Geobacter sulfurreducens is a widely applied microorganism for the reduction of toxic metal salts, as an electron source for bioelectrochemical devices, and as a reagent for the synthesis of nanoparticles. In order to understand the influence of metal salts, and of electron transporting, multiheme c-cytochromes on the electron flux during respiration of G. sulfurreducens, the reduction kinetic of Fe³⁺, Co³⁺, V⁵⁺, Cr⁶⁺, and Mn⁷⁺ containing complexes were measured. Starting from the resting phase, each G. sulfurreducens cell produced an electron flux of 3.7 × 10⁵ electrons per second during the respiration process. Reduction rates were within ± 30% the same for the 6 different metal salts, and reaction kinetics were of zero order. Decrease of c-cytochrome concentrations by downregulation and mutation demonstrated that c-cytochromes stabilized respiration rates by variation of their redox states. Increasing Fe²⁺/heme levels increased electron flux rates, and induced respiration flexibility. The kinetic effects parallel electrochemical results of G. sulfurreducens biofilms on electrodes, and might help to optimize bioelectrochemical devices.

Membrane-bound catechol-O-methyltransferase (MBCOMT), present in the brain and involved in the main pathway of the catechol neurotransmitter deactivation, is linked to several types of human dementia, which are relevant pharmacological targets for new potent and nontoxic inhibitors that have been developed, particularly for Parkinson’s disease treatment. However, the inexistence of an MBCOMT 3D-structure presents a blockage in new drugs’ design and clinical studies due to its instability. The enzyme has a clear tendency to lose its biological activity in a short period of time. To avoid the enzyme sequestering into a non-native state during the downstream processing, a multi-component buffer plays a major role, with the addition of additives such as cysteine, glycerol, and trehalose showing promising results towards minimizing hMBCOMT damage and enhancing its stability. In addition, ionic liquids, due to their virtually unlimited choices for cation/anion paring, are potential protein stabilizers for the process and storage buffers. Screening experiments were designed to evaluate the effect of distinct cation/anion ILs interaction in hMBCOMT enzymatic activity. The ionic liquids: choline glutamate [Ch][Glu], choline dihydrogen phosphate ([Ch][DHP]), choline chloride ([Ch]Cl), 1- dodecyl-3-methylimidazolium chloride ([C12mim]Cl), and 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) were supplemented to hMBCOMT lysates in a concentration from 5 to 500 mM. A major potential stabilizing effect was obtained using [Ch][DHP] (10 and 50 mM). From the DoE 146% of hMBCOMT activity recovery was obtained with [Ch][DHP] optimal conditions (7.5 mM) at −80 °C during 32.4 h. These results are of crucial importance for further drug development once the enzyme can be stabilized for longer periods of time.

Cellulose micro/nanomaterials (CMNM), comprising cellulose microfibrils (CMF), nanofibrils (CNF), and nanocrystals (CNC), are being recognized as promising bio-nanomaterials due to their natural and renewable source, attractive properties, and potential for applications with industrial and economical value. Thus, it is crucial to investigate their potential toxicity before starting their production at a larger scale. The present study aimed at evaluating the cell internalization and in vitro cytotoxicity and genotoxicity of CMNM as compared to two multi-walled carbon nanotubes (MWCNT), NM-401 and NM-402, in A549 cells. The exposure to all studied NM, with the exception of CNC, resulted in evident cellular uptake, as analyzed by transmission electron microscopy. However, none of the CMNM induced cytotoxic effects, in contrast to the cytotoxicity observed for the MWCNT. Furthermore, no genotoxicity was observed for CNF, CNC, and NM-402 (cytokinesis-block micronucleus assay), while CMF and NM-401 were able to significantly raise micronucleus frequency. Only NM-402 was able to induce ROS formation, although it did not induce micronuclei. Thus, it is unlikely that the observed CMF and NM-401 genotoxicity is mediated by oxidative DNA damage. More studies targeting other genotoxicity endpoints and cellular and molecular events are underway to allow for a more comprehensive safety assessment of these nanocelluloses.

Exoelectrogenic microorganisms are in the spotlight due to their unique respiratory mechanisms and potential applications in distinct biotechnological fields, including bioremediation, bioenergy production and microbial electrosynthesis. These applications rely on the capability of these microorganisms to perform extracellular electron transfer, a mechanism that allows the bacteria to transfer electrons to the cell’s exterior by establishing functional interfaces between different multiheme cytochromes at the inner membrane, periplasmic space, and outer membrane. The multiheme cytochrome CbcL from Geobacter sulfurreducens is associated to the inner membrane and plays an essential role in the transfer of electrons to final electron acceptors with a low redox potential, as Fe(III) oxides and electrodes poised at −100 mV. CbcL has a transmembranar di-heme b-type cytochrome domain with six helices, linked to a periplasmic cytochrome domain with nine c-type heme groups. The complementary usage of ultraviolet-visible, circular dichroism and nuclear magnetic resonance permitted the structural and functional characterization of CbcL’s periplasmic domain. The protein was found to have a high percentage of disordered regions and its nine hemes are low-spin and all coordinated by two histidine residues. The apparent midpoint reduction potential of the CbcL periplasmic domain was determined, suggesting a thermodynamically favorable transfer of electrons to the putative redox partner in the periplasm − the triheme cytochrome PpcA. The establishment of a redox complex between the two proteins was confirmed by probing the electron transfer reaction and the molecular interactions between CbcL and PpcA. The results obtained show for the first time how electrons are injected into the periplasm of Geobacter sulfurreducens for subsequent transfer to the cell’s exterior.

Iron is the most versatile of all biochemically active metals, with variability encompassing its electronic configuration, number of unpaired electrons, type of ligands and iron-complexes stability. The versatility of iron properties is transposed to the proteins it can be associated to, especially relevant in the case of heme proteins. In this Review, the structural and functional properties of heme proteins are revisited, with particular focus on c-type cytochromes. The genome of Geobacter bacteria encodes for an unusually high number of assorted c-type cytochromes and, for this reason, they are used in this Review as a showcase of the cytochrome diversity. In the last decades, a vast portfolio of cytochromes has been revealed in these bacteria, with most of them defining new classes, ranging from monoheme to the recently identified polymeric assembly of multiheme cytochromes that forms micrometer-long electrically conductive filaments. These discoveries were on pace with the development of modern NMR equipment and advances in protein isotopic labeling methods, which are also revisited in this Review. Finally, following the description of the current state of the art of Geobacter cytochromes, examples on how the available structural and functional information was explored to structurally map protein–protein and protein–ligand interacting regions in redox complexes, and hence elucidate Geobacter’s respiratory pathways, are presented.

(1) Background: Nanocellulose is an innovative engineered nanomaterial with an enormous potential for use in a wide array of industrial and biomedical applications and with fast growing economic value. The expanding production of nanocellulose is leading to an increased human exposure, raising concerns about their potential health effects. This study was aimed at assessing the potential toxic and genotoxic effects of different nanocelluloses in two mammalian cell lines; (2) Methods: Two micro/nanocelluloses, produced with a TEMPO oxidation pre-treatment (CNFs) and an enzymatic pre-treatment (CMFs), and cellulose nanocrystals (CNCs) were tested in osteoblastic-like human cells (MG-63) and Chinese hamster lung fibroblasts (V79) using the MTT and clonogenic assays to analyse cytotoxicity, and the micronucleus assay to test genotoxicity; (3) Results: cytotoxicity was observed by the clonogenic assay in V79 cells, particularly for CNCs, but not by the MTT assay; CNF induced micronuclei in both cell lines and nucleoplasmic bridges in MG-63 cells; CMF and CNC induced micronuclei and nucleoplasmic bridges in MG-63 cells, but not in V79 cells; (4) Conclusions: All nanocelluloses revealed cytotoxicity and genotoxicity, although at different concentrations, that may be related to their physicochemical differences and availability for cell uptake, and to differences in the DNA damage response of the cell model.

Electroactive Geobacter bacteria can perform extracellular electron transfer and present a wide metabolic versatility. These bacteria reduce organic, toxic and radioactive compounds, and produce electric current while interacting with electrodes, making them interesting targets for numerous biotechnological applications. Their global electrochemical responses rely on an efficient interface between the inside and the cell's exterior, which is driven by the highly abundant periplasmic triheme PpcA-family cytochromes. The functional features of these cytochromes have been studied in G. sulfurreducens and G. metallireducens, and although they share a high degree of structural homology and sequence identity, their properties are quite distinct. In this work, the heme axial ligand geometries and the magnetic properties of PpcF from G. metallireducens were determined. The data obtained constitute important constraints for the determination of its solution structure in the oxidized state and indicate that the (i) heme core architecture; (ii) axial ligands geometries and (iii) magnetic properties of the cytochrome are conserved compared to the other members of the PpcA-families. Furthermore, the results also indicate that the heme arrangement is crucial to maintain an intrinsic regulation of the protein's redox properties and hence its electron transfer efficiency and functionality.

Abstract Relative humidity is simultaneously a sensing target and a contaminant in gas and volatile organic compound (VOC) sensing systems, where strategies to control humidity interference are required. An unmet challenge is the creation of gas-sensitive materials where the response to humidity is controlled by the material itself. Here, humidity effects are controlled through the design of gelatin formulations in ionic liquids without and with liquid crystals as electrical and optical sensors, respectively. In this design, the anions [DCA]− and [Cl]− of room temperature ionic liquids from the 1-butyl-3-methylimidazolium family tailor the response to humidity and, subsequently, sensing of VOCs in dry and humid conditions. Due to the combined effect of the materials formulations and sensing mechanisms, changing the anion from [DCA]− to the much more hygroscopic [Cl]−, leads to stronger electrical responses and much weaker optical responses to humidity. Thus, either humidity sensors or humidity-tolerant VOC sensors that do not require sample preconditioning or signal processing to correct humidity impact are obtained. With the wide spread of 3D- and 4D-printing and intelligent devices, the monitoring and tuning of humidity in sustainable biobased materials offers excellent opportunities in e-nose sensing arrays and wearable devices compatible with operation at room conditions.

{Aiming to rationalize the release profile of an incorporated pharmaceutical drug in terms of its mobility, driven by guest-host interactions, the poorly water-soluble ibuprofen drug was loaded in a mesoporous inorganic silica matrix with unmodified (MCM-41) and modified surface (MCM-41sil) by post-synthesis silylation, both having pore sizes similar to 3 nm. The single calorimetric detection of a broad glass transition step for both ibuprofen com-posites indicates full drug amorphization, confirmed by the only appearance of an amorphous halo in the powder XRD patterns. Moreover, a gradient profile is disclosed by the heat flux derivative plot in the glass transition, in coherence with the thermogravimetric profile that shows a multi-step decomposition trace for confined ibuprofen in these matrixes. While identical guest dynamics, as probed by dielectric relaxation spectroscopy, were found in both dehydrated composites, a significant molecular population with faster relaxation exists in the hydrated state for the drug inside the unmodified matrix. This was rationalized as the concurrence of true confinement effects, which manifest under nanometer dimensions, and greater water affinity of the unmodified matrix, forcing the drug molecules to be placed mostly in the pore core. Finite size effects are also felt in both dehydrated composites, however guest-host interactions give origin to a dominant population with slowed down mobility that governs the overall guest dynamics. In spite of an inferior number of active sites for drug adsorption in the silylated matrix, a faster ibuprofen delivery in phosphate buffer (pH = 6.8) was observed when the drug is released from unmodified MCM-41 in the hydrated state. Therefore, our results suggest that a relevant role is played by water molecules, which impair a strong guest adsorption in the host surface more efficiently than the limited surface modification, influence the higher ratio of a faster population in the pore core and facilitate the diffusion of the aqueous releasing media inside pores.}

{Understanding the behavior of a chemical compound at a molecular level is fundamental, not only to explain its macroscopic properties, but also to enable the control and optimization of these properties. The present work aims to characterize a set of systems based on the ionic liquids {[}Aliquat]{[}Cl] and {[}Aliquat]{[}FeCl4] and on mixtures of these with different concentrations of DMSO by means of H-1 NMR relaxometry, diffusometry and X-ray diffractometry. Without DMSO, the compounds reveal locally ordered domains, which are large enough to induce order fluctuation as a significant relaxation pathway, and present paramagnetic relaxation enhancement for the {[}Aliquat]{[}Cl] and {[}Aliquat]{[}FeCl4] mixture. The addition of DMSO provides a way of tuning both the local order of these systems and the relaxation enhancement produced by the tetrachloroferrate anion. Very small DMSO volume concentrations (at least up to 1%) lead to enhanced paramagnetic relaxation without compromising the locally ordered domains. Larger DMSO concentrations gradually destroy these domains and reduce the effect of paramagnetic relaxation, while solvating the ions present in the mixtures. The paramagnetic relaxation was explained as a correlated combination of inner and outer-sphere mechanisms, in line with the size and structure differences between cation and anion. This study presents a robust method of characterizing paramagnetic ionic systems and obtaining a consistent analysis for a large set of samples having different co-solvent concentrations.}

An innovative pilot installation and eddy current testing (ECT) inspection system for laser-brazed joints is presented. The proposed system detects both surface and sub-surface welding defects operating autonomously and integrated with a robotized arm. Customized eddy current probes were designed and experimentally validated detecting pore defects with 0.13 mm diameter and sub-surface defects buried 1 mm deep. The integration of the system and the manufacturing process towards an Industry 4.0 quality control paradigm is also discussed.

Methyl-substituted 8-hydroxyquinolines (Hquin) were successfully used to synthetize five-coordinated oxovanadium(IV) complexes: [VO(2,6-(Me)2-quin)2 ] (1), [VO(2,5-(Me)2-quin)2 ] (2) and [VO(2-Me-quin)2 ] (3). Complexes 1–3 demonstrated high catalytic activity in the oxidation of hydrocarbons with H2 O2 in acetonitrile at 50◦ C, in the presence of 2-pyrazinecarboxylic acid (PCA) as a cocatalyst. The maximum yield of cyclohexane oxidation products attained was 48%, which is high in the case of the oxidation of saturated hydrocarbons. The reaction leads to the formation of a mixture of cyclohexyl hydroperoxide, cyclohexanol and cyclohexanone. When triphenylphosphine is added, cyclohexyl hydroperoxide is completely converted to cyclohexanol. Consideration of the regioand bond-selectivity in the oxidation of n-heptane and methylcyclohexane, respectively, indicates that the oxidation proceeds with the participation of free hydroxyl radicals. The complexes show moderate activity in the oxidation of alcohols. Complexes 1 and 2 reduce the viability of colorectal (HCT116) and ovarian (A2780) carcinoma cell lines and of normal dermal fibroblasts without showing a specific selectivity for cancer cell lines. Complex 3 on the other hand, shows a higher cytotoxicity in a colorectal carcinoma cell line (HCT116), a lower cytotoxicity towards normal dermal fibroblasts and no effect in an ovarian carcinoma cell line (order of magnitude HCT116 > fibroblasts > A2780).

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Automobile laser brazing remains a complex process whose results are affected by several process variables that may result in nonacceptable welds. A multisensory customized inspection system is proposed, with two distinct non-destructive techniques: the potential drop method and eddy current testing. New probes were designed, simulated, produced, and experimentally validated in automobile's laser-brazed weld beads with artificially introduced defects. The numerical simulations allowed the development of a new four-point probe configuration in a non-conventional orthogonal shape demonstrating a superior performance in both simulation and experimental validation. The dedicated inspection system allowed the detection of porosities, cracks, and lack of bonding defects, demonstrating the redundancy and complementarity these two techniques provide.