Stronger together: Analytical techniques for recombinant adeno associated virus.

With recent FDA approval of two recombinant adeno-associated virus (rAAV)-based gene therapies, these vectors have proven that they are suitable to address monogenic diseases. However, rAAVs are relatively new modalities, and their production and therapy costs significantly exceed those of conventional biologics. Thus, significant efforts are made to improve the processes, methods, and techniques used in manufacturing and quality control (QC). Here, we evaluate transmission electron microscopy (TEM), analytical ultracentrifugation (AUC), and two modes of capillary electrophoresis (CE) for their ability to analyze the DNA encapsidated by rAAVs. While TEM and AUC are well-established methods for rAAV, capillary gel electrophoresis (CGE) has been just recently proposed for viral genome sizing. The data presented reflect that samples are very complex, with various DNA species incorporated in the virus, including small fragments as well as DNA that is larger than the targeted transgene. CGE provides a good insight in the filling of rAAVs, but the workflow is tedious and the method is not applicable for the determination of DNA titer, since a procedure for the absolute quantification (e.g., calibration) is not yet established. For estimating the genome titer, we propose a simplified capillary zone electrophoresis approach with minimal sample preparation and short separation times (<5 min/run). Our data show the benefits of using the four techniques combined, since each of them alone is prone to delivering ambiguous results. For this reason, a clear view of the rAAV interior can only be provided by using several analytical methods simultaneously.

Magnetic field effect on ion pair dynamics upon bimolecular photoinduced electron transfer in solution.

The dynamics of the ion pairs produced upon fluorescence quenching of the electron donor 9,10-dimethylanthracene (DMeA) by phthalonitrile have been investigated in acetonitrile and tetrahydrofuran using transient absorption spectroscopy. Charge recombination to both the neutral ground state and the triplet excited state of DMeA is observed in both solvents. The relative efficiency of the triplet recombination pathway decreases substantially in the presence of an external magnetic field. These results were analyzed theoretically within the differential encounter theory, with the spin conversion of the geminate ion pairs described as a coherent process driven by the hyperfine interaction. The early temporal evolution of ion pair and triplet state populations with and without magnetic field could be well reproduced in acetonitrile, but not in tetrahydrofuran where fluorescence quenching involves the formation of an exciplex. A description of the spin conversion in terms of rates, i.e., incoherent spin transitions, leads to an overestimation of the magnetic field effect.

Ultrafast Photophysics of a Dinitrogen-Bridged Molybdenum Complex.

Among the many metal-dinitrogen complexes synthesized, the end-on bridging (µ2, η1, η1-N2) coordination mode is notoriously unreactive for nitrogen fixation. This is principally due to the large activation energy for ground-state nitrogen-element bond formation and motivates exploration of the photoexcited reactivity of this coordination mode. To provide the foundation for this concept, the photophysics of a dinitrogen-bridged molybdenum complex was explored by ultrafast electronic spectroscopies. The complex absorbs light from the UV to near-IR, and the transitions are predominantly of metal-to-ligand charge transfer (MLCT) character. Five excitation wavelengths (440, 520, 610, 730, and 1150 nm) were employed to access MLCT bands, and the dynamics were probed between 430 and 1600 nm. Despite the large energy space occupied by electronic states (ca. 1.2 eV), the dynamics were independent of the excitation wavelength. In the proposed kinetic model, photoexcitation from a Mo-N═N-Mo centered ground state populates the π*-state delocalized over two terpyridine ligands. Due to a large terpyridine-terpyridine spatial separation, electronic localization occurs within 100 fs, augmented by symmetry breaking. The subsequent interplay of internal conversion and intersystem crossing (ISC) populates the lowest 3MLCT state in 2-3 ps. Decay to the ground state occurs either directly or via a thermally activated metal-centered (3MC) trap state having two time constants (10-15 ps, 23-26 ps [298 K]; 103 ps, 612 ps [77 K]). ISC between 1MLCT and 3MLCT involves migration of energized electron density from the terpyridine π* orbitals to the Mo-N═N-Mo core. Implication of the observed dynamics for the potential N-H bond forming reactivity are discussed.

Looking at Photoinduced Charge Transfer Processes in the IR: Answers to Several Long-Standing Questions.

Because of its crucial role in many areas of science and technology, photoinduced electron transfer is the most investigated photochemical reaction. Despite this, several important questions remain open. We present recent efforts to answer some of them, which concern both inter- and intramolecular processes. The decisive factor that allowed these issues to be successfully addressed was the use of time-resolved infrared (TRIR) spectroscopy. Many different transient species, such as tight and loose ion pairs (TIPs and LIPs) and exciplexes, have been invoked to explain the dynamics of intermolecular photoinduced charge separation reactions (i.e., electron transfer between two neutral species) and the production of free ions. However, their structures are essentially unknown, and their exact roles in the reaction mechanism are unclear. Indeed, the commonly used transient electronic absorption spectroscopy does not give much structural insight and cannot clearly distinguish ion pairs from free ions, at least in the visible region. Unambiguous spectral signatures of TIPs, LIPs, and exciplexes could be observed in the IR using electron donor/acceptor (D/A) pairs with adequate vibrational marker modes. The ability to spectrally distinguish these intermediates allowed their dynamics to be disentangled and their roles to be determined. Structural information could be obtained using polarization-resolved TRIR spectroscopy. Our investigations reveal that moderately to highly exergonic reactions result in the formation of both TIPs and LIPs. TIPs are not only generated upon direct charge-transfer excitation of DA complexes, as usually assumed, but are also formed upon static quenching with reactant pairs at distances and orientations enabling charge separation without diffusion. On the other hand, dynamic quenching produces primarily LIPs. In the case of highly exergonic reactions, strong indirect evidence for the generation of ion pairs in an electronic excited state was found, accounting for the absence of an inverted region. Finally, weakly exergonic reactions produce predominantly exciplexes, which can evolve further into ion pairs or recombine to the neutral ground state. The high sensitivity of specific vibrational modes to the local electronic density was exploited to visualize the photoinduced charge flow in symmetric A-(π-D)2- and D-(π-A)2-type molecules developed for their two-photon absorption properties. The electronic ground state and Franck-Condon S1 state of these molecules are purely quadrupolar, but the strong solvatochromism of their fluorescence points to a highly dipolar relaxed S1 state. This has been explained in terms of excited-state symmetry breaking induced by solvent and/or structural fluctuations. However, real-time observation of this process was missing. Direct visualization of symmetry-breaking charge transfer was achieved using TRIR spectroscopy by monitoring vibrations localized in the two arms of these molecules. A transition from a purely quadrupolar state to a symmetry-broken state on the timescale of solvent relaxation could be clearly observed in polar solvents, indicating that symmetry breaking occurs primarily via solvent fluctuations. In the case of the D-(π-A)2 molecule, this breaking results in different basicities at the two A ends and consequently in different affinities for H-bonds, which in turn leads to the formation of an asymmetric tight H-bonded complex in highly protic solvents.

Charge Localization after Ultrafast Photoexcitation of a Rigid Perylene Perylenediimide Dyad Visualized by Transient Stark Effect.

The intramolecular charge-transfer (CT) dynamics of a rigid and strongly conjugated perylenediimide-bridge-perylene dyad (PDIPe) has been investigated in dichloromethane using ultrafast transient electronic absorption spectroscopy and quantum chemical calculations. The strong electronic coupling between the dyad units gives rise to a CT band. Its photoexcitation forms a delocalized CT state with well-preserved ion bands despite the strong coupling. In the dyad, the electronic transition dipole moment of the electron donor perylene is aligned along the axis of the electric field vector with respect to the CT species. This alignment makes the donor sensitive to the Stark effect and thus charge density fluctuations in the CT state. Charge localization on the picosecond time scale manifests as a time-dependent Stark shift in the visible region. Quantum chemical calculations reveal a twist around the acetylene bridging unit to be the responsible mechanism generating a partial to an almost complete CT state. An estimate of the electric field strength in the CT state yields approximately 25 MV/cm, which increases to around 31 MV/cm during charge localization. Furthermore, the calculations illustrate the complexity of electronic structure in this strongly delocalized superchromophore and reflect the complications in the interpretation of transient absorption results when compared to steady-state approaches such as spectroelectrochemistry and model chromophore experiments such as photoinduced bimolecular charge transfer.

Exciplex formation in bimolecular photoinduced electron-transfer investigated by ultrafast time-resolved infrared spectroscopy.

The dynamics of bimolecular photoinduced electron-transfer reactions has been investigated with three donor/acceptor (D/A) pairs in tetrahydrofuran (THF) and acetonitrile (ACN) using a combination of ultrafast spectroscopic techniques, including time-resolved infrared absorption. For the D/A pairs with the highest driving force of electron transfer, all transient spectroscopic features can be unambiguously assigned to the excited reactant and the ionic products. For the pair with the lowest driving force, three additional transient infrared bands, more intense in THF than in ACN, with a time dependence that differs from those of the other bands are observed. From their frequency and solvent dependence, these bands can be assigned to an exciplex. Moreover, polarization-resolved measurements point to a relatively well-defined mutual orientation of the constituents and to a slower reorientational time compared to those of the individual reactants. Thanks to the minimal overlap of the infrared signature of all transient species in THF, a detailed reaction scheme including the relevant kinetic and thermodynamic parameters could be deduced for this pair. This analysis reveals that the formation and recombination of the ion pair occur almost exclusively via the exciplex.

Real-time observation of the formation of excited radical ions in bimolecular photoinduced charge separation: absence of the Marcus inverted region explained.

Unambiguous evidence for the formation of excited ions upon ultrafast bimolecular photoinduced charge separation is found using a combination of femtosecond time-resolved fluorescence up-conversion, infrared and visible transient absorption spectroscopy. The reaction pathways are tracked by monitoring the vibrational energy redistribution in the product after charge separation and subsequent charge recombination. For moderately exergonic reactions, both donor and acceptor are found to be vibrationally hot, pointing to an even redistribution of the energy dissipated upon charge separation and recombination in both reaction partners. For highly exergonic reactions, the donor is very hot, whereas the acceptor is mostly cold. The asymmetric energy redistribution is due to the formation of the donor cation in an electronic excited state upon charge separation, confirming one of the hypotheses for the absence of the Marcus inverted region in photoinduced bimolecular charge separation processes.

Tetrathiafulvalene-benzothiadiazoles as redox-tunable donor-acceptor systems: synthesis and photophysical study.

Electrochemical and photophysical analysis of new donor-acceptor systems 2 and 3, in which a benzothiadiazole (BTD) unit is covalently linked to a tetrathiafulvalene (TTF) core, have verified that the lowest excited state can be ascribed to an intramolecular-charge-transfer (ICT) π(TTF)→π*(benzothiadiazole) transition. Owing to better overlap of the HOMO and LUMO in the fused scaffold of compound 3, the intensity of the (1)ICT band is substantially higher compared to that in compound 2. The corresponding CT fluorescence is also observed in both cases. The radical cation TTF(+·) is easily observed through chemical and electrochemical oxidation by performing steady-state absorption experiments. Interestingly, compound 2 is photo-oxidized under aerobic conditions.

Tetrathiafulvalene-1,3,5-triazines as (multi)donor-acceptor systems with tunable charge transfer: structural, photophysical, and theoretical investigations.

Palladium-catalyzed cross-coupling reactions between chlorinated 1,3,5-triazines (TZ) and tetrathiafulvalene (TTF) trimethyltin derivatives afford mono- and C3 symmetric tris(TTF)-triazines as donor-acceptor compounds in which the intramolecular charge transfer (ICT) is modulated by the substitution scheme on TTF and TZ and by chemical or electrochemical oxidation. The TTF-TZ-Cl2 and (SMe)2TTF-TZ-Cl2 derivatives show fully planar structures in the solid state as a consequence of the conjugation between the two units. Electrochemical and photophysical investigations, supported by theoretical calculations, clearly demonstrate that the lowest excited state can be ascribed to the intramolecular charge transfer (ICT) π(TTF)→π*(TZ) transition. The tris(TTF) compound [(SMe)2TTF]3-TZ shows fluorescence when excited in the ICT band, and the emission is quenched upon oxidation. The radical cations TTF(+•) are easily observed in all of the cases through chemical and electrochemical oxidation by steady-state absorption experiments. In the case of [(SMe)2TTF]3-TZ, a low energy band at 5000 cm(-1), corresponding to a coupling between TTF(+•) and TTF units, is observed. A crystalline radical cation salt with the TTF-TZ-Cl2 donor and PF6(-) anion, prepared by electrocrystallization, is described.

Ultrafast excited-state dynamics of donor-acceptor biaryls: comparison between pyridinium and pyrylium phenolates.

The excited-state dynamics of two donor-acceptor biaryls that differ by the strength of the acceptor, a pyridinium or a pyrylium moiety, have been investigated using a combination of steady-state solvatochromic absorption, ultrafast fluorescence, as well as visible and infrared transient absorption spectroscopies. The negative solvatochromic behavior of pyridinium phenolate indicates that the permanent electric dipole moment experiences a decrease upon S1 ← S0 excitation, implying that the ground state possesses more zwitterionic character than the excited state. In contrast, pyrylium phenolate exhibits a weakly positive solvatochromic behavior corresponding to a small increase in the dipole moment upon excitation, implying more zwitterionic character in the excited than the ground state. Both compounds are therefore situated at different sides of the cyanine-limit structure, which has equally polar ground and excited states. Despite these differences, both molecules exhibit qualitatively similar excited-state properties. They are characterized by a very short fluorescence lifetime, increasing from about 1 to 20 ps, when varying solvent viscosity from 0.4 to 11 cP. There are, however, characteristic differences between the two compounds: The excited-state lifetimes of the pyrylium dye are shorter and also depend somewhat on polarity. The ensemble of spectroscopic data can be explained with a model where the emitting Franck-Condon excited state relaxes upon twisting around the single bond between the aryl units to a point where the excited- and ground-state surfaces are very close or intersect. After internal conversion to the ground state, the distorted molecule relaxes back to its equilibrium planar configuration, again largely dependent upon solvent viscosity. However, in this case, the kinetics for the pyrylium dye are slower than for the pyridinium dye and the polar solvent-induced acceleration is significantly stronger than in the excited state. This difference of kinetic behavior between the two compounds is a direct consequence of the change of the electronic structure from a normal to an overcritical merocyanine evidenced by steady-state spectroscopy.

How to Make Outpatient Healthcare Data in Germany Available for Research in the Dynamic Course of Digital Transformation.

INTRODUCTION: There is increasing interest on re-use of outpatient healthcare data for research, as most medical diagnosis and treatment is provided in the ambulatory sector. One of the early projects to bring primary data from German ambulatory care into clinical research technically, organizationally and in compliance with legal demands has been the RADAR project, that is based on a broad consent and has used the then available practice information system's interfaces to extract and transfer data to a research repository. In course of the digital transformation of the German healthcare system, former standards are abandoned and new interoperability standards, interfaces and regulations on secondary use of patient data are defined, however with slow adoption by Health-IT systems. Therefore, it is of importance for all initiatives that aim at using ambulatory healthcare data for research, how to access this data in an efficient and effective way. METHODS: Currently defined healthcare standards are compared regarding coverage of data relevant for research as defined by the RADAR project. We compare four architectural options to access ambulatory health data through different components of healthcare and health research data infrastructures along the technical, organizational and regulatory conditions, the timetable of dissemination and the researcher's perspective. RESULTS: A high-level comparison showed a high degree of semantic overlap in the information models used. Electronic patient records and practice information systems are alternative data sources for ambulatory health data - but differ strongly in data richness and accessibility. CONCLUSION: Considering the compared dimensions of architectural routes to access health data for secondary research use we conclude that data extraction from practice information systems is currently the most promising way due to data availability on a mid-term perspective. Integration of routine data into the national research data infrastructures might be enforced by convergence of to date different information models.

Bimolecular photoinduced electron transfer in imidazolium-based room-temperature ionic liquids is not faster than in conventional solvents.

The fluorescence quenching of 3-cyanoperylene upon electron transfer from N,N-dimethylaniline in three room-temperature ionic liquids (RTILs) and in binary solvent mixtures of identical viscosity has been investigated using steady-state and time-resolved fluorescence spectroscopy. This study was stimulated by previous reports of bimolecular electron transfer reactions faster by one or several orders of magnitude in RTILs than in conventional polar solvents. These conclusions were usually based on a comparison with data obtained in low-viscous organic solvents and extrapolated to higher viscosities and not by performing experiments at similar viscosities as those of the RTILs, which we show to be essential. Our results reveal that (i) the diffusive motion of solutes in both types of solvents is comparable, (ii) the intrinsic electron transfer step is controlled by the solvent dynamics in both cases, being slower in the RTILs than in the conventional organic solvent of similar viscosity, and (iii) the previously reported reaction rates much larger than the diffusion limit at low quencher concentration in RTILs originate from a neglect of the static and transient stages of the quenching, which are dominant in solvents as viscous as RTILs.

Spurious observation of the Marcus inverted region in bimolecular photoinduced electron transfer.

The effect of viscosity on the bimolecular electron transfer quenching of a series of coumarins by N,N-dimethylaniline was investigated using steady-state and time-resolved fluorescence spectroscopy. The data reveal that the static and transient stages of the quenching become dominant as viscosity increases. When extracting the quenching rate constants using a simple Stern-Volmer analysis, a decrease of the rate constant with increasing driving force is observed above ~2 cP. However, this apparent Marcus inverted region, already reported several times with the same system in micelles and room temperature ionic liquids, totally vanishes when analyzing the data with a model accounting for the static and transient stages of the quenching. It appears that the apparent Marcus inverted region arises from the neglect of these quenching regimes together with the use of fluorophores with different excited-state lifetimes.

Identification and Classification of Off-Vertex Critical Points for Contour Tree Construction on Unstructured Meshes of Hexahedra.

The topology of isosurfaces changes at isovalues of critical points, making such points an important feature when building contour trees or Morse-Smale complexes. Hexahedral elements with linear interpolants can contain additional off-vertex critical points in element bodies and on element faces. Moreover, a point on the face of a hexahedron which is critical in the element-local context is not necessarily critical in the global context. Weber et al. (2002) introduce a method to determine whether critical points on faces are also critical in the global context, based on the gradient of the asymptotic decider (G. M. Nielson and B. Hamann) (1991) in each element that shares the face. However, as defined, the method of Weber et al. contains an error, and can lead to incorrect results. In this work we correct the error.

Photoinduced electron transfer reactions: from the elucidation of old problems in bulk solutions towards the exploration of interfaces.

The activities of our research group in the field of photoinduced electron transfer reactions are discussed and illustrated by several examples.

Salt Effect in Ion-Pair Dynamics after Bimolecular Photoinduced Electron Transfer in a Room-Temperature Ionic Liquid.

Bimolecular photoinduced electron transfer between perylene and two quenchers was investigated in an imidazolium room-temperature ionic liquid (RTIL) and in a dipolar solvent mixture of the same viscosity using transient absorption on the subpicosecond to submicrosecond time scales. Whereas charge separation dynamics were similar in both solvents, significant differences were observed in the temporal evolution of the ensuing radical ions: although small, the free-ion yield is significantly larger in the RTIL, and recombination of the ion pair to the triplet state of perylene is more efficient in the dipolar solvent. The temporal evolution of reactant, ion, and triplet state populations could be well reproduced using unified encounter theory. This analysis reveals that the observed differences can be explained by the strong screening of the Coulomb potential in the ion pair by the ionic solvent. In essence, RTILs favor free ions compared to highly dipolar solvents of the same viscosity.

Bimodal Exciplex Formation in Bimolecular Photoinduced Electron Transfer Revealed by Ultrafast Time-Resolved Infrared Absorption.

The dynamics of a moderately exergonic photoinduced charge separation has been investigated by ultrafast time-resolved infrared absorption with the dimethylanthracene/phthalonitrile donor/acceptor pair in solvents covering a broad range of polarity. A distinct spectral signature of an exciplex could be identified in the -C≡N stretching region. On the basis of quantum chemistry calculations, the 4-5 times larger width of this band compared to those of the ions and of the locally excited donor bands is explained by a dynamic distribution of exciplex geometry with different mutual orientations and distances of the constituents and, thus, with varying charge-transfer character. Although spectrally similar, two types of exciplexes could be distinguished by their dynamics: short-lived, "tight", exciplexes generated upon static quenching and longer-lived, "loose", exciplexes formed upon dynamic quenching in parallel with ion pairs. Tight exciplexes were observed in all solvents, except in the least polar diethyl ether where quenching is slower than diffusion. The product distribution of the dynamic quenching depends strongly on the solvent polarity: whereas no significant loose exciplex population could be detected in acetonitrile, both exciplex and ion pair are generated in less polar solvents, with the relative population of exciplex increasing with decreasing solvent polarity. These results are compared with those reported previously with donor/acceptor pairs in different driving force regimes to obtain a comprehensive picture of the role of the exciplexes in bimolecular photoinduced charge separation.