The X-ray structure of a nonselective sodium potassium ion channel reveals structural asymmetry in the selectivity filter of an integral membrane protein responsible for ion permeation. NMR studies showed the dynamical nature of the protein structure in the membrane, and molecular dynamics suggest different degrees of conformational plasticity are required for conducting potassium and sodium. + permeation that is unique to NaK. NMR studies and molecular dynamics simulations confirmed the dynamical nature of the top part of the selectivity filter and the inner helix in NaK as also observed in the crystal structure. Taken together, these results indicate that the structural plasticity of the selectivity filter combined with the dynamics of the inner helix of NaK are vital for the efficient conduction of different ions through the non-selective ion channel of NaK.The sodium potassium ion channel (NaK) is a nonselective ion channel that conducts both sodium and potassium across the cellular membrane. A new crystallographic structure of NaK reveals conformational differences in the residues that make up the selectivity filter between the four subunits that form the ion channel and the inner helix of the ion channel. The crystallographic structure also identifies a side-entry, ion-conduction pathway for Na The sodium potassium ion channel (NaK) is a nonselective, monovalent cation channel that conducts both sodium and potassium ions equally efficiently  are also highly prone to either perfect or partial merohedral twining . After transformation and plating of E. coli BL21 cells on LB agar plates, colonies were resuspended in liquid LB medium, grown at 37°C to an A600 of approximately 0.8, and induced with 0.5 mM IPTG for 18 h at 25°C. Cells were then pelleted by centrifugation. For every gram (wet weight) of cells, 5 ml of lysis buffer  containing SIGMAFAST protease inhibitor (Millipore Sigma) and 1 mg ml−1 of lysozyme was added. Cells were resuspended by slow stirring at room temperature for 30 min and further lysed by sonication. Cell debris was removed by centrifugation at 10 000 relative centrifugal force (r.c.f.). NaK was then solubilized by incubation of the supernatant with 40 mM Sol-grade n-decyl-β-d-maltopyranoside (DM) at room temperature for 2 h. Additional debris was removed from the lysate by centrifugation at 21 000 r.c.f. for 30 min. The protein was then purified on a TALON metal affinity resin using buffers containing 4 mM DM. Fractions containing NaK were pooled, and the 6×His-Tag was removed by adding one unit of thrombin per milligram of NaK and incubating at room temperature for 16 h. NaK was then concentrated via ultrafiltration using a 30 kDa MWCO concentrator and further purified on a Superdex 200 Increase 10/300GL column using 20 mM Tris–HCl pH 7.8, 100 mM KCl and 4 mM Anagrade DM.A gene encoding a truncated form of NaK from 2.2.−1 using a 50 kDa MWCO concentrator. The crystals used for data collection were grown in sitting drops prepared by mixing equal volumes of protein solution in buffer  with well solution . Crystals were flash-frozen in liquid nitrogen without any additional cryoprotectant.A solution of NaK protein solution was concentrated via ultrafiltration to 14 mg ml2.3.XDS  from the same crystal using a Dectris PILATUS3 X 6M detector at the Advanced Photon Source (APS) on the ID19 beamline SBC-CAT to 1.53 Å resolution. All data were processed and reduced using 2.4.2.4.1.E. coli NEB express IQ (New England Biolabs). Minimal medium (M9) was supplemented with 13C glucose  and 15N ammonium chloride . At an OD600 of 0.8, the culture was induced with 0.4 mM IPTG  and grown overnight at 25°C. Cells were harvested and resuspended with lysis buffer  and protease inhibitor cocktail . The sample was lysed using a microfluidizer  with five cycles at a working pressure of 15 000 p.s.i. The membrane fraction was isolated by ultracentrifugation at 150 000 r.c.f. for 2 h at 4°C and solubilized in solubilization buffer  at room temperature for 3 h. The protein was batch-purified by immobilized metal affinity chromatography using TALON Superflow beads . Protein concentrations were determined using Bradford reagent supplemented with α-cyclodextrin . The protein sample was mixed with E. coli total lipid extract  in a ratio of 2:1 (w:w protein to lipid) for 1 h at 4°C. Reconstitution into liposomes was performed by dialysis at 4°C for 8 d against 20 mM Tris (pH 8.0), 50 mM NaCl and 50 mM KCl with 5× buffer exchange and a 100× dilution factor. The proteoliposome pellet was collected by ultracentrifugation at 300 000 r.c.f. for 4 h at 4°C. The sample was filled into a 3.2 mm magic angle spinning (MAS) rotor (Bruker BioSpin) and used for measurements, sample A with mixed-ion conditions.NaKΔ19 was expressed in M NaCl and 50 mM KCl conditions to only 50 mM KCl. For this, the sample was removed from the rotor and resuspended in 1 ml buffer with 20 mM Tris (pH 8.0) and 50 mM KCl. The buffer was refreshed twice, after ultracentrifugation for 30 min at 150 000 r.c.f. and 4°C, and the total incubation time was 14 d. After this time, the sample was ultracentrifuged at 300 000 r.c.f. and 4°C for 2 h and again filled into a 3.2 mm MAS rotor. Sample B contained only K+ buffer conditions.The sample was subsequently washed from 50 m2.4.2.15Ni–13Cαi (NCA) and 13C–13C correlation spectra. The carbon–carbon correlated proton-driven spin diffusion (PDSD) spectra were collected with a mixing time of 20 ms. The NCA experiment employed a SPECIFIC CP step with a contact time of 2.8 ms for sample A and 3 ms for sample B.Solid-state NMR spectra were recorded on a 16.4 T wide-bore spectrometer  equipped with a 3.2 mm triple-resonance Efree MAS probe. Spectra were recorded at 11 kHz MAS and referenced with external DSS . The sample temperature was kept constant at approximately 10°C as measured by the temperature-dependent position of the water resonance. The 90° pulse radio frequency amplitudes were 83.3, 50 and 35.7 kHz for proton, carbon and nitrogen, respectively. High-power proton decoupling was performed using the SPINAL-64 sequence during the evolution and detection periods. The samples were characterized using 2D 2.5.GROMACS software package  membrane and solvated in water with K+ or Na+ and Cl− ions, corresponding to a salt concentration of 600 mM. Instead of a WT-NaK channel, the F92A mutant was used in all simulations in order to increase the current as an experimentally increased ion flux was reported for this mutant  of 2 and 4 was maintained between two compartments in the computational electrophysiology setup , we used distance restraints between CA atoms of the A92 residue, similar to our recent work ]. We calculated the RMSD for alpha carbon atoms between the two polypeptide chains (A and B) in the dimer within our structure (1.40 Å) using the least-squares fit function (LSQ) in Coot ]. We also used Coot to calculate the RMSD for all atoms in the A and B polypeptide chains (1.96 Å).Within the asymmetric unit of our structure is a dimer that contains two of the four polypeptides that make up the complete NaK ion channel  in the A polypeptide has a refined occupancy of around 50%. In one conformation, the ND2 atom of the Asn68 side chain interacts with the backbone carbonyl of Asn68 on the B polypeptide (2.87 Å), whereas in the other conformation the ND2 atom interacts with the side-chain hydroxyl OG atom of Ser70 on the B polypeptide chain (3.01 Å). The conformation of the Asn68 side chain in the original NaKΔ19 structure is similar to the conformation that interacts with the backbone carbonyl of Asn68 on the B polypeptide. However, it is not a particularly close match. The backbone carbonyl of Asn68 is also present in dual conformations, although the difference is minor compared with that of Asp66 in the B polypeptide chain.During refinement, it became evident that the entire Asn68 residue in the A polypeptide was present in dual conformations and that the entire Asp66 residue in the B polypeptide was also present in dual conformations. Each of the two conformations of Asn68 [Fig. 3b)] also has a refined occupancy of around 50%. In one conformation its main chain carbonyl is parallel with the SF where it interacts with a water molecule (2.30 Å) that hydrogen bonds to one of four water molecules (2.30 Å) involved in coordinating the bound sodium ion at the second ion binding site within the vestibule of the SF. This conformation of the main-chain carbonyl of Asp66 is the one observed in previous NaKΔ19 structures . However, in the previous structure, the water–sodium coordination distance was an unrealistic 4.02 Å, compared with the much more favorable 2.60 Å distance  is 2.94 Å, which is very similar to the 3.00 Å distance observed in our structure. This coordination distance is somewhat longer than might be expected for a sodium ion but may reflect a mixture of ions within the vestibule (site 2).Within our ion channel structure, a potassium ion is bound at the external site found at the top of the SF Fig. 5. This po3.3.13C-15N]-labeled NaKΔ19 in proteoliposomes with 50 mM potassium and 50 mM sodium present. The 2D 15N–13Cα correlation spectrum can be seen in Fig. 6et al., 2018To further investigate the influence of mixed sodium and potassium conditions on NaKΔ19, we performed solid-state NMR measurements, which had previously only been performed under pure sodium or potassium conditions. We prepared uniformly , which is in good agreement with the X-ray structure showing the same pattern of conformational flexibility.To investigate whether the asymmetric X-ray structure represents the conductive state of the NaK channel, we performed molecular dynamics based computational electrophysiology simulations  ]: (i) the canonical pathway, where the ion transverses through the entire SF, similar to K+ permeation in selective K+ channels like KcsA  before leaving the SF. This result is different from our previous simulations of NaK based on the fourfold symmetric structure, where only the canonical conduction pathway was observed during K+ conduction through the SF ], similar to our earlier simulations of NaK starting from the fourfold symmetric structure  which was suggested by the ssNMR data. This leads to a considerably wider SF to accommodate solvated Na+ ions during permeation. In the current study, the channel was crystallized when both K+ and Na+ are present in the buffer solution. The new structure reveals that K+ ions bind at sites 3 and 4, whereas the Na+ ion is found in the vestibule as well as at the side-entry binding site. This result is in line with the ssNMR data, showing that the lower part of the SF  is stabilized by K+ at the mixed ionic condition. Therefore, we conclude that the current crystal structure with only conformational asymmetry at the top part of the SF represents only the K+-conducting state that enables ion permeations from two different pathways. In contrast, the Na+-conducting state of NaK is likely to exhibit a much larger conformational plasticity at the entire SF.In strong contrast to the K+ permeation can be regulated by relatively minor alterations of the SF width, that is, in turn, affected by the degree of opening of inner helices . The average opening at S4 reached slightly lower levels to those observed for increased current in NaK2K (ca 0.855–0.860 nm) in the CHARMM force fields, suggesting that the coupling between the inner helices and the SF might be weaker in NaK. Nevertheless, we observed that the larger opening could indeed enhance outward K+ conduction, especially with the CHARMM force field. In contrast, we did not observe any Na+ ion permeation events, similarly to smaller openings sampled in unrestrained simulations  revealed that outward Ks Table 2, further4.et al., 2018Our X-ray structure, NMR data and molecular dynamics simulations have revealed the dynamical nature of the inner helix in NaK and the different conformations of the inner helix below the gating hinge indicate the flexibility of this region. The asymmetry between polypeptides observed within the dimer in our asymmetric unit and the different conformations of Asp66 and Asn68 observed in the two polypeptides within the asymmetric subunit indicate the structural plasticity of the SF in NaK. The observed asymmetry reveals a so-far unseen sodium ion binding site adjacent to the SF that enables the side entry of ions into the ion channel. The presence of this side-entry binding site agrees with earlier MD simulations (Shi 6v8y.The structure and corresponding structure factors have been deposited into the Protein Data Bank with the PDB entry 10.1107/S205225252100213X/mf5049sup1.pdfSupplementary Table and Figure. DOI: sodium potassium ion channel NaK, 6v8yPDB reference:


The structural stability, electronic state, and interfacial thermal conductance of the interface between Bi2Te3 and MASnI3 were evaluated. Among the interface structure models, we found stable interface structures and revealed their specific electronic states. Around the Fermi energy, the interface structures with TeII and Bi terminations exhibited interface levels attributed to the overlapping electron densities for Bi2Te3 and MASnI3 at the interface. Calculation of the interfacial thermal conductance using the diffuse mismatch model suggested that construction of the interface between Bi2Te3 and MASnI3 could reduce the thermal conductivity. The obtained value was similar to the experimental value for the inorganic/organic interface.To develop high-performance thermoelectric devices that can be created using printing technology, the interface of a composite material composed of MASnI Madan et al.4 and Navone et al.5 reported early composite printing of thermoelectric devices. Navone et al.5 also noted that a previous study6 attempted to achieve this in 1990 using screen printing. For thermoelectric devices, it is necessary to improve the energy conversion efficiency in addition to reducing costs. The performance of thermoelectric conversion materials is described as the figure of merit, ZT = S2σT/(κel + κlat), which is proportional to the square of the Seebeck coefficient (S), electrical conductivity (σ), and absolute temperature (T), and is inversely proportional to the thermal conductivity (κ)9. Many studies have been conducted on the doping of heterogeneous elements to modify the electronic structure directly; this technique is effective for preventing an increase in the Seebeck coefficient and/or the electrical conductivity12.Printed electronics are attractive next-generation technologies that make full use of printing technology and are used to manufacture various electronic devices, such as organic EL TVs, electronic paper, and solar cells. In addition to making the devices thinner and lighter and increasing the area, printed electronics also save resources, allow for reduced process temperatures, and are more environmentally friendly. In the field of printed electronics, the thermoelectric conversion demand has increased with the development of energy-saving technology, which has a crucial role in the achievement of energy harvesting13. This broadened the outlook of the field because the previous strategy improved semiconductor characteristics by controlling impurities, but new characteristics arose in the obtained composites. To decrease the lattice thermal conductivity, many researchers have introduced crystal grain boundaries and nanostructures, which led to phonon scattering between crystal grains19. These techniques also affect improving the electrical conductivity and Seebeck coefficient, and their mechanisms are complex. As mentioned above, the interfacial thermal conductance has attracted attention for enhancing ZT, and this enhancement is attributed to the difference in lattice vibrations between heterogeneous materials21. Considering the fabrication of thin and flexible thermoelectric materials, hybridization of organic and inorganic materials is a critical technique for improving ZT owing to the formation of many interfaces24. Generally, to maintain the electric conduction ability, the organic materials used are conductive polymers such as poly  doped with poly (4-styrenesulfonate) (PEDOT:PSS)26. Lu et al. developed flexible PEDOT:PSS/Cu2Se nanocomposite films on nylon, and the resulting material exhibited a relatively high power factor of ~ 389.7 µW/(m·K2) at 418 K27. Kumar et al. fabricated a PEDOT:PSS/Te composite material, which reduced the thermal conductivity owing to the enhanced phonon–phonon scattering in the polymer matrix28. Many researchers have also studied the combination of Bi2Te3 and PEDOT:PSS32. Du et al. fabricated Bi2Te3 based alloy nanosheet/PEDOT:PSS composite films, which exhibited high electrical conductivity (1295.21 S/cm) relative to BI2Te3-based alloy bulk materials (850–1250 S/cm), and a power factor of ~ 32.26 µW/(m·K2) was obtained30. In a Te-Bi2Te3/PEDOT:PSS hybrid film synthesized through a solution-phase reaction at low temperature, a power factor of 60.05 µW/(m·K2) with a Seebeck coefficient of 93.63 µV/K and an electrical conductivity of 69.99 S/cm were reported by Bae et al31. Based on these results, it can be concluded that the electronic properties of the interface between the organic and inorganic materials play a critical role in improving the ZT of organic–inorganic hybrid materials.On the other hand, a thousand-fold reduction in thermal conductivity was reported with the use of organic/inorganic interfaces3) have previously demonstrated relatively high values as a printable thermoelectric material (ZT > 0.1 at room temperature)34. Organic–inorganic hybrid perovskites, ABX3 (A: methylammonium cation (CH3NH3+), B: lead or tin, X: iodide) have been investigated as candidate thermoelectric materials and are well known in the field of thin-film solar cells36. Regarding the thermoelectric properties of organic–inorganic perovskites, Pisoni et al. reported that CH3NH3PbI3 exhibited an ultra-low thermal conductivity of 0.3–0.5 W/(m·K) at room temperature due to the slowly rotating CH3NH3+ cations within the crystal structure37. Theoretical studies also predicted that CH3NH3PbI3 would have a low thermal conductivity of ~ 1 W/(m·K) compared with other perovskites such as CsPbI3, CH3NH3Br3, and CH3NH3PbCl340. On the other hand, CH3NH3SnI3 (MASnI3) is expected to exhibit low thermal conductivity compared with CH3NH3PbI3, with improved thermal properties obtained through chemical doping41. The advantage of perovskite compounds such as MASnI3 over the organic materials PEDOT: PSS is that they have a variety of constituent elements, which enable the system elemental substitution. It is possible to change the energy level near the Fermi level, and it is expected that the electric conductivity and Seebeck coefficient will be improved. Such electronic state control can be performed more easily with perovskite than with PEDOT:PSS.Here, we focus on halide perovskites instead of PEDOT:PSS to fabricate a printable thermoelectric material. The thermoelectric properties of inorganic halide perovskite (CsSnI2Te3 and organic–inorganic perovskite (MASnI3) to improve the thermoelectric conversion properties. We previously reported the structural stability and electronic properties of different Bi2Te3 (001) termination surfaces based on first-principles calculations42. Based on the results, we prepared three structures with different Bi2Te3 termination structures and explored statically stable structures through structural optimization. Additionally, we calculated the electronic states and distribution of the charge density near the Fermi energy. The calculation of the diffuse mismatch model (DMM)44 obtained from the results of phonon dispersion in Bi2Te3 and MASnI3 confirmed a decrease in the interfacial thermal conductance at the interface.In this study, we aimed to understand the interface structure of hybrid materials composed of Bi2Te3 was transformed from a rhombohedral lattice to an orthorhombic lattice, and the lattice parameter of MASnI3 was reduced to fit the lattice parameter of Bi2Te3. The interface models consisted of orthorhombic Bi2Te3 (001) and tetragonal MASnI3 (001), and a vacuum layer of ~ 15 Å was inserted. For simplicity, the termination structure of MASnI3 was fixed as SnI2 at the interface. For the structure of Bi2Te3 in contact with MASnI3, three termination structures were considered: TeI, TeII, and Bi terminations, which are relatively stable surface structures that were described in our previous study42 , and charge distribution, we considered the spin–orbit coupling (SOC).To create the interface structure, the crystal structure of BiBi2Te3 00 and tetr2Te3 and MASnI3 is the most direct calculation method. However, for an interface structure, the number of atomic displacement patterns are required to obtain the highly accurate atomic force, and it is impossible to calculate by the first-principles calculation. Therefore, in this paper, we used DMM44, which is often used as a simple method for evaluating interfacial thermal conductance.For the thermal conductance calculation, phonon calculation of the interface structure between BiG, as follows:k and phonon mode I, and n to the interface of A to B. Calculations of the transmission probability of A to B and the phonon frequency and group velocity of A and B obtained from phonon dispersion are required. Here, the transmission probability is calculated from the group velocities of A and B as follows:50.The interfacial thermal conductance  obtained by the DMM is defined as the ratio of the heat current density to the temperature differential. To estimate the thermal boundary conductance for hybrid materials A/B, Reddy et al. defined the thermal boundary conductance, 2Te3 and MASnI3 were evaluated using first-principles phonon calculations, and the group velocity was calculated from the results. To calculate the phonon dispersion, we used the finite displacement method with a displacement distance of 0.01 Å. The supercell sizes of Bi2Te3 and MASnI3 were 2 × 2 × 2 for the rhombohedral cells and 1 × 1 × 1 for the orthorhombic cells, respectively  and group velocity for both structures, the reciprocal space was sampled using 10 × 10 × 10 meshes.The phonon dispersions of Bi2Te3 termination structures: Bi2Te3(TeI)/MASnI3, Bi2Te3(TeII)/MASnI3, and Bi2Te3(Bi)/MASnI3 /MASnI3 structure, as indicated in Table d2/d1 – 1) × 100, where d2 and d1 represent the lattice distance of the transformed or optimized structure and the lattice distance of the bulk structure, respectively. On the other hand, the Bi2Te3(Bi)/MASnI3 structure exhibited a high lattice deformation ratio among the three interface models; the lattice of Bi2Te3 in the interface structure was particularly expanded. A low lattice deformation ratio is expected in the case of easy formation and relatively high stability of the interface structure experimentally.Table es Table . This re2Te3 with the TeII and Bi termination structures, and this phenomenon was prominent for the Bi termination. This result suggests that Bi and Sn atoms can move easily into each structure, and the TeII and Bi termination structures form an interaction between Bi2Te3 and MASnI3 compared with the TeI termination. The relationship between the reconstruction of atoms in the structural optimization, lattice parameters, and lattice deformation ratio was not observed.After structural optimization, the atoms in the interface moved strongly with an incomplete structure for Bi2Te3 and MASnI3, we calculated the binding energy using the following equation: Ebind = Etotal – Ep – Eb, where Etotal, Ep, and Eb denote the energies of Bi2Te3/MASnI3, the MASnI3 (001) surface, and the Bi2Te3 (001) surface, respectively  surface structures, we used the lattice constant of the ground state structure. A positive binding energy value indicates low stability of the interface structure, which makes formation of the interface difficult. Hence, Bi2Te3(TeI)/MASnI3 is the most unstable interface structure; in contrast, Bi2Te3(Bi)/MASnI3 is the most stable interface structure, with a binding energy of − 1.7 eV. Similar to the Bi termination model, Bi2Te3(TeII)/MASnI3 exhibited a negative binding energy of − 0.3 eV. Note that the corresponding interfacial energy, which is represented by energy/area, are 1.59 eV/nm2 for Bi2Te3(TeI)/MASnI, − 0.42 eV/nm2 for Bi2Te3(TeII)/MASnI3, and − 2.36 eV/nm2 for Bi2Te3(Bi)/MASnI3. The optimized structures suggest that the stability of the interface structure is attributable to the reconstruction of atoms in the interface.To evaluate the interface stability between Bi2Te3/MASnI3 interfaces are shown in Fig. 2Te3 in each termination structure were similar to that of the bulk structure. However, the energy levels of the DOS changed with the termination structure in Bi2Te3, which is attributed to the difference in the ratio of Bi and Te atoms. The partial DOS of MASnI3 in each interface structure exhibited different electronic states from the bulk structure over a range of − 0.5 eV to 0 eV; these states are attributed to the I p-orbital in MASnI3 in contact with the vacuum layer. In the DOS around the Fermi energy in the TeII and Bi termination structures  exhibited a similar shape despite the different interface structures. However, the shapes of the DOS for Sn and Bi changed significantly near the interface, and they exhibited a different electronic state with the variation in Bi2Te3 termination. In particular, on the Bi2Te3(Bi)/MASnI3 interface, the conduction band of MASnI3-1L moved to near the Fermi energy. This result is attributed to the large change in the atomic positions of Sn and I at the interface. On the other hand, the shape of the DOS for Bi2Te3 depended on its termination structure; in particular, it changed significantly in the layer in contact with the interface. This phenomenon originates from the different ratios of Bi and Te atoms in the incomplete Bi2Te3 structure. The Bi2Te3(Bi)/MASnI3 interface also had the potential to be affected by the movement of Bi atoms.To investigate the additional electronic state, the decomposed DOS for each layer near the interface is shown in Fig. 2Te3 and MASnI3, the additional electronic state is observed at the same energy level in the layer near the interface between Bi2Te3 and MASnI3. Figure 2Te3(TeII)/MASnI3 and Bi2Te3(Bi)/MASnI3 structures. These results also suggest that the incomplete structure of Bi2Te3, such as the TeII and Bi terminations, plays an important role in the formation of interface states. The TeI termination did not produce an overlap in the DOS between Bi2Te3 and MASnI3 at the interface.Focusing on the first layers from the interface of BiI termination, which is localized at the MASnI3 side, and is not observed at the interface between Bi2Te3 and MASnI3. This suggests a decreasing affinity of Bi2Te3 and MASnI3. On the other hand, the interface structure with the TeII termination possesses a localized charge distribution at the near interface and an overlapping charge density between Sn and Te atoms in the energy range of 0.2 to 0.5 eV /MASnI3 is localized at the interface, similar to Bi2Te3(TeII)/MASnI3. In this case, the overlap of the charge density appears in the valence band of − 0.5 to − 0.2 eV. These results suggest that the overlapping of the charge distribution increases the affinity of the interface and assists in charge transfer between Bi2Te3 and MASnI3. For all interface structures, the charge density is localized at the surface of MASnI3 at − 0.2 eV to 0.2 eV. From the perspective of a thermoelectric material, the material requires a high electrical conductivity and Seebeck coefficient and a low thermal conductivity. The Seebeck coefficient is related to the shape of the DOS around the Fermi energy53, and the formation of interface levels may lead to a change in its value. Further work is needed to clarify the thermoelectric properties of the Bi2Te3/MASnI3 interface structure.The charge densities of each interface structure around the Fermi energy are shown in Fig.  eV Fig. c. Figure2Te3/MASnI3 interface. Figure 2Te3 and (b) MASnI3. Bi2Te3 exhibited low energy phonon modes below 150 cm−1, and MASnI3 had low (f < 120 cm−1) and high (f > 120 cm−1) energy phonon modes. Because of the difference in atomic mass, the vibrations of Sn and I appeared at low energies, and the vibrations of C, N, and H appeared at high energies. Therefore, the phonon dispersion of MASnI3 showed a low-energy mode in the same range as Bi2Te3 until approximately 150 cm−1. Based on the phonon dispersion results for Bi2Te3 and MASnI3, these structures are dynamically stable at T = 0 owing to the lack of observation of the imaginary mode. Our calculated phonon dispersions for both structures are similar to those reported in previous studies for Bi2Te354 and MAPbI3 (not MASnI3)56.Next, we estimated the interfacial thermal conductance of the Bi2Te3 and a-, b-, and c-axes in MASnI3. The group velocity (speed of sound) was estimated from the three low-energy phonon modes within the phonon dispersion; hence, it corresponds to a gradient of the phonon dispersion. In calculated results, Bi2Te3 exhibited a high group velocity at under 10 cm−1, whereas MASnI3 had a high group velocity above 10 cm−1. Moreover, we found that the distribution of the group velocity with respect to the frequency differed between Bi2Te3 and MASnI3. For MASnI3, the group velocity was not dependent on the direction of the crystal axis. It has been experimentally reported that Bi2Te3 exhibits a group velocity of 1750 m/s based on nuclear resonant inelastic scattering57, whereas MAPbI3 has exhibited group velocities of acoustic modes of 2400 or 1200 m/s based on neutron scattering58. Although there is a difference in the structures between our calculations (MASnI3) and the previous experiments (MAPbI3), our calculated values are approximately consistent with the experimental data.The group velocities of phonons are necessary for the evaluation of the interfacial thermal conductance using DMM, as shown in Eqs.  and 2)..2). Figu2Te3/MASnI3 interfaces was estimated by DMM using the group velocities of both structures. Figure 2Te3/MASnI3 interface; the combination of all axes of MASnI3 and the c-axis of Bi2Te3 was evaluated. The obtained values for the interfacial thermal conductance with different Bi2Te3/MASnI3 interfaces were 1.5–2.0 MW/m2K. This result indicates that the interfacial thermal conductance was not affected by the orientation of MASnI3 because the differences between different directions were small, as shown in the phonon dispersion curve )59 and PEDOT:PSS-Bi2Te3 heterostructure (~ 10 MW/(m2·K))60 Therefore, these results suggest that the Bi2Te3/MASnI3 interface has a low interfacial thermal conductance, and we expect that the application of this interface to thermoelectric materials can reduce the thermal conductivity. An extremely low thermal conductance is expected even for a stable structure at the Bi2Te3/MASnI3 interface, although the morphological effects are not included in the DMM model. Direct numerical simulations, such as molecular dynamics, may be necessary for further discussion.Moreover, it was as low as the experimental values for inorganic/organic interfaces such as a graphene-Bi61, is too complex to explore here, we have aimed to discuss the effect of the interfacial thermal conductance of Bi2Te3/MASnI3 on the effective thermal conductivity, κ using a simple composite model. Here, a one-dimensional model is used, in which Bi2Te3 layers and MASnI3 layers with a thickness of D µm are alternately arranged, as shown in the inset of Fig. 2Te3/MASnI3 asymptotically approaches 0.17 W/(m·K) at a film thickness sufficiently larger than 1 µm. This value is calculated from the experimental values of Bi2Te3 and MASnI3: 2.11 W/(m·K)62 and 0.09 W/(m·K)41, respectively. This upper limit does not depend on the interfacial thermal conductance because the influence of the interface is negligible in the limit of large D. In contrast, when the film thickness is sufficiently smaller than 1 µm, the interfacial thermal conductance significantly influences the effective thermal conductivity. The blue line in the figure shows the effective thermal conductivity estimated from the calculated interfacial thermal conductance of 1.75 MW/(m2·K). The smaller the film thickness, the more effectively the interfacial thermal conductance of Bi2Te3/MASnI3 can be utilized. Thus, it is expected that the thermal conductivity of the Bi2Te3/MASnI3 composite, which consists of small Bi2Te3 grains in MASnI3, will be significantly reduced.Although the actual thermal transport mechanism, such as superlattices with very short periodicity2Te3 (001) and MASnI3 (001), and the thermal conductance of the interface between Bi2Te3 and MASnI3 along the (001) direction was estimated. In the structural optimization, the termination of MASnI3 was fixed with SnI2 at the interface and surface, whereas for the structure of Bi2Te3 in contact with MASnI3, three termination structures were considered: TeI, TeII, and Bi termination. After structural optimization, around the Fermi energy, the interface structures with TeII and Bi termination resulted in the formation of interface levels attributed to the overlapping electron densities for both Bi2Te3 and MASnI3 at the interface. It is believed that the formation of interface levels enhances the affinity for the interface structure of Bi2Te3 and MASnI3, and the binding energies for these interface structures are negative. Based on the calculation of the interfacial thermal conductance using DMM, it is expected that the Bi2Te3/MASnI3 interface can significantly reduce the thermal conductivity. These results indicate that the Bi2Te3/MASnI3 composite material is a possible candidate for an excellent thermoelectric material because it has the potential to decrease the thermal conductivity.In this study, we evaluated the stability and electronic state of interface structures of BiSupplementary Information.


Angioedemas localized in the upper airway are potentially life threatening, and without proper treatment, they may lead to death by suffocation. Upper airway edemas (UAE) in bradykinin‐mediated angioedemas can even be the first symptoms of the disease.Our survey was performed with a retrospective long‐term follow‐up method from the medical history of 197 hereditary (C1‐INH‐HAE) and 20 acquired C1‐inhibitor deficiency (C1‐INH‐AAE), 3 factor XII and 3 plasminogen gene mutation  patients treated at our center between 1990 and 2020. The UAE group included edemas localized to the mesopharynx, hypopharynx, and larynx, as narrowing of these anatomical regions can lead to suffocation.98/197 C1‐INH‐HAE (47 families) and 13/20 C1‐INH‐AAE, 1/3 PLG‐HAE, 1/3 FXII‐HAE patients had experienced UAE at least once according to their medical history. In case of C1‐INH‐HAE patients, in 6/47 families who had undiagnosed ancestors had 13 members who died of suffocation. After the diagnosis, 1‐1 member of two families died of UAE. 44/64 C1‐INH‐HAE patients did not smoke, 20/64 did. The occurrence of UAE was significantly higher in smoker patients. We analyzed 7607 HAE attacks of 56/98 patients. Out of all attacks, the incidence of UAE in the C1‐INH‐HAE group was 4%, and 9.5% in the C1‐INH‐AAE group, respectively.Early diagnosis is key in bradykinin‐mediated angioedemas cases, since the patient must be provided with adequate treatment; and also it is essential to inform patients about the importance of avoiding the trigger factors and the early symptoms of UAE, as these measures could significantly decrease the incidence of lethal UAEs. ACEI‐AAEacquired angioedema related to angiotensinconverting enzyme inhibitorsAEangioedemaANGPT1‐HAEhereditary angioedema with angiopoietin 1 gene mutationC1‐INHC1‐inhibitorC1‐INH‐AAEacquired angioedema with C1‐inhibitory deficiencyC1‐INH‐HAEhereditary angioedema with C1‐inhibitor deficiencyFXII‐HAEhereditary angioedema with factor XII gene mutationHAEhereditary angioedemaHS3ST6‐HAEheparan sulfate‐glucosamine 3‐O‐sulfotransferase 6 mutationInH‐AAEidiopathic nonhistaminergic acquired angioedemaKNG1‐HAEhereditary angioedema with kininogen 1 gene mutationMYOF‐HAEhereditary angioedema with myoferlin gene mutationnC1‐INH‐HAEhereditary angioedema with normal C1‐inhibitorPLG‐HAEhereditary angioedema with plasminogen gene mutationUAupper airwayUAEupper airway angioedemaU‐HAEhereditary angioedema of unknown origin1The upper respiratory tract is the anatomical structure connecting the nostrils and the lips with the trachea, at which any pathological change can cause a stenosis. In clinical practice, however, upper airway (UA) stenoses are classified as emergencies that risk suffocation that can affect parts of the mesopharynx, hypopharynx, and the larynx. Classical symptoms of UA obstruction include lump sensation, dysphagia, hoarseness, aphonia, tachypnea, dyspnea, and stridor. One of the causes of UA stenosis can be airway obstruction caused by the edema. The upper airway edema (UAE) can be caused by inflammation due to UA infections, mechanical/chemical trauma of the larynx/pharynx, malignant tumors, or they can occur independently as well. In the last case, fluid from the intravascular space flows into the extracellular space (caused by vasoactive mediators); this process leads to the development of a local edema which can occur in the subcutaneous regions or the mucosa of the UA or the gastrointestinal tract.SERPING1 gene, which results in the activation of the contact‐kinin‐kallikrein system, and which subsequently causes the liberation of bradykinin. Two types can be differentiated: the more frequent C1‐INH‐HAE type I (85% of cases), where the serum concentration level of C1‐INH is decreased; and the less frequent C1‐INH type II (15% of cases), where the antigenic level of C1‐INH is normal, but its functional activity is decreased. Approximately 25% of HAE patients have a negative family history, since the disease is developed due to a de novo mutation.Bradykinin‐mediated AEs have two types: hereditary and acquired ones. Hereditary angioedemas (HAE) can be divided into two groups: C1‐inhibitor (C1‐INH) deficient (C1‐INH‐HAE) ones and those where the level of C1‐INH is normal or slightly deficient (nC1‐INH‐HAE). C1‐INH deficiency can be proved with a complete complement laboratory testing .AE is a rare side effect of ACEI therapy, which could occur either separately or as a provoking factor of C1‐INH deficient forms. In the Caucasian population, the incidence of AE in patients taking ACEI is approximately between 0.1% and 0.7%, but it is more common in Black people.The purpose of the study was to analyze the clinical expression of UA angioedematous attacks in HAE and C1‐INH‐AAE patients treated in our center during the long‐term follow‐up.2Our survey was performed with a retrospective long‐term follow‐up method using the medical history of 197 C1‐INH‐HAE , 3 FXII‐HAE and 3 PLG‐HAE, and 20 C1‐INH‐AAE patients treated at the Hungarian Angioedema Center of Reference and Excellence (HACRE) between 1990 and 2020  or a recombinant C1‐inhibiror concentrate . Moreover, icatibant  was made available for all hereditary and acquired C1‐INH deficient, PLG‐HAE and XII‐HAE patients. For already diagnosed patients, family tree research and family screening were performed.p < 0.05. We used Mann–Whitney's U‐test, and considered p < 0.05 statistically significant for comparing baseline (at the time of diagnosis) C1‐INH functional level and the incidence of UAE. Data of AE symptoms and complement parameters were recorded in the National Angioedema Register.We analyzed the incidence, clinical properties, and the role of provoking factors. The connection between smoking and UAE was determined with Fisher exact test, with the significance level being The study protocol was approved by the institutional review board of Semmelweis University of Budapest, and informed consent was obtained from the participants in accordance with the Declaration of Helsinki.33.13.1.1156/197 patients were treated at HACRE who and/or whose family member(s) have experienced UAE; these patients were from 47 families.Out of 156 patients, 32 (from six families) had 13 relatives who were not diagnosed and died of suffocation.Relatives of 41/197 patients (from 20 families) treated at the Center did not have UAE; in this group, 2 members from 1 family had 2 not diagnosed ancestors who died of suffocation.After C1‐INH‐HAE diagnosis, we had 2 patients who died of UAE  out of 13 patients from two families, due to inadequate compliance. The human plasma derived C1‐esterase inhibitor concentrate on‐demand treatment was available for both patients, but they did not use it. For one of the patients, it was her first UAE; this patient had C1‐INH‐HAE type II  appeared on the lips and the tongue, 463 (1.6%) on the face, 975 (3.4%) appeared on the UAs, 987 (3.4%) appeared on the genitalia, 9288 (32%) on the gastrointestinal tract, and 17,065 (58.8%) attacks appeared in the subcutis.p = 0.6672).During the follow‐up period, there were 98 patients  who experienced an UAE during their lifetime. Out of those 98 patients, 90 patients had C1‐INH‐HAE type I while 8 patients had C1‐INH‐HAE type II. The median of the occurrence of the first UAE was at the age of 22 years . There was no difference between the sexes regarding the incidence of UAE: it occurred in 56/111 women and 42/86 men . At the time of the appearance of the UAE, this group either did not get treatment, or they received conventional  therapy. Four patients  had tracheotomy, one patient had tracheotomy four times, because he had not responded to conventional treatment. Only 10/98 patients had UAE as their first angioedematous attack.In case of 8/56 women and 7/42 men, the first UAE was provoked with a dental procedure or an operation in the mouth or the pharynx, and in case of one patient, it was caused by taking ACEI prior to the establishment of the C1‐INH‐HAE diagnosis.3.1.3p < 0.0001)  cases among patients experiencing UAE, while in the group without UAE we found this type of mutation in 9/97 (9.3%) patients. The splice mutation was confirmed in 9/98 (9.2%) and 22/97 (22.7%) patients, respectively. The missense mutation was confirmed in 29/98 (29.6%) and 22/97 (22.7%).We examined the level of C1‐INH functional activity, during which we found a significant difference between the UAE  and non‐UAE (median: 44%) groups  Figure . We also3.1.4p = 0.1607).From 2012, we have regular information about the smoking habits of our patients, so we were able to analyze 68/98 patients regarding their smoking habits. Four patients often changed their habits so they were excluded from the analysis. Forty‐four out of 64 patients were non‐smokers, while 20 smoked regularly. Examining the relationship between gender and smoking, we found no significant difference (n = 3), 75% experienced UAE more than once.Out of the 44 non‐smoking patients, 50% only had UAE once during the examined period, while for smoking patients, this rate was 15% , 262 face (3.4%), 301 UAE (4%), 2464 abdominal (32.4%), and 4533 subcutaneous attacks (60%) took place  resulted from upper respiratory infections. Mechanical trauma  could be named in case of 11/301 attacks (in seven patients). The UAE attack was provoked by dental procedures in four cases (in four patients), while menstruation provoked six attacks in four patients. 23/301 UAE attacks (14 patients) were provoked by stress and physical exhaustion. ACEI provoked an UAE in 1 patient.We examined the frequency of the appearance of UAE attacks: 16/56 patients (28.6%) had one UAE attack in 10 years; 32/56 patients (57.1%) had 2–10 attacks; 4 patients (7.1%) had 11–20 attacks, and 4 patients (7.1%) had experienced more than 20 UAE attacks.In case of 149/301 UAE attacks, our patients accurately documented when the attack happened. Based on this, four spikes can be observed in the frequency of UAE attacks: in January, April, July, and November.The patients were divided into 10‐year‐long age groups and the localization of the attacks was also compared Figure . The 453The age distribution of UAE showed the following distribution: 1 patient (2 attacks) between 9 and 10 years, 9 patients (37attacks) between 11 and 20 years, 17 patients (100 attacks) between 21 and 30 years, 11 patients (73 attacks) between 31 and 40 years, 15 patients (46 attacks) between 41 and 50 years, 7 patients (22 attacks) between 51 and 60 years, and 7 patients (21 attacks) at 60+ years.3.2Out of 20 C1‐INH‐AAE patients treated at the HACRE, 13 have already experienced UAE. The family history regarding angioedematous attacks was negative for all patients. The median of the appearance of the first UAE was at the age of 60 years . In 5/13 patients, the first AE attack was localized as an UAE. For the first UAE, all 13 patients received conventional treatment. 3/13 patients  were either intubated or had tracheotomy. In case of 8/13 patients, ACEI could be observed as a provoking factor. Out of 20 patients, ACEI provoked an AE attack in case of nine patients. One patient experienced UAE after a dental procedure.10/13 patients had their C1‐INH‐AAE diagnosis established within 1 year after the first UAE. In case of three patients, the time between the first UAE and the diagnosis was 2, 7 and 10 years. In case of the first patient, chronic lymphocytic leukemia was the underlying disease, while the latter two patients have not had an underlying disease diagnosed since. Due to the small number of patients, all the data of C1‐INH‐AAE patients was analyzed focusing on the localization of the attack. The 20 patients had 187 subcutaneous 27.4%), 157 lip and tongue (23%), 146 face (21.4%), 124 abdominal (18.2%) and 69 UAE (10.1%) attacks altogether  are treated due to the mutation of the K330E plasminogen gene. From the 3 PLG‐HAE patients, two experienced AE: one patient at the age of 53 years had a tongue edema, the other patient had a face edema at the age of 17 and multiple tongue and pharynx edemas at the age of 40 years, which were probably provoked by taking ACEI (perindopril). We also treated three patients  due to the 18 base pair duplication in the 9th exon of factor XII (c894_911dup). Out of them, only one patient had AE: they experienced an attack that affected their face, eyelid, lip, pharynx, and abdomen at the age of 35. None of the patients smoked, and we do not know about anyone in their families who died of suffocation.4Currently, the first appearance and the frequency of UAE cannot be predicted, it occurred in all four examined patient groups , and it caused a life‐threatening condition and suffocation for the undiagnosed relatives of the patients in the C1‐INH‐HAE group, based on the family history. We examined nine families whose members have experienced suffocation due to UAE, and in case of 7/9 families, a total of 15 ancestors died of suffocation prior to the diagnosis of C1‐INH deficiency. This is in line with the findings of Bork et al., published in 2012, in which they pointed out the fact that death by suffocation is higher in undiagnosed patients. Almost 30% of the undiagnosed ancestors died of suffocation, and the rate of the death by suffocation in undiagnosed C1‐INH‐HAE patients compared to diagnosed patients was 9:1; the reason for this was presumed to be the lack of awareness in undiagnosed patients .It was an interesting observation that although HAE attacks occur more frequently and in more severe forms in women, the incidence of UAE attacks showed no difference between the two sexes. We found a similar result among men and women who smoked.The incidence of UAE in our patients is substantially higher than in earlier studies, where the incidence of UAE was less than 1% of HAE attacks. UAE covers 4% of all attacks in C1‐INH‐HAE patients, while it makes up 10% of all the attacks in C1‐INH‐AAE patients.The UAE group had significantly lower baseline functional C1‐INH levels, which consistent with the observations of our previous study which demonstrated a significant correlation between the level of functional C1‐INH and disease severity.SERPING1 mutations were detected in a considerable number of patients who ever suffered from UAE (16.3%), compared to those who never had an UAE attack (9.3%), which is in line with our previous observation, where missense SERPING1 mutations were associated with a less severe disease course while the nonsense and splice mutations resulted in a more severe course.SERPING1 occurred less frequently in patients suffering from UAE (9.2%) compared to those who never had an UAE (22.7%). Many trigger factors are known which could provoke UAE, for example, upper respiratory tract infections, operations in the mouth‐pharynx‐larynx area, dental procedures, any kind of mechanical trauma in this region , or taking certain medications (ACEI).Interestingly, nonsense Since the area of the larynx is highly exposed to pathogens and irritative agents, it is presumed that the larynx has an important immunological function in the airways; this is also supported by the mucosa‐associated lymphoid tissue specific to the larynx found on the laryngeal surface of the epiglottis, the false vocal cords.It was an important observation that UAE attacks in C1‐INH deficient patients show a different pattern. The time elapsed between the first UAE attack and the diagnosis was longer in case of C1‐INH‐HAE patients.10/13 patients had the C1‐INH‐AAE diagnosis established within 1 year after the first UAE, which correlates with the diagnostic window in literature.In case of bradykinin‐mediated angioedemas, establishing an early diagnosis is fundamental, as UAE has a crucial role in both hereditary and acquired AEs, being the only edema localization that can be lethal without proper treatment.For the first time, hereditary types usually occur in young adulthood, but they can occur at any age; hence, patients must be informed and trained, which does not only include the early recognition of the starting symptoms of UAE ,Zsuzsanna Balla has participated in clinical trials of CSL Behring, Pharming, Pharvaris, and Takeda. Beáta Visy has participated in clinical trials of Takeda. Lilian Varga has received travel grants from CSL Behring and Shire Human Genetic Therapies Inc. Henriette Farkas received research grants from CSL Behring, Takeda, and Pharming and served as an advisor for these companies and Kalvista and Biocryst, and has participated in clinical trials/registries for BioCryst, CSL Behring, Pharming, Kalvista, Pharvaris, and Takeda. The other authors have declared that no conflict of interest exists.GraphPad Prism 5.0 .


Comparison with the mono‐triarylboranes reveals the large influence of the bridging unit on the properties of the bis‐triarylboranes, especially those of the cationic compounds. Based on these preliminary investigations, the interactions of 1+Cat, 2+Cat, 2+Cat(i), and 3+Cat with DNA, RNA, and DNApore were investigated in buffered solutions. The same compounds were investigated for their ability to enter and localize within organelles of human lung carcinoma (A549) and normal lung (WI38) cells showing that not only the number of charges but also their distribution over the chromophore influences interactions and staining properties.The synthesis, photophysical, and electrochemical properties of selectively  Four selectivelymono‐,bis‐ andtris‐trimethylammonium‐substituted, bithiophene‐bridged bis‐triarylboranes were synthesized and their potential biological applications were investigated including live cell imaging and photo‐induced cell death. It is demonstrated that their photophysical and electrochemical properties and their interactions with DNAs, RNA and cells, are strongly influenced by the number and distribution of charges within the compounds. Additionally, we demonstrate that biomolecule binding and staining selectivity for these compounds depends on the number and distribution of the cationic charges. Thus, the neutral compounds Neut1‐Neut3 and the resulting selectively charged compounds 1+Cat–3+Cat 2, and Neut3 was made possible via a synthetic route to unsymmetrically‐substituted triarylboranes which was previously not well developed,The synthesis of Neut4 and 4+Cat.mono‐ and di‐halogenated bithiophenes  as well as three different, borylated triarylboranes 4b, 5b, and 6b were required , BF3 ⋅ OEt2, and 2,6‐dimethylphenyl lithium using the general approach developed by Grisdale and co‐workers.2BFMes and subsequent reaction with 2,6‐dimethylphenyl lithium gave the triarylborane 4a in 76 % yield over two steps. C−H borylation with [Ir(COD)(μ‐OMe)]2 as the precatalyst, 4,4′‐di‐tert‐butyl‐2,2′‐dipyridyl (dtbpy) as the ligand, and bis‐pinacolato diboron (B2pin2) gave 4b in 90 % yield. The 1H NMR spectrum of 4b matches that reported in the literature.3 using Pd2(dba)3 ⋅ CHCl3 as the precatalyst, 2‐dicyclohexylphosphino‐2′,6′‐dimethoxybiphenyl (SPhos) as the ligand, and potassium hydroxide as the base gave Neut0 in 31 % yield (Scheme Neut(i)2 was synthesized from 5b and 5,5’‐dibromo‐2,2’‐bithiophene 3 in 53 % yield.For the synthesis of Neut1, Neut2, and Neut3, 5‐bromo‐2,2’‐bithiophene 2 was cross‐coupled with 4b, 5b, and 6b, respectively, in Suzuki‐Miyaura reactions using the conditions described above to give 7a, 8a, and 9a in 71 %, 93 %, and 80 % yields, respectively . However, the first reaction sequence gave higher yields in all steps, especially the last one (60 % vs. 6 % yield).To obtain Neut1–3 in hand, their cationic counterparts were synthesized using methyl triflate in CH2Cl2 similar to previous reports[PF6] as the electrolyte and a scan rate of 250 mV s‐1. Each measurement is referenced to the ferrocene/ferrocenium (Fc/Fc+) couple. The redox potentials of uncharged and cationic triarylboranes 4a–6a, 5c, and 6c were examined as well as those of the neutral and cationic bithiophene bis‐triarylboranes Neut0–4 and 1+Cat–2+Cat. Potentials of the neutral compounds were measured in THF, and those of the cationic compounds in MeCN. All cyclic voltammograms are shown in the Supporting Information  together with an extended discussion thereof. The electrochemical data are summarized in Table 4a, 5c and 6c, within the electrochemical window of the solvent used. In the case of 5c and 6c, the presence of ca. 0.5 % starting material was confirmed by 1H NMR spectra. Thus, the minor oxidation waves observed for both compounds can be attributed to the presence of non‐ or partially methylated starting materials.Electrochemical measurements were performed in the respective solvent (THF or acetonitrile) with [4a, 5a, 6a, 5c, and 6c one partially reversible 1e− reduction potential for the boron center and one 1e− oxidation potential per amino group was found. The reduction potentials decrease with increasing electron density at the boron center which increases in the order 6c<5c<4a<5a<6a. The electrochemical data are in good agreement with values reported by Gabbaï and coworkers for very similar trimethylammonium‐substituted triarylboranes. However, the trend that each trimethylammonium group leads to a decrease of 0.26 V in MeCN5c vs. 6c) but not for the addition of the first one (4a vs. 5c).For the triarylboranes bis‐triarylboranes in THF  suggesting negligible delocalization between the two boron atoms. Furthermore, a second (irreversible) 1e− reduction at ca. −3.05 V was observed. For all dimethylamino‐substituted bis‐triarylboranes, an irreversible oxidation occurred at ca. 0.35 V; only Neut3 has an additional irreversible oxidation at 0.49 V.For all neutral F Figure , one parbis‐triarylboranes  and 4+Cat display only one reduction potential. These result from two simultaneous 1e− reductions (Table S20), as the two boron centers in the latter are the same on both sides of the molecule. It was not possible to improve the resolution of the quasi‐reversible reduction waves of 1+Cat by pulse voltammetry experiments .For all cationic s Figure , two fulcalc, ΔEopt, ΔECV) were obtained from DFT calculations and experimental values. The absorption spectra were used to determine the optical bandgap ΔEopt and ΔECV was calculated from the HOMO and LUMO energies according to a literature procedure (see Supporting Information).[opt does not change significantly within a series of neutral or cationic bis‐triarylboranes but decreases by ca. 0.5 eV upon introduction of the strong dimethylamino electron donor in the mono‐triarylboranes, due to destabilization of the HOMO more than the LUMO, as supported by DFT calculations. The values of ΔEopt and ΔECV are of the same magnitude and display the same trends. For the cationic compounds, only the cationic moiety was used in the calculations, so ion‐ion‐interactions were not considered.HOMO‐LUMO gaps (ΔErmation).48, 492‐saturated acetonitrile solutions was investigated for the triarylboranes 4a, 6c, and 5c as well as for the charged bis‐triarylboranes 1+Cat–4+Cat by monitoring the weak 1O2 phosphorescence emission at 1275 nm vs. that obtained from a solution of the known sensitizer perinaphthenoneThe ability to sensitize singlet oxygen in O4a, 6c, and 5c sensitize 1O2 with efficiencies of 0.3, 0.5, and 0.6, respectively,  which increases with increasing number of trimethylammonium substituents (Table S17). The corresponding fluorescence quantum yields in acetonitrile decrease in the same order from 0.34 to 0.11 and 0.10. Thus, the sum of singlet oxygen efficiency ΦΔ and fluorescence quantum Φf yield for these compounds is ca. 0.6 (Table S17).Triarylboranes bis‐triarylboranes sensitize 1O2 with efficiencies of 0.6, 0.8, 0.7, 0.6, and 0.6 for 1+Cat, 2+Cat, 2+Cat(i), 3+Cat, and 4+Cat, respectively , which is the reason for the raised baseline between 1230 and 1260 nm in Figures ΦΔ of ca. 0.6 is estimated for all charged bis‐triarylboranes.The charged y Figure , Table 41 excited states of 1+Cat–4+Cat thus decay ca. 40 % by fluorescence and ca. 60 % by intersystem crossing to the triplet state, which very efficiently sensitizes 1O2. Attaching two triarylboranes to a bithiophene motif leads to a more efficient energy transfer to triplet oxygen than for the mono‐triarylboranes. Thus, interactions with DNA, RNA and DNApore and cell imaging studies were only examined for the cationic bis‐triarylboranes.The Sbis‐triarylboranes 1+Cat–3+Cat and naturally occurring calf thymus DNA  and AT‐(52 %) base pairs), double stranded (ds) poly rA–poly rU  and DNApore .The stability of 1+Cat–3+Cat upon addition of increasing amounts of DNA, or RNA were studied at pH 7 and/or pH 8. In combination with the results of thermal denaturation (Tm) and circular dichroism (CD) experiments of the polynucleotides, which are known to be unique for the respective molecule, conclusions about the mode of interaction  can be drawn.[bis‐triarylboranes 2+Cat–3+Cat, suggest non‐covalent binding of the latter, characterized by the type of interactions which can increase the stability of DNA/RNA . However, the emission of 1+Cat showed negligible changes in the presence of any polynucleotide at any pH , and 3+Cat, Tm is increased compared to pure ctDNA or pApU at pH 7. Thus, these three compounds efficiently bind to ctDNA and pApU and stabilize these polynucleotides , and 3+Cat are lower than that reported for 4+Cat.In the presence of 2+Cat–3+Cat increases depending on the structure of the polynucleotide and the number of charges. Using the Scatchard equation2+Cat, 2+Cat(i), and 3+Cat bind in the minor groove of DNA or the major groove of RNA.Upon addition of ctDNA or pApU, the emission of 2+Cat, 2+Cat(i), and 3+Cat to ctDNA or pApU, a small decrease of the intensity of the CD spectra between 230 nm and 300 nm  was observed. This can be attributed to an unwinding of the double helix of the polynucleotides upon insertion of the bis‐triarylboranes. In contrast to the previously reported tetra‐cationic compound 4+Cat,2+Cat, 2+Cat(i), and 3+Cat might be non‐uniformly oriented with respect to the chiral axis of the polynucleotide, but it was not possible to derive direct structural information. Nevertheless, in analogy with our previous binding studies,[2+Cat, 2+Cat(i), and 3+Cat can be assumed.Upon addition of ‐1 Tris‐HCl, 300 mmol L−1 KCl) were increased in the presence of 2+Cat, 2+Cat(i), 3+Cat, and 4+Cat <3+Cat<4+Cat. The unusual drop of absorbance observed for 2+Cat(i) upon addition of DNA is likely due to precipitation of a 2+Cat/DNA complex upon complete unwinding.Similarly, as described above for sodium cacodylate solutions, thermal denaturation temperatures of ctDNA and DNApore in Tris‐buffer at pH 8 (15 mmol L+ Table . The den2+Cat and 2+Cat(i) upon addition of ctDNA at pH 7, at pH 8, the emission of these compounds decreases , 3+Cat, and 4+Cat decreases  and 3+Cat, a negative band at ca. 425 nm was observed, indicating a perpendicular orientation of the transition moments to the chiral axis of ctDNA at pH 8.Addition of bis‐triarylboranes studied herein, and the same experimental procedures applied, the results of these measurements can be used for comparisons within this series of compounds. For 2+Cat, weak, negative ICD bands were observed which is characteristic for a perpendicular orientation of the transition moment of the chromophore with respect to the chiral axis of the DNApore. In contrast to the CD measurements at pH 7, which did not change at wavelengths >300 nm, addition of 2+Cat(i) to DNApore at pH 8 induced strong ICD bands at 400–450 nm , 3+Cat, and 4+Cat interact with all polynucleotides investigated. Although there are some similarities, e. g. interaction with DNApore leads to emission decrease of the compounds, the number and distribution of charges affects the interaction of bis‐triarylboranes with different polynucleotides. In particular, the induced CD bands of various derivatives , 3+Cat, and 4+Cat are useful probes for further studies of the DNApore structure and interactions with novel ligands.Thus, s Figure  can be ubis‐triarylboranes to see whether they penetrate cell membranes, stain specific organelles therein, or if they have anti‐proliferative effects to A549 and WI38 cells. Confocal microscopy studies showed that the mono‐cationic compound 1+Cat does not penetrate the cells, in contrast to 2+Cat–3+Cat. Thus, this compound was not further investigated in biological studies. For 2+Cat, only preliminary results were obtained, suggesting localization at the cellular membrane and certain compartments in the cell nucleus. However, due to the low stability of this compound in Dulbecco Modified Eagle's Medium (DMEM) containing 10 % fetal bovine serum (FBS), only the staining patterns of 2+Cat(i) and 3+Cat were further investigated.Studies were conducted on the cationic 2+Cat(i) and 3+Cat was tested using the MTT assay against human lung carcinoma (A549) and normal lung (WI38) cell lines . In the dark, 2+Cat(i) is not cytotoxic in all concentrations tested . However, when irradiating the incubated cells of both cell lines in the presence of 1 μM 2+Cat(i) at 400–700 nm for 5 min, the viability of the cells was reduced drastically with increasing concentration of 2+Cat(i).The cytotoxicity of 3+Cat is not cytotoxic to both cell lines in concentrations of 0.1 μM and 1 μM. However, at 10 μM concentrations, 3+Cat is cytotoxic to A549 cells. This toxicity is even more pronounced for WI38 cells. For both cell lines, the toxicity increases under irradiation (400–700 nm) at 1 μM and 10 μM concentrations.In the dark, 2+Cat, 2+Cat(i), or 3+Cat by confocal microscopy while irradiating at 405 nm at the full power of the light source (LED). Within 2 min, significant changes in the cell morphology were observed  suggesting strong cellular damage. Simultaneously, the emission of the dyes bleached rapidly within 30 s to 1 min . The stability of the emission increases with the number of cationic charges. After the emission is quenched completely, no additional cell damage was observed. Control experiments on untreated cells did not show any sensitivity to light exposure. This is in good agreement with the cell toxicity of 2+Cat(i) and 3+Cat under UV irradiation found during the MTT assay.The increased toxicity of these compounds when irradiated with UV light results most likely from their ability to sensitize singlet oxygen (see above) as the latter is a highly reactive compound leading to cell death.2) with 2+Cat(i), or 3+Cat and exciting with light , the cells show strong green emission due to the accumulation of the compounds in some organelles in the cytoplasm. The staining patterns of both compounds were further analyzed by co‐localization experiments 2+Cat(i) (0.54) and 3+Cat (0.46), the best overlap was obtained for co‐localization with LAMP1, a staining antibody selective for lysosomes. However, the staining pattern of the bis‐triarylboranes suggests further interactions with early endosomes and golgi apparati . Thus, these new compounds are not specific for one organelle but are distributed between the organelles mentioned with some selectivity for lysosomes.However, when incubating A549 cells for 90 min , 3+Cat) and their neutral precursors 2, Neut3) are described. The compounds were isolated in good yields and the influence of different numbers and locations of charges on electrochemical and photophysical properties and cellular imaging was investigated. Photophysical investigations reveal expected behavior for D‐A compounds wherein the boron center is the electron‐acceptor in all compounds with differing electron‐donating moieties, as supported by DFT and TD‐DFT calculations. For the cationic bis‐triarylboranes, an increasing hypsochromic shift of the emission spectra with increasing dipole moment was observed in acetonitrile. CV measurements of the neutral and cationic bis‐triarylboranes show that the π‐conjugation, and therefore the communication between the boron centers, is improved upon methylation of the dimethylamino groups. Efficient singlet oxygen sensitization was demonstrated for the cationic bis‐triarylboranes 1+Cat, 2+Cat, 2+Cat(i), and 3+Cat. For comparison with the properties of the bis‐triarylboranes, selected properties were also investigated for the mono‐triarylboranes 4a‐6a, 5c, and 6c.The syntheses of selectively trimethylammonium‐substituted, bithiophene‐bridged, 1+Cat, 2+Cat, 2+Cat(i), and 3+Cat in pure water, photophysical investigations and biological studies were performed in aqueous environments containing less than 1 % acetonitrile and 0.1 % DMSO, respectively. Studies with DNA and RNA in buffered solutions show negligible binding for the mono‐cationic compound 1+Cat but strong binding for 2+Cat, 2+Cat(i), and 3+Cat within the minor groove of DNA or the major groove of RNA exhibiting a strong fluorescence increase under physiological conditions. The compounds also bind strongly to DNApore, most likely inside the pore. Thus, they could potentially act as a “stopper” of ubiquitous material flow, which DNApore enables when inserted in, e. g., bacterial membranes.2+Cat, 2+Cat(i), and 3+Cat might be useful to study the fine structure of DNApore and the interaction with other ligands. Studies with A549 and WI38 cells show all compounds to be cell permeable except for the mono‐cation 1+Cat. Under intense irradiation (400–700 nm), the emission of 2+Cat, 2+Cat(i), and 3+Cat bleaches rapidly with simultaneous changes of cell morphology, most likely due to in situ formation of singlet oxygen. Thus, the cytotoxicity of the compounds is strongly increased upon irradiation with light (400–700 nm) as demonstrated using the MTT assay. Without irradiation, 2+Cat(i) and 3+Cat are not cytotoxic up to concentrations of 10 and 1 μM, respectively, for both cell lines. Co‐localization experiments of both compounds in A549 cells demonstrate the lysosome as the main but not only accumulation site, since 2+Cat(i) and 3+Cat also localize in other organelles. Thus, the cationic bis‐triarylboranes are promising theranostic agentsbis‐triarylborane chromophores for biological applications, the number and distribution of charges should be considered as well as the bridging unit.Due to the moderate solubilities of 4a) and 2072402 (for 4b)2072401 (for  contain(s) the supplementary crystallographic data for this paper. These data are provided free of charge by the joint Cambridge Crystallographic Data Centre and Fachinformationszentrum Karlsruhe Access Structures service.”“Deposition Number(s) The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


The butyrate groups in FeC10(HgX)10 (X=O2CC3H7) can be exchanged by treatment with trifluoro‐ or trichloroacetic acid . Substitution of the trifluoroacetate groups by halides  proceeds easily in aqueous THF. The completeness of metalation was confirmed by NMR and vibrational spectroscopy, mass spectrometry, as well as elemental analysis. Additionally, the first crystal structures of permetallated metallocenes are presented: FeC10(HgX)10 .The permercuration of ferrocene was achieved by reacting ferrocene with 10 equivalents of mercury(II) butyrate Hg(O That's so metal: All ten C−H bonds of ferrocene can be metalated in a simple one‐pot reaction by using mercury butyrate to give permercurated ferrocene, an air‐ and water stable compound. The completeness of metalation is confirmed by NMR and vibrational spectroscopy, mass spectrometry, as well as elemental analysis. Even unreactive C−H bonds such as methane can be brought to reaction using Hg(NTf2)2.The functionalization of unreactive C−H bonds is a constant challenge in organometallic chemistry due to their high intrinsic stability.nBuLi for four days and subsequent quenching with D2O yields only to relatively low degrees of deuteration . The degree of lithiation can be slightly improved by addition of tetramethylethylenediamine (TMEDA).2+ proceeds mechanistically via an electrophilic substitution pathway.Ferrocene derivatives have found numerous applications, for example, in material science, medicinal chemistry, and catalysis, which can be explained by its unusual stability towards moisture and oxygen as well as its unique redox properties.10(HgO2CC3H7)10 (2) was synthesized by reaction of ferrocene with mercury(II) butyrate (1) in 1,2‐dichloroethane (DCE) under reflux (Scheme 2 with trifluoro‐ or trichloroacetic acid in THF yielding compounds FeC10(HgO2CCF3)10 (3 a) and FeC10(HgO2CCCl3)10 (3 b), respectively. The trifluoroacetate derivative 3 a could be converted to the insoluble halide derivatives FeC10(HgX)10 (4)  by reaction of NaF/NaCl in aqueous THF mixtures. In contrast to the literature‐known permercurated metallocenes, compounds 2 and 3 are soluble in DMSO and tetrahydrothiophene (THT). THF adducts of 3 are even soluble in dichloromethane and methanol. This makes these compounds attractive starting materials for further reactions.FeC1, 3 a, 3 b, and 4 b were characterized via single crystal XRD. A detailed description of the crystal structure of mercury(II) butyrate Hg(O2CC3H7)21 can be found in the Supporting Information. Single crystals of compounds 3 a⋅4 THF⋅2 Et2O and 3 b⋅10 THF⋅Et2O were obtained by diffusion of pentane into solutions of 3 a and 3 b in THF/Et2O mixtures, respectively. The permercurated metallocene with trifluoroacetate groups 3 a (X=O2CCF3) crystallizes in monoclinic space group P21/n and 3 b (X=O2CCl3) in orthorhombic space group Pbca. The structures are shown in Figure 5(HgO2CCX3)5] unit. Due to a center of inversion located at the iron atom the overall formula in both cases is FeC10(HgO2CCX3)10. A staggered conformation of the two permercurated cyclopentadienyl rings is observed. The cyclopentadienyl‐iron distances are similar to ferrocene (Table rvdW=1.54 ÅrvdW=1.75 ÅCompounds 10(HgCl)10 (4 b) were found in a decomposed sample of the trichloroacetate (3 b). 4 b⋅9 DMSO crystallizes in the triclinic space group P1‾. In contrast to 3 a and 3 b the center of inversion is located outside of the metallocene moiety. Therefore, the asymmetric unit contains the whole molecule. All mercury atoms exhibit a distorted linear symmetry along the C−Hg−Cl axis, which is again a result of Hg−O(solvent) contacts of 2.708(6)–3.119(6) Å. A remarkable feature of the crystal structure is the presence of significant intra‐ and intermolecular mercurophilic Hg+II−Hg+II interactions  Å  which might be related to this d10–d10 interaction. Additionally, intermolecular interactions between Hg3 and Hg4 as well as Hg6 and Hg7 of 3.353(1) and 3.369(1) Å are observed, which are even shorter than the intramolecular interaction. As a consequence, no isolated ferrocene moieties are observed in the solid‐state structure, but a polymeric chain of ferrocene units connected by Hg−Hg contacts , which could explain the insolubility of the compound. In contrast to this, the well soluble derivatives 3  exhibit no significant Hg+II−Hg+II contacts. Taking all interactions into account the coordination number of Hg4, Hg6, and Hg7 is five and of Hg3 and Hg8 six for FeC10(HgCl)10 (4 b). At this point it should be noted that short Hg+II−Hg+II interactions are usually found when the coordination number of the metal center is small.+II−Hg+II contacts is surprising, since the coordination number of the mercury atoms is relatively high. So far, examples of strong Hg+II−Hg+II interactions are relatively rare.+ . Hence, we conclude that the ten‐fold electrophilic substitution of ferrocene by mercury(II) butyrate was successful.The soluble compounds ivatives.38, 51−1 are present in all spectra which is characteristic for the metallocene backbone and correspond to the vibration of the cyclopentadienyl rings. The bands between 100 and 120 cm−1 correspond to Hg−C stretch vibrations. Other bands are associated to vibrations of the Hg‐bonded substituents (X). The frequencies of the Hg−X bonds between 300 and 500 cm−1 are similar to the analogous vibrations in HgX2.[While the IR spectra of all permercurated metallocenes show only bands which correspond to the mercury‐bound carboxylates (see the Supporting Information), Raman spectra contain more information especially with respect to Hg−Cp bonds Figure . The ban in HgX2.56, 573 a at Ep=+0.87 V in THF  while the less stable trichloroacetate 3 b visibly decomposed during the measurements under formation of insoluble material. The butyrate derivative 2 did not have enough solubility in THF. When tetrahydrothiophene was used instead to increase the solubility, no oxidation process was observed in the corresponding electrochemical window.Cyclovoltammetric measurements of the permercurated ferrocene derivatives only revealed one irreversible oxidation process for the trifluoroacetate 10(HgX)10 with mercury(II) carboxylate  and halide substituents . These compounds can be prepared in facile one‐pot reactions in multi‐gram scale and high yields without the need for inert conditions. The complete metalation as well as the purity of the samples was demonstrated by a variety of spectroscopic methods. Additionally, we present the first crystal structures of permercurated metallocenes. FeC10(HgO2CCF3)10, FeC10(HgO2CCCl3)10, and FeC10(HgCl)10. Depending on the substituent, different conformers are observed in the solid‐state structures. The permercurated ferrocenes with carboxylate groups show good solubility in organic solvents but no significant Hg−Hg interactions. In contrast, FeC10(HgCl)10 is completely insoluble. Its crystal structure displays relatively short intra‐ and intermolecular Hg+II−Hg+II contacts.In summary, we demonstrated the synthesis of ten‐fold metallated ferrocene derivatives FeC2+ in C−H functionalization reactions. However, it might be worthwhile to further investigate this unique behavior with the hope of finding ways to mimic this reactivity with other, less toxic elements.Beside their aesthetic appearance, the permercurated ferrocenes might be very useful starting materials for further functionalization or transmetalation reactions. From a more general point of view, one can only be amazed about the extreme reactivity of HgThe authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Nos2 mRNA expression. In contrast, incubation with LPS and PPARβ/δ agonists leads to a significant increase in Pdk−4 and Angptl−4 mRNA expression, which is significantly decreased in the presence of PPARβ/δ antagonists. Docking using computational chemistry methods indicates that PPARβ/δ agonists form polar bonds with His287, His413 and Tyr437, while antagonists are more promiscuous about which amino acids they bind to, although they are very prone to bind Thr252 and Asn307. Dual binding in the PPARβ/δ binding pocket indicates the ligands retain similar binding energies, which suggests that co-incubation with both agonist and antagonist does not prevent the specific binding of each other to the large PPARβ/δ binding pocket. To our knowledge, this is the first time that the possibility of binding two ligands simultaneously into the PPARβ/δ binding pocket has been explored. Agonist binding followed by antagonist simultaneously switches the PPARβ/δ mode of action from induction to transrepression, which is linked with an increase in Nos2 mRNA expression and nitrite production.Peroxisome proliferator activated receptor beta/delta (PPARβ/δ) is a nuclear receptor ubiquitously expressed in cells, whose signaling controls inflammation. There are large discrepancies in understanding the complex role of PPARβ/δ in disease, having both anti- and pro-effects on inflammation. After ligand activation, PPARβ/δ regulates genes by two different mechanisms; induction and transrepression, the effects of which are difficult to differentiate directly. We studied the PPARβ/δ-regulation of lipopolysaccharide (LPS) induced inflammation (indicated by release of nitrite and IL-6) of rat pulmonary artery, using different combinations of agonists (GW0742 or L−165402) and antagonists (GSK3787 or GSK0660). LPS induced release of NO and IL-6 is not significantly reduced by incubation with PPARβ/δ ligands (either agonist or antagonist), however, co-incubation with an agonist and antagonist significantly reduces LPS-induced nitrite production and  PPARβ/δ are ligand dependent transcription factors that belong to the nuclear receptor family . They arIn vivo studies  indicate that PPARβ/δ agonists induce several favorable pharmacological effects: reduced weight gain, increased metabolism in the skeletal muscle and cardiovascular function, suppression of atherogenic inflammation as well as improvement of the blood lipid profile, all of which are common abnormalities in patients with metabolic syndrome ,6,7. TheNevertheless, the interest in PPARβ/δ continued, and in the last few years several compounds targeting PPARβ/δ were developed and entered clinical trials. The Phase II clinical trial on the PPARβ/δ agonist MBX−8025 for treatment of non-alcoholic steatohepatitis and primary sclerosing cholangitis was terminated early when patients developed early signs of liver damage . This lePPARβ/δ can be activated by numerous endogenous ligands such as eicosanoids, fatty acid, metabolites derived from arachidonic acid and linoleic acid ,14,15 asThe PPARβ/δ endogenous and exogenous ligands control cellular function through changes in very small concentration range. Added to this, in any cell or tissue, the activity of PPARβ/δ may also depend on its promoter activity and relative expression, as well as presence and activity of co-repressor and co-activator proteins. It has been shown that GW0742 is capable of behaving as an agonist activating the transcription pathway at lower concentrations (nM) and antagonist inhibiting this effect at higher concentrations (μM) . In the After ligand activation, PPARβ/δ regulates genes by two different mechanisms, induction and transrepression. In the induction mode, PPARβ/δ forms a complex with the retinoid X receptor (RXR) and together, as a heterodimer, binds the promoter of the target genes (PPRE). In the absence of ligand, co-repressor proteins and histone deacetylases (HDACs) are bound to the heterodimer which tightens the chromatin and prevent it from binding to the PPRE sites . The preIn the transrepression mode PPARβ/δ regulates gene expression in a PPRE-independent manner through the regulation (mostly suppression) of other transcription factors, including nuclear factor-κB (NF-κB) , activatIn order to isolate the transrepression mode of PPARβ/δ, a novel approach was taken in this project. Tissues were co-incubated with both agonist; this will alter the conformational shape of PPARβ/δ and bind to co-activators and place the PPARβ/δ predominantly into gene induction mode. In theory, addition of an antagonist will then prevent PPRE binding and induction of genes, revealing the effects of PPARβ/δ transrepression.Studies have generally focussed on the effects of agonists and antagonists separately, and often results in conflicting theories of how PPARβ/δ controls gene expression. Here we show for the first time that co-incubation of agonists and antagonists to PPARβ/δ leads to a significant decrease in LPS-induced inflammation in rat pulmonary artery compared to single applications of each drug type. The mechanism of action may be explained by the binding studies indicated by docking studies of the agonists and antagonists with PPARβ/δ, which confirms that agonist and antagonist co-binding can occur.Expression of PPARβ/δ was confirmed by ELISA and calculated to 0.04 pg/mL/μg protein. Rat lung pulmonary arteries were incubated with LPS for different periods of time  in order to ascertain the minimum time required to significantly increase NO production. LPS induced a significant increase in NO at 24 h in all tissues tested , subsequPulmonary arteries exposed to LPS for 24 h significantly increased production of NO and IL-6, markers of innate inflammation . IncubatProduction of nitrite is a measure of NO release from cells and tissues; the iNOS specific inhibitor 1400W was used to indicate the proportion of NO originating from iNOS as opposed to eNOS or iNOS. In all experiments, nitrite production following LPS incubation was significantly decreased by incubation with 1400W A,B.Pdk−4 mRNA by 7-fold  was compared to the real GW0742 present in the crystal structure (pink) A. The moGSK3787 binds in a slightly different place than GW0742, although there is some overlapping of the binding sites A. Also, The most stable L−165041 orientation predicted by Autodock Vina binds in the same physical place as GW0742 A and theGSK0660 binds very close but not in the same binding site as GW0742 A. The amIn order to investigate the docking of two ligands simultaneously, the first ligand was bound in the most stable orientation first. The best hit from previous docking was assigned Ligand 1, and then a second molecule was docked, assigned Ligand 2. The aim was to mimic the conditions of the experiments performed in this study and predict what could have happened at the molecular level. When the tissues were treated with only one ligand there is only one option for two ligands to bind, but when two different ligands are present at the same time either of them can bind first into the binding pocket. All these ligand-binding possibilities were considered and summarized in Once GW0742 is bound in the most stable orientation within the PPARβ/δ-LBD, GW0742 and GSK3787 can still bind at favorable energies , although at very different binding sites to the most stable one and forming polar interactions with different residues, as shown in GW0742 and GSK3787 can also bind into the binding pocket after GSK3787 at favorable energies . The binding site is also different to the most stable ones , but intWhen L−165041 is bound to the ligand binding pocket first, a second molecule of L−165041 or GSK0660 can bind with favorable energies  and again, forming polar interactions with different residues. The most interesting finding is that GSK0660, although still in the PPARβ/δ-LBD, has the potential to bind outside the binding pocket .L−165041 and GSK0660 can also bind within the binding pocket at favorable energies  after GSK0660 is bound in the most stable orientation , but agaThe study confirms previous studies that PPARβ/δ is expressed in pulmonary arteries . Here weOther in vivo studies in whole mouse have indicated that GW0742 treatment attenuates inflammation, which is significantly reduced by GSK0660 ,32,33. TAngptl−4 and Pdk−4. Here in this study, qRT-PCR of pulmonary artery showed that the activation of PPARβ/δ with GW0742 increases the transcription of Pdk−4 and Angptl−4 mRNA indicating that agonist activation of PPARβ/δ triggers induction, which was inhibited by co-incubation with GSK3787.Marker genes were selected to represent the induction mode of action of PPARβ/δ. Khozoie  and AdhiNos2 mRNA is regulated by PPARβ/δ [Nos2 mRNA expression is not altered by the presence of either GW0742 or GSK3787 which indicates that direct induction of genes is not the predominant mode of control PPARβ/δ has on Nos2 mRNA expression. However, Nos2 mRNA expression is significantly reduced when co-incubated with both agonist and antagonist. With the activation of the receptor followed by inactivation by antagonist, any reduction in expression and activity of iNOS must be due to transrepression of PPARβ/δ on other nuclear receptors.It has been shown in several studies that  PPARβ/δ ,36,37, aIt has been suggested that the large ligand binding pocket of PPARβ/δ can accommodate more than one ligand, resulting in unusual PPAR:ligand stoichiometries that could trigger inactive receptor conformations , a possiThis finding agrees with previous results were GW0742 was docked to another PPARβ/δ crystal structure (PDB: 3GZ9) using another docking software (Glide), and the same three amino acids bound to GW0742 . Further1) and another one that did not activate PPARβ/δ (Compound 2). The docking and MD simulation results for the Compound 1 showed an interaction with His287, His413 and Tyr437, and the results for Compound 2 showed an interaction with Thr252 [It is worth mentioning another study where the authors selected 5 compounds that potentially bound PPARβ/δ and performed a luciferase transactivation assay to biologically test if these compounds activate PPARβ/δ. They further analyzed two of them by docking and molecular dynamics (MD) simulation, one compound that activated PPARβ/δ . If a second molecule of GW0742 binds to PPARβ/δ, this molecule is predicted to bind not too far from the most stable binding site and with the same binding affinity and residue interaction (Arg258) than the 8th best position predicted for the first molecule. Similarly, when the irreversible antagonist GSK3787 is present, a second molecule of GSK3787 is predicted to bind also not far away from its most stable binding site and with favorable binding affinity. In addition, it is predicted that GSK3787 will still form polar bonds with Thr252, an amino acid that is predicted to bind GSK3787 in five out of eight most stable poses predicted by docking.When investigating dual occupancy of the PPARβ/δ LBD with both agonist (GW0742) and antagonist (GSK3787) all the options mentioned above still apply but two more options are available: GW0742 binds first and GSK3787 after or GSK3787 binds first and GW0742 after. When GW0742 binds first, GSK3787 can still bind very close to its most stable binding site with a very favorable binding affinity, and what is more, still binds the residue Thr252. If GSK3787 binds first, GW0742 is predicted to bind very far away from the most stable binding site, at the entrance of the binding pocket, and as a consequence it will have a very different binding profile forming polar bonds with Trp228 and Lys229, two residues that did not show any interaction with ligands before. This suggests that if the PPARβ/δ receptor is inhibited by GSK3787, the agonist cannot reverse this inhibition. Similar analysis can be done with the other pair of agonist and antagonist L−165041 and GSK0660.Taking into account the docking scores and molecular poses of the ligands, all possibilities described above have very favorable energies for it to happen. That opens a whole new scenario of possibilities that could dramatically change the 3D conformation of PPARβ/δ in ways that have not been thought of before, resulting in the binding of different co-regulators, which ultimately could change the PPARβ/δ response from induction to transrepression or vice versa.To our knowledge, this is the first time that the possibility of binding two ligands simultaneously into the PPARβ/δ binding pocket has been explored. The results suggest that this possibility is very likely to happen with very favorable affinity energies, and it is worth considering when designing and interpreting experiments where PPARβ/δ is ligand-activated and high concentrations of ligands are used. In regards to the conditions set in our study, docking indicates that the co-incubation with both agonist and antagonist does not prevent the specific binding of each other to the large PPARβ/δ binding pocket.In summary, this is a multidisciplinary approach of the study of PPARβ/δ that provides novel information about its functioning at molecular level. In the model used here, the simultaneous co-incubation of pulmonary with both agonist and antagonist potentially opens a window to understand the alternative transrepression PPARβ/δ mode of action as compared to the induction mode of action. Here we show for the first time that there is a characteristic PPARβ/δ-ligand binding profile for agonists and antagonists even in combination; PPARβ/δ agonists form polar bonds with His287, His413 and Tyr437, while antagonists are more promiscuous about which amino acids they bind to, although they are very prone to bind Thr252 and Asn307. In dual predictive docking studies, all the options studied seem feasible with favorable binding energies, suggesting the need for caution when designing and interpreting the results of experiments using PPARβ/δ ligands.The PPARβ/δ ligands GW0742, GSK3787, L−165042 and GSK0660 as well as 1400W, LPS O55:B5, sulfanilamide and naphthylethylenediamine dihydrochloride were purchased from Sigma . Sodium nitrate, orthophosphoric acid and DMSO were purchased from Fisher Scientific . Primers from Applied Biosystem : β-actin (Rn00667869_m1), Pdk−4 (Rn00585577_m1), Angptl−4 (Rn015228817_m1), Nos2 (Rn00561646_m1).Male Wistar rats (350–450 g) were sourced from Charles River  and housed in pairs in standard cages (Tecniplast 2000P) with sawdust (Datesand grade 7 substrate) and shredded paper wool bedding with water and food (5LF2 10% protein LabDiet) in the Biological Services Unit at the University of Hertfordshire. The housing environment was maintained at a constant temperature of 22 ± 2 °C, under a 12 h light/dark cycle (lights on: 07:00 to 19:00 h). All testing was conducted under the light phase of the animals’ light/dark cycle, and care was taken to randomize treatment sequences to control for possible order effects.2 asphyxiation followed by cervical dislocation. Lungs were removed and immediately placed in physiological saline solution (PSS) buffer . Following dissection, tissues were incubated in 1% v/v penicillin/streptomycin in DMEM (no serum) under 5% v/v CO2 at 37 °C in the required treatment  Act 1986 and European directive 2010/63/EU. Rats were euthanized according to schedule 1 procedure by CO2 using a mortar and a pestle, and the proteins were extracted in ice-cold phosphate buffered saline (PBS) with proteinase inhibitor cocktail in a ratio 9 mL PBS  per g tissue. The samples were then sonicated 3 × 30 s and centrifuged for 5 min at 5000 g. The supernatant was collected, and the protein concentration was quantified by BCA assay. Then, 50 µL of standards and samples were added in duplicate to the plate containing pre-coated anti-PPARβ/δ and incubated 45 min at 37 °C. The wells were washed and incubated with 50 µL of the horseradish peroxidase (HRP)-conjugated detection antibody for another 30 min at 37 °C. The wells were washed again and incubated in the dark with the chromogen solution for 15 min at 37 °C. Finally, the reaction was stopped by adding 50 µL of Stop solution and the plate was immediately measured in a microplate reader. The concentrations of PPARβ/δ were determined by comparison of OD450 to a standard curve (0–8 ng/mL).The expression of PPARβ/δ in pulmonary artery from naïve rats was measured using rat PPARβ/δ ELISA kit  according to the manufacturer’s instructions. Briefly, the dissected tissues were homogenized with liquid Nw/v + orthophosphoric acid 5% v/v and naphthylethylenediamine dihydrochloride 0.5% w/v respectively). The concentration was determined by comparison of the OD540 to a standard curve of solutions of sodium nitrite (0–1 mM).An aliquot of the culture medium (50 µL) thawed on ice was mixed with an equal volume of Griess reagent  according to the manufacturer’s instructions. Briefly, all samples and standards were conducted in duplicate to a microtiter plate containing the capture antibody. After two hours of incubation the wells were washed and incubated with the detection antibody for another two hours. The wells were washed again and incubated in the dark with Streptavidin-HRP for 20 min. The wells were washed once more and incubated in the dark with substrate solution. After 20 min the stop solution was added and immediately measured in a microplate reader. The readings at 540 nm were subtracted from the readings at 450 nm and the concentrations of samples were determined by comparison with the standard curve (0–8 ng/mL).2 and the RNA was then extracted following the manufacturer’s instruction and stored at −80 °C until use. The quality and concentration of the RNA was measured using Nanodrop  at a wavelength of 260 nm. Further to that, the degradation of RNA was checked in a 1% w/v agarose gel, and the DNA contamination of the RNA was checked by PCR. To do that, genomic DNA was also extracted from the tissues using PureLink Genomic DNA mini kit from Invitrogen following the manufacturer’s instructions and used as a positive control of the PCR, and two primers for the housekeeping gene β-actin were designed . The termociclator Mastercycler Nexus gradient  was set with the following PCR protocol: 95 °C for 10 min, 40 cycles of 95 °C for 30 s, 56 °C for 30 s and 72 °C for 30 s, 72 °C for 10 min and hold at 4 °C.Total RNA was extracted from tissues using RNeasy Fibrous Tissue Mini Kit . The tissues were first pulverized in a pestle with liquid NcDNA was obtained by reverse transcription (RT) using iScript cDNA synthesis kit (Bio-Rad) following the manufacturer’s instructions. The RT was performed using a thermociclator Stratagene Mx3005P  with the following steps: 5 min at 25 °C, 20 min at 46 °C, 5 min at 95 °C, and hold at 4 °C.qRT-PCR was performed to analyze mRNA expression using a Taqman System. Briefly, 10 µL of reaction mix containing the primers and cDNA was incubated in a 96 well-plate following the cycle conditions: 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min.The ability of drugs to bind into protein active sites was investigated using AutoDock/Vina with Pymol and Ligplot+ as a graphical user interface. For the docking simulations, the PPARβ/δ crystal structure 3TKM was selected for having one of the highest resolutions (1.95 Å). The PDB file was downloaded from the Protein Data Bank. Water molecules, ligands and other hetero atoms were removed from the protein structure, and the addition of hydrogen atoms to the protein was performed using AutoDock Tools version 1.5.6. The grid was set manually to cover the active site. The file was saved as a pdbqt file.scripps.edu) (accessed on 23 February 2021)).The ligand molecule structures were drawn in ChemSketch, the energy was minimized and saved in PDB format, and converted into a pdbqt file with AutoDock Tools version 1.5.6  is less than 0.05 (*), less than 0.01 (**) or less than 0.001 (***), the effect of the difference was regarded as significance.Values of