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The experiment was conducted during two consecutive seasons (years 2016 and 2017) in an organic apple orchard of the cultivar Jonathan. Several biostimulants were tested (10 in total), including humic acids, macro and micro seaweed extracts, alfalfa protein hydrolysate, amino acids alone or in combination with zinc, B-group vitamins, chitosan and a commercial product containing silicon. Treatments were performed at weekly intervals, starting from the end of May until mid-August. The macroseaweed extract was effective in stimulate tree growth potential in both years, as shown by a significantly larger leaf area (+20% as compared to control) and by an higher chlorophyll content and leaf photosynthetic rate in year 2016. As for the yield performances and apples quality traits at harvest (average fruit weight, soluble solids content, titratable acidity, and flesh firmness), they were generally affected by the different climatic conditions that characterized the two growing seasons (year 2017 being characterized by higher maximal and average temperatures and by limited rainfalls at the beginning of the season). Treatments with macroseaweed extract, B-group vitamins and alfalfa protein hydrolysate were able to significantly improve the intensity and extension of the red coloration of apples at harvest. Correspondingly, the anthocyanin content in the skin of apples treated with the same biostimulants resulted significantly higher than control, highlighting the potential influence of these substances on the synthesis of secondary metabolites in apple. The incidence of physiological disorders was also monitored during apple storage period. Amino acids plus zinc application was effective in reducing (more than 50%) the incidence of the \"Jonathan spot,\" the main post-harvest disorder for this cultivar.

Biostimulants have been found effective in enhancing plant resistance toward stressful conditions. The aim of the present study was to evaluate the efficacy of selected biostimulants to overcome the negative effects of nutrient limitation on the growth performances and on the fruit quality of soilless cultivated strawberry plants. The condition of nutrient limitation was imposed by supplying the plants with only a single fertilization at transplantation and by excluding any further nutrient supply for the entire duration of the experiment (three months, from May to July). Strawberry plants were treated seven times during the period from preflowering up to berry maturation with different classes of biostimulants (humic acids, alfalfa hydrolysate, macroseaweed extract and microalga hydrolysate, amino acids alone or in combination with zinc, B-group vitamins, chitosan, and a commercial product containing silicon) at commercial dosages. The use of alfalfa hydrolysate, vitamins, chitosan, and silicon was able to promote biomass accumulation in roots (four to seven folds) and fruits (+20%) of treated plants, whereas the total leaf area increased by 15%–30%. Nutrient concentrations in leaves and roots showed variations for microelements (e.g., Fe, B, Zn, and Si) in response to biostimulant applications, whereas no significant differences were observed for macronutrient contents among treatments. Final berry yield was found around 20% higher in chitosan- and silicon-treated plants. Chitosan treatment significantly increased pulp firmness (by 20%), while a high nutritional value (e.g., phenolic compounds concentration) was observed in alfalfa- and seaweed-treated fruits (+18%–20% as compared to control). The overall outcomes of the present experiment show that selected biostimulants can be considered as a valid agronomic tool able to contrast the negative consequence of growing crops under insufficient nutritional conditions.

Cell-penetrating peptides are widely used as molecular transporters for the internalization inside cells of various cargo, including proteins and nucleic acids. A special role is played by arginine-rich peptides and oligoarginines covalently linked or simply mixed with the cargo. Here we report cell-penetrating agents in which arginine units are clustered on a macrocyclic scaffold. Instead of using long peptides, four single arginine units were covalently attached to either the upper or lower rim of a calix[4]arene, kept in the cone conformation building a ‘parallel’ cyclic array. These new macrocyclic carriers show high efficiency in DNA delivery and transfection in a variety of cell lines. Arginine-rich peptides act as delivery systems for the internalization of cargoes in cells. Here, the clustering of arginine units in a parallel array on a macrocyclic scaffold produces a vector with high efficiency in DNA delivery and transfection.

Background Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory condition. Given patients with COPD continue to experience exacerbations despite the availability of effective therapies, anti-inflammatory treatments targeting novel pathways are needed. Kinases, notably the phosphoinositide 3-kinases (PI3K), are thought to be involved in chronic airway inflammation, with this pathway proposed as a critical regulator of inflammation and oxidative stress response in COPD. CHF6523 is an inhaled PI3Kδ inhibitor that has shown positive preclinical results. This manuscript reports the results of a study of CHF6523 in patients with stable COPD (chronic bronchitis phenotype), and who had evidence of type-2 inflammation. Methods This randomised, double-blind, placebo-controlled, two-way crossover study comprised two 28-day treatment periods separated by a 28-day washout. Patients ( N  = 44) inhaled CHF6523 in one period, and placebo in the other, both twice daily. The primary objective was to assess the safety and tolerability of CHF6523; the secondary objective was to assess CHF6523 pharmacokinetics. Exploratory endpoints included target engagement (the relative reduction in phosphatidylinositol (3,4,5)-trisphosphate [PIP 3 ]), pharmacodynamic evaluations such as airflow obstruction, and hyperinflation, and to identify biomarker(s) of drug response using proteomics and transcriptomics. Results CHF6523 plasma pharmacokinetics were characterised by an early maximum concentration (C max ), reached 15 and 10 min after dosing on Days 1 and 28, respectively, followed by a rapid decline. Systemic exposure on Day 28 showed limited accumulation, with ratios < 1.6 for C max and area under the curve from 0 to 12 h post-dose, and with steady state achieved on Day 20. Target engagement was confirmed by a significant 29.7% reduction from baseline in induced sputum PIP 3 (29.5% reduction vs. placebo; adjusted ratio 0.705 [0.580, 0.856]; p  = 0.001), but this did not translate into an anti-inflammatory pharmacodynamic effect, as assessed through measures including biomarkers and multi-omics. Additionally, although CHF6523 was generally well-tolerated, 95.2% of patients reported cough as an adverse event, most mild to moderate and resolving within one-hour post-dose. Conclusions These data, together with those from other PI3K inhibitors, suggest that PI3Kδ is not a suitable pathway for the management of COPD, as the achieved target engagement did not translate into any pharmacodynamic anti-inflammatory effect. Trial registration ClinicalTrials.gov (NCT04032535); posted 23rd July 2019.

Background Although phosphodiesterase-4 (PDE4) inhibitors have been shown to reduce COPD exacerbation rate, their biological mechanism of action is not completely elucidated at the molecular level. We aimed to characterise the whole genome gene expression profile of the inhaled PDE4-inhibitor CHF6001 on top of triple therapy in sputum cells and whole blood of patients with COPD and chronic bronchitis. Methods Whole genome gene expression analysis was carried out by microarray in 54 patients before and after 32 days treatment with CHF6001 800 and 1600 μg and placebo twice daily (BID) in a randomised crossover study. Results CHF6001 had a strong effect in sputum, with 1471 and 2598 significantly differentially-expressed probe-sets relative to placebo (p-adjusted for False Discovery Rate < 0.05) with 800 and 1600 μg BID, respectively. Functional enrichment analysis showed significant modulation of key inflammatory pathways involved in cytokine activity, pathogen-associated-pattern-recognition activity, oxidative stress and vitamin D with associated inhibition of downstream inflammatory effectors. A large number of pro-inflammatory genes coding for cytokines and matrix-metalloproteinases were significantly differentially expressed for both doses; the majority (> 87%) were downregulated, including macrophage inflammatory protein-1-alpha and 1-beta, interleukin-27-beta, interleukin-12-beta, interleukin-32, tumour necrosis factor-alpha-induced-protein-8, ligand-superfamily-member-15, and matrix-metalloproteinases-7,12 and 14. The effect in blood was not significant. Conclusions Inhaled PDE4 inhibition by CHF6001 on top of triple therapy in patients with COPD and chronic bronchitis significantly modulated key inflammatory targets and pathways in the lung but not in blood. Mechanistically these findings support a targeted effect in the lung while minimising unwanted systemic class-effects. Trial registration ClinicalTrial.gov, EudraCT, 2015–005550-35 . Registered 15 July 2016.

In this study, a metabolomic investigation was presented to correlate single polyphenolic compounds in apple pulp with quality characteristics such as antioxidant activity and content of phenolic compounds and anthocyanins in apple skin. Since the concentration of these compounds is influenced by environmental factors, the twenty-two apple cultivars originate from the same site. The polyphenolic compounds were analyzed by ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS/MS). The antioxidant activity, phenolic content, and anthocyanins were evaluated on the sunny and the shady sides of apple skin by spectrometric assays. In old apple varieties, the measured parameters were higher than in the commercial and red-fleshed varieties. By contrast, the profile of flavan-3-ols and anthocyanins was variable amongst commercial and red-fleshed varieties. The partial least square (PLS) method was applied to investigate the association between the skin proprieties and the metabolic profile of the pulp. The highest coefficients of determination in prediction (Q2) were obtained for compounds quantified in old cultivars. These results provided information to define the old apple varieties as a reliable group based on the pathway of the antioxidant compounds and anthocyanins content. Our results show the possibility to find cultivars with promising health features based on their content of polyphenols suitable for commercialization or breeding.

Background: Assessments of airways inflammation in patients with chronic obstructive pulmonary disease (COPD) require semi-invasive procedures and specialized sample processing know-how. In this study we aimed to set up and validate a novel non-invasive processing-free method for RNA sequencing (RNAseq) of spontaneous sputum samples collected from COPD patients. Methods: Spontaneous sputum samples were collected and stabilized, with or without selection of plugs and with or without the use of a stabilizer specifically formulated for downstream diagnostic testing (PrimeStore® Molecular Transport Medium). After 8 days storage at ambient temperature RNA was isolated according to an optimized RNAzol® method. An average percentage of fragments longer than 200 nucleotides (DV 200 ) >30% and an individual yield >50 ng were required for progression of samples to sequencing. Finally, to assess if the transcriptome generated would reflect a true endotype of COPD inflammation, the outcome of single-sample gene-set enrichment analysis (ssGSEA) was validated using an independent set of processed induced sputum samples. Results: RNA extracted from spontaneous sputum using a stabilizer showed an average DV 200 higher than 30%. 70% of the samples had a yield >50 ng and were submitted to downstream analysis. There was a straightforward correlation in terms of gene expression between samples handled with or without separation of plugs. This was also confirmed by principal component analysis and ssGSEA. The top ten enriched pathways resulting from spontaneous sputum ssGSEA were associated to features of COPD, namely, inflammation, immune responses and oxidative stress; up to 70% of these were in common within the top ten enriched pathways resulting from induced sputum ssGSEA. Conclusion: This analysis confirmed that the typical COPD endotype was represented within spontaneous sputum and supported the current method as a non-invasive processing-free procedure to assess the level of sputum cell inflammation in COPD patients by RNAseq analysis.

Palmitoylethanolamide (PEA) has antinflammatory and antinociceptive properties widely exploited in veterinary and human medicine, despite its poor pharmacokinetics. Looking for prodrugs that could progressively release PEA to maintain effective plasma concentrations, we prepared carbonates, esters and carbamates at the hydroxyl group of PEA. Chemical stability (pH 7.4) and stability in rat plasma and liver homogenate were evaluated by in vitro assays. Carbonates and carbamates resulted too labile and too resistant in plasma, respectively. Ester derivatives, prepared by conjugating PEA with various amino acids, allowed to modulate the kinetics of PEA release in plasma and stability in liver homogenate. L-Val-PEA, with suitable PEA release in plasma, and D-Val-PEA, with high resistance to hepatic degradation, were orally administered to rats and plasma levels of prodrugs and PEA were measured at different time points. Both prodrugs showed significant release of PEA, but provided lower plasma concentrations than those obtained with equimolar doses of PEA. Amino-acid esters of PEA are a promising class to develop prodrugs, even if they need further chemical optimization.

Chronic obstructive pulmonary disease (COPD) patients with higher eosinophil counts are associated with increased clinical response to phosphodiesterase‐4‐inhibitors (PDE4i). However, the underlying inflammatory mechanisms associated with this increased response is not yet elucidated. This post hoc analysis focused on sputum gene expression in patients with chronic bronchitis who underwent 32‐day treatment with two doses of the inhaled PDE4i CHF6001 (tanimilast) or placebo on top of triple therapy. Biological characterization and treatment effects were assessed between patients with different sputum eosinophil levels (eosinophilhigh ≥ 3%; eosinophillow < 3%) at baseline (primary samples) or at the end of the treatment of the placebo arm (validation samples). Forty‐one genes were differentially expressed in primary samples (p‐adjusted for false discovery rate < 0.05); all up‐regulated in eosinophilhigh patients and functionally enriched for type‐2 and PDE4 inflammatory processes. Eleven out of nineteen genes having immune system biological processes annotations including IL5RA, ALOX15, IL1RL1, CLC, GATA1 and PDE4D were replicated using validation samples. The expression of a number of these inflammatory mediators was reduced by tanimilast treatment, with greater effects observed in eosinophilhigh patients. These findings suggest that type‐2 and PDE4 overexpression in COPD patients with higher sputum eosinophil counts contribute to the differential clinical response to PDE4i observed in previous clinical trials.

The function of monoacylglycerol lipase (MGL), a key actor in the hydrolytic deactivation of the endocannabinoid 2-arachidonoyl- sn -glycerol (2AG), is tightly controlled by the cell’s redox state: oxidative signals such as hydrogen peroxide suppress MGL activity in a reversible manner through sulfenylation of the peroxidatic cysteines, C201 and C208. Here, using as a starting point the crystal structures of human MGL (hMGL), we present evidence from molecular dynamics and metadynamics simulations along with high-resolution mass spectrometry studies indicating that sulfenylation of C201 and C208 alters the conformational equilibrium of the membrane-associated lid domain of MGL to favour closed conformations of the enzyme that do not permit the entry of substrate into the active site.