent cohort, and in the most simple case, their funding resources are given by x(t + 1) = LD(t) A(t) x(t) + fDG + RW. (2.11) (2.11) 2.7. Parameter estimation and assumptions To simulate the trends that would occur under these assumptions requires that we select appropriate parameters (these are detailed in table 1), which are used in all simulations unless otherwise stated in the text. It can be seen through inspection that discovery rate per unit resource DR cancels in the analysis for x(t), y(t), z(t) and T(t), and accordingly this can be ascribed any real positive value without skewing analysis. When there is no fraud detection funding penalization (η = 0), journal carrying capacity J also cancels in the analysis and does not impact results. Initially we assume also that G = 0 so that funding levels remain constant. Estimation of fraudulent submission fraction per unit resource δ requires some elaboration, as this is notoriously difﬁcult to ascertain and ﬁeld-speciﬁc. A 1996 analysis by Fuchs & Westervelt [33] extrapolated from known cases to estimate that approximately 0.01% of published papers were fraudulent, though this is considered exceptionally conservative [27]. Empirical estimates of plagiarism vary markedly from 0.02 to 25% of all publications [26]. The frequency of paper retractions from the PubMed database for misconduct is about 0.02%, suggesting that fraud might be present in 0.02–0.2% of papers therein [34]. An investigation in the Journal of Cell Biology found inappropriate image- manipulation occurring in 1% of papers [35]. More alarmingly perhaps, a 1992 data audit by the United States Food and Drug Administration found deﬁciencies a in 10–20% of studies published between 1977 and 1990, with 2% of investigators deemed guilty of severe scientiﬁc misconduct [23,36,37]. on April 18, 2018 http://rsos.royalsocietypublishing.org/ Downloaded from on April 18, 2018 http://rsos.royalsocietypublishing.org/ Downloaded from 6 Table 1. Parameters for initial simulations. Values in this table comprise the default initial assumptions, which are varied to investigate different conditions, as outlined in the respective relevant section. 6 Table 1. Parameters for initial simulations. Values in this table comprise the default initial assumptions, which are varied to investigate different conditions, as outlined in the respective relevant section. 6 rsos.royalsocietypublishing.org R.Soc. opensci. 5: 171511 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . parameter value journal carrying capacity (J) 120/cycle . . . . . . . . . . 2.7. Parameter estimation and assumptions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . total discovery rate per unit funding (DR) 15 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Parameter estimation and assumptions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . initial proportion diligent researchers (fD) 0.65 [23] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . initial proportion careless researchers (fC) 0.33 [23] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Parameter estimation and assumptions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . initial proportion unethical researchers (fU) 0.02 [23] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . reasonable error rate (ϵ) 0.05 [9] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Parameter estimation and assumptions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . fraudulent submission rate per unit resource (δ) 0.0574 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . positive publication bias (B) 0.9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Parameter estimation and assumptions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . multiplicative factor for careless cohort—(c) 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Parameter estimation and assumptions null/negative submission rates—(βD/βC/βU) 0.40 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . resource growth rate—(G) 0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Parameter estimation and assumptions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . fraud detection proportion—(η) 0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . field-specific true positive fraction (pT) 0.2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Parameter estimation and assumptions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . field-specific false positive fraction (pF) 0.2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . For the purposes of this work, we will assume that FFP violations are present in 1% of the published literature so that JF = J/100. Deﬁning xo = x(0), yo = y(0), zo = z(0) and νpO = νp(0), then for any selected values of DR and J, we can readily deﬁne the required rate by δ = JF(xoSD+ + yoSC+ + zoDR(pT + ϵpF)) zoDR(BJ −JF) . 3.1. Impact of the field-specific false positive rate Figure 1 shows the change in funding proportions with time for each cohort in a ﬁeld with a low rate of false positives (pF/(pT + pF) = 0.25) and a ﬁeld with a high rate of false positives (pF/(pT + pF) = 0.8). What is immediately evident is that in ﬁelds where false positives comprise the bulk of positive results, the trustworthiness of published science suffers markedly, and careless and unethical cohorts are disproportionately rewarded at the expense of diligent researchers. Simulation results suggests that the trustworthiness of published science in any given ﬁeld is strongly dependent on the false positive rate in that ﬁeld under a publish or perish paradigm. As evidenced by the ﬁgure, outputs from diligent research and the number of trustworthy results still decline even when FPR is low with publish or perish incentives, as suggested by Smaldino & McElreath [24]. 2.7. Parameter estimation and assumptions (2.14) (2.14) This is dependent on the true/false positive of the ﬁeld, and we initially take an optimistic assumption that the 1% of published fraud occurs in ﬁelds with high levels of false positives, and will be less in ﬁelds with less ambiguity in results, so that the same value of δ is used for all simulations. This is calculated assuming pF = 0.32 and pT = 0.08 so that δ = 0.057 as per table 1. The reasonable error rate is taken from p- value for signiﬁcance, as discussed in Colquhoun [9]. Strictly speaking, Prof. Colquhoun puts forward an eloquent argument in the cited work that p < 0.05 is a frequently abused metric, leading to false positives. For simplicity, however, we will presume that ϵ = 0.05 reﬂects best reasonable practice in this simulation. 3.2. Impact of funding growth rate 7 (a) funding cycles 5 10 funding cycles 5 10 fraction of funding 0 0.1 0.2 0.3 0.4 0.5 0.6 0 0.1 0.2 0.3 0.4 0.5 0.6 diligent cohort careless cohort unethical cohort diligent cohort careless cohort unethical cohort funding cycles 0 1 2 3 4 5 6 7 8 9 10 replicable science proportion 0.65 0.70 0.75 0.80 0.85 0.90 0.95 trustworthiness low pF high pF (c) fraction of funding ng.org R.Soc. opensci. 5: 171511 funding cycles funding cycles funding cycles 0 1 2 3 4 5 6 7 8 9 10 replicable science proportion 0.65 0.70 0.75 0.80 0.85 0.90 0.95 trustworthiness low pF high pF (c) Figure1. Theimpactoffield-specificfalsepositiverateonresourcesallocatedandsciencetrustworthiness.Panel(a)depictstheprojected funding allocations in a field where false positives are relatively rare (pT = 0.32, pF = 0.08). By contrast, (b) shows the impact on resources consumed when false positives are the norm (pT = 0.08, pF = 0.32). In (c), the trustworthiness (proportion of reproducible science)forbothscenariosaredepicted,indicatingfalsepositiveratehighlydrivesthetrustworthinessofscientificpublicationinagiven field. Conversely, reducing funding increases the publication pressure and results in increased selection of suspect works and a fall in scientiﬁc reproducibility. The implications of this require some elaboration and are considered in the Discussion section. 3.2. Impact of funding growth rate As depicted in ﬁgure 2. The increasing of available funds (G) has the net effect of reducing publication pressure by bring down the average number of publications expected per unit funding, provided journal capacity stays roughly constant, reducing the likelihood of dubious publications being selected. on April 18, 2018 http://rsos.royalsocietypublishing.org/ Downloaded from on April 18, 2018 http://rsos.royalsocietypublishing.org/ Downloaded from 7 rsos.royalsocietypublishing.org R.Soc. opensci. 5: 171511 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . funding cycles 5 10 funding cycles 5 10 fraction of funding 0 0.1 0.2 0.3 0.4 0.5 0.6 0 0.1 0.2 0.3 0.4 0.5 0.6 proportion of resources (low pF) diligent cohort careless cohort unethical cohort proportion of resources (high pF) diligent cohort careless cohort unethical cohort funding cycles 0 1 2 3 4 5 6 7 8 9 10 replicable science proportion 0.65 0.70 0.75 0.80 0.85 0.90 0.95 trustworthiness low pF high pF (b) (c) (a) Figure1. Theimpactoffield-specificfalsepositiverateonresourcesallocatedandsciencetrustworthiness.Panel(a)depictstheprojected funding allocations in a field where false positives are relatively rare (pT = 0.32, pF = 0.08). By contrast, (b) shows the impact on resources consumed when false positives are the norm (pT = 0.08, pF = 0.32). In (c), the trustworthiness (proportion of reproducible science)forbothscenariosaredepicted,indicatingfalsepositiveratehighlydrivesthetrustworthinessofscientificpublicationinagiven field. 7 rsos.royalsocietypublishing.org R.Soc. opensci. 5: 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . funding cycles 5 10 0 0.1 0.2 0.3 0.4 0.5 0.6 proportion of resources (high pF) diligent cohort careless cohort unethical cohort (b) funding cycles 5 10 fraction of funding 0 0.1 0.2 0.3 0.4 0.5 0.6 proportion of resources (low pF) diligent cohort careless cohort unethical cohort (a) 7 rsos.royalsocietypublishing.org R.Soc. opensci. 5: 171511 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Impact of increased fraud detection 5: 171511 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . funding cycles 0 5 10 funding cycles 0 5 10 funding cycles 5 10 fraction of funding 0.35 0.40 0.45 0.50 0.55 0.60 0.65 diligent G = 0 G = +5 G = –5 0.34 0.36 0.38 0.40 careless 0 0.05 0.10 0.15 0.20 0.25 unethical funding cycles 0 1 2 3 4 5 6 7 8 9 10 replicable science proportion 0.90 0.91 0.92 0.93 0.94 trustworthiness (b) (a) (c) (d) Figure 2. The impact of funding increases and decreases on funding allocation and science trustworthiness. Panels (a–c) depict the absolute proportion of funding resources allocated to diligent, careless and unethical cohorts, respectively, when funding changes at 8 rsos.royalsocietypublishing.org R.Soc. opensci. 5: 171511 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 funding cycles 0 1 2 3 4 5 6 7 8 9 10 replicable science proportion 0.90 0.91 0.92 0.93 0.94 trustworthiness (d) Figure 2. The impact of funding increases and decreases on funding allocation and science trustworthiness. Panels (a–c) depict the absolute proportion of funding resources allocated to diligent, careless and unethical cohorts, respectively, when funding changes at rates of 0, 5 and −5 per cycle. funding fraction 0.02 0.04 0.06 0.08 0.10 0.12 0.14 absolute funding proportion (unethical cohort) h = 0 h = 0.25 h = 0.75 funding cycles 0 2 4 6 8 10 funding cycles 0 2 4 6 8 10 replicable science proportion 0.910 0.915 0.920 0.925 0.930 0.935 0.940 trustworthiness (a) (b) Figure 3. The impact of strict fraud detection/penalization. (a) Increasing the rate at which fraud is detected limits the amount of resources garnered by unethical cohorts, but (b) high values of η are required to markedly improve the trustworthiness of published science. 3.3. Impact of increased fraud detection Figure 3 depicts the impact of aggressive fraud detection (η) and punishment. Increased fraud detection seems to improve science trustworthiness, but η has to be very high in practice to have a substantial impact on the proportion of funding allocated to unethical cohorts. Negating growth, the funding allocation to this group would only be expected to decrease with time if η > 1 DRδνP LU A −1 . (3.1) (3.1) In practice, this is quite high, and for the values in table 1, a value of η > 0.7688 would be required to fully diminish funding to this cohort in time. In practice, this is quite high, and for the values in table 1, a value of η > 0.7688 would be required to fully diminish funding to this cohort in time. on April 18, 2018 http://rsos.royalsocietypublishing.org/ Downloaded from on April 18, 2018 http://rsos.royalsocietypublishing.org/ Downloaded from 8 rsos.royalsocietypublishing.org R.Soc. opensci. 5: 171511 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . funding cycles 0 5 10 funding cycles 0 5 10 funding cycles 5 10 fraction of funding 0.35 0.40 0.45 0.50 0.55 0.60 0.65 diligent G = 0 G = +5 G = –5 0.34 0.36 0.38 0.40 careless 0 0.05 0.10 0.15 0.20 0.25 unethical funding cycles 0 1 2 3 4 5 6 7 8 9 10 replicable science proportion 0.90 0.91 0.92 0.93 0.94 trustworthiness (b) (a) (c) (d) Figure 2. The impact of funding increases and decreases on funding allocation and science trustworthiness. Panels (a–c) depict the absolute proportion of funding resources allocated to diligent, careless and unethical cohorts, respectively, when funding changes at rates of 0, 5 and −5 per cycle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . funding cycles 0 5 10 funding cycles 0 5 10 funding cycles 5 10 fraction of funding 0.35 0.40 0.45 0.50 0.55 0.60 0.65 diligent G = 0 G = +5 G = –5 0.34 0.36 0.38 0.40 careless 0 0.05 0.10 0.15 0.20 0.25 unethical (b) (a) (c) 8 rsos.royalsocietypublishing.org R.Soc. opensci. 3.4. Impact of rewarding diligence By inspection, it is straightforward to show that for the amount of funding held by the diligent cohort to stay the same or increase, then the condition on RW is RW ≥ 1 −LD(t) A(t) x(t) −fDG, (3.2) (3.2) though in practice for most situations, RW will have to be much greater than this minimum value. For the example depicted in ﬁgure 4, a large reward for diligence (RW = 10) substantially increases the funds awarded to the diligent cohort. However, reproducibility still falls slowly if the unethical cohort is not removed. It is possible to both reward diligence and punish fraud, which can improve trustworthiness, as illustrated in ﬁgure 4. on April 18, 2018 http://rsos.royalsocietypublishing.org/ Downloaded from 9 rsos.royalsocietypublishing.org R.Soc. opensci. 5: 171511 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 2 4 6 8 10 0 2 4 6 8 10 fraction of funding 0.45 0.50 0.55 0.60 0.65 0.70 0.75 diligent proportion funding cycles replicable science proportion 0.91 0.92 0.93 0.94 trustworthiness RW = 0, h = 0 RW = 10, h = 0 RW = 5, h = 0.8 (a) (b) 0 2 4 6 8 10 0 2 4 6 8 10 fraction of funding 0.45 0.50 0.55 0.60 0.65 0.70 0.75 diligent proportion funding cycles replicable science proportion 0.91 0.92 0.93 0.94 trustworthiness RW = 0, h = 0 RW = 10, h = 0 RW = 5, h = 0.8 (a) (b) Figure 4. The impact of rewarding researcher for diligence. This improves the proportion of funding allocated to diligent researchers, but to improve science trustworthiness still requires non-zero values of η under this schema. 0 2 4 6 8 10 fraction of funding 0.45 0.50 0.55 0.60 0.65 0.70 0.75 diligent proportion RW = 0, h = 0 RW = 10, h = 0 RW = 5, h = 0.8 (a) 9 rsos.royalsocietypublishing.org R.Soc. opensci. 5: 171511 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Figure 4. The impact of rewarding researcher for diligence. This improves the proportion of funding allocated to diligent researchers, but to improve science trustworthiness still requires non-zero values of η under this schema. 3.5. Impact of the positive publication weighing To simulate how published science fares under the rather artiﬁcial ﬁxation of top-tier journals with positive novel results, ﬁgure 5 depicts how funding is allocated and science trustworthiness changes with varying values for B. In this simulation, βD = βC = βD = 0.5 when publications were B ≫0.5 (skewed towards positive publication). For the scenario where null and positive results were equally likely to be published, null results were as likely to be published so all were submitted and thus the fraction of null results submitted, respectively, were βD = βC = βD = 1. Higher values of B lead to perverse rewarding of false positives and fraudulent results at the expense of diligent science. Best outcome for science trustworthiness was observed when journals were simulated as completely agnostic to ﬁndings. 3.3. Impact of increased fraud detection funding cycles 0 2 4 6 8 10 replicable science proportion 0.910 0.915 0.920 0.925 0.930 0.935 0.940 trustworthiness (b) funding fraction 0.02 0.04 0.06 0.08 0.10 0.12 0.14 absolute funding proportion (unethical cohort) h = 0 h = 0.25 h = 0.75 funding cycles 0 2 4 6 8 10 (a) Figure 3. The impact of strict fraud detection/penalization. (a) Increasing the rate at which fraud is detected limits the amount of resources garnered by unethical cohorts, but (b) high values of η are required to markedly improve the trustworthiness of published science. on April 18, 2018 http://rsos.royalsocietypublishing.org/ Downloaded from 3.6. Impact of initial unethical funding proportion Figure 6 depicts the sensitivity of trustworthiness to different assumptions of initial unethical funding proportion zo. As might be expected, increasing zo has negative implications for published trustworthiness. on April 18, 2018 http://rsos.royalsocietypublishing.org/ Downloaded from on April 18, 2018 http://rsos.royalsocietypublishing.org/ Downloaded from 1 rsos.royalsocietypublishing.org R.Soc. opensci. 5: 171511 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . funding cycles 2 4 6 8 10 funding cycles 2 4 6 8 10 fraction of funding 0 0.2 0.4 0.6 0.8 proportion of resources (B = 0.95) diligent cohort careless cohort unethical cohort 0 0.2 0.4 0.6 0.8 proportion of resources (B = 0.80) funding cycles 2 4 6 8 10 fraction of funding 0 0.2 0.4 0.6 0.8 proportion of resources (B = 0.65) funding cycles 2 4 6 8 10 0 0.2 0.4 0.6 0.8 proportion of resources (B-independent) funding cycles 0 1 2 3 4 5 6 7 8 9 10 replicable science proportion 0.90 0.92 0.94 0.96 0.98 1.00 trustworthiness B-independent B = 0.65 B = 0.80 B = 0.95 (b) (a) (d) (e) (c) Fi 5 Th i f i i bli i i hi h hi f bli h d i ( )Sh f di ll i 95% on April 18, 2018 http://rsos.royalsocietypublishing.org/ Downloaded from 10 rsos.royalsocietypublishing.org R.Soc. opensci. 5: 171511 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Impact of initial unethical funding proportion funding cycles 2 4 6 8 10 funding cycles 2 4 6 8 10 fraction of funding 0 0.2 0.4 0.6 0.8 proportion of resources (B = 0.95) diligent cohort careless cohort unethical cohort 0 0.2 0.4 0.6 0.8 proportion of resources (B = 0.80) funding cycles 2 4 6 8 10 fraction of funding 0 0.2 0.4 0.6 0.8 proportion of resources (B = 0.65) funding cycles 2 4 6 8 10 0 0.2 0.4 0.6 0.8 proportion of resources (B-independent) funding cycles 0 1 2 3 4 5 6 7 8 9 10 replicable science proportion 0.90 0.92 0.94 0.96 0.98 1.00 trustworthiness B-independent B = 0.65 B = 0.80 B = 0.95 (b) (a) (d) (e) (c) Figure5. Theimpactofpositivepublicationweighingonthetrustworthinessofpublishedscience.(a–c)Showfundingallocation95%, 80%, 65% of published results are positive, respectively, while (d) depicts the situation when publications are completely agnostic. Science trustworthiness of all these scenarios in shown in figure (e), suggesting best trustworthiness obtained when journals were completely agnostic to whether a result was positive or null. 0 94 funding cycles 2 4 6 8 10 0 0.2 0.4 0.6 0.8 proportion of resources (B = 0.80) (b) funding cycles 2 4 6 8 10 fraction of funding 0 0.2 0.4 0.6 0.8 proportion of resources (B = 0.95) diligent cohort careless cohort unethical cohort (a) 10 rsos.royalsocietypublishing.org R.Soc. opensci. 5: 171511 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 fraction of funding funding cycles 2 4 6 8 10 fraction of funding 0 0.2 0.4 0.6 0.8 proportion of resources (B = 0.65) (c) funding cycles 2 4 6 8 10 0 0.2 0.4 0.6 0.8 proportion of resources (B-independent) (d) . . . . . . . . . . . . . . . . funding cycles 2 4 6 8 10 fraction of funding 0 0.2 0.4 0.6 0.8 funding cycles 2 4 6 8 10 0 0.2 0.4 0.6 0.8 funding cycles 0 1 2 3 4 5 6 7 8 9 10 replicable science proportion 0.90 0.92 0.94 0.96 0.98 1.00 trustworthiness B-independent B = 0.65 B = 0.80 B = 0.95 (e) fraction of funding Figure5. 11 rsos.royalsocietypublishing.org R.Soc. opensci. 5: 171511 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . This is a natural consequence of the model, but requires careful interpretation. Crucially, it is important to note that there is no mechanism in the model for unethical or careless researchers to transition into diligent scientists. Rather, decreasing funding increases competition, and ampliﬁes the career advantages of questionable ﬁndings. Conversely, if global funding rates are increased, then competition for resources decreases and the advantage of suspect ﬁndings is somewhat mitigated. While beyond the scope of this work, such a prediction could be empirically tested by analysing situations when research budgets change markedly, such as the doubling of the NIH budget from 1998 to 2003. The model presented pivots on the assumption of a scenario that publication is the dominant metric upon which scientists are rewarded, and elucidates the potential consequences of such a situation. It is important to note this is a substantial simpliﬁcation, and there are other metrics by which scientists are assessed, including other measures of impact, awards and citations. However, the number of publications attributed to a scientist has a marked effect on their career success, with more publications associated with principal investigator status, and acquisition of funding [40]. The average number of authors per paper is increasing over time, and this is not just due to more interdisciplinary work, but also due to a greater demand for having more papers in one’s CV [41]. The model also implicitly assumes that output is an approximately linear function of funding in a given ﬁeld. The exact applicability of this assumption may vary across ﬁelds. For example, wet-lab sciences require a certain threshold of continuous funding just to operate, whereas computational or theoretical sciences may be able to operate with comparatively little funding. Presuming direct comparison of researchers and their teams across a given ﬁeld, however, the assumption of direct correspondence between resources and outputs is reasonable, although outliers are to be expected. One curious result persistently seen in the model was that diligent researchers are unfairly affected by careless or unethical conduct, with avoidable false positives or unethical publications garnering disproportionate reward at their expense. Simply increasing fraud detection does not do much to stop this, as careless researchers beneﬁt from the gap in the market, out-producing their diligent colleagues, as shown in ﬁgure 3. This appears to be an unfortunate and seemingly unavoidable consequence of a ‘publish or perish’ system. 11 rsos.royalsocietypublishing.org R.Soc. opensci. 5: 171511 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The model presented is a simpliﬁcation of a complex ecosystem, but gives some insight into what factors shape scientiﬁc trustworthiness. The model suggests that a ﬁxation in top-tier journals on signiﬁcant or positive ﬁndings tends to drive trustworthiness of published science down, and is more likely to select for false positives and fraudulent results. In our simulations, best outcome was obtained by simply paying no heed to whether a result was signiﬁcant or not. This is akin to the model used by many emerging open access peer-reviewed journals such as PLoS ONE, who have a policy of accepting any work provided it is scientiﬁcally rigorous. Our simulation suggests this model of publishing should improve science trustworthiness, and it is encouraging that many other publishers are taking this approach too, including Royal Society Open Science and Nature Scientiﬁc Reports. As of 2017, Scientiﬁc Reports has surpassed PLoS ONE as the world’s biggest mega-journal [38]. However, there is an important point to consider in the form of the parameter J (the publication carrying-capacity). This can be highly ﬁeld-speciﬁc, comprising the top-tier journals in that speciﬁc ﬁeld. In general, these publications are focused on prestige rather than rapid dissemination of science and it is unlikely these journals would move to replicate the approach of rapid open-access publishers. Accordingly, the suggestion that top-tier journals might aspire to treat all studies, regardless of their results, as equally worthy of publication is likely to be an unworkable ideal. g q y y p y Indeed, there is still a perception that such journals are for ‘trivial’ or unimportant results, and that positive or important results should still go to a few journals with extreme competition for space. Empirical evaluations show that small studies published in top-impact journals have markedly exaggerated results on average compared with similar studies on the same questions published in journals of lesser impact factor [39]. This suggests that the pressure to publish in these ﬂagship journals may still be very real, despite the option of publishing in less competitive journals. The analysis here suggests that science trustworthiness is affected too by changes in funding resources, and that when an increase of funding improves the overall trustworthiness of science, as depicted in ﬁgure 2. Conversely when this is diminished, the increased competition on scientists appears to create conditions when false positives and dubious results are more likely to be selected for and rewarded. 3.6. Impact of initial unethical funding proportion Theimpactofpositivepublicationweighingonthetrustworthinessofpublishedscience.(a–c)Showfundingallocation95%, 80%, 65% of published results are positive, respectively, while (d) depicts the situation when publications are completely agnostic. Science trustworthiness of all these scenarios in shown in figure (e), suggesting best trustworthiness obtained when journals were completely agnostic to whether a result was positive or null. funding cycles 0 2 4 6 8 10 replicable science proportion 0.90 0.91 0.92 0.93 0.94 zo = 2% zo = 1% zo = 3% Figure6. ThetrustworthinessofscienceinafieldwherepT = pF = 0.2,withvaryingvaluesofzO—thelessfundsareinitiallyallocated to unethical cohorts, the better the resultant science trustworthiness. funding cycles 0 2 4 6 8 10 replicable science proportion 0.90 0.91 0.92 0.93 0.94 zo = 2% zo = 1% zo = 3% Figure6. ThetrustworthinessofscienceinafieldwherepT = pF = 0.2,withvaryingvaluesofzO—thelessfundsareinitiallyallocated to unethical cohorts, the better the resultant science trustworthiness. Figure6. ThetrustworthinessofscienceinafieldwherepT = pF = 0.2,withvaryingvaluesofzO—thelessfundsareinitiallyallocated to unethical cohorts, the better the resultant science trustworthiness. on April 18, 2018 http://rsos.royalsocietypublishing.org/ Downloaded from 4. Discussion 11 rsos.royalsocietypublishing.org R.Soc. opensci. 5: 171511 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Finally, measures that can be adopted to begin changing the culture of ﬁxation on novel positive results include the establishment of awards by academic societies designed to recognize methodological rigour rather than positive results, as well as the explicit recognition of material published in online repositories as relevant material in university tenure and promotion guidelines. It is also worth considering how the positive publication weighing might impact on the ‘ﬁle- drawer’ problem [42]. This was the observation ﬁrst articulated by Rosenthal in 1979 that researchers tended to not invest their energy trying to publish null ﬁndings, instead burying them in a ﬁle- drawer. The great tragedy of this is that essential null results are often disregarded by the scientists who discover them, meaning others labour down fruitless avenues. In the model, we have implicitly assumed a version of this by assuming researchers submit only a portion of their negative ﬁndings (β) for consideration. It would be useful to know precisely how much is never submitted, and to gauge the extent of the ﬁle-drawer problem. Certainly estimates have been made in some ﬁelds, notably by Franco et al. [43], who determined that in one study of publications in social sciences, only 35% of the null results were ever written up (in good agreement with our estimates for β in table 1) and ultimately, just over 20.8% of these ﬁndings were published. Also for NIH-funded clinical trials, 32% remained unpublished a median of 51 months after their completion [44]. Whether these patterns apply also in other ﬁelds remains to be seen. One approach might be to consider the issue from an energy- expenditure point of view or game-theory approach which could be coupled with the model to estimate how much vital science never reaches the public domain, though this is beyond the scope of this investigation. A more sophisticated future analysis might include variables that respond to the available funding. For example, the fraudulent publication rate δ is treated as a constant in this work for the most part, but it is easy to imagine a situation where this increases with shrinking funding, or where the number of investigators willing to engage in such practices is a function of available funding. This is not considered here, but the model presented could be easily adapted to probe this further. rsos.royalsocietypublishing.org R.Soc. opensci. 5: 171511 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . While a penalty for carelessness has intuitive appeal, distinguishing between honest and careless errors is fraught with difﬁculty. As has been argued elsewhere [31,32], rewarding diligence is perhaps a better way to ensure researchers do not suffer for good conduct. A simple model of this is shown in ﬁgure 4, and indeed this suggests rewarding diligence improves the proportion of funding allocated to diligent groups. However, it requires some penalty for bad conduct to keep unethical cohorts from beneﬁting at the expense of others. In practice, this level of detection appears to have to be relatively high, which of course would require considerable resources to achieve. It should be noted too that the false positive rate of a ﬁeld has a signiﬁcant impact on science trustworthiness, as illustrated in ﬁgure 1. A high type II error rate provides ample cover for the small minority of unethical researchers to cheat without overt fear of detection [23,27], perhaps explaining the elevated prevalence of dubious practice in biomedical science [23], in particular. The model presented is a much simpliﬁed picture of reality, but it allows us to examine how different factors might inﬂuence the trustworthiness of published science, and potentially suggest strategies to improve it. As the motivations of and pressures on scientists are incredibly complex, it is important to recognize the limitations in the model too. The three cohorts presented here would in reality constitute a spectrum. The sub-divisions in this work are relatively arbitrary and informed by the available data on researcher populations, though it would be easily possible to extend this to consider more subpopulations if desired. Scientiﬁc conduct is notoriously difﬁcult to quantify, and the assumptions we have used in this work reﬂect the best estimates to date [23]. We can also envision a situation where authors are awarded solely on the basis of positive ﬁndings, so that negative ﬁndings have no funding beneﬁt. We can apply the model to these circumstances too, with the realization that under such a scheme, there would be no incentive for authors to submit negative results. In this case, B = 1 and all β terms reduce to zero. Essentially then, one gets a similar result to the one shown in ﬁgure 5a, with even further reduction in trustworthiness. 11 rsos.royalsocietypublishing.org R.Soc. opensci. 5: 171511 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . However, in good scientiﬁc environments carelessness would be sooner or later detected and potentially penalized. We can estimate how much of a penalty for carelessness or reward for diligence we need so as to inverse the worsening trends that we observe, by manipulating equations similar to the manner outlined for unethical conduct. However, this approach risks being on April 18, 2018 http://rsos.royalsocietypublishing.org/ Downloaded from ruthlessly punitive, punishing honest mistakes with the same severity reserved for the most egregious abuses of scientiﬁc trust. ruthlessly punitive, punishing honest mistakes with the same severity reserved for the most egregious abuses of scientiﬁc trust. 12 rsos.royalsocietypublishing.org R.Soc. opensci. 5: 171511 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . David Colquohoun FRS for his thoughtful discussion and advice. The authors would like to thank both the reviewers, Prof. Paul Smaldino and the BioRxiv community for helpful input and constructive criticism. Acknowledgements. D.R.G. thanks Dr Alex G. Fletcher and Dr David Basanta for their useful feedback and thoughtful imbibing. D.R.G. also thanks Prof. David Colquohoun FRS for his thoughtful discussion and advice. The authors would like to thank both the reviewers, Prof. Paul Smaldino and the BioRxiv community for helpful input and constructive criticism. References Chalmers I. 2014 Increasing value and reducing waste in biomedical research regulation and management. Lancet 383, 176–185. (doi:10.1016/ S0140-6736(13)62297-7) 12. Begley CG, Ioannidis JPA. 2015 Reproducibility in science: improving the standard for basic and preclinical research. Circ.Res. 116, 116–126. (doi:10.1161/CIRCRESAHA.114.303819) 13. Editorial. 2017 Fostering reproducible fMRI research. Nat.Neurosci. 20, 298. (doi:10.1038/nn.4521) 14. Begley CG, Ellis LM. 2012 Drug development: raise standards for preclinical cancer research. Nature 483, 531–533. (doi:10.1038/483531a) 15. Ritchie SJ, Wiseman R, French CC. 2012 Failing the future: three unsuccessful attempts to replicate Bem’s ‘retroactive facilitation of recall’ effect. PLoSONE 7, e33423. (doi:10.1371/journal.pone. 0033423) Chalmers I. 2014 Increasing value and reducing waste in biomedical research regulation and management. Lancet 383, 176–185. (doi:10.1016/ S0140-6736(13)62297-7) 12. Begley CG, Ioannidis JPA. 2015 Reproducibility in science: improving the standard for basic and preclinical research. Circ.Res. 116, 116–126. (doi:10.1161/CIRCRESAHA.114.303819) 13. Editorial. 2017 Fostering reproducible fMRI research. Nat.Neurosci. 20, 298. (doi:10.1038/nn.4521) 14. Begley CG, Ellis LM. 2012 Drug development: raise standards for preclinical cancer research. Nature 483, 531–533. (doi:10.1038/483531a) 15. Ritchie SJ, Wiseman R, French CC. 2012 Failing the future: three unsuccessful attempts to replicate Bem’s ‘retroactive facilitation of recall’ effect. PLoSONE 7, e33423. (doi:10.1371/journal.pone. 0033423) Chalmers I. 2014 Increasing value and reducing waste in biomedical research regulation and management. Lancet 383, 176–185. (doi:10.1016/ S0140-6736(13)62297-7) 1. Fanelli D. 2010 Do pressures to publish increase scientists’ bias? An empirical support from US states data. PLoSONE 5, e10271. (doi:10.1371/journal. pone.0010271) 6. Chavalarias D, Wallach JD, Li AHT, Ioannidis JPA. 2016 Evolution of reporting P values in the biomedical literature, 1990–2015. JAMA 315, 1141–1148. (doi:10.1001/jama.2016.1952) 6. Chavalarias D, Wallach JD, Li AHT, Ioannidis JPA. 2016 Evolution of reporting P values in the biomedical literature, 1990–2015. JAMA 315, 1141–1148. (doi:10.1001/jama.2016.1952) 2. Neill US. 2008 Publish or perish, but at what cost? J.Clin.Investig. 118, 2368–2368. (doi:10.1172/ JCI36371) 12. 7. Halsey LG, Curran-Everett D, Vowler SL, Drummond GB. 2015 The fickle P value generates irreproducible results. Nat.Methods 12, 179–185. (doi:10.1038/ nmeth.3288) 12. Begley CG, Ioannidis JPA. 2015 Reproducibility in science: improving the standard for basic and preclinical research. Circ.Res. 116, 116–126. (doi:10.1161/CIRCRESAHA.114.303819) 3. Bornmann L, Mutz R. 2015 Growth rates of modern science: a bibliometric analysis based on the number of publications and cited references. J.Assoc.Inf.Sci.Technol. 66, 2215–2222. (doi:10.1002/asi.23329) 8. Altman N, Krzywinski M. 2016 Points of significance: P values and the search for significance. Nat. Methods 14, 3–4. (doi:10.1038/nmeth.4120) 9. Colquhoun D. rsos.royalsocietypublishing.org R.Soc. opensci. 5: 171511 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . hing.org R.Soc. opensci. 5: 171511 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . We accordingly need to address alternative ways to assess researchers, and to encourage judicious diligence over dubious publishing. The model outlined here is far from complete, but yields some insights into the factors that shape the trustworthiness of published science. There is already evidence that pressure to publish is driving researcher burn-out and cynicism in published research [46], negatively affecting both research and the researchers themselves [47,48]. Other studies have not found a clear association of some productivity incentives with bias [49], but these incentives may be confounded in that sometimes they coexist with other features and research practices that tend to increase also quality of research, rather than just quantity of publications. Crucially, bogus ﬁndings risk undermining public conﬁdence in science. Among notable examples [50–52], the fraudulent Lancet MMR-Autism paper [53] is especially infamous, remaining a cornerstone of anti-vaccine narratives [54]. Scientiﬁc publishing is not intrinsically ﬂawed, and complete, unbiased publication is essential for scientiﬁc progress. This work illuminates potential consequences of a system where publication is the dominating measure of academic success, and strongly suggests we should consider the consequences of our incentives, and look at changing how academics are evaluated. This is key not only to appreciating the exceptional pressures wrought upon researchers by a strict publish or perish imposition, but to improving science itself. This would not only beneﬁt those working in the ﬁeld, but is crucial if public trust in science is to be maintained. Data accessibility. A demonstration version of the model coded is available at https://github.com/drg85/Publish-or- Perish, coded for MATLAB and as a windows stand-alone application. Authors’ contributions. D.R.G. derived the model with major contributions by C.T.B. and J.P.A.I. All authors co to the manuscript and gave ﬁnal approval for publication. Authors’ contributions. D.R.G. derived the model with major contributions by C.T.B. and J.P.A.I. All authors contributed to the manuscript and gave ﬁnal approval for publication. to the manuscript and gave ﬁnal approval for publication. Competing interests. We declare we have no competing interests. nding. We received no funding for this study. Acknowledgements. D.R.G. thanks Dr Alex G. Fletcher and Dr David Basanta for their useful feedback and thoughtful imbibing. D.R.G. also thanks Prof. on April 18, 2018 http://rsos.royalsocietypublishing.org/ Downloaded from on April 18, 2018 http://rsos.royalsocietypublishing.org/ Downloaded from It is also important to note that the model results pivot explicitly on the assumption that scientists are forced to operate under a ‘publish or perish’ regime, and rewarded solely on output. Thus, there is another way to improve the trustworthiness of published science—while publications are indeed one measure of productivity, they are not necessarily the sole measure. While a much harder aspect to gauge, trustworthiness is more fundamentally important. For their part, scientiﬁc journals should realize that issues such as replication and null ﬁndings are equally vital to good science as eye-catching ‘new’ results. This is slowly beginning to be recognized, with some groups coming to the forefront of championing reproducible research methods [45]. The consequences detailed in this manuscript only arise when publishing quantity is the dominant measure of an academic’s worth, but in reality this should only be one consideration among many. The model suggests that if publishing is the sole criteria under which academics are judged, then dubious conduct can thrive. 13 rsos.royalsocietypublishing.org R.Soc. opensci. 5: 171511 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future work with more sophisticated models could explore how best to implement these and other possible interventions designed to improve science trustworthiness. For instance, trustworthiness as a function of positive publication bias (B) and fraud detection rate (η) could be computed and optimization approaches could be applied to determine the optimal combination of B and η to improve science trustworthiness. These parameters can be somewhat inﬂuenced by large academic societies, government agencies or independent foundations for instance, who could fund efforts to detect fraud in published work and support research concerning null results. 14 rsos.royalsocietypublishing.org R.Soc. opensci. 5: 171511 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30. Fang FC, Steen RG, Casadevall A. 2012 Misconduct accounts for the majority of retracted scientific publications. Proc.NatlAcad.Sci.USA 109, 17 028–17 033. 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https://openalex.org/W4362649285	https://www.scielo.br/j/rgo/a/ND9c9RFgBXphPDbfyt6yVdD/?lang=en&format=pdf	English	null	Effect of different materials for temporary removable partial denture: an in vitro study on surface roughness and fracture resistance	RGO - Revista Gaúcha de Odontologia	2,023	cc-by	4,385	ABSTRACT The use of removable partial dentures is considered a common and inexpensive treatment option to rehabilitate edentulous areas. The aim of this study was to evaluate the effect of different materials for provisional removable partial dentures, an in vitro study on surface roughness and fracture resistance. Thermopolymerizable acrylic resin and thermoplastic resin specimens with dimensions of 10 x 10 x 2 mm (blocks) (n=10) and with dimensions of 65 x 20 x 3 mm (bars) (n=10 were analyzed for their surface roughness (Ra) and three-point flexural strength test. All specimens were subjected to oven aging for 60 days at 37°C±0.5°C in distilled water. The statistical analysis was performed with a significance level of 5%, and the surface roughness data were submitted to the Two-way Anova statistical test (P < 0.05) and the mechanical strength data were subjected to One-way Anova statistical test (P < 0.05). For roughness, the factor resin type (P = 0.000) and the factor aging (P = 0.000) were statically significant, as well as showing interaction (P = 0.228). The Thermopolymerizable acrylic resin had the lowest mean roughness values compared to the thermoplastic resin and showed no statistical difference after aging. The same scenario was repeated for flexural strength values; Thermopolymerizable acrylic resin was statistically superior to thermoplastic resin (P = 0.000). Thermopolymerized acrylic resin showed lower surface roughness values and higher flexural strength values compared to thermoplastic resin. Indexing terms: Dental prosthesis. Flexural strength. Polymers. Effect of different materials for temporary removable partial denture: an in vitro study on surface roughness and fracture resistance Effect of different materials for temporary removable partial denture: an in vitro study on surface roughness and fracture resistance Efeito de diferentes materiais para confecção de prótese parcial removível provisória: um estudo in vitro sobre rugosidade superficial e resistência à fratura Luiza Oliveira de ALMEIDA1 0000-0003-2763-7274 Manassés Tercio Vieira GRANGEIRO2 0000-0003-0955-0968 Viviane Maria Gonçalves de FIGUEIREDO1 0000-0003-4657-0984 RESUMO O uso de próteses parciais removíveis é considerado uma opção de tratamento comum e de baixo custo para reabilitar áreas edêntulas. O objetivo deste estudo foi avaliar o efeito de diferentes materiais para próteses parciais removíveis provisórias, um estudo in vitro sobre rugosidade superficial e resistência à fratura. Espécimes de resina acrílica termopolimerizável e de resina termoplástica com NICAL \| CLÍNIC \| ORIGINAL NICAL \| CLÍNIC \| ORIGINAL http://dx.doi.org/10.1590/1981-86372023000620210100 1 Universidade Federal de Pernambuco, Departamento de Prótese e Cirurgia Buco Facial. Av. Prof. Moraes Rego, 1235, Cidade Universitária, 50670-901, Recife, PE, Brasil. E-mail: <vivi_mfigueiredo@yahoo.com.br>. 2 Universidade Estadual Paulista, Instituto de Ciência e Tecnologia Júlio Mesquita Filho, ICT UNESP. São José dos Campos, SP, Brasil. How to cite this article Almeida LO, Grangeiro MTV, Figueiredo VMG. Effect of different materials for temporary removable partial denture: an in vitro study on surface roughness and fracture resistance. RGO, Rev Gaúch Odontol. 2023;71:e20230006. http://dx.doi.org/10.1590/1981- 86372023000620210100 ▼ ▼ ▼ ▼ ▼ ▼ ▼ ▼ ▼ ▼ Almeida LO, Grangeiro MTV, Figueiredo VMG. Effect of different materials for temporary removable partial denture: an in vitro study on surface roughness and fracture resistance. RGO, Rev Gaúch Odontol. 2023;71:e20230006. http://dx.doi.org/10.1590/1981- 86372023000620210100 ▼ ▼ ▼ ▼ ▼ 1 Universidade Federal de Pernambuco, Departamento de Prótese e Cirurgia Buco Facial. Av. Prof. Moraes Rego, 1235, Cidade Universitária, 50670-901, Recife, PE, Brasil. E-mail: <vivi_mfigueiredo@yahoo.com.br>. Almeida LO, Grangeiro MTV, Figueiredo VMG. Effect of different materials for temporary removable partial denture: an in vitro study on surface roughness and fracture resistance. RGO, Rev Gaúch Odontol. 2023;71:e20230006. http://dx.doi.org/10.1590/1981- 86372023000620210100 CC BY CC BY RGO, Rev Gaúch Odontol. 2023;71:e20230006 LO ALMEIDA et al. LO ALMEIDA et al. dimensões de 10 x 10 x 2 mm (blocos) (n = 10) e de 65 x 20 x 3 mm (barras) (n = 10) foram analisados em rugosidade superficial média (Ra) e teste de resistência à flexão de três pontos. Todos os espécimes foram submetidos ao envelhecimento em estufa por 60 dias a 37 ° C ± 0,5 ° C em água destilada. A análise estatística se deu com nível de significância de 5%, e os dados de rugosidade superficial foram submetidos ao teste estatístico Anova dois fatores (P <0,05) e os dados de resistência mecânica foram submetidos ao teste estatístico Anova um fator (P <0,05). Para rugosidade, o fator tipo resina (P = 0,000) e o fator envelhecimento (P = 0,000) foram estatisticamente significantes, além de apresentar interação (P = 0,228). A resina acrílica termopolimerizável apresentou menores valores médios de rugosidade em relação à resina termoplástica e não apresentou diferença estatística após envelhecimento. O mesmo cenário foi repetido para valores de resistência à flexão; a resina acrílica termopolimerizável foi estatisticamente superior à resina termoplástica (P = 0,000). A resina acrílica termopolimerizada apresentou menores valores de rugosidade superficial e maiores valores de resistência à flexão em relação à resina termoplástica. Termos de indexação: Prótese dentária. Resistência à flexão. Polímeros. Termos de indexação: Prótese dentária. Resistência à flexão. Polímeros. Termos de indexação: Prótese dentária. Resistência à flexão. Polímeros. INTRODUCTION The use of removable partial dentures (RPDs) is considered a common and inexpensive treatment option to rehabilitate edentulous areas [1]. Temporary RPDs are a transitional therapy for patients who have suffered the immediate loss of dental elements, as in cases where final metal-framed RPPs cannot be made. Thermally cured polymethyl methacrylate resin (PMMA), called Thermopolymerizable resin, has been used for many years to make the denture base of a final and a temporary RPD [2]. This material is rigid, easy to be relined, repaired, adjusted and polished, but its disadvantages are its toxicity, which is harmful to patients with allergic reactions to the monomer, and its high porosity, which may cause changes in the patient’s mucosa due to accumulation of debris [3]. In an attempt to solve the disadvantages presented by thermopolymerizable acrylic resin, to promote greater comfort for the patient and esthetics, thermoplastic resins have emerged for the construction of flexible temporary prostheses [3,4]. Flexible prostheses have been widely used in some countries and due to the growing popularity in the media; the thermoplastic (flexible) resin presents impact strength values similar to those of conventional resins (PMMA), thus showing that this material can be adopted in the long term [5]. Besides the promising results regarding mechanical strength [2], microhardness, solubility and sorption [6]. There are no clinical researches or studies on the long-term effects of flexible resins [3]. The literature is scarce regarding researches on the use of thermoplastic resins, there are many publications of clinical cases and literature reviews. Therefore, scientific evidence is required for the safe indication of this material by the dentist for the fabrication of temporary RPDs. Based on the above, this study aimed to evaluate the effect of different materials for fabrication of provisional partial dentures, an in vitro study on surface roughness and fracture resistance. The expected results for this research are: Null Hypothesis (H0): There will be no statistically significant differences between different materials for making provisional removable partial dentures, regarding the values of surface roughness and mechanical strength; Alternative Hypothesis 1 (H1): There will be statistically significant differences between different materials for making provisional removable partial dentures, regarding the values of surface roughness and mechanical strength. RGO, Rev Gaúch Odontol. 2023;71:e20230006 Specimen preparation Specimens were made using thermopolymerizable acrylic resin (Conventional) (VIPI Produtos Odontológicos, Pirassununga, São Paulo, Brazil) and thermoplastic resin (Flexible, polypropylene) (Odontoflex, Osasco, São Paulo, Brazil) with the aid of addition reaction silicone molds (3M ESPE, Bayern, Germany), with dimensions of 10 x 10 x 2 mm (blocks) and dimensions of 65 x 20 x 3 mm (bars). The blocks were used for surface analysis and the bars according to ADA specification 12.2 for mechanical testing. Wax patterns 7 were made in the silicone matrices, so that it was possible to RGO, Rev Gaúch Odontol. 2023;71:e20230006 2 Effect of different materials for temporary removable partial denture Effect of different materials for temporary removable partial denture produce the specimens, according to the polymerization recommendations of the manufacturers; for acrylic resin it was the conventional polymerization and for flexible resin it was the injection polymerization. produce the specimens, according to the polymerization recommendations of the manufacturers; for acrylic resin it was the conventional polymerization and for flexible resin it was the injection polymerization. After finishing the polymerization process, the lateral excesses were removed with a handpiece bur, and the dimensions of the specimens were checked with the aid of a digital pachymeter. Polishing was performed on only one face of the specimens, with siliconized rubbers (Acrylic Polisher Exa-Technique PM; Edenta - Labordental, São Paulo-SP), in the green, black and yellow sequence, each one acting on the surface for 1 min; followed by a brush for scratch removal (Brithe Red Coarse Scotch Brush PM, American Burrs, Palhoça -SC); brush for polishing (Suede brush, Derfla - Labordental, São Paulo-SP, Brazil) and brush for polishing and final brightness (Felt Brush for Polishing PM, American Burrs, Palhoça -SC), each brush acting on the material for 1 min. The other side remained unpolished, because when a temporary RPD is made, polishing occurs externally, and if there is an internal change of the piece, it will promote its maladaptation in the mouth. The unpolished side was marked with a permanent pen. Experimental design As there was only one variable under study, the types of resins, two experimental groups were formed, as shown in Table 1. Each experimental group had 10 block specimens for surface analysis and 10 bar specimens for mechanical testing (table 1). The sample size of this study was calculated based on the standard deviation of similar research, the study by Singh et al. [2], thus N=10 exceeds the sample power of 80.0% compared to maximums. The prepared specimens were stored in distilled water and after 24 hours the surface roughness analysis was initiated for the block specimens and the bar specimens followed in the oven aging. Table 1. Experimental groups, description, surface roughness test, fracture resistance test, and polymerization. Experimental groups Description Surface roughness test Fracture resistance test Polimerization RAT Thermopolymerizable Acrylic resin (conventional) N= 10 especimen Block N= 10 especimen Bar Conventional RTP Thermoplastic resin (flexible) N= 10 especimen Block N= 10 especimen Bar Injection Table 1. Experimental groups, description, surface roughness test, fracture resistance test, and polymerization. Experimental groups Description Surface roughness test Fracture resistance test Polimerization RAT Thermopolymerizable Acrylic resin (conventional) N= 10 especimen Block N= 10 especimen Bar Conventional RTP Thermoplastic resin (flexible) N= 10 especimen Block N= 10 especimen Bar Injection Table 1. Experimental groups, description, surface roughness test, fracture resistance test, and polymerization. Aging All study specimens were subjected to oven aging (FANEM, Orion Culture Greenhouse 502) for 60 days at 37°C±0.5°C in distilled water. After aging, the block specimens were subjected again to the surface roughness test and the bar specimens were subjected to the mechanical strength test. Surface roughness Specimens were evaluated for mean surface roughness (Ra) using a contact roughness (Mitutoyo surftest SJ-400, Mitutoyo Corporation, Japan); with a spherical diamond tip, 0.5 mm in radius size, which moves at a speed of 0.5 mm/s. The tip was programmed to travel a distance of 4 mm (cut-off sampling length = 0.8 mm). Three readings were taken on each specimen, polished side. The measurements in longitudinal direction, counting 3 readings per specimen and the roughness value was obtained by the arithmetic mean of the readings in each specimen and on each side. Analysis of results The results were tabulated and analyzed in Minitab software (version 16.1 for windows, Pennsylvania, USA), with a significance level of 5%. The surface roughness data were submitted to the Two-way Anova statistical test (P < 0.05), to evaluate the influence of materials and aging on topographic patterns. The mechanical strength data were submitted to the One-way Anova statistical test (P < 0.05), to identify the influence of the relationship between the supported stress and the materials until fracture. Tukey’s Test was applied when a P < 0.05 was observed. Mechanical resistance The three-point flexural strength test was performed in a universal testing machine EMIC model DL-1000 (EMIC DL 1000, São José dos Pinhais, Brazil). The bar specimens were submitted to tension until fracture, with the polished RGO, Rev Gaúch Odontol. 2023;71:e20230006 3 LO ALMEIDA et al. side facing down. The machine was programmed with a 100Kgf load cell, for a constant speed of 5mm/min, the flexural strength values were obtained in megapascals (Mpa) using the formula below (ISO 1567:1999). γ=3FD/2LH2 F - Maximum Force (N) D - Distance on test device (50 mm) L - Specimen length (65 mm) H - Specimen thickness (3 mm) γ=3FD/2LH2 F - Maximum Force (N) D - Distance on test device (50 mm) L - Specimen length (65 mm) H - Specimen thickness (3 mm) Qualitative analysis Stereomicroscope images (Discovery V20, Zeiss, Gottingen, Germany) were taken of the surfaces of the resin materials as well as an image after the mechanical strength test. RESULTS The study data showed statistical difference between experimental groups. For roughness, the Two-way Anova Test identified that the resin type factor (P = 0.000) and the aging factor (P = 0.000) were statically significant, and also showed interaction (P = 0.228), as shown in table 2. The Thermopolymerizable acrylic resin had the lowest mean roughness values compared to the thermoplastic resin and showed no statistical difference after aging, according to Tukey’s Test. The values of flexural strength, the thermopolymerizable acrylic resin was statistically superior to the thermoplastic resin (P = 0.000), according to the One-way Anova Test (table 3). Table 2. Experimental groups (resin type and aging), Mean Ra in µm (Standard Deviation - SD) and Grouping. Experimental groups Mean Ra in µm (SD) Grouping Resin Type Aging RAT Dry 0.03477 (0.003916) A RAT Aging 0.04283 (0.006962) A RTP Dry 0.05860 (0.006560) B RTP Aging 0.07200 (0.009080) C Note: * Tukey’s test shows difference between groups with different letters. Table 2. Experimental groups (resin type and aging), Mean Ra in µm (Standard Deviation - SD) and Grouping. Note: Tukey’s test shows difference between groups with different letters. Note: * Tukey’s test shows difference between groups with different letters. Note: * Tukey’s test shows difference between groups with different letters. 4 RGO, Rev Gaúch Odontol. 2023;71:e20230006 RGO, Rev Gaúch Odontol. 2023;71:e20230006 Table 3. Experimental groups (Resin Type and Aging), Mean in Mpa (Standard Deviation - SD) and P-value. Experimental groups Mean in Mpa (SD) P-valor Resin Type Aging RAT Aging 44.85 (6.482) 0.000 RTP Aging 27.22 (3.837) Note: * One-way Anova test is significant when P < 0.05. Table 3. Experimental groups (Resin Type and Aging), Mean in Mpa (Standard Deviation - SD) and P-value. RAT Aging 44.85 (6.482) 0.000 RTP Aging 27.22 (3.837) Note: One-way Anova test is significant when P < 0.05. Figure 1. Defects and Pores in the thermoplastic resin specimens. Figure 2. Polished and unpolished surface of the resins under study, A-RAT polished (15x magnification stereomicroscope image), B-RAT unpolished (15x magnification stereomicroscope image), C-RTP polished (12x magnification stereomicroscope image), D-RTP unpolished (12x magnification stereomicroscope image). Figure 1. Defects and Pores in the thermoplastic resin specimens. Figure 2. Polished and unpolished surface of the resins under study, A-RAT polished (15x magnification stereomicroscope image), B-RAT unpolished (15x magnification stereomicroscope image), C-RTP polished (12x magnification stereomicroscope image), D-RTP unpolished (12x magnification stereomicroscope image). Figure 2. RESULTS Polished and unpolished surface of the resins under study, A-RAT polished (15x magnification stereomicroscope image), B-RAT unpolished ( magnification stereomicroscope image), C-RTP polished (12x magnification stereomicroscope image), D-RTP unpolished (12x magnificat stereomicroscope image). Figure 2. Polished and unpolished surface of the resins under study, A-RAT polished (15x magnification stereomicroscope image), B-RAT unpolished (15x magnification stereomicroscope image), C-RTP polished (12x magnification stereomicroscope image), D-RTP unpolished (12x magnification stereomicroscope image). RGO, Rev Gaúch Odontol. 2023;71:e20230006 5 LO ALMEIDA et al. After polymerization of the thermoplastic resin, large amounts of defects and pores were identified in some specimens (figure 1). The polished and unpolished surface of the specimens for both resins, through stereomicroscope imaging, no surface changes are observed between the materials (figure 2). During the flexural strength test, the specimens of thermoplastic resin suffered deformation and did not return to the initial state, but no destructive fracture occurred as with the thermopolymerizable acrylic resin (figure 3). Figure 3. A) Bar specimens of the RTP group after the flexural strength test, the arrow shows the permanent change of the specimen; B) Arrow points are where the chisel was positioned; C) Stereomicroscope image (13x magnification) shows the stress area (arrow) suffered by the specimens during th mechanical test. Figure 3. A) Bar specimens of the RTP group after the flexural strength test, the arrow shows the permanent change of the specimen; B) Arrow points area where the chisel was positioned; C) Stereomicroscope image (13x magnification) shows the stress area (arrow) suffered by the specimens during the mechanical test. RGO, Rev Gaúch Odontol. 2023;71:e20230006 DISCUSSION Replacing lost teeth with RPD by present a considerable demand in the coming years, therefore, it is essential to promote health and training of professionals involved in the treatment, as well as research that shows the performance of alternative materials in the long term. Therefore, it is necessary to study dental materials, their physical and chemical characteristics, and how much they influence, beneficially or not, the final product. The selection of the right material largely depends on the scenario, in which it is to be used, and erroneous selection can lead to failure of the prosthetic rehabilitation [2]. Currently, there is not enough information about RPD without metal clampless and with thermoplastic resin. Professional should be well aware of the properties of these resins, in order to choose a suitable resin for each patient [7]. The research findings indicate that the thermoplastic resin after aging showed higher mean roughness values, while the thermopolymerizable resin showed lower roughness values and did not undergo the action of aging. These results are in agreement with the literature surveyed [2,8,9]. Importantly, a study found that immersion of both resins in red wine and instant coffee did not significantly alter surface roughness values [9]. As for biofilm growth, polyamide resin (flexible) showed greater growth of Candida spp. compared to PMMA (conventional), and its surface roughness was standardized (0.34 ± 0.02 μm) [10]. In the study by Abuzar et al. [8], the surface roughness of polyamide is smoothed after mechanical lathe polishing, and is within the clinically accepted limit of 0.2 μm. 6 Effect of different materials for temporary removable partial denture Effect of different materials for temporary removable partial denture The flexural strength of conventional resin was statistically superior to flexible resin, and the literature shows similar [5,6,11-13] and distinct [2,14] results, i.e., higher mechanical strength of flexible resins. The mechanical performance of resins is affected by material properties, polymerization technique, and aging [7,14]. Different types of artificial aging have tested properties of these resins and proved to be significant for flexural strength values [7,9,11-13,15]. Perhaps, the reason for the low mechanical strength of the flexible resin is due to the high number of defects and pores on the surface of the specimens by the injection technique. Thus, it promoted an increase in water sorption since the bars were aged for 60 days in distilled water. According to Hamanaka et al. DISCUSSION [15], water sorption significantly reduced the mechanical strength and modulus of elasticity of Polyamide (flexible) and PMMA (conventional). An important point to be registered is that fracture analysis, after the flexural strength test, has been little addressed in the literature [7,12]. The present research observed that all thermoplastic resin specimens did not fracture after the mechanical test, a situation that corroborates other published studies [7,12]. In contrast, several studies do not state the fracture condition of the specimens [2,5,11,13-15]. The fracture behavior of a material is relevant to the dental decision in the treatment of his patient [12]. The study by Hamanaka et al. [7] did not obtain any fractured specimens (Thermopolymerizable resin and thermoplastic resins), however the methodology applied was deflection observed through mechanical cycling (5,000 cycles). In the research of Ucar et al. [12] all polyamide specimens (flexible) did not fracture during the flexural test and showed a high risk of deflection, corroborating the findings of this study. So why are flexible resins not extensively applied in prosthetics in daily practice? Since this material does not present fracture after mechanical testing. This behavior is due to the plastic deformation property of these resins, when it exceeds the proportional limit there will be a permanent change and the high or similar, to conventional resin, impact resistance. Therefore, this behavior is unacceptable for denture base materials, as it interferes in the retention and stability of the Removable Partial Denture without clamp [7]. Denture materials must have a high proportional limit to avoid permanent deformation, low elasticity modulus to avoid stress of insertion and removal of the prosthesis, and high impact resistance [7]. The flexibility of these resins is favorable for clamps, but not for making a larger connector, as there is a need for rigidity [12]. Flexible resins are an option for prosthetic materials for individuals allergic to polymethylmethacrylate or cobalt chromium, in cases of constant fractures of conventional prostheses, in patients with reduced manual dexterity, and also in conditions of torus, cleft palate or knife-edge ridge [3,4]. Based on the roughness and mechanical strength results of this research, extrapolating to clinical practice the use of flexible resin as a material should be performed with limitations, since there is an increase in surface roughness after aging, a factor that favors biofilm accumulation. REFERENCES 9. Wieckiewicz M, Opitz V, Richter G, Boening KW. Physical properties of polyamide-12 versus PMMA denture base material. BioMed Res Int. 2014;2014:150298. https://doi. org/10.1155/2014/150298 1. Moreno A, Haddad MF, Goiato MC, Rocha EP, Assunção WG, Gennari Filho H, et al. Epidemiological Data and Survival Rate of Removable Partial Dentures. J Clin Diagn Res. 2016;10(5):84- 7. https://doi.org/10.7860/JCDR/2016/16638.7816 10. Fernandes FSF, Pereira-Cenci T, Silva WJ, Ricomini Filho AP, Straioto FG, Cury ADB. Efficacy of denture cleansers on Candida spp. biofilm formed on polyamide and polymethyl methacrylate resins. J Prosthet Dent. 2011;105(1):51-58. https://doi.org/10.1016/S0022-3913(10)60192-8 2. Singh R, Chawla PS, Shaw E, AV R, Mehrotra A, Pahndey V. Comparison of flexural strenght and surface roughness of two different flexible and heat cure denture base material: An in vitro study. J Contemp Dent Pract. 2018;19(10):1214-1220. https://doi.org/10.5005/jp-journals-10024-2407 11. Jagini AS, Marri T, Jayyarapu D, Kumari R, D V, K M. Effect of long-term immersion in water and artificial saliva on the flexural strenght of two heat cure denture base resins. J Contemp Dent Pract. 2019;20(3):341-346. https://doi. org/10.5005/jp-journals-10024-2520 3. Patrocínio BMG, Antenor AM, Haddad MF. Prótese Parcial Removível Flexível – revisão de literatura. Arch Health Invest. 2017;6(6):258-263. https://doi.org/doi.org/10.21270/archi. v6i6.2065 4. Vojdani M, Giti R. Polyamide as a denture base material: A literature review. J Dent (Shiraz). 2015;16(1):1-9. 12. Ucar Y, Akova T, Aysan I. Mechanical properties of polyamide versus different PMMA denture base materials. J Prosthodont. 2012;21(3):173-6. https://doi.org/10.1111/j.1532- 849X.2011.00804.x 5. Abhay PN, Karishma S. Comparative evaluation of impact and flexural strength of four commercially available flexible denture base materials: An in vitro study. J Indian Prosthodont Soc. 2013;13(4):499-508. https://doi.org/10.1007/s13191- 012-0203-0 13. Shah VR, Shah DN, Chauhan CJ, Doshi PJ, Kumar A. Evaluation of flexural strenght and color stability of different denture base materials including flexible materials after using different denture cleansers. J Indian Prosthodont Soc. 2015;15(4):367- 373. https://doi.org/10.4103/0972-4052.164908 6. Shah J, Bulbule N, Kulkarni S, Shah R, Kakade D. Comparative evaluation of sorption, solubility and microhardness of heat cure polymethylmethacrylate denture base resin & flexible denture base resin. J Clin Diagn Res. 2014;8(8):1-4. https:// doi.org/10.7860/JCDR/2014/8707.4770 14. Takahashi Y, Hamanaka I, Shimizu H. Effect of thermal shock on mechanical properties of injection-molded thermoplastic denture base resins. Acta Odontol Scand. 2012;70(4):297-302. https://doi.org/10.3109/00016357.2011.600719 7. Hamanaka I, Iwamototo M, Lassila L, Vllittu P, Shimizu H, Takahashi Y. Influence of water sorption on mechanical properties of injection-molded thermoplastic denture base resins. Acta Odontol Scand. 2014;72(8):859-65. https://doi. org/10.3109/00016357.2014.919662 15. CONCLUSION Thermopolymerized acrylic resin showed lower surface roughness values and higher flexural strength values compared to thermoplastic resin. The effect of aging showed no change in mean roughness values for the conventional resin, but was significant for the flexible resin. DISCUSSION Besides the low flexural strength, which although it does not promote destructive fracture of the material, there is permanent deformation, which may interfere with retention and stability of the prosthesis? Considering this material as a provisional for partially edentulous arches, perhaps it could be adopted, although studies with more robust scientific evidence are still needed. Among the limitations of the study was the presence of defects in the thermoplastic resin specimens due to the injection technique, thereby increasing roughness and reducing flexural strength. It seems that this situation occurred due to the technique being indicated for making prostheses with less thickness, the use of geometric specimens in the research made the polymerization of flexible resins more difficult. New studies should seek to evaluate the mechanical aging of these resins and adopt specimens that are close to the real patterns of the prostheses in order to evaluate the survival of this material. Try to test different possibilities of polishing, adopted in the dental office, in order to reduce surface roughness. Randomized clinical studies on retention, stability and patient satisfaction with the use of thermoplastic resin prostheses are also desirable. Thus, the null hypothesis of the study was rejected and the alternative hypothesis was accepted. Because there was a statistically significant difference between different materials for making temporary partial dentures, regarding the values of surface roughness and mechanical strength. RGO, Rev Gaúch Odontol. 2023;71:e20230006 LO ALMEIDA et al. Collaborators LO Almeida, analysis, data interpretation and article writing. MTV Grangeiro, conception and design, analysis and data interpretation. VMG Figueiredo, conception and design, analysis and data interpretation and article writing. REFERENCES Hamanaka I, Iwamototo M, Lassila L, Vallittu PK, Hiroshi S, Takahashi Y. The effect of cycling deflection on the injectionmolded thermoplastic denture base resins. Acta Odontol Scand. 2015;74(1):67-72. https://doi.org/10.3109/0 0016357.2015.1042039 8. Abuzar MA, Bellur S, Duong N, Kim BB, Lu P, Palfreyman N, et al. Evaluating surface roughness of a polyamide denture base material in comparison with poly (methyl methacrylate). J Oral Sci. 2010;52(4):577-581. https://doi.org/10.2334/ josnusd.52.577 Received on: 1/9/2021 Final version resubmitted on: 20/5/2022 Assistant editor: Fabiana Mantovani Gomes França Received on: 1/9/2021 Final version resubmitted on: 20/5/2022 Assistant editor: Fabiana Mantovani Gomes França Received on: 1/9/2021 Final version resubmitted on: 20/5/2022 RGO, Rev Gaúch Odontol. 2023;71:e20230006 8
https://openalex.org/W3083341292	https://pubs.rsc.org/en/content/articlepdf/2020/tb/d0tb01546f	English	null	Tuneable peptide cross-linked nanogels for enzyme-triggered protein delivery	Journal of materials chemistry. B	2,020	cc-by	11,611	Journal of Materials Chemistry B View Article Online View Journal \| View Issue Open Access Article. Published on 03 September 2020. Downloaded on 10/24/2024 5:42:34 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Tuneable peptide cross-linked nanogels for enzyme-triggered protein delivery† Tuneable peptide cross-linked nanogels for enzyme-triggered protein delivery† Lucia Massi,‡a Adrian Najer, ‡a Robert Chapman, b Christopher D. Spicer, §a Valeria Nele, a Junyi Che,a Marsilea A. Booth,a James J. Doutchc and Molly M. Stevens a y gg p y† Lucia Massi,‡a Adrian Najer, ‡a Robert Chapman, b Christopher D. Spicer, §a Valeria Nele, a Junyi Che,a Marsilea A. Booth,a James J. Doutchc and Molly M. Stevens a Cite this: J. Mater. Chem. B, 2020, 8, 8894 Cite this: J. Mater. Chem. B, 2020, 8, 8894 Cite this: J. Mater. Chem. B, 202 8, 8894 Received 22nd June 2020, Accepted 22nd August 2020 DOI: 10.1039/d0tb01546f rsc.li/materials-b ess Article. Published on 03 September 2020. Downloaded on 10/24/2024 5:42:34 This article is licensed under a Creative Commons Attribution 3.0 Unported Lucia Massi,‡a Adrian Najer, ‡a Robert Chapman, b Christopher D. Spicer, §a Valeria Nele, a Junyi Che,a Marsilea A. Booth,a James J. Doutchc and Molly M. Stevens a Many diseases are associated with the dysregulated activity of enzymes, such as matrix metalloproteinases (MMPs). This dysregulation can be leveraged in drug delivery to achieve disease- or site-specific cargo release. Self-assembled polymeric nanoparticles are versatile drug carrier materials due to the accessible diversity of polymer chemistry. However, eﬃcient loading of sensitive cargo, such as proteins, and introducing functional enzyme-responsive behaviour remain challenging. Herein, peptide-crosslinked, temperature-sensitive nanogels for protein delivery were designed to respond to MMP-7, which is overexpressed in many pathologies including cancer and inflammatory diseases. The incorporation of N-cyclopropylacrylamide (NCPAM) into N-isopropylacrylamide (NIPAM)-based copolymers enabled us to tune the polymer lower critical solution temperature from 33 to 44 1C, allowing the encapsulation of protein cargo and nanogel-crosslinking at slightly elevated temperatures. This approach resulted in nanogels that were held together by MMP-sensitive peptides for enzyme- specific protein delivery. We employed a combination of cryogenic transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS), small angle neutron scattering (SANS), and fluorescence correlation spectroscopy (FCS) to precisely decipher the morphology, self-assembly mechanism, enzyme-responsiveness, and model protein loading/release properties of our nanogel platform. Simple variation of the peptide linker sequence and combining multiple diﬀerent crosslinkers will enable us to adjust our platform to target specific diseases in the future. Received 22nd June 2020, Accepted 22nd August 2020 DOI: 10.1039/d0tb01546f rsc.li/materials-b Received 22nd June 2020, Accepted 22nd August 2020 Received 22nd June 2020, Accepted 22nd August 2020 rsc.li/materials-b a Department of Materials, Department of Bioengineering, and Institute of Biomedical Engineering, Imperial College London, London SW7 2AZ, UK. E-mail: m.stevens@imperial.ac.uk b Centre for Advanced Macromolecular Design (CAMD) and the Australian Centre for Nanomedicine (ACN), School of Chemistry, UNSW Sydney, Australia c ISIS Neutron and Muon Source, STFC, Rutherford Appleton Laboratory, Didcot OX11 ODE, UK † Electronic supplementary information (ESI) available. See DOI: 10.1039/ d0tb01546f ‡ Contributed equally. § Current address: Department of Chemistry and York Biomedical Research Institute, University of York, Heslington, YO10 5DD, UK. This journal is © The Royal Society of Chemistry 2020 ‡ Contributed equally. Open Access Article. Published on 03 September 2020. Downloaded on 10/24/2024 5:42:34 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Our strategy uses temperature changes as a simple and fast organic solvent-free nanogel formation method to encapsulate biomolecules. Poly(N-cyclopropylacrylamide) (PNCPAM), closely related to PNIPAM, consists of pendant cyclopropylamine groups instead of the isopropylamine groups in PNIPAM and possesses a LCST of B49 1C.10 We propose herein that the copolymerisation of NCPAM and NIPAM to form block copolymers together with a hydrophilic block—consisting of a short block with functional reactive handles and polyethylene glycol (PEG)—provides an eﬃcient method to precisely control the LCST, and hence the self-assembly temperature, from 32 up to 49 1C. Fine-tuning of the transition temperature using a library of copolymers, with precise control of the LCST around body temperature, enables application-dependent selection of a certain copolymer from the library to create customised vehicles for loading and release of sensitive therapeutic protein cargoes. A cross-linking strategy was further included to (i) stabilise the nanocarrier, (ii) avoiding premature release of cargo, and (iii) incorporating stimuli-responsiveness. This additional cross-linking strategy serves as an additional trigger besides temperature for cargo release, through degradation of the responsive cross-links. These are important considerations when taking into account the immensely complex environment faced by nanocarriers upon in vivo administration. We employed a copper-catalysed azide–alkyne cycloaddition (CuAAC) cross- linking strategy, which remains one of the most popular ‘‘click’’ reactions for cross-linking and functionalisation in the bio- material and biomedical fields due to high efficiency, specifi- city, and simplicity, to incorporate the necessary functional handles.15–17 Our covalent cross-linking strategy provided the fundamental advantage of keeping our nanogels intact above and below the LCST, which in turn allows fast and simple loading of cargo and subsequent purification. g A considerable challenge when deciphering the exact mor- phology of copolymer assemblies is the structural ambiguity that is provided by certain routine characterisation techniques such as conventional dynamic light scattering (DLS) and nega- tive stain transmission electron microscopy (TEM). However, accurate information on morphology is of great importance since it directly aﬀects choice of suitable disease targets, drug cargo, delivery route, stability and release mechanism. Thus, the use of additional complementary characterisation techniques is needed to characterise the morphology of soft self-assembled nanostructures. Introduction which include facile functionalisation with targeting ligands, in addition to triggerable drug release via stimuli-responsive copolymers.2–5 Typical morphologies of polymer-based structures used for drug delivery include micelles (spherical and worm-like), vesicles (polymersomes), capsules, polyion complex (PIC) micelles and vesicles, nanoparticles, and nanogels. The key advantages offered by nanogels are their ease of preparation that yields uniform and tuneable sizes, relatively high encapsulation effi- ciency, stability in the presence of serum, and the possibility of simple introduction of stimuli responsiveness.6 The properties and formation method of nanogels make them suitable candi- dates for protein delivery.7,8 The development of smart and controlled nanocarriers for targeted and triggered drug delivery provides therapeutic opportunities for the treatment of major diseases such as infections, inflammation, and cancer.1 Synthetic amphiphilic copolymers, able to undergo self-assembly into nanostructures, are ideal carriers for drug delivery due to the flexibility and diversity of polymer chemistry. Various carrier morphologies can be engineered with tuneable stabilities and surface chemistries, To yield high level of control of drug loading and triggered release, temperature and/or enzyme-responsive motifs can be incorporated in polymer-based drug delivery.9 With respect to temperature-triggered systems, poly(N-isopropylacrylamide) (PNIPAM) remains one of the most studied polymers incorpo- rated in the design of temperature-responsive materials for biomedical applications due to its fast temperature-induced phase transition at a lower critical solution temperature (LCST) of B32 1C.10,11 Copolymers of PNIPAM with a temperature- independent hydrophilic block allow rapid assembly of 8894 \| J. Mater. Chem. B, 2020, 8, 8894--8907 This journal is © The Royal Society of Chemistry 2020 View Article Online Paper Paper Journal of Materials Chemistry B B nanostructures upon heating either after or during the poly- merisation because of the thermally induced collapse of the PNIPAM block at temperatures above the LCST.12 However, PNIPAM-based nanoassemblies are limited to transition around 32 1C; the LCST cannot be changed easily. Copolymerisation of NIPAM with other co-monomers is one option to modulate the LCST.13,14 However, temperature is not an ideal trigger for cargo release given our fixed body temperature. Hence, modulation of the self-assembly temperatures (LCST) using a range of diﬀerent copolymers, combined with a suitable stimuli-responsive cross- linking strategy, represents an interesting alternative platform that could be employed both above and below the LCST. This journal is ©The Royal Society of Chemistry 2020 J. Mater. Chem. B, 2020, 8, 8894--8907 \| 8895 Introduction associated with several disease conditions, including infections, inflammation, cardiovascular diseases and cancer.23 Over- expression of MMPs at disease sites can be exploited to release drugs at the target site by selective rupture of engineered MMP-responsive delivery platforms.22,24 Herein, we successfully synthesised libraries of PNIPAM-, PNCPAM- and P(NIPAM-co-NCPAM)-based triblock copolymers with tuneable compositions via reversible addition–fragmentation chain-transfer (RAFT) polymerisation. The structure of the final block copolymers was carefully designed to include these temperature-responsive blocks, a functional block for nanogel cross-linking and a hydrophilic outer block. The thermo- responsive block is hydrophilic below its LCST and turns hydrophobic above the LCST, thus switching the block copolymer from hydrophilic to amphiphilic, which promotes the formation of self-assembled nanostructures. The choice over amphiphilicity or hydrophilicity of degradation products at body temperature, through selecting a copolymer with a transition temperature below or above 37 1C, offers means of potentially modulating elimination pathways upon in vivo administration in the future. Choosing a suitable NIPAM– NCPAM ratio in the thermo-responsive block will further allow one to adjust the LCST to only slightly below body temperature, which could be a means of speeding up cargo release at the disease site, when close to the surface, through locally applied cold patches. To increase nanoparticle stability and incorporate responsiveness to a model MMP enzyme (MMP-7), azido-bi- functionalised peptides were synthesised and used to cross-link the nanoparticles via the alkyne groups present in the functional middle block using CuAAC. Results and discussion The first step consisted of the synthesis of the RAFT agent 1, following a procedure previously reported in the literature,41 which was then conjugated to a PEG block (113 units) via esterification to form a macroRAFT agent. Chain extension of this macroRAFT agent with a random copolymer of MAA and TMSPMA enabled the introduction of carboxylic groups and terminal alkyne functionalities (in their protected form) as functional handles. Good control over the polymerisation of PEG-b-P(MAA-co-TMSPMA) 3 was evidenced by low dispersity Ð = 1.07, and a clean shift in the gel permeation chromato- graphy traces (GPC, Fig. S1a, ESI†). The same polymer (with 113 PEG units, 5 MAA units and 19 TMSPMA units) was used throughout the remainder of the study and chain extended to produce three diﬀerent libraries of triblock copolymers: (i) a library of PNIPAM-based block copolymers (5a1–5a5) with increasing number of NIPAM units; (ii) a library of PNCPAM- based block copolymers (5b1–5b5) with increasing number of NCPAM units; (iii) a library of P(NIPAM-co-NCPAM)-based block copolymers (5c1–5c3) with different NIPAM/NCPAM (NI/NC) ratios. We hypothesised that the incorporation of cyclopropyl moieties (NCPAM) would lead to an increase in the LCST providing a Tcp (cloud point temperature as measured by DLS) Open Access Article. Published on 03 September 2020. Downloaded on 10/24/2024 5:42:34 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. TEM at cryogenic temperatures (cryo-TEM)25,26 and small-angle neutron scattering (SANS)27 are two complemen- tary methods to study these types of nanostructures in their native hydrated state.28–30 A third technique, fluorescence correlation spectroscopy (FCS) is a diffusion-based single-molecule analysis technique.31,32 FCS allows accurate characterisation of nano- particle assembly/disassembly—including in the presence of enzymatic triggers—surface functionalisation, cargo loading, and release.33–35 Our thoroughly characterised nanogel system is shown to serve as a versatile protein delivery platform with a simple and gentle temperature induced formation and loading protocol, and triggered release through stimuli-responsiveness provided by enzymatically cleavable cross-links. This modular platform can be adjusted for disease-specific applications by choosing the appropriate copolymer, peptide cross-linker sequence, and therapeutic protein cargo. With respect to cross-linker backbone chemistry, peptides are particularly attractive due to their simple solid phase synthesis, incorporation of non-canonical functional groups for the introduction of ‘‘clickable’’ handles,18,19 and the possi- bility of using enzyme-specific cleavable peptide sequences.19,20 Due to their intrinsic involvement in biological and metabolic processes, enzymes have emerged as suitable triggers for drug delivery.21 Diﬀerent classes of enzymes (e.g. proteases, phos- pholipases, oxidoreductases) have been leveraged for the purpose of triggered drug release from nanocarriers.22 A specific class of proteolytic enzymes, matrix metalloproteinases (MMPs), are J. Mater. Chem. B, 2020, 8, 8894--8907 \| 8895 Paper View Article Online View Article Online Journal of Materials Chemistry B Journal of Materials Chemistry B Journal of Materials Chemistry B Paper Synthesis of peptide cross-linkable block copolymers with fine-tuned temperature-responsiveness We designed triblock copolymers to confer the final self- assembled nanogels with favourable properties for enzyme- triggered drug delivery applications (Scheme 1). Conceptually, an inert hydrophilic block (Scheme 1, dark blue), which we expected to form the corona of the assembled nanogels, was connected to a thermo-sensitive block (Scheme 1, light blue/green) which would drive self-assembly at temperatures above its LCST. We chose to use PEG as the hydrophilic block and either PNIPAM, PNCPAM or copolymers of the two P(NIPAM-co-NCPAM) as the thermo- responsive block. Between these two blocks we incorporated a functional block (Scheme 1, red) composed of a mixture of methacrylic acid (MAA) and trimethylsilylpropargylmethacrylate (TMSPMA) to provide a latent handle for cross-linking with azide- bi-functionalised peptide after self-assembly and desilylation. Open Access Article. Published on 03 September 2020. Downloaded on 10/24/2024 5:42:34 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. The small diﬀerence in the number mean size between 5a4 (69.0 2.7 nm, n = 3, N = 3) and 5c1 (98.1 9.6 nm, n = 3, N = 3) above the LCST is significant (p = 0.043, Student’s t-test, n = 3, N = 3) when comparing three indepen- dent samples. The evolution of Mn and Ð versus DP is shown in Fig. S1b (ESI†). We also determined the block copolymer cloud point tempera- tures Tcp via temperature sweeps and monitoring by dynamic light scattering (DLS). The corresponding sizes of the self- assembled nanostructures resulting from heating the block copolymer solutions above the Tcp are included in Table 1 and Table S6 (ESI†). The small diﬀerence in the number mean size between 5a4 (69.0 2.7 nm, n = 3, N = 3) and 5c1 (98.1 9.6 nm, n = 3, N = 3) above the LCST is significant (*p = 0.043, Student’s t-test, n = 3, N = 3) when comparing three indepen- dent samples. Successful synthesis of the three block copolymer libraries was confirmed by 1H NMR and GPC yielding degree of poly- merisation (DP), number average molecular weight (Mn), and dispersity (Ð) (Table 1). Full composition and characterisation details of selected 5a4/5c1–3 block copolymers are reported in Table 1, whilst data for the remaining block copolymers can be found in Table S6 of the ESI.† For the P(NIPAM-co-NCPAM)-based copolymers (5c1–3), full chain extension was observed for all the copolymers with a minimal amount of macro-RAFT agent left behind and minimal shouldering in the GPC traces (Fig. S1a, ESI†). Due to the structural similarity and similar degree of reactivity between NIPAM and NCPAM, the formation of a random block stem- ming from their simultaneous polymerisation rather than two distinct blocks was assumed. In general, the dispersity was Ð o 1.5. The length of the thermo-responsive block was successfully modulated in the separate NIPAM and NCPAM libraries (Table S6, ESI†), whilst the copolymers shown in Table 1 were of similar length, as shown by NMR and GPC. Open Access Article. Published on 03 September 2020. Downloaded on 10/24/2024 5:42:34 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 03 September 2020. Downloaded on 10/24/2024 5:42:3 This article is licensed under a Creative Commons Attribution 3.0 Unported We used RAFT polymerisation for the synthesis of these polymers, due to the ease of preparing well-defined multi-block copolymers and PNIPAM via this route.11,36–40 The detailed synthetic procedure is shown in Scheme 2. Scheme 1 Illustration of copolymer design, and biomolecule loading and release mechanism of the dual-responsive nanogel platform. Thermo- responsive triblock copolymers self-assemble above the LCST (Tcp: cloud point temperature as measured by DLS), which was used to encapsulate biomolecule cargo. Subsequent crosslinking via enzyme-responsive peptides increases the nanogel stability below and above the LCST and provides enzyme-triggered cargo release. Scheme 1 Illustration of copolymer design, and biomolecule loading and release mechanism of the dual-responsive nanogel platform. Thermo- responsive triblock copolymers self-assemble above the LCST (Tcp: cloud point temperature as measured by DLS), which was used to encapsulate biomolecule cargo. Subsequent crosslinking via enzyme-responsive peptides increases the nanogel stability below and above the LCST and provides enzyme-triggered cargo release. 8896 \| J. Mater. Chem. B, 2020, 8, 8894--8907 This journal is © The Royal Society of Chemistry 2020 View Article Online Journal of Materials Chemistry B Paper Scheme 2 Synthetic route for P(NIPAM)-, P(NCPAM)- and P(NIPAM-co-NCPAM)-based triblock copolymers. Copolymer names correspond to the specific thermo-responsive block repeating units: (a) for NIPAM, (b) for NCPAM and (c) for NIPAM/NCPAM copolymers. Open Access Article. Published on 03 September 2020. Downloaded on 10/24/2024 5:42:34 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Scheme 2 Synthetic route for P(NIPAM)-, P(NCPAM)- and P(NIPAM-co-NCPAM)-based triblock copolymers. Copolymer names correspond to the specific thermo-responsive block repeating units: (a) for NIPAM, (b) for NCPAM and (c) for NIPAM/NCPAM copolymers. for nanogel self-assembly/disassembly at temperatures just above body temperature. The evolution of Mn and Ð versus DP is shown in Fig. S1b (ESI†). We also determined the block copolymer cloud point tempera- tures Tcp via temperature sweeps and monitoring by dynamic light scattering (DLS). The corresponding sizes of the self- assembled nanostructures resulting from heating the block copolymer solutions above the Tcp are included in Table 1 and Table S6 (ESI†). This journal is ©The Royal Society of Chemistry 2020 J. Mater. Chem. B, 2020, 8, 8894--8907 \| 8897 Modulation of self-assembly temperature via P(NIPAM-co-NCPAM)-based copolymers (b) Temperature trend from 25 to 60 1C of P(NIPAM-co-NCPAM)-based copolymers measured by DLS demonstrating the evolution of normalised derived count rate versus increasing temperature (the inflection point of each curve indicates Tcp). (c) Normalised size distribution based on dissolved copolymers in PBS below (dashed line) and above (continuous line) the LCST (Tcp) as measured by DLS (number %). (d) Eﬀect of heating rate on self-assembled nanogel hydrodynamic diameter for 5a4 and 5c1 block copolymers as shown by measuring the hydrodynamic diameter (number mean SD in nm, n = 3) by DLS. Journal of Materials Chemistry B Paper y p Open Access Article. Published on 03 September 2020. Downloaded on 10/24/2024 5:42:34 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 1 Thermo-responsive behaviour of P(NIPAM-co-NCPAM)-based triblock copolymers. (a) Schematic representation of temperature-triggered self- assembly of acrylamide-based block copolymers. (b) Temperature trend from 25 to 60 1C of P(NIPAM-co-NCPAM)-based copolymers measured by DLS demonstrating the evolution of normalised derived count rate versus increasing temperature (the inflection point of each curve indicates Tcp). (c) Normalised size distribution based on dissolved copolymers in PBS below (dashed line) and above (continuous line) the LCST (Tcp) as measured by DLS (number %). (d) Eﬀect of heating rate on self-assembled nanogel hydrodynamic diameter for 5a4 and 5c1 block copolymers as shown by measuring the hydrodynamic diameter (number mean SD in nm, n = 3) by DLS. the Tcp of the 5a4 copolymer with 0% NCPAM content, to a Tcp of about 47 1C for the 5c3 copolymer with 60% NCPAM content. It can be concluded that by increasing the number of cyclopropyl units in the copolymers, the self-assembly temperature increases proportionally. However, when the NCPAM content exceeds 50%, the stability of the resulting self-assembled structures is compromised. Formation of large particles/aggregates, which led to sedimentation was observed (Fig. 1b). The self-assembly process of PNIPAM- and P(NIPAM- co-NCPAM)-based copolymers can be reversed by cooling the solution below the transition temperature (down to 25 1C) causing complete nanogel dissolution yielding free polymer chains in solution (Fig. 1b and Fig. S1c, ESI†). The slight hysteretic behaviours in Fig. 1b could be explained by a slightly slower globule-to-coil transition compared to the coil-to-globule transition. According to this initial DLS analysis, the selected block copolymers (5a4/5c1–3) revealed an LCST-type, reversible thermo-responsive behaviour that yielded nanogels at temperatures above their LCST. Modulation of self-assembly temperature via P(NIPAM-co-NCPAM)-based copolymers Temperature-responsive assembly/disassembly of nanostruc- tures around body temperature is a valuable means of control- ling encapsulation/release of therapeutic cargo. Modulation of the transition temperature allows for application-specific optimisation of cargo loading and delivery, which are critical considerations in the drug delivery field. The synthesised P(NIPAM-co-NCPAM)-based triblock copolymers were designed to exhibit increasing transition temperatures. DLS was used to confirm successful modulation of the self-assembly tempera- ture for the P(NIPAM-co-NCPAM)-based triblock copolymers containing increasing amounts of the cyclopropyl-based repeating unit (NCAPM from 0 to 60%) (5a4/5c1–3) (Fig. 1). By dissolving these copolymers in PBS at 25 1C and increasing the temperature to 60 1C (above the LCST), a clear increase in the turbidity of the solution could be observed by eye and by light scattering (derived count rate, kcps) recorded using DLS, which suggests a coil-to- globule transition (Fig. 1b). Since larger structures scatter more light than dissolved copolymers, the self-assembly of the copolymers could be followed by monitoring the scattering intensity. The inflection points of each curve represent the Tcp of the corresponding copolymers. DLS measurements at tem- peratures below/above the LCST indicate formation of stable Table 1 Characterisation of selected triblock copolymers from the synthesised libraries # NI/NC ratioa DP (NMR) Mn (Da) (NMR) Mn (Da) (GPC) Ð (Mw/Mn) (GPC) DH (nm) (DLS) Tcp (1C) 5a4 NI1.0NC0.0 421 55 800 66 600 1.41 74 7 33 5c1 NI0.8NC0.2 421 55 600 61 900 1.31 80 2 37 5c2 NI0.5NC0.5 395 52 500 77 800 1.34 179 20 44 5c3 NI0.4NC0.6 357 48 100 58 700 1.29 295 74 — a NI/NC ratio is NIPAM to NCPAM ratio; DP is degree of polymerisation calculated by 1H NMR; Mn is the number average molecular weight calculated by GPC and NMR; Mw/Mn represents the dispersity obtained by GPC; DH in nm represent the hydrodynamic diameters of the resulting self-assembled nanostructures (T above Tcp: 5a4 = 34 1C, 5c1 = 60 1C, 5c2 = 55 1C, 5c3 = 55 1C) reported by DLS (number/%) and Tcp is the cloud point temperature as measured by DLS. This journal is ©The Royal Society of Chemistry 2020 J. Mater. Chem. B, 2020, 8, 8894--8907 \| 8897 8897 Paper View Article Online View Article Online Journal of Materials Chemistry B Paper Fig. 1 Thermo-responsive behaviour of P(NIPAM-co-NCPAM)-based triblock copolymers. (a) Schematic representation of temperature-triggered self- assembly of acrylamide-based block copolymers. 8898 \| J. Mater. Chem. B, 2020, 8, 8894--8907 Characterisation of morphology and self-assembly process by cryo-TEM and SANS This indicates formation of dense, polymer-filled, non-vesicular spherical nanogels. The sizes of the nanogels (Fig. 2b and e) made from both copolymers were measured via image analysis and were in good agreement with the hydrodynamic diameters obtained by DLS. The increase in size of the self-assembled structures when NCPAM is present, as measured by DLS (Fig. 1c and d), was confirmed by cryo-TEM. We additionally studied the zeta potential of self-assembled nanostructures above the LCST (45 1C), which yielded slightly negative values (5a4 = 5.5 0.2 mV; 5c1 = 16.0 0.5 mV). Accurate shape, size, and morphology characterisation of drug delivery systems is crucial for understanding their behaviour, especially for dynamic self-assembled systems. This will help to define their potential as drug delivery vehicles. As an example, morphology and shape can aﬀect cargo loading and cellular uptake.43 Conventional DLS and TEM can give an indication of nanostructure size, polydispersity, and shape, but complemen- tary techniques are needed to decipher the internal morphology of self-assembled particles. SANS allows more detailed struc- tural information to be acquired in an artefact-free manner to obtain bulk properties such as morphology and polydispersity. Cryo-TEM is a useful tool for imaging self-assembled nano- structures to obtain a snapshot in their native hydrated state. Often, the drying step in the sample preparation procedure needed for negative stain TEM introduces drying artefacts, which may prevent accurate morphology determination.44 A combination of SANS and cryo-TEM is often used when characterising block copolymer assemblies in solution, e.g. when studying vesicle shape transformations at the nano- scale.28–30 We used these two techniques to further analyse the self-assembly process by first studying the copolymers before (‘coil’ state) and after self-assembly (‘globule’ state) (Fig. 2). We chose the copolymers 5a4 and 5c1 for this cryo-TEM and SANS analysis because these two copolymers could be cross-linked successfully using our peptide cross-linker (see next section). Open Access Article. Published on 03 September 2020. Downloaded on 10/24/2024 5:42:34 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. SANS enabled us to capture the morphology change from coil-to-globule (Fig. 2c and f), which is induced by the temperature-triggered transition in agreement with literature on other PEG-b-PNIPAM-based block copolymer systems.45–47 Neutron scattering data of the two selected block copolymers below and above the LCST (25 and 45 1C, respectively) are shown (Fig. 2c and f). Modulation of self-assembly temperature via P(NIPAM-co-NCPAM)-based copolymers We also tested all the other copolymers from the separate PNIPAM (5a1 to 5a5) and PNCPAM (5b1 to 5b5) series for the formation of nanogels at temperatures above the LCST (Table S6, ESI†). All copolymers except 5b1 assembled into nanogels, however, no clear trends regarding polymer lengths and corresponding nanostructure sizes could be found. and well-defined nanostructures when heated above the LCST (Fig. 1c), whilst bigger structures were formed with increasing amounts of NCPAM (mean hydrodynamic diameters between 74 and 295 nm). Due to the formation of large structures/ aggregates that led to some precipitation for 5c3 when heated/cooled slowly (1 1C temperature ramp with 5 min equilibration time), the intensity curves are not smooth (Fig. 1b) and no accurate Tcp can be given for this copolymer. However, it appears to be between 45 to 50 1C when estimating based on both curves (up/down, heating and cooling). The dependence of particle size on the heating rate during the self-assembly process was also confirmed for two copoly- mers that form smaller nanoparticles in general (5a4 and 5c1, Fig. 1d). The final assembled particles tended to be larger when subjected to slower heating. Comparing the transi- tion temperature of 5a4 (33 1C) with previously reported PEG-b-PNIPAM (33.7 1C),42 it can be concluded that the short functional middle block in our copolymers did not aﬀect the LCST. The cyclopropyl-dependent size increase might be caused by the higher rigidity and lower rotational freedom of the cyclo- propyl group when compared to the isopropyl group, which might aﬀect the packing parameters to yield larger structures. The self-assembly temperature of the resulting triblock copoly- mers was successfully tuned from 33 1C, corresponding to 8898 \| J. Mater. Chem. B, 2020, 8, 8894--8907 This journal is © The Royal Society of Chemistry 2020 View Article Online Paper Paper Journal of Materials Chemistry B Characterisation of morphology and self-assembly process by cryo-TEM and SANS This journal is ©The Royal Society of Chemistry 2020 Open Access Article. Published on 03 September 2020. Downloaded on 10/24/2024 5:42:34 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Three of the block copolymers (5a4/5c1/5c2) were used for the development of peptide cross-linked nanogels because these copolymers formed stable structures upon heating above their LCST (Fig. 1b and c). The peptide cross-linker was prepared by solid phase peptide synthesis inserting an azido-lysine residue at both the C- and N-termini according to literature procedures.18 An MMP-7-cleavable sequence (K(N3)PLELRAK(N3), see Fig. S2a for the structure, ESI†) was installed into the peptide backbone enabling enzyme-triggered nanogel degradation. LC-MS spectra of the functionalised MMP-7-cleavable peptide after purification is shown in Fig. S2b (ESI†). In order to prepare the cross-linked nanogels, we developed a two-step protocol. First, the formation of self-assembled nanogels was induced by heating the block copolymer solution to 45 1C (above the LCST). A solution of the cross-linker was then added, together with small amounts of CuSO45H2O and (+)-sodium-L-ascorbate, leading to covalent crosslinking; the mixture was subsequently purified by size exclu- sion chromatography. Between the diﬀerent samples we expect similar cross-linking due to the availability of the same number of alkynes per chain for all the copolymers and use of same equivalents of all the reagents for cross-linking. Upon cooling below the LCST, our nanogels therefore retained their structure, despite the solubilisation of the polymer chains. Details of the cross-linking strategy are schematically shown in Fig. 3a. None of our synthesised block copolymers self-assembled into vesicular morphologies, which would have been observed as membranous structures in the cryo-TEM micrographs and distinct SANS curves that could be fitted with a Hollow sphere model as shown previously.28 This is in disagreement with previous studies on similar PEG-b-PAA-b-PNIPAM block copolymer assemblies,40,51 which highlights the challenge of using theoretical rules to predict the final structure and encourages the use of various complementary techniques to precisely evaluate morphology. A combination of techniques such as DLS, cryo-TEM and scattering techniques like SANS, small-angle X-ray scattering (SAXS) and combining dynamic and static light scattering (DLS/SLS) is necessary to obtain a clear picture of the size and morphology of self-assembled nanostructures, which allows identification of the type of nanostructure formed. After the cross-linking reaction, the resulting polymer nano- gels were analysed by DLS, both above and below the LCST, which is shown in Fig. 3b and c. A small diﬀerence in the hydrodynamic diameter was observed between the cross-linked (93 2 nm) and self-assembled 5a4 nanogels (74 7 nm, before cross-linking) above the LCST. Characterisation of morphology and self-assembly process by cryo-TEM and SANS Both copolymers, either without cyclo- propyl units (5a4) or including cyclopropyl units (20%, 5c1) in the thermo-responsive block, revealed a drastic change in the scattering profiles at the two different temperatures. Below the LCST (at 25 1C), SANS confirmed that the block copolymers are present as chains in solution. Two shape-independent models, Guinier–Porod (5a4; Porod exponent of 1.77 0.04; radius of gyration Rg of 4.6 0.7 nm; dimension variable s is 0.86 0.07) and power law (5c1; power law exponent of 1.96 0.01) were used to fit the scattering profiles at 25 1C. Scattering data of copolymer 5a4 revealed a clear Guinier region, therefore we could fit the data with the Guinier–Porod model to obtain accurate Rg values. The data obtained for 5a4 suggests that this block copolymer has fully swollen chains in good solvent conditions at 25 1C (below the LCST) in agreement with Cryo-TEM of self-assembled nanostructures made from copolymers 5a4 and 5c1 revealed a spherical morphology with homogenous contrast across whole particles (Fig. 2a and d). Fig. 2 Self-assembled nanogel morphology characterisation. (a) Representative cryo-TEM image of self-assembled 5a4 copolymer (scale bar: 200 nm), (b) size distribution histogram from representative images, (c) SANS analysis below and above the LCST. (d) Cryo-TEM image of self-assembled 5c1 copolymer (scale bar: 200 nm), (e) size distribution histogram from representative images, (f) SANS analysis below and above the LCST. Fig. 2 Self-assembled nanogel morphology characterisation. (a) Representative cryo-TEM image of self-assembled 5a4 copolymer (scale bar: 200 nm), (b) size distribution histogram from representative images, (c) SANS analysis below and above the LCST. (d) Cryo-TEM image of self-assembled 5c1 copolymer (scale bar: 200 nm), (e) size distribution histogram from representative images, (f) SANS analysis below and above the LCST. J. Mater. Chem. B, 2020, 8, 8894--8907 \| 8899 This journal is ©The Royal Society of Chemistry 2020 View Article Online Journal of Materials Chemistry B Paper allows triggered structure disassembly and controlled payload delivery. Herein, we leveraged natural enzymes as triggers for cargo release due to their inherent involvement in disease progression. The functional alkyne handles, introduced into the copolymers as described above, were used as cross-linking points using azido-bi-functionalised peptides bearing a protease sensitive sequence. Open Access Article. Published on 03 September 2020. Downloaded on 10/24/2024 5:42:34 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. In contrast, DLS measure- ments at 25 1C (below the LCST) revealed successful cross- linking had taken place. The non-cross-linked polymer nano- gels were disassembled when the temperature was lowered below the LCST, as described above (Fig. 1b and c), but nanogels remained stable when cross-linked. When comparing the DLS traces of the cross-linked 5a4 nanogels at 37 1C and 25 1C, a size increase can be observed (93 2 nm compared to 133 9 nm). The slight size increase with decreasing tempera- ture can be attributed to the thermo-responsive phase transi- tion of the PNIPAM block, which switches from hydrophobic nature above the LCST to hydrophilic below the LCST. This could cause relaxation and water-induced swelling of the polymer chains, as previously observed in the literature.40,52 These networks are expected to swell and relax up to a limit This journal is © The Royal Society of Chemistry 2020 Characterisation of morphology and self-assembly process by cryo-TEM and SANS A model matrix metalloproteinase was selected as our initial target due to their involvement in many diseases.23 Matrix metalloproteinase 7 (MMP-7) was chosen due to its small size with a Mw of only 19 kDa in its active form,54 which minimises steric hinderance to allow the enzyme to access the cross-links in the nanogels. literature on similar copolymers.46 A power law exponent of 1.96 0.01 for block copolymer 5c1 is explained by the presence of polymer chains in theta conditions with equally favoured polymer–polymer and polymer–solvent interactions. This might also explain the bimodal distribution in the DLS data below the LCST (Fig. 1c), which was not observed in case of 5a4 (monomodal DLS) and is supported by the SANS data (see discussion above). When heated above the LCST (45 1C), the copolymers are expected to transition from hydrated and flexible coils to collapsed globules.48–50 SANS data for copolymers 5a4 and 5c1 above the LCST were successfully fitted with a Sphere model. This indicates formation of dense spherical polymer nanogels rather than vesicles (polymersomes), which confirms the con- clusions from the cryo-TEM images (Fig. 2a and d). The inser- tion of 20% cyclopropyl repeating units (5c1) did not affect the final morphology and the scattering data could again be fitted with the simple Sphere model. The best-fit diameters were 47.6 1.0 nm (5a4) and 78.0 0.5 nm (5c1), respectively. Data for size obtained by SANS, cryo-TEM and DLS are similar and the formation of larger structures for the NCPAM-containing copolymer 5c1 compared to 5a4 was consistent across all the measurements. SANS typically shows the smallest diameter of all the three methods, because it does not account for the hydration shell in the measurement. Nanogel cross-linking via peptides to increase stability and introduce enzyme-responsiveness (d) Study of long-term stability of cross-linked 5a4- and 5c1-based nanogels in PBS incubated at 37 1C and measured by DLS at 25 1C (open symbols) and 37 1C (closed symbols) at each timepoint (N = 1, n = 3). to 45 1C and from 32 to 42 1C, respectively (Fig. 4 and Tables S7, S8, ESI†). determined by constraints imposed by the crosslinking, which will be dependent on the length and chemical structure of the peptide, and the cross-linking density. SANS data of 5c1 block copolymers at temperatures between 25 and 34 1C (Fig. 4a and b) were fitted with the power law model (power law exponent of B1.9 obtained for all), which suggests the presence of Gaussian polymer chains in theta solvent conditions and the absence of self-assembly. Upon heating to 35 1C, the scattering data was better fitted using the Unified power Rg model. The obtained power law exponent of 2.40 0.03 indicates a transition from Gaussian chains to a more entangled polymer network (Rg of 52.0 0.6 nm) indicating the start of the coil-to-globule transition. At higher temperatures (36 and 45 1C) data were fitted using a triaxial ellipsoid model, which suggests the formation of non-perfect spherical nanogels, with an observable trend of ellipse transitioning to more sphere shape with increasing temperature (Table S7, ESI†). From these experiments a Tcp of 35–36 1C was obtained, which was similar to the value found by DLS (37 1C). Multiplying the scattering intensity by Q2 allowed us to obtain the Kratky plot with linear x- and y-axes (Fig. 4b and d). This plot is useful for studying nanogel degree of globularity, flexibility and compactness.29,55 The peak appearing in the Kratky plot at higher temperatures suggests the formation of globular particles above 35 1C. As the temperature further increases, the peak becomes more defined suggesting an increase in globularity. g y The developed cross-linking protocol was then applied to the preparation of cross-linked polymer nanogels composed of the other P(NIPAM-co-NCPAM)-based block copolymers. Fig. 3c shows DLS characterisation of 5c1 (NI0.8NC0.2)-based nanogels after cross-linking. 5c1-based nanogels could be cross-linked successfully, yielding larger sizes compared to 5a4-based nano- gels when measured below the LCST (181 4 nm vs. 133 9 nm). Cross-linking of 5c2-based nanogels could not be achieved due to severe aggregation and precipitation. Nanogel cross-linking via peptides to increase stability and introduce enzyme-responsiveness Several cross-linking strategies have been adopted in the drug delivery field to increase the stability of self-assembled nano- structures and introducing stimuli-responsive properties to the delivery system.19,40,52 The dynamic nature of self-assembled nanostructures can compromise their stability in biological environments due to the presence of other entities (e.g. proteins) and dilution upon injection in the body.16,53 This can lead to premature payload leakage. Stabilisation of amphiphilic assem- blies by covalent bonds is a versatile method to address the above- mentioned challenges, whilst stimuli-responsive motifs can be incorporated simultaneously. A stimuli-responsive cross-linker domain that is cleaved in the presence of the desired stimuli 8900 \| J. Mater. Chem. B, 2020, 8, 8894--8907 This journal is © The Royal Society of Chemistry 2020 8900 View Article Online Paper Paper Journal of Materials Chemistry B Journal of Materials Chemistry B Journal of Materials Chemistry B Fig. 3 Nanogel cross-linking via enzyme-responsive peptides using azide-bi-functionalised peptides via CuAAC. (a) Schematic representation of the cross-linking strategy showing how cross-linking enhances nanogel stability below the LCST. (b) DLS traces comparing size distribution of 5a4-based self-assembled (before cross-linking from Fig. 1c) and cross-linked nanogels below (dashed line) and above (solid line) the LCST (33 1C for 5a4). (c) DLS traces comparing size distribution of 5a4- and 5c1-based cross-linked nanogels at 25 1C (below the LCST). (d) Study of long-term stability of cross-linked 5a4- and 5c1-based nanogels in PBS incubated at 37 1C and measured by DLS at 25 1C (open symbols) and 37 1C (closed symbols) at each timepoint (N = 1, n = 3). s licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 03 September 2020. Downloaded on 10/24/2024 5:42:34 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 3 Nanogel cross-linking via enzyme-responsive peptides using azide-bi-functionalised peptides via CuAAC. (a) Schematic representation of the cross-linking strategy showing how cross-linking enhances nanogel stability below the LCST. (b) DLS traces comparing size distribution of 5a4-based self-assembled (before cross-linking from Fig. 1c) and cross-linked nanogels below (dashed line) and above (solid line) the LCST (33 1C for 5a4). (c) DLS traces comparing size distribution of 5a4- and 5c1-based cross-linked nanogels at 25 1C (below the LCST). This journal is ©The Royal Society of Chemistry 2020 J. Mater. Chem. B, 2020, 8, 8894--8907 \| 8901 Nanogel cross-linking via peptides to increase stability and introduce enzyme-responsiveness 4c, d and Table S8, ESI†) when compared to the purely self-assembled nanogels (Fig. 2f, 4a, b and Table S7, ESI†). We measured a narrower temperature range around the LCST, because we already knew the transition temperature from the non-cross-linked nanogels (Fig. 4a and b). The power law exponent value below 2 for the non-cross-linked copolymer compared to a value of B2.4 for the cross-linked nanogels, reveals the diﬀerence of the two populations at lower tempera- tures. As expected, by cross-linking the polymer chains a stable polymer network was formed, and the chains remained inter- connected between each other also at temperatures below the LCST. When comparing the two Kratky plots (Fig. 4b and d) it can be concluded that the self-assembled and cross-linked nanogels both transition from coil to globule at 36 1C, whilst the cross-linked particles were slightly smaller after the transi- tion as seen by the shift of the peak to the higher Q region. This trend is also confirmed by cryo-TEM (Fig. 2d and Fig. S3, ESI†). An explanation for this smaller size could be that the peptide cross-linkers restrict chain flexibility, keeping the polymer chains tightly connected.56 observed, whilst the higher concentrations (9 nM, 90 nM, 900 nM) completely disassembled the nanogels. The inter- mediate concentration of 0.9 nM marks the transition point where a size increase was measured. This can be explained by the partial cleavage of the cross-links, which caused the nano- gels to relax into a larger overall structure (still partially cross- linked) because the measurement was conducted below the LCST. The kinetics of MMP-7-responsive nanogel disassembly at a fixed enzyme concentration (90 nM) were subsequently studied by DLS (normalised derived count rate (in kcps) vs. incubation time). The kinetic study (Fig. 5a) reveals a drop in the scattering intensity over time only in the presence of the enzyme, indicating a decrease in nanogel concentration and/or size. The kcps value remained unvaried in the control nanogel sample without enzyme (red symbols). A further control, incubating the cross-linked nanogels at 37 1C in 5% (v/v) fetal bovine serum (FBS) revealed very high stability of the nanogels in the presence of proteins over at least two days (Fig. S5, ESI†). This confirms the ability of the nanogels to undergo enzyme- triggered degradation. From the evolution of scattering intensity in Fig. Nanogel cross-linking via peptides to increase stability and introduce enzyme-responsiveness Hence, detailed morphological analysis was restricted to the two copolymers that could also be cross-linked successfully (5a4 and 5c1). Long-term stability of cross-linked nanogels made from these two block copolymers when incubated in PBS at 37 1C was demonstrated by DLS (Fig. 3d). The hydrodynamic diameters did not decrease when observed over 9 days, both below and above the LCST, which demonstrates integrity of the structure and the absence of nanogel disassembly. Besides obtaining morphological information at fixed tem- peratures above and below the LCST, as also demonstrated previously with similar PNIPAM-based block copolymers,46,47 SANS was applied herein to dynamically monitor the transition using one selected copolymer. During SANS measurements, 5c1 block copolymers and the corresponding cross-linked nanogels were subjected to a temperature ramp from 25 The eﬀect of cross-linking can be seen in the temperature- dependent SANS data of peptide cross-linked 5c1 nanogels J. Mater. Chem. B, 2020, 8, 8894--8907 \| 8901 8901 View Article Online Fig. 4 Temperature-dependent SANS analysis of self-assembled versus cross-linked 5c1 block copolymers. (a) Evolution of neutron scattering data collected for 5c1 block copolymers at temperatures from 25 to 45 1C (lines are fitted curves; data points are symbols; errors are SD). (b) Kratky plot for (a) showing an increase of globularity as the temperature rises from 25 to 45 1C. (c) Evolution of neutron scattering data collected for 5c1-based cross- linked nanogels at temperatures from 32 to 42 1C. (d) Kratky plot for (c) showing an increase of globularity as the temperature rises from 32 to 42 1C. Journal of Materials Chemistry B Paper Journal of Materials Chemistry B Open Access Article. Published on 03 September 2020. Downloaded on 10/24/2024 5:42:34 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 4 Temperature-dependent SANS analysis of self-assembled versus cross-linked 5c1 block copolymers. (a) Evolution of neutron scattering data collected for 5c1 block copolymers at temperatures from 25 to 45 1C (lines are fitted curves; data points are symbols; errors are SD). (b) Kratky plot for (a) showing an increase of globularity as the temperature rises from 25 to 45 1C. (c) Evolution of neutron scattering data collected for 5c1-based cross- linked nanogels at temperatures from 32 to 42 1C. (d) Kratky plot for (c) showing an increase of globularity as the temperature rises from 32 to 42 1C. (Fig. Nanogel cross-linking via peptides to increase stability and introduce enzyme-responsiveness 5a it is apparent that degradation has already occurred to some extent after 1 h incubation with MMP-7. The number mean size of the nanogels has already decreased (measured below the LCST), which suggests the presence of diﬀerently sized fragments and partial degradation. Within the next few hours of incubation with enzyme, a mixture of diﬀerently sized particles/fragments/ polymers (large errors) was detected. After 24 h incubation, a clear size diﬀerence between the control and enzyme incubated sample became apparent (Fig. 5b) and the small error indicates This journal is © The Royal Society of Chemistry 2020 Enzyme-triggered nanogel degradation In the case of MMP-7 degraded nanogels, no more nanogels of the original size were detected; a two-component fit revealed only free Cy5-polymer diﬀusion (fixed diﬀusion time, 54% of diﬀusing species) and possibly entangled debris of slightly intermediate sizes between intact nanogels and free chains (46%, shown in graph). Values are mean of 30 curves SD. yielded diﬀusion times similar to free dye (Fig. S6, ESI†), which confirms successful specific labelling in the case of Cy5-azide (Fig. 5c and d). that an endpoint has been reached. Indeed after 24 h incuba- tion, the fragment size (27 9 nm) is much closer to that of the corresponding free block copolymer chains (10 2 nm). Following DLS analysis, we also monitored nanogel degra- dation by a single-molecule detection method called fluorescence correlation spectroscopy (FCS). We previously used FCS for a detailed study of MMP-responsive degradation using another peptide-based nanostructure for diagnostic applications.35 Herein, FCS was employed to first observe nanogel degradation and subsequently to quantitatively analyse model biomolecule loading and release. In order to study MMP-7 triggered degra- dation of nanogels by FCS, cross-linked nanogels were labelled with Cy5-azide and incubated with excess MMP-7 (9 mM). Successful labelling of nanogels with Cy5-azide can be seen when calculating the hydrodynamic diameters from the FCS autocorrelation analysis (Fig. 5c) that yielded nanogel size rather than free dye (Fig. 5d). Further, non-crosslinked Cy5- azide labelled copolymers in non-assembled form revealed an intermediate diﬀusion time between free dye and nanogels (Fig. S6, ESI†). FCS curves of control experiments to check for non-specific interactions of dyes with nanogels/copolymers After MMP-7 incubation, the corresponding autocorrelation curves shifted to faster diﬀusion times indicating cleavage of the peptide bonds (Fig. 5c). In the presence of MMP-7, a two- component fit of the autocorrelation curves revealed complete disappearance of any structures with original nanogel sizes. We only detected the presence of free Cy5-polymer chains (54% of total diﬀusing species) and some bigger, possibly entangled degradation products (DH value of 35 6 nm, 46%, shown in Fig. 5d), demonstrating MMP-7-dependent peptide cleavage and complete nanogel disassembly below the LCST. By comparing the nanogel signal intensity (counts per particle, CPP in kHz) to the dye intensity, the number of dye molecules per single nanogel could be determined. Enzyme-triggered nanogel degradation MMP-triggered nanogel disassembly was analysed using MMP-7 as a model MMP. We first confirmed that the free peptide in solution can be cleaved by MMP-7 as shown using LC-MS (Fig. S2b, ESI†). We next established the necessary concentra- tions of MMP-7 to successfully degrade the cross-links of the nanogels, which revealed that physiologically relevant nanomolar concentrations are suﬃcient for degradation (Fig. S4, ESI†). At the lowest concentrations (9 pM, 90 pM), no degradation was 8902 \| J. Mater. Chem. B, 2020, 8, 8894--8907 This journal is © The Royal Society of Chemistry 2020 View Article Online Paper Paper Journal of Materials Chemistry B DLS and FCS analysis of MMP-7 triggered degradation of cross-linked 5a4-based nanogels. (a) Evolution of normalised derived count rate upon on with 90 nM MMP-7 over 24 hours indicating degradation kinetics as monitored by DLS. (b) Evolution of cross-linked nanogel average size r mean) over time upon incubation with 90 nM MMP-7. (c) Normalised autocorrelation curves (coloured lines) and corresponding fits (black lines) CS data of Cy5-labelled nanogels and MMP-7-degraded (9 mM) Cy5-labelled nanogels in enzyme buﬀer. (d) Hydrodynamic diameters of elled nanogels and degraded Cy5-labelled nanogels with controls (Cy5-azide and Cy5-polymer) as calculated from FCS data. In the case of degraded nanogels, no more nanogels of the original size were detected; a two-component fit revealed only free Cy5-polymer diﬀusion (fixed n time, 54% of diﬀusing species) and possibly entangled debris of slightly intermediate sizes between intact nanogels and free chains (46%, shown ). Values are mean of 30 curves SD. Open Access Article. Published on 03 September 2020. Downloaded on 10/24/2024 5:42:34 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 5 DLS and FCS analysis of MMP-7 triggered degradation of cross-linked 5a4-based nanogels. (a) Evolution of normalised derived count rate upon incubation with 90 nM MMP-7 over 24 hours indicating degradation kinetics as monitored by DLS. (b) Evolution of cross-linked nanogel average size (number mean) over time upon incubation with 90 nM MMP-7. (c) Normalised autocorrelation curves (coloured lines) and corresponding fits (black lines) from FCS data of Cy5-labelled nanogels and MMP-7-degraded (9 mM) Cy5-labelled nanogels in enzyme buﬀer. (d) Hydrodynamic diameters of Cy5-labelled nanogels and degraded Cy5-labelled nanogels with controls (Cy5-azide and Cy5-polymer) as calculated from FCS data. This journal is ©The Royal Society of Chemistry 2020 J. Mater. Chem. B, 2020, 8, 8894--8907 \| 8903 Biomolecule loading and release from enzyme-responsive nanogels After confirming the possibility of enzyme-triggered degrada- tion of the herein developed peptide-cross-linked nanogels using a disease-relevant enzyme of the MMP class, loading and release properties of a model protein cargo (fluorescent Oregon Green-labelled bovine serum albumin, OG-BSA) was studied using one selected copolymer (5a4). UV-vis spectroscopy revealed a loading eﬃciency of 18 8% (average SD of three independent batches) after purification by size exclusion chromatography. This represents better loading eﬃciencies for nanogels compared to polymersomes made via standard film rehydration, that often have low loading eﬃciencies of a few percent for hydrophilic cargo such as proteins.57 Further, the film rehydration method for polymersome formation can take several days, whilst solvent injection methods might harm sensitive protein cargo during self-assembly. Our rapid procedure Successful loading of OG-BSA into peptide cross-linked 5a4 nanogels was observed by FCS. Free dye, OG-BSA and OG-BSA incorporated in nanogels can already be distinguished clearly in the fitted autocorrelation curves (Fig. 6a). When calculating the corresponding diﬀusion times (tD) and hydrodynamic diameters (DH) the size diﬀerence between the diﬀusing species was obtained (Fig. 6b). In the case of the nanogel measurements, the diﬀusing species correspond to the nanogel hydrodynamic diameter obtained in other measurements (Fig. 3c and 5c, d), which indicates successful incorporation of OG-BSA. Comparing the signal intensity per nanogel (counts per particle, CPP in kHz) with that of free OG-BSA, the number of loaded OG-BSA per single nanogel was determined and shows that multiple protein cargos were loaded per nanogel (Fig. 6c). Release studies were performed Open Access Article. Published on 03 September 2020. Downloaded on 10/24/2024 5:42:3 This article is licensed under a Creative Commons Attribution 3.0 Unported Fig. 6 Study of model protein (OG-BSA) loading and MMP-7-triggered release by FCS. (a) Normalised autocorrelation curves (coloured lines) and corresponding fits (black lines) from FCS data of three nanogel batches (A–C). (b) Hydrodynamic diameter of the three different batches of OG-BSA loaded nanogels with controls (free OG and free OG-BSA) as calculated from FCS data. (c) Number of OG-BSA per nanogel for the three nanogel batches as calculated from FCS data. (d) Cumulative release (%) of OG-BSA over time. This graph shows the release from loaded nanogels A, B and C upon incubation with increasing concentration of MMP-7 (0.9 nM, 9 nM and 90 nM) as compared with diffusion-mediated release (no MMP-7). Enzyme-triggered nanogel degradation Upon enzyme-mediated nanogel degradation, the calculated number of dyes per particle changed from about 54 dyes per particle before degradation, to about 2 dyes per particle for the degradation products. This is a further indication of nanogel disassembly. Together with the DLS analysis (Fig. 5a and b), J. Mater. Chem. B, 2020, 8, 8894--8907 \| 8903 This journal is ©The Royal Society of Chemistry 2020 View Article Online Journal of Materials Chemistry B Paper Paper enzyme-responsiveness of our nanogel system was successfully demonstrated. enzyme-responsiveness of our nanogel system was successfully demonstrated. to yield protein-loaded, well-defined nanogels at high polymer concentrations without the use of any organic solvent represents a suitable strategy to load sensitive therapeutic proteins for bio- medical applications in the future. Loading and release of our model protein OG-BSA was further studied in detail by FCS (Fig. 6). Biomolecule loading and release from enzyme-responsive nanogels Alternatively, nanogels with LCST just slightly below body tem- perature could be used to achieve MMP7-responsive release that could be accelerated by applying e.g. cold patches locally. Simply changing the peptide cross-linker sequence will further allow application of our nanogel platform using other enzymes as triggers, as relevant in specific diseases. FCS curves at the diﬀerent time points were fitted using a two-component fit to account for both diﬀusing species (loaded nanogels and released OG-BSA). Further, a correction was included to account for the disproportional eﬀect of bright slow diﬀusing species (loaded nanogels) compared to the less bright free OG-BSA.60 We propose herein (see Materials and methods in ESI†) to simply combine the fraction of free OG-BSA (directly from the two-component fit) with the decrease in the number of cargo for the remaining nanogels (via CPP), which is a second indicator for OG-BSA release (number of proteins per nanogel is decreasing). This correction is necessary because nanogels with several loaded proteins overlap with the free protein signal, as demonstrated in the literature for mixtures of diﬀerently sized nanoparticles of varying brightness.60 This finally yielded the percentage of free OG-BSA and allowed us to further calculate the cumulative release profile over time (Fig. 6d). A clear diﬀerence between 90 nM MMP-7-triggered release and diﬀusion-based profiles was detected, whilst the release profile in the presence of 9 and 0.9 nM MMP-7 gave intermediate results as expected for an enzyme-triggered release. Diﬀusion-based release (no MMP-7) reached 87 4% after 39 h (N = 3, n = 30). This successful enzyme-triggered release profile justifies the further study of these copolymer libraries using therapeutic cargo and studying release beha- viour at various temperatures, accompanied by corresponding delivery data at the cell and tissue level but this is beyond the scope of this paper. It is also clear that MMP-7 triggered release profiles will vary significantly in vivo compared to the in vitro data, due to a much higher level of environmental complexity that will cause diﬀerences in cross-linker degradation and hence release. Reasons for these diﬀerences include the presence of other substrates and specific cofactors, as well as continuous turn- over of enzymes. Biomolecule loading and release from enzyme-responsive nanogels These results are calculated from the FCS autocorrelation curves displayed in Fig. S7 (ESI†). Error bars are SEM (N = 3, n = 30). Fig. 6 Study of model protein (OG-BSA) loading and MMP-7-triggered release by FCS. (a) Normalised autocorrelation curves (coloured lines) and corresponding fits (black lines) from FCS data of three nanogel batches (A–C). (b) Hydrodynamic diameter of the three different batches of OG-BSA loaded nanogels with controls (free OG and free OG-BSA) as calculated from FCS data. (c) Number of OG-BSA per nanogel for the three nanogel batches as calculated from FCS data. (d) Cumulative release (%) of OG-BSA over time. This graph shows the release from loaded nanogels A, B and C upon incubation with increasing concentration of MMP-7 (0.9 nM, 9 nM and 90 nM) as compared with diffusion-mediated release (no MMP-7). These results are calculated from the FCS autocorrelation curves displayed in Fig. S7 (ESI†). Error bars are SEM (N = 3, n = 30). Fig. 6 Study of model protein (OG-BSA) loading and MMP-7-triggered release by FCS. (a) Normalised autocorrelation curves (coloured lines) and corresponding fits (black lines) from FCS data of three nanogel batches (A–C). (b) Hydrodynamic diameter of the three different batches of OG-BSA loaded nanogels with controls (free OG and free OG-BSA) as calculated from FCS data. (c) Number of OG-BSA per nanogel for the three nanogel batches as calculated from FCS data. (d) Cumulative release (%) of OG-BSA over time. This graph shows the release from loaded nanogels A, B and C upon incubation with increasing concentration of MMP-7 (0.9 nM, 9 nM and 90 nM) as compared with diffusion-mediated release (no MMP-7). These results are calculated from the FCS autocorrelation curves displayed in Fig. S7 (ESI†). Error bars are SEM (N = 3, n = 30). This journal is © The Royal Society of Chemistry 2020 8904 \| J. Mater. Chem. B, 2020, 8, 8894--8907 8904 View Article Online Paper Journal of Materials Chemistry B in triplicate by incubating OG-BSA loaded nanogels (batches A–C) with various MMP-7 concentrations in a physiologically relevant nanomolar range.58,59 FCS curves recorded at different time points in the presence or absence of MMP-7 revealed the release kinetics (Fig. 6d and Fig. S7, ESI†). This allowed us to compare diffusion-driven release (non-specific release from the nanogels over time) to MMP-7 triggered release. Open Access Article. Published on 03 September 2020. Downloaded on 10/24/2024 5:42:34 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. In this work, we took advantage of the versatility of polymer chemistry for developing a tuneable, temperature-responsive, peptide-crosslinked polymeric nanogel platform for enzyme- triggered biomolecule delivery. Via copolymerisation of NIPAM with NCPAM we could tune the thermal behaviour of the resulting copolymers resulting in a range of Tcp values around body temperature (from 33 to 44 1C). A peptide-based cross- linking approach was developed to covalently constrain the nanogel-forming copolymer chains to increase stability and equip the nanocarrier platform with enzyme-responsiveness. We obtained high-quality morphological information on the self-assembled structures, successfully studied dynamic beha- viours upon temperature changes and cross-linking, as well as model protein loading and enzyme-triggered release by employing a combination of cryo-TEM, SANS and FCS. This detailed analysis yielded a morphology of filled spheres (imaged with cryo-TEM) that relaxed to an interconnected polymer network upon cross- linking and lowering the temperature below the LCST as found by SANS measurements. Even further, the coil-to-globule transition of one selected copolymer was successfully monitored in real-time by SANS. Model enzyme-triggered (MMP-7) particle disassembly and accompanying increase in cargo release kinetics was deter- mined by employing FCS. Our modular platform features the following key characteristics: reproducible, simple and fast temperature-induced nanogel assembly, modulation of the self-assembly temperature via copolymerisation of NIPAM and NCPAM in the thermo-sensitive block, containing two distinct handles (carboxylic groups and alkynes) for cross-linking and functionalisation, as well as enzyme-triggered release mechanism through specific peptide cross-links, which can be adapted to a specific disease when choosing appropriate peptide sequences and drug cargo. Biomolecule loading and release from enzyme-responsive nanogels Release studies in more complex environments, including in the presence of cells and tissue, are needed in the future to choose an optimal copolymer system for the delivery of a specific drug for a chosen disease that involves upregulation of MMP enzymes at the disease site. Cytocompatibility of new materials is one critical aspect for moving towards biomedical applications. We have tested cytotoxicity of cross-linked nanogels (5a4) in a macrophage cell line (RAW 264.7 cells) (Fig. S8, ESI†), where we found no cytotoxic eﬀects. D h i l f MMP h MMP i l This journal is ©The Royal Society of Chemistry 2020 Conflicts of interest The authors declare no competing interests. J. Mater. Chem. B, 2020, 8, 8894--8907 \| 8905 Open Access Article. Published on 03 September 2020. Downloaded on 10/24/2024 5:42:34 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. 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https://openalex.org/W4389358912	https://thejhms.org/index.php/JHMS/article/download/56/53	English	null	The Relationship Of The Quality Of Administrative Services And Bpjs Patient Satisfaction In The Inpatient Room X Rumkit Tk Ii Dr. Soepraoen Kesdam V/Brawijaya Malang In Malang City	Journal of Hospital Management and Services	2,023	cc-by-sa	3,281	Keywords : BPJS Patients, Quality Of Administrative Services, Satisfaction Keywords : BPJS Patients, Quality Of Administrative Services, Satisfaction Keywords : BPJS Patients, Quality Of Administrative Services, Satisfaction The Relationship Of The Quality Of Administrative Services And Bpjs Patient Satisfaction In The Inpatient Room X Rumkit Tk Ii Dr. Soepraoen Kesdam V/Brawijaya Malang In Malang City Sutarji 1, Ratna Wardani 2, Ardi Bastian 3, Panca Radono 4 1 Student Institut Ilmu Kesehatan Strada Indonesia 2,3,4 Lecturer Institut Ilmu Kesehatan Strada Indonesia C di th ji @ il Sutarji 1, Ratna Wardani 2, Ardi Bastian 3, Panca Radono 4 1 Student Institut Ilmu Kesehatan Strada Indonesia 2,3,4 Lecturer Institut Ilmu Kesehatan Strada Indonesia Corresponding author: tarji.casper@gmail.com Journal of Hospital Management and Services p-ISSN: 2829-842X (print); e-ISSN: 2830-1056 (online) Vol. 5 No. 2 November 2023 Page.55-60 Journal of Hospital Management and Services p-ISSN: 2829-842X (print); e-ISSN: 2830-1056 (online) Vol. 5 No. 2 November 2023 Page.55-60 Journal of Hospital Management and Services p-ISSN: 2829-842X (print); e-ISSN: 2830-1056 (online) Vo ABSTRACT Quality of service is one of the factors that people consider when choosing a health facility to use. Service quality is a measure to measure patient satisfaction. The aim of the research is to analyze the relationship between the quality of administrative services and BPJS patient satisfaction at Rumkit Tk.II dr. Soepraoen Kesdam V/Brawijaya Malang City Malang City. This research uses a quantitative research design with a cross-sectional approach. The population is all patients or BPJS patient waiters in Inpatient Room X Rumkit Tk.II dr. Soepraoen Kesdam V/Brawijaya Malang City with a sample of 78 respondents using a purposive sampling technique. The independent variable is the quality of administrative services and the dependent variable is BPJS patient satisfaction. Data were collected using a questionnaire and analyzed by the Spearman Rho test with the help of SPSS. The research results showed that the majority of respondents (56.41%) were good (44 people). the majority (65.3%) of respondents were satisfied (51 people). The cross tabulation results showed that the majority expressed satisfaction and good quality, namely 28 people (54.9%). The Spearman's rho statistical test obtained a P-value of 0.000 < 0.05 and a Correlation Coefficient of 0.340, which means there is a relationship between the quality of administrative services and patient satisfaction. At TK II Hospital, dr. Soepraoen Kesdam V/Brawijaya Malang City. The better the quality of administrative services, the more patient satisfaction will increase, so it is hoped that hospital bureaucracy can improve the quality of administrative services for patient satisfaction. Journal of Hospital Management and Services, its website, and the articles published there in are licensed under a Commons Attribution- ShareAlike 4.0 International License. Website: https://thejhms.org/index.php/JHMS \| Email: publikasistrada@gmail.com INTRODUCTION Quality of service is one of the factors that people consider when choosing a health facility to use. Manoppo and Gurning in their research explained that there are several factors that influence the quality of service in a hospital, these factors are timeliness in providing services, competence of staff, and cleanliness of the room. One of the conditions that indicates a problem with the quality of service in a hospital is complaints from health service users who are targeted, namely the attitudes and actions of doctors, nurses, administrative staff, as well as slow service, drug supplies, inadequate infrastructure and medical equipment. Health services that do not meet patient expectations are expected to be an input for health service organizations to try to fulfill them. If the performance of health services received by patients at a health service facility matches their expectations, the patient will always come for treatment at that health service facility. Patients will always look for health services in facilities whose health service performance can meet expectations or not disappoint patients. Website: https://thejhms.org/index.php/JHMS \| Email: publikasistrada@gmail.com Factor Analysis Of The Implementation Of The Occupational Safety And Health Management System At The Lirboyo General Hospital, Kediri City Factor Analysis Of The Implementation Of The Occupational Safety And Health Management System At The Lirboyo General Hospital, Kediri City Hospitals are run on the basis of Pancasila and are based on human values, ethics, professionalism, justice, equal rights and anti-discrimination, equality, patient safety, and have a social function. The aim of hospitals as mandated by Law Number 44 of 2009, in Article 2 and Article 3, is that individual health services are provided in a comprehensive manner by health service institutions and must be able to provide curative and preventive health services, as well as providing outpatient and inpatient services. Hospital services are a measure of patient satisfaction, whether from health services provided by hospital workers or administrative services carried out by the bureaucracy before running health services. Patient satisfaction as a service user is one indicator in assessing the quality of services in a hospital. High patient satisfaction will indicate the success of the hospital in providing health services. Providing quality services will influence patients in receiving treatment. Patients will tend to obey advice, be loyal and obedient to the agreed treatment plan. INTRODUCTION y , y g p According to the Indonesian Ministry of Health, one of the conditions that indicates a problem with the quality of service in hospitals is complaints from health service users who are targeted, namely the attitudes and actions of doctors, nurses, administrative staff as well as slowness in service, drug supplies, inadequate infrastructure and equipment. medical. According to the 2016 RI Minister of Health Regulation which was explained in a scientific journal by Bagus Zulfana regarding Minimum Service Standards for patient satisfaction, namely above 95%. According to WHO, patient satisfaction is above 98%, but in Indonesia the patient satisfaction level still reaches 42.8%. Based on the information from the Ministry of Health above, we can see that if health services are found with a patient satisfaction level below 95%, then it is considered that the health services provided do not meet minimumstandards or are of low quality. And conversely, if a health service is found with a patient satisfaction level above 95%, it is considered that the service meets the standards and is of course of high quality. Based on the background above, researchers are interested in conducting research related to the relationship between the quality of administrative services and BPJS patient satisfaction at Rumkit Tk.II dr. Soepraoen Kesdam V/Brawijaya Malang City Malang City. METHODS This research uses a quantitative research design with a cross-sectional approach. The population is all patients or BPJS patient waiters in Inpatient Room X Rumkit Tk.II dr. Soepraoen Kesdam V/Brawijaya Malang City with a sample of 78 respondents using a purposive sampling technique. The independent variable is the quality of administrative services and the dependent variable is BPJS patient satisfaction. Data were collected using a questionnaire and analyzed by the Spearman Rho test with the help of SPSS. RESULT Respondent Characteristics Respondent Characteristics Respondent Characteristics A. Age Table 1. Frequency Distribution of Respondents Based on Age at TK.II Dr. Hospital. Soepraoen No Umur (Tahun) Total Presentase 1 20-30 Tahun 22 28.20 2 30-40 Tahun 26 33.33 3 40-50 Tahun 22 28.20 4 50-60 Tahun 8 10.25 Total 78 100 Source: Primary Data 2023 Frequency Distribution of Respondents Based on Age at TK.II Dr. Hospital. Journal of Hospital Management and Services 56 Journal of Hospital Management and Services 56 Journal of Hospital Management and Services Journal of Hospital Management and Services Journal of Hospital Management and Services 56 Factor Analysis Of The Implementation Of The Occupational Safety And Health Management System At The Lirboyo General Hospital, Kediri City Factor Analysis Of The Implementation Of The Occupational Safety And Health Management System At The Lirboyo General Hospital, Kediri City Factor Analysis Of The Implementation Of The Occupational Safety And Health Management System At The Lirboyo General Hospital, Kediri City Based on table 1, it can be seen that the distribution of respondents based on the age of inpatients at Tk Hospital. II dr. Soepraoen Kesdam V/Brawijaya, Malang city, is mostly aged 30 - 40 years, namely 26 people (33.33%). B Gender Table 2. Frequency Distribution of Respondents Based on Gender at TK.II Dr. Hospital. Soepraoen No Jenis Kelamin Jumlah Presentase 1 Laki – Laki 22 20.20 2 Perempuan 56 71.80 Total 78 100 Source: Primary Data 2023 Table 2. Frequency Distribution of Respondents Based on Gender at TK.II Dr. Hospital. Source: Primary Data 2023 In table 2, it can be seen that the distribution of respondents based on gender of inpatients at Rumkit Tk.II dr. The majority of Soepraoen Kesdam V/Brawijaya in Malang are women, 56 people (71.80%). C. Education Table 3. Frequency Distribution of Respondents Based on Education at TK.II Dr. Hospital. Soepraoen No Pendidikan Jumlah Presentase 1 Tidak Sekolah 8 10.25 2 SD 20 25.64 3 SMP 19 24.35 4 SMA 22 28.20 5 Perguruan Tinggi 9 11.53 Total 78 100 Source: Primary Data 2023 Frequency Distribution of Respondents Based on Education at TK.II Dr. oen Table 3. Frequency Distribution of Respondents Based on Education at TK.II Dr. . Soepraoen Source: Primary Data 2023 Based on table 3, it can be seen that the distribution of respondents based on the last education of inpatients at Tk.II Dr. Hospital. In table 4, it can be seen that the distribution of respondents based on the quality of service for inpatients at Tk.II Dr. Hospital. Soepraoen Kesdam V/Brawijaya Malang city showed that the majority perceived the quality of service as good, namely 44 people (56.41%). Journal of Hospital Management and Services RESULT Soepraoen Kesdam V/Brawijaya, Malang city, has the highest number of senior high school students with 22 people (28.20%). Univariate Analysis A. Service quality Table 4. Frequency Distribution of Respondents Based on Service Quality at TK.II Dr. Hospital. Soepraoen No Kualitas Pelayanan Jumlah Presentase 1 Kurang 6 7.69 2 Cukup 28 35.89 3 Baik 44 56.41 Total 78 100 Source: Primary Data 2023 q y Frequency Distribution of Respondents Based on Service Quality at TK.II Dr. In table 4, it can be seen that the distribution of respondents based on the quality of service for inpatients at Tk.II Dr. Hospital. Soepraoen Kesdam V/Brawijaya Malang city showed that the majority perceived the quality of service as good, namely 44 people (56.41%). 57 Journal of Hospital Management and Services Factor Analysis Of The Implementation Of The Occupational Safety And Health Management System At The Lirboyo General Hospital, Kediri City Factor Analysis Of The Implementation Of The Occupational Safety And Health Management System At The Lirboyo General Hospital, Kediri City B. Patient Satisfaction Table 5. Frequency Distribution of Respondents Based on Patient Satisfaction at TK.II Dr. Hospital. Soepraoen No Kepuasan Pasien Jumlah Presentase 1 Cukup 21 27 2 Puas 51 65.3 3 Kurang 6 7.69 Total 78 100 Source: Primary Data 2023 action ncy Distribution of Respondents Based on Patient Satisfaction at TK.II Dr. Source: Primary Data 2023 In table 5, it can be seen that the distribution of respondents based on the satisfaction of inpatient patients at Tk.II Dr. Hospital. Soepraoen Kesdam V/Brawijaya Malang city showed that the most people expressed satisfaction, namely 51 people (65.3%) Bivariate Analysis A C T b l ti f Ad i i t ti S i Q lit BPJS P ti t S ti f ti Bivariate Analysis y A. Cross Tabulation of Administrative Service Quality on BPJS Patient Satisfaction y A. Cross Tabulation of Administrative Service Quality on BPJS Patient Satisfaction Kualitas Kurang 1 16,7% 2 9,6% 3 5,9% Cukup 1 16,7% 7 33,3% 20 39,2% Baik 4 66,6% 12 57,1% 28 54,9 Kurang Cukup Puas Kepuasan Table 6. Frequency Distribution of Respondents Based on Service Quality and Patient Satisfaction at TK.II Dr. Hospital Soepraoen Source: Primary Data 2023 y In table 6, it can be concluded that BPJS patient satisfaction in Inpatient Room X Rumkit TK II dr. Soepraoen Kesdam V/Brawijaya Malang City expressed the most satisfaction and good quality, namely 28 people (54.9%). B. DISCUSSION A. Quality of Administrative Services in Inpatient Room X Rumkit Tk.II dr. Soepraoen. A. Quality of Administrative Services in Inpatient Room X Rumkit Tk.II dr. Soepraoen. Quality of Service for inpatients at Tk.II Dr. Hospital. Soepraoen Kesdam V/Brawijaya Malang city showed that the majority perceived the quality of service as good, namely 44 people (56.41%). ( ) One of the factors that causes good service quality is being patient-centered, always providing responsive service, communicating well, and understanding & respecting the patient's wishes. Parasuraman, Zeithaml, and Berry in the Andalas Dental Journal stated that the quality of services can be measured using the Servqual method. The Servqual method has five main dimensions, namely tangibles (physical evidence), reliability (reliability), responsiveness (responsiveness), assurance (guarantee), and empathy (empathy), where in these dimensions there are several indicators. A person will feel satisfied if the results they experience are the same/exceed their expectations and conversely, they will feel disappointed or dissatisfied if the results they experience do not match their expectations. Good service is service that can meet patient expectations and needs, one of which is administrative services in hospitals. In line with Armiati's research, 2019 explains that service quality can improve all aspects of service quality together. This can increase patient satisfaction for BPJS Health users at Lubuk Basung Regional Hospital. Another research is that administrative services are related to patient satisfaction (Herlini, 2022). This research concludes that good administrative services have a 3.273 times higher chance of patients feeling satisfied than poor administrative services. In conclusion, there is a significant relationship between administrative services and patient satisfaction at the ASRI Medical Center Palembang Dental Clinic. Researchers are of the opinion that having qualified staff, providing promising, fast and excellent service in providing service can improve service quality. Someone will feel satisfied with the quality of service if the results they experience are the same/exceed their expectations. B. Bpjs Patient Satisfaction in Inpatient Room X Rumkit Tk.II dr. Soepraoen pj p p Satisfaction of inpatients at Tk.II Dr. Hospital. Soepraoen Kesdam V/Brawijaya Malang city showed that the most people expressed satisfaction, namely 51 people (65.3%). This means that more respondents expressed satisfaction in inpatient room X Hospital Tk.II dr. Soepraoen Kesdam V/Brawijaya Malang City. Patient satisfaction is a level of patient feeling that arises as a result of the performance of health services obtained after the patient compares it with what he expected. RESULT Analysis results y Table 7. Results of Analysis of Administrative Service Quality with Patient Satisfaction At TK.II Hospital dr. Soepraoen Hipotesis P-value Correlation Coefficient There is a relationship between the quality of administrative services and patient satisfaction in Inpatient Room X Rumkit TK II dr. Soepraoen Kesdam V/ Brawijaya Malang City 0,000 0,340 Source: Primary Data 2023 Results of Analysis of Administrative Service Quality with Patient Satisfaction Hospital dr. Soepraoen Source: Primary Data 2023 y Based on table 7, analysis of the quality of administrative services and patient satisfaction, the results of the Spearman's rho statistical test show a Pvalue (Sig. 2 tailed) of 0.000 < 0.05 Correlation Coefficient of 0.340, which means the relationship between the two variables is unidirectional (unidirectional type of relationship), or It can be interpreted that the better the quality of service, the better the patient satisfaction survey results. 58 Journal of Hospital Management and Services Factor Analysis Of The Implementation Of The Occupational Safety And Health Management System At The Lirboyo General Hospital, Kediri City Journal of Hospital Management and Services CONCLUSION There is a relationship between the quality of administrative services and patient satisfaction at TK II Dr. Hospital. Soepraoen Kesdam V/Brawijaya Malang City. The better the quality of service provided, the greater patient satisfaction will increase. It is hoped that the hospital bureaucracy can improve the quality of service both in terms of administration and health workers to increase patient satisfaction at TK II Dr. Hospital. Soepraoen Kesdam V/Brawijaya Malang City. DISCUSSION According to Nurachmah, patient satisfaction is defined as a post-consumption evaluation that a selected product at least meets or exceeds expectations. Patient satisfaction is the level of satisfaction with patient services from the perception of the patient/closest family. Soedjadi explained that patient satisfaction will be achieved if optimal results are obtained for each patient and health services pay attention to the patient's or family's abilities, pay attention to complaints, physical environmental conditions and prioritize patient needs, so that the best possible balance is achieved between the level of satisfaction or results. and the suffering and toil that has been experienced to obtain these results. Researchers are of the opinion that providing optimal service to patients and their families greatly influences the satisfaction of patients and their families. It is hoped that every health facility, including hospitals, health centers and clinics, can provide the best service in order to maintain the quality and quality of health facilities by upholding high patient satisfaction. 59 Factor Analysis Of The Implementation Of The Occupational Safety And Health Management System At The Lirboyo General Hospital, Kediri City Factor Analysis Of The Implementation Of The Occupational Safety And Health Management System At The Lirboyo General Hospital, Kediri City C. Analyzing Service Quality with Bpjs Patient Satisfaction in Inpatient Room X Rumkit Tk.II dr. Soepraoen C. Analyzing Service Quality with Bpjs Patient Satisfaction in Inpatient Room X Rumkit Tk.II dr. Soepraoen Based on the analysis table for the quality of administrative services and patient satisfaction, the results of the Spearman's rho statistical test showed a P-value of 0.000 < 0.05 and a Correlation Coefficient of 0.340, which means there is a relationship between the quality of administrative services and patient satisfaction. At TK II Hospital, dr. Soepraoen Kesdam V/Brawijaya Malang City. j y g y Tjiptono explained that quality has a close relationship with customer satisfaction. Quality provides an incentive for customers to establish strong relationships with the company. In the long term, this type of bond allows the company to thoroughly understand customer expectations and their needs. A hospital is considered good if it provides services that pay more attention to the needs of patients and other people who visit the hospital. Satisfaction arises from the patient's first impression of the nursing services provided, for example: fast service, responsiveness and friendliness in providing nursing services. Kotler explained that patient satisfaction with service is a comparison between his perception of the service received and his expectations before receiving the service. If expectations are met, it means that the service has provided extraordinary quality and will also create high breadth. REFERENCES … Febrian. 2018. Hubungan Dimensi Kualitas Pelayanan Petugas Administrasi Bpjs Kesehatan Dengan Kepuasan Pasien Bpjs Kesehatan Terhadap Pelayanan Administrasi Di Poliklinik Rawat Jalan Rsud Dr. Rasidin Padang. http://scholar.unand.ac.id/33564/1/Abstrak.pdf. Masruroh, Vivianti. 2016. Hubungan Kualitas Pelayanan Administrasi BPJS Dengan Kepuasan Pasien Di Rawat Jalan Rumah Sakit Petrokimia Gresik. https://repository.unair.ac.id/29625/. Novita, Lestari. 2022. Hubungan Kualitas Pelayanan Kesehatan dan Administrasi dengan Kepuasan Pasien BPJS Kelas III Rawat Inap. http://repository.radenintan.ac.id/19600/. Sari, Rihdu. 2020. Pengaruh Kualitas Pelayanan Administrasi Tehadap Kepuasan Pasien Pengguna BPJS Kesehatan di RSUD Lubuk Basung Kabupaten Agam. http://ejournal.unp.ac.id/students/index.php/pek/article/view/7409. Febrian. 2018. Hubungan Dimensi Kualitas Pelayanan Petugas Administrasi Bpjs Kesehatan Dengan Kepuasan Pasien Bpjs Kesehatan Terhadap Pelayanan Administrasi Di Poliklinik Rawat Jalan Rsud Dr. Rasidin Padang. http://scholar.unand.ac.id/33564/1/Abstrak.pdf. Masruroh, Vivianti. 2016. Hubungan Kualitas Pelayanan Administrasi BPJS Dengan Kepuasan Pasien Di Rawat Jalan Rumah Sakit Petrokimia Gresik. https://repository.unair.ac.id/29625/. i i 2022 b l l h d d d Febrian. 2018. Hubungan Dimensi Kualitas Pelayanan Petugas Administrasi Bpjs Kesehatan Dengan Kepuasan Pasien Bpjs Kesehatan Terhadap Pelayanan Administrasi Di Poliklinik Rawat Jalan Rsud Dr. Rasidin Padang. http://scholar.unand.ac.id/33564/1/Abstrak.pdf. Masruroh, Vivianti. 2016. Hubungan Kualitas Pelayanan Administrasi BPJS Dengan Kepuasan Pasien Di Rawat Jalan Rumah Sakit Petrokimia Gresik. https://repository.unair.ac.id/29625/. Novita, Lestari. 2022. Hubungan Kualitas Pelayanan Kesehatan dan Administrasi dengan Kepuasan Pasien BPJS Kelas III Rawat Inap. http://repository.radenintan.ac.id/19600/. Sari, Rihdu. 2020. Pengaruh Kualitas Pelayanan Administrasi Tehadap Kepuasan Pasien Pengguna BPJS Kesehatan di RSUD Lubuk Basung Kabupaten Agam. http://ejournal.unp.ac.id/students/index.php/pek/article/view/7409. 60 Journal of Hospital Management and Services
https://openalex.org/W4313363312	https://journals.iaepan.pl/jue/article/download/2996/2996	Polish	null	Architektura w turbokapitalizmie. Przypadek biurowca Walkie Talkie w Londynie	Journal of Urban Ethnology	2,022	cc-by	9,580	Journal of Urban Ethnology 20/2022 PL ISSN 1429-0618 e-ISSN: 2719-6526 DOI: 10.23858/JUE20.2022.004 Journal of Urban Ethnology 20/2022 PL ISSN 1429-0618 e-ISSN: 2719-6526 DOI: 10.23858/JUE20.2022.004 Journal of Urban Ethnology 20/2022 PL ISSN 1429-0618 e-ISSN: 2719-6526 DOI: 10.23858/JUE20.2022.004 Joanna Krajewska ORCID: https://orcid.org/0000-0002-1449-8132 Politechnika Wrocławska Wydział Architektury Journal of Urban Ethnology 20/2022 PL ISSN 1429-0618 e-ISSN: 2719-6526 DOI: 10.23858/JUE20.2022.004 Abstract The contemporary form of capitalism, which became the dominant system in the age of globalization, gained the turbo prefix in the literature. Nowadays, the architecture is shaped by the market mechanisms to the unprecedented degree. In the context of turbo-capitalism actions and the phenomena characteristic of the architecture of the late 20th and early 21st centuries, the case study was conducted for which I chose an office building known as the Walkie Talkie, opened in 2014 in a global city, London. The undertaken topic makes it possible to analyse individual participants in the design development and operation of an office building as separate interest groups. It also makes it possible to trace some social and cultural processes that constitute the context of activities around this building. Architecture emerges as a commodity in the hands of developers and corporations, and as an element of a strategy implemented by the city’s decision-makers wishing to attract international capital. Key words: turbo-capitalism, iconic building, the Walkie Talkie, London architecture * * * Współczesna forma kapitalizmu, która w czasach globalizacji stała się na świecie systemem dominującym, w literaturze przedmiotu zyskała przedrostek turbo. Architekturę współczesną kształtują mechanizmy ryn kowe w niespotykanym dotąd stopniu. W kontekście działań turbokapitalizmu oraz zjawisk charaktery stycznych dla architektury przełomu XX i XXI wieku dokonałam studium przypadku, na który wybrałam biurowiec znany jako Walkie Talkie, otwarty w 2014 roku w Londynie − mieście globalnym. Podjęty temat pozwala dokonać analizy poszczególnych uczestników procesu projektowego jako odrębnych grup 56 Journal of Urban Ethnology 20/2022 interesów oraz prześledzić procesy społeczne i kulturowe, które stanowią kontekst działań wokół opisywa nego budynku. Z dokonanych analiz wyłania się obraz architektury jako towaru w rękach deweloperów i korporacji, a także elementu strategii miejskich decydentów, pragnących przyciągnąć międzynarodowy kapitał. owa kluczowe: turbokapitalizm, budynek-ikona, wieżowiec Walkie Talkie, architektura Londynu Odebrano / Received: 16.01.2022 Zaakceptowano / Accepted: 14.08.2022 Wraz z globalizacją1 system ekonomiczny gospodarki światowej wszedł w fazę tur bo. Określenie turbokapitalizm2 (Luttwak 2000, Szahaj 2019) dobrze oddaje rozmach siły napędowej współczesnej gospodarki oraz tempo i dynamikę jej procesów. Działania 1 W pracach z zakresu nauk społecznych za początek globalizacji, rozumianej w kategorii procesów skut kujących zaistnieniem sieci wszechobecnych wzajemnych powiązań obejmujących cały świat, najczęściej przyjmuje się koniec lat 80. XX wieku (Czerny 2005: 21, za: Müller-Mahn 2002). 2 Przytoczę w tym miejscu dwie definicje pojęcia kapitalizm. Według wyjaśnienia encyklopedycznego oznacza ono „system społeczno-gospodarczy oparty na własności prywatnej, wolności osobistej i swobo dzie zawierania umów”, w którym procesy gospodarcze, począwszy od rewolucji przemysłowej, są regulo wane przez rynki; cechą charakterystyczną tego systemu jest akumulacja kapitału (PWN). Zgodnie z tym rozumieniem gospodarka krajów rozwiniętego kapitalizmu jest formą ukształtowanej gospodarki rynko wej (zob. Milewski 2012: 88-93); rynek, w ogólnym sensie, to „samoczynnie działający mechanizm wpły wający na zachowania (w tym decyzje) podmiotów gospodarczych” (Milewski 2012: 90). Historyk Fernand Braudel pisał natomiast, że „[a]by zdyscyplinować, zdefiniować pojęcie kapitalizmu (...), trzeba je wstawić między dwa pojęcia, które leżą u jego podstawy i nadają mu sens: między pojęcia kapitał i ka pitalista. Kapitał, realność uchwytna, masa łatwo identyfikowalnych środków nieustannie w ruchu; kapi talista, ktoś zarządzający lub próbujący zarządzać wprowadzeniem kapitału w nieustający proces produkcji, na który wszystkie społeczeństwa są skazane; kapitalizm to ogólnie (ale tylko ogólnie) sposób, w jaki prowadzi się, zwykle w celach mało altruistycznych, tę stałą grę inwestowania” (Braudel 2013: 63). Braudel nie traktował synonimicznie pojęć kapitalizmu i gospodarki rynkowej − ta ostatnia funkcjonowała według niego już wcześniej, systemowi kapitalistycznemu zaś dała początek dopiero rewolucja przemy słowa; wskazywał, by kapitalizm datować dopiero od początku XX wieku (Braudel 2013: 60). 1 W pracach z zakresu nauk społecznych za początek globalizacji, rozumianej w kategorii procesów skut kujących zaistnieniem sieci wszechobecnych wzajemnych powiązań obejmujących cały świat, najczęściej przyjmuje się koniec lat 80. XX wieku (Czerny 2005: 21, za: Müller-Mahn 2002). Stosując ten termin, używam go przede wszystkim zgodnie z tą definicją oraz w znaczeniu liberalizacji dotyczącej procesu międzynarodowej integracji gospodarczej, zgodnie z jedną z pięciu definicji globalizacji wyodrębnionych przez Jana Aarte Scholte (Czerny 2005: 19, za: Scholte 2000). Abstract Stosując ten termin, używam go przede wszystkim zgodnie z tą definicją oraz w znaczeniu liberalizacji dotyczącej procesu międzynarodowej integracji gospodarczej, zgodnie z jedną z pięciu definicji globalizacji wyodrębnionych przez Jana Aarte Scholte (Czerny 2005: 19, za: Scholte 2000). 2 Przytoczę w tym miejscu dwie definicje pojęcia kapitalizm. Według wyjaśnienia encyklopedycznego oznacza ono „system społeczno-gospodarczy oparty na własności prywatnej, wolności osobistej i swobo dzie zawierania umów”, w którym procesy gospodarcze, począwszy od rewolucji przemysłowej, są regulo wane przez rynki; cechą charakterystyczną tego systemu jest akumulacja kapitału (PWN). Zgodnie z tym rozumieniem gospodarka krajów rozwiniętego kapitalizmu jest formą ukształtowanej gospodarki rynko wej (zob. Milewski 2012: 88-93); rynek, w ogólnym sensie, to „samoczynnie działający mechanizm wpły wający na zachowania (w tym decyzje) podmiotów gospodarczych” (Milewski 2012: 90). Historyk Fernand Braudel pisał natomiast, że „[a]by zdyscyplinować, zdefiniować pojęcie kapitalizmu (...), trzeba je wstawić między dwa pojęcia, które leżą u jego podstawy i nadają mu sens: między pojęcia kapitał i ka pitalista. Kapitał, realność uchwytna, masa łatwo identyfikowalnych środków nieustannie w ruchu; kapi talista, ktoś zarządzający lub próbujący zarządzać wprowadzeniem kapitału w nieustający proces produkcji, na który wszystkie społeczeństwa są skazane; kapitalizm to ogólnie (ale tylko ogólnie) sposób, w jaki prowadzi się, zwykle w celach mało altruistycznych, tę stałą grę inwestowania” (Braudel 2013: 63). Braudel nie traktował synonimicznie pojęć kapitalizmu i gospodarki rynkowej − ta ostatnia funkcjonowała według niego już wcześniej, systemowi kapitalistycznemu zaś dała początek dopiero rewolucja przemy słowa; wskazywał, by kapitalizm datować dopiero od początku XX wieku (Braudel 2013: 60). Pojęciem turbokapitalizm amerykański ekonomista Edward Luttwak określa współczesną formę kapitali zmu, której charakterystycznymi cechami są: deregulacja, prywatyzacja i globalizacja (zob. Luttwak 2000). W polskiej literaturze przedmiotu posługuje się nim m.in. Andrzej Szahaj, dla którego turbokapitalizm zrodził się z tryumfu neoliberalizmu (zob. Szahaj 2019: 54–55). Niektórzy eksperci dla określenia współczesnej fazy kapitalizmu stosują termin późny kapitalizm (np. Crary 2015); był on wielokrotnie używany przez różnych badaczy w XX wieku. Zrezygnowałam z jego użycia, ponieważ nie jest dość precyzyjny i sugeruje wejście w końcową fazę kapitalizmu, o czym zbyt wcześnie nam przesądzać. Podobnie zrezygnowałam z określenia nowy kapitalizm proponowanego przez Richarda Sennetta (Sennett 2010). 57 oanna Krajewska \| Architektura w turbokapitalizmie. Przypadek biurowca Walkie Talkie w Londy te pozostają nie bez wpływu na przestrzeń miejską, co jest zwłaszcza widoczne w mia stach globalnych3 (zob. Węcławowicz 2007: 89). Nie jest właściwe pisanie o architekturze bez ujmowania kontekstu, w jakim po wstaje. 3 Miasto globalne to kompleks urbanistyczny, który pełni m.in. następujące funkcje: jest centrum władzy politycznej w skali krajowej i międzynarodowej, stanowi centrum bankowości, ubezpieczeń oraz powią zanych z nim usług finansowych, stanowi centrum wytwarzania i wykorzystania wiedzy, technologii oraz centrum gromadzące specjalistów, centrum zbierania, przetwarzania i upowszechniania informacji, a tak że centrum demonstracyjnej konsumpcji dóbr luksusowych przez elitę; na wszystkie wymienione funkcje znaczący wpływ mają globalizacja i informatyzacja gospodarki. 4 Nadmierna konsumpcja, mająca charakteryzować współczesne społeczeństwa, na którą wskazują badacze nauk społecznych, jest wynikiem nadmiernego wytwarzania towarów i usług, oraz kreowania zapotrzebo wania na nie. Osią turbokapitalizmu jest maksymalizacja zysków, często za wszelką cenę, czemu sprzyja pozbawiona kontroli efektywność rynkowa. 3 Miasto globalne to kompleks urbanistyczny, który pełni m.in. następujące funkcje: jest centrum władzy politycznej w skali krajowej i międzynarodowej, stanowi centrum bankowości, ubezpieczeń oraz powią zanych z nim usług finansowych, stanowi centrum wytwarzania i wykorzystania wiedzy, technologii oraz centrum gromadzące specjalistów, centrum zbierania, przetwarzania i upowszechniania informacji, a tak że centrum demonstracyjnej konsumpcji dóbr luksusowych przez elitę; na wszystkie wymienione funkcje znaczący wpływ mają globalizacja i informatyzacja gospodarki 4 Nadmierna konsumpcja, mająca charakteryzować współczesne społeczeństwa, na którą wskazują badacze nauk społecznych, jest wynikiem nadmiernego wytwarzania towarów i usług, oraz kreowania zapotrzebo wania na nie. Osią turbokapitalizmu jest maksymalizacja zysków, często za wszelką cenę, czemu sprzyja pozbawiona kontroli efektywność rynkowa. Abstract Będąc materialnym wyrazem kultury, architektura odzwierciedla ducha czasu. Fred Inglis wskazał, iż „[k]ultura opiera się na wartościowaniu praktyk dnia codzienne go” (Inglis 2007: 206). Współczesna forma kapitalizmu przenika wszystkie sfery ludz kiego życia, a te charakteryzuje konsumpcja na niespotykaną dotąd skalę (np. Ritzer 2004, Crary 2015), która „jest coraz bardziej konsumpcją znaków i wrażeń, a nie samych rzeczy” (Szahaj 2019: 60). W systemie zorientowanym na turbozysk pieniądze są osią, wokół której koncentrują się wszelkie działania4. Jest dość interesujące, że pisząc o współczesnej architekturze, rzadko wspomina się o inwestorach, którzy są kluczowymi elementami procesu projektowego, ponieważ od nich pochodzą wytyczne dla planowanych budynków. Jak napisał kilka lat temu krytyk architektury Tom Dyckhoff, autor poczytnej książki Epoka spektaklu: „By naprawdę zro zumieć architekturę, trzeba pojąć związki władzy, które za nią stoją. By naprawdę zrozu mieć dzisiejszą architekturę, trzeba sprawdzać, kto za nią płaci” (Dyckhoff 2018: 29). Niniejszy artykuł ma na celu ukazanie procesów, w które uwikłana jest tzw. architektura ikoniczna; pokazanie mechanizmów − kto i w jakim celu wpływa na to, jakie budynki powstają w centrach współczesnych metropolii. Szerokie tło dla tych mechanizmów stanowi gospodarka turbokapitalistyczna. Wybrana metoda badawcza − studium przy padku − pozwala na dogłębną analizę i umożliwia wzajemne powiązanie zachodzących zjawisk. Przyjęta struktura tekstu służy prezentacji poszczególnych uczestników procesu tworzenia i eksploatacji opisywanego budynku, traktowanych jako odrębne grupy inte resów, a także pozwala zwrócić uwagę na znaczenie formy oraz funkcji obiektu architek tonicznego w kontekście potrzeb współczesnego systemu ekonomicznego, jak i na głosy krytyki. Najbardziej znamiennym fenomenem, jaki zaistniał w architekturze czasów globa lizacji, było pojawienie się tzw. budynku-ikony. To obiekt o charakterystycznym, łatwo rozpoznawalnym kształcie, mający wpływ na wizerunek miasta. Może on pełnić dowol ną funkcję, często stanowi dominantę wysokościową. Budynek taki jest nieraz pokazem 58 Journal of Urban Ethnology 20/2022 aktualnych możliwości budowlanych, manifestem estetyki, a także wyrazem finansowe go potencjału inwestora. To architektura sukcesu, czytelny symbol nowoczesności. g p j y y y W obecnych czasach, w których kultura podporządkowana jest prawom (i sztucz nie kreowanym potrzebom) rynku, architektura, „uwolniona” z pierwotnych znaczeń, często jest sprowadzana do roli kolejnego pożądanego towaru. Daje się zaobserwować zjawisko gadżetyzacji architektury5 (Kwiatkowski 2010: 213), w którym budynek został sprowadzony do roli przedmiotu-obrazu nie niosącego głębszych treści, pełniącego funkcję znaku (miasta, firmy bądź instytucji) (Jencks 2005). 5 Kwiatkowski, powołując się na Bruno Latoura, wskazuje, że gadżety − służące często bezmyślnej zabawie, wikłają użytkowników w powierzchowne relacje, zaś współczesne wytwory architektoniczne nierzadko odgrywają właśnie rolę gadżetów. Abstract Jak zauważyli Scott Lash i Celia Lury: „W globalnym przemyśle kulturowym produkcja odbywa się w ramach postfordystycznego, intensywnie kreatywnego (design-intensive) modelu produkcji róż nicy” (Lash, Lury 2011: 16). Choć ich stwierdzenie dotyczy kreowania marek, w archi tekturze daje się zaobserwować podobne tendencje, a celem (jak się wydaje), jest wyróżnienie obiektu na tle pozostałych. „Ostentacyjność i widowiskowość są łatwym do przewidzenia produktem systemu polityczno-ekonomicznego, który wspiera indywidu alizm i rywalizację” − pisze Tom Dyckhoff (Dyckhoff 2018: 22) i dodaje: „Dziś prestiżo wymi architektonicznymi bibelotami (...) handluje się na rynkach międzynarodowych jak złotem (...). Jak każdym innym towarem” (Dyckhoff 2018: 44). f Znany krytyk architektury Charles Jencks, który scharakteryzował budowle iko niczne, widział je jako „nieuchronną konsekwencję rozwoju cywilizacji obrazkowej, showbiznesu i ekonomicznego wzrostu” (Tokajuk 2008, za: Jencks 2005), przyznając jednocześnie, że tego rodzaju budynki stanowią wyraz rozwoju globalnego kapitalizmu i niszczą miasto (Jencks 2005). Tom Dyckhoff wpisał je w nurt „architektury spektaklu” (Dyckhoff 2018), „dziwacznej”, mającej robić na odbiorcy oszałamiające wrażenie. Spek takl zaś, w ujęciu pisarza i filozofa Guya Deborda „to kapitał, który osiągnął taki sto pień akumulacji, że stał się obrazem” (Debord 2006: 44). Wielu projektantów również wskazuje na szkodliwość powstawania budynków-i kon. Według architekta Andrzeja Tokajuka zaistnienie takiego obiektu powoduje „nie ciągłość przestrzeni miejskiej” (Tokajuk 2008: 556); zalicza je do nurtu „architektury regresywnej” (Tokajuk 2008: 555). Inny architekt, Krzysztof Kwiatkowski, w kontekście „ikon” pisze: „Poszczególne budynki stanowią niczym leibnizowskie monady zamknięte całości nie łączące się w układy urbanistyczne” (Kwiatkowski 2010: 213). Słynny archi tekt i krytyk Rem Koolhaas wyodrębnił we współczesnej architekturze zjawisko Bigness (Wielkości) − problem jej przeskalowania. Sam rozmiar budynków stawia je poza moralnością, − wskazuje Koolhaas − poza dobrem i złem. Ich wpływ nie zależy od ich jakości. (...) architektura Bigness nie jest już częścią 5 Kwiatkowski, powołując się na Bruno Latoura, wskazuje, że gadżety − służące często bezmyślnej zabawie, wikłają użytkowników w powierzchowne relacje, zaś współczesne wytwory architektoniczne nierzadko odgrywają właśnie rolę gadżetów. 5 Joanna Krajewska \| Architektura w turbokapitalizmie. Przypadek biurowca Walkie Talkie w Londynie na Krajewska \| Architektura w turbokapitalizmie. Przypadek biurowca Walkie Talkie w Londynie 59 tkanki miejskiej./ Bigness istnieje w niej, co najwyżej współistnieje./ A w podtekście mówi pieprzyć kontekst (Koolhaas 2017: 50). Choć w 1994 roku, w którym pisał swój tekst, Koolhaas twierdził, że miasta euro pejskie uchroniły się przed tym problemem, w początkach XXI wieku zjawisko to moż na już zaobserwować. 6 Oficjalna nazwa budynku odnosi się wprost do jego adresu. i 7 W wyniku najnowszych badań nad znaczeniem miast, Londyn wraz z Nowym Jorkiem, otrzymały w 2020 r. najwyższą kategorię w rankingu miast światowych: Alpha ++. 8 City of London stanowi suwerenną jednostkę administracyjną o prawach miejskich w granicach Londynu. Studium przypadku − biurowiec Walkie Talkie w Londynie Do analizy przypadku wieżowca-ikony wybrałam biurowiec 20 Fenchurch Street6 znany jako Walkie Talkie, otwarty w 2014 roku w obrębie Londynu. O wyborze budyn ku zadecydowało kilka czynników − zarówno tych, które czynią go typowym przykła dem wieżowca-ikony: charakterystyczny kształt wraz z wyróżniającym przydomkiem oraz dominująca funkcja biurowa, jak i te, które świadczą o tym, że jest to przypadek szczególny (szczególnie wyrazisty): lokalizacja, połączenie funkcji biurowca z publicz nym parkiem oraz rekordowa kwota, za jaką został sprzedany nowemu właścicielowi. Nie bez znaczenia było również moje osobiste doświadczenie tej architektury Nie bez znaczenia było również moje osobiste doświadczenie tej architektury. Londyn należy do miast globalnych pierwszej kategorii7 (GaWC 2020). Analizo wany budynek mieści się w centralnie położonej dzielnicy biznesu The City8, przez Toma Dyckhoffa określonej „jednym z najbardziej bezwzględnie konkurencyjnych ob szarów planety” (Dyckhoff 2018: 132). Londyńskie City do dziś pozostaje miejscem globalnych finansów, nie jest już ono jednak brytyjską instytucją − pisał Richard Sennett na początku XXI wieku − Banki działające na rzecz przedsiębiorców skupiały się coraz bardziej na fuzjach i przejęciach, a te również straciły wszelkie związki z jakimkolwiek interesem państw narodowych (Sennett 2010: 33). Już wówczas lokalni obserwatorzy kwestionowali potrzebę budowy kolejnych wie żowców, które miały niszczyć krajobraz miasta (zob. Weaver 2007). Tom Dyckhoff bar dzo krytycznie odniósł się do zachodzących tam przeobrażeń: City, od wielu stuleci dzielnica finansowa Londynu, za sprawą spekulacyjnych inwestycji w biurowce we wszystkich możliwych kształtach od jakiegoś czasu zmienia swoją panora mę. (...) Gdy się na nie patrzy z tej strony Tamizy [z południowego brzegu − przyp. J.K.], nowe cudaczne nabytki w panoramie miasta prezentują się nierealnie, jak wycięte, wklejone i podkręcone w Photoshopie, jak tandetne durnostoje na kominku lub rząd butelek z i 7 W wyniku najnowszych badań nad znaczeniem miast, Londyn wraz z Nowym Jorkiem, otrzymały w 2020 r. najwyższą kategorię w rankingu miast światowych: Alpha ++. 8 City of London stanowi suwerenną jednostkę administracyjną o prawach miejskich w granicach Londynu. 60 Journal of Urban Ethnology 20/2022 kosmetykami na półce nad wanną. Wydaje się, że można po którąś nieomal sięgnąć ręką (Dyckhoff 2018: 37). kosmetykami na półce nad wanną. Wydaje się, że można po którąś nieomal sięgnąć ręką (Dyckhoff 2018: 37). Podczas pobytów w Londynie w latach 2007–2018 odbywałam nieśpieszne space ry, między innymi południowym brzegiem Tamizy, wspomnianym w cytowanym tek ście, a także kilkakrotnie w samej dzielnicy biznesu. Miałam okazję zaobserwować, jak zmienia się sylweta tej metropolii − m.in. Studium przypadku − biurowiec Walkie Talkie w Londynie jak charakterystyczny wieżowiec w kształcie ogórka, Korniszon − „ikona” Londynu XXI wieku, znika z pola widzenia, przysłonięty nowymi biurowcami. Pośród nich najbardziej wyróżniał się budynek wyglądający tak, jakby zaraz miał się przewrócić, przypominający ogromną słuchawkę Walkie Talkie. Obiekt ten odwiedziłam wiosną 2015 roku, po wcześniejszej rejestracji mojej wizyty na stronie internetowej o nazwie w swobodnym tłumaczeniu brzmiącej: „Podniebny Ogród. Londyn”. Wówczas mogłam nie tylko przyjrzeć mu się z bliska, ale także doświadczyć przyjemności przebywania na szczycie budynku, w otoczeniu zieleni, z panoramicznym widokiem na miasto. Zastanawiało mnie jednak, dlaczego ktoś pozwala, aby w metropo lii przepełnionej „ikonami” stawały kolejne obiekty tak mocno ingerujące w krajobraz, a jednocześnie tak pospolite z uwagi na pełnioną funkcję. Wysoki na 160 metrów wieżowiec 20 Fenchurch Street, zrealizowany według pro jektu urugwajskiego architekta Rafaela Viñoly, zyskał swój przydomek ze względu na kształt, podobnie jak inne charakterystyczne biurowce Londynu. Jak zauważa Dyckhoff: Kiedyś nadanie budynkowi nazwy było oznaką jego wejścia do kultury popularnej, akcep tacji przez masowego odbiorcę. Dziś jednak budowle, zwłaszcza jeśli istnieje podejrzenie, jak w przypadku drapaczy chmur, że mogą się okazać kontrowersyjne, często już rodzą się z przydomkami − dzięki działowi marketingu dewelopera (Dyckhoff 2018: 37). Jednak według Petera Reesa, ówczesnego głównego urbanisty City, nazwę nadali budynkowi dziennikarze magazynu architektonicznego „Architect’s Journal”, po rozmo wie telefonicznej, jaką z nim odbyli na długo przed realizacją biurowca. Rees, poproszo ny wówczas o opis mającego powstać obiektu, porównał go spontanicznie do starego modelu telefonu krótkofalowego, w następstwie czego budynek mianowano Walkie Tal kie i tak przedstawiono w artykule (Crosland 2015). Zaprojektowany w 2004 roku wie żowiec uzyskał pozwolenie na budowę w lipcu 2007 roku, a jego potoczna nazwa już funkcjonowała w prasie (zob. Weaver 2007); ponadto media często określały go jako „ikoniczny”. Wypowiedź Reesa wskazywałaby, iż pośrednio uczestniczył w znalezieniu chwytliwego przydomka dla planowanego wieżowca. Nadanie nazwy wraz z prezentacją wizualizacji pozwoliło „uobecnić” biurowiec w świadomości masowego odbiorcy i „oswo ić” z jego powstaniem w mieście. Na rynku deweloperskim przydomek pozwala stworzyć i wprowadzić nieruchomość jako produkt, który łatwiej wypromować i sprzedać. 61 oanna Krajewska \| Architektura w turbokapitalizmie. Przypadek biurowca Walkie Talkie w Londy Walkie Talkie sprawia wrażenie „niezgrabnego”. Krawędzie biurowca nie są do sie bie równoległe − przeszklona z wszystkich stron bryła (również dach) pokryta rastrem żaluzji (poziomych od południa, i pionowych na bocznych elewacjach) rozszerza się ku górze. Południowa elewacja jest zwrócona ku Tamizie, od której dzielą ją zaledwie dwie ulice. 10 Wypowiedź została jednak udzielona kilka lat po realizacji Walkie Talkie, trudno więc jednoznacznie ocenić, czy podobną postawę architekt prezentował wcześniej, czy w ten sposób tłumaczy swoje minione wybory. 9 Londyńska centrala biura powstała, aby nadzorować rozwój projektów wybranych w tamtym czasie do realizacji drogą konkursu: Visual Arts Facility w Colchester oraz Leicester Performing Arts Centre w Wielkiej Brytanii (Viñoly). Studium przypadku − biurowiec Walkie Talkie w Londynie W przeciwnym kierunku, nieco ponad 100 metrów w linii prostej, znajduje się słynny gmach Lloyd’s of London z pracowni Richard Rogers and Partners, otwarty w 1986 roku − siedziba jednego z wiodących rynków ubezpieczeń i reasekuracji na świe cie. Aby zrealizować 20 Fenchurch Street rozebrano 91-metrowy biurowiec − siedzibę prywatnego banku Kleinwort Benson z 1968 roku, powstałego w stylu międzynarodo wym, który w początkach XXI wieku reprezentował już przeszłość i był jednym z wielu obiektów, jakie nie przetrwały próby czasu. Architekt Firma Rafael Viñoly Architects, założona w 1983 roku, prowadzi działalność na skalę globalną, mając główne siedziby w Nowym Jorku (od początku) oraz w Londynie9 (od 2000 roku), a także biura w Buenos Aires i Cupertino w Kalifornii (USA). W swoim dorobku posiada projekty zlokalizowane na kilku kontynentach, łącznie w trzynastu państwach (zob. Viñoly). Pracownia, wielokrotnie nagradzana za swoje projekty, zdoby ła międzynarodowe uznanie realizując w Japonii projekt Tokyo International Forum w 1997 roku. Tom Dyckhoff zaliczył Rafaela Viñoly do grona star-architektów − projek tantów-celebrytów tworzących w nurcie architektury spektaklu, których sławne nazwi ska mają gwarantować powodzenie inwestycji (Dyckhoff 2018). j g p y j yf W 2014 roku biurowiec 20 Fenchurch Street był dla urugwajskiego architekta pierw szym zrealizowanym projektem budynku w Londynie (zob. Viñoly). Wieżowiec w cen trum miasta globalnego, w dzielnicy biznesu o światowym znaczeniu, jest niezwykle prestiżowym zleceniem, zaś prestiż i potencjalny sukces inwestycji determinuje powierze nie jej uznanej pracowni (zwłaszcza star-architektowi) − zatem zarówno projektantowi, jak i inwestorowi mogło szczególnie zależeć na współpracy przy tej realizacji. Peter Rees wy mienił Rafaela Viñoly’ego jako jednego ze star-architektów „zdesperowanych, by zbudo wać coś w City”, gdyż byli świadomi, że taka realizacja to dla nich „architektoniczna wizytówka” (Crosland 2015, tłum. J.K.). O tym, iż Viñoly nie boi się odważnych decyzji projektowych może świadczyć jego wypowiedź z 2018 roku10, gdy zapytany o wyróżnik „sukcesu” architektury wyznał: „Najlepsza architektura redefiniuje to, jak będzie wyglądać, 9 Londyńska centrala biura powstała, aby nadzorować rozwój projektów wybranych w tamtym czasie do realizacji drogą konkursu: Visual Arts Facility w Colchester oraz Leicester Performing Arts Centre w Wielkiej Brytanii (Viñoly). 62 Journal of Urban Ethnology 20/2022 zamiast powtarzać to, co zostało zaakceptowane lub to, co już jest częścią normy” (Blatter 2018, tłum. J.K.). Uważa on ponadto, iż najważniejsze jest to, czy „rozwiązanie problemu [architektonicznego − przyp. J.K.] było inteligentne” (Ibidem). 12 Brytyjska firma deweloperska powstała w 1993 r. z myślą o rozwoju dawnej dzielnicy portowej Canary Wharf, którą przekształcono w jeden z ośrodków światowego biznesu, co uczyniło ją deweloperem najwięk szego projektu rewitalizacyjnego w Europie; na stronie internetowej pracowni Rafaela Viñoly nie widnieje jako inwestor, jednak jako taka jest wymieniana w publikacjach dotyczących wieżowca Walkie Talkie. 11 Stan na 30.09.2021 r.i 11 Stan na 30.09.2021 r. 12 Brytyjska firma deweloperska powstała w 1993 r. z myślą o rozwoju dawnej dzielnicy portowej Canary Wharf, którą przekształcono w jeden z ośrodków światowego biznesu, co uczyniło ją deweloperem najwięk szego projektu rewitalizacyjnego w Europie; na stronie internetowej pracowni Rafaela Viñoly nie widnieje jako inwestor, jednak jako taka jest wymieniana w publikacjach dotyczących wieżowca Walkie Talkie. 13 Jedyna europejska siedziba firmy mającej swoje centrale w Azji i Ameryce Północnej, mieści się w londyń skiej dzielnicy biznesu Canary Wharf na terenie dawnych doków i obejmuje przedstawicielstwo na Europę i Rosję (zob. Corporate LKK). 14 W latach 1902–1932 siedziba firmy znajdowała się w Makau, a w 1932 r. została przeniesiona do Hongkongu, który do 1997 r. był w posiadaniu Wielkiej Brytanii (w moc 99-letniej „dzierżawy”). 15 Wartość marki w 2017 r. przekroczyła 65,8 miliarda RMB (Corporate LKK). Majątek zmarłego w 2021 r. miliardera szacowano na 17,4 miliardów dolarów netto (Forbes). g g y y p j y j y 15 Wartość marki w 2017 r. przekroczyła 65,8 miliarda RMB (Corporate LKK). Majątek zmarłego w 2021 r. miliardera szacowano na 17,4 miliardów dolarów netto (Forbes). 13 Jedyna europejska siedziba firmy mającej swoje centrale w Azji i Ameryce Północnej, mieści się w londyń skiej dzielnicy biznesu Canary Wharf na terenie dawnych doków i obejmuje przedstawicielstwo na Europę i Rosję (zob. Corporate LKK). 14 W latach 1902–1932 siedziba firmy znajdowała się w Makau, a w 1932 r. została przeniesiona do p j p 14 W latach 1902–1932 siedziba firmy znajdowała się w Makau, a w 1932 r. została przen Hongkongu, który do 1997 r. był w posiadaniu Wielkiej Brytanii (w moc 99-letniej „dzierżaw Inwestor i właściciel Budowa wieżowca kosztowała ponad 220 milionów funtów (zob. Visitlondon). Deweloperem obiektu była jedna z wiodących firm na brytyjskim rynku nieruchomości − Land Securities PLC (zał. w 1944 r.) z tzw. portfelem szacowanym w styczniu 2022 roku na 11 miliardów funtów11 (zob. Landsec), przy finansowym wsparciu Canary Wharf Group12. Ta druga firma na swojej stronie internetowej szczyci się przekształce niem Londynu w „miasto 24/7, gdzie ludzie mogą mieszkać/ pracować/ bawić się” (zob. CWG). Ukośniki obecne w cytowanym tekście wydają się być znamienne − obecne sta dium kapitalizmu cechuje przemieszanie czasu wolnego (w tym czasu przeznaczonego na sen) i pracy, splatające je w niedookreślone kontinuum, tak aby system mógł stale czerpać zyski z funkcjonowania jednostki (zob. Crary 2015, por. Florida 2010). p y j j y p W 2017 roku budynek Walkie Talkie kupiło przedsiębiorstwo spożywcze Lee Kum Kee International Holdings Ltd. z Hongkongu13 specjalizujące się w produkcji sosów azjatyckich i chińskim ziołolecznictwie. Ten globalny koncern − rodzinna firma założo na w 1888 roku w chińskiej prowincji Guangdong (wówczas Nanshui)14 − w 2017 roku kontrolowana przez 88-letniego miliardera Lee Man Tat15 (Butler 2017), posiada rów nież gałąź inwestującą na rynku nieruchomości: Infinitus Property Investment (Hong Kong) Limited. Azjatycki konglomerat nabył wieżowiec Walkie Talkie za 1,28 miliarda funtów, bijąc rekord kwoty, za jaką kiedykolwiek sprzedano pojedynczy budynek w Lon dynie, a fakt ten szeroko opisała krajowa i zagraniczna prasa, posługując się chwytliwą nazwą biurowca (m.in. ”The Guardian”, ”The Times”, ”Chinadaily”). Brytyjski deweloper Land Securities miał otrzymać ze sprzedaży wieżowca 641 milionów funtów, z których 475 milionów zamierzał zwrócić akcjonariuszom, a resztę sumy wykorzystać na spłatę zadłużenia (Reuters 2017), co pozwoliło ocenić inwestorowi projekt wieżowca jako „niezmiernie udany” (Butler 2017, tłum. J.K.). James Bekham, szef londyńskich rynków 63 oanna Krajewska \| Architektura w turbokapitalizmie. Przypadek biurowca Walkie Talkie w Londy kapitałowych w jednej z firm doradczych, ocenił, iż „[t]a bijąca rekord transakcja poka zuje ogromny apetyt inwestorów w Londynie oraz reputację City jako globalnego miej sca do prowadzenia interesów” (Butler 2017, tłum. J.K.). Można dodać, iż był to kolejny zakup dokonany w tamtym czasie na rynku nieruchomości w stolicy Wielkiej Brytanii przez firmę z Hongkongu − przykładowo, nieco wcześniej inny charakterystyczny wie żowiec w City − 122 The Leadenhall Building (zwany „Tarka do sera”), również z 2014 roku − zakupiła firma C C Land Holdings za 1,15 miliarda funtów (Mclean 2017). 16 Ulrich Beck przedstawił koncepcję „społeczeństwa ryzyka” w 1986 roku; odnosiła się ona „do pewnej epoki nowoczesnego społeczeństwa, które nie tylko porzuca tradycyjne formy życia, lecz przede wszyst kim zmaga się ze skutkami ubocznymi udanej modernizacji: z niepewnością życia i trudno uchwytnymi zagrożeniami, które dotyczą wszystkich i przed którymi nikt nie potrafi się już należycie zabezpieczyć” (Beck 2012: 21). W pierwszej dekadzie XXI wieku, w obliczu globalnego terroryzmu oraz grożącej świa tu katastrofy klimatycznej, Beck zdecydował się sformułować tezy dla „społeczeństwa światowego ryzy ka”, według których „światowe ryzyko jest inscenizacją realności światowego ryzyka” (Beck 2012: 23). Typologizując postaci globalnego ryzyka, obok zagrożeń terroryzmem i kryzysów ekonomicznych, wyod rębnia globalne kryzysy finansowe (Beck 2012: 28–29). Inwestor i właściciel Wcześniej przedsiębiorstwo Lee Kum Kee bezskutecznie próbowało nabyć Kor niszon (Butler 2017), co świadczy o jego wielkich ambicjach biznesowych i traktowaniu architektury w kategoriach towaru o znaczeniu wizerunkowym. Po zakupie Walkie Talkie portal Chinadaily donosił, iż jest to „najnowsza chińska inwestycja w londyński sektor nieruchomości komercyjnych napędzana deprecjacją fun ta w związku z niepewnością wynikającą z Brexitu” (Liu 2017, tłum. J.K.). Sytuacja po lityczna od razu przełożyła się zatem na transakcje na rynku nieruchomości w Londynie. Wpisuje się to w teorię Ulricha Becka, według której we współczesnym świecie nie ustannie szacowane są poziomy globalnego ryzyka16 (Beck 2012), w tym również moż liwe zyski i straty finansowe. James Bekham przekonywał, że „[o]d czasu głosowania za opuszczeniem UE [przez Wielką Brytanię − przyp. J.K.] kapitał z regionu Azji i Pacy fiku ukierunkowany na Londyn wzrósł do rekordowych poziomów”, co „wskazuje na długoterminowe zaufanie do Londynu i strategie inwestycyjne, których nie zakłóca krótkoterminowa niepewność polityczna” (Butler 2017, tłum. J.K.).i Wieżowiec był pierwszym budynkiem, jaki firma nabyła w Europie (Forbes). Sam my Lee, prezes LKK Health Products Group wskazał cel zakupu, mówiąc: „Cieszymy się, że możemy przejąć 20 Fenchurch Street w centrum Londynu, aby rozszerzyć naszą globalną obecność poprzez strategiczne inwestycje” (Butler 2017, tłum. J.K.). Wydane oświadczenie precyzowało zamierzenie przedsiębiorstwa: „Przejęcie umożliwia Grupie nie tylko osiągnięcie rozsądnego zwrotu z przychodów z tytułu najmu, ale także rozsze rzenie portfolio nieruchomości do dużego zagranicznego centrum finansowego w celu trwałego i stabilnego wzrostu wartości kapitału” (Isaacson 2017, tłum. J.K.). Firma LKK na swojej stronie internetowej informuje, iż osiągnęła swoją wizję: „Gdzie są ludzie, tam jest Lee Kum Kee” (zob. Corporate LKK) − motto to podkreśla zarówno jej 64 Journal of Urban Ethnology 20/2022 egalitarystyczny charakter, jak i globalny zasięg. Można jednak stwierdzić, że powszech na obecność na światowych rynkach już jej nie wystarczała. Akumulacja kapitału musia ła znaleźć swoją materializację w obiekcie spektakularnym, dając wyraz potęgi firmy. g p y , p y ęp g p p y p hałasu. 18 W pracy projektowej nad budynkiem komercyjnym już na wstępnym etapie zwraca się uwagę, by zmak symalizować GLA. Uzyskanie możliwie największej powierzchni najmu staje się znacznym atutem pro jektu, nieraz przesądzającym o otrzymaniu przez daną pracownię zlecenia. 19 Autor Epoki spektaklu posuwa się jeszcze dalej, pisząc o Rafaelu Viñoly’m: „cwany, najbardziej komercyj nie myślący z architektów” (Dyckhoff 2018: 143). 20 Jest to parafraza słynnej zasady projektowej sformułowanej pod koniec XIX w. przez architekta Louisa Sullivana: „forma podąża za funkcją” (ang. form follows function). Maksymę tę swoim credo uczynili moderniści. 17 Z uwagi na lepszy widok, lepszy dostęp do światła naturalnego oraz prawdopodobnie niższy poziom hałasu. 18 W pracy projektowej nad budynkiem komercyjnym już na wstępnym etapie zwraca się uwagę, by zmak symalizować GLA. Uzyskanie możliwie największej powierzchni najmu staje się znacznym atutem pro jektu, nieraz przesądzającym o otrzymaniu przez daną pracownię zlecenia. 19 Autor Epoki spektaklu posuwa się jeszcze dalej, pisząc o Rafaelu Viñoly’m: „cwany, najbardziej komercyj nie myślący z architektów” (Dyckhoff 2018: 143). 18 W pracy projektowej nad budynkiem komercyjnym już na wstępnym etapie zwraca się uwagę, by zmak symalizować GLA. Uzyskanie możliwie największej powierzchni najmu staje się znacznym atutem pro jektu, nieraz przesądzającym o otrzymaniu przez daną pracownię zlecenia. f 20 Jest to parafraza słynnej zasady projektowej sformułowanej pod koniec XIX w. przez architekta Louisa Sullivana: „forma podąża za funkcją” (ang. form follows function). Maksymę tę swoim credo uczynili moderniści. Forma i skala budynku Nietypowy kształt budynku o łącznej powierzchni użytkowej 104 520 m2 (Viñoly) powstał przez powiększenie powierzchni kondygnacji zlokalizowanych wysoko nad te renem, mających większą wartość ekonomiczną17 − informacji tej nie krył sam architekt, a powtarzały ją liczne publikacje. W ten sposób uzyskano większą powierzchnię najmu brutto (ang. gross leasable area, GLA), tak cenną dla inwestorów18. Taki sposób ukształ towania bryły zdaje się wynikać wprost z kontrowersyjnej decyzji Viñoly’ego, dla które go mogło to być jedno z „inteligentnych rozwiązań” projektowych z przywołanego wcześniej cytatu. Tom Dyckhoff opisuje to w ten sposób: (...) skoro cena za wynajem wyższych pięter jest większa − z powodu widoków − to klasycz ne formy budynków, mimo ograniczeń związanych z grawitacją, nie mają ekonomicznego sensu w czasach obsesji zysku. Dlatego Viñoly, architekt wyjątkowo rozgarnięty w kwe stiach komercyjnych19, odwrócił konwencję: jego wieżowiec wybrzusza się u szczytu, aby najdroższej przestrzeni na wynajem było jak najwięcej (Dyckhoff 2018: 38). Jednak inaczej tę kwestię przedstawia Peter Rees, mówiąc o bryle Walkie Talkie: Jednak inaczej tę kwestię przedstawia Peter Rees, mówiąc o bryle Walkie Talk (...) wiem, że ludzie wydają się być zdenerwowani tym budynkiem, ponieważ jest większy na szczycie. Straciłem rachubę, ile razy próbowałem wyjaśnić; nie jest, jest mniejszy na dole. A to dlatego, że musieliśmy usunąć sekcję od dołu, aby powiększyć przestrzeń chodnika (...) (Crosland 2015, tłum. J.K.). Forma osobowa „my”, której używa, wskazuje, iż czuje się współautorem tego roz wiązania.h W artykule do „The Guardian” krytyk architektury Oliver Wainwright stwierdził, iż tak powstała bryła „jest dowodem na to, że forma podąża za finansami”20 (Wainwright 17 Z uwagi na lepszy widok, lepszy dostęp do światła naturalnego oraz prawdopodobnie niższy poziom hałasu. 65 oanna Krajewska \| Architektura w turbokapitalizmie. Przypadek biurowca Walkie Talkie w Londy 2012). Aby najlepiej zobrazować problem i wskazać winnego, czyli agresywny kapitalizm, opisując budynek użył obrazowej metafory: „Wisi nad swoimi niskimi sąsiadami niczym barczysty bankier w tanim prążkowanym garniturze. I robi się coraz grubszy, gdy się wzno si (...), tworząc dosłowny diagram chciwości” (Wainwright 2015, tłum. J.K.). Architekt próbował jednak w inny sposób przekonać odbiorców do swojej koncepcji − według niego kształt budynku ma uzasadnienie urbanistyczne i koresponduje z rzeką (zob. Viñoly), a także: powiększenie przestrzeni u góry pozwoliło stworzyć na poziomie ulicy dwa parki kieszonkowe (Ibidem) (w istocie kilka rachitycznych drzew po bokach budynku). Istotnym argumentem miała być chęć zapewnienia maksymalnych widoków na miasto oraz zacho wania osi widokowych. Forma i skala budynku Sam Viñoly zauważył: „Widoki mogą być towarem komercyjnym lub dobrem publicznym” (Heathcote 2011) − wykorzystał zatem wszystkie możliwości, powiększając cenną przestrzeń komercyjną z panoramicznym widokiem i tworząc pu blicznie dostępny ogród na najwyższej kondygnacji. W ten sposób ikoniczność, o której zdecydował charakterystyczny kształt obiektu, można uznać za wtórną wobec zabiegu maksymalizacji najdroższej powierzchni najmu oraz uwarunkowań lokalizacji. y j j j p j j Zakrzywiona bryła budynku powodowała uciążliwości dla użytkowników prze strzeni publicznej w najbliższym sąsiedztwie. Jej oddziaływanie było szeroko opisywane przez prasę, czyniąc Walkie Talkie sztandarowym przykładem niekorzystnego wpływu architektury na jej otoczenie. Jeszcze przed ukończeniem wieżowca, w 2013 roku, połu dniowa elewacja, skupiając promienie słoneczne, roztopiła plastikowe elementy zapar kowanego nieopodal luksusowego samochodu i zniszczyła witryny sklepowe. Skutkiem tego, w 2014 roku na fasadzie założono żaluzje. Kolejnym problemem był efekt tunelu aerodynamicznego powstającego na poziomie ulicy i odczuwanego w postaci zwartego strumienia powietrza, który niemal zdmuchiwał przechodniów. Zjawisko to skłoniło planistów do wprowadzenia ostrzejszych wytycznych w tym zakresie i nalegań na nieza leżne studia nad wiatrem (Wainwright 2015). Powszechnej krytyce poddana została nie tylko forma, ale również skala obiektu. Panoramy miast z historycznie cennymi dominantami przestrzennymi lub zachowanym spójnym układem urbanistycznym są postrzegane w kategoriach dziedzictwa kulturo wego, a kwestie wysokości nowej zabudowy są regulowane prawnie (np. w Paryżu czy Krakowie). Sylweta Londynu wyrastająca nad Tamizą z widoczną kopułą Katedry św. Pawła, a także widoki Pałacu Westminsterskiego lub Tower of London od strony rzeki to część tożsamości tego miasta. Po II wojnie światowej, gdy zaczęły powstawać budyn ki wysokie, objęto ochroną prawną szczególnie cenne korytarze widokowe − ich sieć jest nałożona na tkankę miejską Londynu, by chronić widoki na zabytkowe budowle z pew nych strategicznych punktów (Heathcote 2011, por. Crosland 2015). Plan urbanistyczny z 2004 roku, w którym powstał projekt wieżowca Rafaela Viñoly’ego, spowodował możliwość zawężenia potencjalnych widoków oraz zapewniał mniejszą ochronę tła (Heathcote 2011). Można się zastanawiać, czy inwestor i projektanci wiedzieli zawczasu 66 Journal of Urban Ethnology 20/2022 o mających wejść w życie nowych regulacjach21. Wzdęta forma Walkie Talkie wyrosła niemal nad samą Tamizą, zastępując budynek nie rzucający się w oczy, niższy i prostopadłościenny. Projekt nowego biurowca skrytykowały organizacje powołane między innymi w celu ochrony dziedzictwa materialnego. English Heritage22 określił go jako „brutalnie dominujący wyraz powierzchni komercyjnej” (Wainwright 2012, tłum. J.K.). 21 W 2007 r. zostały ustalone nowe wytyczne (Weaver 2007). 22 Organizacja pozarządowa założona w 1983 r., opiekująca się angielskimi zabytkami należącymi do „zbio ru dziedzictwa narodowego”. Forma i skala budynku Organizacja wskazywała, iż „nowa wieża zniszczy historyczny charakter Londynu, zepsuje widoki na miasto i zniszczy otoczenie światowego dziedzictwa wokół Tower of London” (Weaver 2007, tłum. J.K.). UNESCO za groziła dodaniem znajdującej się w pobliżu Tower of London do listy Światowego Dziedzic twa w Zagrożeniu (ang. World Heritage in Danger) ze względu na szkodliwy wpływ, jaki wieżowiec miałby mieć na jej scenerię (Weaver 2007, Wainwright 2012). Obiekt poddał kry tyce również ówczesny dyrektor Victorian Society23, Christopher Costelloe, który ocenił, że „jego «bulwiasty kształt» czyni go «prawdopodobnie najbrzydszym budynkiem w Londynie, który odwraca uwagę od innych zabytkowych budynków»” (Dangerfield 2015, tłum. J.K.). Projekt nie podobał się również księciu Karolowi (Evening Standard 2009), nie raz krytyku jącemu współczesną architekturę miasta − w jego opinii zasada „«wolne dla wszystkich» mia ła zostawić Londyn z «podziobaną panoramą»” (Weaver 2008). Historyk Tristram Hunt24 uznał, iż „cenna panorama stolicy jest świadomie przekształcana w wyraz korporacyjnej do minacji” (Weaver 2007, tłum. J.K.). Efekt, którego się obawiano, jest widoczny między innymi w zestawieniu z niegdyś monumentalnym Tower Bridge (Fot. 1). Fot. 1 Tower Bridge widziany z bulwaru Butlers Wharf Pier na tle nowo powstałych wieżowców, 2018 r., fot. Joanna Krajewska Fot. 1 Tower Bridge widziany z bulwaru Butlers Wharf Pier na tle nowo powstałych wieżowców, 2018 r., fot. Joanna Krajewska 22 Organizacja pozarządowa założona w 1983 r., opiekująca się angielskimi zabytkami należącymi do „zbio ru dziedzictwa narodowego”. 22 Organizacja pozarządowa założona w 1983 r., opiekująca się angielskimi zabytkami należącymi do „zbio ru dziedzictwa narodowego”. g 23 Brytyjska organizacja członkowska założona w 1958 r., zajmująca się zachowaniem architektury i dzie dzictwa z epok wiktoriańskiej i edwardiańskiej oraz promowaniem zainteresowania na ich temat. g 23 Brytyjska organizacja członkowska założona w 1958 r., zajmująca się zachowaniem architektury i dzie dzictwa z epok wiktoriańskiej i edwardiańskiej oraz promowaniem zainteresowania na ich temat. 24 A także dziennikarz i polityk, a od 2017 r. dyrektor Muzeum Wiktorii i Alberta w Londynie. 67 Joanna Krajewska \| Architektura w turbokapitalizmie. Przypadek biurowca Walkie Talkie w Londynie Fot. 2 Wieżowiec zwany Walkie Talkie wyłaniający się zza północnej pierzei ulicy Eastcheap w Londynie, fot. Joanna Krajewska Fot. 2 Wieżowiec zwany Walkie Talkie wyłaniający się zza północnej pierzei ulicy Eastcheap w Londynie, fot. Joanna Krajewska Wieżowiec jednak powstał, mimo licznych krytycznych głosów autorytetów, co po raz kolejny dowodzi siły ekonomii. Jego niedopasowana i przeskalowana forma razi nie tylko widziana z odległości. Forma i skala budynku Również z poziomu sąsiednich ulic, wśród historycznej za budowy, wygląda jak obiekt z innego świata (Fot. 2). Nietrudno odczytać w jego powsta niu materializację fenomenu Bigness, o którym pisał Rem Koolhaas (2017). Poprzez swoją wielkość budynek zdaje się ostentacyjnie ignorować otoczenie. A może to mate rializacja mechanizmów samego turbokapitalizmu? Jeśli sama bryła budynku wygląda tak, że zdaje się nie szanować kontekstu i historycznego dziedzictwa miasta, trzeba mieć na uwadze, że za decyzją o realizacji inwestycji stały konkretne osoby. Obiekt uzyskał kilka nagród przyznawanych budynkom komercyjnym (zob. Viñoly), jednak w opinii publicznej zasłużył na miano najbrzydszego budynku roku w Wielkiej Brytanii, o czym świadczyła nagroda Carbuncle Cup przyznana w 2015 roku Journal of Urban Ethnology 20/2022 68 przez magazyn architektoniczny „Building Design”25. Jednemu z jurorów budynek sko jarzył się z wieżą czarnego charakteru z filmów o Jamesie Bondzie (Butler 2017), co pokazuje możliwe bliskie związki architektury z kulturą popularną, a przede wszystkim w dowcipny sposób naświetla skalę problemu: antagonista agenta 007 zazwyczaj stawia się ponad prawem, nie liczy się z nikim i z niczym, a jego działania, podejmowane z chę ci zysku, zagrażają ludzkości. 25 Budynki są nominowane do niechlubnej nagrody przez czytelników; zwycięzcę wyłania jury złożone z architektów i pisarzy. 26 Peter Rees pełnił tę funkcję w latach 1985–2014. Obecnie jest profesorem renomowanej Szkoły Architektury Bartlett. W październiku 2014 roku otrzymał Honorowy Doktorat Nauk uczelni London South Bank University (LSBU). 27 Komisja Architektury i Środowiska Zabudowanego, ang. Commission for Architecture and the Built Environment − organ doradczy rządu w zakresie architektury. 28 Z licznych artykułów i wywiadów dostępnych na jego temat, wyłania się postać decydenta-indywidualisty, który nie boi się szokować otoczenie, czego przykładem mogą być burgundowe, skórzane buty sięgające kolan, w których przyszedł na przyjęcie prasowe (Morris 2014), a także wypowiedź podczas międzynaro dowej konferencji, w której wyznał, iż Londyn jest wyjątkowy, gdyż „oferuje najlepszy darmowy seks na świecie” (Ibidem). 29 Tzw. „Wielki Wybuch” (ang. Big Bang) nastąpił w 1986 roku i przypadał na okres liberalnej polityki go spodarczej Margaret Thatcher sprawującej urząd premiera Wielkiej Brytanii w latach 1979–1990. 25 Budynki są nominowane do niechlubnej nagrody przez czytelników; zwycięzcę wyłania jury złożone z architektów i pisarzy. 29 Tzw. „Wielki Wybuch” (ang. Big Bang) nastąpił w 1986 roku i przypadał na okres liberalnej polityki go spodarczej Margaret Thatcher sprawującej urząd premiera Wielkiej Brytanii w latach 1979–1990. Decydent W opisach niektórych komentatorów wydanie pozwolenia na realizację 20 Fenchurch Street jawi się jako niemal absolutystyczna decyzja Petera Reesa, wieloletniego głównego urbanisty City26 (Wainwright 2012, por. Dyckhoff 2018: 39). Uważał on, że miasto musi rozwijać się w duchu „nowoczesności” i odpierał ataki tradycjonalistów, nazywając ich „ta libami dziedzictwa” (Wainwright 2012). Oliver Wainwright sugerował, że urzędnicy po parli projekt, będąc „zawsze w niewoli odurzającego koktajlu wielkiego biznesu, gwiazdorskich architektów i dziwacznego przezwiska [nadanego budynkowi − przyp. J.K.]” (Ibidem, tłum. J.K.). Piszący dla „The Guardian” Matthew Weaver wskazał na pe wien schemat działania w wydawaniu pozwolenia na budowę nowych londyńskich wie żowców: „English Heritage ma tendencje do ich dezaprobaty (...). Burmistrz Londynu Ken Livingstone i [komisja − przyp. J.K.] Cabe27 często je popierają, zwłaszcza jeśli doty czą znanych architektów. Następnie zostają zatwierdzone” (Weaver 2007, tłum. J.K.). Warto przyjrzeć się bliżej roli czynnika ludzkiego w tym procesie. Kiedy Peter Rees28 objął funkcję głównego urbanisty w 1985 roku, jego zadaniem szybko stało się przekształcenie City po tzw. „Wielkim Wybuchu”, czyli deregulacji sek tora usług finansowych29, która skutkowała napływem do stolicy zewnętrznego kapitału wraz z zagranicznymi bankami, chcącymi otworzyć swoje siedziby w Londynie (LSBU). Historyczna dzielnica biznesu wkrótce zaczęła konkurować z rozwijającym się szybko nowym obszarem wieżowców Canary Wharf, w który przekształcono teren dawnych 25 Budynki są nominowane do niechlubnej nagrody przez czytelników; zwycięzcę wyłania jury złożone z architektów i pisarzy. 29 Tzw. „Wielki Wybuch” (ang. Big Bang) nastąpił w 1986 roku i przypadał na okres liberalnej polityki go spodarczej Margaret Thatcher sprawującej urząd premiera Wielkiej Brytanii w latach 1979–1990. 29 Tzw. „Wielki Wybuch” (ang. Big Bang) nastąpił w 1986 roku i przypadał na okres liberalnej polityki go spodarczej Margaret Thatcher sprawującej urząd premiera Wielkiej Brytanii w latach 1979–1990. 69 oanna Krajewska \| Architektura w turbokapitalizmie. Przypadek biurowca Walkie Talkie w Londy londyńskich doków zlokalizowany poza ścisłym centrum miasta30. Oba obszary rozpo częły rywalizację o przyciągnięcie kapitału, czemu towarzyszyły deregulacje planistycz ne31 w City (Morris 2014), mające kluczowe znaczenie dla możliwości kształtowania tam nowej architektury. Rees przyjął strategię rozmów z deweloperami − chciał poznać ich zamierzenia, by móc potem przedstawić konkretne propozycje komisji urbanistycz nej (Evening Standard 2014). To on przekonywał inwestorów do zatrudniania uznanych architektów (Crosland 2015). W efekcie tych zabiegów komisja zaakceptowała blisko 100% jego rekomendacji (Morris 2014). 30 Rewitalizacja obszaru Canary Wharf rozpoczęła się w 1981 roku; dziesięć lat później otwarto tam naj wyższy wówczas budynek w Wielkiej Brytanii − One Canada Square (235,1 m) zwieńczony charaktery styczną piramidą. 31 Jednym ze środków planistycznych, jakie zastosowali urbaniści City, było zwiększenie wskaźnika działki − dopuszczalnego stosunku powierzchni biurowej do powierzchni terenu − na 5:1 (Evening Standard 2014). Decydent Kiedy główny urbanista City rozpoczął pracę na tym stanowisku, dzielnica nie dysponowała ofertą dla finansistów, która zatrzymałaby ich w City po godzinach pracy − choć, według Reesa, miejsce to „powinno być centrum imprezowym” (Morris 2014, tłum. J.K.). Według niego było to „marnotrawieniem zasobów”, gdyż, jak powiedział: „[t]am, gdzie masz świetne zaplecze biurowe, masz potencjał do życia nocnego” (Ibidem, tłum. J.K.). Jego wizja obejmowała stworzenie międzynarodowego centrum finansowe go, atrakcyjnego na tyle, by przyciągnąć „najzdolniejsze dzieciaki z całego świata” (Crosland 2015, tłum. J.K.). Uważał, że „[l]udzie przyjeżdżają do City, aby być blisko siebie, plotkować i mówić rzeczy, których nikt nie zrobiłby przez telefon ani nie napisał by w e-mailu” (Morris 2014, tłum. J.K.). Wzmianka o „miejscach do plotkowania” poja wia się również w wywiadzie, którego Rees udzielił Harry’emu Croslandowi − urbanista uważał je za kluczowe dla dalszego rozwoju miasta na globalnej arenie finansowej (zob. Crosland 2015); wskazywał przy tym, iż między innymi firmy ubezpieczeniowe zaczęły się kształtować właśnie w kawiarniach (ang. coffee houses) (Ibidem). Sukcesem City w no wym, zglobalizowanym świecie, miała być zatem nie tylko nowa atrakcyjna architektura komercyjna, lecz także dywersyfikacja funkcji w obrębie dzielnicy, a nawet samej archi tektury, tak by odejść od modelu klasycznego biurowca i stworzyć przestrzenie pozwala jące na poufne, nieformalne kontakty między przedstawicielami branży finansowej. 32 Koncepcja pochodzi z książki Narodziny klasy kreatywnej, którą Richard Florida opublikował w 2002 roku. Według niego „[k]lasę kreatywną tworzą ludzie, którzy zwiększają wartość ekonomiczną poprzez swoją kreatywność” (Florida 2010:83); co więcej, „tworzą [oni − przyp. J.K.] wspólną tożsamość w oparciu głównie o funkcję ekonomiczną. I to z funkcji ekonomicznej wypływają ich preferencje społeczno-kultu ralne, nawyki konsumpcyjne i nabywcze oraz tożsamość społeczna” (Ibidem). Autor pojęcia w tzw. rdzeniu klasy kreatywnej sytuuje ludzi, „których funkcją ekonomiczną jest tworzenie nowych idei i pomysłów, nowych technologii i nowych treści kreatywnych” (Florida 2010: 31); zalicza do nich: inżynierów, archi tektów i projektantów, ludzi pracujących w szkolnictwie i rozrywce, artystów oraz muzyków (Ibidem). Wokół rdzenia lokalizuje grupę tzw. kreatywnych profesjonalistów, m.in. w zakresie biznesu i finansów (Ibidem). Swoją koncepcję Floria stworzył na podstawie badań społeczeństwa amerykańskiego, szybko jednak zyskała ona popularność poza Stanami Zjednoczonymi. Krytyk architektury Tom Dyckhoff pisał z ironią, iż „[u]rzędnicy z ratuszów całego świata rzucili się na książkę Richarda Floridy (...), jakby to była czerwona książeczka Mao. Narodziny... to elementarz dotyczący oświeconego, przynoszącego dochód miasta przyszłości (...)” (Dyckhoff 2018: 161). Funkcja budynku Biurowiec składa się z dwukondygnacyjnego lobby wejściowego, 32 kondygnacji przestrzeni biurowych na wynajem kategorii A oraz trzykondygnacyjnej przestrzeni re kreacyjnej wieńczącej budynek, ze słynnym Podniebnym Ogrodem. Piętra powtarzają sprawdzony model: wokół trzonu komunikacyjno-sanitarnego jest rozmieszczona prze strzeń biurowa o możliwości elastycznej aranżacji (zob. Floorplans). W 2017 roku po wierzchnię tę wynajmowało łącznie 50 firm ubezpieczeniowych (zob. Landmarkspace). 70 Journal of Urban Ethnology 20/2022 Ich nagromadzenie, związane z sąsiedztwem Lloyd’s (Ibidem), dobrze odzwierciedla ce chę czasów globalizacji, którą wskazał Ulrich Beck, diagnozując społeczeństwo świato wego ryzyka (Beck 2012). W świecie pełnym niepewności ubezpieczyciele są pożądaną grupą zawodową. Ekonomista Richard Florida według swojej definicji klasy kreatyw nej32 zalicza finansistów do grona „twórczych profesjonalistów pracujących w dziedzi nach wymagających zaawansowanej wiedzy”, i umieszcza ich w drugiej grupie pod względem potencjału kreatywności (Florida 2010: 83-84). Kreatywność jest według nie go podstawowym czynnikiem napędzającym współczesną gospodarkę (Florida 2010). Miastu powinno zatem zależeć na przyciągnięciu jak największej liczby osób z takim potencjałem. W tym kontekście właśnie potencjału dopatrzono się w publicznie dostęp nym ogrodzie na najwyższych kondygnacjach biurowca Walkie Talkie. Ich nagromadzenie, związane z sąsiedztwem Lloyd’s (Ibidem), dobrze odzwierciedla ce chę czasów globalizacji, którą wskazał Ulrich Beck, diagnozując społeczeństwo świato wego ryzyka (Beck 2012). W świecie pełnym niepewności ubezpieczyciele są pożądaną grupą zawodową. Ekonomista Richard Florida według swojej definicji klasy kreatyw nej32 zalicza finansistów do grona „twórczych profesjonalistów pracujących w dziedzi nach wymagających zaawansowanej wiedzy”, i umieszcza ich w drugiej grupie pod względem potencjału kreatywności (Florida 2010: 83-84). Kreatywność jest według nie go podstawowym czynnikiem napędzającym współczesną gospodarkę (Florida 2010). Miastu powinno zatem zależeć na przyciągnięciu jak największej liczby osób z takim potencjałem. W tym kontekście właśnie potencjału dopatrzono się w publicznie dostęp nym ogrodzie na najwyższych kondygnacjach biurowca Walkie Talkie. Po wcześniejszym zarejestrowaniu wizyty w Sky Garden wjeżdża się windą do ogólnie dostępnej, przeszklonej, kilkupoziomowej przestrzeni z egzotycznymi gatunka mi roślin, tarasem widokowym od strony Tamizy oraz barem i restauracjami (Fot. 3). Spacer wzdłuż fasad pozwala zobaczyć 360-stopniową panoramę Londynu. Pielęgnacja zieleni (m.in. paproci drzewiastych) w postaci spryskiwania jej wilgotną mgiełką, przy daje wrażenia pobytu w oranżerii. j p y Jednak publicznie dostępna przestrzeń różni się od tej, którą przed realizacją obiek tu pokazywano na wizualizacjach, sugerując, iż powstanie tam park; po otwarciu spotka ła się ona z krytyką odbiorców, co znalazło swoje odbicie w mediach (zob. Dangerfield 2015). Funkcja budynku Większość powierzchni zlokalizowanej od południa jest utwardzona, na niej zaś są porozrzucane liczne stoliki z krzesłami; panuje spory ruch i gwar. Osobiście miałam wątpliwość, gdzie wolno mi się znaleźć, jeśli nie korzystam z otwartego baru, oraz czy mogę usiąść. Wiadomo było, że w wydzielonej w osobnym „boksie” restauracji mogą przebywać goście jedynie po wcześniejszej rezerwacji stolika. Wainwright porównał tę przestrzeń z terminalem lotniczym (Dangerfield 2015), co można odczytać jako odnie sienie do przestrzeni anonimowej komunikacji. 71 Joanna Krajewska \| Architektura w turbokapitalizmie. Przypadek biurowca Walkie Talkie w Londynie 71 Fot. 3 Fragment przestrzeni Sky Garden na szczycie wieżowca Walkie Talkie w Londynie, fot. Joanna Krajewska Fot. 3 Fragment przestrzeni Sky Garden na szczycie wieżowca Walkie Talkie w Londynie, fot. Joanna Krajewska Kontrola dostępu do pozornie publicznej przestrzeni oficjalnie jest podyktowana limitem dostępnej wolnej powierzchni. Georg Ritzer zwraca jednak uwagę, że tłumnie odwiedzana przestrzeń komercyjna jest stale monitorowana, a jednostki, które mogłyby zakłócić jej funkcjonowanie − zawczasu niedopuszczane są do wstępu (por. Ritzer 2004). Zatem kontroluje się przepływ ludzi i „czyści” przestrzeń z niepożądanych gości, rów nież w celach zapewnienia bezpieczeństwa. W ujęciu Rafaela Viñoly bezpłatny ogród na dachu otwarty dla publiczności jest re kompensatą w zamian za „wtargnięcie [z wieżowcem] do miasta” (Wainwright 2012, por. Heathcote 2011). Architekt podkreślał, iż „możliwość oferowania miejskiego doświadcze nia na wysokości jest dość niezwykła” (Wainwright 2012, tłum. J.K.). Trudno uważać tę przestrzeń za prawdziwie publiczną − krytycy podawali to stwierdzenie w wątpliwość (zob. Dangerfield 2015), zwłaszcza że po godzinie 18. (w tygodniu) i 21. (w weekendy) korzy stać z niej mogą jedynie klienci baru i restauracji, a bilet wejściowy ma określoną ważność (nie więcej niż kilka godzin). Kluczem do rozumienia funkcji tego miejsca jest sformuło wanie użyte przez samego architekta: „oferowanie doświadczenia”. Według kategoryzacji Georga Ritzera Sky Garden jest środkiem konsumpcji (Ritzer 2004), która dokonuje się tam na kilka sposobów: konsumowany jest widok na 72 Journal of Urban Ethnology 20/2022 miasto i samo wrażenie, jakie wywołuje (po zmroku dostępna panorama iluminowanej metropolii czyni tę przestrzeń miejscem „magicznym” (por. Ibidem)); oferuje się także możliwość korzystania z kilku lokali gastronomicznych, a ponadto − wynajęcie całej strefy na prywatne imprezy. Firma współpracująca z właścicielem obiektu, będąca orga nizatorem różnego typu przyjęć okolicznościowych, jest wyspecjalizowanym projektan tem wrażeń, który na swojej stronie internetowej reklamuje się słowami: „jesteśmy czymś więcej niż firmą cateringową. Funkcja budynku Przypadek biurowca Walkie Talkie w Londy W metropolii tętniącej aktywnością przez całą dobę, w której architektura uczestniczy w procesie pomnażania zysków, materializują się jego siły. * * * Studium przypadku biurowca Walkie Talkie w Londynie, mieście globalnym, pozwala prześledzić procesy mające wpływ na powstawanie i eksploatację tzw. budynów-ikon. Cen tralną osią tych procesów są działania związane z przepływem i kumulacją kapitału w warun kach globalizacji. Dynamika tych działań i ekspansywność sił, które za nimi stoją, uzasadnia stosowanie przedrostka turbo- wobec współczesnego stadium kapitalizmu. Prawa rynku poddanego deregulacji rządzą architekturą − zarówno jej formą, funkcją, jak i sposobem użytkowania. Multiplikacja „ikoniczności” wynika z faktu uczestniczenia du żej liczby korporacji na arenie międzynarodowego biznesu i wiąże się z ich zapotrzebowa niem na wyróżniające się budynki (stanowiące lokatę kapitału). Jednocześnie charakterystyczna bryła może być efektem godzenia wymogów inwestora oraz lokalnych uwarunkowań. W warunkach turbokapitalizmu ocena estetyki w tradycyjnym ujęciu pozo staje bez znaczenia, w myśl zasady: „nieważne, co mówią [o budynku − przyp. J.K.], ważne, żeby mówili”. Architektura podległa utowarowieniu w niespotykanym dotąd stopniu, a po przez charakterystyczną formę i możliwość nadania budynkowi nazwy o charakterze etykiet ki, funkcjonuje jako znak potencjału finansowego przedsiębiorstwa, wzmacniając jego markę. i Jak pokazały analizy, w procesie realizacji inwestycji niezwykle istotną rolę odgrywa czynnik ludzki, który nieraz determinuje strategie postępowania. Choć budowanie w prze strzeni miejskiej zawsze było działalnością uprzywilejowanych, obecnie wymknęło się spod oceny niezależnych autorytetów. Są bezsilni wobec deweloperów i miejskich decydentów chcących zapewnić napływ do miasta jak największego kapitału, by utrzymać konkurencyjność (nie tylko na globalnej arenie biznesowej, lecz także na gruncie lokalnym). Charakterystyczny biurowiec z atrakcyjną strefą wypoczynkową ułatwia przyciągnięcie zarówno wielkiego bizne su, jak i klasy kreatywnej, co pozwala miastu utrzymać status miasta globalnego. Istotne dla opisywanego procesu są również wstępne wymagania inwestora, o czym przekonałam się, pracując w zawodzie architekta. Zlecając projekt, deweloper ma już oszacowane warunki opłacalności inwestycji. W przypadku obiektu komercyjnego okre śla je minimalna powierzchnia GLA, której wartość jest znana projektantom na począt kowym etapie projektowym. Zdobyte doświadczenie pozwala mi twierdzić, iż zazwyczaj obiekt komercyjny buduje się tak duży, jak tylko pozwalają ramy prawne i lokalne uwa runkowania. Zadaniem architekta jest bowiem takie ukształtowanie bryły, aby dewelo per mógł osiągnąć możliwie maksymalne zyski z gruntu, w który zainwestował. Funkcja budynku Wiemy, jak wyróżnić wydarzenie i sprawić, by było na prawdę spektakularne” (RHC).ff Tom Dyckhoff wpisał wieżowiec w nurt architektury spektaklu (zob. Dyckhoff 2018). Można jednak stwierdzić, iż spektakl został osiągnięty nie poprzez wyraz archi tektoniczny, a głównie poprzez symulację ogrodu, oferowanie oszałamiającego widoku i kreowanie wrażeń. Ritzer zwraca uwagę, iż „[p]raktycznie każdy środek konsumpcji jest miejscem symulowanym lub są w nim symulowane elementy (...)” (Ritzer 2004: 295). Symulacja odwraca uwagę od rzeczywistego celu, jakim jest właśnie konsumpcja. y j gę y g j j p j Komentatorzy podkreślali, iż „podniebny ogród” był głównym powodem, jaki prze sądził o pozwoleniu na budowę wieżowca. Sky Garden, jak pisano, stał się „przynętą”, wartościową zwłaszcza dla „branży, która polega na kontaktach twarzą w twarz” (Wainwright 2012). W tym miejscu ujawniła się strategia biznesowa City (i samego Petera Reesa), polegająca na przyciąganiu młodych, „najlepszych umysłów” poprzez stworzenie bogatej oferty dla życia nocnego (Wainwright 2012) oraz tworzeniu „miejsc do plotkowania” (Crosland 2015). Atrakcyjna propozycja miała się stać środkiem do przyciągnięcia klasy kreatywnej. Richard Florida pisał o „zbieraniu [przez nią] doznań”, między innymi poprzez uczestniczenie w nocnym życiu miasta; wskazywał znaczenie „przestrzeni hybrydowych” (a taką jest połączenie biurowca z ogrodem i miejscem noc nej rozrywki), oraz potencjał przyciągania, jaki mają przestrzenie zielone (Florida 2010). Oto dzielnice biurowe, niegdyś nieaktywne i opustoszałe po zmroku, w XXI wieku stają się nie tylko miejscem pracy, ale również wypoczynku. Urzędnicy − mówił Rees − wyko rzystują „każdą okazję, by stworzyć [w Londynie] imprezowe miasto na niebie” (Wainwright 2012, tłum. J.K.). Jednocześnie nie ukrywał: „[d]la naszej oferty bizneso wej bardzo ważne jest, aby ludzie mogli bawić się jak najbliżej biurka” (Ibidem). W ten sposób podział dnia na czas pracy i czas wolny ulega zatarciu (zob. Florida 2010), i prze mienia w kontinuum 24/7. Zarządzanie czasem wolnym staje się kolejną okazją do po mnażania dochodów (Ibidem). Pisze o tym również Jonathan Crary w książce Późny kapitalizm i koniec snu: „Trwające dwieście lat dopasowywanie czasu życia człowieka do działania rynków unieważniło różnice między czasem pracy a czasem wolnym, sferą publiczną a prywatną (...). W tych warunkach bez żadnych ograniczeń zachodzi nie ustanna finansjalizacja niegdyś niezależnych domen aktywności społecznej” (Crary 2015: 123). Szczególnie zwraca uwagę na zawłaszczanie czasu przeznaczonego na sen przez mechanizmy „zachłannego współczesnego kapitalizmu” (Crary 2015: 23). 73 oanna Krajewska \| Architektura w turbokapitalizmie. Funkcja budynku Obecnie mieszkańcom Londynu, niezadowolonym z obecności wieżowca Walkie Talkie w panoramie stolicy, pozostaje skorzystać z platformy widokowej na szczycie budynku i podziwiać widoki, mając w pamięci to, co francuski piosenkarz i aktor Maurice Chevalier powiedział o Paryżu i jego „ikonie” − „[miasto] najpiękniej wygląda z wieży Eiffla, chociażby dlatego, że jej stamtąd nie widać” (Aforyzmy). 74 Journal of Urban Ethnology 20/2022 awowicz G. 2007. Geografia społeczna miast. Warszawa: Wydawnictwo Naukowe PWN Bibliografia gi Beck U. 2012. Społeczeństwo światowego ryzyka. W poszukiwaniu utraconego bezpieczeństwa. Warszawa: Wydawnictwo Naukowe SCHOLAR. Beck U. 2012. Społeczeństwo światowego ryzyka. W poszukiwaniu utraconego bezp Beck U. 2012. Społeczeństwo światowego ryzyka. W poszukiwaniu utraconego bezpieczeństwa. Warszawa: Wydawnictwo Naukowe SCHOLAR. Braudel F. 2013. Dynamika kapitalizmu. Warszawa: Wydawnictwo Aletheia. Braudel F. 2013. Dynamika kapitalizmu. Warszawa: Wydawnictwo Aletheia. Crary J. 2015. Późny kapitalizm i koniec snu. Kraków: Karakter. Czerny M. 2005. Globalizacja a rozwój. Wybrane zagadnienia geografii społeczno-gospodarczej świa ta. Warszawa: Wydawnictwo Naukowe PWN. i ta. Warszawa: Wydawnictwo Naukowe PWN. Debord G. 2006. Społeczeństwo spektaklu oraz Rozważania o społeczeństwie spektaklu. Warszawa: Państwowy Instytut Wydawniczy. Debord G. 2006. Społeczeństwo spektaklu oraz Rozważania o społeczeństwie spektaklu. Warszawa: Państwowy Instytut Wydawniczy. Państwowy Instytut Wydawniczy. Dyckhoff T. 2018. Epoka spektaklu. Perypetie architektury i miasta w XXI wieku. Kraków yf p p yp y Florida R. 2010. Narodziny klasy kreatywnej oraz jej wpływ na przeobrażenia w charakterze pracy, wypoczynku społeczeństwa i życia codziennego. 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The Walkie-Talkie: battle of the bulge on Fenchurch Street. ”The Guardian”. https://www.theguardian.com/artanddesign/architecture-design-blog/2012/dec/ 12/walkie-talkie-fenchurch-street-architecture, 28.12.2021. Wainwright O. 2.09.2015. Carbuncle Cup: Walkie Talkie wins prize for worst building of the year. ”The Guardian”. https://www.theguardian.com/artanddesign/architecture-design-blog/ 2015/sep/02/walkie-talkie-london-wins-carbuncle-cup-worst-building-of-year, 5.01.2022. Weaver M. 18.07.2007. Does London need more skyscrapers?. ”The Guardian”. https://www.thegu ardian.com/artanddesign/artblog/2007/jul/18/doeslondonneedmoreskyscrap?INTCM P=SRCH, 5.01.2022.h Weaver M. 19.02.2008. Livingstone’s towering legacy. ”The Guardian”. https://www.theguardian. com/politics/2008/feb/18/london08.london1, 8.05.2022 Weaver M. 19.02.2008. Livingstone’s towering legacy. ”The Guardian”. https://www.theguardian. com/politics/2008/feb/18/london08.london1, 8.05.2022 Autorka: Dr inż. arch. Joanna Krajewska Wydział Architektury Politechnika Wrocławska ul. B. Prusa 53/55, Wrocław e-mail: joanna.krajewska@pwr.edu.pl
https://openalex.org/W2914213901	https://www.itm-conferences.org/10.1051/itmconf/20192402008/pdf	English	null	Computer simulation of complex of lysine dendrigraft with molecules of therapeutic KED peptide	ITM web of conferences	2,019	cc-by	2,988	Computer simulation of complex of lysine dendrigraft with molecules of therapeutic KED peptide Igor Neelov 1,, Valerii Bezrodnyi1, Anna Marchenko1, Emil Fatullaev1 and Sofia Miktaniuk1 1ITMO University, Saint-Petersburg, 197101, Russian Federation 1ITMO University, Saint-Petersburg, 197101, Russian Federation Abstract. Lysine dendrimers and dendrigrafts are often used in biomedicine for drug and gene delivery to different target organs or cells. In present paper the possibility of complex formation by lysine dendrigraft and 16 molecules of therapeutic KED peptide was investigated using molecular dynamics simulation method. A system containing of one dendrigraftt and 16 KED peptides in water were studied. It was shown that stable complex consisting of the dendrigraft and the peptide molecules formed and structure of this complex was studied. Similar complexes could be used in future for delivery of other therapeutic peptides to target organs. 2 Method and Materials Interest to macromolecules with regular brunched structuregrows every year [1]. One of the most well- known polymers with dendritic structures are dendrimers. The use of different types of dendrimers in drug delivery research is discussed in many papers [2, 3]. Dendrigraft is also a brunched polymer. Dendrigrafts could be described from one hand as dendrimers with short linear chain in their core or from another hand as dendritic brush with short main chain and long side chains. Lysine dendrigrafts consists entirely of lysine aminoacid residues (that are biocompatible) [4, 5]. At the same time their terminal groups could be functionalized by other aminoacids or by other active groups or molecules [6]. Lysine dendrigrafts are polymers that are rich with amines. Due to this reason they could be used as antibacterial [7] or antiviral agents [8]. Also they could make complexes with oppositely charged peptides due to strong electrostatic interaction between their positively charged groups (NH3+) and negatively charged aminoacid side groups (COO-) of peptides. Hydrogen bonds between dendrigraft and peptide and hydrophobic interactions between their nonpolar groups are also important for creation of such complexes. Due to these ability to make complexes thedendrigrafts like other dendritic molecules could be used as. Interest to macromolecules with regular brunched structuregrows every year [1]. One of the most well- known polymers with dendritic structures are dendrimers. The use of different types of dendrimers in drug delivery research is discussed in many papers [2, 3]. Dendrigraft is also a brunched polymer. Dendrigrafts could be described from one hand as dendrimers with short linear chain in their core or from another hand as ITM Web of Conferences 24, 02008 (2019) AMCSE 2018 ITM Web of Conferences 24, 02008 (2019) AMCSE 2018 https://doi.org/10.1051/itmconf/20192402008 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). Corresponding author: i.neelov@mail.ru 2.1 Molecular dynamics method y Molecular dynamics (MD) method is currently the main method for simulation of polymer and biopolymer systems. The method consists in numerical solution of the classical Newton equations of motion for all atoms of the all molecules in the system. It was used first in the mid-fifties of the last century [12] for two-dimensional modeling of hard disks system (2D-model of a monoatomic gas), and then was used tosimulate a variety of liquids, including water [13-14]. In 1972 this method was first applied to the simulation of a simplemodel of a linear polymer chain consisting of atoms connected by rigid bonds [15]. In 1975 the dynamics of short n- alkanes was studied [16]. In subsequent years MD was used for detailed study of many specific molecules using both coarse-grained and detailed full-atomic models. The potential energy of these models usually include valence bonds, valence angles and dihedral angle energies as well as van der Waals and electrostatic energies. The definition of parameters set adequately describing the test molecule properties (force-field) is challenging and requires the experimental data for these molecules, quantum chemical calculations as well as iterative procedures and a very large amount of machine time. These calculations can be made only by large groups of specialists. Due to this reason several packages of standard computer programs, in which these parameters are defined for a fairly wide range of molecules become widely used in recent years. Currently the most popular molecular modeling packages are GROMACS, AMBER, CHARMM, and some others. Our simulation was performed by molecular dynamics method using the Therapeutic KED peptide (Lys-Glu-Arg) [10-11] was selected for our study as a model peptide. This peptide belongs to a class of regulatory peptides and has an protective properties. The goal of this paper is to demonstrate the possibility of complex formation between lysine dendrigraft of second generation and therapeutic KED peptide using molecular dynamics simulation method. * Corresponding author: i.neelov@mail.ru ITM Web of Conferences 24, 02008 (2019) AMCSE 2018 ITM Web of Conferences 24, 02008 (2019) https://doi.org/10.1051/itmconf/20192402008 GROMACS 4.5.6 software package [17] and one of the most modern AMBER_99SB-ildn force fields [18]. GROMACS 4.5.6 software package [17] and one of the most modern AMBER_99SB-ildn force fields [18]. Fig. 1. Stages of the complex formation (initial and final) between dendrigraft and 16 molecules of KED peptide at time: t = 0 (a); t = 100 ns (b).. 2.2 Model and details of calculations Modeling was performed using the molecular dynamics method for systems consisting of one lysine dendrigraft of second generation with positively charged NH3+ end groups, 16 molecules of KED peptide (with charge -1 each), water molecules and chlorine counter- ions in a cubic cell with periodic boundary conditions. The initial conformation for peptide with internal rotation angles of fi = –135º, psi = 135º, theta = 180º was modelled by Avogadro chemical editor. The structures were optimized in vacuum using molecular mechanics of AMBER force field. Further energy minimizations and simulations were performed using the GROMACS 4.5.6 software package and AMBER_99SB-ildn force fields. The potential energy of this force field consists of valence bonds and angles deformation energy, internal rotation angles, van der Waals and electrostatic interactions. The procedure of molecular dynamics simulation used for lysine dendrigraft and peptides has been described earlier (for dendrimers and linear polyelectrlytes) in [19-29]. In all calculations the normal conditions (temperature 300 K, pressure 1 ATM) were used. Computing resources on supercomputers “Lomonosov” were provided by supercomputer center of Moscow State University [30]. Fig. 1. Stages of the complex formation (initial and final) between dendrigraft and 16 molecules of KED peptide at time: t = 0 (a); t = 100 ns (b).. 3.1. Complex formation The time dependence of gyration radius Rg at the beginning of calculation describes the process of compaction of subsystem consisting of dendrigraft and peptide molecules during formation of complex (see Fig. 2). Fig. 2. Time dependence Rg(t) for dendrigraft DG2+16 molecules of KED peptides (a) and for dendrigraft DG2 only (b). Table 2. Formatting sections, subsections and subsubsections. Table 2. Formatting sections, subsections and subsubsections. Font Spacing numbering Section 12-point Arial bold 6 mm before 3 mm after 1, 2, 3, etc. subsection 10-point Arial bold 6 mm before 3 mm after 1.1, 1.2, 1.3, etc. subsubsection 10-point Arial Italic 6 mm before 3 mm after 1.1.1, 1.1.2, 1.1.3, etc. Fig. 2. Time dependence Rg(t) for dendrigraft DG2+16 molecules of KED peptides (a) and for dendrigraft DG2 only (b). From Fig. 2 it can be seen that complex of dendrigraft with 16 molecules of KED peptide forms within 20 ns. After this time the complexes size Rg fluctuate slightly, but its average value practically does not change with time. Therefore, we can assume that the systems become close to equilibrium state after 20ns. 3 Results and Discussion Snapshots of a system consisting of dendrigraft of second generation, peptides, ions and water during simulation are shown on Fig. 1 (water molecules are not shown for clarity). It is clearly seen that at the beginning of process (Fig. 1a) peptide molecules are far from dendrigraft. In the end of simulation (Fig. 1b) all peptide molecules are on dendrigraft surface. Atoms of dendrigraft molecule is shown as beads with diameter equal to their van der Waals radii. Valence bonds of various peptides are shown with lines of different colors (backbone of each peptide is shown by thick line of the same color as valence bonds). To characterize the size of the systems during the equilibration the mean squared gyration radius Rg(t) was used. This function was calculated using g_gyrate function of GROMACS. Another quantity that can characterize the rate of complex formation is the total number of hydrogen bonds (N) between dendrigraft and peptide molecules. The dependence of this value on time is shown on Fig. 3 and demonstrates how the number of specific contacts between them increases during complex formation. This function was calculated using g_hbonds function of GROMACS. From Fig. 3 it can be concluded that the number of Hbonds between dendrigraft and 16 molecules of KED peptide reaches equilibrium (plateau) for the first time after 20 ns. It correlates very well with the time dependence of the inertia radius shown in Fig. 2. 2 2 https://doi.org/10.1051/itmconf/20192402008 ITM Web of Conferences 24, 02008 (2019) AMCSE 2018 ITM Web of Conferences 24, 02008 (2019) Fig. 4. Radial distribution p(r) for: dendrigraft DG2 and 16 molecules of KED peptide. Distribution curves for: peptide atoms (1); dendrigraft atoms (2); all atoms of complex (3). Fig. 3. Time dependence of hydrogen bond (Hbonds) number between DG2 dendrigraft and 16 molecules of KED peptide (b) during complex formation. Fig. 4. Radial distribution p(r) for: dendrigraft DG2 and 16 molecules of KED peptide. Distribution curves for: peptide atoms (1); dendrigraft atoms (2); all atoms of complex (3) Fig. 3. Time dependence of hydrogen bond (Hbonds) number between DG2 dendrigraft and 16 molecules of KED peptide (b) during complex formation. Fig. 3. Time dependence of hydrogen bond (Hbonds) number between DG2 dendrigraft and 16 molecules of KED peptide (b) during complex formation. Fig. 3. Time dependence of hydrogen bond (Hbonds) number between DG2 dendrigraft and 16 molecules of KED peptide (b) during complex formation. Fig. 3.2 Equilibrium characteristics of complex Information about the internal structure of the equilibrium complex could be obtained using radial density distribution of atoms relatively center of inertia of system. These radial distribution functions (not normalized) are shown on Fig. 4. They were calculated using g_rdf function of GROMACS. Fig. 4 demonstrates that atoms of dendrigraft (curve 2) are located mainly in the center (close to distance r=0) of the complexes and atoms of peptides (curve 3) mainly on the surface of complex. They could only slightly penetrate into outer part of dendrigraft but not to its inner part (see curve 3 in Fig. 4). Distribution of all atoms belonging to complex (curve 1) is between distribution for dendrigraft atoms (curve2) and peptide atoms (curve 1). The sizes Rg of complex and dendrigraft in equilibrium state are evaluated by mean square of inertia radius averaged through the time t after equilibration (t>20ns). This values are given in table 1. Table 1.Size Rg, main components of inertia tensor Rg11, Rg22 and Rg33 and axial ratio Rg33/Rg11 Table 1.Size Rg, main components of inertia tensor Rg11, Rg22 and Rg33 and axial ratio Rg33/Rg11 Table 1.Size Rg, main components of inertia tensor Rg11, Rg22 and Rg33 and axial ratio Rg33/Rg11 Система Rg 11, (nm) Rg 22, (nm) Rg 33, (nm) Rg, (nm) Rg 33/Rg 11 DG2+16KED 1,56 1,85 2,23 2,2 1,43 DG2 1,11 1,33 1,59 1,43 1,43 The research is carried out using the equipment of the shared research facilities of HPC computing resources at Lomonosov Moscow State University [20]. I.M.N. and O.V.S. are thankful for the support from the grant of the Government of Russian Federation 08-08. In equilibrium state the sizes of the complex consisting of DG2 dendrigraft and 16 molecules of KED peptides (see Table 1) is greater than size of dendrigraft itself. It is quite natural, since it correlates with the molecular weight of the complexes increase compared to the molecular weight of the individual dendrigraft. 3 Results and Discussion 4. Radial distribution p(r) for: dendrigraft DG2 and 16 molecules of KED peptide. Distribution curves for: peptide atoms (1); dendrigraft atoms (2); all atoms of complex (3). References 1. J.M.J Frechet and D.A. Tomalia, Dendrimers and Other Dendriric Polymers, (John Wiley&Sons Ltd.:West Sussex, 2001) 2. R.G. Denkewalter, J. Kolc, W.J. Lukasavage, Macromolecular Highly Branched Homogeneous Compound, 1983, US Patent № 4410688. The shape of complex can be characterized by main components (Rg11, Rg22 and Rg33) of inertia tensor from Table 1 where Rg11 is minimal and Rg33 are largest and smallest eigenvalues of inertia tensor. The rough evaluation of anisotropy of dendrigraft and complex could be done using axial ratio Rg 33 / Rg11. 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https://openalex.org/W4282588421	https://zenodo.org/record/6637550/files/Josanu_Cristina%20%281%29.%20RECONCEPTUALIZAREA%20IMPLIC%C4%82RII%20REPREZENTAN%C8%9AILOR%20SOCIET%C4%82%C8%9AII%20CIVILE%20%C3%8EN%20PROCESUL%20DE%20AUTOADMINISTRARE%20A%20JUSTI%C8%9AIEI.pdf	Romanian, Moldavan	null	RECONCEPTUALIZAREA IMPLICĂRII REPREZENTANȚILOR SOCIETĂȚII CIVILE ÎN PROCESUL DE AUTOADMINISTRARE A JUSTIȚIEI	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	2,748	RECONCEPTUALIZING THE INVOLVEMENT OF CIVIL SOCIETY REPRESENTATIVES IN THE PROCESS OF SELF-ADMINISTRATION OF JUSTICE JOSANU Cristina doctorandă, USM DOI: 10.5281/zenodo.6637550 CZU: 347.971:347.471:106.2 Summary The new constitutional regulations, contemporary social and legal trends require a reassessment of the status of civil society representatives in the architecture of self-ad ministration of justice. The issue in question is of particular importance not only for the national legal reality. The organization of civil society representatives involved in the process of self-administration of justice in the format of an association - an interaction platform, manifested through internal communication and collaboration with relevant professional organizations and civil society concerned, would make an essential contri bution to building social trust in justice. Keywords: civil society, judicial authority, self-administration of justice, magis trate, good governance Noile reglementări constituționale, tendințele sociale și juridice contemporane impun o reevaluare a statutului reprezentanților societății civile încadrați în arhi tectura autoadministrării justiției. Problematica în cauză este de o importanță deo sebită nu numai pentru realitatea juridică națională. Organizarea reprezentanților societății civile implicați în procesul de autoadministrare a justiției în formatul unei asociații – platformă de interacțiune, manifestată printr-o comunicare inter nă și printr-o colaborare cu organizații profesionale de resort și ale societății civile interesate, ar avea o contribuție esențială pentru fortificarea încrederii sociale în justiție. Statutul reprezentanților societății civile implicați în autoadministrarea justiției în realitatea juridică națională este și în prezent subiectul multiplelor po lemici. Articolul 122 al Constituției Republicii Moldova (redacția veche) menționa anume statutul – profesori titulari, chiar fără precizarea apartenenței acestora la societatea civilă [1]. Legea pentru modificarea Constituției Republicii Moldova, prin alin (1) al aceluiași articol menționează: „Consiliul Superior al Magistraturii este format din 12 membri: șase judecători aleși de Adunarea Generală a Judecătorilor, reprezentând toate nivelurile instanțelor judecătorești, și șase persoane care se bu cură de o înaltă reputație profesională și integritate personală, cu experiență în do meniul dreptului sau în alt domeniu relevant, care nu activează în cadrul organelor 225 puterii legislative, executive sau judecătorești și nu sunt afiliate politic.” [2] Legea cu privire la Consiliul Superior al Magistraturii [3] este nevoită să reglementeze în domeniul vizat numai în limitele normei constituționale. Articolul 123 alin. (1) se completează cu următoarea propoziție: „Consiliul Superior al Magistraturii își exercită atribuțiile în mod direct sau prin intermediul organelor sale specializa te”. Astfel, urmează a fi ajustate și celelalte legi din domeniu, deși autoadminis trarea justiției este realizată prin intermediul altor colegii, la care reglementarea constituțională nu se referă. Summary Astfel, Legea cu privire la răspunderea disciplinară a judecătorilor [4], Legea cu privire la colegiul de evaluare și selecție a judecătorilor [5], normele din Legea cu privire la Procuratură [6], actele normative similare care reglementează activitatea Consiliului Superior al Procurorilor [7], inclusiv norme le constituționale deja utilizează formulare generică, referitoare la reprezentanții societății civile. Deși într-o formă expresă nu întotdeauna rezultă, totuși, vorbim despre reprezentanții societății civile cu studii și experiențe profesionale în dome niul juridic. Importanța deosebită, complexitatea subiectelor abordate în procesul de autoadministrare a justiției invocă un ansamblu de cerințe și instruiri profesi onale speciale. Altfel spus, nu orice reprezentant al societății civile va fi capabil de o monitorizare și implicare în adoptarea hotărârilor de administrare din domeniul justiției. Fiind de acord că termenul „profesor titular” este unul prea îngust, utilizat incorect și abuziv și care nu poate reprezenta întreaga societate civilă, este necesar a menționa că, la etapa inițială de accedere în componența Consiliului Superior al Magistraturii, a Consiliului Superior al Procurorilor și a colegiilor corespunzătoa re, este necesară realizarea unor instruiri în domeniu. Identificarea statutului reprezentanților societății civile este deosebit de re levantă; or, aceștia devin persoane cu funcții de demnitate publică în domeniul justiției, implicați în procesul decizional al administrării justiției, independenți în capacitatea decizională și fiind supuși numai legii. Statutul în cauză este determi nat de normele constituționale și legale din sistemul legislativ național și se referă în exclusivitate la procedurile operaționale de luare a deciziilor de natură indivi duală sau normativă. Statutul reprezentanților societății civile, implicați în struc turile de autoadministrare a justiției necesită o reevaluare fundamentală, în scopul determinării expresiei axiologice a societății civile și a implicării ei în procesul de autoadministrare a justiției. j ț Pornind de la anumite începuturi, istoria încadrării reprezentanților societății civile în acest proces – de autoadministrare a justiției, a început evoluția în pe rioada interbelică în statele și sistemele juridice europene. „Frica” aceasta, mai mult mimată de „juristocrație”, de „dictatura judiciară”, a impus anumite ten tative incipiente de control, monitorizare din partea societății civile în procesul de autoadministrare a justiției, care, actualmente sunt îndreptățite în aspect de integritate. În Republica Moldova, ca și în toate statele postsocialiste, instituțiile democratice din domeniul justiției și-au găsit dezvoltarea după destrămarea la gărului socialist și a U.R.S.S.. Necesitatea identificării unor cerințe de calitate cu 226 referință la reprezentanții societății civile este evidentă. Summary Evidențiem următoare le: profesionalismul, integritatea, caracterul apolitic al activităților profesiona le anterioare. Complexitatea autoadministrării justiției la etapa contemporană impune o anumită pregătire profesională destul de detaliată și implicarea unor reprezentanți ai societății civile care nu posedă o pregătire profesională detaliată stabilește o eficiență iluzorie a activității acestora. Or, încadrarea în procesul de au toadministrare a justiției prezumă atât o monitorizare a judecătorilor implicați în autoadministrare, cât și în monitorizarea întregii arhitecturi juridice de autoadmi nistrare a justiției, elementele cărora pot, în limitele competenței, stabili și aplica o sancțiune disciplinară, organiza și realiza o promovare în carieră ș.a. Evident, cea mai eficientă monitorizare poate fi realizată printr-o nemijlocită participare. Cu trecerea timpului, procedurile operaționale, devenind tot mai sofisticate, impun o specializare mai profundă. Dacă la o anumită etapă se discuta despre implicarea reprezentanților societății civile pe principii de voluntariat, la această experiență s-a renunțat. Mai mult, cerințele de integritate, prin necesitățile declarării venitu rilor și a conflictelor de interese suplimentate de salarizarea din finanțele publice, justifică toate cerințele față de o persoană cu funcție de demnitate publică. Astfel se conturează statutul reprezentanților societății civile – deținător al funcției de demnitate publică în domeniul justiției; cu toate că un atare statut, prin formularea dată, lipsește, realitatea îl identifică. Mai mult, garanțiile de orice natură, caracte ristice statutului de magistrat (judecător sau procuror) la fel sunt de acoperire și pentru reprezentanții societății civile; or, ar fi o lipsă de sens implicarea în acti vitatea decizională a unui organ colegial cu competențe statuare limitate, strâns corelate cu independența și imparțialitatea, ca principii ce se referă la întreg corpul de magistrați. Menționăm o abordare conceptuală absolut nouă a statutului reprezentan tului societății civile în domeniul justiției, ce apare în rezultatul unei evoluții a perceperii fenomenului justiției în societatea contemporană democratică. Odată promovat într-o așa funcție, el (reprezentantul) devine subiect ce deține o funcție de demnitate publică, înzestrată cu anumite calități importante: profesionalism și integritate, ce are drept obiectiv major monitorizarea autorității judecătorești și a justiției în ansamblu. Se referă nu numai nemijlocit la membrii Consiliului Superior al Magistraturii, dar și la membrii – reprezentanți ai societății civile implicați în activitatea colegiilor funcționale pe lângă Consiliu. Or, drepturile ce rezultă din obiectivele formulate implică și anumite obligațiuni de natură juridi că ce completează statutul în cauză. Summary În funcție de legislația națională, numărul funcționarilor în cauză ar putea varia și ar include și reprezentanții Consiliului Superior al Procurorilor, dacă sistemul legislativ prevede o organizare aparte pentru procurori. Menționăm că sistemele juridice europene prevăd o implicare numerică diferită a reprezentanților societății civile în Consiliile Judiciare, însă tendințele sunt orientate spre o creștere a numărului de membri în toate structurile (colegiile) respective, cu respectarea, însă, a pct.1.3. din Carta europeană cu privire 227 la statutul judecătorului [8], ce stabilește un coraport minim clar între numărului de magistrați și cel al reprezentanților societății civile în componența Consiliului Judiciar – cel puțin jumătate, fiind aleși de Adunarea Profesională. Inspirându-ne din obiectivul generic plasat în competența reprezentanților societății civile – de monitorizare prin implicare în procesul de autoadministrare a justiției, orientată spre asigurarea independenței magistraților, identificăm un sistem de obiective concrete: a) transparența funcțională; b) integritatea corpu lui magistraților. Astfel, intenția noastră de valorificare a rolului reprezentanților societății civile în arhitectura autoadministrării justiției o putem exemplifica astfel: a) Prezența argumentată a reprezentanților societății civile în structurile adminis trative ale justiției contemporane constă în asigurarea transparenței procedurilor operaționale ale acestor componente structurale și preîntâmpinarea instaurării unei „juristocrații” închise și selecte în administrarea justiției; b) Implicarea activă în realizarea procedurilor operaționale cu referință la accederea în carieră, pro movarea, sancționarea ș.a., prin asigurarea respectării principiului meritocrației, care înglobează și profesionalismul și integritatea candidaților. Valoarea și rolul reprezentanților societății civile, ce rezultă din aceste obiective interpretate, ne permit, la rândul lor, a identifica sarcinile plasate și modul de organizare a acestor subiecți cu funcție de demnitate publică în domeniul justiției. ț ț p j ț Prima sarcină. Organizarea unei comunicări constructive a tuturor reprezentanților societății civile, implicați în diferite structuri orientate spre admi nistrarea justiției; or, implicarea în diferite componente structurale identifică dife rite probleme ce urmează a fi depășite numai cu participarea societății civile, prin reprezentanții din toate componentele arhitecturale ale administrării. O asociație a reprezentanților societății civile implicați în administrarea justiției va fi mai so noră, opinia ei va avea autoritate, prin profesionalism și transparență, atât pentru celelalte ramuri ale puterii de stat, cât și pentru societate în ansamblu. Summary Dacă con form Legii cu privire la Consiliul Superior al Magistraturii, președintele Consiliului Superior al Magistraturii prezintă raportul de activitate pentru anul precedent în fața Parlamentului[9], care ar fi logic inițial să fie apreciat de Adunarea Generală a Judecătorilor (Adunarea Generală a Procurorilor) [10], atunci o eventuală Asociație a reprezentanților societății civile ar putea elabora un raport de alternativă, atât pentru Adunarea Generală, cât și pentru Parlament. Transparența procedurilor operaționale ar fi apreciată anume prin consistența și complexitatea acestor ra poarte alternative. Anume abordarea critică a problemelor apărute în domeniul justiției va aduce beneficii atât activității Consiliului Superior al Magistraturii, a Consiliului Superior al Procurorilor, cât și a altor componente structural-adminis trative din domeniu. A doua sarcină. Concomitent, menționăm oportunitatea implicării reprezentanților societății civile în anumite proceduri operaționale printr-o pon dere mai consistentă. Anume reprezentanții societății civile necesită a gestiona comunicarea autorităților și instituțiilor implicate cu presa. Astfel, ar 228 fi valorificată o comunicare calitativă cu societatea pe diferite probleme stringen te legate de funcționalitatea autorităților de autoadministrare a justiției. În mod firesc, comunicatele de presă urmează a fi trecute printr-un filtru de transparență și integritate, fiind, astfel, fortificată încrederea societății în justiție și asigura tă transparența funcționalității, prin monitorizarea severă și corectă din partea societății civile. Procedurile operaționale menționate urmează a fi reglementate prin normă legală, în scopul asigurării unei stabilități a raporturilor juridice invoca te și a asigurării unei comunicări trilaterale – politicul, societatea civilă și justiția. A treia sarcină. O altă sarcină, realizată printr-o procedură operațională ce ar impune implicarea nemijlocită a societății civile în formatul Asociației menționate, este cea referitoare la apărarea reputației profesionale a jude cătorilor și procurorilor. Nu este suficient Consiliul Superior al Magistraturii să se limiteze la o constatare a adresării. Reglementările naționale nici nu pre văd anumite proceduri operaționale în domeniu. Urmează elaborarea unei analize minuțioase a situației de fapt și adoptarea unei hotărâri complexe prin interpretă rile de fapt și de drept a cauzei. Or, hotărârea este adresată societății, prin comen tarea, afirmarea sau infirmarea unor acuzații publice în adresa judecătorului sau procurorului din partea unei persoane publice sau a oricărui subiect din societate. Summary Monitorizarea calitativă a acestor categorii de hotărâri sau emiterea comunicatelor din partea reprezentanților societății civile implicați în autoadministrarea justiției, care ar confirma sau infirma acuzațiile menționate, ar contribui la sporirea calității comunicării Consiliului Superior al Magistraturii cu societatea, la fortificarea în crederii în justiție, dar și a responsabilității acuzatorilor civici și politici. Concluzii. Anume sistemul trilateral de comunicare propus și ar asigura capacitatea de independență a justiției în ansamblu, a transparenței funcționale a administrării justiției, a creșterii rolului societății civile în asigurarea unei monitorizări sociale în domeniu și sporirea încrederii în justiție, în calitatea și eficienței ei și a formării unui sistem real de garanții pentru preîntâmpinarea unui control din partea politicului în activitatea justiției. Asociația reprezentanților societății civile din domeniul justiției, având un esențial potențial intelectual, ar contribui eficient la dezbaterile publice organizate de sectorul public (Ministerul Justiției) cu referință la proiectele de legi din domeniu, fără a deține un monopol în domeniu. Mai mult, componența asociației menționate ar putea fi completa tă și cu foști membri ai Consiliului Superior al Magistraturii (Consiliul Superior al Procurorilor) – reprezentanți ai societății civile, asigurându-se, astfel, o con tinuitate a memoriei sociale și profesionale în domeniul vizat. Fiind, în esență, o componentă calitativ-profesională a reprezentanților societății civile, Asociația reprezentanților societății civile din domeniul justiției ar putea avea un dialog so cial, public, nu numai cu reprezentanții ramurilor politice ale puterii de stat, dar și cu asociațiile profesionale ale judecătorilor și procurorilor, în scopul asigurării calității și eficienței actului de justiție și fortificării încrederii în justiție. Vorbim de o comunicare activă cu Uniunea avocaților, Uniunea executorilor judecătorești, 229 alte uniuni ale profesiilor libere din domeniul realizării justiției. Anume Asociația în cauză necesită a se poziționa drept o platformă de comunicare dintre diferiți subiecți, implicați în procesul de realizare a justiției; or, important este a înțelege că principalele impedimente în domeniu pornesc de la lipsa de comunicare, lipsa unui schimb profesionist de opinii, finalitatea cărora ar putea fi proiecte bune de legi, reglementări eficiente și proceduri operaționale calitative – toate acestea încadrându-se în conceptul unei bune guvernări. Or, societatea civilă, în manifes tările actuale nu mai corespunde acelor cerințe de integritate, profesionalism și transparență, înaintate justiției contemporane, dar poate fi capabilă de a se auto organiza în intențiile de democratizare. Summary În caz contrar, în formatul actual, societa tea civilă se poate prezenta numai în calitate de element ce stagnează reformele în domeniul justiției, stagnarea respectivă fiind un factor nociv capabil a afecta toate bunele intenții de modernizare a mecanismului de stat și a societății în ansamblu. Nu este sufucuent de a avea intenții de reformă în domeniul justiției, înaintând anumite cerințe de calitate domeniului de reformare. Este foarte important ca și cine inițiază reformele, și cine le realizează a se impune printr-o autoritate rea lă în fața societății în ansamblu, autoritate caracterizată prin aceleași condiții de calitate intenționate ăn reformele preconizate. Or, societatea civilă, fiind un feno men de natură socială, la fel necesită modernizări și reconceptualizări de formă și conținut și va fi foarte bine dacă ea înseși este capabilă de a se autotransforma, demonstrând astfel capacitatea implicării sale în procesul ulterior de autoadmi nistrare a justiției reformate. 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Disponibil: http://legislatie.just.ro/ Public/DetaliiDocument/106139 Legea cu privire la Consiliul Superior al Magistraturii, nr. 947-XIII din 9. 19.07.96. În: Monitorul Oficial al Republicii Moldova, nr. 64/641 din 03.10.1996. Republicată: Monitorul Oficial al Republicii Moldova , nr. 186-188/752, din 22.08.2003 Legea cu privire la Consiliul Superior al Magistraturii, nr. 947-XIII din 10. 19.07.96. În: Monitorul Oficial al Republicii Moldova, nr. 64/641 din 03.10.1996. Republicată: Monitorul Oficial al Republicii Moldova, nr. 186-188/752, din 22.08.2003. 231 231
https://openalex.org/W2009680880	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0125091&type=printable	English	null	Conjugated Linoleic Acid Supplementation under a High-Fat Diet Modulates Stomach Protein Expression and Intestinal Microbiota in Adult Mice	PloS one	2,015	cc-by	7,616	RESEARCH ARTICLE Conjugated Linoleic Acid Supplementation under a High-Fat Diet Modulates Stomach Protein Expression and Intestinal Microbiota in Adult Mice Alice Chaplin, Pilar Parra, Francisca Serra, Andreu Palou Laboratory of Molecular Biology, Nutrition and Biotechnology, University of the Balearic Islands and CIBER de Fisiopatología de la Obesidad y Nutrición (CIBEROBN), Palma de Mallorca, Spain francisca.serra@uib.es * francisca.serra@uib.es * francisca.serra@uib.es Abstract The gastrointestinal tract constitutes a physiological interface integrating nutrient and micro- biota-host metabolism. Conjugated linoleic acids (CLA) have been reported to contribute to decreased body weight and fat accretion. The modulation by dietary CLA of stomach pro- teins related to energy homeostasis or microbiota may be involved, although this has not been previously analysed. This is examined in the present study, which aims to underline the potential mechanisms of CLA which contribute to body weight regulation. Adult mice were fed either a normal fat (NF, 12% kJ content as fat) or a high-fat (HF, 43% kJ content as fat) diet. In the latter case, half of the animals received daily oral supplementation of CLA. Expression and content of stomach proteins and specific bacterial populations from caecum were analysed. CLA supplementation was associated with an increase in stomach protein expression, and exerted a prebiotic action on both Bacteroidetes/Prevotella and Akkerman- sia muciniphila. However, CLA supplementation was not able to override the negative ef- fects of HF diet on Bifidobacterium spp., which was decreased in both HF and HF+CLA groups. Our data show that CLA are able to modulate stomach protein expression and exert a prebiotic effect on specific gut bacterial species. OPEN ACCESS Citation: Chaplin A, Parra P, Serra F, Palou A (2015) Conjugated Linoleic Acid Supplementation under a High-Fat Diet Modulates Stomach Protein Expression and Intestinal Microbiota in Adult Mice. PLoS ONE 10 (4): e0125091. doi:10.1371/journal.pone.0125091 Academic Editor: Marià Alemany, Faculty of Biology, SPAIN Received: February 3, 2015 Accepted: March 13, 2015 Published: April 27, 2015 Copyright: © 2015 Chaplin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2015 Chaplin et al. This is an open access article distributed under the terms of the Copyright: © 2015 Chaplin 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. Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Introduction Funding: This work was supported by the grant AGL2012-33692 and the EU-funded project (BIOCLAIMS FP7-244995). The authors' group receives financial support from Instituto de Salud Carlos III, Centro de Investigación Biomédica en Red Fisiopatología de la Obesidad y Nutrición, CIBERobn. Authors belong to the Nutrigenomics-group, awarded as “Group of Excellence” of CAIB and supported by “Direcció General d’Universitats, Recerca i Transferència del Coneixement” of Regional Government (CAIB) and FEDER funds (EU). AC is Obesity is currently growing at an epidemic rate, considered a major health threat around the world, and resulting in an increased risk of diabetes mellitus type II, some types of cancer, fatty liver disease, hypertension, cardiovascular disease and increased mortality. Despite large re- search efforts on the effects of diet, exercise, education, surgery or drug therapies, there is still no long-term solution to efficiently prevent or counteract obesity [1]. Dietary conjugated linoleic acids (CLA) refer to a mixture of geometric and positional iso- mers of linoleic acid with conjugated double bonds found mainly in ruminant meat and dairy products [2]. Growing research has shown that the isomers cis-9, trans-11-CLA and trans-10, 1 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0125091 April 27, 2015 Modulation of Microbiota by Conjugated Linoleic Acid cis-12-CLA in particular have a major role in the regulation of body weight and body fat in both animal [3–9] and human [10–13] studies, supported by a PhD fellowship by Conselleria d'Educació, Cultura i Universitats, Govern de les Illes Balears, a project which is cofinanced by the European Social Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The present study was carried out to further characterise the effects of CLA on body weight management, by addressing certain aspects that to our knowledge have not been studied be- fore. The gastrointestinal tract is the largest endocrine of the body and is responsible for the conversion of food into energy, is metabolically highly active and home to trillions of microbes [14, 15]. The stomach is one of the first sites in the gastrointestinal tract that responds to food intake. It synthesises proteins which have an important role in energy balance and have been shown to be modulated by diet [16–21]. Another interesting component is caecum content, which harbours a large amount of bacteria that carry out a number of functions involved in en- ergy regulation, such as the processing of non-digestible polysaccharides, metabolism of pro- teins, synthesis of vitamins and production of energy [22–25]. Emerging evidence suggests that the gut microbiota may be involved in obesity [26–28], and that high-fat diet in particular could contribute to the modulation of this bacterial community [29–33]. Therefore, the role of food in modifying gut microbiota towards a more beneficial profile is of great interest [34]. Competing Interests: The authors have declared that no competing interests exist. Overall, this highlights the importance of the interplay between food and the different com- ponents of the gastrointestinal tract. The aim was to study the potential of CLA in the regula- tion of both stomach protein expression and specific gut bacterial species in obese mice under a HF diet. PLOS ONE \| DOI:10.1371/journal.pone.0125091 April 27, 2015 Quantification of gastric leptin and ghrelin Stomach mucosa was homogenized at 4°C in 1:3 (w/v) PBS (mM: 137 NaCL, 2.7 KCl, 10 phos- phate buffer, pH 7.4) and centrifuged at 7000 x g for 2min at 4°C. Total protein was determined after 5-fold dilution of the supernatant with PBS using the Bradford method [36]. Gastric leptin was determined in the initial homogenate with a mouse leptin enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN). Ghrelin determination in stomach was carried out according to [37]. Stomach homogenate was mixed with 10 volumes 1 M acetic acid containing 20 mM HCl, boiled for 20 min and centrifuged at 7000 x g for 2 min at 4°C. The supernatant was lyophilized and resuspended in PBS. Ghrelin concentration in stomach was then determined using a mouse ghrelin enzyme immunosorbent assay (EIA) kit (Phoenix Europe, Karlsruhe, Germany). RNA isolation, retrotranscription and real-time qPCR Total RNA extraction from stomach was carried out with Tripure Reagent (Roche Diagnostic Gmbh, Mannheim, Germany) according to the manufacturer’s instructions. Isolated RNA was quantified using NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc., Wil- mington, DE, USA) and its integrity was confirmed using agarose gel electrophoresis. Samples were retrotranscribed and real-time PCR was carried out for the analysis of stom- ach proteins. Briefly, 0.25 μg of total RNA (in a final volume of 5 μl) were denatured at 65°C for 10 min and then reverse-transcribed to cDNA using MuLV reverse transcriptase (Applied Biosystems, Madrid, Spain) at 20°C for 15 min, 42°C for 30 min and a final step of 5 min at 95°C in a thermal cycler (Applied Biosystems 2720 Thermal Cycler, Madrid, Spain). Each PCR was performed with diluted cDNA template, forward and reverse primers (5μM each) and Power SYBR Green PCR Master Mix (Applied Biosystems, CA, USA). Primers were designed and obtained from Sigma Aldrich Química SA (Madrid, Spain) and sequences are de- scribed in Table 1. Real-time PCR was performed using the Applied Biosystems StepOnePlus Real-Time PCR Systems (Applied Biosystems) with the following template: 10 min at 95°C fol- lowed by a total of 42 temperature cycles (15 s at 95°C and 1 min at 60°C). In order to verify the purity of the products amplified, a melting curve was produced after each run according to the manufacturer's instructions. The threshold cycle (Ct) was calculated by the instrument's software (StepOne Software v2.0), and the relative expression of each gene was calculated as a percentage of NF mice using the 2−ΔΔCt method [38]. Beta-actin was used as the reference gene. Animals Male C57BL/6J mice from Charles River (Barcelona, Spain) weighing 21 ± 0.1 g (5 weeks-old) were housed under standard conditions in cages in groups of 4–5 and kept in a 12-h light:dark cycle at 22°C with food and water ad libitum. Cages were Makrolon type III (Tecniplast, Biosis Biologic Systems S.L.) and bedding was Ultrasorb fir shavings (Panlab S.L.U). Bedding was changed weekly. After reception, animals were allowed to acclimatize for a week and divided into groups ensuring equal weight average. Food was changed twice a week, and intake and body weight were recorded every three days throughout the experiment [35]. The animal pro- tocol followed in this study was reviewed and approved by the Bioethical Committee of the University of the Balearic Islands (approval 13th February 2006) and University guidelines for the use and care of laboratory animals were strictly followed. All efforts were made to minimize suffering. Mice were divided into three treatment groups (n = 8). All diets were prepared by Research Diets (Inc, New Brunswick) and presented as pellets to the animals. Detailed composition of these diets can be found in S1 Table. Mice received one of the following diets for 54 days: a standard normal-fat diet (NF), containing 12% kJ content as fat, used as control, or a high-fat diet (HF), which contained 43% kJ content as fat. Diets were based on the standard rodent diet AIN-76A. Therefore, both diets contained equal proportion of protein (20% kJ content) and carbohydrate was used to adjust the energy content. Thus, NF diet contained 40% (w/w) of su- crose and HF 35% (w/w). Then, a daily dose of CLA was given to half of the animals receiving the HF diet. Tonalin (kindly provided by Cognis) was used as the CLA supplement, providing 6 mg of CLA/day (21.4 nmol/isomer/day), given as an oral gavage. Tonalin TG 80, derived from safflower oil, is composed of triacylglycerols containing approximately 80% CLA with a 50:50 ratio of the active CLA isomers cis-9, trans-11 and trans-10, cis-12. At the end of the ex- periment, body weight did not differ between HF and CLA group, whereas body fat was statis- tically lower in CLA animals. Complete set of data, including weight of animals, body fat (day 40) and estimated food intake have been previously published [35]. PLOS ONE \| DOI:10.1371/journal.pone.0125091 April 27, 2015 2 / 13 Modulation of Microbiota by Conjugated Linoleic Acid Sacrifice and sample collection Sacrifice of all animals was carried out within the animal facilities, at the beginning of the light cycle and after 10h of food deprivation. Animals were anaesthetized with an intraperitoneal in- jection made up of a mixture of xilacine (10 mg/kg body weight) and ketamine (100 mg/kg body weight). Organs and samples of interest were excised and weighed (stomach and caecum). Stomach was opened and the inside was scraped to collect the mucosa. Caecum was also cut open and content collected. All tissues were rinsed with saline containing 0.1% diethyl pyrocar- bonate (Sigma, Madrid, Spain) and snap-frozen at -80°C. Bacterial profiling by qPCR Total bacterial DNA was extracted from approximately 50 mg of caecal samples using the E.Z. N.A. Stool DNA kit (Omega Biotek, GA, USA). DNA concentration was determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA) and its integrity confirmed by agarose gel. Assessment of the presence and relative 3 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0125091 April 27, 2015 Modulation of Microbiota by Conjugated Linoleic Acid Table 1. Nucleotide sequences of primers used for qPCR amplification in mouse stomach. Mouse genes Forward primer (5’ to 3’) Reverse primer (3’ to 5’) Amplicon size (pb) Beta-actin tacagcttcaccaccacagc tctccagggaggaagaggat 120 Leptin ttgtcaccaggatcaatgaca gacaaactcagaatggggtgaag 186 Ghrelin cagaaagcccagcagagaaa gaagggagcattgaacctga 144 Mboat4 ttgtgaagggaaggtggag gagagcagggaaaaagagca 115 Retn ttccttttcttccttgtccctg ctttttcttcacgaatgtccc 246 Gpr39 ctgctgattggctttgtatgg cggttggagaggttcgtg 188 Gcg tctgacgagatgagcacca tgactggcacgagatgttg 136 Gcgr gcacccgaaactacatcca acacgccctctaccagca 231 Sst accccagactccgtcagtt agcctcatctcgtcctgct 169 Sstr catcgtcaacatcgtcaacc catcctccacaccgtatcct 194 Forward and reverse sequences designed for qPCR ampliﬁcation in stomach samples of mice. doi:10 1371/journal pone 0125091 t001 Table 1. Nucleotide sequences of primers used for qPCR amplification in mouse stomach. amount of bacterial species was determined by measuring DNA abundance of the 16S rRNA gene sequences by qPCR with the Applied Biosystems StepOnePlus Real-Time PCR System (Applied Biosystems), following previously described protocols [39, 40]. Specific primers for Clostridium coccoides, Clostridium leptum and Lactobacillus spp. (Firmicutes representatives); Bacteroides/Prevotella (Bacteroidetes); Bifidobacterium spp. (Actinobacteria); Akkermansia muciniphila (Verrucomicrobia); Enterobacteriaceae (Proteobacteria); and Total Bacteria were obtained from Sigma (Madrid, Spain). Total Bacteria refers to a broad-range universal primer that recognizes the conserved region of the 16S rRNA encoding gene for a wide range of bacte- rial species, and was used to normalize the assay to total bacterial DNA. Sequences are de- scribed in Table 2. The threshold cycle was calculated using the 2−ΔΔCt method [38], and relative bacterial content and fold change (FC) were calculated (Log22−ΔΔCt). Values were nor- malized with the average of the NF group. Caecum microbiota is modulated by CLA To further look into the effect of CLA, caecum content of mice was analysed in order to deter- mine bacterial species potentially associated to obesity and energy metabolism. A significant increase in bacterial DNA caecum content was observed in mice fed a HF diet (3-fold vs. NF, p = 0.032), which decreased with CLA supplementation and showed no differences compared to NF animals (Fig 2A). This was accompanied by differences in gut microbiota composition. No statistically significant changes were found concerning the Firmicutes content under the different dietary treatments (Fig 2B). HF feeding was mainly associated to a decrease in Bifido- bacterium spp. (p = 0.009 vs. NF; with a fold change (Log2 FC) of -3.07). CLA supplementation did not counteract this reduction, presenting similar values (p = 0.034 vs. NF; -2.18 Log2 FC) (Fig 2C). In contrast, animals receiving CLA showed a significant increase in two bacterial spe- cies of interest: Bacteroides/Prevotella (p = 0.021 vs. NF; 1.30 Log2 FC) (Fig 2D) and A. mucini- phila, which dramatically increased compared to both NF (p = 0.014; 4.33 Log2 FC) and HF (p = 0.002) (Fig 2E). No significant changes on Enterobacteriaceae profile were seen (Fig 2F). CLA supplementation modulates the expression of regulatory proteins in the stomach Expression of proteins associated to energy metabolism and regulation of food intake was de- termined in mouse stomach. Leptin mRNA expression in the HF group was not altered, how- ever leptin protein was increased in these animals (2-fold vs. NF, p = 0.003). On the other hand, supplementation with CLA caused a 6-fold higher expression of leptin mRNA (in com- parison with NF, p = 0.001), which was in accordance with higher content of gastric leptin (2-fold, p = 0.014 vs. NF) (Fig 1A). CLA-fed animals also exhibited increased ghrelin mRNA expression (3-fold, p = 0.006 vs.NF), but no changes were seen amongst groups regarding ghre- lin protein (Fig 1B). Furthermore, CLA supplementation was associated to increased expression of all stomach proteins analysed and statistical significance was attained in the case of resistin (Retn) (3-fold, p = 0.026), G protein-coupled receptor 39 (Gpr39) (4-fold, p = 0.001), Glucagon (Gcg) (2-fold, 0.001) and Glucagon Receptor (Gcgr) (5-fold, p = 0.011) (Fig 1C). Statistical analysis Data are presented as means ± SEM. Equality of variances between groups was assessed by Levene’s test. When homogeneity of variances was assumed, one-way ANOVA was used to de- termine the significance of the different parameters between groups. If there was a significant difference, a Bonferroni test was used to determine where the difference lay and to correct for Table 2. Sequence of primers used for bacterial profiling in caecum content. Phylum Bacterial Species Forward primer (5’ to 3’) Reverse primer (3’ to 5’) Proteobacteria Enterobacteriaceae cattgacgttacccgcagaagaagc ctctacgagactcaagcttgc[39] Actinobacteria Biﬁdobacterium spp. cgcgtcyggtgtgaaag ccccacatccagcatcca[39] Firmicutes Clostridium coccoides actcctacgggaggcagc gcttcttagtcargtaccg[39] Clostridium leptum gcacaagcagtggagt cttcctccgtttgtcaa[39] Lactobacillus spp. gaggcagcagtagggaatcttc ggccagttactacctctatccttcttc[39] Bacteroidetes Bacteroides/Prevotella tcctacgggaggcagcagt caatcggagttcttcgtg[39] Verrucomicrobia Akkermansia muciniphila cagcacgtgaaggtggggac ccttgcggttggcttcagat[40] Housekeeping Total Bacteria Total bacteria actcctacgggaggcag gtattaccgcggctgctg[39] Forward and reverse sequences for qPCR ampliﬁcation in mouse caecal content. doi:10.1371/journal.pone.0125091.t002 PLOS ONE \| DOI:10.1371/journal.pone.0125091 April 27, 2015 4 / 13 Table 2. Sequence of primers used for bacterial profiling in caecum content. PLOS ONE \| DOI:10.1371/journal.pone.0125091 April 27, 2015 4 / 13 Modulation of Microbiota by Conjugated Linoleic Acid multiple testing. When homogeneity of variances was not assumed, data were log transformed. Linear relationships between key variables were tested using Pearson's correlation coefficients. Threshold of significance was set at P<0.05. The analysis was performed using the SPSS pro- gram for Windows version 21.0 (SPSS, Chicago, IL, USA). Caecum microbiota correlates with body weight and body fat We next tested the hypothesis that abundance of specific bacterial species in mouse caecum contents could be associated to modulation of body weight and body fat. On one hand, C. coc- coides (r = .433, p = 0.044) and C. leptum (r = .488, p = 0.021), both belonging to the Firmicutes’ group, showed positive correlations with body weight. Bacteroides/Prevotella also showed a positive correlation (r = .581, p = 0.006) with body weight. On the other, body fat was negative- ly correlated with Bifidobacterium spp. (r = -.547, p = 0.008). The correlation matrix is pre- sented in Table 3. 5 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0125091 April 27, 2015 Modulation of Microbiota by Conjugated Linoleic Acid Fig 1. Effect of CLA supplementation on mRNA expression and protein levels of gastric proteins in mice. Expression of stomach proteins were analysed in mice after 54 days of supplementation with CLA. (A) Leptin mRNA (%) expression was increased by CLA, and protein content (pg) was higher in both HF and CLA groups. (B) Ghrelin mRNA (%) expression was also higher in CLA animals, whereas protein levels (ng) did not show significant difference amongst groups. (C) Gastric resistin (Retn), G protein-coupled receptor 39 (Gpr39), glucagon (Gcg) and glucagon receptor (Gcgr) expression increased by CLA supplementation, whereas ghrelin o-acyltransferase (Mboat4) did not. Data are the mean ± SEM of 8 animals/group. Letters indicate differences amongst groups; one-way ANOVA followed by Bonferroni test (p<0.05). d i 10 1371/j l 0125091 001 Fig 1. Effect of CLA supplementation on mRNA expression and protein levels of gastric proteins in mice. Expression of stomach proteins were analysed in mice after 54 days of supplementation with CLA. (A) Leptin mRNA (%) expression was increased by CLA, and protein content (pg) was higher in both HF and CLA groups. (B) Ghrelin mRNA (%) expression was also higher in CLA animals, whereas protein levels (ng) did not show significant differences amongst groups. (C) Gastric resistin (Retn), G protein-coupled receptor 39 (Gpr39), glucagon (Gcg) and glucagon receptor (Gcgr) expression increased by CLA supplementation, whereas ghrelin o-acyltransferase (Mboat4) did not. Data are the mean ± SEM of 8 animals/group. Letters indicate differences amongst groups; one-way ANOVA followed by Bonferroni test (p<0.05). doi:10.1371/journal.pone.0125091.g001 doi:10.1371/journal.pone.0125091.g001 Discussion Letters indicate differences amongst groups; one-way ANOVA followed by Bonferroni test (p<0.05). When homogeneity of variances was not assumed, data were log transformed. doi:10.1371/journal.pone.0125091.g002 proteins involved in energy metabolism, both being secreted in relevant amount by the gastric mucosa [42–47]. In accordance with leptin mRNA levels, higher gastric protein content was also observed, which is in accordance with the increased plasma leptin levels described in these animals [35]. It has been previously described that high-fat feeding stimulates the gastric leptin signalling pathway [44], an effect which would not be counteracted by CLA and would contrib- ute to partially explain why no differences were seen regarding food intake in this experimental set up [35]. On the other hand, ghrelin gene is a gut orexigenic hormone which is mainly regu- lated by feeding [48], and although mRNA expression increased with CLA, protein ghrelin lev- els were not changed. The discrepancies observed between mRNA expression and protein levels would be associated to the presence of diurnal rhythms described for both ghrelin and leptin in the gastric environment which allow for a better metabolic control[49]. In addition to these main gastric proteins, we analysed ghrelin o-acyltransferase (Mboat4), resistin and Gpr39, as well as glucagon, somatostatin and their receptors, proteins which are also involved in energy balance and have been described in the gastric mucosa [50–56], although their poten- tial in weight management has not been thoroughly studied. In this context, Mboat4 was of particular interest as it is the enzyme responsible for the acylation of ghrelin [56] and is activat- ed by dietary lipids which act as acylation substrates [57]. Interestingly, all of the proteins ana- lysed showed increased expression with CLA supplementation, suggesting that CLA isomers are specifically sensed by the genes encoding gastric proteins which respond to food cues. Additionally to the abovementioned effects, CLA promoted changes in the gut microbiota of the lower part of the gastrointestinal tract. In recent years, it has been proposed that in obese states there is an increased ratio of Firmicutes to Bacteroidetes as well as a loss of bacterial di- versity [26–28] although currently more focus is being put on bacterial species for host metabo- lism characterization as new data emerge (discussed in a recent review [58]). Neither CLA supplementation nor HF diet alone was associated with changes amongst bacterial species in the Firmicutes’ phylum. Discussion The present study provides evidence that CLA supplementation under a HF diet has a notice- able effect on particular sites of the gastrointestinal tract in mouse, by increasing gastric protein expression and by promoting a prebiotic effect on gut microbiota. Considering that the gastro- intestinal tract integrates the interplay of food, microbiota and metabolic effects on the host, these results may be relevant for the development of weight management strategies, since CLA are compounds used for the reduction of fat mass in humans [10–13]. Gut hormones are secreted in the stomach in response to food intake and play a key role in signalling food intake to the brain [41]. Leptin and ghrelin constitute two of the most studied PLOS ONE \| DOI:10.1371/journal.pone.0125091 April 27, 2015 6 / 13 Modulation of Microbiota by Conjugated Linoleic Acid Fig 2. DNA levels of representative bacterial species in mice caecum are altered by diet. Bacterial species from caecum content was analysed in all groups. (A) Bacterial DNA levels in caecum content (μg bacterial DNA/g caecum content) was increased significantly by HF diet. DNA abundance of 16S rRNA gene of representative bacterial species (B) Firmicutes, (C) Actinobacteria, (D) Bacteroidetes, (E) Verrucomicrobia erial species in mice caecum are altered by diet. Bacter Fig 2. DNA levels of representative bacterial species in mice caecum are altered by diet. Bacterial species from caecum content was analysed in all groups. (A) Bacterial DNA levels in caecum content (μg bacterial DNA/g caecum content) was increased significantly by HF diet. DNA abundance of 16S rRNA gene of representative bacterial species (B) Firmicutes, (C) Actinobacteria, (D) Bacteroidetes, (E) Verrucomicrobia PLOS ONE \| DOI:10.1371/journal.pone.0125091 April 27, 2015 7 / 13 Modulation of Microbiota by Conjugated Linoleic Acid and (F) Proteobacteria was analysed in mouse caecum and normalised with the average of the NF group (2ΔΔCt). Fold change respect to NF group was calculated (Log2 FC) and is indicated below each column. Data are mean ± SEM of 6–8 animals/group. Letters indicate differences amongst groups; one-way ANOVA followed by Bonferroni test (p<0.05). When homogeneity of variances was not assumed, data were log transformed. doi:10.1371/journal.pone.0125091.g002 and (F) Proteobacteria was analysed in mouse caecum and normalised with the average of the NF group (2ΔΔCt). Fold change respect to NF group was calculated (Log2 FC) and is indicated below each column. Data are mean ± SEM of 6–8 animals/group. Modulation of Microbiota by Conjugated Linoleic Acid correlation with Bifidobacterium spp., an association which has also been previously found in both animal models and human studies [60–63]. In contrast, CLA induced a prebiotic effect in supplemented animals. Bacteroides/Prevotella is a bacterial species known to use dietary polysaccharides in a prebiotic fashion [65]. A re- markable increase was found under CLA supplementation suggesting that CLA was able to confer a prebiotic effect. This is supported by a very high induction of A. muciniphila growth by CLA. The presence of this mucin-degrading bacterial species, which resides in the mucus layer, is associated to a healthy mucosa and is generally reduced in obese states [66–68]. Ever- ard et al. [69] have recently demonstrated that oligofructose restores A. muciniphila content in obese animals, and this is associated with an improvement of their metabolic profile. Therefore, the increased caecal content of A. muciniphila found under CLA supplementation suggests that this compound was exerting a prebiotic action on this bacterial species too. To our knowl- edge, this is the first evidence of a CLA-prebiotic effect favouring the specific growth of poten- tially beneficial bacterial species in the gut. However, this increase in both Bacteroides/ Prevotella and A. muciniphila was not enough to allow a full recovery since these animals re- main obese [35]. We cannot rule out that a higher CLA dose and/or longer treatment, which is generally associated with a leaner phenotype [70], would cause an even higher increase in A. muciniphila content and have more significant effects on metabolic parameters. This would fit with the lower induction of A. muciniphila found in the present study in comparison with oth- ers [69, 71]. Overall, our data show that gastrointestinal tract is a first site for action of the bioactive iso- mers of CLA, able to modulate gastric responses as well as microbiota-host metabolism. We cannot rule out the potential interplay between gastric environment and bacterial growth asso- ciated to food cues accompanying CLA intake. However, CLA induced the expression of genes encoding gastric proteins related with regulation of energy balance and exerted a prebiotic ef- fect on selected bacterial species. Growth of potentially beneficial bacterial species, specifically Bacteroidetes/Prevotella and A. muciniphila, suggests CLA confers a prebiotic effect, which could contribute to a healthier metabolic profile. Supporting Information S1 Table. Detailed composition of diets. Composition of normal and high fat diets used throughout the experiment. (DOCX) Acknowledgments We thank Sarah Laraichi (Laboratory of Calorimetry and Materials, Faculty of Sciences, Abdel- malek Essaâdi University, 93030 Tétouan, Morocco) for her help with the animal care and sample collection during her stay in our lab. Further and thorough research of how specific dietary treatments influence specific physiological components, such as the gastrointestinal tract, will help elucidate their impact on specific conditions such as obesity, and develop effi- cient body weight management strategies. Discussion However, HF feeding lowered Bifidobacterium spp., in accordance with previous animal studies [59–62], a reduction which was not counteracted by CLA. Con- sidering supplementation was not able to re-establish normal body weight [35], a positive asso- ciation with body weight was found for both C. coccoides and C. leptum, in agreement with the potential adverse role of these bacterial species on obesity [33, 63, 64], as well as a negative Table 3. Correlations between bacterial species in caecum contents with body weight and body fat of mice. Bacterial Species Body Weight Body Fat R P R P Clostridium coccoides .433* 0.044 .415 0.055 Clostridium leptum .488* 0.021 .165 0.464 Bacteroidetes .581 0.006 .203 0.378 Biﬁdobacterium spp. -.407 0.060 -.547 0.008 Linear relationships were tested using Pearson’s correlation coefﬁcients (R). Signiﬁcant correlations are marked as follows: * = p<0.05, le 3. Correlations between bacterial species in caecum contents with body weight and body fat of mice. PLOS ONE \| DOI:10.1371/journal.pone.0125091 April 27, 2015 8 / 13 References 1. Swinburn BA, Sacks G, Hall KD, McPherson K, Finegood DT, Moodie ML, et al. The global obesity pan- demic: shaped by global drivers and local environments. Lancet. 2011; 378(9793):804–14. Epub 2011/ 08/30. doi: 10.1016/S0140-6736(11)60813-1 PMID: 21872749. 2. Trigueros L, Pena S, Ugidos AV, Sayas-Barbera E, Perez-Alvarez JA, Sendra E. 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https://openalex.org/W3154038130	https://orca.cardiff.ac.uk/id/eprint/150608/1/ejn.15726.pdf	English	null	Place Cells in the Claustrum Remap Under NMDA Receptor Control	bioRxiv (Cold Spring Harbor Laboratory)	2,021	cc-by	11,481	Funding information g This work was supported by Science Foundation Ireland grant SFI 13/IA/2014. Edited by: Thomas Klausberger R E S E A R C H R E P O R T R E S E A R C H R E P O R T Emanuela Rizzello1 \| Sean K. Martin1 \| Jennifer Rouine1 \| Charlotte Callaghan1 \| Mathias L. Mathiasen2 \| Shane M. O’Mara1 Emanuela Rizzello1 \| Sean K. Martin1 \| Jennifer Rouine1 \| Charlotte Callaghan1 \| Mathias L. Mathiasen2 \| Shane M. O’Mara1 1School of Psychology and Institute of Neuroscience, Trinity College Dublin, The University of Dublin, Dublin, Ireland 2School of Psychology, Cardiff University, Cardiff, Wales, UK 1School of Psychology and Institute of Neuroscience, Trinity College Dublin, The University of Dublin, Dublin, Ireland 2School of Psychology, Cardiff University, Cardiff, Wales, UK 1School of Psychology and Institute of Neuroscience, Trinity College Dublin, The University of Dublin, Dublin, Ireland Abstract Place cells are cells that exhibit location-dependent responses; they have mostly been studied in the hippocampus. Place cells have also been reported in the rat claustrum, an underexplored paracortical region with extensive corto-cortical connectivity. It has been hypothesised that claustral neuronal responses are anchored to cortical visual inputs. We show rat claustral place cells remap when visual inputs are eliminated from the environment, and that this remapping is NMDA-receptor-dependent. Eliminating visual input decreases claustral delta-band oscillatory activity, increases theta-band oscilla- tory activity, and increases simultaneously recorded visual cortical activity. We conclude that, like the hippocampus, claustral place field remapping might be mediated by NMDA receptor activity, and is modulated by visual cortical inputs. 2School of Psychology, Cardiff University, Cardiff, Wales, UK Received: 21 April 2021 Accepted: 18 May 2022 DOI: 10.1111/ejn.15726 Received: 21 April 2021 Accepted: 18 May 2022 DOI: 10.1111/ejn.15726 Received: 21 April 2021 Accepted: 18 May 2022 DOI: 10.1111/ejn.15726 Correspondence Emanuela Rizzello and Shane O’Mara, School of Psychology and Institute of Neuroscience, Trinity College Dublin, The University of Dublin, College Green, Dublin D02 PN40, Ireland. Present address Mathias L. Mathiasen, Department of Veterinary and Animal Sciences, University of Copenhagen, Frederiksberg, Denmark Funding information This work was supported by Science Foundation Ireland grant SFI 13/IA/2014. Eur J Neurosci. 2022;1–14. 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. © 2022 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd. RIZZELLO ET AL. 2 claustral cells also responded to the rotation of visual dis- tal cues in the environment. T A B L E 1 Electrophysiological classification of claustral units (mean SEM) T A B L E 1 Electrophysiological classification of claustral units (mean SEM) T A B L E 1 Electrophysiological classification of claustral units (mean SEM) (mean SEM) Place cells Bursting cells Width (μs) 249.20 9.01 253.00 84.33 Amplitude (μV) 134.89 19.76 126.03 42.01 ISI (ms) 3869.03 968.24 1514.05 618.10 Spike freq. (Hz) 0.35 0.09 3.06 1.02 Spatial Skaggs 2.54 0.29 1.48 0.49 Note: Summary statistics for mean spike width, mean spike amplitude, mean inter-spike interval (ISI), mean spike frequency, and spatial information content (Skaggs) (mean SEM) for all claustral place and bursting cells recorded. Compared with the hippocampus, the claustrum is an under-investigated paracortical region; it is adjacent to the orbitofrontal cortex (anteriorly), insular cortex (laterally), and the caudate nucleus of the striatum (medially). The claustrum is a thin sheet of neurons, possessing a complex dorsoventral and rostrocaudal topography, largely span- ning the rostral half of the telencephalon (Dillingham et al., 2017). There are extensive connections between the claustrum and visual cortex (Pearson et al., 1982), includ- ing dense ipsilateral inputs from the secondary visual cor- tex (Miller & Vogt, 1984). We therefore reasoned claustral place field remapping might be affected by visual inputs. We manipulated visual inputs during freely moving navi- gation by foraging rats to explore potential mechanisms underpinning claustral place fields. We also measured oscillatory activity in the claustrum and visual cortex dur- ing light/dark manipulation. Place fields remained present but rapidly remapped by about 50 when visual inputs were eliminated by turning off the room lights. Previous experiments have shown place field remapping in the hip- pocampus is under NMDA receptor control (Kentros et al., 1998). We therefore modulated NMDA receptor activity via systemic D-serine administration (an NMDA- receptor agonist that readily passes the blood–brain bar- rier). First, we determine the D-SER influences on local claustral network (LFP), finding an increase in total power following the drug administration. Second, we found the expected 50 remapping of claustral place cells when ambient visual inputs are eliminated was absent when D-serine was administered. We also found exposure to darkness altered claustral oscillations in the theta and delta-bands, while increasing visual cortical activity. We conclude that, like the hippocampus, claustral place field remapping might be mediated by NMDA receptor activity, and is modulated by visual cortical inputs. Note: Summary statistics for mean spike width, mean spike amplitude, mean inter-spike interval (ISI), mean spike frequency, and spatial information content (Skaggs) (mean SEM) for all claustral place and bursting cells recorded. Note: Summary statistics for mean spike width, mean spike amplitude, mean inter-spike interval (ISI), mean spike frequency, and spatial information content (Skaggs) (mean SEM) for all claustral place and bursting cells recorded. session; Brotons-Mas et al., 2010). In the light condition, the arena was indirectly lit by four symmetrically posi- tioned spotlights. The animal navigated in a square arena for 30 min; visual inputs changed every 10 min from light (L1) to dark (D) and back to light (L2). At the end of each session, the arena was cleaned to remove odours. The next day (Day 2), rats received a subcutaneous injection of D-SER (n = 6 rats) 30 min before recording. Record- ings were repeated at the same time as Day 1 following the same procedure (L1D-SER, DD-SER, and L2D-SER). This protocol was used to measure the place field stability/ remapping between different conditions and their modu- lation by NMDA receptor activity. The D-SER effect was also assessed via oscillatory power (total power, sub- group, n = 3), before and after drug administration. 2.1 \| Claustral place cells remap in darkness We found place fields were stable between the two ses- sions in light (before and after dark exposure). When visual inputs were eliminated from the environment, place fields remapped their position consistently and significantly. The measure of the distance between the centre of mass (COM) of each place field in the three different conditions (L1, D, and L2) revealed a shift of the place field from light to dark (L1–D). The place field returned to a similar position in the first light exposure during the last recording in light (L2) (ΔL1–D (cm) = 12.75 2.06, ΔL1–L2 (cm) 10.00 1.92, ΔD– L2 (cm) = 9.44 2.26, mean SEM, Figure 1d). A consistent angle was formed by the three place fields (L1, D, and L2) in the arena (Figure 1b). We used the centre of mass of the place fields (x and y coordinates) to measure potential differences between the three con- ditions (see Section 4 for details). Notably, we found claustral place cells consistently remapped by about 50 in darkness (L1DL2 angle [degrees]: 47.84 6.82, 1 \| INTRODUCTION animal’s position in an environment (see Grieves & Jeffery, 2017; OMara & Aggleton, 2019 for review). Place cells have been mostly investigated in the hippocampus, but there are reports of extra-hippocampal place cells (Grieves & Jeffery, 2017; OMara & Aggleton, 2019). Jankowski and OMara (2015) reported spatially coding neurons responding to position, boundaries, object loca- tion, and head direction, across light and dark conditions in the claustrum; moreover, these spatially responsive In mammals, several brain regions (including the hippo- campus, anterior thalamus, and claustrum) contain cells whose firing is localized to ‘place fields’, which code the neurons responding to position, boundaries, object loca- tion, and head direction, across light and dark conditions in the claustrum; moreover, these spatially responsive Abbreviations: COM, centre of mass; D-SER, D-serine; ISI, inter-spike interval; LFP, local field potential; V2L, lateral secondary visual area cortex. 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. © 2022 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd. Abbreviations: COM, centre of mass; D-SER, D-serine; ISI, inter-spike interval; LFP, local field potential; V2L, lateral secondary visual area cortex. wileyonlinelibrary.com/journal/ejn wileyonlinelibrary.com/journal/ejn 1 Eur J Neurosci. 2022;1–14. 2 \| RESULTS (b) Examples of six claustral place cells recorded over consecutive sessions with changes in the environmental conditions; when the light was eliminated from the environment, a shift of the claustral place field occurred with a L1DL2 angle (c) of about 50. (d) The measure of the distance between the centre of mass (COM) of each place field in three different conditions (L1, D, and L2) revealed a shift of the place field in the dark. (e) Spatial coherence was statistically different between light and dark conditions, and there were no differences between the first and the last light exposure F I G U R E 1 Claustral place cells remap in darkness. (a) On Day 1, claustral place cells were recorded over consecutive sessions (10 min each), during which animals navigated in the square arena (80 80 cm) with light manipulation (light [L1]/dark [D]/light [L2]); the figure shows an example of the animal’s path recorded during navigation, with superimposed firing activity of the unit (black line with blue dots), the firing rate map (blue square), and a single unit waveform (corresponding to a claustral place cell). On completion of the recording experiments, positions of recorded cells were estimated by histological analyses (the red circle shows tetrodes in the claustrum). (b) Examples of six claustral place cells recorded over consecutive sessions with changes in the environmental conditions; when the light was eliminated from the environment, a shift of the claustral place field occurred with a L1DL2 angle (c) of about 50. (d) The measure of the distance between the centre of mass (COM) of each place field in three different conditions (L1, D, and L2) revealed a shift of the place F I G U R E 1 Claustral place cells remap in darkness. (a) On Day 1, claustral place cells were recorded over consecutive sessions (10 min each), during which animals navigated in the square arena (80 80 cm) with light manipulation (light [L1]/dark [D]/light [L2]); the figure shows an example of the animal’s path recorded during navigation, with superimposed firing activity of the unit (black line with blue dots), the firing rate map (blue square), and a single unit waveform (corresponding to a claustral place cell). On completion of the recording experiments, positions of recorded cells were estimated by histological analyses (the red circle shows tetrodes in the claustrum). 2 \| RESULTS After post-mortem histological verification, 49 well- isolated units recorded in 13 rats were assigned to the claustrum. Based on their electrophysiological and spatial properties, 40 units were classified as place cells (81.63%), and 9 as bursting cells without spatially related firing (18.37%; Table 1). We performed recordings (see Section 4 and Figure 1a) in which rats navigated in a square arena while foraging for food pellets. Additionally, we investi- gated the effects of light/dark transitions to explore how claustral places are modulated by changes in ambient visual inputs (i.e., light or dark during the recording RIZZELLO ET AL. R E 1 Claustral place cells remap in darkness. (a) On Day 1, claustral place cells were recorded over consecutive session uring which animals navigated in the square arena (80 80 cm) with light manipulation (light [L1]/dark [D]/light [L2]); n example of the animal’s path recorded during navigation, with superimposed firing activity of the unit (black line with b g rate map (blue square), and a single unit waveform (corresponding to a claustral place cell). On completion of the record ents, positions of recorded cells were estimated by histological analyses (the red circle shows tetrodes in the claustrum). mples of six claustral place cells recorded over consecutive sessions with changes in the environmental conditions; when th minated from the environment, a shift of the claustral place field occurred with a L1DL2 angle (c) of about 50. (d) The mea ance between the centre of mass (COM) of each place field in three different conditions (L1, D, and L2) revealed a shift of t the dark. (e) Spatial coherence was statistically different between light and dark conditions, and there were no differences and the last light exposure F I G U R E 1 Claustral place cells remap in darkness. (a) On Day 1, claustral place cells were recorded over consecutive sessions (10 min each), during which animals navigated in the square arena (80 80 cm) with light manipulation (light [L1]/dark [D]/light [L2]); the figure shows an example of the animal’s path recorded during navigation, with superimposed firing activity of the unit (black line with blue dots), the firing rate map (blue square), and a single unit waveform (corresponding to a claustral place cell). On completion of the recording experiments, positions of recorded cells were estimated by histological analyses (the red circle shows tetrodes in the claustrum). 2.3 \| Single unit firing properties are modulated by NMDA receptor activation We next investigated whether the drug effect on place field remapping in darkness underlies the firing changes of single place cells in the claustrum. To this end, we measured both the action potential and firing properties in controls and following drug administration. Previous studies demonstrated that place cell firing properties change between light and dark in the hippocampus (Zhang et al., 2014). However, we observed no statisti- cally significant differences in the control recordings between L1/D/L2 transition for spike amplitude (L1CTRL [μV] = 134.90 19.76 vs. DCTRL = 153.38 21.73, p = 0.22, n = 9; DCTRL = 153.38 21.73 vs. L2CTRL = 122.51 12.46, p = 0.06, n = 9; L1CTRL = 134.90 19.76 vs. L2CTRL = 122.51 12.46, p = 0.31, n = 9, mean SEM, two-tailed t test paired) and spike width (L1CTRL [μs] = 249.20 9.01 vs. DCTRL = 245.48 19.04, p = 0.82, n = 9; DCTRL = 245.48 19.04 vs. L2CTRL = 257.11 14.55, p = 0.34, n = 9; L1CTRL = 249.20 9.01 vs. L2CTRL = 257.11 14.55, p = 0.46, n = 9, mean SEM, two-tailed t test paired). Interest- ingly, spike frequency was significantly lower in the dark, compared with the preceding recording in light (L1CTRL [Hz] = 0.35 0.09 vs. DCTRL = 0.23 0.09, p = 0.04, n = 9, mean SEM, two-tailed t test paired, Figure 3a), as well as the following recording in light (DCTRL [Hz] = We measured spatial (Skaggs) information content in controls, and following drug administration, in each con- dition. Consistent with Jankowski and OMara (2015), we found that the spatial information content was signifi- cantly higher in the dark (DCTRL = 3.09 0.32, mean SEM), compared with the preceding recording in light (L1CTRL = 2.54 0.29, p ≤0.01, mean SEM, two- tailed t test paired). Contrarily, we found a difference in spatial information content between the recordings in the dark (DCTRL = 3.09 0.32, mean SEM) and the fol- lowing recordings in the light (L2CTRL = 2.36 0.34, p < 0.01, mean SEM, two-tailed t test paired). There were no significant differences in spatial information content between the first and the last recording in light (L1CTRL vs. L2CTRL p = 0.06, two-tailed t test). 2.2 \| NMDA receptor modulation sharply changes claustral place cell remapping We tested whether the modulation of NMDA receptor activity decreased remapping in the dark. First, 30 min before the recording session of Day 2, we subcutaneously injected the NMDA receptor co-agonist D-SER (see Section 4). The experimental procedure of Day 2 (day in which rats received the drug) was the same as Day 1 (L1/D/L2), except for the drug pre-injection administra- tion (during a time window where the drug shows its highest peak of activity; Figure 2a). We found again the claustral place cells consistently remapped by about 50 degrees in darkness, but that the angle was signifi- cantly increased following D-SER injection in each cell recorded (L1DL2 angle CTRL [degrees] = 47.57 11.39 vs. L1DL2 angle D SER = 104.13 17.82, p = 0.003, n = 9, mean SEM, two-tailed t test for paired two sam- ples for means, Figure 2c). The increase in the angle indi- cates that the three place fields were formed at a similar location. In D-SER treated rats, the measure of the differ- ence in terms of distance between the place field CoM in L1, D, and L2 revealed the three place fields at a similar location (ΔL1–D (cm) = 13.82 4.84, ΔL1–L2 (cm) 20.30 4.81, ΔD–L2 (cm) = 14.74 3.47, mean SEM, Figure 2d). 3.14 0.29, p = 0.93; DD-SER = 3.14 0.29 vs. L2D-SER = 3.12 0.31, p = 0.92; L1D-SER = 3.17 0.43 vs. L2D- SER = 3.12 0.31, p = 0.92, n = 9, mean SEM, two- tailed t test paired). n = 19, mean SEM, Figure 1c), compared with light conditions. Overall, our results show that when visual inputs are removed the claustral place map moves to a new, con- stant, location in the arena in when the rat is navigating in darkness. The measure of spatial coherence across conditions revealed a significant difference between the first light and dark (L1CTRL = 0.30 0.05 vs. DCTRL = 0.39 0.04, p = 0.05, Figure 1e) and between dark and the last light (DCTRL = 0.39 0.04 vs. L2CTRL = 0.31 0.03, p ≤0.01). There was no difference in spatial coherence between first and last light exposure (L1CTRL = 0.30 0.04 vs. L2CTRL = 0.31 0.03, p = 0.73, mean SEM, two-tailed t test paired, Figure 1e). The difference in the spatial coherence disappeared following D-SER injection between the first light and dark (L1D-SER = 0.32 0.02 vs. DD-SER = 0.35 0.04, p = 0.52, n = 9, mean SEM, two-tailed t test paired, Figure 2e) and between dark and the last light (DD-SER = 0.35 0.04 vs. L2D-SER = 0.35 0.04, p = 0.94, n = 9, mean SEM, two-tailed t test paired). D-SER administration had no effect in the spatial coherence similarity between the first and last light (L1D- SER = 0.32 0.02 vs. L2D-SER = 0.35 0.04, p = 0.42, n = 9, mean SEM, two-tailed t test paired). 2 \| RESULTS (b) Examples of six claustral place cells recorded over consecutive sessions with changes in the environmental conditions; when the light was eliminated from the environment, a shift of the claustral place field occurred with a L1DL2 angle (c) of about 50. (d) The measure of the distance between the centre of mass (COM) of each place field in three different conditions (L1, D, and L2) revealed a shift of the place field in the dark. (e) Spatial coherence was statistically different between light and dark conditions, and there were no differences between the first and the last light exposure 4 RIZZELLO ET AL. RIZZELLO ET AL. 4 RIZZELLO ET A U R E 2 NMDA receptor activation reduces claustral place cells remapping. (a) Day 2 replicated Day 1 (but included subcutaneo nistration of D-SER; 10 mg/kg). (b) Example of three claustral place cells in control, and following D-SER administration; place cel pping in dark was reduced following D-SER administration. (c) L1DL2 angle and (d) distance increased following D-SER nistration. (e) Spatial coherence was not statistically different between light and dark following D-SER administration F I G U R E 2 NMDA receptor activation reduces claustral place cells remapping. (a) Day 2 replicated Day 1 (but included subcutaneous administration of D-SER; 10 mg/kg). (b) Example of three claustral place cells in control, and following D-SER administration; place cell remapping in dark was reduced following D-SER administration. (c) L1DL2 angle and (d) distance increased following D-SER administration. (e) Spatial coherence was not statistically different between light and dark following D-SER administration RIZZELLO ET AL. 2.3 \| Single unit firing properties are modulated by NMDA receptor activation Interest- ingly, there were no significant differences in spatial information content between L1, D, and L2 following D- SER administration (L1D-SER = 3.17 0.43 vs. DD-SER = F I G U R E 3 Claustral place field remap underlies a change of firing. (a) The exposur darkness significantly decreased claustral pl cell firing. (b) D-SER eliminated differences the spike frequency between light and dark 6 RIZZELLO 0.23 0.09 vs. L2CTRL = 0.39 0.08, p = 0.02, n = 9, mean SEM, two-tailed t test paired). There were no significant differences in the spike frequency between the first and final recording in light (L1CTRL [Hz] = 0.35 0.09 vs. L2CTRL = 0.39 0.08, p = 0.08, n = 9, mean SEM, two-tailed t test paired). Th fi di i h i I h d i 40.10 vs. L2 = 124.81 L1 = 126.04 42.01 vs. L2 = n = 9, mean SEM, two-tail dark contrasts had no effect bursting cells (L1 [Hz] = 3 1.00, p = 0.93, n = 9; D = 3 00 0 9 9 L 3 F I G U R E 3 underlies a chan darkness signific cell firing. (b) D- the spike freque 6 F I G U R E 3 Claustral place field rem underlies a change of firing. (a) The expo darkness significantly decreased claustral cell firing. (b) D-SER eliminated differenc the spike frequency between light and da 6 RIZZEL 6 RIZZELLO ET AL. F I G U R E 3 Claustral place field remapping underlies a change of firing. (a) The exposure to darkness significantly decreased claustral place cell firing. (b) D-SER eliminated differences in the spike frequency between light and dark F I G U R E 3 Claustral place field remapping underlies a change of firing. (a) The exposure to darkness significantly decreased claustral place cell firing. (b) D-SER eliminated differences in the spike frequency between light and dark 40.10 vs. L2 = 124.81 41.60, p = 0.28, n = 9; L1 = 126.04 42.01 vs. L2 = 124.81 41.60, p = 0.69, n = 9, mean SEM, two-tailed t test paired). The light/ dark contrasts had no effect on the spike frequency of bursting cells (L1 [Hz] = 3.06 1.02 vs. 2.4 \| Visual manipulations do not interfere with bursting cell activity Place cell firing rate is positively correlated with the ani- mals’ running speed. To determine whether the firing rate modulation by D-SER correlates with changes in running speed, we measured average running speeds before and after drug administration (L1CTRL vs. L1D- SER). Our data suggest that D-SER did not influence the animal’s behaviour during navigation (L1CTRL [cm/s] = 10.05 4.10 vs. L1D-SER = 9.47 3.87, n rats = 6, p = 0.71, n.s. mean SEM, two-tailed t test paired), suggesting D-SER did not affect running speed. We next investigated whether the influence of the visual input elimination was extended to other cells observed in the claustrum. We found that the 18.37% of the cell popu- lation in the claustrum were bursting cells without spatially-related firing. Following their characterization (Table 1), we analysed spike properties and firing rate of bursting cells over the L1/D/L2 sequence. There were no differences in the spike width over the three conditions (L1 [μs] = 253.00 84.33 vs. D = 254.13 84.71, p = 0.78, n = 9; D = 254.13 84.71 vs. L2 = 251.94 83.98, p = 0.58, n = 9; L1 = 253.00 84.33 vs. L2 = 251.94 83.98, p = 0.79, n = 9, mean SEM, two-tailed t test paired). Similarly, no statistically signifi- cant differences were observed in the spike amplitude over the three conditions (L1 [μV] = 126.04 42.01 vs. D = 120.31 40.10, p = 0.23, n = 9; D = 120.31 2.3 \| Single unit firing properties are modulated by NMDA receptor activation D = 3.01 1.00, p = 0.93, n = 9; D = 3.01 1.00 vs. L2 = 3.03 1.00, p = 0.97, n = 9; L1 = 3.06 1.02 vs. L2 = 3.03 1.00, p = 0.91, n = 9, mean SEM, two-tailed t test paired). Finally, we measured the total number of burst events for each cell and found visual stimuli did not affect bursting activity (L1 = 60.33 20.11 vs. D = 45.22 15.07, p = 0.45, n = 9; D = 45.22 15.07 vs. L2 = 65.38 23.11, p = 0.29, n = 9; L1 = 60.33 20.11 vs. L2 = 65.38 23.11, p = 0.42, n = 9, mean SEM, two-tailed t test paired). Thus, the manipulation of visual stimuli in the environment does not affect spike and firing properties of claustral bursting cells. 0.23 0.09 vs. L2CTRL = 0.39 0.08, p = 0.02, n = 9, mean SEM, two-tailed t test paired). There were no significant differences in the spike frequency between the first and final recording in light (L1CTRL [Hz] = 0.35 0.09 vs. L2CTRL = 0.39 0.08, p = 0.08, n = 9, mean SEM, two-tailed t test paired). These findings raise the question: Is the decrease in remapping by D-SER caused by a change of firing? To address this issue, we analysed spike frequency following D-SER injection. Notably, D-SER removed the statistical difference between L1/D/L2 in spike frequency (L1D-SER (Hz) = 0.33 0.13 vs. DD-SER = 0.31 0.11, p = 0.75, n = 9, mean SEM, two-tailed t test paired, Figure 3b; DD-SER = 0.31 0.11 vs. L2D-SER = 0.44 0.12, p = 0.13, n = 9, mean SEM, two-tailed t test paired; L1D-SER = 0.33 0.13 vs. L2D-SER = 0.44 0.12, p = 0.27, n = 9, mean SEM, two-tailed t test paired, Figure 3b), suggesting the decrease in place field remapping following D-SER injection might result from an increase in place cell firing in darkness. 2.6 \| Claustral theta oscillations are not affected by changes to visual inputs To determine how theta oscillations are modulated by dark exposure, we measured the relative power of theta oscillations over the L1/D/L2 sequence, and compared RIZZELLO ET AL. DD-SER = 0.33 0.02, p = 0.074, n = 9; L2CTRL = CTRL , p , ; 1CTRL = 0.36 0.02 vs. L2CTRL = 0.36 0.02, , p , ; CTRL vs. DD-SER = 0.33 0.02, p = 0.074, n = 9; L2CTRL = I G U R E 4 Theta oscillations following light and NMDAR manipulation. (a) Theta power did not change following visual anipulation; D-SER administration did not affect theta power oscillations along the L1/D/L2 sequence. (b) Example of raw LFP traces and the theta range (1 s selected from 10 min of LFP recording) in control and following D-SER administration during recordings in L2. ) Periodogram in L1/D/L2 in control and following D-SER administration (theta range is highlighted by the black dashed) F I G U R E 4 Theta oscillations following light and NMDAR manipulation. (a) Theta power did not cha manipulation; D-SER administration did not affect theta power oscillations along the L1/D/L2 sequence. (b in the theta range (1 s selected from 10 min of LFP recording) in control and following D-SER administrati (c) Periodogram in L1/D/L2 in control and following D-SER administration (theta range is highlighted by p = 0.87, n = 9, mean SEM, two-tailed t test paired, Figure 4a; L1D-SER = 0.34 0.02 vs. DD-SER = 0.33 0.02, p = 0.53, n = 9; DD-SER = 0.33 0.02 vs. L2D- SER = 0.32 0.02, p = 0.27, n = 9; L1D-SER = 0.34 0.02 vs. L2D-SER = 0.32 0.02, p = 0.21, n = 9, mean SEM, two-tailed t test paired, Figure 4a). Although a visible theta oscillation reduction was found by compar- ing each subset of visual stimuli input before and after drug administration, the overall difference was not statis- tically significant (L1CTRL = 0.36 0.02 vs. L1D-SER = 0.34 0.02, p = 0.074, n = 9; DCTRL = 0.37 0.03 vs. DD-SER = 0.33 0.02, p = 0.074, n = 9; L2CTRL = how NMDA receptor activation by the co-agonist D-SER influences the claustral local circuit and the neuronal response to light/dark transition. Specifically, we com- pared theta oscillations for control (Day 1), following D- SER administration (Day 2), and across the 2 days of recording (Day 1 vs. Day 2). Visual input manipulations had no effect on oscillations. Likewise, D-SER adminis- tration did not affect the relative power of theta oscilla- tions over the L1/D/L2 sequence. (L1CTRL = 0.36 0.02 vs. DCTRL = 0.37 0.03, p = 0.72, n = 9; DCTRL = 0.37 0.03 vs. L2CTRL = 0.36 0.02, p = 0.63, n = 9; L1CTRL = 0.36 0.02 vs. L2CTRL = 0.36 0.02, p = 0.87, n = 9, mean SEM, two-tailed t test paired, Figure 4a; L1D-SER = 0.34 0.02 vs. DD-SER = 0.33 0.02, p = 0.53, n = 9; DD-SER = 0.33 0.02 vs. L2D- SER = 0.32 0.02, p = 0.27, n = 9; L1D-SER = 0.34 0.02 vs. L2D-SER = 0.32 0.02, p = 0.21, n = 9, mean SEM, two-tailed t test paired, Figure 4a). Although a visible theta oscillation reduction was found by compar- ing each subset of visual stimuli input before and after drug administration, the overall difference was not statis- tically significant (L1CTRL = 0.36 0.02 vs. L1D-SER = 0.34 0.02, p = 0.074, n = 9; DCTRL = 0.37 0.03 vs. 8 Dtot [μV2] = 7326.231 1016.661, p = 0.031, n samples = 6, Figure 6b; Wilcoxon signed-rank test). The V2L relative powers in the θ, δ, and γ bands were not different between light and dark (L1θ = 0.526 0.014 vs. Dθ = 0.530 0.032, p = 1.000; L1δ = 0.150 0.006 vs. Dδ = 0.152 0.006, p = 1.000; L1γ = 0.086 0.012 vs. Dγ = 0.085 0.012, p pp g Then, to determine how effectively D-SER influences the local claustral network, we examined total power fol- lowing drug administration. The administration of D- SER significantly increased the total power in the claus- trum (total power saline [μV2] = 4710.67 269.51 vs. total power D-SER [μV2] = 5325.92 339.12, p < 0.01, n LFP traces = 24 [n = 4 LFP traces for each rat, n of rats = 3, n sessions = 2 saline vs. 2 D-SER], mean SEM, two-tailed t test paired, Figure 5). These results show that the dose of D-SER used for place cell recordings effectively activated NMDAR receptors, increasing claustral neuronal firing. F I G U R E 4 Theta oscillations following light and NMDAR manipulation. (a) Theta power did not change followin manipulation; D-SER administration did not affect theta power oscillations along the L1/D/L2 sequence. (b) Example o F I G U R E 4 Theta oscillations following light and NMDAR manipulation. (a) Theta power did not change following visual manipulation; D-SER administration did not affect theta power oscillations along the L1/D/L2 sequence. (b) Example of raw LFP traces and in the theta range (1 s selected from 10 min of LFP recording) in control and following D-SER administration during recordings in L2. (c) Periodogram in L1/D/L2 in control and following D-SER administration (theta range is highlighted by the black dashed) 8 RIZZELLO ET AL. 0.36 0.02 vs. L2D-SER = 0.32 0.02, p = 0.055, n = 9, mean SEM, Wilcoxon matched-pairs signed rank test). These results suggest that the theta oscillations do not correlate with claustral place cell remapping in darkness. back to these areas. We therefore recorded oscillatory activity in secondary visual cortex and claustrum during navigation in light and dark. navigation in light and dark. The first question was to investigate whether the pro- tocol that showed us the remapping caused changes in both visual and claustral oscillatory activity. Following tetrode implantation in these two brain areas, animals navigated in the square arena moving from light (L1) to dark (D) (Figure 6a) while the local field potential was simultaneously recorded. The total power of raw LFP oscillations in the V2L (lateral secondary visual area) cor- tex was different between light and dark (L1tot [μV2] = 7061.288 1044.802 vs. Dtot [μV2] = 7326.231 1016.661, p = 0.031, n samples = 6, Figure 6b; Wilcoxon signed-rank test). The V2L relative powers in the θ, δ, and γ bands were not different between light and dark (L1θ = 0.526 0.014 vs. Dθ = 0.530 0.032, p = 1.000; L1δ = 0.150 0.006 vs. Dδ = 0.152 0.006, p = 1.000; L1γ = 0.086 0.012 vs. Dγ = 0.085 0.012, p = 0.563; Wilcoxon signed-rank test). In the claustrum, the total power of raw LFP oscillations and the relative power of γ oscillations were not different between light and dark, while θ and δ oscillations substantially changed (L1tot [μV2] = 5235.302 534.739 vs. Dtot [μV2] = 4971.775 508.535, p = 0.084, n samples = 10; L1γ = 0.099 0.008 vs. Dγ = 0.099 0.007, p = 0.695; L1θ = 0.274 0.014 vs. Dθ = 0.314 0.015, p = 0.002; L1δ = The first question was to investigate whether the pro- tocol that showed us the remapping caused changes in both visual and claustral oscillatory activity. Following tetrode implantation in these two brain areas, animals navigated in the square arena moving from light (L1) to dark (D) (Figure 6a) while the local field potential was simultaneously recorded. The total power of raw LFP oscillations in the V2L (lateral secondary visual area) cor- tex was different between light and dark (L1tot [μV2] = 7061.288 1044.802 vs. 2.7 \| Claustral delta and theta oscillations change from light to dark In our study, we used adult rats with a weight of about 300–350 g. For these reasons, we chose D-SER as the more effectively co-agonist of NMDA recep- tors, with an expectation of a reduction in place field remapping. Accordingly, we found D-SER strongly reduced remapping (Day 2 vs. Day 1). Moreover, after D- SER administration, we observed an increase in neuronal firing in darkness. Indeed, NMDA receptor activation increased firing rates in claustral place cells when the rats navigated in darkness. We conclude that D-SER affects place fields in darkness, perhaps by increasing claustral place cell firing. 0.312 0.023 vs. Dδ = 0.280 0.022, p = 0.002, Figure 6c; Wilcoxon signed-rank test). These results sug- gest that the switch from light to dark modulated the oscillatory activity of both visual cortex and claustrum leading to place cells remapping. 2.7 \| Claustral delta and theta oscillations change from light to dark (1998) observed that blocking NMDA receptors abolished the long-term stabil- ity of place field remapping in the hippocampus. Thus, to explore the mechanisms underlying claustral place field remapping in darkness, we administered the NMDA receptor co-agonist D-SER before performing the light/ dark/light recording. In addition to glutamate, NMDA receptor activation needs the binding of a co-agonist at the GluN1 subunit (Oliet & Mothet, 2009). Recently, it has emerged that only D-SER gates NMDA receptors at the synaptic level, whereas extra-synaptic receptors are (2001) concluded NMDA receptor antagonists prevent the establishment of stable place fields in the hippocam- pus. Furthermore, Kentros et al. (1998) observed that blocking NMDA receptors abolished the long-term stabil- ity of place field remapping in the hippocampus. Thus, to explore the mechanisms underlying claustral place field remapping in darkness, we administered the NMDA receptor co-agonist D-SER before performing the light/ dark/light recording. In addition to glutamate, NMDA receptor activation needs the binding of a co-agonist at the GluN1 subunit (Oliet & Mothet, 2009). Recently, it has emerged that only D-SER gates NMDA receptors at the synaptic level, whereas extra-synaptic receptors are gated by glycine (Papouin et al., 2012; Sullivan & Miller, 2012). Furthermore, D-SER gates NMDA recep- tors only in the mature brain, whereas glycine gates NMDA receptor activity in the immature brain (Ferreira et al., 2017). In our study, we used adult rats with a weight of about 300–350 g. For these reasons, we chose D-SER as the more effectively co-agonist of NMDA recep- tors, with an expectation of a reduction in place field (2001) concluded NMDA receptor antagonists prevent the establishment of stable place fields in the hippocam- pus. Furthermore, Kentros et al. (1998) observed that blocking NMDA receptors abolished the long-term stabil- ity of place field remapping in the hippocampus. Thus, to explore the mechanisms underlying claustral place field remapping in darkness, we administered the NMDA receptor co-agonist D-SER before performing the light/ dark/light recording. In addition to glutamate, NMDA receptor activation needs the binding of a co-agonist at the GluN1 subunit (Oliet & Mothet, 2009). Recently, it has emerged that only D-SER gates NMDA receptors at the synaptic level, whereas extra-synaptic receptors are gated by glycine (Papouin et al., 2012; Sullivan & Miller, 2012). Furthermore, D-SER gates NMDA recep- tors only in the mature brain, whereas glycine gates NMDA receptor activity in the immature brain (Ferreira et al., 2017). 2.7 \| Claustral delta and theta oscillations change from light to dark (2001) concluded NMDA receptor antagonists prevent the establishment of stable place fields in the hippocam- pus. Furthermore, Kentros et al. (1998) observed that blocking NMDA receptors abolished the long-term stabil- ity of place field remapping in the hippocampus. Thus, to explore the mechanisms underlying claustral place field remapping in darkness, we administered the NMDA receptor co-agonist D-SER before performing the light/ dark/light recording. In addition to glutamate, NMDA receptor activation needs the binding of a co-agonist at the GluN1 subunit (Oliet & Mothet, 2009). Recently, it has emerged that only D-SER gates NMDA receptors at the synaptic level, whereas extra-synaptic receptors are gated by glycine (Papouin et al., 2012; Sullivan & Miller, 2012). Furthermore, D-SER gates NMDA recep- tors only in the mature brain, whereas glycine gates NMDA receptor activity in the immature brain (Ferreira et al., 2017). In our study, we used adult rats with a weight of about 300–350 g. For these reasons, we chose D-SER as the more effectively co-agonist of NMDA recep- tors, with an expectation of a reduction in place field remapping. Accordingly, we found D-SER strongly reduced remapping (Day 2 vs. Day 1). Moreover, after D- SER administration, we observed an increase in neuronal firing in darkness. Indeed, NMDA receptor activation increased firing rates in claustral place cells when the rats navigated in darkness. We conclude that D-SER affects F I G U R E 6 Claustral delta and theta oscillations change from light to dark. (a) The animals navigated in a square arena which changed from light (L1) to dark (D); tetrodes and electrodes positions were estimated with histological analyses (the red circle shows tetrodes in the claustrum (left) and electrodes in V2L (right). Total and relative powers in V2L (b) and claustrum (c) from L1 to D RIZZELLO ET AL. 9 F I G U R E 6 Claustral delta and theta oscillations change from light to dark. (a) The animals navigated in a square arena which changed from light (L1) to dark (D); tetrodes and electrodes positions were estimated with histological analyses (the red circle shows tetrodes in the claustrum (left) and electrodes in V2L (right). Total and relative powers in V2L (b) and claustrum (c) from L1 to D RIZZELLO ET AL. 9 RIZZELLO ET AL. (2001) concluded NMDA receptor antagonists prevent the establishment of stable place fields in the hippocam- pus. Furthermore, Kentros et al. 2.7 \| Claustral delta and theta oscillations change from light to dark (1998) observed that blocking NMDA receptors abolished the long-term stabil- ity of place field remapping in the hippocampus. Thus, to explore the mechanisms underlying claustral place field remapping in darkness, we administered the NMDA receptor co-agonist D-SER before performing the light/ dark/light recording. In addition to glutamate, NMDA receptor activation needs the binding of a co-agonist at the GluN1 subunit (Oliet & Mothet, 2009). Recently, it has emerged that only D-SER gates NMDA receptors at the synaptic level, whereas extra-synaptic receptors are gated by glycine (Papouin et al., 2012; Sullivan & Miller, 2012). Furthermore, D-SER gates NMDA recep- tors only in the mature brain, whereas glycine gates NMDA receptor activity in the immature brain (Ferreira et al., 2017). In our study, we used adult rats with a weight of about 300–350 g. For these reasons, we chose D-SER as the more effectively co-agonist of NMDA recep- tors, with an expectation of a reduction in place field remapping. Accordingly, we found D-SER strongly reduced remapping (Day 2 vs. Day 1). Moreover, after D- SER administration, we observed an increase in neuronal firing in darkness. Indeed, NMDA receptor activation increased firing rates in claustral place cells when the rats navigated in darkness. We conclude that D-SER affects place fields in darkness, perhaps by increasing claustral 9 0.312 0.023 vs. Dδ = 0.280 0.022, p = 0.002, Figure 6c; Wilcoxon signed-rank test). These results sug- gest that the switch from light to dark modulated the oscillatory activity of both visual cortex and claustrum leading to place cells remapping. 3 \| DISCUSSION Here, we explored how claustral place cells remap when visual inputs are eliminated from the environment, find- ing they remap by a constant angle; this remapping is likely to be NMDA-receptor-dependent. Furthermore, eliminating visual inputs enhances theta- and reduces delta-band oscillatory activity in the claustrum, while increasing simultaneously recorded visual cortical activ- ity. We conclude that, like the hippocampus, claustral place field remapping might be mediated by NMDA receptor activity, and is modulated by visual cortical inputs. We also investigated claustral unit firing changes to explore the mechanism underlying claustral place field remapping in darkness. We established there was an absence of rate remapping between the two light condi- tions (i.e., before and following the dark condition). Intriguingly, there was a reduction in firing rates when visual inputs were eliminated from the environment, accompanied by place remapping in the dark. 2.7 \| Claustral delta and theta oscillations change from light to dark p = 0.563; Wilcoxon signed-rank test). In the claustrum, the total power of raw LFP oscillations and the relative power of γ oscillations were not different between light and dark, while θ and δ oscillations substantially changed (L1tot [μV2] = 5235.302 534.739 vs. Dtot [μV2] = 4971.775 508.535, p = 0.084, n samples = 10; L1γ = 0.099 0.008 vs. Dγ = 0.099 0.007, p = 0.695; L1θ = 0.274 0.014 vs. Dθ = 0.314 0.015, p = 0.002; L1δ = The constant, reliable angle formed by remapping claus- tral place fields in darkness led us to hypothesize that remapping arose from a loss of inputs from visual cortices to the claustrum, and therefore a loss of visual calibration to support place field stability. The claustrum receives dense ipsilateral inputs from visual cortices and projects F I G U R E 5 Total power increases following D-SER administration. (a) LFPs were recorded following a subcutaneous injection of normal saline (Day 1) or D-SER (Day 2) in a box (24h 38w 38l cm). (b) D-SER significantly increases the total power in the claustrum. (c) Representative traces (1 s segments extracted from 10 min recording) of the total power in saline (top) and following D-SER administration (bottom) F I G U R E 5 Total power increases following D-SER administration. (a) LFPs were recorded following a subcutaneous injection of normal saline (Day 1) or D-SER (Day 2) in a box (24h 38w 38l cm). (b) D-SER significantly increases the total power in the claustrum. (c) Representative traces (1 s segments extracted from 10 min recording) of the total power in saline (top) and following D-SER administration (bottom) F I G U R E 5 Total power increases following D-SER administration. (a) LFPs were recorded following a subcutaneous injection of normal saline (Day 1) or D-SER (Day 2) in a box (24h 38w 38l cm). (b) D-SER significantly increases the total power in the claustrum. (c) Representative traces (1 s segments extracted from 10 min recording) of the total power in saline (top) and following D-SER administration (bottom) 02, ug- he um en nd- is re, ces ile iv- ral DA cal ges eld an di- n). en nt, ud- (2001) concluded NMDA receptor antagonists prevent the establishment of stable place fields in the hippocam- pus. Furthermore, Kentros et al. 4.3.1 \| Place cells recordings in light/dark The experimenter entered the recording room at the start and the end of each sequence of a session to clean rat urine traces in the arena, for sugar pellet throwing and to disentangle the recording cable. Electrophysiological recordings were performed from rats navigating an 80 80 cm square and black arena (see Figure 1a). The arena was in the middle of an opaque, black-curtained, square enclosure. The curtains were open/closed during recordings in light/dark respectively. All the equipment and devices (including the recording setup and com- puters) of the room were covered with black bags to reduce visual cues. During the recording in light the apparatus was indirectly lit by four symmetrically posi- tioned spotlights. During foraging, a small amount of pel- let (TestDiet™, 5 TUL, USA) was thrown in the arena at random locations. 4.1 \| Subjects A total of 13 male Lister-hooded rats (Envigo, United Kingdom) weighing 300–350 g were used. Upon arrival, they were housed two per cage and kept in a temperature-controlled laminar airflow unit with a 12/12 h light/dark cycle beginning at 8:00 AM with ad libitum access to food and water. They were handled daily for 10 days before electrode implantation. Rats were observed and handled during a recovery period of 10 days following electrode implantation. Before starting with electrophysiological recordings, animals underwent a food deprivation procedure until they reached 85% of free-feeding body weight; they were maintained at this weight during the entire study. In three of the animals, a preliminary screening was performed using a single recording of 10 min where rats navigated in a square arena to ensure the presence of place cells in the claus- trum. In 13 animals, we performed electrophysiological recordings following microdrive (Axona, Ltd, UK) 10 behaviour) revealed place field remapping in darkness was not driven by changes in the local network in the claustrum. Theta waves, which are present during vari- ous types of locomotor activities, have been assumed to be generated by the activation of NMDA receptors. Spe- cifically, a combination of NMDA receptor blockers abolished all theta activity in the hippocampus (Buzsaki, 2002). Here, we observed theta power was not modulated by NMDA receptor activation in the claustrum. implantation in the claustrum using a light/dark/light protocol. In six animals, D-SER was systemically admin- istrated. LFP recordings were performed in three animals to determine how effectively the 10 mg/kg dose D-SER influences the local claustral network. Finally, in three animals, we performed electrophysiological recordings following microdrive implantation in both the claustrum and secondary visual cortex. 4.2 \| Compliance with ethical standards The constant angle formed in darkness by claustral place cells and its substantial reduction following D-SER administration led us to hypothesize that synaptic plastic- ity might mask an underestimated input to the claus- trum. Claustral place cell responses may have visual cortical inputs; interestingly, transitioning from light to dark resulted in changes in activity in secondary visual cortex, and increased theta- while reduced delta-band claustral oscillations. Delta oscillations may support attentional dynamics (Harmony, 2013); perhaps claustral remapping involves processes related to attention, rather than direct visual inputs. All experimental procedures were in accordance with the ethical, welfare, legal, and other requirements of the Healthy Products Regulatory Authority regulations and were compliant with the Health Products Regulatory Authority (IrishMedicines Board Acts, 1995 and 2006) and European Union directives on Animal Experimenta- tion (86/609/EEC and Part 8 of the EU Regulations 2012, SI 543). All experimental procedures were approved by the Comparative Medicine/Bioresources Ethics Commit- tee, Trinity College Dublin, Ireland, before conduct and were conducted in accordance with LAST Ireland and international guidelines of good practice. Overall, our findings show that NMDA receptor acti- vation stabilizes claustral place fields in darkness, per- haps by increasing the strength of synaptic transmission. Our findings of differential visual cortical and claustral activity during the light/dark protocol, expand on previ- ous findings suggesting that claustral place cell remapping might be due to attentional dynamics. RIZZELLO ET AL. 3 \| DISCUSSION Here, we explored how claustral place cells remap when visual inputs are eliminated from the environment, find- ing they remap by a constant angle; this remapping is likely to be NMDA-receptor-dependent. Furthermore, eliminating visual inputs enhances theta- and reduces delta-band oscillatory activity in the claustrum, while increasing simultaneously recorded visual cortical activ- ity. We conclude that, like the hippocampus, claustral place field remapping might be mediated by NMDA receptor activity, and is modulated by visual cortical inputs. We also investigated claustral unit firing changes to explore the mechanism underlying claustral place field remapping in darkness. We established there was an absence of rate remapping between the two light condi- tions (i.e., before and following the dark condition). Intriguingly, there was a reduction in firing rates when visual inputs were eliminated from the environment, accompanied by place remapping in the dark. Several lines of evidence suggest spatial maps (includ- ing their stability and expression) are influenced by plas- ticity dependent mechanisms. NMDA receptor- dependent synaptic plasticity is necessary for establishing stable neuronal representations in the hippocampus (Kentros et al., 1998; Shapiro, 2001). Indeed, Shapiro Next, to assess the role of NMDA receptor co- activation in the local circuit, we investigated claustral oscillations following drug administration. Measurement of theta power (oscillations active during exploratory 10 4.5 \| Electrode implantation Following the acclimation period, animals underwent surgery for electrode implantation 25 μm above the claustrum to avoid tissue damages. Rats were implanted with bundles of eight tetrodes of ø 25 μm platinum- iridium wires with impedance 150–350 KΩ (California Fine Wire Ltd., CA, USA). Microdrives were built using eight tetrodes and implanted after craniotomy in the cau- dal part of the anterior claustrum, according to the atlas coordinates (Paxinos and Watson, 5th Edition). The claustral coordinates used relative to bregma were as fol- lows: anterior-posterior (AP) +2.52 mm, medial-lateral (ML) 2.00 mm, dorso-ventral (DV) 4.6 mm from top of the cortex, at an angle of 13. Coordinates for stereo- taxic implantation of a bipolar electrode (Stainless steel wire, SS-5T 127 μm, Science Products GmbH, Hofheim, DE) in secondary visual cortex relative to bregma were as follows: anterior-posterior (AP) 5.28 mm, medial-lateral (ML) 4.00 mm, dorso-ventral (DV) 1.4 mm from top of the cortex, at an angle of 24.8. The bipolar electrode was connected to two microdrive electrodes. 4.7.1 \| Spike units Graphical, cluster-cutting, Tint software (Axona Ltd., UK) was used to cluster-cut the waveforms selected. Place cells were considered as those cells with specific electrophysiological properties (ref., Table 1) that visually and statistically showed a place field. Based on their activity, unit identification involved further criteria: All included as place cells had to have a spatial information content (Skaggs et al., 1993) index ≥0.5, a spatial coher- ence ≥0.25, and a mean firing rate ≥0.25. The spatial path of the subject and the spike train were used to pro- duce a locational firing rate map. A percentage (16%) of the recorded place cells disappeared after the first session (L1), possibly due to positional instability of the elec- trodes and/or the cell. In this case, the cell was counted as a place cell according to its properties, but without the following D/L2 sessions. Statistical analyses for mean spike amplitude, mean spike-width, mean firing fre- quency, mean inter-spike interval (ISI), spatial informa- tion content (Skaggs) and spatial coherence (mean SEM) was performed (Table 1). The spike amplitude (μV) was measured by the difference between the first negative peak and first positive peak, spike-width (μs) was taken at 25% of the spike amplitude; firing fre- quency was considered as the number of spikes per sec- ond (Hz), the ISI (ms) was represented by the time between subsequent action potentials. Spatial informa- tion content represented how much information the fir- ing rate of a cell contained about the spatial location of the rat. It was calculated using the methods from (Skaggs et al., 1993). Spatial coherence represented the correla- tion between the raw firing map and smoothed firing map. The measure of the total burst as the total number of burst events in a cell was used for bursting cells. Neu- roChaT (Islam et al., 2019) was used to analyse the inter- spike interval (ISI), frequency and cell bursting properties from the spike train of bursting cells. The rhythmic effect of the cell was amplified by the autocor- relation measure of ISI histogram. Indeed, bursting cells were discriminated among other cells on account of their fast firing profile, a higher firing frequency and a lower ISI compared to place cells (Table 1), an ISI of minimum 6 ms, a minimum interburst interval (IBI) of 50 ms. Additionally, the total number of bursts during a 11 injected with normal saline (Day 1, NaCl, 0.9% 10 mg/kg) and D-SER (Day 2, 10 mg/kg, Sigma-Aldrich Ireland) and placed in a box (24h 38w 38l cm). The LFP record- ing started 30 min following the substance administration (Figure 5a). LFPs were recorded for 30 min during which sugar pellets were thrown in the box every 5 min. The experimenter was outside the room for the entire record- ing, except every 5 min for pellet throwing. The oscilla- tory activity from the secondary visual cortex and claustrum was simultaneously recorded during light/dark while animals navigated in a square arena. detection. A camera was located above the arena all- owing tracking of the animal’s head position. The ani- mal’s path was visualized and recorded by the software DacqUSB (Axona Ltd., UK). 4.4 \| LFP spectra recording After a recovery period from tetrode implantation in the claustrum, the head stage was connected to the micro- drive for recordings. Three rats were subcutaneously RIZZELLO ET AL. 4.9 \| Drugs Rats were subcutaneously injected with NMDA receptor co-agonist D-SER (s.c. D-SER 10 mg/kg, Sigma-Aldrich, Ireland) in vehicle. D-SER was mixed in 0.9% of normal saline (NaCl) to achieve 10 mg/ml solution. Control rats received no injections. For LFP recordings, rats were injected with normal saline (NaCl, 0.9% w/x Sodium Chloride BP, Braun, Dublin) as a control for the follow- ing day when D-SER was administered. where DL1 and DL2 (numerators) are the vectors from the place field centroid in D to the place field centroid in L1 and from the place field centroid in D to the place field centroid in L2, respectively. DL1.DL2 is the dot product of the vectors and it is divided by the product between the norm of the vectors DL1 and DL2. The equa- tion below was used to measure the difference in terms of distance (cm) between the place field centroids (x and y coordinates) in L1 and D, L1 and L2, and D and L2. The difference (cm) allowed to measure the shift in the place field between light and dark in control and follow- ing drug injection. 4.7.3 \| Spatial firing stability To assess whether place cells had a stable place field between successive sessions, the equation L1DL2 (angle) was used to assess the measure of the angle (degrees) between the place field in darkness (D) and the two place fields in light (L1 and L2) using the COM of each place field: L1DL2 angle ð Þ ¼ cos1 DL1:DL2 jDL1j j j DL2 L1DL2 angle ð Þ ¼ cos1 DL1:DL2 jDL1j j j DL2 4.8 \| Histological analyses Coronal brain sections were made in the coronal plane with a freezing microtome (50 μm sections in four series). One series was directly mounted on gelatin-subbed slides, while the other was stored in a cryoprotective solution. The mounted sections were dried overnight, followed by rehydration in a series of ethanol solutions of decreasing concentrations (2 100%, 90%, 70%). The sections were then stained with cresyl violet after 2 min in deionized water. Following cresyl violet staining, sections were placed in deionized water and dehydrated in a series of ethanol solutions (70%, 90%, 2 100% series). Finally, mounted sections were defatted in xylene and coverslipped with DPX (ThermoFisher, Waltham, MA). The sections were imaged with a Leica DM5000B micro- scope with a Leica DFC310FX digital camera and Leica Application Suite image acquisition software. Tetrode and bipolar electrode tracks were identified in the acquired images. 4.7.2 \| Local field potential DacqTrack (Axona, Ltd, UK) includes various methods for tracking the x and y coordinates of a moving target within the image. During L1/D/L2 and LFP recordings, two-spot (big/small) tracking method was used to track the rat’s position. The average running speed of the rat during navigation was measured in cm/sec. LFP traces obtained from claustrum were referenced to the animal ground, and power was calculated using a random channel for each tetrode. Relative theta power (theta/total) was calculated for each session (L1, D, and L2) by calculating Welch’s periodogram for the LFP sig- nal and then integrating bands of the periodogram (5– 11 Hz for theta), using Simpson’s method. Total power was calculated by integrating the periodogram between 1.5 and 90 Hz. This range was chosen to eliminate very low and very high frequency noise present in the LFP recording. Relative powers in these studies were calcu- lated by dividing the power in a band giving a measure of how much of the overall LFP activity is accounted for by this frequency band. The range of relative powers was 1.5–4 Hz for delta, 5–11 Hz for theta, and 30–90 Hz for gamma oscillations. 12 RIZZELLO ET AL. where x and y are the coordinates of the place field cen- troid, and the equation represents the distance formula. where x and y are the coordinates of the place field cen- troid, and the equation represents the distance formula. recording session, the number of spikes in the bursting cluster, number of spikes per burst, burst duration and propensity to burst were examined. 4.6 \| Screening and recording After the recovery period, tetrodes were lowered about 25 μm to the upper layer of the claustrum. A delay of 24 h was interposed between this stage and the screening sessions. During screening sessions, a cable connecting the recording system to the head-stage (Axona Ltd., UK) was plugged into the microdrive (Axona Ltd., UK). Dur- ing rat navigation, a screening/recording was performed by using Axona system and software DacqUSB (Axona Ltd., UK). Tetrodes were lowered slowly through the brain (25 μm/day) until single unit signals were consid- ered of sufficient amplitude, with spike properties typical of place cells (Table 1) and displaying a place field. Sig- nals were amplified between 3000 and 12,000 and ban- dpass filtered between 380 Hz and 7 kHz for single-unit 12 Shane M. O’Mara https://orcid.org/0000-0001-8087- 8531 Shane M. O’Mara https://orcid.org/0000-0001-8087- 8531 Shane M. O’Mara https://orcid.org/0000-0001-8087- 8531 Shane M. O’Mara https://orcid.org/0000-0001-8087- 8531 PEER REVIEW The peer review history for this article is available at https://publons.com/publon/10.1111/ejn.15726. DATA AVAILABILITY STATEMENT Single-unit, LFP, and positional information recorded in this study are freely available on OSF at https://doi.org/ 10.17605/OSF.IO/3S4MH. REFERENCES Brotons-Mas, J. R., Montejo, N., O’Mara, S. M., & Sanchez- Vives, M. V. (2010). 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The representation of space in the brain. Behavioural Processes, 135, 113–131. https://doi. org/10.1016/j.beproc.2016.12.012 CONFLICT OF INTEREST Jankowski, M. M., & OMara, S. M. (2015). Dynamics of place, boundary and object encoding in rat anterior claustrum. Fron- tiers in Behavioral Neuroscience, 9, 250. https://doi.org/10. 3389/fnbeh.2015.00250 The authors declare no competing financial interests. Second, the pro- tocol was repeated the following day (Day 2) at the same time as the previous day (Day 1). Day 1 was con- sidered as the control for Day 2. In Day 2, a subgroup of rats (n rats = 6, n place cells = 9) received (subcuta- neously) 10 mg/kg of NMDA receptor co-agonist D-SER 30 min before the recording session (see Section 4.9 for further details). Third, an additional group of rats (n = 3) were used to test the total power in the LFP and compared with saline. In this case, rats were sub- cutaneously injected with saline (Day 1) and D-SER (Day 2). Following the injection, rats were placed into a box where 30 min of LFP recording was performed (see Section 4.4 for more details). For the larger part of the study, when the normal distribution was present, we ran parametric tests (t tests, paired), whereas nonpara- metric tests (Wilcoxon signed-rank test for paired data) were used otherwise. The Wilcoxon signed-rank test for paired data was used to compare theta power over the 2 days of recordings before and after the drug. The soft- ware GraphPad Prism 8 was used for statistical analysis (t test and Wilcoxon signed-rank test) and graphs. Neu- roChaT (Islam et al., 2019) was used to analyse spike properties and relative theta power (see Sections 4.7.1 and 4.7.2, respectively). The analysis for the total power in the 30 min LFP recording was written in Python (v3.7), extending NeuroChaT. The analysis for the total power, the relative delta, theta, and gamma powers between visual cortex and claustrum was written in Python (v3.7). 1 main sections. First, we investigated place cell activity when rats navigated in a square arena with manipula- tion of visual stimuli. In this case, the experimental protocol comprised three sessions of 10 min each where rats (n rats = 10, n place cells = 19) moved from light (L1) to dark (D) and back to light (L2). Second, the pro- tocol was repeated the following day (Day 2) at the same time as the previous day (Day 1). Day 1 was con- sidered as the control for Day 2. In Day 2, a subgroup of rats (n rats = 6, n place cells = 9) received (subcuta- neously) 10 mg/kg of NMDA receptor co-agonist D-SER 30 min before the recording session (see Section 4.9 for further details). Third, an additional group of rats (n = 3) were used to test the total power in the LFP and compared with saline. In this case, rats were sub- cutaneously injected with saline (Day 1) and D-SER (Day 2). Following the injection, rats were placed into a box where 30 min of LFP recording was performed (see Section 4.4 for more details). For the larger part of the study, when the normal distribution was present, we ran parametric tests (t tests, paired), whereas nonpara- metric tests (Wilcoxon signed-rank test for paired data) were used otherwise. The Wilcoxon signed-rank test for paired data was used to compare theta power over the 2 days of recordings before and after the drug. The soft- ware GraphPad Prism 8 was used for statistical analysis (t test and Wilcoxon signed-rank test) and graphs. Neu- roChaT (Islam et al., 2019) was used to analyse spike properties and relative theta power (see Sections 4.7.1 and 4.7.2, respectively). The analysis for the total power in the 30 min LFP recording was written in Python (v3.7), extending NeuroChaT. The analysis for the total power, the relative delta, theta, and gamma powers between visual cortex and claustrum was written in Python (v3.7). MLM performed histology. SOM supervised and acquired funding. main sections. First, we investigated place cell activity when rats navigated in a square arena with manipula- tion of visual stimuli. In this case, the experimental protocol comprised three sessions of 10 min each where rats (n rats = 10, n place cells = 19) moved from light (L1) to dark (D) and back to light (L2). ACKNOWLEDGEMENTS Harmony, T. (2013). The functional significance of delta oscillations in cognitive processing. Frontiers in Integrative Neuroscience, 7, We thank our funding agency, Science Foundation Ire- land, for making this work possible, and the Comparative Medicine Unit at Trinity College Dublin, The University of Dublin, for technical assistance. Open access funding provided by IReL. Open access funding provided by IReL. 83. https://doi.org/10.3389/fnint.2013.00083 Islam, M. N., Martin, S. K., Aggleton, J. P., & O’Mara, S. M. (2019). NeuroChaT: A toolbox to analyse the dynamics of neuronal encoding in freely-behaving rodents in vivo. Wellcome Open Research, 4. https://wellcomeopenresearch.org/articles/4- 196/v1 4.10 \| Experimental design and statistical analysis ΔL1D ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ yDyL1 ð Þ2 þ xDxL1 ð Þ2 q The experimental designs are illustrated in Figures 1a, 2a, 5a, and 6a. The present study is divided into four RIZZELLO ET AL. 13 AUTHOR CONTRIBUTIONS Kentros, C., Hargreaves, E., Hawkins, R. D., Kandel, E. R., Shapiro, M., & Muller, R. V. (1998). Abolition of long-term sta- bility of new hippocampal place cell maps by NMDA receptor blockade. Science, 280(5372), 2121–2126. https://doi.org/10. 1126/science.280.5372.2121 SOM and ER conceptualized and designed the experi- ments. ER drafted the initial manuscript, and created fig- ures and tables. SOM, SKM and ER wrote, reviewed, and edited the manuscript. ER and SKM analysed data. SKM coded and developed analysis software. CC performed biochemical analyses. JR performed drug administration. Miller, M. W., & Vogt, B. A. (1984). Direct connections of rat visual cortex with sensory, motor, and association cortices. Journal of Comparative Neurology, 226(2), 184–202. https://doi.org/10. 1002/cne.902260204 14 RIZZELLO ET AL. 14 hippocampal code. In S. J. Hanson, J. D. Cowan, & C. L. Giles (Eds.), Advances in neural information processing systems 5 (pp. 1030–1037). Morgan-Kaufmann. Oliet, S. H., & Mothet, J. P. (2009). Regulation of N-methyl-D- aspartate receptors by astrocytic D-serine. Neuroscience, 158(1), 275–283. https://doi.org/10.1016/j.neuroscience.2008.01.071 Sullivan, S. J., & Miller, R. F. (2012). AMPA receptor-dependent, light-evoked D-serine release acts on retinal ganglion cell NMDA receptors. Journal of Neurophysiology, 108(4), 1044–1051. https://doi.org/10.1152/jn.00264.2012 OMara, S. M., & Aggleton, J. P. (2019). Space and memory (far) beyond the hippocampus: Many subcortical structures also support cognitive mapping and mnemonic processing. Fron- tiers in Neural Circuits, 13, 52. https://doi.org/10.3389/fncir. 2019.00052 Zhang, S., Schönfeld, F., Wiskott, L., & Manahan-Vaughan, D. (2014). Spatial representations of place cells in darkness are supported by path integration and border information. Fron- tiers in Behavioral Neuroscience, 8, 222. https://doi.org/10. 3389/fnbeh.2014.00222 Papouin, T., Ladépêche, L., Ruel, J., Sacchi, S., Labasque, M., Hanini, M., Groc, L., Pollegioni, L., Mothet, J. P., & Oliet, S. H. R. (2012). Synaptic and extrasynaptic NMDA receptors are gated by different endogenous coagonists. Cell, 150(3), 633–646. https://doi.org/10.1016/j.cell.2012.06.029 How to cite this article: Rizzello, E., Martin, S. K., Rouine, J., Callaghan, C., Mathiasen, M. L., & O’Mara, S. M. (2022). Place cells in the claustrum remap under NMDA receptor control. European Journal of Neuroscience, 1–14. https://doi.org/10. 1111/ejn.15726 Pearson, R., Brodal, P., Gatter, K. C., & Powell, T. P. S. (1982). The organization of the connections between the cortex and the claustrum in the monkey. Brain Research, 234(2), 435–441. https://doi.org/10.1016/0006-8993(82)90883-6 Shapiro, M. (2001). Plasticity, hippocampal place cells, and cogni- tive maps. Archives of Neurology, 58(6), 874–881. https://doi. org/10.1001/archneur.58.6.874 Skaggs, W. E., McNaughton, B. L., & Gothard, K. M. (1993). AUTHOR CONTRIBUTIONS An information-theoretic approach to deciphering the
https://openalex.org/W2325993118	https://tos.org/oceanography/assets/docs/19-1_muller.pdf	English	null	BOOK REVIEW \| Baroclinic Tides: Theoretical Modeling and Observational Evidence	Oceanography	2,006	cc-by	2,349	USAGE Permission is granted to copy this article for use in teaching and research. Republication, systematic reproduction, or collective redistribution of any portion of this article by photocopy machine, reposting, or other means is permitted only with the approval of The Oceanography Society. Send all correspondence to: info@tos.org or The Oceanography Society, PO Box 1931, Rockville, MD 20849-1931, USA. Permission is granted to copy this article for use in teaching and research. Republication, systematic reproduction, or collective redistribution of any portion of this article by photocopy machine, reposting, or other means is permitted only with the approval of The Oceanography Society. Send all correspondence to: info@tos.org or The Oceanography Society, PO Box 1931, Rockville, MD 20849-1931, USA. COPYRIGHT COPYRIGHT This article has been published in Oceanography, Volume 19, Number 1, a quarterly journal of The Oceanography Society. Copyright 2006 by The Oceanography Society. All rights reserved. USAGE Oceanography The Official Magazine of the Oceanography Society CITATION P. Müller. 2006. Review of Baroclinic Tides: Theoretical Modeling and Observational Evidence, by V. Vlasenko, N. Stashchuck, and K. Hutter. Oceanography 19(1):195–197, http://dx.doi. org/10.5670/oceanog.2006.107. REVIEWED BY PETER MÜLLER This is a timely book. Baroclinic tides have emerged at the forefront of research in physical oceanography as they might provide a link between large-scale cir- culation and small-scale mixing. This book summarizes the theories, numeri- cal models, and observations that form the basis of our current understanding of the generation and evolution of baro- clinic tides. It is required reading for anyone who wants to get involved in this exciting field of research. energy of the water column, the break- ing internal waves must provide this energy. Where do the internal waves get their energy from? One major source is the conversion of barotropic tidal energy to baroclinic tidal energy at topographic slopes.1 Thus, the astronomically forced barotropic tides convert energy to the baroclinic tides. The baroclinic tides then transfer their energy to other internal waves, which eventually break and pro- vide the energy for ocean mixing, which affects the oceanic general circulation, which in turn affects climate, with abun- dant interesting feedback mechanisms. The connection between baroclinic tides and climate makes this book about baro- clinic tides all the more important. Similarly, the study of barotropic tides has also been isolated from the rest of physical oceanography for a long time. In contrast to most other oceanographic phenomena, the forcing field (the gravi- tational potential of the moon and sun) is extremely well known and the oceanic response (the barotropic tide) is nearly linear. Fairly accurate predictions had been achieved by solving the Laplace tidal equation. Only recently, issues such as tidal loading, earth tides, and gravita- tional self-attraction have increased the complexity of tidal modeling. Baroclinic tides are internal gravity waves of tidal frequency. They are gen- erated when the astronomically forced barotropic tidal currents encounter topographic slopes and displace density surfaces. For a long time these baroclinic tides were regarded as an odd part of the oceanic internal wave field. Much of this field is a statistically homogeneous and stationary superposition of many waves that have different frequencies and wave numbers and that propagate in all horizontal and vertical directions. This part is well described by the celebrated Garrett and Munk spectrum. In con- trast, baroclinic tides exhibit strong geo- which in turn affects climate, with abun- dant interesting feedback mechanisms. The connection between baroclinic tides and climate makes this book about baro- clinic tides all the more important. By Vasiliy Vlasenko, Nataliya Stashchuck, and Kolumban Hutter, Cambridge University Press, 2005, 351 pages, ISBN 0521843952, Hardcover, $120 US graphic variations, are highly directional, and are often confined to a few low modes. Many of the past efforts were de- voted to finding a dynamical explanation for the Garrett and Munk part, while the baroclinic tidal part was largely ignored. Only the acoustic community had an active stake in it, because baroclinic tides are often the major internal wave signal that affects acoustic propagation. By Vasiliy Vlasenko, Nataliya Stashchuck, and Kolumban Hutter, Cambridge University Press, 2005, 351 pages, ISBN 0521843952, Hardcover, $120 US 1The other important source is the generation of inertial oscillations in the surface mixed layer by changes in the atmospheric wind stress. Part of this energy leaks into the ocean interior as near-inertial internal gravity waves. REVIEWED BY PETER MÜLLER This all changed when Walter Munk and Carl Wunsch pushed issues of ocean energetics to the forefront of physical oceanography; barotropic tides, baro- clinic tides, mixing, and the general cir- culation all became entangled. Consider the pycnocline. It is viewed as being maintained by a balance of upwelling and vertical mixing caused by breaking internal gravity waves, ignoring mixing by double diffusive processes. Because vertical mixing increases the potential In writing the book, the authors could draw on more than twenty years of ac- tive research in developing analytic and numerical models of baroclinic tides and in applying these models to concrete oceanic situations. The book covers most of our conceptual knowledge about the generation of linear and nonlinear baro- clinic tides over variable bottom topogra- Oceanography Vol. 19, No. 1, Mar. 2006 195 topography must be regarded as “steep” or supercritical. The Froude number is the ratio of the barotropic tidal cur- rent to the horizontal phase speed of the generated baroclinic waves. For small Froude numbers, the generation process and subsequent evolution are essentially linear. If the Froude number increases, nonlinear effects come into play. The critical latitude delineates the free from the forced wave response. Equatorward of it, linear baroclinic tides can propagate as free waves; poleward of it, they cannot. mode baroclinic tides are generated at flat topography, and multimodal baro- clinic tidal beams at steep topography. When the Froude number increases, a whole suite of new phenomena appears, including bores, nonlinear wave packets, solitary waves, solibores, and wave break- ing. If you are interested in the theories, numerical models, and observations that lead to the results in the table, you have to consult the book. There it is all laid out: analyses of the general wave equa- tion, the Korteweg-deVries equation, and Long’s equation; modal, level, and layer models with often excruciating algorith- mic details; and observations in the Bar- ents Sea, on the Portuguese shelf, and at Oporto Seamount. Details of the results depend, of course, on additional param- eters as well, such as the height of the topographic obstacle to the water depth (adjusted for stratification) and the ra- tio of the barotropic tidal excursion to phy and their subsequent evolution. Figure 1. Schematic representation of the generation and evolution of baroclinic tides for different oceanic conditions. Fr denotes the Froude number and φc the critical latitude. Reproduced with permission. Figure 1. Schematic representation of the generation and evolution of baroclinic tides for different oceanic conditions. Fr denotes the Froude number and φc the critical latitude. Reproduced with permission. REVIEWED BY PETER MÜLLER This conceptual knowledge is all based on a simplified geometric configuration: only two spatial dimensions (the vertical and one horizontal) and localized topograph- ic obstacles, either an underwater ridge or a slope shelf configuration. One can thus neatly define reflected and trans- mitted waves. Some of these simplified models can be solved analytically, but the book spends much effort to define the limits of these analytic solutions and ex- tend them by numerical means. It identi- fies the three most important parameters of the problem: the ratio of the topo- graphic slope to the slope of the internal wave group velocity, the Froude number, and the critical latitude. If the topo- graphic slope is much smaller than the slope of the group velocity, the topog- raphy can be regarded as “flat” or sub- critical. If the topographic slope is larger than the slope of the group velocity, the All this conceptual knowledge is de- rived from basic equations using dif- ferent analytic and numerical tools and substantiated by selected observations. It is neatly summarized in Baroclinic Tides in a table, which is reproduced here as Figure 1. This table contains in concise form most of what we know about the generation and evolution of baroclinic tides. At small Froude numbers, first Linear theory Linear theory c > : no solution baroclinic tidal beam; multimodal solution. Non-linear theory Non-linear theory Non-linear theory Non-linear theory for any latitude weak baroclinic bores, weak unsteady lee waves Flat bottom Steep bottom Fr<<1 Fr~1 Fr>1 Generation regime Geometry everywhere at some region c < : dx dH N f >> − − 2 1 2 2 2 2 σ σ dx dH N f ~ 2 1 2 2 2 2 − − σ σ c > : no solution first-mode harmonic baroclinic tides c < : c > :weak unsteady lee waves first-mode baroclinic tides, evolution into bore, nonlinear wave packets, solitary internal waves c < : c > : multiple harmonics, cnoidal and lee waves multimodal baroclinic tides, evolution to 1-st and 2-nd mode wave trains and SIW, mixed unsteady lee waves c < : for any latitude strong unsteady lee waves, solibores, water mixing, solitary internal waves Figure 1. Schematic representation of the generation and evolution of baroclinic tides for different oceanic conditions. Fr denotes the Froude number and φc the critical latitude. Reproduced with permission. REVIEWED BY PETER MÜLLER Non-linear theory weak baroclinic bores, weak unsteady lee waves Linear theory c > : no solution first-mode harmonic baroclinic tides c < : c > :weak unsteady lee waves first-mode baroclinic tides, evolution into bore, nonlinear wave packets, solitary internal waves c < : first-mode harmonic baroclinic tides c < : weak baroclinic bores, weak unsteady lee waves Non-linear theory for any latitude strong unsteady lee waves, solibores, water mixing, solitary internal waves Non-linear theory c > : multiple harmonics, multimodal baroclinic tides, evolution to 1-st and 2-nd mode wave trains and SIW, mixed unsteady lee waves c < : Linear theory > : no solution baroclinic tidal beam; multimodal solution. c < : > : multiple harmonics multimodal baroclinic tides, evolution to 1-st and 2-nd mode wave trains and SIW, mixed unsteady lee waves c < : baroclinic tidal beam; multimodal solution. c : strong unsteady lee waves, solibores, water mixing, solitary internal waves c > : no solution Oceanography Vol. 19, No. 1, Mar. 2006 196 the Journal of Physical Oceanography on the Hawaiian Ocean Mixing Experiment (HOME), which will appear at about the same time that this book review will ap- pear. HOME is a multi-faceted study of tidal energy conversion and related pro- cesses along the Hawaiian Ridge. In their articles, the HOME investigators make abundant use of the concepts in the table in order to understand what exactly is happening at the Hawaiian Ridge. In summary, I would like to congrat- ulate the authors on putting together this book at this time. It will be of great value to researchers in this new and ex- citing field. the horizontal scale of the topography. Nevertheless, this table represents in a nutshell most of what we know about the generation and evolution of baro- clinic tides and what we use as concep- tual models when we must analyze more complex situations, where the topog- raphy is not two-dimensional and the oceanic environment is more variable. This can be seen in the special issue of In summary, I would like to congrat- ulate the authors on putting together this book at this time. It will be of great value to researchers in this new and ex- citing field. Peter Müller (pmuller@hawaii.edu) is Professor of Oceanography, University of Hawaii, Honolulu, HI, USA. Peter Müller (pmuller@hawaii.edu) is Professor of Oceanography, University of Hawaii, Honolulu, HI, USA. Computer Modelling in Atmospheric and Oceanic Sciences By Peter Müller and Hans von Storch, Springen-Verlag, 2004, 304 pages, ISBN 3540404783, $89.95 US By Peter Müller and Hans von Storch, Springen-Verlag, 2004, 304 pages, ISBN 3540404783, $89.95 US and Data, (4) The Dynamics of Tides and Climate, (5) Modeling in Applied Environmental Sciences, (6) Modeling in Fundamental Environmental Sciences, and (7) Issues and Conclusions. Reviewed by James J. O’Brien The Introduction sets the tone for the book with a review of tide and climate modeling. Chapter 2 reviews the fun- damental laws and the classical closure problem for a turbulent fluid. Unless the readers have studied these concepts before, they will have a difficult job of understanding this section. In addition, this chapter includes a short discus- sion of models as dynamical systems and stochastic systems as well as limits to predictability. Chapter 3, Models and Data, contains a useful discussion of validation and assimilation. These con- cepts are difficult for the new scientist to grasp and they will need additional material. Chapter 4 reviews the tidal problem and modeling the complex cli- mate system. Here we really begin to see This book is sewed together into seven chapters of 200 pages and another 100 pages of appendices. Because this re- viewer knows both of these eminent oceanographers, it is straightforward to assign an author to each section. Com- puter Modelling contains much useful information, but it would not work well as a textbook in an Earth system model- ing class either for the atmosphere or the ocean. Experienced numerical model- ers will find some nice discussion of old and modern concepts. The text contains lengthy discussions of the authors’ phi- losophies with regard to models. It is unusual to find such opinions in a text. some shortcomings of the text. Already in the previous chapters, many concepts are introduced without definitions (e.g., isentropic coordinate system). It seems as if the authors have not decided who their readers are to be. They explain many concepts from fundamental prin- ciples, but often skip over “new” words. In discussing tides, the authors reduce the discussion to a very simple problem to show computer details. Then, they include incorrect finite difference equa- tions. An expert who knows this will not be harmed, but a novice will be lost. In The seven chapters are: (1) Introduc- tion, (2) Computer Models, (3) Models Oceanography Vol. 19, No. 1, Mar. 2006 1
https://openalex.org/W2127391962	https://bmcbioinformatics.biomedcentral.com/counter/pdf/10.1186/1471-2105-8-209	English	null	CRISPR Recognition Tool (CRT): a tool for automatic detection of clustered regularly interspaced palindromic repeats	BMC bioinformatics	2,007	cc-by	8,425	BioMed Central BioMed Central BioMed Central Software Address: 1Department of Computer Science, Jackson State University, Jackson, MS 39217, USA, 2DOE Joint Genome Institute, Walnut Creek, CA 94598, USA and 3Department of Computer Science and Engineering, University of Nebraska-Lincoln, Lincoln, NE 68504, USA Email: Charles Bland* - charles.bland@jsums.edu; Teresa L Ramsey - tramsey@cse.unl.edu; Fareedah Sabree - fareedah.sabree@jsums.edu; Micheal Lowe - michael.l.lowe@jsums.edu; Kyndall Brown - kyndall.d.brown@jsums.edu; Nikos C Kyrpides - NCKyrpides@lbl.gov; Philip Hugenholtz - phugenholtz@lbl.gov * Corresponding author Received: 13 October 2006 Accepted: 18 June 2007 Published: 18 June 2007 BMC Bioinformatics 2007, 8:209 doi:10.1186/1471-2105-8-209 BMC Bioinformatics 2007, 8:209 doi:10.1186/1471-2105-8-209 This article is available from: http://www.biomedcentral.com/1471-2105/8/209 This article is available from: http://www.biomedcentral.com © 2007 Bland et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Clustered Regularly Interspaced Palindromic Repeats (CRISPRs) are a novel type of direct repeat found in a wide range of bacteria and archaea. CRISPRs are beginning to attract attention because of their proposed mechanism; that is, defending their hosts against invading extrachromosomal elements such as viruses. Existing repeat detection tools do a poor job of identifying CRISPRs due to the presence of unique spacer sequences separating the repeats. In this study, a new tool, CRT, is introduced that rapidly and accurately identifies CRISPRs in large DNA strings, such as genomes and metagenomes. Results: CRT was compared to CRISPR detection tools, Patscan and Pilercr. In terms of correctness, CRT was shown to be very reliable, demonstrating significant improvements over Patscan for measures precision, recall and quality. When compared to Pilercr, CRT showed improved performance for recall and quality. In terms of speed, CRT proved to be a huge improvement over Patscan. Both CRT and Pilercr were comparable in speed, however CRT was faster for genomes containing large numbers of repeats. Conclusion: In this paper a new tool was introduced for the automatic detection of CRISPR elements. This tool, CRT, showed some important improvements over current techniques for CRISPR identification. CRT's approach to detecting repetitive sequences is straightforward. It uses a simple sequential scan of a DNA sequence and detects repeats directly without any major conversion or preprocessing of the input. This leads to a program that is easy to describe and understand; yet it is very accurate, fast and memory efficient, being O(n) in space and O(nm/l) in time. Open Acc Software CRISPR Recognition Tool (CRT): a tool for automatic detection of clustered regularly interspaced palindromic repeats Charles Bland*1, Teresa L Ramsey3, Fareedah Sabree1, Micheal Lowe1, Kyndall Brown1, Nikos C Kyrpides2 and Philip Hugenholtz2 Open Access Searching for exact k-mer matches The algorithm begins its search for repeats with a left-to- right scan of a sequence using a small sliding search win- dow of length k. The value in the search window represents a candidate repeat, and each time the window reads a new k-mer, the algorithm searches forward for exact k-mer matches. When searching for each successive match, the search space can be restricted to a small range, called search range. Given a k-mer that begins at position i, any exact k-mer match, if one exists, should occur in the range: This study centers on a recently recognized family of repeats known as Clustered Regularly Interspaced Palin- dromic Repeats (CRISPRs). Since their description by Mojica et al. [8], CRISPRs have attracted a great deal of interest [9-16]. CRISPRs have been found only in the genomes of prokaryotes and are composed of short direct repeats currently known to range in sizes from 21 – 47 base pairs. This family of repeats is unique in that they are interspaced by non-repeating sequences of similar size. CRISPRs were found in approximately 40% of bacterial genomes investigated [14]. Of those genomes with CRISPRs present, about one half contained multiple CRISPR loci. The average number of repeats per loci was found to be 27, with an average repeat length of 32 base pairs. Although knowledge of the characteristics of CRISPRs continues to grow, their complete function is still not yet known. One recently verified hypothesis, however, is that they defend against invading viruses [16]. [i + minR + minS .. i + maxR + maxS + k] Here, minR and maxR refer to the lengths of the smallest and largest repeats to be detected. The lengths of spacers, which are the similarly sized non-repeating regions between repeats, are referred to by minS and maxS (See Figure 1). Since CRISPRs are to some degree evenly spaced, the distance between the initial repeats can be used to approximate the spacing between subsequent exact k-mer matches. Thus the size of the search range can be reduced further, resulting in faster processing time. Several software applications are available for identifying various forms of repeats. However, because the focus on CRISPR elements is recent, only one CRISPR-specific tool has been published for their automatic detection [17]. Identification based on generic repeat searching applica- tions such as Patscan [18] require considerable manual post-processing. If exact k-mer matches are found The search described above detects a succession of simi- larly spaced repeats of length k. Since these repeats do not represent the true length of the repeating pattern, they must be extended (left and right) to the actual repeat length. Any method for extending repeats must consider that mutations occur in DNA sequences, so, repeats may not be exact. The approach taken is this paper is to read the characters to the left or right of all repeats and com- pute occurrence percentages for each base, ACGT. If there is a character that has an occurrence percentage greater An occurrence of a CRISPR Figure 1 An occurrence of a CRISPR. Repetitive sequences are detected by reading a small search window and then scanning ahead for exact k-mer matches separated by a similar dis- tance. k-mer match search window repeat repeat spacer search range Page 2 of 8 (page number not for citation purposes) An occurrence of a CRISPR Figure 1 An occurrence of a CRISPR. Repetitive sequences are detected by reading a small search window and then scanning ahead for exact k-mer matches separated by a similar dis- tance. k-mer match search window repeat repeat spacer search range k-mer match search window repeat repeat spacer search range Searching for exact k-mer matches In this study, a new tool for the auto- matic detection of CRISPR elements is presented. This software program, CRISPR Recognition Tool (CRT), uses a simple sequential search technique that detects repeats directly from a DNA sequence. Unlike most repeat detec- tion techniques, the algorithm presented in this paper does not rely on the use of the suffix tree or alignment matrix as a central data structure. Instead, repeats are dis- covered directly from the DNA. As a result, this technique is very efficient in terms of memory usage, and it is much easier to understand and implement than most other methods. Despite its simplicity, the presented algorithm is able to achieved impressive execution speed when com- pared to other repeat detection tools. The size of the search range has a direct effect on the processing time of the algorithm, with smaller ranges being more desirable. Thus, the algorithm runs fastest when there is little variation between the sizes of the smallest/largest repeats and the smallest/largest spacers. Background various forms/sizes and may be found dispersed through- out a genome, clustered in close proximity or arranged contiguously. The identification of repeats has proven to g Repetitive sequences are abundant in bacteria and archaea, accounting for close to 5% of the genome size in many organisms [1,2]. These repetitive sequences come in Page 1 of 8 (page number not for citation purposes) Page 1 of 8 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/8/209 http://www.biomedcentral.com/1471-2105/8/209 http://www.biomedcentral.com/1471-2105/8/209 BMC Bioinformatics 2007, 8:209 be of significance, as they provide insight into the func- tional and evolutionary roles of various organisms [3-7]. be of significance, as they provide insight into the func- tional and evolutionary roles of various organisms [3-7]. Implementation CRT's search for CRISPRs is based on finding a series of short exact repeats of length k that are separated by a sim- ilar distance and then extending these exact k-mer matches to the actual repeat length. The value of k should be small and less than the length of the shortest repeat to be detected. By making k small, string comparison is faster and the likelihood of finding exact matches between approximate repeats is increased. Once actual repeats are found, they are filtered to remove those that do not meet CRISPR specific requirements. An occurr Figure 1 Results CRT (version 1.0), Pilercr (version 1.0) [17] and Patscan [18] were compared based on execution speed and ability to correctly identify CRISPRs. Patscan is a generic pattern discovery application that identifies repetitive sequences given a user-specified input pattern. The number of repeats that Patscan detects must be predefined, and the tool has no mechanism for distinguishing repeating and non-repeating regions of CRISPRs. Thus, considerable manual processing of the output is required in order to remove unwanted results and to extend repetitive sequences beyond the fixed size limit set by the input pat- If no exact k-mer matches are found The final part of program checks the left and right flanks of a CRISPR in case repeats were missed because of too many mismatches. The flank check is less strict than the initial search for repeats in that it does not look for short exact matches. Instead, the discovered repeats within the CRISPR are used for comparison (using hamming dis- tance) to detect any nearby approximate repeats. The flank check is important for two reasons. The likelihood of missing repeats with mismatches increases when advanc- ing the search window in intervals. Furthermore, accord- ing to Jansen et al. [10], the last or last few repeats of CRISPRs contain mutations in most organisms, and about one-third of CRISPRs have the last repeat truncated. If no exact k-mer matches are found, the search window advances forward and the process described above is repeated. The search window can actually advance for- ward in intervals greater than one without missing any repeats. The size of this interval is one of the major factors contributing to the speed of the presented algorithm. The key to being able to advance at greater intervals is guaranteeing that the search window will never skip any repetitive sequence during its traversal of the DNA sequence. That is, the interval at which the search window advances must be small enough that the entire window will (at some point) fall entirely within each repeat. The length of this interval is dependent on the size of the search window, k, and the length of the smallest repeats to be identified, minR. It can be computed as follows. An occurr Figure 1 g An occurrence of a CRISPR. Repetitive sequences are detected by reading a small search window and then scanning ahead for exact k-mer matches separated by a similar dis- tance. g An occurrence of a CRISPR. Repetitive sequences are detected by reading a small search window and then scanning ahead for exact k-mer matches separated by a similar dis- tance. Page 2 of 8 (page number not for citation purposes) BMC Bioinformatics 2007, 8:209 http://www.biomedcentral.com/1471-2105/8/209 http://www.biomedcentral.com/1471-2105/8/209 http://www.biomedcentral.com/1471-2105/8/209 than or equal to some preset value, p, the repeats are extended. For example, if extending left, a p value of 100% extends exact k-mer matches to exact (k+1)-mer matches only if the character to the left of all repeats within the CRISPR is the same. Thus, for p = 100%, exact repeats are detected, while lower values allow for the detection of approximate repeats. This method of extending repeats works well for CRISPRs, give an appropriate value for p (CRT uses a default value of 75%). repeats with incorrect starting and/or ending positions. To remove unwanted repetitive sequences, filters are applied. The first filter checks that the candidate CRISPR is com- posed of short repeats (between minR and maxR in length). If that condition is met, the spacers are checked for being non-repeating and similarly sized. Filtering is fast because most repetitive sequences do not make it deep into the process. Also, when testing for similarly sized/non-repeating spacers, it is only necessary to check the first few spacers of the CRISPR. interval = max {minR - (2k - 1), 1} Longer repeats produce larger intervals, as do smaller search windows. Larger intervals result in significant improvements in speed because less data is analyzed. For example, for minR = 21 and k = 6, the search window can skip 10 positions each time it advances. Thus, processing a DNA sequence of length 1,000,000, for the most part, becomes equivalent to processing a sequence of length 1,000,000/10 (or 100,000). Although smaller search windows improve processing speed, if continuing to reduce their size, the speed of the algorithm may at some point worsen. This is because smaller search windows increase the likelihood of the pro- gram finding short repetitive sequences that are not really part of a true CRISPR element, but happen by chance. This will cause the program to spend more time processing repeats that are actually false positives. As an example, for a search window of length k = 3, there is a 1/43 chance that any 3-mer will be a match to the search window. This assumes that all four bases are equally likely to appear at any position. Time and Space The CRT algorithm moves a search window through a sequence in intervals, at each step scanning the search range for the pattern in the search window. Searching for a pattern in a text can be done using any fast search algo- rithm. The Boyer-Moore [19] string-matching algorithm is used here. It is linear in time (on average, the algorithm has a sublinear behavior). Thus, the running time of the algorithm for finding CRISPRs as described in this paper is O(nm/l), where n is the length of the DNA sequence, m is the length of the search range and l is the interval at which the search window advances. (The actual behavior of the algorithm is linear and is supported by empirical evidence in the following section.) The algorithm is also linear in space, since repeats are detected directly from the input sequence with no additional major structures required. interval = max {minR - (2k - 1), 1} interval = max {minR - (2k - 1), 1} Running tim 37 and spa Figure 2 Figures 2, 3 and 4 show the execution times of the three tools. The x-axis contains the accession number for the analyzed organisms followed by their approximate number of base pairs in millions (Mbp). As the number of repeats may affect execution time, only genomes with similar repeats counts were used (between 70 and 80). Figures 2 and 3 are based on a search with repeat size 21 – 37, spacer size 19 – 48, and minimum number of CRISPR repeats 3. CRT required an additional setting for search window length. It was tested for values 6 and 8. Fig- ure 4 shows results when searching for longer repeats of size 19 – 50 and spacer size 19 – 60 (this is beyond the range of any CRISPRs found in any previous work). Pats- can is not included in this figure, or any subsequent fig- ures, because it's extended running times flattens the other graph lines, making it difficult to compare the other tools (see Figure 2). Running time based on genome size, using repeat length 21–37 and spacer length 19–48. Running times for the three compared search tools, based on genome size (CRT is listed twice, once for windows size 6 and once for window size 8). The y-axis represents time in seconds. The x-axis lists the genome accession numbers, followed by their sizes in million base pairs (Mbp). As the size of the genomes increase, it can be seen that running times of the search tools increase at different rates. Below, the corresponding organ- ism names are given. [IMG:AE015450] Mycoplasma gallisepti- cum (strain R(low)) [IMG:AE004439] Pasteurella multocida (strain Pm70) [IMG:AE017282] Methylococcus capsulatus (strain Bath/NCIMB 11132) [IMG:AP006627] Bacillus clausii (strain KSM-K16) [IMG:BX470251] Photorhabdus lumines- cens (subsp. laumondii, strain TT01). ent of the size range of repeats, CRT's speed is independ- ent of the number of repeats contained in a genome. Actually, CRT improves slightly in processing time as the number of repeats increases. This is because it is able to process sections of a sequence containing repeats very fast, as explained in the previous section. The speed of CRT and Pilercr is very impressive and a huge improvement over the previous technique of CRISPR detection using Patscan, as shown in Figures 2 and 3. Retrieval Evaluation In order to assist in determining the effectiveness of the three tools in identifying CRISPR elements, three evalua- tion measures were used: quality, precision and recall. Figure 4 shows a slight decrease in the performance of CRT as the range in the size of repeats to be detected is increased (see the previous section). The performance of Pilercr, however, appears to be independent of the size range of repeats. For these settings, the speed of CRT(8) and Pilercr are about the same, with CRT(6) performing best. Running tim 37 and spa Figure 2 CRT achieves the best performance, being able to process a DNA sequence of nearly 6 million bases in about 3 sec- onds using a search window of size 8, CRT(8), and in about 2 seconds for a search window of size 6, CRT(6). Filtering g Many of the candidate CRISPRs found from the process described above will either be contiguous repeats or Page 3 of 8 (page number not for citation purposes) Page 3 of 8 (page number not for citation purposes) BMC Bioinformatics 2007, 8:209 http://www.biomedcentral.com/1471-2105/8/209 Running time based on genome size, using repeat length 21– 37 and spacer length 19–48 Figure 2 Running time based on genome size, using repeat length 21–37 and spacer length 19–48. Running times for the three compared search tools, based on genome size (CRT is listed twice, once for windows size 6 and once for window size 8). The y-axis represents time in seconds. The x-axis lists the genome accession numbers, followed by their sizes in million base pairs (Mbp). As the size of the genomes increase, it can be seen that running times of the search tools increase at different rates. Below, the corresponding organ- ism names are given. [IMG:AE015450] Mycoplasma gallisepti- cum (strain R(low)) [IMG:AE004439] Pasteurella multocida (strain Pm70) [IMG:AE017282] Methylococcus capsulatus (strain Bath/NCIMB 11132) [IMG:AP006627] Bacillus clausii (strain KSM-K16) [IMG:BX470251] Photorhabdus lumines- cens (subsp. laumondii, strain TT01). 0 20 40 60 80 100 120 AE015450 1.0 Mbp AE004439 2.3 Mbp AE017282 3.3 Mbp AP006627 4.3 Mbp BX470251 5.7 Mbp Number of bases (millions) PilerCR CRT(8) CRT(6) Patscan Time (seconds) 0 20 40 60 80 100 120 AE015450 1.0 Mbp AE004439 2.3 Mbp AE017282 3.3 Mbp AP006627 4.3 Mbp BX470251 5.7 Mbp Number of bases (millions) PilerCR CRT(8) CRT(6) Patscan Time (seconds) 0 20 40 60 80 100 120 AE015450 1.0 Mbp AE004439 2.3 Mbp AE017282 3.3 Mbp AP006627 4.3 Mbp BX470251 5.7 Mbp Number of bases (millions) PilerCR CRT(8) CRT(6) Patscan Time (seconds) tern. Pilercr is a recently developed tool designed specifi- cally for the automatic detection of CRISPR elements. It is based on the Piler [20] program, which utilizes alignment matrices for detecting contiguous repeats. Time (seconds) Time (seconds) Both Patscan and Pilercr were implemented in the C pro- gramming language. CRT was developed using Java. All tools were tested on finished microbial genomes available in the IMG version 1.5 database [21]. Each was run under Cygwin version 1.5.21 on a PC having the following spec- ifications: Windows XP operation system, Pentium 3.4 GHz processor, 1.0 GB RAM. Number of bases (millions) Number of bases (millions) Speed Evaluation Running tim 37 and spac Figure 2 Quality Below, the corresponding organism names and the number of CRISPR loci are given. All genomes are close in size (2.7 – 3.8 Mbp). [IMG:BA000031] Vibrio parahaemolyti- cus (serovar O3:K6, strain RIMD 2210633) loci: 0 [IMG:CR628337] Legionella pneumophila (strain Lens) loci: 2 [IMG:AP006840] Symbiobacterium thermophilum (strain IAM 14863/T) loci: 3 [IMG:AE017180] Geobacter sulfurredu- cens (strain ATCC 51573/PCA) loci: 2 [IMG:AE008691] Thermoanaerobacter tengcongensis (strain MB4/JCM 11007) loci: 3 [IMG:AE006641] Sulfolobus solfataricus P2 loci: 7 [IMG:BA000023] Sulfolobus tokodaii str. 7 DNA loci: 7. 0 2 4 6 8 10 12 14 BA000031 0 CR628337 66 AP006840 117 AE017180 183 AE008691 309 AE006641 400 BA000023 450 Number of repeats PilerCR CRT(8) CRT(6) Time (seconds) Running time based on genome size, excluding Patscan Figure 3 Running time based on genome size, excluding Pats- can. Running times for the search tools, excluding Patscan. The parameter values and organisms are the same as that in Figure 2. However, by removing Patscan, a better compari- son of the execution speeds of PilerCR and CRT can be achieved. 0 2 4 6 8 10 12 14 AE015450 1.0 Mbp AE004439 2.3 Mbp AE017282 3.3 Mbp AP006627 4.3 Mbp BX470251 5.7 Mbp Number of bases (millions) PilerCR CRT(8) CRT(6) Time (seconds) Running time based on genome size, excluding Patscan Figure 3 Running time based on genome size, excluding Pats- can. Running times for the search tools, excluding Patscan. The parameter values and organisms are the same as that in Figure 2. However, by removing Patscan, a better compari- son of the execution speeds of PilerCR and CRT can be achieved. 0 2 4 6 8 10 12 14 AE015450 1.0 Mbp AE004439 2.3 Mbp AE017282 3.3 Mbp AP006627 4.3 Mbp BX470251 5.7 Mbp Number of bases (millions) PilerCR CRT(8) CRT(6) Time (seconds) Running time based on number of repeats, using repeat length 21–37 and spacer length 19–48 Figure 5 Running time based on number of repeats, using repeat length 21–37 and spacer length 19–48. Running times for two of the compared search tools based on number of repeats processed. CRT is listed twice, once for windows size 6 and once for window size 8. The y-axis rep- resents time in seconds. The x-axis lists the genome acces- sion numbers, followed by the number of repeats detected in the genome. Running tim length 21– Figure 5 [IMG:BA000031] Vibrio parahaemolyti- cus (serovar O3:K6, strain RIMD 2210633) loci: 0 [IMG:CR628337] Legionella pneumophila (strain Lens) loci: 2 [IMG:AP006840] Symbiobacterium thermophilum (strain IAM 14863/T) loci: 3 [IMG:AE017180] Geobacter sulfurredu- cens (strain ATCC 51573/PCA) loci: 2 [IMG:AE008691] Thermoanaerobacter tengcongensis (strain MB4/JCM 11007) loci: 3 [IMG:AE006641] Sulfolobus solfataricus P2 loci: 7 [IMG:BA000023] Sulfolobus tokodaii str. 7 DNA loci: 7. g g g g Running time based on genome size, excluding Pats- can. Running times for the search tools, excluding Patscan. The parameter values and organisms are the same as that in Figure 2. However, by removing Patscan, a better compari- son of the execution speeds of PilerCR and CRT can be achieved. beginning with TTTAC. In this case, it can be seen that the reporting tool is off by one position. Type III inconsisten- cies occur when a CRISPR is split. For example, a single CRISPR containing 10 repeats may be reported as two CRISPRS, each containing 5 repeats. beginning with TTTAC. In this case, it can be seen that the reporting tool is off by one position. Type III inconsisten- cies occur when a CRISPR is split. For example, a single CRISPR containing 10 repeats may be reported as two CRISPRS, each containing 5 repeats. Let quality represent the likelihood that a CRISPR reported by a search tool does not contain an inconsist- ency of Type I, Type II or Type III. Based on this definition, there is no distinction between a CRISPR with one incon- Running time based on genome size, using repeat length 19– 50 and spacer length is 19–60 Figure 4 Running time based on genome size, using repeat length 19–50 and spacer length is 19–60. Running times for two of the compared search tools, based on genome size (CRT is listed twice, once for windows size 6 and once for window size 8). This figure is the same as Figure 3, except the ranges of the repeat length and spacer length to be detected are increased. 0 2 4 6 8 10 12 14 AE015450 1.0 Mbp AE004439 2.3 Mbp AE017282 3.3 Mbp AP006627 4.3 Mbp BX470251 5.7 Mbp Number of bases (millions) PilerCR CRT(8) CRT(6) Time (seconds) 0 2 4 6 8 10 12 14 AE015450 1.0 Mbp AE004439 2.3 Mbp AE017282 3.3 Mbp AP006627 4.3 Mbp BX470251 5.7 Mbp Number of bases (millions) PilerCR CRT(8) CRT(6) Time (seconds) sistency and a CRISPR with three inconsistencies. Running tim length 21– Figure 5 Given the set of CRISPRs resulting from a search, let a be the total number of CRISPRs reported and b be the total number of CRISPRs containing at least one inconsistency. Assuming a > 0, quality (q) for a search tool can be com- puted as follows. Time (seconds) q b a = − 1 Number of bases (millions) Running tim length 21– Figure 5 Running tim Figure 3 Running time based on number of repeats, using repeat length 21 37 and spacer length 19 48 Figure 5 Running time based on number of repeats, using repeat length 21–37 and spacer length 19–48. Running times for two of the compared search tools based on number of repeats processed. CRT is listed twice, once for windows size 6 and once for window size 8. The y-axis rep- resents time in seconds. The x-axis lists the genome acces- sion numbers, followed by the number of repeats detected in the genome. As the size of the genomes increase, it can bee seen that running times of the search tools increase at differ- ent rates. Below, the corresponding organism names and the number of CRISPR loci are given. All genomes are close in size (2.7 – 3.8 Mbp). [IMG:BA000031] Vibrio parahaemolyti- cus (serovar O3:K6, strain RIMD 2210633) loci: 0 [IMG:CR628337] Legionella pneumophila (strain Lens) loci: 2 [IMG:AP006840] Symbiobacterium thermophilum (strain IAM 14863/T) loci: 3 [IMG:AE017180] Geobacter sulfurredu- cens (strain ATCC 51573/PCA) loci: 2 [IMG:AE008691] Thermoanaerobacter tengcongensis (strain MB4/JCM 11007) loci: 3 [IMG:AE006641] Sulfolobus solfataricus P2 loci: 7 [IMG:BA000023] Sulfolobus tokodaii str. 7 DNA loci: 7. g g , g g Running time based on genome size, excluding Pats- can. Running times for the search tools, excluding Patscan. The parameter values and organisms are the same as that in Figure 2. However, by removing Patscan, a better compari- son of the execution speeds of PilerCR and CRT can be achieved. g p g p g p g g Running time based on number of repeats, using repeat length 21–37 and spacer length 19–48. Running times for two of the compared search tools based on number of repeats processed. CRT is listed twice, once for windows size 6 and once for window size 8. The y-axis rep- resents time in seconds. The x-axis lists the genome acces- sion numbers, followed by the number of repeats detected in the genome. As the size of the genomes increase, it can bee seen that running times of the search tools increase at differ- ent rates. Below, the corresponding organism names and the number of CRISPR loci are given. All genomes are close in size (2.7 – 3.8 Mbp). Quality As the size of the genomes increase, it can bee seen that running times of the search tools increase at differ- ent rates. Below, the corresponding organism names and the number of CRISPR loci are given. All genomes are close in size (2.7 – 3.8 Mbp). [IMG:BA000031] Vibrio parahaemolyti- cus (serovar O3:K6, strain RIMD 2210633) loci: 0 [IMG:CR628337] Legionella pneumophila (strain Lens) loci: 2 [IMG:AP006840] Symbiobacterium thermophilum (strain IAM 14863/T) loci: 3 [IMG:AE017180] Geobacter sulfurredu- cens (strain ATCC 51573/PCA) loci: 2 [IMG:AE008691] Thermoanaerobacter tengcongensis (strain MB4/JCM 11007) loci: 3 [IMG:AE006641] Sulfolobus solfataricus P2 loci: 7 [IMG:BA000023] Sulfolobus tokodaii str. 7 DNA loci: 7. 0 2 4 6 8 10 12 14 BA000031 0 CR628337 66 AP006840 117 AE017180 183 AE008691 309 AE006641 400 BA000023 450 Number of repeats PilerCR CRT(8) CRT(6) Time (seconds) 0 2 4 6 8 10 12 14 AE015450 1.0 Mbp AE004439 2.3 Mbp AE017282 3.3 Mbp AP006627 4.3 Mbp BX470251 5.7 Mbp Number of bases (millions) PilerCR CRT(8) CRT(6) Time (seconds) 0 2 4 6 8 10 12 14 BA000031 0 CR628337 66 AP006840 117 AE017180 183 AE008691 309 AE006641 400 BA000023 450 Number of repeats PilerCR CRT(8) CRT(6) Time (seconds) Time (seconds) Number of repeats Number of repeats Number of bases (millions) Quality Detected CRISPRs are sometimes inconsistent with their actual form in a sequence. This generally results because DNA repeats are not always exact, and consequently are often difficult to correctly identify. Three common types of inconsistencies were identified in this study. Type I inconsistencies occur when a tool reports a CRISPR that is incomplete (that is, the CRISPR does not contain all of the repetitive sequences). Type II inconsistencies occur when repeats within a CRISPR do not begin and/or end at the correct position. For example, A CRISPR that actually begins with the sequence GTTTAC may be reported as In the previous example, execution speed was analyzed based on increasing genome size. In Figure 5, speed is ana- lyzed for increasing number of repeats. Only genomes of similar sizes were used (2.7 – 3.8 Mbp). The repeat size is 21 – 37 and spacer size is 19 – 48. The figures show that CRT performs better than Pilercr for larger number of repeats. Like Pilercr, whose speed appears to be independ- Page 4 of 8 (page number not for citation purposes) Page 4 of 8 (page number not for citation purposes) BMC Bioinformatics 2007, 8:209 http://www.biomedcentral.com/1471-2105/8/209 beginning with TTTAC. In this case, it can be seen that the reporting tool is off by one position. Type III inconsisten- cies occur when a CRISPR is split. For example, a single CRISPR containing 10 repeats may be reported as two CRISPRS, each containing 5 repeats. Let quality represent the likelihood that a CRISPR reported by a search tool does not contain an inconsist- ency of Type I, Type II or Type III. Based on this definition, there is no distinction between a CRISPR with one incon- Running time based on number of repeats, using repeat length 21–37 and spacer length 19–48 Figure 5 Running time based on number of repeats, using repeat length 21–37 and spacer length 19–48. Running times for two of the compared search tools based on number of repeats processed. CRT is listed twice, once for windows size 6 and once for window size 8. The y-axis rep- resents time in seconds. The x-axis lists the genome acces- sion numbers, followed by the number of repeats detected in the genome. As the size of the genomes increase, it can bee seen that running times of the search tools increase at differ- ent rates. Precision and Recall The descriptions given here are expressed in terms of the tools evaluated in this study. false positives and false negatives. The descriptions given here are expressed in terms of the tools evaluated in this study. True positive (TP): the number of instances retrieved that were CRISPRs, False positive (FP): the number of instances retrieved that were not CRISPRs, False negative (FN): the number of instances not retrieved that were CRISPRs. Determining FN can be problematic because it requires the total number of CRISPRs in the dataset to be known. As is often done in IR, in this study FN is estimated using the composite result sets from all of the available searches tools. A comparison of the three search tools, based on measures quality, precision and recall. The higher scores for CRT and Pilercr show that automatic detection of CRISPRs can be very reliable, even more so than with the use manual post-processing as is done with Patscan. The results in the left half of the table are for CRISPRs containing Cas genes. Because the authors suspect that CRISPRs with Cas genes have fewer mutations, and are thus easier to detect, a second experiment was performed using randomly selected finished genomes. The results of this second experiment are shown in the right half of the table. As expected, slightly lower scores resulted, and they should better reflect the effectiveness of the tools. Using the definitions above, precision (p) and recall (r) can be computed as follows. p TP TP FP r TP TP FN = + = + with Patscan. However, it is not clear whether the lower scores for Patscan were mostly from the human involve- ment in the detection process or from the Patscan algo- rithm. Precision is the ratio of the number of instances correctly identified to all the instances retrieved. Given an instance from the result set, it represents the likelihood of that instance being a CRISPR. Thus, precision can be used to answer the question, "Did the retrieval system identify a lot of junk (or instances that were not CRISPRs)?" The quality score was highest for CRT. The lower score for Patscan was due entirely to Type I inconsistencies. The cat- egories of inconsistencies for Pilercr were evenly spread, with Type I and Type II inconsistencies usually missing by only small amounts. Precision was highest with Pilercr, while CRT had the best recall score. Precision and Recall In this application of precision/recall, recall is more significant as it gives an indication of the number of CRISPRs that were missed by a search tool. Although precision is important, a more sensitive tool that detects most CRISPRs but also reports a few repetitive sequences that are not really CRISPRs is more desirable than a less sensitive tool that misses sev- eral CRISPRs but reports very few false positives. Recall is the ratio of the number of instances correctly identified to the total number of instances that are CRISPRs (whether retrieved or missed). Thus, recall can be used to answer the question, "Were all of the CRISPRs retrieved?" In [14], Godde and Bickerton documented CRISPRs in 101 species with the use of Patscan. From that set, a ran- dom sample of size 27 was selected for comparison with results from CRT and Pilercr (using default parameter set- tings). Between Patscan, CRT and Pilercr, a total of 83 dis- tinct CRISPRs were identified. Using the collective information, quality, precision and recall were computed for each tool. The results are presented in Table 1 under the heading CRISPRs with Cas genes. Note that precision is not applicable for Patscan, because false positives are removed during manual post-processing. Also, the results for CRT are based on a search window length of 8. A search window length of 6 would produce similar preci- sion/recall results, but would have a slightly lower quality score, because the likelihood of Type III inaccuracies is slightly increased. As mentioned above, in order to include Patscan in retrieval evaluations, results were used from Godde and Bickerton. However, they reported CRISPRs only for spe- cies that had CRISPR-associated (Cas) genes [10]. The authors of this study suspect that CRISPRs with Cas genes may have fewer mutations, thus they are easier for search tools to detect. As a result, the tools have higher evalua- tion scores. For this reason, a second experiment was undertaken using 80 randomly selected finished genomes from the IMG version 1.5 database. Using CRT and Pilercr, a total of 51 distinct CRISPR elements were iden- tified within the 80 genomes. The evaluation scores are shown in Table 1 under the heading CRISPRs with/with- out Cas genes. These results should be more reflective of the performance of the tools for a typical search. Almost all measures show a reduction in performance. Precision and Recall Quality alone is insufficient for measuring performance, as it does not consider the cost of failing to retrieve rele- vant CRISPRs or the cost of mistakenly retrieving instances that are not CRISPRs. For evaluating inconsist- encies of these types, precision and recall are used. Running tim 50 and spa Figure 4 g g g p g p g g Running time based on genome size, using repeat length 19–50 and spacer length is 19–60. Running times for two of the compared search tools, based on genome size (CRT is listed twice, once for windows size 6 and once for window size 8). This figure is the same as Figure 3, except the ranges of the repeat length and spacer length to be detected are increased. Precision and recall are measures commonly used in the field of information retrieval (IR) when evaluating search algorithms. Their definitions are based on true positives, Page 5 of 8 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/8/209 BMC Bioinformatics 2007, 8:209 Table 1: Performance evaluation measures for the examined tools. CRISPRs with Cas genes CRISPRs with/without Cas genes Quality Precision Recall Quality Precision Recall CRT .95 .99 .99 .90 .89 1 Pilercr .77 1 .95 .75 1 .86 Patscan .74 n/a .89 -- -- -- A comparison of the three search tools, based on measures quality, precision and recall. The higher scores for CRT and Pilercr show that automatic detection of CRISPRs can be very reliable, even more so than with the use manual post-processing as is done with Patscan. The results in the left half of the table are for CRISPRs containing Cas genes. Because the authors suspect that CRISPRs with Cas genes have fewer mutations, and are thus easier to detect, a second experiment was performed using randomly selected finished genomes. The results of this second experiment are shown in the right half of the table. As expected, slightly lower scores resulted, and they should better reflect the effectiveness of the tools. Table 1: Performance evaluation measures for the examined tools. CRISPRs with Cas genes CRISPRs with/without Cas genes Quality Precision Recall Quality Precision Recall CRT .95 .99 .99 .90 .89 1 Pilercr .77 1 .95 .75 1 .86 Patscan .74 n/a .89 -- -- -- Table 1: Performance evaluation measures for the examined tools. false positives and false negatives. Page 6 of 8 (page number not for citation purposes) Authors' contributions Unlike most repeat detection techniques, the algorithm presented in this paper does not rely on the use of the suf- fix tree or alignment matrix as a central data structure. No major conversion or preprocessing of the input is required. Instead, repeats are discovered directly from the DNA sequence using a simple left-to-right skip search technique with localized iterative extensions of identified repeat arrays in order to find exact boundaries. As a result, CRT is very efficient in terms of memory usage, at O(n), and O(nm/l) in time. Thus, a standard desktop machine is sufficient for processing large prokaryotic genomes, usu- ally in a matter of seconds. CB implemented CRT and wrote the manuscript. NCK and PH guided the research and revised the manuscript. FS, TLR, ML and KDB participated in background research, data collection and tool comparison. Conclusion In this paper a new tool was introduced for the automatic detection of CRISPR elements. This tool, CRT, was shown to be a significant improvement over the current tech- nique for CRISPR identification using Patscan. CRT's approach detects repeats directly from a DNA sequence. This leads to a program that is easy to describe and under- stand, yet it is very fast and memory efficient. In terms of retrieval performance, CRT was shown to be very reliable in detecting CRISPRs, based on measures quality, preci- sion and recall. For performance measures tested, CRT outperformed Patscan in all cases. Additionally, when compared to a recently developed CRISPR detection pro- gram, Pilercr, CRT showed improved performance under some important conditions. However, using CRT and Pilercr for detecting CRISPRs is recommended, as both are fast and have complementary strengths for precision and recall. Precision and Recall The most The high scores for CRT and Pilercr show that automatic detection of CRISPRs can be very reliable, even more so than with the use of manual post-processing as is done Page 6 of 8 (page number not for citation purposes) Page 6 of 8 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/8/209 http://www.biomedcentral.com/1471-2105/8/209 http://www.biomedcentral.com/1471-2105/8/209 BMC Bioinformatics 2007, 8:209 noticeable difference is a decrease in precision for CRT and a decrease in recall for Pilercr. noticeable difference is a decrease in precision for CRT and a decrease in recall for Pilercr. Acknowledgements This work was performed under the auspices of the US Department of Energy's Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Livermore National Labora- tory under Contract No. W-7405-Eng-48, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231 and Los Alamos National Laboratory under contract No. W-7405-ENG-36. Also, Lawrence Berkeley National Lab's CSEE and Jackson State University's LSMAMP pro- grams are kindly acknowledged for their support. Future research plans are to modify the presented algo- rithm so that it is also able to identify contiguous repeats. Because of the nature of the CRT algorithm, the tool would not be practical for detecting very short patterns of sizes 2 – 4 nucleotides, for example. CRT is fastest when identifying longer repeats, and when there is little varia- tion between the sizes of the smallest and largest repeats to be detected. Also, the tool is fast when processing genomes with large numbers of repeats; so, CRT may be useful for detecting contiguous repeats in eukaryotes, which tend to have more repetitive sequences than prokaryotic genomics. Operating system(s): Platform independent Programming language: Java Availability and requirements Project name: CRISPR Recognition Tool (CRT) y q Project name: CRISPR Recognition Tool (CRT) Project home page: http://www.room220.com/crt Project home page: http://www.room220.com/crt Another technique frequently used for detecting repeats involves computing alignment matrices from DNA sequences [27,28]. Once implemented, the matrix can be used to find repeated regions in the sequence using one of several algorithms [22,29-31]. These algorithms, however, can be problematic because of extended processing times. Operating system(s): Platform independent Discussion The importance of identifying repetitive sequences is clear; however, the considerable size of many genomes makes fast and efficient repeat detection very challenging. Consequently, many detection techniques convert sequences to an alternative representation in an attempt to make analysis more efficient. A frequently used repre- sentation is the suffix tree [22]. Here, a DNA sequence is converted into a tree structure containing indices to all suffixes in the original sequence. By traversing the tree, an algorithm is able to find all occurrences of any pattern in time proportional to the size of the pattern. Because of the impressive speed of suffix trees, they have been widely used in DNA repeat detection [23-26]. The increased speed, however, comes at a cost. First, even before the search for repeats can begin, the suffix tree must be con- structed from the sequence data. 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https://openalex.org/W4212996365	https://hal.inrae.fr/hal-03615662/document	English	null	Waste Biopolymers for Eco-Friendly Agriculture and Safe Food Production	Coatings	2,022	cc-by	8,702	To cite this version: Elio Padoan, Enzo Montoneri, Giorgio Bordiglia, Valter Boero, Marco Ginepro, et al.. Waste Biopolymers for Eco-Friendly Agriculture and Safe Food Production. Coatings, 2022, 12 (2), pp.239. 10.3390/coatings12020239. hal-03615662 Distributed under a Creative Commons Attribution 4.0 International License Article Elio Padoan 1 , Enzo Montoneri 2,, Giorgio Bordiglia 1, Valter Boero 1, Marco Ginepro 3 , Philippe Evon 4, , Carlos Vaca-Garcia 4 , Giancarlo Fascella 5 and Michéle Negre 1 1 Dipartimento di Scienze Agrarie, Forestali e Alimentari, Università di Torino, Largo P. Braccini 2, 10095 Grugliasco, Italy; elio.padoan@unito.it (E.P.); giorgio.bordiglia@unito.it (G.B.); valter.boero@unito.it (V.B.); negre.michele@gmail.com (M.N.) 1 Dipartimento di Scienze Agrarie, Forestali e Alimentari, Università di Torino, Largo P. Braccini 2, 10095 Grugliasco, Italy; elio.padoan@unito.it (E.P.); giorgio.bordiglia@unito.it (G.B.); lt b @ it it (VB ) i h l @ il (M N ) 2 Dipartimento di Scienze delle Produzioni Agrarie e Alimentari, Università di Catania, Via S. Soﬁa 98, 95123 Catania, Italy y 3 Dipartimento di Chimica, Università di Torino, Via P. Giuria 7, 10125 Torino, Italy; marco.ginepro@unito.it 4 Dipartimento di Chimica, Università di Torino, Via P. Giuria 7, 10125 Torino, Italy; marco.ginepro@unito.it 4 Laboratoire de Chimie Agro-Industrielle (LCA), Université de Toulouse, ENSIACET, INRAE, Toulouse INP, 4 Allée Emile Monso, 31030 Toulouse, France; carlos.vacagarcia@toulouse-inp.fr , , ; g p 5 CREA Research Centre for Plant Protection and Certiﬁcation, 90011 Bagheria, Italy; giancarlo.fascella@crea.gov.it g g * Correspondence: enzo.montoneri@gmail.com (E.M.); philippe.evon@toulouse-inp.fr (P.E.) Abstract: This work addresses environmental problems connected with biowaste management, the chemical industry, and agriculture. These sectors of human activity cause greenhouse gas (GHG) emissions in the air, climate change, leaching of excess mineral fertilizers applied to soil into ground water, and eutrophication. To mitigate this problem in agriculture, controlled release fertilizers (CRFs) are made by coating mineral fertilizers granules with synthetic polymers produced from the fossil-based chemical industry. This strategy aggravates GHG emission. In the present work, six formulations containing sunﬂower protein concentrate (SPC) and a new biopolymer (BP) obtained from sunﬂower oil cake and by hydrolysis of municipal biowaste, respectively, and commercial urea were tested as CRFs for spinach cultivation against the control growing substrate Evergreen TS and commercial Osmocote®. The results show large differences in plants’ nitrate concentration due to the different treatments, although the same nitrogen amount is added to the substrate in all trials. BP is the key component mitigating nitrate accumulation in plants. HAL Id: hal-03615662 https://hal.inrae.fr/hal-03615662v1 Submitted on 21 Mar 2022 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License coatings Citation: Padoan, E.; Montoneri, E.; Bordiglia, G.; Boero, V.; Ginepro, M.; Evon, P.; Vaca-Garcia, C.; Fascella, G.; Negre, M. Waste Biopolymers for Eco-Friendly Agriculture and Safe Food Production. Coatings 2022, 12, 239. https://doi.org/10.3390/ coatings12020239 Keywords: biopolymers; municipal bio-waste; spinach; agriculture; nitrates Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Article The plants grown in the substrates containing BP together with SPC and/or urea, although exhibiting relatively high total N uptake (47–52 g kg−1), have signiﬁcantly lower nitric to total N ratio (9.6–12.0) than that (15.3–16.5) shown by the plants grown in the substrates containing SPC and/or urea, but no BP. The data conﬁrm that all composites containing BP yield the safest crop coupled with high biomass production. Replication of BP effects for the cultivation of different plants will contribute to the development of a biobased chemical industry exploiting biowastes as feedstock. Citation: Padoan, E.; Montoneri, E.; Bordiglia, G.; Boero, V.; Ginepro, M.; Evon, P.; Vaca-Garcia, C.; Fascella, G.; Negre, M. Waste Biopolymers for Eco-Friendly Agriculture and Safe Food Production. Coatings 2022, 12, 239. https://doi.org/10.3390/ coatings12020239 1. Introduction The augmentation of human population, along with its concentration in cities and increasing consumption habits, is causing several problems connected with biowaste management, the chemical industry, and agriculture. For example, municipal biowaste (MBW) production in Europe is 100 Mt yr−1 [1], with more than half still landﬁlled releasing 25,000 Mm3 yr−1 GHG [2]. To satisfy the food demand, crop production is boosted by applying mineral fertilizer doses higher than those adsorbed by soil and plants. Excess nutrients accumulate in soil, leach into ground water, and cause eutrophication [3]. In this context, for a long time the Universities of Torino and Toulouse focused their research on the valorization of biowaste from urban (MBW) and agro-industrial sources as feedstock Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/coatings Coatings 2022, 12, 239. https://doi.org/10.3390/coatings12020239 Coatings 2022, 12, 239 2 of 12 for the production of value added biobased chemical specialties and materials to use in place of commercial products obtained from fossil sources [4]. for the production of value added biobased chemical specialties and materials to use in place of commercial products obtained from fossil sources [4]. With speciﬁc reference to the agriculture sector, very recently the authors of the present work have reported the manufacturing and thermomechanical properties of a composite material made by twin-screw extrusion followed by injection-molding for use as controlled release fertilizer (CRF) [5]. This material contains urea and two polymers, herein after named sunﬂower proteins concentrate (SPC) and biopolymer (BP). These are obtained from sunﬂower oil cake and by hydrolysis of the anaerobic fermentation digestate of municipal unsorted food wastes, respectively. All three components contain nitrogen, have soil fertilizing power, have different solubility in water and, therefore, can release nitrogen in soil. Their fertilizing properties depend on the nitrogen release rate. In addition to the fertilizing property, SPC is well known for its good processability and is suitable to be used as a thermoplastic matrix for manufacturing the CRF composite pellets by twin-screw extrusion followed by injection-molding [5]. 1. Introduction To test and assess the performance of the SPC and BP biopolymers, the experimental plant reported in the present work included three key elements, i.e., urea as widely used fertilizer, spinach as a sensitive probe to measure SPC/BP effects, and Osmocote® as a reference of widely used commercial CRF, representing a typical material made by conventional coating technology. Urea is one of the most important N-fertilizers. Its world consumption is 51 Mt yr−1 [6]. Urea has environmental drawbacks deriving from the release of excess nitrogen over the plant uptake rate. Urea is highly soluble in water. In soil, it is hydrolyzed to ammonia [7,8] and then, transformed into nitrates. These are adsorbed by the plant roots and transferred to the leaves. Nitrates in leaves are reduced to ammonia and then converted to proteins. In the presence of excess ammonia, proteins’ production is slowed down. Nitrates accumulate in leaves and soil. They may impact the environment negatively, leaching from soil into ground water and causing eutrophication [9]. Nitrates in food plants may also have carcinogenic effects for humans [10]. In the human body, nitrate can be reduced to nitrite, which may cause methemoglobinemia, and the possibility of gastric cancer and other diseases [11,12]. Leaves of spinach are a typical food that can exhibit the nitrates’ accumulation ef- fects [13]. Spinach is a high value crop that requires sufﬁcient N fertilizer to ensure optimal growth and to meet high quality criteria [14]. It absorbs NO3−from the soil efﬁciently but is known to be relatively inefﬁcient in NO3−reduction [15]. The European Commission issued the regulation No. 1258/2011, stating the maximum acceptable concentration of nitrates in spinach [16]. These are 3.5 g kg−1 in fresh spinach, 2 g kg−1 in preserved, frozen spinach, and 0.2 g kg−1 in baby food. Spinach leaves should have high nutritional value from protein content and no toxicity from nitrates. p y Commercial Osmocote® is a typical material belonging to the family of CRFs con- taining urea granules coated with synthetic polymers [17,18]. These are used to mitigate nitrate accumulation and effects in the environment [19]. The drawbacks of these CRFs are those typical of the fossil-based chemical industry. To substitute synthetic organic materials derived from fossil sources with natural or biobased materials [20], other CRFs have been investigated, based on natural polymers derived from plants [21]. 1. Introduction No CRF is known to contain biopolymers derived from biowastes as SPC and BP. In the previous work [5], the mechanical properties’ advantages of the injection- molding technology, compared to conventional coating processes, were discussed. The inclusion of Osmocote® in the experimental plan of the present work has allowed the vis-à-vis direct comparison of the twin-screw extruded/injection-molded SPC composite and the coated Osmocote® composite for their performance as CRF for the cultivation of spinach. 3 of 12 Coatings 2022, 12, 239 2.2. Fabrication and Composition of SPC Composites The SPC composites were fabricated and characterized for their elemental composition (Table 1) in the previous work [5]. Granules containing urea and/or BP dispersed into the SPC matrix were made by twin-screw extrusion. These were converted into dense dark pellets, all having the same (10 mm × 10 mm × 4 mm) dimension, by injection-molding. The tested SPC composites had the following compositions: neat SPC, SPC-U containing 10% urea, SPC-BP containing 10% BP, and SPC-BP-U containing 5% BP and 5% urea. For the neat components and the composite materials, Table 1 reports the C and soil/plant N, P, and K nutrient contents. Table 1. Total C, N, P, and K concentrations (%, w w−1) in neat SPC, BP, urea (U), and in the SPC pellets’ samples 1. Table 1. Total C, N, P, and K concentrations (%, w w−1) in neat SPC, BP, urea (U), and in the SPC pellets’ samples 1. Formulation C N P2O5 K2O BP 39.6 6.6 1.1 5.5 U 20.0 46.6 - - SPC 38.7 ± 5.2 a 6.9 ± 0.9 a 2.5 ± 0.1 a 1.5 ± 0.1 a SPC-U 37.7 ± 0.2 a 10.5 ± 0.1 c 2.3 ± 0.1 a 1.3 ± 0.1 a SPC-BP 39.5 ± 2.6 a 7.0 ± 0.6 a 2.6 ± 0.3 a 1.8 ± 0.1 b SPC-BP-U 38.6 ± 0.4 a 8.5 ± 0.0 b 2.1 ± 0.1 a 1.5 ± 0.1 a 1 Values in the same column followed by different letters are signiﬁcantly different (p < 0.05). 2.3. Plant Growth Trials 2. Materials and Methods 2.1. Materials The components to fabricate the SPC composites were available from previous work [5]. SPC was obtained by sieving a sunﬂower oil cake (SOC) using a Ritec (Signes, France) 600 vibrating sieve shaker ﬁtted with a 1 mm grid. The undersize (SPC) was enriched with the smaller particles coming from the kernel of the seed, thus having a high protein content (51%, in proportion to the SPC dry weight). The rest (oversize) was constituted mainly by particles from the seed hull, and it was therefore rich in lignocellulosic ﬁbers and minerals. BP was obtained by hydrolysis at pH 13 and 60 ◦C of the anaerobic digestate of MBW [4]. The anaerobic digestate was collected from the MBW waste treatment plant owned by the Acea Pinerolese Industriale SpA (ACEA) company in Pinerolo (TO). This plant processes, by fermentation, urban food wastes from separate source collection to produce biogas and solid anaerobic digestate containing the recalcitrant lignocellulosic fraction of the pristine MBW. To prepare the BP, the ACEA anaerobic digestate was taken up in water to yield a slurry at 4 water/digestate (w/w) ratio. Potassium hydroxide was added to the slurry until it reached pH 13. The alkaline slurry was heated up to 60 ◦C for 4 h. Afterwards, the slurry was allowed to settle until the liquid hydrolysate separated from the insoluble phase. The liquid hydrolysate was ﬁltered through a 5 kDa polysulphone membrane to separate the retentate containing BP and the permeate containing the excess unreacted alkali reagent. The retentate was ﬁnally dried at 60 ◦C to yield the solid BP, which was used in the present work. Major components of BP were 45% protein, 13% lignin, and 15% minerals. Urea was a common commercial product. 2.3. Plant Growth Trials Spinacia oleracea L. “Gigante d’inverno” seedlings, purchased from Beltrame Roberto nursery, strada Sanda, Moncalieri, Italy, were transplanted on the commercial growing sub- strate Evergreen TS (Turco S.a.s, Moncalieri, Italy) in 2 L pots with 13 cm × 13 cm × 13 cm size (5 plants per pot, and 5 pots per trial). The plants were grown during November and December 2019 in an unheated greenhouse at an average temperature of 18 ◦C and 90% relative humidity under natural lighting conditions. Surface irrigation was performed manually every three days. After three days, the neat components and the composites were added to the substrate in the following amounts: 4 of 12 Coatings 2022, 12, 239 • Trial 0: control (growing substrate) containing 25 mg organic N. • Trial 1: growing substrate + SPC (4.1 g per pot, 283 mg N). • Trial 2: growing substrate + SPC-U (2.7 g per pot, 284 mg N). • Trial 3: growing substrate + SPC-BP (4.0 g per pot, 280 mg N). • Trial 4: growing substrate + SPC-BP-U (3.3 g per pot, 281 mg N). • Trial 5: growing substrate + urea (0.6 g per pot, 280 mg N). • Trial 6: growing substrate + BP (4.3 g per pot, 284 mg N). • Trial 7: growing substrate + urea (0.3 g per pot, 140 mg N) + BP (2.2 g per pot, 145 mg N). l ® ( ) g g g p p g • Trial 6: growing substrate + BP (4.3 g per pot, 284 mg N). g g g p p g • Trial 7: growing substrate + urea (0.3 g per pot, 140 mg N) + BP (2.2 g per pot 145 mg N). g g g p p g • Trial 7: growing substrate + urea (0.3 g per pot, 140 mg N) + BP (2.2 g per pot, 145 mg N). • Trial 8: Osmocote® (1.8 g per pot, 282 mg N). The trials’ plan included the control (trial 0) without added N fertilization, trials 1–7 in the presence of added SPC, BP, and urea products, and trial 8 in the presence of commercial slow-release N, P, K fertilizer Osmocote® [22], which was used as reference material. 2.4. Analyses Total N was determined by the ﬂash combustion method (i.e., “Dumas method”) with thermal conductivity detection using the UNICUBE elementar analyzer (Elementar, Langenselbold, Germany) according to the CSN EN 13654-2 standard method. Nitrate concentrations in leaves and roots at the end of the trial have been analyzed on the fresh material. For each plant, 100 mg of fresh tissue was ground in liquid nitrogen and sus- pended in 10 mL of deionized water. Suspensions were incubated for 1 h at 45 ◦C and then centrifuged at 5000 rpm for 15 min. The extract was ﬁltered, and the nitrate N concentration was determined spectrophotometrically by the Griess reaction [23]. The determination of chlorophyll a and b and of carotenoids was performed on each plant by extraction of 300 mg fresh foliar tissue ground in liquid nitrogen with 10 mL 96% (v/v) ethanol. The samples were kept in the dark for 2 days at 4 ◦C, and the extracts were ﬁltered and then analyzed by spectrophotometry using a Hitachi (Tokyo, Japan) U-2000 spectrophotometer. The ab- sorbance readings were performed at 665 nm for chlorophyll a, at 649 nm for chlorophyll b, and at 470 nm for total carotene. Chlorophyll a, b, and total carotenoid concentrations were calculated according to the literature [24]. 2.5. Statistical Treatment of Data The data were evaluated by one-way ANOVA (p < 0.05 or 0.01) followed by the Tukey’s test for multiple comparison procedures. 2.3. Plant Growth Trials The amount of SPC materials (trial 1–4) and of urea and BP (trial 5–7) added in each pot was calculated to provide, in each pot, nearly the same total N amount (280–285 mg N) as in trial 8. After 55 days, the shoots and roots were collected, and their respective fresh weights were measured. Two plants per pot were dried at 60 ◦C for 3 days for the determination of the dry weight of leaves and roots. 3. Results Fresh and dry weight of spinach leaves and roots (mean value ± standard deviation) 1 cultivated in trials 0–8. Table 2. Fresh and dry weight of spinach leaves and roots (mean value ± standard deviation) 1 cultivated in trials 0–8. Table 3. Chlorophyll and carotenoids content in spinach leaves expressed as mg kg−1 fresh weight (mean value ± standard deviation) 1 cultivated in trials 0–8. Trial Chlorophyll a Chlorophyll b Carotenoids 0 242 ± 49.0 a 123 ± 28.9 a 19 ± 0.9 a 1 330 ± 32.3 b 176 ± 11.0 b 8 ± 0.3 a 2 323 ± 46.6 ab 172 ± 15.0 b 8 ± 0.4 a 3 258 ± 41.0 ab 125 ± 26.9 a 16 ± 0.8 a 4 321 ± 29.4 ab 158 ± 11.0 ab 7 ± 0.3 a 5 341 ± 29.9 b 172 ± 34.1 ab 18 ± 0.6 a 6 241 ± 55.4 a 138 ± 23.3 ab 24 ± 0.7 a 7 309 ± 22.1 ab 158 ± 19.4 ab 11 ± 0.4 a 8 289 ± 42.2 ab 168 ± 34.5 ab 17 ± 0.6 a 1 Within columns, mean values followed by the same letter are not signiﬁcantly different (p < 0.01). Table 4 reports the total N concentration in the plants. The experimental data in Table 4 show that the nitrogen uptake per plant is much lower in roots than in leaves. This is expected since most of the nitrate absorbed by the roots is transported in the xylem to the leaves for further reduction to ammonia, which is then used to synthesize the leaf proteins. The results also point out some signiﬁcant differences between the trials. In trials 1–5, and 7 and 8, the total N concentration in both leaves and roots is higher than that for the control (trial 0). The highest total N concentration values are shown in the leaves of the plants grown on SPC-based fertilizers (trials 1–4) and urea (trial 5). g The data in Table 5 point out large differences in nitrate concentration among the trials, although the same nitrogen amount was added to the substrate in all trials. In all trials, the nitrate concentration in fresh spinach is well below the 3.5 g kg−1 safe limit recommended by the European Commission [18]. 3. Results For the spinach cultivated in the different trials, Table 2 reports the fresh and dry weights of leaves and roots. At the end of the experiment, the plants were healthy and reached the marketable size without apparent differences between the trials, except for the fresh weight of the leaves as higher values were recorded in trials 2 and 3, compared to trial 6. Overall, the experimental values suggest that the control substrate provides a sufﬁcient amount of nutrients to allow a regular and constant growth of the plants. Table 3 reports the chlorophyll a, chlorophyll b, and carotenoids concentrations in the fresh spinach leaves. It shows that the chlorophyll a content in the plants fertilized with SPC and urea (trials 1 and 5) is signiﬁcantly higher than that of the control (trial 0). The plants fertilized with SPC and SPC-U (trials 1 and 2) exhibit signiﬁcantly higher concentrations of chlorophyll b than the control (trial 0). The treatments appear not to signiﬁcantly affect the carotenoid concentration compared to the control (trial 0). Coatings 2022, 12, 239 5 of 12 Table 2. Fresh and dry weight of spinach leaves and roots (mean value ± standard deviation) 1 cultivated in trials 0–8. Trial Leaves Roots Fresh Weight (g) Dry Weight (g) Fresh Weight (g) Dry Weight (g) 0 18.7 ± 3.2 ab 1.6 ± 0.55 a 0.86 ± 0.29 a 0.11 ± 0.04 a 1 25.6 ± 5.3 ab 2.3 ± 0.56 a 1.16 ± 0.22 a 0.13 ± 0.02 a 2 27.3 ± 4.5 a 2.4 ± 0.39 a 1.08 ± 0.23 a 0.12 ± 0.03 a 3 27.7 ± 6.1 a 2.3 ± 0.61 a 0.99 ± 0.26 a 0.10 ± 0.03 a 4 23.5 ± 5.1 ab 2.2 ± 0.54 a 0.99 ± 0.18 a 0.11 ± 0.03 a 5 24.5 ± 5.0 ab 2.3 ± 0.60 a 0.98 ± 0.16 a 0.12 ± 0.03 a 6 18.1 ± 1.9 b 1.8 ± 0.20 a 0.89 ± 0.10 a 0.12 ± 0.02 a 7 23.2 ± 3.4 ab 2.3 ± 0.54 a 1.08 ± 0.14 a 0.13 ± 0.03 a 8 22.8 ± 2.7 ab 2.2 ± 0.27 a 1.04 ± 0.08 a 0.13 ± 0.01 a 1 Within columns, mean values followed by the same letter are not signiﬁcantly different (p < 0.01). Table 2. 3. Results The highest concentration is found in the spinach fertilized with urea (2816 mg kg−1 in trial 5) and in the spinach fertilized with the sunﬂower proteins-based fertilizer (1890–2559 mg kg−1 in trials 1, 2, and 4). The lowest nitrate concentration is found in the plants grown on the control substrate (101 mg kg−1 in trial 0) and in the plants fertilized with BP (247 mg kg−1 in trial 6). However, these plants exhibit a rather low total nitrogen concentration (Table 4), probably due to the low level of nitrogen mineralization in the substrate. This is conﬁrmed by the low nitric to total N ratio (1.0 and 2.2 in trial 0 and trial 6, respectively), which suggests that most of the absorbed inorganic nitrogen is promptly transformed into amino acids and proteinaceous matter. In all other cases, the nitric to total N ratio ranges from 9.6 to 16.5. Coatings 2022, 12, 239 6 of 12 Table 4. Total nitrogen concentration and N uptake for spinach plants (mean value ± standard deviation) 1 cultivated in trials 0–8. Trial Total N (g kg−1 Dry Matter) N Uptake (mg Plant−1) Leaves Roots Leaves Roots 0 34.0 ± 1.82 a 20.9 ± 2.15 a 54.4 ± 21.6 a 2.3 ± 1.01 a 1 51.9 ± 1.32 bcd 29.8 ± 1.72 bc 117.6 ± 32.0 b 3.7 ± 0.88 a 2 52.7 ± 1.31 bd 30.4 ± 2.09 bc 128.3 ± 23.6 bc 3.6 ± 1.09 a 3 48.5 ± 2.04 bce 28.2 ± 0.81 bcd 113.0 ± 34.3 ab 2.9 ± 1.01 a 4 52.1 ± 2.99 bcd 29.1 ± 1.77 bc 114.5 ± 34.5 b 3.3 ± 1.12 a 5 55.5 ± 1.71 d 30.7 ± 2.36 b 129.7 ± 37.4 bc 3.6 ± 1.20 a 6 34.3 ± 3.44 a 20.7 ± 3.44 a 63.1 ± 13.3 ab 2.6 ± 0.87 a 7 47.2 ± 2.55 ce 26.9 ± 2.14 bc 106.2 ± 31.1 ab 3.5 ± 0.96 a 8 45.1 ± 3.47 e 26.5 ± 2.11 c 99.8 ± 19.8 ab 3.3 ± 0.57 a 1 Within columns, mean values with the same letter are not signiﬁcantly different (p < 0.01). Table 5. Nitric nitrogen (mean value ± standard deviation) 1 in spinach leaves cultivated in trials 0–8. Table 5. Nitric nitrogen (mean value ± standard deviation) 1 in spinach leaves cultivated in trials 0–8. 3. Results Trial NO3−N (g kg−1 Dry Matter) NO3−/Total N (w w−1) NO3−N (mg kg−1 Fresh Matter) 0 1.1 ± 0.60 a 1.0 ± 0.58 a 101 ± 53.7 a 1 26.0 ± 4.9 bcd 15.3 ± 3.3 c 2281 ± 410 bcd 2 28.6 ± 2.8 bc 16.5 ± 2.0 c 2559 ± 297 bc 3 15.4 ± 7.5 e 9.6 ± 5.1 b 1373 ± 575 e 4 20.5 ± 7.0 bde 12.0 ± 4.8 bc 1890 ± 593 bde 5 29.8 ± 1.9 c 16.3 ± 1.5 c 2816 ± 125 c 6 2.5 ± 1.4 a 2.2 ± 1.5 a 247 ± 122 a 7 16.4 ± 5.1 de 10.5 ± 3.9 b 1541 ± 398 de 8 14.9 ± 4.0 e 10.1 ± 3.5 b 1438 ± 323 e 1 Within columns, mean values followed the same letter are not signiﬁcantly different (p < 0.01). 4. Discussion 4.1. Effect of BP on Plant Performances 4.1. Effect of BP on Plant Performances The data obtained from the present experiment show signiﬁcant differences only between trials 2, 3, and 6 for the leaf fresh biomass, but not the dry one (Table 2). The average dry weight of the leaves (2.2 g per plant) is consistent with literature data for cultivated spinach under different conditions: 0.6–2.0 g per plant for hydroponic cultures [15,25], 0.2–1.2 g per plant in pot experiments, depending on the amount of added nitrogen [26], and 4.3–5.4 g per plant in trials where different composts were used as fertilizers [27]. g p p p The chlorophyll content of spinach leaves was also affected by BP application (Table 3). The concentration values reported in Table 3 fall in the range of values reported by other workers for spinach cultivated under various operational conditions. For example, for chlorophyll a, the following values are reported: 300–400 mg kg−1 in spinach cultivated in ﬁeld experiments at different N and water applied levels [28], and 860 mg kg−1 in spinach cultivated under hydroponic conditions at 105 mg L−1 N applied doses [29]. The data for the group of trials 1–7, in which SPC, BP, and/or urea were used, show that trials 3 and 6 exhibit lower contents of chlorophyll and higher contents of carotenoids than the other 5 trials. The statistical analysis of these groups of data does not prove signiﬁcant differences between most of the trials. Yet, speciﬁcally for chlorophyll, the apparent lower chlorophyll concentration recorded in leaves from trials 3 and 6 may be correlated to the lower total N content and uptake (Table 4) measured in leaves. Indeed, it is well known that leaf chlorophyll concentration is linked to leaf N content due to the presence of N atoms in chlorophyll molecules [30]. Some authors observed that BP application to plants enhanced the availability of N requested for chlorophyll formation [31], with positive effects on photosynthetic activity. The apparent higher carotenoid content and lower chlorophyll content in trials 3 and 6 can be related to a trend that plants show in Coatings 2022, 12, 239 7 of 12 7 of 12 some growing conditions (particularly under stress), i.e., when chlorophyll values decrease, carotenoids tend to increase, and vice versa [32,33]. some growing conditions (particularly under stress), i.e., when chlorophyll values decrease, carotenoids tend to increase, and vice versa [32,33]. 4.1. Effect of BP on Plant Performances The BP application also inﬂuenced the total nitrogen content in spinach leaves and roots (Table 4). To be assimilated by plants, nitrogen must be in nitric or ammonia forms. Therefore, nitrogen present in organic forms in soil or substrate must be made available through mineralization by microorganisms. The prevailing form is the nitric one because ammonium ion is promptly oxidized and made less available by adsorption on the soil surfaces. Once absorbed by the roots, nitrates are transferred to the shoots where they are reduced to ammonium and used to ﬁrst synthesize glutamate and glutamine through the enzymatic nitroreductase and GOGAT systems, and then other amino acids and N- containing compounds. The total N concentration values are consistent with those reported in literature for spinach leaves (22.7–51.5 g kg−1) [15,27]. The low nitrogen concentration found in the plants grown on the BP-added substrate (trial 6) suggests that BP is recalcitrant to the biochemical attack by microorganisms, in accordance with the high lignin-like chemical moieties present in its macromolecular structure [4]. On the other hand, the low nitrogen uptake of the plants grown in trial 6 does not seem to negatively affect the leaf biomass yield as shown in Table 2. This is consistent with previous results reported by other authors, who used BPs in ﬂoriculture trials [30,31,34]. These authors agreed that the positive effects exhibited by the investigated biopolymers were due to the biopolymers’ chemical structure interacting with the microorganisms and stimulating the plant metabolism, more than to their fertilizing power stemming from their contribution of organic nitrogen as soil fertilizer. On the other hand, although a high N content in the leaves, as shown in Table 4, can be considered a positive result because it is correlated to a high protein concentration, it should not be accompanied with a high nitrate concentration. Accumulation of nitrates in plants generally occurs when the plant uptakes more NO3−than the NO3−assimilable in protein form [35]. Most absorbed nitrate is stored in the vacuole until release for reduction in the cytosol [36]. In the present work, this issue is addressed by the collected data shown in Table 5. Actually, BP application also affected nitric nitrogen content in spinach leaves (Table 5). Accumulation of nitrates in the vacuoles of the cells occurs when the enzymatic systems leading to the reduction of nitrates and further synthesis of protein matter are inhibited by excessive nitrate uptake. 4.1. Effect of BP on Plant Performances In this scenario, it is noteworthy that the plants grown in trials 3, 4, and 7 substrates, all containing BP together with SPC and/or urea, although exhibiting a high total N uptake (Table 4), have signiﬁcantly lower nitric to total N ratio (9.6–12.0) than that (15.3−16.5) shown in the other trials (1, 2, and 5) containing SPC and/or urea but no BP. The best fertilizers in terms of high total N content and low nitrates accumulation were the SPC-BP (trial 3) and SPC-BP-U (trial 4) composites, the mix of urea and BP in trial 7, and Osmocote® (trial 8). In all these trials, the nitrate concentration in the spinach leaves is even below the limit of 2 g kg−1 recommended for preserved frozen spinach by the European Commission [16]. The data conﬁrm that all composites containing BPs yield the safest crop coupled with high biomass production. 4.2. Plausible Explanation of the BP Effects The experimental data point out that BP can mitigate nitrates’ accumulation in spinach plants. The fertilizer used in trial 3 was SPC loaded with 10% BP. About 90% of the total nitrogen present in this specimen comes from SPC. Therefore, most of the N uptake of the plants in trial 3 is to be attributed to SPC nitrogen. Indeed, trial 6 has demonstrated that BP nitrogen is relatively less available for the plant to take up. In trial 7, in which urea was the nitrogen source and the same amount of BP was added, high nitrogen uptake is also accompanied by a relatively low nitrate content. The results suggest that BP, although not supplying to the plant as much nitrogen as SPC and urea, strongly affects the pathways responsible for the mineralization of organic nitrogen. A similar effect was observed in the previous work [5] reporting the kinetics of ammonia and organic N release rates of the SPC composites in water. BP was shown to retard the formation of ammonia from urea Coatings 2022, 12, 239 8 of 12 hydrolysis and enhance the release of organic nitrogen from SPC. In this case, the effect might be due to a plausible interaction of BP functional groups with urea and SPC. g p g p BP belongs to a family of biopolymers obtained by the same hydrolysis process from fermented lignocellulose biowastes of different sources [4]. All these biopolymers keep the memory of the sourcing lignocellulose materials, are constituted by a mix of macromolecules containing the same carbon types and various acid and basic functional groups capable of interacting with other molecules by protonation and donor-acceptor complexing reactions. The relative ratios of the chemical functionalities in these families of biopolymers depend on the sourcing materials. As a consequence, they have the same multiple properties and performances, although at different levels depending on the sourcing materials. Other BPs, obtained from composted green wastes or composted mixes of green wastes and MBW anaerobic digestates, have been investigated as auxiliaries in the anaerobic fermentation of MBW [37,38] carried out in bioreactors dedicated to the production of biogas. These biopolymers can reduce the ammonia and/or the nitrate level in the process digestate. Other workers have used BPs as animal diet supplements [39]. They have shown that BPs reduce the proteolysis occurring in the caecum intestine of pigs with consequent reduction of ammonia formations. 4.2. Plausible Explanation of the BP Effects Biagini and coworkers [40] tested the biopolymer obtained from composted mixes of green wastes and MBW anaerobic digestates as a supplement for rabbits’ protein diet. They demonstrated that the rabbits fed with the biopolymer-supplemented diet produced manure with signiﬁcantly lower ammonia and GHG emissions compared to the animals fed with the control diet. While the conﬁrmation and replicability of the effects of the above biopolymers under different operational conditions and of the BP used in the present work are unquestionable, the role of these biopolymers is not yet clear. So far, it cannot be established deﬁnitely whether the biopolymer effects involve pure chemical reactions or biochemical processes with participation of a microorganism. The second hypothesis is the most likely, according to Baglieri and coworkers [41]. These researchers cultivated bean plants using a biopolymer obtained from the hydrolysis of exhausted tomato plants as fertilizers. The biopolymer sourced from the agriculture biowastes bears strong chemical similarities with BP sourced from MBW. The former was found [41] to signiﬁcantly enhance nitrate reductase, glutamine synthetase, and glutamate synthase activities, and to increase soluble proteins’ concentra- tion in shoots and roots, compared to the control. Based on the lack of differences between the concentrations of mineral nitrogen in the control and treated cultivation substrate, as opposed to the signiﬁcant differences observed for enzymatic activity and soluble proteins’ concentration in the plants, Baglieri and coworkers [41] concluded that the biopolymer acts as plant biostimulant with a possible auxin-like effect, more than as soil fertilizer. On the other hand, a possible action of BP as chemical catalyst must be considered. This is also in view of previous work reporting the property of BPs to catalyze oxidation reactions in the absence of any microorganism [42]. According to the current view of the behavior of urea in soil and the fate of the produced ammonia [7,8,35] for the system investigated in the present work, the following reaction scheme may help clarify the BPs effects: CO(NH2)2 + H2O ⇆2 NH3 + CO2 (1) NH3 + 2 O2 ⇆HNO3 + H2O (2) R-CH(OH)-COOH + CH3-COOH + NH3 ⇆R-CH[NH-(C = O)-CH3]-COOH + 2 H2O (3) (2) (3) The scheme shows that urea is hydrolyzed to ammonia (reaction (1)) and then, trans- formed into nitrates (reaction (2) forward). These are adsorbed by the plant roots and transferred to the leaves. 4.2. Plausible Explanation of the BP Effects Nitrates in leaves are reduced to ammonia (reaction (2) backward) and then converted to proteins (reaction (3)). A recent work [38] investigated the BPs’ assisted anaerobic fermentation of MBW in 150 mL shake ﬂasks. It demonstrated that BPs, Coatings 2022, 12, 239 9 of 12 in the investigated MBW fermentation system comprising organic, nitrate and ammonia N, catalyze the following chemical redox reaction: in the investigated MBW fermentation system comprising organic, nitrate and ammonia N, catalyze the following chemical redox reaction: (4) HNO3 + NH3 ⇆N2 + 2 H2O + 1/2 O2 (4) HNO3 + NH3 ⇆N2 + 2 H2O + 1/2 O2 The calculated Gibbs free energy from literature data [43] for this reaction, i.e., −360 kJ/N2 mole, shows that reaction (4) is thermodynamically favored. Occurrence of the chemical catal- ysis by BP in the system investigated in the present work, and the consequent reduction of nitrates by reaction (4), may be a plausible explanation for the lower nitric to total N ratio reported in Table 4 for the composite materials containing BPs (trials 3, 4 and 7), compared to the other materials in trials 1, 2, and 5 that contain SPC and/or urea but no BP. 4.3. Perspectives for a New Biowaste-Based Chemical Industry The spinach case study reported in the present work shows that, for use in the agricul- ture sector, materials obtained from urban and agro-industrial biowastes are competitive with materials obtained from fossil sources. Particularly, the BP material obtained from MBW exhibits unique properties that allow modulating the N release rate and fate in soil and in the plant crop. These properties are very important to safeguard the quality of soil, water, and crops. More ambitiously, the present article has relevance also for the sectors of waste management, pollution, and the chemical industry. Montoneri [4] and Tabasso and coworkers [44] have reviewed the sustainability of the hydrolysis process to produce BPs, as well as the BPs’ multipurpose performance and related economic, environmental, and social beneﬁts for several sectors of the chemical industry and agriculture. The engineered composite materials tested in the present work disclose a further beneﬁt offered by the tested BP for developing safe agriculture and food. They prove a new property of BP capable of reducing fertilizer nitrate leaching through soil and eutrophication effects, and at the same time diminish nitrate accumulation in crops. They add further important incentives for valorizing MBW as feedstock for the production of BPs and their use at commercial scale. They also prospect the feasibility of substituting products from fossil sources with products from biowastes. Particularly, because of their origin and special properties, BPs have high potential for developing a sustainable, waste-based industry integrating chemical and biochemical processes. MBW treatment plants are the ideal settings to this end. At present, they are service providers for citizens, as they perform the collection, disposal, and recycling of urban biowastes. To reduce landﬁll disposal, the most advanced plants process MBW by anaer- obic fermentation yielding biogas and digestate, and by aerobic fermentation producing compost. The value of these products is not enough to cover the plant operational costs [4]. The missing revenue is covered by citizens’ taxes. No MBW plant applies chemical pro- cesses. Yet, MBWs are a potential source of valuable renewable organic C, which could be recycled in the form of valued-added, biobased products for consumer use. 6. Patent The French patent application submitted 16 October 2020 under number FR2010597 to Institut National de la Propriété Industrielle (INPI) in France and entitled “Produit pour l’agriculture, et procédé de préparation” results from both [5] and the work reported in the present manuscript. The inventors of this patent are Philippe Evon, Carlos Vaca-Garcia, Laurent Labonne, and Antoine Rouilly. The owners of this patent are Institut National Polytechnique de Toulouse (INPT) and Institut National pour l’Agriculture, l’Alimentation et l’Environnement (INRAE). Author Contributions: E.P., E.M., G.B., V.B., M.G., P.E., C.V.-G., G.F. and M.N. contributed equally to this research article. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported partly by endowed funds of the authors’ institutions, and partly funded by the European Commission in order to support the implementation of the actions pursued in the LIFE16 ENV/IT/000179-LIFECAB and the LIFE19 ENV/IT/000004-LIFEEBP projects. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data is contained within the article. Data Availability Statement: Data is contained within the article. 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. 5. Conclusions It has been demonstrated that composite controlled release fertilizers made by twin- screw extrusion followed by injection-molding can achieve the same performance in terms of spinach growth, nitrogen uptake, and nitrate accumulation as the commercial Osmocote® controlled release fertilizer, which contains urea coated with synthetic polymers. This result is achieved thanks to the municipal biowaste derived biopolymer. This component can control the nitrogen release rate of urea and from the sunﬂower protein concentrate, and reduce nitrogen accumulation by the plant while maintaining the same biomass growth and nitrogen uptake of Osmocote®. This ﬁnding poses a worthwhile scope for testing the twin-screw extruded/injection-molded biopolymer composites in the cultivation of other plant species in order to contribute to the replacement of current commercial materials coated with synthetic polymers from fossil sources. However, the ambition of the authors of the present work is far beyond developing controlled release fertilizers. Coatings 2022, 12, 239 10 of 12 10 of 12 Previous work [4] has demonstrated that the hydrolysis of municipal biowaste from different sources is a cost-effective, feasible process that yields a family of biopolymers for sustainable use in different sectors of agriculture and the chemical industry. The demonstration of the BP biopolymer properties in the present work is a new ﬁnding. It widens the ﬁelds of application and the potential beneﬁts of BPs. 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https://openalex.org/W4298113106	https://www.emerald.com/insight/content/doi/10.1108/MBE-01-2022-0015/full/pdf?title=facilitating-performance-measurement-and-management-through-digital-business-strategy	English	null	Facilitating performance measurement and management through digital business strategy	Measuring business excellence	2,022	cc-by	8,747	Mira Holopainen, Minna Saunila and Juhani Ukko Mira Holopainen, Minna Saunila and Juhani Ukko Mira Holopainen, Minna Saunila and Juhani Ukko Abstract Purpose – This study aims to focus on the connection between digital business strategy and performance measurement and management (PMM). Purpose – This study aims to focus on the connection between digital business strategy and performance measurement and management (PMM). Design/methodology/approach – The implications of digital business strategy and its dimensions with regard to PMM were investigated through a survey. The survey questionnaire provided 202 valid cases with a focus on senior management of small- and medium-sized enterprises. Strategic dimensions were identified from the literature on management in the context of digitalization to build a theoretical framework that highlights the mechanisms that companies should focus on when managing and implementing digital technologies successfully. Findings – The aspects that comprise digital business strategy are grouped into five major dimensions: technological understanding, goals, resources, management and responsibilities. The study reveals a direct and positive relationship between goals and management related to digital business strategy and PMM. Research limitations/implications – The study contributes to the existing PMM literature in the context of digitalization. Practical implications – The results indicate that if a company has excellent goals and management with regard to its digital business strategy, it uses PMM in a more successful and effective way. Originality/value – To the best of the authors’ knowledge, this study is among the first to examine PMM in terms of managing digital business strategy by trying to determine the extent to which the elements of digital business strategy can be integrated effectively into PMM. Keywords Performance management, Performance measurement, PMM, Digital, Business strategy Paper type Research paper © Mira Holopainen, Minna Saunila and Juhani Ukko. Published by Emerald Publishing Limited. This article is published under the Creative Commons Attribution (CC BY 4.0) licence. Anyone may reproduce, distribute, translate and create derivative works of this article (for both commercial and non-commercial purposes), subject to full attribution to the original publication and authors. The full terms of this licence may be seen at http://creativecommons. org/licences/by/4.0/legalcode Mira Holopainen, Minna Saunila and Juhani Ukko are all based at the Department of Industrial Engineering and Management, School of Engineering Science, LUT University, Lahti, Finland. Received 24 January 2022 Revised 22 June 2022 Accepted 10 September 2022 Ej VOL. 27 NO. 2 2023, pp. 246-260, Emerald Publishing Limited, ISSN 1368-3047 1. Introduction Performance measurement and management (PMM) has been impacted by ongoing digitalization and the changes it has forced in the management of companies’ operations (Nudurupati et al., 2020). In the context of digitalization, PMM can be viewed as a system of systems (Bourne et al., 2013; Sardi et al., 2020) that “need to interrelate complex subsystems, different in technology, context, operation, geography, and conceptual frame” (Sardi et al., 2020), thereby affecting the use of PMM in complex and turbulent environments (Bourne et al., 2013). Digitalization built on the utilization of digital technologies is consequently affecting PMM. Digital technologies can cause transformations resulting in improvements in companies’ offerings and customer satisfaction (Tortorella et al., 2020). Such major transformations require companies to adopt a strategy that connects their technologies and businesses (Bharadwaj et al., 2013). This type of digital business strategy and its management require companies to implement success metrics, that is, PMM (Bharadwaj et al., 2013; Nasiri et al., 2020). Such PMM is PAGE 246j MEASURING BUSINESS EXCELLENCEj VOL. 27 NO. 2 2023, pp. 246-260, Emerald Publishing Limited, ISSN 1368-3047 j VOL. 27 NO. 2 2023, pp. 246-260, Emerald Publishing Limited, ISSN 1368-3047 DOI 10.1108/MBE-01-2022-0015 challenged by the turbulence of the external environment (Nudurupati et al., 2016) and, therefore, requires the capability to gather information that is highly volatile and fast- changing. Despite the abundant literature on PMM practices, little research has been conducted on how the current highly volatile, uncertain and ambiguous digital environment has been affecting how organizations measure and manage performance (Nasiri et al., 2020; Nudurupati et al., 2020). Specifically, while digital transformation’s influence on companies’ strategies is widely recognized, its influence on PMM has elicited scant attention. Presumably, digital transformation and its alignment with a company’s strategic objectives exert a meaningful effect on PMM. The phenomenon – digitalization’s influence on PMM – is acknowledged in the literature, but little research has been conducted on the specific factors that influence PMM. If the company’s strategic goal is to digitalize its operations, for example by moving to e-commerce, they must find the most suitable possible solution in terms of costs, quality and goals. 1. Introduction It is assumed that the better a company manages the various aspects of a digital business strategy, such as understanding technology, the various costs of investment and maintenance and the goals set for it, the better they will be able to link digital investment goals to company-wide strategic goals and decision-making in their PMM. This in turn positively contributes to the efficient use of PMM. Thus, the aim of the study is to answer the question of whether digital business strategy positively influences PMM. The aspects that comprise digital business strategy are grouped into five major dimensions: technological understanding, goals, resources, management and responsibilities. The research is based on an analysis of survey results from 202 respondents. The research contributes to recent literature and practices as follows. First, it reveals the influence of the specific dimensions of digital business strategy on PMM. The results explain which digital business strategy dimensions are more effective in this sense. According to the results, goals and management related to digital business strategy should be considered with regard to supporting PMM in digital transformation. Second, this study offers a holistic framework for indicative research on digital business strategy’s influence on PMM. A better understanding of the dimensions of digital business strategy related to PMM will assist the development of a comprehensive picture of performance management in line with digital business strategy. The paper proceeds as follows. In the literature review, prior research on PMM in the context of digitalization is reviewed. Then, in Section 2, hypotheses are developed, and a research model is presented. After this, the methodology and results of the study are outlined in Sections 3 and 4, respectively. Finally, the research results are discussed in Section 5, and a conclusion is presented in Section 6. 2.1 Theoretical underpinnings 2.1.1 Performance measurement and management. Despite the abundant literature on PMM practices, little research has been conducted on how the current highly volatile, uncertain and ambiguous operating environment has been affecting how organizations measure and manage performance (Nudurupati et al., 2020). Similarly, limited effort has been made to construct a theoretical framework to understand the interplay between performance measurement and performance management, or their impact on employee engagement and performance (Bourne et al., 2013; Smith and Bititci, 2017). To understand the differences between the studied concepts, the key ideas must be defined. Ittner et al. (2003) touted a performance measurement system as a system that “provides financial and nonfinancial information that allows the firm to identify the strategies offering the highest potential for achieving the firm’s objectives, and aligns management processes, such as target setting, decision-making, and performance evaluation with the achievement of the VOL. 27 NO. 2 2023 j MEASURING BUSINESS EXCELLENCEj PAGE 247 VOL. 27 NO. 2 2023 chosen strategic objectives.” Hall (2008) defined a performance measurement system as one that “translates business strategies into deliverable results by combining financial, strategic, and operating business measures to gauge how well a company meets its targets.” From an organizational control perspective, PMM can be examined through technical control and social control mechanisms (Child, 1973; Cardinal et al., 2004; Smith and Bititci, 2017). As a technical control, performance measurement refers to a process (or processes) of setting goals, developing a set of performance measures and collecting, analyzing, reporting, interpreting, reviewing and acting on performance data (Neely et al., 1995; Smith and Bititci, 2017). Performance management, in turn, refers to social controls that are conceptualized as cultural and behavioral routines that define how we use a performance measurement system to manage an organization’s performance (Bititci, 2015; Smith and Bititci, 2017). Summing up, it is suggested that the research around PMM could focus more on the co-existence and interplay between the technical and social control aspects of PMM in changing business environments. PMM systems have also been demonstrated to be complex, adaptive systems that are evolving and constantly adjusting to transformations within an internal company environment (Okwir et al., 2018). Koufteros et al. (2014) suggested that in changing business environments, organizations can use these systems as orchestration mechanisms, not only to control and evaluate internal organizational behavior, but also to adapt to the needs of organizational renewal activities. 2.1 Theoretical underpinnings Digital technologies foster transformations that can result in major improvements in offerings and customer satisfaction (Tortorella et al., 2020), thereby requiring a strategy that reflects a fusion between technologies and the business (Bharadwaj et al., 2013). This type of strategy is called a digital business strategy. To manage such a strategy, companies need success metrics, that is, PMM (Bharadwaj et al., 2013; Nasiri et al., 2020). A challenge associated with this is coping with turbulence from the external environment (Nudurupati et al., 2016). Thus, PMM should possess the capability to gather information that is highly volatile and fast-changing. In response, contemporary research has defined PMM in the digital context as a system of systems (Bourne et al., 2013; Sardi et al., 2020). Consequently, it “need(s) to interrelate complex subsystems, different in technology, context, operation, geography, and conceptual frame” (Sardi et al., 2020), thereby facilitating the use of PMM in complex and turbulent environments (Bourne et al., 2013). According to this perspective, novel directions for the utilization of PMM to foster digital business strategy are evident. These are summarized in Table 2 and described next. Contemporary PMM puts greater emphasis on evaluation, rather than on traditional measurement (Nudurupati et al., 2016; Ukko et al., 2020). This requires deep understanding about the entire process directing digital business strategy. Ravelomanantsoa et al. (2019) suggested that prior technological changes have led to the view that a company is not understood as one company, but rather as a network of companies with interconnected performances. According to them, this view elicits major changes in how PMM is constructed. Further, PMM success strongly depends on different technological capabilities, as they facilitate process transformations and promote decision-making (Vallurupalli and Bose, 2018). The importance of goal alignment in PMM has been highlighted in prior studies (Reinking et al., 2020; Sardi et al., 2020). Nudurupati et al. (2016) concluded that companies should integrate technological developments into their strategic and performance expectations. Further, PMM is a powerful tool that can translate strategy to the lower levels of a company and assess performance against strategy. Strategic alignment is also critical to the utilization of specific PMM tools, such as dashboards. Reinking et al. (2020) studied dashboards as a tool to align company performance with strategy and found that the strategic alignment of digital dashboards enhances performance (Reinking et al., 2020). In addition to individual PMM tools, Sardi et al. 2.1 Theoretical underpinnings For example, companies experience internal and external pressures to implement digital transformation, which forces them to retool their functions and operations. Renewal activities are strongly related to PMM’s social control aspect with regard to, for example, people’s behavior and organizational capabilities. Franco-Santos et al. (2012) noted that people’s behavior refers to consequences related to employees’ actions or reactions (e.g. motivation, participation) and their underlying cognitive mechanisms (e.g. perceptions). They also suggested that organizational capabilities refer to consequences associated with specific processes, activities, or competencies that enable an organization to perform and gain competitive advantages (e.g. strategic alignment, organizational learning). Nudurupati et al. (2020) also highlighted the use of more subjective, perception-based evaluation measures, in which both the multi- tier supply chain and customers are asked to evaluate an organization, often using social media and other information technology-enabled platforms. Similarly, Nasiri et al.’s (2020) findings showed that when companies digitize their operations and functions, social controls (digital-related human and collaboration capabilities) positively affect technical controls (i.e. performance measurement systems) and, further, financial performance. This indicates that changes in people’s behavior and organizational capabilities (social controls) influence PMM (technical controls) and vice versa. The discussion above indicates the need for parallel assessments in terms of both the social and technical control mechanisms of PMM, as well as the need to consider in which case (e.g. social controls) the notion of performance evaluation, rather than measurement, would be a viable alternative (cf. Bititci et al., 2012). Therefore, PMM is, all in all, an essential means of handling digital transformation. 2.1.2 Performance management and digital business strategy. To survive the intensifying competition, companies need a digital business strategy that reflects a fusion between technologies and business (Bharadwaj et al., 2013). Bharadwaj et al. (2013) defined digital business strategy as “organizational strategy formulated and executed by leveraging digital resources to create differential value”. Mithas et al. (2013) concluded that “digital business strategy is not solely a matter of optimizing firm operations internally or of responding to one or two focal competitors, but also arises strikingly from awareness and responsiveness to the digital business competitive environment”. The successful implementation of such a digital business strategy certainly causes some changes to PMM as well (Bharadwaj et al., 2013; Nasiri et al., 2020). PAGE 248j MEASURING BUSINESS EXCELLENCEj VOL. 27 NO. 2 2023 Comprehensive PMM facilitates the interplay between company operations and strategy (Chenhall, 2005). 2.1 Theoretical underpinnings (2020) highlighted the importance of defining and managing the entire PMM process in relation to utilizing digital technologies and resources in line with a company’s strategic goals. In digital transformation, PMM requires continuous updates, as the tools implemented need to keep up with changes in the external environment (Kamble et al., 2020; Chanias et al., 2019). Targeting different types of resources is also indicated as being crucial in PMM. Nudurupati et al. (2016) stated that PMM, in the digital era, is characterized by a balance between traditional measures, with an emphasis on financial aspects and the evaluation of behavioral metrics. Kamble et al. (2020) reported similar findings in their study of smart manufacturing systems’ PMM. They concluded that PMM should incorporate effects beyond traditional performance dimensions, such as cost, quality and productivity. Thus, in addition to financial aspects, human resources should be targeted to facilitate digital business strategy. Further, Ukko et al. (2020) studied PMM in the context of digital services and highlighted the role of relational mechanisms in such a system. They stated that behavioral factors, along with technical ones, should be considered in contemporary PMM. Thus, it should not focus merely on design and data analysis, but also engage humans in the process (Ukko et al., 2020). This makes human resources crucial. Digital transformation has elicited a greater emphasis on management facilitating PMM processes (Kamble et al., 2020; Nasiri et al., 2020). According to Nudurupati et al. (2016), VOL. 27 NO. 2 2023 j MEASURING BUSINESS EXCELLENCEj PAGE 249 VOL. 27 NO. 2 2023 j MEASURING BUSINESS EXCELLENCEj PAGE 24 VOL. 27 NO. 2 2023 contemporary PMM should aim to enhance and support different skills and capabilities, as it is the way forward for affecting behavior and further driving cultural change. Similarly, Seele (2016) stated that the implementation of digitally unified measurement changes company processes and routines. Further, different types of management capabilities are required to manage these changes. Nasiri et al. (2020) found that companies can improve their financial performance with the assistance of PMM-facilitated human and collaboration capabilities. PMM is also an important tool for facilitating decision-making. Sahlin and Angelis (2019) studied the opportunities for the digitalization of real-time PMM and found that it can serve as a useful tool for decision-making in turbulent environments, as digitalization has facilitated much faster data access for managers. Finally, balancing and defining different responsibilities is crucial in PMM. 2.1 Theoretical underpinnings Ukko et al. (2020) demonstrated that inter-organizational trust and know-how development via knowledge sharing act as relational mechanisms that facilitate the relationships governed by social interactions that drive PMM. Their study, within the digital service context, classified the criticality and personalization of services to guide exploited relational mechanisms (Ukko et al., 2020). Engaging with different parties via performance-based collaboration also facilitates innovation (Nudurupati et al., 2016). Other benefits from involving actors in PMM include enhanced trust and a willingness to share information and knowledge (Ukko et al., 2020). The above evidence indicates that PMM is influenced by different perspectives of digital business strategy. Digital business strategy is an essential motivator of actions and also acts as a determinant for firms to transform their processes and routines to manage performance. Next, hypotheses are developed by examining how PMM is driven by digital business strategy. H1. Digital business strategy positively influences PMM. Technological understanding in relation to digital business strategy describes whether a firm strives to gain a competitive edge through digital technology by recognizing the importance of such technology and by implementing projects based on it. McAdam et al. (2017) stated that transformations in technology challenge the connection between operations and strategy. They continued by recognizing that avoiding such misalignment requires developing dynamic capabilities built on PMM. These capabilities assist in balancing business strategy, technological strategy and technological practices (McAdam et al., 2017). This is likely to transform PMM as well. Nudurupati et al. (2016) suggested that adopting PMM in digital economies requires firms to understand the potential of technologies in terms of creating competitive advantage via strategy. Technological developments lead to greater amounts of data, and therefore, the adoption of new easy-to- use methods facilitates decision-makers (Nudurupati et al., 2016), which affects PMM. Accordingly, we hypothesize the following: H1a. Technological understanding related to digital business strategy positively influences PMM. Goals related to digital business strategy refer to the objectives pursued by the new strategic technologies. The importance of goal alignment that also includes the ambitions of the new PMM technologies has been highlighted in prior studies (Reinking et al., 2020; Sardi et al., 2020). Nudurupati et al. (2016) emphasized how companies need to integrate technological development expectations into their strategic and performance goals. Kamble et al. (2020) studied the interplay between PMM and smart manufacturing systems and found that aligning manufacturing goals with performance targets guides the adoption of new technologies. Sardi et al. (2020), in turn, emphasized the importance of defining and managing the entire PMM process for utilizing digital technologies and resources in line with a company’s strategic goals. Thus, in line with the considerations above, the following hypothesis is proposed: H1b. Goals related to digital business strategy positively influence PMM. H1b. Goals related to digital business strategy positively influence PMM With regard to digital business strategy, resources refer to financial resources as well as the time and skills that employees need to implement digital technologies in line with the objectives. Nasiri et al. (2020) showed that human capabilities significantly contribute to PMM. Also, Nudurupati et al. (2016) found that the workforce is crucial in the digital era in terms of managing recurring inter-organizational business turbulence and related PMM transformations. Similarly, Beer and Micheli (2018) recognized that PMM should cover both the objects (e.g. 2.2 Hypothesis development The objective of this study was to examine the effect of digital business strategy on PMM. A digital business strategy is a multidimensional entity that requires the consideration of various factors. In our study, digital business strategy consists of the subdimensions of technological understanding, goals, resources, management and responsibilities, which are identified from previous literature on management in the context of digitalization. Figure 1 shows the conceptual model and hypotheses guiding our research. The model suggests that digital business strategy offers the potential to influence PMM. Thus, in the digital business environment, a firm lacking strong emphasis on the five dimensions of digital business strategy is not necessarily capable of improving its PMM. In addition, control Figure 1 Research model Digital business strategy Goals Resources Understanding PMM Industry H1a-e Control variable Management Responsibilities Customers Control variable PAGE 250j MEASURING BUSINESS EXCELLENCEj VOL. 27 NO. 2 2023 Figure 1 Research model Digital business strategy Goals Resources Understanding PMM Industry H1a-e Control variable Management Responsibilities Customers Control variable Figure 1 Research model Digital business strategy Goals Resources Understanding PMM Industry H1a-e Control variable Management Responsibilities Customers Control variable PAGE 250j MEASURING BUSINESS EXCELLENCEj VOL. 27 NO. 2 2023 variables are added to the research model. We propose the following hypothesis and five sub-hypotheses that are explained in more detail in the following section: H1. Digital business strategy positively influences PMM. H1. Digital business strategy positively influences PMM. 3.1 Sample and data collection This research focuses on the connection between digital business strategy and PMM. The implications of strategic dimensions for PMM were investigated through a survey. The survey was conducted in September–October 2021. The questionnaire was sent to 5,665 companies by e-mail and was answered by 205 representatives. The responses were received in four waves. One week after the first mailing of the link, reminder messages were sent. The last two reminders were circulated a week after the previous reminder. Of the responses, 202 were considered to be valid cases, which provided a sufficient sample size. The research was limited to Finland with a focus on small and medium-sized enterprises. In total, 95.6% of the respondents were senior management, and the rest were middle management or lower-level employees. After the data collection, statistical analyses were conducted using SPSS and Excel software to test the hypotheses. H1. Digital business strategy positively influences PMM. cost or quality) being measured and the mechanisms that are created and used by the subjects involved in the process. Based on these, the following hypothesis is proposed: H1c. Resources related to digital business strategy positively influence PMM. The management of digital business strategy is considered as crucial and refers to managers’ strategic capabilities, decision-making capabilities and willingness to implement changes, in addition to their experience and skills developed through technology projects (Matt et al., 2015). Managerial capabilities have been highlighted as one of the main dimensions of a digital business strategy, along with launching new technologies (El Sawy et al., 2016; Li et al., 2018). Managerial capabilities also benefit PMM (Horvath and Szabo, 2019; Frederico et al., 2020). For example, Nasiri et al. (2020) showed how personal capabilities, such as adaptable mind-sets and skill sets, together with digital know-how (El Sawy et al., 2016), have been linked to the better utilization of PMM. This new way of using PMM enables diagnoses of digitalized processes with real-time information and reveals VOL. 27 NO. 2 2023 j MEASURING BUSINESS EXCELLENCEj PAGE 251 VOL. 27 NO. 2 2023 opportunities for improving financial performance. Horvath and Szabo (2019) suggested that an increased managerial emphasis on PMM enables increased control and permits real-time performance measurement, which in turn, promote the adoption of new technologies. Based on the notions above, managerial capabilities related to digital business strategy can influence both the adaption of new technologies and PMM by changing the culture and enabling real-time performance measurement. Thus, the following hypothesis is put forward: H1d. Management-related aspects of digital business strategy positively influence PMM. Responsibilities related to digital business strategy include the implementation of digital strategy functions and involve having sufficient experience in managing digital technology projects. These may require the development of joint platforms to share pertinent resources and skills to encourage shared continuous improvement activity via PMM (Nudurupati et al., 2016). The way responsibilities and roles are divided between individuals and groups has a significant impact on how a company operates (Tardieu et al., 2020). As distributing responsibilities in a meaningful way affects company performance, it will require changes in PMM as well. Thus, the following hypothesis is proposed: H1e. Responsibilities related to digital business strategy positively influence PMM. 3.2 Survey measures and their validity and reliability (2019) Interconnected performances Technological understanding Source of higher performance Goals Nudurupati et al. (2016), Chanias et al. (2019), Kamble et al. (2020), Reinking et al. (2020), Sardi et al. (2020) Technological developments aligned with organizational strategic objectives Deploying strategy to lower levels Continuous review of performance measures Resources Nudurupati et al. (2016), Kamble et al. (2020), Ukko et al. (2020) Effects beyond traditional performance dimensions (cost, quality, productivity, etc.) Behavioral factors concurrent with technical ones Human involvement Management Nudurupati et al. (2016), Seele (2016), Sahlin and Angelis (2019), Kamble et al. (2020), Nasiri et al. (2020) Promoting behavior and collaboration Driving cultural change Transforming processes and routines Potential for decision-making Responsibilities Nudurupati et al. (2016), Ukko et al. (2020) Need for personalization Subjects’ engagement Trust between partners Information sharing financial resources, human resources and employee skills related to the implementation of digital projects. The management scale included three items that assessed management’s awareness of the digital technologies, its strategic capability in relation to the digital technologies and its ability to seek new opportunities through digital technologies. Finally, the responsibility scale was formed by two items that investigated the appointment and experience of the person in charge of managing digital transformation projects. Every item was assessed with a seven-tier Likert scale (with the responses varying from totally disagree to totally agree). Control variables were used in the models to examine the effect of the variables on the results. The control variables included the customer group and industry of the respondent’s company. The customer variable included two items: business-to-business and business-to-customer. The industry variable was composed of the following items: manufacturing and service. Respondents chose one of the alternatives for each of the control variables. The survey dimensions and item reliability and validity were tested at various stages and through different analyses. First, we tested that all the constructs were unidimensional and that all the item loadings were above the minimum limit using factor analysis (Hair et al., 2014). The loadings for all the items exceeded the required limit (> 0.50). In addition, the composite reliability (CR) and average variance extracted (AVE) were calculated to evaluate the validity and reliability of the construct of the model. All the AVE values were greater than the threshold (0.50), and the CR values were higher than 0.70, confirming the validity. Finally, the reliability of the variables was analyzed by calculating Cronbach’s alphas. 3.2 Survey measures and their validity and reliability All the study scales were developed based on previous studies. The dependent variable was PMM. A scale was informed by previous research to reflect the impact of PMM on company operations (Child, 1973; Cardinal et al., 2004; Smith and Bititci, 2017; Neely et al., 1995; Bititci, 2015). It included a total of seven items, each of which was assessed with a seven-tier Likert scale (with the responses varying from totally disagree to totally agree) to find out how companies use PMM to support their businesses and operations (Chenhall, 2005). The seven items that constituted the PMM scale are presented in Table 2. Digital business strategy, as the independent variable, was divided into five different dimensions: technological understanding, goals, resources, management and responsibilities (Table 1). All five dimensions consisted of two or three items based on recent studies (see Section 2.1.2). Technological understanding was composed of three items: the technological development of the industry, the existence of digital projects and the achievement of a competitive advantage through digital technology. The goals dimension included a company’s ability to define goals related to the use of digital technologies, to monitor the achievement of such goals and to use digital technology to achieve the defined goals. The items in the resource dimension focused on dealing with PAGE 252j MEASURING BUSINESS EXCELLENCEj VOL. 27 NO. 2 2023 Table 1 Summary of contemporary perspectives on PMM related to digital business strategy Perspective References Features Understanding Vallurupalli and Bose (2018), Ravelomanantsoa et al. (2019) Interconnected performances Technological understanding Source of higher performance Goals Nudurupati et al. (2016), Chanias et al. (2019), Kamble et al. (2020), Reinking et al. (2020), Sardi et al. (2020) Technological developments aligned with organizational strategic objectives Deploying strategy to lower levels Continuous review of performance measures Resources Nudurupati et al. (2016), Kamble et al. (2020), Ukko et al. (2020) Effects beyond traditional performance dimensions (cost, quality, productivity, etc.) Behavioral factors concurrent with technical ones Human involvement Management Nudurupati et al. (2016), Seele (2016), Sahlin and Angelis (2019), Kamble et al. (2020), Nasiri et al. (2020) Promoting behavior and collaboration Driving cultural change Transforming processes and routines Potential for decision-making Responsibilities Nudurupati et al. (2016), Ukko et al. (2020) Need for personalization Subjects’ engagement Trust between partners Information sharing Table 1 Summary of contemporary perspectives on PMM related to digital business strategy Perspective References Features Understanding Vallurupalli and Bose (2018), Ravelomanantsoa et al. 3.2 Survey measures and their validity and reliability All the six dimensions had Cronbach’s alpha values higher than the proposed limit (> 0.60). Thus, it can be stated that the construct of the model is reliable (De Vellis, 1991). Thus, the reliability and validity of the model construct are ensured. VOL. 27 NO. 2 2023 j MEASURING BUSINESS EXCELLENCEj PAGE 253 VOL. 27 NO. 2 2023 Table 2 Survey dimensions and items and their validity and reliability Dimension Format of the items Items Loadings CR AVE a Understanding The company considers and understands the impact of digital technology on the following (Totally disagree (1) – Totally agree (7)) 1 Industry development 0.882 0.92 0.79 0.86 2 Digital projects 0.867 3 Competitive advantage 0.923 Goals The company has set goals related to the utilization of digital technology (Totally disagree (1) – Totally agree (7)) 1 Setting goals 0.934 0.94 0.84 0.91 2 Monitoring goals 0.921 3 Reaching goals 0.901 Resources The company’s resources support digital projects (Totally disagree (1) – Totally agree (7)) 1 Financial resources 0.880 0.88 0.70 0.79 2 Human resources 0.873 3 Employee skills 0.757 Management Management-related aspects related to digital technology (Totally disagree (1) – Totally agree (7)) 1 Awareness 0.936 0.92 0.78 0.85 2 Capability 0.882 3 Activity 0.837 Responsibilities Ability of person in charge of digital projects (Totally disagree (1) – Totally agree (7)) 1 Appointment 0.903 0.90 0.82 0.77 2 Experience 0.903 PMM The company utilizes PMM as follows (Totally disagree (1) – Totally agree (7)) 1 To support management 0.919 0.95 0.75 0.94 2 To provide business information 0.892 3 To link measures to strategic objectives 0.887 4 To report regularly on measurement information 0.857 5 To support strategic decisions 0.839 6 To develop ideas etc. 0.834 7 To impact the activities of the staff 0.824 PAGE 254j MEASURING BUSINESS EXCELLENCEj VOL. 27 NO. 2 2023 3.3 Bias To address the problem of non-response bias, an analysis of variance (ANOVA) test was performed. The respondents were distributed into two groups: first respondents and late respondents. The results revealed that there was no significant difference (at the level of p 0.05) between the two groups. Therefore, non-response bias does not have an effect. Other sampling biases were additionally assessed to make sure that the sample was representative. The sample was randomly selected to decrease the potential of voluntary response bias. Random sampling is likely to ensure that sufficient numbers of distinct types of small- and medium-sized companies (SMEs) were included in the sample. Also, this procedure was adopted to make sure that only one response was received from each firm and respondent. As proposed by Podsakoff et al. (2003), several procedural remedies were utilized to decrease the influence of common method bias. The execution of the survey allowed anonymous responses, and the cover letter urged the respondents to reply to the PAGE 254j MEASURING BUSINESS EXCELLENCEj VOL. 27 NO. 2 2023 questions as truthfully as possible. This is likely to decrease social desirability bias. Common method bias can also be minimized by the careful construction of the questions (taking into account the question wording and ease of understanding). The questions were reviewed by researchers familiar with the theme. Also, statistical remedies were performed to minimize the potential common method bias (Harman’s one-factor test). All the questions utilized in the analyses were loaded into the exploratory factor analysis. The unrotated solutions factor solution did not indicate the presence of one factor or that the first factor corresponded to most of the variance (Podsakoff et al., 2003). Thus, common method variance is not a problem in this case. 4. Results The objective of this study was to examine the effect of digital business strategy on PMM. We examined digital business strategy through five dimensions: technological understanding, goals, resources, management and responsibilities. Table 3 shows the means, standard deviations and correlations of the construct. The regression results of the relationships are shown in Table 4. The research model is significant at the p < 0.01 level (R2 = 0.27). In the model, the goals dimension is significant at the p < 0.05 level, and the management dimension is significant at the p < 0.01 level, and thus, they positively influence PMM. Technological understanding, resources and responsibilities are insignificant. Thus, the results provide support for H1b and H1d, but not for H1a, H1c and H1e. The results of the study reveal that if a company has excellent goals and management with regard to digital business strategy, it uses PMM in a more successful and effective way. A company’s main industry and customer group, which acted as the control variables, did not influence the implementation of PMM. influence the implementation of PMM. Table 3 Correlation matrix Dimension Mean SD 1 2 3 4 5 6 1 Understanding 5.84 1.25 1 2 Goals 5.18 1.45 0.808 1 3 Resources 4.44 1.32 0.488 0.656 1 4 Management 5.11 1.31 0.703 0.761 0.629 1 5 Responsibilities 4.29 1.77 0.481 0.586 0.579 0.654 1 6 PMM 5.08 1.41 0.418 0.467 0.273 0.582 0.272 1 Note: Correlation is significant at the 0.01 level (two-tailed) Table 4 Regression results Model b SE St. b R2 Adj. R2 SE F Controls Customer group 0.217 0.186 0.078 Industry –0.220 0.262 –0.055 Main effects Understanding 0.032 0.125 0.028 Goals 0.294 0.125 0.300 Resources –0.118 0.094 –0.111 Management 0.406 0.118 0.375 Responsibilities –0.066 0.069 –0.083 Summary (Constant) 2.044 0.687 0.272 0.245 1.217 10.055 Notes: p 0.001, 0.001 < p 0.01, 0.01 < p 0.05 VOL. 27 NO. 2 2023 j MEASURING BUSINESS EXCELLENCEj PAGE 255 Table 3 Correlation matrix Dimension Mean SD 1 2 3 4 5 6 1 Understanding 5.84 1.25 1 2 Goals 5.18 1.45 0.808 1 3 Resources 4.44 1.32 0.488 0.656 1 4 Management 5.11 1.31 0.703 0.761 0.629 1 5 Responsibilities 4.29 1.77 0.481 0.586 0.579 0.654 1 6 PMM 5.08 1.41 0.418 0.467 0.273 0.582 0.272 1 Note: Correlation is significant at the 0.01 level (two-tailed) Table 4 Regression results Model b SE St. 5. Discussion The objective of this study was to examine the effect of digital business strategy on PMM. Digital transformation has been found to be effective in terms of transforming PMM across industries (Nudurupati et al., 2016; Ravelomanantsoa et al., 2019; Kamble et al., 2020; Nasiri et al., 2020; Sardi et al., 2020). Digital business strategy, in particular, is influencing PMM as it is the key to managing such transformations connecting technology and business (Bharadwaj et al., 2013; Nasiri et al., 2020). This study contributes to the research in the intersection of PMM and digital transformation by demonstrating that several dimensions of digital business strategy are important factors that influence PMM. The results indicate that if a company has excellent goals and management related to digital business strategy, it uses PMM in a more successful and effective way. On the other hand, understanding, resources and responsibilities related to digital business strategy do not directly influence PMM. The main findings are discussed next. First, the study shows that management-related aspects of digital business strategy positively influence PMM. This supports the view that the ability of companies to adopt new digital technologies has become increasingly important for competitiveness, but it is not an easy task for management due to their inherent complexity and uncertainty. The results of this study also support the idea that companies should understand how digital technology tied to a strategy can create a competitive advantage and should exploit it using relevant behavioral measures (Nudurupati et al., 2016). This requires new capabilities from management, as it should be aware of new technological opportunities, possess the necessary strategic and decision-making capabilities and actively lead technological change projects (Matt et al., 2015; Chanias et al., 2019). The study reveals that, when business leaders possess these leadership skills, they are positively reflected in PMM. According to the results, the management of digital business strategy has the greatest impact on PMM during digital transformation. This is in line with previous studies, for example, Nasiri et al. (2020), who showed that personal capabilities, such as adaptable mind-sets and skill sets together with digital know-how, have been linked to the better utilization of PMM. 4. Results b R2 Adj. R2 SE F Controls Customer group 0.217 0.186 0.078 Industry –0.220 0.262 –0.055 Main effects Understanding 0.032 0.125 0.028 Goals 0.294 0.125 0.300 Resources –0.118 0.094 –0.111 Management 0.406 0.118 0.375 Responsibilities –0.066 0.069 –0.083 Summary (Constant) 2.044 0.687 0.272 0.245 1.217 10.055 Notes: p 0.001, 0.001 < p 0.01, 0.01 < p 0.05 6.1 Theoretical contributions The study contributes to the existing PMM literature in the context of digitalization by examining the connection between digital business strategy and PMM. It is among the first studies to examine PMM in terms of managing digital business strategy by trying to determine the extent to which the elements of digital business strategy can be integrated effectively into PMM. Specifically, the study contributes to recent literature as follow. First, it shows the influence of the specific dimensions of digital business strategy on PMM. The results explain which digital business strategy dimensions are more effective at influencing PMM. According to the results, goals and management related to digital business strategy directly influence PMM in digital transformation. Understanding, resources and responsibilities related to digital business strategy do not directly shape PMM in digital transformation. Second, this study offers a holistic framework for indicative research on digital business strategy’s influence on PMM. A better understanding of the dimensions of digital business strategy related to PMM will assist the development of a more comprehensive picture of performance management in line with digital business strategy. 5. Discussion In addition, Horvath and Szabo (2019) suggested that one of the driving forces for adopting digital technologies was increased managerial emphasis on PMM that enables increased control and permits real-time performance measurement, which in turn, promote the adoption of new technologies and improve decision-making and performance appraisal. Also, Sahlin and Angelis (2019) found that PMM can serve as a useful tool for management in turbulent environments, as digitalization has facilitated much faster data access to support decision-making. Thus, the higher the level of management capabilities related to digital business strategy, the more effectively a company can leverage PMM to support its digital transformation. Second, the study reveals that goals related to digital business strategy positively influence PMM. Thus, the study supports the notion that digital technology alone does not add much value to a company’s operations without a connection to the company’s strategic visions and goals. Alongside management, the importance of goal alignment in PMM has been highlighted in prior studies as well (Reinking et al., 2020; Sardi et al., 2020). Nudurupati et al. (2016) concluded that companies should integrate technological developments into their strategic and performance expectations. Sardi et al. (2020) highlighted the importance of defining and managing the entire PMM process for using digital technologies and resources in line with a company’s strategic goals. In digital transformation, it is important to set goals and update them as and when required, which means that PMM will need continuous updates, as it has to keep up with changes in the external environment (Kamble et al., 2020; Chanias et al., 2019). Our research is in line with these studies, as it shows that if a company sets goals for the utilization of digital technology, monitors the achievement of these goals and strives to achieve them, it can use PMM more effectively to support digital transformation. PAGE 256j MEASURING BUSINESS EXCELLENCEj VOL. 27 NO. 2 2023 Finally, the results indicated no direct effect of technological understanding, resources and responsibilities with regard to digital business strategy on the way companies use PMM. This is somewhat inconsistent with previous studies, which suggest that adopting PMM in digital transformation requires understanding the potential of technologies, as well as the creation of joint platforms to share pertinent resources and skills to create competitive advantage via digital business strategy (Nudurupati et al., 2016; Tardieu et al., 2020). 5. Discussion Because the impact of all the five dimensions of digital business strategy was studied in the same model, it may be that the management and goals dimensions diluted the impact of the other three. It may also be that the dimensions of technological understanding, resources and responsibilities do not have an impact without an adequate level of the management and goals dimensions. In the future, it will be interesting to explore in more detail the technological understanding, resources and responsibilities dimensions and their relation to PMM. References Beer, H.A. and Micheli, P. 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(2016), “How LEGO built the foundations and enterprise capabilities for digital leadership”, MIS Quarterly Executive, Vol. 15 No. 2, pp. 141-166. Franco-Santos, M., Lucianetti, L. and Bourne, M. (2012), “Contemporary performance measurement systems: a review of their consequences and a framework for research”, Management Accounting Research, Vol. 23 No. 2, pp. 79-119. Frederico, G.F., Garza-Reyes, J.A., Kumar, A. and Kumar, V. (2020), “Performance measurement for supply chains in the industry 4.0 era: a balanced scorecard approach”, International Journal of Productivity and Performance Management, Vol. 70 No. 4, pp. 789-807. Hair, J.F., Black, W.C., Babin, B.J. and Anderson, R.E. (2014), Multivariate Data Analysis, 7th ed., Pearson New International ed., Pearson Education, London. Hall, M. 6.2 Practical contributions Digital transformation has been found to be effective in terms of transforming PMM across industries. Digital business strategy, in particular, is influencing PMM, as it is the key to managing such transformations connecting technology and business. First, this study contributes to practice by showing that management-related aspects of digital business strategy positively influence PMM. This supports the view that the ability of companies to adopt new digital technologies has become increasingly important for competitiveness, but it is not an easy task for management due to their inherent complexity and uncertainty. Thus, the study contributes to practice by highlighting the role of managers in digital transformation. The ability of management to adapt and understand new technologies and integrate them with objectives, performance metrics and decision-making is critical to digital business strategy to be effectively integrated into PMM. Second, the study shows that if a company sets goals for the utilization of digital technology, monitors the achievement of these goals and strives to achieve them, it can use PMM more effectively to support digital transformation. Setting goals, in forming a digital business strategy, requires the company to be able to assess the significance of technology and digital transformation for its operations. Thus, to succeed in digital transformation, companies should integrate technological developments into their strategic and performance expectations, allowing PMM to serve as an effective tool to translate digital strategy to the lower levels of a company and evaluate performance against strategy. MEASURING BUSINESS EXCELLENCEj PAGE 2 VOL. 27 NO. 2 2023 6.3 Research limitations This study’s limitations are as follows. First, this study adopted a cross-sectional design. Future studies could use a longitudinal design to gain an in-depth assessment of the interconnections of the study variables over time. Second, the sample covers SMEs in one geographical district. Future research could collect data from specific industries, larger companies, or other geographical districts. This study did not target a specific type of PMM and instead looked at PMM in general. A more specific approach could be used in future studies to examine the types of PMM used. Different dimensions of digital business strategy may drive different types of PMM. References (2008), “The effect of comprehensive performance measurement systems on role clarity, psychological empowerment and managerial performance”, Accounting, Organizations and Society, Vol. 33 Nos 2/3, pp. 141-163. Horvath, D. and Szabo, R.Z. (2019), “Driving forces and barriers of industry 4.0: do multinational and small and medium-sized companies have equal opportunities?”, Technological Forecasting and Social Change, Vol. 146, pp. 119-132. PAGE 258j MEASURING BUSINESS EXCELLENCEj VOL. 27 NO. 2 2023 VOL. 27 NO. 2 2023 Ittner, C.D., Larcker, D.F. and Randall, T. (2003), “Performance implications of strategic performance measurement in financial services firms”, Accounting, Organizations and Society, Vol. 28 Nos 7/8, pp. 715-741. Kamble, S.S., Gunasekaran, A., Ghadge, A. and Raut, R. (2020), “A performance measurement system for industry 4.0 enabled smart manufacturing system in SMMEs-a review and empirical investigation”, International Journal of Production Economics, Vol. 229, p. 107853. Koufteros, X., Verghese, A.J. and Lucianetti, L. (2014), “The effect of performance measurement systems on firm performance: a cross-sectional and a longitudinal study”, Journal of Operations Management, Vol. 32 No. 6, pp. 313-336. Li, L., Su, F., Zhang, W. and Mao, J.Y. (2018), “Digital transformation by SME entrepreneurs: a capability perspective”, Information Systems Journal, Vol. 28 No. 6, pp. 1129-1157. McAdam, R., Bititci, U. and Galbraith, B. (2017), “Technology alignment and business strategy: a performance measurement and dynamic capability perspective”, International Journal of Production Research, Vol. 55 No. 23, pp. 7168-7186. Matt, C., Hess, T. and Benlian, A. (2015), “Digital transformation strategies”, Business & Information Systems Engineering, Vol. 57 No. 5, pp. 339-343. Mithas, S., Tafti, A. and Mitchell, W. (2013), “How a firm’s competitive environment and digital strategic posture influence digital business strategy”, MIS Quarterly, Vol. 37 No. 2, pp. 511-536. Nasiri, M., Ukko, J., Saunila, M., Rantala, T. and Rantanen, H. (2020), “Digital-related capabilities and financial performance: the mediating effect of performance measurement systems”, Technology Analysis & Strategic Management, Vol. 32 No. 12, pp. 1393-1406. Neely, A., Gregory, M. and Platts, K. (1995), “Performance measurement system design: a literature review and research agenda”, International Journal of Operations & Production Management, Vol. 15 No. 4, pp. 80-116. Nudurupati, S.S., Garengo, P. and Bititci, U.S. (2020), “Impact of the changing business environment on performance measurement and management practices”, International Journal of Production Economics, Vol. 232, p. 107942. Nudurupati, S.S., Tebboune, S. and Hardman, J. (2016), “Contemporary performance measurement and management (PMM) in digital economies”, Production Planning & Control, Vol. 27 No. 3, pp. References 31 Nos 2/3, pp. 233-244. Vallurupalli, V. and Bose, I. (2018), “Business intelligence for performance measurement: a case based analysis”, Decision Support Systems, Vol. 111, pp. 72-85. References 226-235. Okwir, S., Nudurupati, S.S., Ginieis, M. and Angelis, J. (2018), “Performance measurement and management systems: a perspective from complexity theory”, International Journal of Management Reviews, Vol. 20 No. 3, pp. 731-754. Podsakoff, P.M., MacKenzie, S.B., Lee, J.Y. and Podsakoff, N.P. (2003), “Common method biases in behavioral research: a critical review of the literature and recommended remedies”, Journal of Applied Psychology, Vol. 88 No. 5, p. 879. Ravelomanantsoa, M.S., Ducq, Y. and Vallespir, B. (2019), “A state of the art and comparison of approaches for performance measurement systems definition and design”, International Journal of Production Research, Vol. 57 Nos 15/16, pp. 5026-5046. Reinking, J., Arnold, V. and Sutton, S.G. (2020), “Synthesizing enterprise data to strategically align performance: the intentionality of strategy surrogation”, International Journal of Accounting Information Systems, Vol. 36, p. 100444. Sahlin, J. and Angelis, J. (2019), “Performance management systems: reviewing the rise of dynamics and digitalization”, Cogent Business & Management, Vol. 6 No. 1, p. 1642293. Sardi, A., Sorano, E., Cantino, V. and Garengo, P. (2020), “Big data and performance measurement research: trends, evolution and future opportunities”, Measuring Business Excellence. Seele, P. (2016), “Digitally unified reporting: how XBRL-based real-time transparency helps in combining integrated sustainability reporting and performance control”, Journal of Cleaner Production, Vol. 136, pp. 65-77. Smith, M. and Bititci, U.S. (2017), “Interplay between performance measurement and management, employee engagement and performance”, International Journal of Operations & Production Management, Vol. 37 No. 9, pp. 1207-1228. VOL. 27 NO. 2 2023 j MEASURING BUSINESS EXCELLENCEj PAGE 259 VOL. 27 NO. 2 2023 Tardieu, H., Daly, D., Esteban-Lauzan, J., Hall, J. and Miller, G. (2020), “Organization structure – allocating roles and responsibilities to support business strategy”, Deliberately Digital, Springer, Cham, pp. 107-121. Tardieu, H., Daly, D., Esteban-Lauzan, J., Hall, J. and Miller, G. (2020), “Organization structure – allocating roles and responsibilities to support business strategy”, Deliberately Digital, Springer, Cham, pp. 107-121. Tortorella, G.L., Vergara, A.M.C., Garza-Reyes, J.A. and Sawhney, R. (2020), “Organizational learning paths based upon industry 4.0 adoption: an empirical study with Brazilian manufacturers”, International Journal of Production Economics, Vol. 219, pp. 284-294. Ukko, J., Saunila, M. and Rantala, T. (2020), “Connecting relational mechanisms to performance measurement in a digital service supply chain”, Production Planning & Control, Vol. 31 Nos 2/3, pp. 233-244. Ukko, J., Saunila, M. and Rantala, T. (2020), “Connecting relational mechanisms to performance measurement in a digital service supply chain”, Production Planning & Control, Vol. For instructions on how to order reprints of this article, please visit our website: www.emeraldgrouppublishing.com/licensing/reprints.htm Or contact us for further details: permissions@emeraldinsight.com About the authors About the authors Mira Holopainen (MSc Tech) is a Project Researcher and a Doctoral Student in the School of Engineering Science at LUT University, Finland. Her research is related to performance measurement and management as well as digital transformation of industrial companies. She has previously published in the journals of Technology Analysis & Strategic Management and Information Technology and People. Mira Holopainen is the corresponding author and can be contacted at: mira.holopainen@lut.fi Minna Saunila (DSc Tech) is an Associate Professor at LUT University, School of Engineering Science, Department of Industrial Engineering and Management. She received a DSc degree from LUT in 2014 in the field of Industrial Management. Her research covers topics related to performance management, innovation, service operations, as well as sustainable value creation. Recently, her research projects have been related to digitization of services and production. She has previously published in Technovation, Computers in Industry, Journal of Engineering and Technology Management and Technology Analysis and Strategic Management among others. Since 2018, she is also a docent of the University of Jyva¨skyla¨ School of Business and Economics. Juhani Ukko (DSc Tech.) is a Professor at LUT University, School of Engineering Science, Department of Industrial Engineering and Management. He is also an adjunct professor at Tampere University. His current research focuses on performance measurement, operations management, digital transformation, digital services and corporate sustainability performance. In recent years, he has managed and participated in research projects related to digital transformation in companies and society. His work has been published in journals such as Information Systems Frontiers, Computers in Industry, International Journal of Operations and Production Management and International Journal of Production Economics. PAGE 260j MEASURING BUSINESS EXCELLENCEj VOL. 27 NO. 2 2023
https://openalex.org/W4321097821	https://portal.findresearcher.sdu.dk/files/223493388/ijms_24_03937.pdf	English	null	Staying below the Radar: Unraveling a New Family of Ubiquitous “Cryptic” Non-Tailed Temperate Vibriophages and Implications for Their Bacterial Hosts	International journal of molecular sciences	2,023	cc-by	17,536	Download date: 24. Oct. 2024 Citation for pulished version (APA): Kalatzis, P. G., Mauritzen, J. J., Winther-Have, C. S., Michniewski, S., Millard, A., Tsertou, M. I., Katharios, P., & Middelboe, M. (2023). Staying below the Radar: Unraveling a New Family of Ubiquitous “Cryptic” Non-Tailed Temperate Vibriophages and Implications for Their Bacterial Hosts. International Journal of Molecular Sciences , 24(4), Article 3937. https://doi.org/10.3390/ijms24043937 University of Southern Denmark Staying below the Radar Unraveling a New Family of Ubiquitous “Cryptic” Non-Tailed Temperate Vibriophages and Implications for Their Bacterial Hosts Kalatzis, Panos G.; Mauritzen, Jesper Juel; Winther-Have, Caroline Sophie; Michniewski, Slawomir; Millard, Andrew; Tsertou, Maria Ioanna; Katharios, Pantelis; Middelboe, Mathias Document license: CC BY Citation for pulished version (APA): Kalatzis, P. G., Mauritzen, J. J., Winther-Have, C. S., Michniewski, S., Millard, A., Tsertou, M. I., Katharios, P., & Middelboe, M. (2023). Staying below the Radar: Unraveling a New Family of Ubiquitous “Cryptic” Non-Tailed Temperate Vibriophages and Implications for Their Bacterial Hosts. International Journal of Molecular Sciences , 24(4), Article 3937. https://doi.org/10.3390/ijms24043937 Go to publication entry in University of Southern Denmark's Research Portal Panos G. Kalatzis 1 , Jesper Juel Mauritzen 1 , Caroline Sophie Winther-Have 1 , Slawomir Michniewski 2, Andrew Millard 2 , Maria Ioanna Tsertou 3, Pantelis Katharios 3 and Mathias Middelboe 1,4,* 1 Marine Biological Section, Department of Biology, University of Copenhagen, 3000 Elsinore, Denmark 2 Department of Genetics and Genome Biology, University of Leicester, University Road, Leicester LE1 7RH, UK 3 Institute of Marine Biology, Biotechnology and Aquaculture, Hellenic Centre for Marine Research, Former American Base of Gournes, 71500 Heraklion, Greece 4 Department of Biology, University of Southern Denmark, 5230 Odense, Denmark * Correspondence: mmiddelboe@bio.ku.dk 1 Marine Biological Section, Department of Biology, University of Copenhagen, 3000 Elsinore, Denmark 2 Department of Genetics and Genome Biology, University of Leicester, University Road, Leicester LE1 7RH, UK 3 Institute of Marine Biology, Biotechnology and Aquaculture, Hellenic Centre for Marine Research, Former American Base of Gournes, 71500 Heraklion, Greece 4 Department of Biology, University of Southern Denmark, 5230 Odense, Denmark * Correspondence: mmiddelboe@bio.ku.dk 1 Marine Biological Section, Department of Biology, University of Copenhagen, 3000 Elsinore, Denmark 2 Department of Genetics and Genome Biology, University of Leicester, University Road, Leicester LE1 7RH, UK 3 Institute of Marine Biology, Biotechnology and Aquaculture, Hellenic Centre for Marine Research, Former American Base of Gournes, 71500 Heraklion, Greece 4 4 Department of Biology, University of Southern Denmark, 5230 Odense, Denmark * Correspondence: mmiddelboe@bio ku dk Abstract: Bacteriophages are the most abundant biological entities in the oceans and play key roles in bacterial activity, diversity and evolution. While extensive research has been conducted on the role of tailed viruses (Class: Caudoviricetes), very little is known about the distribution and functions of the non-tailed viruses (Class: Tectiliviricetes). The recent discovery of the lytic Autolykiviridae family demonstrated the potential importance of this structural lineage, emphasizing the need for further exploration of the role of this group of marine viruses. Here, we report the novel family of temperate phages under the class of Tectiliviricetes, which we propose to name “Asemoviridae” with phage NO16 as a main representative. These phages are widely distributed across geographical regions and isolation sources and found inside the genomes of at least 30 species of Vibrio, in addition to the original V. anguillarum isolation host. Genomic analysis identiﬁed dif-like sites, suggesting that NO16 prophages recombine with the bacterial genome based on the XerCD site-speciﬁc recombination mechanism. The interactions between the NO16 phage and its V. anguillarum host were linked to cell density and phage–host ratio. Citation: Kalatzis, P.G.; Mauritzen, J.J.; Winther-Have, C.S.; Michniewski, S.; Millard, A.; Tsertou, M.I.; Katharios, P.; Middelboe, M. Staying below the Radar: Unraveling a New Family of Ubiquitous “Cryptic” Non-Tailed Temperate Vibriophages and Implications for Their Bacterial Hosts. Int. J. Mol. Sci. 2023, 24, 3937. https://doi.org/10.3390/ ijms24043937 Keywords: non-tailed phages; NO16; Vibrio; ubiquitous presence; integration; dif sites; spontaneous induction; lysogenic conversion; Asemoviridae Received: 5 December 2022 Revised: 1 February 2023 Accepted: 2 February 2023 Published: 15 February 2023 Panos G. Kalatzis 1 , Jesper Juel Mauritzen 1 , Caroline Sophie Winther-Have 1 , Slawomir Michniewski 2, Andrew Millard 2 , Maria Ioanna Tsertou 3, Pantelis Katharios 3 and Mathias Middelboe 1,4,* High cell density and low phage predation levels were shown to favor the temperate over the lytic lifestyle for NO16 viruses, and their spontaneous induction rate was highly variable between different V. anguillarum lysogenic strains. NO16 prophages coexist with the V. anguillarum host in a mutualistic interaction by rendering ﬁtness properties to the host, such as increased virulence and bioﬁlm formation through lysogenic conversion, likely contributing to their global distribution. Terms of use Terms of use This work is brought to you by the University of Southern Denmark. Unless otherwise specified it has been shared according to the terms for self-archiving. If no other license is stated, these terms apply: This work is brought to you by the University of Southern Denmark. Unless otherwise specified it has been shared according to the terms for self-archiving. If no other license is stated, these terms apply: • You may download this work for personal use only. y p y • You may not further distribute the material or use it y y p • You may freely distribute the URL identifying this open access version at this document breaches copyright please contact us providing details and we will investigate your claim. enquiries to puresupport@bib.sdu.dk If you believe that this document breaches copyright please contact us providing details and we will in Please direct all enquiries to puresupport@bib.sdu.dk International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences 1. Introduction Bacteriophages are the most abundant biotic agents in the ocean [1], having a major impact on bacterial mortality, diversity and global biogeochemical cycling [2]. While some phages seem to be endemic to speciﬁc environments [1], other phages are cosmopolitan [3,4] and distributed across large distances and among different biomes [5]. Therefore, phage diversity can be equally high when examined on a local scale as on a broader, regional or global scale [6]. A bacteriophage infection may either result in lysis of the host (lytic infec- tion) and release of new virions or in the integration of the phage genomes as a prophage in the host (lysogenic infection) [7,8]. Since prophage-encoded genes may improve host ﬁtness [9], the outcome of phage infection as lytic or lysogenic is important for the impli- cations of phage infections on their host communities. Bacteriophages are traditionally grouped according to their morphology and genome type (e.g., RNA, DNA) [10,11], while Staying below the Radar: Unraveling a New Family of Ubiquitous “Cryptic” Non-Tailed Temperate Vibriophages and Implications for Their Bacterial Hosts Panos G. Kalatzis 1 , Jesper Juel Mauritzen 1 , Caroline Sophie Winther-Have 1 , Slawomir Michniewski 2, Andrew Millard 2 , Maria Ioanna Tsertou 3, Pantelis Katharios 3 and Mathias Middelboe 1,4,* 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/). https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2023, 24, 3937. https://doi.org/10.3390/ijms24043937 Int. J. Mol. Sci. 2023, 24, 3937 2 of 23 more modern molecular tools were suggested for a more uniﬁed taxonomic classiﬁca- tion [12]. Classiﬁcation of structural lineages is the most reliable method since it better illustrates the evolutionary pathway of the viruses [13–15]. y p y The lineage of tailed dsDNA viruses is mainly represented by prokaryotic viruses such as the entire class of Caudoviricetes. The class of Tectiliviricetes are clustered in the non-tailed structural lineage [13,16]. However, exceptions were reported as, for instance, in the case of ﬂavophage FLiP, which is a member of the non-tailed lineage based on its DJR architecture even though it has ssDNA genome [17]. The non-tailed phages PRD1 with a 15 kb dsDNA linear genome [18] and PM2 with a 10 kb dsDNA circular genome [19] are representative members of Tectiliviricetes. For the Tectiviridae family (Order: Kalamavirales), 12 different species in ﬁve genera have been described [20,21]. However, for Corticoviridae (Order: Vinavirales), phage PM2 has been the main focus for research in protein composition, penetration process, etc. [22–24], and in the 50 years since its original isolation, only one additional 10 kb Pseudoalteromonas-speciﬁc potential Corticoviridae family member has been sequenced [25]. However, references of PM2-like morphology viruses can be found even for phylogenetically distant isolation hosts such as Microbulbifer and Pseudanabaena with genomes of 48.5 and 137 kb, respectively [26,27]. g p y The recent work of Kauffman et al. [28] unraveled a new family (Autolykiviridae) of lytic, 10 kb non-tailed Vibrio predators, whose presence and role in the oceans have so far been overlooked. Coupling that with the previously reported wide presence of putatively inducible PM2-like prophages in the genomes of aquatic bacteria [29] suggested that a signiﬁcant proportion of the non-tailed viral elements may still go unnoticed from the general analyses of marine phages. Certain lab-related biases were identiﬁed as the main reasons for the systematic omission from the studies of environmental phages. Sensitivity to chloroform, low buoyant density, requirement for protease treatment and long incubation time for plaque formation are features that characterize non-tailed viruses [28]. These features are typically not compatible with standard protocols for phage isolation, and likely part of the explanation for the limited data available on prokaryotic non-tailed viruses from the marine environment [30,31]. Although electron microscopy of non-tailed vibriophages and virus-like particles (VLPs) are sporadically reported in the literature, little work has been implemented on characterizing further their life traits and genome [32–36]. Sequencing is of utmost importance in order to conﬁdently detect novel DJR viral elements and avert false positive results, as almost happened in the case of lactic acid bacteria (LAB) starter cultures. Large numbers of spontaneously induced tailless prophages were observed in LAB cultures; however, it was later shown that those were just phenotypic variants of the P335 siphovirus morphotype with disrupted phage tail encoding genes [37,38]. In the current study, we identify and characterize a novel family of inducible non-tailed prophages, designated as “Asemoviridae” (Asemos (Greek, ´ασηµoς): Unknown, fameless, obscured), which are prevalent in the Vibrio genus on a global scale. Furthermore, using the recently published, representative phage NO16 [39] and its ﬁsh pathogenic V. anguillarum host, we assess in vitro the variability, dynamics and mechanism of integration, as well as the spontaneous induction of the NO16 phages in the phage–host system, and evaluate the role of cell density and phage predation in the process. Furthermore, exploring the mutually beneﬁcial coexistence with the bacterial host through lysogenic conversion and the functional role of NO16 in the community demonstrated that the prophages contribute to host cell functional properties, such as virulence and bioﬁlm formation. 2.1. Characterization of NO16 Screening a collection of 25 different V. anguillarum strains [40] showed that only strain A023 was sensitive to bacteriophage NO16, and this strain was, therefore, used as phage proliferation host. The morphology of phage NO16 under transmission electron microscopy (Figure 1A,B) revealed non-tailed virions of approximately 80 nm in diameter, which re- sembled the corticovirus PM2 [41] and the recently described phages of Autolykiviridae Int. J. Mol. Sci. 2023, 24, 3937 3 of 23 3 of 23 family [28]. The viability of the NO16 phages was signiﬁcantly affected by chloroform treatment with titers of chloroform-treated NO16 phages that were three orders of magni- tude lower than untreated controls (Supplementary Figure S1A). From the one-step growth curve of NO16, latency time and burst size of 30 min and 31 virions per cell, respectively, were calculated (Supplementary Figure S1B). Increasing the multiplicity of infection (MOI) also increased the initial inhibition of the V. anguillarum strain A023 host (Supplementary Figure S1C), whereas the total duration of inhibition (~5 h) and total viral production (~108 PFU mL−1) was unaffected by the initial MOI (Supplementary Figure S1D). Figure 1. (A) Successful infection and consequent cell lysis of V. anguillarum strain A023 by phage NO16 under TEM, (B) Morphology of the NO16 non-tailed virions under TEM, (C) Genomic map of bacteriophage NO16 (GenBank accession no. MH730557) illustrating the 23-gene content of the novel phage. The viral core genes, DJR MCP and ATPase, are slightly shaded while the attributed functions of all 23 genes are listed on the side. (D) Dendrogram generated by GRAViTy (Database selection: DB-B: Baltimore Group Ib—Prokaryotic and archaeal dsDNA viruses). The major viral families are color coded and the phylogenetic clade, which includes the novel phage NO16, is highlighted and magniﬁed in order to illustrate its single monophyletic taxon with only one member, marked with an asterisk . Figure 1. (A) Successful infection and consequent cell lysis of V. anguillarum strain A023 by phage NO16 under TEM, (B) Morphology of the NO16 non-tailed virions under TEM, (C) Genomic map of bacteriophage NO16 (GenBank accession no. MH730557) illustrating the 23-gene content of the novel phage. The viral core genes, DJR MCP and ATPase, are slightly shaded while the attributed functions of all 23 genes are listed on the side. (D) Dendrogram generated by GRAViTy (Database selection: DB-B: Baltimore Group Ib—Prokaryotic and archaeal dsDNA viruses). 2.2. Genetic Diversity and Distribution of NO16 Viral Elements in Vibrio Genomes An initial analysis of phage NO16 distribution in the GenBank generated certain hits (E-value: 0) in chromosome 2 of V. anguillarum strains 87-9-116 and NB10, isolated from Sweden and Finland, respectively [40], with identical query coverage and identity of 98% and 99.65%, respectively. This indicated that NO16 is a prophage, which is integrated in bacterial chromosomes and was not identiﬁed by regular prophage ﬁnding tools e.g., PHASTER [45]. A third hit in chromosome 2 of V. anguillarum VIB18 with 99.91% identity yet with a low coverage of 52% raised additional questions regarding the potential existence of phage NO16 elements in more V. anguillarum strains, which may have been accidentally omitted from the submitted genomes. Subsequent access to the original contigs unraveled assembly gaps in the chromosomes submitted to NCBI and identiﬁed NO16 prophages in genomic parts of 10 genomes that were not included in the submitted versions. All available contigs of V. anguillarum genomes [40] were re-submitted and the new accession numbers JAHGUG000000000-JAHGVD000000000 are now available in the GenBank. The in silico results were conﬁrmed by the spontaneous induction experiments (Supplementary Figure S3A) where the supernatants from overnight cultures of 10 bacte- rial strains with prophage-containing genomes could both inhibit and produce plaques on lawns of A023. Isolation, proliferation and sequencing of the induced NO16 prophages validated their genome size and content (Supplementary Figure S3B). The induced and sequenced NO16-like phages had an identical number of genes and genomic organization as NO16 [39] but showed some nucleotide polymorphisms in particularly 2 genomic regions (NO16 genes 1–6 and 22–23), which clustered them in four closely related subgroups (Supplementary Figure S3B). However, no particular differences were detected in the generated coding regions. The sequences of the spon- taneously induced NO16 phages were submitted to the GenBank under the accession numbers MZ230992-MZ231001. The wide presence of inducible NO16-like phages in the V. anguillarum collection moti- vated the examination for this phage among the Vibrio genus. Searches using HMMs built on the three key phage genes ATPase, MCP DJR and SAH hydrolase generated a total of 1231, 860 and 1214 unique hits (min E-value: 0.01), respectively, in the entire Vibrio genome database (Supplementary Table S4). Based on the overlapping hits from all three models combined to maximize the conﬁdence of the prophage predictions, 630 NO16-like prophage elements were found in 30 different Vibrio species, in addition to V. anguillarum (Figure 2, Supplementary Table S4). 2.1. Characterization of NO16 The major viral families are color coded and the phylogenetic clade, which includes the novel phage NO16, is highlighted and magniﬁed in order to illustrate its single monophyletic taxon with only one member, marked with an asterisk . Figure 1. (A) Successful infection and consequent cell lysis of V. anguillarum strain A023 by phage NO16 under TEM, (B) Morphology of the NO16 non-tailed virions under TEM, (C) Genomic map of bacteriophage NO16 (GenBank accession no. MH730557) illustrating the 23-gene content of the novel phage. The viral core genes, DJR MCP and ATPase, are slightly shaded while the attributed functions of all 23 genes are listed on the side. (D) Dendrogram generated by GRAViTy (Database selection: DB-B: Baltimore Group Ib—Prokaryotic and archaeal dsDNA viruses). The major viral families are color coded and the phylogenetic clade, which includes the novel phage NO16, is highlighted and magniﬁed in order to illustrate its single monophyletic taxon with only one member, marked with an asterisk . The genome size of vibriophage NO16 is 10,594 bp (GenBank accession no. MH730557) [39], which makes it one of the shortest dsDNA bacteriophages that has been described in the literature (Figure 1C). Although the majority of the predicted phage genes are of unknown function, in silico predicted roles were attributed to ﬁve out of the 23 genes by combining RAST [42] with phyre2 [43], as previously reported [39]. DNA-binding protein (gene 6,fNO16_0006), S-adenosylhomocysteine hydrolase (gene 7, fNO16_0007), phage hydrolase (gene 11, fNO16_0011), double-jelly roll major capsid protein (DJR MCP) (gene 19, fNO16_0019) and ATPase (gene 21, fNO16_0021) were the genes with a predicted Int. J. Mol. Sci. 2023, 24, 3937 4 of 23 function (Supplementary Table S3), and their potential implications for the phage biology were examined. function (Supplementary Table S3), and their potential implications for the phage biology were examined. In order to identify the taxonomy of NO16, a phylogenetic dendrogram against the Baltimore Group Ib—Prokaryotic and archaeal dsDNA viruses was constructed (Figure 1D). The phylogenetic position of NO16 is most related with, yet very signiﬁcantly distinct from, the sole non-tailed corticoviruses that are included in the database. This suggests that vibrio- phage NO16 is a member of a novel family, which is classiﬁed as a cohesive, monophyletic taxon in a major proteome-based clustering tool such as GRAViTy, following the main prin- ciples that were recently suggested as the roadmap for genome-based phage taxonomy [44]. 2.1. Characterization of NO16 Based on the concatenated aminoacid sequences of DJR and ATPase encoding genes, a ML-based phylogenetic tree conﬁrmed that the NO16 family bacteriophages form a distinct, monophyletic clade that is clearly separated from other non-tailed phage representatives: corticoviruses, autolykiviruses and tectiviruses (Supplementary Figure S2). 2.2. Genetic Diversity and Distribution of NO16 Viral Elements in Vibrio Genomes 2.2. Genetic Diversity and Distribution of NO16 Viral Elements in Vibrio Genomes The genomic structure of the cryptic phage elements is quite syntenic across representative strains from 30 Vibrio species (Figure 2). The characterization of “naturally excising integrated prophages”, which was previously documented in the Vibrionaceae family [28], i.e., Vibrio sp. 10N.222.52.B12, V. lentus strain 10N.261.48.B11 and V. kanaloae strain 10N.261.46.F4, ﬁt the description of the novel NO16-like viral elements while unravelling their global and intraspeciﬁc prevalence. 5 of 23 of 25 Int. J. Mol. Sci. 2023, 24, 3937 Int. J. Mol. Sci. 2023, 24, x FO Figure 2. (Left): Successful HMM hits of DJR and ATPase and DJR, ATPase and SAH in 42 and 30 different Vibrio species (including the unidentified Vibrio sp. group), respectively, other than V. anguillarum. The asterisk indicates the species where NO16 phages were detected using only the DJR and ATPase core genes. (Right): Genomic organization of representative strains of the species where NO16 prophages were detected. The synteny of NO16-like prophage genomes are vertically illustrated, while the key genes (DJR MCP, ATPase and SAH) are consistently present and high- lighted (blue: SAH, brown: DJR MCP, gray: ATPase). Figure 2. (Left): Successful HMM hits of DJR and ATPase and DJR, ATPase and SAH in 42 and 30 different Vibrio species (including the unidentiﬁed Vibrio sp. group), respectively, other than V. anguillarum. The asterisk indicates the species where NO16 phages were detected using only the DJR and ATPase core genes. (Right): Genomic organization of representative strains of the species where NO16 prophages were detected. The synteny of NO16-like prophage genomes are vertically illustrated, while the key genes (DJR MCP, ATPase and SAH) are consistently present and highlighted (blue: SAH, brown: DJR MCP, gray: ATPase). Figure 2. (Left): Successful HMM hits of DJR and ATPase and DJR, ATPase and SAH in 42 and 30 different Vibrio species (including the unidentified Vibrio sp. group), respectively, other than V. anguillarum. The asterisk indicates the species where NO16 phages were detected using only the DJR and ATPase core genes. (Right): Genomic organization of representative strains of the species where NO16 prophages were detected. The synteny of NO16-like prophage genomes are vertically illustrated, while the key genes (DJR MCP, ATPase and SAH) are consistently present and high- lighted (blue: SAH, brown: DJR MCP, gray: ATPase). Figure 2. (Left): Successful HMM hits of DJR and ATPase and DJR, ATPase and SAH in 42 and 30 different Vibrio species (including the unidentiﬁed Vibrio sp. 2.2. Genetic Diversity and Distribution of NO16 Viral Elements in Vibrio Genomes group), respectively, other than V. anguillarum. The asterisk indicates the species where NO16 phages were detected using only the DJR and ATPase core genes. (Right): Genomic organization of representative strains of the species where NO16 prophages were detected. The synteny of NO16-like prophage genomes are vertically illustrated, while the key genes (DJR MCP, ATPase and SAH) are consistently present and highlighted (blue: SAH, brown: DJR MCP, gray: ATPase). Our entire Vibrio database contained 106 different species, of which 23 accounted for 97% of the included sequences (8507 out of 8768). Overall, 19 out of the 23 most prevalent Vibrio species (83%) carried the NO16-like prophages, representing all available sample types: water, human, seafood and even terrestrial animal for two V. parahaemolyticus sequences. Moreover, the NO16-like prophages showed a global occurrence without any ob- vious patterns in geographical distribution or isolation source (Supplementary Figure S4). The Vibrio database is heavily skewed towards V. cholerae (5804 biosamples) and V. para- haemolyticus (1428 biosamples). Remarkably, fewer than 1% of the V. cholerae biosamples, the most abundant species in the database, were associated with NO16-like prophages (49/5804). On the contrary, the vibrios of merely marine origin harbored NO16-like phages in 13–73% of the available sequences. The vast majority of hits were recorded in the USA, in agreement with prior signiﬁcant work on marine vibrios [28], representing samples from marine water, cultured seafood or humans (Figure 3). The high occurrence of NO16 in V. parahaemolyticus genomes unfolds its global presence and multiple isolation sources, as more than half of the total hits (350/630) were found in this species, despite it only com- prising 16.5% of the Vibrio database. Based on this analysis, previously observed non-tailed phages such as VP2, VP7 and VP9 lytic against V. parahaemolyticus could presumptively belong to the NO16-like phages family, yet their sequences and characterization on the molecular level are still incomplete [32]. Representation for the rest of the marine species also demonstrated a relatively high incidence of NO16-prophage elements in their genomes. V. coralliilyticus is the species with the highest fraction of NO16-like prophages, with the Our entire Vibrio database contained 106 different species, of which 23 accounted for 97% of the included sequences (8507 out of 8768). Overall, 19 out of the 23 most prevalent Vibrio species (83%) carried the NO16-like prophages, representing all available sample types: water, human, seafood and even terrestrial animal for two V. parahaemolyticus sequences. 2.2. Genetic Diversity and Distribution of NO16 Viral Elements in Vibrio Genomes Moreover, the NO16-like prophages showed a global occurrence without any ob- vious patterns in geographical distribution or isolation source (Supplementary Figure S4). The Vibrio database is heavily skewed towards V. cholerae (5804 biosamples) and V. para- haemolyticus (1428 biosamples). Remarkably, fewer than 1% of the V. cholerae biosamples, the most abundant species in the database, were associated with NO16-like prophages (49/5804). On the contrary, the vibrios of merely marine origin harbored NO16-like phages in 13–73% of the available sequences. The vast majority of hits were recorded in the USA, in agreement with prior signiﬁcant work on marine vibrios [28], representing samples from marine water, cultured seafood or humans (Figure 3). The high occurrence of NO16 in V. parahaemolyticus genomes unfolds its global presence and multiple isolation sources, as more than half of the total hits (350/630) were found in this species, despite it only com- prising 16.5% of the Vibrio database. Based on this analysis, previously observed non-tailed phages such as VP2, VP7 and VP9 lytic against V. parahaemolyticus could presumptively belong to the NO16-like phages family, yet their sequences and characterization on the molecular level are still incomplete [32]. Representation for the rest of the marine species also demonstrated a relatively high incidence of NO16-prophage elements in their genomes. V. coralliilyticus is the species with the highest fraction of NO16-like prophages, with the Int. J. Mol. Sci. 2023, 24, 3937 6 of 23 phage elements being present in 11 out of 15 genomes (73%). Although the low number of available sequences hampers a generalized conclusion, the previous indications of 10 kb non-tailed viral elements associated with V. coralliilyticus obtained from gel electrophoresis studies of phage YB2 [33] or electron microscopy of phage RYC [34] imply that these phages could potentially be viruses related to the NO16 family. However, lack of genomic data renders such a conclusion rather speculative. p The inducible, cryptic NO16 bacteriophages that are widely present in V. anguillarum provide insights on a novel bacteriophage family with the suggested name of “Asemoviri- dae”. To our knowledge, this is the ﬁrst sequenced and characterized temperate 10 kb non-tailed bacteriophage whose naïve host is also isolated and fully characterized. The ability to handle the original host in the lab allowed further studies on the regulation of lysis/lysogeny switch and the potential properties that the prophages may render to their hosts (lysogenic conversion). 2.3. Lysogenization and Development of Resistance The prevalence of cryptic 10 kb non-tailed prophage elements in vibrios indicates a looming mobility for NO16, which can be assessed by the integration potential of the viruses in V. anguillarum. In order to assess the lysogenization rates of the NO16 phage, as well as the development of resistance against the bacterial host over time, a large-scale bacterial isolation effort of roughly 1500 clones during a phage exposure experiment was conducted, including determination of potential differences in lysogenization between free-swimming and aggregate-forming subpopulations. In both sample types, lysogenization began very early in the infection process, reaching the highest rate at 4 h incubation (Figure 4A). Both lysogenized (40.9%) and resistant (52.4%) fractions during the early stage of infection were similar among free-swimming and aggregate fractions. Moreover, most of the lysogenized bacteria were resistant to NO16, pointing to prophage-mediated immunity against re- infection [46]. However, 45% and 38% of the lysogens after 4 and 24 h, respectively, regardless of habitat, were still susceptible to the phage. The lysogenization rate at that stage was 20% higher in the free-swimming bacteria compared to the aggregates. During the following stages of the infection, however, no differences between bacterial habitats were reported. Lysogenization continuously decreased over time, reaching 1% after 72 h, whereas resistance reached 95% by the end of the experiment (Figure 4A). The decrease in lysogenization levels over time in the NO16-infected V. anguillarum cultures seemed to follow the pfu counts; however, a substantial number of lysogens were maintained during the infection (Figure 4B,C). Hence, during the stationary phase, there seemed to be a selection for resistance mechanisms other than prophage-mediated immunity, as the fraction of lysogens was decreasing while the isolates maintained resistance. Interestingly, the phage-sensitive clones of the bacterial population isolated during the experiment had reduced phage susceptibility upon phage exposure, relative to the wild type. According to EOP, 75 to 98% at the early stage (4 h) and 50 to 65% after 24 h of the sensitive strains had at least a 1000-fold decrease in sensitivity to NO16 infection compared to the wild type (Figure 4D). Out of the entire experiment, 306 clones were sensitive and only 7 of them recorded a less than 10-fold decrease in sensitivity, indicating that a subpopulation of sensitive clones allows for the proliferation of new virions. 7 of 23 7 of 25 Int. J. Mol. Sci. 2023, 24, 3937 Int. J. Mol. Sci. 2.3. Lysogenization and Development of Resistance 2023, 24, x FOR Figure 3. Fifteen different Vibrio species with more than one strain. The percentage in the colo piece of each pie chart corresponds to the NO16-containing sequences and geographic locatio painted on the map. Information on isolation sources (marine, human, seafood, terrestrial anim or non available) are also reported in the legend Figure 3. Fifteen different Vibrio species with more than one strain. The percentage in the colore piece of each pie chart corresponds to the NO16-containing sequences and geographic location painted on the map. Information on isolation sources (marine, human, seafood, terrestrial animals non-available) are also reported in the legend. Figure 3. Fifteen different Vibrio species with more than one strain. The percentage in the colored piece of each pie chart corresponds to the NO16-containing sequences and geographic location i painted on the map. Information on isolation sources (marine, human, seafood, terrestrial animal or non-available) are also reported in the legend. Figure 3. Fifteen different Vibrio species with more than one strain. The percentage in the colored piece of each pie chart corresponds to the NO16-containing sequences and geographic location is painted on the map. Information on isolation sources (marine, human, seafood, terrestrial animals or non-available) are also reported in the legend. 8 of 23 9 of 25 8 of 23 9 of 25 Int. J. Mol. Sci. 2023, 24, 3937 Int. J. Mol. Sci. 2023, 24, x FOR Figure 4. (A). Progress of lysogenization and resistance development over 72 h where potential re- sponses between free-swimming and aggregate-forming subpopulations were assessed, (B). Num- ber of lysogenized CFUs in the NO16-infected V. anguillarum strain A023 cultures, (C). Gradual decrease in free viruses over the 72 h experiment, (D). EOP percentages of the 306 NO16-sensitive bacterial clones over the 72 h experiment, in both free-swimming and aggregate-forming subpopu- lations. The values are means ± standard deviation of the three replicates. 2.4. Prophage Integration and Spontaneous Induction Dynamics The integration of NO16 into the host genome and the spontaneous induction of NO16 prophages was examined at various MOIs and cell density levels, respectively, in order to assess the potential role of phage predation pressure or cell density related fac Figure 4. (A). Progress of lysogenization and resistance development over 72 h where potential re sponses between free-swimming and aggregate-forming subpopulations were assessed, (B). Number of lysogenized CFUs in the NO16-infected V. anguillarum strain A023 cultures, (C). 2.3. Lysogenization and Development of Resistance Gradual decrease in free viruses over the 72 h experiment, (D). EOP percentages of the 306 NO16-sensitive bacterial clones over the 72 h experiment, in both free-swimming and aggregate-forming subpopulations. The values are means ± standard deviation of the three replicates. 2.4. Prophage Integration and Spontaneous Induction Dynamics The integration of NO16 into the host genome and the spontaneous induction of NO16 prophages was examined at various MOIs and cell density levels, respectively, in order Figure 4. (A). Progress of lysogenization and resistance development over 72 h where potential re- Figure 4 (A) Progress of lysogenization and resistance development over 72 h where potential re- Figure 4. (A). Progress of lysogenization and resistance development over 72 h where potential re- sponses between free-swimming and aggregate-forming subpopulations were assessed, (B). Num- ber of lysogenized CFUs in the NO16-infected V. anguillarum strain A023 cultures, (C). Gradual decrease in free viruses over the 72 h experiment, (D). EOP percentages of the 306 NO16-sensitive bacterial clones over the 72 h experiment, in both free-swimming and aggregate-forming subpopu- lations. The values are means ± standard deviation of the three replicates. Figure 4. (A). Progress of lysogenization and resistance development over 72 h where potential re- sponses between free-swimming and aggregate-forming subpopulations were assessed, (B). Number of lysogenized CFUs in the NO16-infected V. anguillarum strain A023 cultures, (C). Gradual decrease in free viruses over the 72 h experiment, (D). EOP percentages of the 306 NO16-sensitive bacterial clones over the 72 h experiment, in both free-swimming and aggregate-forming subpopulations. The values are means ± standard deviation of the three replicates. 2.4. Prophage Integration and Spontaneous Induction Dynamics Th i t ti f NO16 i t th h t d th 2.4. Prophage Integration and Spontaneous Induction Dynamics During early stages of infection (9 h), the two NO16 genes (genes 6 and 7), which encode DNA-binding domain-containing proteins and are transcribed in opposite direction to the rest of the genome, were shown to be 44- and 21-fold more expressed, respectively, in low than in high MOI conditions (Figure 5C), in agreement with the higher lysogenization rates at low MOI, supporting their tentative function as repressors of the lysis/lysogeny switch. The effect of cell density on lysogenization in response to the addition of spent su- pernatant from the naïve V. anguillarum strain A023 culture at MOI 0.1 showed that the accumulation of infective phages decreased with the increasing age of the added super- natant (Figure 5B), suggesting that a conditioned medium from high cell density (HCD) cultures promoted the integration of the free phages. Overall, assuming that the decrease in accumulation of free phages reﬂects integration and lysogenization of susceptible hosts (Figure 5A,B), these results suggest that low MOI (i.e., high relative cell density) and matured conditioned medium (i.e., high concentrations of metabolites/signal molecules present at a high absolute cell density) promote the integration of NO16 phages. The spontaneous induction dynamics of phage NO16 were examined both in the V. anguillarum strains in which NO16-like phages were present originally (Supplementary Figure S3A) and also in seven A023 experimentally lysogenized clones. In the former group, prophage induction varied considerably between strains with differences in observed pfu production ranging from 1 × 102 to 3 × 107 pfu mL−1 (Figure 6A). The presence of prophages in lysogenized A023 clones cl5, cl13, cl15, cl26, #20, #28 and #40 was initially de- tected both by PCR on the integrated prophages and by pfu formation on the V. anguillarum strain A023 bacterial lawn. The seven NO16 lysogenized clones were sequenced and submit- ted in the GenBank under the accession numbers JAHKER000000000-JAHKEX000000000. The dynamics of the spontaneous induction among different lysogens were also variable, supporting the observed strain-speciﬁc variability of prophage induction on the environ- mental strains. Based on the induction pattern, the seven lysogenized clones clustered in two groups: Group 1 including strains cl5, cl13, cl15, #28 and cl26 with a high induction rate (4 × 105–3.5 × 108 phages produced per mL) and group 2 with strains #20, #28 and #40 with an initial decrease in phage abundance, and only 4.5 × 103–4 × 105 phages produced mL−1 (Figure 6B). 2.4. Prophage Integration and Spontaneous Induction Dynamics Th i t ti f NO16 i t th h t d th 2.4. Prophage Integration and Spontaneous Induction Dynamics 2.4. Prophage Integration and Spontaneous Induction Dynamics Th i t ti f NO16 i t th h t d th 2.4. Prophage Integration and Spontaneous Induction Dynamics The integration of NO16 into the host genome and the spontaneous induction of NO16 prophages was examined at various MOIs and cell density levels, respectively, in order to assess the potential role of phage predation pressure or cell density-related fac- tors on prophage mobility. Using a moderate MOI of 0.1 as reference point to quantify the relative integration frequency in the host, NO16 was found to integrate at significantly higher rates at a lower MOI of 0.01, with 8-, 1.5-, 5- and 3-fold higher integration at time points 9, 24, 48 and 72 h, respectively. On the contrary, higher MOI (MOI: 10) consistently led to lower integration rates compared with MOI: 0.1 (Figure 5A). The accumulation of phages 48 h post infection decreased with increasing initial MOI, and >100-fold more free ha e e e dete ted at MOI 10 tha at MOI 0 01 (Fi u e 5B) Thi u e ted a hi he The integration of NO16 into the host genome and the spontaneous induction of NO16 prophages was examined at various MOIs and cell density levels, respectively, in order to assess the potential role of phage predation pressure or cell density-related factors on prophage mobility. Using a moderate MOI of 0.1 as reference point to quantify the relative integration frequency in the host, NO16 was found to integrate at signiﬁcantly higher rates at a lower MOI of 0.01, with 8-, 1.5-, 5- and 3-fold higher integration at time points 9, 24, 48 and 72 h, respectively. On the contrary, higher MOI (MOI: 10) consistently led to lower integration rates compared with MOI: 0.1 (Figure 5A). The accumulation of phages 48 h post infection decreased with increasing initial MOI, and >100-fold more free phages were Int. J. Mol. Sci. 2023, 24, 3937 9 of 23 9 of 23 detected at MOI 10 than at MOI 0.01 (Figure 5B). This suggested a higher fraction of phages integrating in the host genome at a low compared to a high initial MOI, as was also evident from the integration experiment (Figure 5A). 2.4. Prophage Integration and Spontaneous Induction Dynamics Th i t ti f NO16 i t th h t d th 2.4. Prophage Integration and Spontaneous Induction Dynamics The lysogenized strains cl15 and #20 were selected as representatives of the high and low induction group, respectively, for further examination of their response under various cell density conditions, emphasizing their different spontaneous induction behavior. In the case of low-induction strain #20, the addition of the conditioned medium did not affect prophage induction, and a decrease in induced phages similar to the initial induction experiment was observed in all treatments (Figure 6C). For the high-induction strain cl15, however, the presence of the conditioned medium inhibited the release of new virions by at least 2 log units (Figure 6D), leading to a response in prophage induction similar to the Group 2. Overall, these strain-speciﬁc differences in prophage induction suggested that the regulation vary between strains and may involve quorum sensing or other cell-density related factors. 2.5. Genomic Mechanism of Integration and Induction According to comparative genomic analysis among the NO16-like sequenced prophages and the resubmitted contigs of lysogenized V. anguillarum strains, it seems that the mecha- nism of phage integration in the bacterial host genome may be driven by a XerCD (chro- mosomally encoded tyrosine recombinases) site-speciﬁc recombination mechanism. In Escherichia coli, XerCD recombinases recognize a 28-bp motif, called dif site, and through site-speciﬁc recombination, they untangle circularized chromosomal dimers [47]. By car- rying dif-like sites, ﬁlamentous phages such as CTXϕ utilize host-encoded XerCD to recombine with its host genome, and similar dif-like sites were also found in the V. an- guillarum ﬁlamentous phage VAIϕ [48–50] (Supplementary Figure S5). The XerCD-dif Int. J. Mol. Sci. 2023, 24, 3937 10 of 23 site-speciﬁc recombination process is facilitated by DNA translocases through ATP hydrol- ysis, and homologs of those enzymes are typically found in the vast majority of sequenced bacteria [47]. fraction of phages integrating in the host genome at a low compared to a high initial MOI, as was also evident from the integration experiment (Figure 5A). During early stages of infection (9 h), the two NO16 genes (genes 6 and 7), which encode DNA-binding domain- containing proteins and are transcribed in opposite direction to the rest of the genome, were shown to be 44- and 21-fold more expressed, respectively, in low than in high MOI conditions (Figure 5C), in agreement with the higher lysogenization rates at low MOI, supporting their tentative function as repressors of the lysis/lysogeny switch. Figure 5. (A) Relative integrated NO16 prophage genomes (prophage per bacteria genomes) among different MOI (0.01, 0.1, 10) according to the quantitative assessment of the prophage–host integra- tion sites, (B) Abundance of free phages 48 h post infection at different MOI (0.1, 1, 10) and cell densities (addition of 6 h, 12 h and 24 h host’s spent supernatant) conditions, (C) Expression levels of NO16-related genes at the early stage of infection (9 h) under different MOI (0.01, 10). The values are means ± standard deviation of the three replicates. (a, b, c, d: different groups of statistical sig- nificance, p-value < 0.05; statistical significance, p-value < 0.05) Figure 5. 2.5. Genomic Mechanism of Integration and Induction 11 of 23 f 25 Int. J. Mol. Sci. 2023, 24, 3937 Int. J. Mol. Sci. 2023, 24, Figure 6. (A) Spontaneous induction of NO16 phages from 10 different V. anguillarum strains with considerable variations over 72 h, (B) Seven different lysogenized A023 clones that form two differ- ent groups based on their spontaneous induction rates over 72 h: the upper box corresponds to the high whereas the lower box to the low induction rate clones, (C) Spontaneous induction of lyso- genized A023 clone #20 remains unaffected by different cell density levels (addition of 6 h, 12 h and 24 h host’s spent supernatant), (D) Spontaneous induction of lysogenized A023 clone cl15 is inhib- ited by increased cell density (addition of 6 h, 12 h and 24 h host’s spent supernatant). The values are means ± standard deviation of the three replicates. 2.5. Genomic Mechanism of Integration and Induction According to comparative genomic analysis among the NO16 like sequenced pro Figure 6. (A) Spontaneous induction of NO16 phages from 10 different V. anguillarum strains wi considerable variations over 72 h, (B) Seven different lysogenized A023 clones that form two differe groups based on their spontaneous induction rates over 72 h: the upper box corresponds to the hig whereas the lower box to the low induction rate clones, (C) Spontaneous induction of lysogenize A023 clone #20 remains unaffected by different cell density levels (addition of 6 h, 12 h and 24 host’s spent supernatant), (D) Spontaneous induction of lysogenized A023 clone cl15 is inhibite by increased cell density (addition of 6 h, 12 h and 24 h host’s spent supernatant). The values a means ± standard deviation of the three replicates. 2 6 R l f NO16 P h f B i l F i l P i Fi 6 (A) S i d i f NO16 h f 10 diff V ill i i h Figure 6. (A) Spontaneous induction of NO16 phages from 10 different V. 2.5. Genomic Mechanism of Integration and Induction anguillarum strains with considerable variations over 72 h, (B) Seven different lysogenized A023 clones that form two differ- ent groups based on their spontaneous induction rates over 72 h: the upper box corresponds to the high whereas the lower box to the low induction rate clones, (C) Spontaneous induction of lyso- genized A023 clone #20 remains unaffected by different cell density levels (addition of 6 h, 12 h and 24 h host’s spent supernatant), (D) Spontaneous induction of lysogenized A023 clone cl15 is inhib- ited by increased cell density (addition of 6 h, 12 h and 24 h host’s spent supernatant). The values are means ± standard deviation of the three replicates. 2.5. Genomic Mechanism of Integration and Induction Figure 6. (A) Spontaneous induction of NO16 phages from 10 different V. anguillarum strains with considerable variations over 72 h, (B) Seven different lysogenized A023 clones that form two different groups based on their spontaneous induction rates over 72 h: the upper box corresponds to the high whereas the lower box to the low induction rate clones, (C) Spontaneous induction of lysogenized A023 clone #20 remains unaffected by different cell density levels (addition of 6 h, 12 h and 24 h host’s spent supernatant), (D) Spontaneous induction of lysogenized A023 clone cl15 is inhibited by increased cell density (addition of 6 h, 12 h and 24 h host’s spent supernatant). The values are means ± standard deviation of the three replicates. g y y ( , 24 h host’s spent supernatant), (D) Spontaneous induction of lysogenized A023 clone cl15 is inhib- ited by increased cell density (addition of 6 h, 12 h and 24 h host’s spent supernatant). The values are means ± standard deviation of the three replicates. 2.5. Genomic Mechanism of Integration and Induction whereas the lower box to the low induction rate clones, (C) Spontaneous induction of lysogenized A023 clone #20 remains unaffected by different cell density levels (addition of 6 h, 12 h and 24 h host’s spent supernatant), (D) Spontaneous induction of lysogenized A023 clone cl15 is inhibited by increased cell density (addition of 6 h, 12 h and 24 h host’s spent supernatant). The values are means ± standard deviation of the three replicates. 2.5. Genomic Mechanism of Integration and Induction (A) Relative integrated NO16 prophage genomes (prophage per bacteria genomes) among different MOI (0.01, 0.1, 10) according to the quantitative assessment of the prophage–host integration sites, (B) Abundance of free phages 48 h post infection at different MOI (0.1, 1, 10) and cell densi- ties (addition of 6 h, 12 h and 24 h host’s spent supernatant) conditions, (C) Expression levels of NO16-related genes at the early stage of infection (9 h) under different MOI (0.01, 10). The values are means ± standard deviation of the three replicates. (a, b, c, d: different groups of statistical signiﬁcance, p-value < 0.05; * statistical signiﬁcance, p-value < 0.05). Int. J. Mol. Sci. 2023, 24, 3937 10 of 23 stages of domain- 10 of 23 stages of domain- site-speciﬁc recombination process is facilitated by DNA translocases through ATP hydrol- ysis, and homologs of those enzymes are typically found in the vast majority of sequenced bacteria [47]. p , p y, g conditions (Figure 5C), in agreement with the higher lysogenization rates at low MOI, supporting their tentative function as repressors of the lysis/lysogeny switch. Figure 5. (A) Relative integrated NO16 prophage genomes (prophage per bacteria genomes) among different MOI (0.01, 0.1, 10) according to the quantitative assessment of the prophage–host integra- tion sites, (B) Abundance of free phages 48 h post infection at different MOI (0.1, 1, 10) and cell densities (addition of 6 h, 12 h and 24 h host’s spent supernatant) conditions, (C) Expression levels of NO16-related genes at the early stage of infection (9 h) under different MOI (0.01, 10). The values are means ± standard deviation of the three replicates. (a, b, c, d: different groups of statistical sig- nificance, p-value < 0.05; * statistical significance, p-value < 0.05) Figure 5. (A) Relative integrated NO16 prophage genomes (prophage per bacteria genomes) among different MOI (0.01, 0.1, 10) according to the quantitative assessment of the prophage–host integration sites, (B) Abundance of free phages 48 h post infection at different MOI (0.1, 1, 10) and cell densi- ties (addition of 6 h, 12 h and 24 h host’s spent supernatant) conditions, (C) Expression levels of NO16-related genes at the early stage of infection (9 h) under different MOI (0.01, 10). The values are means ± standard deviation of the three replicates. (a, b, c, d: different groups of statistical signiﬁcance, p-value < 0.05; * statistical signiﬁcance, p-value < 0.05). According to comparative genomic analysis among the phages and the resubmitted contigs of lysogenized V anguillar 2.6. Role of NO16 Prophages for Bacterial Functional Properties phages and the resubmitted contigs of lysogenized V. anguillarum strains, it seems that the mechanism of phage integration in the bacterial host genome may be driven by a XerCD (chromosomally encoded tyrosine recombinases) site-specific recombination mechanism. In Escherichia coli, XerCD recombinases recognize a 28-bp motif, called dif site, and through site-specific recombination, they untangle circularized chromosomal dimers [47]. By car- rying dif-like sites, filamentous phages such as CTXφ utilize host-encoded XerCD to The wide prevalence and mobility of NO16 phages raises questions about their po- tential role in aquatic bacterial communities, and their potential contribution to bioﬁlm formation and virulence was assessed. Bioﬁlm formation was quantiﬁed for the seven lysogens (cl5, cl13, cl15, cl26, #20, #28 and #40) but also for seven more strains, which were exposed to phage predation but were not lysogenized (#8, #9, #10, #11, #16, #17 and cl23). The control wild type strain A023 was a poor bioﬁlm former, and there was a statistically signiﬁcant increase in bioﬁlm formation for all the lysogens with the exceptions of cl15 and cl26 at 48; however, all strains were signiﬁcantly better bioﬁlm formers in at least one sampling point (Figure 7A). The addition of the phage NO16 signiﬁcantly increased the bioﬁlm formation for A023 strain at 48 and 72 h at both low and high MOI. In addition, while the supernatant that was harvested from naïve A023 culture (A023 sn) had little effect Int. J. Mol. Sci. 2023, 24, 3937 12 of 23 12 of 23 on bioﬁlm formation, the phage-free supernatant that was harvested from the A023/NO16- infected culture (p-f sn) signiﬁcantly increased bioﬁlm formation (Figure 7B). No direct link between prophage induction rates and bioﬁlm formation was observed. Overall, the pres- ence or previous exposure of NO16 in the bacterial population seemed to promote bioﬁlm formations regardless of it being integrated in the chromosomal genome or not. In addition to bioﬁlm formation, we assessed if lysogenized strains had obtained potential virulent properties from the prophages by challenging Gilthead seabream (Sparus aurata) larvae with the lysogenized strains cl13 and cl15 and the naïve A023 host. Over a two-week experiment, both lysogens were signiﬁcantly more virulent compared to their naïve counterpart and the bacteria-free control treatment (Figure 7C). Strain A023 was previously characterized as low virulence among several tested V. According to comparative genomic analysis among the phages and the resubmitted contigs of lysogenized V anguillar 2.6. Role of NO16 Prophages for Bacterial Functional Properties anguillarum strains [40], and together with the enhanced bioﬁlm potential, our current results suggest that lysogenic conversion by NO16 does indeed affect host functional properties. R PEER REVIEW 14 of 25 oes ee ec os c o p ope es Figure 7. (A) Biofilm formation of lysogenized (#20, #28, #40, cl5, cl13, cl15, cl26) and non-lyso- genized (#8, #9, #10, #11, #16, #17, cl23), but exposed to NO16, A023 clones after 48 h. A023 was used as control. (B) Biofilm formation of A023 under low (MOI: 0.01) and high (MOI: 10) phage predation, as well as in the presence of host’s spent supernatant (A023 sn) and supernatant from bacteria– phage lysis event (p-f sn). A023 was used as control. (C) In vivo mortality rates of S. aurata larvae infected by A023 lysogens (cl13 and cl15), wild type (A023) and non-infected (bacteria-free) condi- tion as negative control, over a 7-day period. Letters a and b indicate statistically significant groups (Dunn’s method versus control). The values are means ± standard deviation of the three replicates. 3. Discussion The interactions between bacteriophages and their hosts play key roles in bacterial l di i d l i i h i i h ill k Figure 7. (A) Bioﬁlm formation of lysogenized (#20, #28, #40, cl5, cl13, cl15, cl26) and non-lysogenized (#8, #9, #10, #11, #16, #17, cl23), but exposed to NO16, A023 clones after 48 h. A023 was used as control. (B) Bioﬁlm formation of A023 under low (MOI: 0.01) and high (MOI: 10) phage predation, as well as in the presence of host’s spent supernatant (A023 sn) and supernatant from bacteria–phage lysis event (p-f sn). A023 was used as control. (C) In vivo mortality rates of S. aurata larvae infected by A023 lysogens (cl13 and cl15), wild type (A023) and non-infected (bacteria-free) condition as negative control, over a 7-day period. Letters a and b indicate statistically signiﬁcant groups (Dunn’s method versus control). The values are means ± standard deviation of the three replicates. Figure 7. (A) Biofilm formation of lysogenized (#20, #28, #40, cl5, cl13, cl15, cl26) and non-lyso- genized (#8, #9, #10, #11, #16, #17, cl23), but exposed to NO16, A023 clones after 48 h. A023 was used as control. 3. Discussion The interactions between bacteriophages and their hosts play key roles in bacterial ecology, diversity and evolution in the marine environment; however, we still know very little about their diversity and distribution. The current discovery and characterization of a new family of non-tailed marine phages with a global distribution as a prophage among 30 Vibrio species emphasizes the vast importance of these particular phages in shaping the genetic and functional properties of their bacterial hosts and illustrates their dissemination potential across bacterial species and large spatial scales. The NO16 phages (“Asemoviridae”), which were originally isolated from V. anguillarum and, subsequently, shown to be present as a prophage in the entire Vibrionaceae family, have so far been undetected by traditional bioinformatic prophage detection tools [45,51,52]. The recent discovery of a new family of lytic non-tailed DJR capsid vibriophages, Autolykiviridae, has demonstrated that DJR viruses are far more diverse than previously recognized, represent- ing important killers of marine Vibrios [28]. The “Asemoviridae” show very little genetic similarity to the Autolykiviridae, even among their signature core genes, such as gene 19 (MCP DJR) and gene 21 (packaging ATPase). Kauffman et al. [28] have already addressed the presence of naturally excising prophages in Vibrionaceae, which were not classiﬁed in the Autolyikiviridae family, corroborating the existence of more than just three recognized non-tailed families. BLAST failed to generate any substantial genomic overlap between any genes of the two viral families, and we, therefore, proposed that the “Asemoviridae” represents a novel family of phages. Due to the underrepresentation of non-tailed phages in the database, there is a special reference to them in the recently published roadmap for genome-based phage taxonomy, according to which we need new criteria to be tailor made for such special groups [44]. However, the fundamental principle that rules the phage taxonomy roadmap is conﬁrmed in this case for “Asemoviridae”, since the generated GRAViTy classiﬁes them as a distinct, cohesive and monophyletic taxonomic unit. The statistical robustness of the phylogenetic tree that was constructed, including representative viruses from the class of Tectiliviricetes, corroborates the distinct taxonomic position of this novel phage family. The low genomic and amino acid similarity among the DJR protein among Autolykiviridae, Corticoviridae, Tectiviridae and “Asemoviridae” [39] dictates that the distinction of families is directly reﬂected on the structural level and further expanded to the rest of the genomic content. According to comparative genomic analysis among the phages and the resubmitted contigs of lysogenized V anguillar 2.6. Role of NO16 Prophages for Bacterial Functional Properties (B) Biofilm formation of A023 under low (MOI: 0.01) and high (MOI: 10) phage predation, as well as in the presence of host’s spent supernatant (A023 sn) and supernatant from bacteria– phage lysis event (p-f sn). A023 was used as control. (C) In vivo mortality rates of S. aurata larvae infected by A023 lysogens (cl13 and cl15), wild type (A023) and non-infected (bacteria-free) condi- tion as negative control, over a 7-day period. Letters a and b indicate statistically significant groups (Dunn’s method versus control). The values are means ± standard deviation of the three replicates. 3. Discussion Th i t ti b t b t i h d th i h t l k l i b t i l Figure 7. (A) Bioﬁlm formation of lysogenized (#20, #28, #40, cl5, cl13, cl15, cl26) and non-lysogenized (#8, #9, #10, #11, #16, #17, cl23), but exposed to NO16, A023 clones after 48 h. A023 was used as control. (B) Bioﬁlm formation of A023 under low (MOI: 0.01) and high (MOI: 10) phage predation, as well as in the presence of host’s spent supernatant (A023 sn) and supernatant from bacteria–phage lysis event (p-f sn). A023 was used as control. (C) In vivo mortality rates of S. aurata larvae infected by A023 lysogens (cl13 and cl15), wild type (A023) and non-infected (bacteria-free) condition as negative control, over a 7-day period. Letters a and b indicate statistically signiﬁcant groups (Dunn’s method versus control). The values are means ± standard deviation of the three replicates. Int. J. Mol. Sci. 2023, 24, 3937 13 of 23 All exposed but not lysogenized clones were also sequenced and submitted in the GenBank under the accession numbers JAHKEJ000000000-JAHKEQ000000000. 3. Discussion Although demarcation criteria for non-tailed viruses are not yet as standardized as they are for their tailed counterparts, the articulate and recent roadmap for genome-based phage taxonomy infers that common principles are to be shared among bacterial viruses with the additional consideration for case-speciﬁc amendments [44]. Furthermore, their temperate lifestyle and wide dispersal among Vibrios originating from environmental, human, seafood, terrestrial animal sources suggest that this family of phages is not only an important killer of marine Vibrios but also contributes to host performance when integrated into their genome. This suggestion was strongly supported by our experimental veriﬁcation that the “Asemoviridae” phages are dynamic components of 40% of the ﬁsh pathogenic V. anguillarum strains in our collection, with positive implications for key functional properties, such as virulence and bioﬁlm formation by the lysogenized strains. d h ib i h l f k l f l d h Beyond the Vibrio genus, there are only a few known examples of non-tailed phage isolates, such as the lytic phages PM2 [41], Cr39582 [25] and FLiP [17], which infect Pseu- doalteromonas and Flavobacterium, respectively, with poor to no genomic similarities to the new family of “Asemoviridae”. The identiﬁcation of viral “self” genes was useful for exploring genetic relatedness among DJR phages [29]. The analysis of these genes have supported the presence of major capsid protein and packaging ATPase as essential genes for all corticoviral elements, such as PM2 and revealed hidden PM2-like elements inside the genomes of aquatic bacteria [29]. Although these elements were previously reported Int. J. Mol. Sci. 2023, 24, 3937 14 of 23 14 of 23 as presumably circular, it was not determined if they are inducible, as demonstrated for “Asemoviridae” in the present study. Previous observations of inducible DJR elements from V. kanaloae and V. cyclotrophicus by Kauffman et al. [28] suggested that these phages were indeed mobile. In the current study, however, the availability of a naïve, susceptible V. anguillarum strain A023 host was key to validate circularity and examine induction and integration potential of the “Asemoviridae” phages. Coupling current and previous ﬁndings suggests that also other non-tailed DJR phages such as PM2, which has long been characterized as a lytic phage [19,41], may have a so far undetected lysogenic lifestyle under the right infection conditions of a susceptible Pseudoaltermonas host. The in silico synteny of the PM2 genome with chromosomal parts of P. piscicida and P. 3. Discussion lipolytica are indications supporting an undiscovered temperate PM2 lifestyle, as was shown for the temperate PM2-like phage Cr39582 [25]. A presumptive 28-bp dif site was found in the genome of PM2 (CGTGCTTACGATT-TATA-TTATGTTAAAT), though with 8 mismatches compared to the NO16 dif site. p f The wide prevalence of NO16 phages was validated by the construction of HMMs, which were based on the viral “self” genes of the non-tailed elements. Probabilistic models for protein homology and structure prediction were previously used for both the tailless dsDNA corticoviruses and tectiliviruses [29,53,54], while core genes such as DJR MCP were recently used as bait to successfully unravel their diversity and prevalence in bacterial genomes and metagenomes [54,55]. In the current study, we used the overlapping results of three individual HMMs to identify the presence of NO16 viruses in Vibrio genomes to ensure high conﬁdence of the prediction, resulting in 630 genomes containing all three genes. If single individual models were used, the estimated prevalence exceeded 1200 genomes, and we, therefore, believe that the 630 prophage hits represent a conservative estimate. From an evolutionary perspective, such probabilistic models have signiﬁcant value because although the majority of viral “self” genes encode structurally conserved proteins, detection of homology in both their amino acid and genomic level is rather challenging. Our results add to the emerging consensus of a high prevalence of lysogeny in marine bacteria [56,57]. A recent screening of 1778 marine bacterial genomes demonstrated that on average 17.7% were lysogenized with at least one prophage across all phyla, and within the Vibrio genus, 29% of the 91 published genomes were lysogenized [57]. In our study, 180 of the 679 analyzed Vibrio genomes of conﬁrmed marine origin (26.5%) contained the NO16 prophage, suggesting that the frequency of marine vibrios, which contain the “Asemoviridae” prophage is similar to the total frequency of lysogenized vibrios detected using traditional prophage-ﬁnder tools detected. Wide geographical coexistence patterns of bacteria and their corresponding bacterio- phages were previously described, not only for Vibrio phages [3,58] but also for phages infecting other genera [4]. The global presence of asemoviruses as prophages strongly supports previous suggestions that DJR bacteriophages are highly overlooked [13,28] and emphasizes the potential for the discovery of new viral families within the DJR viral lineage. In the V. 3. Discussion anguillarum strain A023 and phage NO16 system, lysogenization during the early stages of infection affected almost half of the population, with a 20% higher lysoge- nization rate in the free swimming bacteria compared to the aggregates. This suggested that aggregate-associated bacteria were partially protected from infection, as previously reported for V. anguillarum [59]. Over time, there was a strong selection for phage-resistant bacteria at the expense of the lysogens, and phage production was likely maintained by in- fection of a small fraction of susceptible bacteria and by induction of prophages. The results demonstrate that phage exposure drives a diversiﬁcation of the host population and leads to co-existence of phage-sensitive non-lysogens, phage-resistant and sensitive lysogens and phage-resistant non-lysogens, which likely contributes to the successful dissemination of the phage. p g Interestingly, phage integration was negatively correlated to the multiplicity of in- fection, as both frequency of lysogenization and expression of selected prophage genes in the host were signiﬁcantly higher at low than at high MOI. Accordingly, the reduced Int. J. Mol. Sci. 2023, 24, 3937 15 of 23 15 of 23 accumulation of free phages at low MOI in the infection experiment indicated a higher integration rate at low MOI than at high MOI. The reduced accumulation of phages at low MOI could also reﬂect an increased rate of superinfection or decay of the produced phages in these cultures. However, apart from the initial differences in phage abundance the experimental settings were identical. Together these results point to phage integration and prophage induction being favored at low and high MOI, respectively. However, an alternative hypothesis that the MOI dependent lysogeny rate could be a matter of the host being able to “capture” a low number of phages via XerCD, whereas a high number of phages may overwhelm the system, cannot be ruled out. Previous knowledge on the lysis–lysogeny regulation mainly stems from experiments with temperate phages infecting E. coli, such as λ, Mu, P1 or N1 [60]. These have generally reported the opposite trend, i.e., high MOI favors lysogeny in these phages, which was interpreted as a strategy to reduce the risk of unsuccessful lytic infection of the next phage generation during condi- tions, where phages outnumber their bacterial hosts (i.e., high MOI) [61–63]. In the marine environments, however, lysogeny is considered to be less prevalent in oligotrophic waters with high virus–bacteria ratios (VBR) [56]. 3. Discussion The large range in VBR (from 3 to 160) observed across marine environments [64] and decline in VBR at increasing microbial densities have led to the so-called Piggyback-the-Winner model [65], suggesting an increased prevalence of lysogeny at high cell densities as the key driver of this relationship between VBR and cell density. The current study also indicated a correlation between cell density and the induction of NO16 prophages, since the accumulation of free phages from induction in V. anguillarum was negatively correlated with age of the culture supernatant added to the lysogens. Addition of cell-free supernatant from high cell-density cultures (24 h and 6 h cultures of then non-lysogen) led to signiﬁcantly lower accumulation of free phage particles than cultures without supernatant or supernatant from low cell density (LCD) cultures, suggesting that extracellular metabolites, such as QS signal molecules, in the spent medium could reduce prophage induction [66]. Previous studies have reported that host cells through QS may regulate the lysis–lysogeny switch and trigger prophage induction [67,68]. Although it is not known if QS regulates NO16 prophage induction, our results support that NO16-like prophages favor lysogeny at high cell density conditions in V. anguillarum, presumably to promote host ﬁtness. [66]. This hypothesis was further supported in the current study by the observed signiﬁcant increase in bioﬁlm formation and virulence against ﬁsh larvae in the lysogenic strain compared with the non-lysogen. Together, these data support that the interaction between host and prophage is regulated both by the host density through QS pathways and by the phage–host ratio (MOI), resulting in relatively high induction and low integration frequency of prophages at LCD and high phage–host ratio, and vice versa (i.e., low induction and high integration) at HCD and low phage–host ratio. Interestingly, the response to cell-free supernatant from HCD cultures of the non-lysogenic V. anguillarum wild type A023 seemed to vary between prophage-bearing clones, as indicated by the differences in prophage induction in the lysogenic strains cl15 and #20 after exposure to spent medium. While this may reﬂect clone-speciﬁc differences in QS response, other cell-density factors, which are not yet known, may play signiﬁcant roles as well. These differences in how the regulation of the lysis–lysogeny switch ﬁts into dif- ferent phage and host strategies, cell density and environmental conditions highlights the need for a deeper understanding of this process and its ecological and disease implications. 4. Materials and Methods Abridged Methods are provided below; details and additional information are pro- vided in the extended Methods in the SI. All experiments were conducted in triplicate. 4.1. Bacterial Strains and Growth Conditions 4.3. Characterization of Bacteriophages The morphology of the phage NO16 was observed under a JEM-1010 transmission electron microscope (Jeol, Tokyo, Japan) by mounting them on negatively stained copper grids as previously described [48]. Phage concentrates were prepared by the addition of poly-ethylene glycol 8000 (PEG-8000) and sodium chloride (ﬁnal concentration 10% w/v and 1 M, respectively), and the phage solutions were incubated overnight at 4 ◦C. Finally, the suspensions were centrifuged (10,000× g, 30 min, 4 ◦C) and the phage pellet was resuspended in 200 mL of SM buffer [72]. Latency time and burst size of the novel virions were calculated from their one-step growth curve. Phage NO16 was added to 1 mL early exponential phase V. anguillarum A023 culture in marine broth using low multiplicity of infection (MOI: 0.001) and incubated with agitation for 15 min at room temperature. After centrifugation at 6000× g for 10 min, the bacteria–phage pellet was resuspended in 20 mL fresh marine broth, samples were taken every 10 min for a total of 100 min, and infective phages were quantiﬁed as above. To evaluate chloroform sensitivity of phage NO16, chloroform was added to the phage stock in a ratio of 1:5 followed by quantiﬁcation of infective phages. Mixtures of phage and chloroform were vortexed for 6 s and incubated at room temperature for 2 h. Following centrifugation at 5000× g for 5 min, phage viability was tested using serial dilutions mixed with the bacterial hosts and poured into marine-agar-containing 6-well plates and compared with control incubations without chloroform [28]. The host range of the NO16 phage was assessed by spotting 10-µL drops of the phage stock on the bacterial lawns of 23 clinical V. anguillarum strains that were previously characterized [40] using the double agar layer method [71] (Supplementary Table S1). 4.2. Isolation, Puriﬁcation and Proliferation of Bacteriophages Samples for phage isolation were collected at a coastal area of the Central East main- land of Greece, and the presence of bacteriophages was assessed by the classic enrichment method [70] using a combination of four V. anguillarum strains (A023, 261/91, 178/90 and 601/91) [40]. The water was supplemented with 10× marine broth adjusting the nutrient concentrations according to the ﬁnal volume, and the enrichment culture was incubated overnight at 25 ◦C with constant agitation. Samples of 1 mL were then centrifuged at 6000× g for 10 min, ﬁltered through 0.22 µm pore-size syringe ﬁlters and the supernatant was spotted on the bacterial lawn of each candidate host using the double agar layer method [71]. Samples producing clearing zones on the bacterial lawn were serially diluted in SM buffer (sodium magnesium; 100 mM NaCl, 8 mM MgSO4·7H2O, 50 mM Tris-Cl, 0.01% gelatin, pH: 7.5), and 10 µL of each dilution were mixed with 90 µL of mid-log-phase bacterial culture and left for 5 min to allow bacteria and phage binding. Then, 1 mL melted soft agar (0.4% agar and 1% Sigma-Aldrich sea salts) was added, and it was poured into 6-well plates with marine agar. Following overnight incubation at room temperature, the wells were examined for the presence of plaque forming units (pfu). Individual pfu were picked and puriﬁed by repeating the process ﬁve times in order to create clonal phage stocks. 4.1. Bacterial Strains and Growth Conditions The strains used in this study were collected from clinical incidents of vibriosis out- breaks at different ﬁsh farms in numerous countries around the world. All bacterial strains were previously sequenced and classiﬁed to the species of V. anguillarum, and their vir- Int. J. Mol. Sci. 2023, 24, 3937 16 of 23 ulence and pathogenicity traits were characterized [40,69]. Marine broth (0.5% tryptone, 0.1% yeast extract and 2% sea salts) was used for the liquid bacterial cultures, with the addition of 1.5% agar for growth on solid medium. ulence and pathogenicity traits were characterized [40,69]. Marine broth (0.5% tryptone, 0.1% yeast extract and 2% sea salts) was used for the liquid bacterial cultures, with the addition of 1.5% agar for growth on solid medium. 4.4. Genomic Analysis and HMM Construction DNA extraction of both bacteria and phage genomes was performed with Wizard Genomic DNA puriﬁcation kit (Promega, Madison, WI, USA) following the manufacturer’s protocol. Puriﬁed DNA samples were sequenced by Beijing Genomics Institute (BGI) using Illumina HiSeq platform (BGI, Shenzhen, China) with paired-end read sizes of 100 bp. Library construction (TruSeq), sequencing and data pipelining were performed in accordance with the manufacturer’s protocol. Int. J. Mol. Sci. 2023, 24, 3937 17 of 23 17 of 23 The V. anguillarum strains that were sequenced by Rønneseth et. al. [40] were assem- bled using V. anguillarum strain 775 as reference [69]. Although the assembly was successful, the genomic variability of vibrios led to the generation of several assembly gaps and, hence, omission of sequenced genomic information. Therefore, all previously sequenced data were re-submitted to the GenBank, as contigs, in order to include all the missing data. The bacterial genomes were annotated by NCBI Prokaryotic Genome Automatic Annotation Pipeline (PGAAP) [73]. p ( ) The genome of NO16 bacteriophages was recently sequenced [39] and de novo as- sembled using the Geneious assembler at high sensitivity, which is included in Geneious Prime 2020.2 software bioinformatics platform. Genomic comparisons among NO16-like phages were conducted by using the NO16 phage’s genome as reference (GeneBank ac- cession no. MH730557). BLAST https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 13 March 2020) was used as the ﬁrst tool in order to screen for similar genomes in the non-redundant nucleotide database. Geneious Prime 2020.2 software bioinformatics plat- form was also used to visualize and in silico search the sequenced genomes, construct the genomic map of NO16 and explore the phylogenetic relationships with the spontaneously induced NO16-like phages from the different strains of V. anguillarum. Furthermore, a ML-based phylogenetic tree (RAxML; gamma blosum62; 1000 bootstrap replicates) using the concatenated aminoacid sequences of major capsid protein and ATPase encoding genes was constructed in order to determine the speciﬁc relationships among autolykiviruses, corticoviruses, tectiviruses and the group of NO16-like bacteriophages. g p p g The detection of NO16-like prophages in the publicly available Vibrio genome database was conducted by constructing three individual Hidden Markov Models (HMM) [74] for the three key NO16 phage genes DJR MCP (gene 19), ATPase (gene 21) and SAH (gene 7). DJR MCP and ATPase are the viral “self” core genes [29], which are the signature genes of all the hitherto described 10 kb non-tailed bacteriophages. 4.4. Genomic Analysis and HMM Construction The third gene is a BLAST-predicted S-adenosylhomocysteine (SAH) hydrolase (gene 7), which is one out of only two NO16-genes transcribed in the opposite direction to the rest of the prophage genes and, according to its predicted protein structure, contains a DNA-binding domain. Analytical details for both genomic analysis and HMM construction are included in the Methods supplementary section. 4.6. Bacteriophage Dynamics: Prophage Integration and Spontaneous Induction 4.6. Bacteriophage Dynamics: Prophage Integration and Spontaneous Induction The temperate life cycle of NO16 phage [39] was characterized by assessing dynamics of integration and induction of the phage, respectively. Phage integration was assessed at various MOI and cell density conditions to examine the role of phage and cell density on the lysis/lysogeny switch. V. anguillarum strain A023 was infected at early exponential phase (OD600 ~ 0.2), 3 h post inoculation from an overnight culture, at 3 different MOIs: low (0.01), moderate (0.1) and high (10), prepared in triplicate in 50-mL centrifuge tubes. ( ) ( ) g ( ) p p p g To examine the role of signal molecules or other extracellular metabolites produced during bacterial growth, additional experiments were established with conditioned media with cell-free supernatant from bacterial host cultures grown at different densities. The conditioned medium was prepared by growing 0.2 L liquid cultures of the V. anguillarum strain A023 for 6, 12 and 24 h, respectively, followed by centrifugation (6000× g; 10 min) and 0.22 µm ﬁltration to obtain supernatant from cultures that had reached early exponential, late exponential and stationary phase growth, respectively. A mixture of conditioned media and regular marine broth at 1:1 ratio in triplicate 50-mL centrifuge tubes was used to generate three different cell density conditions, and phages were added at an initial MOI of 0.1. An additional incubation where V. anguillarum strain A023 was growing in regular marine broth without phages served as control. Samples were collected after 9, 24, 48 and 72 h, respectively (6, 21, 45, 69 h post phage infection) for quantiﬁcation of lysogenic cells and phage production. p g p Identiﬁcation of the phage integration site was evaluated at the molecular level after extracting the bacterial genome using the boiling method [75]. Speciﬁc primers were de- signed for measuring the relative integration site DNA copies and calibrating them to the DNA copies of a bacterial reference gene, recA (Supplementary Table S2, and Supplemen- tary Methods). Liquid culture V. anguillarum strain A023 infected with the temperate phage NO16, led to the formation of lysogenized clones. For further examination of clonal diver- sity among lysogens, seven clones were randomly selected from 200 lysogenized colonies isolated after 24 h and 48 h incubations. 4.6. Bacteriophage Dynamics: Prophage Integration and Spontaneous Induction Detection of lysogenized bacteria was performed from observations of spontaneous induction measured by PFU and by phage-targeted PCR using speciﬁc primers, which targeted signature phage genes (e.g., DJR MCP), and the integration sites where prophages were integrated (Supplementary Table S2). Negative controls of uninfected V. anguillarum strain A023 were always included in the PCR. Spontaneous induction dynamics were evaluated for the seven conﬁrmed lysogens. The strains were cultured on marine agar, and fresh colonies were picked and grown overnight in marine broth and, subsequently, inoculated in marine broth. Samples for optical density, pfu and cfu counts were obtained at 0, 6, 24, 48 and 72 h post inoculation to examine potential clone-speciﬁc variabilities in prophage induction. Based on the induction proﬁles, two representative strains were chosen for further analysis, where cell density and MOI were examined to evaluate their role in prophage induction. Regarding cell density, conditioned media from three different cell densities were applied for each strain as described above, and samples were collected for optical density, cfu counts, pfu counts and gene expression analysis after 9, 24, 48 and 72 h. 4.7. Quantiﬁcation of Phage and Bacteria Dynamics At each sampling point in the experiments above, several samples were taken and processed from all replicates in different conditions to assess optical density, bacterial cell counts, infective phage counts, prophage integration, and selected gene expression levels. Details of the applied methods are described in the Methods supplementary section. 4.5. Resistance Development and Lysogenization Assay The rates of resistance and lysogenization, as well as potential factors that may affect them, were explored during NO16 phage and V. anguillarum strain A023 infection process, following an extensive clone picking experiment. V. anguillarum strain A023 was infected at the early exponential phase by NO16 phage (MOI: 0.1) in triplicate, and four samplings at 4, 24, 48 and 72 h post infection were performed. To separate the aggregated cells from the free-living cells, mild centrifugation (500× g for 2 min) was applied to determine the role of the bacterial habitat (aggregated or free-living) on the fraction of resistant and lysogenic cells. At each sampling point, bacteria from both swimming and aggregate samples were spread on marine agar plates for isolation of the individual colonies. A total of ~1500 isolates were transferred to 96-well plates with marine broth for subsequent screening for lysogenization and resistance against phage NO16. Prophage integration was assessed by examining the supernatant of overnight cultures of each isolate for lytic activity against a fresh bacterial lawn of V. anguillarum strain A023, using the double agar layer method [71]. Resistance of each individual isolate was likewise tested with spot assay using NO16 phage. All phage-sensitive isolates were recultured, and the efﬁciency of plating (EOP) was determined [71] as a measure of the various sensitivity levels that NO16 may cause in the bacterial population. Furthermore, samples were collected for optical density, pfu counts, cfu counts and light electron microscopy at all time points. Int. J. Mol. Sci. 2023, 24, 3937 18 of 23 18 of 23 4.6. Bacteriophage Dynamics: Prophage Integration and Spontaneous Induction 4.10. Statistics Statistical analysis was performed to assess signiﬁcant differences among different conditions and treatments that were tested in gene expression analysis, bioﬁlm formation, in vitro and in vivo challenges. One-way and two-way ANOVA were performed following assessment of normality of the data distribution (signiﬁcance: p-value < 0.05). All statistical analyses were performed with SigmaPlot, version 14.0, Systat Software, Inc., San Jose, CA, USA. 4.9. In Vivo Challenges of Fish Larvae to Assess Lysogenic Conversion In vivo trials were performed using a previously described ﬁsh larvae method, ideal for bacterial virulence assays [40]. Brieﬂy, good quality eggs of gilthead seabream, Sparus au- rata, were collected and placed individually in 24-well plates (Cellstar, Greiner Bio-One, Kremsmünster, Austria). A total of 336 eggs in 14 plates were prepared and divided into three treatments (3 plates × 24 healthy eggs) and one control (5 plates × 24 healthy eggs). In the three treatments, the eggs were challenged with 106 cells per ml (high bacterial dose) fresh overnight culture of the wild type V. anguillarum A023 and two lysogenized clones, respectively, whereas sterile SM buffer was added to the control plates. The plates were incubated at 18 ◦C and egg and larval mortality were monitored for seven consecutive days post infection (d.p.i.). Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijms24043937/s1. References [43,58,79–90] are cited in the sup- plementary materials. 4.8. Bioﬁlm Formation in Lysogens and Non-Lysogens 4.8. Bioﬁlm Formation in Lysogens and Non-Lysogens The potential for bioﬁlm formation was tested in 15 different strains: the wild type V. anguillarum strain A023, and seven lysogenized and seven non-lysogenized clones obtained after NO16 infection. Additionally, bioﬁlm formation was also examined when V. anguillarum strain A023 was exposed to four different conditions: low and high MOI (0.01 Int. J. Mol. Sci. 2023, 24, 3937 19 of 23 19 of 23 and 10), bacteria-free supernatant and phage-free supernatant of lysed culture, to assess the role of potential signal molecules or other extracellular metabolites that may be produced during bacteria and phage proliferation [76]. Bacteria-free supernatant was retrieved after the 0.22 µm ﬁltration of an overnight V. anguillarum strain A023. Phage-free supernatant was harvested after ﬁltering overnight the V. anguillarum strain A023 and phage NO16 infected culture, ﬁrst through 0.22 µm and then through Amicon (3 kDa, Milipore, Burlington, VT, USA). The bioﬁlm formation protocol was based on the previously reported crystal violet method [59] modiﬁed to 96-well plates with 8 replicate wells for each condition. The ﬁnal absorbance was measured at 595 nm using a TECAN Inﬁnite Pro 200 microplate reader. 5. Conclusions The prevalence of vibriophages that coexist inside virulent Vibrio genomes was ex- tensively documented in several important human and animal pathogens [3,48,58,69], highlighting the importance of temperate phages for the functional and genetic diversity of this bacterial genus. The increased bioﬁlm formation and virulence of NO16 prophage lysogens are direct evidence for lysogenic conversion [77], being properties that are af- fected by phage exposure [78]. The discovery of a new family of temperate phages, the “Asemoviridae”, with a global high prevalence and ubiquitous distribution across the Vibrio genus, having signiﬁcant implications for host functional properties, have further emphasized the importance of DJR phages as mobile elements in marine microbial com- munities. The observed indications of the cell density dependent regulation of phage–host interactions, lysogenic conversion through bioﬁlm formation and virulence, as well as the intra- and interspeciﬁc variabilities, represent key topics for further investigation to resolve the ecological and evolutionary role of the asemoviruses across marine bacterial genera. Updating viral genomic and metagenomic databases, isolation and cultivation of more “Asemoviridae” phage–host systems, and more targeted approaches on host and prophage transcriptome under speciﬁc infection conditions are future work that could provide crucial insights and further elucidate the role of these widespread “cryptic” viral elements. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijms24043937/s1. References [43,58,79–90] are cited in the sup- plementary materials. 20 of 23 20 of 23 Int. J. Mol. Sci. 2023, 24, 3937 Author Contributions: Conceptualization, P.G.K. and M.M.; Methodology, P.G.K., J.J.M., S.M., A.M., M.I.T., P.K. and M.M.; Software, P.G.K. and A.M.; Validation, P.G.K.; Formal analysis, P.G.K., J.J.M., C.S.W.-H., S.M., A.M. and M.M.; Investigation, P.G.K., J.J.M., C.S.W.-H., M.I.T., P.K. and M.M.; Resources, A.M., P.K. and M.M.; Data curation, P.G.K.; Writing—original draft, P.G.K. and M.M.; Writing—review & editing, P.G.K., J.J.M., C.S.W.-H., S.M., P.K. and M.M.; Visualization, P.G.K.; Supervision, M.M.; Project administration, M.M.; Funding acquisition, P.G.K. and M.M. All authors have read and agreed to the published version of the manuscript. Funding: The study was supported by grants from the Danish Council for Independent Research (Project # DFF-7014–00080), Danish National Research Foundation through the Danish Center for Hadal Research, HADAL (No. DNRF14), and from the University of Copenhagen, Department of Biology as a PhD stipend granted to JJM. Experimental work on ﬁsh larvae took place at the facilities of Hellenic Center for Marine Research in the frames of AquaExcel 2020 project (https: //www.aquaexcel2020.eu/) with PID: AE130019. 5. Conclusions Data Availability Statement: All generated and analyzed data are presented in the manuscript, its supplementary materials and are publicly available in open access databases. Acknowledgments: We thank Sachia Jo Traving for help with strain isolation and quantiﬁcation of lysogens, Sine Lo Svenningsen for providing the spike-in strain, and Øjvind Moestrup for support with the TEM facility. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References Bacteriophage lysis: Mechanism and regulation. Microbiol. Rev. 1992, 56, 430–481. [CrossRef] 8. Oppenheim, A.B.; Kobiler, O.; Stavans, J.; Court, D.L.; Adhya, S. Switches in Bacteriophage Lambda Development. Annu. Rev. 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https://openalex.org/W3208425325	https://europepmc.org/articles/pmc8555205?pdf=render	English	null	RaDiCo, the French national research program on rare disease cohorts	Orphanet journal of rare diseases	2,021	cc-by	9,787	Abstract Background: Rare diseases (RDs) affect nearly 3 million people in France and at least 26–30 million people in Europe. These diseases, which represent a major medical concern, are mainly of genetic origin, often chronic, progressive, degenerative, life threatening and disabling, accounting for more than one third of all deaths occurring during infancy. In this context, there are needs for coordinated information on RDs at national/international levels, based on high quality, interoperable and sharable data. The main objective of the RaDiCo (Rare Disease Cohorts) program, coor- dinated by Inserm, was the development of RD e-cohorts via a national platform. The cohort projects were selected through a national call in 2014. The e-cohorts are supported by an interoperable platform, equivalent to an infrastruc- ture, constructed on the "cloud computing" principle and in compliance with the European General Data Protection Regulation. It is dedicated to allow a continuous monitoring of data quality and consistency, in line with the French Health Data Hub. Results: Depending on cohorts, the objectives are to describe the natural history of the studied RD(s), identify the underlying disease genes, establish phenotype-genotype correlations, decipher their pathophysiology, assess their societal and medico-economic impact, and/or identify patients eligible for new therapeutic approaches. Inclusion of prevalent and incident cases started at the end of 2016. As of April 2021, 5558 patients have been included within 13 RD e-cohorts covering 67 diseases integrated in 10 European Reference Networks and contributing to the European Joint Program on RDs. Several original results have been obtained in relation with the secondary objectives of the RaDiCo cohorts. They deal with discovery of new disease genes, assessment of treatment management, deciphering the underlying pathophysiological mechanisms, diagnostic approaches, genotype–phenotype relationships, develop- ment and validation of questionnaires relative to disease burden, or methodological aspects. Conclusion: RaDiCo currently hosts 13 RD e-cohorts on a sharable and interoperable platform constructed on the “cloud computing” principle. New RD e-cohorts at the European and international levels are targeted. Keywords: Rare diseases, e-Cohorts, Mutualized platform, Cloud computing, Infrastructure-as-a service, GDPR, Interoperability, Health Data Hub, European Reference Networks, Rare Disease European Joint Program used, it is estimated that there are 7000 to 8000 RDs. Taken as a whole, RDs affect at least 26–30 million people in Europe. These diseases involve children in about 75% of cases. © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract They are often chronic, progressive, degenerative, life threatening and disabling, accounting for more than one third of all deaths occurring during infancy. A large number of these diseases lead to a signifi- cant decrease of life expectancy and most of them have a Open Access Background Rare Diseases (RDs) represent a major health care issue. A disease is called rare in Europe when it affects fewer than 5 in 10,000 persons. Depending on the definition *Correspondence: paul.landais@umontpellier.fr 1 RaDiCo, Inserm, Trousseau Hospital, Paris, France Full list of author information is available at the end of the article Amselem et al. Orphanet J Rare Dis (2021) 16:454 https://doi.org/10.1186/s13023-021-02089-5 Amselem et al. Orphanet J Rare Dis (2021) 16:454 https://doi.org/10.1186/s13023-021-02089-5 Coordinated care and research on RDs appear critical for patients and families, in order to better describe the natural history of the disease, to improve the diagnostic procedures, to decipher the underlying pathophysiologi- cal mechanisms and to better stratify patients for tar- geted clinical trials and treatments through personalized medical approaches. It is also expected that research on RDs will increase our understanding of the pathophysi- ology of common chronic diseases, as RDs often repre- sent a “model of dysfunction” severely affecting a limited number of biological pathways. One main objective of the 3rd RD Plan (PNMR3, (2018–2022) [5], in line with the International Rare Dis- eases Research Consortium [6], was to provide an accu- rate diagnosis within a year of the first specialty medical consultation. The PNMR3, created 109 RD Coordinating Centers (RDCC) associated to 386 constitutive centers and 1840 competence centers, and reinforced the links with European research initiatives on RDs. It also aims at strengthening the role of RaDiCo in integrating RDs research data (PNMR3, Action 11.4). A need for launching RD cohorts. In this fast-mutating context, RD professionals highlighted the critical need to implement nation-wide, multidisciplinary, high-qual- ity cohort studies in order to address key scientific and medico-economic questions. In this context, the request was high to get access to appropriate resources, meth- ods and tools for collecting RD data at the (inter)national level. Given the specificities of RDs—limited number of patients per country, scarcity of relevant knowledge and expertise, and fragmentation of research—they have been considered as a distinctive domain of very high national and European added value. A support- ing research program was therefore required to provide essential information on disease history and characteris- tics, and to foster the identification of underlying molec- ular mechanisms, genotype/phenotype correlations. New knowledge would ultimately lead to better targeted care and treatments. The structuring boost given by the PNMR2 was considered as an opportunity to make col- lective efforts for building RD cohorts and therefore to propose the RaDiCo (Rare Disease Cohorts) project to the national call on cohorts of the first Investments for the Future Program (https://anr.fr/en/investments-for- the-future/investments-for-the-future/) launched by the French Ministry of Research in 2010 [7]. Identifying the locks is essential. © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Amselem et al. Orphanet J Rare Dis (2021) 16:454 Amselem et al. Orphanet J Rare Dis (2021) 16:454 Page 2 of 12 Twenty University genetic laboratories were equipped with the Next Generation Sequencing technology for clinical use. In 2014, RD Reference and Competence Centers were grouped into 23 thematic RD Healthcare Networks (RDHNs) (https://solidarites-sante.gouv.fr/ soins-et-maladies/prises-en-charge-specialisees/malad ies-rares/article/l-offre-de-soins). This move anticipated the 23 RD European Reference Networks (ERNs) that were launched at the end of 2016 (https://ec.europa.eu/ health/ern_en). The RDHNs coordinate diagnosis, pro- vision of health, social care and training; they collect healthcare data, develop research programs and write National Protocols for Diagnosis and Care. significant impact on patients’ quality of life and health care systems. This is a major medical concern since, for most of these diseases, there is no available cure. RDs are mainly of genetic origin. At the date of writing this article, 4425 genes underlying 6868 disease phenotypes have been discovered (https://www.omim.org/statistics/ geneMap), but the genes underlying a number of known Mendelian phenotypes are still unknown, and addi- tional Mendelian conditions have yet to be recognized. Thus, there are needs for high quality, interoperable and sustainable creation and monitoring of (inter)national cohorts of patients with rare diseases. Then the analysis of such observational data has to take into account prospective as well as retrospective information. As already outlined, retrospective data are more bias-sensitive than prospective data and this must be considered in the analysis (https://www.strobe-state ment.org). On the other hand, to reach these scientific objectives, another goal was to build a national opera- tional platform, equivalent to an Infrastructure as a Ser- vice, for implementing a potentially unlimited number of e-cohorts consisting of prevalent and incident cases. genes; (iii) Establish genotype–phenotype correlations; (iv) Decipher the underlying pathophysiological mecha- nisms; (v) Identify new therapeutic avenues; (vi) Estimate their societal and medico-economic impact; (vii) Identify patients eligible for new therapeutic approaches; (viii) Define a methodological strategy of analysis for cohorts which recruit both prevalent and incident cases, that require adapted modeling and bias analyses. In a rare disease setting, cases are either prevalent (already known with a past history) or incident (recently discovered). Most patients are prevalent since diseases by definition are rare. Then the analysis of such observational data has to take into account prospective as well as retrospective information. As already outlined, retrospective data are more bias-sensitive than prospective data and this must be considered in the analysis (https://www.strobe-state ment.org). On the other hand, to reach these scientific objectives, another goal was to build a national opera- tional platform, equivalent to an Infrastructure as a Ser- vice, for implementing a potentially unlimited number of e-cohorts consisting of prevalent and incident cases. PNMR2 also fostered the development of the National RD Data Bank (“Banque Nationale de Données Maladies Rares” (BNDMR), Fig. 1) built as part of the CEMARA initiative [8], which started in 2007 at Necker Enfants- Malades Hospital (AP-HP).The BNDMR, which is dedi- cated to public health issues, aims at collecting general epidemiological and public health data on all patients with a RD in France, on the basis of a common RD mini- mum data set [9] and a unique identifier [10] for each RD patient.h PNMR2 also fostered the development of the National RD Data Bank (“Banque Nationale de Données Maladies Rares” (BNDMR), Fig. 1) built as part of the CEMARA initiative [8], which started in 2007 at Necker Enfants- Malades Hospital (AP-HP).The BNDMR, which is dedi- cated to public health issues, aims at collecting general epidemiological and public health data on all patients with a RD in France, on the basis of a common RD mini- mum data set [9] and a unique identifier [10] for each RD patient.h The RD cohorts also had to integrate, whenever appro- priate, non-French RD expert centers and patients, in order to overcome the low number of patients in one sin- gle country with respect to the sample sizes needed for proper statistical power, thereby anticipating the emer- gence of the future RD European Research Networks. Yet, RD clinical man- agement and research in France and Europe have been hampered by a lack of resources at several levels: few sci- entists work on only one given disease; few patients per disease and patients are scattered over large geographic areas, causing difficulties in gathering data on their dis- ease; existing databases as well as biological collections, when existing, are usually local, small, incomplete, not always quality-controlled, of heterogeneous formats and contents, and are rarely accessible or standardized for allowing interoperability; phenomes are often complex and partially described along with time, with insufficient interdisciplinary cooperation.h y The need to promote networks of expertise for RDs in order to improve both RD clinical care and research has been considered a priority in France since 1995. Indeed, RDs occupy an important place in public health in France: Orphanet, an informational RD website and a directory of expert services was launched in 1997 [1] by the Health Ministry; the French federation of patients’ organizations (“Alliance Maladies Rares”), was launched in February 2000 [2]; and the RD Scientific Interest Group was created to fund research in the field of RDs. The creation of the first French National Rare Disease Plan (PNMR1, 2005–2008) [3] allowed access to high quality care, and treatment was facilitated by the crea- tion, at the national level, of RD Reference Centers and Competence Centers. The second Plan (PNMR2, 2011– 2016) consolidated previous achievements, aiming at reinforcing national and international cooperation [4]. The objective of the RaDiCo project was twofold. On one hand, the scientific objective was to set up several RD e-cohorts with the following aims according to the idiosyncrasy of each cohort: (i) Describe the natural his- tory of the targeted RDs; (ii) Identify the disease-causing Amselem et al. Orphanet J Rare Dis (2021) 16:454 Amselem et al. Orphanet J Rare Dis (2021) 16:454 Page 3 of 12 genes; (iii) Establish genotype–phenotype correlations; (iv) Decipher the underlying pathophysiological mecha- nisms; (v) Identify new therapeutic avenues; (vi) Estimate their societal and medico-economic impact; (vii) Identify patients eligible for new therapeutic approaches; (viii) Define a methodological strategy of analysis for cohorts which recruit both prevalent and incident cases, that require adapted modeling and bias analyses. In a rare disease setting, cases are either prevalent (already known with a past history) or incident (recently discovered). Most patients are prevalent since diseases by definition are rare. Resultsh Thirty-three letters of intent were received after the pub- lication of the RaDiCo call for RD cohorts (see Material and Methods). This call led to select 16 national and/or European RD cohort projects on July 15th, 2014. Among these, 3 have been discontinued, secondary to decisions Such cohort projects had to be closely articulated with the above-mentioned established networks on RDs. The Fig. 1 French RD Healthcare Networks, BNDMR (RD National Databank) and RaDiCo Fig. 1 French RD Healthcare Networks, BNDMR (RD National Databank) and RaDiCo Fig. 1 French RD Healthcare Networks, BNDMR (RD National Databank) and RaDiCo Amselem et al. Orphanet J Rare Dis (2021) 16:454 Page 4 of 12 of the Scientific and Plenary committees of the RaDiCo program (see Material and Methods): after demand from the principal investigators (PIs) for two of them, since the cohorts had not started, and another in 2019 because it could not start in due time. The groups of RDs targeted by the 13 current cohorts appear in Table 1. the inclusions and the inclusion targets are mentioned. All cohorts are multicentric, mainly national but also European (SEDVasc and ECYSCO cohorts, see Table 2) and international (GenIDA). For all the cohorts, a total of 11,650 included patients are expected at the end of the inclusion period (July 2027). As of April 2021, 5558 patients had been included into 13 RD e-cohorts, cov- ering 67 diseases from ~ 300 affiliated expert centers (Fig. 2). Each implementation step of the e-cohorts is presented in Fig. 3. the inclusions and the inclusion targets are mentioned. All cohorts are multicentric, mainly national but also European (SEDVasc and ECYSCO cohorts, see Table 2) and international (GenIDA). For all the cohorts, a total of 11,650 included patients are expected at the end of the inclusion period (July 2027). As of April 2021, 5558 patients had been included into 13 RD e-cohorts, cov- ering 67 diseases from ~ 300 affiliated expert centers (Fig. 2). Each implementation step of the e-cohorts is presented in Fig. 3. The general framework of the RaDiCo cohorts is presented below (Table 2), with or without associ- ated biocollections at each site, together with the planned inclusion and follow-up period. The start of planned inclusion and follow-up period. The start of presented in Fig. 3. Resultsh Table 1 The 13 ongoing RaDiCo e-cohorts (acronyms in alphabetic order) a RDNH: Rare Disease Healthcare Network b ERN: European Research Network Cohorts’ acronyms Diseases RDHNa Corresponding ERNb AC-ŒIL Congenital eye defects SENSGENE AnDDI-Rares ERN-Eye AcoStill Still’s disease FAi2R ERN-Rita COLPAC LPAC syndrome FILFOIE RARE-LIVER DCP Primary ciliary dyskinesia RESPIFIL ERN-Lung ECYSCO Cystinosis ORKiD ERKnet EURBIO-Alport Alport syndrome ORKiD ERKnet FARD Rare skin diseases burden FIMARAD ERN Skin GenIDA Intellectual deficiency and Autism spectrum disorders DéfiScience AnDDi-Rares ERN ITHACA IDMet Imprinting disorders FIRENDO OSCAR DéfiScience ENDO-ERN MPS Mucopolysaccharidoses G2M MetabERN PID Idiopathic interstitial pneumonia RESPIFIL ERN-Lung PP Periodic paralysis FILNEMUS ERN euro-NMD SEDVasc Vascular Ehlers-Danlos syndrome FAVA-Multi VASCERN Table 2 RaDiCo cohorts: general framework RaDiCo-Cohort Acronyms Biocollections Inclusion period (years) Follow-up period (years) Start of inclusions Inclusion targets AC-ŒIL Diagnosis 10 10 Jul.-2017 800 AcoStill Yes 5 5 Dec.-2017 500 COLPAC Yes 3 5 Nov.-2017 550 DCP Diagnosis 5 5 May-2017 700 ECYSCO Diagnosis 2 2 Apr.-2017 400 EURBIO Alport Yes 2 3 May-2017 1000 FARD Yes 4 5 Mar.-2018 900 GenIDA No 5 5 Nov.-2016 1000 IDMet Yes 5 10 Mar.-2018 2000 MPS Yes 2 5 Dec.-2017 1000 PID Diagnosis 5 5 Nov.-2017 2400 PP No 1 1.5 Jun.-2019 60 SEDVasc Diagnosis 4 3 Dec.-2016 340 Expected total inclusions 11,650 Table 1 The 13 ongoing RaDiCo e-cohorts (acronyms in alphabetic order) a RDNH: Rare Disease Healthcare Network b ERN: European Research Network Cohorts’ acronyms Diseases RDHNa Corresponding ERNb AC-ŒIL Congenital eye defects SENSGENE AnDDI-Rares ERN-Eye AcoStill Still’s disease FAi2R ERN-Rita COLPAC LPAC syndrome FILFOIE RARE-LIVER DCP Primary ciliary dyskinesia RESPIFIL ERN-Lung ECYSCO Cystinosis ORKiD ERKnet EURBIO-Alport Alport syndrome ORKiD ERKnet FARD Rare skin diseases burden FIMARAD ERN Skin GenIDA Intellectual deficiency and Autism spectrum disorders DéfiScience AnDDi-Rares ERN ITHACA IDMet Imprinting disorders FIRENDO OSCAR DéfiScience ENDO-ERN MPS Mucopolysaccharidoses G2M MetabERN PID Idiopathic interstitial pneumonia RESPIFIL ERN-Lung PP Periodic paralysis FILNEMUS ERN euro-NMD SEDVasc Vascular Ehlers-Danlos syndrome FAVA-Multi VASCERN Table 1 The 13 ongoing RaDiCo e-cohorts (acronyms in alphabetic order) Table 2 RaDiCo cohorts: general framework Table 2 RaDiCo cohorts: general framework RaDiCo-Cohort Acronyms Biocollections Inclusion period (years) Follow-up period (years) Start of inclusions Inclusion targets AC-ŒIL Diagnosis 10 10 Jul.-2017 800 AcoStill Yes 5 5 Dec.-2017 500 COLPAC Yes 3 5 Nov.-2017 550 DCP Diagnosis 5 5 May-2017 700 ECYSCO Diagnosis 2 2 Apr.-2017 400 EURBIO Alport Yes 2 3 May-2017 1000 FARD Yes 4 5 Mar.-2018 900 GenIDA No 5 5 Nov.-2016 1000 IDMet Yes 5 10 Mar.-2018 2000 MPS Yes 2 5 Dec.-2017 1000 PID Diagnosis 5 5 Nov.-2017 2400 PP No 1 1.5 Jun.-2019 60 SEDVasc Diagnosis 4 3 Dec.-2016 340 Expected total inclusions 11,650 Amselem et al. Resultsh Moreover, it contributed to the design of the Information System of the Inserm “France Cohortes” program designed to support not only RD e-cohorts, but also cohorts of patients with common multifactorial disorders, as well as population-based epi- demiological cohorts. development and validation of questionnaires relative to the diseases burden, or methodological aspects (Table 3). the diseases burden, or methodological aspects (Table 3). Discovery of new disease genes—Four genes, i.e., TTC12 [11], GAS2L2 [12], DNAH9 [13] and DNAJB13 [14], whose mutations are responsible for Primary Ciliary Dyskinesia (PCD) have been identified through molecu- lar and cellular studies performed in the framework of the RaDiCo-DCP cohort. This cohort has been built on the deep phenotyping of the patients, which includes the ultrastructural defects of the microtubule-based struc- ture of motile cilia and sperm flagella (i.e., the ciliary and flagellar axonemes). These organelles contain sev- eral dynein arms, each of them consisting of multipro- tein complexes that carry an ATPase activity required for ciliary/flagellar motility. Specific phenotypes have been associated with mutations in these new genes that code for different classes of proteins. TTC12 is believed to be a co-chaperone involved in the cytoplasmic pre-assem- bly of dynein arms [11]; the lack of GAS2L2 causes PCD by impairing cilia orientation and mucociliary clear- ance [12], whereas DNAH9 encodes one of the axone- mal dynein chains [13]. As for DNAJB13, it encodes an HSP40 family member involved in the proper building of the ciliary and flagellar axoneme [14]. In addition, besides This platform fulfills the necessary requirements of an Information System. It has been positively assessed by an independent external auditor. Moreover, it contributed to the design of the Information System of the Inserm “France Cohortes” program designed to support not only RD e-cohorts, but also cohorts of patients with common multifactorial disorders, as well as population-based epi- demiological cohorts. Primary objectives of the RD e-cohorts are not yet achieved. This situation mainly concerns the description of the natural history of the targeted RD(s). According to the study protocol of each cohort, this objective relies on the follow-up of each patient enrolled in a given cohort. Thus we have to wait for the data freeze procedure to be allowed to analyze the data collected in the purpose of exploring the primary endpoint. In contrast, this is not the case for the secondary objectives. Resultsh Orphanet J Rare Dis (2021) 16:454 Page 5 of 12 Fig. 2 Geographical distribution of RDs Coordinating Centres contributing to RaDiCo e-cohorts (France (blue); other European countries (orange) Fig. 2 Geographical distribution of RDs Coordinating Centres contributing to RaDiCo e-cohorts (France (blue); other European countries (orange) Fig. 3 RaDiCo cohorts’ preparatory work and 2014–2021 follow-up (CCTIRS, «Comité Consultatif pour le Traitement de l’Information en Recherche en Santé»; CNIL «Commission Nationale Informatique et Libertés») Fig. 3 RaDiCo cohorts’ preparatory work and 2014–2021 follow-up (CCTIRS, «Comité Consultatif pour le Traitement de l’Information en Recherche en Santé»; CNIL «Commission Nationale Informatique et Libertés») Amselem et al. Orphanet J Rare Dis (2021) 16:454 Page 6 of 12 Amselem et al. Orphanet J Rare Dis (2021) 16:454 development and validation of questionnaires relative to the diseases burden, or methodological aspects (Table 3). Discovery of new disease genes—Four genes, i.e., TTC12 [11], GAS2L2 [12], DNAH9 [13] and DNAJB13 [14], whose mutations are responsible for Primary Ciliary Dyskinesia (PCD) have been identified through molecu- lar and cellular studies performed in the framework of the RaDiCo-DCP cohort. This cohort has been built on the deep phenotyping of the patients, which includes the ultrastructural defects of the microtubule-based struc- ture of motile cilia and sperm flagella (i.e., the ciliary and flagellar axonemes). These organelles contain sev- eral dynein arms, each of them consisting of multipro- tein complexes that carry an ATPase activity required for ciliary/flagellar motility. Specific phenotypes have been associated with mutations in these new genes that code for different classes of proteins. TTC12 is believed to be a co-chaperone involved in the cytoplasmic pre-assem- bly of dynein arms [11]; the lack of GAS2L2 causes PCD by impairing cilia orientation and mucociliary clear- ance [12], whereas DNAH9 encodes one of the axone- mal dynein chains [13]. As for DNAJB13, it encodes an HSP40 family member involved in the proper building of the ciliary and flagellar axoneme [14]. In addition, besides The electronic cohorts (e-cohorts) were supported by a platform on which RaDiCo’s work system was developed. Its activities was devoted to capturing, transmitting, stor- ing, retrieving, manipulating and displaying information. This platform fulfills the necessary requirements of an Information System. It has been positively assessed by an independent external auditor. Resultsh They deal with discovery of new disease genes, assessment of treatment management, deciphering the pathophysiology and diag- nostic approaches, genotype–phenotype relationships, Table 3 International publications so far associated to the RaDiCo program Discovery of new disease genes TTC12 loss-of function mutations cause primary ciliary dyskinesia and unveil distinct dynein assembly in motile cilia vs. flagella [11] Lack of GAS2L2 causes primary ciliary dyskinesia by impairing cilia orientation and mucociliary clearance [12] Mutations in outer dynein arm heavy chain DNAH9 cause motile cilia defects and situs inversus [13] Mutations in DNAJB13, encoding an HSP40 family member, cause primary ciliary dyskinesia and male infertility [14] de novo missense variants in FBXW11, a gene that encodes an F-box protein involved in ubiquitination and proteosomal degradation [15] Assessment of treatment management Vascular Ehlers-Danlos syndrome – Long-term observational study [16] Pathophysiology and diagnostic approaches Accuracy of clinical diagnostic criteria for patients with vascular Ehlers-Danlos syndrome in a tertiary referral centre [17] Functional assessment and phenotypic heterogeneity of SFTPA1 and SFTPA2 mutations in interstitial lung diseases and lung cancer [18] Pulmonary fibrosis in children [19] Chronic interstitial lung diseases in children: diagnosis approaches [20] Pulmonary hemosiderosis in children with Down syndrome: a national experience [21] Paediatric sarcoidosis [22] Genetic causes and clinical management of pediatric interstitial lung diseases [23] Genotype–phenotype relationships Infertility in an adult cohort with primary ciliary dyskinesia: phenotype-gene association [24] Primary ciliary dyskinesia gene contribution in Tunisia: Identification of a major Mediterranean allele [25] Alport syndrome: a unified classification of genetic disorders of collagen IV α345 [26] Genetics of anophthalmia and microphthalmia. Discovery of new disease genes Resultsh Part 1: Non-syndromic anophthalmia/microphthalmia [27] Development and validation of burden questionnaires and Quality of life Burden of albinism: development and validation of a burden assessment tool [28] Burden of adult neurofibromatosis 1: development and validation of a burden assessment tool [29] Health-related quality of life in infants and children with interstitial lung disease [30] Methodological aspects Federating patients identities: the case of rare diseases [10] Cerberus, an access control scheme for enforcing least privilege in patient cohort study platforms: [31] National registries of rare diseases in Europe: an overview of current situation and experiences [32] Recommendations for improving the quality of rare disease registries [33] Data quality in rare diseases registries [34] Table 3 International publications so far associated to the RaDiCo program Burden of albinism: development and validation of a burden assessment tool [28] Burden of adult neurofibromatosis 1: development and validation of a burden assessment tool [29] Health-related quality of life in infants and children with interstitial lung disease [30] Federating patients identities: the case of rare diseases [10] Cerberus, an access control scheme for enforcing least privilege in patient cohort study platforms: [31] National registries of rare diseases in Europe: an overview of current situation and experiences [32] Recommendations for improving the quality of rare disease registries [33] Data quality in rare diseases registries [34] Amselem et al. Orphanet J Rare Dis (2021) 16:454 Page 7 of 12 identities of RD patients [10]. Patient information in RD registries is generally collected as de-identified data from numerous sources, requiring the data to be federated. Transforming nominative data into de-identified data is thus a key issue, while minimizing the number of iden- tity duplicates. We proposed a method enabling patient identity federation and RD data de-identification while preserving the pertinence of the provided data with a RD patient identifier. the identification of those new molecular causes of PCD, one of the key results obtained through functional stud- ies performed on both patients’ primary airway epithelial cells (AECs) and CRISPR-Cas9-edited human primary AECs is the existence of distinct dynein assembly mech- anisms in human motile cilia versus flagella [11]. As for the developmental eye defects reported in patients from the RaDiCo-AC-OEIL cohort, de novo missense variants have been identified in FBXW11, a gene that encodes an F-box protein involved in ubiquitination and proteoso- mal degradation [15]. Resultsh i A critical issue for cohort information platforms is to enforce strict control on access privileges and particu- larly for patients themselves. Cerberus, a comprehen- sive access control Scheme was designed to provide an access control scheme that covers design, implementa- tion, deployment and maintenance operations [31]. It enables a targeted access to the RaDiCo GENIDA infor- mation platform by patients themselves who can cap- ture their own data, or via an access to targeted medical data through explicit authorization by the study sponsor, when required, to practitioners and/or researchers. Assessment of treatment management—From a thera- peutic viewpoint, as shown in the RaDiCo-SEDVasc cohort, which is dedicated to patients with a rare genetic connective tissue disorder called vascular Ehlers-Danlos syndrome, the assessment of treatment management has revealed the impact of different therapies on morbid- ity and mortality. Indeed, in this disease condition due to mutations in COL3A, it has been shown that patients treated with celiprolol —a beta blocker— had a better survival than those not treated with celiprolol and that the observed reduction in mortality was dose-dependent [16]. Together with other European colleagues, we proposed an overview of the current situation and experiences of the national RD registries in Europe [32] and suggested recommendations for improving the quality of RD reg- istries [33, 34]. These two papers illustrated part of the design of our e-Health approach. Moreover, RaDiCo is involved in the French PNMR3, notably in its 11.4 Action that reinforces the role of RaDiCo related to the integra- tion of RD research data while developing operational links with the national RD databank by offering techni- cal and ethical-regulatory support for the setting up by the RD health networks of new interoperable data ware- houses and by studying the possibility of accommodating them on the RaDiCo platform. We also established oper- ational links with the data platform of the European Joint Program on RD [35] and the ERNs currently being set up. Resultsh Pathophysiology and diagnostic approaches—Several aspects were explored: Accuracy of clinical diagnostic criteria for patients with vascular Ehlers-Danlos syn- drome in a tertiary reference center for the RaDiCo-SED- Vasc cohort [17]; In the RaDiCo-PID cohort, functional assessment and phenotypic heterogeneity of SFTPA1 and SFTPA2 mutations in interstitial lung diseases and lung cancer [18]; Pulmonary fibrosis in children [19]; Diagno- sis approaches of chronic interstitial lung diseases in chil- dren: [20–22]; or genetic causes and clinical management of pediatric interstitial lung diseases [23]. Genotype–phenotype relationships – For the RaDiCo- DCP cohort, infertility in an adult cohort with primary ciliary dyskinesia [24]; Identification of a major Mediter- ranean allele in primary ciliary dyskinesia gene [25]. For the RaDiCo-EURBIO Alport cohort, a unified classifica- tion of genetic disorders of collagen IV α345 in Alport syndrome [26]. For the RaDiCo AC-OEIL cohort, review of the genetics of non-syndromic anophthalmia/micro- phthalmia [27]. Discussionh the ethical and regulatory authorizations, as well as the evolving regulations both at the EU level, with the new European regulation on personal data safety and security (GDPR) [36], and at the national level, with modifications of the law on research involving human subjects (French «Jardé law») [37]. Moreover, a consortium agreement had to be signed between the partners for formalizing legal links between them, setting the modes of governance of the project on scientific, strategic and operational plans, and outlining the scientific valorization in terms of intel- lectual property, publications and citations of the pro- ject. A set of 19 Key Performance Indicators (KPIs) was implemented to enforce the cohorts’ follow-up (Table 4). Once initiated, the 13 ongoing RD cohorts progressed appropriately and the first results related to the second- ary objectives of several cohorts have been published in international peer-reviewed journals. to collect data from deceased children. We implemented solutions through identifying key data and prioritiza- tion of the fields related to the main objectives to be completed by the clinical research technicians with sup- port from residents for filling the medical information. Clinical research technicians have been recruited by the RDHNs and specific resources have been obtained through targeted research projects supported by Public– Private Partnerships or applications to specific grants. i We explored the impact of RaDiCo for the medical community. Based on current information provided by all the cohorts’ participating teams, the program should contribute to significant improvements in patients’ care and outcome. Indeed, the design of the cohorts is made in such a way that it allows structured collection of dis- ease symptoms at various stages of the pathological pro- cesses. Expected benefits include better knowledge of the natural history of all the investigated RDs, recognition of relevant comorbidities, novel proposals for healthcare management, and, ultimately, better quality of life for the patients and their families. In line with the objectives specifically defined for each cohort, other expectations are: identification of relevant disease biomarkers for diag- nosis, disease severity and exacerbations; patient selec- tion for clinical and therapeutic trials; and production of novel quality of life questionnaires. The development of electronic health tools is also among the secondary objectives of several cohorts. These tools are designed to help patients to self-report their symptoms as well as The costs and management of informatics providers and Clinical Research Organizations are important. Discussionh The RaDiCo program is intended to promote, through the support of RD e-cohorts’ projects, the collection of phenotypic data for epidemiological and clinical research purposes in connection with basic and translational research. To this end, we implemented an operational team (cf. Material and Methods section) with the mis- sion to establish a centralized platform of expert services and tools to ensure installation and follow-up of the RD e-cohorts, via consulting services, for instance legal & regulatory services, or clinical research quality tools, development and provision of innovative information technology tools (electronic case report forms (eCRF), interoperability solutions, or access to e-health tools).h Development and validation of burden questionnaires and quality of life study—Two questionnaires were vali- dated in order to support the RaDiCo–FARD cohort: for albinism [28], and adult neurofibromatosis 1 [29]: development and validation of burden assessment tools. Health-related quality of life was explored in infants and children with interstitial lung disease [30]. Methodological aspects – Several tools for harmoniz- ing our approaches in our own country and in Europe have been developed. We defined a way to federating the The preparatory period of the cohorts was slowed down by the uncontrollable delays required to obtain all Amselem et al. Orphanet J Rare Dis (2021) 16:454 Amselem et al. Orphanet J Rare Dis (2021) 16:454 Page 8 of 12 Page 8 of 12 the ethical and regulatory authorizations, as well as the evolving regulations both at the EU level, with the new European regulation on personal data safety and security (GDPR) [36], and at the national level, with modifications of the law on research involving human subjects (French «Jardé law») [37]. Moreover, a consortium agreement had to be signed between the partners for formalizing legal links between them, setting the modes of governance of the project on scientific, strategic and operational plans, and outlining the scientific valorization in terms of intel- lectual property, publications and citations of the pro- ject. A set of 19 Key Performance Indicators (KPIs) was implemented to enforce the cohorts’ follow-up (Table 4). Once initiated, the 13 ongoing RD cohorts progressed appropriately and the first results related to the second- ary objectives of several cohorts have been published in international peer-reviewed journals. Discussionh Pub- lic–Private Partnerships helped to build our sustainabil- ity plan. Legal contracts were set up for consortium and collaboration agreements, enabling data collection and sharing. At the cohort level, we industrialized our production processes, as well as tooling methods such as standard- ized operating procedures or documents shared online. Recurring difficulties are the shortage of manpower for data entry, the difficulties in identifying cases in the dif- ferent hospital information systems, or the legal barriers Table 4 RaDiCo e-cohorts key performance indicators (KPI) KPI 1 Methodology/biostatistics expertise as SC member KPI 2 One Scientific Committee meeting per year with reporting KPI 3 One yearly global RaDiCo progress report KPI 4 Yearly cohort-specific progress reports KPI 5 Reinforcement of the biostatistical expertise KPI 6 Number of Scientific/Steering Committee meeting repoarts according to the predefined scientific agenda of each cohort (protocol and time- lines) KPI 7 Cohorts’ progress reports including accrual rate of prevalent and incident cases recruited KPI 8 Number of recruitment sites in France/Europe per cohort KPI 9 Number of per site actual recruitments per cohort, according to pre-defined six-month objectives KPI 10 Loss to follow-up of included patients/death KPI 11 Completeness of data collection KPI 12 Per cohort data management status KPI 13 Whenever relevant, number of bio samples collected/associated to clinical data collected KPI 14 Integration to the National RD Plan (PNMR3), RD European Joint Program (EJP) and European Reference Networks activities KPI 15 Scientific publications/communications produced by each cohort and the RaDiCo platform KPI 16 Number of Specific Research Projects exploiting each cohort’s resources (data and biocollections) KPI 17 Number of EU projects (H2020/COST/ERN) valorising/integrating RaDiCo cohorts KPI 18 Implementation and follow-up of the Infrastructure as a Service, also proposed as a service to other cohorts of non-rare diseases; conformity to the European General Data Protection Regulation through yearly security audit results KPI 19 Building an exchange framework with the French Health Data Hub Services (https://www.health-data-hub.fr/) Table 4 RaDiCo e-cohorts key performance indicators (KPI) Amselem et al. Orphanet J Rare Dis (2021) 16:454 Page 9 of 12 production of the RDs National Protocols for Diagnosis and Care [42]. their medical management, and ultimately to assist the health care providers to make appropriate changes to medication use. The ERNs are networks of RD centers of expertise, healthcare providers and laboratories that are organized across borders. Discussionh Most of the medical teams associated with the RaDiCo program are involved in the activities of the ERN in their field. They participate in the implemen- tation of European data registries, in the production of clinical practice recommendations, and in the setting of epidemiological and economic indicators. Altogether, the cohort studies should help progress- ing towards a more personalized medicine, particularly for appropriate investigations and therapeutic strategies. They should contribute to decrease the burden of these chronic diseases and to improve their socio-economic impact. p The RD cohorts are multidisciplinary and, for most of them, include molecular diagnostics and research labo- ratories. Organization and standardization of biological sample collections (biobanks) are part of the program. Strong interactions and collaborations between clinicians and basic scientists are developed to identify genetic and environmental determinants in well-defined groups of patients, to establish genotype/phenotype correlations, and to progress in the understanding of the underlying molecular and cellular mechanisms. RaDiCo’s national call for implementing RD cohorts In 2014, RaDiCo launched a national call for RD Cohort proposals organized as a two-stage procedure (letter of intention/full application). Guidelines and templates for candidate cohorts were designed and made accessible on line to applicants. The full dossiers were evaluated by independent international experts (at least 3 per project), with pre-established evaluation criteria. Conclusionh The RaDiCo program promotes the collection of RD phe- notypic data of different types for epidemiological and clinical research purposes in connection with basic and translational research. The RaDiCo platform offers the cohorts an information system based on the cloud prin- ciple (Information as a Service) together with a common core of services and specific procedures for some cohorts to ensure installation and follow-up of the RD e-cohorts. The information system of RaDiCo can drive a virtually unlimited number of RD e-cohorts, a major strength compared to other IS that usually deal with only one cohort. Currently, 13 RD e-cohorts are implemented and other national, European and international cohorts are targeted. Rare diseases carry high morbidity and mortality; they represent a significant burden for the health care sys- tems. Evaluation of the economic impact of RDs is there- fore critical. This implicates a dedicated focus on the cost-effectiveness analyses, which evaluate both the costs and results of the health care systems and organizations applied in RDs. The final goal is to appropriately allocate the health care resources to the RD patients all over the country. This implies that RD are adequately traceable in the national health information systems. In line with these needs, the Ministry of Health has set up the RD national data bank. All the reference centers and their network of teams in France have the obligation to imple- ment this data bank. Standardized and detailed patient information collected through the cohort programs support the production of economic indicators. Moreo- ver, access to the French Health Data Hub [38] that is dedicated to cross-referencing the health databases and facilitating their use by research teams with full respect of user privacy of the health system, will open access to patients’ health expenditures from three main databases: the National Health Insurance Fund [39], hospital stays from the French hospital discharge database (PMSI) [40] and to the causes of death from the Inserm Centre for Epidemiology on the Medical Causes of Death, in charge of the national statistics of medical causes of death [41]. Economic indicators aim at monitoring the development and implementation of national strategies for RDs, which imply homogeneity and standardization of patient man- agement by the medical structures dedicated to RDs. Material and methods RaDiCo’s national call for implementing RD cohorts The RaDiCo platformh The RaDiCo platform is managed by the IS team together with the e-Health team. It uses exchange for- mat and data security in compliance with the European directive on the General Data Protection Regulation. The RaDiCo work plan for each cohort is structured to achieve four levels of interoperability: technological, semantic, syntactic, and institutional. This allows net- working and optimising the use of existing RD patient cohorts at the EU level, while allowing integration of new types of data and technologies. The data of each RaDiCo cohort and associated services have to be com- pliant with the FAIR principles (Findable, Accessible, Interoperable and Reusable) (https://www.go-fair.org/ Acknowledgements Acknowledgements Inserm is the sponsor of the RaDiCo cohorts. Study data were collected and managed using REDCap (Research Electronic Data Capture). We warmly acknowledge the RaDiCo team and the PIs of the RaDiCo cohorts: C. Bodemer, P. Calvas, N. Chassaing, A. Clement, C. Corpechot, V. Cottin, E. Escudier, B. Fautrel, S. Georgin-Lavialle, L. Heidet, B. Héron, X. Jeunemaître, B. Knebelmann, A. Linglart, B. Maitre, J.L. Mandel, I. Netchine, A. Servais, and S. Vicart, as well as their colleagues and teams, who contribute to the RaDiCo program (https:// www.radico.fr/en/accueil) Authors’ contributions Conceptualization, SA, PL and AC; methodology, SA, PL, AC, JW and SG; validation, SA, PL, AC, SG and JW; resources, SA, PL, AC, JW, SG; data curation, SG; writing original draft preparation, SA and PL; review and editing, SA, PL, AC, JW and SG; supervision, SA, PL; project administration, SA, PL and SG; funding acquisition, SA, AC, SG and PL All authors read and approved the final manuscript. Conceptualization, SA, PL and AC; methodology, SA, PL, AC, JW and SG; validation, SA, PL, AC, SG and JW; resources, SA, PL, AC, JW, SG; data curation, SG; writing original draft preparation, SA and PL; review and editing, SA, PL, AC, JW and SG; supervision, SA, PL; project administration, SA, PL and SG; funding acquisition, SA, AC, SG and PL All authors read and approved the final manuscript. RaDiCo’s governance An Institutional Committee is composed of the founding institutions including the following: Inserm (coordinat- ing institution), 6 Universities: Paris Descartes and Paris Diderot now merged in the University of Paris, as well as Sorbonne, Paris-Est Créteil, Aix-Marseille and Montpel- lier universities; and the « Entreprises du Médicament», together with the Alliance for Research and Innovation of the Industries of Health. These bodies are linked to the RaDiCo program by an agreement established with the National Research Agency. Among other missions, the Institutional Commit- tee regularly follows the implementation of the project including the budget implementation. It validates, on proposal of the Scientific Committee, the annual pro- gram of activities. It decides the creation, composition, All the teams of the RaDiCo cohorts are members of the RD Coordinating Centers in France. Among their tasks, through their organizations and their multidisci- plinary teams, all the cohorts actively participate in the Amselem et al. Orphanet J Rare Dis (2021) 16:454 Page 10 of 12 missions and the functioning of all ad-hoc governing bodies. fair-principles/) by both people and computers. An important step in the FAIR data approach is to publish existing and new datasets from RaDiCo cohorts in a semantically interoperable format that can be under- stood by computer systems. The RaDiCo Scientific Committee for the whole Pro- gram is composed of 18 members with complementary expertise associating coordinators of RD Coordinating Centers, RD Healthcare Networks, epidemiologists, bio- statisticians, experts in Information Systems, molecular geneticists, directors of Inserm Research Units in the field of RDs, and representatives of Inserm Thematic Institutes. Its role is to support the Executive Committee concerning strategic issues and scientific governance of the program, including management of the call for pro- jects and specific follow-up of cohorts; and to contribute to the national call for RD cohort projects launched by RaDiCo in 2014. The RaDiCo clinical research unit RaDiCo Clinical research unit helps the PIs of the cohorts to finalize their project according to the SPIRIT recommendations (https://www.equator-network.org/ reporting-guidelines/spirit-2013-statement-defining- standard-protocol-items-for-clinical-trials/), to sub- mit the protocol to the ethical and legal authorities and obtain the corresponding agreements; design the e-CRF (electronic Case Report Form); implement the e-CRF on the IS platform; organize the data capture and the data quality control; monitor the follow-up according to 19 Key Performance Indicators; perform the data management; data freeze the data; prepare and perform the data analysis. An Executive Committee is shared by all the cohorts. It is composed of 4 members with complementary exper- tise (SG, AC, PL and SA). Its role is to ensure the imple- mentation, development and monitoring of the entire program. Its main missions are operational management and deployment planning of the sustainability and inter- nationalization of the RaDiCo platform, the links with each RD cohort, and factorization of the shares. It is responsible for the scientific management of the Program which is implemented at different levels: by the Execu- tive Committee, the Scientific Committee, the Inserm Thematic Institutes of “Public health”, “Technology for Health”, “Genetics, Genomics and Bioinformatics”, as well as by the 3 scientific governance bodies of each RD cohort. Indeed, each cohort has its own governance, on a scheme comparable to that of the whole program, with a Plenary Committee, a Scientific Committee and an Exec- utive Committee. Patients are recruited through RD clinical sites for 12 cohorts. One cohort (GENIDA) proposes self-enrollment and self-reporting according to a pre-defined chart. Col- lected data for a given cohort were defined in an e-CRF. They are collected along with time (according to each cohort design, either quarterly, semi-annual or annual) and managed via the RaDiCo platform through REDCap (Research Electronic Data Capture) which is a secure, web-based application from the Vanderbilt University, USA. It was designed to support data capture for research studies, providing an intuitive interface for validated data entry; audit trails for tracking data manipulation and export procedures; automated export procedures for seamless data downloads to common statistical packages; and procedures for importing data from external sources. The RaDiCo team is directed by a Scientific & Opera- tional Director who leads 24 employees in close collab- oration with the Executive Committee. The team acts within 3 different sub-units: Clinical research, Biometry, and Information System & e-Health. Acknowledgements Inserm is the sponsor of the RaDiCo cohorts. Study data were collected and managed using REDCap (Research Electronic Data Capture). We warmly acknowledge the RaDiCo team and the PIs of the RaDiCo cohorts: C. Bodemer, P. Calvas, N. Chassaing, A. Clement, C. Corpechot, V. Cottin, E. Escudier, B. Fautrel, S. Georgin-Lavialle, L. Heidet, B. Héron, X. Jeunemaître, B. Knebelmann, A. Linglart, B. Maitre, J.L. Mandel, I. Netchine, A. Servais, and S. Vicart, as well as their colleagues and teams, who contribute to the RaDiCo program (https:// www.radico.fr/en/accueil) References 18. Legendre M, Butt A, Borie R, et al. Functional assessment and phenotypic heterogeneity of SFTPA1 and SFTPA2 mutations in interstitial lung dis- eases and lung cancer. Eur Respir J. 2020. https://doi.org/10.1183/13993 003.02806-2020. 1. https://www.orpha.net/consor/cgi-bin/index.php?lng=EN. Accessed May 30 2021. y 2. http://alliant.cluster028.hosting.ovh.net/. Accessed May 30 2021. http://alliant.cluster028.hosting.ovh.net/. 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Armand ML, Taieb C, Bourgeois A, Bourlier M, Bennani M, Bodemer C, Wolkenstein P; along with the French national network on rare skin diseases (FIMARAD). Burden of adult neurofibromatosis 1: develop- ment and validation of a burden assessment tool. Orphanet J Rare Dis. 2019;14(1):94. https://doi.org/10.1186/s13023-019-1067-8. 13. Funding g RaDiCo is funded by the French National Research Agency under the specific programme “Investments for the Future”, Cohort grant agreement ANR-10-COHO-0003. Page 11 of 12 Amselem et al. Orphanet J Rare Dis (2021) 16:454 Availability of data and materials 14. El Khouri E, Thomas L, Jeanson L, Bequignon E, Vallette B, Duquesnoy P, Montantin G, Copin B, Dastot-Le Moal F, Blanchon S, Papon JF, Lorès P, Yuan L, Collot N, Tissier S, Faucon C, Gacon G, Patrat C, Wolf JP, Dulioust E, Crestani B, Escudier E, Coste A, Legendre M, Touré A, Amselem S. Muta- tions in DNAJB13, encoding an HSP40 family member, cause primary cili- ary dyskinesia and male infertility. Am J Hum Genet. 2016;99(2):489–500. https://doi.org/10.1016/j.ajhg.2016.06.022. The data of the cohorts are not available presently since they are still running and primary objectives have not been analyzed yet. The datasets analyzed will be available from the PIs of the corresponding cohorts on reasonable request. Declarations g j j g 15. Holt RJ, Young RM, Crespo B, Ceroni F, Curry CJ, Bellacchio E, Bax DA, Ciolfi A, Simon M, Fagerberg CR, van Binsbergen E, De Luca A, Memo L, Dobyns WB, Mohammed AA, Clokie SJH, Zazo Seco C, Jiang YH, Sørensen KP, Andersen H, Sullivan J, Powis Z, Chassevent A, Smith-Hicks C, Petrovski S, Antoniadi T, Shashi V, Gelb BD, Wilson SW, Gerrelli D, Tartaglia M, Chassaing N, Calvas P, Ragge NK. De novo missense variants in FBXW11 cause diverse developmental phenotypes including brain, eye, and digit anomalies. Am J Hum Genet. 2019;105(3):640–57. https://doi.org/10. 1016/j.ajhg.2019.07.005. Author details 1 RaDiCo, Inserm, Trousseau Hospital, Paris, France. 2 Sorbonne Université, Inserm U933, Childhood Genetic Disorders, Trousseau Hospital, 26 rue du Dr. Arnold Netter, 75012 Paris, France. 3 Department of Paediatric Respiratory Medicine, Trousseau Hospital, Assistance Publique Hôpitaux de Paris, Paris, France. 4 EA2415, University Clinical Research Institute, Montpellier University, Montpellier, France. 5 Present Address: Direction Générale de La Santé, Ministry of Health, Paris, France. 16. Frank M, Adham S, Seigle S, Legrand A, Mirault T, Henneton P, Albuisson J, Denarié N, Mazzella JM, Mousseaux E, Messas E, Boutouyrie P, Jeunemai- tre X. Vascular ehlers-danlos syndrome: long-term observational study. J Am Coll Cardiol. 2019;73(15):1948–57. https://doi.org/10.1016/j.jacc.2019. 01.058. Received: 11 June 2021 Accepted: 17 October 2021 Received: 11 June 2021 Accepted: 17 October 2021 17. Henneton P, Albuisson J, Adham S, Legrand A, Mazzella JM, Jeunemaitre X, Frank M. Accuracy of clinical diagnostic criteria for patients with vascu- lar ehlers-danlos syndrome in a tertiary referral centre. Circ Genom Precis Med. 2019;12(3):e001996. Competing interests The authors declare that they have no competing interests. References Fassad MR, Shoemark A, Legendre M, Hirst RA, Koll F, le Borgne P, Louis B, Daudvohra F, Patel MP, Thomas L, Dixon M, Burgoyne T, Hayes J, Nicholson AG, Cullup T, Jenkins L, Carr SB, Aurora P, Lemullois M, Aubusson-Fleury A, Papon JF, O’Callaghan C, Amselem S, Hogg C, Escudier E, Tassin AM, Mitchison HM. Mutations in outer dynein arm heavy chain DNAH9 cause motile cilia defects and situs inversus. Am J Hum Genet. 2018;103(6):984– 94. https://doi.org/10.1016/j.ajhg.2018.10.016. 30. 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https://openalex.org/W3210206841	https://dc.etsu.edu/cgi/viewcontent.cgi?article=11098&context=etsu-works	English	null	New Look of EBV LMP1 Signaling Landscape	Cancers	2,021	cc-by	18,610	East Tennessee State University East Tennessee State University Digital Commons @ East Tennessee State University Digital Commons @ East Tennessee State University East Tennessee State University East Tennessee State University Faculty Works Follow this and additional works at: https://dc.etsu.edu/etsu-works Citation Information Citation Information Wang, Ling; and Ning, Shunbin. 2021. New Look of EBV LMP1 Signaling Landscape. Cancers. Vol.13(21). https://doi.org/10.3390/cancers13215451 Citation Information Citation Information Wang, Ling; and Ning, Shunbin. 2021. New Look of EBV LMP1 Signaling Landscape. Cancers. Vol.13(21). https://doi.org/10.3390/cancers13215451 This Review is brought to you for free and open access by the Faculty Works at Digital Commons @ East Tennessee State University. It has been accepted for inclusion in ETSU Faculty Works by an authorized administrator of Digital Commons @ East Tennessee State University. For more information, please contact digilib@etsu.edu. New Look of EBV LMP1 Signaling Landscape New Look of EBV LMP1 Signaling Landscape Copyright Statement Copyright Statement 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/). This review is available at Digital Commons @ East Tennessee State University: https://dc.etsu.edu/ This review is available at Digital Commons @ East Tennessee State University: https://dc.etsu.edu/etsu-works/9832 Ling Wang 1,2,* and Shunbin Ning 1,2,* Ling Wang 1,2,* and Shunbin Ning 1,2,* Ling Wang 1,2,* and Shunbin Ning 1,2,* Ling Wang 1,2,* and Shunbin Ning 1,2,* 1 Department of Internal Medicine, Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA 1 Department of Internal Medicine, Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA 1 Department of Internal Medicine, Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA J y, , 2 Center of Excellence for Inﬂammation, Infectious Diseases and Immunity, Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA * Correspondence: wangl3@etsu.edu (L.W.); nings1@etsu.edu (S.N.); Tel.: +1-423-439-6411 (L.W.); +1-423-439-8232 (S.N.); Fax: +1-423-439-7010 (L.W. & S.N.) J y, , 2 Center of Excellence for Inﬂammation, Infectious Diseases and Immunity, Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA * Correspondence: wangl3@etsu.edu (L.W.); nings1@etsu.edu (S.N.); Tel.: +1-423-439-6411 (L.W.); +1-423-439-8232 (S.N.); Fax: +1-423-439-7010 (L.W. & S.N.) J y 2 Center of Excellence for Inﬂammation, Infectious Diseases and Immunity, Quillen College of Medicin East Tennessee State University, Johnson City, TN 37614, USA East Tennessee State University, Johnson City, TN 37614, USA * Correspondence: wangl3@etsu.edu (L.W.); nings1@etsu.edu (S.N.); Tel.: +1-423-439-6411 (L.W.); +1-423-439-8232 (S.N.); Fax: +1-423-439-7010 (L.W. & S.N.) Simple Summary: Epstein-Barr Virus (EBV) infection is associated with various lymphomas and carcinomas as well as other diseases in humans. The transmembrane protein LMP1 plays versatile roles in EBV life cycle and pathogenesis, by perturbing, reprograming, and regulating a large range of host cellular mechanisms and functions, which have been increasingly disclosed but not fully understood so far. We summarize recent research progress on LMP1 signaling, including the novel components LIMD1, p62, and LUBAC in LMP1 signalosome and LMP1 novel functions, such as its induction of p62-mediated selective autophagy, regulation of metabolism, induction of extracellular vehicles, and activation of NRF2-mediated antioxidative defense. A comprehensive understanding of LMP1 signal transduction and functions may allow us to leverage these LMP1-regulated cellular mechanisms for clinical purposes. Abstract: The Epstein–Barr Virus (EBV) principal oncoprotein Latent Membrane Protein 1 (LMP1) is a member of the Tumor Necrosis Factor Receptor (TNFR) superfamily with constitutive activity. Academic Editors: Naoki Mori, Lorenzo Leoncini and Lucia Mundo Received: 22 July 2021 Accepted: 26 October 2021 Published: 29 October 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Keywords: EBV; LMP1; LIMD1; LUBAC; p62 Keywords: EBV; LMP1; LIMD1; LUBAC; p62 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/). Creative Commons License Creative Commons License This work is licensed under a Creative Commons Attribution 4.0 International License. cancers cancers cancers Ling Wang 1,2,* and Shunbin Ning 1,2,* LMP1 shares many features with Pathogen Recognition Receptors (PRRs), including the use of TRAFs, adaptors, and kinase cascades, for signal transduction leading to the activation of NFκB, AP1, and Akt, as well as a subset of IRFs and likely the master antioxidative transcription factor NRF2, which we have gradually added to the list. In recent years, we have discovered the Linear UBiquitin Assembly Complex (LUBAC), the adaptor protein LIMD1, and the ubiquitin sensor and signaling hub p62, as novel components of LMP1 signalosome. Functionally, LMP1 is a pleiotropic factor that reprograms, balances, and perturbs a large spectrum of cellular mechanisms, including the ubiquitin machinery, metabolism, epigenetics, DNA damage response, extracellular vehicles, immune defenses, and telomere elongation, to promote oncogenic transformation, cell proliferation and survival, anchorage-independent cell growth, angiogenesis, and metastasis and invasion, as well as the development of the tumor microenvironment. We have recently shown that LMP1 in- duces p62-mediated selective autophagy in EBV latency, at least by contributing to the induction of p62 expression, and Reactive Oxygen Species (ROS) production. We have also been collecting evidence supporting the hypothesis that LMP1 activates the Keap1-NRF2 pathway, which serves as the key antioxidative defense mechanism. Last but not least, our preliminary data shows that LMP1 is associated with the deregulation of cGAS-STING DNA sensing pathway in EBV latency. A comprehensive understanding of the LMP1 signaling landscape is essential for identifying potential targets for the development of novel strategies towards targeted therapeutic applications. Citation: Wang, L.; Ning, S. New Look of EBV LMP1 Signaling Landscape. Cancers 2021, 13, 5451. https://doi.org/10.3390/ cancers13215451 Academic Editors: Naoki Mori, Lorenzo Leoncini and Lucia Mundo Received: 22 July 2021 Accepted: 26 October 2021 Published: 29 October 2021 1. Introduction Human herpesviruses are of particular importance in medical research in that they are associated with severe diseases including cancers in immunocompromised popula- tions [1,2]. Epstein–Barr Virus (EBV), known as human herpesvirus 4 (HHV4), serves as https://www.mdpi.com/journal/cancers Cancers 2021, 13, 5451. https://doi.org/10.3390/cancers13215451 Cancers 2021, 13, 5451 2 of 22 a fascinating paradigm for the study of herpesvirus infection, replication, latency, and associated diseases, as well as host–pathogen interactions due to its ability to establish lifelong persistent infection in normal immunocompetent healthy hosts [3]. EBV establishes lifetime persistence in both IgD+CD27+ non-class switched and IgD-CD27+ class switched memory B cells, but not in naïve B cells [4]. EBV entry receptors include CD21, CR1, and CR2, and MHCII in B cells, and αvβ integrins and the newly identiﬁed Ephrin receptor A2 in epithelial cells [5,6]. p As the ﬁrst identiﬁed human cancer virus, EBV infection is associated with various lymphomas and carcinomas, which most often occur in immunocompromised patients [7]. EBV replication has two modes: one is lytic replication that produces virions and lyses the host cell, and the other is proliferative replication that occurs when the infected B cell proliferates. EBV lytic replication seems to be the cause of Hodgkin lymphomas (HL) [3]. Its latent infection in B cells results in the development of endemic Burkitt’s Lymphoma (BL), Post-Transplant Lymphoproliferative Disorder (PTLD), and Non-Hodgkin Lymphomas (NHL), and in epithelial cells results in Nasopharyngeal Carcinoma (NPC) and some forms of gastric carcinoma [8,9]. In its latency, EBV resides in the infected cell in either integrated or episomal form, with three distinct processes: viral persistence, controlled viral latent gene expression, and the potential to be reactivated from latency [10]. EBV, the other gammaherpesvirus Kaposi’s Sarcoma-Associated Herpesvirus (KSHV/HHV8), and Human papillomavirus (HPV), are three oncogenic viruses causally involved in HIV/AIDS- associated malignancies [11,12]. More than 50% of AIDS-related lymphomas (ARLs), which are a leading cause of HIV/AIDS-related cancer deaths even in the era of combined antiretroviral therapy [13], are associated with EBV infection [14,15]. EBV infection is also associated with autoimmune diseases, including Infectious Mononucleosis (IM), Multiple Sclerosis (MS), Rheumatoid Arthritis (RA), and Systemic Lupus Erythematosus (SLE) [16,17], underscored by the fact that EBNA1 was initially identiﬁed as a seropositive EBV antigen in RA patients [18]. 1. Introduction More recently, EBV has been detected in astrocytes and microglia in the brain of more than 90% (91/101) of MS patients versus 24% (5/21) of non-MS neuropathologic patients [19], which may represent a new mechanism that underlies the association of EBV with Mild Cognitive Impairment (MCI) or Alzheimer’s Disease (AD), supporting the hypothesis that EBV infection promotes cognitive dysfunction [20]. The EBV Latent membrane protein 1 (LMP1) is a pleiotropic factor, which is consti- tutively active without the need of ligand stimulation and plays diverse roles through the whole EBV life cycle, including lytic, reactivation, and latency. As the principal on- coprotein of EBV, LMP1 is essential for transformation and proliferation in multiple cell backgrounds in vitro as well as in transgenic mice, through regulating a vast scale of cellu- lar functions such as proliferation and survival, apoptotic resistance, immune modulation, anchorage-independent growth, metabolism, angiogenesis, metastasis, and invasion, in a context-dependent manner. Other EBV products, however, play complementary roles in regulating these processes to promote oncogenesis [21,22]. In this review, we summarize recent research progress on LMP1 signaling, focusing on the novel components of LMP1 signalosome, including LIMD1, p62, and LUBAC, and the novel functions of LMP1 signaling, including induction of p62-mediated selective autophagy, regulation of metabolism, induction of extracellular vehicles, and activation of Keap1-NRF2-mediated antioxidative defense. 2. LMP1 Structure and Novel Components of LMP1 Signalosome 2. LMP1 Structure and Novel Components of LMP1 Signalosome The LMP1 protein N-terminus has six Transmembrane Domains (TMs) that are majorly responsible for LMP1 membrane tethering and oligomerization. The long C-terminus in the cytoplasm can be functionally divided into two well-known domains, C-Terminal Activation Region 1 (CTAR1) and CTAR2, and a third less-understood domain CTAR3. Decades have been spent on the study of LMP1 signaling pathway, with numerous essential intermediators, such as TRAFs and MAPKs, being identiﬁed for the activation Cancers 2021, 13, 5451 3 of 22 of NFκB, AP1, and Akt. LMP1 CTAR1 directly interacts with TRAF1, -2, -3 and -5, and indirectly interacts with TRAF6 via TRAF2 and -5; CTAR2 directly interacts with TRAF2, and indirectly interacts with TRAF6 that is in a complex containing TRAF2, TRADD, RIP1, and other potentially unidentiﬁed components [23–25]. As a member of the tumor necrosis factor receptor (TNFR) superfamily, LMP1 shares many signaling components with TNFR and CD40, as well as with pathogen recognition receptors (PRRs), and both LMP1 and PRRs activate a subset of Interferon Regulatory Factors (IRFs), which, along with NFκB, AP1, and Akt, all contribute to EBV-mediated pathogenesis [24–29]. LMP1 also shares a spectrum of signaling components with Human T-cell Leukemia Virus-1 (HTLV1) Tax for NFκB/AP1 activation [30,31]. Recent efforts on the study of LMP1 signaling include the identiﬁcation of BS69, an adaptor containing PHD, Bromo, PWWP, and MYND domains, which recruits TRAF6 to LMP1 leading to AP1 activation [32]. Later, BS69 was shown to compete with TRADD for LMP1 binding, and therefore represses LMP1 CTAR2 activation of canonical NFκB [33]; BS69 also represses LMP1 CTAR1 activation of noncanonical NFκB through its direct interaction with TRAF3 [34]. Furthermore, we have identiﬁed several novel components of LMP1 signalosome in recent years, including the linear ubiquitin (Ub) assembly complex (LUBAC), the adaptor protein LIMD1, and the Ub sensor and signaling adaptor p62. 2.1. The Linear Ubiquitin Assembly Complex LUBAC Post-Translational Modiﬁcations (PTMs) by ubiquitin (Ub) or Ub-like small regula- tory proteins are a pervasive topic important to the regulation of activation, stability, and functions of many transcription factors and other proteins in various processes [35,36]. It is clear that targeting of cellular proteins for K48 ubiquitination-mediated proteaso- mal degradation is an important aspect of infection and cell transformation by tumor viruses including EBV [37]. Further, atypical ubiquitination, represented by K63-linked nonproteolytic polyubiquitination, has become a key mechanism in a myriad of biological functions [38–40]. EBV encodes several viral proteins that exploit the host ubiquitin system to regulate its latency and persistence in the host cell [37,41,42]. Both regulatory and degradative ubiquitination forms are involved in LMP1 activation of downstream transcription factors. We have shown that K63-linked regulatory ubiquitination contributes to LMP1 activation of IRF7, with the requirement of RIP1 and TRAF6 [43,44]. Later, we have further identiﬁed TNFAIP3 (also known as A20) as an important de-ubiquitinating enzyme (DUB) that negatively regulates IRF7 ubiquitination stimulated by LMP1, and therefore plays an important role in balancing IRF7-mediated functions in EBV latency [45]. These intriguing ﬁndings indicate that EBV utilizes both ubiquitination and deubiquitination machinery to subvert the host cellular pathways. LUBAC is a ternary ubiquitin ligase complex composed of HOIP (RNF31), HOIL1L (RNF54), and SHARPIN (Figure 1), with RNF31 being the central component [46]. LUAC complex is constitutively formed under normal physiological conditions [47–49]. LUBAC- mediated linear polyubiquitination has come into focus in the past years due to its emerging role in speciﬁc activation of NFκB, but not JNK, by conjugating linear polyubiquitin chains onto NEMO and RIP1 [50,51], in response to diverse signaling stimuli [50,52–56], including apoptotic and immune stimuli such as TNFα [47,49], IL1β [57], genotoxic stress [58], CD40 [59], Toll-Like Receptors (TLRs) [60], NOD2 [61], and NLRP3 [62]. However, LUBAC negatively regulates RIG-I-mediated innate immune responses by targeting RIG-I, TRIM25, and IRF3 for degradation [63,64], and by disrupting the TRAF3-MAVS complex [65]. The involvement of LUBAC in EBV infection is also emerging [66,67]. A high through- put screen identiﬁed the association of LUBAC with TRAF1, which is induced by LMP1 in EBV latency [66]. Further investigation has revealed that LMP1 CTAR1 induces K63- linked ubiquitination of TRAF1 in the TRAF1:TRAF2 complex, which further facilitates the recruitment of LUBAC to LMP1 [67]. 4 of 22 Cancers 2021, 13, 5451 Figure 1. A domain scheme of the LUBAC complex. 2.1. The Linear Ubiquitin Assembly Complex LUBAC The LUBAC complex includes three proteins, RNF31, SHARPIN and HOIL1, with RNF31 being the core. PUB: PNGase/UBA or UBX-containing proteins; NZF: Npl4-type zinc ﬁnger domain; UBA: ubiquitin-associated domain; IBR: in-between RING domain; UBL: ubiquitin-like domain. Figure 1. A domain scheme of the LUBAC complex. The LUBAC complex includes three proteins, RNF31, SHARPIN and HOIL1, with RNF31 being the core. PUB: PNGase/UBA or UBX-containing proteins; NZF: Npl4-type zinc ﬁnger domain; UBA: ubiquitin-associated domain; IBR: in-between RING domain; UBL: ubiquitin-like domain. Importantly, we have shown that the key LUBAC component RNF31 interacts with LMP1 and IRF7 in EBV-transformed cells. Consequently, LUBAC stimulates linear ubiqui- tination of NEMO and IRF7, promoting NFκB activity but inhibiting IRF7 activity down- stream of LMP1 signaling [68]. These ﬁndings implicate the importance of LUBAC in controlling LMP1-mediated pathways for its pathogenic functions and may have broad signiﬁcance on IRF7-mediated IFN response in antiviral immunity. Likewise, a later study has shown that HTLV1 Tax also invokes LUBAC-mediated linear ubiquitination for NFκB activation [69]. 2.2. The Adaptor Protein LIMD1 LIMD1 is a member of the ZYXIN gene family that includes AJUBA, TRIP6, LPP, WTIP, migﬁlin, and ZYXIN. As an adapter, LIMD1 possesses multiple protein-interacting domains (Figure 2A), which are able to interact with various proteins, including Rb [70], TRAF6 [71], p62/SQSTM1 [72], VHL and PHD [73], to regulate different cellular processes [72]. For example, the interaction of LIMD1 with TRAF6 enhances the ability of TRAF6 to ac- tivate AP1 and negatively regulates the canonical Wnt receptor signaling pathway in osteoblasts [71]. LIMD1 also positively regulates microRNA (miRNA)-mediated gene silencing by interacting with the microRNA-Induced Silencing Complex (miRISC) [74]. Interestingly, our GEO high throughput proﬁling has revealed that LIMD1 is associ- ated with IRF4 at the transcriptional level in B-cell lymphomas, including EBV-associated lymphomas [75]. Further analyses have demonstrated that IRF4 and NFκB, both onco- genic transcription factors activated by EBV LMP1 and HTLV1 Tax signaling pathways, transcriptionally upregulate LIMD1 expression in EBV- or HTLV1-transformed cells [76]. More importantly, LIMD1, in turn, interacts with TRAF6 and participates in LMP1 signal transduction for the activation of downstream NFκB and AP1. LIMD1 depletion impairs LMP1 signaling and functions, potentiates ionomycin-induced DNA damage and apopto- sis, and inhibits p62-mediated selective autophagy that we have later demonstrated to be induced by oxidative stress in oncoviral latency [76,77]. Thus, LIMD1, known as a tumor suppressor in several tumors such as lung, gastric, and breast cancers [78–80], oppositely acts as a tumor promoter in viral hematomalignancies. 5 of 22 Cancers 2021, 13, 5451 Figure 2. Domain schemes of LIMD1, p62 and TRAF6. The domains of LIMD1 (A), p62 (B) and TRAF6 (C) for their interactions are shown. NES: nuclear export signal; NLS: nuclear localization signal; PB1: Phox/Bem 1p protein–protein binding domain; AID: atypical PKC interacting domain; RL: regulatory linker; ZNF: Zinc ﬁnger; LB: LIM protein binding; TB: TRAF6 binding; LIR: LC3-interacting region that mediates interaction with ATG8 family; UBA: Ubiquitin-binding re-gion that binds speciﬁcally to K63-linked polyubiquitin chains of polyubiquitinated substrates; NES: nuclear export signal. NLS: nuclear localization signal; ZnF: Zinc ﬁnger. Figure 2. Domain schemes of LIMD1, p62 and TRAF6. The domains of LIMD1 (A), p62 (B) and TRAF6 (C) for their interactions are shown. 2.2. The Adaptor Protein LIMD1 NES: nuclear export signal; NLS: nuclear localization signal; PB1: Phox/Bem 1p protein–protein binding domain; AID: atypical PKC interacting domain; RL: regulatory linker; ZNF: Zinc ﬁnger; LB: LIM protein binding; TB: TRAF6 binding; LIR: LC3-interacting region that mediates interaction with ATG8 family; UBA: Ubiquitin-binding re-gion that binds speciﬁcally to K63-linked polyubiquitin chains of polyubiquitinated substrates; NES: nuclear export signal. NLS: nuclear localization signal; ZnF: Zinc ﬁnger. 3.1. LMP1 Induces Both Random and Selective Autophagy Programs Autophagy, with either non-selective or selective form, is one of the two major in- tracellular protein degradation mechanisms, which are essential for proteostasis in eu- karyotes [93,94]. Although debates currently exist on the segregation of these two forms, selective autophagy, such as mitophagy, is mediated by an increasing pool of receptors, including p62 [82,95,96]. The two major degradation mechanisms, autophagy and Ub- Proteasome System (UPS), have reciprocal crosstalks; however, they are different in many ways. UPS targets K48-linked ubiquitinated, short-life, soluble misfolded proteins to protea- somes for degradation, whereas autophagy targets non-ubiquitinated (random autophagy) or ubiquitinated (selective autophagy), long-life, insoluble misfolded proteins or whole organelles to lysosomes for degradation [97,98]. g y g Autophagy plays a dual role in cancers; as either tumor suppressor at early stage or promotor at late stage [99–104]. Oncogenic viruses, including EBV, are known to inhibit autophagy, which serves as an immune defense strategy [105,106], at their early stage of infection for optimal replication and oncogenic transformation [107–111], but induce autophagy in their latency to facilitate their persistence and tumorigenesis through different mechanisms, including regulation of metabolism and DDR [112–114]. g g High physiologic levels of LMP1 is able to induce the Unfolded Protein Response (UPR)/ Endoplasmic Reticulum (ER) stress response via the N-terminal six TMs by ac- tivating all three branches of the UPR (PERK, ATF6, and IRE-1) in a sequential man- ner [115–117], which further induce autophagy, and in turn, high levels of autophagy promote LMP1 degradation [118,119]. Moreover, higher levels of LMP1 induce reactive oxygen/nitrogen species (ROS/RNS, referred to ROS hereafter), which trigger DDR that can also induce autophagy [120]. LMP1 also induces the expression of autophagy-related genes such as Bcl2A1, and might induce the formation of autophagosomes via induction of PI3K/mTOR that is activated by LMP1 CTAR1 [121]. Interestingly, we have collected preliminary data suggesting that LIMD1, which is induced by LMP1 [76], is also involved in autophagosome formation in response to ROS in EBV latency. In agreement with this, our recent study shows that LIMD1 is associated with a panel of proteins involved in membrane trafﬁcking at the transcriptional level in non-small-cell lung carcinoma [78]. Furthermore, our recent study shows that LMP1 induces p62 expression in EBV la- tency, licensing the induction of p62-mediated selective autophagy (Figure 3) [77]. 2.3. The Ubiquitin Sensor and Signaling Hub p62 p62 (also named EBIAP, ZIP3, SQSTM1/Sequestosome-1), a human homolog of mouse Zeta PKC-interacting proteins (ZIPs), functions as a signaling hub that comprises multi- ple protein-interacting domains and is able to interact with different proteins in distinct signaling pathways including LIMD1, TRAF6, LC3b, Keap1, etc. (Figure 2B), to control myriad cellular processes, including osteoclastogenesis, obesity, cancer development, DNA damage response (DDR), aging, inﬂammation and immunity, autophagy, and oxidative stress [81,82]. It is known that p62 is upregulated in several types of cancer, such as breast, lung, and liver tumors [83,84]. Of note, p62 C-terminus is a ubiquitin-binding region (UBA) that enables its function as a “ubiquitin sensor” (Figure 2B). The UBA binds to K63 ubiquitin chains to facilitate NFκB activation in diverse contexts [81,85]. Like LIMD1, as a signaling hub protein, p62 also has a TRAF6-binding domain (Figure 2B), which speciﬁcally recognizes TRAF6, but not TRAF5 or TRAF2. Binding of p62 to TRAF6 facilitates TRAF6 K63-linked polyu- biquitination, in which the N-terminal dimerization domain and the UBA domain of p62 are also required [86–88]. Thus, p62 has at least two roles in activating NFκB. First, p62 probably favors the phosphorylation of IKKβ by functioning as a ubiquitin receptor that facilitates the recruitment of ubiquitinated signal intermediators. Second, p62 facili- tates TRAF6 K63-linked polyubiquitination through interacting with TRAF6. In addition, the PB1 domain of p62 interacts with MEKK3 to regulate NFκB activity [89]. Our most recent study has identiﬁed p62 as another novel component of LMP1 sig- nalosome, like LIMD1, also through its interaction with TRAF6, with LIMD1 binding to the N-terminal RING domain and p62 to the C-terminal TRAF domains (Figure 2C). Consequently, p62-TRAF6 interaction potentiates TRAF6 ubiquitination, further promoting activation of downstream NFκB, AP1, and Akt [90]. Cancers 2021, 13, 5451 6 of 22 3. New Insights into LMP1-Mediated Pathogenesis LMP1 is well known to activate the transcription factors NFκB and AP1, as well as HIF1α, T-Cell Factor (TCF) and STATs. We have expanded this list by adding IRF7 [43,44] and IRF4 [91,92]. Moreover, we have collected evidence suggesting that the master antioxi- dant transcription factor NRF2 can also be activated by LMP1 pathway (to be published). LMP1 exerts most of its known functions via these transcription factors, which transcrip- tionally regulate the expression of unique targets in distinct biological processes, with some functions being context dependent. Besides the means of activation of transcription factors, LMP1 also takes many other strategies to perturb, reprogram, and regulate a large range of host cellular mechanisms and functions, which have been increasingly disclosed. 3.1. LMP1 Induces Both Random and Selective Autophagy Programs As such, endogenous p62(S403) phosphorylation, p62-LC3b interaction, and p62-autophagosome colocalization, which are unique requirements for the induction of p62-mediated selec- tive autophagy in addition to p62 transcriptional upregulation and p62(K420) ubiqui- tination [122–125], are all readily detectable in EBV type 3 latency in association with endogenous ROS levels [77]. These ﬁndings indicate that EBV latent infection induces p62-mediated selective autophagy via ROS-mediated mechanism, in which LMP1 at least contributes to p62 expression and ROS production. LMP1 may be also required for PTM- mediated p62 activation (i.e., phosphorylation and ubiquitination), which is under our further investigation. 7 of 22 Cancers 2021, 13, 5451 Figure 3. An updated LMP1 signaling pathway. The involvement of LUBAC, LIMD1 and p62 in the LMP1 signal transduction is shown. LIMD1 expression is induced by NFκB and IRF4, and p62 expression is induced by NFκB and NRF2. The induction of p62-mediated selective autophagy and the activation of NRF2-mediated antioxidative defense by LMP1 signaling are shown. Functionally, our results show that p62-mediated selective autophagy protects viru transformed cells from oxidative stress-induced DNA damage at least by conﬁning p62 Figure 3. An updated LMP1 signaling pathway. The involvement of LUBAC, LIMD1 and p62 in the LMP1 signal transduction is shown. LIMD1 expression is induced by NFκB and IRF4, and p62 expression is induced by NFκB and NRF2. The induction of p62-mediated selective autophagy and the activation of NRF2-mediated antioxidative defense by LMP1 signaling are shown. Figure 3. An updated LMP1 signaling pathway. The involvement of LUBAC, LIMD1 and p62 in the LMP1 signal transduction is shown. LIMD1 expression is induced by NFκB and IRF4, and p62 expression is induced by NFκB and NRF2. The induction of p62-mediated selective autophagy and the activation of NRF2-mediated antioxidative defense by LMP1 signaling are shown. Functionally, our results show that p62-mediated selective autophagy protects virus- transformed cells from oxidative stress-induced DNA damage at least by conﬁning p62 in Cancers 2021, 13, 5451 8 of 22 the cytoplasmic compartment. Inhibition of autophagy causes p62 translocation to and accumulation in the nucleus, where p62 inhibits DNA damage repair through its ability to target DNA repair proteins CHK1 and RAD51 for proteasomal degradation and its interaction with RNF168 [77], a key Ub E3 ligase for chromatin ubiquitination for activation of both Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ) mechanisms for repairing Double-Strand Breaks (DSBs) [126]. 3.2. LMP1 Reprograms Multiple Metabolism Pathways Cancer metabolism is an old theme, but it has attracted intensive attention in recent years with deeper mechanistic insights. Not only ER-mediated UPR, which can be induced by high levels of LMP1, is involved in regulating lipid and glucose metabolism [127,128], but also LMP1 regulates mitochondrial NAD (NAD+ and NADH) and NADP (NADP+ and NADPH) energy metabolism as well as other metabolism pathways [129–131]. gy p y Mitochondrial oxidative phosphorylation (OxPhos) and cytoplasmic glycolysis are the two major pathways for energy production in a normal cell, but cancer cells usually take the latter strategy to produce a large amount of lactate even in the presence of oxygen, which is termed aerobic glycolysis (Warburg effect), a cancer hallmark characterized by increased glucose consumption and decreased OxPhos. LMP1 promotes the Warburg effect by directly potentiating the expression, stability, and plasma localization of GLUT1, and consequently induces the expansion of Myeloid Derived Suppressor Cells (MDSCs) in NPC [132,133]. LMP1 also promotes glycolysis by constitutively activating the FGF2- FGFR1 signaling pathway, and by upregulating the glycolysis-related hexokinase 2 (HK2) and IDH2, and HIF1α-mediated PDK1 and PKM2, and by repressing the expression of HOX genes [131]. Furthermore, in EBV lymphomas, LMP1 promotes glucose metabolism through stabilization of c-Myc, promoting c-Myc activity, and also through upregulation of c-Myc expression via STAT3 [129]. y p LMP1 activates PI3K, which further activates Akt and mTOR1, the master regulators of glucose metabolism. In fact, the PI3K subunit, PIK3CA, is hyperactivated by somatic mutation in epithelial cancers, including >80% EBV-positive gastric carcinomas. More recently, it has been revealed that metabolic rewiring is beyond the Warburg effect in many cancer cells. Mitochondrial NAD/NADP metabolism, nucleotide and fatty acid synthesis, glutaminolysis, and mitochondrial one carbon metabolism pathways are also deregulated in cancer cells to support proliferation, malignant outgrowth, and survival [130,131]. Myc, upregulated by EBNA2 and LMP1 in EBV lymphomas, promotes one carbon metabolism that contributes to nucleotide synthesis, NADPH production, and antioxidative defense [134]. LMP1 also regulates NAD/NADP metabolism by upregulating the NAD(P)H oxidase NOX [135]. 3.3. LMP1 Regulates Epigenetics by Promoting Histone and DNA Methylation Apart from regulating individual genes via phosphorylation-mediated activation of a panel of transcription factors, LMP1 is also able to regulate global gene expression by promoting chromatin and DNA methylation. g y PARP1 is a crucial player in DNA damage repair, NAD metabolism, and chromosome 3D architecture conformation. 3.1. LMP1 Induces Both Random and Selective Autophagy Programs The selective targets other than p62 for p62-mediated selective autophagy in EBV latency remain to be identiﬁed. 3.4. LMP1 Promotes Formation of Extracellular Vehicles LMP1 has been long recognized as a membrane protein tethering to the lipid rafts with its N-terminal TM domains, of which the region 38-FWLY-41 in TM1/2 is critical for membrane tethering, LMP1 dimerization, and signaling [142]. In EBV-transformed B lymphocytes, a small portion of LMP1 protein undergoes phosphorylation, which is preferentially associated with vimentin in the cytoskeleton network likely mediated by TRAF3 that interacts with CTAR1 [143,144]. Opposite to the conventional doctrine that LMP1 signals from the plasm membrane, later studies have shown that LMP1 principally signals from intracellular compartments containing lipid rafts in different cell types [145]. Tethering LMP1 to the lipid rafts and the vimentin cytoskeleton facilitates PI3K localization and consequent signal transduction to Akt and ERK activation [146]. LMP1 also undergoes palmitoylation at cysteine 78, but palmitoylation is not required for its localization to the membrane [143,147]. p y q Besides intracellular compartments, interestingly, LMP1 is also enriched in exosomes isolated from EBV-infected B cells and epithelial cells, and can be transferred to uninfected cells by these extracellular vesicles [148]. Moreover, LMP1 stimulates biogenesis and secretion of extracellular vesicles, with the requirement of CD63 [121,149]. In this regard, LMP1 utilizes extracellular vesicles to promote cell growth, invasion, and metastasis. Extracellular vesicles are also believed to associate with the immune modulation function of LMP1, underscored by the fact that they play a role in antigen transfer [148–152]. 3.5. LMP1 Regulates Antiviral and Antitumor Immune Responses 3.2. LMP1 Reprograms Multiple Metabolism Pathways LMP1 promotes PARP1 activation that requires high levels of NAD+, likely mediated by ERK, in B lymphocytes and epithelial cells [136]. Upon activation, PARP1 promotes histone PARylation to alter chromosome 3D architecture in cooperation with CTCF, which is important for maintenance of EBV latency [136,137]. PARP1 also promotes replication stress at oriP, in a manner depending on the cell cycle [138]. Global CpG DNA hypermethylation (CIMP) is a unique feature of epithelial can- cers. LMP1 transcriptionally upregulates the DNA methyltransferase I (DNMT1) and potentiates its activity [139], and in turn, DNMT1 and its partner UHRF1 restricts the expression of LMPs and EBNAs through CIMP [140], in addition to a pool of cellular tumor-suppressor genes as week as B-cell activation antigens that are also downregulated Cancers 2021, 13, 5451 9 of 22 9 of 22 through CIMP [141]. LMP1 also regulates DNMT1 mitochondrial localization, which consequently silences pTEN gene expression and downregulates the OxPhos complexes by promoting hypermethylation of mitochondrial DNA (mtDNA), leading to metabolic reprogramming in NPC [139]. The universal methyl donor S-adenosylmethionine (SAMe) for DNA, protein, and lipid methylation is produced by metabolism from ATP, serine, methionine, and vitamins B9 and B12. Thus, high metabolic rates, which are partially contributed by LMP1, are required for epigenetic regulation in EBV latency. EBV induces high levels of MTHFD2 and SHMT2, two key enzymes in methionine and serine metabolism, respectively, during B-cell immortalization [134]. 3.5. LMP1 Regulates Antiviral and Antitumor Immune Responses Accumulating evidence has shown that LMP1 regulates both innate and adaptive immune responses at discrete stages and multiple layers [16,153–155]. As stated above, LMP1 induces autophagy that, as an intrinsic immune defense mechanism, intimately crosstalks with the immune system, for instance, enhancing antigen presentation to expose the infected cells to the immune system [156–158], and inducing the formation of exosomes that plays a role in antigen transfer. Expression of LMP1 in mouse B cells enhances antigen presentation and costimulation through CD70 and OX40L [159], consequently driving potent cytotoxic CD4+ and CD8+ T cell responses [160]. Moreover, LMP1 plays a crucial role in the regulation of apoptosis, which also serves as a defense strategy in antiviral and antitumor responses [161]. LMP1, like PRRs, activates NFκB, AP1, and Akt by promoting their site-speciﬁc phosphorylation, and we have added IRF7 and IRF4 to this list of its activated tar- gets (Figure 3) [43,44,92], all of which participate in the regulation of innate immune re- sponse [162,163]. As to IRF4, it is known to inhibit type I IFN-mediated response, and inhibits IRF5 expression and activity in the context of EBV infection [164,165]. We and others have also shown that LMP1 induces the expression of IRF7 and IRF4 via NFκB in EBV-infected cell lines as well as in LMP1 transgenic mice [159,165–167]. Moreover, LMP1 CTAR3 stabilizes IRF7 and limits its transcriptional activity by promoting its sumoy- lation (Figure 3) [168]. We have further shown that IRF5, the dominant-negative mutant of which is induced by TLR7 signaling in EBV latency, interacts with IRF7 and inhibits its Cancers 2021, 13, 5451 10 of 22 10 of 22 activity stimulated by LMP1 [169]. In addition, LMP1 is a potent inhibitor of TLR9 tran- scription [170], and promotes RIG-I proteasomal degradation [171]. activity stimulated by LMP1 [169]. In addition, LMP1 is a potent inhibitor of TLR9 tran- scription [170], and promotes RIG-I proteasomal degradation [171]. g The type I IFN Jak-STAT pathway, which is responsible for the robust production of type I IFNs, is substantially compromised if not completely disabled in EBV latency, while the complex mechanisms are poorly understood. We have conducted high throughput phosphoproteomics to proﬁle the deregulated phosphorylation in Jak-STAT pathways, and the results have shown that STAT2 Y690 phosphorylation was most downregulated and barely detected in EBV latency. 3.5. LMP1 Regulates Antiviral and Antitumor Immune Responses IFNα treatment or virus infection restored STAT2 Y690 phos- phorylation in cell lines lacking LMP1 but did not or only weakly restored in cell lines expressing high levels of LMP1, strongly suggesting a role for LMP1 in repressing the type I IFN Jak-STAT pathway. Consistent with these preliminary results, it has been reported that LMP1 N-terminal TM interacts with Tyk2, and consequently, suppresses phospho- rylation of both STAT1 and -2 and subsequently blocks type I IFN-mediated antiviral responses [172]. Our phosphoproteomics results imply that all other Jak-STAT pathways, however, are active in EBV latency [173]. In addition, we have preliminary data in an ongoing project showing that LMP1 plays a role in the regulation of the cGAS-STING DNA sensing pathway. In summary, LMP1 regulates both innate and adaptive immunity in both positive and negative manners, thus playing multiple roles in the ﬁne balance of the complicated host– virus interaction for EBV long-term persistence and pathogenesis. Further investigation to disclose important but unknown strategies that LMP1 employs to deactivate type I IFN Jak-STAT pathway is one of our current plans. 3.6. LMP1 Promotes Chronic Inﬂammation and the Tumor Microenvironment 3.6. LMP1 Promotes Chronic Inﬂammation and the Tumor Microenvironment It is also known that LMP1 functions as an inﬂammation-promoting factor, at least by inducing a panel of pro-inﬂammatory cytokines, such as TNFα, IL6, IP10, among many others, via NFκB, AP1, and STAT3 [174–176]. Activation of NFκB and AP1 by LMP1 also induces miRNAs including miR-155, miR-146a, and miR-21, which play key roles in the regulation of inﬂammation [177–179]. We have shown that LMP1 induction and activation of IRF4 contributes to BIC/miR-155 expression in EBV latency [92,180]. Several novel mechanisms accounting for LMP1-mediated chronic inﬂammation are emerging, including its ability to balance oxidative stress and NRF2-mediated antioxidant defense, to balance DNA damage and DNA repair at least via induction of autophagy, to transfer to extracellular compartments via extracellular vehicles, and to reprogram extra- mitochondrial glycolysis metabolism by promoting the expression of multiple glycolytic genes (e.g., GLUT1), which further promotes MDSC expansion to facilitate immune escape in the Tumor Microenvironment (TME) and promotes the expression of NLRP3 inﬂamma- some [133]. All these events contribute to the development of chronic inﬂammation, as discussed elsewhere in this review. As such, the indispensable roles of LMP1 in regulating redox homeostasis, chronic inﬂammation, and immune response render its ability to contribute to the development of the TME, as discussed elsewhere in the review. 3.8. LMP1 Facilitates Telomere Integrity and Represses Premature Cell Senescence 3.8. LMP1 Facilitates Telomere Integrity and Represses Premature Cell Senescence One of the mechanisms accounting for EBV oncogenesis is the ability of the virus to repress premature cell senescence in its latency, which involves LMP1 regulation of telomere/telomerase and cell cycle regulators such as p16INK4a, p21CIP1/WAF1, and CDKN2A/B [21,197–199]. Telomere integrity is a prerequisite for the cell to escape senescence during EBV immortalization [21]. LMP1 stable overexpression in EBV-negative BL cells causes telomere de-protection and dysfunction, as well as multinucleation, by negatively modulating telomerase-independent telomere elongation named alternative lengthening of telomere (ALT) through downregulation of shelterin components including the telomere repeat binding factors TRF1, TRF2 and POT1, which were also found in newly EBV-transformed B cells involving distinct mechanisms mediated by several viral proteins other than LMP1, including BNRF1 and EBNA1 [200,201]. Consequently, these deregulations may favor chromosomal instability, cell immortalization, and oncogenic transformation. However, in LCLs, LMP1 promotes telomere elongation and protection through upregulation of telomerase activity that involves distinct mechanisms at both the transcriptional and translational levels [21,199]. Of note, LMP1 transcriptionally activates the TERT gene to induce telomerase expression, which involves NFκB, MAPK, and ERK pathways, but the involvement of c-Myc is controversial that may be cell type-dependent [202,203]. y y yp p Consistent with its ability to promote malignant transformation and immortalization, LMP1 represses mouse ﬁbroblast replicative senescence in vitro by promoting CRM1- dependent nuclear export of Ets2 that is a transcription factor for p16INK4a, consequently inhibiting p16INK4a expression [204,205], in which LMP1 CTAR2 TRADD-binding domain is required [206], and also by blocking downstream mediators of the p16INK4a-Rb path- way [205]. Counterintuitively, chronic NFκB activity induced by HTLV1 Tax was reported to promote p21/p27-dependent senescence response, which is overcome at least by the viral antisense protein HBZ [207,208]. 3.7. LMP1 Inhibits DNA Damage Response and Promotes Genomic Instability 3.7. LMP1 Inhibits DNA Damage Response and Promotes Genomic Instability DNA damage is directly linked to many human diseases including cancer. Thus, eukaryotic organisms have developed sophisticated mechanisms to repair damaged DNA to secure genomic integrity. HR and NHEJ are two major mechanisms responsible for repairing DSBs [181]. Most cancers, if not all, harbor deﬁcient traditional DNA repair mechanisms [182]. Viruses can manipulate host DDR machinery in that infected cells recognize viral replication as DNA damage stress [183–186]. In the absence of active viral replication, ROS produced in viral persistence are the major cause of different levels of endogenous DNA lesions [187]. It is well documented that LMP1 inhibits DNA repair to promote genome Cancers 2021, 13, 5451 11 of 22 11 of 22 instability and renders DNA damage resistance in different cell contexts [188–191]. So does HTLV1 Tax [30,192,193]. However, the underlying mechanisms are not fully understood. As one of these mechanisms, p62-mediated selective autophagy plays an alternative and indispensable role in DNA repair in cancer cells [126,194–196]. Importantly, our recent discovery of the induction of p62-mediated selective autophagy by LMP1-ROS in EBV latency has deﬁned two distinct roles for the autophagy-p62 interplay in DDR in this setting: cytoplasmic p62, which is partially induced by LMP1 [90], mediates selective autophagy (in addition to mediating LMP1 signal transduction [90]), and nuclear p62 accumulated upon autophagy inhibition represses DNA repair. These original ﬁndings indicate that a well-controlled autophagy-p62 interplay renders EBV-positive cells with the ability to balance pro-survival DNA damage resistance and pro-mutagenic genomic perturbation under oxidative stress [76,77]. 3.9. Potential Role of LMP1 in Activation of the Master Antioxidant Defense 3.9. Potential Role of LMP1 in Activation of the Master Antioxidant Defense The discovery of the cellular mechanism of oxygen sensing has been awarded the 2019 Nobel Prize in Physiology and Medicine, highlighting the importance of oxygen homeostasis in cellular functions. ROS at low or moderate levels act as important second messengers for normal cellular functions but are harmful at high levels. Thus, ROS levels are strictly balanced by several detoxiﬁcation processes mediated by antioxidant enzymes to ensure normal cellular functions, and this balance is called “redox homeostasis”. Loss of this balance is called “oxidative stress” [209,210]. ROS at abnormally high levels are the major cause of endogenous DNA damage [211–213], which can incite inﬂammation, and excess inﬂammation, in turn, causes oxidative stress, ultimately resulting in tissue damage [211]. DNA damage also perturbs genomic instability that promotes malignant transformation under certain conditions [187,214]. Abnormal ele- vation of ROS levels and chronic inﬂammation are the most common features of persistent Cancers 2021, 13, 5451 12 of 22 12 of 22 (chronic and latent) viral infections [215,216]. Aberrant redox homeostasis is one of the hallmarks of cancer [217,218]. ROS represent a crucial component of tumor niches [219,220], and activate various transcription factors such as NFκB, AP1, HIF1α, and STAT3 that are essential for cancer initiation and development. ROS also control the expression of various tumor suppressor genes such as p53, Rb, and pTEN [221]. Moreover, ROS promote cancer development by inducing autophagy [222]. Elevated ROS levels exist in almost all cancers, if not all, as well as in EBV latency [223]. It is well known that LMP1, among several other EBV products, contributes to the majority of ROS production in EBV latency, at least via JNK-p38/AP1 [223,224]. Mitochondria produce the majority of ROS [225]. The mitochondria in malignant cells are functionally and structurally deregulated and are able to overproduce ROS, thus playing a central role in ROS-caused DNA damage [226]. p p y g g The transcription factor NRF2, a member of the basic leucine zipper (bZIP) family, is the master regulator of oxidative and inﬂammatory stresses by transactivating about 250 genes, of which many are involved in antioxidant defense including p62, Keap1, Cox2, iNOS, PRDX1, HIF1, NQO1, HMOX1, GSTs, etc., as well as NRF2 itself [227,228]. 3.9. Potential Role of LMP1 in Activation of the Master Antioxidant Defense NRF2 tar- get genes are also involved in the regulation of immune responses and inﬂammation, metabolism, cell proliferation, survival and anti-apoptosis, angiogenesis, cell migration and invasion, all of which contribute to cancer development [229–232]. Under normoxia, NRF2 is constitutively synthesized but quickly ubiquitinated for proteasome-mediated degradation by the Ub E3 ligase complex Keap1/Cul3/RBX1. ROS trigger autophagic degradation of Keap1, consequently resulting in the accumulation and activation of NRF2 (phosphorylation at S40) [233]. Due to its importance in redox homeostasis, quick acti- vation of NRF2 is a hallmark in response to ROS under various stresses [234]. As the master antioxidant defense mechanism, the Keap1-NRF2 pathway plays a central role in the regulation of inﬂammation, and has a close connection to NFκB-mediated inﬂammatory regulation [228,230]. However, deregulation of the Keap1-NRF2 pathway by EBV infection has never been reported. Of note, our recent ﬁndings strongly suggest that the Keap1-NRF2 path- way is activated in association with LMP1 in EBV latency [76,77]. The contributions of LMP1 to its activation at least include the ROS production that triggers autophagic degradation of Keap1, and the induction of p62 expression that positively regulates the Keap1-NRF2 pathway activity in a feedback loop. The details underlying the activation of the Keap1-NRF2 pathway in EBV latency and whether EBV primary infection and reactiva- tion also activate this pathway are among the priorities of our current investigation. This study will add NRF2 as another crucial transcription factor that is activated downstream of LMP1 signaling and will disclose a crucial role for the LMP1-NRF2 pathway in redox homeostasis in EBV latency and pathogenesis. Since we have shown that cytoplasmic p62 also mediates LMP1 signal transduction, p62 plays multiple roles in virus-mediated oncogenesis in both cytoplasmic and nuclear compartments. It is a challenge to separate its cytoplasmic roles in LMP1 and autophagy pathways in that they share the same functional domains of p62, in particular, the C- terminal UBA domain for ubiquitin-mediated process is required for enabling both roles. Nevertheless, the PB domain may be speciﬁc to p62’s role for autophagy induction since p62 without this domain still interacts with LMP1, and we have shown that the TB domain, which interacts with TRAF6, is speciﬁc to LMP1 signal transduction [90]. In addition, site- speciﬁc phosphorylation and ubiquitination of p62, which are essential for p62 function in autophagy, may not be required for LMP1 signal transduction. 3.9. Potential Role of LMP1 in Activation of the Master Antioxidant Defense These details of mechanism to discriminate p62’s roles are under our investigation. 4. Perspectives Identiﬁcation of novel components is the key to decipher the complicated LMP1 sig- naling and functions, which would advance mechanistic insights into EBV infection and pathogenesis. It is our understanding that LMP1 engages different signaling components to connect with and manipulate diverse cellular processes. For example, LMP1 employs Cancers 2021, 13, 5451 13 of 22 13 of 22 p62 to enable its function for manipulating cellular selective autophagy and antioxidative stress, employs LIMD1 to repress the anti-oncogenic Hippo pathway, besides their roles in activation of NFκB, and employs UCHL1 to regulate Wnt/β-Catenin/TCF proliferation pathway [235]. Thus, continuing efforts are still needed to fully proﬁle the components of LMP1 signalosome, which would involve high throughput screening, followed by molecu- lar conﬁrmation and functional validation. Further, it will be of great interest to employ the CryoEM technique to study the structure of LMP1 signalosome. This technique has been recently applied to the capsids of EBV and other herpesviruses [236–239]. LMP1 itself has poor immunogenicity, which restricts it to produce a substantial humoral or cellular immune response and renders it with the ability to escape from CD8+ T cell targeting in EBV-positive healthy individuals. Strategies directly targeting LMP1 for therapeutic purposes seem difﬁcult, although some have been made with promising progress, including LMP1-targeting vaccines and antibodies as well as small peptides and molecules [175]. Targeting LMP1 signal transduction and functions may serve as a viable strategy that could discover unique targets with potential clinical applications [240]. For example, autophagy-mediated degradation is a promising strategy to target non-enzymatic proteins for therapy [241]. A comprehensive understanding of LMP1 signaling pathway, such as the identiﬁcation of novel components of LMP1 signalosome, is thus essential towards this end. Disclosing how LMP1 regulates unique immune cells and molecules represents a promising strategy for immunotherapy. For example, LMP1 modulates the activities of stroma, NK, and T cells in the TME, to promote cancer cell growth, survival, metastasis, angiogenesis, and Epithelial–Mesenchymal Transition (EMT) [175,176]. We have been collecting evidence showing that LMP1 is associated with the deregulation of the cGAS- STING DNA-sensing pathway, which has potent anti-tumor activity. As such, we have several ongoing projects, aiming to identify novel components of the LMP1 signalosome and its unknown or underappreciated functions. These studies may reveal potential targets for designing clinical strategies to treat EBV-associated cancers and diseases. 5. Conclusions LMP1 signalosome contains a pool of cellular proteins, with many being unidentiﬁed, which furnish LMP1 with diverse roles to deregulate a large spectrum of cellular events in different stages of the whole EBV life cycle as well as in different cell contexts. Some components of LMP1 signalosome may compete with each other to differentiate LMP1’s abilities to regulate different cellular mechanisms in the same context. As such, some of the components likely vary depending on EBV infection stages and cell contexts, which result in context-dependent outcomes, making it possible to design speciﬁc strategies for clinical applications. Author Contributions: Writing—review and editing, L.W. and S.N. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the NIH grants CA252986 (L.W.), DE029621 (S.N.), and DE027314 (S.N.), an ASH grant (S.N.), and in part by the NIH grant C06RR0306551. Acknowledgments: This publication is the result of work supported with resources and the use of facilities at the James H. Quillen Veterans Affairs Medical Center. The contents in this publication do not represent the views of the Department of Veterans Affairs or the United States Government. Conﬂicts of Interest: The authors declare that they have no competing interests in this paper. 1. Damania, B.; Münz, C. Immunodeﬁciencies that predispose to pathologies by human oncogenic γ-herpesviruses. FEMS Microbiol. Rev. 2019, 43, 181–192. [CrossRef] [PubMed] 2. Pagano, J.S.; Blaser, M.; Buendia, M.A.; Damania, B.; Khalili, K.; Raab-Traub, N.; Roizman, B. Infectious agents and cancer: 1. Damania, B.; Münz, C. Immunodeﬁciencies that predispose to pathologies by human oncogenic γ-herpesviruses. FEMS Microbiol. Rev. 2019, 43, 181–192. [CrossRef] [PubMed] 2. Pagano, J.S.; Blaser, M.; Buendia, M.A.; Damania, B.; Khalili, K.; Raab-Traub, N.; Roizman, B. 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https://openalex.org/W2315327657	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0152931&type=printable	English	null	Enhancing Beta-Catenin Activity via GSK3beta Inhibition Protects PC12 Cells against Rotenone Toxicity through Nurr1 Induction	PloS one	2,016	cc-by	7,997	Abstract Received: December 13, 2015 Accepted: March 20, 2016 Published: April 5, 2016 Copyright: © 2016 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. RESEARCH ARTICLE OPEN ACCESS Citation: Zhang L, Cen L, Qu S, Wei L, Mo M, Feng J, et al. (2016) Enhancing Beta-Catenin Activity via GSK3beta Inhibition Protects PC12 Cells against Rotenone Toxicity through Nurr1 Induction. PLoS ONE 11(4): e0152931. doi:10.1371/journal.pone.0152931 Limin Zhang1,2☯, Luan Cen1☯, Shaogang Qu3☯, Lei Wei1,4, Mingshu Mo1, Junmin Feng3, Congcong Sun5, Yousheng Xiao1, Qin Luo6, Shaomin Li7, Xinling Yang6, Pingyi Xu1,8* 1 Department of Neurology, The First Affiliated Hospital of Sun Yat-sen University, Guangdong 510080, China, 2 Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan Province, 450052, China, 3 Department of Blood Transfusion, The Fifth Affiliated Hospital, Southern Medical University, Guangzhou, Guangdong, 510900, China, 4 Department of Neurology, The Third Affiliated Hospital of Sun Yat-sen University, Guangdong, 510080, China, 5 Department of Neurology, Qilu Affiliated Hospital of Shandong University, Jinan, Shandong, 250001, China, 6 Department of Neurology, The Third Affiliated Hospital of Xinjiang Medical University, Urumqi, 830011, China, 7 Ann Romney Center for Neurologic Disease, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, 02115, United States of America, 8 Department of Neurology, The First Affiliated Hospital of Guangzhou Medical University, Guangdong, 510080, China ☯These authors contributed equally to this work. * pingyixu@sina.com (PX); shaomin_li@hms.harvard.edu (SL) Abstract Parkinson’s disease (PD) is characterized by progressive degeneration of dopaminergic (DA) neurons in the substantial nigra pars compacta. Increasing evidence showed that Wnt/ β-catenin pathway and the orphan nuclear receptor Nurr1 play crucial roles in the survival and functional maintenance of DA neurons in the midbrain and GSK-3β antagonists LiCl and SB216763 were used to activate Wnt/β-catenin pathway experimentally. However, the detail mechanism underlying the neuroprotection against apoptosis on DA neuron is still unclear and the interaction between Wnt/β-catenin and Nurr1 remains undisclosed. In this study, using cell biological assay we investigated the function of Wnt/β-catenin and its crosstalk with Nurr1 on the course of PC12 cell degeneration in vitro. Our data showed that PC12 cell viability was inhibited by rotenone, but attenuated by GSK-3β antagonists LiCl or SB216763. The activity of Wnt/β-catenin pathway was deregulated on exposure of rote- none in a concentration-dependent manner. After the interference of β-catenin with siRNA, LiCl or SB216763 failed to protect PC12 cells from apoptosis by the rotenone toxicity. Our data confirmed that Wnt/β-catenin signaling activated by LiCl or SB216763 enhanced Nurr1 expression to 2.75 ± 0.55 and 4.06 ± 0.41 folds respectively compared with control detected by real-time PCR and the interaction of β-catenin with Nurr1 was identified by co-immu- noprecipitate analysis. In conclusion, the data suggested that Wnt/β-catenin and Nurr1 are crucial factors in the survival of DA neurons, and the activation of Wnt/β-catenin pathway exerts protective effects on DA neurons partly by mean of a co-active pattern with Nurr1. This finding may shed a light on the potential treatment of Parkinson disease. Editor: Weidong Le, Institute of Health Science, CHINA Editor: Weidong Le, Institute of Health Science, CHINA Received: December 13, 2015 Accepted: March 20, 2016 Published: April 5, 2016 Copyright: © 2016 Zhang 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. CHINA Received: December 13, 2015 Accepted: March 20, 2016 Published: April 5, 2016 Copyright: © 2016 Zhang 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. Enhancing Beta-Catenin Activity via GSK3beta Inhibition Protects PC12 Cells against Rotenone Toxicity through Nurr1 Induction Limin Zhang1,2☯, Luan Cen1☯, Shaogang Qu3☯, Lei Wei1,4, Mingshu Mo1, Junmin Feng3, Congcong Sun5, Yousheng Xiao1, Qin Luo6, Shaomin Li7, Xinling Yang6, Pingyi Xu1,8* Cell Culture and Treatments Differentiated PC12 cells, which were obtained 5 hours after the addition of NGF, were pur- chased from the Cell Library of the Chinese Academy of Science (Shanghai, China), main- tained in Dulbecco's Modified Eagle's Medium (DMEM) with high glucose (Invitrogen, Carlsbad, California, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen, Carls- bad, California, USA), 100 U/mL benzyl penicillin, and 100 mg/L streptomycin (Gibco, Grand Island, NY, USA) and cultured in a humidified incubator with 5% CO2 at 37°C. Cells were seeded on 96-well plates, 6-well plates or 25cm2 plastic flasks at a density of 1×105 cells/mL for 24 hours. The culture of HEK-293T cells was based on the instruction of Invitrogen. To investigate Wnt/β-catenin signaling pathway, 2 selective GSK-3β inhibitors LiCl and SB216763 were used. LiCl (Sigma, St. Louis, MO, USA) was added into the cultures for 72 hours followed by rotenone treatment for 24 h. SB216763 (Sigma, St. Louis, MO, USA) was added into the cultures 30 minutes prior to rotenone 24hours-exposure (Sigma, St. Louis, MO, USA). In β-catenin gene silencing tests, differentiated PC12 cells were treated with the siRNA for 6 hours, and then the medium was replaced and added with LiCl for another 72 hours, fol- lowed by treatment with 1 μmol/L rotenone for last 24 hours. Similarly, following the siRNA protocol, differentiated PC12 cells were treated with SB216763 30 minutes prior to 24 hours of rotenone. Introduction Competing Interests: The authors have declared that no competing interests exist. Parkinson’s disease (PD) is characterized by progressive degeneration of dopaminergic (DA) neurons in the substantial nigra pars compacta (SNc) [1–2]. While host genetics account for < 10% of cases, environmental neurotoxins, such as 1-methyl-4-phenyl-1, 2, 3, 6-tetrahy- dropyridine (MPTP), rotenone, and 6-hydroxydopamine (6-OHDA), play a significant role on the disease onset and/or progression of PD [3–4]. However, the molecular mechanism underly- ing the degeneration of dopaminergic neurons remains to disclose. Wnt and Nurr1 are important factors in the survival and functional maintenance of DA neurons in midbrain [5–12]. The Wnt pathway is a part of highly-conserved antocrine-para- crine signaling cascades [13–15], and the canonical Wnt pathway, known as Wnt/β-catenin signaling pathway, is an important signaling branch for cell survival. Studies have revealed that Wnt/β-catenin pathway was deregulated in cellular and animal models of toxin-induced PD [5,9,16–17] and the related genes express abnormally in DA neurons in midbrain of PD patients [18]. As for Nurr1, it is highly expressed in DA neurons for cell differentiation [19–20] and neonate Nurr1-/- mice died after 24 hours without DA neurons in the midbrain[21]. The polymorphisms of Nurr1 have been reported to result in a marked decrease of Nurr1 transcrip- tion in lymphocytes of affected individuals with PD[22]. Meanwhile, reduction of Nurr1 in adult mice gives rise to the vulnerability of DA neurons to oxidative stress [23] and accumula- tion of α-synuclein[24]. However, so far what is the detail mechanism of Wnt/β-catenin inter- acting with Nurr1 to protect DA neurons from apoptosis remains unclear. In this study, using rotenone toxic cell model of PD we investigated the function of Wnt/β-catenin and Nurr1 against neurodegeneration and try to explore the mutual regulation related to their potential crosstalk in differentiated PC12 cells. β-Catenin Crosstalk with Nurr1 Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by research grants from the State Key Development Program for Basic Research of China (2011CB510000), the National Natural Science Foundation of China (81271428, 81471292, U1503222 and 81430021) the Science Foundation of Guangdong of China (2015A030311021), a grant supported by technology project of Guangzhou (20151260) and a grant supported by assisting research project of science and technology for Xinjiang (201591160). 1 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0152931 April 5, 2016 RNA Extraction, Reverse Transcription and Real-Time PCR Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, California, USA) and reverse transcribed into first-strand complementary DNA (cDNA) using reverse transcription kit (TaKaRa, Dalian, China). Real-time polymerase chain reaction (PCR) was performed in a fluorescent temperature cycler (ABI-Prism 7700 Sequence Detection System, Applied Biosys- tems) using a Light Cycler FastStart DNA Master Plus SYBR Green kit (Roche Diagnostics GmbH) with the following primes. β-catenin forward, 5’-GTCTGAGGACAAGCCACAGGA CTAC-3’; β-catenin reverse, 5’- AATGTCCAGTCCGAGATCA GCA-3’; Nurr1 forward, 5’- C CAATCCGGCAATGACCAG-3’; Nurr1 reverse, 5’-TGATGATCTCCAT AGAGCCAGTCA G -3’. Rat GAPDH gene was used as an internal control. After 94°C for 4 min, the experimental reaction consisted of 39 cycles of 94°C for 15 s and 61°C for 45 s. Fluorescent readings from real-time PCR reactions were quantitatively analyzed by determining the Ct difference (delta Ct) between β-catenin/Nurr1 and the internal GAPDH control. The mRNA expression levels were determined by the formation of 2-delta Ct. Caspase-3 Activity Assay Caspase-3 activity was determined using the Caspase-3/CPP32 Fluor metric Assay Kit (Bio vision, USA). For each assay, 50 μg of cell lysate was loaded and read on the fluorescence microliter (Spectra Max Gemini EM, Molecular Devices, USA) with 400 nm excitation and 505 nm emission filter. Caspase-3 activity in sample was expressed as folds comparing to control. Gene Silencing with Small Interfering RNA (siRNA) B-catenin siRNA or control siRNA were chemically synthesized by Guangzhou Riborio CO. LTD (Guangzhou, China). β-catenin siRNA: sense strand: 5’ -GCACCAUGCAGAAUACAAA dTdT -3’, Antisense strand: 5’ -UUUGUAUUCUGCAUGGUGCTdTd -3’. Cells were trans- fected with 100 nmol/L siRNA at 60% confluence using Lipofectamine2000 (Invitrogen, Carls- bad, CA, USA) according to the transfection protocol. The cells were harvested for analysis the transcription and protein of β-catenin at 24 h or 72 h following transfection, respectively. Dur- ing the transfection, 2 mmol/L LiCl or 5 μmmol/L SB216763 was added to determine whether these chemicals protect PC12 cells from rotenone toxicity under depletion of β-catenin. At that time, total RNA was collected to detect the relative mRNA level of the target genes including β- catenin and Nurr1. β-Catenin Crosstalk with Nurr1 per 100 μL of medium was added to the wells of 96-well plate and incubated at 37°C for 2 hour. Cell viability was assessed by measuring the absorbance at 450nm with ELISA plate reader. The results were expressed as a percentage of the control group. Each treatment group was repli- cated in three wells. Cell Counting Kit-8 Assay The cell counting kit-8 (CCK-8) assay was performed to measure PC12 cells viability following rotenone, LiCl, and SB216763 treatments, respectively. Briefly, 10 μL of the CCK-8 kit reagent 2 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0152931 April 5, 2016 GST Pull Down Assay The GST fusion proteins used for GST pull down assay were produced by cloning Nurr1 into pGEX6p1 (Amersham Pharmacia Biotech, UK). The primers used for the clone were: AF1 for Nurr1 (Forward, 5’-CG CCTTGTGTTCAGGCGCAGTATG -3’. reverse, 5’- CTCGAGCTAG AAGAGTGAAAGGCGG GAGA C-3’), DBD for Nurr1 (Forward, 5’- CG GGCTCCCTTCA CAACTTCCAC -3’. reverse, 5’- CTC GAGCTAATCCTGTGGGCTCTTCGGTTT-3’), AF2 for Nurr1 (Forward, 5’-CGGGGCCCGAAAGG TAAGGTGTCCAGGAAA-3’. reverse, 5’-CT CGAGCTAAAACCGAAGAGCCCACAGGAT-3’). Nuclear proteins containing β-catenin were obtained by transfecting PC12 cells with β-catenin vectors for 24 hours and incubated with GST-fused mutants of Nurr1, AF1, DBD, AF2. And washed with Wash buffer containing 50mM TrisHCl(pH7.3), 150mM NaCl, 3mM MgCl2, 1mM EDTA, 1mM DTT, 0.5%Triton X-100 β-catenin immobilized on glutathione-Sepharose beads prior to analysis by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Western Blotting Immunoblotting was performed in accordance with a standard procedure. Briefly, cells were homogenized with ice-cold lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, and 0.5 mM Triton X-100, pH 7.4) containing protease inhibitor cocktail tablets (Roche, Basel, Switzer- land). BCA protein assay kit (Thermo Fisher Scientific Inc., IL, USA) was used to measure protein concentrations. Both 20μg proteins and in each group and 5ug protein marker were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore, USA). Positive and negative controls also were used for the first time to detect the specificity antibodies. After the membranes were blocked in 5% skim milk, 0.05% Tween 20, and Tris-buffered saline (TBS) for 1 hour, they were incubated with primary antibodies: rabbit anti-Wnt3a (1:1,000 dilution, 09–162, Millipore, USA), rabbit anti-β-catenin (1:200 dilution, Cell Signaling Technology Inc., 8480, Danvers, MA, USA), rabbit anti-non-phospho-β-catenin (1:200 dilution, Cell Signaling Technology Inc., 8814, Danvers, MA, USA), rabbit anti-Nurr1 (1:1,000 dilution, Sigma, N6413, USA) and rabbit anti- GAPDH (1:8,000 dilution, R&D, AF5718, USA) overnight at 4°C. The second horseradish peroxi- dase (HRP)-labeled antibody (1:3000 dilution, Cell Signaling Technology Inc., Danvers, MA, USA) were used in the next day. The bands were visualized using enhanced chemiluminescences (ECL) method (Millipore, USA). Membranes probed for GAPDH used as an internal control. The protein bands were quantified using image analysis software (Image J, V.1.42, National Insti- tutes of Health, Bethesda, MD) and the protein levels were expressed as percent (%) of controls. β-Catenin Crosstalk with Nurr1 cocktail (Nacalai Tesque). The supernatant was pre-cleared with protein-A-Sepharose beads and incubated overnight at 4°C. After incubation, protein-A-Sepharose beads were added for 4 h to make the immune complex and washed in the washing buffer I (50 mM Tris-Cl pH7.6 with PI, 150 mM NaCl, 1% NP40, 0.5% Sodium deoxycholate, 4% protease inhibitor cocktail tablets), Washing buffer II (50 mM Tris-Cl pH7.6, 50 mM NaCl, 0.1% NP40, 0.05% Sodium deoxycho- late), Washing buffer III (50 mM Tris-Cl pH7.6, 0.1% NP40, 0.05% Sodium deoxycholate, ddH2O) at 4°C. And then 500 μg of cell lysate was used to co-immunoprecipitation with rabbit anti-β-catenin (1:1,000 dilution, Cell Signaling Technology Inc., Danvers, MA, USA) and mouse Nurr1 antibodies (1:1,000 dilution, sigma, USA). Western blotting was carried out to detect Nurr1 and β-catenin respectively. Normal IgG (rabbit, Santa Cruz Biotechnology) was used as a control. Plasmid Construction and Co-Immunoprecipitation Full-length human Nurr1 and β-catenin genes were inserted into pShuttle-IRES-hrGFP-2 vec- tors (Novagen, USA). HEK-293T cells or PC12 cells were transfected with vectors carrying Nurr1, β-catenin gene, or control vector with EndoFectinTM reagent Plus (GeneCopoeia Inc., USA). At 72 h after transfection, cell lysates were prepared for co-immunoprecipitation. A Pierce Immunoprecipitation Kit (Thermo Fisher Scientific, USA) was used to detect the immunoprecipitation of Nurr1 with β-catenin proteins in transfected HEK-293T cells or PC12 cells. Briefly, cells were lysed in ice-cold immunoprecipitation (IP) buffer containing 50 mmol/ L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 1% NP-40, 1 mmol/L Na3VO4, and protease inhibitor 3 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0152931 April 5, 2016 Chromatin Immunoprecipitation Sequence (ChIP-seq) Assay Soluble chromatin from PC12 cells was prepared with an acetyl-histone H4 immunoprecipita- tion assay kit (Upstate Biotechnology) and immunoprecipitated with antibodies against β- PLOS ONE \| DOI:10.1371/journal.pone.0152931 April 5, 2016 4 / 14 β-Catenin Crosstalk with Nurr1 Fig 1. Rotenone affected the cell viability and Wnt/β-catenin of PC12 cells. A. cells were treated with 0~100 μmol/L (μM) rotenone for 24 hours. Cell viability was measured as percentage relative to vehicle (P < 0.05; P < 0.01). B. Rotenone deregulated Wnt3a, β-catenin, and non-phospho β-catenin in a concentration-dependent manner. The relative intensity of protein band was normalized to GAPDH by Image J software (P < 0.05; *P < 0.01). All data presented as means ± SD from three independent experiments. doi:10.1371/journal.pone.0152931.g001 Fig 1. Rotenone affected the cell viability and Wnt/β-catenin of PC12 cells. A. cells were treated with 0~100 μmol/L (μM) rotenone for 24 hours. Cell viability was measured as percentage relative to vehicle (P < 0.05; *P < 0.01). B. Rotenone deregulated Wnt3a, β-catenin, and non-phospho β-catenin in a concentration-dependent manner. The relative intensity of protein band was normalized to GAPDH by Image J software (P < 0.05; *P < 0.01). All data presented as means ± SD from three independent experiments. doi:10.1371/journal.pone.0152931.g001 doi:10.1371/journal.pone.0152931.g001 catenin in the presence, absence of LiCl and β-catenin siRNA. Specific primer pairs were designed to amplify the promoter region of Nurr1 (Forward 5’-ACAATGTTTGGCTTCCTT GG-3’, reverse 5’- AACCACCAGCCTCCATACAC -3’). The CHIP resulting DNA samples were amplified by real time quantitative using SYBR Green master Mix (Applied Biosystems, Saint-Aubin, France). Statistical Analysis All results were presented as means ± standard deviation (SD). One-way analysis of variance (ANOVA) followed by Student–Newman–Keuls test were used to compare differences between means in more than two groups. The level of significance was set at P < 0.05. All the statistical analyses were performed with SPSS 21.0 software for Windows (SPSS Inc., Chicago, IL, USA). Rotenone Induced Cell Injury and Deregulated Wnt/β-Catenin Pathway LiCl inhibited caspase-3 activity in PC12 cells (P< 0.05; #P< 0.05 compared to rotenone treated group). C. LiCl reversed the down-regulation of Wnt3a, β-catenin and non-phospho-β-catenin induced by rotenone (P < 0.05; P < 0.01 compared to vehicle; #P < 0.05 compared to rotenone treated group). All data are presented as means ± SD from three independent experiments. doi:10.1371/journal.pone.0152931.g002 Remarkable decreases were observed in the protein levels of β-catenin and non-phospho-β- catenin, molecules representing the activity of Wnt/β-catenin signaling, in a rotenone concen- tration-dependent manner (Fig 1B). Our results demonstrate that Wnt/β-catenin signaling was deregulated in the toxic cell model induced by rotenone. Remarkable decreases were observed in the protein levels of β-catenin and non-phospho-β- catenin, molecules representing the activity of Wnt/β-catenin signaling, in a rotenone concen- tration-dependent manner (Fig 1B). Our results demonstrate that Wnt/β-catenin signaling was deregulated in the toxic cell model induced by rotenone. LiCl Attenuated Rotenone-Induced Cell Injury We further ask whether interfering of Wnt/β-catenin signaling would affect the outcome of rote- none-treated cells. Pretreatment of 2 mM LiCl for 72h attenuated rotenone-induced cell injury when the concentration of rotenone was in the range of 0.1~10μM (Fig 2A). Consistently, using Caspase-3/CPP32 Fluor metric Assay Kit, the caspase-3 activity was detected significantly up to 204.51 ± 6.44% in PC12 cells treated with 1 μmol/L rotenone compared with non-treated control (Fig 2B). However, when LiCl was pre-added 72 hours before rotenone, the activity of caspase-3 was down to 141.09 ± 4.67% (P < 0.01) in comparison to rotenone treatment alone. To further investigate Wnt/β-catenin signaling in rotenone-induced cell injury, we examined Wnt3a, β-cate- nin and non-phospho-β-catenin by Western blotting. After the cells were exposed to 1 μmol/L rotenone, a significant decrease in β-catenin and non-phospho-β-catenin was found in PC12 cells (68.89 ± 3.84%, 66.39 ± 4.14% respectively, P < 0.05). Pretreatment of 2 mmol/L LiCl signif- icantly attenuated those changed (Fig 2C). Collectively, our data here demonstrate that the acti- vation of Wnt/β-catenin signaling by LiCl functions as a rescue of rotenone-induced cell injury. Rotenone Induced Cell Injury and Deregulated Wnt/β-Catenin Pathway As showed in Fig 1A, treatment of 0.1~100 μmol/L rotenone for 24 hours caused a concentra- tion-dependent reduction of PC12 cell viability. 1 μmol/L rotenone reduced the cell viability to nearly half (49.68 ± 8.24%) (Fig 1A), which was referenced to the subsequent experiments. 5 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0152931 April 5, 2016 β-Catenin Crosstalk with Nurr1 Fig 2. LiCl activated Wnt/β-catenin pathway for the survival of PC12 cells. PC12 Cells were pretreated with 2 mmol/L LiCl for 72 hours or 7 days prior to 1 μmol/L (μM) rotenone exposure, respectively. A. LiCl attenuated rotenone-induced cell injury. The cell viability was assessed as a percentage relative to vehicle group (n = 6 per group) and means ± SD (P< 0.05; #P< 0.01 compared to equivalent rotenone treated group). B. LiCl inhibited caspase-3 activity in PC12 cells (P< 0.05; #P< 0.05 compared to rotenone treated group). C. LiCl reversed the down-regulation of Wnt3a, β-catenin and non-phospho-β-catenin induced by rotenone (P < 0.05; *P < 0.01 compared to vehicle; #P < 0.05 compared to rotenone treated group). All data are presented as means ± SD from three independent experiments. doi:10 1371/journal pone 0152931 g002 Fig 2. LiCl activated Wnt/β-catenin pathway for the survival of PC12 cells. PC12 Cells were pretreated Fig 2. LiCl activated Wnt/β-catenin pathway for the survival of PC12 cells. PC12 Cells were pretreated with 2 mmol/L LiCl for 72 hours or 7 days prior to 1 μmol/L (μM) rotenone exposure, respectively. A. LiCl attenuated rotenone-induced cell injury. The cell viability was assessed as a percentage relative to vehicle group (n = 6 per group) and means ± SD (P< 0.05; #P< 0.01 compared to equivalent rotenone treated group). B. LiCl inhibited caspase-3 activity in PC12 cells (P< 0.05; #P< 0.05 compared to rotenone treated group). C. LiCl reversed the down-regulation of Wnt3a, β-catenin and non-phospho-β-catenin induced by rotenone (P < 0.05; *P < 0.01 compared to vehicle; #P < 0.05 compared to rotenone treated group). All data are presented as means ± SD from three independent experiments. g β p y p with 2 mmol/L LiCl for 72 hours or 7 days prior to 1 μmol/L (μM) rotenone exposure, respectively. A. LiCl attenuated rotenone-induced cell injury. The cell viability was assessed as a percentage relative to vehicle group (n = 6 per group) and means ± SD (P< 0.05; #P< 0.01 compared to equivalent rotenone treated group). B. SB216763 Activated Wnt/β-Catenin and Attenuated Rotenone-Induced Cell Injury Combined with LiCl result, we conclude that Wnt/β-catenin pathway was deregulated by rotenone treatment, and re-activation of it might alleviate rotenone-induced cell injury. SB216763 Activated Wnt/β-Catenin and Attenuated Rotenone-Induced Cell Injury Similarly, GSK3β antagonist SB216763 was used to activate Wnt/β-catenin pathway to see its effect on rotenone-induced cell injury. Our data showed that SB216763 (0.5 to 10μM) attenuate rotenone-induced cell injury in a concentration-dependent manner (Fig 3A). Therefore 6 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0152931 April 5, 2016 β-Catenin Crosstalk with Nurr1 Fig 3. SB216763 activated Wnt/β-catenin pathway for the survival of PC12 cells. A. SB216763 attenuated rotenone-induced cell injury (P< 0.05 compared to vehicle; #, P < 0.05 compared to rotenone). B. SB216763 inhibited the caspase-3 activity. Cells pretreated with 0~10 μM SB216763 in 30 minutes prior to 24 hours treatment of 1 μM rotenone. Caspase-3 activity was assessed by Caspase-3/CPP32 Fluor metric Assay Kit, and expressed as percentages relative to vehicle (n = 6 per group) ( P < 0.05; #P < 0.05 compared to rotenone treated group). doi:10 1371/journal pone 0152931 g003 Fig 3. SB216763 activated Wnt/β-catenin pathway for the survival of PC12 cells. A. S # Fig 3. SB216763 activated Wnt/β-catenin pathway for the survival of PC12 cells. A. SB216763 attenuated rotenone-induced cell injury (P< 0.05 compared to vehicle; #, P < 0.05 compared to rotenone). B. SB216763 inhibited the caspase-3 activity. Cells pretreated with 0~10 μM SB216763 in 30 minutes prior to 24 hours treatment of 1 μM rotenone. Caspase-3 activity was assessed by Caspase-3/CPP32 Fluor metric Assay Kit, and expressed as percentages relative to vehicle (n = 6 per group) ( P < 0.05; #P < 0.05 compared to rotenone treated group). Fig 3. SB216763 activated Wnt/β-catenin pathway for the survival of PC12 cells. A. SB216763 attenuated rotenone-induced cell injury (P< 0.05 compared to vehicle; #, P < 0.05 compared to rotenone). B. SB216763 inhibited the caspase-3 activity. Cells pretreated with 0~10 μM SB216763 in 30 minutes prior to 24 hours treatment of 1 μM rotenone. Caspase-3 activity was assessed by Caspase-3/CPP32 Fluor metric Assay Kit, and expressed as percentages relative to vehicle (n = 6 per group) ( P < 0.05; #P < 0.05 compared to rotenone treated group). doi:10.1371/journal.pone.0152931.g003 5 μmol/L SB216763 was used to the follow-up experiments for its protective effect on cell via- bility. Further experiment showed that 5 μmol/L SB216763 was sufficient to reverse the rote- none-increased caspase-3 activity from 212.63 ± 22.58% to 158.79 ± 21.64% (P < 0.05) (Fig 3B). β-Catenin Protected PC12 Cells from Rotenone-Induced Injury To look into the association between β-catenin and rotenone induced cell death, a siRNA strat- egy was used to knock down β-catenin gene. As shown in Fig 4, when PC12 cells were trans- fected with 100 nmol/L β-catenin siRNA, the level of β-catenin mRNA was reduced to 16.15 ± 1.36% within 24 hours which was detected by real-time PCR while the protein level was down to 30.79 ± 2.81% after 72 hours by Western blotting. PC12 cell viability was also sig- nificantly reduced to 59.85 ± 9.13% with the interference of 100 nmol/L β-catenin siRNA, while the cell viability was further worsened to 27.96 ± 1.59% by the treatment of β-catenin siRNA plus 1 μmol/L rotenone. Under the β-catenin siRNA condition, pretreatment with 2 7 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0152931 April 5, 2016 β-Catenin Crosstalk with Nurr1 Fig 4. β-catenin prevented PC12 cells from death induced by rotenone. A. Real-time PCR for measuring the mRNA level of β-catenin which was down-regulated by siRNA (P < 0.05). B. the protein level of β- catenin was inhibited in PC12 cells transfected with β-catenin siRNA (P < 0.05). C. Under transfection of β- catenin siRNA, pretreatment with LiCl or SB216763 failed to attenuate the cell loss induced by rotenone. (P< 0.05 compared to control, #P< 0.05 compared to rotenone. &P< 0.05 compared to β-catenin siRNA). doi:10.1371/journal.pone.0152931.g004 Fig 4. β-catenin prevented PC12 cells from death induced by rotenone. A. Real-time PCR for measuring the mRNA level of β-catenin which was down-regulated by siRNA (P < 0.05). B. the protein level of β- catenin was inhibited in PC12 cells transfected with β-catenin siRNA (P < 0.05). C. Under transfection of β- catenin siRNA, pretreatment with LiCl or SB216763 failed to attenuate the cell loss induced by rotenone. (P< 0.05 compared to control, #P< 0.05 compared to rotenone. &P< 0.05 compared to β-catenin siRNA). doi:10.1371/journal.pone.0152931.g004 Fig 4. β-catenin prevented PC12 cells from death induced by rotenone. A. Real-time PCR for measuring the mRNA level of β-catenin which was down-regulated by siRNA (P < 0.05). B. the protein level of β- catenin was inhibited in PC12 cells transfected with β-catenin siRNA (P < 0.05). C. Under transfection of β- catenin siRNA, pretreatment with LiCl or SB216763 failed to attenuate the cell loss induced by rotenone. (P< 0.05 compared to control, #P< 0.05 compared to rotenone. &P< 0.05 compared to β-catenin siRNA). β-Catenin Protected PC12 Cells from Rotenone-Induced Injury doi:10.1371/journal.pone.0152931.g004 mmol/L LiCl or 5 μmol/L SB216763 for 72 hours failed to attenuate the cell loss induced by rotenone and the cell viability were dropped to 28.83 ± 4.35% and 28.42 ± 5.73% respectively, which showed no significant difference compared with 1 μmol/L rotenone only (P > 0.05) (Fig 4C). The Regulation of Wnt/β-Catenin on Nurr1 Expression in PC12 Cells Previous studies have demonstrated that both β-catenin and Nurr1 have protective effects on DA neurons. But little is known about whether there is a synergistic effect between β-catenin and Nurr1 and whether one regulates the expression of the other. So the interaction between β- catenin and Nurr1 was investigated. As our results showed, LiCl or SB216763 both significantly raised β-catenin and Nurr1 mRNA levels (Fig 5A). Similarly, the data of western blotting showed that treatment of rotenone caused a significant decrease of Nurr1 level, while pretreat- ment of LiCl reversed this change (Fig 5B). The co-immunoprecipitation of Nurr1 with β-cate- nin was detected in PC12, or HEK 293T cells which were transfected with Nurr1 or β-catenin plasmids, suggesting that Nurr1 and β-catenin were interacted with each other in the PLOS ONE \| DOI:10.1371/journal.pone.0152931 April 5, 2016 8 / 14 β-Catenin Crosstalk with Nurr1 Fig 5. Crosstalk between β-catenin and Nurr1. A. LiCl or SB216763 activated the Wnt/β-catenin to induce Nurr1 transcription, while β-catenin siRNA had no effect on Nurr1 expression, which was detected by Real- time PCR (P< 0.05 β-catenin compared to control; #P < 0.05 Nurr1 compared to control). B. LiCl reversed the down-regulation of Nurr1 expression induced by rotenone in vitro. The relative band intensities of Nurr1 were normalized to GAPDH (*P < 0.01 compared to control; # P < 0.05 compared to rotenone). C. Interaction of Nurr1 with β-catenin. Cells were transfected with plasmids carrying Nurr1 or β-catenin gene respectively. Collected lysates from HEK 293T and PC12 were subjected to co-immunoprecipitation by Nurr1 or β-catenin antibodies to detect their crosstalk. doi:10 1371/journal pone 0152931 g005 Fig 5. Crosstalk between β-catenin and Nurr1. A. LiCl or SB216763 activated the Wnt/β-catenin to induce Nurr1 transcription, while β-catenin siRNA had no effect on Nurr1 expression, which was detected by Real- time PCR (P< 0.05 β-catenin compared to control; #P < 0.05 Nurr1 compared to control). B. LiCl reversed the down-regulation of Nurr1 expression induced by rotenone in vitro. The relative band intensities of Nurr1 were normalized to GAPDH (*P < 0.01 compared to control; # P < 0.05 compared to rotenone). C. Interaction of Nurr1 with β-catenin. Cells were transfected with plasmids carrying Nurr1 or β-catenin gene respectively. Collected lysates from HEK 293T and PC12 were subjected to co-immunoprecipitation by Nurr1 or β-catenin antibodies to detect their crosstalk. doi:10.1371/journal.pone.0152931.g005 transfected cells (Fig 5C). The Regulation of Wnt/β-Catenin on Nurr1 Expression in PC12 Cells To summary, our data suggest that β-catenin might directly interact with Nurr1 to execute its function of cell survival against rotenone. The Analysis of the Interaction between β-Catenin and Nurr1 The crosstalk between β-catenin with Nurr1 was further studied based on the hypothesis above in both PC12 cells and rat midbrains. As shown in Fig 6A and 6B, in PC12 cells and the mid- brain of rat, the interaction between β-catenin and the AF1 domain of Nurr1 was detected by the GST pull down assay. Chromatin immunoprecipitation assay also confirmed that β-catenin directly bound to the promoter region of Nurr1 gene (Fig 6C). GSK3β inhibitor LiCl and SB216763 enhanced the bindings of β-catenin to 1.686±0.13 and 2.128±0.05 respectively com- pared with control level: 1.213±0.10. Consistent with previous result, knockdown of β-catenin with siRNA decreased the enrichment level of β-catenin on the promoter region of Nurr1 down to 0.473±0.04 (P < 0.05). These data clearly showed that mechanistically β-catenin not only directly regulates the gene expression of Nurr1 but also interacts with Nurr1 protein to execute their cell survival function against rotenone. Discussion This study demonstrated the protection effect of Wnt/β-catenin and Nurr1 on the differenti- ated PC12 cells against rotenone-induced DA neurotoxin. Differentiated PC12 cells are widely used as a suitable model to explore the mechanisms of degeneration of DA neuron in Parkin- son’s disease [25] and the function of the Wnt/β-catenin signaling pathway[26–28]. Here we 9 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0152931 April 5, 2016 β-Catenin Crosstalk with Nurr1 Fig 6. The interaction of β-catenin with Nurr1. The binding site was at AF1, the domain of Nurr1 with β- catenin detected by GST pull down assay. A. PC12 cells. B. Midbrain of rat. C. β-catenin bonded to the up- stream promoter of Nurr1, resulting in enhanced transcription of Nurr1 (P < 0.05 compared to control). doi:10.1371/journal.pone.0152931.g006 Fig 6. The interaction of β-catenin with Nurr1. The binding site was at AF1, the domain of Nurr1 with β- catenin detected by GST pull down assay. A. PC12 cells. B. Midbrain of rat. C. β-catenin bonded to the up- stream promoter of Nurr1, resulting in enhanced transcription of Nurr1 (*P < 0.05 compared to control). doi:10.1371/journal.pone.0152931.g006 doi:10.1371/journal.pone.0152931.g006 confirmed that rotenone caused a concentration-dependent reduction in the cell viability, and 50% of the cell viability affected by 1 μmol/L rotenone was set as a desired cellular model for follow-up tests. We also identified that β-catenin and non-phospho β-catenin of Wnt/β-catenin signaling pathway were expressed in differentiated PC12 cells. As an environment factor, rote- none selectively inhibits mitochondrial complex I, causing mitochondrial impairment via oxi- dative stress, and resulting in an increase of apoptotic markers of neurons such as caspase-3 activity, caspase-9 activity, Bax expression, cytochrome c, lactate dehydrogenase release and microtubule destabilization in rotenone-evoked Parkinsonism [29–34]. In our experiment, rotenone was demonstrated to increase caspase-3 activity, reduce the PC12 cell viability, dereg- ulate the activity of Wnt/β-catenin signaling pathway in a concentration-dependent manner. The relative level of Wnt3a, β-catenin and non-phospho-β-catenin was found at a similar reduction pattern. Similarly many reports showed that MPTP/MPP+ or 6-OHDA resulted in dysfunction of Wnt/β-catenin signaling pathway both in mice and cellular models of Parkinson disease [30, 35–36]. confirmed that rotenone caused a concentration-dependent reduction in the cell viability, and 50% of the cell viability affected by 1 μmol/L rotenone was set as a desired cellular model for follow-up tests. We also identified that β-catenin and non-phospho β-catenin of Wnt/β-catenin signaling pathway were expressed in differentiated PC12 cells. PLOS ONE \| DOI:10.1371/journal.pone.0152931 April 5, 2016 β-Catenin Crosstalk with Nurr1 Our data indicated that Wnt/β-catenin signaling pathway can be activated by GSK3β antag- onists LiCl or SB216763 in PC12 cells. LiCl or SB216763 was found to increase the level of β- catenin and non-phospho-β-catenin (a form of active β-catenin) and attenuate the cell death affected by rotenone, but either of them failed to protect β-catenin siRNA transfected PC12 cells from the cell death induced by rotenone, indicating β-catenin might be a key molecule in Wnt/β-catenin pathway for PC12 cells to survive. Consistently, our previous data showed that 6-OHDA decreased β-catenin level, but activation of Wnt/β-catenin pathway by exogenous wnt1 attenuated 6-OHDA-induced neurotoxicity through restoring mitochondria and endo- plasmic reticulum function [30]. In addition, activation of the Wnt/β-catenin signaling path- way by exogenous wnt1 was reported to exert robust neuroprotection on the caspase-3 activation. Besides, canonical Wnt/β-catenin pathway is required for the survival of adult DA neurons in aged mice following MPTP insult [9], and this pathway was demonstrated to play a critical role in promoting neuronal survival in Parkinson disease and Alzheimer disease [37– 44]. In summary, our data provide one more piece of solid evidence on the critical survival effect of Wnt/β-catenin pathway for DA neuronal toxin resistance. β p y The mechanisms underlying the protection of the Wnt/β-catenin signaling pathway on DA neurons remain elusive. It involves in multiple mechanisms including the regulation of pro- survival process or anti-apoptosis [5,9], plasticity of neuroprogenitors of subventricular zone in adult brain [35], enhanced DA differentiation potential of multipotent clonogenic neural stem/progenitor cells (mNPCs) in the adult midbrain aqueduct periventricular regions (Aq- PVRs) [45], synaptic plasticity [46–47], inhibition of oxidative stress and inflammation[30,45], and so on. In this study, we demonstrated the interaction between β-catenin and AF1 region of Nurr1 as the underlying mechanism. Furthermore, using CHIP PCR assay, we found that LiCl or SB216763 promoted β-catenin binding on the upstream promoter region of Nurr1 and increased the transcription of Nurr1. Thus we hypothesis that as a downstream of Wnt/β-cate- nin signaling pathway, β-catenin not only directly regulates Nurr1 gene expression but also interacts with Nurr1 protein to improve the anti-apoptosis capability of PC 12 cells and dopa- minergic neurons. PLOS ONE \| DOI:10.1371/journal.pone.0152931 April 5, 2016 Discussion As an environment factor, rote- none selectively inhibits mitochondrial complex I, causing mitochondrial impairment via oxi- dative stress, and resulting in an increase of apoptotic markers of neurons such as caspase-3 activity, caspase-9 activity, Bax expression, cytochrome c, lactate dehydrogenase release and microtubule destabilization in rotenone-evoked Parkinsonism [29–34]. In our experiment, rotenone was demonstrated to increase caspase-3 activity, reduce the PC12 cell viability, dereg- ulate the activity of Wnt/β-catenin signaling pathway in a concentration-dependent manner. The relative level of Wnt3a, β-catenin and non-phospho-β-catenin was found at a similar reduction pattern. Similarly many reports showed that MPTP/MPP+ or 6-OHDA resulted in dysfunction of Wnt/β-catenin signaling pathway both in mice and cellular models of Parkinson disease [30, 35–36]. 10 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0152931 April 5, 2016 References 1. Dauer W, Przedborski S. Parkinson's disease: mechanisms and models. Neuron 2003; 39: 889–909. PMID: 12971891. 2. Gasser T. Molecular pathogenesis of Parkinson disease: insights from genetic studies. Expert Rev Mol Med 2009; 11: e22. PMID: 19631006. doi: 10.1017/S1462399409001148 3. Morale MC, Serra PA, Delogu MR, Migheli R, Rocchitta G, Tirolo C, et al. Glucocorticoid receptor defi- ciency increases vulnerability of the nigrostriatal dopaminergic system: critical role of glial nitric oxide. Faseb J 2004; 18: 164–166. PMID: 14630699. 4. Warner TT, Schapira AH. Genetic and environmental factors in the cause of Parkinson's disease. Ann Neurol 2003; 53 Suppl 3: S16–S23, S23-S25. PMID: 12666095. 5. L'Episcopo F, Tirolo C, Testa N, Caniglia S, Morale MC, Cossetti C, et al. Reactive astrocytes and Wnt/ β-catenin signaling link nigrostriatal injury to repair in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease. Neurobiol. Dis 2011; 41: 508–527. PMID: 21056667. doi: 10.1016/j.nbd. 2010.10.023 6. Wurst W, Prakash N. Wnt1-regulated genetic networks in midbrain dopaminergic neuron development. J Mol Cell Biol2014; 6: 34–41. PMID: 24326514. doi: 10.1093/jmcb/mjt046 7. Backman C, Perlmann T, Wallen A, Hoffer BJ, Morales M. A selective group of dopaminergic neurons express Nurr1 in the adult mouse brain. Brain Res 1999; 851: 125–132. PMID: 10642835. 8. Joksimovic M, Awatraman R. Wnt/beta-catenin signaling in midbrain dopaminergic neuron specification and neurogenesis. J Mol Cell Biol 2014; 6: 27–33. PMID: 24287202. doi: 10.1093/jmcb/mjt043 9. L'Episcopo F, Serapide MF, Tirolo C, Testa N, Caniglia S, Morale MC, et al. A Wnt1 regulated Frizzled- 1/beta-Catenin signaling pathway as a candidate regulatory circuit controlling mesencephalic dopami- nergic neuron-astrocyte crosstalk: Therapeutical relevance for neuron survival and neuroprotection. Mol Neurodegener 2011; 6: 49. PMID: 21752258. doi: 10.1186/1750-1326-6-49 10. Saucedo-Cardenas O, Quintana-Hau JD, Le WD, Smidt MP, Cox JJ, De Mayo F, et al. Nurr1 is essen- tial for the induction of the dopaminergic phenotype and the survival of ventral mesencephalic late dopaminergic precursor neurons. Proc Natl Acad Sci U S A 2008; 95: 4013–4018. PMID: 9520484. 11. Zetterstrom RH, Solomin L, Jansson L, Hoffer BJ, Olson L, Perlmann T. Dopamine neuron agenesis in Nurr1-deficient mice. Science 1997; 276: 248–250. PMID: 9092472. 12. Zetterstrom RH, Williams R, Perlmann T, Olson L. Cellular expression of the immediate early transcrip- tion factors Nurr1 and NGFI-B suggests a gene regulatory role in several brain regions including the nigrostriatal dopamine system. Brain Res Mol Brain Res 1996; 41: 111–120. PMID: 8883941. 13. Freese JL, Pino D, Pleasure SJ. Author Contributions Conceived and designed the experiments: LZ SQ PX. Analyzed the data: LZ LW PX. Contrib- uted reagents/materials/analysis tools: LZ LW ML PX. Wrote the paper: LZ SL PX. Contributed to cell culture: MM YX. Contributed to immunofluorescence, RT-PCR, western blotting and co-immunoprecipitation: LZ LC CS QL JF XY. β-Catenin Crosstalk with Nurr1 could, in part, affect the activity of Nurr1 in the maintenance of dopaminergic neurons function. could, in part, affect the activity of Nurr1 in the maintenance of dopaminergic neurons function. PLOS ONE \| DOI:10.1371/journal.pone.0152931 April 5, 2016 In agreement with our discovery, Wnt/β-catenin signaling pathway was reported to enhance the transcription of Nurr1 in HEK293 cells [48] and Nurr1 mRNA level was markedly increased in BATGAL mice and 10-month-old 4Cat mice, two models of con- stitutive β-catenin activation mice[45,49]. Moreover, β-catenin activated by BIO, a known inhibitor of GSK3β, was reported to increase production of aldosterone by stimulating Nurr1 expression in mouse Y1 adrenocortical cells in vitro [49]. In HEK293T cells, β-catenin facili- tated Nurr1 mediated transactivation of the NBRE3tk-LUC reporter regulated by three binding sites for the NR4A receptors. Furthermore, knocking down β-catenin resulted in a significant decrease in both Nurr1 mRNA and protein in H295R cells [49]. However, in U2-OS and HeLa cells, β-catenin was reported to repress Nurr1 mediated transactivation of the NBRE3tk-LUC reporter [48]. These inconsistent observations suggested that Nurr1 expression regulated by β- catenin may vary with different cell types. In short, β-catenin inducing Nurr1 expression or Nurr1 affecting the activity of β-catenin suggests that a positive regulatory loop may exist between β-catenin (activation of Nurr1) and Nurr1 itself (reciprocal inhibition of β-catenin). For our results, it should be kept in mind that although differentiated PC12 cells adopt a neuro- nal-like phenotype, the cells might not completely represent typical DA neurons[50–52]. Fur- ther studies need to be done in animal models of PD. In conclusion, our data supported that the enhancement of Wnt/β-catenin signaling pathway protect dopaminergic cells against rote- none-toxicity. In term of mechanism, Wnt/β-catenin signaling play a role in restoring mito- chondria and endoplasmic reticulum function by interacting partly with Nurr1 on its dopaminergic regulation effects. 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https://openalex.org/W2143229392	https://bmcpublichealth.biomedcentral.com/counter/pdf/10.1186/1471-2458-13-20	English	null	Outdoor advertising, obesity, and soda consumption: a cross-sectional study	BMC public health	2,013	cc-by	7,129	RESEARCH ARTICLE Open Access * Correspondence: LesserL@pamfri.org 1Department of Health Policy, Palo Alto Medical Foundation Research Institute, Palo Alto, CA, USA Full list of author information is available at the end of the article © 2013 Lesser 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. Outdoor advertising, obesity, and soda consumption: a cross-sectional study Lenard I Lesser1*, Frederick J Zimmerman2 and Deborah A Cohen3 Lesser et al. BMC Public Health 2013, 13:20 http://www.biomedcentral.com/1471-2458/13/20 Lesser et al. BMC Public Health 2013, 13:20 http://www.biomedcentral.com/1471-2458/13/20 Abstract Background: Recent research has shown that neighborhood characteristics are associated with obesity prevalence. While food advertising in periodicals and television has been linked to overweight and obesity, it is unknown whether outdoor advertising is related to obesity. Methods: To test the association between outdoor food advertising and obesity, we analyzed telephone survey data on adults, aged 18–98, collected from 220 census tracts in Los Angeles and Louisiana. We linked self-reported information on BMI and soda consumption with a database of directly observed outdoor advertisements. Results: The higher the percentage of outdoor advertisements promoting food or non-alcoholic beverages within a census tract, the greater the odds of obesity among its residents, controlling for age, race and educational status. For every 10% increase in food advertising, there was a 1.05 (95% CI 1.003 - 1.093, p<0.03) greater odds of being overweight or obese, controlling for other factors. Given these predictions, compared to an individual living in an area with no food ads, those living in areas in which 30% of ads were for food would have a 2.6% increase in the probability of being obese. Conclusions: There is a relationship between the percentage of outdoor food advertising and over Conclusions: There is a relationship between the percentage of outdoor food advertising and overweight/obesity. Keywords: Obesity, Sugar-sweetened beverages, Advertising Keywords: Obesity, Sugar-sweetened beverages, Advertising Keywords: Obesity, Sugar-sweetened beverages, Advertising Keywords: Obesity, Sugar-sweetened beverages, Advertising Background even counter the effect of health promotion messages. For instance, in response to the growing public health concern over cholesterol and a decrease in butter sales, a marketing campaign to promote butter in Canada had a positive effect on butter demand [11]. Obesity is one of the world’s most intractable health pro- blems [1]. While the causes of obesity are multifactorial, a growing body of evidence implicates food marketing as a major contributor to the epidemic [2,3]. Recent com- prehensive reviews leave no doubt that a variety of mar- keting strategies increases food consumption in the laboratory environment and in natural settings [4-6]. Yet the public health community does not know how large an effect food marketing has on population-level obesity status. Additionally, there is limited research on advertis- ing’s effect on consumption and obesity in adults outside the laboratory setting. p Many researchers have focused on the content of food television advertising and its association with consump- tion and obesity [12-16]. The public health community has focused little attention to the role of outdoor adver- tising on food consumption and obesity. One important driver of differences in obesity rates among socioeco- nomic and racial and ethnic groups [17] may be the vari- ation in their exposure to outdoor food advertising. A recent study directly observed outdoor advertisements related to food and physical activity in several diverse zip codes in New York, Philadelphia, Los Angeles, and Austin [18]. The density of advertising in zip codes whose residents were predominantly African American was highest; Latino zip code areas had slightly lower densities; zip code areas with predominantly white resi- dents had the lowest densities of all. This study further There is ample evidence that, in the words of the Insti- tute of Medicine, “advertising works” [7]. Marketing re- search demonstrates that as marketing expenditures to promote a particular food increase, so do purchases of those foods [8-10]. Industry marketing campaigns can Page 2 of 7 Lesser et al. BMC Public Health 2013, 13:20 http://www.biomedcentral.com/1471-2458/13/20 found that living in a high SES zip code, regardless of its residents' predominant race or ethnicity, was generally protective against exposure to obesity-promoting out- door advertising (food, fast food, sugary beverages, sed- entary entertainment, and motorized transportation). separate concurrent observations in approximately 10% of the selected census tracts in both sites to ensure the reliability of observations. Data sources We analyzed outdoor advertisement data collected from a study on alcohol consumption that was geographically limited to allow site visits [20]. The areas selected were densely populated (>2000 residents per square mile) tracts within a one-hour drive from Drew Medical Cen- ter in Los Angeles and within 2 hours from Tulane Uni- versity in New Orleans. From those areas (1328 tracts in Los Angeles; 381 tracts in Louisiana), a random sample of 114 census tracts in Los Angeles County and 114 census tracts in Southeastern Louisiana were selected. Observers in two-person teams visited sampled census tracts from September 2004 to August 2005 in Southern Louisiana and from October 2004 to November 2005 in Los Angeles County. They systematically surveyed each tract once, first following the perimeter of each tract and then going through each street in the tract from north to south and from east to west. Observers recorded each outdoor adver- tisement’s latitude and longitude using a GPS monitor. Background The supervisor’s results were compared to the field staff’s to verify agreement, which was routinely greater than 0.8. Given that food marketing predominantly promotes foods that discouraged by the Dietary Guidelines for Americans [19] differences in exposure to outdoor food advertising by SES and race/ethnicity may partly explain observed racial/ethnic disparities in obesity rates. This study investigated whether individuals living in areas with higher proportions of outdoor food advertising, compared to those in living in areas with lower amounts, have greater odds of obesity and a higher rate of soda consumption. During the same time period and in the same census tracts in which the outdoor advertisements were sur- veyed, telephone interviews were conducted with a sys- tematic sample of adults from geographically referenced telephone-listed households. Calling was halted early in New Orleans due to Hurricane Katrina, when 106 tracts had been completed. Participants were offered $15 to complete a 15–20 minute interview. The interview asked many health questions, and included the participants’ height, weight, and how many 12-oz sodas they con- sumed in the past 24 hours. In Louisiana the average response rate per census tract was 37.9%; in Los Angeles, it was 34.4%. For comparison, the response rate for California and Louisiana in the 2005 wave of the Behavioral Risk Factor Surveillance Survey was 29.2% and 36.5%, respectively [21]. There were 2,881 respondents in our study. We excluded adults who were underweight (BMI<18.5, n=48), because we were concerned that this could have reflected an illness, rendering them less susceptible to obesity. Our main analysis and demographic data contain information on the 2589 participants without missing data. Statistical methods O i Our main outcome was self-reported BMI categorized according to National Institutes of Health criteria (nor- mal weight, overweight, or obese) [22]. A secondary out- come variable was the number of 12-oz sodas consumed in the previous 24 hours. Advertisements were coded into one of several mutu- ally exclusive categories, including entertainment, food/ beverage (not including alcohol), alcohol, and other pro- ducts. We calculated the percentage of the advertise- ments in each census tract that promoted all types of food or non-alcoholic beverages. We did not distinguish between healthy and unhealthy food advertisements, as fewer than 5% of the ads were for vegetables or fruits. Most were for drinks, snacks, and restaurants. Addition- ally, both healthy and unhealthy food advertisements are likely to cue the body to eat and stimulate hunger [23]. g g Each team then followed a standard protocol and used a standard data collection instrument to document the type of the product advertised. This information was used to classify all the outdoor advertisements into 4 major product subcategories: alcohol, tobacco, food and/or restaurants, and other products. Observers also recorded the format of media used: 1) posters, flyers, flags, banners, or transit shelters or benches, 2) small billboards (larger than poster or banners but smaller than 12’ × 24’), 3) average size billboards (12’ × 24’), and 4) extra-large billboards (14’ × 48’). The surveyors included all types of outdoor advertisements, except for storefront adver- tisements, as they were part of a separate study. In order to better capture double-sided billboards and multiple banners, posters, and flyers at the same adver- tising location, observers coded the frequency with which the ad appeared: Once, 2–4 times, 5–10 times, 11+ times. A quality control supervisor conducted As a measure of effective exposure to food ads, we used the percentage of total advertising that was for food. Other authors have used this measure when ana- lyzing outdoor advertising [18]. This percentage-based measure is useful because the effect of advertising is reduced when it must compete with other advertising. That is, a tract with 50 food ads out of 100 ads total will have an effective exposure to food ads that is much higher than a tract that has 50 food ads out of 1,000 Lesser et al. BMC Public Health 2013, 13:20 http://www.biomedcentral.com/1471-2458/13/20 Page 3 of 7 amounts of food advertising. Statistical methods O i Doing so permits the com- parison of estimated obesity rates in a hypothetical cen- sus tract with no food advertising to one with high levels. To perform this simulation, we used our regres- sion model and held all covariates at their mean level. Then, using the regression equation, we predicted the probability of obesity at food advertising levels of 0% and 30%. The values of 0% (no food advertising) and 30% correspond to the 10th and 90th percentile values of per- centage of all advertisements promoting food in the sampled census tracts. These values also correspond to two representative regions in the regions we studied: West Los Angeles or Old Aurora in New Orleans (which have generally higher median incomes and few minor- ities) and South Los Angeles or an urban area of the Sev- enth Ward in New Orleans (which have lower median incomes and more minorities). total ads. Accordingly the percentage reflects the strength of food advertising exposure, while also controlling for zoning laws that may limit the total number of all types of advertisements in an area. Additionally, the percentage- based measure was used instead of looking at percentage of sheet space because the former adjusts for physical proximity to the ad and for the speed with which consu- mers view ads. For instance, billboards have more sheet space, but are often viewed while driving, and thus are viewed for a shorter period of time. Bus stop ads have less sheet space; individuals view them for a longer period of time, while waiting for a bus or driving by them at slower speeds. We coded those census tracts with no advertisements at all as having 0% of ads related to food. To enhance interpretability of the scale of the association, we divided the food-ad percentage measure by 10, so that a 1-unit increase in the main independent variable represents a 10-percentage-point increase in the per- cent of ads devoted to food. Because previous work has shown that advertisements for sugary drinks were one of the most prevalent forms of outdoor advertising [18], we hypothesized that the amount of general advertising would likely be associated soda drinking. We used count data models to assess the association of the number of soda drinks per day with food advertising. As above, we conducted simulations to compare predicted soda consumption in two hypothet- ical census tracts. Statistical methods O i We included several covariates in our model includ- ing age in years and years-squared (to increase the fit of our model), education, race, and ethnicity as poten- tial confounders. Education was dummy coded, with college education as the baseline. We included race and ethnicity in the model as exclusive categories. For respondents that indicated more than one race category (approximately 2%), we used the first one recorded by the surveyor. To control for the total number of advertise- ments in each census tract, we included the total count of all advertisements for each census tract in the model. We accounted for the respondents’ clustering in census tracts by using STATA’s svy command. All analyses were performed using STATA, version 11. The RAND Human Subjects Protection Committee approved the study. Results BMC Public Health 2013, 13:20 Page 4 o http://www.biomedcentral.com/1471-2458/13/20 Table 1 Demographic information on sample used in main analysis (n=2589) Number Percent of Sample or Mean (SD) Age 43.5 (13.5) Weight Status Normal Weight 1028 40% Overweight 909 35% Obese 652 25% Gender Male 975 38% Female 1614 62% Highest Grade Completed Less Than High School 88 3% Less than 12th Grade 210 8% High School Graduate 630 24% Some College Education 647 25% College Graduate 1010 39% Ethnicity Not Hispanic 1796 81% Hispanic 497 19% Race White 1306 50% Black 793 31% Asian 92 4% Other Race 398 15% Servings of soda in the last day 1.3 (1.9) Table 1 Demographic information on sample used in main analysis (n=2589) obesity. (Table 3) For every 10% increase in food adver- tisements, the odds of being obese increased by 5% [OR 1.05 (95% CI: 1.00, 1.10), p<0.03]. Advertising clutter (the total number of advertisements in the census tract) did not significantly confound this relationship. g y p Using a simulation, and controlling for individual fac- tors such as race/ethnicity and education, we predicted that in an area with no food ads, 37.1% of adults would be overweight and 22.6% would be obese. In an area with 30% food ads, 38.0% would be overweight and 25.2% would be obese. Given these predictions, com- pared to an individual living in an area with no food ads (e.g. West Los Angeles or Old Aurora), those living in areas with 30% food ads (e.g., South Los Angeles or an urban area of the Seventh Ward) would have a 2.6% increase in the probability of being obese. In a census tract with 5000 people, if 30% of ads promote food, we would expect to find an additional 100–150 people who are obese. We then tested the association between soda drinking and food advertisements, using a count model. We per- formed several tests before picking the appropriate count model. The likelihood test for the assumption of equidispersion was significant, rejecting the poisson model and suggesting a negative binomial model is pre- ferred. Results The Vuong test was not significant, failing to r ject the assumption of no excess zeros, suggesting tha Table 1 Demographic information on sample used in main analysis (n=2589) Number Percent of Sample or Mean (SD) Age 43.5 (13.5) Weight Status Normal Weight 1028 40% Overweight 909 35% Obese 652 25% Gender Male 975 38% Female 1614 62% Highest Grade Completed Less Than High School 88 3% Less than 12th Grade 210 8% High School Graduate 630 24% Some College Education 647 25% College Graduate 1010 39% Ethnicity Not Hispanic 1796 81% Hispanic 497 19% Race White 1306 50% Black 793 31% Asian 92 4% Other Race 398 15% Servings of soda in the last day 1.3 (1.9) Table 2 Predictors of having any food advertisements in a census tracta Odds Ratio 95% CI Socio-Economic Characteristics High Income White 1.00 - Low Income White 1.52 0.52-4.41 High Income Black 2.94 0.83-10.35 Low Income Black 2.59 1.04-6.48 High Income Latino 0.93 0.21-4.21 Low Income Latino 3.10 1.03-9.20 High Income Asian 6.34 0.61-66.2 Low Income Asian 2.15 0.38-12.19 aRegression also controls for percent female, age and age-squared (not shown). Table 3 Odds of obesity in relation to percentage of foo advertisementsa Odds Ratio 95% C Food Ad Percentage (in 10% increments) 1.05 1.00-1. Total Number of Ads 1.00 1.00-1. Demographic Control Variables Female Gender 0.65 0.56-0. Education Less Than High School 1.99 1.29-3. Some High School 1.54 1.16-2. High School 1.38 1.14-1. Some College 1.33 1.09-1. College 1.00 - Ethnicity Not Hispanic 1.00 - Hispanic 1.38 1.08-1. Race White 1.00 - Black 2.22 1.83-2. Asian 0.57 0.36-0. Other Race 1.31 0.99-1. aModel also controls for age and age-squared (not shown). bp<0.03: CI overlaps 1.00 due to rounding. Lesser et al. Results Table 1 shows the demographic characteristics of the sample used in our ordered BMI analysis. Twenty-five percent (25%) of the adults were obese, while 35% were overweight and 40% were of normal weight. Adults drank, on average, 1.3 (sd 1.9) 12-ounce sodas per day. We also examined which census-tract level factors were associated with the percentage of food advertise- ments. We used 2000 census data and defined census tracts based on predominant race/ethnicity and income. Each census tract was labeled with the race that corre- sponded to the most prevalent race or ethnicity in that census tract. We further categorized the census tracts as low or high income by whether their median incomes were below or above the median income for the sampled census tracts in their region (i.e. Louisiana or Los Angeles). We categorized the census tracts instead of trying to model a linear relationship because the former is recommended on efficiency grounds when the under- lying relationship is non-linear. We used logistic regres- sion to determine which factors were associated with a census tract had any food advertisements. We conducted an ordered logistic regression to test the hypothesized association between overweight/obesity and advertising in a fully adjusted model. We included 219 census tracts in the analysis. (One tract was deleted due to its being an industrial area, with only one respondent). The average number of outdoor advertisements in each census tract was 10.2 (sd=17.3, median=4). The average percentage of the advertise- ments related to food or beverages was 10.4% (sd=17.5, median=0). Among the census tracts, 67 (30.6%) had no outdoor advertisements, and 122 (55.7%) had no food advertisements. Los Angeles had significantly more ads per census tract than New Orleans (14 vs. 6, p<0.01), but had a lower percentage of food ads than New Orleans (6% vs. 15%, p<0.05). The median household income in the sampled tracts in Louisiana was $34,930; in Los Angeles it was $41,957. Census tracts that were low-income with the plurality of their population designated as Black or Latino had sig- nificantly greater odds of having any food ads compared to high-income white census tracts. (Table 2) We found To facilitate interpretation, we simulated the probabil- ities of obesity for adults living in areas with different Page 4 of 7 Lesser et al. Results BMC Public Health 2013, 13:20 http://www.biomedcentral.com/1471-2458/13/20 no significant relationship between census tract charac- teristics and percent of outdoor advertisements promot- ing food. The ordered logistic regression revealed that those who lived in areas with a greater percentage of food advertisements had increased odds of overweight and obesity. (Table 3) For every 10% increase in food adve tisements, the odds of being obese increased by 5% [O 1.05 (95% CI: 1.00, 1.10), p<0.03]. Advertising clut (the total number of advertisements in the census tra did not significantly confound this relationship. Using a simulation, and controlling for individual fa tors such as race/ethnicity and education, we predict that in an area with no food ads, 37.1% of adults wou be overweight and 22.6% would be obese. In an ar with 30% food ads, 38.0% would be overweight a 25.2% would be obese. Given these predictions, com pared to an individual living in an area with no fo ads (e.g. West Los Angeles or Old Aurora), those livi in areas with 30% food ads (e.g., South Los Angeles an urban area of the Seventh Ward) would have a 2.6 increase in the probability of being obese. In a cens tract with 5000 people, if 30% of ads promote food, w would expect to find an additional 100–150 peop who are obese. We then tested the association between soda drinki and food advertisements, using a count model. We p formed several tests before picking the appropria count model. The likelihood test for the assumption equidispersion was significant, rejecting the poiss model and suggesting a negative binomial model is pr ferred. aModel also controls for age and age-squared (not shown). bp<0.03: CI overlaps 1.00 due to rounding. p between census tract charac- utdoor advertisements promot- egression revealed that those a greater percentage of food eased odds of overweight and y ( ) tisements, the odds of be 1.05 (95% CI: 1.00, 1.10 (the total number of adve did not significantly confo Using a simulation, and tors such as race/ethnicit that in an area with no fo be overweight and 22.6% with 30% food ads, 38. 25.2% would be obese. pared to an individual l ads (e.g. West Los Angel in areas with 30% food a an urban area of the Sev increase in the probabili tract with 5000 people, i would expect to find a who are obese. We then tested the ass and food advertisements, formed several tests be count model. The likelih equidispersion was sign model and suggesting a n ferred. The Vuong test w ject the assumption of no p Number Percent of Sample or Mean (SD) 43.5 (13.5) 1028 40% 909 35% 652 25% 975 38% 1614 62% 88 3% 210 8% 630 24% 647 25% 1010 39% 1796 81% 497 19% 1306 50% 793 31% 92 4% 398 15% 1.3 (1.9) ng any food advertisements in Odds Ratio 95% CI 1.00 - 1.52 0.52-4.41 2.94 0.83-10.35 2.59 1.04-6.48 0.93 0.21-4.21 3.10 1.03-9.20 6.34 0.61-66.2 2.15 0.38-12.19 t female, age and age-squared (not Table 3 Odds of obesity in advertisementsa Food Ad Percentage (in 10% inc Total Number of Ads Demographic Control Variables Female Gender Education Less Than High School Some High School High School Some College College Ethnicity Not Hispanic Hispanic Race White Black Asian Other Race aModel also controls for age and ag overlaps 1 00 due to rounding Results The Vuong test was not significant, failing to re- ject the assumption of no excess zeros, suggesting that a Table 3 Odds of obesity in relation to percentage of food advertisementsa Odds Ratio 95% CI Food Ad Percentage (in 10% increments) 1.05 1.00-1.10b Total Number of Ads 1.00 1.00-1.00 Demographic Control Variables Female Gender 0.65 0.56-0.76 Education Less Than High School 1.99 1.29-3.07 Some High School 1.54 1.16-2.02 High School 1.38 1.14-1.67 Some College 1.33 1.09-1.61 College 1.00 - Ethnicity Not Hispanic 1.00 - Hispanic 1.38 1.08-1.76 Race White 1.00 - Black 2.22 1.83-2.71 Asian 0.57 0.36-0.89 Other Race 1.31 0.99-1.76 aModel also controls for age and age-squared (not shown). bp<0.03: CI overlaps 1.00 due to rounding. Table 3 Odds of obesity in relation to percentage of food advertisementsa no significant relationship between census tract charac- teristics and percent of outdoor advertisements promot- ing food. The ordered logistic regression revealed that those who lived in areas with a greater percentage of food advertisements had increased odds of overweight and Table 2 Predictors of having any food advertisements in a census tracta Odds Ratio 95% CI Socio-Economic Characteristics High Income White 1.00 - Low Income White 1.52 0.52-4.41 High Income Black 2.94 0.83-10.35 Low Income Black 2.59 1.04-6.48 High Income Latino 0.93 0.21-4.21 Low Income Latino 3.10 1.03-9.20 High Income Asian 6.34 0.61-66.2 Low Income Asian 2.15 0.38-12.19 aRegression also controls for percent female, age and age-squared (not shown). Table 2 Predictors of having any food advertisements in a census tracta Table 2 Predictors of having any food advertisements in a census tracta Odds Ratio 95% CI Lesser et al. BMC Public Health 2013, 13:20 http://www.biomedcentral.com/1471-2458/13/20 Page 5 of 7 Angeles, likely reflects the city’s focus on entertainment, as documented in another study [18]. negative binomial model is preferred to a zero-inflated negative binomial model. Thus, our final model used the negative binomial model. Most research on the effect of outdoor advertising has focused on tobacco and alcohol. For instance, many studies have shown that African-American neighbor- hoods (regardless of community income) have more al- cohol and tobacco outdoor ads than White areas [29,30]. In a study similar to ours, researchers found that expos- ure to outdoor alcohol advertising was associated with problem drinking in women [31]. The percent of food ads was also associated with the number of 12-ounce sodas the respondents drank in the last day (Table 4). Discussion Advertising can influence food consumption by directly promoting food purchasing and indirectly by influencing social norms. Food advertising is associated with obesity in children [24-27] and marketing more generally has been identified as one of the major factors contributing to the obesity epidemic [2]. Our research confirms the observations of previous studies, which documented that unhealthy food advertise- ments are more prevalent in low income and minority communities [18,28]. We also found different amounts of advertising in the two cities we surveyed. This higher number of ads, but lower percentage of food ads in Los Table 4 Rate of soda consumption in relation to food advertisementsa Incidence Rate Ratio 95% CI Food Ad Percentage (in 10% increments) 1.06 1.03-1.09 Total Number of Ads 1.00 0.99-1.03 Demographic Control Variables Female Gender 0.76 0.68-0.85 Education Less Than High School 1.18 0.89-1.56 Some High School 1.61 1.29-2.01 High School 1.32 1.15-1.51 Some College 1.09 0.95-1.25 College 1.00 - Ethnicity Not Hispanic 1.00 - Hispanic 0.71 0.59-0.85 Race White 1.00 - Black 0.97 0.85-1.11 Asian 0.42 0.30-0.58 Other Race 0.85 0.72-1.01 aModel also controls for age, age-squared (not shown). Table 4 Rate of soda consumption in relation to food advertisementsa Thus, the summary of research in other areas points to an effect of outdoor advertising on the intentions of the viewers of those ads. This analysis finds parallel results to the previous research on alcohol, tobacco, and food: those who live in areas with higher percentages of food advertising have greater odds of obesity than those living in areas with a lower percentage of food ads. While we controlled for several factors that were likely to confound the association, there may be other unmeasured variables (e.g. individual preferences, urbanization of census tract, roadway structure, exposure to television advertisements) that could explain the association. Our study has limitations. We used self-reported in- formation on height, weight, and soda drinking. Self- reports of dietary practices are always subject to recall bias, but 24-hour recalls seem to be better than longer-term food frequency questionnaires [37]. BMI data are likely under-reported by about 1 unit (kg/m2) [38], which could have shifted the BMI distribution in our study downward. If respondents in our study reported a lower BMI than was true, we would be less likely to find an association between obesity and adver- tisements. Thus, the actual association may be stronger than the one we report. Results Each 10% increase in food advertise- ments was associated with a 6% increase in the number of sodas consumed [IRR=1.06 (95% CI: 1.03-1.09, p<0.01)]. A simulation showed that compared to a tract with 0% food advertising, people in a tract with 30% ad- vertising would be drinking 0.2 more sodas per day. This translates to about 16.8 fluid ounces, or about 196 kilo- calories per week. Most of the other evidence around the effect of adver- tising has looked at children, who are likely more suscep- tible to advertising [7]. Alcohol advertisements have been found to cluster around schools and predicted alco- hol consumption intentions by children at those schools [32]. Children exposed to tobacco advertising are more likely to smoke [33]. Other research has shown that un- healthy ads (including food, tobacco, and alcohol) have clustered around child-serving institutions [28]. In the area of obesity, many studies have documented an associ- ation between various types of advertising and consump- tion of unhealthy foods and obesity [12,24,34,35]. One other study looked at restaurant and food store signage and its relationship to obesity in adults living in the sur- rounding area [36]. The authors found that while adver- tisements for unhealthy foods around restaurants were associated with obesity, signage around convenience stores was not associated with obesity. aModel also controls for age, age-squared (not shown). Discussion Our data is also limited to two metropolitan areas. Fur- ther studies should try to replicate these results in Lesser et al. BMC Public Health 2013, 13:20 http://www.biomedcentral.com/1471-2458/13/20 Page 6 of 7 Page 6 of 7 would be to reduce outdoor food advertising in certain neighborhoods and determine whether obesity rates change. Given the health crisis associated with obesity, such measures may be warranted. different geographic settings. The survey data were col- lected by phone surveys. While the response rates were similar to those in another large national survey, our results could be biased by lack of response from those without landlines. However, one study found that the odds of obesity were similar in those with landlines and wireless phones [39]. While those without any phone service may have a higher odds of obesity than those with a landline, the proportion of the population in this category is less than 2% [39]. If those without phone ser- vice have a higher prevalence of obesity and are likely to live in areas where minorities predominate, and thus advertisements are prevalent, our effect estimates are likely to be biased towards the null hypothesis. If the above associations are confirmed by additional research, policy approaches may be important to reduce the amount of food advertising in urban areas. Bans on certain kinds of alcohol ads have reduced consumption in many countries [41]. Although efforts to control the placement of a particular type of outdoor advertising are likely to be deemed unconstitutional in the United States, requiring warnings on those advertisements are likely to be constitutionally acceptable [42]. Innovative strategies, such as warning labels, counter-advertising, or a tax on obesigenic advertising should be tested as pos- sible public health interventions for reducing the preva- lence of obesity. Furthermore, research on outdoor advertising is lim- ited by the difficulty in determining who is actually exposed to the advertisements over a particular time period. We assumed that people were exposed to adver- tisements in their own neighborhoods. However, people may also be exposed to advertisements on their way to work and other destinations; advertisements and expo- sures may also change over time. Our findings indicate that the exposure to food ads around a person’s home does measure some aspect of the food environment that is associated with obesity. Authors’ contributions LL l d h d f LL led the study, performed the analysis, and wrote the manuscript. FZ provided statistical input and did extensive editing of the manuscript. DC provided the dataset and did extensive editing of the manuscript. All authors read and approved the final manuscript. Author details 1D f 1Department of Health Policy, Palo Alto Medical Foundation Research Institute, Palo Alto, CA, USA. 2Department of Health Services, School of Public Health, University of California, Los Angeles, USA. 3RAND Corporation, Santa Monica, CA, USA. Our independent variable, percent of food advertise- ments devoted to food, has some limitations. In census tracts where the total number of ads is small, but all (100%) of the ads are for food, we may have overestimated the impact of food ads. However, we controlled for the total number of ads in a tract, which may also be a meas- ure of census tract geographic size. Even if there are only two ads in a geographically small census tract, they could be highly impactful in promoting consumption. Received: 28 June 2012 Accepted: 20 December 2012 Published: 10 January 2013 Received: 28 June 2012 Accepted: 20 December 2012 Published: 10 January 2013 Acknowledgements l d Dr. Lesser completed this work while at the University of California, Los Angeles. The Robert Wood Johnson Foundation funded this study. The Foundation had no role in the design, analysis, or manuscript preparation. References Chang H-S, Green R: The Effects of Advertising on Food Demand Elasticities. Can J Agric Econ/Rev Can Agroecon 1989, 37:481–494. 9. Ward RW, Dixon BL: Effectiveness of fluid milk advertising since the Dairy and Tobacco Adjustment Act of 1983. Am J Agric Econ 1989, 71:730–740. 10. Brester GW, Schroeder TC: The impacts of brand and generic advertising on meat demand. Am J Agric Econ 1995, 77:969–979. 6. Chandon P, Chandon P, Wansink B, Wansink B: Does food marketing need to make us fat? A review and solutions. Nutr Rev 2012, 70:571–593. Discussion Future studies taking into ac- count work location, time spent at work/home, and means of transportation to work may find a stronger as- sociation. Additionally, future studies could use actual locations of respondents to evaluate whether distance to advertisements matters. Competing interest Th h d l h Competing interest The authors declare that they have no competing interests. References 1. Swinburn BA, Sacks G, Hall KD, McPherson K, Finegood DT, Moodie ML, Gortmaker SL: The global obesity pandemic: shaped by global drivers and local environments. Lancet 2011, 378:804–814. 1. Swinburn BA, Sacks G, Hall KD, McPherson K, Finegood DT, Moodie ML, Gortmaker SL: The global obesity pandemic: shaped by global drivers and local environments. Lancet 2011, 378:804–814. 1. Swinburn BA, Sacks G, Hall KD, McPherson K, Finegood DT, Moodie ML, Gortmaker SL: The global obesity pandemic: shaped by global drivers and local environments. Lancet 2011, 378:804–814. 2. 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https://openalex.org/W107252767	https://www.cifor.org/publications/pdf_files/WPapers/WP127CIFOR.pdf	Portuguese	null	An&aacute;lise de aprova&ccedil;&otilde;es para empresas chinesas investirem nos setores de minera&ccedil;&atilde;o, agricultura e silvicultura da &Aacute;frica	null	2,013	cc-by	17,027	D O C U M E N T O D E T R A B A L H O Análise de aprovações para empresas chinesas investirem nos setores de mineração, agricultura e silvicultura da África D O C U M E N T O D E T R A B A L H O Análise de aprovações para empresas chinesas investirem nos setores de mineração, agricultura e silvicultura da África Análise de aprovações para empresas chinesas investirem nos setores de mineração, agricultura e silvicultura da África Huang Wenbin Andreas Wilkes Análise de aprovações para empresas chinesas investirem nos setores de mineração, agricultura e silvicultura da África Huang Wenbin Andreas Wilkes Documento de Trabalho 127 Documento de Trabalho 127 Documento de Trabalho 127 © 2013 Centro de Pesquisa Florestal Internacional (CIFOR) O conteúdo desta publicação é licenciado sob Creative Commons Attribution-NonCommercial- NoDerivs 3.0 Unported License http://creativecommons.org/licenses/by-nc-nd/3.0/ Huang W e Wilkes A. 2013. Análise de aprovações para empresas chinesas investirem nos setores de mineração, agricultura e silvicultura da África. Documento de Trabalho 127. Bogor, Indonésia: CIFOR. Traduzido de: Huang W and Wilkes A. 2011. Analysis of approvals for Chinese companies to invest in Africa’s mining, agriculture and forestry sectors. Working Paper 81. Bogor, Indonesia: CIFOR. Foto da capa por Iain Ding Complexo do Ministério do Comércio (MOFCOM) em Chang’an Avenue, em Pequim. O MOFCOM serve os interesses das empresas chinesas no país e no exterior e seu apoio está por trás de muitos dos recentes esforços de expansão de negócios internacionais da China. CIFOR Jl. CIFOR, Situ Gede Bogor Barat 16115 Indonésia T +62 (251) 8622-622 F +62 (251) 8622-100 E cifor@cgiar.org CIFOR Jl. CIFOR, Situ Gede Bogor Barat 16115 Indonésia T +62 (251) 8622-622 F +62 (251) 8622-100 E cifor@cgiar.org Análise de aprovações para empresas chinesas investirem nos setores de mineração, agricultura e silvicultura da África Huang Wenbin Centro Mundial Agroflorestal, Escritório para a China e o Leste da Ásia, Pequim Huang Wenbin Centro Mundial Agroflorestal, Escritório para a China e o Leste da Ásia, Pequim Andreas Wilkes Centro Mundial Agroflorestal, Escritório para a China e o Leste da Ásia, Pequim Andreas Wilkes Centro Mundial Agroflorestal, Escritório para a China e o Leste da Ásia, Pequim Documento de Trabalho 127 cifor.org Gostaríamos de agradecer a todos os os doadores que apoiaram esta pesquisa através de suas contribuições ao Fundo do CGIAR. Para uma lista dos doadores do Fundo, veja: https://www.cgiarfund.org/FundDonors Todos os argumentos expostos nesta publicação são atribuídos aos autores e não necessariamente representam a posição do CIFOR, instituições representadas pelos autores ou financiadores desta publicação. Índice Agradecimentos v Resumo vi Summary vii 1 Introdução 1 2 Aprovações para investimentos de empresas chinesas na África 2 2.1 Tendências na aprovação de investimentos 2 2.2 Destinos dos investimentos nas aprovações para a África 2 2.3 Investimentos previstos por tipo e setor 3 3 Aprovações para empresas chinesas que investem nos setores de mineração, silvicultura e agricultura da África 3 3.1 Visão geral 3 3.2 Aprovações de investimentos no setor de mineração na África 4 3.3 Aprovações de investimento no setor florestal na África 4 3.4 Aprovações de investimentos no setor agrícola na África 6 4 Aprovações de investimentos da China no setor de mineração, silvicultura e agricultura em ecorregiões 6 4.1 Visão geral 6 4.2 Aprovações de investimentos no setor de mineração em ecorregiões 7 4.3 Aprovações de investimentos no setor florestal em ecorregiões 7 4.4 Aprovação de investimentos agrícolas nas ecorregiões 8 5 Atividades de fusão e aquisição de empresas chinesas na África 8 6 Discussão 9 7 Referências 10 Anexo 1 Lista de empresas chinesas aprovadas para investir em mineração, silvicultura e agricultura na África 12 2 Principais eventos de aquisições e fusões da China no exterior 27 5 Atividades de fusão e aquisição de empresas chinesas na África Anexo 1 Lista de empresas chinesas aprovadas para investir em mineração, silvicultura e agricultura na África 12 2 Principais eventos de aquisições e fusões da China no exterior 2 Principais eventos de aquisições e fusões da China no exterior Tabela Tabela 1 Distribuição setorial de aprovações de investimentos da China no setor de mineração 5 Figuras 1 Número de empresas que receberam aprovação para investir na África (1988-2010) 2 2 Os 10 principais destinos para investimentos previstos na África 2 3 Composição da aprovação de investimentos na África, por setor econômico (1988-2010) 3 4 Principais destinos para investimentos chineses aprovados para o setor de mineração na África 4 5 Distribuição setorial de aprovações de investimentos chineses no setor de mineração 4 6 Os cinco principais destinos na África para a realização de investimentos no setor florestal por empresas chinesas 5 7 Distribuição setorial das aprovações de investimento chinês no setor florestal 5 8 Principais destinos para investimentos chineses aprovados para o setor agrícola 6 9 Distribuição setorial das aprovações de investimentos chineses no setor agrícola 6 10 Principais destinos para investimentos chineses no setor de mineração nas ecorregiões 6 11 Distribuição setorial de aprovações para investimentos chineses no setor de mineração nas ecorregiões 7 13 Distribuição setorial do investimento chinês no setor florestal nas ecorregiões 7 12 Principais destinos para investimentos chineses no setor florestal nas ecorregiões 7 14 Principais destinos de investimentos chineses no setor agrícola nas ecorregiões 8 15 Eventos de fusões e aquisições chinesas no exterior (de 2003 a meados de 2010) 8 16 Composição setorial de fusões e aquisições chinesas no exterior por número de eventos de M&A (de 2003 a meados de 2010) 9 Tabela 1 Distribuição setorial de aprovações de investimentos da China no setor de mineração Figuras Siglas BMZ Ministério Federal de Cooperação Econômica e Desenvolvimento, Alemanha CIFOR Centro de Pesquisa Florestal Internacional GIZ Cooperação Internacional da Alemanha ICRAF Centro Mundial Agroflorestal IED Investimento Estrangeiro Direto M&A Fusões e aquisições MOFCOM Ministério do Comércio, República Popular da China MOFTEC Ministério do Comércio Exterior e Cooperação Econômica, República Popular da China NPC Comissão Nacional de Planejamento RDC República Democrática do Congo Resumo Os impactos do investimento e comércio chinês com a África tornaram-se objeto de muita especulação. O projeto do CIFOR “Comércio e investimento chinês na África” busca entender os impactos do papel da China na mudança de fluxos comerciais globais e nos padrões de investimento em florestas em duas ecorregiões da África, a bacia do Congo e as florestas do sul da África, com foco em setores com impactos diretos potenciais sobre as florestas: mineração, silvicultura e agricultura. foram para a extração mineral e um terço foi para prospecção. As aprovações para investimento em mineração nas duas ecorregiões foram responsáveis por 45% de todos os registros de mineração para a África. A República Democrática do Congo, Zâmbia e Tanzânia, representaram 90% das aprovações para o setor de mineração, a grande maioria (86%) foi para extração mineral e atividades de prospecção. No setor florestal, houve 34 aprovações de investimentos em 14 países. Os principais destinos de investimento destinado às empresas florestais chineses na África são o Gabão, Zâmbia, Gana, Nigéria e República do Congo. No geral, o Gabão responde por quase 23% das aprovações do setor florestal. Dezoito das 34 aprovações foram nas duas ecorregiões, e mais de 40% destas foram no Gabão. A extração de madeira e a manufatura de madeira serrada juntas representam a maioria (84%) dos registros de aprovação. A falta de um banco de dados abrangente sobre os investimentos chineses na África torna difícil construir um contexto e analisar as tendências e padrões. A análise deste documento de trabalho baseia-se em um banco de dados (abrangendo de 1983 a 2010) de aprovações pelo Ministério do Comércio da República Popular da China (MOFCOM) para que empresas chinesas desenvolvam investimentos no exterior. Registros no banco de dados indicam a intenção das empresas de investir no exterior, mas não registram os investimentos reais que foram realizados. Fontes adicionais são utilizadas para caracterizar as tendências nas atividades de fusões e aquisições chinesas nos setores de interesse na África. Houve 35 registros para atividades relativas à agricultura. Zâmbia, Tanzânia, Sudão, Gana e Etiópia foram responsáveis por quase 60% dos registros de aprovação. A maioria das aprovações (74%) foi para a produção agrícola, com menor número para atividades de atacado e varejo (20%) e manufatura (5,7%). No geral, 43% das aprovações agrícolas estavam nas duas ecorregiões, quase metade das quais foram para o investimento na Zâmbia. Agradecimentos Este documento de trabalho é um produto do projeto “Comércio e investimento chinês na África: Avaliação e monitoramento de trade-offs para economias nacionais, meios de subsistência locais e ecossistemas florestais”. O projeto foi apoiado por um financiamento do Ministério Federal de Cooperação Econômica e Desenvolvimento, Alemanha (BMZ) através da Sociedade Alemã de Cooperação Internacional (GIZ) para CIFOR, e é implementado pelo CIFOR, o Centro Mundial Agroflorestal e da Universidade de Leipzig. A pesquisa para este documento de trabalho foi conduzida pelo Escritório para a China e o Leste da Ásia do Centro Mundial Agroflorestal, Pequim. 1. Introdução base na dispersa documentação disponível. Uma parte desse projeto é desenvolver a documentação sistemática de eventos de investimento na Bacia do Congo e nas florestas do sul da África. O projeto “Comércio e investimento chinês na África: Avaliação e monitoramento de trade-offs para economias nacionais, meios de subsistência locais e ecossistemas florestais”, lançado em março de 2010, tem como objetivo promover a compreensão dos impactos sociais, econômicos e ambientais do investimento chinês em mercadorias ou setores que afetam as florestas e meios de subsistência na África (por exemplo, madeira, mineração, agricultura), e fortalecer a capacidade dos tomadores de decisão do governo, da sociedade civil e do setor privado para aprovar reformas que maximizem os benefícios sociais e econômicos e, ao mesmo tempo, minimizem os efeitos adversos. Na ausência de um banco de dados completo, a análise deste documento de trabalho baseia-se em um banco de dados de solicitações de permissões por empresas chinesas para investir no exterior. Para tal foi utilizado o Banco de Dados de Empresas Chinesas que Investem no Exterior, do Ministério do Comércio da República Popular da China (MOFCOM), o qual contém uma lista de empresas chinesas que receberam aprovação para realizar novos investimentos no exterior durante o período de 1983-2010. O banco de dados foi construído para cumprir com as regulamentações do governo chinês, emitidas pela primeira vez em 1983 e, posteriormente, atualizadas várias vezes, as quais exigem que as empresas chinesas que pretendem fazer investimentos não comerciais no exterior devem solicitar a aprovação do MOFCOM (Conselho de Estado 1983, MOFTEC 1984, 1985, 1992, 1999, NPC 1991, MOFCOM, 2003, 2007, 2009). A listagem no banco de dados só pode ser tomada como uma indicação da intenção de uma empresa de investir no exterior. A listagem não implica que todas essas empresas, ao fim do processo, realizaram os investimentos na África ou nos setores em que elas solicitaram. Há duas outras ressalvas a serem observadas. Primeiro, o banco de dados não inclui fusões e aquisições (M&A) ou outros investimentos de capital minoritário. Uma vez que o banco de dados não inclui as empresas envolvidas apenas em fusões e aquisições de investimento, este documento de trabalho descreve algumas tendências em M&A chinesas (ver Seção 5). Em segundo lugar, o banco de dados não registra o valor dos investimentos previstos. Resumo O banco de dados contém 1.346 registros de aprovações para empresas chinesas investirem na África de 1988 a 2010, o que representa 8,47% de todas as aprovações para investimento no exterior concedido a empresas chinesas. Antes de 2002, menos de 10 aprovações foram concedidas por ano. Depois de 2002, as aprovações cresceram a uma taxa média anual de 250%, chegando a 388 em 2010. Dessas, 278 aprovações foram para os setores de mineração, silvicultura e agricultura, dos quais 128 (44%) estavam nas duas ecorregiões de interesse para o projeto do CIFOR. Globalmente, as fusões e aquisições (M&A) por empresas chinesas têm aumentado nos últimos anos. A África é o segundo destino mais frequente para eventos de M&A em mineração, e a pesquisa mostra um crescente interesse em atividades de fusões e aquisições no continente. A maioria das atividades de M&A é nos setores de mineração e petróleo. Mais pesquisas são necessárias para produzir um banco de dados abrangente das atividades reais de investimento chineses na África. As M&A, por serem uma forma crescente de investimentos no exterior por empresas chinesas, também merecem investigação. No setor de mineração, foram 209 aprovações, com 70% concentradas em 10 países: República Democrática do Congo (RDC), Zâmbia, Nigéria, Argélia, África do Sul, Sudão, Tanzânia, Etiópia, Gana e no Chade. Mais da metade das aprovações 1. Introdução A segunda fase é de 2002-2010, período em que o registro de solicitações de investimento aumentou dramaticamente, com uma taxa de crescimento médio anual de 250%. Em 2010, houve 388 registros de aprovações, quase 40 vezes mais do que em 2002. Camarões, República Democrática do Congo, Guiné Equatorial, Gabão, Moçambique, Tanzânia, Zâmbia e Zimbábue). A Seção 5 analisa os dados disponíveis sobre investimentos de M&A nesses setores com base em outras fontes de dados. A seção final discute algumas questões para futuras pesquisas levantadas pela presente análise. Camarões, República Democrática do Congo, Guiné Equatorial, Gabão, Moçambique, Tanzânia, Zâmbia e Zimbábue). A Seção 5 analisa os dados disponíveis sobre investimentos de M&A nesses setores com base em outras fontes de dados. A seção final discute algumas questões para futuras pesquisas levantadas pela presente análise. 1. Introdução As agências governamentais chinesas pertinentes registram os dados do valor de investimento estrangeiro direto (IED) separadamente e os divulgam usando outros sistemas. Este projeto visa compreender o papel da China na mudança de fluxos comerciais globais e padrões de investimento; entender o que é único sobre o comércio da África com a China (em relação a outros parceiros de desenvolvimento) no setor florestal; e identificar como as tendências do comércio e investimento do setor florestal estão se desenvolvendo na Bacia do Congo e as florestas do sul da África. Essas regiões foram escolhidas devido à importância de suas florestas. Os países de interesse nessas ecorregiões são a República do Congo, Camarões, República Democrática do Congo (RDC), Guiné Equatorial, Gabão, Moçambique, Tanzânia, Zâmbia e Zimbábue. Juntos, esses países têm uma área total de 6.300.000 km2, abrangendo mais de um quinto do total da massa terrestre africana. O projeto está identificando os trade-offs (impactos positivos e negativos) econômicos, sociais e ambientais para uma seleção de investimentos chineses que têm um impacto sobre as florestas no continente (por exemplo, madeira, cultivos agrícolas, biocombustíveis e mineração). Nos últimos anos, em linha com a estratégia “Going Out” da China anunciada em 2000 (MOFCOM 2001), muitas empresas chinesas começaram a explorar e fazer investimentos na África. Os impactos dessa tendência – tanto positivos como negativos – têm gerado muita especulação em todo o mundo. Um número crescente de relatórios acadêmicos e de organizações da sociedade civil documentam algumas dessas atividades e, em alguns casos, os impactos específicos sobre a ecologia, os meios de subsistência e a economia local. No entanto, eventos específicos de investimento não foram sistematicamente reunidos em nenhum outro lugar, o que torna difícil construir uma imagem e analisar tendências e padrões com Baseando-se principalmente no banco de dados do MOFCOM, este documento de trabalho analisa as tendências das intenções de investimento e os principais destinos de investimentos previstos de empresas chinesas em três setores importantes do uso da terra que podem ter impactos ambientais e sociais diretos: silvicultura, mineração e agricultura. A análise centra-se em dados para empresas que pretendem investir nesses setores em países da bacia do Congo e de florestas do sul da África (Congo, Huang Wenbin e Andreas Wilkes 2 10 empresas obtiveram a aprovação do MOFCOM a cada ano. 2. Aprovações para investimentos de empresas chinesas na África Os 1.346 registros de investimento encontrados referem-se a 45 países africanos, ou cerca de 85% 2.2 Destinos dos investimentos nas aprovações para a África 2. Aprovações para investimentos de empresas chinesas na África 2.3 Investimentos previstos por tipo e setor Para analisar os investimentos previstos por setor, adotamos a classificação no Código e Classificação da Indústria da Economia Nacional Chinesa (GB / T 4754-2002), um padrão nacional aplicado na China. A aplicação desse método de classificação revela que as empresas chinesas pretendem investir principalmente em oito setores econômicos: manufatura, serviços de locação e negócios, atacado e varejo, construção, mineração, pesquisa, serviços técnicos e prospecção geológica, agricultura, silvicultura, criação de animais e pesca, e mercado imobiliário1 (Figura 3). Atacado e varejo 14.0% Figura 3. Composição da aprovação de investimentos na África, por setor econômico (1988-2010) Comentário: Para entender melhor a composição dos investimentos da China na África, a análise nesta figura baseia- se no primeiro nível de “Classificação e Código”. Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores Entre esses setores, a meta de investimento mais frequente é a manufatura, representando cerca de 27,6% de todas as aprovações. Esta categoria abrange a indústria de fabricação em todos os setores, incluindo têxteis, produtos químicos, mineração, processamento de madeira e processamento de alimentos. O setor de serviços de locação e negócios é o segundo maior setor de investimentos previstos por empresas chinesas, representando mais de 21% do número total de registros. Esse setor inclui um grande número de escritórios de representação que pretendem fazer investimentos ou expandir a comercialização de seus produtos, muitos dos quais têm o propósito de procurar oportunidades de negócios e clientes potenciais na África. As empresas envolvidas no comércio internacional são categorizadas como pertencentes ao setor de atacado e varejo, o qual responde por quase 14% de todos os registros de investimento. A indústria da construção, que inclui atividades tais como moradias, estradas, ferrovias e pontes, corresponde a pouco mais de 12 % dos registros. O setor de mineração também é um campo de investimento importante para as empresas chinesas, representando mais de 9% dos registros. Deve-se notar, contudo, que no presente método de classificação, a mineração inclui apenas atividades de extração de minerais, tais como a extração de petróleo, gás natural, minerais metálicos e não metálicos. Outras atividades relacionadas à mineração são classificadas em outros setores, por exemplo: pesquisa, serviços técnicos e prospecção geológica. A classificação que inclui a agricultura, criação de animais, pesca e silvicultura em uma categoria pode ser considerada “agrícola” em seu sentido mais amplo. 2.1 Tendências na aprovação de investimentos Figura 2. Os 10 principais destinos para investimentos previstos na África Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores Nigéria 12.6% África do Sul 8.1% Zâmbia 6.2% Egito 5.9% Etiópia 5.6% Angola 4.8% Tanzânia 4.8% República Democrática do Congo 4.7% Argélia 4.6% Gana 4.5% Outros 38.2% Nigéria 12.6% África do Sul 8.1% Zâmbia 6.2% Egito 5.9% Etiópia 5.6% Angola 4.8% Tanzânia 4.8% República Democrática do Congo 4.7% Argélia 4.6% Gana 4.5% Outros 38.2% A análise do banco de dados do MOFCOM identificou 1.346 registros de empresas chinesas declarando sua intenção de investir na África, abrangendo o período entre 1988 e 2010. Isso representa 8,47% do número total (15.895) dos registros no banco de dados do MOFCOM. Embora o registro do primeiro investimento no banco de dados date de 1983, o primeiro registro de investimento na África é de 1988, quando a estatal Jiangxi International Economy e Technology Cooperation Corporation submeteu uma solicitação para estabelecer um escritório de representação na Zâmbia. Figura 2. Os 10 principais destinos para investimentos previstos na África Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores Para ajudar a compreender as tendências sobre as aprovações de investimentos, o período de 1988- 2010 pode ser dividido em duas fases. A primeira fase é de 1988-2001, período no qual menos de Figura 2. Os 10 principais destinos para investimentos previstos na África Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores Figura 1. Número de empresas que receberam aprovação para investir na África (1988-2010) Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores 0 50 1988 1992 1993 1996 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 100 150 200 250 300 350 400 450 Figura 1. Número de empresas que receberam aprovação para investir na África (1988-2010) Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores 0 50 1988 1992 1993 1996 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 100 150 200 250 300 350 400 450 Figura 1. 2.1 Tendências na aprovação de investimentos Número de empresas que receberam aprovação para investir na África (1988-2010) Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores de aprovações para empresas chinesas investirem nos setores de mineração, agricultura e silvicultura da África 3 3 Processamento e manufatura 27.6% Serviços de locação e negócios 21.2% Atacado e varejo 14.0% Indústria da construção civil 12.0% Mineração 9.3% Pesquisa, serviços técnicos e prospecção geológica 6.5% Agricultura, silvicultura, criação de animais e pesca 4.2% Setor imobiliário 3.9% Outros 1.4% Figura 3. Composição da aprovação de investimentos na África, por setor econômico (1988-2010) Comentário: Para entender melhor a composição dos investimentos da China na África, a análise nesta figura baseia- se no primeiro nível de “Classificação e Código”. Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores Processamento e manufatura 27.6% Serviços de locação e negócios 21.2% Atacado e varejo 14.0% Indústria da construção civil 12.0% Mineração 9.3% Pesquisa, serviços técnicos e prospecção geológica 6.5% Agricultura, silvicultura, criação de animais e pesca 4.2% Setor imobiliário 3.9% Outros 1.4% do continente. Os 10 principais destinos previstos para investimentos de empresas chinesas são Nigéria, África do Sul, Zâmbia, Egito, Etiópia, Angola, Tanzânia, República Democrática do Congo, Argélia e Gana. Os registros referentes a estes 10 países correspondem a 61,8 % do número total de empresas aprovadas para investir na África. 1 A China National Economy Industry Classification and Code (GB/T 4754–2002) é uma lista hierárquica de classificação industrial de três níveis, com itens mais detalhados em cada nível principal. 3. Aprovações para empresas chinesas que investem nos setores de mineração, silvicultura e agricultura da África 3. Aprovações para empresas chinesas que investem nos setores de mineração, silvicultura e agricultura da África 2.3 Investimentos previstos por tipo e setor Esse setor é responsável por pouco mais de 4 % do número total de registros. Distribuição por setor Os registros relacionados à mineração são distribuídos principalmente em cinco setores da classificação padrão: extração mineral, prospecção geológica, manufatura, serviços de locação e negócios, e atacado e varejo. A extração mineral é o mais comum deles, representando 55% de todos os registros relacionados à mineração. A prospecção geológica também é uma atividade importante, representando cerca de 31% dos registros. Alguns desses registros referem- se a empresas que prestam serviços de prospecção mineral para outras empresas, enquanto outros são para empresas que pretendem extrair os minerais descobertos através de prospecção. Manufatura e serviços de locação e negócios representam, cada um, cerca de 6% do total de registros de mineração; e o setor de atacado e varejo representa pouco menos de 2% dos registros (Figura 5). Figura 5. Distribuição setorial de aprovações de investimentos chineses no setor de mineração Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores Figura 5. Distribuição setorial de aprovações de investimentos chineses no setor de mineração Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores A indústria de mineração também inclui 14 empresas de petróleo, das quais 12 obtiveram aprovações para envolver-se em extração de petróleo e duas em manufatura (Tabela 1). 3.1 Visão geral Como mencionado acima, muitas aprovações para empresas de mineração, silvicultura e agricultura investirem na África são classificadas em outros setores da economia, tais como manufatura, atacado e varejo, e prospecção geológica. Nesta seção, Huang Wenbin e Andreas Wilkes 4 Figura 4. Principais destinos para investimentos chineses aprovados para o setor de mineração na África Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores República Democrática do Congo 18.2% Zâmbia 14.8% Nigéria 7.7% Argélia 5.3% África do Sul 4.8% Sudão 4.8% Tanzânia 4.8% Outros 39.7% República Democrática do Congo 18.2% Zâmbia 14.8% Nigéria 7.7% Argélia 5.3% África do Sul 4.8% Sudão 4.8% Tanzânia 4.8% Outros 39.7% separamos mineração, silvicultura e agricultura da categoria geral “agrícola”, e incluímos registros relativos a esses três setores que são alocados para outros setores ao utilizar a classificação padrão apresentada acima. No setor de mineração, os registros referentes a minerais metálicos e não metálicos (por exemplo, pedreiras) são algumas vezes difíceis de distinguir, e algumas aprovações de investimentos no setor de mineração de não metálicos podem ter sido incluídas no setor de mineração. Essa recategorização resulta em 278 registros relativos aos setores de mineração, silvicultura e agricultura, os quais representam mais de um quinto do total de registros de solicitação de investimentos. Existem 209, 34 e 35 registros nos setores de mineração, silvicultura e agricultura, respectivamente, ou cerca de 15%, 2,6 % e 2,6 % de todos os registros de solicitação de investimentos. Figura 4. Principais destinos para investimentos chineses aprovados para o setor de mineração na África Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores Distribuição por país O banco de dados recategorizado mostra um total de 209 registros no setor de mineração, distribuídos em 33 países africanos. Os 10 principais destinos previstos para investimentos por essas empresas são a República Democrática do Congo, Zâmbia, Nigéria, Argélia, África do Sul, Sudão, Tanzânia, Etiópia, Gana e o Chade. Registros referentes a estes 10 países correspondem a menos de 70% de todos os registros do setor de mineração. Outros 0.5% Serviços de locação e de negócios 6.2% Extração mineral 55.0% 3.2 Aprovações de investimentos no setor de mineração na África Figura 5. Distribuição setorial de aprovações de investimentos chineses no setor de mineração Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores Extração mineral 55.0% Pesquisa, serviços técnicos e prospecção geológica 30.6% Serviços de locação e de negócios 6.2% Processamento e manufatura 5.7% Atacado e varejo 1.9% Outros 0.5% Extração mineral 55.0% Pesquisa, serviços técnicos e prospecção geológica 30.6% Serviços de locação e de negócios 6.2% Processamento e manufatura 5.7% Atacado e varejo 1.9% Outros 0.5% Distribuição por país Os principais destinos para investimentos de empresas florestais chinesas na África são Gabão, Zâmbia, Gana, Nigéria e República do Congo, entre os quais o Gabão é o mais importante, representando quase 40% do total de registros. O Gabão é um importante fornecedor de madeira africana para a China, que por seus ricos recursos florestais e pelo ambiente político relativamente estável, tem atraído empresas florestais chinesas (Forest Trends 2007). As exportações de madeira em tora do Gabão para a China somam cerca de 40% das exportações de madeira em tora da África para a China nos últimos anos. Figura 6. Os cinco principais destinos na África para a realização de investimentos no setor florestal por empresas chinesas Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores 3.3 Aprovações de investimento no setor florestal na África Trinta e quatro registros de investimentos no setor florestal distribuídos em 14 países africanos abrangem Tabela 1. Distribuição setorial de aprovações de investimentos da China no setor de mineração Setor Registro de investimentos No. de registros de investimentos em petróleo Extração mineral 115 12 Pesquisa, serviços técnicos e prospecção geológica 64 0 Serviços de locação e de negócios 13 0 Processamento e fabricação 12 2 Atacado e varejo 4 0 Outros 1 0 Total 209 14 Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores Tabela 1. Distribuição setorial de aprovações de investimentos da China no setor de mineração e investimentos da China no setor de mineração Registro de investimentos No. de registros de investimentos em petróleo 115 12 64 0 13 0 12 2 4 0 1 0 209 14 tos no exterior, organizado pelos autores Figura 6. Os cinco principais destinos na África para a realização de investimentos no setor florestal por empresas chinesas Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores Gabão 22.9% Gana 11.4% Zâmbia 8.6% Nigéria 8.6% Congo (Brazzaville) 8.6% Outros 40.0% Figura 7. Distribuição setorial das aprovações de investimento chinês no setor florestal Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores Extração de recursos forestais 45.7% Processamento de madeira 22.9% Papel e produtos derivados de papel 20.0% Fabricação de móveis 11.4% Gabão 22.9% Gana 11.4% Zâmbia 8.6% Nigéria 8.6% Congo (Brazzaville) 8.6% Outros 40.0% o manejo florestal, a exploração madeireira, o processamento de madeira, a fabricação de papel e móveis e materiais para piso de madeira. Distribuição por país Os principais destinos para investimentos de empresas florestais chinesas na África são Gabão, Gabão 22.9% Gana 11.4% Outros 40.0% o manejo florestal, a exploração madeireira, o processamento de madeira, a fabricação de papel e móveis e materiais para piso de madeira. Gabão 22.9% Outros 40.0% o manejo florestal, a exploração madeireira, o processamento de madeira, a fabricação de papel e móveis e materiais para piso de madeira. 22.9% Outros 40 0% Distribuição por setorl ç p No setor florestal, cerca de 45,7% das aprovações de investimentos foram direcionados para a extração de recursos florestais (manejo florestal e exploração madeireira). Outras atividades incluíram o processamento de produtos madeireiros (painéis à base de madeira, etc.), fabricação de papel e produtos derivados de papel e móveis, os quais representam, respectivamente, cerca de 22,9%, 20,0% e 11,4% do total (Figura 7). Papel e produtos derivados de papel 20.0% Extração de recursos forestais 45.7% Processamento de madeira 22.9% Quando a exploração madeireira e o processamento de madeira serrada são categorizados como “processamento primário” e outras atividades como “tratamento secundário”, 18 registros (52,9%) correspondem ao setor de processamento primário e 16 (47,1%) ao setor de processamento secundário. Figura 7. Distribuição setorial das aprovações de investimento chinês no setor florestal Figura 7. Distribuição setorial das aprovações de investimento chinês no setor florestal Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores Huang Wenbin e Andreas Wilkes 6 Distribuição por país Os 35 registros de investimentos estão distribuídos em 17 países africanos. Zâmbia, Sudão, Tanzânia, Etiópia e Gana são os principais destinos de investimentos, sendo responsáveis por quase 60% do total de registros de aprovação de investimentos. 4. Aprovações de investimentos da China no setor de mineração, silvicultura e agricultura em ecorregiões Figura 8. Principais destinos para investimentos chineses aprovados para o setor agrícola Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores Zâmbia 20.0% Sudão 11.4% Tanzânia 11.4% Etiópia 8.6% Gana 8.6% Outros 40.0% Zâmbia 20.0% Sudão 11.4% Tanzânia 11.4% Etiópia 8.6% Gana 8.6% Outros 40.0% Zâmbia 20.0% Distribuição por setor A maioria das aprovações no setor agrícola é para a produção agrícola. Os outros setores registrados são atacado e varejo e processamento, que são responsáveis por 20,0% e 5,7% das aprovações, respectivamente (Figura 9). 3.4 Aprovações de investimentos no setor agrícola na África Dentre esses cinco países, Zâmbia é o mais representado, sendo responsável por quase 20% do número total de registros. Nesta seção, consideramos a agricultura definida em sentido estrito como cultivos, ou seja, excluindo silvicultura, criação de animais e pesca. Aplicando esta definição, 35 registros foram encontrados nesse setor. Distribuição por país Há um total de 18 registros de aprovação de investimento para o setor florestal nas ecorregiões da Bacia do Congo e das florestas do sul, que correspondem a quase 53% do total de registros florestais na África. O Gabão é o destino mais frequente de investimento, sendo responsável por mais de 40% do total de registros de investimento nas ecorregiões. contribuem com menos de 22% do total de registros para a África, o que não é muito. No entanto, destes, 44% (128 registros) referem-se à mineração, silvicultura e agricultura. Isso indica que esses setores nesses destinos de investimentos são relativamente significativos para as empresas chinesas. Distribuição por setor Quando os registros são analisados por setor econômico, a extração mineral é vista como responsável por uma grande parcela (64%). A extração e a prospecção geológica, juntas, respondem por 86% do total de registros de investimentos no setor de mineração. Não foram encontrados registros relacionados a petróleo nas ecorregiões alvo. Extração de recurso forestal 66.6% Figura 13. Distribuição setorial do investimento chinês no setor florestal nas ecorregiões 4.3 Aprovações de investimentos no setor florestal em ecorregiões Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores Distribuição por setor Quando olhamos para a distribuição setorial do investimento florestal da China nos países sob estudo, notamos que o processamento primário (ou seja, a extração de madeira e manufatura de madeira serrada) é responsável pela maioria (84%) dos registros (Figura 13). 4.1 Visão geral Distribuição setorial do investimento chinês no setor florestal nas ecorregiões Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores Extração de recurso forestal 66.6% Processamento de madeira 16.7% Papel e fabricação de produtos derivados de papel 11.1% Fabricação de móveis 5.6% Fabricação de móveis 5.6% Papel e fabricação de produtos derivados de papel 11.1% 4.1 Visão geral Distribuição setorial de aprovações para investimentos chineses no setor de mineração nas ecorregiões Fonte: Banco de dados do MOFCOM sobre investimentos no exterior organizado pelos autores Extração mineral 64.2% Pesquisa, serviços técnicos e prospecção geológica 22.1% Processamento e manufatura 8.4% Atacado e varejo 2.1% Serviços de locação e de negócios 2.1% Outros 1.1% Figura 11. Distribuição setorial de aprovações para investimentos chineses no setor de mineração nas ecorregiões Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores Extração mineral 64.2% Pesquisa, serviços técnicos e prospecção geológica 22.1% Processamento e manufatura 8.4% Atacado e varejo 2.1% Serviços de locação e de negócios 2.1% Outros 1.1% Figura 13. Distribuição setorial do investimento chinês no setor florestal nas ecorregiões Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores Extração de recurso forestal 66.6% Processamento de madeira 16.7% Papel e fabricação de produtos derivados de papel 11.1% Fabricação de móveis 5.6% Figura 12. Principais destinos para investimentos chineses no setor florestal nas ecorregiões Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores Gabão 44.4% Zâmbia 16.7% Congo (Brazzaville) 16.7% Guiné Equatorial 11.1% Tanzânia 5.6% Camarões 5.6% Extração mineral 64.2% Pesquisa, serviços técnicos e prospecção geológica 22.1% Processamento e manufatura 8.4% Atacado e varejo 2.1% Serviços de locação e de negócios 2.1% Outros 1.1% Gabão 44.4% Zâmbia 16.7% Congo (Brazzaville) 16.7% Guiné Equatorial 11.1% Tanzânia 5.6% Camarões 5.6% Camarões 5.6% Tanzânia 5.6% Extração mineral 64.2% Zâmbia 16.7% Zâmbia 16 7% Figura 12. Principais destinos para investimentos chineses no setor florestal nas ecorregiões Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores Figura 12. Principais destinos para investimentos chineses no setor florestal nas ecorregiões Figura 11. Distribuição setorial de aprovações para investimentos chineses no setor de mineração nas ecorregiões Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores são República Democrática do Congo, Zâmbia e Tanzânia. Juntos esses três países representam mais de 90% dos registros de investimento em mineração nas ecorregiões (Figura 10). Figura 13. 4.1 Visão geral Este projeto de pesquisa centra-se na Bacia do Congo e nas florestas do sul da África, devido à importância de seus remanescentes florestais. Os países dessas duas ecorregiões que levantam preocupavam são Camarões, República Democrática do Congo, Guiné Equatorial, Gabão, Moçambique, República do Congo, Tanzânia, Zâmbia e Zimbábue. Juntos, esses países representam 6,3 milhões de km2, mais de um quinto da área total africana. Figura 8. Principais destinos para investimentos chineses aprovados para o setor agrícola O banco de dados do MOFCOM inclui 293 registros de investimento nesses países, os quais Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores Figura 10. Principais destinos para investimentos chineses no setor de mineração nas ecorregiões Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores República Democrática do Congo 40.0% Zâmbia 32.6% Tanzânia 10.5% Congo (Brazzaville) 5.3% Guiné Equatorial 4.2% Gabão 4.2% Camarões 3.2% República Democrática do Congo 40.0% Zâmbia 32.6% Tanzânia 10.5% Congo (Brazzaville) 5.3% Guiné Equatorial 4.2% Gabão 4.2% Camarões 3.2% Figura 9. Distribuição setorial das aprovações de investimentos chineses no setor agrícola Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores Agricultura (cultivos) 74.3% Atacado e varejo 5.7% Processamento 20% Figura 10. Principais destinos para investimentos chineses no setor de mineração nas ecorregiões Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores República Democrática do Congo 40.0% Zâmbia 32.6% Tanzânia 10.5% Congo (Brazzaville) 5.3% Guiné Equatorial 4.2% Gabão 4.2% Camarões 3.2% Figura 9. Distribuição setorial das aprovações de investimentos chineses no setor agrícola Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores Agricultura (cultivos) 74.3% Atacado e varejo 5.7% Processamento 20% Guiné Equatorial 4.2% Processamento 20% Camarões 3.2% Atacado e varejo 5.7% Tanzânia 10.5% Zâmbia 32.6% Figura 10. Principais destinos para investimentos chineses no setor de mineração nas ecorregiões Figura 9. Distribuição setorial das aprovações de investimentos chineses no setor agrícola Figura 9. Distribuição setorial das aprovações de investimentos chineses no setor agrícola Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores lise de aprovações para empresas chinesas investirem nos setores de mineração, agricultura e silvicultura da África 7 7 Figura 11. Distribuição por país Um total de 95 registros para o setor de mineração nesses países corresponde a mais de 45% do total de registros de mineração para o continente africano. Os principais destinos para investimentos em mineração Huang Wenbin e Andreas Wilkes 8 8 é responsável por mais de 73% de todos os investimentos nas duas ecorregiões. é responsável por mais de 73% de todos os investimentos nas duas ecorregiões. Figura 14. Principais destinos de investimentos chineses no setor agrícola nas ecorregiões Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores Zâmbia 46.7% Tanzânia 26.7% Camarões 6.7% República Democrática do Congo 6.7% Guiné Equatorial 6.7% Gabão 6.7% Zâmbia 46.7% Tanzânia 26.7% Camarões 6.7% República Democrática do Congo 6.7% Guiné Equatorial 6.7% Gabão 6.7% Guiné Equatorial 6.7% Destes, 13 registros (87%) são para investimentos previstos na produção agrícola, e dois registros são para o setor de processamento. Destes, 13 registros (87%) são para investimentos previstos na produção agrícola, e dois registros são para o setor de processamento. Zâmbia 46.7% 5. Atividades de fusão e aquisição de empresas chinesas na África Tanzânia 26.7% Além dos novos investimentos registrados no banco de dados do MOFCOM, as empresas chinesas têm estado envolvidas em um grande número de atividades de M&A no exterior nos últimos anos, muitas das quais estão na África. As aprovações de M&A não estão incluídas na base de dados do MOFCOM. Para contribuir com as tendências nessa área, contamos com dois relatórios compilados pela Deloitte (2010a, 2010b). Estes relatórios incluem apenas atividades de M&A completadas, e não levam em conta o provável grande número de iniciativas de M&A que não foram completadas. Figura 14. Principais destinos de investimentos chineses no setor agrícola nas ecorregiões Figura 14. Principais destinos de investimentos chineses no setor agrícola nas ecorregiões Fonte: Banco de dados do MOFCOM sobre investimentos no exterior, organizado pelos autores 4.4 Aprovação de investimentos agrícolas nas ecorregiões O banco de dados do MOFCOM inclui 15 registros para agricultura, nas ecorregiões, respondendo por mais de 42% do total de registros no setor agrícola para a África. Isso indica que essas regiões são importantes destinos de investimentos agrícolas chineses. De 2003 a meados de 2010, a atividade de M&A da China no exterior aumentou consideravelmente, tanto em termos de valor como em números (Figura 15). Entre os países das duas ecorregiões, Zâmbia é o destino mais frequente de investimento, sendo responsável por quase metade de todos os registros. O potencial de investimento na Zâmbia torna o sul da África um importante destino de investimento agrícola. Junto com a Tanzânia, essa sub-região Entre essas fusões e aquisições, um grande número (tanto em termos de quantidade como valor) ocorreram nos setores de petróleo e mineração. Essas Quantidade Quantidade 40 35 30 25 20 15 10 5 Q1 Q2 2003 2004 2005 2006 2007 2008 2009 2010 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 0 25 000 20 000 15 000 10 000 5 000 0 Valor em milhões de USD Valor em milhões de USD Figura 15. Eventos de fusões e aquisições chinesas no exterior (de 2003 a meados de 2010) Fonte: Deloitte (2010a) Figura 15. Eventos de fusões e aquisições chinesas no exterior (de 2003 a meados de 2010) Fonte: Deloitte (2010a) Análise de aprovações para empresas chinesas investirem nos setores de mineração, agricultura e silvicultura da África Análise de aprovações para empresas chinesas investirem nos setores de mineração, agricultura e silvicultura da África 9 Figura 16. Composição setorial de fusões e aquisições chinesas no exterior por número de eventos de M&A (de 2003 a meados de 2010) Fonte: Deloitte (2010a) Energia, mineração e serviços públicos 39% Manufatura e indústria química 20% Telecomunicações, mídia e tecnologia 14% Bens de consumo 7% Serviços fnanceiros 7% Bens de lazer 4% Agricultura 3% Médica e biotecnologia 3% Negócios 2% Outros 1% atividades de M&A representam 39% do total de registros agrupados pelos relatos da Deloitte. Negócios 2% Outros 1% Entre os mercados alvos de M&A das empresas de mineração chinesas, a Australásia é o destino mais importante, sendo responsável por um quarto do total de eventos de M&A. 4.4 Aprovação de investimentos agrícolas nas ecorregiões Por setor, energia, petróleo e mineração são importantes indústrias alvo para atividades de M&A por empresas chinesas. 4.4 Aprovação de investimentos agrícolas nas ecorregiões A África é o segundo continente mais importante, responsável por 15% do total de registros. Deve-se notar que empresas da Australásia e algumas outras empresas podem ter sido assumidas ou compradas por empresas chinesas, que também operam na África. A Deloitte (2010b) apresenta uma análise de possíveis tendências futuras em atividades de M&A da China no setor de mineração no exterior. Utilizando dados a partir de entrevistas com 26 empresas mineradoras chinesas com participação anterior em atividades de M&A no exterior, a Deloitte identificou 33 eventos de M&A da China no setor de mineração no exterior, em 2009, representando USD 9,2 bilhões de investimento. A maioria dos eventos de M&A envolveu a compra de participações majoritárias ou minoritárias da empresa no exterior. A maioria dos entrevistados revelou que eles esperavam que a atividade de M&A da China aumentasse em 2010- 2011. A força motriz mais citada dessa tendência esperada era o objetivo de garantir o fornecimento de recursos. O aumento da participação no mercado, o alcance da economia de escala e o poder de barganha de preços também foram forças motrizes frequentemente mencionadas. Preocupações com a proteção ambiental foram o terceiro obstáculo mais citado para atividades de M&A, depois de regulamentações monetárias e a instabilidade do mercado financeiro. De longe, a África foi o destino potencial mais citado para M&A no exterior. As razões dadas para isso incluem a complementaridade entre os recursos abundantes da África e a capacidade das empresas chinesas a investir, os baixos custos de mão de obra e, a ameaça de imposição de um imposto sobre superlucros na Austrália nesse momento (antes de julho de 2010) que tornaram aquisições em outros países relativamente mais atraentes. Em Zhang (2011) são analisadas as forças motrizes que explicam a necessidade das empresas chinesas de garantir o abastecimento de matérias- primas no setor de mineração. Manufatura e indústria química 20% Figura 16. Composição setorial de fusões e aquisições chinesas no exterior por número de eventos de M&A (de 2003 a meados de 2010) investimentos no exterior mostra 1.346 registros de intenção de investir na África. Isso é equivalente a menos de 8,5% do número total de aprovações para empresas chinesas investirem no exterior. 4.4 Aprovação de investimentos agrícolas nas ecorregiões Entre os setores específicos de interesse para este projeto de pesquisa, o investimento em mineração na África é um campo importante para as empresas chinesas que investem na África, correspondendo a 15% do número total de registros de investimento. A República Democrática do Congo, Zâmbia, Nigéria, Argélia e África do Sul são importantes destinos de investimentos no setor de mineração, representando quase a metade de todos os registros de aprovação de investimento. As participações da silvicultura e da agricultura são muito menores (cerca de 2,6% cada). Os países da ecorregião da África central e do sul da África são importantes destinos de investimentos no setor de mineração, silvicultura e agricultura destinos de investimento para as empresas chinesas – uma grande parte de todos os registros de investimento para estes três setores em todo o continente Africano está concentrada em países nessas duas ecorregiões. A África também é responsável por uma parcela razoável das atividades de M&A no exterior realizadas por empresas chinesas, sendo o segundo destino de investimento mais frequente. Por setor, energia, petróleo e mineração são importantes indústrias alvo para atividades de M&A por empresas chinesas. investimentos no exterior mostra 1.346 registros de intenção de investir na África. Isso é equivalente a menos de 8,5% do número total de aprovações para empresas chinesas investirem no exterior. Entre os setores específicos de interesse para este projeto de pesquisa, o investimento em mineração na África é um campo importante para as empresas chinesas que investem na África, correspondendo a 15% do número total de registros de investimento. A República Democrática do Congo, Zâmbia, Nigéria, Argélia e África do Sul são importantes destinos de investimentos no setor de mineração, representando quase a metade de todos os registros de aprovação de investimento. As participações da silvicultura e da agricultura são muito menores (cerca de 2,6% cada). Os países da ecorregião da África central e do sul da África são importantes destinos de investimentos no setor de mineração, silvicultura e agricultura destinos de investimento para as empresas chinesas – uma grande parte de todos os registros de investimento para estes três setores em todo o continente Africano está concentrada em países nessas duas ecorregiões. A África também é responsável por uma parcela razoável das atividades de M&A no exterior realizadas por empresas chinesas, sendo o segundo destino de investimento mais frequente. 6. Discussão A análise deste documento de trabalho baseia-se principalmente em um banco de dados que registra as aprovações do MOFCOM para empresas chinesas A análise dos registros de aprovação do governo chinês para a participação de empresas chinesas em Huang Wenbin e Andreas Wilkes 10 proposals and feasibility reports for overseas investments. [Jiwaizi (1991) No. 1271]. investirem no exterior. O banco de dados não registra quais dessas empresas realmente realizaram os investimentos depois. Além disso, uma vez que o banco de dados registra apenas as intenções das empresas de investirem no exterior, é possível que algumas empresas com autorização para investirem no exterior, mais tarde, mudem o país ou setor de investimento com base no seu escopo original e atividades de planejamento. Por isso, o banco de dados não pode ser visto com uma indicação dos investimentos que realmente foram realizados por empresas chinesas. Conselho de Estado, República Popular da China 1983 Circular on assignment of responsibility in foreign economy and trade. [Guofa (1982) No. 13]. Deloitte 2010a Rising sun: a new chapter in China’s overseas merger and acquisitions. Deloitte, Pequim, China (em chinese). Deloitte 2010b Mining for growth: a review of outbound mining M&A activity from China. Deloitte, Pequim, China. Isto pode ser ilustrado com referência ao setor florestal. O banco de dados do MOFCOM registra 18 empresas pretendendo investir no setor florestal nas ecorregiões do centro e sul da África. Usando a lista de empresas obtida a partir do banco de dados do MOFCOM e cruzando referências com a lista de empresas registradas pela alfândega da China como importadoras de madeira em tora da África para a China em 2008 e 2009 para realizar a busca on-line por relatos da mídia sobre eventos de investimento, 10 empresas informaram ou declararam em seus próprios produtos de mídia que fizeram investimentos no setor de manejo florestal, e quatro empresas informaram que fizeram investimentos no processamento de produtos de madeira na África (Huang et al. 2011). No entanto, pesquisas realizadas nos países (por exemplo, Putzel e Kabuyaya 2011) revelaram que alguns investimentos reivindicados são, na verdade, não operacionais, como ocorre quando há desistência das concessões ou quando outros fatores impedem as atividades de investimento. Mais pesquisas seriam necessárias para identificar se as empresas de fato realizaram os investimentos. 6. Discussão A pesquisa sobre as razões para o sucesso ou fracasso na realização dos investimentos previstos aumentaria a compreensão dos fatores que afetam as decisões de investimento por parte de empresas chinesas. Forest Trends 2007 Forest products trade between China and Africa: an analysis of import and export statistics. Forest Trends, Washington, D.C. Huang, W.B., Canby, K. e Sun, X.F. 2011 China forest products trade and investment. Unpublished report submitted by World Agroforestry Centre China & East Asia Node to CIFOR, Bogor, Indonésia. Ministério do Comércio Exterior e Cooperação Econômica, República Popular da China (MOFTEC) 1992 Approval and temporary management regulation for setting up non-trade overseas companies. Ministério do Comércio Exterior e Cooperação Econômica, República Popular da China (MOFTEC) 1984 Circular about the approving authority and procedure of establishing the non- trade joint venture overseas. Ministério do Comércio Exterior e Cooperação Econômica, República Popular da China (MOFTEC) 1985 Approval procedure and administrative method for setting up non-trade management joint ventures overseas (temporary) [Waijingmaohefa (1985) No. 19]. Ministério do Comércio, República Popular da China (MOFCOM) 2001 China commerce yearbook 2001. MOFCOM, Pequim, China. Atores do setor industrial preveem que as atividades de M&A da China aumentarão no curto prazo. Outra questão decorrente do aumento da atividade de M&A é se as empresas internacionais, mas de propriedade chinesa ou parcialmente de propriedade chinesa, também são ativas nos setores alvo na África. Ministério do Comércio, República Popular da China (MOFCOM) 2003 Circular about implementing well the approval reform pilots for overseas investments [Shanghezi (2003) No. 16]. Ministério do Comércio, República Popular da China (MOFCOM) 2007 Circular on adjusting the authorization of overseas investments [Shanghezi (2007) No. 112]. Putzel, L. e Kabuyaya, N. 2011 Forest-related impacts of Chinese aid, trade and investment in the Democratic Republic of Congo: preliminary observations. Trabalho não publicado, CIFOR, Bogor, Indonésia. 7. Referências Comissão Nacional de Planejamento (NPC) 1991 Regulation on developing and approving project Ministério do Comércio, República Popular da Ministério do Comércio, República Popular da 11 China (MOFCOM) 2009 Management method for overseas investments [Shangwubuling (2009) No. 5]. China (MOFCOM) 2009 Management method for overseas investments [Shangwubuling (2009) No. 5]. Zhang, H. 2011 Trends in Chinese trade and investment in Africa’s mining sector. Trabalho não publicado apresentado pelo Escritório para a China e o Leste da Ásia do Centro Mundial Agroflorestal ao CIFOR, Bogor, Indonésia. Putzel, L. e Kabuyaya, N. 2011 Forest-related impacts of Chinese aid, trade and investment in the Democratic Republic of Congo: preliminary observations. Trabalho não publicado, CIFOR, Bogor, Indonésia. Anexo 1. Lista de empresas chinesas aprovadas para investir em mineração, silvicultura e agricultura na África Comentário: Transliteração e tradução do nome das empresas foram feitas pelos autores, na versão original em inglês. Fonte: Banco de dados do MOFCOM sobre investimentos no exterior Tabela 1. Empresas chinesas aprovadas para investir em mineração na África País/região Empresa controladora (China) Nome da empresa Tipo de empresa Data da aprovação Tanzânia Anhui Geoscientific Investment Co. Ltd Fucheng Mining Co. Ltd Exploração, produção e processamento de minérios 10 de dezembro de 2010 Argélia Anhui Shuangcheng Electric Wire and Electric Cable Co. Ltd Incerto Exploração e comércio de minérios 7 de setembro de 2009 África do Sul Baosteel Resources Co. Ltd Baosteel Resources South Africa Co. Ltd Exploração e comércio de minérios 26 de março de 2010 Zâmbia Beijing Huayihuisheng Trade Co. Ltd Huayi Zambia Co. Ltd Exploração e extração de minérios 22 de junho de 2007 República Democrática do Congo Beijing Huayihuisheng Trade Co. Ltd Incerto Exploração, produção e processamento de minérios 25 de dezembro de 2009 Zâmbia Beijing Yongtongchang Mining Co. Ltd Zâmbia Yongtongchang Mining Co. Ltd Exploração e extração de minérios 28 de setembro de 2009 Sudão Beijing Yongxinghongsheng Co. Ltd Huatuo Mining Co. Ltd Exploração e extração de minérios 29 de julho de 2009 Nigéria Beijing Yuhongxing Co. Ltd Zhongni Gold Stat Mining Co. Ltd Exploração e extração de minérios 5 de dezembro de 2008 Nigéria Changzhou Pangu Economy and Technology Development Co. Ltd Changzhou Pangu Nigeria Co. Ltd Exploração e comércio de minérios 28 de fevereiro de 2006 República Democrática do Congo Chenzhou Jinshan Metallurgy Chemical Co. Ltd Jinshan Africa Mining Co. Ltd Exploração mineral 3 de junho de 2010 Tanzânia Diying Tian Mining Development Co. 7. Referências Ltd Diying Mining Co. Ltd Exploração, produção e processamento de minérios 23 de novembro de 2010 Nigéria Dongguan Zhongzhan Mining Co. Ltd Kenyang Industry Exploração, produção e processamento de minérios 17 de novembro de 2009 Madagascar Foshan Ruima Mining Investment Hi Dam Resources Investimento em mineração 12 de junho de 2010 República Democrática do Fujian Benyuan Metal Material Co. Ltd Benyuan DRC Co. Ltd Exploração e comércio de minérios 27 de janeiro de 2008 se de aprovações para empresas chinesas investirem nos setores de mineração, agricultura e silvicultura da África 13 País/região Empresa controladora (China) Nome da empresa Tipo de empresa Data da aprovação Botsuana Fujian Zhongyu Investment Development Co. Ltd Botswana Zhongyu Mining Development Co. Ltd Exploração e comércio de minérios 15 de dezembro de 2009 República Democrática do Congo Fuyang Shenneng Incerto Exploração, produção e processamento de minérios 27 de junho de 2008 Zâmbia Guangdong Chemical Industry Geology Exploration Institute Yuedi Zambia Mining Co. Ltd Exploração mineral 11 de fevereiro de 2010 República Democrática do Congo Guangzhou Longyuan Metal Co. Ltd JMT Mining Co. Ltd Exploração e comércio de minérios 17 de dezembro de 2007 Tanzânia Guizhou Geo-mineral Resource Development Co. Ltd Guishan Tanzania Mining Co. Ltd Exploração e comércio de minérios 1 de setembro de 2010 Camarões Harbin Long Engineer Co. Ltd C&K Exploração, produção e processamento de minérios 27 de março de 2007 República Democrática do Congo Han International Resources Group Kingrui Mining Investment Co. Ltd Exploração, produção e processamento de minérios 29 de junho de 2010 Madagascar Hainan Non-ferrous Mining Development Co. Ltd Hainan Mining Madagascar Co. Ltd Exploração mineral 11 de junho de 2010 República Democrática do Congo Haining Droin Imp & Exp. Co. Ltd DRC Chenfei Mining Co. Ltd Processamento 23 de junho de 2008 Nigéria Wuhan Industrial Co. Ltd Wuhan Global Mining Nigeria Company Exploração, produção e processamento de minérios 6 de setembro de 2010 Zâmbia Hebei Jidong Construction Incerto Exploração e comércio de minérios 21 de junho de 2010 República Democrática do Congo Henan Qianli Machinery Co. Ltd Jinjucheng Mining Co. Ltd Exploração e comércio de minérios 24 de janeiro de 2008 República Democrática do Congo Henan Rebecca Holding Co. Ltd Incerto Exploração, produção e processamento de minérios 8 de janeiro de 2008 República Democrática do Congo Henan Rebecca Holding Co. Ltd Rebecca DRC Mining Co. 7. Referências Ltd Exploração mineral 15 de outubro de 2008 Tanzânia Henan Geology and Mapping General Institute Henan International Mining Co. Ltd Exploração mineral 2 de agosto de 2010 ç continua na próxima página Huang Wenbin e Andreas Wilkes 14 País/região Empresa controladora (China) Nome da empresa Tipo de empresa Data da aprovação Namíbia Henan Bureau of Geo-exploration and Mineral Development Henan Asia-Africa Geology Engineer Namibia Co. Ltd Exploração mineral 27 de outubro de 2006 Tanzânia Henan Bureau of Geo-exploration and Mineral Development Henan Asia-Africa Geology Engineer Tanzania Co. Ltd Exploração mineral 9 de agosto de 2007 Tanzânia Henan Coal Field Geology Institute Sushan Geological Engineer Tanzânia Co. Ltd Exploração mineral 9 de dezembro de 2010 Nigéria Henan Coal Field Geology Institute Sushan Geological Engineer Co. Ltd Exploração mineral 17 de agosto de 2007 Argélia Henan Hydrogeology Geology Exploration Institute Incerto Exploração, produção e processamento de minérios 15 de janeiro de 2009 Sudão Henan Hydrogeology Geology Exploration Institute Incerto Exploração mineral 27 de setembro de 2008 Argélia Henan Bureau of Geo-exploration and Mineral Development Sarl Shaolin Mines Exploração e comércio de minérios 10 de maio de 2010 Etiópia Henan Zhengzhou Geology & Mineral Exploration Institute Henan Zhengzhou Geology & Mineral Exploration Institute Etiópia Office Extração 2 de agosto de 2010 Gana Henan Geologi av Asia-afrikanske teknisk samarbeid med International Ltd Henan Geologi av Asia-afrikanske teknisk samarbeid med International Ltd Exploração mineral 11 de junho de 2008 Botsuana Henan Zhonglian Mining Co. Ltd Zhonglian Mining Co. Ltd Exploração mineral 23 de janeiro de 2009 Nigéria Hegang City DiMei Co. Ltd Incerto Exploração e comércio de minérios 19 de dezembro de 2008 Zâmbia Longjiang Non-ferrous Zambia Mining Co. Ltd Exploração mineral 10 de maio de 2010 Etiópia Hubei Geo-mineral Exploration Co. Ltd Zhonge Mining Investment Co. Ltd Exploração mineral 22 de fevereiro de 2008 República Democrática do Congo Hunan Jiahe Mining Investment Co. Ltd Incerto Processamento 23 de agosto de 2010 República Democrática do Congo Hunan Jiahe Mining Investment Co. Ltd Ruixiang DRC Co. Ltd Exploração e comércio de minérios 23 de agosto de 2010 Gana JUSSK Ghana JUSSK Geo-mineral Resource Co. Ltd Exploração e processamento de minérios 9 de fevereiro de 2010 Tabela 1. 7. Referências Continuação i ó i á i se de aprovações para empresas chinesas investirem nos setores de mineração, agricultura e silvicultura da África 15 País/região Empresa controladora (China) Nome da empresa Tipo de empresa Data da aprovação República Democrática do Congo Hunan Kaitong Metal Co. Ltd DRC Kaixin Smelt Co. Ltd Exploração e comércio de minérios 15 de setembro de 2009 África do Sul Hunan Ruixiang New Material Co. Ltd Ruixiang Mining Resources África do Sul Co. Ltd Processamento 23 de dezembro de 2009 República Democrática do Congo Hunan Shanshan New Material Co. Ltd Shanshan Congo Co. Ltd Exploração e comércio de minérios 17 de setembro de 2010 Zâmbia Hunan Geological & Mining Organization for Non-Ferrous Metals Zhong Xiang Zambia Mining Co. Ltd Exploração, produção e processamento de minérios 7 de junho de 2010 República Democrática do Congo Liaoning Nonferrous Geological Exploration Institute Xinxiang Ruixiang Congo Mining Co. Ltd Exploração mineral 3 de junho de 2010 Zâmbia Hunan Tianxiong Industry Tianxiong Mining Investment Co. Ltd. Exploração, produção e processamento de minérios 9 de agosto de 2010 África do Sul Hunan Zexin Imp & Exp Co. Ltd ZERO-BITS CC Processamento 4 de dezembro de 2008 Zâmbia Jilin Nonferrous Metal Geological Exploration Bureau Jilin Nonferrous Zambia Mining Co. Ltd. Exploração mineral 22 de fevereiro de 2010 Nigéria Jiangsu Pan Pacific Development Co. Ltd KEPXING NIG LTD Exploração e comércio de minérios 24 de setembro de 2009 Namíbia East China Mineral Exploration and Development Bureau Namíbia China East China Mineral Exploration Co. Ltd. Exploração, produção, processamento e comércio de minérios 19 de agosto de 2009 Namíbia East China Mineral Exploration and Development Bureau Namibia China East China Mineral Exploration Investment Co. Ltd Exploração e comércio de minérios 25 de agosto de 2009 República Democrática do Congo Jiangsu Bureau of Coal Geology ICAM Processamento 21 de maio de 2010 República Democrática do Congo Jiangxi Gold Fuda Mining Co. Ltd. Hai Lun Mining Co. Ltd. Exploração e comércio de minérios 5 de março de 2009 Marrocos Jiangxi Kingan Hi-Tech Co. Ltd. Kingan Morocco Mining Co. Ltd. Exploração mineral 16 de setembro de 2009 Nigéria Jiangxi Baofeng Mining Industry Co. Ltd. Fengyuan Mining Nigeria Co. Ltd. 7. Referências Exploração e comércio de minérios 23 de novembro de 2007 Tanzânia Jiangxi Geo-engineering (Group) Corporation Jiangdi Africa Exploração mineral 9 de julho de 2010 ç Huang Wenbin e Andreas Wilkes 16 País/região Empresa controladora (China) Nome da empresa Tipo de empresa Data da aprovação Namíbia Jiangxi Geo-mineral Development General Corporation Incerto Exploração mineral 5 de maio de 2010 Nigéria Jiangxi Western Africa Mining Development Co. Ltd Bowang Mining Investment Co. Ltd Exploração e comércio de minérios 10 de agosto de 2009 Zâmbia JCHX Mining Construction Group JCHX Africa Development Co. Ltd Exploração e comércio de minérios 13 de outubro de 2009 África do Sul Jinchuan Group Limited (JNMC) South Africa JNMC Resource Co. Ltd. Exploração e comércio de minérios 30 de junho de 2008 República Democrática do Congo Jinchuan Group Limited (JNMC) JNMC DRC Office Exploração e comércio de minérios 17 de abril de 2006 República Democrática do Congo Jinchuan Group Limited (JNMC) Incerto Exploração e comércio de minérios 7 de dezembro de 2009 Sudão Jinzhou Xintai trading Co. Ltd. Sudão JinYu International Investment and Trade Co. Ltd Exploração, produção e processamento de minérios 20 de agosto de 2010 África do Sul Jiuquan Iron & Steel (Group) Co. Ltd. Incerto Exploração e processamento de minérios 30 de outubro de 2007 Zâmbia Liaoning Huazan Foreign Trade Co. Ltd. CHIMAN Manufacture Co. Ltd. Exploração e comércio de minérios 2 de março de 2005 República Democrática do Congo Liaoning Jinding Magnesite Group Runding Industrial Congo Co. Ltd. Exploração, produção e processamento de minérios 29 de setembro de 2007 Zâmbia Liaoning Sanhe Mining Investment Co. Ltd. Sanhe Mining Investment Co. Ltd. Zâmbia Company Exploração e comércio de minérios 5 de janeiro de 2010 Zâmbia Liaoning Nonferrous Geological Exploration Institute Liaoning Nonferrous Geological Exploration Institute Zambia Company Exploração e extração mineral 22 de dezembro de 2010 Zâmbia Liaoning Xin Xing Mining Development Co. Ltd. Incerto Exploração e processamento de minérios 20 de dezembro de 2007 Zâmbia Liaoning Non-ferrous Jubao Mining Co. Ltd. Liaoning Non-ferrous Zambia Company Exploração mineral 31 de julho de 2009 Zâmbia Liaoning Zhongrui Mining Co. Ltd. Zhongrui Mining Investment Co. Ltd Exploração e extração mineral 20 de dezembro de 2010 Nigéria Linyi Jiuli Food Co. Ltd. Jiuli Nigeria Co. Ltd. Exploração e comércio de minérios 27 de novembro de 2008 abe a . 7. Referências Co t uação se de aprovações para empresas chinesas investirem nos setores de mineração, agricultura e silvicultura da África 17 País/região Empresa controladora (China) Nome da empresa Tipo de empresa Data da aprovação África do Sul Naijing Hairui Cobalt South Africa Shengbao Mining Co. Ltd Exploração e comércio de minérios 5 de novembro de 2007 República Democrática do Congo Naijing Hairui Cobalt Metal Mines SPRL Exploração, produção e processamento de minérios 14 de julho de 2009 República Democrática do Congo Naijing Hairui Cobalt Congo Shengbao Mining Co. Ltd. Exploração e comércio de minérios 23 de novembro de 2006 República Democrática do Congo Nantong Xiongfeng Metal Material Co. Ltd. ZHX International Metal Co. Ltd. Exploração, produção e processamento de minérios 28 de junho de 2006 Gabão Hwazhou Group Hwazhou Mining Gabon Co. Ltd. Exploração, produção e processamento de minérios 28 de abril de 2006 Gabão Hwazhou Group Hwazhou Mining Gabon Trading Co. Ltd. Exploração, produção e processamento de minérios 8 de novembro de 2005 Gabão Ningbo Mining Investment Co. Ltd. Zhoonghui Mining Investment Development Co. Ltd. Exploração e comércio de minérios 20 de março de 2009 Gabão Ningbo Mining Investment Co. Ltd. Zhoonghui Mining Industry and Trade Co. Ltd. Exploração e comércio de minérios 22 de dezembro de 2008 Congo Ningbo Xinglong Vehicle Industry Co. Ltd. Ningbo Xinglong Vehicle Industry Co. Ltd. Congo Company Exploração e comércio de minérios 29 de novembro de 2004 República Democrática do Congo Ningbo Xinglong Investment Co. Ltd. Xinglong Africa Mining Co. Ltd. Exploração e comércio de minérios 11 de dezembro de 2007 Tanzânia Rizhao Jialin Trading Co. Ltd. Jialin Group Tanzania Co. Ltd. Exploração e comércio de minérios 26 de outubro de 2009 Congo Shandong Luyuan Investment Co. Ltd. Congo Luyuan Mining Co. Ltd. Exploração mineral 26 de outubro de 2009 Tanzânia Shandong Sheng Stem Heavy Mining Equipment Ltd. Sheng Stem Investment Tanzania Co. Ltd. Exploração e comércio de minérios 25 de novembro de 2010 Etiópia Shandong Geo-mineral Corporation Joint Mining Industry Co. Ltd. Exploração mineral 8 de maio de 2009 Congo Shandong Geo-mineral Institute Congo Huadi Mining Co. Ltd. Exploração mineral 2 de junho de 2009 Namíbia Shandong Deep Base Construction Engineering Corporation Namíbia Huadi Mining Investment Co. Ltd. Exploração mineral 20 de julho de 2010 Eritreia Shandong No. 4 Institute of Geological Mineral Survey Eritreia Shandong Mining Co. Ltd. 7. Referências Exploração mineral 12 de novembro de 2010 ç Huang Wenbin e Andreas Wilkes 18 País/região Empresa controladora (China) Nome da empresa Tipo de empresa Data da aprovação Zâmbia Shandong Zheng Yuan Geology Resources Exploration Co. Ltd. Zambia Zheng Yuan Co. Ltd. Exploração e comércio de minérios 24 de junho de 2009 Argélia Shaanxi Geological Engineering Corporation Shaanxi Geological Engineering Corporation Overseas Company Exploração e comércio de minérios 4 de março de 2008 Gana Shaanxi Mining Development Co. Ltd. Shanxi Mining (Gana) Co. Ltd. Exploração e comércio de minérios 6 de novembro de 2009 República Democrática do Congo Shanghai Overseas Company Exploração e comércio de minérios 7 de abril de 2006 República Democrática do Congo Shanghai Hongbang Development Co. Ltd. Hongbang Mining (DRC) Co. Ltd. Exploração mineral 4 de janeiro de 2006 República Democrática do Congo Shanghai Huangpu Cereals, Oils and Foodstuffs Development Co. Ltd. Maofa Mining Co. Ltd. Exploração e comércio de minérios 17 de dezembro 2007 República Democrática do Congo Shanghai Jiachuang Group Jia Cuang Mining Investment Co. Ltd. Exploração, produção e processamento de minérios 17 de março de 2008 República Democrática do Congo Shanghai Kuntai Group Kuntai Congo Mining Co. Ltd. Exploração e comércio de minérios 5 de novembro de 2008 República Democrática do Congo Shanghai Kuntai Group Congo Union Mining Co. Ltd. Exploração e processamento de minérios 27 de outubro de 2010 República Democrática do Congo Shanghai Kuntai Group China-Africa International Mining Co. Ltd Processamento 11 de maio de 2010 Zâmbia Shanghai Tongyi Steel Material Co. Ltd. Tongyi Lead-Zinc Mining Zambia Co. Ltd Processamento 24 de maio de 2010 Tanzânia Sichuan Hongda Group Hongda International Mining Resources Co. Ltd. Exploração, produção e processamento de minérios 14 de agosto de 2007 Zâmbia Sichuan Hua Suo Automation Information Engineering Co. Ltd. Sichuan Hua Suo (Zambia) Mining Co. Ltd. Exploração e comércio de minérios 22 de outubro de 2007 África do Sul Suzhou Kaiyuan Group South Africa De Feng Mining Co. Ltd. Exploração, produção e processamento de minérios 20 de setembro de 2010 Tabela 1. Continuação ti ó i á i se de aprovações para empresas chinesas investirem nos setores de mineração, agricultura e silvicultura da África 19 continua na próxima página suite page suivante País/região Empresa controladora (China) Nome da empresa Tipo de empresa Data da aprovação Argélia Tianjin Hua Kan Hua Kan Mining Algeria Co. Ltd. Exploração mineral 2 de dezembro de 2009 Ruanda Xiamen Tungsten Co. Ltd. 7. Referências (XTC) Lu Xiang Mining Co. Ltd. Exploração e comércio de minérios 4 de março de 2009 Zâmbia Xiangtan Hong Xin Investment Co. Ltd. Zambia Fu Xin Investment Co. Ltd. Exploração e processamento de minérios 8 de julho de 2008 Zâmbia Xiangtan Liquefied Petroleum Gas (LPG) Co. Ltd. Xing Xiang Co. Ltd. Exploração e comércio de minérios 31 de dezembro de 2008 Zâmbia Xinxing Ductile Iron Pipes Co. Ltd Zambia Maosheng Mining Co. Ltd 26 de novembro de 2010 Zâmbia Xinxing Ductile Iron Pipes Co. Ltd Xinxing Mining Zambia Co. Ltd Comércio 26 de novembro de 200 República Democrática do Congo Yixing Mingyue Ceramics Co. Ltd Ming Yue DRC Mining Co. Ltd Exploração e extração mineral 16 de maio de 2008 Zâmbia Yinkou BL Mining Co. Ltd Jia Xing Mining (Zambia) Co. Ltd Processamento 6 de maio de 2009 República Democrática do Congo Yinkou BL Mining Co. Ltd Jia Xing Mining (Congo) Co. Ltd Exploração, produção e processamento de minérios 6 de março de 2009 África do Sul Zhejiang Huayou Cobalt Dong Fang International Mining Spr Comércio 19 de agosto de 2009 República Democrática do Congo Zhejiang Huayou Cobalt Congo Dong Fang International Mining Spr Exploração, produção e processamento de minérios 5 de maio de 2009 República Democrática do Congo Zhejiang Jiu Lun Garment Co. Ltd. Congo Jiu Lun Mining Co. Ltd. Exploração e comércio de minérios 16 de janeiro de 2007 Argélia Zhejiang Qianjiang Group Algeria Shao Lin Mining Co. Ltd. Exploração e comércio de minérios 10 de fevereiro de 2009 Zâmbia Zhejiang Zi Hua Mining Co. Ltd. China-Africa Mining Co. Ltd. Exploração e comércio de minérios 23 de setembro de 2009 Eritreia Zhong Lu Resources Investment Co. Ltd. Zhong Lu Mining Development Co. Ltd. Exploração mineral 21 de outubro de 2010 Zâmbia CGCOC Hansom (Beijing) Trading Co. Ltd. TPI Industrial Co. Ltd. Exploração, produção e processamento de minérios 21 de maio de 2008 Guiné China Power Investment Corporation (CPI) International Mining Investment Co. Ltd. CPI International Mining Investment Co. Ltd. Guinea Company Pesquisa de mercado 29 de julho de 2010 ç Huang Wenbin e Andreas Wilkes 20 País/região Empresa controladora (China) Nome da empresa Tipo de empresa Data da aprovação Eritreia China-Africa North China Geological Exploitation Investment Co. Ltd. China–Africa North China Geological Exploitation Investment Co. Ltd. Exploração, produção e processamento de minérios 8 de fevereiro de 2010 Sudão China-Africa North China Geological Exploitation Investment Co. Ltd. 7. Referências HAMADI Resource Co. Ltd Exploração e comércio de minérios 23 de setembro de 2009 Sudão China-Africa North China Geological Exploitation Investment Co. Ltd. Incerto Exploração mineral 5 de março de 2010 Sudão China-Africa Investment Development Co. Ltd Incerto Exploração mineral 14 de setembro de 2010 Zâmbia China Geo-Engineering Corporation (CGC) CGC Southern Africa Co. Ltd. Extração de recursos 10 de junho de 2009 Costa do Marfim China National Geological & Mining Corp. (CGM) Bin Hai Mining Co. Ltd. Exploração e comércio de minérios 23 de março de 2010 Namíbia China National Nuclear Corporation (CNNC) CNNC Resource Extraction Co. Ltd. Exploração e comércio de minérios 18 de junho de 2009 República Democrática do Congo China Overseas Engineering Group Co. Ltd. DRC Luishia Mining Co. Ltd. Extração e processamento de minérios 25 de junho de 2007 Namíbia China Henan International Cooperation Group Co. Ltd. (CHICO) China Henan International Cooperation Group Co. Ltd. (CHICO) Namibia Company Exploração e comércio de minérios 6 de março de 2009 Serra Leoa China Qing Hua Group Serra Leoa Qing Hua Investment Co. Ltd. Exploração mineral 26 de agosto de 2010 República Democrática do Congo Sinohydro Sicomines Sarl Exploração mineral 16 de março de 2009 Botsuana Central South China Bureau of Metallurgical Geology Botswana Central-south Resource Co. Ltd. Exploração, produção e processamento de minérios 29 de dezembro de 2009 Zâmbia China Nonferrous Metal Mining (Group) Co. Ltd. (CNMC) Sino-Metals Leach Zambia Limited Processamento 12 de dezembro de 2005 Zâmbia China Nonferrous Metal Mining (Group) Co. Ltd. (CNMC) Incerto Processamento 12 de dezembro de 2005 Zâmbia China Nonferrous Metal Mining (Group) Co. Ltd. (CNMC) Luanshya Copper Mines Exploração, produção e processamento de minérios 7 de setembro de 2009 continua na próxima página nálise de aprovações para empresas chinesas investirem nos setores de mineração, agricultura e silvicultura da Áf 21 País/região Empresa controladora (China) Nome da empresa Tipo de empresa Data da aprovação Zâmbia China Nonferrous Metal Mining (Group) Co. Ltd. (CNMC) NCFA Mining Exploração e comércio de minérios 20 de abril de 2006 África do Sul Sinosteel Corporation Tubatse Chrome Minerals Pty. Ltd. Exploração e comércio de minérios 31 de março de 2008 Camarões Sinosteel Corporation Sinosteel Corporation Camarões Co. Ltd. Exploração e processamento de minérios 17 de julho de 2008 Zâmbia Hunan Geological Prospecting Institute of China Chemical Geology and Mine Bureau China Chemical Geology and Mine Bureau (Zambia) Co. Ltd. 7. Referências Exploração mineral 14 de maio de 2010 Namíbia Shandong Geological Prospecting Institute of China Chemical Geology and Mine Bureau China Chemical Geology and Mine Bureau (Namibia) Co. Ltd. Exploração mineral 27 de maio de 2010 Zâmbia Sinomine Resource Exploration Co. Ltd. (Beijing Sinomine Construction Engineering Co. Ltd.) Zambia Sinomine Mining Resource Co. Ltd. Exploração, produção e processamento de minérios 7 de outubro de 2008 Zâmbia Sinomine Resource Exploration Co. Ltd. (Beijing Sinomine Construction Engineering Co. Ltd.) Zambia Sinomine Construction Engineering Co. Ltd. Exploração, produção e processamento de minérios 30 de outubro de 2007 Zâmbia Zhongrunhualong Group Zambia Kun lun Gu Ming Co. Ltd. Exploração e comércio de minérios 14 de julho de 2010 Zâmbia CNMC International Trade Co. Ltd. CNMIC Africa Logistic and Trade Co. Ltd. Exploração e comércio de minérios 17 de setembro de 2009 Angola Beijing Donia Resources Co. Ltd. Angola Duo Li Mining Co. Ltd. Exploração mineral 25 de dezembro de 2008 Eritreia Beijing Donia Resources Co. Ltd. Beijing Southeast Asia Resource and Technology Co. Ltd. Exploração, produção e processamento de minérios 22 de maio de 2009 Etiópia Beijing Donia Resources Co. Ltd. Rui Feng Mining Co. Ltd. Exploração, produção e processamento de minérios 25 de dezembro de 2008 Etiópia Beijing Donia Resources Co. Ltd. Ethiopia Southeast Mining Co. Ltd. Exploração, produção e processamento de minérios 27 de julho de 2007 Zâmbia Chongqing Bureau of Geology and Mineral Exploitation Yu Zambia Mining Co. Ltd. Exploração mineral 13 de junho de 2010 País/região Empresa controladora (China) Nome da empresa Tipo de empresa Data da aprovação Zâmbia China Nonferrous Metal Mining (Group) Co. Ltd. (CNMC) NCFA Mining Exploração e comércio de minérios 20 de abril de 2006 África do Sul Sinosteel Corporation Tubatse Chrome Minerals Pty. Ltd. Exploração e comércio de minérios 31 de março de 2008 Camarões Sinosteel Corporation Sinosteel Corporation Camarões Co. Ltd. Exploração e processamento de minérios 17 de julho de 2008 Zâmbia Hunan Geological Prospecting Institute of China Chemical Geology and Mine Bureau China Chemical Geology and Mine Bureau (Zambia) Co. Ltd. Exploração mineral 14 de maio de 2010 Namíbia Shandong Geological Prospecting Institute of China Chemical Geology and Mine Bureau China Chemical Geology and Mine Bureau (Namibia) Co. Ltd. Exploração mineral 27 de maio de 2010 Zâmbia Sinomine Resource Exploration Co. Ltd. (Beijing Sinomine Construction Engineering Co. Ltd.) Zambia Sinomine Mining Resource Co. Ltd. 7. Referências Exploração, produção e processamento de minérios 7 de outubro de 2008 Zâmbia Sinomine Resource Exploration Co. Ltd. (Beijing Sinomine Construction Engineering Co. Ltd.) Zambia Sinomine Construction Engineering Co. Ltd. Exploração, produção e processamento de minérios 30 de outubro de 2007 Zâmbia Zhongrunhualong Group Zambia Kun lun Gu Ming Co. Ltd. Exploração e comércio de minérios 14 de julho de 2010 Zâmbia CNMC International Trade Co. Ltd. CNMIC Africa Logistic and Trade Co. Ltd. Exploração e comércio de minérios 17 de setembro de 2009 Angola Beijing Donia Resources Co. Ltd. Angola Duo Li Mining Co. Ltd. Exploração mineral 25 de dezembro de 2008 Eritreia Beijing Donia Resources Co. Ltd. Beijing Southeast Asia Resource and Technology Co. Ltd. Exploração, produção e processamento de minérios 22 de maio de 2009 Etiópia Beijing Donia Resources Co. Ltd. Rui Feng Mining Co. Ltd. Exploração, produção e processamento de minérios 25 de dezembro de 2008 Etiópia Beijing Donia Resources Co. Ltd. Ethiopia Southeast Mining Co. Ltd. Exploração, produção e processamento de minérios 27 de julho de 2007 Zâmbia Chongqing Bureau of Geology and Mineral Exploitation Yu Zambia Mining Co. Ltd. Exploração mineral 13 de junho de 2010 Huang Wenbin e Andreas Wilkes 22 Tabela 2 Empresas chinesas aprovadas para investir em silvicultura na África País/região Empresa controladora (China) Nome da empresas Tipo de empresa Data da aprovação Angola Jiangsu Kaitong Plastics Industry Xun Tong International Industrial Co. Ltd. Processamento de madeira 13 de agosto de 2009 Benin Nanjing Mai Jie Kai Fei Trading Co. Ltd. Shunfeng Wood. Co. Ltd. Extração de recurso florestal 22 de novembro de 2010 Benin Sehnzhen Yilin Wood Co. Ltd. Benin Yilin Wood Co. Ltd. Fabricação de piso de madeira 20 de novembro de 2009 Camarões Hangzhou Sfeicom Imp & Export Co. Ltd. Cameroon Sfeicom Stock Co. Ltd. Investimento em projeto florestal 17 de setembro de 2010 Congo Zhejiang Jiashan Xianghua Wood Work Co. Ltd. Incerto Compensado 18 de abril de 2008 Congo Dejia Wood Co. Ltd. Congo Dejia Wood Co. Ltd. Extração e processamento de madeira 22 de janeiro de 2008 Congo Bengbu Fuda Material Co. Ltd. Fareast Wood Congo Co. Ltd. Comércio de madeira rara 22 de maio de 2006 Guiné Equatorial Jiangsu Shengyang Industry Equator Sijifu Co. Ltd. 7. Referências Extração de toras de madeira, produção de compensados 9 de agosto de 2004 Etiópia Yongxing Cooperation Ltd Yun Long Paper Mill Todos os tipos de papel 25 de fevereiro de 2009 Gabão Jiangsu Shengyang Industry Gabon Shengyang International Forestry Industry Co. Ltd. Manejo florestal e produção de compensado 14 de julho de 2010 Gabão Shandong Longsheng Import & Export Co. Ltd. International Timber Transportation and Trade Corporation Exploração madeireira e processamento de madeira 22 de dezembro de 2009 Gabão Xianju Jiade Arts & Crafts Co. Ltd. Gabon Jia De Timber Co. Ltd. Processamento de madeira 10 de dezembro de 2009 Gabão Wenzhou Timber Group Co. Gabon Forest Development Co. Ltd. Manejo florestal 22 de setembro de 2008 Gabão Guangzhou Dingtian Wood Co. Ltd. Incerto Manejo florestal 9 de maio de 2008 Gabão Zhangjiagang Free Trade Zone Ganglin International Trade Co. Ltd. Gabon Loulin Co. Ltd. Exploração madeireira e processamento de madeira 4 de janeiro de 2008 Gabão Ahlers (Zhangjiagang Free Trade Zone) International Trading Co. Ltd. Merry Gabon Co. Ltd. Exploração madeireira e processamento de madeira 17 de dezembro 2007 Gabão Shaoxing Wandi Wood Co. Ltd Shaoxing Wandi Co. Ltd. Serviço de vendas 29 de novembro de 2006 Gabão Shen Zhen Qiyang Industrial Development Co. Ltd. Hua Peng Wood. Co. Ltd Produção e comércio de madeira 7 de maio de 2003 Gana Xiangtan High-Tech Zone Zhi Yao Metallurgical Materials Co. Ltd. Hua Wei Furniture Co. Ltd. Fabricação de movéis 23 de dezembro 2009 Gana Huasheng Jiangquan Group Incerto Fabricação de papel 19 de agosto de 2009 continua na próxima página Incerto nálise de aprovações para empresas chinesas investirem nos setores de mineração, agricultura e silvicultura da Áf 23 País/região Empresa controladora (China) Nome da empresas Tipo de empresa Data da aprovação Guiné Jiangsu Jiaya International Investment Group Co. Ltd. Jiangsu Jiaya International Investment Group Co. Ltd. Guinea Company Extração de madeira e produtos madeireiros 22 de julho de 2008 Libéria Ningbo Jujin Investment Co. Ltd. Yu Shi International Stock Co. Ltd. Exploração e comércio de madeira 19 de janeiro de 2009 Libéria Ningbo Ningdian Imp & Export Co. Ltd.. The Atlantic International Investment Co. Ltd. Exploração e comércio de madeira 2 de dezembro 2009 Maurícia Shanxi Grain Trade Center Incerto Fabricação de papel 13 de fevereiro de 2004 Nigéria Hongkong Longgang Furniture Ltd. Incerto Fabricação de movéis 22 de setembro de 2009 Nigéria Zhengzhou Redsun Furniture Company Ltd. 7. Referências Incerto Fabricação de movéis 16 de junho de 2009 Nigéria Linyi Jiuli Food Co. Ltd. Jiu Li Wood (Nigeria) Co. Ltd. Produção de compensado 28 de novembro de 2008 Nigéria Linyi Millioner Bio-Industry Co. Ltd. Wanguo Wood (Nigeria) Co. Ltd Produção de compensado 7 de novembro de 2007 Nigéria Linyi Zhongba Food Co. Ltd. Zhongyang Industrial (Nigeria) Co. Ltd. Produção de compensado 27 de novembro de 2006 África do Sul Huaqiao Fenghuang Group Incerto Silvicultura e paisagem 14 de setembro de 2010 Zâmbia Guangzhou Huiyu Trade Ltd. Zambia Hui Yu Paper Mill Fabricação de papel 24 de novembro de 2010 Zâmbia Suzhou Golden Ocean Co. Ltd. Afrizam Timber Limited Fabricação de movéis 30 de novembro de 2009 Zâmbia Suzhou Golden Ocean Co. Ltd. Incerto Venda de madeira e produtos madeireiros 21 de maio de 2009 País/região Empresa controladora (China) Nome da empresas Tipo de empresa Data da aprovação Guiné Jiangsu Jiaya International Investment Group Co. Ltd. Jiangsu Jiaya International Investment Group Co. Ltd. Guinea Company Extração de madeira e produtos madeireiros 22 de julho de 2008 Libéria Ningbo Jujin Investment Co. Ltd. Yu Shi International Stock Co. Ltd. Exploração e comércio de madeira 19 de janeiro de 2009 Libéria Ningbo Ningdian Imp & Export Co. Ltd.. The Atlantic International Investment Co. Ltd. Exploração e comércio de madeira 2 de dezembro 2009 Maurícia Shanxi Grain Trade Center Incerto Fabricação de papel 13 de fevereiro de 2004 Nigéria Hongkong Longgang Furniture Ltd. Incerto Fabricação de movéis 22 de setembro de 2009 Nigéria Zhengzhou Redsun Furniture Company Ltd. Incerto Fabricação de movéis 16 de junho de 2009 Nigéria Linyi Jiuli Food Co. Ltd. Jiu Li Wood (Nigeria) Co. Ltd. Produção de compensado 28 de novembro de 2008 Nigéria Linyi Millioner Bio-Industry Co. Ltd. Wanguo Wood (Nigeria) Co. Ltd Produção de compensado 7 de novembro de 2007 Nigéria Linyi Zhongba Food Co. Ltd. Zhongyang Industrial (Nigeria) Co. Ltd. Produção de compensado 27 de novembro de 2006 África do Sul Huaqiao Fenghuang Group Incerto Silvicultura e paisagem 14 de setembro de 2010 Zâmbia Guangzhou Huiyu Trade Ltd. Zambia Hui Yu Paper Mill Fabricação de papel 24 de novembro de 2010 Zâmbia Suzhou Golden Ocean Co. Ltd. Afrizam Timber Limited Fabricação de movéis 30 de novembro de 2009 Zâmbia Suzhou Golden Ocean Co. Ltd. Incerto Venda de madeira e produtos madeireiros 21 de maio de 2009 Huang Wenbin e Andreas Wilkes 24 Tabela 3. 7. Referências Empresas chinesas aprovadas para investir em agricultura na África País/região Empresa controladora (China) Nome da empresa Tipo de empresa Data da aprovação Angola Tanan Liren Imp & Export Co. Ltd. Angola Taishan Industrial Development Co. Ltd. Produção e venda de hortícolas 11 de julho de 2007 Camarões Shijiazhuang Lion Feed Group Cameroon Lion Animal Husbandry Co. Ltd. Produção avícola 26 de junho de 2002 Camarões Shanxi Construction Engineering Group (Co.) Cameroon Huan Yu Animal Husbandry Company Produção avícola 22 de junho de 2009 Camarões Shanxi Land Reclamation Co. Ltd. Sino- Cameroon Iko Agriculture Development Co. Ltd. Cultivo de arroz 23 de junho de 2010 Guiné Equatorial Tian Jin Hai Xin Da Commercial Co. Ltd. China-Guinea Equatorial Agriculture Development Co. Ltd. Produção e venda de hortícolas e frutícolas 31 de agosto de 2005 Etiópia Shenyang Jiang Hong Foreign Trade Corp. Ltd. North China Farm Produção hortícola orgânica 29 de janeiro de 2007 Gabão Guangzhou Shi Mao Scientific Industrial and Trading Co. Ltd. Akok Farm Development Co. Ltd. Cultivo agrícola, criação de animais e processamento relacionado 29 de outubro de 2007 Gana Yancheng County Si Kai Er Daily-Use Chemical Industry Co. Ltd. Ghana New Oriental High- Tech Agriculture Research and Development Center Cultivo agrícola e de plantas 29 de julho de 2008 Gana Sichuan Sanherb Biotech Inc. Ghana G.C. Bio-Tec Inc. Cultivo de plantas medicinais 23 de dezembro de 2009 Quênia Linying Fangyuan Foreign Trade Co. Ltd. Kenya Shun Mao Co. Ltd. Produção e venda de produtos nativos 12 de janeiro de 2010 Maurícia Shanxi Tian Li Enterprises Co. Ltd. Tianli Spinning (Mauritius) Ltd. Produção de algodão 4 de novembro de 2005 Maurícia Wuhan Xin Neng Group Qianlima Investment Limited Company Produção de uva 2 de setembro de 2004 Nigéria CGCOC Group Co. Ltd. Green Agricultural Development Nigeria Limited Produção agrícola 17 de novembro de 2009 Nigéria CGCOC Group Co. Ltd. Green West Africa Limited Produção agrícola 16 de fevereiro de 2007 Nigéria Zhengzhou Cangxi Industry and Commerce Development Co. Ltd. Abia Palm Nigeria Limited Dendê 18 de setembro de 2008 África do Sul Jiangxi Yaxing Textile Industry Co. Ltd. Jiangxi Yaxing Textile Industry Co. Ltd. 7. Referências (Subsidiária na África do Sul) Produção de algodão 23 de novembro de 2009 África do Sul Anhui Wanling Rare Animal Breed Limited Liability Company Elaborates Shen Investment Company Cultivo agrícola, criação de animais 27 de novembro de 2006 se de aprovações para empresas chinesas investirem nos setores de mineração, agricultura e silvicultura da África 25 País/região Empresa controladora (China) Nome da empresa Tipo de empresa Data da aprovação Sudão Fujian Ude Electrical Machinery Co. Ltd. Sudan-China Joint Development Corporation Processamento de produtos agrícolas 18 de novembro de 2009 Sudão Binzhou Feng Hui Trading Co. Ltd. Sudan Agricultural Development Co. Ltd. Produção agrícola e hortícola 12 de dezembro de 2008 Sudão Zhongneng Enterprise Development (Group) Co. Ltd. Sun Neng Co. Ltd. Produção de produtos agrícolas 19 de março de 2010 Sudão Shandong Tianyuan Home Textile Co. Ltd. Zhong Tian International Industrial Corporation Produção de algodão 13 de abril de 2010 Tanzânia Hainan Qi Lin Technology Co. Ltd. Qi Lin (Tanzania) Co. Ltd. Produção de sisal de cânhamo 28 de dezembro de 2010 Tanzânia Shijiazhuang Hui Heng Fu De Run Imp & Exp Co. Ltd. Fa Da Agriculture Development Co. Ltd. Produção de produtos agrícolas 20 de fevereiro de 2008 Tanzânia China-Africa Agriculture Investment Co. Ltd. Tanzania China-Africa Agriculture Investment Co. Ltd. Produção e processamento de fibras de sisal 9 de agosto de 2010 Tanzânia China-Africa Agriculture Investment Co. Ltd. China Agricultural Reclamation (Group) Tanzania Limited Produção e processamento de fibras de sisal 20 de dezembro de 2010 Togo Hunan Zhulu Ramie Textile Co. Ltd. Togo 5 Star Textile Co. Ltd. Produção de produtos de rami 20 de dezembro de 2010 Uganda Hebei Hanhe Investment Co. Ltd. Hanhe Uganda Hebei Farm Cultivo agrícola, silvicultura e criação de animais 20 de novembro de 2008 Zâmbia Qingdao Textiles Group Chipata Cotton Co. Ltd. Produção de algodão e seus subprodutos 7 de setembro de 2009 Zâmbia Qingdao Textiles Group The Zambia China Mulungushi Textiles (ZCMT) Produção de algodão e de óleo comestível 9 de maio de 2006 Zâmbia Kaifeng China-Zambia Agriculture & Forestry Development Co. Ltd. Kaifeng Agriculture Investment Co. Ltd. Produção avícola 9 de maio de 2006 Zâmbia Huayong Overseas Agri-Scientific Technology (Beijing) Co. Ltd. Hua Feng Investment Co. Ltd. Cultivo agrícola e de plantas 9 de outubro de 2006 Zâmbia Hangzhou Changrun Plastic Products Co. Ltd. Feng Run Investment Zambia Co. Ltd. 7. Referências Produção de produtos agrícolas 7 de dezembro de 2006 Zâmbia Giant Hui Autonomous County Shuailin Comprehensive Farms Hua Fei Livestock (Zambia) Co. Ltd. Criação de animais e produção de forragem 15 de agosto de 2008 ç /região Empresa controladora (China) Nome da empresa Tipo de empresa Data da aprovação ão Fujian Ude Electrical Machinery Co. Ltd. Sudan-China Joint Development Corporation Processamento de produtos agrícolas 18 de novembro de 2009 ão Binzhou Feng Hui Trading Co. Ltd. Sudan Agricultural Development Co. Ltd. Produção agrícola e hortícola 12 de dezembro de 2008 ão Zhongneng Enterprise Development (Group) Co. Ltd. Sun Neng Co. Ltd. Produção de produtos agrícolas 19 de março de 2010 ão Shandong Tianyuan Home Textile Co. Ltd. Zhong Tian International Industrial Corporation Produção de algodão 13 de abril de 2010 ânia Hainan Qi Lin Technology Co. Ltd. Qi Lin (Tanzania) Co. Ltd. Produção de sisal de cânhamo 28 de dezembro de 2010 ânia Shijiazhuang Hui Heng Fu De Run Imp & Exp Co. Ltd. Fa Da Agriculture Development Co. Ltd. Produção de produtos agrícolas 20 de fevereiro de 2008 ânia China-Africa Agriculture Investment Co. Ltd. Tanzania China-Africa Agriculture Investment Co. Ltd. Produção e processamento de fibras de sisal 9 de agosto de 2010 ânia China-Africa Agriculture Investment Co. Ltd. China Agricultural Reclamation (Group) Tanzania Limited Produção e processamento de fibras de sisal 20 de dezembro de 2010 o Hunan Zhulu Ramie Textile Co. Ltd. Togo 5 Star Textile Co. Ltd. Produção de produtos de rami 20 de dezembro de 2010 nda Hebei Hanhe Investment Co. Ltd. Hanhe Uganda Hebei Farm Cultivo agrícola, silvicultura e criação de animais 20 de novembro de 2008 bia Qingdao Textiles Group Chipata Cotton Co. Ltd. Produção de algodão e seus subprodutos 7 de setembro de 2009 bia Qingdao Textiles Group The Zambia China Mulungushi Textiles (ZCMT) Produção de algodão e de óleo comestível 9 de maio de 2006 bia Kaifeng China-Zambia Agriculture & Forestry Development Co. Ltd. Kaifeng Agriculture Investment Co. Ltd. Produção avícola 9 de maio de 2006 bia Huayong Overseas Agri-Scientific Technology (Beijing) Co. Ltd. Hua Feng Investment Co. Ltd. Cultivo agrícola e de plantas 9 de outubro de 2006 bia Hangzhou Changrun Plastic Products Co. Ltd. Feng Run Investment Zambia Co. Ltd. Produção de produtos agrícolas 7 de dezembro de 2006 bia Giant Hui Autonomous County Shuailin Comprehensive Farms Hua Fei Livestock (Zambia) Co. Ltd. 7. Referências Criação de animais e produção de forragem 15 de agosto de 2008 ç i ó i á i Huang Wenbin e Andreas Wilkes 26 País/região Empresa controladora (China) Nome da empresa Tipo de empresa Data da aprovação Zâmbia Kaifeng The Shelter-Forest Virescence Management Co. Ltd. Daliang Agriculture Investment Co. Ltd. Criação de animais e cultivo agrícola 20 de agosto de 2008 Zâmbia Kaifeng Huazhong Wan Tou Cultivation Co. Ltd. Xinghua Agricultural Investment Company Criação de animais e cultivo agrícola 12 de setembro de 2008 Zâmbia Huasheng Jiangquan Group Jiang Quan International Corporation Cultivo e processamento agrícola 12 de setembro de 2008 Zâmbia Zhongying Changjiang International Investment Guarantee Co. Ltd. Heidi Biomass Zambia Co. Ltd. Produção de bioenergia 12 de dezembro de 2008 País/região Empresa controladora (China) Nome da empresa Tipo de empresa Data da aprovação Zâmbia Kaifeng The Shelter-Forest Virescence Management Co. Ltd. Daliang Agriculture Investment Co. Ltd. Criação de animais e cultivo agrícola 20 de agosto de 2008 Zâmbia Kaifeng Huazhong Wan Tou Cultivation Co. Ltd. Xinghua Agricultural Investment Company Criação de animais e cultivo agrícola 12 de setembro de 2008 Zâmbia Huasheng Jiangquan Group Jiang Quan International Corporation Cultivo e processamento agrícola 12 de setembro de 2008 Zâmbia Zhongying Changjiang International Investment Guarantee Co. Ltd. Heidi Biomass Zambia Co. Ltd. Produção de bioenergia 12 de dezembro de 2008 Anexo 2. Principais eventos de aquisições e fusões da China no exterior Tabela 1. Vinte principais eventos de fusões e aquisições chinesas no exterior (de 2003 a meados de 2010) Data de anúncio Empresa alvo Localização da empresa alvo Comprador Vendedor País vendedor Valor (milhões de USD) 1 de fevereiro de 2008 Direito a 12% das ações da Rio Tinto PLC RTP Reino Unido Alcoa Inc. & Aluminium Corporation of China 14.000 13 de agosto de 2009 Phoenix Resources Co. Ltd. Austrália Yanzhou Coal Mining Company Limited 2.568 3 de julho de 2009 Direito a 17,2% das ações da Tektronix Resources Co. Ltd. Canadá Fullbloom Investment 1.508 14 de abril de 2009 OZ Mineral Co. Ltd. (parcialmente) Austrália China Minmetals Non-ferrous Metals Co. Ltd. OZ Mineral Co. Ltd. Austrália 1.386 14 de março de 2008 Midwest Corporation Ltd. 7. Referências (direito a 80,31% das ações) Austrália Sinosteel Group 879 10 de novembro de 2006 Anglo American Plc (direito a 1,01% das ações) Reino Unido China Vision Resources E Oppenheimer & Son África do Sul 812 11 de junho de 2007 Peru Copper Inc. Canadá Aluminium Corporation of China 779 24 de março de 2006 Ashapura Minechem Ltd. (direito a 50% das ações em uma fábrica de Alumina) India China Power Investment Corporation Ningxia Qingtongxia Energy Aluminum Group Co. Ltd. China 651 29 de agosto de 2007 Bellavista Holding Group Ltd. (direito a 60% das ações) Chile China Elegance Resources Ltd. Ceasers Development Ltd. Hong Kong 600 28 de dezembro de 2009 Corriente Resources Inc. Canadá China Railway Construction Corporation Limited 549 6 de dezembro de 2007 Northern Peru Copper Company of Canada Canadá China Minmetals Corp. & Jiangxi Copper Corporation 411 24 de fevereiro de 2009 Ford Basescu Metals Group (direito a 9,07% das ações) Austrália Hunan Valin Iron & Steel Group Co. Ltd. Harbinger Capital Partners Special Situations Fund, L.P., Harbinger Capital Partners Master Fund I Ltd Estados Unidos 408 continua na próxima página Huang Wenbin e Andreas Wilkes 28 Data de anúncio Empresa alvo Localização da empresa alvo Comprador Vendedor País vendedor Valor (milhões de USD) 30 de novembro de 2009 MMX Mineração e Metálicos SA (21,52% do capital) Brasil Wuhan Iron and Steel Co. Ltd. 400 18 de abril de 2010 Sul Americana de Metais S.A. Brasil Hongqiao Group Votorantim Novos Negócios Ltda, Lit Mining Cooperatief U.A. Brasil 390 24 de fevereiro de 2009 Ford Basescu Metals Group (direito a 42% das ações) Austrália Hunan Valin Iron & Steel Group Co. Ltd. 363 27 de outubro de 2009 Krom Maden (direito a 50% das ações), Kop Krom (direito a 50% das ações), Guney Krom (direito a 50% das ações) Turquia Taiyuan Iron and Steel Group Co. Ltd. CVK Group Corporation Turquia 300 26 de setembro de 2007 Zimasco (direito a 92% das ações) Zimbábue Steel Trading Company 292 14 de novembro de 2008 Langfeld Enterprises Limited (direito a 90% das ações) Chipre Grandvest International Ltd. Cordia Global Limited Chipre 253 6 de janeiro de 2010 African Minerals Limited (direito a 12,5% das ações) Ilhas do Canal China Railway Materials Corporation 244 28 de agosto de 2009 Aquila Resources Limited (direito a 15% das ações) Austrália Baosteel Group 241 ç Tabela 1. 7. Referências Continuação nálise de aprovações para empresas chinesas investirem nos setores de mineração, agricultura e silvicultura da Áfr 29 Tabela 2. Principais eventos de M&A no setor de mineração no exterior Data de anúncio Empresa alvo Localização da empresa alvo Comprador Vendedor País vendedor Valor (milhões de USD) Fevereiro de 2008 Rio Tinto (12% de participação) Reino Unido Alcoa Inc; Chinalco 14.000 Agosto de 2009 Felix Resources Ltd. Austrália Yangzhou Coal Mining Co. Ltd. 2.568 Julho de 2009 Teck Resources Ltd. (17,2% de participação) Canadá Fullbloom Investment Corp 1.508 Abril de 2009 OZ Minerals (alguns ativos excluindo a proeminete Hill & Martabe) Austrália China Minmetals Non-Ferrous Metals Co. Ltd. OZ Minerals Ltd. Austrália 1.386 Março de 2008 Midwest Corp. Ltd. (80,31% de participação) Austrália Sinosteel Corp 879 Novembro de 2006 Anglo American Plc (1,01% de participação) Reino Unido China Vision Resources E Oppenheimer & Son África do Sul 812 Junho de 2007 Peru Copper Inc. Canadá Chinalco 779 Março de 2006 Ashapura Minecham Ltd. (fábrica de Alumina em Kutch, 50% de participação) Índia Qingtongxia Aluminium Group Co. Ltd. Sichuan Aostar Aluminum Co. Ltd. China 651 Agosto de 2007 Bellavista Holding Group Ltd. (60% de participação) Chile China Elegance Resources Ltd. Ceasers Development Ltd. Hong Kong 600 Dezembro de 2009 Corriente Resources Inc. Canadá CRCC-Tongguan Investment Co. Ltd. 549 Fevereiro de 2009 Fortescue Metals Group Ltd. (7,42% ou 9,07% de participação) Austrália Hunan Valin Iron & Steel Group Co. Ltd Harbinger Capital Partners Master Fund, HCP Special Situations Fund Estados Unidos 363 ou 408 Novembro de 2009 MMX Mineração e Metálicos SA (21,52% de participação) Brasil Wuhan Iron & Steel Co. Ltd. 400 Abril de 2010 Sul Americana de Metais SA Brasil Honbridge Holdings Ltd. Lit Mining Cooperatief UA; Votorantim Novos Negocios Ltd. Brasil 390 Março de 2010 Spring Vast Ltd. Rússia FinTronics Holding Co. Ltd. Truffle Rich Holdings Ltd Ilhas Virgens Britânicas 300 Mars 2010 Spring Vast Ltd Russie FinTronics Holding Co Ltd Truffle Rich Holdings Ltd Îles Vierges britanniques 300 i ó i á i Tabela 2. Principais eventos de M&A no setor de mineração no exterior Data de anúncio Empresa alvo Localização da empresa alvo Comprador Vendedor País vendedor Valor (milhões de USD) Fevereiro de 2008 Rio Tinto (12% de participação) Reino Unido Alcoa Inc; Chinalco 14.000 Agosto de 2009 Felix Resources Ltd. Austrália Yangzhou Coal Mining Co. Ltd. 2.568 Julho de 2009 Teck Resources Ltd. 7. Referências de anúncio Empresa alvo Localização da empresa alvo Comprador Vendedor País vendedor Valor (milhões de USD) bre 2009 Guney Krom (50 % de participation) Turquie Taiyuan Iron & Steel Group Co Ltd Turquie 300 Kop Krom (50 % de participation) Krom Maden (50 % de participation) CVK group Corp mbre 2007 Northern Peru Copper Corp Canada Northern Peru Acquisition Co 411 2009 Consolidated Thompson Iron Mines Ltd (19,9 % de participation) Canada Wuhan Iron & Steel Co Ltd 240 mbre 2008 Langfeld Enterprises Ltd (90 % de participation) Chypre Grandvest International Ltd Cordia Global Ltd Chypre 253 mbre 2007 Zimasco Consolidated Enterprises Ltd (92 % de participation) Zimbabwe Sinosteel Trading Co 292 mbre 2006 Samacor Chrome (mine de chrome & usine métallurgique, 50 % de participation) Afrique du Sud Sinosteel Corp Samacor Chrome Ltd Afrique du Sud 200 bro de 2009 Guney Krom (50% de participação) rom de participação) Maden (50% de ipação) Turquia Taiyuan Iron & Steel Group Co Ltd CVK group Corp Turquia 300 mbro de 2007 Northern Peru Copper Corp Canadá Northern Peru Acquisition Co 411 o de 2009 Consolidated Thompson Iron Mines Ltd (19.9% de participação) Canadá Wuhan Iron & Steel Co Ltd 240 mbro de 2008 Langfeld Enterprises Ltd (90% de participação) Chipre Grandvest International Ltd Cordia Global Ltd Chipre 253 mbro de 2007 Zimasco Consolidated Enterprises Ltd (92% de participação) Zimbábue Sinosteel Trading Co 292 mbro de 2006 Samacor Chrome (fábrica de minério de cromo e metalurgia, 50% de participação) África do Sul Sinosteel Corp Samacor Chrome Ltd África do Sul 200 tário: Alguns negócios anunciados podem ainda não terem sido concluídos 2. 7. Referências (17,2% de participação) Canadá Fullbloom Investment Corp 1.508 Abril de 2009 OZ Minerals (alguns ativos excluindo a proeminete Hill & Martabe) Austrália China Minmetals Non-Ferrous Metals Co. Ltd. OZ Minerals Ltd. Austrália 1.386 Março de 2008 Midwest Corp. Ltd. (80,31% de participação) Austrália Sinosteel Corp 879 Novembro de 2006 Anglo American Plc (1,01% de participação) Reino Unido China Vision Resources E Oppenheimer & Son África do Sul 812 Junho de 2007 Peru Copper Inc. Canadá Chinalco 779 Março de 2006 Ashapura Minecham Ltd. (fábrica de Alumina em Kutch, 50% de participação) Índia Qingtongxia Aluminium Group Co. Ltd. Sichuan Aostar Aluminum Co. Ltd. China 651 Agosto de 2007 Bellavista Holding Group Ltd. (60% de participação) Chile China Elegance Resources Ltd. Ceasers Development Ltd. Hong Kong 600 Dezembro de 2009 Corriente Resources Inc. Canadá CRCC-Tongguan Investment Co. Ltd. 549 Fevereiro de 2009 Fortescue Metals Group Ltd. (7,42% ou 9,07% de participação) Austrália Hunan Valin Iron & Steel Group Co. Ltd Harbinger Capital Partners Master Fund, HCP Special Situations Fund Estados Unidos 363 ou 408 Novembro de 2009 MMX Mineração e Metálicos SA (21,52% de participação) Brasil Wuhan Iron & Steel Co. Ltd. 400 Abril de 2010 Sul Americana de Metais SA Brasil Honbridge Holdings Ltd. Lit Mining Cooperatief UA; Votorantim Novos Negocios Ltd. Brasil 390 Março de 2010 Spring Vast Ltd. Rússia FinTronics Holding Co. Ltd. Truffle Rich Holdings Ltd Ilhas Virgens Britânicas 300 Mars 2010 Spring Vast Ltd Russie FinTronics Holding Co Ltd Truffle Rich Holdings Ltd Îles Vierges britanniques 300 i ó i á i continua na próxima página Tabela 2. Principais eventos de M&A no setor de mineração no exterior Huang Wenbin e Andreas Wilkes 30 (50% de participação) Krom Maden (50% de participação) Turquia Taiyuan Iron & Steel Group Co Ltd CVK group Corp Turquia 300 Dezembro de 2007 Northern Peru Copper Corp Canadá Northern Peru Acquisition Co 411 Março de 2009 Consolidated Thompson Iron Mines Ltd (19.9% de participação) Canadá Wuhan Iron & Steel Co Ltd 240 Novembro de 2008 Langfeld Enterprises Ltd (90% de participação) Chipre Grandvest International Ltd Cordia Global Ltd Chipre 253 Setembro de 2007 Zimasco Consolidated Enterprises Ltd (92% de participação) Zimbábue Sinosteel Trading Co 292 Novembro de 2006 Samacor Chrome (fábrica de minério de cromo e metalurgia, 50% de participação) África do Sul Sinosteel Corp Samacor Chrome Ltd África do Sul 200 Comentário: Alguns negócios anunciados podem ainda não terem sido concluídos. 7. Referências Continuação Data de anúncio Empresa alvo Localização da empresa alvo Comprador Vendedor País vendedor Valor (milhões de USD) Octobre 2009 Guney Krom (50 % de participation) Turquie Taiyuan Iron & Steel Group Co Ltd Turquie 300 Kop Krom (50 % de participation) Krom Maden (50 % de participation) CVK group Corp Décembre 2007 Northern Peru Copper Corp Canada Northern Peru Acquisition Co 411 Mars 2009 Consolidated Thompson Iron Mines Ltd (19,9 % de participation) Canada Wuhan Iron & Steel Co Ltd 240 Novembre 2008 Langfeld Enterprises Ltd (90 % de participation) Chypre Grandvest International Ltd Cordia Global Ltd Chypre 253 Septembre 2007 Zimasco Consolidated Enterprises Ltd (92 % de participation) Zimbabwe Sinosteel Trading Co 292 Novembre 2006 Samacor Chrome (mine de chrome & usine métallurgique, 50 % de participation) Afrique du Sud Sinosteel Corp Samacor Chrome Ltd Afrique du Sud 200 Tabela 2. Continuação Os Documentos de Trabalho do CIFOR contêm resultados preliminares ou avançados de pesquisas sobre questões florestais tropicais que precisam ser publicados em tempo hábil para informar e promover o debate. O conteúdo destes documentos é revisado internamente, mas não passa pela revisão por pares externos. Os impactos do investimento chinês no comércio com a África tornaram-se objeto de muita especulação. O projeto do CIFOR “Comércio e investimento chinês na África” objetiva entender o impacto do papel da China na mudança dos fluxos comerciais globais e os padrões de investimento em florestas em duas regiões ecológicas da África, a bacia do Congo e as florestas do sul da África, com foco em setores com impactos diretos potenciais sobre as florestas: mineração, silvicultura e agricultura. Globalmente, as fusões e aquisições (M&A) por empresas chinesas têm aumentado nos últimos anos. A África é o segundo destino mais frequente para eventos de M&A em mineração, e a pesquisa indica um crescente interesse em atividades de M&A na África. A maioria das atividades de M&A é nos setores de mineração e petróleo. Mais pesquisas são necessárias para produzir um banco de dados abrangente das reais atividades de investimento chineses na África. As M&A, por serem uma forma crescente de investimentos no exterior por empresas chinesas, também merecem investigação. blog.cifor.org cifor.org cifor.org Centro de Pesquisa Florestal Internacional (CIFOR) O CIFOR contribui para o bem-estar humano, a conservação ambiental e a equidade, realizando pesquisas para servir de base para as políticas e práticas que afetam as florestas nos países em desenvolvimento. O CIFOR é um membro do Consórcio do CGIAR. Nossa sede fica em Bogor, na Indonésia, com escritórios na Ásia, África e América Latina. Centro de Pesquisa Florestal Internacional (CIFOR) Centro de Pesquisa Florestal Internacional (CIFOR) O CIFOR contribui para o bem-estar humano, a conservação ambiental e a equidade, realizando pesquisas para servir de base para as políticas e práticas que afetam as florestas nos países em desenvolvimento. O CIFOR é um membro do Consórcio do CGIAR. Nossa sede fica em Bogor, na Indonésia, com escritórios na Ásia, África e América Latina. q ( ) O CIFOR contribui para o bem-estar humano, a conservação ambiental e a equidade, realizando pesquisas para servir de base para as políticas e práticas que afetam as florestas nos países em desenvolvimento. O CIFOR é um membro do Consórcio do CGIAR. Nossa sede fica em Bogor, na Indonésia, com escritórios na Ásia, África e América Latina.
https://openalex.org/W1980691438	https://europepmc.org/articles/pmc3356870?pdf=render	English	null	Implication of Crystal Water Molecules in Inhibitor Binding at ALR2 Active Site	Computational and mathematical methods in medicine	2,012	cc-by	6,831	Correspondence should be addressed to M. Elizabeth Sobhia, mesophia@niper.ac.in Received 23 September 2011; Revised 30 December 2011; Accepted 13 January 2012 Academic Editor: Joti Yasumasa Copyright © 2012 Hymavati et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Water molecules play a crucial role in mediating the interaction between a ligand and a macromolecule. The solvent environment around such biomolecule controls their structure and plays important role in protein-ligand interactions. An understanding of the nature and role of these water molecules in the active site of a protein could greatly increase the eﬃciency of rational drug design approaches. We have performed the comparative crystal structure analysis of aldose reductase to understand the role of crystal water in protein-ligand interaction. Molecular dynamics simulation has shown the versatile nature of water molecules in bridge H bonding during interaction. Occupancy and life time of water molecules depend on the type of cocrystallized ligand present in the structure. The information may be useful in rational approach to customize the ligand, and thereby longer occupancy and life time for bridge H-bonding. Hindawi Publishing Corporation Computational and Mathematical Methods in Medicine Volume 2012, Article ID 541594, 11 pages doi:10.1155/2012/541594 Hindawi Publishing Corporation Computational and Mathematical Methods in Medicine Volume 2012, Article ID 541594, 11 pages doi:10.1155/2012/541594 Hindawi Publishing Corporation Computational and Mathematical Methods in Medicine Volume 2012, Article ID 541594, 11 pages doi:10.1155/2012/541594 Hindawi Publishing Corporation Computational and Mathematical Methods in Medicine Volume 2012, Article ID 541594, 11 pages doi:10.1155/2012/541594 Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, Punjab 160 062, S.A.S Nagar, India Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, Punjab 160 062, S.A.S Nagar, India Correspondence should be addressed to M. Elizabeth Sobhia, mesophia@niper.ac.in Hymavati, Vivek Kumar, and M. Elizabeth Sobhia Hymavati, Vivek Kumar, and M. Elizabeth Sobhia Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, Punjab 160 062, S.A.S Nagar, India 1. Introduction glucose [6]. Under diabetic hyperglycaemia, excess glucose is metabolized by the polyol pathway (Figure 1). This pathway comprised of two enzymes, namely, ALR2 which reduces excess D-glucose into D-sorbitol, and sorbitol dehydrogenase which converts sorbitol to fructose. Diabetic complications have been linked to excessive accumulation of sorbitol, and targeting the polyol pathway by inhibiting ALR2 oﬀers an option for treatment [7]. Diabetes is a debilitating disease leading to severe compli- cations and a shortened life expectancy. Diabetes-speciﬁc microvascular disease leads to blindness, renal failure and nerve damage, and diabetes-accelerated atherosclerosis which increase risk of myocardial infarction, stroke, and limb amputation [1]. The insulin therapy for tissues that do not require insulin does not prevent complications such as neuropathy, retinopathy, nephropathy, and cataracts [2]. Large prospective clinical studies show a strong relationship between glycaemia and diabetic microvascular complications in both type 1 and type 2 diabetes [3]. Hyperglycaemia and insulin resistance are suggested to play important roles in the pathogenesis of macrovascular complications [4]. In normal physiological conditions, ALR2 is involved in osmoregulation while under hyperglycaemic conditions it contributes to the onset and development of severe compli- cations in diabetes [8]. The elevated glucose level enhances the activity of ALR2 by directly increasing the glucose ﬂux through this pathway and indirectly by forming reactive oxy- gen species (ROS), which activates ALR2 [9]. The increased activity of ALR2 results in decreased NADPH/NADP+ ratio, and it aﬀects other NADPH-dependent enzymes, such as nitric oxide (NO) synthase and glutathione reductase [10]. The reduction of NO level leads to decreases nerve conduc- tion and microvascular derangement. The retarded activity of the antioxidative enzyme glutathione reductase causes Aldose reductase 2 (ALR2, alditol: NAD(P) 1-oxidor- eductase, EC 1.1.1.21) is the ﬁrst enzyme in the polyol pathway that catalyses the NADPH-dependent reduction of D glucose to D sorbitol [5]. It is a cytosolic, monomeric oxidoreductase that catalyses the NADPH-dependent reduc- tion of wide variety of carbonyl compounds including Computational and Mathematical Methods in Medicine 2 Hyperglycemia Polyol pathway Glucose ALR2 Sorbitol SDH Fructose NADPH NO GSH Oxidative stress AGEs Glycation 3-deoxycholate Fructose-3-phosphate Figure 1: Hyperglycemia pathway. Polyol pathway Fructose-3-phosphate 3-deoxycholate Figure 1: Hyperglycemia pathway. oxidative stress under diabetic conditions [11]. Inhibiting ALR2 would provide a way of avoiding diabetic complica- tions, and, therefore, identifying inhibitors is an important pharmaceutical goal. loop residues. The large and highly hydrophobic active site is located at the carboxy terminus of (β/α)8 barrel. 1. Introduction To date, there are 95 crystals of ALR2 deposited in Protein Data Bank [13]. The ligand-binding site is a large, deep, elliptical pocket with the nicotinamide ring of NADPH cofactor lying at the base. The enzyme has anion-binding pocket and speciﬁcity pocket (Figure 2). The anion-binding pocket has Tyr 48, His110, Trp111, Trp 20, Phe122, and Trp 219 while the speciﬁcity pocket is lined by residues Trp111, Thr113, Phe122, Ala 299, and Leu 300. Epalrestat is the only inhibitor of aldose reductase that is successfully marketed in Japan for treatment of diabetic neuropathy. Many promising compounds from diﬀerent in vitro and in vivo studies have failed to proceed beyond clinical trials. The lack of eﬃcacy or adverse eﬀects as a result of less-speciﬁc inhibitor, and a likely inhibition of the related aldehyde reductases are the major bottlenecks in the development of ALR2 inhibitors. The knowledge of catalytic mechanism and available structures information of current inhibitors coupled with ALR2 can help in accelerating the discovery or designing of speciﬁc inhibitors. A limited number of currently available drugs for the treatment of diabetic complications only suggests the importance of research for new ALRIs [12]. We present here molecular dynamics simulation study on the crystal structure of ALR2 enzyme. Moreover, the goal of our study is to evaluate the importance of the crystal water molecule in the active site of the ALR2. These water molecules mediate the hydrogen bond and help ALRIs to bind at the active site. 2. Materials and Methods ALR2 is a monomer (35.8 kDa), encoded by a single gene located on chromosome region 7q35.ALR2, made up of a single polypeptide chain with 315 residues. The crystal is a single domain structure folded into an eight-stranded parallel α/β motif. The substrate-binding site is located in a cleft at the carboxy terminal end of the β-barrel and involves 2.1. Comparative ALR Crystal Structure Analysis. The 87 crystal structures of human ALR were obtained from the Protein Data Bank (PDB) [13]. The crystals were aligned by “Align by homology” program in Biopolymer module in Sybyl7.1, taking the apoenzyme (PDB id: 1ADS) as the Computational and Mathematical Methods in Medicine 3 Trp79 HN HN Val47 Phe122 Phe115 Tyr48 N N H H NH Trp111 Thr113 Cys303 Tyr309 OH OH Speciﬁcity pocket Anion- binding pocket Val297 Cys298 Ala299 Leu300 O O O O O N N N N N N HS Trp219 Trp20 HO SH His110 NADP+ H2N N+ Figure 2: Schematic drawing of ALR2 binding pocket. Trp79 Val47 HN Phe122 His110 Tyr48 Phe115 Trp111 Thr113 Speciﬁcity pocket Cys303 Leu300 Ala299 Figure 2: Schematic drawing of ALR2 binding pocket. was solvated using orthorhombic TIP3P water box, distance between the edge of the box, and periphery of the protein is 8 Angstrom [17]. Solvated systems were neutralized by adding required number of counter ions. A two-stage approach was adopted for minimizing the protein. In the ﬁrst stage, the protein, ligand, and cofactor were kept ﬁxed with weak restraint (10 kcal/mol- ˚A2), and only solvent was allowed to minimized. Then, in the second stage, the entire system was minimized. Initially, minimization of 1000 steps of steepest descent was carried out and this was followed by another 500 steps of conjugate gradient minimization. Thereafter, minimizing solvent, the entire system consisting water, protein, and ligand cofactor was minimized for 2500 steps without restraint. reference structure [14]. The crystals were analyzed for the presence of ordered/conserved water molecules in the active site using PyMol [15]. 2.2. Preparation of Input Files for MD Simulation. Among all available crystal structures of human ALR2, ﬁve crystal structures having diﬀerent ligand (PDB id: 1T40, 1Z89, 2FZ8, 3H4G, and 3G5E) were selected to understand the role of water molecules in the binding of ligands with human ALR2 [13]. Coordinates of crystal structures were taken from the PDB. Computational and Mathematical Methods in Medicine Computational and Mathematical Methods in Medicine 4 Table 1: Table showing PDB ID of complexes and their inhibitors and water molecules in the active site. PDB ID Ligand Ligand atom ID Water molecule ID 1AZ1 Alrestatin O21 HOH669 1PWL Minalrestat O HOH2323 1PWM Fidarestat F17 N21 HOH1654 HOH1552 & 1774 1IEI Zenarestat O34 HOH477 1T40 IDD552 O16 HOH738 1T41 IDD552 O16 HOH5004 1Z89 Sulfonyl pyridazinone O18 HOH1147 1Z8A Sulfonyl pyridazinone O18 HOH1179 & 1268 2ACQ G6P G6P317 HOH460 2ACU CIT CIT317 HOH319 2FZ8 Zopolrestat O1 HOH1082 2FZD Tolrestat S1 F3 HOH355 & 389 HOH487 2PD5 Zopolrestat O1 HOH748 2PD9 Fidarestat N21 HOH624&816 2PDF Zopolrestat O1 HOH815 2PDI Zopolrestat O1 HOH736 & 687 2PDM Zopolrestat O1 HOH782 2PDW Fidarestat N21 HOH 750 & 615 2PDX Zopolrestat O1 HOH 691 2PDY Fidarestat N21 HOH 617 3H4G Fidarestat N21 HOH 2275 3G5E Q74 N24 HOH 698 Table 1: Table showing PDB ID of complexes and their inhibitors and water molecules in the active site. hydrogen atoms [19]. The van der Waals cutoﬀwas kept 8 angstrom and long range electrostatic interactions were treated using the Particle Mesh Ewald (PME) method. Co- ordinates were saved after each 1000 step, which were ﬁnally used for analysis. module in Sybyl7.1, taking the apoenzyme, 1ADS as a ref- erence structure. This module aligns proteins on the basis of their sequence similarity. The tailor variables control the gap penalty, the number of jumbles, and similarity matrix used. The sequences were compared by superimposing the Cα, backbone, side chain, and all atoms. The crystal structures having a RMSD value greater than 0.5 ˚A were rejected. The selected 28 crystal structures were analyzed using Accelrys Discovery Studio 2.0 [22] and Pymol to visualize and identify atoms participating in the ligand binding including the water molecules in the active site. After analyzing the atoms, 23 crystal structures were found to have water molecules in the active site which showed hydrogen bond interactions. y All simulations were carried out using SANDER program of AMBER10 package. Analysis was performed using VMD and Ptraj module of Amber tools [20]. Additionally, Chimera and Pymol were used for molecular visualization [21]. 2.3. MM-PB (GB) SA Calculations. MMPBSA calculations were performed on 5 complexes: 1T4O, 1Z89, 2FZ8, 3H4G and 3G5E. Computational and Mathematical Methods in Medicine The interaction energy and salvation-free energy for the complex, receptor, and ligand were calculated with the help of the snapshots extracted at each 10 ps from the 1.5 ns to 5 ns run. The average of the results was calculated to get an estimate of the binding free energy. The binding energy calculation was carried out with MM PBSA and MM GBSA method for the sake of comparison. Additionally, binding energies were also estimated by MM-PBSA program of Prime module of Schrodinger maestro package. The criterion in selecting the hydrogen bonds was the bond length that should not be greater than 3 ´˚A. The active site was observed to have more number of bridging water molecules within the anionic pocket of NADPH rather than the ligand (Table 1). We assumed that the phosphate groups present in the NADPH are responsible for it. The ligands were found to interact with at least one water molecule in the active site. The water molecules are said to be bridging if they simultaneously make hydrogen bonds with both ligand and amino acid residues. 2. Materials and Methods Missing residues were added, and mutated residues were corrected to wild-type residues using Schrodinger maestro package [16]. Missing hydrogen atoms of ligands and cofactors were added using Schrodinger maestro package. AM1-BCC charges were used for the ligands as well as cofactor NADPH, calculated using Antechamber program. Parameters for the ligands were generated using the general amber force ﬁeld (GAFF) and Antechamber module of AMBER while ﬀ99SB force ﬁeld was used for protein. Missing hydrogen atoms of the protein structures were added using LeaP module of AMBER. Amino acids were kept in their default ionization states. Crystal water molecules were kept as such while preparing the system. Complex After minimization process, the system was heated from 0 to 300 K. In order to avoid any instability during the MD production run, an initial MD run were carried out in NPT ensemble (pressure = 1 bar, temperature = 300 Kel) to equilibrate the system for 20 ps with weak restraints on the solute. Production run were carried out for 5 ns time, using NVT ensemble and Langevin dynamics used to control the temperature through a collision frequency of 1.0 ps−1 [18]. Step size was kept 2 fs for the throughout simulations. A SHAKE algorithm was applied to constrain bonds involving 3. Results and Discussion 3.1. Comparative Crystal Structure Analysis of ALR2 Com- plexes. All the 87 crystal structures of human ALR2 were aligned by “Align by homology” program in Biopolymer 3.2. Analysis of Ligand-Binding Site. The crystal structures, 1T40, 1Z89, 2FZ8, 3H4G, and 3G5E, were selected for further studies. The active sites were analyzed around 5 ˚A region 5 Computational and Mathematical Methods in Medicine (b) (a) (b) Figure 3: Water molecule interacting with ligand in crystal structure (a) 2FZ8 and (b) 3H4G. The ligand and the amino acid residues in the active site are represented in stick. The yellow broken line represents the hydrogen bond between the ligand and the active site residues. (a) (a) (b) Figure 3: Water molecule interacting with ligand in crystal structure (a) 2FZ8 and (b) 3H4G. The ligand and the amino acid residues in the active site are represented in stick. The yellow broken line represents the hydrogen bond between the ligand and the active site residues. of ligand to identify the number of water molecules and hydrogen bond interactions with corresponding ligand. It was noted that most of the structures have at least one water molecule in the active site which shows hydrogen bond interactions with the ligand. In Figure 3, the ligands of 2FZ8 and 3H4G make a hydrogen bond with the water molecule at the active site. All the ligands, except in 1Z89, displaced the 9 ordered water molecules in the active site of the apoenzyme, whereas the ligand in 1Z89 displaced 6 water molecules. These diﬀerences can be attributed to the size and conformation of the ligand in the active site. The 5 complexes, upon superimposing on the apoenzyme, showed their ligands, displacing the 6 ordered water molecules in the active site of the apoenzyme. The size of the ligand from 3H4G is exactly equal to the size occupied by the 6 ordered water molecules in the apoenzyme; whereas, the other ligands have an extended conformation in both the anionic and speciﬁcity site. contribution to the solvation free energy due to cavity formation and van der Waals interactions, between the solute and the solvent, was estimated by a solvent-accessible surface area. The total binding energy was negative in all the com- plexes, and it signiﬁes a favorable protein-ligand complexes. The results for the binding free energy after performing simulation for 2 ns are shown in Table 2. Computational and Mathematical Methods in Medicine Computational and Mathematical Methods in Medicine 6 Table 2: IC50 and calculated binding free energy for ALR2 crystal structure complexes. S.No. PDB ID Structure IC50(μM) ΔGbind 1 1T40 O HO O O HN S N F F F F 0.0110 −33.88 2 1Z89 N NH O S O O O Cl 0.0010 −22.24 3 2FZ8 N N O OH S N F F F O 0.0031 −35.22 4 3H4G O HN NH O O F O NH2 0.5700 −31.02 5 3G5E O HO N N S N F F F 0.0050 −29.17 reasonable. The binding energy of the ALR2 complexes without crystal water was observed low by 3–7 kcal/mol as compared to complexes with water. It clearly indicated that l l l h l d i ki d bl crystal structures—1Z89, 2FZ8 and 3H4G (Figure 4). The crystallized water molecules were retained in this simulation. The notion of crystal waters is tricky in MD simulation i i hi hl bil d h f h h Table 2: IC50 and calculated binding free energy for ALR2 crystal structure complexes. Table 2: IC50 and calculated binding free energy for ALR2 crystal structure complexes. Table 2: IC50 and calculated binding free energy for ALR2 crystal structure complexes. Table 2: IC50 and calculated binding free energy for ALR2 crystal structure complexes. S.No. PDB ID Structure IC50(μM) ΔGbind 1 1T40 O HO O O HN S N F F F F 0.0110 −33.88 2 1Z89 N NH O S O O O Cl 0.0010 −22.24 3 2FZ8 N N O OH S N F F F O 0.0031 −35.22 4 3H4G O HN NH O O F O NH2 0.5700 −31.02 5 3G5E N N S N F F F 0.0050 −29.17 S.No. Structure PDB ID Structure 1T40 O HO O O HN S N F F F F O HO O O HN S N F F F F 2 2FZ8 N N O OH S N F F F O 0.0031 2FZ8 O 3H4G O HN NH O O F O NH2 0.5700 −31.02 3G5E O HO N N S N F F F 0.0050 −29.17 HN 5 reasonable. The binding energy of the ALR2 complexes without crystal water was observed low by 3–7 kcal/mol as compared to complexes with water. It clearly indicated that crystal water molecules helped in making strong and stable protein-ligand complexes (Table 3). 3.4. Molecular Dynamics Simulation with Crystallized Water Molecules. Explicit simulations were carried out for the 3. Results and Discussion Although the result shows zopolrestat, the second potent ligand (2FZ8) has the highest binding aﬃnity, while sulfonyl pyridazinone (1Z89) has the lowest binding aﬃnity. The inhibitors have maximum potency diﬀerence of 2.8 between most and least potent ligand at scale of pIC50. It indicates coinciding nature of biological activity which merely expected to be demarcated by correlation with predicted binding free energy due to lack of wide range. The electrostatic contribution to solvation free energy in the 5 complexes is shown in Table 3. The ΔGele and ΔGvdW are electrostatic (ELE) and van der Waals (VDW) contribution, calculated by the MM force ﬁeld respectively. The ΔGele-int is the internal energy (INT) arising from bond, angle, and dihedral terms in the MM force ﬁeld. The sum of ELE, VDW, and INT is known as total gas phase energy. The ΔGnon-polar is the non-polar contribution to the solvation free energy calculated by an empirical model while ΔGele-MM, is sum of the electrostatic solvation free energy and MM electrostatic energy. 1Z89 makes the highest contribution to the solvation free energy while 3G5E makes the lowest. The nonpolar contributions to the solvation are similar in all the ﬁve complexes. Electrostatic component of the solvation free energy was calculated by GB model. Polar contributions to the solvation free energy were computed by applying linear PB model and an extension to the GB model. It disfavors binding of the protein ligand complexes in ALR2. PB and GB calculations give very similar results in that respect. 3.3. Binding Free Energy Calculations. Explicit solvent MD simulations were carried out for all complexes under study. The absolute binding free energy of the complex formation was estimated from energetic and entropic contributions, and calculated for snapshots extracted from the trajectories. The snapshots of the unbound proteins and complexes were taken from molecular dynamics (MD) and were further processed for 2 ns using periodic boundary conditions. The snapshots of the unbound molecules were extracted only from the trajectory of the solvated complex. A total of 1000 snapshots were extracted from the 2 ns trajectory, with 200 snapshots for each of the solvated complex. Solvation free energies were computed as the sum of polar and nonpolar contribution, using a continuum representation of the solvent. The polar contribution was calculated by solving the Poisson equation. 3. Results and Discussion The nonpolar Moreover, the ΔGbind calculated for the ALR2 crystal structure with and without crystal molecules was found Computational and Mathematical Methods in Medicine Computational and Mathematical Methods in Medicine crystal structures—1Z89, 2FZ8 and 3H4G (Figure 4). The crystallized water molecules were retained in this simulation. The notion of crystal waters is tricky in MD simulation since water is highly mobile and therefore, they exchange rapidly. Even at tight interfaces, such as a protein-nucleic acid interface, the lifetimes of bound water are on the nanosecond time scale. Hence, the waters in the solvation shell around the ligand were calculated after processing of the trajectories with ptraj module of AMBER. The output for this functionality 3.4. Molecular Dynamics Simulation with Crystallized Water Molecules. Explicit simulations were carried out for the Computational and Mathematical Methods in Medicine 7 Table 3: Thermodynamics of binding for the 5 complexes of ALR2 calculated using MM-PBSA. PDB ID ΔGele ΔGvdW ΔGele-int ΔGnon-polar ΔGele-MM ΔGbind ∗ Gbind ∗ (With water) (Without water) 1T40 5.97 −48.31 14.96 −5.67 20.93 −25.24 −21.29 1Z89 −6.92 −44.85 36.83 −4.94 29.91 −53.71 −49.73 2FZ8 5.66 −51.96 21.49 −6.07 27.16 −18.77 −16.23 3H4G 11.81 −45.37 17.53 −5.69 29.34 −44.16 −36.60 3G5E 22.7 −46.89 4.79 −5.44 27.49 −17.55 −14.22 ∗Estimated by MM-GBSA program of Prime module implemented in Schrodinger maestro package. Table 3: Thermodynamics of binding for the 5 complexes of ALR2 calculated using MM-PBSA. (a) (b) Figure 4: Water molecules present in the apoenzyme (red dot) displaced by the ligand in crystal structure (green dot) (a) 2FZ8 and (b) 3H4G. The ligand in the active site is represented in stick. (b) (a) (a) (b) Figure 4: Water molecules present in the apoenzyme (red dot) displaced by the ligand in crystal structure (green dot) (a) 2FZ8 and (b) 3H4G. The ligand in the active site is represented in stick. throughout the simulation. The water molecule is stable for the location of the oxygen atom, but it tumbles freely with the hydrogen atoms jumping between several positions of the hydrogen bond network. The plot for the number of water molecules ﬂuctuating throughout the simulation during the explicit 5 ns is shown in Figure 5(a). contains number of waters in the ﬁrst shell and second shell. The ﬁrst solvation shell represents a distance of 3.4 ˚A from the solute, that is, ligand of interest while the second solvation shell represents a distance of 5 ˚A from the center of mass of the ligand. The analysis is presented separately for each complex. Computational and Mathematical Methods in Medicine The number of water molecules in the ﬁrst solvation shell was found to be 1 on an average throughout the 5 ns simulation. However, in the second solvation shell, there were 3 water molecules on an average. To understand the water association, visual analysis of the areas of high occupancy or search, a speciﬁc water interaction is useful (Table 4). The occupancy is deﬁned as the percentage over the whole trajectory in which both the distance and the angle criteria are satisﬁed. 3.4.1. Analysis for the Complex 1Z89. 1Z89 is a human ALR2, coupled with novel sulfonyl-pyridazinone (62P), having a resolution of 0.95 ˚A. The pyridazinone group of the inhibitor occupies the catalytic site, whereas the chlorobenzofuran moiety penetrates the open speciﬁcity pocket. The pyridazi- none exhibits a binding aﬃnity similar to that of tolrestat and sorbinil, showing slightly reduced aﬃnity compared to IDD594. The 62P displaces the 6 ordered water molecules which are present in the apoenzyme 1ADS, whereas other ligands replaced 9 water molecules in the apoenzyme. From the analysis of the crystal, it was observed that O18 of the ligand made hydrogen bond interactions with HOH1147. The other hydrogen bond interactions were observed for O7 with OH of Tyr48, and NE2 of His110 and N4 with NE1 of Trp111. As the simulation progressed the positions of water molecules ﬂuctuated as indicated by graph from Figure 5(a). The graph shows the movement of water molecules within the ﬁrst and second solvation shell A grid of 100 angstroms was generated around the solute. The structure was ﬁtted to a common reference frame, by RMS ﬁt to the ﬁrst frame, to all the solute molecules in the protein. If the molecule is tumbling in space that ﬁxed water will move with the molecule but the grid does not, it is ﬁxed at the ﬁrst frames location. If the water in question is in the same position with respect to the biomolecule, then the water molecules will move with the side chain. Since the grid did not move, the density can be smeared across multiple grid elements. Computational and Mathematical Methods in Medicine This will give an idea of the probable involvement 8 Computational and Mathematical Methods in Medicine Computational and Mathematical Methods in Medicine 8 Number of waters around 62P in 1Z89 during 5 ns simulation 0 1 2 3 4 5 Time (ns) Number of water molecules Outer shell Inner shell 18 16 14 12 10 8 6 4 2 0 (a) (b) Figure 5: (a) Number of water molecules ﬂuctuating in the solvation shell of 62P during 5 ns simulation and (b) ligand 62P interacting with HOH1147. (b) (a) Figure 5: (a) Number of water molecules ﬂuctuating in the solvation shell of 62P during 5 ns simulation and (b) ligand 62P interacting with HOH1147. Table 4: Hydrogen bonds data showing occupancy with distance cutoﬀof 5 ˚A along the MD simulations for 1Z89. also. This shows that positions of the water molecules are conserved in all the complexes belonging to this family. Table 4: Hydrogen bonds data showing occupancy with distance cutoﬀof 5 ˚A along the MD simulations for 1Z89. Donor H-X Acceptor atom of water 1Z89 Occupancy (%) Distance ( ˚A) 62P@O18 8106@O 16.50 3.517 62P@S8 8106@O 14.50 3.753 62P@O19 8106@O 14.00 3.723 62P@N4 8106@O 11.50 3.986 62P@C3 8106@O 11.00 3.730 3.4.2. Analysis for the Complex 2FZ8. The bound zopolrestat occupies almost the entire active site pocket at the C-terminal end of the β barrel. The inhibitor makes an unusually large number of contacts with the active site. It made a total of 132 contacts within ≤4 ˚A, 110 with 15 residues, 13 with the nicotinamide moiety of the coenzyme, and 9 with four ordered water molecules coenzyme. This contributes favorable entropic eﬀect to the tight binding of the inhibitor. From our analysis, we found out that the ligand displaces 9 water molecules present in the apoenzyme. The O1 of the ligand makes a hydrogen bond with HOH1082. The other water molecules were also present in the active site, but they did not make any interactions with the ligand. The other hydrogen bond interactions were made between O3 of the ligand with OH of Tyr 48, and NE2 of His110; O2 of ZST with NE1 of Trp111, and N3 with N of Leu300. of water molecules in the active site of the protein as the simulation progresses. The hydration site was constructed from the water den- sity by using the coordinate system local to each hydration site. Computational and Mathematical Methods in Medicine The water structure is broken down into hydration sites constructed from water density from the protein surface. This gives the structural and dynamic properties of the water molecules from the explicit simulations. Properties studied include site occupancy, number of neighboring waters and hydrogen bonds. The occupancy of water around the ligand sulfonyl pyridazinone is analyzed by Chimera. From the density map, it was observed that the density is concentrated around O18 and O7 of the ligand, which in the native crystal was making a hydrogen bond with the water molecule. The neighboring water molecules in the active site were studied. The number of waters in the ﬁrst solvation shell, around the ligand in 2FZ8, was 6 throughout the simulation, whereas, in the second solvation shell it was 13 on an average. The number of water molecules in the ﬁrst and second solvation shell, is plotted as the number of water molecules against the number of frames in the graph (Figure 6). around the ligand in 2FZ8, was 6 throughout the simulation, whereas, in the second solvation shell it was 13 on an average. The number of water molecules in the ﬁrst and second solvation shell, is plotted as the number of water molecules against the number of frames in the graph (Figure 6). The occupancy of water oxygen atom with the ligand throughout 5 ns simulation per frame is shown in Table 5. The highest occupancy was approximately 53% for 2FZ8 in diﬀerent frames during the simulation, and it gradually reduced to 15% as the simulation progressed. It is shown in the surface representation of the protein that ligand is making a hydrogen bond with a water molecule, HOH 1147 (Figure 5(b)). In this simulation, the crystal waters were removed, but the water density around the ligand shows the positions of the water molecule being retained, that is, they were occupied by the water molecules which were added explicitly. The density was at the same position where the ligand was making a bond with the water molecule, and the same pattern was observed in the remaining complexes 3.4.3. Analysis for the Complex 3H4G. 3H4G is an ALR crystal structure with Fidarestat (FID), which was shown to have implications in inhibitor binding and selectivity. Computational and Mathematical Methods in Medicine The ligand displaces 9 water molecules in the active site of the Computational and Mathematical Methods in Medicine 9 0 1 2 3 4 5 Time (ns) Number of water molecules Outer shell Inner shell 40 30 20 10 0 Number of waters in solvation shell of ZST in 2FZ8 in simulation 5 ns (a) (b) Figure 6: (a) Number of water molecules ﬂuctuating in the solvation shell of ZST during 5 ns simulation, and (b) ligand ZST interacting with HOH1082. (b) (a) (b) Figure 6: (a) Number of water molecules ﬂuctuating in the solvation shell of ZST during 5 ns simulation, and (b) ligand ZST interacting with HOH1082. Table 5: Hydrogen bonds data showing occupancy with a distance cutoﬀof 5 ˚A for 2FZ8 during the MD simulations. Table 5: Hydrogen bonds data showing occupancy with a distance cutoﬀof 5 ˚A for 2FZ8 during the MD simulations. Donor H-X Acceptor atom of Water 2FZ8 Occupancy (%) Distance ( ˚A) ZST@C9 328@O 53.55 3.730 ZST@N3 328@O 49.73 4.378 ZST@C10 328@O 46.45 4.435 ZST@N1 328@O 36.09 4.614 ZST@O1 328@O 15.00 4.747 Table 6: Hydrogen bonds data showing occupancy with a distance cutoﬀof 5 ˚A in the MD simulations for 3H4G. y g g p y cutoﬀof 5 ˚A in the MD simulations for 3H4G. Donor H-X Acceptor atom of water 3H4G Occupancy (%) Distance ( ˚A) FID@C9 346@O 98.82 3.683 FID@O10 346@O 97.55 3.765 FID@C11 346@O 95.27 4.047 FID@C19 346@O 91.91 4.299 FID@O6I 346@O 89.64 3.423 Concurrently, decreasing numbers of water molecules are observed in both the hydration shell (Figures 5(a), 6(a), and 7(a)). Although the active site of the ALR2 is highly hydrophobic, and water molecules are observed present at the opening of the active site cavity, the region included in the hydration shell. The water molecules are randomly moved away with progress of simulation while the average number of solvent water molecules remained similar to water molecules present in the crystal structures. The bridge H- bonding interaction is believed to keep these water molecules in the highly perturb site. Moreover, no severe structural changes are observed as the average backbone root mean square deviation (RMSD) is found <0.72 ˚A and <0.89 ˚A for active site residues (5 ˚A regions around cocrystal ligand) and whole protein respectively (Figure 8). apoenzyme. Computational and Mathematical Methods in Medicine A bridging water molecule HOH2275 is present between the ligand and the protein, making hydrogen bond interactions with N21, O10 of FID, and HOH 2015. The O6I of FID makes a hydrogen bond with NE1 of Trp114. Also, O10 of FID makes a hydrogen bond with HOH 2196, and O3 of the ligand with OH of Tyr 50. The extent of solvent exposure for each site is measured by the average number of neighboring waters within the cut- oﬀdistance 3.5 ˚A and 5 ˚A, of all waters that exist in the ligand active site, as shown in the graph (Figure 7). The number of water molecules in the ﬁrst solvation shell around the ligand in 3H4G was 3 throughout the simulation, whereas it was 6 at an average in the second solvation shell. Figure 6(b) shows a picture of the protein surface, and the ligand in stick representation, interacting with water molecules displayed as red spheres. Here, the ligand is interacting with more than one water molecule in the active site. 4. Conclusions The traditional approaches for absolute free energy esti- mation in the MM-PB(GB)SA approach, no “nonphysical” annihilation or decoupling of the ligand in solution and bound to its receptor is necessary, nor need one simulate the partially unbound states that would be required for a potential of mean force estimate using umbrella sampling. Hence, binding free energy calculations with MM-PB(GB)SA use physical states at both end points of the binding reaction Table 6 shows the occupancy of water oxygen atoms with the ligand throughout the simulation for diﬀerent frames. The occupancy and the hydrogen bond distances were monitored throughout the simulation. Initially, the highest occupancy was 98%, and it got reduced to 89% as the simulation progressed. The occupancy of water in this protein was the highest when compared to other proteins in this study, and this also conﬁrmed the sharp hydration sites. 10 Computational and Mathematical Methods in Medicine 0 1 2 3 4 5 Time (ns) Number of water molecules Outer shell Inner shell 40 35 30 25 20 15 10 5 0 Number of waters in solvation shell of FID in 3H4G (a) (b) Figure 7: (a) Number of water molecules ﬂuctuating in the solvation shell of FID during 5 ns simulation and, (b) ligand FID interacts with HOH2275, HOH2015 and HOH2196. 0 1 2 3 4 5 Time (ns) Number of water molecules Outer shell Inner shell 40 35 30 25 20 15 10 5 0 Number of waters in solvation shell of FID in 3H4G (a) Number of waters in solvation shell of FID in 3H4G (b) (a) (b) Figure 7: (a) Number of water molecules ﬂuctuating in the solvation shell of FID during 5 ns simulation and, (b) ligand FID interacts with HOH2275, HOH2015 and HOH2196. 1.2 1 0.8 0.6 0.4 0.2 0 0 1 2 3 4 5 Time (ns) 1.4 RMSD ( ˚A) (a) 0 1 2 3 4 5 Time (ns) 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0 RMSD ( ˚A) (b) 1.2 1 0.8 0.6 0.4 0.2 0 0 1 2 3 4 5 Time (ns) RMSD ( ˚A) (c) Figure 8: RMSD of backbone is shown for whole protein (blue) and 5 ˚A regions around the active site of cocrystal ligand (red). The structures, 1Z89 (a) and 3H4G (c), have lower RMSD for active site than whole protein, while 2FZ8 (b) has similar RMSD for both throughout the trajectory. 4. Conclusions 1.2 1 0.8 0.6 0.4 0.2 0 0 1 2 3 4 5 Time (ns) RMSD ( ˚A) (c) 0 1 2 3 4 5 Time (ns) 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0 RMSD ( ˚A) (b) 1.2 1 0.8 0.6 0.4 0.2 0 0 1 2 3 4 5 Time (ns) 1.4 RMSD ( ˚A) (a) (b) (a) (c) Figure 8: RMSD of backbone is shown for whole protein (blue) and 5 ˚A regions around the active site of cocrystal ligand (red). The structures, 1Z89 (a) and 3H4G (c), have lower RMSD for active site than whole protein, while 2FZ8 (b) has similar RMSD for both throughout the trajectory. only, thereby, avoiding the need to devote computer time on intermediate states. Because the accuracy of absolute binding free energy calculations depends on a delicate balance of diﬀerent energetic and entropic contributions, the calculated in silico binding free energy can be still considered close to the experimentally determined data. has a low binding energy. Moreover, binding energy pattern of the ALR2 complexes with and without water signify the importance of the water molecules in protein-ligand interaction. This study conﬁrmed the stability of structural bridge water molecules that are always present in simulations and also revealed the presence of other structural water molecules, with lower residence time and occupancy in the complexes. The water density around the ligand shows areas of high occupancy of water at these positions. They closely reproduce the position of the water molecules which were observed in the crystal structure. The life time and occupancies of water molecules also varies depending on the type of inhibitor with 3H4G having occupancy of 98.92%, 53.55% for 2FZ8, and only 16.5% for 1Z89. The occupancy ratios are also in agreement with the observed density, in which 3H4G have more density around the ligand when compared to other inhibitors. The contributions toward electrostatic free energy of solvation are very low for 3H4G as compared to other complexes. So the energy required to desolvate the binding particles in this complex is very low, while the highest energy required to desolvate is seen in 1Z89 which Computational and Mathematical Methods in Medicine Computational and Mathematical Methods in Medicine [4] G. M. Reaven, “Banting lecture 1988. Role of insulin resistance in human disease,” Diabetes, vol. 37, no. 12, pp. 1595–1607, 1988. [5] A. E. Heesom, M. L. Hibberd, A. Millward, and A. G. 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Rosenqvist, “The eﬀect of long-term intensiﬁed insulin treatment on the development of microvascular complications of diabetes mellitus,” New England Journal of Medicine, vol. 329, no. 5, pp. 304–309, 1993. 11 11 Computational and Mathematical Methods in Medicine [19] J. P. Ryckaert, G. Ciccotti, and H. J. C. Berendsen, “Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes,” Journal of Computational Physics, vol. 23, no. 3, pp. 327–341, 1977. [20] W. Humphrey, A. Dalke, and K. Schulten, “VMD: visual molecular dynamics,” Journal of Molecular Graphics, vol. 14, no. 1, pp. 33–38, 1996. [21] E. F. Pettersen, T. D. Goddard, C. C. Huang et al., “UCSF Chimera—a visualization system for exploratory research and analysis,” Journal of Computational Chemistry, vol. 25, no. 13, pp. 1605–1612, 2004. [22] D. Studio, version 2.0, Accelrys Inc., San Diego, Calif, USA, 2007.
https://openalex.org/W3204246644	https://www.frontiersin.org/articles/10.3389/fevo.2021.697511/pdf	English	null	Big Bird Plots: Benchmarking Neotropical Bird Communities to Address Questions in Ecology and Conservation in an Era of Rapid Change	Frontiers in ecology and evolution	2,021	cc-by	14,522	Big Bird Plots: Benchmarking Neotropical Bird Communities to Address Questions in Ecology and Conservation in an Era of Rapid Change W. Douglas Robinson1, Dan Errichetti1, Henry S. Pollock2, Ari Martinez3, Philip C Stouffer4,5, Fang-Yu Shen1 and John G. Blake6,7 1 Oak Creek Lab of Biology, Department of Fisheries, Wildlife and Conservation Sciences, Oregon State University, Corvallis, OR, United States, 2 Department of Natural Resources and Environmental Sciences, University of Illinois at Urbana-Champaign, Urbana, IL, United States, 3 Department of Biological Sciences, California State University, Long Beach, CA, United States, 4 School of Renewable Natural Resources, Louisiana State University AgCenter and Louisiana State University, Baton Rouge, LA, United States, 5 Biological Dynamics of Forest Fragments Project, Instituto Nacional de Pesquisas da Amazônia, Manaus, Brazil, 6 Department of Wildlife Ecology and Conservation, University of Florida, Gainesville, FL, United States, 7 College of Biological and Environmental Sciences, Universidad San Francisco de Quito, Cumbayá, Ecuador ORIGINAL RESEARCH published: 05 October 2021 doi: 10.3389/fevo.2021.697511 Extensive networks of large plots have the potential to transform knowledge of avian community dynamics through time and across geographical space. In the Neotropics, the global hotspot of avian diversity, only six 100-ha plots, all located in lowland forests of Amazonia, the Guianan shield and Panama, have been inventoried sufﬁciently. We review the most important lessons learned about Neotropical forest bird communities from those big bird plots and explore opportunities for creating a more extensive network of additional plots to address questions in ecology and conservation, following the model of the existing ForestGEO network of tree plots. Scholarly impact of the big bird plot papers has been extensive, with the papers accumulating nearly 1,500 citations, particularly on topics of tropical ecology, avian conservation, and community organization. Comparisons of results from the plot surveys show no single methodological scheme works effectively for surveying abundances of all bird species at all sites; multiple approaches have been utilized and must be employed in the future. On the existing plots, abundance patterns varied substantially between the South American plots and the Central American one, suggesting different community structuring mechanisms are at work and that additional sampling across geographic space is needed. Total bird abundance in Panama, dominated by small insectivores, was double that of Amazonia and the Guianan plateau, which were dominated by large granivores and frugivores. The most common species in Panama were three times more abundant than those in Amazonia, whereas overall richness was 1.5 times greater in Amazonia. Despite these differences in community structure, other basic information, including uncertainty in population density estimates, has yet to be quantiﬁed. Results from existing plots may inform drivers of differences in community structure and create baselines for detection of long-term regional changes in bird abundances, but supplementation of the small number of plots is needed to increase generalizability Edited by: Edited by: Umesh Srinivasan, Indian Institute of Science (IISc), India Edited by: Umesh Srinivasan, Indian Institute of Science (IISc), India Umesh Srinivasan, Indian Institute of Science (IISc), India Reviewed by: Andres Garcia, National Autonomous University of Mexico, Mexico William Ernest Magnusson, National Institute of Amazonian Research (INPA), Brazil Correspondence: W. Douglas Robinson douglas.robinson@oregonstate.edu Reviewed by: Andres Garcia, National Autonomous University of Mexico, Mexico William Ernest Magnusson, National Institute of Amazonian Research (INPA), Brazil *Correspondence: W. Douglas Robinson douglas.robinson@oregonstate.edu Specialty section: This article was submitted to Conservation and Restoration Ecology, a section of the journal Frontiers in Ecology and Evolution Received: 19 April 2021 Accepted: 10 September 2021 Published: 05 October 2021 Citation: Robinson WD, Errichetti D, Pollock HS, Martinez A, Stouffer PC, Shen F-Y and Blake JG (2021) Big Bird Plots: Benchmarking Neotropical Bird Communities to Address Questions in Ecology and Conservation in an Era of Rapid Change. Front. Ecol. Evol. 9:697511. Specialty section: This article was submitted to Conservation and Restoration Ecology, a section of the journal Frontiers in Ecology and Evolution Specialty section: This article was submitted to Conservation and Restoration Ecology, Received: 19 April 2021 Accepted: 10 September 2021 Published: 05 October 2021 INTRODUCTION Logistical issues have also hindered the establishment of benchmark tropical bird surveys. Recognition that most species, at least in tropical forests, occur at very low abundances, established a logistical hurdle because population densities of most species could only reasonably be estimated in large (suggested to be at least 100-ha) plots (Terborgh et al., 1990). Surveys of large plots require substantial and consistent sampling eﬀort that may not be feasible in tropical countries where obtaining funding for long-term research has been challenging (Barlow et al., 2018). In addition, most sites selected for such large plots have been placed in relatively low-elevation and accessible terrain to facilitate plot access. No large plots yet exist in mountainous terrain, which may bias perspectives and limit generalizations about the structure of Neotropical bird communities. The Neotropics is the global hotspot of avian diversity (Harvey et al., 2020), but its bird communities generally lack suﬃcient benchmark measurements of bird species composition and abundance. Establishment of such baselines provides the historical context required for eﬀective evaluation of change through time and is becoming increasingly important in a rapidly changing world (Magurran et al., 2010; McNellie et al., 2020). Patterns of abundance across species are a fundamental characteristic of any community, and the absence of such data presents a formidable impediment to advancement of ecological knowledge. The combination of abundance data and species traits also permits analysis of functional diversity and evaluation of ecosystem services (¸Sekercio˘glu, 2012). The need for foundational data from the world’s richest biomes can be remedied with solutions for the methodological and logistical challenges associated with thorough characterization of its rich and diverse communities. The current focus by tropical ornithologists on conservation of at-risk landscapes has probably also contributed to the lack of benchmark surveys in undisturbed forest (Robinson et al., 2004). Neotropical forests, for example, have been experiencing some of the fastest rates of deforestation and habitat conversion worldwide (Kim et al., 2015; Giam, 2017). Thus, most previous research has focused on conservation-relevant topics such as the impacts of fragmentation and habitat loss on Neotropical forest bird communities (Boyle and Sigel, 2015; Stouﬀer, 2020). Yet, the establishment of reliable biodiversity benchmarks from intact forests has provided the opportunity to quantify long-term, gradual changes in bird communities from relatively undisturbed areas. Citation: Robinson WD, Errichetti D, Pollock HS, Martinez A, Stouffer PC, Shen F-Y and Blake JG (2021) Big Bird Plots: Benchmarking Neotropical Bird Communities to Address Questions in Ecology and Conservation in an Era of Rapid Change. Front. Ecol. Evol. 9:697511. doi: 10.3389/fevo.2021.697511 October 2021 \| Volume 9 \| Article 697511 Frontiers in Ecology and Evolution \| www.frontiersin.org 1 Robinson et al. Benchmarking Tropical Birds on Plots of results and reveal the texture of geographic variation. We propose fruitful avenues of future research based on our current synthesis of the big bird plots. Collaborating with the large network of ForestGEO tree plots could be one approach to improve understanding of linkages between plant and bird diversity. Careful quantiﬁcation of bird survey effort, recording of exact locations of survey routes or stations, and archiving detailed metadata will greatly enhance the value of benchmark data for future repeat surveys of the existing plots and initial surveys of newly established plots. Keywords: Neotropical forests, bird community structure, biogeography, foraging guild, species richness, bird survey methods Frontiers in Ecology and Evolution \| www.frontiersin.org Cocha Cashu, Peru — The history of large plots used to study Neotropical bird communities is paradoxically both long and brief, being initiated in the 1980s but including only a few big plots surveyed, mostly, for short, discrete time periods (Table 1). Perhaps inspired by the North American network of plots surveyed by volunteers (Johnston, 1990), mostly via spot-mapping, Karr recognized the absence of similar data from the Neotropics (Karr, 1971). He established a 2-ha plot in Soberanía National Park, Panama, in the late 1960s, apparently creating the ﬁrst eﬀort to go beyond simple listing of species occurrences to estimation of abundances. Observing that, at least in Amazonia, most bird species were rare and had very large home-ranges, Terborgh et al. (1990) concluded that any plot-based eﬀort aimed at estimating population densities of most tropical forest bird species would have to be much larger. Consequently, they established the 97- ha Cocha Cashu plot near the Manú River in southeastern Peru. Completing surveys in the late 1980s, they discovered that, indeed, most species occurred at densities of less than 2.5 pairs/100 ha and had home ranges many times larger than the 2- ha Soberanía plot, validating the need for large plots. Even from surveys of nearly 100 ha, the Cocha Cashu plot was still too small to allow reliable estimates for a quarter of the species detected (Terborgh et al., 1990), but nearly all of those were assumed to be quite rare or transients. Furthermore, they realized that multiple methods were needed to survey Neotropical birds given their tremendous ecological and life-history diversity, often low rates and amplitudes of vocalizations, and variation in detectability (Robinson et al., 2000b, 2018). Cocha Cashu, Peru — Situated alongside an oxbow lake for which it is named, Cocha Cashu Biological Station was established in 1968 within the meander belt of the Manú River in southeastern Peru. Consequently, the vegetation throughout the meander belt represents a mix of successional stands reﬂecting the periodic ﬂooding dynamics of the river. The 97-ha plot itself is in a mature ﬂoodplain forest that remains above the normal annual ﬂooding level of the river. The plot is surrounded by the oxbow lake, ﬁg swamps, and is contiguous to a larger tract of mature ﬂoodplain forest. An extensive trail system became the basis for a study grid system which encompasses the original 97-ha plot and has expanded to approximately 10 km2. INTRODUCTION As a guide to the potential of creating a larger network of bird plots, we look to the extensive conceptual advances created through the global October 2021 \| Volume 9 \| Article 697511 Frontiers in Ecology and Evolution \| www.frontiersin.org Frontiers in Ecology and Evolution \| www.frontiersin.org 2 Benchmarking Tropical Birds on Plots Robinson et al. TABLE 1 \| Summary of the ∼100-ha plots used to characterize Neotropical bird communities, including their locations, basic environmental characteristics, geographic coordinates, range of years sampled, and species richness. TABLE 1 \| Summary of the ∼100-ha plots used to characterize Neotropical bird communities, including their locations, basic environmental characteristics, geographic coordinates, range of years sampled, and species richness. Source Plot Location Coordinates Elevation (m) Annual rainfall (mm) Years sampled Species richness Terborgh et al., 1990 Cocha Cashu Manú National Park, Peru 11◦54′ S, 71◦18′ W 400 2,000 1982, 2018 319 Robinson et al., 2000b Limbo Soberanía National Park, Panama 9◦9′ N, 79◦44′ W 35–80 2,600 1994–1996 252 Thiollay, 1994 Nouragues French Guiana 4◦5′ N, 52◦41′ W 40–400 3,500 1990–1992 248 Blake and Loiselle, 2015, 2016 Tiputini (2 plots) Yasuní Biosphere Reserve, Ecuador 0◦37′ S, 76◦10′ W 190–270 3,100 2001–2020 318, 320 (349 total) Johnson et al., 2011 Manaus Brazil 2◦30′ S, 60◦0′W 110–150 2,714 2006–2008 228 the basic measurements of richness and abundance with species traits opened the door to test for diﬀerences in functional diversity and community structure between tropical and temperate bird communities. The idea that 100 ha is a useful plot size in which to study tropical forest birds gained traction and led to the eventual establishment of analogous plots in French Guiana (1986), Panama (1994), Ecuador (2001), and Brazil (2008) to improve and evaluate the generalizability of the Cocha Cashu results, as well as address other particular questions of interest detailed in the individual plot histories below. network of tree plots established by the ForestGEO group (Davies et al., 2021). We then connect the potential gain in knowledge from expanding the network of big bird plots with the challenges of establishing such a network. Finally, we identify and address challenges that need to be addressed if we are to reap potential beneﬁts of an expanded network of tropical bird plots. INTRODUCTION For example, even in remote Tiputini, Ecuador, and Brazilian Amazonia, evidence for subtle changes in the avifauna over the last two decades, perhaps driven by climate change, has been found (Blake and Loiselle, 2015; Stouﬀer et al., 2021). Methodological challenges have included basic aspects of species identiﬁcation in diverse and poorly studied communities, a lack of standardized counting protocols, and issues accounting for interspeciﬁc variation in detectability, which can impede accurate estimation of species’ abundances (Banks-Leite et al., 2014). Reasonably complete community inventories require reliable information on taxonomy, which has greatly improved in the last few decades despite a growing appreciation that many cryptic species continue to lack formal recognition (Bickford et al., 2007). Current information, however, is certainly suﬃcient to produce reliable identiﬁcations for most species. Our abilities to identify birds by their vocalizations have greatly improved recently with the proliferation of freely available online sound recording archives (https://www.xeno-canto.org/ and eBird.org). Many Neotropical bird species inhabit structurally complex habitats such as forests, and are more often heard than seen, so detection from vocal cues is critical during community surveys (Celis-Murillo et al., 2012). Furthermore, even when vocalizations are learned exhaustively, low vocalization rates and population densities may hamper detectability of certain species and impede accurate abundance estimation (Anderson et al., 2015). As new techniques for handling sampling diﬃculties continue to be developed, we anticipate wider application of modern approaches to estimate detectability and generate reliable estimates of population density (e.g., Gómez et al., 2018). Here, we review the history of the six big bird plots surveyed in the Neotropics (Table 1). We summarize the motivations for establishment of these plots, the primary methodological approaches used to inventory Neotropical forest bird communities, as well as the key ecological questions addressed from the resultant datasets. After comparing the major ﬁndings from the plots, we brieﬂy evaluate and interpret diﬀerences in community structure and organization. Because scope of inference has been limited by the small number of big plots in exclusively lowland forests, we suggest that a more extensive network of plots is both needed and feasible. Frontiers in Ecology and Evolution \| www.frontiersin.org Cocha Cashu, Peru — The trail system was converted into a grid by mapping trail markers every 25 m along all trails. At the time of the establishment of the plot, ornithological knowledge was on the cusp of allowing reliable identiﬁcation of most Neotropical bird species by sight and sound. The development of these identiﬁcation criteria, which had long been a cornerstone of studies in temperate systems, allowed for the incorporation of survey techniques such as spot-mapping to evaluate the organization and structure of rich Neotropical bird communities. Multiple techniques were employed to overcome the challenges of counting the variety of species with diverse life histories and behaviors, which included traditional methods such as spot-mapping, and also employed mist-netting and visual counts to estimate the density of ﬂocking and colonial species. Even some radio-tracking was implemented to map territories of woodcreepers. Based upon survey work of forest surrounding the Cocha Cashu plot in which just three additional species were detected, Terborgh et al. (1990) estimated that the plot The successful accomplishment of a nearly complete inventory of species alongside population density estimates allowed assessment of guild structure and biomass distribution. Linking October 2021 \| Volume 9 \| Article 697511 Frontiers in Ecology and Evolution \| www.frontiersin.org 3 Benchmarking Tropical Birds on Plots Robinson et al. encompassed 99% of the bird community. It should be noted, however, that although the plot was estimated to be large enough to detect 99% of the bird community, this was still not adequate for estimating population density for a quarter of the species. encompassed 99% of the bird community. It should be noted, however, that although the plot was estimated to be large enough to detect 99% of the bird community, this was still not adequate for estimating population density for a quarter of the species. Collectively, J. L. Dujardin and M. Jullien mapped 6,658 individuals comprised of 248 resident species on the 100-ha plot (Thiollay, 1994). 220 species had a density of ≥0.50 pair/100 ha and 157 species had a density of ≥1 pair/100-ha. The estimated density on the entire plot was 829 pairs/100 ha (about 1,658 individuals), quite similar to the 1,910 individuals/100 ha at Cocha Cashu (Terborgh et al., 1990). According to Thiollay (1994), 441 resident species occurred in the 80,000 km2 of the interior primary forest of French Guiana. Of these, more than half (58%) had an average density under one pair/100 ha. Nouragues, French Guiana — g The Nouragues ﬁeld station, established in 1986, was located in continuous primary lowland rainforest of the Eastern Amazonian interior of French Guiana. The Nouragues bird plot was located deep in forest interior to facilitate bird population and behavioral studies, as well as to provide a comparison of community structure with the Cocha Cashu plot. Standardized surveying commenced at the 100-ha plot in 1990 (Thiollay, 1994). The plot’s location was designed to avoid habitat-edge eﬀects, but an eﬀort to describe internal spatial heterogeneity created by treefall gaps was included. On a central, 24-ha subplot, 78 treefall gaps that accounted for 3.7% of the area were mapped in 1991, one of the primary years of the bird counting work. Spot- mapping and mist-netting were the primary approaches used to generate population density estimates during 8 months of surveys between February 1990 and November 1992. The plot was systematically searched from dawn to dusk to map residents using a grid of 1-ha quadrats. A subset of 1-ha quadrats were scrutinized in nine 33 m × 33 m sub-squares to facilitate territory mapping and estimation of typical territory sizes. Mist-netting took place within a core 24-ha quadrat in September 1991 and March and October 1992. Twenty 12-m mist nets were placed along seven 400-m parallel trails and operated for 5880 net-hours resulting in 694 marked individuals. Data were supplemented by an earlier study (5 years of eﬀort) where 1,353 mist-netted birds of 99 species were followed and spot-mapped. Supplementary surveys such as the use of acoustic playback, nest locations, nocturnal surveys, radio-telemetry, color-band resighting and canopy observations were conducted to quantify community composition and home-range sizes. Limbo, Panama The Limbo plot (104 ha), located in Soberanía National Park on the isthmus of central Panama, was established in 1994 following scouting for site placement in 1993 (Robinson et al., 2000b). The site had a prior history of mist-netting studies extending back to the 1960s. Those eﬀorts were centered at the Limbo Hunt Club, a former camp site and small cabin used by military personnel during hunting trips along the Pipeline Road (Karr, 1971). A 2-ha plot was established at the site in 1968 by Karr (1971), following the success of similarly sized plots in measuring bird community structure across North America (e.g., Short, 1979). The community was studied via mist-netting and mapping observations of color-marked birds to estimate territory sizes and densities. Cocha Cashu, Peru — The species found at Nouragues included 77% (234/305) of the most forest- restricted species of the region. The community was dominated by two species that had 28 and 38 pairs on the plot and ten “subdominant” species with 14–18 pairs in the 100 ha. Those dozen species made up 31% of the estimated bird density on the plot. Deﬁning rare species as those with <2 pairs/100 ha, Thiollay (1994) suggested the Nouragues plot had 137 rare species representing every guild, family, and ecological niche of the region; 64 were species considered to occur at densities <1 pair/100 ha. By deﬁnitions used in Terborgh et al. (1990), 37% of the 248 species were rare (≤1 pair/100 ha), including species of large body mass with large home ranges as well as patchily distributed species. Distributions on the Nouragues plot were often patchy, also a characteristic of birds in Cocha Cashu, Peru (Terborgh et al., 1990). Thiollay (1994) hypothesized that the local absences from the Nouragues plot of some common French Guianan species could be attributed simply to patchiness of distributions. Overall, the community structure was remarkably similar to that at Cocha Cashu. In sum, the original survey included >15,000 spot-map registrations which yielded an average of ∼15 detections per territory. The use of mist-nets on the plot was employed on 6 separate mist net lines and captured ∼755 diﬀerent birds representing ∼80 species. This was supplemented by focal studies on Yellow-rumped Caciques (Cacicus cela) which yielded greater than 1,000 color-banded individuals (Robinson, 1986), and 1,173 individual group counts of monospeciﬁc ﬂocks of parrots. Combining all these diﬀerent survey eﬀorts yielded 1,920 birds/100 ha. In total, 319 species were detected and density estimates were derived for 245 species that held territories on the plot. Most species tended to be somewhat rare, with a median of 2.5 pairs/100 ha. Among foraging guilds, insectivores had the highest species richness but accounted for the smallest fraction of overall biomass (18%), whereas granivores comprised the largest portion of biomass (43%) of any given foraging guild. Thus, as the ﬁrst exhaustive survey of a Neotropical bird community at a relatively large spatial scale, the results showed the exceptional richness, domination of the community rank-abundance curve by a long tail of rare species, concentration of most avian biomass among granivores and frugivores, and a large diversity of lifestyles requiring implementation of multiple survey techniques. Frontiers in Ecology and Evolution \| www.frontiersin.org Nouragues, French Guiana — The points were surveyed 8 times each in 1994–1996 to facilitate density measurements and also to establish an easily repeatable survey method to allow future re-surveys. Extensive mist-netting to color-mark birds was conducted largely by two observers at 8 diﬀerent routes across the plot. Point count data and mapping of color-marked bird observations were used to spot-map, along with discoveries of nests for a subset of species (Robinson et al., 2000a,c), then clusters were identiﬁed to enumerate density. For wide-ranging and patchily distributed species, transects and encounter rates were used to estimate densities. We took two approaches to sample the birds: mist nets and visual observations. Mist nets (12 × 2.6 m, 36-mm mesh) set at ground level were arranged in a series of eight sets of 12 nets on each plot (96 sites per plot). Each set of 12 nets formed a rectangle (100 × 200 m) with nets set ∼50 m apart; maximum distance between nets on a plot was approximately 920 m. Each set of nets was run for one day (∼0600–1200 h) in January (peak of breeding for many species) and one day in March (late breeding season for many species), starting in March 2001. March samples have largely been discontinued during the last few years, primarily because heavy rains precluded netting. Captured birds were identiﬁed and most were banded with aluminum leg bands. Most manakins were also marked with color bands. Blood samples were collected from many species during the ﬁrst years of the project and were used to investigate occurrence of blood parasites (e.g., Svensson-Coelho et al., 2014). Altogether, more than 30,000 bird observations were mapped on the 104-ha Limbo plot, representing 252 species. Of those, 152 resident species were present in densities of at least 0.5 pairs/100 ha. The original 2-ha plot results suggested that the Panama and Amazonian bird communities were structured quite diﬀerently, a conclusion conﬁrmed by the 104-ha plot results. The Limbo community was dominated by eight very common species whose abundances were several times greater than the most common species at the Cocha Cashu and Nouragues plots. To obtain a more complete picture of the community, JGB has conducted transect observations that started in 2005. Locations of all birds seen or heard were noted on scale maps of each plot while walking along transects; unknown songs were recorded for later identiﬁcation. Nouragues, French Guiana — The station and nearby areas are dominated by terra ﬁrme forest and also include várzea forest, palm swamps, and various successional habitats. Two 100-ha plots (ca 1 km × 1 km each) were established in terra ﬁrme forest during 2001. Plots are approximately 1.5 km apart at the closest point. Both plots are gridded (100 × 200-m grid lines) and marked with 1.5-m PVC tubes. The Harpia plot is characterized by more dissected upland forest while the Puma plot is ﬂatter overall. Both areas experience partial inundation when small streams back up as the Tiputini River rises; Puma has more areas that ﬁll with persistent standing water during the rainy season. Dominant vegetation on both plots is tall, evergreen forest. The 104-ha plot was positioned to overlap the original 2-ha plot. It encompassed tropical moist forest ranging in age from 250 or more years old (largely on and near the original plot) to less than 15 years old along the margins of Pipeline Road and at large treefall gaps created by windstorms. Aside from the one- lane unimproved road passing through it, the plot was more than 3.5 km from edges at the eastern park boundary and Gatun Lake. Its permanently ﬂowing Rio Limbo, a small creek arcing through the northern and eastern portions of the plot, attracted some riparian species. Otherwise, most of plot was terra ﬁrme forest in a relatively ﬂat basin. We had several diﬀerent objectives for long-term studies. At the most basic level, we wanted to investigate spatial patterns of species distribution at within-and-between-plot scales and how those patterns might change over time. By employing capture-mark-recapture analyses we wanted to be able to estimate survival rates for a diverse set of species (Blake and Loiselle, 2008, 2013). A second major focus was on behavior, spatial distribution, genetic relatedness, and seed dispersal by manakins (family Pipridae; e.g., Loiselle et al., 2007; Blendinger et al., 2008, 2011; Ryder et al., 2009), with the majority of the studies based on the two plots. The Limbo plot was constructed of north-south transects spaced at 100-m intervals and three east-west transects, one each at the northern and southern plot boundaries and one through the plot center. Surveys were conducted largely by one observer (WDR) who walked each transect, stopping every 100 m to conduct 8-min point counts where the direction and distance of each bird was noted. Nouragues, French Guiana — Richness on the original 2-ha Limbo plot was 140 resident species (Karr, 1971). Extrapolation of abundances to 100-ha suggested a combined density of 1,800 pairs. The richness and density values per 100 ha were eventually questioned when the Cocha Cashu plot, nearly 50 times larger (97 ha), discovered much diﬀerent community structure, with many more rare species and lower maximum abundances. Importantly, territory sizes of most Amazonian species averaged 4.5 ha, more than twice the size of the original Limbo plot (Terborgh et al., 1990). The Cocha Cashu results, therefore, suggested the Limbo 2-ha plot may have been too small to adequately characterize the community if species were too wide-ranging or if their distributions were too patchy, as reported from French Guiana (Thiollay, 1994), to be sampled by a small plot. Because of October 2021 \| Volume 9 \| Article 697511 4 Benchmarking Tropical Birds on Plots Robinson et al. these concerns and how they might inﬂuence interpretation of geographical diﬀerences in community structure, a larger plot was established at Limbo (104 ha) to facilitate fairer comparisons with South American plots. In addition to increasing plot size, a wider variety of survey methods, focused on extensive spot- mapping of territories, was also used. Thus, the primary questions posed by Robinson et al. (2000b) were: (1) Are results from the original 2-ha Limbo plot inﬂuenced by the small spatial scale of the initial study? (2) Is organization of the Panama bird community fundamentally diﬀerent from communities in Amazonia and, if so, why? these concerns and how they might inﬂuence interpretation of geographical diﬀerences in community structure, a larger plot was established at Limbo (104 ha) to facilitate fairer comparisons with South American plots. In addition to increasing plot size, a wider variety of survey methods, focused on extensive spot- mapping of territories, was also used. Thus, the primary questions posed by Robinson et al. (2000b) were: (1) Are results from the original 2-ha Limbo plot inﬂuenced by the small spatial scale of the initial study? (2) Is organization of the Panama bird community fundamentally diﬀerent from communities in Amazonia and, if so, why? surrounded by large expanses of forest, and reasonably accessible, where we could establish two replicate 100-ha plots, something that had not been done at other sites. Two plots provide the opportunity to compare community composition at a relatively small spatial scale. Nouragues, French Guiana — Approximately 1–1.4 km of transects were covered during a morning with starting positions distributed throughout the plots. Each plot took ∼12–13 days to cover. Transects covered the entire plot but were not repeated during a given sample, precluding the more traditional spot-map analyses. From 2013 to 2017, passive acoustic monitors were deployed on both plots to evaluate their eﬀectiveness as a sampling tool (Blake, 2021). Results from the recordings were compared to transect counts conducted during the same periods. Finally, a long-term Tiputini, Ecuador — Tiputini Biological Station (TBS) is located adjacent to Yasuní National Park and within Yasuní Biosphere Reserve, one of the most diverse regions of the world (Bass et al., 2010). Although TBS itself is only ∼700 ha, it is surrounded by extensive areas of intact forest. An initial site visit was made to Tiputini Biodiversity Station (TBS) in 2000 to determine the feasibility of establishing long-term study plots. The goal was to ﬁnd a site that was diverse, October 2021 \| Volume 9 \| Article 697511 Frontiers in Ecology and Evolution \| www.frontiersin.org 5 Benchmarking Tropical Birds on Plots Robinson et al. camera trapping project has provided additional information on some of the larger, terrestrial species, such as tinamous (family Tinamidae) and trumpeters (family Psophidae; Blake et al., 2017). provided a research base for projects in unfragmented forest. At a larger spatial scale, deforestation mostly ceased by the late 1980s, with the overall BDFFP area remaining ∼90% forested to the present, maintaining unbroken connection to vast rainforest with minimal disturbance (Rutt et al., 2019). a dae) a d t u pete s ( a y sop dae; a e et a ., 0 7). To date (2001–2020), 180 species have been captured in mist nets (16,883 captures) on the two plots combined, including 160 on Harpia (8307 captures) and 155 on Puma (8576 captures). A total of 336 species have been detected during transect counts, with 302 on Harpia (34,249 records) and 304 on Puma (29,719 records). With both captures and observations combined, 320 species have been recorded on Harpia, 318 on Puma, and 349 with both plots combined. Patterns of species accumulation, capture rates, and observation rates are generally similar on both plots. Family and overall species composition also are similar on the two plots; the same species are the most dominant on both plots (Blake, 2007; Blake and Loiselle, 2009). Despite the overall similarities, many species showed diﬀerences in abundance (captures or observations) across the plots with diﬀerences often related to small-scale variation in topography and habitat between the plots. For example, Screaming Piha (Lipaugus vociferans) has a large lek on the Harpia plot but is absent from Puma. In contrast, Wire-tailed Manakin (Pipra ﬁlicauda) is common on Puma but has no leks on Harpia. Tiputini, Ecuador — Comparisons with other sites (e.g., Cocha Cashu, French Guiana, Panama) showed stronger similarities between Cocha Cashu than with other sites, particularly Panama (Blake and Loiselle, 2009). Several projects at KM41 set the stage for the big-plot survey. Standardized mist-netting, beginning in the late 1980s, contributed to the BDFFP bird capture database. We described mixed-species ﬂock structure and space use (Develey and Stouﬀer, 2001). We spot-mapped and radio-tagged terrestrial insectivores over 10 years (Stouﬀer, 2007). In the days before GPS could provide accurate locational information under the rainforest canopy, the accuracy of spatial data for these studies hinged on the 100 m × 100 m trail grid. During work at KM41 and elsewhere at the BDFFP we identiﬁed criteria for aging birds and assembled audio recordings of almost all bird species, eventually producing two important resources for the big plot survey (Naka et al., 2008; Johnson and Wolfe, 2017). In 2008, we had accumulated the necessary experience and resources to conduct a community-wide survey at the scale of a 100-ha plot, with metrics that could be compared to the handful of other studies that estimated space use and absolute abundance (Terborgh et al., 1990; Thiollay, 1994; Robinson et al., 2000b). Our objectives were to determine: species richness; density, biomass, and territory size of individual species; and the distribution of these metrics by foraging guild (Johnson et al., 2011). Plots at Tiputini have been surveyed annually since 2001, something that has not been done at the other big plots. As such, we have a more detailed picture of temporal ﬂuctuations in bird numbers than at the other plots. Capture rates and observations ﬂuctuated over the ﬁrst years – 2001–2009 – but showed no consistent pattern of change. Since 2009, however, captures and observations have declined by approximately 50% on both plots, in the absence of any change in local anthropogenic inﬂuences (e.g., logging, hunting) (Blake and Loiselle, 2015, 2016). Declines have occurred across many guilds and species, with insectivores particularly hard-hit. Some terrestrial insectivores (e.g., Formicarius antthrushes, Sclerurus leaftossers) largely disappeared for some years, although numbers have increased slightly in the last few years. Initial declines coincided with some strong La Niña events, which bring periods of heavy rains. Despite a lack of such strong events in the last few years, numbers of captures and observations have remained low. Manaus, Brazil— Research with birds has been an integral part of the Biological Dynamics of Forest Fragments Project (BDFFP) since the project began in 1979 (Stouﬀer, 2020). The original intent of the BDFFP was to follow biological processes, including bird communities, in fragments of rainforest that would be isolated by agricultural development in an area of undisturbed rainforest about 80 km north of Manaus, Amazonas, Brazil (Bierregaard et al., 2001). During the 1980s, research fragments were isolated, and the BDFFP established a control site of continuous forest at KM 41 of the ZF3 road, at the far eastern end of the BDFFP, connected to nearly unbroken forest to the north and east. With the gradual construction of what became a permanent camp and some 500 ha of continuous forest gridded with 100 m × 100 m trails, KM41 Tiputini, Ecuador — The plot was within the gridded network at KM41, in an area that we considered representative terra ﬁrme forest. As is typical at the BDFFP, the topography included steep ascents and descents along old stream beds. One small stream passed through the plot. We sampled with mist nets from June to November 2008, with spot-mapping concentrated in June and July. Our methods for spot-mapping and interpretation of spot-map data were generally concordant with Terborgh et al. (1990). We improved abundance estimates for three of the most common species in the mist-net sample by estimating density of adults. Color-banded birds and radio-tagged birds helped us discover territory boundaries. Overall, we recorded 5,581 unique observations (sometimes of multiple individuals, as of a pair together or a monospeciﬁc ﬂock) of 228 species and found community structure to be very similar to that at Cocha Cashu. Lower species richness compared to other Amazonian plots can be mostly attributed to the homogeneity of the surrounding landscape, which precluded the wandering birds that contribute to species richness without being part of the core resident avifauna (Johnson et al., 2011). Frontiers in Ecology and Evolution \| www.frontiersin.org Inﬂuence in the Scientiﬁc Literature To explore inﬂuence of the plot studies on the scientiﬁc literature, we examined citation patterns by compiling all sources citing the bird plot papers from Web of Science and utilizing Vos Viewer (van Eck and Waltman, 2010) to conduct network analyses of keywords. For each citation, we also extracted metadata including the year of publication, whether or not the citing source collected data from the same geographic location (binary yes vs. no), the range of latitudes at which the study was conducted (temperate, tropical, subtropical, global), the research theme (biogeography, conservation, ecology, evolution, natural history), and the taxa studied. Across the six plots, ﬁfty-four bird families were recorded. Cocha Cashu, Peru, had the highest species richness, followed by the two Tiputini plots, Nouragues, Limbo and Manaus (Table 1). Migrant species were minor components of most communities except for Limbo (Figure 1). Overall, plots were quite similar with respect to the number of species per family, with a few notable exceptions (Supplementary Figure 1). At Cocha Cashu, species richness of certain families was disproportionately high, including ovenbirds (Furnariidae), antbirds (Thamnophilidae), parrots (Psittacidae), and tinamous (Tinamidae). Conversely, at the only Central American plot (Limbo, Panama), species richness was disproportionately low for ovenbirds, antbirds and parrots. With respect to diet, Cocha Cashu had more granivores and insectivores than the other plots, whereas Panama had the most omnivorous species (Supplementary Table 1). Through the end of 2020, the six big bird plot papers had been cited 1,443 times (Google Scholar, accessed 18 Feb 2021). Overall, citations peaked between 2005 and 2010. Citations of the Cocha Cashu paper, in particular, have declined in recent years. The primary inﬂuences of the papers included topics focused on ornithology, ecological, biogeographical, and conservation- related themes. The papers have had extensive inﬂuence on speciﬁc topics ranging from forest fragmentation to community dynamics and from community organization to frugivory and dispersal (Figure 3). Studies citing the papers have focused almost exclusively on birds and been largely conducted at tropical latitudes. Most of the citing research took place at diﬀerent study sites than the original studies. Importantly, most plots have also spawned numerous additional studies conducted on the plots, illustrating the value of providing logistical access for researchers and advancing scientiﬁc knowledge more broadly. Inﬂuence in the Scientiﬁc Literature Aside from the present collection of big bird plots being developed at least partly as a consequence of establishment of the Cocha Cashu plot, we did not see evidence that publication of the plot results has spawned production of additional tropical bird plots despite the strong citation rates. In addition, while most of the big bird plot publications interpret results in the context of ﬁndings from the other plots, no eﬀort to analyze in depth the results across plots or coordinate development of additional plots has emerged. Therefore, looking to other plot networks, such as the global collection of tree plots, could provide a useful guide. p pp y Population density distributions varied substantially among the plots (Figure 2). Limbo supported an average density of 7 versus 2.5 pairs/100 ha at the Amazonian plots. Total number of birds was estimated to be 3,230/100 ha at Limbo, nearly twice the densities in Amazonia. Yet, the total bird biomass was quite similar. Distributions of body masses across size categories were also remarkably similar across the four plots for which body mass data were available and pairwise comparisons did not reveal any signiﬁcant diﬀerences among the plots (Kolmogorov- Smirnov tests, all p > 0.05; Supplementary Figure 2). Body size distributions favored many small insectivores at Limbo, versus larger granivores and frugivores in Amazonia. Collectively, the South American sites were most similar to one another while the structure of the Panama community exhibited lower richness and higher number of migratory species. Diﬀerences in species identities across the plots are obvious as are diﬀerences in richness and abundance values, suggesting diﬀerent mechanisms structuring the communities. But, similarities in body mass distributions, in particular, suggest that some mechanisms determining community structure are common to all the plots. The biogeographic and human history of the Panamanian isthmus might be responsible for the diﬀerences between Limbo and the Amazonian plots owing to disturbances associated with changing sea levels and human alteration of forest structure and, to some extent, hunting of large- bodied birds. However, the small number of plots limits our ability to evaluate hypotheses rigorously. Furthermore, the Community-Level Comparisons The common theme across the Neotropical big plot studies was to characterize the species composition in the bird communities as fully as possible, and for most of them, to estimate plot-level densities of as many species as possible October 2021 \| Volume 9 \| Article 697511 Frontiers in Ecology and Evolution \| www.frontiersin.org 6 Benchmarking Tropical Birds on Plots Robinson et al. using a variety of methods (Supplementary Table 1). We explored diﬀerences in community structure across the plots by compiling data on a suite of ecological traits including categorical (taxonomic family, migratory tendency and diet) and continuous variables (average body mass, population density; Supplementary Table 2). Population densities and body masses were unavailable for the Tiputini plots. To identify major diﬀerences across the plots in our categorical variables, we visually compared cross-plot diﬀerences using bar charts. For continuous variables, we compared distributions across the plots using pairwise Kolmogorov-Smirnov tests. We used a Bonferroni correction to generate adjusted p-values that corrected for multiple pairwise comparisons among the four plots. We used logarithmic transformations to normalize distributions of body masses and densities. degree to which potential errors in estimating densities may inﬂuence determination of community-level abundance patterns cannot yet be explored because of inherent limitations in survey methodologies. FOREST GLOBAL EARTH OBSERVATORY (FORESTGEO) FOREST DYNAMICS PLOTS AS A MODEL A network of tropical tree plots began with the Hubbell- Foster 50-ha plot on Barro Colorado Island, Panama, in 1980. Motivations for creation of that plot were to generate horizontal life table data on tropical trees, quantify change so as to test competing equilibrium and non-equilibrium hypotheses for the creation and maintenance of species diversity, and to map individual trees to facilitate additional research by collaborators (Anderson-Teixeira et al., 2015; Davies et al., 2021). Within the October 2021 \| Volume 9 \| Article 697511 Frontiers in Ecology and Evolution \| www.frontiersin.org 7 Robinson et al. Benchmarking Tropical Birds on Plots FIGURE 1 \| Species richness of migrants and residents at six of the big Neotropical bird plots. FIGURE 1 \| Species richness of migrants and residents at six of the big Neotropical bird plots. data sharing agreements, as well as analyses through freely sharing R code, was implemented. These steps moved scientiﬁc knowledge gained from a collection of case studies, examining data from one plot at a time, to examination of emergent patterns across many sites. By creating the network, more generalizable conclusion about the drivers of species coexistence, creation and maintenance of species diversity, and factors inﬂuencing ecological function of tropical forests were within reach. The incorporation of data from many plots led to creation of new ideas such as neutral theory (Hubbell, 2001), the relative impacts of density-dependence on recruitment (Comita et al., 2010), the inﬂuence of pathogens on diversity and increasingly robust assessments of the intermediate disturbance hypothesis (Wills, 2006; LaManna et al., 2017). We suggest that the ForestGEO network should serve as a model for the development of a pantropical network of big bird plots, creating opportunities to discover mechanisms inﬂuencing the structure of tropical bird communities and to identify factors responsible for long-term drivers of diversity change (Blake and Loiselle, 2015; Stouﬀer et al., 2021). next decade similar plots were established in Malaysia and India. Today, the network includes 71 plots ranging in size from 4 to 120 ha (average of 26 ha) with every individual of more than 7 million trees mapped and identiﬁed to species or morphospecies. Frontiers in Ecology and Evolution \| www.frontiersin.org FOREST GLOBAL EARTH OBSERVATORY (FORESTGEO) FOREST DYNAMICS PLOTS AS A MODEL Two potential challenges might reduce collaborative opportunities. First, any disturbance of plants by ornithological activities, particularly mist-netting that involves clearing of plants or trampling by repeatedly walking the same routes across plots, could negatively inﬂuence plant communities, but carefully constructed collaborative agreements should minimize such challenges. Second, most ForestGEO plots are 50 ha or less (averaging 26 ha), thus are smaller than the current notion of an ideal size for sampling tropical birds (100 ha). However, no critical analysis of optimal tropical bird plot sizes has yet to be conducted. Depending on the questions of interest, more 50 ha plots might be better solutions than fewer 100 ha plots. Current data do indicate that bigger plots sample more species and provide more opportunities to characterize space use (territory sizes) for a greater percentage of the bird community. If, however, primary goals are to benchmark smaller fractions of each community for tracking change in numbers through time, to link bird and plant community data, and to connect bird diversity data with other forms of habitat data (e.g., satellite observations) and predict distributions across geographic space, then smaller plots can be useful. As the ForestGEO network has demonstrated, many research questions can be addressed within single large plots, whereas other questions may be addressed with data gathered from collections of much smaller plots (Condit et al., 2012). Thus, the questions drive and are inﬂuenced by the details of sampling designs. challenges of detecting and attempting to count them accurately can be formidable (Robinson et al., 2018). Nevertheless, we argue that creation of a network of big bird plots, which expands beyond the existing six plots, could provide new insights into tropical, avian and conservation ecology. Formally establishing collaborative agreements so that birds can be sampled on the existing ForestGEO plots could quickly produce ecological insights regarding the roles of birds in tropical forests, especially the linkages between bird and plant diversity. Because plants are identiﬁed and mapped, and in many locations their phenological patterns, herbivorous consumers and fruit production patterns are quantiﬁed, opportunities to measure roles of birds as seed dispersers and in control of herbivorous insects are unprecedented. Two potential challenges might reduce collaborative opportunities. Frontiers in Ecology and Evolution \| www.frontiersin.org FOREST GLOBAL EARTH OBSERVATORY (FORESTGEO) FOREST DYNAMICS PLOTS AS A MODEL Two simple, but logistically challenging, innovations of Hubbell and Foster catalyzed rapid change in our understanding of tropical forest dynamics: (1) establishing plots suﬃciently large to sample most of the tree species present and to contain reasonable sample sizes of most rare species; and (2) including individual trees down to 1 cm diameter at breast height. Previous tree plot studies were normally 1 ha or less and measured only trees 10 cm dbh or greater, missing a substantial portion of species diversity present and thereby inhibiting abilities to provide robust tests of hypotheses about species diversity and to quantify tree life table parameters. The collaborative eﬀorts of hundreds of scientists in the ForestGEO network have produced nearly 1,400 scientiﬁc publications, including dozens in top-tiered journals that have addressed fundamental ideas in ecology, global change, evolution, and forest management (Ashton et al., 1999; Losos and Leigh, 2004). To facilitate collaboration, standardization of protocols for data collection and management, metadata, Would the ForestGEO model be eﬀective for birds? Trees stay where you leave them. The mobility of birds and the October 2021 \| Volume 9 \| Article 697511 8 Robinson et al. Benchmarking Tropical Birds on Plots FIGURE 2 \| Population density (log 2 intervals) across species in the four Neotropical bird communities where density was estimated. The South American sites (Cocha Cashu, Manaus, and Nourages) are largely similar but quite different from the Central American site (Limbo). FIGURE 2 \| Population density (log 2 intervals) across species in the four Neotropical bird communities where density was estimated. The South American sites (Cocha Cashu, Manaus, and Nourages) are largely similar but quite different from the Central American site (Limbo). challenges of detecting and attempting to count them accurately can be formidable (Robinson et al., 2018). Nevertheless, we argue that creation of a network of big bird plots, which expands beyond the existing six plots, could provide new insights into tropical, avian and conservation ecology. Formally establishing collaborative agreements so that birds can be sampled on the existing ForestGEO plots could quickly produce ecological insights regarding the roles of birds in tropical forests, especially the linkages between bird and plant diversity. Because plants are identiﬁed and mapped, and in many locations their phenological patterns, herbivorous consumers and fruit production patterns are quantiﬁed, opportunities to measure roles of birds as seed dispersers and in control of herbivorous insects are unprecedented. FOREST GLOBAL EARTH OBSERVATORY (FORESTGEO) FOREST DYNAMICS PLOTS AS A MODEL Keywords occurring ﬁve or more times in citing literature are included. Colors indicate major associations of co-occurring keywords. these plots can then be used to calibrate abundance estimates derived from point counts, which can be more easily replicated consistently across larger temporal and spatial scales, also then providing opportunities to precisely repeat surveys and monitor temporal changes. Historically, point counts have been limited by diﬃculties detecting and estimating abundances of social species and rare species, in particular (Robinson et al., 2018). The diﬀerences in estimates between spot-mapping and point counts can be used to calibrate the detection probabilities used in statistical models. Furthermore, with the development of N-mixture models which take into account both probabilities of detection and abundance (Gómez et al., 2018), assumptions can be built into the models which will allow for the estimation of densities of rarely detected species. If a goal is reliable estimation of population densities for all or nearly all species occurring on plots, then utilizing combinations of methods, and those currently under development, will improve quantity and quality of data available for analyses. extension of collaborations with the existing ForestGEO plots, should involve collaborations with local experts as foundational aspects of the work. FOREST GLOBAL EARTH OBSERVATORY (FORESTGEO) FOREST DYNAMICS PLOTS AS A MODEL First, any disturbance of plants by ornithological activities, particularly mist-netting that involves clearing of plants or trampling by repeatedly walking the same routes across plots, could negatively inﬂuence plant communities, but carefully constructed collaborative agreements should minimize such challenges. Second, most ForestGEO plots are 50 ha or less (averaging 26 ha), thus are smaller than the current notion of an ideal size for sampling tropical birds (100 ha). However, no critical analysis of optimal tropical bird plot sizes has yet to be conducted. Depending on the questions of interest, more 50 ha plots might be better solutions than fewer 100 ha plots. Current data do indicate that bigger plots sample more species and provide more opportunities to characterize space use (territory sizes) for a greater percentage of the bird community. If, however, primary goals are to benchmark smaller The success of the existing six big bird plots indicates that creation of an expanded network like the ForestGEO forest dynamics plots would produce new insights. For example, even with the small sample of plots currently, clear diﬀerences in richness, abundance patterns and biomasses appear across the plots. Amazonian sites tend to be similar but show obvious variability in abundances even within the same species. Such geographic variation in abundances as well as life history traits remains largely undocumented (Wolfe et al., 2014). The Panama community hosts a larger proportion of migrants than the Amazonian sites. The Amazonian sites have many more large granivores. How do all of these observed diﬀerences translate to meaningful ecological dynamics? Without more coordinated studies across more plots, particularly addition of more Central American sites, it is challenging to draw robust conclusion. Finally, beyond advancement of scientiﬁc knowledge per se, plots can also build capacity for local researchers to develop and share expertise, contribute data, and publish results. The successful development of a big bird plot network, or October 2021 \| Volume 9 \| Article 697511 Frontiers in Ecology and Evolution \| www.frontiersin.org Frontiers in Ecology and Evolution \| www.frontiersin.org 9 Robinson et al. Benchmarking Tropical Birds on Plots FIGURE 3 \| Network analysis of the inﬂuence of the big bird plot papers on the scientiﬁc literature. Keywords occurring ﬁve or more times in citing literature are included. Colors indicate major associations of co-occurring keywords. FIGURE 3 \| Network analysis of the inﬂuence of the big bird plot papers on the scientiﬁc literature. Frontiers in Ecology and Evolution \| www.frontiersin.org IMPEDIMENTS TO OVERCOME The academic culture of ecological science normally values testing of period-speciﬁc hypotheses and publications in peer- reviewed journals. Although we have clearly argued that a network of big bird plots can align with such values, we also note that establishment of such plots with a goal of tracking changes in bird populations over long time periods can be perceived by some as having lower value. Yet, a tension certainly exists between testing modern ecological ideas, addressing pressing conservation issues in landscapes with the world’s most diverse bird communities, and the continuing challenge of ﬁlling information needs concerning basic natural history of species, proper taxonomic identiﬁcation and the iterative development and improvement of reliable sampling methods. We consider that the eﬀect of certain academic philosophies is well-illustrated by the demise of the former, very extensive, North American network of bird spot-mapping plots (e.g., Short, 1979). After several decades of surveys in the 1900s with results being published in ornithological journals, the eﬀort was deemed too unimportant and unproductive to take up valuable journal space. The disappearance of journal support eroded the volunteer base and the network died. If tracking temporal trends in bird numbers is an objective, precise repeatability of sampling methods is important. Stationary, or point, counts provide a high degree of repeatability because the same locations can be monitored, even repeating surveys on the same date and time of day in future years (Robinson and Curtis, 2020). Large plots also provide, obviously, a larger number of points, again improving sample sizes and increasing statistical power to detect trends. Even as new analytical methods for estimating abundances or population densities are developed, the simple elegance of a design where qualiﬁed surveyors or automatic recording units are deployed at exactly the same locations through time creates opportunities to accurately measure temporal change, establishing the benchmarking value of large plots (Robinson and Curtis, 2020). Despite the current academic cultural emphasis on ecological publications in high-impact journals as the yardstick by which “success” and “importance” are measured, some new methods for publishing and archiving big plot data have arisen. For example, electronic archiving of metadata so that they remain available through open access is becoming increasingly common. The opportunities for publishing “data papers” where extensive community inventory observations may be made available also continue to increase. REASONS FOR BUILDING A LARGER NETWORK OF BIG BIRD PLOTS Plots can play a major role in developing methodologies for larger scale eﬀorts to facilitate long-term monitoring of community dynamics as well as quantifying community structure and how it varies according to relative inﬂuences of biotic and abiotic processes (Kraft et al., 2008; Gómez et al., 2020). Knowledge of long-term population trends is essential for eﬀective evaluation of potential regional and global changes on bird populations. Large-scale plots can facilitate ﬁeld-intensive techniques such as spot-mapping or radio telemetry to determine territory sizes and the number of territories for a subsample of species representing a broad range of traits (e.g., body size). By establishing known density estimates for a number of species, For most questions addressing patterns of community structure or species diversity, plots must be suﬃciently large Frontiers in Ecology and Evolution \| www.frontiersin.org October 2021 \| Volume 9 \| Article 697511 10 Benchmarking Tropical Birds on Plots Robinson et al. to include most of the species locally present. Many forest species have large home ranges, patchy distributions, and are rare, being present in densities of less than ﬁve individuals per 100 ha. In addition, the intrinsic scale of many forms of disturbance, which will aﬀect species composition on the local scale, such as treefall gaps, occurs at the spatial extent of 0.5– 5 ha. Thus, plots of 100 ha or more in size are likely to contain multiple individuals of most species and should be less likely to have patterns of species composition driven mostly by local disturbance dynamics. Therefore, large plots increase the chances to more fully characterize local communities while reducing chances that any especially local eﬀects, such as smaller-scale past disturbances, strongly inﬂuence community structure. Even if 100 ha plots are too small to include suﬃcient numbers of most rare species for detection of statistically signiﬁcant trends over time, alternative methods of community composition analysis can characterize patterns of rare species loss. Plot-based studies still face challenges of how to estimate numbers of the most mobile species, such as parrots and many raptors, colonial species that utilize plots but may breed elsewhere, and the most cryptic components of the avifauna that elude detection with traditional methods. Such consequences might be evaluated by establishing plots so that some are in undisturbed sites isolated from easy human access whereas others are less isolated. REASONS FOR BUILDING A LARGER NETWORK OF BIG BIRD PLOTS This approach might also permit detection of global-change-driven temporal changes versus changes inﬂuenced mostly by local landscape eﬀects (e.g., hunting, introduced species). Frontiers in Ecology and Evolution \| www.frontiersin.org IMPEDIMENTS TO OVERCOME Such opportunities may promote the proliferation of further academic specialization where people skilled at identifying and counting birds but uninterested or unable to publish hypothesis-focused papers can share information publicly and concomitantly receive credit for their expertise. Emphasizing the benchmark value of bird population and community studies, additional progress toward recognizing the value of Transgenerational Collaborations, where current community members establish a well-designed survey so that it can be precisely repeated in the future, needs to be made. The insights provided by several famous studies and their recent re-surveys such as the Grinnell project (Tingley et al., 2009) and Forbes’s early 19th century of Illinois birds (Walk et al., 2010) demonstrate the value of well-executed benchmark studies. Expanding opportunities to develop such benchmark datasets outside the traditional academic realm oﬀers to increase inclusivity and build social capital, particularly in Another potential value of large observational plots is that they are less likely to be aﬀected by unpredictable land use, especially if those plots attract other scientists to study various aspects of the site’s biology. Well-constructed plots (for example, with trail networks permitting easier access) allow for coordinated research with scientists addressing a broader array of ecological questions. Such plots also may serve as a focal point for building social capital by engaging local inhabitants in plot construction, maintenance, and data collection, as well as building scientiﬁc literacy. Ideally, plots could and should be established by in-country residents who know the landscapes and their avifauna the best. The involvement of regional scientists as project leaders can contribute to advancing equity in ecological sciences and open more eﬃcient avenues for educating local residents of the value of characterizing dynamics of native biodiversity (Seidler et al., 2021). At the same time, establishment of trail networks near human settlements may have the detrimental eﬀect of elevating harvest for food, particularly cracids and other game species, and for the pet trade (Peres and Lake, 2003; Peres et al., 2006; Ferreguetti et al., 2018). October 2021 \| Volume 9 \| Article 697511 Frontiers in Ecology and Evolution \| www.frontiersin.org Frontiers in Ecology and Evolution \| www.frontiersin.org 11 Benchmarking Tropical Birds on Plots Robinson et al. support infrastructure to provide opportunities to link with existing data on plant communities. Most ForestGEO plots are smaller than 100 ha, so expansion by adding bird surveys around those plots in buﬀers would be necessary (Robinson and Curtis, 2020). RECOMMENDATIONS Without long-term, high-quality data, we have no reliable way to identify mechanisms of population or community change through time and across geography in the world’s richest bird communities. Recent declines of some insectivorous species have occurred in bird communities of mature forests with no evidence of direct impacts from anthropogenic activities (Blake and Loiselle, 2015; Stouﬀer et al., 2021). Presumably, declines in insectivores, particularly terrestrial species, may have some link to subtle shifts in regional precipitation or climatic conditions, but the mechanisms cannot be reliably identiﬁed yet and cannot even be separated from simple stochastic processes that play out over long periods of time. Even basic information on tracking relevant insect populations is generally lacking (Lamarre et al., 2020; Montgomery et al., 2021). In short, the paucity of basic data on bird populations from the world’s richest locations is a glaring deﬁciency in our abilities to understand drivers of change, community structuring mechanisms and the importance of birds as interactants on their ecological stage. Estimated costs of surveying big plots for birds are important to consider. Decisions about intensiveness of survey eﬀort, whether or not plots are already existing or will be established at new locations, plot size and costs of labor and travel will inﬂuence estimated costs per plot. The existing big plots were largely established and surveyed by foreign scientists, elevating travel and labor costs above potential costs if local talent were available. A probable key to long-term success of a big bird plot network would be to enhance local human capacity to establish and monitor plots. Despite potential large variability in costs, it is probably worth suggesting that surveying Neotropical bird plots may not cost much relative to the estimated costs of surveying tree plots. For example, the ForestPlots.Net assemblage of 1105 small plots (normally averaging about 1 ha in size) costs about 27,000 USD to install a plot (ForestPlots.net et al., 2021). Those costs are high because each individual tree is mapped and identiﬁed. Subsequent resurveys of plots have been estimated to cost less, about 18,000 USD or around 30 USD per tree. Because we argue large plots, approximately 100 ha in size, are necessary for adequate surveys of forest bird communities, costs could be prohibitively high per plot if costs of surveying birds were as high as they are for trees. RECOMMENDATIONS However, the spatial precision with which birds can be mapped, given their mobility, is much lower, the total number of birds per plot is much smaller than that of trees, and the taxonomy is much better known so identiﬁcations are not as time- consuming. If the advice of Robinson and Curtis (2020) is heeded to create a simple benchmark survey designed to measure species richness accurately and estimate abundances of most species in a community, they estimated the eﬀort could be accomplished in 4 weeks. Assuming two skilled surveyors are involved, as they recommended, and the plot has already been established, such as with a large ForestGEO plot, the costs for surveying a single plot in one year could be approximately 4,800 USD (320 h times 15 USD/h) notwithstanding consideration of travel and lodging costs. If new plots were established, we estimate at least 4 weeks to create the basic trail system with measured stops along transect routes, which could double initial costs. The costs assume surveyors are already suﬃciently We recommend two major steps in using tropical big bird plots to enhance knowledge of tropical avian ecology. First, assemble metadata and data from existing plots and make them publicly accessible to facilitate future re-surveys. Many options for storage of metadata now exist. The ForestPlots.Net group database could provide a useful model (ForestPlots.net et al., 2021) if big bird plot data were to be managed as a stand-alone resource. Addition of a Neotropical node in Avian Knowledge Network would be appropriate (Robinson and Curtis, 2020) and has the advantage that the diversity of methods used to survey birds are already included. With current eﬀorts to resurvey some plots, now is an appropriate time to establish data archival sites. The Cocha Cashu plot has recently been resurveyed and a planned re-survey of the Limbo plot was postponed by the coronavirus pandemic. Existing plots have the immediate advantage of the initial surveys to facilitate analysis of temporal change. During re-surveys, adopting new methodologies, such as deployment of automated recording units, and utilizing methods that improve precise repeatability of surveys (stationary counts) and help adjust for detectability issues when estimating density are important. IMPEDIMENTS TO OVERCOME Alternatively, ForestGEO could be invited to expand their network to incorporate portions of big bird plots. Addition of plots in other locations should also be relatively straightforward if the main goal is to benchmark current bird populations in a manner that uses highly repeatable survey methods (Robinson and Curtis, 2020). In that case, labor-intensive methods such as mist-netting, which sample portions of tropical communities that may be diﬃcult to accurately measure with auditory surveys, are potentially de-emphasized in favor of eﬃcient and more precisely repeatable sampling methods. If exhaustive surveys of communities are desired, then multiple methods extending beyond the grids of stationary counts and collections of transects advocated by Robinson and Curtis (2020) will need to be employed as we discussed earlier. human communities with less opportunities for advanced academic training. Frontiers in Ecology and Evolution \| www.frontiersin.org DATA AVAILABILITY STATEMENT The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author. RECOMMENDATIONS If questions comparing broad patterns in community organization require less detailed data from each plot, then larger numbers of smaller plots sampled less intensively may be suitable (Rosa et al., 2021). On the other hand, highly detailed data from fewer big plots might be needed to assess geographic variation in patterns of community structure, species rank-abundance proﬁles, and beta diversity, and determine if those patterns are parallel in trees and birds, or at least correlated. Data from the ForestGEO plots might be used to predict the quantity and distribution of big bird plots that could suﬃciently characterize Neotropical forest bird communities. From our simple comparisons of community structure among the existing forest bird plots, it is clear that Limbo, Panama, stands out as being quite diﬀerent. Adding plots in Central America should, therefore, be a priority to better understand diﬀerences between Central and South American bird communities. At this point in history, any plot at any location would be a welcome addition and would promise to expand perspective on geographic variation in community structure. RECOMMENDATIONS Additionally, quantifying other aspects of plot characteristics, such as environmental conditions at the time of surveys, habitat patchiness owing to treefall gaps and other disturbances, and even insect sampling would provide opportunities to link changes in bird numbers or richness with potential mechanisms inﬂuencing change. Training local technicians to contribute can add temporal continuity and social value to re-survey eﬀorts. Second, we recommend expanding the size of the network of plots. This could be done by linking with the ForestGEO network, through development of collaborative agreements to add bird surveys where such data would be logistically feasible and mutually beneﬁcial and building on that network’s standing October 2021 \| Volume 9 \| Article 697511 12 Benchmarking Tropical Birds on Plots Robinson et al. experienced to reliably detect, identify and count birds. Building capacity of local surveyors is also an important cost and contribution (Magnusson et al., 2013). Teaching bird surveying skills to local scientists would add to the time and funds required in the short-term but would save costs in the long-term and also contribute to the development of human capacity for ecological research. We consider our estimate to be at the low end of a potentially large range in costs inﬂuenced by local labor costs, terrain, lodging, and many other considerations including annual plot maintenance and data management. Of the practical aspects aﬀecting creation of a network of big bird plots, establishment of detailed best practices for survey methodology is still necessary. The existing big plots were all surveyed with multiple methods, yet no speciﬁc coordination of common methods was attempted. Given the complexities of tropical bird communities, especially those in forests where many species are cryptic and/or range widely, further attention to survey methodology strengths and weaknesses is still required. It may be possible that cross-plot comparisons of densities or biomass may be reliable for only certain subsets of communities that can be surveyed with precisely repeatable methods. Yet, exhaustive surveys using multiple methods might still permit useful general comparisons of community organization like we have summarized here. Overall, more advances are needed to establish the most reliable set of survey methodologies for consistent and standardized data collection to benchmark such diverse species communities. How many plots should be added and where should they be located? This remains an open question that might be addressed with modeling experiments or considerations of speciﬁc scientiﬁc goals. ACKNOWLEDGMENTS We thank the many technicians and ﬁeld biologists who helped gathering the data on each of the big bird plots, as well as the relevant authorities for permitting such surveys. AUTHOR CONTRIBUTIONS WR and HP conceived the study. DE, HP, and F-YS organized the database. HP and F-YS performed the statistical analysis. WR, DE, HP, AM, JB, and PS wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version. y We also recommend a few speciﬁc priorities for addition of new plots. Add plots in a wider diversity of “undisturbed” forest types and across elevational gradients. Add plots in sites recovering from disturbance to provide opportunities to characterize long-term successional eﬀects on bird communities. Locate plots in threatened habitats that may be most likely to change in the near future, either within the plots themselves or in the surrounding landscapes, to provide before-after perspectives on local and landscape-level inﬂuences on communities, such as the role of mass eﬀects on plot-level diversity (Condit et al., 2012). Use as many methods as possible to characterize entire communities as not all guilds or functional groups are equally responsive to each potential driver of change. Beyond enumeration of richness and density, inclusion of new approaches to sample diets and genetic and genomic diversity can help identify interactions of birds with other forms of biodiversity (Garcia and Robinson, 2021). 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https://openalex.org/W2998871639	https://www.research-collection.ethz.ch/bitstream/20.500.11850/348406/3/document%2853%29.pdf	English	null	Structure and Supersaturation for Intersecting Families	arXiv (Cornell University)	2,018	cc-by	21,931	ETH Library Rights / license: Rights / license: Creative Commons Attribution 4.0 International g Creative Commons Attribution 4.0 International Journal Article Author(s): Balogh, József; Das, Shagnik; Liu, Hong; Sharifzadeh, Maryam; Tran, Tuan Publication date: 2019-05-31 Permanent link: https://doi.org/10.3929/ethz-b-000348406 Rights / license: ∗Partially supported by NSF Grant DMS-1500121 and by the Langan Scholar Fund (UIUC). †Supported by GIF grant G-1347-304.6/2016. ‡Supported by the Leverhulme Trust Early Career Fellowship ECF-2016-523. §Supported by ERC grant 306493 and the European Union’s Horizon 2020 research and innovation programme under the Marie Curie grant agreement No 752426. ¶Supported by the Alexander Humboldt Foundation, and by the GACR grant GJ16-07822Y, with institutional support RVO:67985807. Structure and supersaturation for intersecting families for intersecting families J´ozsef Balogh∗ Department of Mathematical Sciences University of Illinois at Urbana-Champaign Illinois, U.S.A. and Moscow Institute of Physics and Technology 9 Institutskiy per. Dolgoprodny Moscow Region, 141701 Russian Federation jobal@illinois.edu Shagnik Das† Institut f¨ur Mathematik Freie Universit¨at Berlin Berlin, Germany shagnik@mi.fu-berlin.de Hong Liu‡ Maryam Sharifzadeh§ Mathematics Institute University of Warwick Coventry, U.K. {h.liu.9,m.sharifzadeh}@warwick.ac.uk Tuan Tran¶ Department of Mathematics ETH Z¨urich, Switzerland manh.tran@math.ethz.ch Submitted: Feb 22, 2018; Accepted: Sep 17, 2018; Published: May 31, 2019 c⃝The authors. Released under the CC BY-ND license (International 4.0). Shagnik Das† Institut f¨ur Mathematik Freie Universit¨at Berlin Berlin, Germany shagnik@mi.fu-berlin.de Shagnik Das† Institut f¨ur Mathematik Freie Universit¨at Berlin Berlin, Germany shagnik@mi.fu-berlin.de J´ozsef Balogh∗ Department of Mathematical Sciences University of Illinois at Urbana-Champaign Illinois, U.S.A. and Moscow Institute of Physics and Technology 9 Institutskiy per. Dolgoprodny Moscow Region, 141701 Russian Federation jobal@illinois.edu Hong Liu‡ Maryam Sharifzadeh§ Mathematics Institute University of Warwick Coventry, U.K. {h.liu.9,m.sharifzadeh}@warwick.ac.uk Tuan Tran¶ Department of Mathematics ETH Z¨urich, Switzerland manh.tran@math.ethz.ch Submitted: Feb 22, 2018; Accepted: Sep 17, 2018; Published: May 31, 2019 c⃝The authors. Released under the CC BY-ND license (International 4.0). ∗Partially supported by NSF Grant DMS-1500121 and by the Langan Scholar Fund (UIUC). †S d b GIF G 1347 304 6/2016 y y †Supported by GIF grant G-1347-304.6/2016. Originally published in: g y p The Electronic Journal of Combinatorics 26(2), https://doi.org/10.37236/768 The Electronic Journal of Combinatorics 26(2), https://doi.org/10.37236/7683 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. Structure and supersaturation for intersecting families p g g g ¶Supported by the Alexander Humboldt Foundation, and by the GACR grant GJ16-07822Y, with institutional support RVO:67985807. pp y g / ‡Supported by the Leverhulme Trust Early Career Fellowship ECF-2016-523. 1 Introduction Determining the size of intersecting families of discrete objects is a line of research with a long history, originating in extremal set theory. A set family is intersecting if any two of its sets share a common element. A classic result of Erd˝os, Ko and Rado [20] from 1961 states that when n ⩾2k, the size of the largest intersecting k-uniform set family over [n] is n−1 k−1 . Furthermore, when n ⩾2k + 1, the only extremal conﬁgurations are the trivial families, where all edges contain a given element. This fundamental theorem has since inspired a great number of extensions and variations. A recent trend in extremal combinatorics is to study the supersaturation extension of classic results. This problem, sometimes referred to as the Erd˝os–Rademacher problem, asks for the number of forbidden substructures that must appear in a conﬁguration larger than the extremal threshold. We often observe an interesting phenomenon: while the extremal result only requires one forbidden substructure to appear, we usually ﬁnd several. The ﬁrst such line of research extended Mantel’s Theorem [37], which states that an n- vertex triangle-free graph can have at most ⌊n2/4⌋edges. Rademacher (unpublished) showed that one additional edge would force the appearance of at least ⌊n/2⌋triangles. Determining the number of triangles in larger graphs attracted a great deal of attention, starting with the works of Erd˝os [17, 18] and Lov´asz and Simonovits [36] and culminating in the asymptotic solution due to Razborov [42] and the recent exact solution determined by Liu, Pikhurko and Staden [33]. Supersaturation problems have since been studied in various contexts; examples include extremal graph theory [2, 30, 38, 39, 41, 43], extremal set theory [5, 9, 13, 31, 45], poset theory [4, 40, 46], and group theory [8, 27, 47]. The ﬁrst result of our paper concerns supersaturation for the extension of the Erd˝os– Ko–Rado Theorem to families of permutations. A pair of permutations σ, π ∈Sn is said to be intersecting if {i ∈[n] : π(i) = σ(i)} ̸= ∅, and disjoint otherwise. A family F ⊆Sn is intersecting if every pair of permutations in the family is. A natural construction of an intersecting family is to ﬁx some pair i, j ∈[n], and take all permutations that map i to j; we call this a coset, and denote it by T(i,j). Abstract The extremal problems regarding the maximum possible size of intersecting fam- ilies of various combinatorial objects have been extensively studied. In this paper, we investigate supersaturation extensions, which in this context ask for the mini- mum number of disjoint pairs that must appear in families larger than the extremal threshold. We study the minimum number of disjoint pairs in families of permu- tations and in k-uniform set families, and determine the structure of the optimal 1 the electronic journal of combinatorics 26(2) (2019), #P2.34 families. Our main tool is a removal lemma for disjoint pairs. We also determine the typical structure of k-uniform set families without matchings of size s when n ⩾2sk + 38s4, and show that almost all k-uniform intersecting families on vertex set [n] are trivial when n ⩾(2 + o(1))k. Mathematics Subject Classiﬁcations: 05C30, 05C65, 05D05 Mathematics Subject Classiﬁcations: 05C30, 05C65, 05D05 1 Introduction Our ﬁrst result shows, for certain ranges of family sizes s, that these families indeed minimise dp(F) over all families F ⊆Sn with \|F\| = s. Theorem 2. There exists a constant c > 0 such that the following holds. Let n, k and s be positive integers such that k ⩽cn1/2, and s = (k + ε)(n −1)! for some real ε with \|ε\| ⩽ck−3. Then any family F ⊆Sn with \|F\| = s satisﬁes dp(F) ⩾dp(T (n, s)). Theorem 2. There exists a constant c > 0 such that the following holds. Let n, k and s be positive integers such that k ⩽cn1/2, and s = (k + ε)(n −1)! for some real ε with \|ε\| ⩽ck−3. Then any family F ⊆Sn with \|F\| = s satisﬁes dp(F) ⩾dp(T (n, s)). We next consider the supersaturation extension of the original Erd˝os–Ko–Rado Theo- rem, where one seeks to minimise the number of disjoint pairs of sets in a k-uniform family of s subsets of [n]. Bollob´as and Leader [7] provided, for every s, a family of constructions known as the ℓ-balls, and conjectured that for some 1 ⩽ℓ⩽k, an ℓ-ball is optimal for the supersaturation problem. In particular, when ℓ= 1, the construction is an initial segment of the lexicographic ordering. [ ] Denote by [n] k the family of all k-element subsets of [n]. Letting L(n, k, s) be the initial segment of the ﬁrst s sets in [n] k , we write dp(n, k, s) for dp(L(n, k, s)), where again dp(F) is the number of disjoint pairs in a set family F. Das, Gan and Sudakov [10] proved that if n > 108(k3r + k2r2) and s ⩽ n k − n−r k , then for any family F ⊆ [n] k of size s, dp(F) ⩾dp(n, k, s). That is, when n is suﬃciently large and the families are of small size, the initial segments of the lexicographic order minimise the number of disjoint pairs, conﬁrming the Bollob´as–Leader conjecture in this range. g j g Note that, for ﬁxed r, the result in [10] requires k = O(n1/3). Frankl, Kohayakawa and R¨odl [23] showed that initial segments of the lexicographic order are asymptotically optimal even for larger uniformities k. Theorem 3. There is some absolute constant C such that if n ⩾Ck2r3 and s ⩽ n k − n−r k , then any family F ⊆ [n] k with \|F\| = s satisﬁes dp(F) ⩾dp(n, k, s); that is, L(n, k, s) minimises the number of disjoint pairs. 1 Introduction Observe that T(i,j) = (n −1)!, and Deza and Frankl [12] showed that this is the largest possible size of an intersecting family in Sn. In the corresponding supersaturation problem, we seek to determine how many disjoint pairs of permutations must appear in larger families. We write dp(F) for the number of disjoint pairs of permutations in a family F ⊆Sn. By the Deza–Frankl Theorem, when \|F\| ⩽(n −1)!, we need not have any disjoint pairs in F, while for \|F\| > (n −1)!, dp(F) must be positive. One might expect a family of permutations with the minimum number of disjoint pairs 2 the electronic journal of combinatorics 26(2) (2019), #P2.34 to contain large intersecting subfamilies, and a candidate construction is therefore the union of an appropriate number of cosets. However, these unions are not isomorphic, as pairs of cosets can intersect each other diﬀerently. Indeed, given pairs (i1, j1) ̸= (i2, j2) ∈ [n]2, we have T(i1,j1) ∩T(i2,j2) = {π ∈Sn : π(i1) = j1 and π(i2) = j2}, which is empty if i1 = i2 or j1 = j2, and has size (n −2)! otherwise. To ﬁt the family within as few cosets as possible, we should take the cosets to be pairwise-disjoint, motivating the following deﬁnition. Deﬁnition 1 (The family T (n, s)). Writing π = π(1) π(2) . . . π(n) , we can equip Sn with the lexicographic ordering, where π < σ if there is some k ∈[n] such that π(k) < σ(k) and π(i) = σ(i) for all i < k. Then, given any 0 ⩽s ⩽n!, we denote by T (n, s) the ﬁrst s permutations under this ordering. Deﬁnition 1 (The family T (n, s)). Writing π = π(1) π(2) . . . π(n) , we can equip Sn with the lexicographic ordering, where π < σ if there is some k ∈[n] such that π(k) < σ(k) and π(i) = σ(i) for all i < k. Then, given any 0 ⩽s ⩽n!, we denote by T (n, s) the ﬁrst s permutations under this ordering. In particular, if s = ℓ(n −1)! + r for some 0 ⩽ℓ⩽n and 0 ⩽r < (n −1)!, the family T (n, s) contains the pairwise-disjoint cosets T(1,j) for 1 ⩽j ⩽ℓ, together with r further permutations from the disjoint coset T(1,ℓ+1). 1 Introduction In our next result, we extend the exact results to larger k as well, showing that the lexicographic initial segments are still optimal when k = O(n1/2). Theorem 3. There is some absolute constant C such that if n ⩾Ck2r3 and s ⩽ n k − n−r k , then any family F ⊆ [n] k with \|F\| = s satisﬁes dp(F) ⩾dp(n, k, s); that is, L(n, k, s) minimises the number of disjoint pairs. 3 the electronic journal of combinatorics 26(2) (2019), #P2.34 With our next results, we address a diﬀerent variation of classic extremal problems. Rather than considering the supersaturation phenomenon, we describe the typical struc- ture of set families with a given property, showing that almost all such families are sub- families of the trivial extremal constructions. We ﬁrst consider the famous Erd˝os Matching Conjecture concerning the largest k- uniform set families over a ground set of size n that have no matching of size s. There are two constructions that trivially avoid a matching of size s: a clique on ks−1 vertices, and the family of all edges intersecting a set of size s −1. In [19], Erd˝os conjectured that one of these constructions is always optimal. Conjecture 4 (Erd˝os [19], 1965). Given integers n, k and s, let F ⊆ [n] k be a set family with no matching of size s. Then \|F\| ⩽max ks −1 k , n k − n −s + 1 k . Frankl [22] proved the conjecture in the range n ⩾(2s −1)k −s + 1, showing that the extremal families can be covered by s −1 elements. Adapting the methods of Balogh et al. [3], we show that a slightly larger lower bound on n guarantees that almost all families without a matching of size s have a cover of size s −1. Theorem 5. Let n, k ⩾3 and s ⩾2 be integers with n ⩾2sk + 38s4. Then the number of subfamilies of [n] k with no matching of size s is n s−1 + o(1) 2(n k)−(n−s+1 k ), where the term o(1) tends to 0 as n →∞. The s = 2 case corresponds to intersecting families. In this case, Balogh et al. [3] showed that when n ⩾(3 + o(1))k, almost all intersecting families are trivial. the electronic journal of combinatorics 26(2) (2019), #P2.34 1 Introduction Our ﬁnal result improves the required bound on n to the asymptotically optimal n ⩾(2 + o(1))k. Indeed, when n = 2k, then the number of intersecting families is 3 1 2(n k) = 3(n−1 k−1), since we can freely choose at most one set from each complementary pair of k-sets {A, [n] \ A}. Theorem 6. There exists a positive constant C such that for k ⩾2 and n ⩾2k+C √ k ln k, almost all intersecting families in [n] k are trivial. In particular, the number of intersecting families in [n] k is (n + o(1))2(n−1 k−1), where the term o(1) tends to 0 as n →∞. Remark: During the preparation of this paper, Theorem 6 (with a superior constant C = 2) was proven independently by Frankl and Kupavskii [25] using diﬀerent methods. Outline and notation. The rest of the paper is organised as follows. We discuss families of permutations in Section 2, in particular proving the supersaturation result of Theorem 2. Section 3 is devoted to supersaturation for set families and the proof of Theorem 3. In Section 4, we address the typical structure of families, proving Theorems 5 and 6. Section 5 contains some concluding remarks, including a counterexample to the Bollob´as–Leader conjecture. We use standard set-theoretic and asymptotic notation. We write X k for the family of all k-element subsets of a set X. Given two functions f and g of some underlining parameter n, if limn→∞f(n)/g(n) = 0, we write f = o(g). For a, b, c ∈R+, we write a = b ± c if b −c ⩽a ⩽b + c. 4 the electronic journal of combinatorics 26(2) (2019), #P2.34 2 Supersaturation for families of permutations In this section, we study the supersaturation problem concerning the number of disjoint pairs in a family of permutations. Our main tool is a removal lemma for disjoint pairs of permutations, showing that families with relatively few disjoint pairs are close to unions of cosets. We start by collecting some basic facts. 2.1 The derangement graph Let Sn be the symmetric group on [n]. A permutation τ ∈Sn is called a derangement if τ(i) ̸= i for every i ∈[n]. Let Dn be the set of all derangements in Sn. Denote by Γn the derangement graph on Sn, that is, σ ∼π if σ · τ = π for some τ ∈Dn. In other words, σ and τ are adjacent in Γn if and only if they are disjoint. We denote by dn the number of derangements in Sn. By construction, Γn is a dn-regular graph. A standard application of the inclusion–exclusion principle shows dn = \|Dn\| = n! n X i=0 (−1)i i! ∼n! e . dn = \|Dn\| = n! n X i=0 (−1)i i! ∼n! e . We also introduce the notation Dn = dn +dn−1, which we will use to keep track of disjoint pairs in certain subgraphs of the derangement graph. We also introduce the notation Dn = dn +dn−1, which we will use to keep track of disjoint pairs in certain subgraphs of the derangement graph. For instance, consider the subgraph of Γn induced by two disjoint cosets T(i1,j) and T(i2,j). Since the cosets are intersecting families, they are independent sets in Γn, and so Γn[T(i1,j), T(i2,j)] is bipartite. For any σ ∈T(i1,j) and any neighbour π = σ · τ, where τ ∈Dn, we have π ∈T(i2,j) if and only if τ(i2) = i1. It is straightforward to see that there are dn−2 such derangements τ with τ(i1) = i2 and dn−1 derangements with τ(i1) ̸= i2. As a result, every vertex of the bipartite graph has the same degree dn−2 + dn−1 = Dn−1. Repeating this argument for all the cosets of the form T(i′,j) gives the recurrence relation dn = (n −1)Dn−1 = (n −1)(dn−1 + dn−2). (1) (1) For our investigation we shall need some information on the spectrum of the derange- ment graph Γn. Since Γn is dn-regular, its largest eigenvalue λ0 is dn with constant eigenvector ⃗1 = 1 . . . 1 . We shall order the eigenvalues {λ0, λ1, . . . , λn!−1} in a (per- haps non-standard) way, so that dn = \|λ0\| ⩾\|λ1\| ⩾\|λ2\| ⩾. . . ⩾\|λn!−1\|. Rentner [44] showed λ1 = −dn/(n −1), while Ellis [14] proved there is some positive constant K such that \|λ2\| ⩽Kdn/n2. 2.2 A removal lemma For any integer s > 1 2(n −1)!, there are unique k ∈N and ε ∈ −1 2, 1 2 such that s = (k + ε)(n −1)!. For this choice of s, the family T (n, s) from Deﬁnition 1 is a subfamily of Sn consisting of ⌊k + ε⌋pairwise disjoint cosets and (k + ε −⌊k + ε⌋)(n −1)! permutations from another disjoint coset. Hence, dp(T (n, s)) = ⌊k + ε⌋ 2 + ⌊k + ε⌋(k + ε −⌊k + ε⌋) (n −1)!Dn−1 = k 2 + k −1 2 ε + 1 2\|ε\| (n −1)!Dn−1, (3) (3) as dp(F) = e(Γn[F]), and the bipartite subgraphs of Γn induced by disjoint cosets are Dn−1-regular. We will now prove a removal lemma for disjoint pairs of permutations, which states that any family F ⊆Sn of size s ≈k(n −1)! with dp(F) ≈dp(T (n, s)) must be ‘close’ to a union of k cosets. Lemma 7. There exist positive constants C and c such that the following holds for suf- ﬁciently large n. Let 1 ⩽k < n/2 be an integer, and let ε ∈R and β ∈R+ be such that max{\|ε\|, β} ⩽ck. If F ⊆Sn is a family of size s = (k + ε)(n −1)! and dp(F) ⩽dp(T (n, s)) + β(n −1)!Dn−1, then there is some union G of k cosets with the property that \|F∆G\| ⩽Ck2 1 n + r 6(\|ε\| + β) k ! (n −1)!. In the proof of Lemma 7 we shall use a stability result due to Ellis, Filmus and Friedgut [15, Theorem 1]. To state their theorem we need some additional notation. We equip Sn with the uniform distribution. Then, for any function f : Sn →R, the expected value of f is deﬁned by E[f] = 1 n! P σ∈Sn f(σ). The inner product of two functions f, g : Sn →R is deﬁned as ⟨f, g⟩= E[fg] = 1 n! P σ∈Sn f(σ)g(σ); this induces the norm ∥f∥= p ⟨f, f⟩. Given c > 0, let round(c) denote the nearest integer to c. Theorem 8 (Ellis, Filmus and Friedgut). There exist positive constants C0 and δ0 such that the following holds. Let F be a subfamily of Sn with \|F\| = α(n −1)! for some α ⩽n/2. 2.1 The derangement graph (2) (2) Furthermore, as shown by Ellis, Friedgut and Pilpel [16], the span of the λ0- and λ1- eigenspaces is U1 = span{1T(i,j) : i, j ∈[n]}, the span of the characteristic vectors of the cosets. Furthermore, as shown by Ellis, Friedgut and Pilpel [16], the span of the λ0- and λ1- eigenspaces is U1 = span{1T(i,j) : i, j ∈[n]}, the span of the characteristic vectors of the cosets. the electronic journal of combinatorics 26(2) (2019), #P2.34 5 the electronic journal of combinatorics 26(2) (2019), #P2.34 5 2.2 A removal lemma Let f = 1F be the characteristic function of F and let fU1 be the orthogonal projection of f onto U1. If E[(f −fU1)2] = δE[f] for some δ ⩽δ0, then E[(f −g)2] ⩽C0α2(1/n + δ1/2)/n, where g is the characteristic function of a union of round(α) cosets of Sn. where g is the characteristic function of a union of round(α) cosets of Sn. We now derive the removal lemma from Theorem 8. We now derive the removal lemma from Theorem 8. Proof of Lemma 7. Set c = min{ δ0 12, 1 2} and C = 3C0, where δ0 and C0 are the positive constants from Theorem 8. Let f be the characteristic vector of F. Write f = f0+f1+f2, Proof of Lemma 7. Set c = min{ δ0 12, 1 2} and C = 3C0, where δ0 and C0 are the positive constants from Theorem 8. Let f be the characteristic vector of F. Write f = f0+f1+f2, the electronic journal of combinatorics 26(2) (2019), #P2.34 6 where fi is the projection of f onto the λi-eigenspace for i = 0, 1. By the orthogonality of the eigenspaces, ∥f∥2 = ∥f0∥2 + ∥f1∥2 + ∥f2∥2. (4) (4) Since f is Boolean, Since f is Boolean, Since f is Boolean, ∥f∥2 = E[f 2] = E[f] = \|F\| n! = k + ε n and ∥f0∥2 = ⟨f,⃗1⟩2 = E[f]2 = k + ε n 2 . (5) (5) Let A be the adjacency matrix of the derangement graph Γn. Then 2 dp(F) = 2e(Γn[F]) = f TAf = 2 X i=0 f T i Afi (2) ⩾λ0f T 0 f0 + λ1f T 1 f1 −Kdn n2 f T 2 f2. Dividing both sides by n!, we obtain the following inequalities when n ⩾4K: 2 dp(F) n! ⩾ λ0∥f0∥2 + λ1∥f1∥2 −Kdn n2 ∥f2∥2 (4) = λ0∥f0∥2 + λ1(∥f∥2 −∥f0∥2 −∥f2∥2) −Kdn n2 ∥f2∥2 = (λ0 −λ1)∥f0∥2 + λ1∥f∥2 + \|λ1\| −Kdn n2 ∥f2∥2 (5) ⩾ ndn n −1 · k + ε n 2 − dn n −1 · k + ε n + 3dn 4(n −1) · ∥f2∥2 = dn n(n −1) · (k + ε)(k + ε −1) + 3dn 4(n −1) · ∥f2∥2 (1) = 2Dn−1 n · k 2 + k −1 2 ε + ε2 2 + 3 4Dn−1 · ∥f2∥2. where g is the characteristic function of a union of round(α) cosets of Sn. Let F ⊆Sn be an extremal family of size k(n −1)!, and let f = 1F. By (3), we must have dp(F) ⩽dp(T ) = k 2 (n −1)!Dn−1. Hence, as in the proof of Lemma 7, we can use (7) with ε = β = 0, and so ∥f2∥2 = 0. It follows from (6) that dp(F) ⩾ k 2 (n −1)!Dn−1 = dp(T ), showing that T minimises the number of disjoint pairs. Proof. Let F ⊆Sn be an extremal family of size k(n −1)!, and let f = 1F. By (3), we must have dp(F) ⩽dp(T ) = k 2 (n −1)!Dn−1. Hence, as in the proof of Lemma 7, we can use (7) with ε = β = 0, and so ∥f2∥2 = 0. It follows from (6) that dp(F) ⩾ k 2 (n −1)!Dn−1 = dp(T ), showing that T minimises the number of disjoint pairs. 1In the proof of Lemma 7, we used that n was suﬃciently large to bound Kdn n2 ∥f2∥2. However, in Proposition 9, we have ∥f2∥2 = 0, and so do not require n to be large. where g is the characteristic function of a union of round(α) cosets of Sn. (5) ⩾ ndn n −1 · k + ε n 2 − dn n −1 · k + ε n + 3dn 4(n −1) · ∥f2∥2 = dn n(n −1) · (k + ε)(k + ε −1) + 3dn 4(n −1) · ∥f2∥2 (1) = 2Dn−1 n · k 2 + k −1 2 ε + ε2 2 + 3 4Dn−1 · ∥f2∥2. (6) On the other hand, by assumption we have dp(F) ⩽dp(T (n, s)) + β(n −1)!Dn−1 (3) = k 2 + k −1 2 ε + 1 2\|ε\| + β (n −1)!Dn−1. dp(F) ⩽dp(T (n, s)) + β(n −1)!Dn−1 (3) = k 2 + k −1 2 ε + 1 2\|ε\| + β (n −1)!Dn−1. Combined with (6), we get ∥f2∥2 ⩽4 3 · \|ε\| + 2β −ε2 n ⩽3(\|ε\| + β) n . (7) (7) Moreover, E[f] = k+ε n ⩾ k 2n, as \|ε\| ⩽ck ⩽k 2. Therefore, E[(f −fU1)2] = E[(f −f0 −f1)2] = ∥f2∥2 ⩽6(\|ε\| + β) k · E[f]. the electronic journal of combinatorics 26(2) (2019), #P2.34 7 the electronic journal of combinatorics 26(2) (2019), #P2.34 7 the electronic journal of combinatorics 26(2) (2019), #P2.34 Since 6(\|ε\|+β) k ⩽12c ⩽δ0, we may apply Theorem 8 to conclude that there exists a union G of k cosets in Sn such that E[(f −1G)2] ⩽C0(k + ε)2 n 1 n + r 6(\|ε\| + β) k ! ⩽Ck2 n 1 n + r 6(\|ε\| + β) k ! . This gives \|F∆G\| = E[(f −1G)2] · n! ⩽Ck2 1 n + q 6(\|ε\|+β) k (n −1)!, completing our proof. E[(f −1G)2] ⩽C0(k + ε)2 n 1 n + r 6(\|ε\| + β) k ! ⩽Ck2 n 1 n + r 6(\|ε\| + β) k ! . We will use this removal lemma to prove Theorem 2 (a supersaturation result for disjoint pairs in Sn) in Subsection 2.4. However, from the proof above we can immediately deduce that for any1 1 ⩽k ⩽n, the union of k pairwise disjoint cosets minimises the number of disjoint pairs among all families of k(n −1)! permutations. Proposition 9. For any positive integers 1 ⩽k ⩽n, the family T = ∪k j=1T(1,j) minimises the number of disjoint pairs over all families F ⊆Sn of size k(n −1)!. Proof. 2.3 Intersection graphs The removal lemma states that families with relatively few disjoint pairs must be close to unions of cosets. While this describes their large-scale structure, it falls short of deter- mining the ﬁner details of such families. As we have observed previously, certain pairs of cosets are disjoint, while other pairs share a small number of permutations. In order to keep track of this information, we introduce the notion of an intersection graph. Given a union G = T(i1,j1)∪. . .∪T(ik,jk) of k diﬀerent cosets in Sn, its intersecting graph is the graph with vertex set {(i1, j1), . . . , (ik, jk)} and edges between pairs corresponding to cosets with non-empty intersection. As remarked before Deﬁnition 1, we therefore have (i, j) ∼(i′, j′) if and only if i ̸= i′ and j ̸= j′; that is, when these vertices do not lie on an axis-aligned line in Z2. For Theorem 2, we need to show that pairwise disjoint cosets minimise the number of disjoint pairs. To that end, we call a union G of k cosets canonical if at least k −1 of its cosets are pairwise disjoint. In terms of the intersection graph G of G, this means there is an axis-aligned line containing at least v(G) −1 vertices. For example, when s = k(n −1)!, the lexicographic family T (n, s) is canonical, as all the vertices (i, j) of its intersection graph lie on the line i = 1. Our next proposition, central to the proof of Theorem 2, describes how the intersection graph of a union G of cosets can be used to bound the size of G and the number of disjoint the electronic journal of combinatorics 26(2) (2019), #P2.34 8 pairs it is involved in. For this we require some further notation. Given a graph G and an integer t ⩾1, we denote by kt(G) the number of t-cliques in G. In particular, we have k1(G) = v(G) and k2(G) = e(G). When the graph G is clear from context, we write kt for kt(G). Proposition 10. There is some c > 0 such that if, for 2 ⩽k1 ⩽cn1/2, G is the union of k1 cosets in Sn with intersection graph G, then the following hold: (a) \|G\| = k1(n −1)! −k2(n −2)! + k3(n −3)! ± k4(n −4)!, (b) dp(π, G) = k1Dn−1 −k2Dn−2 ± 3k1k2(n −3)! 2.3 Intersection graphs for every π ∈Sn \ G, (c) dp(G) = k1 2 (n −1)!Dn−1 −(k1 −1)k2(n −2)!Dn−1 ± 2k2 1k2(n −1)!(n −3)!, and (d) dp(G) ⩾dp(T (n, \|G\|)), with equality if and only if G is canonical. (d) dp(G) ⩾dp(T (n, \|G\|)), with equality if and only if G is canonical. The proof of Proposition 10, though elementary, is rather technical, involving careful and repeated application of the Bonferroni inequalities to estimate the number of per- mutations in a union of cosets that are disjoint from a given permutation. We therefore defer the proof to Appendix A, and instead proceed to show how the proposition can be combined with Lemma 7 to prove Theorem 2. 2.4 Supersaturation Here we prove Theorem 2. Our strategy is to use Lemma 7 to reduce the statement to the case when F is a union of some cosets in Sn, and then apply Proposition 10 to obtain the desired lower bound on the number of disjoint pairs. of of Theorem 2. Let c7 and C7 be the positive constants from Lemma 7, and set c = min c7, 10−5C−2 7 , 10−2 . Now letting n, k and ε be as in the statement of the theorem, let F ⊆Sn be an extremal family of s = (k + ε)(n −1)! permutations. In the ﬁrst part of our proof, we establish Claim 11, a rough structural result for F. Claim 11. Either F contains k cosets or F is contained in a union of k cosets. Claim 11. Either F contains k cosets or F is contained in a union of k cosets. Proof. Since \|ε\| ⩽ck−3 ⩽c7 and dp(F) ⩽dp(T (n, s)) by the extremality of F, we may apply Lemma 7 to F with β7 = 0 to ﬁnd a union G = Sk i=1 Ti of k cosets in Sn such that \|F∆G\| ⩽C7k2 1 n + r 6\|ε\| k ! (n −1)!. (8) (8) (8) Let A = F \ G and B = G \ F. We may assume that A ̸= ∅and B ̸= ∅, otherwise either F ⊆G or G ⊆F as claimed. We shall show that if the permutations in A are replaced by those in B, the number of disjoint pairs decreases, which then contradicts the 9 the electronic journal of combinatorics 26(2) (2019), #P2.34 extremality of F. Fix two arbitrary permutations σ ∈A and π ∈B. It suﬃces to show that dp(σ, F) > dp(π, F). F). ⩽ck−3, k ⩽cn1/2 and c ⩽10−5C−2 7 , we see that p( , ) p( , ) First, using (8), \|ε\| ⩽ck−3, k ⩽cn1/2 and c ⩽10−5C−2 7 , we see that \|A\| + \|B\| = \|F∆G\| ⩽0.02(n −1)!. (9) (9) Recall that any two cosets in Sn have at most (n −2)! elements in common, and that a permutation is disjoint from Dn−1 other permutations in any coset not containing it. Since Dn−1 = dn−1 + dn−2 = (e−1 + o(1))(n −1)! and k ⩽cn1/2 ⩽10−2n1/2, we have Recall that any two cosets in Sn have at most (n −2)! elements in common, and that a permutation is disjoint from Dn−1 other permutations in any coset not containing it. Since Dn−1 = dn−1 + dn−2 = (e−1 + o(1))(n −1)! and k ⩽cn1/2 ⩽10−2n1/2, we have dp(σ, F) ⩾dp(σ, G \ B) ⩾dp(σ, G) −\|B\| ⩾ k X i=1 dp(σ, Ti) − X i<j \|Ti ∩Tj\| −\|B\| (9) ⩾kDn−1 − k 2 (n −2)! −0.02(n −1)! > (k −0.1)Dn−1. On the other hand, π is contained in G, and thus can have disjoint pairs to at most k −1 of the cosets in G. Hence, dp(π, F) = dp(π, F ∩G) + dp(π, F \ G) = dp(π, G \ B) + dp(π, A) ⩽(k −1)Dn−1 + \|A\| (9) < (k −0.2)Dn−1 < dp(σ, F). Next, we combine this claim with Proposition 10 to bound dp(F) from below and ﬁnish the proof. We consider two cases, depending on the sign of ε. Claim 11. Either F contains k cosets or F is contained in a union of k cosets. Moreover, at the beginning of the process, dp(G) ⩾dp(T (n, t)) by Proposition 10(d). Thus same process for the family T (n, t), we see that the number of disjoint pairs is decreased by exactly (k −1)Dn−1 each time we remove a permutation from the last coset in T (n, t). Moreover, at the beginning of the process, dp(G) ⩾dp(T (n, t)) by Proposition 10(d). Thus dp(F) ⩾dp(G) −(t −s)(k −1)Dn−1 ⩾dp(T (n, t)) −(t −s)(k −1)Dn−1 = dp(T (n, s)), completing the proof in Case 1. completing the proof in Case 1. completing the proof in Case 1. Case 2: ε > 0. This case will be handled rather diﬀerently. Since ε > 0, formula (3) gives p g p Case 2: ε > 0. This case will be handled rather diﬀerently. Since ε > 0, formula (3) gives dp(T (n, s)) = k 2 + kε (n −1)!Dn−1. (10) (10) Also, as \|F\| = (k + ε)(n −1)! > k(n −1)!, Claim 11 shows that F = G ⊔H, where G is a union of k (not necessarily disjoint) cosets in Sn. Also, as \|F\| = (k + ε)(n −1)! > k(n −1)!, Claim 11 shows that F = G ⊔H, where G is a union of k (not necessarily disjoint) cosets in Sn. If G is a union of k disjoint cosets, then dp(F) = dp(G) + dp(H, G) + dp(H) ⩾dp(G) + dp(H, G) = dp(T (n, s)), where equality holds if and only if dp(H) = 0, that is, H is intersecting. It remains to verify that dp(F) ⩾dp(T (n, s)) when the k cosets of G are not pairwise disjoint. In this scenario we in fact have a strict inequality. Indeed, let G be the intersec- tion graph of G. We shall use the inequality dp(F) ⩾dp(G) + dp(H, G) to lower bound dp(F). By Proposition 10(c), we have dp(G) = k 2 (n −1)!Dn−1 −(k −1)k2(n −2)!Dn−1 ± 2k2k2(n −1)!(n −3)!. (11) (11) We next estimate the number of disjoint pairs between H and G. By Proposition 10(b), We next estimate the number of disjoint pairs between H and G. By Proposition 10(b), dp(π, G) = kDn−1 −k2Dn−2 ± 3kk2(n −3)! for every π ∈H. Furthermore, using Proposition 10(a) to estimate \|G\| gives \|H\| = \|F\| −\|G\| = ε(n −1)! + k2(n −2)! ± 2kk2(n −3)!. Claim 11. Either F contains k cosets or F is contained in a union of k cosets. Case 1: ε ⩽0. We have shown in Claim 11 that either F ⊇G or F ⊆G, where G is a Next, we combine this claim with Proposition 10 to bound dp(F) from below and ﬁnish the proof. We consider two cases, depending on the sign of ε. Case 1: ε ⩽0. We have shown in Claim 11 that either F ⊇G or F ⊆G, where G is a union of some k cosets. Let t = \|G\|. Case 1: ε ⩽0. We have shown in Claim 11 that either F ⊇G or F ⊆G, where G is a union of some k cosets. Let t = \|G\|. We ﬁrst treat the case F ⊇G. Observe that since t ⩽s, T (n, t) ⊆T (n, s), and from Proposition 10(d) we have dp(G) ⩾dp(T (n, t)). Since s = (k +ε)(n−1)! ⩽k(n−1)!, the family T (n, s) is contained in a union of k disjoint cosets in Sn. Hence dp(π, T (n, s)) ⩽ (k −1)Dn−1 for every π ∈T (n, s) \ T (n, t), and there are \|F \ G\| such permutations π. M G i i f k t h Moreover, as G is a union of k cosets, we have Moreover, as G is a union of k cosets, we have dp(σ, G) ⩾kDn−1 − k 2 (n −2)! > (k −0.5)Dn−1 for each σ ∈F \ G, and there are again \|F \ G\| such permutations σ. Altogether, we deduce that, as required, for each σ ∈F \ G, and there are again \|F \ G\| such permutations σ. Altogether, we deduce that, as required, dp(F) ⩾dp(G) + X σ∈F\G dp(σ, G) > dp(T (n, t)) + X π∈T (n,s) π /∈T (n,t) dp(π, T (n, s)) ⩾dp(T (n, s)). We next deal with the case F ⊆G. It is convenient to think of F as a family obtained by removing permutations in G one by one. Since G is a union of k cosets in Sn, the number of disjoint pairs is decreased by at most (k −1)Dn−1 each time. Following the 10 the electronic journal of combinatorics 26(2) (2019), #P2.34 same process for the family T (n, t), we see that the number of disjoint pairs is decreased by exactly (k −1)Dn−1 each time we remove a permutation from the last coset in T (n, t). 3 Supersaturation for uniform set systems In this section, we shall prove Theorem 3, but ﬁrst let us examine dp(n, k, s). When n k − n−r+1 k ⩽s ⩽ n k − n−r k , if we write s = n k − n−r+1 k + γ n−r k−1 where γ ∈[0, 1], L(n, k, s) consists of the full stars with centres in [r −1], with a further γ n−r k−1 sets from the star with centre r. Let L(i) = {L ∈L(n, k, s) : i ∈L} and L∗(i) = {L ∈L(n, k, s) : min L = i}. One can then compute the number of disjoint pairs as dp(n, k, s) = r−1 X i=1 dp(∪j>iL∗(j), L∗(i)) = r−1 X i=1 s − n k − n −i k n −i −k k −1 . (13) (13) ( ) This expression is quite unwieldy, so we shall make use of a few estimates. We ﬁrst note that any set outside a star has exactly n−k−1 k−1 disjoint pairs with the star, so dp(n, k, s) ⩽ X 1⩽i<j⩽r−1 dp(L(i), L(j)) + X 1⩽i⩽r−1 dp(L(i), L∗(r)) ⩽ r −1 2 + (r −1)γ n −1 k −1 n −k −1 k −1 . (14) (14) This is only an upper bound as we overcount disjoint pairs involving sets belonging to multiple stars. For an even simpler upper bound, observe that every set belongs to at least one of the r stars, and is not disjoint from any other set in its star. In the worst case, there are an equal number of sets in each star, with each set disjoint from at most a 1 −1 r -proportion of the family. We thus have This is only an upper bound as we overcount disjoint pairs involving sets belonging to multiple stars. For an even simpler upper bound, observe that every set belongs to at least one of the r stars, and is not disjoint from any other set in its star. In the worst case, there are an equal number of sets in each star, with each set disjoint from at most a 1 −1 r -proportion of the family. We thus have dp(n, k, s) ⩽1 2 1 −1 r s2. 3.1 Tools There are two main tools we use in our proof of Theorem 3: a removal lemma for disjoint pairs, and the expander-mixing lemma applied to the Kneser graph. Before proving the theorem, we introduce these tools and explain how we shall use them. 3 Supersaturation for uniform set systems (15) (15) We shall use these upper bounds on the number of disjoint pairs present in any ex- tremal family. Claim 11. Either F contains k cosets or F is contained in a union of k cosets. ing that (n −1)!Dn−2 = (n −2)!Dn−1 ± (n −1)!(n −3)!, we get dp(H, G) = (kDn−1 −k2Dn−2 ± 3kk2(n −3)!) \|H\| = kε(n −1)!Dn−1 + (k −ε)k2(n −2)!Dn−1 ± 3k2k2(n −1)!(n −3)!. (12) (12) Combining (10), (11) and (12), and simplifying gives dp(G) + dp(H, G) −dp(T (n, s)) ⩾(1 −ε)k2(n −2)!Dn−1 −5k2k2(n −1)!(n −3)! > 0, since k2 ⩾1, k ⩽cn1/2 ⩽10−2n1/2 and Dn−1 ⩾(n −1)!/3. Thus dp(F) ⩾dp(G) + dp(H, G) > dp(T (n, s)), completing the proof of Theorem 2. Combining (10), (11) and (12), and simplifying gives Combining (10), (11) and (12), and simplifying gives dp(G) + dp(H, G) −dp(T (n, s)) ⩾(1 −ε)k2(n −2)!Dn−1 −5k2k2(n −1)!(n −3)! > 0, 1/2 2 1/2 dp(G) + dp(H, G) −dp(T (n, s)) ⩾(1 −ε)k2(n −2)!Dn−1 −5k2k2(n −1)!(n −3)! > 0, + dp(H, G) −dp(T (n, s)) ⩾(1 −ε)k2(n −2)!Dn−1 −5k2k2(n −1)!(n −3)! > 0, since k2 ⩾1, k ⩽cn1/2 ⩽10−2n1/2 and Dn−1 ⩾(n −1)!/3. Thus dp(F) ⩾dp(G) + dp(H, G) > dp(T (n, s)), completing the proof of Theorem 2. since k2 ⩾1, k ⩽cn1/2 ⩽10−2n1/2 and Dn−1 ⩾(n −1)!/3. Thus dp(F) ⩾dp(G) + dp(H, G) > dp(T (n, s)), completing the proof of Theorem 2. the electronic journal of combinatorics 26(2) (2019), #P2.34 11 3.1.1 Removal lemma Using a result of Filmus [21], Das and Tran [11, Theorem 1.2] proved the following removal lemma, showing that large families with few disjoint pairs must be close to a union of stars. Lemma 12 (Das and Tran). There is an absolute constant C > 1 such that if n, k and ℓare positive integers satisfying n > 2kℓ2, and F ⊂ [n] k is a family of size \|F\| = 12 the electronic journal of combinatorics 26(2) (2019), #P2.34 (ℓ−α) n−1 k−1 with at most ℓ 2 + β n−1 k−1 n−k−1 k−1 disjoint pairs, where max{2ℓ\|α\| , \|β\|} ⩽ n−2k (20C)2n, then there is a family S that is the union of ℓstars satisfying \|F∆S\| ⩽C ((2ℓ−1)α + 2β) n n −2k n −1 k −1 . Observe that the bound on the number of disjoint pairs in the lemma is very similar to the upper bound given in (14). Thus one may interpret this result as a stability version of our previous calculation: any family with size similar to the union of r −1 stars without many more disjoint pairs can be made a union of r −1 stars by exchanging only a small number of sets. Given this stability, it is not diﬃcult to show that the lexicographic ordering is optimal in this range. Corollary 13. There is some constant c > 0 such that if r, k and n are positive integers satisfying n ⩾2c−1k2r3, and s = n k − n−r+1 k + γ n−r k−1 , where γ ∈[0, c r], any family F ⊂ [n] k of size s has dp(F) ⩾dp(n, k, s). Proof. Let C be the constant from Lemma 12 and choose c = n−2k 2(20C)2n. For the given range of s, the lexicographic initial segment has r −1 full stars with one small partial star, so we wish to apply Lemma 12 with ℓ= r −1. [ ] Let F be a subfamily of [n] k with s sets and the minimum number of disjoint pairs. Note that s = (ℓ+ α) n−1 k−1 , where γ −kr2 2n ⩽α ⩽γ. In particular, we have \|α\| ⩽ c r. the electronic journal of combinatorics 26(2) (2019), #P2.34 3.1.1 Removal lemma By optimality of F, and our calculation in (14), we also have dp(F) ⩽dp(n, k, s) ⩽ ℓ 2 + (r −1)γ n−1 k−1 n−k−1 k−1 , and hence take β = (r −1)γ. 2 k 1 k 1 We thus have \|β\| = (r −1)γ ⩽c < n−2k (20C)2n and 2ℓ\|α\| ⩽2c = n−2k (20C)2n, and may apply Lemma 12. This gives a family S, a union of ℓstars, such that We thus have \|β\| = (r −1)γ ⩽c < n−2k (20C)2n and 2ℓ\|α\| ⩽2c = n−2k (20C)2n, and hence we may apply Lemma 12. This gives a family S, a union of ℓstars, such that \|F∆S\| ⩽C ((2ℓ−1)α + 2β) n n −2k n −1 k −1 ⩽4cC n n −2k n −1 k −1 < 1 200 n −1 k −1 . Hence, we know an optimal family F must be close to a union of ℓstars S. We ﬁrst show that S ⊆F. If not, there is some set F ∈F \ S in our family, as well as a set G ∈S \ F missing from our family (note that \|F\| ⩾\|S\|). For each star in S, there are at most n−1 k−1 − n−k−1 k−1 ⩽k2 n n−1 k−1 sets intersecting F, and hence F intersects at most ℓk2 n n−1 k−1 sets from F ∩S. Even if F intersects every set in F \ S, it can intersect at most 2 ℓk2 n + 1 200 n−1 k−1 < 1 2 n−1 k−1 sets in F. ℓk2 n + 1 200 n−1 k−1 < 1 2 n−1 k−1 sets in F. On the other hand, the set G is in one of the stars of S, which contains at least n−1 k−1 −\|S \ F\| ⩾ 1 − 1 200 n−1 k−1 > 2 3 n−1 k−1 sets of F. Hence replacing F by G in F strictly increases the number of intersecting pairs, thus decreasing the number of disjoint pairs, contradicting the optimality of F. p , g p y Thus we have S ⊆F. Let H = F \ S. We then have dp(F) = dp(S) + dp(S, H) + dp(H). 3.1.2 Expander-mixing lemma The second tool we shall use is the expander-mixing lemma2 of Alon and Chung [1], which relates the spectral gap of a d-regular graph to its edge distribution. Since the graph is d-regular, its largest eigenvalue is trivially d, corresponding to the constant eigenvector. In what follows, an (n, d, λ)-graph is a d-regular n-vertex graph whose largest non-trivial eigenvalue (in absolute value) is λ. Lemma 14 (Alon and Chung). Let G be an (n, d, λ)-graph, and let S, T be two vertex subsets. Then d \|S\| \|T\| e(S, T) −d \|S\| \|T\| n ⩽λ p \|S\| \|T\|. As we are interested in counting disjoint pairs, we shall apply the expander-mixing lemma to the Kneser graph, where the vertices are sets and edges represent disjoint pairs. The spectral properties of the Kneser graph were determined by Lov´asz [35]. In particular, the Kneser graph KG(m, a) for a-uniform sets over [m] is an m a , m−a a , m−a−1 a−1 -graph. We shall combine this with Lemma 14 to obtain a useful corollary. Corollary 15. Given 1 ⩽i < j ⩽n, and k-uniform families F(i) of subsets of [n] containing i and F(j) of subsets of [n] containing j, dp(F(i), F(j)) ⩾ 1 −k2 n \|F(i)\| \|F(j)\| −3k 2n (\|F(i)\| + \|F(j)\|) n −1 k −1 . Proof. Without loss of generality we assume i = n −1 and j = n. Let A = {F \ {n −1} : F ∈F(n −1), n /∈F} and B = {F \ {n} : F ∈F(n), n −1 /∈F}, and observe that dp(F(n −1), F(n)) = dp(A, B). Furthermore, we have A, B ⊆ [n−2] k−1 , with \|A\| ⩾\|F(n −1)\| − n−2 k−2 and \|B\| ⩾\|F(n)\| − n−2 k−2 . Proof. Without loss of generality we assume i = n −1 and j = n. Let A = {F \ {n −1} : F ∈F(n −1), n /∈F} and B = {F \ {n} : F ∈F(n), n −1 /∈F}, and observe that dp(F(n −1), F(n)) = dp(A, B). Furthermore, we have A, B ⊆ [n−2] k−1 , with \|A\| ⩾\|F(n −1)\| − n−2 k−2 and \|B\| ⩾\|F(n)\| − n−2 k−2 . 3.1.1 Removal lemma 13 the electronic journal of combinatorics 26(2) (2019), #P2.34 Since every set outside a union of ℓstars is contained in exactly the same number of disjoint pairs with sets from the stars, the terms dp(S) and dp(S, H) are determined by ℓand s, and independent of the structure of F. It follows that dp(F) is minimised precisely when dp(H) is minimised. As \|H\| = \|F\| −\|S\| = γ n−r k−1 ⩽ n−r k−1 , we may take H to be an intersecting family, and so dp(H) = 0 is possible. Since in L(n, k, s), the set H corresponds to the ﬁnal (intersecting) partial star, it follows that L(n, k, s) is optimal, and so dp(F) ⩾dp(n, k, s) for any family F of s sets. 2This also plays a key role in the proof of Lemma 12, and so in some sense is the foundation for this entire proof. 3.2 Proof of Theorem 3 With the preliminaries in place, we now proceed with the proof of the main theorem Proof of Theorem 3. We prove the result by induction on s. For the base case, if s ⩽ n k − n−1 k = n−1 k−1 , then L(n, k, s) consists of sets that all contain the element 1. Hence dp(L(n, k, s)) = 0, which is clearly optimal. ( ( )) For the induction step, we have s = n k − n−r+1 k + γ n−r k−1 for some r ⩾2 and γ ∈(0, 1]. Letting c be the positive constant from Corollary 13, if γ ∈(0, c r], we are done. Hence we may assume γ ∈( c r, 1]. Let F be a k-uniform set family over [n] of size s with the minimum number of disjoint pairs. In particular, we must have dp(F) ⩽dp(n, k, s). For any set F ∈F, by the induction hypothesis we have dp(F \{F}) ⩾dp(n, k, s−1). Hence dp({F}, F) = dp(F)−dp(F \{F}) ⩽dp(n, k, s)−dp(n, k, s−1), where the right- hand side is the number of disjoint pairs involving the last set L added to L(n, k, s). The set L is in a star of size γ n−r k−1 > γ 2 n−1 k−1 , and hence intersects at least γ 2 n−1 k−1 sets in L(n, k, s). Thus it follows that every set F ∈F must also intersect at least γ 2 n−1 k−1 sets in F. Now suppose that F contains a full star; without loss of generality, assume F(1) consists of all n−1 k−1 sets containing the element 1. Let G = F \ F(1). Since F(1) is intersecting, and every set outside F(1) has exactly n−k−1 k−1 disjoint pairs with sets in F(1), we have dp(F) = dp(F(1), G) + dp(G) = \|G\| n −k −1 k −1 + dp(G). Now G is a k-uniform set family over [n]\{1} of size s′ = s− n−1 k−1 , and so by induction dp(G) is minimised by the initial segment of the lexicographic order of size s′. However, adding back the full star F(1) gives the initial segment of the lexicographic order of size s, and as a result dp(F) ⩾dp(L(n, k, s)) = dp(n, k, s). 3.1.2 Expander-mixing lemma k 2 k 2 Since disjoint pairs between A and B correspond to edges between the corresponding vertex sets in the Kneser graph KG(n −2, k −1), Lemma 14 gives dp(F(n −1), F(n)) = dp(A, B) ⩾ n−k−1 k−1 n−2 k−1 \|A\| \|B\| − n −k −2 k −2 p \|A\| \|B\|. 2This also plays a key role in the proof of Lemma 12, and so in some sense is the foundation for this entire proof. 2This also plays a key role in the proof of Lemma 12, and so in some sense is the foundation for this entire proof. the electronic journal of combinatorics 26(2) (2019), #P2.34 14 We now recall that \|F(n −1)\|− n−2 k−2 ⩽\|A\| ⩽\|F(n −1)\|, with similar bounds holding for B. We shall also remove the square root by appealing to the AM-GM inequality. Also observe that n−k−1 k−1 ⩾ 1 −k2 n n−2 k−1 and n−k−2 k−2 ⩽k n n−1 k−1 . Hence dp(F(n−1), F(n)) is at least 1 −k2 n \|F(n −1)\| − n−2 k−2 \|F(n)\| − n−2 k−2 − k 2n (\|F(n −1)\| + \|F(n)\|) n−1 k−1 . Noting that n−2 k−2 ⩽k n n−1 k−1 , taking the main order term and collecting the negative terms then gives the desired bound. 3.2 Proof of Theorem 3 Hence we may assume that F does not contain any full star. In particular, this means for any set F ∈F and element i ∈[n], we have the freedom to replace F with some set containing i. We shall use such switching operations to show that F, like L(n, k, s), must have a cover of size r, from which the result will easily follow. Relabel the elements if necessary so that for every i ∈[n], i is the vertex of maximum degree in F\|[n]\[i−1]. Let F∗(i) = {F ∈F : min F = i} be those sets containing i that do 15 the electronic journal of combinatorics 26(2) (2019), #P2.34 not contain any previous element. Deﬁne X = x ∈[n] : \|F∗(x)\| ⩾γ 4k n −1 k −1 , and let F1 = {F ∈F : F ∩X ̸= ∅} and F2 = F \ F1 = {F ∈F : F ∩X = ∅}. We shall show that X is a cover for F (that is, F1 = F and F2 = ∅), but to do so we shall ﬁrst have to establish a few claims. The ﬁrst shows that X cannot be too big. Claim 16. \|X\| ⩽4kr γ . Proof. Observe that the families {F∗(x) : x ∈X} partition F1. Hence we have n 1 γ n 1 Proof. Observe that the families {F∗(x) : x ∈X} partition F1. Hence we have r n −1 k −1 ⩾s = \|F\| ⩾\|F1\| = X x∈X \|F∗(x)\| ⩾γ 4k n −1 k −1 \|X\| , from which the claim immediately follows. The next claim asserts that every set in F must intersect many sets in F1. Claim 17. Every set F ∈F intersects at least γ 4 n−1 k−1 sets in F1. Proof. First observe that any element i ∈[n] is contained in fewer than γ 4k n−1 k−1 sets in F2. Indeed, the elements x ∈X have all of their sets in F1, and hence have F2-degree zero. Thus the F2-degree of any element is its degree in F\|[n]\X. If the element of largest F2-degree was contained in at least γ 4k n−1 k−1 sets from F2, then it would have been in X, giving a contradiction. 1 g g Now recall that every set F ∈F must intersect at least γ 2 n−1 k−1 sets in F. 3.2 Proof of Theorem 3 By Claim 17, this quantity must be at least γ 4 n−1 k−1 , which gives \|F∗(i)\| ⩾ γ 4 −13k2r2 cn n −1 k −1 ⩾γ 8 n −1 k −1 , since n > Ck2r3 for some large enough constant C. since n > Ck2r3 for some large enough constant C. Hence for every i ∈X, we in fact have the much stronger bound \|F∗(i)\| ⩾γ 8 n−1 k−1 . Repeating the calculation of Claim 16 with this new bound gives \|X\| ⩽8r γ , as required. Hence for every i ∈X, we in fact have the much stronger bound \|F∗(i)\| ⩾γ 8 n−1 k−1 . Repeating the calculation of Claim 16 with this new bound gives \|X\| ⩽8r γ , as required. Our next claim shows that X is indeed a cover for F. Our next claim shows that X is indeed a cover for F. Claim 19. X is a cover for F; that is, F1 = F and F2 = ∅. Claim 19. X is a cover for F; that is, F1 = F and F2 = ∅. m 19. X is a cover for F; that is, F1 = F and F2 = ∅. Proof. Suppose for contradiction we had some set F ∈F2. By Claim 17, at least γ 4 n−1 k−1 sets in F1 must intersect F. However, each such set must contain at least one element of X, which by Claim 18 has size at most 8r γ , together with one element from F. Hence there are at most k \|X\| n−2 k−2 ⩽ 8k2r γn n−1 k−1 sets in F1 intersecting F. Since γ ⩾ c r and n > Ck2r3 for some large enough constant C, this is less than γ 4 n−1 k−1 , giving the desired contradiction. Now observe that every set in F∗(i) meets X in the element i. If it intersects X in further elements, there are at most \|X\| ⩽8r γ choices from the other element, and at most n−2 k−2 ⩽k n n−1 k−1 choices for the rest of the set. 3.2 Proof of Theorem 3 The number of sets in F2 it can intersect is at most X i∈F \|F2(i)\| ⩽k · γ 4k n −1 k −1 = γ 4 n −1 k −1 . the remaining γ 4 n−1 k−1 intersections must come from sets in F1. ce the remaining γ 4 n−1 k−1 intersections must come from sets in F1. The following claim combines our previous results with the expander-mixing corollary to provide much sharper bounds on the size of X. The following claim combines our previous results with the expander-mixing corollary to provide much sharper bounds on the size of X. 18. \|X\| ⩽8r γ . Claim 18. \|X\| ⩽8r γ . Claim 18. \|X\| ⩽8r γ . Proof. For every i ∈X, we shall estimate dp(F∗(i), F1). Since {F∗(x) : x ∈X} is a par- tition of F1, we have dp(F∗(i), F1) = P j∈X\{i} dp(F∗(i), F∗(j)). Applying Corollary 15, we get Proof. For every i ∈X, we shall estimate dp(F∗(i), F1). Since {F∗(x) : x ∈X} is a par- tition of F1, we have dp(F∗(i), F1) = P j∈X\{i} dp(F∗(i), F∗(j)). Applying Corollary 15, we get dp(F∗(i), F1) = X j∈X\{i} dp(F∗(i), F∗(j)) ⩾ X j∈X\{i} 1 −k2 n \|F∗(i)\| \|F∗(j)\| −3k 2n (\|F∗(i)\| + \|F∗(j)\|) n −1 k −1 ⩾ 1 −k2 n (\|F1\| −\|F∗(i)\|) \|F∗(i)\| −3k 2n (\|X\| \|F∗(i)\| + \|F1\|) n −1 k −1 . the electronic journal of combinatorics 26(2) (2019), #P2.34 16 the electronic journal of combinatorics 26(2) (2019), #P2.34 the electronic journal of combinatorics 26(2) (2019), #P2.34 16 By averaging, some F ∈F∗(i) is disjoint from at least 1 −k2 n (\|F1\| −\|F∗(i)\|) −3k 2n \|X\| + \|F1\| \|F∗(i)\| n −1 k −1 sets in F1. By Claim 16, \|X\| ⩽4kr γ . Since \|F1\| ⩽s ⩽r n−1 k−1 , and \|F∗(i)\| ⩾ γ 4k n−1 k−1 , we can lower bound this expression by dp({F}, F1) ⩾ 1 −k2 n (\|F1\| −\|F∗(i)\|) −12k2r γn n −1 k −1 . Recalling that γ ⩾c r, we ﬁnd that F intersects at most 1\| −dp({F}, F1) ⩽\|F∗(i)\| + k2 n \|F1\| + 12k2r γn n −1 k −1 ⩽\|F∗(i)\| + 13k2r2 cn n −1 k −1 sets from F1. 3.2 Proof of Theorem 3 Hence at most 8kr γn n−1 k−1 < γ 8 n−1 k−1 ⩽\|F∗(i)\| sets in F∗(i) meet X in at least two elements, and thus there must be some set Fi ∈F∗(i) such that Fi ∩X = {i}. We shall use this fact to establish the following claim. Cl i 20 F ll i j X \|F∗(j)\| \|F∗(i)\| 8k2rn 1 Claim 20. For all i, j ∈X, \|F∗(j)\| −\|F∗(i)\| ⩽8k2r γn n−1 k−1 . 17 the electronic journal of combinatorics 26(2) (2019), #P2.34 Proof. Suppose for contradiction \|F∗(j)\| > \|F∗(i)\| + 8k2r γn n−1 k−1 . Let Fi ∈F∗(i) be such that Fi ∩X = {i}. Then Fi intersects only the sets that contain i together with sets containing some other element in X and some element in Fi. This gives a total of at most \|F∗(i)\| + k \|X\| n −2 k −2 ⩽\|F∗(i)\| + 8k2r γn n −1 k −1 < \|F∗(j)\| sets. On the other hand, if we replace Fi by some set G containing j (which we may do, since we assume the family F(j) is not a full star), we would gain at least \|F∗(j)\| intersecting pairs. Hence F ∪{G} \ {Fi} is a family of s sets with strictly fewer disjoint pairs, contradicting the optimality of F. This claim shows that the sets in F are roughly equally distributed over the families F∗(i), i ∈X. To simplify the notation, we let m = \|X\|, and so we have X = [m]. By Claim 18, m ⩽8r γ . We shall now proceed to lower-bound the number of disjoint pairs in F. Note that dp(F) = P 1⩽i<j⩽m dp(F∗(i), F∗(j)). We shall use Corollary 15 to bound these summands. We let si = \|F∗(i)\| n−1 k−1 −1 and set s = s n−1 k−1 −1 = P i si. Note that s = n k − n−r+1 k + γ n−r k−1 and γ ∈ c r, 1 implies r −1 ⩽r −1 + γ −kr2 2n ⩽s ⩽r. the electronic journal of combinatorics 26(2) (2019), #P2.34 3.2 Proof of Theorem 3 the electronic journal of combinatorics 26(2) (2019), #P2.34 18 Now recall we have s = s n−1 k−1 , s ⩾r −1, γ ⩾c r, m ⩽8r γ ⩽8c−1r2 and n ⩾Ck2r3 for some suﬃciently large constant C. Substituting these bounds into (16), we ﬁnd Now recall we have s = s n−1 k−1 , s ⩾r −1, γ ⩾c r, m ⩽8r γ ⩽8c−1r2 and n ⩾Ck2r3 for some suﬃciently large constant C. Substituting these bounds into (16), we ﬁnd dp(F) > 1 2 1 −1 m − 1 r(r + 1) s2. However, by (15), we must have However, by (15), we must have dp(F) ⩽1 2 1 −1 r s2. These two bounds together imply 1 m + 1 r(r+1) > 1 r, which in turn gives m < r + 1. This shows m = r, and X is thus a cover of size r. Hence it follows that F is covered by some r elements, which we may without loss of generality assume to be [r]. We now ﬁnish with a similar argument as in the proof of Corollary 13: let S be the union of the r stars with centres in [r], and let G = S \ F be the missing sets. Then dp(F) = dp(S) −dp(G, S) + dp(G) is minimised when G is an intersecting family of sets that each meet [r] in precisely one element, which is the case for F = L(n, k, s). Hence dp(F) ⩾dp(n, k, s), completing the proof of the theorem. The problem of minimising the number of disjoint pairs can be viewed as an isoperi- metric inequality in the Kneser graph. The following lemma links isoperimetric problems for small and large families (see, for instance, [10, Lemma 2.3]). Lemma 22. Let G = (V, E) be a regular graph on n vertices. Then S ⊂V minimises the number of edges e(S) over all sets of \|S\| vertices if and only if V \ S minimises the number of edges over all sets of n −\|S\| vertices. The following corollary, which is a direct consequence of Theorem 3 and Lemma 22, shows that the complements of the lexicographical initial segments, which are isomorphic to initial segments of the colexicographical order, are optimal when s is close to n k . Corollary 23. 3.2 Proof of Theorem 3 We then have dp(F) = X 1⩽i<j⩽m dp(F∗(i), F∗(j)) ⩾ X 1⩽i<j⩽m 1 −k2 n \|F∗(i)\| \|F∗(j)\| −3k 2n (\|F∗(i)\| + \|F∗(j)\|) n −1 k −1 ⩾ " 1 −k2 n X i<j sisj −3k 2n X i<j (si + sj) # n −1 k −1 2 ⩾ " 1 2 1 −k2 n s2 − X i s2 i ! −3kms 2n # n −1 k −1 2 ⩾1 2 " s2 −k2s2 n − X i s2 i −3kms n # n −1 k −1 2 . 1⩽i<j⩽m ⩾ " 1 −k2 n X i<j sisj −3k 2n X i<j (si + sj) # n −1 k −1 2 ⩾ " 1 2 1 −k2 n s2 − X i s2 i ! −3kms 2n # n −1 k −1 2 ⩾1 2 " s2 −k2s2 n − X i s2 i −3kms n # n −1 k −1 2 . Since P i si = s, there must be some ℓwith sℓ⩽ s m, and Claim 20 then implies that for every i, si ⩽s m + 8k2r γn . Hence P i s2 i ⩽(maxi si) P i si ⩽ s m + 8k2r γn s, giving Since P i si = s, there must be some ℓwith sℓ⩽ s m, and Claim 20 then implies that for every i, si ⩽s m + 8k2r γn . Hence P i s2 i ⩽(maxi si) P i si ⩽ s m + 8k2r γn s, giving dp(F) ⩾1 2 1 −1 m −k2 n −8k2r γsn −3km sn s n −1 k −1 2 . (16) (16) Claim 21. \|X\| = r; that is, F has a cover of size r. Claim 21. \|X\| = r; that is, F has a cover of size r. Proof. Since s > n k − n−r+1 k , F cannot be covered by r −1 elements. Hence we must have m = \|X\| ⩾r. Proof. Since s > n k − n−r+1 k , F cannot be covered by r −1 elements. Hence we must have m = \|X\| ⩾r. 3.2 Proof of Theorem 3 There exists a positive constant C such that the following statement holds. Provided n ⩾Ck2r3 and n−r k ⩽s ⩽ n k , [n] k \ L(n, k, n k −s) minimises the number of disjoint pairs among all systems of s sets in [n] k . the number of families with property P is (T + o(1))2N0. We will apply Lemma 24 with P being the property of avoiding a matching of size s or, equivalently, of not containing s pairwise disjoint sets. To do so, we ﬁrst bound the number of maximal families with no matching of size s. Proposition 25. The number of maximal k-uniform families over [n] with no matching of size s is at most n k (sk k ). Proposition 25. The number of maximal k-uniform families over [n] with no matching f i i n(sk k ) Proposition 25. The number of maximal k-uniform families over [n] with no matching of size s is at most n k (sk k ). Proof. Given F ⊂ [n] k , let I(F) = G ∈ [n] k : F ∪{G} does not have s pairwise disjoint sets}. of size s is at most n k (sk k ). Proof. Given F ⊂ [n] k , let Note that F does not contain a matching of size s if and only if F ⊂I(F), while F is maximal if and only if I(F) = F. Given a maximal family F, we say that G ⊂F is a generating family of F if I(G) = F. Note that F does not contain a matching of size s if and only if F ⊂I(F), while F is maximal if and only if I(F) = F. Given a maximal family F, we say that G ⊂F is a generating family of F if I(G) = F. ( ) Let F0 = {F1, . . . , Fm} ⊂F be a minimal generating family of F. By the minimality of F0, we must have I(F0 \ {Fi}) ⊋F = I(F0), for each 1 ⩽i ⩽m. Hence we can ﬁnd some set Gi,s−1 ∈I(F0 \ {Fi}) \ I(F0). It follows that there exist s −2 sets Gi,1, . . . , Gi,s−2 in F0 such that Fi, Gi,1, . . . , Gi,s−2 and Gi,s−1 are pairwise disjoint, while for every j ̸= i, Fj, Gi,1, . . . , Gi,s−2 and Gi,s−1 are not pairwise disjoint. In other words, if we let Gi = Gi,1 ∪. . . ∪Gi,s−1, then Fi ∩Gi = ∅and Fj ∩Gi ̸= ∅for j ̸= i. Given these conditions, we may apply the Bollob´as set-pairs inequality [6] to bound the size of F0. Theorem 26 (Bollob´as). Let A1, . 4.1 Families with no matching of size s In this section we describe the structure of k-uniform set families without matchings of size s. The following lemma, which follows readily from [3, Lemmas 2.2 and 2.3], gives a suﬃcient condition for the trivial extremal families to be typical. 19 the electronic journal of combinatorics 26(2) (2019), #P2.34 Lemma 24. Let P be a decreasing property. Let N0 denote the size of the extremal (that is, largest) family with property P, N1 the size of the largest non-extremal maximal family, and suppose two distinct extremal families have at most N2 members in common. Suppose further that the number of extremal families is T, and there are at most M maximal families. Provided Lemma 24. Let P be a decreasing property. Let N0 denote the size of the extremal (that is, largest) family with property P, N1 the size of the largest non-extremal maximal family, and suppose two distinct extremal families have at most N2 members in common. Suppose further that the number of extremal families is T, and there are at most M maximal families. Provided 2 log M + max(N1, N2) −N0 →−∞, (17) (17) the number of families with property P is (T + o(1))2N0. the number of families with property P is (T + o(1))2N0. the number of families with property P is (T + o(1))2N0. the number of families with property P is (T + o(1))2N0. . . , Am be sets of size a and B1, . . . , Bm sets of size b such that Ai ∩Bi = ∅and Ai ∩Bj ̸= ∅for every i ̸= j. Then m ⩽ a+b a . Theorem 26 (Bollob´as). Let A1, . . . , Am be sets of size a and B1, . . . , Bm sets of size b such that Ai ∩Bi = ∅and Ai ∩Bj ̸= ∅for every i ̸= j. Then m ⩽ a+b a . We apply this to the pairs {(Ai, Bi)}m i=1, where for 1 ⩽i ⩽m we take Ai = Fi and Bi = Gi. The conditions of Theorem 26 are satisﬁed, and hence we deduce m ⩽ sk k . We apply this to the pairs {(Ai, Bi)}m i=1, where for 1 ⩽i ⩽m we take Ai = Fi and Bi = Gi. The conditions of Theorem 26 are satisﬁed, and hence we deduce m ⩽ sk k . k We map each maximal family F to a minimal generating family F0 ⊂F. This map is injective because I(F0) = F. We have shown that \|F0\| ⩽ sk k , and thus the number of maximal families is bounded from above by P(sk k ) i=0 (n k) i ⩽ n k (sk k ), as desired. Proof of Theorem 5. We shall verify that the condition (17) from Lemma 24 holds. A result of Frankl [22, Theorem 1.1] states that when n ⩾(2s −1)k −s + 1, the extremal families with no s pairwise disjoint sets are isomorphic to n F ∈ [n] k : F ∩[s −1] ̸= ∅ o , 20 the electronic journal of combinatorics 26(2) (2019), #P2.34 and consequently we may take N0 = n k − n−s+1 k and T = n s−1 . Moreover, it is not diﬃcult to see that the intersection of any two extremal families has size at most N2 = n k − n−s+1 k − n−s k−1 . Furthermore, a result due to Frankl and Kupavskii [24, Theorem 5] implies that N1 ⩽ n k − n−s+1 k − 1 s+1 n−k−s+1 k−1 for n ⩾2sk −s. Hence max(N1, N2) ⩽ n k − n−s+1 k − 1 s+1 n−k−s+1 k−1 . the number of families with property P is (T + o(1))2N0. In addition, Proposition 25 shows that we may use the estimate log M ⩽n sk k . Altogether we have 2 log M+ max(N1, N2) −N0 ⩽2n sk k − 1 s + 1 n −k −s + 1 k −1 = 2n sk k − k (s + 1)(n −k −s + 2) n −k −s + 2 k ⩽2n sk k " 1 − k 2(s + 1)(n −k −s + 2)n n −k −s + 2 sk k# . (18) (18) As n ⩾2sk + 38s4 and s ⩾2, we ﬁnd n−k−s+2 sk ⩾3 2 1 + 74s3 3k , and hence n −k −s + 2 sk k−2 ⩾ 3 2 k−2 1 + 74s3 3k k−2 ⩾k 2 · 74(k −2)s3 3k = 37 3 (k −2)s3. This implies This implies k 2(s + 1)(n −k −s + 2)n n −k −s + 2 sk k = n −k −s + 2 2(s + 1)s2kn n −k −s + 2 sk k−2 ⩾37s(k −2)(n −k −s + 2) 6(s + 1)kn ⩾37 36 as s/(s + 1) ⩾2/3, (k −2)/k ⩾1/3 and (n −k −s + 2)/n ⩾3/4. Substituting this inequality into (18), we obtain as s/(s + 1) ⩾2/3, (k −2)/k ⩾1/3 and (n −k −s + 2)/n ⩾3/4. Substituting this inequality into (18), we obtain 2 log M + max(N1, N2) −N0 ⩽−1 18n sk k →−∞. 4.2 Intersecting set systems In this section we shall use the removal lemma for disjoint sets (Lemma 12) to show that intersecting set systems in [n] k are typically trivial when n ⩾2k + C √ k ln k for some positive constant C. Since the number of trivial intersecting families is n · 2(n−1 k−1) ± n 2 · 2(n−2 k−2) = (n + o(1))2(n−1 k−1), it suﬃces to prove that there are o(2(n−1 k−1)) non-trivial intersecting families. it suﬃces to prove that there are o(2(n−1 k−1)) non-trivial intersecting families. f f ﬁ We need a few classic theorems from extremal set theory. The ﬁrst is a theorem of Hilton and Milner [26], bounding the cardinality of a non-trivial uniform intersecting family. 21 the electronic journal of combinatorics 26(2) (2019), #P2.34 Theorem 27 (Hilton and Milner). Let F ⊂ [n] k be a non-trivial intersecting family with k ⩾2 and n ⩾2k + 1. Then \|F\| ⩽ n−1 k−1 − n−k−1 k−1 + 1. The next result we require is a theorem of Kruskal [32] and Katona [28]. For a family F ⊂ [n] r , its s-shadow in [n] s , denoted ∂(s)F, is the family of those s-sets contained in some member of F. For x ∈R and r ∈N, we deﬁne the generalised binomial coeﬃcient x r by setting x r = x(x −1) . . . (x −r + 1) r! . The following convenient formulation of the Kruskal-Katona theorem is due to Lov´asz [34]. Theorem 28 (Lov´asz). Let n, r and s be positive integers with s ⩽r ⩽n. If F is a subfamily of [n] r with \|F\| = x r for some real number x ⩾r, then ∂(s)F ⩾ x s . With these results in hand, we now prove Theorem 6. Proof of Theorem 6. The statement has been established for n ⩾3k+8 ln k in [3, Theorem 1.4], and so we may assume n = 2k + s for some integer s with C √ k ln k ⩽s ⩽k + 8 ln k. For each ℓ∈N, let Nℓdenote the number of maximal non-trivial intersecting families of size n−1 k−1 −ℓ. By Theorem 27, we know Nℓ= 0 for ℓ< n−k−1 k−1 −1. 4.2 Intersecting set systems By taking a simple union bound over the subfamilies of these families, we can bound the number of non-trivial intersecting families by (n−1 k−1) X ℓ=(n−k−1 k−1 )−1 Nℓ2(n−1 k−1)−ℓ= X ℓ Nℓ2−ℓ ! 2(n−1 k−1), so it suﬃces to show P ℓNℓ2−ℓ= o(1). so it suﬃces to show P ℓNℓ2−ℓ= o(1). so it suﬃces to show P ℓNℓ2 o(1). By a result of Balogh et al. [3, Proposition 2.2], we know the total number of maximal intersecting families can be bounded by P ℓNℓ⩽2 1 2 n(2k k ), and so we have By a result of Balogh et al. [3, Proposition 2.2], we know the total number of maximal intersecting families can be bounded by P ℓNℓ⩽2 1 2 n(2k k ), and so we have X ℓ⩾n(2k k ) Nℓ2−ℓ⩽2−n(2k k ) · X ℓ⩾n(2k k ) Nℓ⩽2−1 2 n(2k k ) = o(1). Hence it suﬃces to show Hence it suﬃces to show n(2k k ) X ℓ=(n−k−1 k−1 )−1 Nℓ2−ℓ= o(1). (19) (19) We ﬁx some integer ℓwith n−k−1 k−1 −1 ⩽ℓ⩽n 2k k , and ﬁx some maximal intersecting family F of size n−1 k−1 −ℓ. Let S be the star that minimises \|F∆S\|, and without loss of generality assume that n is the center of S. Let A = F \ S, and t = \|A\|. Let B = S \ F, and note that \|B\| = t + ℓ. the electronic journal of combinatorics 26(2) (2019), #P2.34 22 Let P = {[n −1] \ A : A ∈A}, and observe that P ⊆ [n−1] n−k−1 , since n /∈A for all A ∈A. Let Q = {B \ {n} : B ∈B} ⊆ [n−1] k−1 . We claim that ∂(k−1)P = Q. Let P = {[n −1] \ A : A ∈A}, and observe that P ⊆ [n−1] n−k−1 , since n /∈A for all A ∈A. Let Q = {B \ {n} : B ∈B} ⊆ [n−1] k−1 . We claim that ∂(k−1)P = Q. k 1 Indeed, suppose H ∈∂(k−1)P. Then there is some A ∈A such that H ⊂[n −1] \ A, and so H ∩A = ∅. As n /∈A, this forces ({n} ∪H) ∩A = ∅, and so {n} ∪H /∈F. Hence {n} ∪H ∈B, giving H ∈Q. k Indeed, suppose H ∈∂(k−1)P. 4.2 Intersecting set systems Then there is some A ∈A such that H ⊂[n −1] \ A, and so H ∩A = ∅. As n /∈A, this forces ({n} ∪H) ∩A = ∅, and so {n} ∪H /∈F. Hence {n} ∪H ∈B, giving H ∈Q. For the opposite direction, suppose H /∈∂(k−1)P. Then, following the same argument as above, ({n}∪H)∩A ̸= ∅for all A ∈A. By maximality of F, we must have {n}∪H ∈F, and thus {n} ∪H /∈B, resulting in H /∈Q. We shall show that ℓ⩾2nt. First let us see why this implies (19). For each family F counted by Nℓ, it suﬃces to provide the star S and the family A outside the star3. Indeed, since Q = ∂(k−1)P, we can compute F ∩S, and hence completely determine F. Moreover, \|A\| = t ⩽ℓ/(2n). Thus Nℓ2−ℓ⩽n n −1 k ℓ/(2n) 2−ℓ< n · 2−ℓ/2, and so n(2k k ) X ℓ=(n−k−1 k−1 )−1 Nℓ2−ℓ⩽ 2n √ 2 −12−1 2(n−k−1 k−1 ) = o(1). It remains to show ℓ⩾2nt. Letting P and Q be as above, recall that Q = ∂(k−1)P. According to Theorem 28, if x is a real number so that t = \|P\| = x n−k−1 , then ℓ+ t = \|Q\| ⩾ x k 1 . \|Q\| ⩾ x k−1 . N b k 1 Now observe that by Lemma 12, we have t ⩽C′nℓfor some absolute constant C′. Since ℓ⩽n 2k k , this implies t ⩽C′n22k k . Since n = 2k + s, we have t = x n−k−1 = x k+s−1 . We next show that x < 2k + ⌊3 4s⌋. If not, then t = x k + s −1 ⩾ 2k + ⌊3 4s⌋ k + s −1 = 2k k · 2k+s−1 k+s−1 2k k · 2k+⌊3 4 s⌋ k+s−1 2k+s−1 k+s−1 = 2k k · s−1 Y j=1 2k + j k + j · s−1 Y j=⌊3 4 s⌋+1 k −s + 1 + j 2k + j ⩾ 2k k 2k + s k + s s−1 k −1 4s 2k + 3 4s 1 4 s . he electronic journal of combinatorics 26(2) (2019), #P2.34 4.2 Intersecting set systems C 4 √ k ln k−1 −1 > e 3C 64 ln k −1 ⩾8k > 2n, −1 > e 3C 64 ln k −1 ⩾8k > 2n, as required. This completes the proof. as required. This completes the proof. 5.1 Supersaturation for permutations Theorem 2 shows, for k ⩽cn1/2 and s (very) close to k(n −1)!, one minimises the number of disjoint pairs in a family of s permutations by selecting them from pairwise- disjoint cosets. This leaves large gaps between the ranges where we know the answer to the supersaturation problem, and it would be very interesting to determine the correct behaviour throughout. For instance, which family of 1.5(n −1)! permutations minimises the number of disjoint pairs? Note that the derangement graph is dn-regular, and so we can apply Lemma 22 to determine the optimal families for sizes close to k(n −1)! when k ⩾n −cn1/2 by taking complements. However, the complement of a union of pairwise disjoint cosets is again a union of pairwise disjoint cosets, and hence there may well be a nested sequence of optimal families for this problem. One candidate would be the initial segments of the lexicographic order on Sn, where π < σ if and only if πj < σj for j = min{i ∈[n] : πi ̸= σi}. 5 Concluding remarks We close by oﬀering some ﬁnal remarks and open problems related to the supersaturation problems discussed in this paper. 4.2 Intersecting set systems The bases of the exponential factors are minimised when s is as large as possible; substi- tuting s ⩽k + 8 ln k < 1.1k, we can lower bound the coeﬃcient of 2k k by The bases of the exponential factors are minimised when s is as large as possible; substi- tuting s ⩽k + 8 ln k < 1.1k, we can lower bound the coeﬃcient of 2k k by 2.1 3.1 3.1 2.1 s 0.725 2.825 1 4 s > 2 31.05s > n3 3For every choice of F there is a unique A, but not every A corresponds to a maximal family F 3For every choice of F there is a unique A, but not every A corresponds to a maximal family F the electronic journal of combinatorics 26(2) (2019), #P2.34 23 as s > C √ k ln k ⩾100 ln n, contradicting our upper bound t ⩽C′n22k k . Suppose, then, that x ⩽2k + ⌊3 4s⌋−1. Since t = x k+s−1 and ℓ+ t ⩾ x k−1 , we have as s > C √ k ln k ⩾100 ln n, contradicting our upper bound t ⩽C′n22k k . Suppose, then, that x ⩽2k + ⌊3 4s⌋−1. Since t = x k+s−1 and ℓ+ t ⩾ x k−1 , we have ℓ t ⩾ x k−1 x k+s−1 −1 = k+s−1 Y j=k j x + 1 −j −1. This product is decreasing in x, so we can substitute our upper bound x ⩽2k + ⌊3 4s⌋−1 to ﬁnd This product is decreasing in x, so we can substitute our upper bound x ⩽2k + ⌊3 4s⌋−1 to ﬁnd ℓ t ⩾ k+s−1 Y j=k j 2k + ⌊3 4s⌋−j −1 = s−1 Y j=⌊3 4 s⌋+1 k + j k + ⌊3 4s⌋−j −1 ⩾ 1 + 3s 4k 1 4 s−1 −1 This is increasing in s, so plugging in the lower bound s ⩾C √ k ln k, we have This is increasing in s, so plugging in the lower bound s ⩾C √ k ln k, we have ℓ t ⩾ 1 + 3 4C r ln k k ! 5.2 Set systems of very large uniformity For set families, we improved the range of uniformities for which the small initial segments of the lexicographic order are known to be optimal. In Corollary 13, which applies when 24 the electronic journal of combinatorics 26(2) (2019), #P2.34 n = Ω(r3k2), we handled the case where the family is a little larger than the union of r stars. However, if one restricts the size of the set families even further, one can obtain optimal bounds on n. For instance, Katona, Katona and Katona [29] showed that adding one set to a full star is always optimal. n = Ω(r3k2), we handled the case where the family is a little larger than the union of r stars. However, if one restricts the size of the set families even further, one can obtain optimal bounds on n. For instance, Katona, Katona and Katona [29] showed that adding one set to a full star is always optimal. Proposition 29. Suppose n ⩾2k + 1. Any system F ⊆ [n] k with \|F\| = n−1 k−1 + 1 contains at least n−k−1 k−1 disjoint pairs. Proposition 29. Suppose n ⩾2k + 1. Any system F ⊆ [n] k with \|F\| = n−1 k−1 + 1 contains at least n−k−1 k−1 disjoint pairs. Proposition 29. Suppose n ⩾2k + 1. Any system F ⊆ [n] k with \|F\| = n−1 k−1 + 1 contains at least n−k−1 k−1 disjoint pairs. By applying the removal lemma (Lemma 12), we can extend this exact result to a larger range of family sizes. Proposition 30. For some positive constant c, the following holds. Provided n ⩾2k + 2 and 0 ⩽s ⩽ n−1 k−1 + c · n−2k n n−k−1 k−1 , L(n, k, s) minimises the number of disjoint pairs among all systems of s sets in [n] k . Proof. Let C be the positive constant from Lemma 12 and set c = (20C)−2. Suppose F ⊆ [n] k is a family with \|F\| = n−1 k−1 + t for some 1 ⩽t ⩽c · n−2k n n−k−1 k−1 . Letting s = n−1 k−1 + t, we shall show that dp(F) ⩾dp(Ln,k(s)) = t n−k−1 k−1 . Suppose otherwise that dp(F) < t n−k−1 k−1 . 5.2 Set systems of very large uniformity By Lemma 12, there exists a star S such that \|F∆S\| ⩽1 2 n−k−1 k−1 . It follows that \|F ∩S\| = n−1 k−1 −p for some integer p with 0 ⩽p ⩽ 1 2 n−k−1 k−1 . As \|F\| = n−1 k−1 + t and \|F ∩S\| = n−1 k−1 −p, we must have \|F \ S\| = p + t. Since each set in F \ S is disjoint from exactly n−k−1 k−1 sets in the star S and \|F ∩S\| = n−1 k−1 −p, we conclude dp(F, F ∩S) ⩾ n−k−1 k−1 −p > 0 for all F ∈F \ S. Thus dp(F) ⩾ X F∈F\S dp(F, F ∩S) ⩾\|F \ S\| n −k −1 k −1 −p = (p + t) n −k −1 k −1 −p = t n −k −1 k −1 + p n −k −1 k −1 −p −t ⩾t n −k −1 k −1 , where the last inequality holds since p ⩽1 2 n−k−1 k−1 and t ⩽c · n−2k n n−k−1 k−1 . where the last inequality holds since p ⩽1 2 n−k−1 k−1 and t ⩽c · n−2k n n−k−1 k−1 . 5.3 A counterexample to the Bollob´as–Leader conjecture Finally, it remains to extend the set supersaturation results to larger values of k. Are small initial segments of the lexicographic order still optimal when k > √n? This is not the case when n = 3k −1, as the following construction shows. Let s = n−1 k−1 + 2k−1 k −1. Then L(n, k, s) consists of one full star, and 2k−1 k −1 sets from another star, each of which is disjoint from n−k−1 k−1 = 2k−2 k−1 sets from the full star. Hence dp(L(n, k, s)) = 2k−1 k −1 2k−2 k−1 . ′ Now instead let F′ be the family consisting of the S1, the full star with centre 1, and all but one k-element subset of {2, 3, . . . , 2k}. Since F′ again consists of a full star and 25 the electronic journal of combinatorics 26(2) (2019), #P2.34 an intersecting family of size 2k−1 k −1, we have dp(F′) = dp(L(n, k, s)). Now form the family F from F′ by replacing the set A = {1, 2k + 1, . . . , 3k −1} with the missing k-set B from {2, 3, . . . , 2k}. We lose 2k−1 k −1 disjoint pairs when we remove A, and gain only n−k−1 k−1 −1 = 2k−2 k−1 −1 disjoint pairs when we add B. As 2k−2 k−1 < 2k−1 k , it follows that dp(F) < dp(L(n, k, s)), showing the initial segment of the lexicographic order is not optimal. Bollob´as and Leader [7] conjectured that the solution to the supersaturation problem is always given by an ℓ-ball. Given n, k and s, an ℓ-ball of size s is a family Bℓ(n, k, s) of s sets such that there is some r with F ∈ [n] k : \|F ∩[r]\| ⩾ℓ ⊆Bℓ(n, k, s) ⊆ F ∈ [n] k : \|F ∩[r + 1]\| ⩾ℓ . In particular, the initial segments of the lexicographic order are 1-balls, while their com- plements are isomorphic to k-balls. We have shown that the construction F given above has fewer disjoint pairs than the 1-balls of size s = \|F\|. 5.3 A counterexample to the Bollob´as–Leader conjecture Computer-aided calculations show that for n = 3k −1, s = n−1 k−1 + 2k−1 k −1 and 5 ⩽k ⩽15, the 1-balls have far fewer disjoint pairs than the ℓ-balls for ℓ⩾2, showing that F gives a counterexample to the Bollob´as–Leader conjecture for these parameters. The numerical evidence suggests that F should be a counterexample for all k ⩾5, but it is diﬃcult to estimate the number of disjoint pairs in Bℓ(3k −1, k, s) for ℓ⩾2, and so we have been unable to prove this. 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[45] W. Samotij, Subsets of posets minimising the number of chains, Trans. of AMS 371 (2019), 7259–7274. [46] W. Samotij and B. Sudakov, On the number of monotone sequences, J. Combin. Theory, Ser. B 115 (2015), 132–163. 28 the electronic journal of combinatorics 26(2) (2019), #P2.34 [47] W. Samotij and B. Sudakov, The number of additive triples in subsets of abelian groups, Math. Proc. Cambridge Philos. Soc. 160 (2016), 495–512. A Intersection graphs In this appendix, we prove Proposition 10, which shows how the intersection graph deter- mines various parameters about the corresponding union of cosets, including its size and number of disjoint pairs. A.1 Some preliminaries We start by introducing some further notation we will use throughout this appendix. First, recall that dn denotes the number of derangements in Sn, and that Dn = dn +dn−1. It will also be convenient for us to deﬁne the parameter D′ n = dn + 2dn−1, a quantity that arise later in our proof. Next, given a graph G, kt(G) denotes the number of t-cliques in G. We will further write Kt(G) for the set of these t-cliques. Moreover, given a vertex subset X ⊆V (G), we denote by kt,X(G) the number of t-cliques in G that contain X. In particular, we have kt(G) = kt,∅(G) = \|Kt(G)\|. Again, we will omit G from the notation when the graph is clear from the context. Finally, ¯P3 is the complement of the path on three vertices, which is the union of an edge and an isolated vertex. Let 1 ¯ P3 : V (G) × V (G) 2 →{0, 1} be the function deﬁned by setting 1 ¯ P3(x, {y, z}) = 1 if and only if yz is the only edge of the induced subgraph G[{x, y, z}]. We then denote the number of induced copies of ¯P3 in G by i( ¯P3, G), noting that i( ¯P3, G) = P x∈V (G) P {y,z}∈(V (G) 2 ) 1 ¯ P3(x, {y, z}). {y, } ( 2 ) With this additional notation in place, we close these preliminaries with the following crucial observation, which we shall make repeated use of. Observation 31. If G is the intersection graph of a union of cosets, the following prop- erties hold. (i) For every subset X ⊂V (G), the intersection ∩x∈XTx is non-empty if and only if G[X] is a clique. In this case, \|∩x∈XTx\| = (n −\|X\|)!. If n ⩾10ℓ2, (i1, j1), . . . , (iℓ, jℓ) form an ℓ-clique in G, and π ∈Sn \Sℓ s=1 T(is,js), the (ii) If n ⩾10ℓ2, (i1, j1), . . . , (iℓ, jℓ) form an ℓ-clique in G, and π ∈Sn \Sℓ s=1 T(is,js), then dp π, \ℓ s=1 T(is,js) =dn−ℓ+ ℓ− {i1, . . . , iℓ} ∩{π−1(j1), . . . , π−1(jℓ)} dn−ℓ−1 ± 7ℓ2(n −ℓ−2)!. dp π, \ℓ s=1 T(is,js) =dn−ℓ+ ℓ− {i1, . . . , iℓ} ∩{π−1(j1), . . . , π−1(jℓ)} dn−ℓ−1 ± 7ℓ2(n −ℓ−2)!. Proof. A.1 Some preliminaries (i) Since T(i,j)∩T(i′,j′) = ∅whenever (i, j) and (i′, j′) are not adjacent in G, we have ∩x∈XTx = ∅whenever G[X] is not a clique. Now suppose that X = {(i1, j1), . . . , (iℓ, jℓ)} spans a clique in G. Then we must have \|{i1, . . . , iℓ}\| = \|{j1, . . . , jℓ}\| = ℓ. Hence \|∩x∈XTx\| is the number of bijections from [n] \ {i1, . . . , iℓ} to [n] \ {j1, . . . , jℓ}, which is (n −ℓ)!. Proof. (i) Since T(i,j)∩T(i′,j′) = ∅whenever (i, j) and (i′, j′) are not adjacent in G, we have ∩x∈XTx = ∅whenever G[X] is not a clique. Now suppose that X = {(i1, j1), . . . , (iℓ, jℓ)} spans a clique in G. Then we must have \|{i1, . . . , iℓ}\| = \|{j1, . . . , jℓ}\| = ℓ. Hence \|∩x∈XTx\| is the number of bijections from [n] \ {i1, . . . , iℓ} to [n] \ {j1, . . . , jℓ}, which is (n −ℓ)!. 29 the electronic journal of combinatorics 26(2) (2019), #P2.34 (ii) Fix a permutation π ∈Sn\ T(i1,j1) ∪. . . ∪T(iℓ,jℓ) , that is, a permutation satisfying π(is) ̸= js for all s ∈[ℓ]. If a permutation σ ∈∩ℓ s=1T(is,js) intersects π, then we must have σ(x) = π(x) for some x ∈[n]. Let Ax denote the family of such permutations, and observe Ax = T(x,π(x))∩ ∩ℓ s=1T(is,js) . By part (i), for this intersection to be non-empty, we require (x, π(x)) to be adjacent to each (is, js) in the intersection graph, or, equivalently, x /∈{i1, . . . , iℓ} ∪{π−1(j1), . . . , π−1(jℓ)}. For brevity, set (ii) Fix a permutation π ∈Sn\ T(i1,j1) ∪. . . ∪T(iℓ,jℓ) , that is, a permutation satisfying π(is) ̸= js for all s ∈[ℓ]. If a permutation σ ∈∩ℓ s=1T(is,js) intersects π, then we must have σ(x) = π(x) for some x ∈[n]. Let Ax denote the family of such permutations, and observe Ax = T(x,π(x))∩ ∩ℓ s=1T(is,js) . By part (i), for this intersection to be non-empty, we require (x, π(x)) to be adjacent to each (is, js) in the intersection graph, or, equivalently, x /∈{i1, . . . , iℓ} ∪{π−1(j1), . . . , π−1(jℓ)}. A.1 Some preliminaries For brevity, set c = ℓ− {i1, . . . , iℓ} ∩{π−1(j1), . . . , π−1(jℓ)} and I = [n] \ {i1, . . . , iℓ} ∪{π−1(j1), . . . , π−1(jℓ)} . Note that \|I\| = n −ℓ−c, and ∪x∈IAx is the family of all permutations σ ∈∩ℓ s=1T(is,js) that intersect π. Applying the inclusion-exclusion formula and part (i), we obtain Note that \|I\| = n −ℓ−c, and ∪x∈IAx is the family of all permutations σ ∈∩ℓ s=1T(is,js) that intersect π. Applying the inclusion-exclusion formula and part (i), we obtain dp π, ∩ℓ s=1T(is,js) = ∩ℓ s=1T(is,js) \ ∪x∈IAx = n−ℓ−c X i=0 (−1)i n −ℓ−c i (n −ℓ−i)!. (20) (20) Let αi be the real number such that n−ℓ−c i (n−ℓ−i)! = αi (n−ℓ)! i! . We shall approximate αi by a simple function, and then use it to compute dp π, ∩ℓ s=1T(is,js) . By deﬁnition, Let αi be the real number such that n−ℓ−c i (n−ℓ−i)! = αi (n−ℓ)! i! . We shall approximate αi by a simple function, and then use it to compute dp π, ∩ℓ s=1T(is,js) . By deﬁnition, αi = (n −ℓ−c)!(n −ℓ−i)! (n −ℓ)!(n −ℓ−c −i)! = Qc−1 j=0(n −ℓ−i −j) Qc−1 j=0(n −ℓ−j) = c−1 Y j=0 1 − i n −ℓ−j . Hence αi ⩾ 1 − i n−ℓ−c+1 c ⩾1 − ci n−ℓ−c+1 = 1 − ci n−ℓ− c(c−1)i (n−ℓ)(n−ℓ−c+1). On the other hand, αi ⩽ 1 − i n−ℓ c ⩽exp −ci n−ℓ ⩽1 − ci n−ℓ+ ci n−ℓ 2. Since c ⩽ℓand n ⩾10ℓ2, we may write αi = 1 − ci n−ℓ+ εi with \|εi\| ⩽ c2i2 (n−ℓ)2, providing an eﬀective estimate when i is small. Plugging these estimates into (20), we have ( ) Plugging these estimates into (20), we have ( ) Plugging these estimates into (20), we have dp π, ∩ℓ s=1T(is,js) = n−ℓ−c X i=0 (−1)iαi (n −ℓ)! i! = n−ℓ−c X i=0 (−1)i 1 − ci n −ℓ (n −ℓ)! i! ± n−ℓ−c X i=0 \|εi\| (n −ℓ)! i! = n−ℓ X i=0 (−1)i 1 − ci n −ℓ (n −ℓ)! i! ± n−ℓ X i=n−ℓ−c+1 1 − ci n −ℓ (n −ℓ)! i! ± n−ℓ−c X i=0 c2i2(n −ℓ)! (n −ℓ)2i! . A.2 Proof of Proposition 10(a) Armed with these preliminaries, we may begin to prove the statements in Proposition 10, of which the ﬁrst is by far the simplest. Proof of Proposition 10(a). By the Bonferroni inequalities, we have Proof of Proposition 10(a). By the Bonferroni inequalities, we have \|G\| = [ x∈V (G) Tx = X 1⩽i⩽3 (−1)i−1    X X∈(V (G) i ) \ x∈X Tx   ± X X∈(V (G) 4 ) \ x∈X Tx . By Observation 31(i), ∩x∈XTx is empty unless X induces a clique in G, in which case \|∩x∈XTx\| = (n −\|X\|)!. Hence we have By Observation 31(i), ∩x∈XTx is empty unless X induces a clique in G, in which case \|∩x∈XTx\| = (n −\|X\|)!. Hence we have \|G\| = k1(n −1)! −k2(n −2)! + k3(n −3)! ± k4(n −4)!, as required. as required. A.1 Some preliminaries Expanding the leading term gives Expanding the leading term gives Expanding the leading term gives n−ℓ X i=0 (−1)i(n −ℓ)! i! + c n−ℓ−1 X i=0 (−1)i(n −ℓ−1)! i! = dn−ℓ+ c · dn−ℓ−1. the electronic journal of combinatorics 26(2) (2019), #P2.34 the electronic journal of combinatorics 26(2) (2019), #P2.34 30 30 To bound the ﬁrst error term, observe that if n−ℓ−c+1 ⩽i ⩽n−ℓ(for which we must have c ⩾1), we have 1 − ci n−ℓ ⩽c and (n−ℓ)! i! ⩽nc−1, and so n−ℓ X i=n−ℓ−c+1 1 − ci n −ℓ (n −ℓ)! i! ⩽c2nc−1 ⩽ℓ2nc−1 ⩽ℓ2(n −ℓ−2)!. Finally, using c ⩽ℓ, we bound the second error term by Finally, using c ⩽ℓ, we bound the second error term by n−ℓ−c X i=0 c2i2(n −ℓ)! (n −ℓ)2i! ⩽c2(n −ℓ−2)! X i⩾0 i2 i! ⩽6ℓ2(n −ℓ−2)!. Putting these bounds together gives the desired expression for dp π, ∩ℓ s=1T(is,js) . Putting these bounds together gives the desired expression for dp π, ∩ℓ s=1T(is,js) . A.3 Proof of Proposition 10(b) In the second part of the proposition, we count the number of disjoint pairs between G and an arbitrary permutation π ∈Sn\G. We will in fact prove the more accurate estimate given in the claim below, as this will be required in the proof of part (c). Claim 32. Let G be a union of k1 ⩽n cosets with intersection graph G. Then, for every π ∈Sn \ G, dp(π, G) = k1Dn−1 −k2D′ n−2 +  k3 + X xy∈E(G) {x1, y1} ∩{π−1(x2), π−1(y2)}  dn−3 ± (28k2 + 4k3 + k4)(n −4)!, ± (28k2 + 4k3 + k4)(n −4)!, where the indices x = (x1, x2) and y = (y1, y2) are vertices of G. where the indices x = (x1, x2) and y = (y1, y2) are vertices of G. the electronic journal of combinatorics 26(2) (2019), #P2.34 31 the electronic journal of combinatorics 26(2) (2019), #P2.34 the electronic journal of combinatorics 26(2) (2019), #P2.34 31 We ﬁrst verify that this implies the bound from the proposition. Proof of Proposition 10(b). The leading term is already in the desired form. For the second-order term, observe that D′ n−2 = Dn−2 + dn−3. In the third term, we bound the coeﬃcient of dn−3 above by k3 + 2k2, and recall that we also have a term of −k2dn−3 from the second-order term. Thus, in total, the third-order term is at most (k3+k2)dn−3. Since k3 ⩽k1k2 and dn−3 ⩽(n −3)!, we can bound this from above by 2k1k2(n −3)!. ( ) ( ) With regards to the error term, note that by making the constant c in Proposition 10 suﬃciently small, we may assume n is large. Thus, using the aforementioned bound on k3, and bounding k4 ⩽k2 1k2/12, we have (28k2 + 4k3 + k4)(n −4)! ⩽k1k2(n −3)!. 3, g 4 ⩽ 1 2/ , ( 2 3 4)( ) ⩽ 1 2( ) Substituting these estimates into the equation from Claim 32 gives dp(π, G) = k1Dn−1 −k2Dn−2 ± 3k1k2(n −3)!, as required. We now prove the claim. Proof of Claim 32. Let π be an arbitrary permutation in Sn \ G. It follows from the Bonferroni inequalities and Observation 31(i) that We now prove the claim. Proof of Claim 32. Let π be an arbitrary permutation in Sn \ G. A.3 Proof of Proposition 10(b) It follows from the Bonferroni inequalities and Observation 31(i) that dp(π, G) = 3 X i=1 (−1)i−1   X X∈Ki(G) dp (π, ∩x∈XTx)  ± X X∈K4(G) dp (π, ∩x∈XTx) . Recall from Subsection 2.1 that dp(π, Tx) = Dn−1 for every x ∈V (G). For xy ∈E(G) say x = (x1, x2) and y = (y1, y2), Observation 31(ii) implies dp(π, Tx ∩Ty) = dn−2 + 2 − {x1, y1} ∩{π−1(x2), π−1(y2)} dn−3 ± 28(n −4)! = D′ n−2 − {x1, y1} ∩{π−1(x2), π−1(y2)} dn−3 ± 28(n −4)! as D′ n−2 = dn−2 + 2dn−3. Again using Observation 31(ii) gives dp (π, ∩x∈XTx) = dn−3 ± 4(n −4)! for every X ∈K3(G). For each X ∈K4(G), we may deduce from Observation 31 (i) that dp(π, ∩x∈XTx) ⩽\|∩x∈XTx\| = (n −4)!. Combining these bounds gives the claim. A.4 Proof of Proposition 10(c) In the third part of the proposition, we count the number of disjoint pairs within a union G of cosets, with the result depending on numerous parameters of the intersection graph G. We shall once more prove a more precise estimate that we will need in the proof of part (d). Claim 33. Let G be a union of k1 ⩽cn1/2 cosets with intersection graph G. Then dp(G) = 3 X i=1 ai(n −i)!Dn−1 ± 12k3 1k2(n −1)!(n −4)!, the electronic journal of combinatorics 26(2) (2019), #P2.34 32 the electronic journal of combinatorics 26(2) (2019), #P2.34 the electronic journal of combinatorics 26(2) (2019), #P2.34 32 where a1 = k1 2 , a2 = −(k1 −1)k2, and a3 = 1 2 (2k1 −3)k3 + (k2 −k1 + 1)k2 + i( ¯P3, G) . Let us ﬁrst verify that this claim suﬃces for the proposition. Proof of Proposition 10(c). The ﬁrst two terms in Claim 33 are exactly as required. We need only verify that the sum of the other terms is at most 2k2 1k2(n −1)!(n −3)! in magnitude. This is easily seen to be true, using the bounds (2k1 −3)k3 ⩽2 3k2 1k2, k2 2 ⩽ 1 2k2 1k2, and i( ¯P3, G) ⩽k1k2 ⩽1 2k2 1k2, and recalling that k1 ⩽cn1/2 for some small constant c. We now prove the claim. due to Observation 31 (i). Hence P i⩾4 dp(Mi, G) ⩽\|∪i⩾4Mi\| \|G\| ⩽k1k4(n − 1)!(n −4)!. We next consider permutations in exactly three cosets. Proof of Claim 34(ii). Fix X ∈K3(G) and let π ∈MX. Observe that if σ ∈Tx for x ∈X, then π and σ intersect. Thus, when counting the disjoint pairs between π and G, we need only consider the subfamily of G corresponding to the intersection graph given by G −X. Applying Proposition 10(b) to this subgraph, we see that dp(π, G) = dp(π, ∪x∈V (G)−XTx) = (k1 −3)Dn−1 ± 2k2(n −2)!. On the other hand, it follows from the Bonferroni inequalities and Observation 31 (i) that \|MX\| = (n −3)! ± k4,X(n −4)! for every triangle X ∈K3(G). Combining with the trivial bound k4,X ⩽k1, this gives On the other hand, it follows from the Bonferroni inequalities and Observation 31 (i) that \|MX\| = (n −3)! ± k4,X(n −4)! for every triangle X ∈K3(G). Combining with the trivial bound k4,X ⩽k1, this gives \|M3\| = X X∈K3(G) \|MX\| = k3(n −3)! ± k1k3(n −4)!. Summing dp(π, G) over all permutations π ∈M3 and using the estimate 2k2 ⩽k2 1 gives the desired result. We shall use a similar counting argument to estimate dp(M2, G). We shall use a similar counting argument to estimate dp(M2, G). Proof of Claim 34(iii). Fix X ∈K2(G) and π ∈MX. As before, any disjoint permut tions come from cosets in G −X. Applying Proposition 10(b) to G −X gives We shall use a similar counting argument to estimate dp(M2, G). Proof of Claim 34(iii). Fix X ∈K2(G) and π ∈MX. As before, any disjoint permuta- tions come from cosets in G −X. Applying Proposition 10(b) to G −X gives dp(π, G) = (k1 −2)Dn−1 −k2(G −X)Dn−2 ± 4k1k2(n −3)!. On the other hand, by appealing to the Bonferroni inequalities and Observation 31(i), we see that \|MX\| = (n −2)! −k3,X · (n −3)! ± k4,X(n −4)!. These two bounds, together with the estimates k3,X ⩽k1 < n and k4,X ⩽k2 ⩽k2 1, imply dp(MX, G) =(k1 −2)(n −2)!Dn−1 −(k1 −2)k3,X(n −3)!Dn−1 −k2(G −X)(n −2)!Dn−2 ± 15k1k2(n −1)!(n −4)!. As (n−2)!Dn−2 = (n−3)!Dn−1 ±(n−1)!(n−4)!, this expression can be simpliﬁed further as as dp(MX, G) =(k1 −2)(n −2)!Dn−1 −((k1 −2)k3,X + k2(G −X)) (n −3)!Dn−1 ± 16k1k2(n −1)!(n −4)!. the electronic journal of combinatorics 26(2) (2019), #P2.34 We now prove the claim. We now prove the claim. Proof of Claim 33. The idea behind the proof is to partition the permutations in G based on how many of the cosets they are contained in. For each vertex set X ⊆V (G), let MX be the family of all permutations π ∈G which satisfy {x ∈V (G) : π ∈Tx} = X. We shall use the symbol ˙∪to denote an union of disjoint sets. From Observation 31(i) we ﬁnd Mi := ˙∪\|X\|=iMX = ˙∪X∈Ki(G)MX, and thus dp(Mi, G) = P X∈Ki(G) dp(MX, G). The following claim evaluates these expressions. Claim 34. For G and Mi as deﬁned above, Claim 34. For G and Mi as deﬁned above, (i) P i⩾4 dp(Mi, G) ⩽k1k4(n −1)!(n −4)!, (ii) dp(M3, G) = (k1 −3)k3(n −3)!Dn−1 ± 3k2 1k3(n −1)!(n −4)!, (iii) dp(M2, G) = 3P i=2 ci(n −i)!Dn−1 ± 16k1k2 2(n −1)!(n −4)!, where c2 = (k1 −2)k2 and c3 = −3(k1 −2)k3 − P X∈K2(G) k2(G −X), and (i) P i⩾4 dp(Mi, G) ⩽k1k4(n −1)!(n −4)!, (ii) dp(M3, G) = (k1 −3)k3(n −3)!Dn−1 ± 3k2 1k3(n −1)!(n −4)!, (iv) dp(M1, G) = 3P i=1 bi(n −i)!Dn−1 ± 13k3 1k2(n −1)!(n −4)!, in which b1 = k1(k1 −1), b2 = (−3k1+4)k2, and b3 = (4k1−6)k3−(k1−2)k2+i( ¯P3, G)+ P x∈V (G) k2,{x}k2(G−x). Since G = ˙∪i⩾1Mi, one has dp(G) = 1 2 P 1⩽i⩽k1 dp(Mi, G). Claim 33 thus follows by summing the above, noting that P x k2,{x}k2(G −x) − P X∈K2(G) k2(G −X) = k2(k2 −1). Since G = ˙∪i⩾1Mi, one has dp(G) = 1 2 P 1⩽i⩽k1 dp(Mi, G). Claim 33 thus follows by summing the above, noting that P x k2,{x}k2(G −x) − P X∈K2(G) k2(G −X) = k2(k2 −1). It remains to prove Claim 34. We begin by bounding the contribution from permuta- tions in at least four cosets. the electronic journal of combinatorics 26(2) (2019), #P2.34 33 Proof of Claim 34(i). One has \|G\| ⩽k1(n −1)!, and \|∪i⩾4Mi\| ⩽P X∈K4(G) \|∩x∈XTx\| ⩽ k4(n −4)! due to Observation 31 (i). Hence P i⩾4 dp(Mi, G) ⩽\|∪i⩾4Mi\| \|G\| ⩽k1k4(n − 1)!(n −4)!. Proof of Claim 34(i). One has \|G\| ⩽k1(n −1)!, and \|∪i⩾4Mi\| ⩽P X∈K4(G) \|∩x∈XTx\| ⩽ k4(n −4)! due to Observation 31 (i). Hence P i⩾4 dp(Mi, G) ⩽\|∪i⩾4Mi\| \|G\| ⩽k1k4(n − 1)!(n −4)!. Proof of Claim 34(i). One has \|G\| ⩽k1(n −1)!, and \|∪i⩾4Mi\| ⩽P X∈K4(G) \|∩x∈XTx\| ⩽ k4(n −4)! We now prove the claim. Summing dp(MX, G) over all X ∈K2(G) and using the identity P X∈K2(G) k3,X = 3k3 results in the desired equation. Summing dp(MX, G) over all X ∈K2(G) and using the identity P X∈K2(G) k3,X = 3k3 results in the desired equation. Finally we come to what is, in some sense, the trickiest part of our proof, which is dealing with permutations in a single coset. the electronic journal of combinatorics 26(2) (2019), #P2.34 34 Proof of Claim 34(iv). Fix a vertex x ∈V (G) and a permutation π ∈M{x}. Once again, any disjoint permutations must come from cosets in G −x. Applying Claim 32 to this subgraph, we ﬁnd Proof of Claim 34(iv). Fix a vertex x ∈V (G) and a permutation π ∈M{x}. Once again, any disjoint permutations must come from cosets in G −x. Applying Claim 32 to this subgraph, we ﬁnd dp(π, G) =(k1 −1)Dn−1 −k2(G −x)D′ n−2 +  k3(G −x) + X (y1,y2)(z1,z2)∈E(G−x) {y1, z1} ∩{π−1(y2), π−1(z2)}  dn−3 ± (28k2 + 4k3 + k4)(n −4)!. On the other hand, from the Bonferroni inequalities we have On the other hand, from the Bonferroni inequalities we have M{x} = (n −1)! + X i∈{2,3} (−1)i−1ki,{x}(n −i)! ± k4,{x}(n −4)!. For each edge (y1, y2)(z1, z2) ∈E(G −x), we observe that for all but at most 2(n −2)! permutations π ∈M{x}, we have \|{y1, z1} ∩{π−1(y2), π−1(z2)}\| = 1 ¯ P3(x, {y, z}). Indeed, 1 ¯ P3(x, {y, z}) = 1 if and only if x ∈{(y1, z2), (z1, y2)}, in which case, since π(x1) = x2, we have \|{y1, z1} ∩{π−1(y2), π−1(z2)}\| ⩾1, with equality unless both π(y1) = z2 and π(z1) = y2. When x /∈{(y1, z2), (z1, y2)}, in order for \|{y1, z1} ∩{π−1(y2), π−1(z2)}\| to be positive, we need π(y1) = z2 or π(z1) = y2 in addition to π(x1) = x2, thus giving at most 2(n −2)! exceptions in this case. We now prove the claim. Putting these facts together and summing dp(π, G) over all π ∈M{x} then gives Putting these facts together and summing dp(π, G) over all π ∈M{x} then gives dp(M{x}, G) = (k1 −1)(n −1)!Dn−1 −k2(G −x)(n −1)!D′ n−2 −(k1 −1)k2,{x}(n −2)!Dn−1 + k2,{x}k2(G −x)(n −2)!D′ n−2 + (k1 −1)k3,{x}(n −3)!Dn−1 +  k3(G −x) + X yz∈E(G−x) 1 ¯ P3(x, {y, z})  (n −1)!dn−3 ± 4k2(n −2)!dn−3 ± 10k2 1k2(n −1)!(n −4)!, Putting these facts together and summing dp(π, G) over all π ∈M{x} then gives dp(M{x}, G) = (k1 −1)(n −1)!Dn−1 −k2(G −x)(n −1)!D′ n−2 −(k1 −1)k2,{x}(n −2)!Dn−1 + k2,{x}k2(G −x)(n −2)!D′ n−2 + (k1 −1)k3,{x}(n −3)!Dn−1 +  k3(G −x) + X yz∈E(G−x) 1 ¯ P3(x, {y, z})  (n −1)!dn−3 ± 4k2(n −2)!dn−3 ± 10k2 1k2(n −1)!(n −4)!,  ± 4k2(n −2)!dn−3 ± 10k2 1k2(n −1)!(n −4)!, where in the ﬁnal term we use ki ⩽2ki−2 1 k2/i! and the fact that k1 ⩽cn1/2 to bound the lower-order error terms. Moreover, we have the identities (n −1)!D′ n−2 = (n −2)!Dn−1 + (n −3)!Dn−1 ± (n −1)!(n −4)!, (n −2)!D′ n−2 = (n −3)!Dn−1 ± (n −1)!(n −4)!, and (n −1)!dn−3 = (n −3)!Dn−1 ± (n −1)!(n −4)!. Hence dp(M{x}, G) = 3 X i=1 b′ i(n −i)!Dn−1 ± 13k2 1k2(n −1)!(n −4)!, dp(M{x}, G) = 3 X i=1 b′ i(n −i)!Dn−1 ± 13k2 1k2(n −1)!(n −4)!, where b′ 1 = k1 −1, b′ 2 = −k2(G −x) −(k1 −1)k2,{x}, and b′ 3 = −k2(G −x) + k2,{x}k2(G −x) + (k1 −1)k3,{x} + k3(G −x) + X yz∈E(G) x/∈{y,z} 1 ¯ P3(x, {y, z}). the electronic journal of combinatorics 26(2) (2019), #P2.34 35 the electronic journal of combinatorics 26(2) (2019), #P2.34 35 the electronic journal of combinatorics 26(2) (2019), #P2.34 35 Noting that P x k2(G −x) = (k1 −2)k2, P x k2,{x} = 2k2, P x k3(G −x) = (k1 −3)k3, and P k3,{x} = 3k3, and summing the above estimate for dp(M{x}, G) over all x ∈V (G), we P x ( ) ( ) P x ,{ } P x ( ) ( ) P x k3,{x} = 3k3, and summing the above estimate for dp(M{x}, G) over all x ∈V (G), we get the desired formula for dp(M1, G). A.5 Proof of Proposition 10(d) The ﬁnal part of this appendix is devoted to showing that if G is a union of few cosets in Sn, then G has at least as many disjoint pairs as T (n, s), where s = \|G\|. To bound the gap dp(G) −dp(T (n, s)), we shall use part (III) of the following claim concerning structural properties of intersection graphs. Claim 35. Let G be the intersection graph of a union G of at most cn1/2 cosets in Sn. Then the following properties hold. (I) k2 ⩾max{k1, 2k1 −6} unless one of the following cases occurs: (I) k2 ⩾max{k1, 2k1 −6} unless one of the following cases occurs: (i) G is canonical; (i) G is canonical; (ii) G is isomorphic to 2K2, P4, P5 or C4 ∪K1. (ii) G is isomorphic to 2K2, P4, P5 or C4 ∪K1. (II) If k2 ⩾k1, then k2 (k2 −k1 + 1) ⩾2k3 + 1. (II) If k2 ⩾k1, then k2 (k2 −k1 + 1) ⩾2k3 + 1. (III) If G is not canonical, then k2 (k2 −k1 + 1) + i( ¯P3, G) −k3 ⩾ 1 50k1k2. We note that parts (I) and (II) will only be used to prove part (III). Before proving this claim, we show how it implies the ﬁnal part of the proposition. Proof of Proposition 10(d). If G is canonical, then G is a union of k1 −1 pairwise disjoint cosets and an intersecting family, and so dp(G) = dp(T (n, s)). ( ) ( ( )) Now suppose that G is not canonical. By Proposition 10(a), and since k4 ⩽k2 1k2, ( ) ( ( )) Now suppose that G is not canonical. By Proposition 10(a s = \|G\| = k1(n −1)! −k2(n −2)! + k3(n −3)! ± k2 1k2(n −4)!. So if we write s =: (k1 + ε)(n −1)!, then ε(n −1)! = s −k1(n −1)! = −k2(n −2)! + k3(n −3)! ± k2 1k2(n −4)!, which is non-negative since k3 ⩽k1k2 and k1 ⩽cn1/2. Using (3) with ε ⩽0 yields s = \|G\| = k1(n −1)! −k2(n −2)! + k3(n −3)! ± k2 1k2(n −4)!. So if we write s =: (k1 + ε)(n −1)!, then ε(n −1)! = s −k1(n −1)! = −k2(n −2)! + k3(n −3)! ± k2 1k2(n −4)!, which is non-negative since k3 ⩽k1k2 and k1 ⩽cn1/2. A.5 Proof of Proposition 10(d) Using (3) with ε ⩽0 yields dp(T (n, s)) = X 1⩽i⩽3 bi(n −i)!Dn−1 ± k3 1k2(n −1)!(n −4)!, ε(n −1)! = s −k1(n −1)! = −k2(n −2)! + k3(n −3)! ± k2 1k2(n −4)!, which is non-negative since k3 ⩽k1k2 and k1 ⩽cn1/2. Using (3) with ε ⩽0 yields dp(T (n, s)) = X 1⩽i⩽3 bi(n −i)!Dn−1 ± k3 1k2(n −1)!(n −4)!, which is non-negative since k3 ⩽k1k2 and k1 ⩽cn1/2. Using (3) with ε ⩽0 yields dp(T (n s)) = X b (n i)!D ± k3k (n 1)!(n 4)! dp(T (n, s)) = X 1⩽i⩽3 bi(n −i)!Dn−1 ± k3 1k2(n −1)!(n −4)!, where b1 = k1 2 , b2 = −(k1 −1)k2 and b3 = (k1 −1)k3. We can easily derive from this and Claim 33 that where b1 = k1 2 , b2 = −(k1 −1)k2 and b3 = (k1 −1)k3. We can easily derive from this and Claim 33 that dp(G) −dp(T (n, s)) =1 2 k2(k2 −k1 + 1) + i( ¯P3, G) −k3 (n −3)!Dn−1 ± 13k3 1k2(n −1)!(n −4)!. dp(G) −dp(T (n, s)) =1 2 k2(k2 −k1 + 1) + i( ¯P3, G) −k3 (n −3)!Dn−1 ± 13k3 1k2(n −1)!(n −4)!. dp(G) −dp(T (n, s)) =1 2 k2(k2 −k1 + 1) + i( ¯P3, G) −k3 (n −3)!Dn−1 ± 13k3 1k2(n −1)!(n −4)!. the electronic journal of combinatorics 26(2) (2019), #P2.34 36 Furthermore, we have k2(k2 −k1 + 1) + i( ¯P3, G) −k3(G) ⩾ 1 50k1k2 by Claim 35(III). Therefore, rmore, we have k2(k2 −k1 + 1) + i( ¯P3, G) −k3(G) ⩾ 1 50k1k2 by Claim 35(III). ore, dp(G) −dp(T (n, s)) ⩾ 1 100k1k2(n −3)!Dn−1 −13k3 1k2(n −1)!(n −4)! > 0, dp(G) −dp(T (n, s)) ⩾ 1 100k1k2(n −3)!Dn−1 −13k3 1k2(n −1)!(n −4)! > 0, as Dn−1 = (e−1 + o(1))(n −1)!, 1 ⩽k1 ⩽cn1/2, and, since G is not canonical, k2 ⩾1. Thus to complete the proof of Proposition 10, we need to prove Claim 35. The ﬁrst part shows that, but for a handful of small exceptions, the intersection graph of a non- canonical union of cosets must have many edges. Proof of Claim 35(I). It is not diﬃcult to verify the result for k1 ⩽5. a contradiction. We may therefore assume 3 ⩽d ⩽k1 −2. Since each vertex x ∈V (G) \ ℓis incident to all but at most one vertex in ℓ, we must have k2 ⩾(k1 −d)(d −1) ⩾2(k1 −3), adiction. giving the required contradiction. giving the required contradiction. A.5 Proof of Proposition 10(d) It remains to deal with the case that k1 ⩾6 and k2 < max{k1, 2k1 −6} = 2k1 −6, in which case we wish to show G to be canonical. Let ℓbe an axis-aligned line that maximises d := \|ℓ∩V (G)\|. If d ⩾k1 −1, then G is canonical, as desired. If d ⩽2, then dG(x) ⩾k1 −3 for every x ∈V (G). Hence, as k1 ⩾6, k2 ⩾1 2k1(k1 −3) ⩾3(k1 −3) > 2k1 −6, a contradiction. a contradiction. 4We applaud the reader for making it this far. 5Observe that this is where we require the term i( ¯P3, G); in all other cases we simply use the fact that this is non-negative. giving the required contradiction. giving the required contradiction. The next part of the claim bounds the number of triangles in terms of the number of edges and vertices. Proof of Claim 35(II). We use induction on k1. The cases k1 ⩽6 can be checked by hand. Now suppose k1 ⩾7. If k2 ⩾k1, G cannot be canonical. It then follows from part (I) that (21) k2 ⩾2k1 −6 ⩾k1 + 1. (21) Let x be a vertex of G of minimum degree. We distinguish two cases. Case 1: x is isolated. In this case, vertices of G must lie entirely in the two axis- aligned lines ℓ1 and ℓ2 passing through x, and thus G is bipartite, implying k3 = 0. As a Let x be a vertex of G of minimum degree. We distinguish two cases. Let x be a vertex of G of minimum degree. We distinguish two cases. Case 1: x is isolated. In this case, vertices of G must lie entirely in the two axis- aligned lines ℓ1 and ℓ2 passing through x, and thus G is bipartite, implying k3 = 0. As a consequence, q , k2 (k2 −k1 + 1) (21) ⩾2(k1 + 1) ⩾16 > 2k3 + 1. Case 2: dG(x) ⩾1. Let G′ := G −{x}. Then, as x is of minimum degree in G, k2(G′) ⩾k2(G) −2k2(G) k1(G) (21) > k1(G) −2. q , k2 (k2 −k1 + 1) (21) ⩾2(k1 + 1) ⩾16 > 2k3 + 1. Case 2: dG(x) ⩾1. Let G′ := G −{x}. Then, as x is of minimum de q , k2 (k2 −k1 + 1) (21) ⩾2(k1 + 1) ⩾16 > 2k3 + 1. k2 (k2 −k1 + 1) (21) ⩾2(k1 + 1) ⩾16 > 2k3 + 1. Let G′ := G −{x}. Then, as x is of minimum degree in G, k2(G′) ⩾k2(G) −2k2(G) k1(G) (21) > k1(G) −2. k2(G′) ⩾k2(G) −2k2(G) k1(G) (21) > k1(G) −2. the electronic journal of combinatorics 26(2) (2019), #P2.34 37 combinatorics 26(2) (2019), #P2.34 37 the electronic journal of combinatorics 26(2) (2019), #P2.34 the electronic journal of combinatorics 26(2) (2019), #P2.34 37 Thus k2(G′) ⩾k1(G) −1 = k1(G′), and so the induction hypothesis applies to G′. Note that e(G) (e(G) −v(G) + 1) = (e(G′) + dG(x))(e(G′) −v(G′) + 1 + dG(x) −1) ⩾e(G′)(e(G′) −v(G′) + 1) + dG(x)(dG(x) −1) since dG(x) ⩾1 and e(G′) ⩾v(G′). 4We applaud the reader for making it this far. giving the required contradiction. By the induction hypothesis, e(G′)(e(G′)−v(G′)+1) ⩾ 2k3(G′)+1, and since there are at most dG(x) 2 triangles in G containing x, the right hand side of the above expression is at least 2k3(G) + 1. since dG(x) ⩾1 and e(G′) ⩾v(G′). By the induction hypothesis, e(G′)(e(G′)−v(G′)+1) ⩾ 2k3(G′)+1, and since there are at most dG(x) 2 triangles in G containing x, the right hand side of the above expression is at least 2k3(G) + 1. At long last, this brings us to the ﬁnal proof of this paper,4 the crucial inequality in the proof of Proposition 10(d). Proof of Claim 35(III). If k2 < k1, then, by part (I), G is isomorphic to 2K2, P4, P5 or C4∪K1. We can easily check that k2 (k2 −k1 + 1)+i( ¯P3, G)−k3 ⩾ 1 50k1k2 in these cases.5 Suppose, then, that k2 ⩾k1. If k1 ⩽5, then by part (II) we have y ( ) ( ) 50 Suppose, then, that k2 ⩾k1. If k1 ⩽5, then by part (II) we have k2 (k2 −k1 + 1) + i( ¯P3, G) −k3 ⩾1 ⩾ 1 50k1k2, as desired. It remains to handle the case k2 ⩾k1 ⩾6. Part (I) implies k2 ⩾2k1 −6, and so k2 −k1 + 1 ⩾1 6k1. Combining this estimate with part (II), we ﬁnd as desired. It remains to handle the case k2 ⩾k1 ⩾6. Part (I) implies k2 ⩾2k1 −6, and so k2 −k1 + 1 ⩾1 6k1. Combining this estimate with part (II), we ﬁnd k2 (k2 −k1 + 1) + i( ¯P3, G) −k3 ⩾1 2k2(k2 −k1 + 1) ⩾ 1 12k1k2, ﬁnishing the proof. ﬁnishing the proof. the electronic journal of combinatorics 26(2) (2019), #P2.34 38

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