High-Throughput Screening Method Using Escherichia coli Keio Mutants for Assessing Primary Damage Mechanism of Antimicrobials.

The Escherichia coli Keio mutant collection has been a tool for assessing the role of specific genes and determining their role in E. coli physiology and uncovering novel functions. In this work, specific mutants in the DNA repair pathways and oxidative stress response were evaluated to identify the primary targets of silver nanoparticles (NPs) and their mechanism of action. The results presented in this work suggest that NPs mainly target DNA via double-strand breaks and base modifications since the recA, uvrC, mutL, and nfo mutants rendered the most susceptible phenotype, rather than involving the oxidative stress response. Concomitantly, during the establishment of the control conditions for each mutant, the katG and sodA mutants showed a hypersensitive phenotype to mitomycin C, an alkylating agent. Thus, we propose that KatG catalase plays a key role as a cellular chaperone, as reported previously for the filamentous fungus Neurospora crassa, a large subunit catalase. The Keio collection mutants may also be a key tool for assessing the resistance mechanism to metallic NPs by using their potential to identify novel pathways involved in the resistance to NPs.

Surface Functionalization and Escherichia coli Detection Using Surface-Enhanced Raman Spectroscopy Driven by Functional Organic Polymer/Gold Nanofilm-Based Microfluidic Chip.

In this work, a microfluidic prototype based on polymeric materials was developed to monitor surface processes using surface-enhanced Raman spectroscopy (SERS), keeping the reagents free of environmental contamination. The prototype was fabricated on poly(methyl methacrylic acid) (PMMA). A micrometric membrane of a functional organic polymer (FOP) based on p-terphenyl and bromopyruvic acid monomers was formed on the PMMA surface to promote the formation of metal nanoclusters. Au nanosized film was deposited on the FOP membrane to give rise to the SERS effect. A microchannel was formed on another piece of PMMA using micromachining. A representative 3D model of the prototype layer arrangement was built and simulated in COMSOL Multiphysics® to approximate the electric field distribution and calculate the power enhancement factor as the Au film changes over time. The fabrication process was characterized using UV-visible and Raman spectroscopies and XPS. The prototype was tested using a Raman microscope and liquid solutions of cysteamine and Escherichia coli (E. coli). The simulation results demonstrated that the morphological characteristics of the Au layer give rise to the SERS effect, and the power enhancement factor reaches values as high as 8.8 × 105 on the FOP surface. The characterization results showed the formation of the FOP and the Au film on PMMA and the surface functionalization with amine groups. The Raman spectra of the prototype showed temporal evolution as different compounds were deposited on the upper wall of the microchannel. Characteristic peaks associated with these compounds were detected with continuous monitoring over time. This prototype offers many benefits for applications like monitoring biological processes. Some advantages include timely surface evaluation while avoiding environmental harm, decreased use of reagents and samples, minimal interference with the process by measuring, and detecting microorganisms in just 1 h, as demonstrated with the E. coli sample.

Sargassum natans I Algae: An Alternative for a Greener Approach for the Synthesis of ZnO Nanostructures with Biological and Environmental Applications.

In this work, the influence of the Sargassum natans I alga extract on the morphological characteristics of synthesized ZnO nanostructures, with potential biological and environmental applications, was evaluated. For this purpose, different ZnO geometries were synthesized by the co-precipitation method, using Sargassum natans I alga extract as stabilizing agent. Four extract volumes (5, 10, 20, and 50 mL) were evaluated to obtain the different nanostructures. Moreover, a sample by chemical synthesis, without the addition of extract, was prepared. The characterization of the ZnO samples was carried out by UV-Vis spectroscopy, FT-IR spectroscopy, X-ray diffraction, and scanning electron microscopy. The results showed that the Sargassum alga extract has a fundamental role in the stabilization process of the ZnO nanoparticles. In addition, it was shown that the increase in the Sargassum alga extract leads to preferential growth and arrangement, obtaining well-defined shaped particles. ZnO nanostructures demonstrated significant anti-inflammatory response by the in vitro egg albumin protein denaturation for biological purposes. Additionally, quantitative antibacterial analysis (AA) showed that the ZnO nanostructures synthesized with 10 and 20 mL of extract demonstrated high AA against Gram (+) S. aureus and moderate AA behavior against Gram (-) P. aeruginosa, depending on the ZnO arrangement induced by the Sargassum natans I alga extract and the nanoparticles' concentration (ca. 3200 µg/mL). Additionally, ZnO samples were evaluated as photocatalytic materials through the degradation of organic dyes. Complete degradation of both methyl violet and malachite green were achieved using the ZnO sample synthesized with 50 mL of extract. In all cases, the well-defined morphology of ZnO induced by the Sargassum natans I alga extract played a key role in the combined biological/environmental performance.

Antibacterial and Anti-Inflammatory Properties of ZnO Nanoparticles Synthesized by a Green Method Using Sargassum Extracts.

The present work shows the synthesis of ZnO nanoparticles through a green method, using sargassum extracts, which provide the reducing and stabilizing compounds. The conditions of the medium in which the reaction was carried out was evaluated, that is, magnetic stirring, ultrasound assisted, and resting condition. UV-Vis, FTIR spectroscopy, and X-ray diffraction results confirmed the synthesis of ZnO with nanometric crystal size. The scanning electron microscopy analysis showed that the morphology and size of the particles depends on the synthesis condition used. It obtained particles between 20 and 200 nm in the sample without agitation, while the samples with stirring and ultrasound were 80 nm and 100 nm, respectively. ZnO nanoparticles showed antibacterial activity against Gram-positive S. aureus and Gram-negative P. aeruginosa. A quantitative analysis was performed by varying the concentration of ZnO nanoparticles. In all cases, the antibacterial activity against Gram-positives was greater than against Gram-negatives. Ultrasound-assisted ZnO nanoparticles showed the highest activity, around 99% and 80% for S. aureus and P. aeruginosa, respectively. Similar results were obtained in the study of the anti-inflammatory activity of ZnO nanoparticles; the ultrasound-assisted sample exhibited the highest percentage (93%), even above that shown by diclofenac, which was used as a reference. Therefore, the ZnO nanoparticles synthesized with sargassum extracts have properties that can be used safely and efficiently in the field of biomedicine.

Chitosan-G-Glycidyl Methacrylate/Au Nanocomposites Promote Accelerated Skin Wound Healing.

Herein, we report the synthesis of Au nanoparticles (AuNPs) in chitosan (CTS) solution by chemically reducing HAuCl4. CTS was further functionalized with glycidyl methacrylate (chitosan-g-glycidyl methacrylate/AuNP, CTS-g-GMA/AuNP) to improve the mechanical properties for cellular regeneration requirements of CTS-g-GMA/AuNP. Our nanocomposites promote excellent cellular viability and have a positive effect on cytokine regulation in the inflammatory and anti-inflammatory response of skin cells. After 40 days of nanocomposite exposure to a skin wound, we showed that our films have a greater skin wound healing capacity than a commercial film (TheraForm®), and the presence of the collagen allows better cosmetic ave aspects in skin regeneration in comparison with a nanocomposite with an absence of this protein. Electrical percolation phenomena in such nanocomposites were used as guiding tools for the best nanocomposite performance. Our results suggest that chitosan-based Au nanocomposites show great potential for skin wound repair.

Antibacterial Activity of Electrospun Nanocomposites Fabricated by In Situ Chitosan/Silver Nanoparticles.

The process of disinfection of wastewater must use friendly materials with the environment that achieve the inhibition of bacterial growth, aiming to improve the quality of the water. In this study, electrospun nanocomposites CS (chitosan)/AgNPs (silver nanoparticles) was developed for wastewater disinfection through filtration. First, AgNPs were synthesized by a green synthesis method using aloe vera (Aloe Barbadensis Miller) extract as a reducing agent, and AgNO3 as metal precursor. AgNPs were characterized by UV-Vis spectroscopy with a SPR band at 420 nm. Transmission Electron Microscopy (TEM) demonstrates the formation of semispherical AgNPs with a diameter ca. 16 nm. The nanocomposites fibers CS/AgNPs were analyzed by scanning electron microscopy (SEM), with the presence of uniform fibers (diameter ca. 120 nm) and embedded AgNPs, determined by EDS and elemental mapping. Surface topography of CS/AgNPs fibers was evaluated by Atomic Force Microscopy (AFM), where the surface roughness of fibers is increased with the NPs content. Finally, the antibacterial activity (AA) of CS/AgNPs nanocomposites was evaluated against S. aureus and P. aeruginosa during 1 and 2 h of contact, obtaining an AA of 90 % in CS/AgNPs-2 mM after 2 h with P. aeruginosa. The fabrication of electrospun nanocomposites with silver nanoparticles represents a platform to the fabrication of multifunctional polymer membranes for wastewater disinfection.

Chitosan-BaTiO3 nanostructured piezopolymer for tissue engineering.

Herein we report the synthesis of a piezopolymer composed of chitosan (CS)/hydroxylated BaTiO3 (OH-BTO) nanoparticles with enhanced biocompatibility, non-toxicity, and piezoelectric behavior that can be advantageously used in biomedical applications. Our CS/OH-BTO nanocomposites exhibit piezoelectric coefficient (d33 = 11.29 pC/N) between those of dry skin (0.05-0.19 pC/N) and bone (4-11 pC/N), demonstrating biocompatibility in contact with human fibroblasts (HF) cells after 24 h. SEM, XRD, FTIR and Raman measurements were performed to assess the mechanism of interaction between CS matrix and OH-BTO NPs and their correlation with the biological responses. Cytotoxicity assays with HF cells reveal that hydroxylation of BTO NPs does not affect the cell viability of CS/OH-BTO films with NPs concentration from 1 to 30 wt.%. In contrast, non-hydroxylated BTO NPs showed significant cell damage, which could be traced to uncontrollable NPs agglomeration. This behavior suggests that CS/OH-BTO nanocomposites can act as active material that promotes cell growth and can be used for biomedical purposes.

aeBlue Chromoprotein Color is Temperature Dependent.

BACKGROUND: Marine sessile organisms display a color palette that is the result of the expression of fluorescent and non-fluorescent proteins. Fluorescent proteins have uncovered transcriptional regulation, subcellular localization of proteins, and the fate of cells during development. Chromoproteins have received less attention until recent years as bioreporters. Here, we studied the properties of aeBlue, a a 25.91 kDa protein from the anemone Actinia equina. OBJECTIVE: To assess the properties of aeBlue chromoprotein under different physicochemical conditions. METHODS: In this article, during the purification of aeBlue we uncovered that it suffered a color shift when frozen. We studied the color shift by different temperature incubation and physicochemical conditions and light spectroscopy. To assess the possible structural changes in the protein, circular dichroism analysis, size exclusion chromatography and native PAGE was performed. RESULTS: We uncover that aeBlue chromoprotein, when expressed from a synthetic construct in Escherichia coli, showed a temperature dependent color shift. Protein purified at 4 °C by metal affinity chromatography exhibited a pinkish color and shifts back at higher temperatures to its intense blue color. Circular dichroism analysis revealed that the structure in the pink form of the protein has reduced secondary structure at 4 °C, but at 35 °C and higher, the structure shifts to a native conformation and Far UV- vis CD spectra revealed the shift in an aromatic residue of the chromophore. Also, the chromophore retains its properties in a wide range of conditions (pH, denaturants, reducing and oxidants agents). Quaternary structure is also maintained as a tetrameric conformation as shown by native gel and size exclusion chromatography. CONCLUSION: Our results suggest that the chromophore position in aeBlue is shifted from its native position rendering the pink color and the process to return it to its native blue conformation is temperature dependent.

From Intermolecular Interactions to Texture in Polycrystalline Surfaces of 1,ω-alkanediols (ω = 10-13).

Differences on herringbone molecular arrangement in two forms of long-chain 1,ω-alkanediols (CnH2n+2O2 with n = 10, 11, 12, 13) are explained from the analysis of O-H···O hydrogen-bond sequences in infinite chains and the role of a C-H···O intramolecular hydrogen-bond in stabilization of a gauche defect, as well as the inter-grooving effectiveness on molecular packing. GIXD (Glancing Incidence X-ray Diffraction) experiments were conducted on polycrystalline monophasic samples. Diffracted intensities were treated with the multi-axial March-Dollase method to correlate energetic and geometrical features of molecular interactions with the crystalline morphology and textural pattern of samples. The monoclinic (P21/c, Z = 2) crystals of the even-numbered members (n = 10, 12; DEDOL and DODOL, respectively) are diametrical prisms with combined form {104}/{-104}/{001} and present a two-fold platelet-like preferred orientation, whereas orthorhombic (P212121, Z = 4) odd-numbered members (n = 11, 13; UNDOL and TRDOL, respectively) present a dominant needle-like orientation on direction [101] (fiber texture). We show that crystalline structures of medium complexity and their microstructures can be determined from rapid GIXD experiments from standard radiation, combined with molecular replacement procedure using crystal structures of compounds with higher chain lengths as reference data.

Early Stages of Antibacterial Damage of Metallic Nanoparticles by TEM and STEM-HAADF.

BACKGROUND: Propagation of pathogens has considered an important health care problem due to their resistance against conventional antibiotics. The recent challenge involves the design of functional alternatives such as nanomaterials, used as antibacterial agents. Early stages of antibacterial damage caused by metallic nanoparticles (NPs) were studied by Transmission Electron Microscopy (TEM) and combined Scanning Transmission Electron Microscopy with High Angle Annular Dark Field (STEM-HAADF), aiming to contribute to the elucidation of the primary antibacterial mechanism of metallic NPs. METHODS: We analyze the NPs morphology by TEM and their antibacterial activity (AA) with different amounts of Ag and Cu NPs. Cultured P. aeruginosa were interacted with both NPs and processed by TEM imaging to determine NPs adhesion into bacteria wall. Samples were analyzed by combined STEM-HAADF to determine the NPs penetration into bacterium and elemental mapping were done. RESULTS: Both NPs displays AA depending on NPs concentration. TEM images show NPs adhesion on bacterial cells, which produces morphological changes in the structure of the bacteria. STEM-HAADF also proves the NPs adhesion and penetration by intracellular localization, detecting Ag/Cu species analyzed by elemental mapping. Moreover, the relative amount of phosphorus (P) and sulfur (S) increases slightly in P. aeruginosa with the presence of NPs. These elements are associated with damaged proteins of the outer cell membrane. CONCLUSIONS: Combined microscopy analyses suggest that the early stages of antibacterial damage caused by alteration of bacterial cell wall, and can be considered a powerful tool aiming to understand the primary antibacterial mechanism of NPs.