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Abstract: The development of molecules that interact with G-quadruplex (G4) sequences requires effective evaluation methods. Several techniques are currently available, including nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and mass spectrometry (MS), fluorescence using FRET-melting, G4-fluorescent intercalator displacement assay (G4-FID) and affinity chromatography. Among these, CD spectroscopy is gaining prominence due to its lower material requirements, faster experimentation and quicker data processing. However, conventional CD methods have limitations, such as higher sample volume required and the inability to handle high-throughput analysis efficiently. The use of synchrotron radiation in high-throughput analysis methods (HT-SRCD) has further advanced the investigation of small-molecule interactions with DNA G4 structures in the presence of various monovalent cations. HT-SRCD offers the capability to analyze multiple samples simultaneously, overcoming the limitations of conventional CD methods. To validate this approach, three biologically relevant G4 sequences—HTelo1, G3T3 and T95-2T—were investigated. Their interactions with a library of small tetrazole-based molecules, synthesized via a four-component Ugi reaction, and with a peptide sequence deriving from RHAU helicases (Rhau25), were evaluated. The results demonstrate that this method not only effectively discriminates between different ligands but also provides valuable insights into the selectivity and the modes of interaction of these ligands with the G4 sequences.

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Abstract: The interaction between lipids and proteins impacts on a multitude of cellular processes and may contribute to the onset of several pathologies and ageing. Such processes are frequently linked to oxidative stress, whereby polyunsaturated fatty acids act as substrates for in vivo lipoxidation. The subsequent lipid peroxidation and/or isomerisation is known to affect membrane organization, as well as to modify proteins and DNA, leading to functional alterations. Aim of this study was to evaluate the capacity of UV denaturation experiments to induce lipid modification and to investigate the influence of lipid presence on the conformational stability of selected soluble model proteins. To this end, the UV-denaturation experiment developed at the B23 beamline of the Diamond Light Source (UK) is employed, which high photon flux and brilliance of the incident beamlight induce protein denaturation when repeated consecutive synchrotron radiation circular dichroism spectra are acquired in the far-UV region, diagnostic of protein folding. This allows the estimation of protein photostability. Our findings show that the presence of lipid vesicles (SUVs) significantly impacts the UV-denaturation of proteins, preserving the native structure in proteins with a high helical content. This suggests that lipids may play a protective role against light-induced damage to proteins.

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Abstract: V-domain Ig-containing suppressor of T cell activation (VISTA) is a unique immune checkpoint protein, which was reported to display both receptor and ligand activities. However, the mechanisms of regulation of VISTA activity and functions by factors of tumour microenvironment (TME) remain unclear and understanding these processes is required in order to develop successful personalised cancer immunotherapeutic strategies and approaches. Here we report for the very first time that VISTA interacts with another immune checkpoint protein galectin-9 inside the cell most likely facilitating its interaction with TGF-β-activated kinase 1 (TAK1). This process is required for protection of lysosomes, which is crucial for many cell types and tissues. We found that VISTA expression can be differentially controlled by crucial factors present in TME, such as transforming growth factor beta type 1 (TGF-β) and hypoxia as well as other factors activating hypoxic signalling. We confirmed that involvement of these important pathways modulated by TME differentially influences VISTA expression in different cell types. These networks include: TGF-β-Smad3 pathway, TAK1 (TGF-β-activated kinase 1) or apoptosis signal-regulating kinase 1 (ASK1)-induced activation of activating transcription factor 2 (ATF-2) and hypoxic signalling pathway. Based on this work we determined the five critical functions of VISTA and the role of TME factors in controlling (modulating or downregulating) VISTA expression.

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Abstract: INTRODUCTION: The creation of mesoscale chiroptic materials from nanoscale achiral building blocks has been previously realized through exciton coupling in organic supramolecular assemblies and with plasmons in inorganic metal nanoparticle assemblies, but extending excitonic chirality to inorganic semiconducting systems has remained elusive. Among the many challenges to achieving this goal is the need for degenerate excited electronic states, which are difficult to obtain in nanocrystals with nonzero size distributions, and the requirement for aligned transition dipoles, an unlikely characteristic in spherical nanocrystals that lack a distinct axis. This goal is consequential, as introducing chirality into the band structure of semiconductors enables simultaneous control over light, spin, and charge, key capabilities for driving innovations in next-generation photonic, optoelectronic, and spintronic technologies. RATIONALE: We hypothesized that magic-sized nanoclusters could overcome these obstacles to form an exciton-coupled chiroptic inorganic assembly. Magic-sized nanoclusters are atomically and electronically identical, enabling greater wavefunction overlap and coupling between neighboring nanoclusters. And they need not be spherical, allowing for anisotropic arrangements of their transition dipoles through dipole-dipole interactions during solution processing. Additionally, the bandgap, composition, and size of semiconducting magic-sized clusters can be rationally synthesized to tailor the chiroptic responses over a range of wavelengths. RESULTS: Following our hypothesis, we found that three different species of nanoclusters [cadmium sulfide (CdS), cadmium selenide (CdSe), and cadmium telluride (CdTe)] could be assembled through meniscus-guided evaporative processing into films with strong circular dichroic (CD) responses. Films made from CdS achieved dissymmetry factors, the CD figure of merit, as large as 1.30 for unpatterned, drop-cast films and 1.06 for patterned assemblies, values that approach the theoretical maximum. CD maps collected with Mueller matrix polarimetry showed that controlling the evaporative processing geometry enabled the creation of millimeter-scale homochiral domains that continuously transition between left- and right-handed, with their spatial organization adjustable through processing parameters. The mechanism behind these organized assemblies is elucidated through linear dichroism (LD), small-angle x-ray scattering (SAXS), and in situ microscopy, which reveal that the meniscus guides highly concentrated fibrous solutions, aligning their transition dipoles within fibrous bands as they deposit on a substrate. These bands are subsequently twisted by fluid flows, generating homochiral domains. CONCLUSION: We report on a method to form chiral films of three different inorganic semiconductor nanocrystals, with near-limit dissymmetry factors and large homochiral domains. Our results for the three different semiconducting nanocluster systems demonstrate the generality of the method, suggesting the applicability to other nanocluster species or colloidal nanoplatelets. Our experiments uncovered the key mechanisms for achieving these chiroptic films by the meniscus-guided deposition process, including the alignment of transition dipoles of the constituents and the fluid flows responsible for twisting the fibers, and make a connection to emergent chiral properties. Harnessing methods to form chiral films from achiral, solution-processable semiconductors offers an opportunity in the design and fabrication of complex chiroptical metamaterials in ways that are both scalable and versatile. Beyond nanocluster films, this study provides valuable insights into the complexities of hierarchical assembly found in nature and offers a pathway to extend these principles to other chiral molecules and nanomaterials for engineering sophisticated, twisted structures.

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Abstract: Circularly polarized luminescence (CPL) in purely organic materials is limited by the almost exclusively electric nature of electronic transitions. Resolving this problem is possible through structuring organic materials at dimensions comparable to the wavelength of visible light. Here, the study explores the use of thin films made of chiral organic nanotubes for the enhanced induction of CPL. The study first performs multi-scale modeling of the chiroptical properties of organic nanotubes using the T-matrix method. Combined with chiroptical measurements, including Mueller matrix polarimetry, the study discusses the chiroptical properties of organic materials within the frames of their multi-scale structuring. When embedding aggregation-induced fluorogens (AIEgens) into the structured films, composites featured with gigantic glum factors reaching ≈10−1 are obtained. Importantly, a series of control experiments is performed to exclude common parasitic effects that can lead to the apparent CPL signals. The enhanced chiroptical properties of the composite films of organic nanotubes and AIEgens enable visual discrimination of their handedness both in the absorption and emission realms. The uncovered multi-mode enhancement of CPL directs future endeavors for anticounterfeiting or holography applications.