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Abstract: Insulin mucoadhesive buccal films (MBF) are a noninvasive insulin delivery system that offers an advantageous alternative route of administration to subcutaneous injection. One major concern in the formulation of insulin MBF is the preservation of an insulin secondary structure in the presence of the other film components. Buccal films were formulated using chitosan, glycerin, and L-arginine. The MBF-forming solutions (MBF-FS) and the films (MBF) were examined for their chemical and structural stability and for their in vivo activity. Enzyme-Linked Immunosorbent Assay (ELISA) of the insulin-loaded MBF showed that each individualized unit dose was at least loaded with 80% of the insulin theoretical dose. Results of Synchrotron Radiation Circular Dichroism (SRCD) measurements revealed that MBF-FS retained the α-helices and β–sheets conformations of insulin. Fourier transform infrared (FTIR)-microspectroscopy (FTIR-MS) examination of insulin MBF revealed the protective action of L-arginine on insulin structure by interacting with chitosan and minimizing the formation of an unordered structure and β-strand. A blood glucose-lowering effect of insulin MBF was observed in comparison with subcutaneous (S.C) injection using a rat model. As a result; chitosan-based MBFs were formulated and characterized using SRCD and FTIR-MS techniques. Furthermore, the results of in vivo testing suggested the MBFs as a promising delivery system for insulin.

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Abstract: The first example of uniformly chiral thin films of silicon phthalocyanines (SiPcs) are reported. The local domains of the films are mapped using circular dichroism (CD) imaging (CDi) technique available at the Diamond B23 beamline. The CDi allowed us to increase the spatial resolution up to 525× when compared with benchtop spectrometers. The results indicate formation on-surface of chiral and stable supramolecular assemblies with homogenous distribution. Chemical functionalization and solvent choice for deposition allow controllable chiroptical properties to be obtained. The method and technique reported in this work could be applied to prepare and characterize a wide variety of chiral thin films.

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Abstract: Background: Human Exonuclease1 (hExo1) participates in the resection of DNA double-strand breaks by generating long 3′-single-stranded DNA overhangs, critical for homology-based DNA repair and activation of the ATR-dependent checkpoint. The C-terminal region is essential for modulating the activity of hExo1, containing numerous sites of post-translational modification and binding sites for partner proteins. Methods: Analytical Ultracentrifugation (AUC), Dynamic Light Scattering (DLS), Circular Dichroism (CD) spectroscopy and enzymatic assays. Results: AUC and DLS indicates the C-terminal region has a highly extended structure while CD suggest a tendency to adopt a novel left-handed β-sheet structure, together implying the C-terminus may exhibit a transient fluctuating structure that could play a role in binding partner proteins known to regulate the activity of hExo1. Interaction with 14–3-3 protein has a cooperative inhibitory effect upon DNA resection activity, which indicates an allosteric transition occurs upon binding partner proteins. Conclusions: This study has uncovered that hExo1 consist of a folded N-terminal nuclease domain and a highly extended C-terminal region which is known to interact with partner proteins that regulates the activity of hExo1. A positively cooperative mechanism of binding allows for stringent control of hExo1 activity. Such a transition would coordinate the control of hExo1 by hExo1 regulators and hence allow careful coordination of the process of DNA end resection.

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Abstract: Apolipoprotein A-I (ApoA-I) of high-density lipoprotein (HDL) is essential for the transportation of cholesterol between peripheral tissues and the liver. However, specific mutations in Apolipoprotein A-I (ApoA-I) of high-density lipoprotein (HDL) are responsible for a late-onset systemic amyloidosis, the pathological accumulation of protein fibrils in tissues and organs. Carriers of these mutations do not exhibit increased cardiovascular disease risk despite displaying reduced levels of ApoA-I/ HDL-cholesterol. To explain this paradox, we show that the HDL particle profile of patients carrying either L75P or L174S ApoA-I amyloidogenic variants a higher relative abundance of the 8.4 nm vs 9.6 nm particles, and that serum from patients, as well as reconstituted 8.4 and 9.6 nm HDL particles (rHDL), possess increased capacity to catalyze cholesterol efflux from macrophages. Synchrotron radiation circular dichroism and hydrogen-deuterium exchange revealed that the variants in 8.4 nm rHDL have altered secondary structure composition and display a more flexible binding to lipids compared to their native counterpart. The reduced HDL-cholesterol levels of patients carrying ApoA-I amyloidogenic variants are thus balanced by higher proportion of small, dense HDL particles and better cholesterol efflux due to altered, region-specific protein structure dynamics.

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Abstract: Acute myeloid leukemia (AML), a blood/bone marrow cancer, is a severe and often fatal malignancy. AML cells are capable of impairing the anti-cancer activities of cytotoxic lymphoid cells. This includes the inactivation of natural killer (NK) cells and killing of T lymphocytes. Here we report for the first time that V-domain Ig-containing suppressor of T cell activation (VISTA), a protein expressed by T cells, recognizes galectin-9 secreted by AML cells as a ligand. Importantly, we found that soluble VISTA released by AML cells enhances the effect of galectin-9, most likely by forming multiprotein complexes on the surface of T cells and possibly creating a molecular barrier. These events cause changes in the plasma membrane potential of T cells leading to activation of granzyme B inside cytotoxic T cells, resulting in apoptosis.