Gene-Delivery Ability of New Hydrogenated and Partially Fluorinated Gemini bispyridinium Surfactants with Six Methylene Spacers

The pandemic emergency determined by the spreading worldwide of the SARS-CoV-2 virus has focused the scientific and economic efforts of the pharmaceutical industry and governments on the possibility to fight the virus by genetic immunization. The genetic material must be delivered inside the cells by means of vectors. Due to the risk of adverse or immunogenic reaction or replication connected with the more efficient viral vectors, non-viral vectors are in many cases considered as a preferred strategy for gene delivery into eukaryotic cells. This paper is devoted to the evaluation of the gene delivery ability of new synthesized gemini bis-pyridinium surfactants with six methylene spacers, both hydrogenated and fluorinated, in comparison with compounds with spacers of different lengths, previously studied. Results from MTT proliferation assay, electrophoresis mobility shift assay (EMSA), transient transfection assay tests and atomic force microscopy (AFM) imaging confirm that pyridinium gemini surfactants could be a valuable tool for gene delivery purposes, but their performance is highly dependent on the spacer length and strictly related to their structure in solution. All the fluorinated compounds are unable to transfect RD-4 cells, if used alone, but they are all able to deliver a plasmid carrying an enhanced green fluorescent protein (EGFP) expression cassette, when co-formulated with 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE) in a 1:2 ratio. The fluorinated compounds with spacers formed by six (FGP6) and eight carbon atoms (FGP8) give rise to a very interesting gene delivery activity, greater to that of the commercial reagent, when formulated with DOPE. The hydrogenated compound GP16_6 is unable to sufficiently compact the DNA, as shown by AFM images.     

High Glucose Impairs Expression and Activation of MerTK in ARPE-19 Cells

MerTK (Mer Tyrosine Kinase) is a cell surface receptor that regulates phagocytosis of photoreceptor outer segments (POS) in retinal pigment epithelial (RPE) cells. POS phagocytosis is impaired in several pathologies, including diabetes. In this study, we investigate whether hyperglycemic conditions may affect MerTK expression and activation in ARPE-19 cells, a retinal pigment epithelial cellular model. ARPE-19 cells were cultured in standard (CTR) or high-glucose (HG) medium for 24 h. Then, we analyzed: mRNA levels and protein expression of MerTK and ADAM9, a protease that cleaves the extracellular region of MerTK; the amount of cleaved Mer (sMer); and the ability of GAS6, a MerTK ligand, to induce MerTK phosphorylation. Since HG reduces miR-126 levels, and ADAM9 is a target of miR-126, ARPE-19 cells were transfected with miR-126 inhibitor or mimic; then, we evaluated ADAM9 expression, sMer, and POS phagocytosis. We found that HG reduced expression and activation of MerTK. Contextually, HG increased expression of ADAM9 and the amount of sMer. Overexpression of miR-126 reduced levels of sMer and improved phagocytosis in ARPE-19 cells cultured with HG. In this study, we demonstrate that HG compromises MerTK expression and activation in ARPE-19 cells. Our results suggest that HG up-regulates ADAM9 expression, leading to increased shedding of MerTK. The consequent rise in sMer coupled to reduced expression of MerTK impairs binding and internalization of POS in ARPE-19 cells.     

Biotechnological Applications and Health-Promoting Properties of Flavonols: An Updated View

Flavonols are a subclass of natural flavonoids characterized by a remarkable number of biotechnological applications and health-promoting properties. They attract researchers’ attention due to many epidemiological studies supporting their usage. They are phytochemicals commonly present in our diet, being ubiquitous in the plant kingdom and, in particular, relatively very abundant in fruits and vegetables. All these aspects make flavonols candidates of choice for the valorization of products, based on the presence of a remarkable number of different chemical structures, each one characterized by specific chemical features capable of influencing biological targets inside the living organisms in very different manners. In this review, we analyzed the biochemical and physiological characteristics of flavonols focalizing our attention on the most promising compounds to shed some light on their increasing utilization in biotechnological applications in processing industries, as well as their suitable employment to improve the overall wellness of the humankind.     

A Glance into MTHFR Deficiency at a Molecular Level

MTHFR deficiency still deserves an investigation to associate the phenotype to protein structure variations. To this aim, considering the MTHFR wild type protein structure, with a catalytic and a regulatory domain and taking advantage of state-of-the-art computational tools, we explore the properties of 72 missense variations known to be disease associated. By computing the thermodynamic ΔΔG change according to a consensus method that we recently introduced, we find that 61% of the disease-related variations destabilize the protein, are present both in the catalytic and regulatory domain and correspond to known biochemical deficiencies. The propensity of solvent accessible residues to be involved in protein-protein interaction sites indicates that most of the interacting residues are located in the regulatory domain, and that only three of them, located at the interface of the functional protein homodimer, are both disease-related and destabilizing. Finally, we compute the protein architecture with Hidden Markov Models, one from Pfam for the catalytic domain and the second computed in house for the regulatory domain. We show that patterns of disease-associated, physicochemical variation types, both in the catalytic and regulatory domains, are unique for the MTHFR deficiency when mapped into the protein architecture.     

Lignin Nanoparticles Deliver Novel Thymine Biomimetic Photo-Adducts with Antimelanoma Activity

We report here the synthesis of novel thymine biomimetic photo-adducts bearing an alkane spacer between nucleobases and characterized by antimelanoma activity against two mutated cancer cell lines overexpressing human Topoisomerase 1 (TOP1), namely SKMEL28 and RPMI7951. Among them, Dewar Valence photo-adducts showed a selectivity index higher than the corresponding pyrimidine-(6-4)-pyrimidone and cyclobutane counterpart and were characterized by the highest affinity towards TOP1/DNA complex as evaluated by molecular docking analysis. The antimelanoma activity of novel photo-adducts was retained after loading into UV photo-protective lignin nanoparticles as stabilizing agent and efficient drug delivery system. Overall, these results support a combined antimelanoma and UV sunscreen strategy involving the use of photo-protective lignin nanoparticles for the controlled release of thymine dimers on the skin followed by their sacrificial transformation into photo-adducts and successive inhibition of melanoma and alert of cellular UV machinery repair pathways.     

Epoxy resins reinforced with TiO2 generated by nonhydrolytic sol–gel process

Innovative epoxy‐titania nanocomposites were prepared starting from titania nanoparticles suspended in benzyl alcohol (BzOH) generated by nonhydrolytic sol–gel process from TiCl₄. The obtained suspensions were mixed with an epoxy resin (bisphenol A diglycidyl ether, DGEBA) and the formulations were cured in the presence of ytterbium(III) trifluoromethanesulfonate as cationic initiator. The thermally activated cationic ring‐opening polymerization produced a three‐dimensional network in which the suspending medium BzOH was covalently linked to the epoxy network according to the “activated monomer” mechanism during the propagation step. The presence of titania nanoparticles resulted in a reinforcing and stiffening effect due to both their hydrodynamic effect and, most important, a significantly higher cross‐linking density of the composite material with respect to the unfilled epoxy resin. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40470.     

Robust control in presence of parametric uncertainties: Observer-based feedback controller design

This paper proposes a complete procedure for the design of a robust controller for a nonlinear process, taking into account the various issues arising in the design and using the main theoretical results from the Literature about this topic. An extended model is set up, linking performance and robustness to the control law: the H_∞ norm of the extended system in closed loop measures the achievement of the objectives. The result is a state feedback control law which guarantees robust performance. The problem of the design of an observer to estimate the state of the system is also addressed, as the complete knowledge of the state is required to calculate the control action; moreover, the implications of the use of the observer in the design of the controller are pointed out. The methodology is illustrated via simulation of a regulation problem in a continuous stirred tank reactor (CSTR). The application of this methodology to more complex systems will be discussed.     

Focusing on bioactive compounds in grapes: stilbenes in Uvalino cv.

Phenolic compounds belonging to the group of stilbenes have recently received a particular attention as bioactive compounds because of their role in plant physiology and for their antioxidant properties. Monomer stilbenes in grapes are mainly present as glycosides. The aim of this work was the identification and quantification of monomer stilbenes (as both free and glycosilated form) in Uvalino grapes, a grapevine variety known for its significant content in stilbenes. Their content in grapes was determined during ripening and overripening stages, in comparison with other phenolic compounds. Piceid (cis and trans form), piceatannol (cis and trans form), cis resveratrol and trans-pterostilbene were identified and quantified in grape skin. Piceid was the most abundant stilbene as both cis and trans forms. As for anthocyanins, the synthesis and the stilbenes accumulation started during veraison, and then, the content of these molecules remained constant with the exception of piceid, whose content slightly increased until harvest. Therefore, stilbenes accumulation seems not to be affected by anthocyanins synthesis. On the contrary, proanthocyanidins, flavonols and hydroxycinnamyl tartrates decreased during ripening. When withering was applied to grapes as a post-harvest treatment, the content of anthocyanins, proanthocyanidins and of the most abundant stilbenes increased, while flavonols and hydroxycinnamyl tartrates decreased. The results suggest that degradation processes during withering are probably weaker or absent in the case of stilbenes, anthocyanins and proanthocyanidins.     

Time-resolved reflectance spectroscopy for non-destructive assessment of food quality

In the majority of most food and feed, visible, and near infrared light undergoes multiple scattering events and the overall light distribution is determined more by scattering rather than absorption due to the microscopic spatial changes in the refractive index. Conventional steady state reflectance spectroscopy can provide information on light attenuation, which depends both on light absorption and light scattering, but cannot separate these two effects. In contrast, time-resolved reflectance spectroscopy (TRS) allows more detailed optical characterization of diffusive media in terms of their absorption coefficient and reduced scattering coefficient. From the assessment of the absorption and reduced scattering coefficients, information can then be derived on the composition and internal structure of the medium. The main advantages of the technique are the absolute non-invasiveness, the potentiality for non-contact measurements and the capacity to probe internal properties with no influence from the skin. In this work we review the physical and technical issues related to the use of TRS for non-destructive quality assessment of fruit and vegetable. A laboratory system for broadband TRS, based on tunable mode-locked lasers and fast micro-channel plate photomultiplier and a portable set-up for TRS measurements, based on pulsed diode lasers and compact metal-channel photomultiplier, are described. Results on broadband optical characterization of fruits and applications of TRS to the detection of internal defects in pears and to maturity assessment in nectarines are presented. Conference: SPIE OPTICS EAST, 9–12 September 2007, Seaport World Trade Center, Boston, MA USA; Session: Optics for Natural Resources, Agriculture, and Foods II; Conference Chairs: Yud-Ren Chen, USDA Agricultural Research Service; George E. Meyer, Univ. of Nebraska/Lincoln; Shu-I Tu, USDA Agricultural Research Service.