Split Gp41-1 intein splicing as a model to evaluate the cellular location of the oncosuppressor Maspin in an in vitro model of osteosarcoma.

Inteins are proteins involved in the protein splicing mechanism, an autoprocessing event, where sequences (exteins) separated by inteins become ligated each other after recombination. Two kinds of inteins have been described, contiguous inteins and split inteins. The former ones are transcribed and translated as a single peptide along with their exteins, while the latter are fragmented between two different genes and are transcribed and translated separately. The aim of this study is to establish a method to obtain a fluorescent eukaryotic protein to analyze its cellular localization, using the natural split gp41-1 inteins. We chose natural split inteins due to their distribution in all three domains of life. Two constructs were prepared, one containing the N-terminal split intein along with the N-moiety of the Red Fluorescent Protein (RFP) and a second construct containing the C-terminal of split intein, the C-moiety of RFP and the gene coding for Maspin, a tumor suppressor protein. The trans-splicing was verified by transfecting both N-terminal and C-terminal constructs into mammalian cells. The success of the recombination event was highlighted through the fluorescence produced by reconstituted RFP after recombination, along with the overlap of the red fluorescence produced by recombined RFP and the green fluorescence produced by the hybridization of the recombinant Maspin with a specific antibody. In conclusion, we opted to use this mechanism of recombination to obtain a fluorescent Maspin instead to express a large fusion protein, considering that it could interfere with Maspin's structure and function.

Nanostructured Poly-l-lactide and Polyglycerol Adipate Carriers for the Encapsulation of Usnic Acid: A Promising Approach for Hepatoprotection.

The present study investigates the utilization of nanoparticles based on poly-l-lactide (PLLA) and polyglycerol adipate (PGA), alone and blended, for the encapsulation of usnic acid (UA), a potent natural compound with various therapeutic properties including antimicrobial and anticancer activities. The development of these carriers offers an innovative approach to overcome the challenges associated with usnic acid's limited aqueous solubility, bioavailability, and hepatotoxicity. The nanosystems were characterized according to their physicochemical properties (among others, size, zeta potential, thermal properties), apparent aqueous solubility, and in vitro cytotoxicity. Interestingly, the nanocarrier obtained with the PLLA-PGA 50/50 weight ratio blend showed both the lowest size and the highest UA apparent solubility as well as the ability to decrease UA cytotoxicity towards human hepatocytes (HepG2 cells). This research opens new avenues for the effective utilization of these highly degradable and biocompatible PLLA-PGA blends as nanocarriers for reducing the cytotoxicity of usnic acid.

Design of bioactive and biomimetic scaffolds based on chitosan-alginate polyelectrolyte complexes for tissue engineering.

The replacement and regeneration of biological tissues by fabricating three-dimensional functionalized constructs that can improve material interaction with cells is an important challenge of tissue engineering. In this study, bioactive and biomimetic scaffolds based on chitosan-alginate polyelectrolyte complexes (PECs) were fabricated by freeze-drying method and then crosslinked with CaCl2. Various chitosan-alginate (CS-AL) molar ratios were used to obtain PECs with different structural and mechanical properties. The CS1-AL2.3 scaffold showed to possess the best mechanical properties (8 MPa) and good pore morphology with an average size of 100-150 µm. After the crosslinking process, a less porous structure but with higher elastic modulus (30 MPa) was obtained. To make matrix bioactive and biomimetic, the CS1-AL2.3 system was first functionalized with 3,4-dihydroxyhydrocinnamic acid (HCAF) and then with PySO3 or Heparin to introduce groups/molecules mimicking the extracellular matrix. While the antioxidant properties of the scaffolds containing HCAF improved by 3 orders of magnitude, compared to the non-functionalized matrix, the introduction of sulfonic groups into the bioactive scaffold made the structure more porous and hydrophilic with respect to the heparinized one also favoring the penetration and proliferation of fibroblasts into the scaffold. These results indicate the potential of these novel systems for tissue engineering.

Repositioned Natural Compounds and Nanoformulations: A Promising Combination to Counteract Cell Damage and Inflammation in Respiratory Viral Infections.

Respiratory viral diseases are among the most important causes of disability, morbidity, and death worldwide. Due to the limited efficacy or side effects of many current therapies and the increase in antiviral-resistant viral strains, the need to find new compounds to counteract these infections is growing. Since the development of new drugs is a time-consuming and expensive process, numerous studies have focused on the reuse of commercially available compounds, such as natural molecules with therapeutic properties. This phenomenon is generally called drug repurposing or repositioning and represents a valid emerging strategy in the drug discovery field. Unfortunately, the use of natural compounds in therapy has some limitations, due to their poor kinetic performance and consequently reduced therapeutic effect. The advent of nanotechnology in biomedicine has allowed this limitation to be overcome, showing that natural compounds in nanoform may represent a promising strategy against respiratory viral infections. In this narrative review, the beneficial effects of some promising natural molecules, curcumin, resveratrol, quercetin, and vitamin C, which have been already studied both in native form and in nanoform, against respiratory viral infections are presented and discussed. The review focuses on the ability of these natural compounds, analyzed in in vitro and in vivo studies, to counteract inflammation and cellular damage induced by viral infection and provide scientific evidence of the benefits of nanoformulations in increasing the therapeutic potential of these molecules.

The Formulation of the N-Acetylglucosamine as Nanoparticles Increases Its Anti-Inflammatory Activities: An In Vitro Study.

Nanomedicine can represent a new strategy to treat several types of diseases such as those with inflammatory aetiology. Through this strategy, it is possible to obtain nanoparticles with controlled shape, size, and eventually surface charge. Moreover, the use of molecules in nanoform may allow more effective delivery into the diseased cells and tissues, reducing toxicity and side effects of the used compounds. The aim of the present manuscript was the evaluation of the effects of N-acetylglucosamine in nanoform (GlcNAc NP) in an in vitro model of osteoarthritis (OA). Human primary chondrocytes were treated with Tumor Necrosis Factor (TNF)-α to simulate a low-grade inflammation and then treated with both GlcNAc and GlcNAc NP, in order to find the lowest concentrations able to counteract the inflammatory state of the cells and ensure a chondroprotective action. The findings showed that GlcNAc NP was able to decrease the pro-inflammatory mediators, IL-6 and IL-8, which are among the main effectors of inflammation; moreover, the nanoparticles downregulated the production of metalloprotease enzymes. GlcNAc NP was effective at a very low concentration compared to GlcNAc in its native form. Furthermore, GlcNAc NP stimulated an increase in collagen type II synthesis. In conclusion, the GlcNAc in nanoform showed better performance than GlcNAc, at concentrations lower than those reached in the joints after oral administration to patients of 1.5 g/die of glucosamine.

In Vitro Antiviral and Anti-Inflammatory Activities of N-Acetylglucosamine: Development of an Alternative and Safe Approach to Fight Viral Respiratory Infections.

Viral respiratory tract infections (RTIs) are responsible for significant morbidity and mortality worldwide. A prominent feature of severe respiratory infections, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, is the cytokine release syndrome. Therefore, there is an urgent need to develop different approaches both against viral replication and against the consequent inflammation. N-acetylglucosamine (GlcNAc), a glucosamine (GlcN) derivative, has been developed as an immunomodulatory and anti-inflammatory inexpensive and non-toxic drug for non-communicable disease treatment and/or prevention. Recent studies have suggested that GlcN, due to its anti-inflammatory activity, could be potentially useful for the control of respiratory virus infections. Our present study aimed to evaluate in two different immortalized cell lines whether GlcNAc could inhibit or reduce both viral infectivity and the inflammatory response to viral infection. Two different viruses, frequent cause of upper and lower respiratory tract infections, were used: the H1N1 Influenza A virus (IAV) (as model of enveloped RNA virus) and the Human adenovirus type 2 (Adv) (as model of naked DNA virus). Two forms of GlcNAc have been considered, bulk GlcNAc and GlcNAc in nanoform to overcome the possible pharmacokinetic limitations of GlcNAc. Our study suggests that GlcNAc restricts IAV replication but not Adv infection, whereas nano-GlcNAc inhibits both viruses. Moreover, GlcNAc and mainly its nanoformulation were able to reduce the pro-inflammatory cytokine secretion stimulated by viral infection. The correlation between inflammatory and infection inhibition is discussed.

The Nutraceuticals as Modern Key to Achieve Erythrocyte Oxidative Stress Fighting in Osteoarthritis.

Osteoarthritis (OA), the most common joint disease, shows an increasing prevalence in the aging population in industrialized countries. OA is characterized by low-grade chronic inflammation, which causes degeneration of all joint tissues, such as articular cartilage, subchondral bone, and synovial membrane, leading to pain and loss of functionality. Erythrocytes, the most abundant blood cells, have as their primary function oxygen transport, which induces reactive oxygen species (ROS) production. For this reason, the erythrocytes have several mechanisms to counteract ROS injuries, which cause damage to lipids and proteins of the cell membrane. Oxidative stress and inflammation are highly correlated and are both causes of joint disorders. In the synovial fluid and blood of osteoarthritis patients, erythrocyte antioxidant enzyme expression is decreased. To date, OA is a non-curable disease, treated mainly with non-steroidal anti-inflammatory drugs and corticosteroids for a prolonged period of time, which cause several side effects; thus, the search for natural remedies with anti-inflammatory and antioxidant activities is always ongoing. In this review, we analyze several manuscripts describing the effect of traditional remedies, such as Harpagophytum procumbens, Curcumin longa, and Boswellia serrata extracts, in the treatments of OA for their anti-inflammatory, analgesic, and antioxidant activity. The effects of such remedies have been studied both in in vitro and in vivo models, considering both joint cells and erythrocytes.

Harpagophytum procumbens Root Extract Mediates Anti-Inflammatory Effects in Osteoarthritis Synoviocytes through CB2 Activation.

The endocannabinoid system is involved in the nociceptive and anti-inflammatory pathways, and a lowered expression of CB2 receptors has been associated with inflammatory conditions, such as osteoarthritis (OA). This suggests that CB2 modulators could be novel therapeutic tools to treat OA. In the present study, the involvement of Harpagophytum procumbens root extract, a common ingredient of nutraceuticals used to treat joint disorders, in CB2 modulation has been evaluated. Moreover, to clarify the effects of the pure single components, the bioactive constituent, harpagoside, and the main volatile compounds were studied alone or in a reconstituted mixture. Human fibroblast-like synoviocytes, extracted by joints of patients, who underwent a total knee replacement, were treated with an H. procumbens root extract dissolved in DMSO (HPEDMSO). The effectiveness of HPEDMSO to affect CB2 pathways was studied by analyzing the modulation of cAMP, the activation of PKA and ERK MAP kinase, and the modulation of MMP-13 production. HPEDMSO was able to inhibit the cAMP production and MAP kinase activation and to down-regulate the MMP-13 production. Pure compounds were less effective than the whole phytocomplex, thus suggesting the involvement of synergistic interactions. Present findings encourage further mechanistic studies and support the scientific basis of the use of H. procumbens in joint disorders.

Efficacy of Combined Mechanical and Chemical Decontamination Treatments on Smooth and Rough Titanium Surfaces and Their Effects on Osteoconduction: An Ex Vivo Study.

PURPOSE: The aim of this ex vivo study was to assess the ability to remove oral biofilm by different combinations of mechanical and chemical treatments on smooth and rough titanium surfaces, as well as their impact on osteoconduction. MATERIALS AND METHODS: Forty-eight sandblasted acid-etched (SLA) and 48 machined titanium disks were contaminated with oral bacterial biofilm and exposed to the following treatments: (1) titanium brush (TB), (2) TB + 40% citric acid (CA), (3) TB + 5.25% sodium hypochlorite (NaOCl), (4) air polishing with glycine powder (AP), (5) AP + 40% CA, and (6) AP + 5.25% NaOCl. Residual bacteria and chemical contamination were assessed using viable bacterial count assay, scanning electron microscopy (SEM), and x-ray spectroscopy (XPS). Human primary osteoblast (hOB) adhesion and osteocalcin (OC) release were also evaluated. RESULTS: The microbiologic, SEM, and XPS analysis indicate a higher biofilm removal efficiency of combined mechanical-chemical treatments compared with exclusively mechanical approaches, especially on SLA surfaces. SEM analysis revealed significant alterations of surface microtopography on the disks treated with TB, while no changes were observed after AP treatment. OC release by hOBs was mainly decreased on disks treated with CA and NaOCl. CONCLUSION: The combination of mechanical and chemical treatments provides effective oral biofilm removal on both SLA and machined implant surfaces. NaOCl and CA may have a negative effect on osteoblasts cultured on SLA samples.

One-Pot Preparation of Hydrophilic Polylactide Porous Scaffolds by Using Safe Solvent and Choline Taurinate Ionic Liquid.

Polylactides (PLAs) are a class of polymers that are very appealing in biomedical applications due to their degradability in nontoxic products, tunable structural, and mechanical properties. However, they have some drawbacks related to their high hydrophobicity, lack of functional groups able to graft bioactive molecules, and solubility in unsafe solvents. To circumvent these shortcomings, porous scaffolds for tissue engineering were prepared by vigorously mixing a solution of isotactic and atactic PLA in nontoxic ethyl acetate at 70 °C with a water solution of choline taurinate. The partial aminolysis of the polymer ester bonds by taurine -NH2 brought about the formation of PLA oligomers with surfactant activity that stabilized the water-in-oil emulsion. Upon drying, a negligible shrinking occurred, and mechanically stable porous scaffolds were obtained. By varying the polymer composition and choline taurinate concentration, it was possible to modulate the pore dimensions (30-50 µm) and mechanical properties (Young's moduli: 1-6 MPa) of the samples. Furthermore, the grafted choline taurinate made the surface of the PLA films hydrophilic, as observed by contact angle measurements (advancing contact angle: 76°; receding contact angle: 40°-13°). The preparation method was very simple because it was based on a one-pot mild reaction that did not require an additional purification step, as all the employed chemicals were nontoxic.

Role of Caryophyllane Sesquiterpenes in the Entourage Effect of Felina 32 Hemp Inflorescence Phytocomplex in Triple Negative MDA-MB-468 Breast Cancer Cells.

Cannabis sativa L. crops have been traditionally exploited as sources of fibers, nutrients, and bioactive phytochemicals of medical interest. In the present study, two terpene-rich organic extracts, namely FOJ and FOS, obtained from Felina 32 hemp inflorescences collected in June and September, respectively, have been studied for their in vitro anticancer properties. Particularly, their cytotoxicity was evaluated in different cancer cell lines, and the possible entourage effect between nonintoxicating phytocannabinoids (cannabidiol and cannabichromene) and caryophyllane sesquiterpenes (ß-caryophyllene, ß-caryophyllene oxide and α-humulene), as identified at GC/MS analysis, was characterized. Modulation of cannabinoid CB1 and CB2 receptors was studied as a mechanistic hypothesis. Results highlighted marked cytotoxic effects of FOJ, FOS, and pure compounds in triple negative breast cancer MDA-MB-468 cells, likely mediated by a CB2 receptor activation. Cannabidiol was the main cytotoxic constituent, although low levels of caryophyllane sesquiterpenes and cannabichromene induced potentiating effects; the presence in the extracts of unknown antagonistic compounds has been highlighted too. These results suggest an interest in Felina 32 hemp inflorescences as a source of bioactive phytocomplexes with anticancer properties and strengthen the importance of considering the possible involvement of minor terpenes, such as caryophyllane sesquiterpenes, in the entourage effect of hemp-based extracts.

Metabolomic Profiling of Fresh Goji (Lycium barbarum L.) Berries from Two Cultivars Grown in Central Italy: A Multi-Methodological Approach.

The metabolite profile of fresh Goji berries from two cultivars, namely Big Lifeberry (BL) and Sweet Lifeberry (SL), grown in the Lazio region (Central Italy) and harvested at two different periods, August and October, corresponding at the beginning and the end of the maturation, was characterized by means of nuclear magnetic resonance (NMR) and electrospray ionization Fourier transform ion cyclotron resonance (ESI FT-ICR MS) methodologies. Several classes of compounds such as sugars, amino acids, organic acids, fatty acids, polyphenols, and terpenes were identified and quantified in hydroalcoholic and organic Bligh-Dyer extracts. Sweet Lifeberry extracts were characterized by a higher content of sucrose with respect to the Big Lifeberry ones and high levels of amino acids (glycine, betaine, proline) were observed in SL berries harvested in October. Spectrophotometric analysis of chlorophylls and total carotenoids was also carried out, showing a decrease of carotenoids during the time. These results can be useful not only to valorize local products but also to suggest the best harvesting period to obtain a product with a chemical composition suitable for specific industrial use. Finally, preliminary studies regarding both the chemical characterization of Goji leaves generally considered a waste product, and the biological activity of Big Lifeberry berries extracts was also investigated. Goji leaves showed a chemical profile rich in healthy compounds (polyphenols, flavonoids, etc.) confirming their promising use in the supplements/nutraceutical/cosmetic field. MG63 cells treated with Big Lifeberry berries extracts showed a decrease of iNOS, COX-2, IL-6, and IL-8 expression indicating their significant biological activity.

Chitosan scaffolds with enhanced mechanical strength and elastic response by combination of freeze gelation, photo-crosslinking and freeze-drying.

In this study, a new scaffold fabrication method based on the combination of a series of stabilization processes was set up to obtain chitosan scaffolds with improved mechanical properties for regeneration of load-bearing tissues. Specifically, thermally induced phase separation (TIPS) of chitosan solutions was used to obtain an open structure which was then stabilized by freeze-gelation and photo cross-linking. Freeze-gelation combined with freeze-drying permitted to obtain a porous structure with a 95 µm-mean pore size suitable for osteoblast cells' housing. Photo-crosslinking improved by ca. three times the scaffold compressive modulus, passing from 0,8 MPa of the uncrosslinked scaffolds to 2,2 MPa of the crosslinked one. Hydrated crosslinked scaffolds showed a good elastic response, with an 80% elastic recovery for at least 5 consecutive compressive cycles. The herein reported method has the advantage to not require the use of potentially toxic cross-linking agents and may be extended to other soft materials.

Biochemical and Computational Studies of the Interaction between a Glucosamine Derivative, NAPA, and the IKKα Kinase.

The glucosamine derivative 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-ß-D-glucose (NAPA), was shown to inhibit the kinase activity of IKKα, one of the two catalytic subunits of IKK complex, decreasing the inflammatory status in osteoarthritis chondrocytes. In the present work we have investigated the inhibition mechanism of IKKα by NAPA by combining computational simulations, in vitro assays and Mass Spectrometry (MS) technique. The kinase in vitro assay was conducted using a recombinant IKKα and IKKtide, a 20 amino acid peptide substrate derived from IkBα kinase protein and containing the serine residues Ser32 and Ser36. Phosphorylated peptide production was measured by Ultra Performance Liquid Chromatography coupled with Mass Spectrometry (UPLC-MS), and the atomic interaction between IKKα and NAPA has been studied by molecular docking and Molecular Dynamics (MD) approaches. Here we report that NAPA was able to inhibit the IKKα kinase activity with an IC50 of 0.5 mM, to decrease the Km value from 0.337 mM to 0.402 mM and the Vmax from 0.0257 mM·min-1 to 0.0076 mM·min-1. The computational analyses indicate the region between the KD, ULD and SDD domains of IKKα as the optimal binding site explored by NAPA. Biochemical data indicate that there is a non-significant difference between Km and Ki whereas there is a statistically significant difference between the two Vmax values. This evidence, combined with computational results, consistently indicates that the inhibition is non-competitive, and that the NAPA binding site is different than that of ATP or IKKtide.

Pheomelanin Effect on UVB Radiation-Induced Oxidation/Nitration of l-Tyrosine.

Pheomelanin is a natural yellow-reddish sulfur-containing pigment derived from tyrosinase-catalyzed oxidation of tyrosine in presence of cysteine. Generally, the formation of melanin pigments is a protective response against the damaging effects of UV radiation in skin. However, pheomelanin, like other photosensitizing substances, can trigger, following exposure to UV radiation, photochemical reactions capable of modifying and damaging cellular components. The photoproperties of this natural pigment have been studied by analyzing pheomelanin effect on oxidation/nitration of tyrosine induced by UVB radiation at different pH values and in presence of iron ions. Photoproperties of pheomelanin can be modulated by various experimental conditions, ranging from the photoprotection to the triggering of potentially damaging photochemical reactions. The study of the photomodification of l-Tyrosine in the presence of the natural pigment pheomelanin has a special relevance, since this tyrosine oxidation/nitration pathway can potentially occur in vivo in tissues exposed to sunlight and play a role in the mechanisms of tissue damage induced by UV radiation.

Antiarthritic Effects of a Root Extract from Harpagophytum procumbens DC: Novel Insights into the Molecular Mechanisms and Possible Bioactive Phytochemicals.

Harpagophytum procumbens (Burch.) DC. ex Meisn. is a traditional remedy for osteoarticular diseases, including osteoarthritis (OA), although the bioactive constituents and mechanisms involved are yet to be clarified. In the present study, an aqueous H. procumbens root extract (HPE; containing 1.2% harpagoside) was characterized for its effects on synoviocytes from OA patients and phytochemical composition in polyphenols, and volatile compounds were detected. HPE powder was dissolved in different solvents, including deionized water (HPEH2O), DMSO (HPEDMSO), 100% v/v ethanol (HPEEtOH100), and 50% v/v ethanol (HPEEtOH50). The highest polyphenol levels were found in HPEDMSO and HPEEtOH50, whereas different volatile compounds, mainly ß-caryophyllene and eugenol, were detected in all the extracts except for HPEH2O. HPEH2O and HPEDMSO were able to enhance CB2 receptor expression and to downregulate PI-PLC ß2 in synovial membranes; moreover, all the extracts inhibited FAAH activity. The present results highlight for the first time a multitarget modulation of the endocannabinoid system by HPE, likely ascribable to its hydrosoluble compounds, along with the presence of volatile compounds in H. procumbens root. Although hydrosoluble compounds seem to be mainly responsible for endocannabinoid modulation by HPE, a possible contribution of volatile compounds can be suggested, strengthening the hypothesis that the entire phytocomplex can contribute to the H. procumbens healing properties.

Platelet Rich Fibrin (PRF) and Its Related Products: Biomolecular Characterization of the Liquid Fibrinogen.

Liquid fibrinogen is an injectable platelet concentrate rich in platelets, leukocytes, and fibrinogen obtained by blood centrifugation. The aim of this study was to analyze the release of different growth factors in the liquid fibrinogen at different times and to assess possible correlations between growth factors and cell counts. The concentration of transforming growth factor beta 1 (TGF-ß1), platelet-derived growth factor-AB (PDGF-AB), platelet-derived growth factor-BB (PDGF-BB), bone morphogenetic protein 2 (BMP-2), fibroblast growth factor 2 (FGF-2) and vascular endothelial growth factor (VEGF) released by liquid fibrinogen were examined with ELISA at three time points (T0, time of collection; T7, 7 days; T14, 14 days). The cellular content of the liquid fibrinogen and whole blood was also calculated for each volunteer. A mean accumulation of platelets of almost 1.5-fold in liquid fibrinogen compared to whole blood samples was found. An increase of TGF-ß1, PDGF-AB, FGF-2, and VEGF levels was detected at T7. At T14, the level of TGF-ß1 returned to T0 level; PDGF-AB amount remained high; the levels of FGF-2 and VEGF decreased with respect to T7, but remained higher than the T0 levels; PDGF-BB was high at all time points; BMP-2 level was low and remained constant at all time points. TGF-ß1, PDGF-AB, and PDGF-BB showed a correlation with platelet amount, whereas BMP-2, FGF-2, and VEGF showed a mild correlation with platelet amount. Due to the high concentration of platelets, liquid fibrinogen does contain important growth factors for the regeneration of both soft and hard tissue. The centrifugation protocol tested in this study provides a valid solution to stimulate wound healing in oral and periodontal surgery.