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The complex of B- and T-lymphocyte attenuator (BTLA) and herpes virus entry mediator (HVEM) plays a critical role in immune regulation and has emerged as a promising therapeutic target for cancer treatment. In this study, we investigated the potential of the peptide inhibitor HVEM(14-39) to restore peripheral T cell activity in patients with advanced melanoma. In these patients, CD8+ T cells downregulated BTLA expression and increased HVEM expression upon activation. The addition of HVEM(14-39) reduced the percentage of BTLA+ CD8+ T cells and increased the subpopulation of HVEM+ CD8+ T cells. Additionally, HVEM(14-39) enhanced T cell activation, proliferation, and the shift toward effector memory T cell subpopulations. Finally, this peptide affected the proliferation rate and late apoptosis of melanoma cell line in co-culture with T cells. These findings suggest that HVEM(14-39) can overcome T cell exhaustion and improve antitumor responses. Peptide-based immunotherapy targeting the BTLA-HVEM complex offers a promising alternative to monoclonal antibody-based therapies, with the potential for fewer side effects and higher treatment efficacy., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The PD-1/PD-L1 complex belongs to the group of inhibitory immune checkpoints and plays a critical role in immune regulation. The PD-1/PD-L1 axis is also responsible for immune evasion of cancer cells, and this complex is one of the main targets of immunotherapies used in oncology. Treatment using immune checkpoint inhibitors is mainly based on antibodies. This approach has great therapeutic potential; however, it also has major drawbacks and can induce immune-related adverse events. Thus, there is a strong need for alternative, non-antibody-based therapies using small molecules, peptides, or peptidomimetics. In the present study, we designed, synthesized, and evaluated a set of PD-1-targeting peptides based on the sequence and structure of PD-L1. The binding of these peptides to PD-1 was investigated using SPR and ELISA. We also assessed their ability to compete with PD-L1 for binding to PD-1 and their inhibitory properties against the PD-1/PD-L1 complex at the cellular level. The best results were obtained for the peptide PD-L1(111-127) (Y112C-I126C) , named (L11), which displaced PD-L1 from binding to PD-1 in the competitive assay and inhibited the formation of the PD-1/PD-L1 complex. The (L11) peptide also exhibited strong affinity for PD-1. NMR studies revealed that (L11) does not form a well-defined secondary structure; however, MD simulation indicated that (L11) binds to PD-1 at the same place as PD-L1. After further optimization of the structure, the peptide inhibitor obtained in this study could also be used as a potential therapeutic compound targeting the PD-1/PD-L1 axis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Herpes simplex virus 1 (HSV-1) is a well-studied herpesvirus that causes various human diseases. Like other herpesviruses, HSV-1 produces the transmembrane glycoprotein N (gN/UL49.5 protein), which has been extensively studied, but its function in HSV-1 remains largely unknown. The amino-acid sequences and lengths of UL49.5 proteins differ between herpesvirus species. It is, therefore, crucial to determine whether and to what extent the spatial structure of UL49.5 orthologs that are transporter associated with antigen processing (TAP) inhibitors (i.e., of bovine herpesvirus 1; BoHV-1) differ from that of non-TAP inhibitors (i.e., of HSV-1). Our study aimed to examine the 3D structure of the HSV-1-encoded UL49.5 protein in an advanced model of the endoplasmic reticulum (ER) membrane using circular dichroism, 2D nuclear magnetic resonance, and multiple-microsecond all-atom molecular dynamics simulations in an ER membrane mimetic environment. According to our findings, the N-terminus of the HSV-1-encoded UL49.5 adopts a highly flexible, unordered structure in the extracellular part due to the presence of a large number of proline and glycine residues. In contrast to the BoHV-1-encoded homolog, the transmembrane region of the HSV-1-encoded UL49.5 is formed by a single long transmembrane α-helix, rather than two helices oriented perpendicularly, while the cytoplasmic part of the protein (C-terminus) has a short unordered structure. Our findings provide valuable experimental structural information on the HSV-1-encoded UL49.5 protein and offer, based on the obtained structure, insight into its lack of biological activity in inhibiting the TAP-dependent antigen presentation pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Metallic materials for long-term load-bearing implants still do not provide high antimicrobial activity while maintaining strong compatibility with bone cells. This study aimed to modify the surface of Ti13Nb13Zr alloy by electrophoretic deposition of a chitosan coating with a covalently attached Arg-Gly-Asp (RGD) peptide. The suspensions for coating deposition were prepared in two different ways either using hydroxyacetic acid or a carbon dioxide saturation process. The coatings were deposited using a voltage of 10 V for 1 min. The prepared coatings were examined using SEM, EDS, FTIR, and XPS techniques. In addition, the wettability of these surfaces, corrosion resistance, adhesion of the coatings to the metallic substrate, and their antimicrobial activity (E. coli, S. aureus) and cytocompatibility properties using the MTT and LDH assays were studied. The coatings produced tightly covered the metallic substrate. Spectroscopic studies confirmed that the peptide did not detach from the chitosan chain during electrophoretic deposition. All tested samples showed high corrosion resistance (corrosion current density measured in nA/cm 2 ). The deposited coatings contributed to a significant increase in the antimicrobial activity of the samples against Gram-positive and Gram-negative bacteria (reduction in bacterial counts from 99% to, for CS-RGD-Acid and the S. aureus strain, total killing capacity). MTT and LDH results showed high compatibility with bone cells of the modified surfaces compared to the bare substrate (survival rates above 75% under indirect contact conditions and above 100% under direct contact conditions). However, the adhesion of the coatings was considered weak., (© 2023 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials published by Wiley Periodicals LLC.)

Immune checkpoints can be divided into co-stimulatory and co-inhibitory molecules that regulate the activation and effector functions of T cells. The co-inhibitory pathways mediated by ICPs are used by cancer cells to escape from immune surveillance, and therefore the blockade of these receptor/ligand interactions is one of the strategies used in the treatment of cancer. The two main pathways currently under investigation are CTLA-4/CD80/CD86 and PD-1/PD-L1, and the monoclonal Abs targeting them have shown potent immunomodulatory effects and activity in clinical environments. Another interesting target in cancer treatment is the BTLA/HVEM complex. Binding of BTLA protein on T cells to HVEM on cancer cells leads to inhibition of T cell proliferation and cytokine production. In the presented work, we focused on blocking the HVEM protein using BTLA-derived peptides. Based on the crystal structure of the BTLA/HVEM complex and MM/GBSA analysis performed here, we designed and synthesized peptides, specifically fragments of BTLA protein. We subsequently checked the inhibitory capacities of these compounds using ELISA and a cellular reporter platform. Two of these peptides, namely BTLA(35-43) and BTLA(33-64) C58Abu displayed the most promising properties, and we therefore performed further studies to evaluate their affinity to HVEM protein, their stability in plasma and their effect on viability of human PBMCs. In addition, the 3D structure for the peptide BTLA(33-64) C58Abu was determined using NMR. Obtained data confirmed that the BTLA-derived peptides could be the basis for future drugs and their immunomodulatory potential merits further examination., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Self-assembling peptides can be used for the regeneration of severely damaged skin. They can act as scaffolds for skin cells and as a reservoir of active compounds, to accelerate scarless wound healing. To overcome repeated administration of peptides which accelerate healing, we report development of three new peptide biomaterials based on the RADA16-I hydrogel functionalized with a sequence (AAPV) cleaved by human neutrophil elastase and short biologically active peptide motifs, namely GHK, KGHK and RDKVYR. The peptide hybrids were investigated for their structural aspects using circular dichroism, thioflavin T assay, transmission electron microscopy, and atomic force microscopy, as well as their rheological properties and stability in different fluids such as water or plasma, and their susceptibility to digestion by enzymes present in the wound environment. In addition, the morphology of the RADA-peptide hydrogels was examined with a unique technique called scanning electron cryomicroscopy. These experiments enabled us to verify if the designed peptides increased the bioactivity of the gel without disturbing its gelling processes. We demonstrate that the physicochemical properties of the designed hybrids were similar to those of the original RADA16-I. The materials behaved as expected, leaving the active motif free when treated with elastase. XTT and LDH tests on fibroblasts and keratinocytes were performed to assess the cytotoxicity of the RADA16-I hybrids, while the viability of cells treated with RADA16-I hybrids was evaluated in a model of human dermal fibroblasts. The hybrid peptides revealed no cytotoxicity; the cells grew and proliferated better than after treatment with RADA16-I alone. Improved wound healing following topical delivery of RADA-GHK and RADA-KGHK was demonstrated using a model of dorsal skin injury in mice and histological analyses. The presented results indicate further research is warranted into the engineered peptides as scaffolds for wound healing and tissue engineering., (© 2023. The Author(s).)

Due to unique features, proline residues may control protein structure and function. Here, we investigated the role of 52 PPQ 54 residues, indicated by the recently established experimental 3D structure of bovine herpesvirus 1-encoded UL49.5 protein as forming a characteristic proline hinge motif in its N-terminal domain. UL49.5 acts as a potent inhibitor of the transporter associated with antigen processing (TAP), which alters the antiviral immune response. Mechanisms employed by UL49.5 to affect TAP remain undetermined on a molecular level. We found that mutations in the 52 PPQ 54 region had a vast impact on its immunomodulatory function, increasing cell surface MHC class I expression, TAP levels, and peptide transport efficiency. This inhibitory effect was specific for UL49.5 activity towards TAP but not towards the viral glycoprotein M. To get an insight into the impact of proline hinge modifications on structure and dynamics, we performed all-atom and coarse-grained molecular dynamics studies on the native protein and PPQ mutants. The results demonstrated that the proline hinge sequence with its highly rigid conformation served as an anchor into the membrane. This anchor was responsible for the structural and dynamical behavior of the whole protein, constraining the mobility of the C-terminus, increasing the mobility of the transmembrane region, and controlling the accessibility of the C-terminal residues to the cytoplasmic environment. Those features appear crucial for TAP binding and inhibition. Our findings significantly advance the structural understanding of the UL49.5 protein and its functional regions and support the importance of proline motifs for the protein structure., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Defining dynamic protein-protein interactions in the ubiquitin conjugation reaction is a challenging research area. Generating peptide aptamers that target components such as ubiquitin itself, E1, E2, or E3 could provide tools to dissect novel features of the enzymatic cascade. Next-generation deep sequencing platforms were used to identify peptide sequences isolated from phage-peptide libraries screened against Ubiquitin and its ortholog NEDD8. In over three rounds of selection under differing wash criteria, over 13,000 peptides were acquired targeting ubiquitin, while over 10,000 peptides were selected against NEDD8. The overlap in peptides against these two proteins was less than 5% suggesting a high degree in specificity of Ubiquitin or NEDD8 toward linear peptide motifs. Two of these ubiquitin-binding peptides were identified that inhibit both E3 ubiquitin ligases MDM2 and CHIP. NMR analysis highlighted distinct modes of binding of the two different peptide aptamers. These data highlight the utility of using next-generation sequencing of combinatorial phage-peptide libraries to isolate peptide aptamers toward a protein target that can be used as a chemical tool in a complex multi-enzyme reaction., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lisowska, Lickiss, Gil-Mir, Huart, Trybala, Way, Hernychova, Krejci, Muller, Krejcir, Zhukow, Jurczak, Rodziewicz-Motowidło, Ball, Vojtesek, Hupp and Kalathiya.)

DNA methyltransferase inhibitor zebularine was proven to induce regeneration in the ear pinna in mice. We utilized a dorsal skin wound model to further evaluate this epigenetic inhibitor in wound healing. Full-thickness excisional wounds were made on the dorsum of 2 and 10-month-old healthy BALB/c and 3 and 8-month-old diabetic (db/db) mice, followed by topical or intraperitoneal zebularine delivery. Depending on the strain, age, dose, and delivery, the zebularine treatments either had no effect or accelerated or delayed wound closure. In principle, zebularine applied topically moderately promoted wound closure in the healthy but markedly delayed in the diabetic mice, which was in line with decreased viability of cultured keratinocytes from diabetic patients exposed to zebularine. The histological analysis revealed an improvement in the architecture of restored skin in zebularine-treated mice, manifested as a distinct layered pattern resembling panniculus carnosus. The finding corresponds with the zebularine-mediated activation of the Wnt5a gene, an essential regulator of Wnt signaling, the pathway involved in hair follicle development, the process which in turn is connected with regenerative skin healing. Although zebularine did not remarkably accelerate wound healing, zebularine and other epigenetic inhibitors deserve further testing as potential drugs to improve the quality of restored skin., (© 2022 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons Ltd.)

Synthetic implants are used to treat large bone defects that are often unable to regenerate, for example those caused by osteoporosis. It is necessary that the materials used to manufacture them are biocompatible and resorbable. Polymer-ceramic composites, such as those based on poly(L-lactide) (PLLA) and calcium phosphate ceramics (Ca-P), are often used for these purposes. In this study, we attempted to investigate an innovative strategy for two-step (dual) modification of composites and their components to improve the compatibility of composite components and the adhesion between PLA and Ca-P whiskers, and to increase the mechanical strength of the composite, as well as improve osteological bioactivity and prevent bone resorption in composites intended for bone regeneration. In the first step, Ca-P whiskers were modified with a saturated fatty acid namely, lauric acid (LA), or a silane coupling agent γ-aminopropyltriethoxysilane (APTES). Then, the composite, characterized by the best mechanical properties, was modified in the second stage of the work with an active chemical compound used in medicine as a first-line drug in osteoporosis-sodium alendronate, belonging to the group of bisphosphonates (BP). As a result of the research covered in this work, the composite modified with APTES and alendronate was found to be a promising candidate for future biomedical engineering applications.

Over the past few years, many molecules such as monoclonal antibodies, affibodies, nanobodies, and small compounds have been designed and tested as inhibitors of PD-1/PD-L1 complex formation. Some of them have been successfully implemented into clinical oncology practice. However, the majority of these compounds have disadvantages and limitations, such as high production price, potential for immunogenicity and/or prolonged clearance. Thus, new inhibitors of the PD-1/PD-L1 immune checkpoints are needed. Recently, peptides emerged as potential novel approach for blocking receptor/ligand interaction. In the presented studies we have designed, synthesised and tested peptides, which are potential inhibitors of the PD-1/PD-L1 axis. The amino acid sequences of the designed peptides were based on the binding sites of PD-1 to PD-L1, as determined by the crystal structure of the protein complex and also based on MM/GBSA analysis. Interactions of the peptides with PD-L1 protein were confirmed using SPR, while their inhibitory properties were studied using cell-based PD-1/PD-L1 immune checkpoint blockade assays. The characterization of the peptides has shown that the peptides PD-1(119-142) T120C-E141C , PD-1(119-142) C123-S137C and PD-1(122-138) C123-S137C strongly bind to PD-L1 protein and disrupt the interaction of the proteins. PD-1(122-138) C123-S137C peptide was shown to have the best inhibitory potential from the panel of peptides. Its 3D NMR structure was determined and the binding site to PD-L1 was established using molecular modelling methods. Our results indicate that the PD-1 derived peptides are able to mimic the PD-1 protein and inhibit PD-1/PD-L1 complex formation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The UNited RESidue (UNRES) model of polypeptide chains was applied to study the association of 20 peptides with sizes ranging from 6 to 32 amino-acid residues. Twelve of those were potentially aggregating hexa- or heptapeptides excised from larger proteins, while the remaining eight contained potentially aggregating sequences, functionalized by attaching larger ends rich in charged residues. For 13 peptides, the experimental data of aggregation were used. The remaining seven were synthesized, and their properties were measured in this work. Multiplexed replica-exchange simulations of eight-chain systems were conducted at 12 temperatures from 260 to 370 K at concentrations from 0.421 to 5.78 mM, corresponding to the experimental conditions. The temperature profiles of the fractions of monomers and octamers showed a clear transition corresponding to aggregate dissociation. Low simulated transition temperatures were obtained for the peptides, which did not precipitate after incubation, as well as for the H-GNNQQNY-NH 2 prion-protein fragment, which forms small fibrils. A substantial amount of inter-strand β -sheets was found in most of the systems. The results suggest that UNRES simulations can be used to assess peptide aggregation except for glutamine- and asparagine-rich peptides, for which a revision of the UNRES sidechain-sidechain interaction potentials appears necessary.

The ear pinna is a complex tissue consisting of the dermis, cartilage, muscles, vessels, and nerves. Ear pinna healing is a model of regeneration in mammals. In some mammals, including rabbits, punch wounds in the ear pinna close spontaneously; in common-use laboratory mice, they remain for life. Agents inducing ear pinna healing are potential regenerative drugs. We tested the effects of selected bioactive agents on 2 mm ear pinna wound closure in BALB/c mice. Our previous research demonstrated that a DNA methyltransferase inhibitor, zebularine, remarkably induced ear pinna regeneration. Although experiments with two other demethylating agents, RG108 and hydralazine, were unsuccessful, a histone deacetylase inhibitor, valproic acid, was another epigenetic agent found to increase ear hole closure. In addition, we identified a pro-regenerative activity of 4-ketoretinoic acid, a retinoic acid metabolite. Attempts to counteract the regenerative effects of the demethylating agent zebularine, with folates as methyl donors, failed. Surprisingly, a high dose of methionine, another methyl donor, promoted ear hole closure. Moreover, we showed that the regenerated areas of ear pinna were supplied with nerve fibre networks and blood vessels. The ear punch model proved helpful in testing the pro-regenerative activities of small-molecule compounds and observations of peripheral nerve regeneration.

Cancer immunotherapy using blockade of immune checkpoints is mainly based on monoclonal antibodies. Despite the tremendous success achieved by using those molecules to block immune checkpoint proteins, antibodies possess some weaknesses, which means that there is still a need to search for new compounds as alternatives to antibodies. Many current approaches are focused on use of peptides/peptidomimetics to destroy receptor/ligand interactions. Our studies concern blockade of the BTLA/HVEM complex, which generates an inhibitory effect on the immune response resulting in tolerance to cancer cells. To design inhibitors of such proteins binding we based our work on the amino acid sequence and structure of a ligand of HVEM protein, namely glycoprotein D, which possesses the same binding site on HVEM as BTLA protein. To disrupt the BTLA and HVEM interaction we designed several peptides, all fragments of glycoprotein D, and tested their binding to HVEM using SPR and their ability to inhibit the BTLA/HVEM complex formation using ELISA tests and cellular reporter platforms. That led to identification of two peptides, namely gD(1-36)(K10C-D30C) and gD(1-36)(A12C-L25C), which interact with HVEM and possess blocking capacities. Both peptides are not cytotoxic to human PBMCs, and show stability in human plasma. We also studied the 3D structure of the gD(1-36)(K10C-D30C) peptide using NMR and molecular modeling methods. The obtained data reveal that it possesses an unstructured conformation and binds to HVEM in the same location as gD and BTLA. All these results suggest that peptides based on the binding fragment of gD protein represent promising immunomodulation agents for future cancer immunotherapy., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Assessing the toxicity of new biomaterials dedicated to bone regeneration can be difficult. Many reports focus only on a single toxicity parameter, which may be insufficient for a detailed evaluation of the new material. Moreover, published data frequently do not include control cells exposed to the environment without composite or its extract. Here we present the results of two assays used in the toxicological assessment of materials' extracts (the integrity of the cellular membrane and the mitochondrial activity/proliferation), and the influence of different types of controls used on the obtained results. Results obtained in the cellular membrane integrity assay showed a lack of toxic effects of all tested extracts, and no statistical differences between them were present. Control cells, cells incubated with chitosan extract or chitosan-bioglass extract were used as a reference in proliferation calculations to highlight the impact of controls used on the result of the experiment. The use of different baseline controls caused variability between obtained proliferation results, and influenced the outcome of statistical analysis. Our findings confirm the thesis that the type of control used in an experiment can change the final results, and it may affect the toxicological assessment of biomaterial.

In this research, we describe the properties of three-component composite foam scaffolds based on poly(ε-caprolactone) (PCL) as a matrix and hydroxyapatite whiskers (HAP) and L-Lysine as fillers (PCL/HAP/Lys with wt% ratio 50/48/2). The scaffolds were prepared using a thermally induced phase separation technique supported by salt leaching (TIPS-SL). All materials were precisely characterized: porosity, density, water uptake, wettability, DSC, and TGA measurements and compression tests were carried out. The microstructure of the obtained scaffolds was analyzed via SEM. It was found that the PCL/HAP/Lys scaffold has a 45% higher Young's modulus and better wettability compared to the PCL/HAP system. At the same time, the porosity of the system was ~90%. The osteoblast hFOB 1.19 cell response was also investigated in osteogenic conditions (39 °C) and the cytokine release profile of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α was determined. Modification of PCL scaffolds with HAP and L-Lysine significantly improved the proliferation of pre-osteoblasts cultured on such materials.

The glycosylphosphatidylinositol-anchored transmembrane glycoprotein CD160 (cluster of differentiation 160) is a member of the immunoglobulin superfamily. Four isoforms, which differ by the presence or absence of an immunoglobulin-like domain and the mode of anchoring in the cell membrane, have been identified. CD160 has a significant impact on the proper functioning of the immune system by activating natural killer cells and inhibiting T cells. CD160 is a natural ligand for herpes virus entry mediator (HVEM), a member of the tumor necrosis factor superfamily. The CD160-HVEM complex is a rare example of direct interaction between the two different superfamilies. The interaction of these two proteins leads to the inhibition of CD4 + T cells which, in consequence, leads to the inhibition of the correct response of the immune system. Available research articles indicate that CD160 plays a role in various types of cancer, chronic viral diseases, malaria, paroxysmal nocturnal hemoglobinuria, atherosclerosis, autoimmune diseases, skin inflammation, acute liver damage and retinal vascular disease. We present here an overview of the CD160 protein, the general characteristics of the receptor and its isoforms, details of structural studies of CD160 and the CD160-HVEM complex, as well as a description of the role of this protein in selected human diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Ischemic stroke is a disturbance in cerebral blood flow caused by brain tissue ischemia and hypoxia. We optimized a multifactorial in vitro model of acute ischemic stroke using rat primary neural cultures. This model was exploited to investigate the pro-viable activity of cell-penetrating peptides: arginine-rich Tat(49-57)-NH 2 (R 49 KKRRQRRR 57 -amide) and its less basic analogue, PTD4 (Y 47 ARAAARQARA 57 -amide). Our model included glucose deprivation, oxidative stress, lactic acidosis, and excitotoxicity. Neurotoxicity of these peptides was excluded below a concentration of 50 μm, and PTD4-induced pro-survival was more pronounced. Circular dichroism spectroscopy and molecular dynamics (MD) calculations proved potential contribution of the peptide conformational properties to neuroprotection: in MD, Tat(49-57)-NH 2 adopted a random coil and polyproline type II helical structure, whereas PTD4 adopted a helical structure. In an aqueous environment, the peptides mostly adopted a random coil conformation (PTD4) or a polyproline type II helical (Tat(49-57)-NH 2 ) structure. In 30% TFE, PTD4 showed a tendency to adopt a helical structure. Overall, the pro-viable activity of PTD4 was not correlated with the arginine content but rather with the peptide's ability to adopt a helical structure in the membrane-mimicking environment, which enhances its cell membrane permeability. PTD4 may act as a leader sequence in novel drugs for the treatment of acute ischemic stroke.

This work describes the modification of a gold electrode with the BMS-8 compound that interacts with the Programmed Death-Ligand 1 (PD-L1), an immune checkpoint protein. The results show that we can confirm the presence of the sPD-L1 in the concentration range of 10 -18 to 10 -8 M using electrochemical impedance spectroscopy (EIS) with a limit of detection (LOD) of 1.87 × 10 -14 M for PD-L1 (S/N = 3.3) and at a concentration of 10 -14 M via cyclic voltammetry (CV). Additionally, high-resolution X-ray photoelectron spectroscopy (XPS), contact angle, and surface free energy measurements were applied to confirm the functionalization of the electrode. We investigated the selectivity of the electrode for other proteins: Programmed Death-1 (PD-1), cluster of differentiation 160 (CD160), and B- and T-lymphocyte attenuator (BTLA) at concentrations of 10 -8 M. Differentiation between PD-L1 and PD-1 was achieved based on the analysis of the capacitance effect frequency dispersion at the surface of the modified Au electrode with BMS-8 after incubation at various concentrations of PD-L1 and PD-1 proteins in the range of 10 -18 to 10 -8 M. Significant differences were observed in the heterogeneity of PD-L1 and PD-1. The results of the quasi-capacitance studies demonstrate that BMS-8 strongly and specifically interacts with the PD-L1 protein., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Technological developments in the field of biologically active peptide applications in medicine have increased the need for new methods for peptide delivery. The disadvantage of peptides as drugs is their low biological stability. Recently, great attention has been paid to self-assembling peptides that can form fibrils. Such a formulation makes bioactive peptides more resistant to enzymatic degradation and druggable. Peptide fibrils can be carriers for peptides with interesting biological activities. These features open up prospects for using the peptide fibrils as long-acting drugs and are a valid alternative to conventional peptidic therapies. In our study, we designed new peptide scaffolds that are a hybrid of three interconnected amino acid sequences and are: pro-regenerative, cleavable by neutrophilic elastase, and fibril-forming. We intended to obtain peptides that are stable in the wound environment and that, when applied, would release a biologically active sequence. Our studies showed that the designed hybrid peptides show a high tendency toward regular fibril formation and are able to release the pro-regenerative sequence. Cytotoxicity studies showed that all the designed peptides were safe, did not cause cytotoxic effects and revealed a pro-regenerative potential in human fibroblast and keratinocyte cell lines. In vivo experiments in a dorsal skin injury model in mice indicated that two tested peptides moderately promote tissue repair in their free form. Our research proves that peptide fibrils can be a druggable form and a scaffold for active peptides.

Herpesviruses are the most prevalent viruses that infect the human and animal body. They can escape a host immune response in numerous ways. One way is to block the TAP complex so that viral peptides, originating from proteasomal degradation, cannot be transported to the endoplasmic reticulum. As a result, a reduced number of MHC class I molecules appear on the surface of infected cells and, thus, the immune system is not efficiently activated. BoHV-1-encoded UL49.5 protein is one such TAP transporter inhibitor. This protein binds to TAP in such a way that its N-terminal fragment interacts with the loops of the TAP complex, and the C-terminus stimulates proteasomal degradation of TAP. Previous studies have indicated certain amino acid residues, especially the RRE(9-11) motif, within the helical structure of the UL49.5 N-terminal fragment, as being crucial to the protein's activity. In this work, we investigated the effects of modifications within the RRE region on the spatial structure of the UL49.5 N-terminal fragment. The introduced RRE(9-11) variations were designed to abolish or stabilize the structure of the α-helix and, consequently, to increase or decrease protein activity compared to the wild type. The terminal structure of the peptides was established using circular dichroism (CD), 2D nuclear magnetic resonance (NMR), and molecular dynamics (MD) in membrane-mimetic or membrane-model environments. Our structural results show that in the RRE(9-11)AAA and E11G peptides the helical structure has been stabilized, whereas for the RRE(9-11)GGG peptide, as expected, the helix structure has partially unfolded compared to the native structure. These RRE modifications, in the context of the entire UL49.5 proteins, slightly altered their biological activity in human cells., (© 2021 Wiley-VHCA AG, Zurich, Switzerland.)

Objective: This study evaluated the use of novel peptides derived from platelet-derived growth factor (PDGF-BB) as potential wound healing stimulants. One of the compounds (named PDGF2) was subjected for further research after cytotoxicity and proliferation assays on human skin cells. Further investigation included evaluation of: migration and chemotaxis of skin cells, immunological and allergic safety, the transcriptional analyses of adipose-derived stem cells (ASCs) and dermal fibroblasts stimulated with PDGF2, and the use of dorsal skin wound injury model to evaluate the effect of wound healing in mice. Approach: Colorimetric lactate dehydrogenase and tetrazolium assays were used to evaluate the cytotoxicity and the effect on proliferation. PDGF2 effect on migration and chemotaxis was also checked. Immunological safety and allergic potential were evaluated with a lymphocyte activation and basophil activation test. Transcriptional profiles of ASCs and primary fibroblasts were assessed after stimulation with PDGF2. Eight-week-old BALB/c female mice were used for dorsal skin wound injury model. Results: PDGF2 showed low cytotoxicity, pro-proliferative effects on human skin cells, high immunological safety, and accelerated wound healing in mouse model. Furthermore, transcriptomic analysis of ASCs and fibroblasts revealed the activation of processes involved in wound healing and indicated its safety. Innovation: A novel peptide derived from PDGF-BB was proved to be safe drug candidate in wound healing. We also present a multifaceted in vitro model for the initial screening of new compounds that may be potentially useful in wound healing stimulation. Conclusion: The results show that peptide derived from PDGF-BB is a promising drug candidate for wound treatment.

One of the major current trends in cancer immunotherapy is the blockade of immune checkpoint proteins that negatively regulate the immune response. This has been achieved through antibodies blocking PD-1/PD-L1 and CTLA-4/CD80/CD86 interactions. Such antibodies have revolutionized oncological therapy and shown a new way to fight cancer. Additional (negative) immune checkpoints are also promising targets in cancer therapy and there is a demand for inhibitors for these molecules. Our studies are focused on BTLA/HVEM complex, which inhibits T-cell proliferation and cytokine production and therefore has great potential as a new target for cancer treatment. The goal of the presented studies was the design and synthesis of compounds able to block BTLA/HVEM interactions. For that purpose, the N -terminal fragment of glycoprotein D (gD), which interacts with HVEM, was used. Based on the crystal structure of the gD/HVEM complex and MM/GBSA analysis performed on it, several peptides were designed and synthesized as potential inhibitors of the BTLA/HVEM interaction. Affinity tests, ELISA tests, and cellular-based reporter assays were performed on these compounds to check their ability to bind to HVEM and to inhibit BTLA/HVEM complex formation. For leading peptides candidates, all-atom and subsequent docking simulations with a coarse-grained force field were performed to determine their binding modes. To further evaluate their potential as drug candidates, their stability in plasma and their cytotoxicity effects on PBMCs were assessed. Our data indicate that the peptide gD(1-36)(K10C-T29C) is the best candidate as a future drug. It interacts with HVEM protein, blocks the BTLA/HVEM interaction, and is nontoxic to cells. The present study provides a new perspective on the development of BTLA/HVEM inhibitors that disrupt protein interactions.

Human cystatin C (HCC), a cysteine-protease inhibitor, exists as a folded monomer under physiological conditions but has the ability to self-assemble via domain swapping into multimeric states, including oligomers with a doughnut-like structure. The structure of the monomeric HCC has been solved by X-ray crystallography, and a covalently linked version of HCC (stab-1 HCC) is able to form stable oligomeric species containing 10-12 monomeric subunits. We have performed molecular modeling, and in conjunction with experimental parameters obtained from atomic force microscopy (AFM), transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS) measurements, we observe that the structures are essentially flat, with a height of about 2 nm, and the distance between the outer edge of the ring and the edge of the central cavity is ~5.1 nm. These dimensions correspond to the height and diameter of one stab-1 HCC subunit and we present a dodecamer model for stabilized cystatin C oligomers using molecular dynamics simulations and experimentally measured parameters. Given that oligomeric species in protein aggregation reactions are often transient and very highly heterogeneous, the structural information presented here on these isolated stab-1 HCC oligomers may be useful to further explore the physiological relevance of different structural species of cystatin C in relation to protein misfolding disease.

Regeneration and wound healing are vital to tissue homeostasis and organism survival. One of the biggest challenges of today's science and medicine is finding methods and factors to stimulate these processes in the human body. Effective solutions to promote regenerative responses will accelerate advances in tissue engineering, regenerative medicine, transplantology, and a number of other clinical specialties. In this study, we assessed the potential efficacy of a synthetic hexapeptide, RDKVYR, for the stimulation of tissue repair and wound healing. The hexapeptide is marketed under the name "Imunofan" (IM) as an immunostimulant. IM displayed stability in aqueous solutions, while in plasma it was rapidly bound by albumins. Structural analyses demonstrated the conformational flexibility of the peptide. Tests in human fibroblast and keratinocyte cell lines showed that IM exerted a statistically significant ( p < 0.05) pro-proliferative activity (30-40% and 20-50% increase in proliferation of fibroblast and keratinocytes, respectively), revealed no cytotoxicity over a vast range of concentrations ( p < 0.05), and had no allergic properties. IM was found to induce significant transcriptional responses, such as enhanced activity of genes involved in active DNA demethylation ( p < 0.05) in fibroblasts and activation of genes involved in immune responses, migration, and chemotaxis in adipose-derived stem cells derived from surgery donors. Experiments in a model of ear pinna injury in mice indicated that IM moderately promoted tissue repair (8% in BALB/c and 36% in C57BL/6 in comparison to control).

The recent impact of implants on improving the human life quality has been enormous. During the past two decades we witnessed major advancements in both material and structural development of implants. They were driven mainly by the increasing patients' demand and the need to address the major issues that come along with the initially underestimated complexity of the bone-implant interface. While both, the materials and design of implants reached a certain, balanced state, recent years brought a shift in focus towards the bone-implant interface as the weakest link in the increasing implant long-term usability. As a result, several approaches were developed. They aimed at influencing and enhancing the implant osseointegration and its proper behavior when under load and stress. With this review, we would like to discuss the recent advancements in the field of implant surface modifications, emphasizing the importance of chemical methods, focusing on proteins, peptides and peptidomimetics as promising agents for titanium surface coatings., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 Elsevier B.V. All rights reserved.)

It is recognized that interactions between most materials are governed by their surface properties and manifest themselves at the interface formed between them. To gain more insight into this thin layer, several methods have been deployed. Among them, spectroscopic methods have been thoroughly evaluated. Due to their exceptional sensitivity, data acquisition speed, and broad material tolerance they have been proven to be invaluable tools for surface analysis, used by scientists in many fields, for example, implant studies. Today, in modern medicine the use of implants is considered standard practice. The past two decades of constant development has established the importance of implants in dentistry, orthopedics, as well as extended their applications to other areas such as aesthetic medicine. Fundamental to the success of implants is the knowledge of the biological processes involved in interactions between an implant and its host tissue, which are directly connected to the type of implant material and its surface properties. This review aims to demonstrate the broad applications of spectroscopic methods in implant material studies, particularly discussing hard implants, surface composition studies, and surface-cell interactions., Competing Interests: The authors declare no conflict of interest.

Immune checkpoints are crucial in the maintenance of antitumor immune responses. The activation or blockade of immune checkpoints is dependent on the interactions between receptors and ligands; such interactions can provide inhibitory or stimulatory signals, including the enhancement or suppression of T-cell proliferation, differentiation, and/or cytokine secretion. B-and T-lymphocyte attenuator (BTLA) is a lymphoid-specific cell surface receptor which is present on T-cells and interacts with herpes virus entry mediator (HVEM), which is present on tumor cells. The binding of HVEM to BTLA triggers an inhibitory signal which attenuates the immune response. This feature is interesting for studying the molecular interactions between HVEM and BTLA, as they may be targeted for novel immunotherapies. This work was based on the crystal structure of the BTLA/HVEM complex showing that BTLA binds the N-terminal cysteine-rich domain of HVEM. We investigated the amino acid sequence of HVEM and used molecular modeling methods to develop inhibitors of the BTLA/HVEM interaction. We synthesized novel compounds and determined their ability to interact with the BTLA protein and inhibit the formation of the BTLA/HVEM complex. Our results suggest that the HVEM (14-39) peptide is a potent inhibitor of the formation of the BTLA/HVEM protein complex.

Human cystatin C (hCC), a member of the superfamily of papain-like cysteine protease inhibitors, is the most widespread cystatin in human body fluids. This small protein, in addition to its physiological function, is involved in various diseases, including cerebral amyloid angiopathy, cerebral hemorrhage, stroke, and dementia. Physiologically active hCC is a monomer. However, all structural studies based on crystallization led to the dimeric structure formed as a result of a three-dimensional exchange of the protein domains (3D domain swapping). The monomeric structure was obtained only for hCC variant V57N and for the protein stabilized by an additional disulfide bridge. With this study, we extend the number of models of monomeric hCC by an additional hCC variant with a single amino acid substitution in the flexible loop L1. The V57G variant was chosen for the X-ray and NMR structural analysis due to its exceptional conformational stability in solution. In this work, we show for the first time the structural and dynamics studies of human cystatin C variant in solution. We were also able to compare these data with the crystal structure of the hCC V57G and with other cystatins. The overall cystatin fold is retained in the solute form. Additionally, structural information concerning the N terminus was obtained during our studies and presented for the first time. DATABASE: Crystallographic structure: structural data are available in PDB databases under the accession number 6ROA. NMR structure: structural data are available in PDB and BMRB databases under the accession numbers 6RPV and 34399, respectively., (© 2019 Federation of European Biochemical Societies.)

Applications of bioactive peptides and polypeptides are emerging in areas such as drug development and drug delivery systems. These compounds are bioactive, biocompatible and represent a wide range of chemical properties, enabling further adjustments of obtained biomaterials. However, delivering large quantities of peptide derivatives is still challenging. Several methods have been developed for the production of concatemers - multiple copies of the desired protein segments. We have presented an efficient method for the production of peptides of desired length, expressed from concatemeric Open Reading Frame. The method employs specific amplification-expression DNA vectors. The main methodological approaches are described by Skowron et al., 2020 [1]. As an illustration of the demonstrated method's utility, an epitope from the S protein of Hepatitis B virus (HBV) was amplified. Additionally, peptides, showing potentially pro-regenerative properties, derived from the angiopoietin-related growth factor (AGF) were designed and amplified. Here we present a dataset including: ( i ) detailed protocols for the purification of HBV and AGF - derived polyepitopic protein concatemers, ( ii ) sequences of the designed primers, vectors and recombinant constructs, ( iii ) data on cytotoxicity, immunogenicity and stability of AGF-derived polypeptides., (© 2019 The Author(s).)

Herpesvirus entry mediator (HVEM), a member of the TNF-receptor superfamily, plays an important role in the regulation of the immune system. It forms a complex with ligands and can either activate or inhibit the response of the immune system. Furthermore, HVEM can exhibit pro-inflammatory or anti-inflammatory effects in many human diseases. Therefore, understanding the mechanism underlying the interaction of HVEM with other receptors is extremely important to design small therapeutic molecules that can stimulate the response of the immune system. In this study, we attempted to develop the most efficient method for the expression and purification of the extracellular domain of HVEM using Escherichia coli. The soluble fraction constituted only a small portion of the E. coli-expressed protein, whereas majority of the protein was found to be accumulated in the insoluble fraction. Three different protein refolding methods were analyzed: dialysis, dilution, and using chromatographic column. The oligomeric state of the protein was determined by characterizing the obtained fractions using analytical size exclusion chromatography. All the obtained fractions were tested for their ability to form a complex with B- and T-lymphocyte attenuator using enzyme-linked immunosorbent assay. The results of this study provide crucial information regarding the production of HVEM protein in a robust, well-established, and convenient heterologous expression system using E. coli as a host. In addition, it allows for the selection of the most effective method for appropriate refolding of HVEM protein, which gets accumulated in the insoluble fraction., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Most studies on regenerative medicine focus on cell-based therapies and transplantations. Small-molecule therapeutics, though proved effective in different medical conditions, have not been extensively investigated in regenerative research. It is known that healing potential decreases with development and developmental changes are driven by epigenetic mechanisms, which suggests epigenetic repression of regenerative capacity., Methods: We applied zebularine, a nucleoside inhibitor of DNA methyltransferases, to stimulate the regenerative response in a model of ear pinna injury in mice., Findings: We observed the regeneration of complex tissue that was manifested as improved ear hole repair in mice that received intraperitoneal injections of zebularine. Six weeks after injury, the mean hole area decreased by 83.2 ± 9.4% in zebularine-treated and by 43.6 ± 15.4% in control mice (p < 10 -30 ). Combined delivery of zebularine and retinoic acid potentiated and accelerated this effect, resulting in complete ear hole closure within three weeks after injury. We found a decrease in DNA methylation and transcriptional activation of neurodevelopmental and pluripotency genes in the regenerating tissues., Interpretation: This study is the first to demonstrate an effective induction of complex tissue regeneration in adult mammals using zebularine. We showed that the synergistic action of an epigenetic drug (zebularine) and a transcriptional activator (retinoic acid) could be effectively utilized to induce the regenerative response, thus delineating a novel pharmacological strategy for regeneration. The strategy was effective in the model of ear pinna regeneration in mice, but zebularine acts on different cell types, therefore, a similar approach can be tested in other tissues and organs., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Antibacterial peptidyl derivative - Cystapep 1, was previously found to be active both against antibiotic-resistant staphylococci and streptococci as well as antibioticsusceptible strains of these species. Therefore, it is a promising lead compound to search for new antimicrobial peptidomimetics., Objectives: We focused on identifying structural elements that are responsible for the biological activity of Cystapep 1 and its five analogues. We tried to find an answer to the question about the mechanism of action of the tested compounds. Therefore, we have investigated in details the possibility of interacting these compounds with biological membrane mimetics., Methods: The subject compounds were synthesized in solution, purified and characterized by HPLC and mass spectrometry. Then, the staphylococci susceptibility tests were performed and their cytotoxicity was established. The results of Cystapep 1 and its analogues interactions with model target were examined using the DSC and ITC techniques. At the end the spatial structures of the tested peptidomimetics using NMR technique were obtained., Results: Antimicrobial and cytotoxicity tests show that Cystapep 1 and its peptidomimetic V are good drug candidates. DSC and ITC studies indicate that disruption of membrane is not the only possible mechanism of action of Cystapep 1-like compounds. For Cystapep 1 itself, a multi-step mechanism of interaction with a negatively charged membrane is observed, which indicates other processes occurring alongside the binding process. The conformational analysis indicated the presence of a hydrophobic cluster, composed of certain side chains, only in the structures of active peptidomimetics. This can facilitate the anchoring of the peptidyl derivatives to the bacterial membrane., Conclusion: An increase in hydrophobicity of the peptidomimetics improved the antimicrobial activity against S. aureus, however there is no simple correlation between the biological activity and the strength of interactions of the peptidyl with bacterial membrane., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The transporter associated with antigen processing (TAP) directly participates in the immune response as a key component of the cytosolic peptide to major histocompatibility complex (MHC) class I protein loading machinery. This makes TAP an important target for viruses avoiding recognition by CD8+ T lymphocytes. Its activity can be suppressed by the UL49.5 protein produced by bovine herpesvirus 1, although the mechanism of this inhibition has not been understood so far. Therefore, the main goal of our study was to investigate the 3D structure of bovine herpesvirus 1 - encoded UL49.5 protein. The final structure of the inhibitor was established using circular dichroism (CD), 2D nuclear magnetic resonance (NMR), and molecular dynamics (MD) in membrane mimetic environments. In NMR studies, UL49.5 was represented by two fragments: the extracellular region (residues 1-35) and the transmembrane-intracellular fragment (residues 36-75), displaying various functions during viral invasion. After the empirical structure determination, a molecular docking procedure was used to predict the complex of UL49.5 with the TAP heterodimer. Our results revealed that UL49.5 adopted a highly flexible membrane-proximal helical structure in the extracellular part. In the transmembrane region, we observed two short α-helices. Furthermore, the cytoplasmic part had an unordered structure. Finally, we propose three different orientations of UL49.5 in the complex with TAP. Our studies provide, for the first time, the experimental structural information on UL49.5 and structure-based insight in its mechanism of action which might be helpful in designing new drugs against viral infections., (Copyright © 2019 Elsevier B.V. All rights reserved.)

CD160 is a T cell coinhibitory molecule that interacts with the herpes virus entry mediator (HVEM) on antigen-presenting cells to provide an inhibitory signal to T cells. To date, the structure of CD160 and its complex with HVEM are unknown. Here, we have identified the fragments of CD160 interacting with HVEM using ELISA tests, hydrogen/deuterium studies, affinity chromatography and mass spectrometry (MS). By combining hydrogen/deuterium exchange and mass spectrometry (HDX-MS) we obtained key information about the tertiary structure of CD160, predicting the 3D structure of the CD160-HVEM complex. Our results provide insights into the molecular architecture of this complex, serving as a useful basis for designing inhibitors for future immunotherapies., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest.

Amyloidoses are a group of diseases caused by the extracellular deposition of proteins forming amyloid fibrils. The amyloidosis is classified according to the main protein or peptide that constitutes the amyloid fibrils. The most effective methods for the diagnosis of amyloidosis are based on mass spectrometry. Mass spectrometry enables confirmation of the identity of the protein precursor of amyloid fibrils in biological samples with very high sensitivity and specificity, which is crucial for proper amyloid typing. Due to the fact that biological samples are very complex, mass spectrometry is usually connected with techniques such as liquid chromatography or capillary electrophoresis, which enable the separation of proteins before MS analysis. Therefore mass spectrometry constitutes an important part of the so called "hyphenated techniques" combining, preferentially in-line, different analytical methods to provide comprehensive information about the studied problem. Hyphenated methods are very useful in the discovery of biomarkers in different types of amyloidosis. In systemic forms of amyloidosis, the analysis of aggregated proteins is usually performed based on the tissues obtained during a biopsy of an affected organ or a subcutaneous adipose tissue. In some cases, when the diagnostic biopsy is not possible due to the fact that amyloid fibrils are formed in organs like the brain (Alzheimer's disease), the study of biomarkers presented in body fluids can be carried out. Currently, large-scale studies are performed to find and validate more effective biomarkers, which can be used in diagnostic procedures. We would like to present the methods connected with mass spectrometry which are used in the diagnosis of amyloidosis based on the analysis of proteins occurring in tissues, blood and cerebrospinal fluid., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Adipose-derived stem cells (ASCs) have become an important research model in regenerative medicine. However, there are controversies regarding the impact of prolonged cell culture on the ASCs phenotype and their differentiation potential. Hence, we studied 10 clinical ASCs replicates from plastic and oncological surgery patients, in six-passage FBS supplemented cultures. We quantified basic mesenchymal cell surface marker transcripts and the encoded proteins after each passage. In parallel, we investigated the differentiation potential of ASCs into chondrocytes, osteocytes and adipocytes. We further determined the effects of FBS supplementation and subsequent deprivation on the whole transcriptome by comprehensive mRNA and miRNA sequencing. Our results show that ASCs maintain differentiation potential and consistent profile of key mesenchymal markers, with apparent expression of distinct isoforms, in long-term cultures. No significant differences were observed between plastic and oncological surgery cohorts. ASCs in FBS supplemented primary cultures are almost committed to mesenchymal lineages as they express key epithelial-mesenchymal transition genes including early mesenchymal markers. Furthermore, combined mRNA/miRNA expression profiling strongly supports a modulatory role for the miR-30 family in the commitment process to mesenchymal lineages. Finally, we propose improvements to existing qPCR based assays that address alternative isoform expression of mesenchymal markers.