Quantification of Enzymatic Biofilm Removal Using the Sauerbrey Equation: Application to the Case of Pseudomonas protegens.

A methodology for the quantitative analysis of enzymatic removal of biofilms (BF) was developed, based on a quartz crystal microbalance (QCM) under stationary conditions. This was applied to the case of Pseudomonas protegens (PP) BFs, through a series of five enzymes, whose removal activity was screened using the presented methodology. The procedure is based on the following: when BFs can be modeled as rigid materials, QCM can be used as a balance under stationary conditions for determining the BFs mass reduction by enzymatic removal. For considering a BF as a rigid model, energy dissipation effects, associated with viscoelastic properties of the BF, must be negligible. Hence, a QCM system with detection of dissipation (referred to as QCM with dissipation) was used for evaluating the energy losses, which, in fact, resulted in negligible energy losses in the case of dehydrated PP BFs, validating the application of the Sauerbrey equation for the change of mass calculations. The stationary methodology reduces operating times and simplifies data analysis in comparison to dynamic approaches based on flow setups, which requires the incorporation of dissipation effects due to the liquid media. By carrying out QCM, glycosidase-type enzymes showed BF removal higher than 80% at enzyme concentration 50 ppm, reaching removal over 90% in the cases of amylase and cellulase/xylanase enzymes. The highest removal percentage produced a reduction from about 15 to 1 µg in the BF mass. Amylase enzyme was tested from below 50 to 1 ppm, reaching around 60% of removal at 1 ppm. The obtained results were supported by other instrumental techniques such as Raman spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy, atomic force microscopy, high performance anion exchange chromatography, thermogravimetric analysis, and differential scanning calorimetry. The removal quantifications obtained with QCM were compared with those obtained by well-established screening techniques (UV-vis spectrophotometry using crystal violet and agar diffusion test). The proposed methodology expands the possibility of using a quartz microbalance to perform enzymatic activity screening.

Chitosan-Based Nanogels Designed for Betanin-Rich Beetroot Extract Transport: Physicochemical and Biological Aspects.

Nanotechnology has emerged as a possible solution to improve phytochemicals' limitations. The objective of the present study was to encapsulate beetroot extract (BR Ext) within a chitosan (CS)-based nanogel (NG) designed via ionic crosslinking with tripolyphosphate (TPP) for betanin (Bet) delivery, mainly in the ophthalmic environment. BR Ext is rich in betanin (Bet) according to thin layer chromatography (TLC), UV-visible spectroscopy, and HPLC analysis. NG presented a monodisperse profile with a size of 166 ± 6 nm and low polydispersity (0.30 ± 0.03). ζ potential (ζ-Pot) of +28 ± 1 is indicative of a colloidally stable system. BR Ext encapsulation efficiency (EE) was 45 ± 3%. TEM, with the respective 3D-surface plots and AFM, showed spherical-elliptical-shaped NG. The BR Ext release profile was biphasic with a burst release followed by slow and sustained phase over 12 h. Mucoadhesion assay demonstrated interactions between NG with mucin. Moreover, NG provided photoprotection and pH stability to BR Ext. FRAP and ABTS assays confirmed that BR Ext maintained antioxidant activity into NG. Furthermore, in vitro assays using human retinal cells displayed absence of cytotoxicity as well as an efficient protection against injury agents (LPS and H2O2). NGs are a promising platform for BR Ext encapsulation, exerting controlled release for ophthalmological use.

Biofilm Formation of Two Different Marine Bacteria on Modified PDMS Surfaces is Affected by Surface Roughness and Topography.

Different strategies were tested to reduce biofilm formation of the model marine bacteria Cobetia marina and Marinobacter hydrocarbonoclasticus on cross-linked polydimethylsiloxane (PDMS) coated aluminum and cellulose acetate surfaces modified by addition of multi-walled carbon nanotubes (MWCNT) or exposure of the surfaces to bromine vapors in the presence and absence of UV irradiation. The three surface modifications explored led to important reductions in biofilm formation for the two marine bacteria, up to 30% in the case of exposure to Br2(g). Biofouling reduction could be correlated to surface properties in all cases through the introduction of a quantitative theoretical model based on an effective roughness parameter, Raeff, that accounted for the different morphological changes observed. The model considers the possibility of bacterial inclusion into large surface wells, as observed by AFM in the case of Br2(g) + UV light treatment. In addition, a linear relationship was observed between biofouling reduction and the Raeff effective roughness parameter.

Hybrid Antimicrobial Films Containing a Polyoxometalate-Ionic Liquid.

The increasing resistance of pathogenic microorganisms against common treatments requires innovative concepts to prevent infection and avoid long-term microbe viability on commonly used surfaces. Here, we report the preparation of a hybrid antimicrobial material based on the combination of microbiocidal polyoxometalate-ionic liquids (POM-ILs) and a biocompatible polymeric support, which enables the development of surface coatings that prevent microbial adhesion. The composite material is based on an antibacterial and antifungal room-temperature POM-IL composed of guanidinium cations (N,N,N',N'-tetramethyl-N″, N″-dioctylguanidinum) combined with lacunary Keggin-type polyoxotungstate anions, [α-SiW11O39]8-. Integration of the antimicrobial POM-IL into the biocompatible, flexible, and stable polymer poly(methyl methacrylate) (PMMA) results in processable films, which are suitable as surface coatings or packaging materials to limit the proliferation and spread of pathogenic microorganisms (e.g., on public transport and hospital surfaces, or in ready-to-eat-food packaging).

Aromaticity at position 39 in α-synuclein: A modulator of amyloid fibril assembly and membrane-bound conformations.

Recent studies revealed that molecular events related with the physiology and pathology of αS might be regulated by specific sequence motifs in the primary sequence of αS. The importance of individual residues in these motifs remains an important open avenue of investigation. In this work, we have addressed the structural details related to the amyloid fibril assembly and lipid-binding features of αS through the design of site-directed mutants at position 39 of the protein and their study by in vitro and in vivo assays. We demonstrated that aromaticity at position 39 of αS primary sequence influences strongly the aggregation properties and the membrane-bound conformations of the protein, molecular features that might have important repercussions for the function and dysfunction of αS. Considering that aggregation and membrane damage is an important driver of cellular toxicity in amyloid diseases, future work is needed to link our findings with studies based on toxicity and neuronal cell death. BRIEF STATEMENT OUTLINING SIGNIFICANCE: Modulation by distinct sequential motifs and specific residues of αS on its physiological and pathological states is an active area of research. Here, we demonstrated that aromaticity at position 39 of αS modulates the membrane-bound conformations of the protein, whereas removal of aromatic functionality at position 39 reduces strongly the amyloid assembly in vitro and in vivo. Our study provides new evidence for the modulation of molecular events related with the physiology and pathology of αS.

Resveratrol encapsulation in high molecular weight chitosan-based nanogels for applications in ocular treatments: Impact on human ARPE-19 culture cells.

Oxidative stress and inflammation play a pivotal role in ocular diseases. Resveratrol (RSV) is a natural bioactive that has recently attracted attention due to it potent antioxidant and anti-inflammatory properties. However, RSV presents poor aqueous solubility and chemical instability. Besides, effective drug delivery to the posterior segment of the eye is challenge. Nanotechnology emerges as a possible solution to improve both limitations. Here, we developed and characterized nanogels (NG) based on high molecular weight chitosan (HCS) crosslinked with sodium tripolyphosphate. The distribution size of NG presented a major population around 140 nm with a ζ-potential value of 32 ± 2 mV. TEM and AFM images showed that NG exhibited a rounded morphology. RSV encapsulation efficiency was 59 ± 1%. Photodegradation experiments showed that HCS by its own protects RSV from UV light-induced degradation. Biocompatibility assays revealed that NG were not cytotoxic neither inflammatory in human retinal pigment epithelial cells (ARPE-19), which constitutes the outer blood-retinal barrier. After cellular internalization, we report an endo-lysosomal escape of NG, which is crucial for efficient nanocarriers delivery systems. In conclusion, we envision that HCS based NG could constitute novel carriers for RSV, opening the possibility of its application in ocular diseases.

Effect of copper on diesel degradation in Pseudomonas extremaustralis.

Environments co-contaminated with heavy metals and hydrocarbons have become an important problem worldwide, especially due to the effect of metals on hydrocarbon degrading microorganisms. Pseudomonas extremaustralis, a bacterium isolated from a pristine pond in Antarctica, showed high capabilities to cope with environmental stress and a very versatile metabolism that includes alkane degradation under microaerobic conditions. In this work, we analyzed P. extremaustralis' capability to resist high copper concentrations and the effect of copper presence in diesel biodegradation. We observed that P. extremaustralis resisted up to 4 mM CuSO4 in a rich medium such as LB. This copper resistance is sustained by the presence of the cus and cop operons together with other efflux systems and porins located in a single region in P. extremaustralis genome. When copper was present, diesel degradation was negatively affected, even though copper enhanced bacterial attachment to hydrocarbons. However, when a small amount of glucose (0.05% w/v) was added, the presence of CuSO4 enhanced alkane degradation. In addition, atomic force microscopy analysis showed that the presence of glucose decreased the negative effects produced by copper and diesel on the cell envelopes.

Novel role of the LPS core glycosyltransferase WapH for cold adaptation in the Antarctic bacterium Pseudomonas extremaustralis.

Psychrotroph microorganisms have developed cellular mechanisms to cope with cold stress. Cell envelopes are key components for bacterial survival. Outer membrane is a constituent of Gram negative bacterial envelopes, consisting of several components, such as lipopolysaccharides (LPS). In this work we investigated the relevance of envelope characteristics for cold adaptation in the Antarctic bacterium Pseudomonas extremaustralis by analyzing a mini Tn5 wapH mutant strain, encoding a core LPS glycosyltransferase. Our results showed that wapH strain is impaired to grow under low temperature but not for cold survival. The mutation in wapH, provoked a strong aggregative phenotype and modifications of envelope nanomechanical properties such as lower flexibility and higher turgor pressure, cell permeability and surface area to volume ratio (S/V). Changes in these characteristics were also observed in the wild type strain grown at different temperatures, showing higher cell flexibility but lower turgor pressure under cold conditions. Cold shock experiments indicated that an acclimation period in the wild type is necessary for cell flexibility and S/V ratio adjustments. Alteration in cell-cell interaction capabilities was observed in wapH strain. Mixed cells of wild type and wapH strains, as well as those of the wild type strain grown at different temperatures, showed a mosaic pattern of aggregation. These results indicate that wapH mutation provoked marked envelope alterations showing that LPS core conservation appears as a novel essential feature for active growth under cold conditions.

Increased Surface Roughness in Polydimethylsiloxane Films by Physical and Chemical Methods.

Two methods, the first physical and the other chemical, were investigated to modify the surface roughness of polydimethylsiloxane (PDMS) films. The physical method consisted of dispersing multi-walled carbon nanotubes (MWCNTs) and magnetic cobalt ferrites (CoFe2O4) prior to thermal cross-linking, and curing the composite system in the presence of a uniform magnetic field H. The chemical method was based on exposing the films to bromine vapours and then UV-irradiating. The characterizing techniques included scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS), Fourier transform infrared (FTIR) spectroscopy, optical microscopy, atomic force microscopy (AFM) and magnetic force microscopy (MFM). The surface roughness was quantitatively analyzed by AFM. In the physical method, the random dispersion of MWCNTs (1% w/w) and magnetic nanoparticles (2% w/w) generated a roughness increase of about 200% (with respect to PDMS films without any treatment), but that change was 400% for films cured in the presence of H perpendicular to the surface. SEM, AFM and MFM showed that the magnetic particles always remained attached to the carbon nanotubes, and the effect on the roughness was interpreted as being due to a rupture of dispersion randomness and a possible induction of structuring in the direction of H. In the chemical method, the increase in roughness was even greater (1000%). Wells were generated with surface areas that were close to 100 µm² and depths of up to 500 nm. The observations of AFM images and FTIR spectra were in agreement with the hypothesis of etching by Br radicals generated by UV on the polymer chains. Both methods induced important changes in the surface roughness (the chemical method generated the greatest changes due to the formation of surface wells), which are of great importance in superficial technological processes.

Effect of water absorption on pollen adhesion.

Pollens possess a thin liquid coating, pollenkitt, which plays a major role in adhesion by forming capillary menisci at interfaces. Unfortunately, the influence of humidity on pollenkitt properties and capillary adhesion is unknown. Because humidity varies widely in the environment, the answers have important implications for better understanding plant reproduction, allergy and asthma, and pollen as atmospheric condensation nuclei. Here, pollenkitt-mediated adhesion of sunflower pollen to hydrophilic and hydrophobic surfaces was measured as a function of humidity. The results quantify for the first time the significant water absorption of pollenkitt and the resulting complex dependence of adhesion on humidity. On hydrophilic Si, adhesion increased with increasing RH for pollens with or without pollenkitt, up to 200nN at 70% RH. In contrast, on hydrophobic PS, adhesion of pollenkitt-free pollen is independent of RH. Surprisingly, when pollenkitt was present adhesion forces on hydrophobic PS first increased with RH up to a maximum value at 35% RH (∼160nN), and then decreased with further increases in RH. Independent measurement of pollenkitt properties is used with models of capillary adhesion to show that humidity-dependent changes in pollenkitt wetting and viscosity are responsible for this complex adhesion behavior.

Electrochemical promotion of propane oxidation on Pt deposited on a dense ß″-Al2O3 ceramic Ag(+) conductor.

A new kind of electrochemical catalyst based on a Pt porous catalyst film deposited on a ß″-Al2O3 ceramic Ag(+) conductor was developed and evaluated during propane oxidation. It was observed that, upon anodic polarization, the rate of propane combustion was significantly electropromoted up to 400%. Moreover, for the first time, exponential increase of the catalytic rate was evidenced during galvanostatic transient experiment in excellent agreement with EPOC equation.

Effect of diesel oxidation catalysts on the diesel particulate filter regeneration process.

A Diesel Particulate Filter (DPF) regeneration process was investigated during aftertreatment exhaust of a simulated diesel engine under the influence of a Diesel Oxidation Catalyst (DOC). Aerosol mass spectrometry analysis showed that the presence of the DOC decreases the Organic Carbon (OC) fraction adsorbed to soot particles. The activation energy values determined for soot nanoparticles oxidation were 97 ± 5 and 101 ± 8 kJ mol(-1) with and without the DOC, respectively; suggesting that the DOC does not facilitate elementary carbon oxidation. The minimum temperature necessary for DPF regeneration was strongly affected by the presence of the DOC in the aftertreatment. The conversion of NO to NO(2) inside the DOC induced the DPF regeneration process at a lower temperature than O(2) (ΔT = 30 K). Also, it was verified that the OC fraction, which decreases in the presence of the DOC, plays an important role to ignite soot combustion.

Quasi-equilibrium volume changes of polyaniline films upon redox switching. Formal potential distribution and configurational modeling.

The quasi-equilibrium electrochemomechanical behavior of relatively thick polyaniline films in sulfuric acid is investigated through experimental measurements and theoretical modeling. The leucoemeraldine (LE)-emeraldine (EM) conversion, or redox switching, is studied. The dependence of film volume and electrochemical charge is determined as a function of applied potential. It is observed that the film volume follows the charge, showing an expansion during the second half of the LE-EM oxidation. The model postulates the existence of a stable intermediate, protoemeraldine (PE), with a formal potential distribution for the PE-EM reaction. The volume change is modeled statistically considering contributions from mixing, polymer deformation, and electrostatic charge. The model shows very good agreement with the experiments, indicating that, in the conditions studied, the deformation contribution dominates the volume changes as a result of the conformational modifications undergone by the polymer in the PE-EM oxidation.