Twelve biochars from forest and agri-food wastes (pruning of Quercus ilex, Eucalyptus grandis, Pinus pinaster, Quercus suber, Malus pumila, Prunus spinosa, Cydonia oblonga, Eriobotrya japonica, Juglans regia, Actinidia deliciosa, Citrus sinensis and Vitis vinifera) were investigated as potential low-cost and renewable adsorbents for removal of a commonly used pharmaceutical, fluoxetine. Preliminary adsorption experiments allowed to select the most promising adsorbents, Quercus ilex, Cydonia oblonga, Eucalyptus, Juglans regia and Vitis vinifera pruning material. They were characterized by proximate, elemental and mineral analysis, thermogravimetric analysis, Fourier transform infrared spectroscopy, determination of specific surface area and pH at the point of zero charge. Batch and equilibrium studies were performed, and the influence of pH was evaluated. The equilibrium was reached in less than 15 min in all systems. The maximum adsorption capacity obtained was 6.41 mg/g for the Eucalyptus biochar, which also demonstrated a good behavior in continuous mode (packed column).

Nuclear Magnetic Resonance (NMR) spectroscopy is extremely powerful to study distinct biological systems ranging from biomolecules to specific metabolites. This chapter presents the basic concepts of the technique and illustrates its potential to study such systems. Similarly, to other spectroscopic techniques, the theoretical background of NMR is sustained by detailed mathematics and physical chemistry concepts, which were kept to the minimum. The intent is to introduce the fundamentals of the technique to science students from different backgrounds. The basic concepts of NMR spectroscopy are briefly presented in the first section, and the following sections describe applications in the biosciences field, using electron transfer proteins as model, particularly cytochromes. The heme groups endow cytochromes with particular features making them excellent examples to illustrate the high versatility of NMR spectroscopy. The main methodologies underlying protein solution structure determination are discussed in the second section. This is followed by a description of the main experiments explored to structurally map protein-protein or protein-ligand interface regions in molecular complexes. Finally, it is shown how NMR spectroscopy can assist in the functional characterization of multiheme cytochromes.

Abstract Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) is a major cause of bovine mastitis and has been regarded as an animal-restricted pathogen, although rare infections have been described in humans. Previous studies revealed the presence of virulence genes encoded by phages of the human pathogen Group A Streptococcus pyogenes (GAS) in SDSD isolated from the milk of bovine udder with mastitis. The isolates SDSD VSD5 and VSD13 could adhere and internalize human primary keratinocyte cells, suggesting a possible human infection potential of bovine isolates. In this work, the in vitro and in vivo potential of SDSD to internalize/adhere human cells of the respiratory track and zebrafish as biological models was evaluated. Our results showed that, in vitro, bovine SDSD strains could interact and internalize human respiratory cell lines and that this internalization was dependent on an active transport mechanism and that, in vivo, SDSD are able to cause invasive infections producing zebrafish morbidity and mortality. The infectious potential of these isolates showed to be isolate-specific and appeared to be independent of the presence or absence of GAS phage-encoded virulence genes. Although the infection ability of the bovine SDSD strains was not as strong as the human pathogenic S. pyogenes in the zebrafish model, results suggested that these SDSD isolates are able to interact with human cells and infect zebrafish, a vertebrate infectious model, emerging as pathogens with zoonotic capability.

The triheme cytochrome PpcA from Geobacter sulfurreducens is highly abundant under several growth conditions and is important for extracellular electron transfer. PpcA plays a central role in transferring electrons resulting from the cytoplasmic oxidation of carbon compounds to the cell exterior. This cytochrome is designed to couple electron and proton transfer at physiological pH, a process achieved via the selection of dominant microstates during the redox cycle of the protein, which are ultimately regulated by a well-established order of oxidation of the heme groups. The three hemes are covered only by a polypeptide chain of 71 residues and are located in the small hydrophobic core of the protein. In this work, we used NMR and X-ray crystallography to investigate the structural and functional role of a conserved valine residue (V13) located within van der Waals contact of hemes III and IV. The residue was replaced by alanine (V13A), isoleucine (V13I), serine (V13S), and threonine (V13T) to probe the effects of the side chain volume and polarity. All mutants were found to be as equally thermally stable as the native protein. The V13A and V13T mutants produced crystals and their structures were determined. The side chain of the threonine residue introduced in V13T showed two conformations, but otherwise the two structures did not show significant changes from the native structure. Analysis of the redox behavior of the four mutants showed that for the hydrophobic replacements (V13A and V13I) the redox properties, and hence the order of oxidation of the hemes, were unaffected in spite of the larger side chain, isoleucine, showing two conformations with minor changes of the protein in the heme core. On the other hand, the polar replacements (V13S and V13T) showed the presence of two more distinctive conformations, and the oxidation order of the hemes was altered. Overall, it is striking that a single residue with proper size and polarity, V13, was naturally selected to ensure a unique conformation of the protein and the order of oxidation of the hemes, endowing the cytochrome PpcA with the optimal functional properties necessary to ensure effectiveness in the extracellular electron transfer respiratory pathways of G. sulfurreducens.

Mesenchymal stem cells are multipotent adult stem cells capable of generating bone, cartilage and fat, and are thus currently being exploited for regenerative medicine. When considering osteogenesis, developments have been made with regards to chemical induction (e.g. differentiation media) and physical induction (e.g. material stiffness, nanotopography), targeting established early transcription factors or regulators such as runx2 or bone morphogenic proteins and promoting increased numbers of cells committing to osteo-specific differentiation. Recent research highlighted the involvement of microRNAs in lineage commitment and terminal differentiation. Herein, gold nanoparticles that confer stability to short single stranded RNAs were used to deliver MiR-31 antagomiRs to both pre-osteoblastic cells and primary human MSCs in vitro. Results showed that blocking miR-31 led to an increase in osterix protein in both cell types at day 7, with an increase in osteocalcin at day 21, suggesting MSC osteogenesis. In addition, it was noted that antagomiR sequence direction was important, with the 5 prime reading direction proving more effective than the 3 prime. This study highlights the potential that miRNA antagomiR-Tagged nanoparticles offer as novel therapeutics in regenerative medicine.

Two mononuclear NiII and MnII compounds, [Ni(bdtbpza)2(CH3OH)4] (1) and [Mn(bdtbpza)2(CH3OH)2(H2O)2] (2), are afforded by employing a ‘scorpionate’ type precursor [bdtbpza = bis(3,5-di-t-butylpyrazol-1-yl)acetate]. The single crystal X-ray structure reveals that the central metal ion (NiII for 1 and MnII for 2) is surrounded by a pair of Oacetate atoms of two bis(pyrazol-1-yl)acetate units, while four OMeOH donors for 1 and two OMeOH plus two Owater for 2 complete the first coordination sphere. Thus, both compounds exhibit a slightly distorted octahedral geometry possessing an O6 coordination environment. EPR spectra of CuII-doped 1 and of 2 recorded on the polycrystalline solids and in organic solution confirm the octahedral geometry around the metal ions and the binding of six oxygen atoms. The electrochemical study of compounds 1 and 2 shows that one electron reduction of MnII occurs at a more negative potential than NiII, indicating a lower tendency of reduction for Mn than Ni. Both compounds displayed a high cytotoxic activity against A2780 ovarian carcinoma cells and no cytotoxic activity in normal primary human fibroblasts for concentrations up to 55 μM. Notwithstanding, compound 1 is found to be the most cytotoxic towards A2780 cancer cells. The cytotoxic activity of compound 1 is correlated with the induction of apoptosis associated with a higher mitochondria dysfunction and autophagy cell death. In addition, the compounds can induce oxidative damage leading to ROS accumulation. Overall, the data presented here demonstrate that 1 has potential for further in vivo studies aiming at its future application in ovarian cancer therapy.

The heterogenization of the highly active monovacant polyoxotungstate ([PW11O39]7 −, abbreviated as PW11) was achieved by preparing the corresponding long chain quaternary ammonium salt (ODA7PW11, ODA = CH3(CH2)17(CH3)3N). The complete cation exchange confers total heterogeneity to the monovacant catalyst while keeping its oxidative catalytic activity. In fact, the heterogeneous catalyst allowed for the complete desulfurization of a multicomponent model diesel (2000 ppm S) after 40 min of reaction, conciliating extraction (using BMIMPF6 solvent) and oxidation (ECODS process using H2O2 oxidant). The heterogeneous catalyst has shown a superior desulfurization performance when compared with the homogeneous quaternary ammonium TBAPW11 catalyst (TBA = (C4H9)4 N). Both hybrid catalysts have been successfully reused in consecutive ECODS cycles. Additionally, the long carbon chain cations provide a protective environment around the polyoxometalate allowing for ODA7PW11 to retain its heterogeneity and structure after the ECODS process.

Gene knock-out studies on Geobacter sulfurreducens have shown that the monoheme c-type cytochrome OmcF is essential for the extracellular electron transfer pathways involved in the reduction of iron and uranium oxy-hydroxides, as well as, on electricity production in microbial fuel cells. A detailed electrochemical characterization of OmcF was performed for the first time, allowing attaining kinetics and thermodynamic data. The heterogeneous electron transfer rate constant was determined at pH 7 (0.16 ± 0.01 cm s−1) indicating that the protein displays high electron transfer efficiency compared to other monoheme cytochromes. The pH dependence of the redox potential indicates that the protein has an important redox-Bohr effect in the physiological pH range for G. sulfurreducens growth. The analysis of the structures of OmcF allowed us to assign the redox-Bohr centre to the side chain of His47 residue and its pKa values in the reduced and oxidized states were determined (pKox = 6.73; pKred = 7.55). The enthalpy, entropy and Gibbs free energy associated with the redox transaction were calculated, pointing the reduced form of the cytochrome as the most favourable. The data obtained indicate that G. sulfurreducens cells evolved to warrant a down-hill electron transfer from the periplasm to the outer-membrane associated cytochrome OmcF.

Abstract Electrogenic bacteria, such as Geobacter, can couple the oxidation of carbon sources to the reduction of extracellular electron acceptors; such acceptors include toxic and radioactive metals, as well as electrode surfaces, making Geobacter a suitable candidate for applied use in bioremediation and bioenergy generation. Geobacter metallireducens is more promising in this regard than the better studied Geobacter sulfurreducens, as it has more efficient Fe (III) reduction rates and can respire nitrate to ammonia. The operon responsible for nitrate reductase activity in G. metallireducens includes the gene encoding the cytochrome PpcF, which was proposed to exchange electrons with nitrate reductase. In the present work, we perform a biochemical and biophysical characterization of PpcF. Spectroscopic techniques, including circular dichroism (CD), UV-visible, and nuclear magnetic resonance (NMR) revealed that the cytochrome is very stable (Tm > 85 °C), contains three low-spin hemes, and is diamagnetic (S=0) and paramagnetic (S=1/2) in the reduced and oxidized states, respectively. The NMR chemical shifts of the heme substituents were assigned and used to determine the heme core architecture of PpcF. Compared to the PpcA-family from G. sulfurreducens, the spatial disposition of the hemes is conserved, but the functional properties are clearly distinct. In fact, potentiometric titrations monitored by UV-visible absorption reveal that the reduction potential values of PpcF are significantly less negative (-56 and -64 mV, versus the normal hydrogen electrode at pH 7.0 and 8.0, respectively). NMR redox titrations showed that the order of oxidation of the hemes is IV-I-III a feature not observed for G. sulfurreducens. The different redox properties displayed by PpcF, including the small redox-Bohr effect and low reduction potential value of heme IV, were structurally rationalized and attributed to the lower number of positively charged residues located in the vicinity of heme IV. Overall, the redox features of PpcF suggest that biotechnological applications of G. metallireducens may require less negative working functional redox windows than those using by G. sulfurreducens.

Nano-Fe3O4-synthetic talc gel was used as filler in the synthesis of waterborne polyurethane/Fe3O4-synthetic talc nanocomposites. This filler presents numerous edges (Si–O and Mg–O) and OH groups easily forming hydrogen bonds and polar interaction with water conferring hydrophilic character, consequently improving filler dispersion within a water-based matrix. Yet, the use of waterborne polyurethane (WPU) as matrix must be highlighted due to its environmentally friendly characteristics and low toxicity compared to solvent-based product. Fe3O4-synthetic talc-nanofillers were well dispersed into the polyurethane matrix even at high filler content as supported by XRD and TEM analyses. NMR indicates the interaction of filler OH groups with the matrix. For all nanocomposites, one can see a typical ferromagnetic behavior below Curie temperature (about 120 K) and a superparamagnetic behavior above this temperature. The use of Fe3O4-synthetic talc for obtaining magnetic nanocomposites resulted in improved materials with superior mechanical properties compared to solvent-based nanocomposites.

G. metallireducens bacterium has highly versatile respiratory pathways that provide the microorganism an enormous potential for many biotechnological applications. However, little is known about the structural and functional properties of its electron transfer components. In this work, the periplasmic cytochrome PpcA from G. metallireducens was studied in detail for the first time using complementary biophysical techniques, including UV–visible, CD and NMR spectroscopy. The results obtained showed that PpcA contains three low-spin c-type heme groups with His-His axial coordination, a feature also observed for its homologue in G. sulfurreducens. However, despite the high sequence homology between the two cytochromes, important structural and functional differences were observed. The comparative analysis of the backbone, side chain and heme substituents NMR signals revealed differences in the relative orientation of the hemes I and III. In addition, redox titrations followed by visible spectroscopy showed that the redox potential values for PpcA from G. metallireducens (−78 and −93 mV at pH 7 and 8, respectively) are considerably less negative. Overall, this study provides biochemical and biophysical data of a key cytochrome from G. metallireducens, paving the way to understand the extracellular electron transfer mechanisms in these bacteria.

Blends of rice waste streams were submitted to co-gasification assays. The resulting chars (G1C and G2C) were characterized and used in Cr(III) removal assays from a synthetic solution. A Commercial Activated Carbon (CAC) was used for comparison purposes. The chars were non-porous materials mainly composed by ashes (68.3–92.6% w/w). The influences of adsorbent loading (solid/liquid ratio – S/L) and initial pH in Cr(III) removal were tested. G2C at a S/L of 5 mg L−1 and an initial pH of 4.50 presented an uptake capacity significantly higher than CAC (7.29 and 2.59 mg g−1, respectively). G2C was used in Cr(III) removal assays from an industrial wastewater with Cr(III) concentrations of 50, 100 and 200 mg L−1. Cr(III) removal by precipitation (uptake capacity ranging from 11.1 to 14.9 mg g−1) was more effective in G2C, while adsorption (uptake capacity of 16.1 mg g−1) was the main removal mechanism in CAC.

The p53 tumor suppressor is widely found to be mutated in human cancer. This protein is regarded as a molecular hub regulating different cell responses, namely cell death. Compelling data have demonstrated that the impairment of p53 activity correlates with tumor development and maintenance. For these reasons, the reactivation of p53 function is regarded as a promising strategy to halt cancer. In the present work, the recombinant mutant p53R280K DNA binding domain (DBD) was produced for the first time, and its crystal structure was determined in the absence of DNA to a resolution of 2.0 Å. The solved structure contains four molecules in the asymmetric unit, four zinc(II) ions, and 336 water molecules. The structure was compared with the wild-type p53 DBD structure, isolated and in complex with DNA. These comparisons contributed to a deeper understanding of the mutant p53R280K structure, as well as the loss of DNA binding related to halted transcriptional activity. The structural information derived may also contribute to the rational design of mutant p53 reactivating molecules with potential application in cancer treatment.

Abstract This work reports for the first time a new series of promising porous catalytic carbon materials, functionalized with Lewis and Brønsted acid sites useful in the green synthesis of 2,3-dihydro-1H-1,5-benzodiazepine – nitrogen heterocyclic compounds. Benzodiazepines and derivatives are fine chemicals exhibiting interesting therapeutic properties. Carbon materials have been barely investigated in the synthesis of this type of compounds. Two commercial carbon materials were selected exhibiting different textural properties: i) Norit RX3 (N) as microporous sample and ii) mesoporous xerogel (X), both used as supports of ZrO2 (Zr) and ZrO2/SO42− (SZr). The supported SZr led to higher conversion values and selectivities to the target benzodiazepine. Both chemical and textural properties influenced significantly the catalytic performance. Particularly relevant are the results concerning the non-sulfated samples, NZr and XZr, that were able to catalyze the reaction leading to the target benzodiazepine with high selectivity (up to 80 %; 2 h). These results indicated an important role of the carbon own surface functional groups, avoiding the use of H2SO4. Even very low amounts of SZr supported on carbon reveal high activity and selectivity. Therefore, the carbon materials herein reported can be considered an efficient and sustainable alternative bifunctional catalysts for the benzodiazepine synthesis.

The bacterium Geobacter sulfurreducens can transfer electrons to quinone moieties of humic substances or to anthraquinone-2,6-disulfonate (AQDS), a model for the humic acids. The reduced form of AQDS (AH2QDS) can also be used as energy source by G. sulfurreducens. Such bidirectional utilization of humic substances confers competitive advantages to these bacteria in Fe(III) enriched environments. Previous studies have shown that the triheme cytochrome PpcA from G. sulfurreducens has a bifunctional behavior toward the humic substance analogue. It can reduce AQDS but the protein can also be reduced by AH2QDS. Using stopped-flow kinetic measurements we were able to demonstrate that other periplasmic members of the PpcA-family in G. sulfurreducens (PpcB, PpcD and PpcE) also showed the same behavior. The extent of the electron transfer is thermodynamically controlled favoring the reduction of the cytochromes. NMR spectra recorded for 13C,15N-enriched samples in the presence increasing amounts of AQDS showed perturbations in the chemical shift signals of the cytochromes. The chemical shift perturbations on cytochromes backbone NH and 1H heme methyl signals were used to map their interaction regions with AQDS, showing that each protein forms a low-affinity binding complex through well-defined positive surface regions in the vicinity of heme IV (PpcB, PpcD and PpcE) and I (PpcE). Docking calculations performed using NMR chemical shift perturbations allowed modeling the interactions between AQDS and each cytochrome at a molecular level. Overall, the results obtained provided important structural-functional relationships to rationalize the microbial respiration of humic substances in G. sulfurreducens.