Rapid Parallel Synthesis of Bioactive Folded Cyclotides by Using a Tea‐Bag Approach

We report here the first rapid parallel production of bioactive folded cyclotides by using Fmoc‐based solid‐phase peptide synthesis in combination with a “tea‐bag” approach. Using this approach, we efficiently synthesized 15 analogues of the CXCR4 antagonist cyclotide MCo‐CVX‐5c. Cyclotides were synthesized in a single‐pot, cyclization/folding reaction in the presence of reduced glutathione. Natively folded cyclotides were quickly purified from the cyclization/folding crude mixture by activated thiol Sepharose‐based chromatography. The different folded cyclotide analogues were then tested for their ability to inhibit the CXCR4 receptor in a cell‐based assay. The results indicated that this approach can be used for the efficient chemical synthesis of libraries of cyclotides with improved biological properties that can be easily interfaced with solution or cell‐based assays for rapid screening.     

Reinterpretation of the mechanical reinforcement of polymer nanocomposites reinforced with cellulose nanorods

The mechanical reinforcement of nanocomposites containing nanorods‐like fillers such as cellulose nanocrystals (CNCs) is often interpreted by adapting the classical parallel–series model, assuming a simple hyperbolic dependence between the percolation threshold and aspect ratio. However, such assumptions are valid only for nanorods with high aspect ratio and often are misinterpreting the reinforcement obtained at low volume fraction of filler loading. To elucidate this intriguing scenario, we proposed a new approach and validated it by compiling and reinterpreting some of available literature that represent the experimental reinforcement with CNCs. Our approach showed better accuracy, specifically for the cases of CNC nanorods with lower aspect ratio. We conclude that this route permits a more realistic evaluation of the mechanical reinforcement, where a physical parameter accounting the polymer filler association is introduced. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134, 45254.     

Silica nanoparticles inhibit the cation channel TRPV4 in airway epithelial cells

Background

Silica nanoparticles (SiNPs) have numerous beneficial properties and are extensively used in cosmetics and food industries as anti-caking, densifying and hydrophobic agents. However, the increasing exposure levels experienced by the general population and the ability of SiNPs to penetrate cells and tissues have raised concerns about possible toxic effects of this material. Although SiNPs are known to affect the function of the airway epithelium, the molecular targets of these particles remain largely unknown. Given that SiNPs interact with the plasma membrane of epithelial cells we hypothesized that they may affect the function of Transient Receptor Potential Vanilloid 4 (TRPV4), a cation-permeable channel that regulates epithelial barrier function. The main aims of this study were to evaluate the effects of SiNPs on the activation of TRPV4 and to determine whether these alter the positive modulatory action of this channel on the ciliary beat frequency in airway epithelial cells.

Results

Using fluorometric measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_i) we found that SiNPs inhibit activation of TRPV4 by the synthetic agonist GSK1016790A in cultured human airway epithelial cells 16HBE and in primary cultured mouse tracheobronchial epithelial cells. Inhibition of TRPV4 by SiNPs was confirmed in intracellular Ca²⁺ imaging and whole-cell patch-clamp experiments performed in HEK293T cells over-expressing this channel. In addition to these effects, SiNPs were found to induce a significant increase in basal [Ca²⁺]_i, but in a TRPV4-independent manner. SiNPs enhanced the activation of the capsaicin receptor TRPV1, demonstrating that these particles have a specific inhibitory action on TRPV4 activation. Finally, we found that SiNPs abrogate the increase in ciliary beat frequency induced by TRPV4 activation in mouse airway epithelial cells.

Conclusions

Our results show that SiNPs inhibit TRPV4 activation, and that this effect may impair the positive modulatory action of the stimulation of this channel on the ciliary function in airway epithelial cells. These findings unveil the cation channel TRPV4 as a primary molecular target of SiNPs.

     

Identification of a Small‐Molecule Inhibitor of HIV‐1 Assembly that Targets the Phosphatidylinositol (4,5)‐bisphosphate Binding Site of the HIV‐1 Matrix Protein

The development of drug resistance remains a critical problem for current HIV‐1 antiviral therapies, creating a need for new inhibitors of HIV‐1 replication. We previously reported on a novel anti‐HIV‐1 compound, N²‐(phenoxyacetyl)‐N‐[4‐(1‐piperidinylcarbonyl)benzyl]glycinamide ( 14 ), that binds to the highly conserved phosphatidylinositol (4,5)‐bisphosphate (PI(4,5)P₂) binding pocket of the HIV‐1 matrix (MA) protein. In this study, we re‐evaluate the hits from the virtual screen used to identify compound 14 and test them directly in an HIV‐1 replication assay using primary human peripheral blood mononuclear cells. This study resulted in the identification of three new compounds with antiviral activity; 2‐(4‐{[3‐(4‐fluorophenyl)‐1,2,4‐oxadiazol‐5‐yl]methyl})‐1‐piperazinyl)‐N‐(4‐methylphenyl)acetamide ( 7 ), 3‐(2‐ethoxyphenyl)‐5‐[[4‐(4‐nitrophenyl)piperazin‐1‐yl]methyl]‐1,2,4‐oxadiazole ( 17 ), and N‐[4‐ethoxy‐3‐(1‐piperidinylsulfonyl)phenyl]‐2‐(imidazo[2,1‐b][1,3]thiazol‐6‐yl)acetamide ( 18 ), with compound 7 being the most potent of these hits. Mechanistic studies on 7 demonstrated that it directly interacts with and functions through HIV‐1 MA. In accordance with our drug target, compound 7 competes with PI(4,5)P₂ for MA binding and, as a result, diminishes the production of new virus. Mutation of residues within the PI(4,5)P₂ binding site of MA decreased the antiviral effect of compound 7 . Additionally, compound 7 displays a broadly neutralizing anti‐HIV activity, with IC₅₀ values of 7.5–15.6 μM for the group M isolates tested. Taken together, these results point towards a novel chemical probe that can be used to more closely study the biological role of MA and could, through further optimization, lead to a new class of anti‐HIV‐1 therapeutics.     

Does the Neuroprotective Role of Anandamide Display Diurnal Variations?

The endocannabinoid system is a component of the neuroprotective mechanisms that an organism displays after traumatic brain injury (TBI). A diurnal variation in several components of this system has been reported. This variation may influence the recovery and survival rate after TBI. We have previously reported that the recovery and survival rate of rats is higher if TBI occurs at 1:00 than at 13:00. This could be explained by a diurnal variation of the endocannabinoid system. Here, we describe the effects of anandamide administration in rats prior to the induction of TBI at two different times of the day: 1:00 and 13:00. We found that anandamide reduced the neurological damage at both times. Nevertheless, its effects on bleeding, survival, food intake, and body weight were dependent on the time of TBI. In addition, we analyzed the diurnal variation of the expression of the cannabinoid receptors CB1R and CB2R in the cerebral cortex of both control rats and rats subjected to TBI. We found that CB1R protein was expressed more during the day, whereas its mRNA level was higher during the night. We did not find a diurnal variation for the CB2R. In addition, we also found that TBI increased CB1R and CB2R in the contralateral hemisphere and disrupted the CB1R diurnal cycle.     

Assessment of production efficiency,physicochemical properties and storage stability of spray-dried propolis,a natural food additive,using gum Arabic and OSA starch-based carrier systems

The aim of this work was to obtain propolis in a powder, alcohol-free, water-dispersed and shelf-stable form. Propolis extract was spray-dried using gum Arabic and octenyl succinic anhydride (OSA) starch as carriers in two different weight ratios (1:4 and 1:6). Spray-dried propolis samples were evaluated for morphology, moisture, water activity, water dispersibility, hygroscopicity, particle size, particle distribution, entrapping efficiency, stability, isotherms and antioxidant properties. The spray-drying process produced round particles with sizes ranging from 15 to 24 μm. This process preserved the antioxidant activity of propolis and also allowed propolis to be obtained in a powder form, which was stable during storage at room temperature, had low hygroscopicity and was highly dispersible in cold water. The application of this technology could increase the use of propolis in various industrial applications, such as an antimicrobial and as an antioxidant in food.     

Rancidity development during the chilled storage of farmed Coho salmon (Oncorhynchus kisutch)

Coho salmon (Oncorhynchus kisutch) is a fatty fish species whose farming production has greatly increased in recent years. Lipid damage produced during Coho salmon chilled storage was studied for up to 24 d. Lipid hydrolysis (free fatty acids, FFA) and oxidation (conjugated dienes; peroxide value, PV; thiobarbituric acid index, TBA‐i; fluorescent compounds formation, FR; browning development) were determined and compared to lipid composition (polyene index, PI; astaxanthin, AX) changes and sensory assessment (rancid odour development) results. Most lipid damage indices developed slowly during storage; thus, values obtained for FFA, PV, TBA‐i and FR were in all cases under 1.5 g/100 g, 4.0 meq oxygen/kg lipid, 0.40 mg malondialdehyde/kg muscle and 0.40, respectively. Odour assessment showed a significant (p <0.05) rancidity development at day 10, when compared to starting fish material; then, non‐acceptable values were obtained at days 19 and 24. The PI analysis showed not many differences during the storage time, with the lowest mean value at day 19. AX analysis indicated a relatively high content in the white muscle, which was maintained till the end of the experiment. A low oxidation development is concluded for Coho salmon lipids when compared to other fatty fish species under the same chilling conditions. AX was found to contribute to the oxidation stability of Coho salmon lipids, due to its free radical scavenger properties.     

Determination of the predominant polymorphic form of mango (Mangifera indica) almond fat by differential scanning calorimetry and X‐ray diffraction

In this paper, some important properties, from the application point of view, of mango (Mangifera indica) almond fat var. Manila (MAF) were analyzed by differential scanning calorimetry and X‐ray diffraction techniques. The thermal profile, the solid fat content (SFC) and the predominant polymorphism of MAF samples, previously characterized chemically, were studied. The results showed that this fat, from one of the main residues of mango industrialization, had a relatively simple fusion/crystallization behavior. Stabilized samples showed a simple SFC profile with one marked slope between 35 and 40 °C. Different thermal histories demonstrated the existence of at least four polymorphs. The non‐stabilized samples corresponded predominantly to the formation of the crystalline &α form. The stabilized samples, tested under several time and temperature conditions, allowed the formation of two other polymorphs, which are both unstable forms and were formed during &α to β polymorphic transition. The X‐ray diffraction information confirmed the presence of the less and more stable MAF polymorphs, allowing us to conclude that MAF is a β‐stable fat, just as is cocoa butter.