Oligomerization and Nitration of the Grass Pollen Allergen Phl p 5 by Ozone,Nitrogen Dioxide,and Peroxynitrite: Reaction Products,Kinetics, and Health Effects

The allergenic and inflammatory potential of proteins can be enhanced by chemical modification upon exposure to atmospheric or physiological oxidants. The molecular mechanisms and kinetics of such modifications, however, have not yet been fully resolved. We investigated the oligomerization and nitration of the grass pollen allergen Phl p 5 by ozone (O₃), nitrogen dioxide (NO₂), and peroxynitrite (ONOO^–). Within several hours of exposure to atmospherically relevant concentration levels of O₃ and NO₂, up to 50% of Phl p 5 were converted into protein oligomers, likely by formation of dityrosine cross-links. Assuming that tyrosine residues are the preferential site of nitration, up to 10% of the 12 tyrosine residues per protein monomer were nitrated. For the reaction with peroxynitrite, the largest oligomer mass fractions (up to 50%) were found for equimolar concentrations of peroxynitrite over tyrosine residues. With excess peroxynitrite, the nitration degrees increased up to 40% whereas the oligomer mass fractions decreased to 20%. Our results suggest that protein oligomerization and nitration are competing processes, which is consistent with a two-step mechanism involving a reactive oxygen intermediate (ROI), as observed for other proteins. The modified proteins can promote pro-inflammatory cellular signaling that may contribute to chronic inflammation and allergies in response to air pollution.     

Robot training of upper limb in multiple sclerosis: comparing protocols with or without manipulative task components

In this pilot study, we compared two protocols for robot-based rehabilitation of upper limb in multiple sclerosis (MS): a protocol involving reaching tasks (RT) requiring arm transport only and a protocol requiring both objects' reaching and manipulation (RMT). Twenty-two MS subjects were assigned to RT or RMT group. Both protocols consisted of eight sessions. During RT training, subjects moved the handle of a planar robotic manipulandum toward circular targets displayed on a screen. RMT protocol required patients to reach and manipulate real objects, by moving the robotic arm equipped with a handle which left the hand free for distal tasks. In both trainings, the robot generated resistive and perturbing forces. Subjects were evaluated with clinical and instrumental tests. The results confirmed that MS patients maintained the ability to adapt to the robot-generated forces and that the rate of motor learning increased across sessions. Robot-therapy significantly reduced arm tremor and improved arm kinematics and functional ability. Compared to RT, RMT protocol induced a significantly larger improvement in movements involving grasp (improvement in Grasp ARAT sub-score: RMT 77.4%, RT 29.5%, p=0.035) but not precision grip. Future studies are needed to evaluate if longer trainings and the use of robotic handles would significantly improve also fine manipulation.     

Peptides from water buffalo cheese whey induced senescence cell death via ceramide secretion in human colon adenocarcinoma cell line

Scope: Milk proteins are a source of bioactive peptides. Recent studies have indicated that protein‐derived peptides released in buffalo cheese acid whey exert a cytomodulatory effect in human epithelial colon cancer (CaCo2) cells. The aim of the present study was to explain the molecular mechanism involved in the response of CaCo2 cells to oxidative stress in the presence of peptide fractions of buffalo cheese whey, purified and characterized by mass spectrometry. Methods and results: We demonstrated that treatment of CaCo2 treated with H₂O₂ (H‐CaCo2) cells with a partially purified peptide sub‐fraction (f3) from buffalo cheese acid whey induced a reduction of mitochondrial superoxide anion with subsequent decrease in heat shock protein 70 and 90 expression. Moreover, we observed a 5‐fold decrease in cyclin A expression and cell cycle arrest in G1/G0 phases. These responses were associated with increased activity of alkaline phosphatase and beta‐galactosidase, markers of differentiation and senescence respectively. Conclusions: The structural characterization of the active peptide fraction and the elucidation of the effects induced by its treatment on H‐CaCo2 cells in vitro demonstrated an activity of this peptide sub‐fraction in the modulation of cell cycle, thus suggesting potential application for the development of nutraceuticals as well as health‐promoting functional foods.     

Design,Synthesis, Radiosynthesis and Biological Evaluation of Fenretinide Analogues as Anticancer and Metabolic Syndrome-Preventive Agents

Fenretinide (4-HPR) is a synthetic derivative of all-trans-retinoic acid (ATRA) characterised by improved therapeutic properties and toxicological profile relative to ATRA. 4-HPR has been mostly investigated as an anti-cancer agent, but recent studies showed its promising therapeutic potential for preventing metabolic syndrome. Several biological targets are involved in 4-HPR's activity, leading to the potential use of this molecule for treating different pathologies. However, although 4-HPR displays quite well-understood multitarget promiscuity with regards to pharmacology, interpreting its precise physiological role remains challenging. In addition, despite promising results in vitro, the clinical efficacy of 4-HPR as a chemotherapeutic agent has not been satisfactory so far. Herein, we describe the preparation of a library of 4-HPR analogues, followed by the biological evaluation of their anti-cancer and anti-obesity/diabetic properties. The click-type analogue 3 b showed good capacity to reduce the amount of lipid accumulation in 3T3-L1 adipocytes during differentiation. Furthermore, it showed an IC₅₀ of 0.53±0.8 μM in cell viability tests on breast cancer cell line MCF-7, together with a good selectivity (SI=121) over noncancerous HEK293 cells. Thus, 3 b was selected as a potential PET tracer to study retinoids in vivo, and the radiosynthesis of [¹⁸F] 3b was successfully developed. Unfortunately, the stability of [¹⁸F] 3b turned out to be insufficient to pursue imaging studies.     

A2B Adenosine Receptors: When Outsiders May Become an Attractive Target to Treat Brain Ischemia or Demyelination

Adenosine is a signaling molecule, which, by activating its receptors, acts as an important player after cerebral ischemia. Here, we review data in the literature describing A_2BR-mediated effects in models of cerebral ischemia obtained in vivo by the occlusion of the middle cerebral artery (MCAo) or in vitro by oxygen-glucose deprivation (OGD) in hippocampal slices. Adenosine plays an apparently contradictory role in this receptor subtype depending on whether it is activated on neuro-glial cells or peripheral blood vessels and/or inflammatory cells after ischemia. Indeed, A_2BRs participate in the early glutamate-mediated excitotoxicity responsible for neuronal and synaptic loss in the CA1 hippocampus. On the contrary, later after ischemia, the same receptors have a protective role in tissue damage and functional impairments, reducing inflammatory cell infiltration and neuroinflammation by central and/or peripheral mechanisms. Of note, demyelination following brain ischemia, or autoimmune neuroinflammatory reactions, are also profoundly affected by A_2BRs since they are expressed by oligodendroglia where their activation inhibits cell maturation and expression of myelin-related proteins. In conclusion, data in the literature indicate the A_2BRs as putative therapeutic targets for the still unmet treatment of stroke or demyelinating diseases.     

Unpatterned Bioactive Poly(Butylene 1,4-Cyclohexanedicarboxylate)-Based Film Fast Induced Neuronal-Like Differentiation of Human Bone Marrow-Mesenchymal Stem Cells

Herein, we present poly(butylene 1,4-cyclohexanedicarboxylate) (PBCE) films characterized by an unpatterned microstructure and a specific hydrophobicity, capable of boosting a drastic cytoskeleton architecture remodeling, culminating with the neuronal-like differentiation of human bone marrow-mesenchymal stem cells (hBM-MSCs). We have used two different filming procedures to prepare the films, solvent casting (PBCE) and compression-moulding (PBCE*). PBCE film had a rough and porous surface with spherulite-like aggregations (Ø = 10–20 μm) and was characterized by a water contact angle = 100°. PBCE* showed a smooth and continuous surface without voids and visible spherulite-like aggregations and was more hydrophobic (WCA = 110°). Both surface characteristics were modulated through the copolymerization of different amounts of ether-oxygen-containing co-units into PBCE chemical structure. We showed that only the surface characteristics of PBCE-solvent-casted films steered hBM-MSCs toward a neuronal-like differentiation. hBM-MSCs lost their canonical mesenchymal morphology, acquired a neuronal polarized shape with a long cell protrusion (≥150 μm), expressed neuron-specific class III β-tubulin and microtubule-associated protein 2 neuronal markers, while nestin, a marker of uncommitted stem cells, was drastically silenced. These events were observed as early as 2-days after cell seeding. Of note, the phenomenon was totally absent on PBCE* film, as hBM-MSCs maintained the mesenchymal shape and behavior and did not express neuronal/glial markers.     

Serenoa repens and Urtica dioica Fixed Combination: In-Vitro Validation of a Therapy for Benign Prostatic Hyperplasia (BPH)

Benign prostatic hyperplasia (BPH) is an age-related chronic disorder, characterized by the hyperproliferation of prostatic epithelial and stromal cells, which drives prostate enlargement. Since BPH aetiology and progression have been associated with the persistence of an inflammatory stimulus, induced both by Nuclear Factor-kappa B (NF-κB) activation and reactive oxygen species (ROS) production, the inhibition of these pathways could result in a good tool for its clinical treatment. This study aimed to evaluate the antioxidant and anti-inflammatory activity of a combined formulation of Serenoa repens and Urtica dioica (SR/UD) in an in vitro human model of BPH. The results confirmed both the antioxidant and the anti-inflammatory effects of SR/UD. In fact, SR/UD simultaneously reduced ROS production, NF-κB translocation inside the nucleus, and, consequently, interleukin 6 (IL-6) and interleukin 8 (IL-8) production. Furthermore, the effect of SR/UD was also tested in a human androgen-independent prostate cell model, PC3. SR/UD did not show any significant antioxidant and anti-inflammatory effect, but was able to reduce NF-κB translocation. Taken together, these results suggested a promising role of SR/UD in BPH and BPH-linked disorder prevention.