Production of mono and bilayer devices for wound dressing by coupling of electrospinning and supercritical impregnation techniques.

In this paper, electrospinning and supercritical impregnation were coupled to produce polyurethane fibrous membranes loaded with mesoglycan and lactoferrin. The proposed methodology allowed the production of three skin wound healing bilayer systems: a first system containing mesoglycan loaded through electrospinning and lactoferrin loaded by supercritical impregnation, a second system where the use of the two techniques was reversed, and a third sample where the drugs were both encapsulated through a one-step process. SEM analysis demonstrated the formation of microfibers with a homogeneous drug distribution. The highest loadings were 0.062 g/g for mesoglycan and 0.013 g/g for lactoferrin. Then, hydrophilicity and liquid retention analyses were carried out to evaluate the possibility of using the manufacturers as active patches. The kinetic profiles, obtained through in vitro tests conducted using a Franz diffusion cell, proved that the diffusion of the active drugs followed a double-step release before attaining the equilibrium after about 30 h. When the electrospun membranes were placed in contact with HUVEC, HaCaT, and BJ cell lines, as human endothelial cells, keratinocytes, and fibroblasts, respectively, no cytotoxic events were assessed. Finally, the capacity of the most promising system to promote the healing process was performed by carrying out scratch tests on HaCat cells.

The pro-healing effects of heparan sulfate and growth factors are enhanced by the heparinase enzyme: New association for skin wound healing treatment.

Effective treatment strategies for skin wound repair are the focus of numerous studies. New pharmacological approaches appear necessary to guarantee a correct and healthy tissue regeneration. For these reasons, we purposed to investigate the effects of the combination between heparan sulfate and growth factors further adding the heparinase enzyme. Interestingly, for the first time, we have found that this whole association retains a marked pro-healing activity when topically administered to the wound. In detail, this combination significantly enhances the motility and activation of the main cell populations involved in tissue regeneration (keratinocytes, fibroblasts and endothelial cells), compared with single agents administered without heparinase. Notably, using an experimental C57BL/6 mouse model of skin wounding, we observed that the topical treatment of skin lesions with heparan sulfate + growth factors + heparinase promotes the highest closure of wounds compared to each substance mixed with the other ones in all the possible combinations. Eosin/hematoxylin staining of skin biopsies revealed that treatment with the whole combination allows the formation of a well-structured matrix with numerous new vessels. Confocal analyses for vimentin, FAP1α, CK10 and CD31 have highlighted the presence of activated fibroblasts, differentiated keratinocytes and endothelial cells at the closed region of wounds. Our results encourage defining this combined treatment as a new and appealing therapy expedient in skin wound healing, as it is able to activate cell components and promote a dynamic lesions closure.

Targeting USP-7 by a Novel Fluorinated 5-Pyrazolyl-Urea Derivative.

The impact of innovative technologies on the target discovery has been employed here to characterize the interactome of STIRUR 41, a promising 3-fluoro-phenyl-5-pyrazolyl-urea derivative endowed with anti-cancer activity, on neuroblastoma-related cells. A drug affinity responsive target stability-based proteomic platform has been optimized to elucidate the molecular mechanism at the basis of STIRUR 41 action, together with immunoblotting analysis and in silico molecular docking. Ubiquitin Specific Protease 7 (USP-7), one of the deubiquitinating enzymes which protect substrate proteins from proteasomal degradation, has been identified as the most affine STIRUR 41 target. As further demonstrated by in vitro and in-cell assays, STIRUR 41 was able to inhibit both the enzymatic activity of USP-7 and its expression levels in neuroblastoma-related cells, thus laying an encouraging base for the blockade of USP-7 downstream signaling.

Supercritical Impregnation of Mesoglycan and Lactoferrin on Polyurethane Electrospun Fibers for Wound Healing Applications.

Fibrous membranes of thermoplastic polyurethane (TPU) were fabricated through a uni-axial electrospinning process. Fibers were then separately charged with two pharmacological agents, mesoglycan (MSG) and lactoferrin (LF), by supercritical CO2 impregnation. Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Spectroscopy (EDS) analysis proved the formation of a micrometric structure with a homogeneous distribution of mesoglycan and lactoferrin. Besides, the degree of retention is calculated in four liquid media with different pHs. At the same time, angle contact analysis proved the formation of a hydrophobic membrane loaded with MSG and a hydrophilic LF-loaded one. The impregnation kinetics demonstrated a maximum loaded amount equal to 0.18 ± 0.20% and 0.07 ± 0.05% for MSG and LT, respectively. In vitro tests were performed using a Franz diffusion cell to simulate the contact with the human skin. The release of MSG reaches a plateau after about 28 h while LF release leveled off after 15 h. The in vitro compatibility of electrospun membranes has been evaluated on HaCaT and BJ cell lines, as human keratinocytes and fibroblasts, respectively. The reported data proved the potential application of fabricated membranes for wound healing.

A multidisciplinary functional proteomics-aided strategy as a tool for the profiling of a novel cytotoxic thiadiazolopyrimidone.

In recent years, thiadiazolopyrimidine derivatives have been acknowledged for their striking poly-pharmacological framework, thus representing an interesting scaffold for the development of new therapeutic candidates. This paper examines the synthesis and the interactome characterization of a novel bioactive thiadiazolopyrimidone (compound 1), endowed with cytotoxic activity on HeLa cancer cells. In detail, starting from a small set of synthesized thiadiazolopyrimidones, a multi-disciplinary strategy has been carried out on the most bioactive one to disclose its potential biological targets by functional proteomics, using a label-free mass spectrometry based platform coupling Drug Affinity Responsive Target Stability and targeted Limited Proteolysis-Multiple Reaction Monitoring. The identification of Annexin A6 (ANXA6) as compound 1 most reliable cellular partner paved the way to deepen the protein-ligand interaction through bio-orthogonal approaches and to prove compound 1 action on migration and invasion processes governed by ANXA6 modulation. The identification of compund 1 as the first ANXA6 protein modulator represents a relevant tool to further explore the biological role of ANXA6 in cancer, as well as to develop novel anticancer candidates.

The Protecting Activity of RIPACUT®: A New Therapeutic Approach Preserving Epithelial Health Based on the Combination of Iceland Lichen Extract, Silver Salt, and Sodium Hyaluronate.

Epithelial integrity and function must be maintained in a dynamic healthy equilibrium, keeping unaltered the oxidative and inflammatory conditions and the microbiome of the cutaneous layers. Beside the skin, other mucous membranes can be injured, such as the nasal and anal ones, because of the contact with the external environment. Here, we detected the effects of RIPACUT®, a combination of Iceland lichen extract, silver salt and sodium hyaluronate that individually act in diverse biological ways. The findings we obtained on keratinocytes, nasal and intestinal epithelial cells reveal that this combination showed a marked antioxidant activity, further assessed by the DPPH assay. Additionally, by analyzing the release of the IL-1ß, TNF-α and IL-6 cytokines, we proved the anti-inflammatory effect of RIPACUT®. In both cases, the main preserving action was due to Iceland lichen. We also observed a notable antimicrobial activity mediated by the silver compound. These data suggest that RIPACUT® could signify the basis for an attractive pharmacological approach to maintaining healthy epithelial conditions. Interestingly, this may be extended to the nasal and anal areas where it protects against oxidative, inflammatory and infectious insults. Thus, these outcomes encourage the creation of sprays or creams for which sodium hyaluronate can guarantee a surface film-forming effect.

Simvastatin Reduces Doxorubicin-Induced Cardiotoxicity: Effects beyond Its Antioxidant Activity.

This study aimed to evaluate if Simvastatin can reduce, and/or prevent, Doxorubicin (Doxo)-induced cardiotoxicity. H9c2 cells were treated with Simvastatin (10 µM) for 4 h and then Doxo (1 µM) was added, and the effects on oxidative stress, calcium homeostasis, and apoptosis were evaluated after 20 h. Furthermore, we evaluated the effects of Simvastatin and Doxo co-treatment on Connexin 43 (Cx43) expression and localization, since this transmembrane protein forming gap junctions is widely involved in cardioprotection. Cytofluorimetric analysis showed that Simvastatin co-treatment significantly reduced Doxo-induced cytosolic and mitochondrial ROS overproduction, apoptosis, and cytochrome c release. Spectrofluorimetric analysis performed by means of Fura2 showed that Simvastatin co-treatment reduced calcium levels stored in mitochondria and restored cytosolic calcium storage. Western blot, immunofluorescence, and cytofluorimetric analyses showed that Simvastatin co-treatment significantly reduced Doxo-induced mitochondrial Cx43 over-expression and significantly increased the membrane levels of Cx43 phosphorylated on Ser368. We hypothesized that the reduced expression of mitochondrial Cx43 could justify the reduced levels of calcium stored in mitochondria and the consequent induction of apoptosis observed in Simvastatin co-treated cells. Moreover, the increased membrane levels of Cx43 phosphorylated on Ser368, which is responsible for the closed conformational state of the gap junction, let us to hypothesize that Simvastatin leads to cell-to-cell communication interruption to block the propagation of Doxo-induced harmful stimuli. Based on these results, we can conclude that Simvastatin could be a good adjuvant in Doxo anticancer therapy. Indeed, we confirmed its antioxidant and antiapoptotic activity, and, above all, we highlighted that Simvastatin interferes with expression and cellular localization of Cx43 that is widely involved in cardioprotection.

A DR/NIR Hybrid Polymeric Tool for Functional Bio-Coatings: Theoretical Study, Cytotoxicity, and Antimicrobial Activity.

Among modern biomaterials, hybrid tools containing an organic component and a metal cation are recognized as added value, and, for many advanced biomedical applications, synthetic polymers are used as thin protective/functional coatings for medical or prosthetic devices and implants. These materials require specific non-degradability, biocompatibility, antimicrobial, and antiproliferative properties to address safety aspects concerning their use in medicine. Moreover, bioimaging monitoring of the biomedical device and/or implant through biological tissues is a desirable ability. This article reports a novel hybrid metallopolymer obtained by grafting zinc-coordinated fragments to an organic polymeric matrix. This hybrid polymer, owing to its relevant emission in the deep red to near-infrared (DR/NIR) region, is monitorable; therefore, it represents a potential material for biomedical coating. Furthermore, it shows good biocompatibility and adhesion properties and excellent stability in slightly acidic/basic water solutions. Finally, in contact with the superficial layers of human skin, it shows antimicrobial properties against Staphylococcus aureus bacterial strains.

Effects of Hst3p inhibition in Candida albicans: a genome-wide H3K56 acetylation analysis.

Candida spp. represent the third most frequent worldwide cause of infection in Intensive Care Units with a mortality rate of almost 40%. The classes of antifungals currently available include azoles, polyenes, echinocandins, pyrimidine derivatives, and allylamines. However, the therapeutical options for the treatment of candidiasis are drastically reduced by the increasing antifungal resistance. The growing need for a more targeted antifungal therapy is limited by the concern of finding molecules that specifically recognize the microbial cell without damaging the host. Epigenetic writers and erasers have emerged as promising targets in different contexts, including the treatment of fungal infections. In C. albicans, Hst3p, a sirtuin that deacetylates H3K56ac, represents an attractive antifungal target as it is essential for the fungus viability and virulence. Although the relevance of such epigenetic regulator is documented for the development of new antifungal therapies, the molecular mechanism behind Hst3p-mediated epigenetic regulation remains unrevealed. Here, we provide the first genome-wide profiling of H3K56ac in C. albicans resulting in H3K56ac enriched regions associated with Candida sp. pathogenicity. Upon Hst3p inhibition, 447 regions gain H3K56ac. Importantly, these genomic areas contain genes encoding for adhesin proteins, degradative enzymes, and white-opaque switching. Moreover, our RNA-seq analysis revealed 1330 upregulated and 1081 downregulated transcripts upon Hst3p inhibition, and among them, we identified 87 genes whose transcriptional increase well correlates with the enrichment of H3K56 acetylation on their promoters, including some well-known regulators of phenotypic switching and virulence. Based on our evidence, Hst3p is an appealing target for the development of new potential antifungal drugs.

Role of Intracellular and Extracellular Annexin A1 in MIA PaCa-2 Spheroids Formation and Drug Sensitivity.

Among solid tumors, pancreatic cancer (PC) remains a leading cause of death. In PC, the protein ANXA1 has been identified as an oncogenic factor acting in an autocrine/paracrine way, and also as a component of tumor-deriving extracellular vesicles. Here, we proposed the experimental protocol to obtain spheroids from the two cell lines, wild-type (WT) and Annexin A1 (ANXA1) knock-out (KO) MIA PaCa-2, this last previously obtained through CRISPR/Cas9 genome editing system. The use of three-dimensional (3D) models, like spheroids, can be useful to mimic tumor characteristics and for preclinical chemo-sensitivity studies. By using PC spheroids, we have assessed the activity of intracellular and extracellular ANXA1. Indeed, we have proved that the intracellular protein influences in vitro tumor development and growth by spheroids analysis, in addition to defining the modification about cell protein pattern in ANXA1 KO model compared to the WT one. Moreover, we have tested the response to FOLFIRINOX chemotherapy regimen whose cytostatic effect appeared notably increased in ANXA1 KO spheroids. Additionally, this study has highlighted that the extracellular ANXA1 action is strengthened through the EVs supporting spheroids growth and resistance to drug treatment, mainly affecting tumor progression. Thus, our data interestingly suggest the relevance of ANXA1 as a potential therapeutic PC marker.

Drug affinity-responsive target stability unveils filamins as biological targets for artemetin, an anti-cancer flavonoid.

Artemetin is a valuable 5-hydroxy-3,6,7,3',4'-pentamethoxyflavone present in many different medicinal plants with very good oral bioavailability and drug-likeness values, owing to numerous bioactivities, such as anti-inflammatory and anti-cancer ones. Here, a multi-disciplinary plan has been settled and applied for identifying the artemetin target(s) to inspect its mechanism of action, based on drug affinity-responsive target stability and targeted limited proteolysis. Both approaches point to the disclosure of filamins A and B as direct artemetin targets in HeLa cell lysates, also giving detailed insights into the ligand/protein-binding sites. Interestingly, also 8-prenyl-artemetin, which is an artemetin more permeable semisynthetic analog, directly interacts with filamins A and B. Both compounds alter filamin conformation in living HeLa cells with an effect on cytoskeleton disassembly and on the disorganization of the F-actin filaments. Both the natural compound and its derivative are able to block cell migration, expectantly acting on tumor metastasis occurrence and development.

Production of Mesoglycan/PCL Based Composites through Supercritical Impregnation.

The development of targeted therapies for wound repair is knowing a growing interest due to the increasing aging of the population and the incidence of chronic pathologies, mainly pressure ulcers. Among molecules recruiting cell populations and promoting the formation of new vital tissue, sodium mesoglycan (MSG) has been proven to be effective in wound healing. In this work, MSG impregnation of polymer matrices has been attempted by a supercritical carbon dioxide-based process. Polymeric matrices are composed of polycaprolactone blends, where water-soluble polymers, polyethylene glycol, polyvinyl pyrrolidone, gelatin, and thermoplastic starch, have been employed to modulate the MSG release, making the devices potentially suitable for topical administrations. Two different techniques have been used to obtain the films: the first one is compression molding, producing compact and continuous structures, and the second one is electrospinning, producing membrane-like designs. A higher amount of MSG can be loaded into the polymeric matrix in the membrane-like structures since, in these films, the impregnation process is faster than in the case of compression molded films, where the carbon dioxide has firstly diffused and then released the active molecule. The type of water-soluble polymer influences the drug release rate: the blend polycaprolactone-gelatin gives a prolonged release potentially suitable for topical administration.

Plant hairy roots for the production of extracellular vesicles with antitumor bioactivity.

Plant extracellular vesicles (EVs) concentrate and deliver different types of bioactive molecules in human cells and are excellent candidates for a next-generation drug delivery system. However, the lack of standard protocols for plant EV production and the natural variations of their biomolecular cargo pose serious limitation to their use as therapeutics. To overcome these issues, we set up a versatile and standardized procedure to purify plant EVs from hairy root (HR) cultures, a versatile biotechnological system, already successfully employed as source of bioactive molecules with pharmaceutical and nutraceutical relevance. Herewith, we report that HR of Salvia dominica represent an excellent platform for the production of plant EVs. In particular, EVs derived from S. dominica HRs are small round-shaped vesicles carrying typical EV-associated proteins such as cytoskeletal components, chaperon proteins and integral membrane proteins including the tetraspanin TET-7. Interestingly, the HR-derived EVs showed selective and strong pro-apoptotic activity in pancreatic and mammary cancer cells. These results reveal that plant hairy roots may be considered a new promising tool in plant biotechnology for the production of extracellular vesicles for human health.

Cytotoxic abietane diterpenoids from Salvia leriifolia Benth.

The Phytochemical profiling of the root extract of Salvia leriifolia, an endemic plant of Iran, was investigated and 16 abietane diterpenes were isolated, and three were original compounds. 1D and 2D NMR and HRMS performed structural elucidation. The absolute configuration of the previously unreported compounds was determined by circular dichroism (ECD). The cytotoxicity of the isolated compounds was investigated against AGS, MIA PaCa-2, HeLa, and MCF-7 cell lines by the MTT assay. The known diterpene pisiferal possesses high cytotoxicity against all investigated cell lines at a concentration between 9.3 ± 0.6 and 14.38 ± 1.4 µM.

The combination of mesoglycan and VEGF promotes skin wound repair by enhancing the activation of endothelial cells and fibroblasts and their cross-talk.

Skin wound healing requires accurate therapeutic topical managements to accelerate tissue regeneration. Here, for the first time, we found that the association mesoglycan/VEGF has a strong pro-healing activity. In detail, this combination induces angiogenesis in human endothelial cells promoting in turn fibroblasts recruitment. These ones acquire a notable ability to invade the matrigel coating and to secrete an active form of metalloproteinase 2 in presence of endothelial cells treated with mesoglycan/VEGF. Next, by creating intrascapular lesions on the back of C57Bl6 mice, we observed that the topical treatments with the mesoglycan/VEGF promotes the closure of wounds more than the single substances beside the control represented by a saline solution. As revealed by eosin/hematoxylin staining of mice skin biopsies, treatment with the combination mesoglycan/VEGF allows the formation of a well-structured matrix with a significant number of new vessels. Immunofluorescence analyses have revealed the presence of endothelial cells at the closed region of wounds, as evaluated by CD31, VE-cadherin and fibronectin staining and of activated fibroblasts assessed by vimentin, col1A and FAP1α. These results encourage defining the association mesoglycan/VEGF to activate endothelial and fibroblast cell components in skin wound healing promoting the creation of new vessels and the deposition of granulation tissue.

Helicobacter pylori Pathogen-Associated Molecular Patterns: Friends or Foes?

Microbial infections are sensed by the host immune system by recognizing signature molecules called Pathogen-Associated Molecular Patterns-PAMPs. The binding of these biomolecules to innate immune receptors, called Pattern Recognition Receptors (PRRs), alerts the host cell, activating microbicidal and pro-inflammatory responses. The outcome of the inflammatory cascade depends on the subtle balance between the bacterial burn and the host immune response. The role of PRRs is to promote the clearance of the pathogen and to limit the infection by bumping inflammatory response. However, many bacteria, including Helicobacter pylori, evolved to escape PRRs' recognition through different camouflages in their molecular pattern. This review examines all the different types of H. pylori PAMPs, their roles during the infection, and the mechanisms they evolved to escape the host recognition.

Label-Free Quantitative Proteomics to Explore the Action Mechanism of the Pharmaceutical-Grade Triticum vulgare Extract in Speeding Up Keratinocyte Healing.

Plant extracts have shown beneficial properties in terms of skin repair, promoting wound healing through a plethora of mechanisms. In particular, the poly-/oligosaccharidic aqueous extract of Triticum vulgare (TVE), as well as TVE-based products, shows interesting biological assets, hastening wound repair. Indeed, TVE acts in the treatment of tissue regeneration mainly on decubitus and venous leg ulcers. Moreover, on scratched monolayers, TVE prompts HaCat cell migration, correctly modulating the expression of metalloproteases toward a physiological matrix remodeling. Here, using the same HaCat-based in vitro scratch model, the TVE effect has been investigated thanks to an LFQ proteomic analysis of HaCat secretomes and immunoblotting. Indeed, the unbiased TVE effect on secreted proteins has not yet been fully understood, and it could be helpful to obtain a comprehensive picture of its bio-pharmacological profile. It has emerged that TVE treatment induces significant up-regulation of several proteins in the secretome (153 to be exact) whereas only a few were down-regulated (72 to be exact). Interestingly, many of the up-regulated proteins are implicated in promoting wound-healing-related processes, such as modulating cell-cell interaction and communication, cell proliferation and differentiation, and prompting cell adhesion and migration.

The Pyrazolyl-Urea Gege3 Inhibits the Activity of ANXA1 in the Angiogenesis Induced by the Pancreatic Cancer Derived EVs.

The pyrazolyl-urea Gege3 molecule has shown interesting antiangiogenic effects in the tumor contest. Here, we have studied the role of this compound as interfering with endothelial cells activation in response to the paracrine effects of annexin A1 (ANXA1), known to be involved in promoting tumor progression. ANXA1 has been analyzed in the extracellular environment once secreted through microvesicles (EVs) by pancreatic cancer (PC) cells. Particularly, Gege3 has been able to notably prevent the effects of Ac2-26, the ANXA1 mimetic peptide, and of PC-derived EVs on endothelial cells motility, angiogenesis, and calcium release. Furthermore, this compound also inhibited the translocation of ANXA1 to the plasma membrane, otherwise induced by the same ANXA1-dependent extracellular stimuli. Moreover, these effects have been mediated by the indirect inhibition of protein kinase Cα (PKCα), which generally promotes the phosphorylation of ANXA1 on serine 27. Indeed, by the subtraction of intracellular calcium levels, the pathway triggered by PKCα underwent a strong inhibition leading to the following impediment to the ANXA1 localization at the plasma membrane, as revealed by confocal and cytofluorimetry analysis. Thus, Gege3 appeared an attractive molecule able to prevent the paracrine effects of PC cells deriving ANXA1 in the tumor microenvironment.

ANXA1 Contained in EVs Regulates Macrophage Polarization in Tumor Microenvironment and Promotes Pancreatic Cancer Progression and Metastasis.

The tumor microenvironment (TME) is a dynamic system where nontumor and cancer cells intercommunicate through soluble factors and extracellular vesicles (EVs). The TME in pancreatic cancer (PC) is critical for its aggressiveness and the annexin A1 (ANXA1) has been identified as one of the oncogenic elements. Previously, we demonstrated that the autocrine/paracrine activities of extracellular ANXA1 depend on its presence in EVs. Here, we show that the complex ANXA1/EVs modulates the macrophage polarization further contributing to cancer progression. The EVs isolated from wild type (WT) and ANXA1 knock-out MIA PaCa-2 cells have been administrated to THP-1 macrophages finding that ANXA1 is crucial for the acquisition of a protumor M2 phenotype. The M2 macrophages activate endothelial cells and fibroblasts to induce angiogenesis and matrix degradation, respectively. We have also found a significantly increased presence of M2 macrophage in mice tumor and liver metastasis sections previously obtained by orthotopic xenografts with WT cells. Taken together, our data interestingly suggest the relevance of ANXA1 as potential diagnostic/prognostic and/or therapeutic PC marker.

Synthesis, functional proteomics and biological evaluation of new 5-pyrazolyl ureas as potential anti-angiogenic compounds.

Based on biological results of previous synthesized pyrazolyl ureas able to interfere with angiogenesis process, we planned and synthesized the new benzyl-urea derivatives 2-4; some of them showed an interesting anti-proliferative profile and particularly 4e potently inhibited HUVEC proliferation. To shed light on the mechanism of action of 4e, its interactome has been deeply inspected to identify the most prominent protein partners, mainly taking into account kinome and phosphatome, through drug affinity responsive target stability experiments, followed by targeted limited proteolysis analysis. From these studies, PP1γ emerged as the most reliable 4e potential target in HUVEC. Molecular docking simulations on PP1γ were carried out to predict 4e binding mode. To assess its potential anti-angiogenic effect, 4e was tested in vitro to verify interference on kinase and phosphate activities. Overall, our results evidenced for 4e an interesting anti-angiogenic action, probably due to its action at intracellular level on PP1γ signalling pathways.