The bi-thiophene-vinylene-benzothiazole (bTVBT4) ligand developed for Alzheimer’s disease (AD)-specific detection of amyloid tau has been studied by a combination of several theoretical methods and experimental spectroscopies. With reference to the cryo-EM tau structure of the tau protofilament ( 28678775 Nature 2017 547 185 ), a periodic model system of the fibril was created, and the interactions between this fibril and bTVBT4 were studied with nonbiased molecular dynamics simulations. Several binding sites and binding modes were identified and analyzed, and the results for the most prevailing fibril site and ligand modes are presented. A key validation of the simulation work is provided by the favorable comparison of the theoretical and experimental absorption spectra of bTVBT4 in solution and bound to the protein. It is conclusively shown that the ligand–protein binding occurs at the hydrophobic pocket defined by the residues Ile360, Thr361, and His362. This binding site is not accessible in the Pick’s disease (PiD) fold, and fluorescence imaging of bTVBT4-stained brain tissue samples from patients diagnosed with AD and PiD provides strong support for the proposed tau binding site.

Anionic pentameric thiophene acetates can be used for fluorescence detection and diagnosis of protein amyloid aggregates. Replacing the central thiophene unit by benzothiadiazole (BTD) or quinoxaline (QX) leads to large emission shifts and basic spectral features have been reported [Chem. Eur. J. 2015, 21, 15133‐13137]. Here we present new detailed experimental results of solvent effects, time‐resolved fluorescence and examples employing multi‐photon microscopy and lifetime imaging. Quantum chemical response calculations elucidate how the introduction of the BTD/QX groups changes the electronic states and emissions. The dramatic red‐shift follows an increased conjugation and quinoid character of the π‐electrons of the thiophene backbone. An efficient charge transfer in the excited states S1 and S2 compared to the all‐thiophene analogue makes these more sensitive to the polarity and quenching by the solvent. Taken together, the results guide in the interpretation of images of stained Alzheimer disease brain sections employing advanced fluorescence microscopy and lifetime imaging, and can aid in optimizing future fluorescent ligand development.

The binding pocket proposed by König König Chem. Commun.20185430303033 for the biomarker pentameric formyl thiophene acetic acid (p-FTAA) in the fibrillar structure of amyloid-β(1–42) has been put to the test by the comparison of theoretical and experimental optical absorption and fluorescence spectra obtained in a water environment and inside the protein scaffold. The optical absorption/emission properties of this luminescent conjugated oligothiophene were studied by means of classical force field molecular dynamics simulations to account for the sampling of configuration space in conjunction with polarizable embedding time-dependent density functional theory calculations of spectra. The nuclear motions of residues in the β-sheet were found to be modest, and the time dependence of embedding parameters was shown to be negligible so that a time-independent parameter set could be derived and used for all 300 snapshots considered in the spectrum averaging. In regard to linear absorption spectra, the calculated red shift due to protein binding for the dominant S 1 ← S 0 transition in p-FTAA was found to be equal to 23 nm (0.17 eV), which is in excellent agreement with the corresponding experimental result of 18 nm and taken as corroborating evidence for having correctly identified the binding pocket of p-FTAA in the amyloid. The underlying mechanisms for the calculated red shift were disentangled, and it is shown that some 20 nm (0.15 eV) of the total 23 nm (0.17 eV) is associated with increased planarity of p-FTAA in the binding pocket, whereas a mere 3 nm (0.02 eV) is associated with changes in the environment. In regard to emission spectra, we demonstrate that intersystem crossing from the excited S 1 state to the triplet manifold of states is a less likely event for p-FTAA in the binding pocket as compared to in the aqueous solution, and we thereby partly explain the much higher quantum yield of fluorescence for the more rigid p-FTAA in the binding pocket. Two-photon absorption in p-FTAA is shown to predominantly occur to an overall symmetric excited state and be more than twice as strong for the biomarker in the binding pocket as compared to in water. The corresponding red shift, on the other hand, is very small. Earlier experimental two-photon fluorescence imaging using p-FTAA is shown not to target the dominant two-photon state, and ways to reach a higher image quality (lower signal-to-noise ratio) are proposed in terms of tuning the laser wavelength toward the region of 600 nm or the synthesis of asymmetric ligands with S 1 states that are both one- and two-photon allowed.

Objectives To investigate whether hospitalised dogs treated surgically may become culture positive for methicillin‐resistant Staphylococcus pseudintermedius or methicillin‐resistant Staphylococcus aureus. Methods Surgically treated dogs (n=45) were sampled for methicillin‐resistant Staphylococcus pseudintermedius or methicillin‐resistant Staphylococcus aureus on admission, before and after surgery and at the time of removal of surgical stitches. The hospital environment (n=57), including healthy dogs in the veterinary hospital environment (n=34), were sampled for methicillin‐resistant Staphylococcus pseudintermedius or methicillin‐resistant Staphylococcus aureus. Genetic variations among methicillin‐resistant Staphylococcus pseudintermedius or methicillin‐resistant Staphylococcus aureus isolates were identified through detection of restriction fragment polymorphisms. Results No dogs developed a wound infection due to methicillin‐resistant Staphylococcus pseudintermedius or methicillin‐resistant Staphylococcus aureus. However, there was a significant increase in the number of dogs carrying methicillin‐resistant Staphylococcus pseudintermedius after hospitalisation compared to admission (P<0·001). No methicillin‐resistant Staphylococcus aureus was isolated from dogs, but was present in the environment. Methicillin‐resistant Staphylococcus pseudintermedius isolates were recovered from environmental surfaces and hospitalised animals, but not from healthy dogs. Methicillin‐resistant Staphylococcus pseudintermedius isolates representing nine different restriction endonuclease digestion patterns were found, with two of these occurring in both the environment and on dogs. Clinical Significance Dogs may contract methicillin‐resistant Staphylococcus pseudintermedius in association with surgery and hospitalisation. Resistant bacteria may be transmitted between dogs, staff and the environment. Dogs colonised with methicillin‐resistant Staphylococcus pseudintermedius may be a source for hospital‐ and community‐acquired infections.

Squalene–hopene cyclase catalyzes the cyclization of squalene to hopanoids. A previous study has identified a network of tunnels in the protein, where water molecules have been indicated to move. Blocking these tunnels by site-directed mutagenesis was found to change the activation entropy of the catalytic reaction from positive to negative with a concomitant lowering of the activation enthalpy. As a consequence, some variants are faster and others are slower than the wild type (wt) in vitro under optimal reaction conditions for the wt. In this study, molecular dynamics (MD) simulations have been performed for the wt and the variants to investigate how the mutations affect the protein structure and the water flow in the enzyme, hypothetically influencing the activation parameters. Interestingly, the tunnel-obstructing variants are associated with an increased flow of water in the active site, particularly close to the catalytic residue Asp376. MD simulations with the substrate present in the active site indicate that the distance for the rate-determining proton transfer between Asp376 and the substrate is longer in the tunnel-obstructing protein variants than in the wt. On the basis of the previous experimental results and the current MD results, we propose that the tunnel-obstructing variants, at least partly, could operate by a different catalytic mechanism, where the proton transfer may have contributions from a Grotthuss-like mechanism.