Atomic force microscopy measurement of heterogeneity in bacterial surface hydrophobicity

The structure and physicochemical properties of microbial surfaces at the molecular level determine their adhesion to surfaces and interfaces. Here, we report the use of atomic force microscopy (AFM) to explore the morphology of soft, living cells in aqueous buffer, to map bacterial surface heterogeneities, and to directly correlate the results in the AFM force-distance curves to the macroscopic properties of the microbial surfaces. The surfaces of two bacterial species, Acinetobacter venetianus RAG-1 and Rhodococcus erythropolis 20S-E1-c, showing different macroscopic surface hydrophobicity were probed with chemically functionalized AFM tips, terminating in hydrophobic and hydrophilic groups. All force measurements were obtained in contact mode and made on a location of the bacterium selected from the alternating current mode image. AFM imaging revealed morphological details of the microbial-surface ultrastructures with about 20 nm resolution. The heterogeneous surface morphology was directly correlated with differences in adhesion forces as revealed by retraction force curves and also with the presence of external structures, either pili or capsules, as confirmed by transmission electron microscopy. The AFM force curves for both bacterial species showed differences in the interactions of extracellular structures with hydrophilic and hydrophobic tips. A. venetianus RAG-1 showed an irregular pattern with multiple adhesion peaks suggesting the presence of biopolymers with different lengths on its surface. R. erythropolis 20S-E1-c exhibited long-range attraction forces and single rupture events suggesting a more hydrophobic and smoother surface. The adhesion force measurements indicated a patchy surface distribution of interaction forces for both bacterial species, with the highest forces grouped at one pole of the cell for R. erythropolis 20S-E1-c and a random distribution of adhesion forces in the case of A. venetianus RAG-1. The magnitude of the adhesion forces was proportional to the three-phase contact angle between hexadecane and water on the bacterial surfaces.     

Evaluation of different polymerase chain reaction methods for the identification of Vibrio parahaemolyticus strains isolated by cultural methods

Control of contamination by Vibrio parahaemolyticus in fishery products is often hampered by the lack of standardized methods and by the uncertainty associated with biochemical identification of the isolates. In this study, 5 polymerase chain reaction (PCR) methods for the identification of V. parahaemolyticus to the species level were evaluated by using 25 Vibrio reference strains and 163 isolates from fishery products, environmental sources, and clinical samples. Sequence targets of the methods were toxR, gyrB, and tlh genes (tested with 2 protocols), and the fragment pR72H. Isolate identification was confirmed by sequencing of the 16S rRNA gene and by PCR protocols for the identification of other Vibrio species. The PCR assay targeting the toxR gene achieved the highest performance (100% inclusivity and exclusivity). The 2 PCR protocols based on tlh gene detection, although showing the same inclusivity (100%), differed in the exclusivity (50 and 91%, respectively). Finally, the results provided by the PCR assays targeting the gyrB gene and pR72H fragment were less reliable and, in some cases, difficult to assess. According to the results of this study, the characteristics of accuracy expressed by the toxR identification method make it a suitable candidate as a reference method for the molecular identification of V. parahaemolyticus strains.     

A Semi Rigid Novel Hydroxamate AMPED-Based Ligand for 89Zr PET Imaging

In this work, we designed, developed, characterized, and investigated a new chelator and its bifunctional derivative for ⁸⁹Zr labeling and PET-imaging. In a preliminary study, we synthesized two hexadentate chelators named AAZTHAS and AAZTHAG, based on the seven-membered heterocycle AMPED (6-amino-6-methylperhydro-1,4-diazepine) with the aim to increase the rigidity of the ⁸⁹Zr complex by using N-methyl-N-(hydroxy)succinamide or N-methyl-N-(hydroxy)glutaramide pendant arms attached to the cyclic structure. N-methylhydroxamate groups are the donor groups chosen to efficiently coordinate ⁸⁹Zr. After in vitro stability tests, we selected the chelator with longer arms, AAZTHAG, as the best complexing agent for ⁸⁹Zr presenting a stability of 86.4 ± 5.5% in human serum (HS) for at least 72 h. Small animal PET/CT static scans acquired at different time points (up to 24 h) and ex vivo organ distribution studies were then carried out in healthy nude mice (n = 3) to investigate the stability and biodistribution in vivo of this new ⁸⁹Zr-based complex. High stability in vivo, with low accumulation of free ⁸⁹Zr in bones and kidneys, was measured. Furthermore, an activated ester functionalized version of AAZTHAG was synthesized to allow the conjugation with biomolecules such as antibodies. The bifunctional chelator was then conjugated to the human anti-HER2 monoclonal antibody Trastuzumab (Tz) as a proof of principle test of conjugation to biologically active molecules. The final ⁸⁹Zr labeled compound was characterized via radio-HPLC and SDS-PAGE followed by autoradiography, and its stability in different solutions was assessed for at least 4 days.     

Participation of the nucleobases in the regioselective backbone fragmentation of nucleic acids. A molecular dynamics and tandem mass spectrometric investigation on a model dinucleoside phosphotriester

The anions (I–III) obtained from O-methyl 5′-O-(5′-deoxythymidine) 3′-O-(2′,3′-dideoxyuridine) phosphate by the competitive removal of the 3-N-H protons of the nucleobases and of the methyl group from the phosphotriester bond, assume in the gas phase stable conformations as a function of their charge site. The mass-analyzed ion kinetic energy (MIKE) spectra of I and III show that the regioselective backbone cleavage of the internucleotidic linkage is controlled by the 2′-H proton transfer to the nucleobase within the 5′-end nucleoside. Similar pathways are taken by species II when the nucleobase is eliminated as neutral from the 5′-end nucleoside.     

Electrochemical study and MO — modeling of the reduction behaviour of some arylidene substituted derivatives of dibenz [b,e]-thiepin-5,5-dioxide-11-one

The electrochemical reduction of four arylidene substituted derivatives of dibenz-[b,e]-thiepin-5,5-dioxide-11- one has been studied by cyclic voltammetry on platinum (Pt) electrode in aprotic media (DMSO), coupled with spectral EPR techniques. From the analysis of cyclic voltammetry experiments, aided by digital simulations using DigiSim software, the kinetic and thermodynamic parameters were evaluated for each system. The electrochemical behaviour is strongly directed by the nature of the arylidene substituents bound to the central heterocycle. The electrochemical investigation and solvent dependent semiempirical modeling using the PM3 hamiltonian in the frame of AMPAC program package allowed a rationalisation of experimental data regarding the electrochemical reduction and the reactivity of the intermediate species involved.      

Convergence of a finite element/ALE method for the Stokes equations in a domain depending on time

We consider the approximation of the unsteady Stokes equations in a time dependent domain when the motion of the domain is given. More precisely, we apply the finite element method to an Arbitrary Lagrangian Eulerian (ALE) formulation of the system. Our main results state the convergence of the solutions of the semi-discretized (with respect to the space variable) and of the fully-discrete problems towards the solutions of the Stokes system.     

The structure characterization of dinucleoside and nucleoside glucopyranosides lipophilic phosphotriesters by fast-atom bombardment tandem mass spectrometry

Rules for the gas-phase fragmentation mechanism of the negative ions of lipophilic phosphotriester molecules of biological interest have been established by fast-atom bombardment mass spectrometry/mass spectrometry. The mass-analyzed ion kinetic energy spectra of the [M ? H]^? of dinucleoside (1–4) and nucleoside glucopyranoside (5–9) phosphotriesters show that in the absence of charges on the phosphate bridge, the availability of acidic protons on the 5′-end nucleobase drives a preferred reaction path which leads to 5′-O-nucleotide or 6-O-glucopyranoside monophosphate anions.     

Copolymerization of VDF and HFP in Supercritical Carbon Dioxide: A Robust Approach for Modeling Precipitation and Dispersion Kinetics

A kinetic model is developed for the heterogeneous free‐radical copolymerization of vinylidene fluoride and hexafluoropropylene in supercritical CO₂. The model accounts for polymerization in both the dispersed (polymer‐rich) phase and in the continuous (polymer‐free) supercritical phase, for radical interphase transport, diffusion limitations, and chain‐length‐dependent termination in the polymer‐rich phase. A parameter evaluation strategy is developed and detailed to estimate most of the kinetic parameters a priori while minimizing their evaluation by direct fitting. The resulting model predictions compare favorably with the experimental results of conversion and MWD at varying monomer feed composition, monomer concentration, interphase area, and pressure of the system.

     

Immobilization of kojic acid in ZnAl-hydrotalcite like compounds

Kojic acid (KOJ) is a melanin synthesis inhibitor widely used as skin lightening agent in topical preparations. Unfortunately it is easily susceptible to photo-oxidation, phenomenon responsible for chemical and organoleptic modifications. The aim of this work was the intercalation of KOJ in hydrotalcite-like compounds (HTlc) in order to stabilize KOJ and to reduce its photolability. Hydrotalcite containing Zn and Al (ZnAl-HTlc) was used as host to obtain the final compound ZnAl-HTlc-KOJ. The intercalation was carried out, after many attempts, by ionic exchange mechanism by means of the strong base EtO^? in anhydrous ethanol/dimethylsulfoxide (DMSO) mixture as solvent in order to generate KOJ^? anions. The final product was characterized by the X-ray powder diffraction (XRPD), FT-IR spectroscopy, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and elemental analysis. The intercalated compound was formulated in a siliconic water free self-emulsifying ointment and the in vitro release profile was evaluated. All samples (intercalation compound and its formulation) were submitted also to spectrophotometric assays in order to evaluate the matrix protective effect towards ultraviolet rays.     

The Effect of UV-Irradiation on Poly(vinyl alcohol) Composites with Bacterial Cellulose

The development of biodegradable packaging materials, especially from renewable resources is a constant preoccupation of nowadays, because of the environmental problems caused by synthetic polymers. The combination of cellulose with other polymeric materials could be an ecologic alternative and a way to use renewable resources for food packaging. Bacterial cellulose which is produced by microbial fermentation is also a promising material which can be used not only in biomedical application, but also as food packaging material. In this research different composite films between poly(vinyl alcohol)-bacterial cellulose (PVA-BC) were obtained by casting method. The obtained films were UV irradiated for different periods of times from 1 to 10 hours, using a mercury lamp, Philips TUV-30, emitting light mainly at 254 nm. Changes in FT-IR spectra before and after UV irradiation and the modification of transparency and of the swelling characteristics of the films were observed. As it was expected the composites materials are sensitive at UV exposure.