Chronic wounds (CW) are a worldwide concern, causing serious strives on the health and quality of patients’ life. In CW, human neutrophil elastase (HNE) enzyme gets highly expressed during inflammation, reaching abnormally elevated concentrations. Additionally, prevalence of Staphylococcus aureus-induced infections remains very high and difficult to treat. Considering these phenomena, a drug delivery system made of co-axial wet-spun fibers, loaded with the tetrapeptide Ala-Ala-Pro-Val (AAPV, a known inhibitor of HNE activity) and N-carboxymethyl chitosan (NCMC, responsive to neutral-basic pH’s, characteristic of CW and endowed with antibacterial features), was proposed.

AAPV was synthesized by solid-phase peptide synthesis, whereas NCMC was synthesized from low molecular weight chitosan in a chloroacetic acid mixture. HNE inhibition tests were conducted to establish the AAPV IC₅₀ in 1.50 µg/mL and the NCMC minimum bactericidal concentration (MBC) against S. aureus in 6.40 mg/mL. These determinations were used to establish fiber loading amounts. Core-shell structures were produced with 10% w/v polycaprolactone (PCL) at the core and 2% w/v sodium alginate (SA) solutions at the shell. NCMC was mixed with SA at 2xMBC so neutral-basic pH-triggered solubility (characteristic of CW) would allow pores to be opened in the outer layer for accessing the core, where AAPV was combined with PCL.

Fourier-transform infrared spectroscopy and brightfield microscopy were used to confirm the presence of the four components on the fibers and the co-axial architecture, respectively. Fibers presented maximum elongations of over 100%. Release kinetics studies conducted via UV-visible absorption spectroscopy mapped NCMC liberation overtime but were uncapable of detecting AAPV, since polymer degradation masked AAPV absorption peaks. Time-kill kinetics studies against S. aureus demonstrated the effectiveness of NCMC in eliminating this bacterium, particularly after 6 h of incubation. On its turn, AAPV guaranteed HNE inhibition. Data demonstrated the potential of SA-NCMC-PCL-AAPV co-axial systems to work as stepwise, pH-triggered delivery platforms.

The mango (Mangifera indica L.) is one of the most cultivated fruits in the world, however, its processing generates high amounts of waste, called agro-industrial by-products, among which the stone or seed, peel and bagasse stand out. It has been reported that these by-products contain significant amounts of nutrients and phytochemicals, mainly fiber and phenolic compounds, which represent a potential added value for the food industry, which has increased the interest in their study and applications by various authors. One of these booming applications is functional confectionery, which represents a vehicle for taking advantage of the benefits of these by-products. Therefore, the present work seeks to develop and evaluate the effect of a functional confectionery product (gummy) enriched with different concentrations of bagasse and mango peel on the texture profile characteristics (TPA), essential for sensory evaluation, selecting the mixture with greater similarity to the commercial product for its subsequent proximal characterization. As a result, for mixture 2, which contains a high pectin and bagasse content, added to a low shell content, with respect to the other mixtures, it presents a TPA profile similar to the control commercial gummy. The proximal composition showed 41% carbohydrates, of which 59% belongs to total dietary fiber, divided into 26% soluble and 33% insoluble. When performing the Pearson correlation of mixtures vs. the texture profile, the hardness parameter is negatively correlated with the type of mixture, which in turn is related to both the gumminess and the chewiness of the product. These results suggest that it is possible to incorporate these agro-industrial by-products into food and that it is comparable with that already marketed. In addition, with its high percentage of fiber, it could be considered a potentially prebiotic food.

A method is described for high-quality protein crystal solution growth with the help of localized action of a thermal control field. Two techniques for the nucleation and growth of single crystals of biological macromolecules have been proposed. The first one utilizes a very slow temperature shift at a capillary point where the crystal is to be grown. This allows to suppress an undesirable multiple nucleation. The second technique includes several local rapid temperature changes (a temperature “shock”) forcing the nucleation at the given point.

A mathematical model has been developed and computational investigation has been performed of the processes of protein crystallization from a homogeneous aqueous solution in the crystallization volume. The mathematical model describes crystal nucleation and growth depending on the local supersaturation and temperature as well as heat-and-mass exchange within the entire volume of the solution including the protein crystals.

The temperature was shown to be a factor, capable to initiate and drive the crystallization, and also to influence the nucleation stage and, hence, the protein crystal morphology. This may be useful for obtaining good quality single crystals of biological macromolecules. One or the other technique may be chosen. The stronger the dependence of protein solubility against the temperature, the better chances one has to succeed with any of the approaches, especially with the first one.

The mathematical model developed describes the process of nucleation and growth of protein crystals from solution under the control action of a thermal field based on an intermediate phase concept, the intermediate phase consisting of a mixture of solid and liquid phase fractions. This model was used to calculate an experiment on growing a protein crystal from a homogeneous aqueous solution, with the process of crystal nucleation and growth being acted upon by the precipitant and the thermal field. The calculations showed that this model is adequate to the processes being modeled and can be used for parametric investigations and predictive calculations of protein crystallization processes under thermal control field conditions both under terrestrial and space conditions.

These techniques were successfully tested while growing single crystals of lysozyme, xylanase and human serum albumin (HSA) respectively.

SALS (sarcomere length short), a WH2-domain-containing protein was identified in Drosophila as an important regulator of the assembly of sarcomeric actin structures (Bai et al.⁽¹⁾). It contributes to the establishment of sarcomere length and organization by promoting the lengthening of actin filaments at the pointed end. The absence of SALS is already lethal in the embryonic age. This may be due to the shortening of the length of sarcomeric actin filaments, and/or the disruption of their organization.

SALS is a relatively large protein, consisting of 935 amino acids. According to our bioinformatics analysis, it is an intrinsically disordered protein (IDP). IDPs are biologically active proteins, that, however, do not have a well-defined three-dimensional structure. They possess specific physicochemical properties, different from those of ordered proteins (e.g. hydrophilic/charged:hydrophobic amino acid ratio, thermal stability, electrophoretic mobility).

In the case of SALS previous studies have revealed only two motifs consisting of a few 10 amino acids, called Wiscott-Aldrich syndrome homology 2 (WH2) domains, that are intrinsically disordered protein regions (IDR) of low structural complexity. Considering their role, they possess actin-binding properties. Depending on the number and sequence of domains, proteins containing WH2 show multifunctional properties.

In our previous research we completed the functional analysis of the SALS WH2 domains (SALS-WH2) ⁽²⁾. Based on our results both of the SALS WH2 domains interact with the actin, and through their activities shift the monomer:filament ratio towards monomeric actin. We further aimed to characterize the structural and conformational dynamic properties of SALS-WH2 by using in silico and experimental approaches. Our bioinformatics analysis suggests that the SALS-WH2 domains have IDR elements. Our prediction-based results were experimentally verified by fluorescence spectroscopy and thermal analysis.

(1) Bai J, Hartwig JH, Perrimon N. SALS, a WH2-domain-containing protein, promotes sarcomeric actin filament elongation from pointed ends during Drosophila muscle growth. Dev Cell.2007 Dec;13(6):828-42.

(2) Tóth MÁ, Majoros AK, Vig AT, Migh E, Nyitrai M, Mihály J, Bugyi B. Biochemical Activities of the Wiskott-Aldrich Syndrome Homology Region 2 Domains of Sarcomere Length Short (SALS) Protein. J Biol Chem. 2016 Jan 8;291(2):667-80.

New National Excellence Program of the Ministry for Innovation and Technology ÚNKP-21-3-II-PTE-997 (PG), University of Pécs, Medical School, KA-2021-30 (AV). We thank József Mihály (Institute of Genetics, Biological Research Centre) for the SALS plasmid.

Papain-like cysteine proteases (PLCPs) are widely expressed enzymes, the main function of which is low-specific protein turnover in the acidic conditions of lysosomes. Additionally, these proteases provide specific functions in other compartments such as cytosol, nucleus, and extracellular space. The specificity of each protease to its substrates mainly depends on the patterns of the amino acids in the binding cleft. This specificity is highly regulated by media conditions and the presence of accessory proteins. In this study, we examined structural aspects ensuring pH-dependent substrate specificity of PLCPs. Experiments employing fluorogenic peptide substrates demonstrated that plant PLCPs and human cathepsins possess a pH-dependent specificity for the residue in the P1 position. X-ray crystallographic studies and molecular simulations allowed overall structure determination of the enzymes to predict residues in the S1 binding pocket which can form electrostatic contacts with the substrates. Sequence analysis established variability of these residues among PLCPs. Based on the obtained data we designed a peptide inhibitor for human cathepsin L and described its inhibitory potential. As a conclusion, we state that the S1 binding pocket defines specific pH-dependent recognition of substrates by PLCPs, ensuring multiple physiological functions of these proteases. This work was supported by the Russian Science Foundation (grant No. 22-25-00648).