Growth factors, CD34 positive cells, and fibrin network analysis in concentrated growth factors fraction

An interesting clinical option for optimizing healing tissue is the use of platelet concentrate. Platelets contain high quantities of growth factors, among these TGF-β1 and VEGF, which are known to be implicated in tissue regeneration. CGF is produced by processing blood samples with a special centrifuge device; three layers are formed: top acellular plasma (PPP), middle CGF and bottom red blood cells (RBC) layers. Given that to date there are no data concerning the biological characteristic of CGF, the aim of this study was to evaluate the presence of TGF-β1 and VEGF in CGF and also in PPP and RBC layers. In addition, since circulating stem cells are recruited from blood to injured tissue for healing we also evaluated the presence of CD34 positive cells. Our data show the presence of TGF-β1 and VEGF in CGF and RBC layers. In addition, we show CD34 positive cells in CGF.     

Engineering of poly(ε-caprolactone) microcarriers to modulate protein encapsulation capability and release kinetic

Drug delivery applications using biodegradable polymeric microspheres are becoming an important means of delivering therapeutic agents. The aim of this work was to modulate the microporosity of poly(ε-caprolactone) (PCL) microcarriers to control protein loading capability and release profile. PCL microparticles loaded with BSA (bovine serum albumin) have been de novo synthesized with double emulsion solvent evaporation technique transferred and adapted for different polymer concentrations (1.7 and 3% w/v) and stabilizer present in the inner aqueous phase (0.05, 0.5 and 1% w/v). SEM (scanning electron microscope) and CLSM (confocal laser scanning microscope) analysis map the drug distribution in homogeneously distributed cavities inside the microspheres with dimensions that can be modulated by varying double emulsion process parameters. The inner structure of BSA-loaded microspheres is greatly affected by the surfactant concentration in the internal aqueous phase, while a slight influence of polymer concentration in the oil phase was observed. The surfactant concentration mainly determines microspheres morphology, as well as drug release kinetics, as confirmed by our in-vitro BSA release study. Moreover, the entrapped protein remained unaltered during the protein encapsulation process, retaining its bio-activity and structure, as shown through a dedicated gel chromatographic analytical method.     

Equine Chorionic Gonadotropin as an Effective FSH Replacement for In Vitro Ovine Follicle and Oocyte Development

The use of assisted reproductive technologies (ART) still requires strategies through which to maximize individual fertility chances. In vitro folliculogenesis (ivF) may represent a valid option to convey the large source of immature oocytes in ART. Several efforts have been made to set up ivF cultural protocols in medium-sized mammals, starting with the identification of the most suitable gonadotropic stimulus. In this study, Equine Chorionic Gonadotropin (eCG) is proposed as an alternative to Follicle Stimulating Hormone (FSH) based on its long superovulation use, trans-species validation, long half-life, and low costs. The use of 3D ivF on single-ovine preantral (PA) follicles allowed us to compare the hormonal effects and to validate their influence under two different cultural conditions. The use of eCG helped to stimulate the in vitro growth of ovine PA follicles by maximizing its influence under FBS-free medium. Higher performance of follicular growth, antrum formation, steroidogenic activity and gap junction marker expression were recorded. In addition, eCG, promoted a positive effect on the germinal compartment, leading to a higher incidence of meiotic competent oocytes. These findings should help to widen the use of eCG to ivF as a valid and largely available hormonal support enabling a synchronized in vitro follicle and oocyte development.     

Effects of the Escherichia coli Bacterial Toxin Cytotoxic Necrotizing Factor 1 on Different Human and Animal Cells: A Systematic Review

Cytotoxic necrotizing factor 1 (CNF1) is a bacterial virulence factor, the target of which is represented by Rho GTPases, small proteins involved in a huge number of crucial cellular processes. CNF1, due to its ability to modulate the activity of Rho GTPases, represents a widely used tool to unravel the role played by these regulatory proteins in different biological processes. In this review, we summarized the data available in the scientific literature concerning the observed in vitro effects induced by CNF1. An article search was performed on electronic bibliographic resources. Screenings were performed of titles, abstracts, and full-texts according to PRISMA guidelines, whereas eligibility criteria were defined for in vitro studies. We identified a total of 299 records by electronic article search and included 76 original peer-reviewed scientific articles reporting morphological or biochemical modifications induced in vitro by soluble CNF1, either recombinant or from pathogenic Escherichia coli extracts highly purified with chromatographic methods. Most of the described CNF1-induced effects on cultured cells are ascribable to the modulating activity of the toxin on Rho GTPases and the consequent effects on actin cytoskeleton organization. All in all, the present review could be a prospectus about the CNF1-induced effects on cultured cells reported so far.     

Vascular-confined multi-passage discoidal nanoconstructs for the low-dose docetaxel inhibition of triple-negative breast cancer growth

Taxane efficacy in triple negative breast cancer(TNBC)is limited by insufficient tumor accumulation and severe off-target effects.Nanomedicines offer a unique opportunity to enhance the anti-cancer potency of this drug.Here,1,000 nm×400 nm discoidal polymeric nanoconstructs(DPN)encapsulating docetaxel(DTXL)and the near infrared compound Iipid-Cy5 were engineered.DPN were obtained by filling multiple times cylindrical wells in a poly(vinyl alcohol)template with a polymer mixture comprising poly(lactic-co-glycolic acid)(PLGA)and poly(ethylene glycol)diacrylate(PEG-DA)chains together with therapeutic and imaging agents.The resulting“multi-passage”DPN exhibited higher DTXL loading,Iipid-Cy5 stability,and stiffness as compared to the conventional"single-passage"approach.Confocal microscopy confirmed that DTXL-DPN were not taken up by MDA-MB-231 cells but would rather sit next to the cell membrane and slowly release DTXL thereof.Empty DPN had no toxicity on TNBC cells,whereas DTXL-DPN presented a cytotoxic potential comparable to free DTXL(IC₅₀=2.6 nM±1.0 nM vs.7.0 nM±1.09 nM at 72 h).In orthotopic murine models,DPN accumulated in TNBC more efficiently than free-DTXL.With only 2 mg/kg DTXL,intravenously administered every 2 days for a total of 13 treatments,DTXL-DPN induced tumor regression and were associated to an overall 80%survival rate as opposed to a 30%survival rate for free-DTXL,at 120 days.All untreated mice succumbed before 90 days.Collectively,this data demonstrates that vascular confined multi-passage DPN,biomimicking the behavior of circulating platelets,can efficiently deliver chemotherapeutic molecules to malignant tissues and effectively treat orthotopic TNBC at minimal taxane doses.     

Peptides Derived from Angiogenin Regulate Cellular Copper Uptake

The angiogenin protein (ANG) is one of the most potent endogenous angiogenic factors. In this work we characterized by means of potentiometric, spectroscopic and voltammetric techniques, the copper complex species formed with peptide fragments derived from the N-terminal domain of the protein, encompassing the sequence 1-17 and having free amino, Ang1-17, or acetylated N-terminus group, AcAng1-17, so to explore the role of amino group in metal binding and cellular copper uptake. The obtained data show that amino group is the main copper anchoring site for Ang1-17. The affinity constant values, metal coordination geometry and complexes redox-potentials strongly depend, for both peptides, on the number of copper equivalents added. Confocal laser scanning microscope analysis on neuroblastoma cells showed that in the presence of one equivalent of copper ion, the free amino Ang1-17 increases cellular copper uptake while the acetylated AcAng1-17 strongly decreases the intracellular metal level. The activity of peptides was also compared to that of the protein normally present in the plasma (wtANG) as well as to the recombinant form (rANG) most commonly used in literature experiments. The two protein isoforms bind copper ions but with a different coordination environment. Confocal laser scanning microscope data showed that the wtANG induces a strong increase in intracellular copper compared to control while the rANG decreases the copper signal inside cells. These data demonstrate the relevance of copper complexes’ geometry to modulate peptides’ activity and show that wtANG, normally present in the plasma, can affect cellular copper uptake.     

Combined GC/MS analytical procedure for the characterization of glycerolipid, waxy, resinous, and proteinaceous materials in a unique paint microsample

A novel GC/MS analytical procedure for the identification of lipids, waxes, proteins, and resinous materials in the same microsample from painted works of art has been optimized. It is based on a sample multistep chemical pretreatment (solvent extractions and microwave-assisted chemolysis) that is able to separate the various organic components into different fractions, which are suitably treated and derivatized before analysis. In particular, the procedure allows the complete saponification of wax esters and the completeness of the Cannizzaro type reaction of shellac acids in conditions that are suitable also for glycerides saponification. The method was tested on reference materials for the identification of proteinaceous binders (egg, collagen, casein) on the basis of the quantitative determination of the amino acid profile and the identification of glycerolipids (linseed oil, poppy seed oil, walnut oil, and egg), plant resins (Pinaceae resins, sandarac, mastic, and dammar), animal resins (shellac), tars or pitches, and natural waxes (beeswax, carnauba wax) on the basis of the determination of fatty acid, alcohol, and hydrocarbon profiles and of significant terpenic molecular markers. The procedure was applied to the characterization of three old paint microsamples. Animal glue, egg, linseed oil, beeswax, Pinaceae resin, dammar, and shellac were the identified materials found in mixtures and recognized as original and/or restoration substances.