Nanoscale organization of the pathogen receptor DC-SIGN mapped by single-molecule high-resolution fluorescence microscopy.

DC-SIGN, a C-type lectin exclusively expressed on dendritic cells (DCs), plays an important role in pathogen recognition by binding with high affinity to a large variety of microorganisms. Recent experimental evidence points to a direct relation between the function of DC-SIGN as a viral receptor and its spatial arrangement on the plasma membrane. We have investigated the nanoscale organization of fluorescently labeled DC-SIGN on intact isolated DCs by means of near-field scanning optical microscopy (NSOM) combined with single-molecule detection. Fluorescence spots of different intensity and size have been directly visualized by optical means with a spatial resolution of less than 100 nm. Intensity- and size-distribution histograms of the DC-SIGN fluorescent spots confirm that approximately 80 % of the receptors are organized in nanosized domains randomly distributed on the cell membrane. Intensity-size correlation analysis revealed remarkable heterogeneity in the molecular packing density of the domains. Furthermore, we have mapped the intermolecular organization within a dense cluster by means of sequential NSOM imaging combined with discrete single-molecule photobleaching. In this way we have determined the spatial coordinates of 13 different individual dyes, with a localization accuracy of 6 nm. Our experimental observations are all consistent with an arrangement of DC-SIGN designed to maximize its chances of binding to a wide range of microorganisms. Our data also illustrate the potential of NSOM as an ultrasensitive, high-resolution technique to probe nanometer-scale organization of molecules on the cell membrane.     

Phenolic molecules in virgin olive oils: a survey of their sensory properties, health effects, antioxidant activity and analytical methods. An overview of the last decade

Among vegetable oils, virgin olive oil (VOO) has nutritional and sensory characteristics that to make it unique and a basic component of the Mediterranean diet. The importance of VOO is mainly attributed both to its high content of oleic acid a balanced contribution quantity of polyunsaturated fatty acids and its richness in phenolic compounds, which act as natural antioxidants and may contribute to the prevention of several human diseases. The polar phenolic compounds of VOO belong to different classes: phenolic acids, phenyl ethyl alcohols, hydroxy-isochromans, flavonoids, lignans and secoiridoids. This latter family of compounds is characteristic of Oleaceae plants and secoiridoids are the main compounds of the phenolic fraction. Many agronomical and technological factors can affect the presence of phenols in VOO. Its shelf life is higher than other vegetable oils, mainly due to the presence of phenolic molecules having a catechol group, such as hydroxytyrosol and its secoiridoid derivatives. Several assays have been used to establish the antioxidant activity of these isolated phenolic compounds. Typical sensory gustative properties of VOO, such as bitterness and pungency, have been attributed to secoiridoid molecules. Considering the importance of the phenolic fraction of VOO, high performance analytical methods have been developed to characterize its complex phenolic pattern. The aim of this review is to realize a survey on phenolic compounds of virgin olive oils bearing in mind their chemical-analytical, healthy and sensory aspects. In particular, starting from the basic studies, the results of researches developed in the last ten years will be focused.     

Characterization of Two Light-Harvesting Subunits Isolated from the Brown Alga Pelvetia canaliculata: Heterogeneity of Xanthophyll Distribution

The main light-harvesting fraction from Pelvetia canaliculata was isolated on a sucrose density gradient from digitonin-solubilized chloroplasts. After further solubilization by dodecyl maltoside, the bulk fraction was separated into two subunits by preparative isoelectric focusing. The more acidic brown fraction was mainly composed of 22 kDa polypeptides having an apparent pI of 4.55. Its pigment composition was very simple, containing chlorophyll (Chi) a, Chi c and fucoxanthin. The in vivo spectral properties of fucoxanthin, namely a shift in light absorption to the green and efficient energy transmission to Chi a, were conserved in this subunit. No xanthophyll associated with photoprotection was found in this band, even when obtained from photoinhibited thalli. The less acidic green band contained predominantly 22 kDa polypeptides that were resolved into numerous components by denaturing isoelectric focusing. Its pigment composition was more complex, containing, in addition, pigments of the so-called xanthophyll cycle. In photoinhibited thalli, about half of the violaxanthin was converted into antheraxanthin and zeaxanthin. All the pigments of the xanthophyll cycle were specifically associated with this subunit, and it may thus have a central role in the thermal dissipation of the absorbed light energy as postulated for light-harvesting complex II isolated from green plants.     

Some Novel Cobalt Diphenylphosphine Complexes: Synthesis,Characterization, and Behavior in the Polymerization of 1,3-Butadiene

Some novel cobalt diphenylphosphine complexes were synthesized by reacting cobalt(II) chloride with (2-methoxyethyl)diphenylphosphine, (2-methoxyphenyl)diphenylphosphine, and 2-(1,1-dimethylpropyl)-6-(diphenylphosphino)pyridine. Single crystals suitable for X-ray diffraction studies were obtained for the first two complexes, and their crystal structure was determined. The novel compounds were then used in association with methylaluminoxane (MAO) for the polymerization of 1,3-butadiene, and their behavior was compared with that exhibited in the polymerization of the same monomer by the systems CoCl₂(PⁿPrPh₂)₂/MAO and CoCl₂(PPh₃)₂/MAO. Some significant differences were observed depending on the MAO/Co ratio used, and a plausible interpretation for such a different behavior is proposed.     

Electrosprayed Shrimp and Mushroom Nanochitins on Cellulose Tissue for Skin Contact Application

Cosmetics has recently focused on biobased skin-compatible materials. Materials from natural sources can be used to produce more sustainable skin contact products with enhanced bioactivity. Surface functionalization using natural-based nano/microparticles is thus a subject of study, aimed at better understanding the skin compatibility of many biopolymers also deriving from biowaste. This research investigated electrospray as a method for surface modification of cellulose tissues with chitin nanofibrils (CNs) using two different sources—namely, vegetable (i.e., from fungi), and animal (from crustaceans)—and different solvent systems to obtain a biobased and skin-compatible product. The surface of cellulose tissues was uniformly decorated with electrosprayed CNs. Biological analysis revealed that all treated samples were suitable for skin applications since human dermal keratinocytes (i.e., HaCaT cells) successfully adhered to the processed tissues and were viable after being in contact with released substances in culture media. These results indicate that the use of solvents did not affect the final cytocompatibility due to their effective evaporation during the electrospray process. Such treatments did not also affect the characteristics of cellulose; in addition, they showed promising anti-inflammatory and indirect antimicrobial activity toward dermal keratinocytes in vitro. Specifically, cellulosic substrates decorated with nanochitins from shrimp showed strong immunomodulatory activity by first upregulating then downregulating the pro-inflammatory cytokines, whereas nanochitins from mushrooms displayed an overall anti-inflammatory activity via a slight decrement of the pro-inflammatory cytokines and increment of the anti-inflammatory marker. Electrospray could represent a green method for surface modification of sustainable and biofunctional skincare products.     

Unravelling the Phytochemical Composition and the Pharmacological Properties of an Optimized Extract from the Fruit from Prunus mahaleb L.: From Traditional Liqueur Market to the Pharmacy Shelf

Prunus mahaleb L. fruit has long been used in the production of traditional liqueurs. The fruit also displayed scavenging and reducing activity, in vitro. The present study focused on unravelling peripheral and central protective effects, antimicrobial but also anti-COVID-19 properties exerted by the water extract of P. mahaleb. Anti-inflammatory effects were studied in isolated mouse colons exposed to lipopolysaccharide. Neuroprotection, measured as a blunting effect on hydrogen-peroxide-induced dopamine turnover, was investigated in hypothalamic HypoE22 cells. Antimicrobial effects were tested against different Gram+ and Gram- bacterial strains. Whereas anti-COVID-19 activity was studied in lung adenocarcinoma H1299 cells, where the gene expression of ACE2 and TMPRSS2 was measured after extract treatment. The bacteriostatic effects induced on Gram+ and Gram- strains, together with the inhibition of COX-2, TNFα, HIF1α, and VEGFA in the colon, suggest the potential of P. mahaleb water extract in contrasting the clinical symptoms related to ulcerative colitis. The inhibition of the hydrogen peroxide-induced DOPAC/DA ratio indicates promising neuroprotective effects. Finally, the downregulation of the gene expression of ACE2 and TMPRSS2 in H1299 cells, suggests the potential to inhibit SARS-CoV-2 virus entry in the human host. Overall, the results support the valorization of the local cultivation of P. mahaleb.     

Highly Efficient Trityl-Nitroxide Biradicals for Biomolecular High-Field Dynamic Nuclear Polarization

Dynamic nuclear polarization (DNP) is a powerful method to enhance the sensitivity of solid-state magnetic nuclear resonance (ssNMR) spectroscopy. However, its biomolecular applications at high magnetic fields (preferably>14 T) have so far been limited by the intrinsically low efficiency of polarizing agents and sample preparation aspects. Herein, we report a new class of trityl-nitroxide biradicals, dubbed SNAPols that combine high DNP efficiency with greatly enhanced hydrophilicity. SNAPol-1, the best compound in the series, shows DNP enhancement factors at 18.8 T of more than 100 in small molecules and globular proteins and also exhibits strong DNP enhancements in membrane proteins and cellular preparations. By integrating optimal sensitivity and high resolution, we expect widespread applications of this new polarizing agent in high-field DNP/ssNMR spectroscopy, especially for complex biomolecules.     

Exciplex Formation in Lipid-bound Escherichia coli Flavohemoglobin

Flavohemoglobins have the particular capability of binding unsaturated and cyclopropanated fatty acids as free acids or phospholipids. Fatty acid binding to the ferric heme results in a weak but direct bonding interaction. Ferrous and ferric protein, in presence or absence of a bound lipid molecule, have been characterized by transient absorption spectroscopy. Measurements have been also carried out both on the ferrous deoxygenated and on the CO bound protein to investigate possible long-range interaction between the lipid acyl chain moiety and the ferrous heme. After excitation of the deoxygenated derivatives the relaxation process reveals a slow dynamics (350 ps) in lipid-bound protein but is not observed in the lipid-free protein. The latter feature and the presence of an extra contribution in the absorption spectrum, indicates that the interaction of iron heme with the acyl chain moiety occurs only in the excited electronic state and not in the ground electronic state. Data analysis highlights the formation of a charge-transfer complex in which the iron ion of the lipid-bound protein in the expanded electronic excited state, possibly represented by a high spin Fe III intermediate, is able to bind to the sixth coordination ligand placed at a distance of at 3.5 Å from the iron. A very small nanosecond geminate rebinding is observed for CO adduct in lipid-free but not in the lipid-bound protein. The presence of the lipid thus appears to inhibit the mobility of CO in the heme pocket.     

Exploiting Protein N-Terminus for Site-Specific Bioconjugation

Although a plethora of chemistries have been developed to selectively decorate protein molecules, novel strategies continue to be reported with the final aim of improving selectivity and mildness of the reaction conditions, preserve protein integrity, and fulfill all the increasing requirements of the modern applications of protein conjugates. The targeting of the protein N-terminal alpha-amine group appears a convenient solution to the issue, emerging as a useful and unique reactive site universally present in each protein molecule. Herein, we provide an updated overview of the methodologies developed until today to afford the selective modification of proteins through the targeting of the N-terminal alpha-amine. Chemical and enzymatic strategies enabling the selective labeling of the protein N-terminal alpha-amine group are described.