An integrated design strategy coupling additive manufacturing and matrix-assisted pulsed laser evaporation (MAPLE) towards the development of a new concept 3D scaffold with improved properties for tissue regeneration.

Bioinspired strategies for scaffold design and optimization were improved by the introduction of Additive Manufacturing (AM), thus allowing for replicating and reproducing complex shapes and structures in a reliable manner, adopting different kinds of polymeric and nanocomposite materials properly combined according to the features of the natural host tissues. Benefiting from recent findings in AM, a Matrix-Assisted Pulsed Laser Evaporation (MAPLE) technique was employed for obtaining graphene-like material (GL) uniform coatings on 3D scaffolds for tissue repair strategies, towards the development of a new concept 3D scaffold with controlled morphological/architectural and surface features and mechanical and biological properties. The effect of the material-design combination through an integrated technological approach (i.e., MAPLE deposition of GL on 3D AM PCL scaffolds) was assessed through scanning electron microscopy, atomic force microscopy, contact angle measurements, mechanical measurements and biological analyses (cell viability assay and alkaline phosphatase activity) in conjunction with confocal laser scanning microscopy. The differentiation of hMSCs towards the osteoblast phenotype was also investigated analysing the gene expression profile. The obtained findings provided a further insight into the development of improved strategies for the functionalization or combination of GL with other materials and 3D structures in a hybrid fashion for ensuring a tighter adhesion onto the substrates, improving cell fate over time, without negatively altering the mechanical properties and behaviour of the neat constructs. In particular, the results provided interesting information, making 3D AM GL-coated scaffolds potential candidates for bone tissue engineering.

Multi-step Strategies Toward Regioselectively Sulfated M-Rich Alginates.

Sulfated alginates (ASs), as well as several artificially sulfated polysaccharides, show interesting bioactivities. The key factors for structure-activity relationships studies are the degree of sulfation and the distribution of the sulfate groups along the polysaccharide backbone (sulfation pattern). The former parameter can often be controlled through stoichiometry, while the latter requires the development of suitable chemical or enzymatic, regioselective methods and is still missing for ASs. In this work, a study on the regioselective installation of several different protecting groups on a d-mannuronic acid enriched (M-rich) alginate is reported in order to develop a semi-synthetic access to regioselectively sulfated AS derivatives. A detailed structural characterization of the obtained ASs revealed that the regioselective sulfation could be achieved complementarily at the O-2 or O-3 positions of M units through multi-step sequences relying upon a silylating or benzoylating reagent for the regioselective protection of M-rich alginic acid, followed by sulfation and deprotection.

CO2 Capture and Gas Storage Capacities Enhancement of HKUST-1 by Hybridization with Functionalized Graphene-like Materials.

The role of graphene related material (GRM) functionalization on the structural and adsorption properties of MOF-based hybrids was deepened by exploring the use of three GRMs obtained from the chemical demolition of a nanostructured carbon black. Oxidized graphene-like (GL-ox), hydrazine reduced graphene-like (GL), and amine-grafted graphene-like (GL-NH2) materials have been used for the preparation of Cu-HKUST-1 based hybrids. After a full structural characterization, the hybrid materials underwent many adsorption-desorption cycles to evaluate their capacities to capture CO2 and store CH4 at high pressure. All the MOF-based samples showed very high specific surface area (SSA) values and total pore volumes, but different pore size distributions attributed to the instauration of interactions between the MOF precursors and the specific functional groups on the GRM surface during MOF growth. All the samples showed a good affinity toward both gases (CO2 and CH4) and a comparable structural stability and integrity (possible aging was excluded). The trend of the maximum storage capacity values of the four MOF samples toward CO2 and CH4 was HKUST-1/GL-NH2 > HKUST-1 > HKUST-1/GL-ox > HKUST-1/GL. Overall, the measured CO2 and CH4 uptakes were in line with or higher than those already reported in the open literature for Cu-HKUST-1 based hybrids evaluated in similar conditions.

Pyrolysis and Gasification of a Real Refuse-Derived Fuel (RDF): The Potential Use of the Products under a Circular Economy Vision.

Refuse-Derived Fuels (RDFs) are segregated forms of wastes obtained by a combined mechanical-biological processing of municipal solid wastes (MSWs). The narrower characteristics, e.g., high calorific value (18-24 MJ/kg), low moisture content (3-6%) and high volatile (77-84%) and carbon (47-56%) contents, make RDFs more suitable than MSWs for thermochemical valorization purposes. As a matter of fact, EU regulations encourage the use of RDF as a source of energy in the frameworks of sustainability and the circular economy. Pyrolysis and gasification are promising thermochemical processes for RDF treatment, since, compared to incineration, they ensure an increase in energy recovery efficiency, a reduction of pollutant emissions and the production of value-added products as chemical platforms or fuels. Despite the growing interest towards RDFs as feedstock, the literature on the thermochemical treatment of RDFs under pyrolysis and gasification conditions still appears to be limited. In this work, results on pyrolysis and gasification tests on a real RDF are reported and coupled with a detailed characterization of the gaseous, condensable and solid products. Pyrolysis tests have been performed in a tubular reactor up to three different final temperatures (550, 650 and 750 °C) while an air gasification test at 850 °C has been performed in a fluidized bed reactor using sand as the bed material. The results of the two thermochemical processes are analyzed in terms of yield, characteristics and quality of the products to highlight how the two thermochemical conversion processes can be used to accomplish waste-to-materials and waste-to-energy targets. The RDF gasification process leads to the production of a syngas with a H2/CO ratio of 0.51 and a tar concentration of 3.15 g/m3.

Coating of Flexible PDMS Substrates through Matrix-Assisted Pulsed Laser Evaporation (MAPLE) with a New-Concept Biocompatible Graphenic Material.

In this study, matrix-assisted pulsed laser evaporation (MAPLE) was used to deposit graphene-like materials (GL), a new class of biocompatible graphene-related materials (GRMs) obtained from a controlled top-down demolition of a carbon black, on silicone slices to test their potential use as functional coating on invasive medical devices as indwelling urinary catheters. Results indicate that the relevant chemical-physical features of the deposit (controlled by FTIR and AFM) were maintained after MAPLE deposition. After deposition, GL films underwent a biological survey toward target cellular lines (murine fibroblast NIH3T3, human keratinocytes HaCAT and the human cervical adenocarcinoma epithelial-like HeLa). Results indicate that the GL films did not lead to any perturbations in the different biological parameters evaluated. The presented results and the possibility to further functionalize the GL or combine them with other functional materials in a hybrid fashion to assure a tighter adhesion onto the substrate for use in harsh conditions open the door to practical applications of these new-concept medical devices (drug delivery, next generation flexible devices, multifunctional coatings) paving the way to the prevention of nosocomial infections driven by catheterization through antibiotics-free approaches.

Sulfated lactose-modified chitosan. A novel synthetic glycosaminoglycan-like polysaccharide inducing chondrocyte aggregation.

Lactose-modified chitosan (CTL) is sulfated using SO3·py or SO3·DMF as sulfating agents. The two products are characterized by elemental analysis, FT-IR, 1H,13C-DEPT-HSQC and 1H,13C-HSQC-TOCSY experiments which allow the extent and selectivity of chemical sulfation to be determined. Dynamic Light Scattering shows a pH-dependent association of the sulfated polysaccharides which are described as flexible by the Smidsrød's B parameter and the intrinsic viscosity at infinite ionic strength. Shear viscosity and intrinsic viscosity show that sulfation protocols lead to chain scission which is more pronounced when SO3·DMF is used. The sulfated samples are able to induce aggregation of human bone marrow mesenchymal stem cells, resulting in the formation of smaller nodules compared to the unmodified CTL sample. Over time, the sample with the higher degree of sulfation allows further aggregation between cell clusters while the sample with the lower degree of sulfation shows dissolution of the aggregates.

COVID-19 Vaccine mRNABNT162b2 Elicits Human Antibody Response in Milk of Breastfeeding Women.

OBJECTIVE: The objective of this research is to demonstrate the release of SARS-CoV-2 Spike (S) antibodies in human milk samples obtained by patients who have been vaccinated with mRNABNT162b2 vaccine. METHODS: Milk and serum samples were collected in 10 volunteers 20 days after the first dose and 7 seven days after the second dose of the mRNABNT162b2 vaccine. Anti-SARS-CoV-2 S antibodies were measured by the Elecsys® Anti-SARS-CoV-2 S ECLIA assay (Roche Diagnostics AG, Rotkreuz, Switzerland), a quantitative electrochemiluminescence immunometric method. RESULTS: At first sample, anti-SARS-CoV-2 S antibodies were detected in all serum samples (103.9 ± 54.9 U/mL) and only in two (40%) milk samples with a low concentration (1.2 ± 0.3 U/mL). At the second sample, collected 7 days after the second dose, anti-SARS-CoV-2 S antibodies were detected in all serum samples (3875.7 ± 3504.6 UI/mL) and in all milk samples (41.5 ± 47.5 UI/mL). No correlation was found between the level of serum and milk antibodies; the milk antibodies/serum antibodies ratio was on average 2% (range: 0.2-8.4%). CONCLUSION: We demonstrated a release of anti-SARS-CoV-2 S antibodies in the breast milk of women vaccinated with mRNABNT162b2. Vaccinating breastfeeding women could be a strategy to protect their infants from COVID-19 infection.

Suitability of Nanoparticles to Face Benzo(a)pyrene-Induced Genetic and Chromosomal Damage in M. galloprovincialis. An In Vitro Approach.

Benzo(a)pyrene (B(a)P) is a well-known genotoxic agent, the removal of which from environmental matrices is mandatory, necessitating the application of cleaning strategies that are harmless to human and environmental health. The potential application of nanoparticles (NPs) in the remediation of polluted environments is of increasing interest. Here, specifically designed NPs were selected as being non-genotoxic and able to interact with B(a)P, in order to address the genetic and chromosomal damage it produces. A newly formulated pure anatase nano-titanium (nano-TiO2), a commercial mixture of rutile and anatase, and carbon black-derived hydrophilic NPs (HNP) were applied. Once it had been ascertained that the NPs selected for the work did not induce genotoxicity, marine mussel gill biopsies were exposed in vitro to B(a)P (2 µg/mL), alone and in combination with the selected NPs (50 µg/mL nano-TiO2, 10 µg/mL HNP). DNA primary reversible damage was evaluated by means of the Comet assay. Chromosomal persistent damage was assessed on the basis of micronuclei frequency and nuclear abnormalities by means of the Micronucleus-Cytome assay. Transmission Electron Microscopy (TEM) was performed to investigate the mechanism of action exerted by NPs. Pure Anatase n-TiO2 was found to be the most suitable for our purpose, as it is cyto- and genotoxicity free and able to reduce the genetic and chromosomal damage associated with exposure to B(a)P.

Clinical features of ProMisE groups identify different phenotypes of patients with endometrial cancer.

BACKGROUND: The Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) groups has identified four molecular prognostic groups of endometrial cancer (EC): POLE-mutated (POLE-mt), mismatch repair-deficient (MMR-d), p53-abnormal (p53-abn), p53-wild-type (p53-wt). These groups might have different pathogenesis and risk factors, and might occur in different phenotypes of patients. However, these data are still lacking. OBJECTIVE: To provide a clinical characterization of the ProMisE groups of EC. METHODS: A systematic review and meta-analysis was performed by searching seven electronic databases from their inception to December 2020, for all studies reporting clinical characteristics of EC patients in each ProMisE group. Pooled means of age and BMI and pooled prevalence of FIGO stage I and adjuvant treatment in each ProMisE group were calculated. RESULTS: Six studies with 1, 879 women were included in the systematic review. Pooled means (with standard error) and prevalence values were: in the MMR-d group, age = 66.5 ± 0.6; BMI = 30.6 ± 1.2; stage I = 72.6%; adjuvant treatment = 47.3%; in the POLE-mt group, age = 58.6 ± 2.7; BMI = 27.2 ± 0.9; stage I = 93.7%; adjuvant treatment = 53.6%; in the p53-wt group, age = 64.2 ± 1.9; BMI = 32.3 ± 1.4; stage I = 80.5%; adjuvant treatment = 45.3%; in the p53-abn group, age = 71.1 ± 0.5; BMI = 29.1 ± 0.5; stage I = 50.8%; adjuvant treatment = 64.4%. CONCLUSION: The ProMisE groups identify different phenotypes of patients. The POLE-mt group included the youngest women, with the lower BMI and the highest prevalence of stage I. The p53-wt group included patients with the highest BMI. The p53-abn group included the oldest women, with the highest prevalence of adjuvant treatment and the lowest prevalence of stage I. The MMR-d group showed intermediate values among the ProMisE groups for all clinical features.

Graphene-Like Layers from Carbon Black: In Vivo Toxicity Assessment.

Graphene-like (GL) layers, a new graphene-related material (GRM), possess peculiar chemical, colloidal, optical and transport properties. Considering the very recent promising application of GL layers in biomedical and bioelectronic fields, it is of utmost importance to investigate the toxicological profile of these nanomaterials. This study represents an important first report of a complete in vivo toxicity assessment of GL layers on embryonic zebrafish (Danio rerio). Our results show that GL layers do not lead to any perturbations in the different biological parameters evaluated, indicating their good biocompatibility on a vertebrate model. The new insight into the biosafety of GL layers will expand their applications in nanomedicine.

Biopolymer Skeleton Produced by Rhizobium radiobacter: Stoichiometric Alternation of Glycosidic and Amidic Bonds in the Lipopolysaccharide O-Antigen.

The lipopolysaccharide (LPS) O-antigen structure of the plant pathogen Rhizobium radiobacter strain TT9 and its possible role in a plant-microbe interaction was investigated. The analyses disclosed the presence of two O-antigens, named Poly1 and Poly2. The repetitive unit of Poly2 constitutes a 4-α-l-rhamnose linked to a 3-α-d-fucose residue. Surprisingly, Poly1 turned out to be a novel type of biopolymer in which the repeating unit is formed by a monosaccharide and an amino-acid derivative, so that the polymer has alternating glycosidic and amidic bonds joining the two units: 4-amino-4-deoxy-3-O-methyl-d-fucose and (2'R,3'R,4'S)-N-methyl-3',4'-dihydroxy-3'-methyl-5'-oxoproline). Differently from the O-antigens of LPSs from other pathogenic Gram-negative bacteria, these two O-antigens do not activate the oxidative burst, an early innate immune response in the model plant Arabidopsis thaliana, explaining at least in part the ability of this R. radiobacter strain to avoid host defenses during a plant infection process.

Eumelanin Graphene-Like Integration: The Impact on Physical Properties and Electrical Conductivity.

The recent development of eumelanin pigment-based blends integrating "classical" organic conducting materials is expanding the scope of eumelanin in bioelectronics. Beyond the achievement of high conductivity level, another major goal lays in the knowledge and feasible control of structure/properties relationship. We systematically investigated different hybrid materials prepared by in situ polymerization of the eumelanin precursor 5,6-dihydroxyindole (DHI) in presence of various amounts of graphene-like layers. Spectroscopic studies performed by solid state nuclear magnetic resonance (ss-NMR), x-ray photoemission, and absorption spectroscopies gave a strong indication of the direct impact that the integration of graphene-like layers into the nascent polymerized DHI-based eumelanin has on the structural organization of the pigment itself, while infrared, and photoemission spectroscopies indicated the occurrence of negligible changes as concerns the chemical units. A tighter packing of the constituent units could represent a strong factor responsible for the observed improved electrical conductivity of the hybrid materials, and could be possible exploited as a tool for electrical conductivity tuning.

Diesel exhaust particles induce autophagy and citrullination in Normal Human Bronchial Epithelial cells.

A variety of environmental agents has been found to influence the development of autoimmune diseases; in particular, the studies investigating the potential association of systemic autoimmune rheumatic diseases with environmental micro and nano-particulate matter are very few and contradictory. In this study, the role of diesel exhaust particles (DEPs), one of the most important components of environment particulate matter, emitted from Euro 4 and Euro 5 engines in altering the Normal Human Bronchial Epithelial (NHBE) cell biological activity was evaluated. NHBE cells were exposed in vitro to Euro 4 and Euro 5 particle carbon core, sampled upstream of the typical emission after-treatment systems (diesel oxidation catalyst and diesel particulate filter), whose surfaces have been washed from well-assessed harmful species, as polycyclic aromatic hydrocarbons (PAHs) to: (1) investigate their specific capacity to affect cell viability (flow cytometry); (2) stimulate the production of the pro-inflammatory cytokine IL-18 (Enzyme-Linked ImmunoSorbent Assay -ELISA-); (3) verify their specific ability to induce autophagy and elicit protein citrullination and peptidyl arginine deiminase (PAD) activity (confocal laser scanning microscopy, immunoprecipitation, Sodium Dodecyl Sulphate-PolyAcrylamide Gel Electrophoresis -SDS-PAGE- and Western blot, ELISA). In this study we demonstrated, for the first time, that both Euro 4 and Euro 5 carbon particles, deprived of PAHs possibly adsorbed on the soot surface, were able to: (1) significantly affect cell viability, inducing autophagy, apoptosis and necrosis; (2) stimulate the release of the pro-inflammatory cytokine IL-18; (3) elicit protein citrullination and PAD activity in NHBE cells. In particular, Euro 5 DEPs seem to have a more marked effect with respect to Euro 4 DEPs.

Assessing the Potential of Biochars Prepared by Steam-Assisted Slow Pyrolysis for CO2 Adsorption and Separation.

The potentialities in the use of biochars prepared by steam-assisted slow pyrolysis as adsorbents of gases of strategic interest (N2, CO2, and CH4) and their mixtures were explored. The biochars prepared from Populus nigra wood and cellulose fibers exhibited a narrow microporosity, with average pore sizes ranging between 0.55 and 0.6 nm. The micropore volume increased with the pyrolysis temperature, allowing CO2 and CH4 uptakes at room temperature between 1.5 and 2.5 mmol/g and between 0.1 and 0.5 mmol/g, respectively. These values are in line with those from the literature on biomass-derived carbon-based materials, exhibiting much higher porous features than those reported herein. As for the separation of CO2/N2 and CO2/CH4 gas mixtures, data showed that the prepared biochars exhibited good selectivities for CO2 over both N2 and CH4: between ca. 34 and 119 for a CO2/N2 mixture in typical post-combustion conditions (15:85, v/v) and between 14 and 34 for a CO2/CH4 mixture typical of natural gas upgrading (30:70, v/v).

Supplementing π-systems: eumelanin and graphene-like integration towards highly conductive materials for the mammalian cell culture bio-interface.

Organic (bio)electronics appears to be the first target for competitive exploitation in the materials science of eumelanins, black insoluble photoprotective human biopolymers. Nonetheless, the low conductivity of these pigments is limiting the implementation of eumelanin-based devices. Here we present a novel organic/organic hybrid material (EUGL) by integration of conductive graphene-like (GL) layers within the EUmelanin pigment (EU). GL layers were obtained by a two-step oxidation/reduction of carbon black. The stability of GL layers over a wide pH range and the self-assembling tendency place this material in a leading position for the fabrication of hybrid materials in aqueous media. EUGL was obtained by inducing the polymerization of eumelanin precursors (5,6-dihydroxyindole, DHI and 5,6-dihydroxyindole-2 carboxylic acid, DHICA) in aqueous media containing GL layers. The new material featured promising biocompatibility and an increased conductivity with respect to eumelanin by four orders of magnitude.

Diesel exhaust particle exposure in vitro impacts T lymphocyte phenotype and function.

BACKGROUND: Diesel exhaust particles (DEP) are major constituents of ambient air pollution and their adverse health effect is an area of intensive investigations. With respect to the immune system, DEP have attracted significant research attention as a factor that could influence allergic diseases interfering with cytokine production and chemokine expression. With this exception, scant data are available on the impact of DEP on lymphocyte homeostasis. Here, the effects of nanoparticles from Euro 4 (E4) and Euro 5 (E5) light duty diesel engines on the phenotype and function of T lymphocytes from healthy donors were evaluated. METHODS: T lymphocytes were isolated from peripheral blood obtained from healthy volunteers and subsequently stimulated with different concentration (from 0.15 to 60 µg/ml) and at different time points (from 24 h to 9 days) of either E4 or E5 particles. Immunological parameters, including apoptosis, autophagy, proliferation levels, mitochondrial function, expression of activation markers and cytokine production were evaluated by cellular and molecular analyses. RESULTS: DEP exposure caused a pronounced autophagic-lysosomal blockade, thus interfering with a key mechanism involved in the maintaining of T cell homeostasis. Moreover, DEP decreased mitochondrial membrane potential but, unexpectedly, this effect did not result in changes of the apoptosis and/or necrosis levels, as well as of intracellular content of adenosine triphosphate (ATP). Finally, a down-regulation of the expression of the alpha chain of the interleukin (IL)-2 receptor (i.e., the CD25 molecule) as well as an abnormal Th1 cytokine expression profile (i.e., a decrease of IL-2 and interferon (IFN)-γ production) were observed after DEP exposure. No differences between the two compounds were detected in all studied parameters. CONCLUSIONS: Overall, our data identify functional and phenotypic T lymphocyte parameters as relevant targets for DEP cytotoxicity, whose impairment could be detrimental, at least in the long run, for human health, favouring the development or the progression of diseases such as autoimmunity and cancer.

Wet chemical method for making graphene-like films from carbon black.

Reduction of strongly oxidized carbon black by hydrazine hydrate yields water-insoluble graphene-like sheets that undergo to self-assembling in thin film on surfaces after drying. The height of a drop-casted graphene-like film was determined by atomic force microscopy (AFM) to be around 20 nm, corresponding to approximately 25 graphene-like layers. The oxidized carbon black and the corresponding reduced form were carefully characterized.

Identification and structural determination of the capsular polysaccharides from two Acinetobacter baumannii clinical isolates, MG1 and SMAL.

The structures of the capsular polysaccharides (CPSs) of the two clinical isolates Acinetobacter baumannii SMAL and MG1 were elucidated. Hot phenol/water extractions of the dry biomasses, followed by enzymatic digestions and repeated ultracentrifugations led to the isolation of polysaccharides that were negative in Western blot analysis utilizing an anti-lipid A antibody, thus proving that they were not the LPS O-antigens but CPSs. Their structures were established on the basis of NMR spectroscopy and GC-MS analyses. The A. baumannii MG1 CPS consisted of a linear aminopolysaccharide with acyl substitution heterogeneity at the N-4 amino group of QuipN4N: 4)-α-d-GlcpNAc-(1→4)-α-l-GalpNAcA-(1→3)-ß-d-QuipNAc4NR-(1→ R=3-hydroxybutyrryl or acetyl. The repeating unit of the CPS produced by strain SMAL is a pentasaccharide, already reported for the O-antigen moiety from A. baumannii strain ATCC 17961:

The structure of the carbohydrate backbone of the lipooligosaccharide from an alkaliphilic Halomonas sp.

The structure of the carbohydrate backbone of the core-lipid A region of the lipooligosaccharide (LOS) of the alkaliphilic and slightly halophilic bacterium Halomonas sp. has been elucidated. The LOS was fully deacylated, dephosphorylated, and reduced at the free reducing end. The structure, obtained by means of compositional analysis, 2D NMR spectroscopy, and MALDI mass spectrometry, was determined as the following: [ structure: see text ]. All sugars are d-pyranoses; Hep is l-glycero-d-manno-heptose and Kdo is 3-deoxy-d-manno-oct-2-ulosonic acid.

Insights on the conformational properties of hyaluronic acid by using NMR residual dipolar couplings and MD simulations.

The conformational features of hyaluronic acid, a key polysaccharide with important biological properties, have been determined through the combined used of nuclear magnetic resonance (NMR) spectroscopy and molecular modeling techniques. A decasaccharide fragment of sodium hyaluronate (HA) was submitted to 3.5 ns of molecular dynamics in explicit water environment form. The same decasaccharide was prepared by hyaluronidase digestion for the experimental study. The approach consisted in the measurements of NMR residual dipolar coupling (RDC) which were used to filter the molecular dynamics data by retaining those structures which were in agreement with the experimental observations. Further analysis of the new conformer ensemble (HA(RDC)) and clustering the molecules with respect to their overall length led to seven representative structures, which were described in terms of their secondary motifs, namely the best fitting helix geometry. As a result, this protocol permitted the assessment that hyaluronic acid can adopt two different arrangements, which can be described by a three- or four-folded left-handed helix, with a higher occurrence of the first one.