One More for Light-triggered Conformational Changes in Cryptochromes: CryP from Phaeodactylum tricornutum.

Cryptochromes are a ubiquitously occurring class of photoreceptors. Together with photolyases, they form the Photolyase Cryptochrome Superfamily (PCSf) by sharing a common protein architecture and binding mode of the FAD chromophore. Despite these similarities, PCSf members exert different functions. Photolyases repair UV-induced DNA damage by photocatalytically driven electron transfer between FADH¯ and the DNA lesion, whereas cryptochromes are light-dependent signaling molecules and trigger various biological processes by photoconversion of their FAD redox and charge states. Given that most cryptochromes possess a C-terminal extension (CTE) of varying length, the functions of their CTE have not yet been fully elucidated and are hence highly debated. In this study, the role of the CTE was investigated for a novel subclass of the PCSf, the CryP-like cryptochromes, by hydrogen/deuterium exchange and mass-spectrometric analysis. Striking differences in the relative deuterium uptake were observed in different redox states of CryP from the diatom Phaeodactylum tricornutum. Based on these measurements we propose a model for light-triggered conformational changes in CryP-like cryptochromes that differs from other known cryptochrome families like the insect or plant cryptochromes.

Accurate quantification of DNA content in DNA hydrogels prepared by rolling circle amplification.

Accurate quantification of polymerized DNA in rolling circle amplification (RCA)-based hydrogels is challenging due to the high viscosity of these materials, however, it can be achieved with a photometric nucleotide depletion assay or qPCR. We show that the DNA content strongly depends on the template sequence and correlates with the mechanical properties of the hydrogels.

High-Load Gemcitabine Inorganic-Organic Hybrid Nanoparticles as an Image-Guided Tumor-Selective Drug-Delivery System to Treat Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) has a devastating prognosis without effective treatment options. Thus, there is an urgent need for more effective and safe therapies. Here, inorganic-organic hybrid nanoparticles (GMP-IOH-NPs) are presented as a novel drug-delivery system for the selective delivery of extraordinarily high concentrations of gemcitabine monophosphate (GMP), not only to the primary tumor but also to metastatic sites. GMP-IOH-NPs have a composition of [ZrO]2+ [GMP]2 - with GMP as drug anion (76% of total IOH-NP mass). Multiscale fluorescence imaging confirms an efficient uptake in tumor cells, independent of the activity of the human-equilibrative-nucleoside transporter (hENT1), being responsible for gemcitabine (GEM) transport into cells and a key factor for GEM resistance. Delivering already phosphorylated GMP via GMP-IOH-NPs into tumor cells also allows the cellular resistance induced by the downregulation of deoxycytidine kinase to be overcome. GMP-IOH-NPs show high accumulation in tumor lesions and only minor liver trapping when given intraperitoneally. GMP-IOH-NPs result in a higher antitumor efficacy compared to free GEM, which is further enhanced applying cetuximab-functionalized GMP-CTX-IOH-NPs. By maximizing the therapeutic benefits with high drug load, tumor-specific delivery, minimizing undesired side effects, overcoming mechanisms of chemoresistance, and preventing systemic GEM inactivation, GMP-IOH-NPs are anticipated to have a high chance to significantly improve current PDAC-patient outcome.

Hapten-Decorated DNA Nanostructures Decipher the Antigen-Mediated Spatial Organization of Antibodies Involved in Mast Cell Activation.

The immunological response of mast cells is controlled by the multivalent binding of antigens to immunoglobulin E (IgE) antibodies bound to the high-affinity receptor FcεRI on the cell membrane surface. However, the spatial organization of antigen-antibody-receptor complexes at the nanometer scale and the structural constraints involved in the initial events at the cell surface are not yet fully understood. For example, it is unclear what influence the affinity and nanoscale distance between the binding partners involved have on the activation of mast cells to degranulate inflammatory mediators from storage granules. We report the use of DNA origami nanostructures (DON) functionalized with different arrangements of the haptenic 2,4-dinitrophenyl (DNP) ligand to generate multivalent artificial antigens with full control over valency and nanoscale ligand architecture. To investigate the spatial requirements for mast cell activation, the DNP-DON complexes were initially used in surface plasmon resonance (SPR) analysis to study the binding kinetics of isolated IgE under physiological conditions. The most stable binding was observed in a narrow window of approximately 16 nm spacing between haptens. In contrast, affinity studies with FcεRI-linked IgE antibodies on the surface of rat basophilic leukemia cells (RBL-2H3) indicated virtually no distance-dependent variations in the binding of the differently structured DNP-DON complexes but suggested a supramolecular oligovalent nature of the interaction. Finally, the use of DNP-DON complexes for mast cell activation revealed that antigen-directed tight assembly of antibody-receptor complexes is the critical factor for triggering degranulation, even more critical than ligand valence. Our study emphasizes the significance of DNA nanostructures for the study of fundamental biological processes.

Trait mindfulness, self-efficacy, and coping strategies during COVID-19.

BACKGROUND: The worldwide COVID-19 pandemic may have negative impacts on individuals' mental health. At the same time, protective factors such as mindfulness, i.e., a moment-to-moment awareness of own experiences without judgment, may have positive effects on various psychological outcomes during the pandemic. OBJECTIVES: The current study analyzed the associations between trait mindfulness and psychological outcomes during the COVID-19 pandemic at three measurement points, testing self-efficacy and coping strategies as further potential predictors. DESIGN & METHODS: Altogether 207 students (85% female, mostly between 18 and 25 years old) participated in a longitudinal online survey from May to July 2020. At t1, t2, and t3, trait mindfulness, COVID-19-specific psychological well-being, depressiveness, anxiety, and stress were assessed. In addition, coping strategies were measured at t1 and self-efficacy at t2. RESULTS: Psychological outcomes at t1 were associated with trait mindfulness and coping strategies. The change in psychological outcomes between t2 and t3 was predicted by trait mindfulness and self-efficacy and to some extent by maladaptive coping. In a cross-lagged panel design, trait mindfulness was a better predictor of the psychological outcomes than vice versa. CONCLUSIONS: Our results support the value of trait mindfulness for psychological outcomes during the COVID-19 pandemic.

Structural and Functional Analysis of a Prokaryotic (6-4) Photolyase from the Aquatic Pathogen Vibrio Cholerae†.

Photolyases are flavoproteins, which are able to repair UV-induced DNA lesions in a light-dependent manner. According to their substrate, they can be distinguished as CPD- and (6-4) photolyases. While CPD-photolyases repair the predominantly occurring cyclobutane pyrimidine dimer lesion, (6-4) photolyases catalyze the repair of the less prominent (6-4) photoproduct. The subgroup of prokaryotic (6-4) photolyases/FeS-BCP is one of the most ancient types of flavoproteins in the ubiquitously occurring photolyase & cryptochrome superfamily (PCSf). In contrast to canonical photolyases, prokaryotic (6-4) photolyases possess a few particular characteristics, including a lumazine derivative as antenna chromophore besides the catalytically essential flavin adenine dinucleotide as well as an elongated linker region between the N-terminal α/ß-domain and the C-terminal all-α-helical domain. Furthermore, they can harbor an additional short subdomain, located at the C-terminus, with a binding site for a [4Fe-4S] cluster. So far, two crystal structures of prokaryotic (6-4) photolyases have been reported. Within this study, we present the high-resolution structure of the prokaryotic (6-4) photolyase from Vibrio cholerae and its spectroscopic characterization in terms of in vitro photoreduction and DNA-repair activity.

Orthogonal protein decoration of DNA nanostructures based on SpyCatcher-SpyTag interaction.

We present an efficient and readily applicable strategy for the covalent ligation of proteins to DNA origami by using the SpyCatcher-SpyTag (SC-ST) connector system. This approach showed orthogonality with other covalent connectors and has been used exemplarily for the immobilization and study of stereoselective ketoreductases to gain insight into the spatial arrangement of enzymes on DNA nanostructures.

A topologically distinct class of photolyases specific for UV lesions within single-stranded DNA.

Photolyases are ubiquitously occurring flavoproteins for catalyzing photo repair of UV-induced DNA damages. All photolyases described so far have a bilobal architecture with a C-terminal domain comprising flavin adenine dinucleotide (FAD) as catalytic cofactor and an N-terminal domain capable of harboring an additional antenna chromophore. Using sequence-similarity network analysis we discovered a novel subgroup of the photolyase/cryptochrome superfamily (PCSf), the NewPHLs. NewPHL occur in bacteria and have an inverted topology with an N-terminal catalytic domain and a C-terminal domain for sealing the FAD binding site from solvent access. By characterizing two NewPHL we show a photochemistry characteristic of other PCSf members as well as light-dependent repair of CPD lesions. Given their common specificity towards single-stranded DNA many bacterial species use NewPHL as a substitute for DASH-type photolyases. Given their simplified architecture and function we suggest that NewPHL are close to the evolutionary origin of the PCSf.