Simulation methods for these systemsmust be able to treat relatively large organic molecules, as wellas predict their excited state energies, transition energies, singlet–tripletsplittings, and absorption and emission cross sections with reasonableaccuracy, in order to prove useful in the design process. Here wecompare predictions of TDDFT with M06-2X and ωB97X-D exchange-correlationfunctionals and a GoWo@HF/BSE method for these quantitiesin the well-studied DPTZ-DBTO2 TADF emitter molecule. Geometry optimizationis performed for ground state (GS) and lowest donor–acceptorcharge transfer (CT) state for each functional. Optical absorptionand emission cross sections and energies are calculated at these geometries.Relaxation energies are on the order of 0.5 eV, and the importanceof obtaining excited state equilibrium geometries in predicting delayedfluorescence is demonstrated. There are clear trends in predictionsof GoWo@HF/BSE, and TDDFT/ωB97X-D andM06-2X methods in which the former method favors local exciton (LE)states while the latter favors DA CT states and ωB97X-D makesintermediate predictions. GoWo@HF/BSE suffersfrom triplet instability for LE states but not CT states relevantfor TADF. Shifts in HOMO and LUMO levels on adding a conductor-likepolarizable continuum model dielectric background are used to estimatechanges in excitation energies on going from the gas phase to a solvatedmolecule.Design concepts for organic light emitting diode (OLED)emitters,which exhibit thermally activated delayed fluorescence (TADF) andthereby achieve quantum yields exceeding 25%, depend on singlet–tripletsplitting energies of order  I1 is commonlyperformed using time-dependent density functional theory (TDDFT) usingdensity functionals that include a significant proportion of exactexchange.6 Mostly, vertical optical absorption spectra are calculated for moleculesin their gas phase ground state equilibrium geometries and experimentsare interpreted in light of results of those calculations. TDDFT hasthe advantage of being a relatively inexpensive method, allowing largeorganic systems to be treated using limited basis sets. The GW method,8 in which the electron self-energy,Σ, is evaluated to first order in the screened Coulomb interaction, W, and the one-electron Green function, G, is the state-of-the-art approach for calculating quasiparticleenergies in bulk solids, surfaces, and nanostructures from first principles.These methods have also been used to calculate electron affinities(EA) and ionization potentials, (IP) of organic molecules13 and a combination of GW and Bethe–Salpeterequation (BSE) methods has been used to calculate excited states inorganic systems.16 Both GW and BSE methods depend onscreening of the electron–hole interaction to determine particleand hole energies and electron–hole excitations. Several groupshave focused on the importance of mean-field starting point in a GW calculation and effects of self-consistency in G and W on predicted IP and EA values andexcitation energies in molecules.17Simulation of TADF systemsS,S-dioxide (DPTZ-DBTO2)  1 is one 24 using the M06-2Xfunctional.26 Conclusions drawn from thatwork depend on the specific ordering of low lying excited states ofboth local exciton (LE) and CT and singlet and triplet character.Here we compare TDDFT predictions of the excited states from the M06-2Xfunctional to a range-separated functional, ωB97X-D,28 and GoWo@HF/BSE calculations. It is found thatoverall, both TDDFT/M06-2X and TDDFT/ωB97X-D yield similar predictionsfor excited state energies and absorption and fluorescence spectra,while GoWo@HF/BSE predicts a different orderingof excited state energies. GoWo@HF/BSE alsosuffers from the well-known triplet instability problem in predictingtriplet excited state energies of LE states but not of CT states whereelectron and hole are well separated in space.DAD TADF emitter molecules such as DPTZ-DBTO2 aredesigned to havesmall singlet–triplet splittings by ensuring that their lowestsinglet states have strong donor–acceptor charge transfer (CT)character. This system and its DA counterpart, PTZ-DBTO2, have beenextensively studied31 DAD TADF systems exhibit both local exciton (LE)and charge transfer (CT) excited states, in which an excited electronand hole are both located on the D or A (LE) or are separated on Dand A (CT). Methods for treating TADF systems must be able to treatshort and long-range electron–hole interactions accuratelyin order to predict the relative ordering of LE and CT excited states.While the M06-2X functional performs well in benchmark studies ofLE states in relatively small organic molecules,31 it containsa fraction of the Coulombic electron–hole attraction whichis independent of the electron–hole separation. In contrast,the BSE method incorporates a screened electron–hole attractionwhich is separation dependent. Range-separated functionals such asthe ωB97X-D functional28 include distance-dependentelectron–hole attraction within the parametrization of thefunctional. Here we compare predictions of GoWo@HF/BSE and TDDFT/M06-2X and ωB97X-D methods for bothLE and CT states in the DPTZ-DBTO2 emitter, including the effectsof excited stated geometry.The M06-2X densityfunctional performs well in benchmark studiesof density functionals for TDDFT of both singlet and triplet statesof organic molecules.32 which may be a solvent33 or an amorphousor crystalline host. TADF DA and DAD systems exhibit low lying CTexcited states where the excited state singlet–triplet splitting, ΔEST, is small (of order kT or less) which facilitates reverse intersystem crossing(rISC)34 and an increase in electroluminescencequantum yield.Two important effects which arecommonly neglected in TADF simulationsare the relaxation of the excited state geometry and interactionswith the local environment,36 The case of valence versus CT excitations has been studied by TDDFTand GW/BSE methods16 compared toCASPT2 calculations. The cause of the failure of local TDDFT for CTstates is the omission of long-range, screened, electron–holeattraction by the local functional.35 Thiscan be added to the TDDFT method by replacing the local exchange functionalby an exact exchange hybrid functional with a constant weight factoror by a range-separated functional such as CAM-B3LYP37 or ωB97X-D in which exact exchange is strongly attenuatedat short-range and reaches a fixed fraction of the exact exchangeat long-range. In hybrid functionals such as B3LYP (20% exact exchange)39 or M06-2X (54% exact exchange)26 theweight of exact exchange in SCF calculations and electron–holeattraction in TDDFT calculations is independent of electron–holeseparation.Excitation energies for optical transitions witha strong CT characterare known to be grossly underestimated by TDDFT with local or generalizedgradient exchange-correlation functionals.GW approximation, reductionof the proportionof exact exchange is determined by a screened, distance and state-dependentCoulomb interaction, W. The GoWo@HF approximation yields a relatively small correction to SCFIP and EA levels, which places them in good agreement with gas phaseIP and EA values of organic molecules. Bruneval and co-workers40 showed that GoWo@HF predictedslightly higher HOMO–LUMO (H-L) gaps than CCSD(T) for the Thielset,42 while GoWo@BHLYPpredictedslightly lower gaps than CCSD(T). Density functionals with lower exactexchange weights than BHLYP (50% exact exchange)43 predicted lower H-L gaps and poorer agreement with CCSD(T), suggestingthat the GoWo@HF starting point adopted in this workis a reasonable starting point for gas phase GW calculationsfor singlet excited states. However, it is known that mean absoluteand mean signed errors in GW/BSE predictions of tripletexcitation energies are larger than those for singlets.40 Electrochemical measurements show that gas phase IP and EA valuesare significantly modified in solution owing to screening by solventmolecules, and we use a conductor-like polarizable continuum model(C-PCM)44 to estimate shifts in IP andEA values in solution.In the kT in order for rISC to proceed. Predicted singlet and tripletenergies in solution or condensed phase environments therefore dependon competing factors that determine the degree of inter and intramolecularscreening. This has led to a number of studies that have benchmarkedvarious density functionals48 and the GW/BSEapproximation in their abilities to predict singlet33 and triplet45 excitationenergies and singlet–triplet splittings46 of mainly small organic molecules, mainly against superior but morecomputationally expensive quantum chemistry methods.Linear response methods for excited statesincluding TDDFT, BSE,and configuration interaction singles (CIS) are readily applied toboth singlet and triplet excited states. Singlet state Hamiltonianscontain both electron–hole attraction and electron–holescattering terms, while triplet states contain only the former. Ina single-particle excitation picture, the electron–hole scatteringterm is the exchange integral which determines singlet–tripletsplitting. Inclusion of exact exchange in an SCF calculation increasesthe H-L gap in molecules and results in more localized orbitals. Indonor–acceptor systems, this results in smaller electron–holescattering (exchange) integrals and smaller singlet–tripletsplittings. This is crucial to the TADF systems of interest here asthe singlet–triplet splitting energy must be within GW/BSEmethod used in this work, we review existing benchmark studies forsinglet and triplet excitation energies of organic molecules using GW/BSE and TDDFT methods, and we describe the geometry optimizationprotocols and the basis sets used in this work. Two molecular geometriesare used throughout, denoted GS and CT. GS is the ground state geometryof the DPTZ-DBTO2 molecule, CT is the equilibrium geometry of thelowest energy singlet CT excited state. Single-particle energy levelspredicted by GoWo@HF/BSE and TDDFT/ωB97X-Dand M06-2X methods for the GS and CT geometries are compared, followedby excited state energies and optical absorption and fluorescencespectra at each of the three geometries. This is followed by a discussionof results and a summary.In thefollowing section, we outline the IIII.AGoWo calculations are commonlyperformed using DFT Kohn–Sham states and energy eigenvaluesas a starting point for calculation of G0 and W0. Here, HF states and energy eigenvaluesare used. The bare Fock exchange is included in the starting Hamiltonianin this case and does not replace a DFT exchange-correlation potentialin the self-energy, as when starting from Kohn–Sham DFT. Rostgaardand co-workers9 found that a GoWo@HF approach applied to 34 small molecules yielded the lowestmean absolute error (MAE), 0.4 eV, in the IP, compared to experiment,smaller than the MAE for self-consistent GW(0.5 eV)and the PBE0 functional51 .Working on 13 larger organic molecules, Blase and co-workers10 found that while a DFT-LDA starting point yieldedrelatively large MAE in IP values for those molecules, ,employing “HF-like” eigenvalues in the dielectric functionyielded improved agreement with experimental IP values , similar to self-consistent GW values  and improved prediction of the H-L gap for three acenesand C60 . Application of the Exciton code54 used in this work to acenes from naphthalene to pentacene yieldedMAE of 0.03 eV for the IP values and 0.17 eV for both the EA and H-Lgaps (unpublished results). Recent work17 has shownthat PBE0 starting points for GW calculations yieldedpoor singlet excitation energies in a GoWo@PBE0approximation and that this is improved significantly by partial self-consistencyin the GWself-energy.Perturbative r,r′; ϵ),in many-body theory replaces the exchange correlation potential inDFT or the Fock exchange in HF theory by a dynamically screened exchangeinteraction. The GoWo approximation to the self-energyhas been widely applied to periodic systems where it is conventionalto express the screened interaction, Wo, in terms of the random phase approximation (RPA)inverse dielectric function, and bare Coulomb potential.7 A formally equivalent approach is to express Wo as,v is the bare Coulombpotential and ΠRPA is the RPA polarizability.55The self-energy operator,Σ ψi*(r) intothe density–density response matrix ψi*(r′)v(r – r′), and diagonal matrix elements of thisterm in the self-energy are the HF exchange matrix elements. As notedin the previous section, these are included in the HF Hamiltonianand hence are omitted from the self-energy. Only the latter, dynamicpart of the screened interaction contributes to the self-energy. Theform of the self-energy in 7 However, in the present case, no plasmon poleapproximation is required. Residues of Wd contribute the following diagonal matrix elementsto the self-energy operator for occupied or virtual states,Diagonal elements of the self-energy are obtained from convolutionof the HF Green’s function with the screened interaction alongthe real energy axis,II.CGoWo@HF yields IP and EA values in reasonableagreement with gas phase experimental values, the H-L gap derivedfrom these values is significantly larger than values estimated fromcyclic voltammetry measurements.34 We applya very simple approximation to estimate changes in H and L level positionsdue to additional solvent screening based on the reduction in IP andincrease in EA in the presence of a uniform dielectric with dielectricconstant, ε, and cavity radius, R, which isgiven by,ΔE areperformed using the conductor-like polarizable continuum model (C-PCM)44 in GAMESS.59While II.DB, matrices in A matrix in ab|Wd|ji), are replaced by unscreened matrix elements, (ab|ji), and the GoWo@HF eigenvaluedifferences, (ϵ̃a –ϵ̃i), by HF eigenvalue differences,(ϵa – ϵi). CIS generally overestimates singlet excitationenergies,46 especially in CT states.60 BSE-TDA diagonal element GoWo@HF eigenvalue differences are smaller than HF eigenvalue differences,which reduces excitation energies, while screening of electron–holeattraction matrix elements raises excitation energies. BSE-TDA calculationswere performed using the GoWo@HF/BSE modulein the Exciton code,53 TDDFT-TDA calculations were performedusing the Gaussian16 code.61In this work, both BSE and TDDFT calculations areperformed within the TDA by solving the ordinary eigenvalue problemswhich result when the coupling, GoWo@HF/BSE calculations for the D and Amolecules used all valence and all virtual orbitals in the activespace, and for the DAD molecule, they used 70 valence orbitals and412 virtual orbitals. Increasing the virtual orbital space by an additional200 orbitals reduced the first excited state energy in the CT geometryby 0.04 eV, indicating that the excitation energies are reasonablywell converged with respect to the active space dimension.TheExciton code uses a density fitting method to calculate four-centerintegrals. The implementation of density fitting is described in refs  and 54. II.E39 TPSSh,63 M06-2X,26 andωB97X-D28 functionals with cc-pVDZ64 or KTZVP65 basissets. Excitation energies at these geometries were calculated usingdef2-TZVP66 or cc-pVDZ basis sets. Theeffect of dispersion forces on molecular geometries was investigatedby adding the D3-BJ functional67 to theB3LYP and TPSSh functionals. Bond lengths to heteroatoms in DPTZ-DBTO2predicted using HF, B3LYP, TPSSh, CIS, TDDFT/B3LYP, and TDDFT/ωB97X-Dare given in the Supporting Information, Table S1. HF and M06-2X frontier orbital isosurfaces are shownin the Supporting Information, FiguresS1 and S2. Molecular coordinates for the GS and CT geometries usedare given in the Supporting Information, Tables S2 to S11.Ground (GS) and (CT) excited state equilibriumgeometries were calculated using HF, B3LYP,Supporting Information, Tables S12and S13. Each of these geometries yielded similar singlet and tripletexcitation energies. Excitation energies for the HF ground state anda GW/BSE method are also given in Tables S12 and S13.Singlet and triplet excitation energiesfor the ground state equilibrium geometries with HF, B3LYP, B3LYP+D3-BJ,TPSSh+D3-BJ, ωB97X-D, and M06-2X functionals using M06-2X andωB97X-D functionals in the TDDFT calculation are compared inthe GoWo@HF/BSE singletand triplet excitationenergies were calculated using the TPSSh + D3-BJ GS geometry obtainedusing cc-pVDZ basis sets and def2-TZVP basis sets for the BSE calculationand corresponding def2-TZVP-RI auxiliary basis sets68 for density fitting Coulomb matrix elements in RPA andBSE A matrices. Basis sets were obtained from theEMSL database.70Supporting Information, Tables S12 and S13. Thefirst five excitation energies of each irreducible representationare given for a range of basis sets, inclusion or omission of dispersionforces via the D3-BJ functional and additional functionals used forGS geometry relaxation  are included. Switchingfrom a B3LYP+D3-BJ to a TPSSh+D3-BJ functional for GS geometry relaxationincreased the average excitation energy by 0.003 eV for both ωB97X-Dand M06-2X TDDFT kernels over all irreducible representations (columns5 and 6 and columns 10 and 11 in Table S12). Switching from a cc-pVDZ basis to a def2-TZVP basis for a ωB97X-DTDDFT kernel increased the average excitation energy by 0.061 eV (columns8 and 9). Adding a D3-BJ functional to a B3LYP functional for geometryrelaxation in a TDDFT/M06-2X calculation reduced the average excitationenergy by −0.024 eV (columns 9 and 10). Using the B3LYP equilibriumgeometry versus the HF equilibrium geometry increased the averageexcitation energy by 0.014 eV. Differences in A2 and B1 excitation energies on adding the D3-BJ functional to theB3LYP functional in geometry optimization are up to 0.08 eV  with a MAD of 0.028 eV. Adding the D3-BJ functional to theB3LYP functional in geometry optimization reduced the GoWo@HF/BSE 11A2 excitation energy by 0.14eV . The main determinant of excitation energies, however,is the choice of functional in the TDDFT or BSE calculation.The effects of basis setquality, addition of dispersion forces,and choice of DFT exchange-correlation potential on singlet and tripletexcitation energies in the GS geometry are presented in the 1DD state. A local minimum in thepotential energy surface can be found for this state in CIS geometryoptimization, but not using any of the TDDFT methods. In that casethe optimizing state evolves into a 1DA state. The lowestenergy 1AA acceptor LE state is higher in energy. The CTstate is predominantly of H/L character and the 1DD LEstate is of H/L+2 character. H, L and L+2 orbitals are of a1, b1, and a2 symmetry in the Cv2 GS geometry, and therefore, the 1DA and 1DD states are of B1 and A2 symmetries. There is a nearly degenerate 1DD stateof B1 symmetry in the GS geometry with H-1/L+2 character,since the H and H-1 levels are nearly degenerate. The CT state inthe GS geometry has a nearly degenerate 1DA state of A2 symmetry. Degeneracies of these states are lifted when thegeometry of excited states are optimized and the molecule breaks symmetry.In the CT 1DA state equilibrium geometry, the excited holeand electron localize on D and A, respectively, and have large excitedstate dipole moments.Each of the methods used predicts the lowest singlet excited stateof LE character to be a 29 applied 34 functionals to calculation of triplet excited state energiesusing def2-TZVP basis sets and found the best functionals to be BMK71 and M06-2X with mean signed error (MSE) of 0.18and 0.07 and mean absolute error (MAE) of 0.24 and 0.23 eV, respectively,compared to best theoretical estimates. In comparison, CIS and M06-HFgive −0.06 and 0.12 and 0.44 and 0.56 eV for MSE. Brücknerand Engels investigated the effect of a fraction of the Fock exchangeon the singlet–triplet splitting46 in organic DAD systems using a multistate CASPT2/ccpVTZ method asreference. They concluded that, “the M06-2X functional providesbest estimates for both vertical and adiabatic singlet–tripletgaps when adiabatic linear-response TD-DFT singlet excitations arecombined with TDA triplet values”. More recent assessmentsof hybrid functionals have been performed for singlet and tripletexcitations48 from the QUEST database.72 Grotjahn and Kaupp48 found that the overall best performing global hybrid was M06-2X,with a mean absolute error of 0.20 eV for π to π* tripletvertical excitation energies. Performances of M06-2X and a range ofother functionals over a range of excitation types  were compared by Jacquemin and co-workers.73 In many cases M06-2X performs comparativelywell, although in some cases, such as transitions in Ni(II) complexes,74 it can fail spectacularly.The choice of hybrid density functionalfor this work was basedon benchmark studies of singlet and triplet states of organic moleculedatabases against best theoretical estimate values from superior quantumchemistry methods. Jacquemin and co-workers30 found that theωB97X-D28 and BMK71 functionals gave the best overall performance for 463 vertical excitationenergies, with a root-mean-square error (RMSE) of 0.27 eV. The RMSEfor M06-2X in that study was less than 0.3 eV. Loos and co-workers31 reported reference energies for 17 moleculeswith intramolecular CT states. They found the best performing functionalin TDDFT to be ωB97X-D. Bruneval and co-workers40 applied a GW/BSE approach to singlet andtriplet excitation energies of Thiel’s set of organic molecules42 using a range of DFT, hybrid DFT, and HF starting points. They founda strong, linear correlation between the MSE of the S1andT1excitation energies predicted by BSE and the H-L gap.The H-L gap is determined by the chosen starting point. No BSE@HFresult was reported, but the smallest S1 MSE  was found for BSE@BHLYP, where thestarting hybrid functional has an exact exchange weight of 50%. T1 excitation energies showed a similar correlation with H-Lgap, but the optimal H-L gaps for minimal MSE, in either case, differedby 0.6 eV.Liang and co-workers45 reportedsinglet and triplet excitation energies and singlet–tripletsplittings in 20 organic molecules from TDDFT and GW/BSE calculations in which single-particle eigenvalues appearingin G and W . Bycomparison, the M06-HF functional (100% exact exchange) gave a MSEof 0.20 eV and a MAE of 0.44 eV in TDDFT-TDA. BSE-TDA using the M06-2Xfunctional gave a MSE of −0.31 eV and a MAE of 0.31 eV. Themore localized wave functions in a M06-HF starting point gave correspondingerrors of −0.06 and 0.17 eV, using the self-consistent eigenvalue GW approach.Jacquemin and co-workers G and W 4 were it45 found similar MSE and MAE values for both M06-2Xand M06-HF starting points. The MSE and MAE using a M06-2X startingpoint and TDDFT-TDA were 0.42 and 0.43 eV, respectively, and for BSE-TDAthey were 0.31 and 0.31 eV. These results give some context for thetriplet energies and singlet–triplet splittings reported in GW/BSE with a M06-HF starting point iteratedto self-consistency in the GW calculation had rathersimilar MSE and MAE for triplet excitations (0.00 versus −0.06eV and 0.20 versus 0.17 eV). Errors in singlet–triplet splittingswere smaller by 0.1 eV in BSE-TDA.Turning to singlet–triplet splittingenergies calculatedusing TZVP basis sets and compared to CC3, Jacquemin and co-workersIIIIII.AGoWo@HF, ωB97X-D, and M06-2X to ΔSCF calculations forthose methods. We also consider the effect of polar and nonpolar solventpolarization on single particle levels using the C-PCM model and compareto H and L energies from cyclic voltammetry.Inthis section, we compare single particle energies from GoWo@HF, ωB97X-D,and M06-2X calculations for DPTZ-DBTO2 in the GS and CT geometriesare shown in Supporting Information, Figures S1 and S2, for isosurfaceplots of HF and M06-2X Kohn–Sham orbitals). The first threevirtual orbitals are localized on A and the fourth and fifth on Dfragments in the GS and CT geometries. Supporting Information Figure S1 shows that the L+4 HF orbital in theGS geometry becomes the L+3 orbital in the CT geometry. The L+2 orbitalis mainly localized on the A fragment in the GS and CT geometriesand partly on the D fragments.Single particleenergy levels from 1, b2 orbital pairs (H/H-1 and H-2/H-3) localized on the D fragments.Relaxation of the structure in the CT geometry lifts the near degeneracyof the H/H-1 pair. These MOs localize on either D fragment and thequasi-degenerate H and H-1 a1, b2 pair of levelssplits by 0.5 eV in GoWo@HF and ωB97X-Dand by 0.6 eV in M06-2X. The L+4 and L+5 levels are also localizedon D fragments and are nearly degenerate with L+3. There is reorderingof levels in the GS geometry between HF and DFT. In HF, L+4 is ofb1 symmetry, and in DFT L+5, it is of b1 symmetry.In the GS geometry, weak interaction between linearcombinationsof D orbitals produces quasi-degenerate aGoWo@HF, ωB97X-D,and M06-2X eigenvalues and from ΔSCF calculations are givenin EG gap is overestimated by 1.77 and 2.63 eV, respectively. Thesedifferences are reduced in GoWo@HF. The IPis overestimated by 0.08 and 0.76 eV, the EA is underestimated by0.33 and 0.25 eV and the EG gap is overestimated by 0.41 and 1.00 eV for the GS and CTgeometries, respectively. The ωB97X-D functional overestimatesthe IP by 0.10 and 0.26 eV, underestimates the EA by 0.34 and 0.33eV, and overestimates EG by 0.45 and 0.59 eV for the GS and CT geometries. In contrast, theM06-2X functional underestimates the IP by 0.46 and 0.51 eV and overestimatesthe EA by 0.47 and 0.46 eV, leading to underestimates of the EG gap by 0.93 and 0.98 eV inthe GS and CT geometries, respectively.Ionization potentials (IP), electron affinities (EA), and IP–EAdifferences from SCF HF, r eVÅ–1, where r is the electron–hole separation. Since the electron–holeseparation is significantly larger in a CT transition and the Coulombicinteraction energy is comparable to excitation energies, interactionscreening in GoWo@HF and ωB97X-D or truncationin M06-2X DFT calculations determines the relative cost of CT andLE excitations. GoWo@HF. When electron and hole separate to a larger degree inthe CT state, this advantage overcomes the energetic cost of separatingthe electron and hole to a larger extent in the CT state and resultsin lowest energy CT and LE excitations of similar energies.One of the challenges for prediction of relative excitationenergiesof CT versus LE states is the physical difference in exciton bindingin the CT state versus the LE state, owing to the localization ofelectron and hole on the same or different molecular fragments. Anunscreened Coulombic interaction energy is approximately 14.4/34 from which they estimated the H levels in theD and DAD systems to be −5.2 and −5.4 eV and the L levelsin the A and DAD systems to be −2.9 and −3.1 eV, respectively.Measurements were performed in a 0.1 M solution of Bu4NBF4 in dichloromethane(DCM). We present results of conductor-likePCM calculations using DCM and cyclohexane (CHX) as polar and nonpolarsolvents. Polar and nonpolar solvents differ in that only the formerhave a large screening contribution from permanent dipole reorientation.These calculations are intended to give an indication of the magnitudeof solvent induced shifts.Finally, in this section, we consider the effect ofdielectricscreening by the solvent on H and L levels of DPTZ-DBTO2. Dias etal. reported electrochemical measurements of the redox potentialsof the PTZ donor, DBTO2 acceptor, and DPTZ-DBTO2 DAD system,76 In an equilibriumelectrochemical ionization process, where dipole reorientation andelectronic polarization screen the ion formed lowering of the IP andraising of the EA will be larger than in a fast process, such as opticalexcitation, where dipole reorientation is generally too slow to contributeto screening. Changes in IP and EA values estimated from C-PCM calculationsusing DCM and CHX therefore represent limits where electronic plusdipole orientation and electronic polarization only are included.Redox potentials are successfullyreproduced in first-principlescalculations which use protocols where the free energies of geometry-relaxedsolvated complexes are computed.44 for theD, A, and DAD systems in cyclohexane  and DCM(ε = 8.93) are given in 77 M06-2X H and L levels for the DAD system inthe gas phase and the CT geometry are −6.12 and −2.10eV compared to estimates of these levels from cyclic voltammetry of−5.4 and −3.1 eV.34 Usingthe simple estimates given in Differences in electrostatic interaction energies for solvatedionic and neutral species from conductor-like PCM calculations usingthe M06-2X functional in GAMESSGoWo@HFgas phase H and L levels (−6.56 and −0.61 eV in DAD)and ωB97X-D gas phase H and L levels (−6.81 and −1.09eV) with CHX solvent results in H levels 0.4 and 0.6 eV below theexperimental value and L levels 1.8 and 1.3 eV above the experimentalestimates from cyclic voltammetry.34 Witha DCM solvent the IP value is overestimated by GoWo@HF by 0.3 eV and within 0.1 eV (ωB97X-D). The EA valueis underestimated by 1.2 eV by GoWo@HF and within0.1 eV of experiment according to ωB97X-D.Applying the same estimated shifts to III.BGoWo@HF/BSEat one end and TDDFT/M06-2X at the other. This may reflect the strengthof electron–hole interaction in these methods. This is likelyto be greatest in GoWo@HF/BSE, followed by TDDFT/ωB97X-Dand TDDFT/M06-2X. However, the different ways in which the Coulombicelectron–hole attraction is attenuated by screening approximationsin these methods should be kept in mind when comparing electron–holeinteraction strengths in LE and CT states.In thissection, we consider the character of low-lying singlet and tripletexcited states for the GS and CT geometries, their relative stabilitiesand singlet–triplet splittings. As noted in GoWo@HF/BSE, TDDFT/ωB97X-D, and TDDFT/M06-2Xare given in GoWo@HF, 0.35 eV for ωB97X-D, and 0.40 eV for M06-2X) hasbeen added to the transition energies in The lowest 10 singletand 10 triplet transition energies for the GS and CT geometries predictedby III.B.11DA levelbelow the 1DD level in the GS geometry. The singlet–tripletsplitting for this DA level is too small to be shown in 1AA levellies above these levels. GoWo@HF/BSE placesthe lowest 1DA level just above the lowest 1DD level and the 1AA level above these levels. Changesin excited state wave function character for these levels can be seenin the top half of 1DD excited state wave function differs betweenthe BSE and TDDFT methods, while the wave functions of the 1DA and 1AA states change steadily from BSE to M06-2X.Inspectionof the levels illustrated in 3AA state at 3.38 eV with two 3DD states slightly above it at 3.38 and 3.39 eV and the 3DA state at 3.88 eV. TDDFT/M06-2X predicts the 3DD stateto be the lowest GS excited state at 3.46 eV, with the 3AA state at 3.59 eV and the 3DA state at 3.78 eV. Gibsonand co-workers22 reported a similar orderingof excited state levels at the equilibrium geometry of the PTZ-DBTO2DA system using the same M06-2X TDDFT functional. They found the S1, T1 and T2 energies at 3.55, 3.45,and 3.53 eV. These compare to energies for the GS geometry from TDDFT/ωB97X-D predicts the lowest triplet statein the GSgeometry to be the GoWo@HF/BSE predicts the 3AAstate to be the lowest at 2.50 eV, with two 3DD statesat 2.71 eV and the 3DA state at 4.15 eV. There are smallchanges in wave function coefficients for singlet or triplet statespredicted by TDDFT/ωB97X-D or TDDFT/M06-2X  symmetry and its σh mirror plane is the yz plane, which it shares withthe acceptor in the DAD molecule. Transitions with the transitionmoment parallel or perperdicular to the D σh mirror plane belong to the A′ and A″ irreduciblerepresentations, respectively.III.C.1GoWo@HF/BSE and TDDFT/M06-2X methods are compared toabsorption spectra for the DAD molecule in the GS geometry in GoWo@HF/BSE and TDDFT show good agreement, with the exception ofthe intensity of the second A″ transition, which is strongerin GoWo@HF/BSE. The donor H, H-1, and L MOsare even and the L and L+1 MOs are odd with respect to the σh mirror plane. The H/L+1 (A″) transitionat 4.02 eV transition at 4.37 eV has its transition moment perpendicular to thelong x molecular axis and the H/L+2 (A″) transitionat 4.47 eV has its transition moment along the x axis.The A″ transitions at 5.43 and 5.85 eV are mixtures of theH-1/L+1 and H/L+2 transitions. Most TDDFT transition energies forD are higher than GoWo@HF energies, by an averageof 0.15 eV.The PTZ donor shows six singlet transitions in the range 4 to 6eV. Transition energies are given in 2 singlet absorptions in the range 4 to 6.5 eV . L and L+1 are fully localizedon A and L+2 is partly on D and A. On going from the GS geometry tothe CT geometry, the HF L+4 orbital localized on D switches to L+3.The two largest coefficients in GoWo@HF/BSE or TDDFTeigenvectors for the excited states discussed in The DPTZ-DBTO2 emitter has 17 opticallyactive transitions in the range 4 to 6 eV. The H to H-3 MOs are localizedon D and the H-4 MO is localized on A . with similar weights . Thus,LE DD transition energies for the three methods lie within a rangeof 0.22 eV.In the GS geometry the H-1 → L+4/H →L+2 A is weak 7. These  weights 7. Note t20 reportedoptical absorption peaks in 2,8-DPTZ-DBTO2 in DCM at 3.71, 4.35, and4.87 eV. Dias and co-workers34 reportedthe variation of the S1 B3LYP TDDFT excitation energy asa function of the dihedral angle containing the C–N bond connectingthe D and A fragments of the 2,8-DPTZ-DBTO2 DAD system. They foundthat the S1 energy ranged from about 3.5 eV .There are four DA transitions in  and L+1 6 and 11.1 and A2 excited statetransition energies in TDDFT/ωB97X-D are found at 3.90, 3.91,3.91, and 3.95 eV and in TDDFT/M06-2X they occur at 3.60, 3.61, 3.74,and 3.75 eV, and all are weak . GoWo@HF/BSE predicts the LE 1DD transition to lie below the 1DA transitionswhile the TDDFT methods predict the 1DA transition energiesto be lower.The corresponding Bts again 6. Partic2 symmetryare mainlyAA in character with significant weights of the H-4/L and H-4/L+1transitions ,and the excitation energy reduces sharply going from GoWo@HF/BSE to TDDFT/M06-2X .Absorption spectra for the GS geometry from these three methods in x and y incident lightpolarization.Inspection of GS singlet excitationenergies 6,comparnsitions 11. The rIII.C.3GoWo@HF/BSE and TDDFT/ωB97X-D, and the first fourare given for TDDFT/M06-2X since four states of clear DA charactercan be identified in that case. In the GS geometry the splittingsof the first two DA states reduce on going from GoWo@HF/BSE to TDDFT/M06-2X. GoWo@HF/BSE predictssplittings for these states of nearly 300 meV. This reduces to 17.9meV in TDDFT/ωB97X-D and to around 5 meV in TDDFT/M06-2X. Inthat case it was possible to obtain splittings for the third and fourthstates of DA character and these are around −40 meV. DA statesare linear combinations of transitions from the H and H-1 levels tothe L and L+1 levels. The change in signs of the splittings in thethird and fourth levels is expected to be due to changes in phasesof the excited state wave functions for those cases. Sizes of excitedstate dipole moments also change steadily on going from GoWo@HF/BSE to TDDFT/M06-2X. The dipole moment in the 11DA state changes from 11.85 D in GoWo@HF/BSE to 6.52D in TDDFT/M06-2X.Excitation energies, excited state dipole moments, and singlet–tripletsplittings predicted by each method are given in kT or less. Thelarge splittings predicted by GoWo@HF/BSE in theGS geometry are much reduced, to 25.4 and 9.3 meV for the first twostates of DA character. TDDFT/ωB97X-D predicts splittings of−61.1 and 6.1 meV in this geometry and TDDFT/M06-2X predictslarger splittings for the first two DA states (50.2 and 23.0 meV)and negative splittings (−23.7 and −23.2 meV) for thenext two DA states. Magnitudes of excited state dipole moments arelarger in the CT geometry and show the same magnitude trend from GoWo@HF/BSE to TDDFT/M06-2X as in the GS geometry. Theincrease in magnitude arises as holes localize in one donor fragmentin the CT geometry, leading to dipole moment components in both the y and z directions, whereas there is onlya z component in the GS geometry.The singlet–triplet splitting forthe CT geometry is the more important one, from a TADF point of view.Triplet states in this geometry may undergo rISC and delayed fluorescence,provided the splitting is of order 79 found that underestimationof singlet and triplet excitation energies by the PBE GGA functional,compared to third order coupled cluster values, was corrected usinga range-separated CAM-B3LYP functional, for both LE and CT excitations.They also note that the triplet instability problem affects LE statesto a much greater extent than CT states. Hence it is not surprisingthat GoWo@HF/BSE predicts singlet–tripletsplittings in the expected range for the CT geometry. The larger splittingspredicted by GoWo@HF/BSE in the GS geometrymay arise from greater mixing of DA and DD configurations. In theGS geometry the 11DD, 11DA, and 13DA states are close in energy while the 13DD state lieslower . These results arediscussed in this section as well as changes in excitation energiesin solution or condensed phases.The main variations inpredictions of the IV.AGoWo@HF gaps exceed M06-2Xgaps by over 2 eV, except for the DA gap in the CT geometry. ωB97X-Dgaps lie between those extremes and closer to GoWo@HF gaps. The difference in DD and DA gaps is nearly constantfor each method and rises by 0.2 to 0.3 eV on going from the GS toCT geometry. GoWo@HF/BSE methods. In particular, relative weights of T1/T2 amplitudesin the triplet states could reveal the origin of the triplet instabilityof the GoWo@HF/BSE method in LE states.Low level coupled cluster calculations such as CC2, which includedouble excitations perturbatively, would yield additional insightsinto the relative accuracy of these TDDFT and GoWo@HF/BSE.This observation also holds in DAD where LE excitations of B2 symmetry (mainly AA transitions) and A1 symmetry arepredicted by TDDFT/M06-2X to be 0.15 and 0.02 eV higher in energy,compared to GoWo@HF/BSE . On theother hand, excitations of B1 symmetry (mainly DA transitions)are predicted by TDDFT/M06-2X to be −0.45 eV lower on average.Even though differences in DD and DA gaps according to either methodare roughly the same, TDDFT/M06-2X predicts relatively lower DA transitionenergies compared to GoWo@HF/BSE by around0.5 eV.35 The TDDFT fxc kernel which replaces the Coulombic electron–hole attractionis short-ranged in a nonhybrid density functional, resulting in predictedCT excitation energies which approximate single-particle excitationenergies.35 A heuristic explanation forthe observation that TDDFT local exciton energies are larger than GoWo@HF/BSE energies while CT energies are smaller isthat screening of electron–hole attraction in GoWo@HF/BSE depends on the distance between electron and hole ina manner similar to electron–hole attraction in a range-separatedTDDFT approach. In the latter, it is strongly screened at short-rangeand essentially unscreened beyond a cutoff distance around 8ao.80 On the otherhand, in TDDFT/M06-2X the electron–hole attraction is scaledby 54%, independent of electron–hole separation. Thus, thelatter will underscreen at short-range, leading to more HF-like overestimatesof excitation energies in local excitons and overscreen at long-range,leading to relative underestimation of CT excitons.Underestimation of CT excitation energies by TDDFT methodsanda remedy of adding a fraction of exact exchange were highlighted sometime ago.IV.B1DD state in the GS geometry predicted by GoWo@HF/BSE is close in energy to the 13DA state (3.01 eV) only inthe CT geometry. Hence these splittings must be calculated in therelevant CT geometry and predictions will depend on TDDFT fxc kernel or screened interaction in BSE to some extent.Except for the case just mentioned, singlet–triplet splittigsare positive for the first two pairs of 1,3DA states. Negativesplittings occur for the third and fourth pairs of states, presumablybecause of orthogonality to the lower pairs of these states. In contrastto triplet energies of LE states which are subject to triplet instability, as expectedfrom Work to determine how best to incorporate effects of the dielectricbackground on light emission is in progress. Sun and co-workers23 simulated the solvated 1CT excited state of PTZ-DBTO2 (DA) using both C-PCM and explicitsolvent molecules. In the ground state geometry they found a smallincrease in absorption energy . However, in a state-specific TDDFT/M06-2X calculation,the excitation energy reduced from 2.65 eV in vacuum  geometry and a 1DA charge transfer excited state (CT)geometry. Relative stabilities of specific excited states depend onthe geometry and method used. The lowest singlet found by GoWo@HF/BSE is a LE 1DD state and the CT 1DA state is predicted to be 0.44 eV higher using a TZVP basis.In contrast the lowest singlet found by TDDFT/ωB97X-D and TDDFT/M06-2Xmethods is the CT 1DA state and the LE 1DD stateis 0.33 (0.49) eV higher in energy. This reflects a systematic trendfound in excited states of the isolated donor (D) and acceptor (A)molecules as well as the DAD molecule. TDDFT/M06-2X typically predictslocal exciton states to be 0.1 to 0.2 eV higher in energy than GoWo@HF/BSE, while it predicts charge transfer statesto be 0.4 eV lower in energy. Predictions of the range-separated TDDFT/ωB97X-Dmethod typically lie between the other methods. This accounts forthe reversal in order of the LE 1DD and CT 1DA states by the methods in the GS geometry. Oscillator strengthsfor optical transitions in the GS and CT geometries from all methodsused agree reasonably well, and it is possible to identify correspondingtransitions in each method. Oscillator strengths for the lowest singletexcited state in each geometry and method are very low.In this work, we presenteda comparison of single-particle andexcited state energy levels and optical transitions of the DPTZ-DBTO2molecule predicted by the M06-2X and ωB97X-D functionals inTDDFT and 1DA and 11DD statesreverse in order and all methods predict the 11DA stateto be the lowest excited state. TDDFT/M06-2X predicts this state at2.53 eV, TDDFT/ωB97X-D at 2.95 eV and GoWo@HF/BSE at 3.28 eV. Both GoWo@HF and DFT/ωB97X-Dpredict the IP to lie below the value predicted by a ΔSCF calculationand the EA to be smaller than the ΔSCF values while DFT/M06-2Xpredicts the IP to lie above the ΔSCF value and the EA to belarger than the ΔSCF value. It is therefore likely that TDDFT/M06-2Xunderestimates the 11DA excitation and TDDFT/ωB97X-Dand GoWo@HF/BSE overestimate it. Obtaining thecorrect single-particle gap in the SCF state used for a linear responsecalculation may be crucial in predicting the best excitation energiesand these can be judged using the ΔSCF value.80 It is recommended that singlet–triplet splittingsin TADF emitters be calculated in the relevant CT geometry ratherthan the vertical splitting as predicted values can differ significantlybetween geometries.In theCT geometry, the 11,3DA and 1,3AA states by either methodare generally inagreement. However, anomalies exist which may be explained by mixingof either the singlet or triplet with other states, which can alterthe predicted splitting significantly. 11,3DA splittings,which are critical for TADF applications, are predicted to be 25.4,−61.1, and 50.2 meV, according to GoWo@HF/BSEand TDDFT/ωB97X-D and TDDFT/M06-2X, respectively.Predictions of singlet–triplet splittingfor


A recent study demonstrated that exogenous supplementation of H2S ameliorates functional insufficiency in chronic kidney disease (CKD). However, how the H2S system is impaired by CKD has not been elucidated. The uremic toxin indoxyl sulfate (IS) is known to accumulate in CKD patients and harm the renal tubular cells. This study therefore treated the proximal tubular cells, LLC-PK1, with IS to see how IS affects H2S formation. Our results showed that H2S release from LLC-PK1 cells was markedly attenuated by IS when compared with control cells. The H2S donors NaHS and GYY-4137 significantly attenuated IS-induced tubular damage, indicating that IS impairs H2S formation. Interestingly, IS downregulated the H2S-producing enzymes cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST), and these effects could be reversed by inhibition of the IS receptor, aryl hydrocarbon receptor (AhR). As transcription factor specificity protein 1 (Sp1) regulates the gene expression of H2S-producing enzymes, we further showed that IS significantly decreased the DNA binding activity of Sp1 but not its protein expression. Blockade of AhR reversed low Sp1 activity caused by IS. Moreover, exogenous H2S supplementation attenuated IS-mediated superoxide formation and depletion of the cellular glutathione content. These results clearly indicate that IS activates AhR, which then attenuates Sp1 function through the regulation of H2S-producing enzyme expression. The attenuation of H2S formation contributes to the low antioxidant defense of glutathione in uremic toxin-mediated oxidative stress, causing tubular cell damage.Hydrogen sulfide (H The half-life of H2S in plasma is less than 30 min, and elimination occurs through excretion in exhaled breath , as previously described i) directly, where an increase in [Ca2+]i enhances CSE activity and vice versa [2+]i overload via hyperfunction of the Ca2+-permeable TRPV1 in tubular cells [2+]i overload-mediated by TRPV1 to reduce CSE activity needs further study. Similar to CSE, an increase in [Ca2+]i also attenuates the activity of the 3-MST/CAT pathway for H2S production, and the enzyme regulated by Ca2+ has been suggested to be CAT and not 3-MST iot 3-MST . Furtheroduction . As the 2S has been implicated in various kidney diseases, including renal ischemia/reperfusion injury (IRI) [2S production on the renal tubules. In the IRI model of the rat kidneys, both CSE and CBS protein levels are decreased at 4 days after ischemia, and this persists throughout this period for up to 21 days. Impaired H2S production significantly increases formation of the phosphorylated form of histone H2A (pγH2AX), which indicates that oxidative damage to DNA occurs in renal tubular cells in response to IRI. Instead, exogenous supplementation CSE-deficient mice with H2S attenuates the area of renal cortical necrosis, reduces the quantity of pγH2AX-positive cells, retards the renal function decline, and rescues animal survival [2S donors NaHS or GYY-4137. H2S deficiency, therefore, increases the vulnerability of the renal tubules to oxidative damage.Alteration of Hry (IRI) , hypertery (IRI) ,43, diabry (IRI) , obstrucry (IRI) , and CKDry (IRI) ,8. IRI isurvival . In this2S in IS-mediated tubular damage. In fact, H2S and its dissociated ion HS- act as a powerful one-electron chemical reluctant and have a high capacity to scavenge ROS by directly reacting to superoxide and H2O2 [2S attenuates superoxide formation in the IS-treated tubular cells. In addition to H2S itself, a previous study demonstrated that H2S increases the intracellular GSH level by enhancing the production of the cystine/cysteine transporter and redistributes GSH to the mitochondria, where a large amount of ROS is produced [2S increases the intracellular GSH content in IS-treated tubular cells. Hence, H2S, together with GSH recruitment, act as an overall antioxidant defense against oxidative damage triggered by IS/AhR signaling. Accordingly, impaired H2S production by IS renders tubular cells vulnerable to oxidative stress damage and underlies one of the mechanisms of IS-mediated tubulotoxicity, which inevitably leads to tubulointerstitial inflammation and renal fibrosis.Previous studies showed that the mechanisms underlying IS-induced tubular oxidative damage include increases in NOX4 activity and ROS generation , downregand H2O2 ,52. In tproduced . In this1 is the most widely used model to study the harmful uremic toxins on renal tubular cells [The single cell line used is the limitation of our study. LLC-PKar cells ,49,53,542S formation and induces tubular oxidative damage. Downregulation of the H2S-producing enzymes CBS, CSE, or 3-MST and a decrease in Sp1 DNA binding activity underlie the mechanism behind the decrease in H2S production in the proximal renal tubules. As expected, AhR is involved in IS-mediated defective H2S production and subsequent renal tubular damage. The accumulation of IS during CKD progression impairs H2S formation, rendering tubular cells more susceptible to oxidative injury. Thus, H2S may serve as a potential therapeutic molecule to alleviate renal function decline in CKD.Our results show that treatment of the proximal tubule cells with IS impairs H


This work introduces a novel humidity sensor based on a nanocomposite material comprising graphene decorated with three-dimensional flower-like structures of zinc oxide (Gr/3D–ZnO) fabricated via a hydrothermal method with various weight percentages of graphene. The surface structure and morphology of the Gr/3D–ZnO nanocomposite were analyzed using XRD, EDS, SEM, TEM, and Raman spectroscopy. The influence of humidity on the electrical properties of the nanocomposite was also investigated. Experiment results revealed that the nanocomposite with 70 wt% of graphene provided high sensitivity (S = 446) with rapid response times (120 s) and recovery times (160 s). These results demonstrate the excellent potential of the proposed Gr/3D–ZnO nanocomposite in monitoring atmospheric humidity. A discussion on the mechanism underlying the effects of humidity on the Gr/3D–ZnO nanocomposite is also provided. Atmospheric levels of water vapor  can profoundly affect electronic devices, meteorological phenomena, agriculture, manufacturing, and food storage . Efforts2 nanopowders [3 nanosheets [2O3 nanopowders [2 nanorods [2 nanofibers [2 bonded carbon atoms, has a large surface to volume ratio and excellent conductivity [Numerous types of humidity sensors have been developed using resistive ,5 and caopowders  and nanoopowders , ZnO nanopowders , WO3 nannosheets , Fe2O3 nopowders , graphenopowders , organicopowders , ZrO2 nananorods , and NiOnofibers . Among tnofibers . ZnO cannofibers , nanorodnofibers , nanoshenofibers , nanoparnofibers , nanofibnofibers , hollow nofibers , and nannofibers . Despitenofibers . Nonethenofibers . Graphenuctivity ,35,36, wuctivity ,39,40,41uctivity ,43. The uctivity ,45.Our objective in this study was to fabricate a sensor based on a nanocomposite material comprising graphene decorated with three-dimensional flower-like structures of zinc oxide (Gr/3D–ZnO). The surface structure and morphology of the Gr/3D–ZnO nanocomposite were characterized using XRD, EDS, SEM, TEM, and Raman spectroscopy. We also investigated the influence of humidity on the electrical properties of the nanocomposite. The completed humidity sensor exhibited ultra-high sensitivity with fast recovery and response times over a broad range of relative humidity (RH) levels.3)2·6H2O, 98%), anhydrous alcohol , L-histidine , urea ((NH2)2CO, 99.5%), and polyvinyl alcohol  were purchased from Sigma-Aldrich Co., Inc.Commercial graphene film of high purity (>99%) was purchased from UniRegion Bio-Techn  and Sigma–Aldrich Co., Inc. . Zinc nitrate hexahydrate . The crystal structure of Gr/3D–ZnO was characterized using an XRD-6000 Shimadzu X-ray diffractometer with Cu Kα radiation of 1.5405 Å at 40 kV and 30 mA between 10° and 80° (2θ) at intervals of 2°/min. Infrared spectra were measured using an Agilent Cary spectrophotometer. The characteristic Raman spectrum of the Gr/3D–ZnO nanocomposite was measured at RT under excitation from a laser source at a wavelength of 532 nm .2), whereupon the chips were heated to 80 °C and held at that temperature for 0.5 h before undergoing calcination at 400 °C for 2 h.A solution of 10% PVA was added to promote the binding of the composite. A pair of comb-like gold electrodes were applied to an alumina substrate via dip-coating  and Rh is the resistance under a specific level of humidity [The experiment setup used to assess the performance of the humidity sensor is described in our previous work . Brieflyhumidity . The humHumidity hysteresis error (H) was calculated using the following equation:e output . The rese output . RH was −1 and 1568 cm−1) were presumably due to in-plane bond stretching of sp2 hybridized carbon atoms and the corresponding combinational modes and overtones [−1 are the vibrational modes of ZnO [As shown in vertones ,51. The s of ZnO . The Gr/We assessed the sensitivity of Gr/3D–ZnO nanocomposite materials  to humidity based on impedance measurements obtained using AC voltage (1V) at a frequency of 1 kHz. As shown in Humidity hysteresis is an important factor in evaluating the reliability of humidity sensors . As showResponse and recovery times largely determine the practical applicability of humidity sensors. As shown in 12%/R90% = 446). The response to other gas species was as follows: CO (1.55), NH3 (1.41), NO2 (1.36), and NO (1.18). Overall, the Gr/3D–ZnO nanocomposite exhibited excellent selectivity for humidity.Selectivity is another crucial parameter in assessing the performance of humidity sensors. 2O or H3O+ was on the discontinuous water layer was difficult [2O or H3O+. Grotthuss and Casalbore-Miceli et al. outlined an ion transfer mechanism involving the transfer of H2O or H3O+ on serial water layers: H2O + H3O+ → H3O+ + H2O [At low relative humidity (RH), the probability of contact between water molecules and Gr/3D–ZnO nanocomposite particles was low, so only the outer particles came into contact with the water molecules, as shown in ifficult . TherefoO+ + H2O ,60. ThisIn this work we have succeeded in fabricating Gr/3D–ZnO nanocomposites via a hydrothermal process to create a novel low-cost humidity sensor based on Gr/3D–ZnO nanocomposite material. Experiment results revealed that the nanocomposite with 70 wt% graphene provided a high sensitivity (S = 446) with rapid response times (120 s) and recovery times (160 s). The highest humidity hysteresis error was only 2.32% over a wide range of RH values. These results demonstrate the excellent potential of the proposed Gr/3D–ZnO nanocomposite for the development of low-cost sensors to monitor atmospheric humidity.


Background: The role of purinergic P2X7 receptor (P2X7R) is of interest due to its involvement in inflammation and mediating immune cell responses. P2X7R is particularly implicated in the development of inflammatory bowel disease (IBD). However, the extent of the actions of P2X7R in the gastrointestinal (GI) system under physiological and pathophysiological conditions remains to be elucidated. This systematic review aimed to identify, summarize and evaluate the evidence for a critical role of P2X7R in the GI system.Methods: We searched PubMed, Embase and Scopus with search terms pertained to P2X7R in the GI system in disease or physiological state, including “P2X7 or P2X7 receptor or purinergic signaling” in combination with any of the terms “intestine or colon or gut or gastrointestinal,” “pathology or inflammation or disease or disorder,” and “physiology or expression.” Titles and abstracts were screened for potentially eligible full texts, and animal and human studies published in English were included in this study. Data were extracted from papers meeting inclusion criteria. Meta-analysis was not feasible given the study diversity.Results: There were 48 papers included in this review. We identified 14 experimental colitis models, three sepsis models and one ischemia-reperfusion injury model. Among them, 11 studies examined P2X7R in GI infections, six studies on immune cell regulation, four studies on GI inflammation, two studies on GI malignancies, three studies involving intestinal injury due to various causes, two studies on ATP-activated P2X7R in the GI system and two studies on metabolic regulation.Conclusion: Evidence supports P2X7R mediating inflammation and immune cell responses in GI inflammation, infections and injury due to IBD and other challenges to the intestinal wall. P2X7R inhibition by gene knockout or by application of P2X7R antagonists can reduce tissue damage by suppressing inflammation. P2X7R is also implicated in GI malignancies and glucose and lipid homeostasis. P2X7R blockade, however, did not always lead to beneficial outcomes in the various pathological models of study. Despitechannels  in cell channels . The conchannels , but thichannels .Short- and long-term purinergic signaling is involved in physiological and pathophysiological processes. Short-term signaling mediates neuromodulation, neurotransmission, secretion, chemotaxis and platelet aggregation . Long-tevia purinergic P2 receptors. P2 receptors are classified into P2X and P2Y subfamilies. P2X receptors are ligand-gated ion channels, and P2Y receptors are G protein-coupled receptors. They are further classified into seven P2X subtypes and eight P2Y subtypes (ATP exerts its actions subtypes . Differesubtypes .Among the seven P2X receptors (P2X1-7), the P2X7 receptor (P2X7R) is of particular interest in inflammation, with evidence showing its role in the inflammatory process . ExtraceApart from IBD, P2X7R has also been implicated in other gastrointestinal (GI) pathologies, especially for its role in aberrant inflammation when activated . In infeThe topic of P2X7R in inflammatory diseases has gained growing interest in recent years. To the best of our knowledge, no systematic review concerning this topic has been published. Thus, the aim of this article is to identify, summarize and evaluate the roles and actions of P2X7R in the GI system.We searched PubMed, Embase and Scopus using the terms “P2X7 or P2X7 receptor or purinergic signaling” in combination with any of the terms “intestine or colon or gut or gastrointestinal,” “pathology or inflammation or disease or disorder,” and “physiology or expression.” There were no date limits, but only studies published in English were included. Titles and abstracts were screened, and full-text articles were obtained for potentially eligible studies.Eligible studies were animal or human experimental studies undertaken with a focus on the role of P2X7R. Studies were limited to those involving the GI system. The criteria for methodological rigor of experimental articles included the reporting of species, strain, gender and age of animals , tissue and cell line used, clear description of the experimental protocol with inclusion of proper controls, and reporting the type of statistical analysis undertaken.The search identified a total of 387 records; after eliminating duplicate results, the titles and abstracts of the remaining 265 unique records were screened, of which 78 were selected for full-text review. After a full-text review, 30 did not meet the inclusion criteria and 48 were included in the review. A PRISMA flow chart of the search results is shown in There were 48 papers included in this study. We identified 14 experimental colitis models, three sepsis models and one ischemia-reperfusion injury model. Among them, 11 studies examined P2X7R in GI infections, six studies on immune cell regulation, four studies on GI inflammation, two studies on GI malignancies, three studies involving intestinal injury due to various causes, two studies on ATP-activated P2X7R in the GI system and two studies on metabolic regulation.P2X7R activation has been shown to induce a broad array of cellular responses, including cytokine release, apoptosis, and cell death, which are associated with inflammatory processes. The key actions of P2X7R in the GI system are summarized in In the GI system, it is well established that ATP released from neuronal and non-neuronal cells involves purinergic mechanosensory transduction that modulates enteric reflex activities, ion transport, tissue blood flow and pain transmission . The phyvia caecal ligation and puncture (CLP)  , infectire (CLP)  in both re (CLP) .There is strong supporting evidence that P2X7R plays a key role in GI inflammation by upregulating the production of pro-inflammatory mediators.ad libitum  azoxymethane (ad libitum)  (+ T cells (5 × 105 cells/mouse)  . Two stulibitum)  and 4% (libitum)  in drinkml each)  and one s/mouse) . Twelve s/mouse) , four sts/mouse) , two stus/mouse) , and ones/mouse) .in vitro that P2X7R-mediated activation of mast cells played key roles in initiating and exacerbating intestinal inflammation in colitis by promoting the release of TNF-α, IL-6, leukotrienes and chemokines, and neutrophil infiltration into the colon. The work by P2X7R has been shown to play vital roles in intestinal inflammation in IBD and mediating colitis-induced tissue damage. P2X7R upregulated the production of pro-inflammatory cytokines  and mediThere were many different variables across these papers, including different chemicals to induce colitis, different methods to inhibit P2X7R, different rat or mouse types, making it difficult to compare the significance of their findings in relation to one another. The study by in situ. Different P2X7R inhibitors of various concentrations were used in different chemically-induced colitis experiments across these papers. Overall, inhibition of P2X7R rescued colitis-induced intestinal damage, disruption of the epithelial layer, reduced expression of inflammatory cytokine expression and decreased clinical signs of colitis such as diarrhoea or weight loss.In the studies involving P2X7R antagonists, -/- mast cells had reduced inflammatory responses, including decreased mast cell activation and neutrophil infiltration in the colon compared to mice with P2X7R+/+ mast cells. This implies that P2X7R-mediated activation of mast cells is involved in exacerbated inflammation seen in TNBS-induced colitis  ( colitis . SimilarX7R mAb) . FigliuoNevertheless, Other papers on P2X7R in the GI system that do not involve chemically-induced colitis still highlight important roles of P2X7R when activated. Taken together, P2X7R appears to be highly involved in the development of IBD, which makes P2X7R blockade of particular interest for its therapeutic management. However, the areas of concern regarding P2X7R antagonism are the exacerbated inflammation  and assoin vivo animal studies, the CLP procedure was done by both papers. One of the severe complications of IBD is sepsis. Two sepsis models using rats and mice were reported , one of via the ERK and NF-κB signaling pathways   , lymphocrus (Hp) , C. diffifficile , C. kose. koseri , L. monoytogenes , and LPS typhosa . Modes o typhosa  or intra typhosa  for in v KO mice , pharmac KO mice , P2X7R s KO mice , ARTC2.2 KO mice  and anti KO mice .T. gondii infection  and AZD9056 (10 μM) or transfecting CRC cell lines with P2X7R siRNA reduced CRC proliferation by inhibiting P13/Akt/GSK-3-β/β-catenin pathway which is an important signaling pathway in tumor development (Similar to the findings by elopment . In concelopment , hepatocelopment . These pelopment , 1 monthelopment  and 2 moelopment ], makingP2X7R has been shown to play a role in controlling the microecology of GI bacteria . ATP frovia affecting the composition of gut microbiota (Apart from its role in glucose homeostasis crobiota , it has crobiota . This recrobiota . The fincrobiota . P2X7R Kcrobiota . With inIn a chronic binge alcohol feeding model in mice by Taken together, as summarized in A limitation of this systematic review is that it focuses on homeostatic and pathological conditions of the GI system. P2X7R is highly expressed in various tissues and organs, and its role is not limited to the GI only . SecondlIn conclusion, P2X7R is expressed in many cell types and plays important physiological and pathophysiological roles in the GI system. It can regulate immune cell populations under normal conditions, mediate immune responses when challenged by infection, inflammation or other forms of tissue injury. Differing observations on the roles of P2X7R have also been reported. For instance, although the majority of papers agree on its role in upregulating inflammation, there are reports of P2X7R involved in downregulating inflammation and suppressing colitis. P2X7R can enhance or suppress immune responses in GI infections, and suppress or promote tumor development. P2X7R antagonism has potential for therapeutic uses; however, from differing evidence on its role, P2X7R inhibition can have beneficial or detrimental effects in IBD, sepsis, infections and malignancies. The effects on gut microbiota composition and glucose and lipid metabolism are also important considerations in P2X7R blockade. For a more comprehensive understanding of P2X7R’s actions and the effects of its blockade, further research in GI and other systems is crucial, especially in light of conflicting reports on its role.


Finally, comparing sE21E with sE2 in mouse immunizations, we show similar induction of heterologous NAbs. In summary, we find that C-terminal E2 fusion of E1 or 1E is superior to N-terminal fusion, both in terms of antigenicity and the induction of heterologous NAbs. This has relevance when designing HCV E1E2 vaccine antigens.Yearly, about 1.5 million people become chronically infected with hepatitis C virus (HCV) and for the 71 million with chronic HCV infection about 400,000 die from related morbidities, including liver cirrhosis and cancer. Effective treatments exist, but challenges including cost-of-treatment and wide-spread undiagnosed infection, necessitates the development of vaccines. Vaccines should induce neutralizing antibodies (NAbs) against the HCV envelope (E) transmembrane glycoprotein 2, E2, which partly depends on its interaction partner, E1, for folding. Here, we generated three soluble HCV envelope protein antigens with the transmembrane regions deleted , termed sE1E2 (E1 followed by E2), sE2E1 (E2 followed by E1), and sE21E (E2 followed by inverted E1). The E1 inversion for sE21E positions C-terminal residues of E1 near C-terminal residues of E2, which is in analogy to how they likely interact in native E1/E2 complexes. Probing conformational E2 epitope binding using HCV patient-derived human monoclonal antibodies, we show that sE21E was superior to sE2E1, which was consistently superior to sE1E2. This correlated with improved induction of NAbs by sE21E compared with sE2E1 and especially compared with sE1E2 in female BALB/c mouse immunizations. The deletion of the 27 N-terminal amino acids of E2, termed hypervariable region 1 (HVR1), conferred slight increases in antigenicity for sE2E1 and sE21E, but severely impaired induction of antibodies able to neutralize  Global HCV prevalence varies geographically from about 0.5% to 2.3%, with nearly 2 million new acute infections annually and a chronicity rate of 55–85% . ChronicSix epidemiologically relevant HCV genotypes exist and their RNA genomes differ by 30–33% both at the nucleotide and amino acid level ,6. The hL) between sE1 and sE2, but the antigen still elicited low amounts of HCV NAbs when used for immunization of mice [Studies have indicated that E1 folding is dependent on E2 ,15, wher of mice .in vitro neutralization studies using cell culture infectious HCV (HCVcc), we found that sE21E also induced higher levels of heterologous (cross-genotype reactive) NAbs compared to sE2E1 and especially to sE1E2L in immunized mice. While sE21E did not induce higher levels of heterologous NAbs than sE2, it may be relevant in future HCV subunit vaccine designs to consider C-terminal fusions to sE2 as opposed to N-terminal fusion.Here, we aimed to devise a structurally improved HCV E1E2 protein antigen by generating and comparing three variants of backbone fused, soluble (i.e. transmembrane deleted) envelope protein: N-terminal fusion of sE1 to sE2 termed sE1E2 and C-terminal fusion of sE1 or s1E to sE2 termed sE2E1 and sE21E, respectively . sE21E ede novo (Genscript), including the reintroduction of four N-linked glycosylation sites by the substitution V43T and Serine insertion at position 4, 18 and 115 in the original sE1 sequence . These substitutions restored the canonical N-linked glycosylation site motif (N-X-S/T). The sequences were ligation cloned into a modified pCEP4 vector (ThermoFischer). All constructs that had sE1 at the N-terminus (sE1E2 backbone) had methionine followed by the 35 C-terminal residues of Core as a leader sequence, which includes a signal peptidase cleavage site to ensure proper protein processing. Similarly, all constructs that had sE2 at the N-terminus  had methionine followed by the 35 C-terminal residues of E1 as a leader sequence, which includes a signal peptidase cleavage site to ensure proper protein processing.HCV sequences used for making envelope protein expression plasmids were of the HCV isolate J6 , specifHEK 293T cells were plated at 8 x 10^5 cells per well in 6-well plates 24 h prior to transfection. Cells were transfected with plasmids encoding HCV envelope protein variants using lipofectamine2000 (Invitrogen) as described . Followi5 cells per well in 6-well plates and incubated overnight at 37°C and 5% CO2. Cells were transfected with plasmids encoding the expression of sE1E2L, sE2E1 and sE21E with and without HVR1 using lipofectamine2000 (Invitrogen), as per the manufacturer’s instructions. After 6 hours, medium was replaced with DMEM supplemented with 10% fetal calf serum and penicillin/streptomycin, before overnight incubation at 37°C, 5% CO2. 72 hours post transfection, cells supernatants were harvested, filtered and stored at -80°C.HEK 293-T cells were plated at 6 x 10HEK 293T cell supernatants were diluted in NativePAGE sample buffer with 5% G-250 sample additive (Invitrogen) and loaded in a NativePAGE 4–16% Bis-Tris gel (Invitrogen). Gel electrophoresis was carried out at 150V in Dark Blue Buffer  until the dye front migration reached 1/3 of the gel. Gel electrophoresis was continued in Light Blue Buffer  until protein migration reached the end of the gel. After gel electrophoresis, proteins were transferred to a PVDF membrane by western blotting at 25V for 1 hour. After transfer, the proteins were fixed by incubating the membrane with 8% acetic acid for 15 minutes. The membrane was rinsed with deionized water, air-dried and incubated with ethanol until dye was removed. The membrane was incubated in blocking buffer  for 30 minutes. The proteins were probed using E2-specific AR3A antibody [Cells supernatants were diluted in NuPAGE LDS sample buffer and NuPAGE reducing agent (Invitrogen), heated at 70°C for 10 minutes, and loaded in a NuPAGE 4–12% Bis-Tris Gels (Invitrogen). Gel electrophoresis was carried out at 200V for 50 minutes in MOPS SDS running buffer containing NuPAGE antioxidant (Invitrogen). After gel electrophoresis proteins were transferred to a PVDF membrane by western blotting at 30V for 1 hour. After transfer, the membrane was rinsed with deionized water and incubated in blocking buffer  for 30 minutes. The proteins were probed using E2-specific AP33 antibody  and HRP-2) using FreeStyleTM 293 Expression Medium (Gibco). The transfection reagent was prepared by diluting 100μg endotoxin free plasmid in OptiProTM SFM (Gibco) and subsequently mixing the plasmid with FreeStyle TM Max Reagent (Gibco) that had been diluted in OptiProTM SFM (Gibco). Following a 10 minutes incubation step the transfection reagent was added dropwise to HEK293-F cells (1.0x106 cells/ml). The cells were further incubated in suspension (as above) and proteins were expressed and secreted to the media over a course of 5–6 days. The supernatant was harvested by centrifuging the cells 4000xg for 10 minutes. The media was exchanged to PBS buffer  by dialyzing  Spectrum Labs). Purification was either done using Strep-Tactin column purification or by using Ion Metal Affinity Chromatography (IMAC). Following purification, imidazole or desthiobiotin was removed by dialysis (Spectrum Labs) against PBS buffer.Protein was expressed in HEK293-F or HEK293-EBNA1 cells. For purification purposes, all constructs included either a Tobacco etch virus cleavage sequence (TEV)-Twin-Streptavidin tag (LENLYFQSASWSHPQFEKGGGSGGGSGGGSWSHPQFEK) or a TEV-hexa-Histidine tag (LENLYFQSASHHHHHH) at the C-terminus. HEK293-F cells or HEK293-EBNA1 were cultured in suspension by rotation  were coated overnight using 80 μl/well of a 10 μg/ml PBS suspension of protein of interest and negative wells incubated with PBS only. All wells were blocked by a 1-hour incubation at room temperature using a 1% suspension of bovine serum albumin (Sigma) in PBS with 0.1% Tween20 . Mouse antibodies purified from immunized animals or human monoclonal antibodies AR2A, AR3A, CBH4G, CBH5, CBH7, HC84.26 or IGH520 against the HCV envelope proteins ,32–34 weL, E2E1, E21E or their corresponding constructs without HVR1 , per immunization. In a subsequent study, a total of 16 BALB/C mice were randomly allocated into two groups (n = 8), which received 5μg of either sE21E or sE2  per immunization. Besides gender and age, no other inclusion/exclusion criteria were set for the mice. No protocol for these experiments were registered elsewhere prior to the study. All study data is included in the manuscript and no confounders were controlled for.Mouse immunization studies were conducted in order to evaluate the efficacy of proteins expressed from the different HCV constructs as vaccine antigens. In all studies, 7–10 week old female BALB/c mice were immunized intramuscularly in the thigh at day 0, 21 and 42. Blood samples were taken at day 14, 35 and 56. In the first animal trial, used to screen for the optimal antigen, a total of 18 mice were randomly allocated into six groups (n = 3), which received a dose (4–10μg) of either E1E2In order to conduct the neutralization experiments, mouse antibodies were purified from cardiac bleed sera (day 56). Mouse antibodies were purified using either Amicon pro-affinity purification columns Protein G (Merck) or Polyprep chromatography columns (Bio-Rad) loaded with Gammabind Plus Sepharose , as per the manufacturer’s instructions. Briefly, the columns were washed with PBS and the mouse serum samples were filtered (0.2μm) and passed through columns multiple times for binding. The columns were washed as per the manufacturer’s instructions and mouse antibodies were eluted in weak acid (pH 2.5–2.7). pH was neutralized to a neutral pH using 1M Tris-HCl buffer. Eluted antibodies from the Amicon columns were buffer exchanged on the columns into PBS, concentrated using Vivaspin 6 columns (MWCO 50kDa) and the antibody concentrations were measured using Mouse IgG (Immunoglobulin G) Total Ready-Set-Go (Affymetrix) ELISA as per the manufacturer’s instructions. Eluted antibodies from the Polyprep columns were dialyzed  against PBS and concentrated using Vivaspin 6 columns, MWCO 50kDa, and the antibody concentrations were quantified using the IgG setting on a Nanodrop2000 (Thermo Scientific).3 Huh7.5 cells [ΔHVR1, J4 (genotype 1b), J6 (genotype 2a), J6ΔHVR1 or S52 (genotype 3a) [2. Cells were washed, and fresh medium was added prior to incubation for a total infection time of 48 hours before fixation. Following fixation, the cells were incubated with anti-mouse Fab fragments (Jackson Immuno Research) to block the Fc region of the mouse antibodies. Staining and counting of the number of FFUs were done as described [7x10.5 cells  were platype 3a) –39), corescribed ,41, withAll animal experiments were conducted in accordance with national Danish guidelines and were approved by the National Animal Experiments Inspectorate . Mice were housed in an AAALAC accredited facility in accordance with good animal practice as defined by FELASA. Mice were monitored for adverse effects throughout the study and finally euthanized by cervical dislocation.L, sE2E1L, and sE21EL expression plasmids.We generated plasmids for mammalian protein expression encoding three different permutations of transmembrane domain-deleted (e.g. solubilized ectodomains) HCV envelope proteins, sE1 and sE2 , of the L, sE2E1L, and sE21EL, which include the linker), we transfected HEK 293T cells and stained for correctly folded E2 protein using the HCV NAb, AR3A , the deletion of HVR1 decreased antibody binding. For sE2E1 and sE21E, in which HVR1 is located at the N-terminus, we observed very modest increases in AR3A binding  with each of the six antigens  tag) formulated in Freund’s incomplete adjuvant. Specifically, mice were immunized using a two-week interval prime-boost-boost regimen, and then sacrificed two weeks after the final immunization to obtain a final high-volume bleed. Total mouse Immunoglobulin G (IgG) antibodies were purified from the obtained sera and antibody concentrations were measured. The purified antibodies were initially tested in ELISA for binding to the respective antigens. Although variation was observed, we overall found similar, high levels of antigen-specific antibody responses  or only neutralized HVR1-deleted viruses (sE2E1ΔHVR1 and sE21EΔHVR1), indicating that they primarily targeted epitopes subject to high levels of HVR1-mediated protection. We concluded that sE21E was the most promising antigen for further study.We compared the ability of the three proteins sE1E2esponses , which wnd sE21E , they alnd sE21E . Despitend sE21E  and 0.97nd sE21E . HoweverH77ΔHVR1 . In addiH77ΔHVR1 . The HVRMost humoral vaccine approaches currently being pursued for HCV employ variants of sE2 to induce NAbs. Thus, we produced J6 sE2 as described above for sE21E. To compare the immunogenicity of sE21E with sE2, we immunized groups of BALB/c mice (n = 8) prime-boost-boost with each antigen. Both vaccines were formulated using AddaVax, which has a similar composition  as the clinically approved MF59 adjuvant. Two weeks after the final immunization, animals were sacrificed to obtain a final high-volume bleed. Mouse IgG were purified from the resultant sera and antibody content was quantified. Subsequently, we assessed the quality of the responses by using the mouse antibodies to perform dose-response neutralization against HCVcc of genotype 2a  and genotypes 1a (H77), 1b (J4) and 3a (S52) . On average, sE2 elicited a statistically significant ~3-fold  higher level of autologous NAbs compared with sE21E . HoweverL. Compared with sE2 we find that sE21E induced comparable levels of heterologous NAbs. Our data suggests that C-terminal fusion to sE2 of accessory proteins (in this study sE1 or s1E) is superior to N-terminal fusion. This has relevance for future efforts to develop E1E2 subunit vaccine antigens.The urgent need for developing an HCV vaccine that protects against chronicity hinges on uncovering ways to produce optimally folded HCV envelope proteins that adequately expose cross-genotype conserved NAb epitopes. It seems clear that E1 and E2 folding are mutually dependent in the context of native E1/E2 complexes. However, it has proven technically difficult to extract and purify such E1/E2 complexes and solubilization by TM truncation has, so far, not resulted in E1E2 envelope chimeras able to induce high levels of heterologous NAbs . Here, wThe most common approach to virus vaccine development has historically been whole inactivated or attenuated virus particles, and efforts are ongoing to develop whole particle vaccines for HCV. Studies have shown that mouse immunizations with inactivated particles lead to the induction of NAbs, but NAb titers in immunized animals have so far been modest ,43. In aithe peptide backbone is not symmetrical, which is particularly prominent for proline sidechain that are covalently attached to the backbone at two positions (J6 sE1 contains 9 proline residues); iiGiven that most amino acid sidechains are in the trans configuration, it is likely that even a complete native folding of s1E (compared with sE1) would result in the mirror image structure of sE1; and iiiN-linked glycosylation motifs, N-X-S/T, are directional in the primary sequence (J6 sE1 contains 4 N-linked glycosylation motifs). Most of these effects were not amenable to further manipulation, but we did reestablish N-linked glycosylation motifs by minimal site-directed mutagenesis of the 1E sequence.One known aspect of E1-E2 interactions is their heterodimeric association in the membrane, mediated through their respective TM regions –54. ConsTo our knowledge, there are no studies that involve the inversion of the protein backbone of an entire protein domain or, as done here, the entire ectodomain of a transmembrane protein. However, a number of experimental and modelling studies have suggested that inversion of short and long peptide amino acid backbones can result in folding, which resembles the fold of the original non-inverted peptides ,26,55. Tin vitro [in vivo [Research by us and others showed that the removal of the 27 N-terminal amino acids from E2  led to increased exposure of NAb epitopes on HCVcc both in vitro  and in v[in vivo . Interes[in vivo . However[in vivo . The exp[in vivo  and in f[in vivo ,61. Perf[in vivo ,13. Howe[in vivo , and couIn conclusion, we show that sE21E and sE2E1 are superior to sE1E2, irrespective of the inclusion of an intra-protein linker sequence. This was true both in terms of HCV NAb binding and the ability to induce cross-reactive HCV NAbs, although it should be pointed out that NAb responses will differ between rodents and humans. However, sE21E did not induce higher levels of heterologous NAbs compared with sE2. Thus, our data suggests that sE2 tolerates C-terminal fusion partners (here sE1 and s1E) better than N-terminal fusion partners, which has implications for future E1E2 subunit vaccine approaches.S1 Raw imagesPanels A and B refer to the same SDS PAGE western blot. Panels C and D refer to the same blue native PAGE western blot. A) Blot was exposed for a short duration to visualize marker bands while avoiding over-exposure. B) Blot was exposed for a longer time to better visualize the annotated HCV envelope protein bands. C) As no blue native PAGE western blot marker was available the protein bands from a colorimetric native PAGE marker were marked with a pencil on the blot following transfer, but before ethanol washing. The pencil marks are clear in the colorimetric picture of the blot (panel C) and can be made out as faint white markings in the chemiluminescence capture (panel D). D) folded HCV E2 protein was probed on the blot using the conformational antibody, AR3A.(PDF)Click here for additional data file.