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Abstract: It is common to switch between H2O and D2O when examining peptide-based systems with the assumption being that there are no effects from this change. Here, we describe the effect of changing from H2O to D2O in a number of low molecular weight dipeptide-based gels. Gels are formed by decreasing the pH. In most cases, there is little dif-ference in the structures formed at high pH, but this is not universally true. On lowering the pH, the ki-netics of gelation are affected and, in some cases, the structures underpinning the gel network are dif-ferent. Where there are differences in the self-assembled structures, the resulting gel properties are different. We therefore show isotopic control over gel properties is possible.

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Abstract: Multicopper oxidases oxidize a vast range of aromatic substrates coupled to the reduction of molecular oxygen to water. A vast broad spectrum of applications reflects their high biotechnological importance. The crystal structure of McoA from the hyperthermophilic bacteria Aquifex aeolicus has the most tightly compact and hydrophobic core among its prokaryotic counterparts. A 29-residue long loop enriched in glycines and methionines (Met-loop) close to the active T1 Cu center is not detected in the electron density maps. Accurate prediction of loop structures remains challenging, especially for long segments with sizable conformational space. Therefore, a combination of Rosetta and molecular dynamics simulations with ensemble-based small-angle X-ray scattering analysis was used to probe the conformational landscape of the Met-loop. The results indicate a highly flexible omega-loop, which is nevertheless not random but preferentially follows open-to-close transitions, exposing or occluding the T1 Cu site. Loop-truncated variants maintain wild-type stability and consistently lower and higher catalytic efficiencies (kcat/Km) for organic and metal substrates, respectively. Our results suggest that the loop transient dynamic equilibrium can exert important switch-like regulatory function, defining a role for Met-rich motifs as dynamic gate-gappers. This work provides insights into the dynamics of Met-rich loops essential to understand the molecular determinants of substrate promiscuity and catalytic rates within multicopper oxidases. We anticipate that engineering the Met-loop structural dynamics will unleash important changes in enzyme function and specificity with impact on their applications.

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Abstract: Ethosome represents a smart transdermal vehicle suitable for solubilization and cutaneous application of drugs. Coenzyme Q10 is an endogenous antioxidant whose supplementation can counteract many cutaneous disorders and pathologies. In this respect, the present study describes the production, characterization, and cutaneous protection of phosphatidylcholine based ethosomes as percutaneous delivery systems for coenzyme Q10. CoQ10 entrapment capacity in ethosomes was almost 100%, vesicles showed the typical ‘fingerprint’ structure, while mean diameters were around 270 nm, undergoing an 8% increase after 3 months from production. An ex-vivo study, conducted by transmission electron microscopy, could detect the uptake of ethosomes in human skin fibroblasts and the passage of the vesicles through 3D reconstituted human epidermis. Immunofluorescence analyses were carried on both on fibroblasts and 3D reconstituted human epidermis treated with ethosomes in the presence of H2O2 as oxidative stress challenger, evaluating 4-hydroxynonenal protein adducts which is as a reliable biomarker for oxidative damage. Notably, the pretreatment with CoQ10 loaded in ethosomes exerted a consistent protective effect against oxidative stress, in both models, fibroblasts and in reconstituted human epidermis respectively.

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Abstract: Mislocalisation, cleavage and aggregation of the human protein TDP-43 is found in many neurodegenerative diseases. As is the case with many other proteins that are completely or partially structurally disordered, production of full-length recombinant TDP-43 in the quantities necessary for structural characterisation has proven difficult. We show that the full-length TDP-43 protein and two truncated N-terminal constructs 1-270 and 1-263 can be heterologously expressed in E. coli. Full-length TDP-43 could be prevented from aggregation during purification using a detergent. Crystals grown from an N-terminal construct (1-270) revealed only the NTD domain (residues 1-80) with molecules arranged as parallel spirals with neighbouring molecules arranged in head-to-tail fashion. In order to obtain detergent free, full-length TDP-43 we mutated all six tryptophan residues that provided low-resolution structure of full-length TDP-43 by small angle X-ray scattering and refining the relative positions of individual domains and intrinsically disordered regions in the model using molecular dynamics.

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Abstract: Anti-CD40 monoclonal antibodies (mAbs) comprise agonists and antagonists, which display promising therapeutic activities in cancer and autoimmunity, respectively. We previously showed that epitope and isotype interact to deliver optimal agonistic anti-CD40 mAbs. The impact of Fc engineering on antagonists, however, remains largely unexplored. Here, we show that clinically relevant antagonists used for treating autoimmune conditions can be converted into potent FcγR-independent agonists with remarkable antitumor activity by isotype switching to hIgG2. One antagonist is converted to a super-agonist with greater potency than previously reported highly agonistic anti-CD40 mAbs. Such conversion is dependent on the unique disulfide bonding properties of the hIgG2 hinge. This investigation highlights the transformative capacity of the hIgG2 isotype for converting antagonists to agonists to treat cancer.