In Vitro Selection of Lactobacillus and Bifidobacterium Probiotic Strains for the Management of Oral Pathobiont Infections Associated to Systemic Diseases

The human oral pathobionts Aggregatibacter actinomycetemcomitans, Streptococcus mitis and Streptococcus mutans, in dysbiosis-promoting conditions, lead to oral infections, which also represent a threat to human systemic health. This scenario may be worsened by antibiotic misuse, which favours multi-drug resistance, making the research on pathogen containment strategies more than crucial. Therefore, we aimed to in vitro select the most promising probiotic strains against oral pathogen growth, viability, biofilm formation, and co-aggregation capacity, employing both the viable probiotics and their cell-free supernatants (CFSs). Interestingly, we also assessed probiotic efficacy against the three-pathogen co-culture, mimicking an environment similar to that in vivo. Overall, the results showed that Lactobacillus CFSs performed better than the Bifidobacterium, highlighting Limosilactobacillus reuteri LRE11, Lacticaseibacillus rhamnosus LR04, Lacticaseibacillus casei LC04, and Limosilactobacillus fermentum LF26 as the most effective strains, opening the chance to deeper investigation of their action and CFS composition. Altogether, the methodologies presented in this study can be used for probiotic efficacy screenings, in order to better focus the research on a viable probiotic, or on its postbiotics, suitable in case of infections.     

Modeling and biological investigations of an unusual behavior of novel synthesized acridine-based polyamine ligands in the binding of double helix and G-quadruplex DNA

Three novel 2,7-substituted acridine derivatives were designed and synthesized to investigate the effect of this functionalization on their interaction with double-stranded and G-quadruplex DNA. Detailed investigations of their ability to bind both forms of DNA were carried out by using spectrophotometric, electrophoretic, and computational approaches. The ligands in this study are characterized by an open-chain (L1) or a macrocyclic (L2, L3) framework. The aliphatic amine groups in the macrocycles are joined by ethylene (L2) or propylene chains (L3). L1 behaved similarly to the lead compound m-AMSA, efficiently intercalating into dsDNA, but stabilizing G-quadruplex structures poorly, probably due to the modest stabilization effect exerted by its protonated polyamine chains. L2 and L3, containing small polyamine macrocyclic frameworks, are known to adopt a rather bent and rigid conformation; thus they are generally expected to be sterically impeded from recognizing dsDNA according to an intercalative binding mode. This was confirmed to be true for L3. Nevertheless, we show that L2 can give rise to efficient π-π and H-bonding interactions with dsDNA. Additionally, stacking interactions allowed L2 to stabilize the G-quadruplex structure: using the human telomeric sequence, we observed the preferential induction of tetrameric G-quadruplex forms. Thus, the presence of short ethylene spacers seems to be essential for obtaining a correct match between the binding sites of L2 and the nucleobases on both DNA forms investigated. Furthermore, current modeling methodologies, including docking and MD simulations and free energy calculations, provide structural evidence of an interaction mode for L2 that is different from that of L3; this could explain the unusual stabilizing ability of the ligands (L2>L3>L1) toward G-quadruplex that was observed in this study.     

NMR structure of the (+)-CPI-indole/d(GACTAATTGAC)-d(GTCAATTAGTC) covalent complex

We report the NMR solution structure of (+)-CPI-indole (CPI, 1,2,8,8a-tetrahydrocyclopropa[c]pyrrolo[3,2-e]indol-4(5H)-one), an agent belonging to the CC-1065/duocarmycin family of antitumor compounds. This (+)-CPI-indole structure is covalently bound to d(G(1)ACTAATTGTC(11))-d(G(12)TCAATTAGTC(22)), a synthetic DNA duplex containing a high-affinity binding site. The three-dimensional structure has been determined by several cycles of restrained molecular dynamics calculations with a total of 563 NMR-derived constraints, both in vacuo and by using the generalized Born solvent continuum model. In-depth analysis of the structure of this ligand-DNA complex led to a detailed knowledge of the bound state conformation of the CPI-indole, the most simplified agent related to CC-1065 and duocarmycins, the parent members of a family of extremely potent antitumor compounds. Comparison of the CPI-indole bound conformation with those previously found for (+)-duocarmycin SA (DSA), its unnatural enantiomer (-)-DSA, and the demethoxylated analogue (+)-DSI in their DNA complexes provided additional evidence of the tight correlation between the catalytic effect exerted by DNA on the alkylation reaction and the extent of angular twist between the two planar heteroaromatic subunits of these agents. Additionally, comparison of the structural features of the DNA-bound state of a "naked" ligand, such as CPI-indole, with those of various other duocarmycin agents provided useful information for the interpretation of the observed effects on chemical reactivity of the different substitution patterns at the hemispheres of these types of complex.     

A cyclic CCK8 analogue selective for the cholecystokinin type A receptor: design, synthesis, NMR structure and binding measurements

A cyclic CCK8 analogue, cyclo(29,34)[Dpr(29),Lys(34)]-CCK8 (Dpr=L-2,3-diaminopropionic acid), has been designed on the basis of the NMR structure of the bimolecular complex between the N-terminal fragment of the CCK(A) receptor and its natural ligand CCK8. The conformational features of cyclo(29,34)[Dpr(29),Lys(34)]-CCK8 have been determined by NMR spectroscopy in aqueous solution and in water containing DPC-d(38) micelles (DPC=dodecylphosphocholine). The structure of the cyclic peptide in aqueous solution is found to be in a relaxed conformation, with the backbone and Dpr29 side chain atoms making a planar ring and the N-terminal tripeptide extending approximately along the plane of this ring. In DPC/water, the cyclic peptide adopts a "boat-shaped" conformation, which is more compact than that found in aqueous solution. The cyclic constraint between the Dpr29 side chain and the CCK8 carboxyl terminus (Lys34) introduces a restriction in the backbone conformational freedom. However, the interaction of cyclo(29,34)[Dpr(29),Lys(34)]-CCK8 with the micelles still plays an important role in the stabilisation of the bioactive conformation. A careful comparison of the NMR structure of the cyclic peptide in a DPC micelle aqueous solution with the structure of the rationally designed model underlines that the turn-like conformation in the Trp30-Met31 region is preserved, such that the Trp30 and Met31 side chains can adopt the proper spatial orientation to interact with the CCK(A) receptor. The binding properties of cyclo(29,34)[Dpr(29),Lys(34)]-CCK8 to the N-terminal receptor fragment have been investigated by fluorescence spectroscopy in a micellar environment. Estimates of the apparent dissociation constant, K(d), were in the range of 70-150 nM, with a mean value of 120+/-27 nM. Preliminary nuclear medicine studies on cell lines transfected with the CCK(A) receptor indicate that the sulfated-Tyr derivative of cyclo(29,34)[Dpr(29),Lys(34)]-CCK8 displaces the natural ligand with an IC(50) value of 15 microM.     

Semicontinuous heterophase copolymerization of vinyl acetate and butyl acrylate

Vinyl acetate (VAc) and butyl acrylate (BuA) were copolymerized in heterophase by a semicontinuous process (unseeded) and compared with the seeded semicontinuous microemulsion polymerization of the same monomers. A mixture of sodium dodecyl sulfate (SDS) and poly(ethylene oxide) dodecyl ether (Brij‐35®) were used as surfactants. The effects of monomer addition rate (R_a) and surfactants concentrations (4 or 1 wt % with respect to the initial mixture of reaction) on polymer and latex properties were studied. High copolymer content latexes (24–36 wt %) with average particle diameters (D_p) from 38 to 55 nm and relatively narrow particle size distributions, high polymerization rates, weight ratios of polymer to surfactant (P/S) from 13.3 to 32.8 were obtained. The number‐average molecular weights (M_n) were between 96,000 and 188,000 g/mol. Homogeneous copolymer compositions were obtained throughout the reaction for both, seeded and unseeded processes, which is not possible by the usual batch microemulsion process. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

New Methodology for Quantifying Polycyclic Aromatic Hydrocarbons (PAHs) Using High-Resolution Aerosol Mass Spectrometry

This article presents a new methodology to potentially quantify polycyclic aromatic hydrocarbon (PAH) isomers using high-resolution time of flight aerosol mass spectrometer (HR-AMS). The fragmentation of PAHs within the HR-AMS is such that significant signal remains at the molecular ion. After quantifying the molecular ion signal and taking into account potential interferences, the amount of the parent PAH in the aerosol may be inferred once its fragmentation pattern is also known. The potential of this approach was evaluated using mixed gasoline and diesel engine exhaust sampled under varying conditions. This dataset led to the identification and quantification within the aerosol mass spectra of the molecular ions associated with 53 PAH isomers, including both unsubstituted and functionalized species. An evaluation of anticipated interferences shows that interferences from larger molecular weight PAHs (i.e., PAH/PAH interferences) could be constrained based on the fragmentation behavior of PAHs from existing HR-AMS laboratory spectra. Other signal interferences for this data set are typically less than 5% of the total signal or, for ¹³C isotopic interferents, are well constrained by measurements of the dominant isotope. The experimental data reveal that the fractional PAH molecular ion signal remained stable despite dramatic temporal variability of the total particulate organic signal. The fractional contributions of the molecular ions for grouped PAH species and even individual compounds were remarkably consistent across experiments. The distribution of PAHs showed no apparent dependence on engine load or exhaust type. Full application of this approach will require a greater number of standard HR-AMS spectra for PAHs, so that the relationship between compounds and their molecular ions may be understood more precisely.

Copyright 2015 American Association for Aerosol Research     

Single Scattering Albedo Monitor for Airborne Particulates

We describe a robust, compact, field deployable instrument (the CAPS PM_ssa) that simultaneously measures airborne particle light extinction and scattering coefficients and thus the single scattering albedo (SSA) on the same sample volume. With an appropriate change in mirrors and light source, measurements have been made at wavelengths ranging from 450 to 780 nm. The extinction measurement is based on cavity attenuated phase shift (CAPS) techniques as employed in the CAPS PM_ex particle extinction monitor; scattering is measured using integrating nephelometry by incorporating a Lambertian integrating sphere within the sample cell. The scattering measurement is calibrated using the extinction measurement. Measurements using ammonium sulfate particles of various sizes indicate that the response of the scattering channel with respect to measured extinction is linear to within 1% up to 1000 Mm^?1 and can be extended further (4000 Mm^?1) with additional corrections. The precision in both measurement channels is less than 1 Mm^?1 (1s, 1σ). The truncation effect in the scattering channel, caused by light lost at extreme forward/backward scattering angles, was measured as a function of particle size using monodisperse polystyrene latex particles (n = 1.59). The results were successfully fit using a simple geometric model allowing for reasonable extrapolation to a given wavelength, particle index of refraction, and particle size distribution, assuming spherical particles. For sub-micron sized particles, the truncation corrections are comparable to those reported for commercial nephelometers. Measurements of the optical properties of ambient aerosol indicate that the values of the SSA of these particles measured with this instrument (0.91 ± 0.03) using scattering and extinction agreed within experimental uncertainty with those determined using extinction measured by this instrument and absorption measured using a multi-angle absorption photometer (0.89 ± 0.03) where the uncertainties are derived from best estimates of the accuracy of the two approaches.

Copyright 2015 American Association for Aerosol Research     

Peptide-Lipid Interactions: Experiments and Applications

The interactions between peptides and lipids are of fundamental importance in the functioning of numerous membrane-mediated cellular processes including antimicrobial peptide action, hormone-receptor interactions, drug bioavailability across the blood-brain barrier and viral fusion processes. Moreover, a major goal of modern biotechnology is obtaining new potent pharmaceutical agents whose biological action is dependent on the binding of peptides to lipid-bilayers. Several issues need to be addressed such as secondary structure, orientation, oligomerization and localization inside the membrane. At the same time, the structural effects which the peptides cause on the lipid bilayer are important for the interactions and need to be elucidated. The structural characterization of membrane active peptides in membranes is a harsh experimental challenge. It is in fact accepted that no single experimental technique can give a complete structural picture of the interaction, but rather a combination of different techniques is necessary.     

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.     

The influence of drying air temperature on the physical properties of dried and rehydrated eggplant

Drying processes generally cause volume and surface change of foodstuffs. Information on the porous structure and the mechanical properties of dried food products is needed for determining food quality, process design and estimating properties such as density and moisture diffusivity.In this work we investigated the structural changes induced in eggplant by convective air drying at four different temperatures (40, 50, 60 and 70 °C) and their effect on the subsequent rehydration process. Drying and rehydration kinetic curves were also measured.The changes in physical properties, such as porosity, pore-size distribution and bulk density were determined by Hg porosimetry, scanning electron microscopy and optical microscopy while their effect on the textural characteristics by dynamometric measurements.As expected, the increase of the drying air temperature causes shorter drying times. The drying temperature influences strongly the microstructure of dried samples: the porosity increases with the air temperature, but the structure is better preserved at intermediate temperature (60 °C) as confirmed by the lower firmness values with respect to the other dehydrated samples (40, 50 and 70 °C). In these latter, the longer drying time and the higher temperature, respectively, causes the development of a wrinkled structure. In particular, at 70 °C the structure of dehydrated samples appears totally broken with a consequent faster water uptake during rehydration.