The developments in the use of plasmid DNA (pDNA) in gene therapy and vaccines have motivated the search and improvement of optimized purification processes. In this context, dipeptides l-tyrosine-l-tyrosine and l-tyrosine-l-arginine are synthetized to explore their application as affinity ligands for supercoiled (sc) plasmid DNA (pDNA) purification. The synthesis is based on the protection of N-Boc-l-tyrosine, followed by condensation with l-tyrosine or l-arginine methyl esters in the presence of dicyclohexylcarbodiimide (DCC), which after hydrolysis and acidification give the afforded dipeptides. The supports are then obtained by coupling l-tyrosine, l-tyrosine-l-tyrosine and l-tyrosine-l-arginine to epoxy-activated Sepharose and are characterized by high resolution magic angle spinning (HR-MAS) NMR and Fourier transform infrared spectroscopy (FTIR). Surface plasmon resonance (SPR) biosensor is used to establish the promising ligand to be used in the chromatographic experiments and ascertain experimental conditions. Sc isoform showed the highest affinity to the dipeptides, followed by linear (ln) pDNA, being the open circular (oc) the one that promoted the lowest affinity to l-tyrosine-l-arginine. Saturation transfer difference (STD)-NMR experiments show that the interaction is mainly hydrophobic with the majority of the 5'-mononucleotides, except for 5'-GMP with l-tyrosine-l-arginine Sepharose that is mainly electrostatic. The support l-tyrosine Sepharose used in chromatographic experiments promotes the separation of native pVAX1-LacZ and pcDNA3-FLAG-p53 samples (oc+sc) by decreasing the salt concentration. The results suggest that it is possible to purify different plasmids with the l-tyrosine Sepharose, with slight adjustments in the gradient conditions.

Electric vehicles (EVs) are considered alternatives to internal combustion engines due to their energy efficiency and contribution to CO2 mitigation. The adoption of EVs depends on consumer preferences, including cost, social status and driving habits, although it is agreed that current and expected costs play a major role. We use a partial equilibrium model that minimizes total energy system costs to assess whether EVs can be a cost-effective option for the consumers of each EU27 member state up to 2050, focusing on the impact of different vehicle investment costs and CO2 mitigation targets. We found that for an EU-wide greenhouse gas emission reduction cap of 40% and 70% by 2050 vis-à-vis 1990 emissions, battery electric vehicles (BEVs) are cost-effective in the EU only by 2030 and only if their costs are 30% lower than currently expected. At the EU level, vehicle costs and the capability to deliver both short- and long-distance mobility are the main drivers of BEV deployment. Other drivers include each state’s national mobility patterns and the cost-effectiveness of alternative mitigation options, both in the transport sector, such as plug-in hybrid electric vehicles (PHEVs) or biofuels, and in other sectors, such as renewable electricity.

Gene knockout studies on Geobacter sulfurreducens (Gs) cells showed that the outer membrane cytochrome OmcF is involved in respiratory pathways leading to the extracellular reduction of Fe(III) citrate and U(VI) oxide. In addition, microarray analysis of OmcF-deficient mutant versus the wild-type strain revealed that many of the genes with decreased transcript level were those whose expression is upregulated in cells grown with a graphite electrode as electron acceptor. This suggests that OmcF also regulates the electron transfer to electrode surfaces and the concomitant electrical current production by Gs in microbial fuel cells. Extracellular electron transfer processes (EET) constitute nowadays the foundations to develop biotechnological applications in biofuel production, bioremediation and bioenergy. Therefore, the structural characterization of OmcF is a fundamental step to understand the mechanisms underlying EET. Here, we report the complete assignment of the heme proton signals together with (1)H, (13)C and (15)N backbone and side chain assignments of the OmcF, excluding the hydrophobic residues of the N-terminal predicted lipid anchor.

Gene knock-out studies on Geobacter sulfurreducens (Gs) cells showed that the periplasmic triheme cytochrome PpcD is involved in respiratory pathways leading to the extracellular reduction of Fe(III) and U(VI) oxides. More recently, it was also shown that the gene encoding for PpcD has higher transcript abundance when Gs cells utilize graphite electrodes as sole electron donors to reduce fumarate. This sets PpcD as the first multiheme cytochrome to be involved in Gs respiratory pathways that bridge the electron transfer between the cytoplasm and cell exterior in both directions. Nowadays, extracellular electron transfer (EET) processes are explored for several biotechnological applications, which include bioremediation, bioenergy and biofuel production. Therefore, the structural characterization of PpcD is a fundamental step to understand the mechanisms underlying EET. However, compared to non-heme proteins, the presence of numerous proton-containing groups in the redox centers presents additional challenges for protein signal assignment and structure calculation. Here, we report the complete assignment of the heme proton signals together with 1H, 13C and 15N backbone and side chain assignments of the reduced form of PpcD.

Adenoviruses are important platforms for vaccine development and vectors for gene therapy, increasing the demand for high titers of purified viral preparations. Monoliths are macroporous supports regarded as ideal for the purification of macromolecular complexes, including viral particles. Although common monoliths are based on synthetic polymers as methacrylates, we explored the potential of biopolymers processed by clean technologies to produce monoliths for adenovirus purification. Such an approach enables the development of disposable and biodegradable matrices for bioprocessing. A total of 20 monoliths were produced from different biopolymers (chitosan, agarose, and dextran), employing two distinct temperatures during the freezing process (−20 °C and −80 °C). The morphological and physical properties of the structures were thoroughly characterized. The monoliths presenting higher robustness and permeability rates were further analyzed for the nonspecific binding of Adenovirus serotype 5 (Ad5) preparations. The matrices presenting lower nonspecific Ad5 binding were further functionalized with quaternary amine anion-exchange ligand glycidyltrimethylammonium chloride hydrochloride by two distinct methods, and their performance toward Ad5 purification was assessed. The monolith composed of chitosan and poly(vinyl) alcohol (50:50) prepared at −80 °C allowed 100% recovery of Ad5 particles bound to the support. This is the first report of the successful purification of adenovirus using monoliths obtained from biopolymers processed by clean technologies.

Chitin, the second most abundant polymer in nature, is a renewable, nontoxic, biodegradable, and antibacterial polysaccharide. This semicrystalline biopolymer exhibits hierarchical structure from nano to micro-scale and is responsible for interesting living tissue properties. Recently, the scientific interest in chitin nanofibrils for applications in biomedical and tissue engineering fields has increased due to their particular capabilities such as matrix reinforcements, bioactivity and morphology similar to natural tissues. This chapter is focused on composite materials reinforced with chitin nanofibrils and their biomedical applications.

Composite ferrogels were obtained by encapsulation of magnetic nanoparticles at two different concentrations (2.0 and 5.0 % w/v) within mixed agarose/chitosan hydrogels having different concentrations of agarose (1.0, 1.5 and 2.0% (w/v)) and a fixed concentration of chitosan (0.5% (w/v)). The morphological characterization carried out by scanning electron microscopy showed that dried composite ferrogels present pore sizes in the micrometer range. Thermogravimetric measurements showed that ferrogels present higher degradation temperatures than blank chitosan/agarose hydrogels without magnetic nanoparticles. In addition, measurements of the elastic moduli of the composite ferrogels evidenced that the presence of magnetic nanoparticles in the starting aqueous solutions prevents to some extent the agarose gelation achieved by simply cooling chitosan/agarose aqueous solutions. Finally, it is shown that composite chitosan/agarose ferrogels are able to heat in response to the application of an alternating magnetic field so that they can be considered as potential biomaterials to be employed in magnetic hyperthermia treatments.

Gum arabic (GA) is a hydrophilic composite polysaccharide derived from exudates of Acacia senegal and Acacia seyal trees. It is biocompatible, possesses emulsifying and stabilizing properties and has been explored as coating agent of nanomaterials for biomedical applications, namely magnetic nanoparticles (MNPs). Previous studies focused on the adsorption of GA onto MNPs produced by co-precipitation methods. In this work, MNPs produced by a thermal decomposition method, known to produce uniform particles with better crystalline properties, were used for the covalent coupling of GA through its free amine groups, which increases the stability of the coating layer. The MNPs were produced by thermal decomposition of Fe(acac)3 in organic solvent and, after ligand-exchange with meso-2,3-dimercaptosuccinic acid (DMSA), GA coating was achieved by the establishment of a covalent bond between DMSA and GA moieties. Clusters of several magnetic cores entrapped in a shell of GA were obtained, with good colloidal stability and promising magnetic relaxation properties (r2 /r1 ratio of 350). HCT116 colorectal carcinoma cell line was used for in vitro cytotoxicity evaluation and cell-labeling efficiency studies. We show that, upon administration at the respective IC50 , GA coating enhances MNP cellular uptake by 19 times compared to particles bearing only DMSA moieties. Accordingly, in vitro MR images of cells incubated with increasing concentrations of GA-coated MNP present dose-dependent contrast enhancement. The obtained results suggest that the GA magnetic nanosystem could be used as a MRI contrast agent for cell-labeling applications.

Gum arabic (GA) is a hydrophilic composite polysaccharide derived from exudates of Acacia senegal and Acacia seyal trees. It is biocompatible, possesses emulsifying and stabilizing properties and has been explored as coating agent of nanomaterials for biomedical applications, namely magnetic nanoparticles (MNPs). Previous studies focused on the adsorption of GA onto MNPs produced by co-precipitation methods. In this work, MNPs produced by a thermal decomposition method, known to produce uniform particles with better crystalline properties, were used for the covalent coupling of GA through its free amine groups, which increases the stability of the coating layer. The MNPs were produced by thermal decomposition of Fe(acac)3 in organic solvent and, after ligand-exchange with meso-2,3-dimercaptosuccinic acid (DMSA), GA coating was achieved by the establishment of a covalent bond between DMSA and GA moieties. Clusters of several magnetic cores entrapped in a shell of GA were obtained, with good colloidal stability and promising magnetic relaxation properties (r2 /r1 ratio of 350). HCT116 colorectal carcinoma cell line was used for in vitro cytotoxicity evaluation and cell-labeling efficiency studies. We show that, upon administration at the respective IC50 , GA coating enhances MNP cellular uptake by 19 times compared to particles bearing only DMSA moieties. Accordingly, in vitro MR images of cells incubated with increasing concentrations of GA-coated MNP present dose-dependent contrast enhancement. The obtained results suggest that the GA magnetic nanosystem could be used as a MRI contrast agent for cell-labeling applications.

Geobacter bacteria have a remarkable respiratory versatility that includes the dissimilatory reduction of insoluble metal oxides in natural habitats and electron transfer to electrode surfaces from which electricity can be harvested. In both cases, electrons need to be exported from the cell interior to the exterior via a mechanism designated as extracellular electron transfer (EET). Several c-type cytochromes from G. sulfurreducens (Gs) were identified as key players in this process. Biochemical and biophysical data have been obtained for ten Gs cytochromes, including inner-membrane associated (MacA), periplasmic (PpcA, PpcB, PpcC, PpcD, PpcE and GSU1996) and outer membrane-associated (OmcF, OmcS and OmcZ). The redox properties of these cytochromes have been determined, except for PpcC and GSU1996. In this perspective, the reduction potentials of these two cytochromes were determined by potentiometric redox titrations followed by visible spectroscopy. The data obtained are taken together with those available for other key cytochromes to present a thorough overview of the current knowledge of Gs EET mechanisms and provide a possible rationalization for the existence of several multiheme cytochromes involved in the same respiratory pathways.

The influence of Ag nanoparticles (Ag NPs) on the luminescence of electrospun nonwoven mats made of polyvinylpyrrolidone (PVP) has been studied in this work. The PVP fibers incorporating 2.1–4.3 nm size Ag NPs show a significant photoluminescence (PL) band between 580 and 640 nm under 325 nm laser excitation. The down conversion luminescence emission is present even after several hours of laser excitation, which denotes the durability and stability of fibers to consecutive excitations. As so these one-dimensional photonic fibers made using cheap methods is of great importance for organic optoelectronic applications, fluorescent clothing or counterfeiting labels.

The influence of Ag nanoparticles (Ag NPs) on the luminescence of electrospun nonwoven mats made of polyvinylpyrrolidone (PVP) has been studied in this work. The PVP fibers incorporating 2.1–4.3 nm size Ag NPs show a significant photoluminescence (PL) band between 580 and 640 nm under 325 nm laser excitation. The down conversion luminescence emission is present even after several hours of laser excitation, which denotes the durability and stability of fibers to consecutive excitations. As so these one-dimensional photonic fibers made using cheap methods is of great importance for organic optoelectronic applications, fluorescent clothing or counterfeiting labels.

The eutrophication of surface waters caused by cyanobacteria is a worldwide problem, leading to expensive
water treatment costs [1]. In addition, the production of microcystins by these microalgae may cause many
health problems to humans and animals (e.g. liver cancer) and even death [2]. Therefore, a variety of
methods have been developed to control cyanobacteria blooms, including physical and chemical treatments.
However, they have negative impacts on other species of (micro) algae and on other aquatic biota. As a
consequence, ultrasonic algae treatment has been proposed as a clean approach to controlling the blooms of
some algae species and microcystins degradation [3]. Still, the specific effects of ultra-sonication on
cyanobacteria are not well known. The present work aimed to study the effects of ultra-sonication on the
cyanobacteria structure under different ultrasound conditions (changing frequency and power) by using
conventional histology and electron microscopy methods.
Microcystis spp. were harvested in a lake from Azores (Portugal) and stored in the cool and dark until
transported to the laboratory. Cyanobacteria were cultured in liquid BG-11 axenic medium at 22ºC in an
incubator chamber, under continuous illumination (fluorescent cold white light).
Samples were collected and suspensions of cells (1ml each) were subjected to ultrasonic irradiation using
diverse ultrasonic equipment (UP100H; UP200S, sonoreactor UTR 200 and ultrasonic bath) and testing
different exposure times. All the experimental algal suspensions were exposed for 5 min to ultrasonication
(on ice for periods of 10s to avoid heating). After ultrasonication cyanobacteria growth was assessed for a
period of 14 days and structural changes in cells were evaluated by light (LM) and scanning electron
microscopy (SEM) examination. The results show growth inhibition of the cyanobacteria according to
intensity and power used in each ultrasonic device. The use of the most powerful devices (sonoreactor and
UP200S) resulted in a massive disrupting of cell walls with consequent cell death (Fig. 1e,f). Similar results
were obtained by Ahan et al. [1] and Nakano et al. [4] and showing cell wall disruption. However, even
after exposure to the most powerful instrumentation it was possible to detect some viable cells and after 14
days colonies were already visible. The results from light and electron microscopy showed noticeable
changes at the structural level such as disruption of cell gas vacuoles (arrowhead), colony disaggregation and
damage of cell walls of cells (Fig. 1c-f).
As a consequence, the use of ultrasounds to improve water quality from eutrophic waters must be considered
with careful in terms of efficiency and other complementary methods should be considered to assure good
water quality criteria. In addition, the effects of ultrasonication in other aquatic organisms require further
studies before using this technology to control algae blooms.

X-ray intensity ratios, such as the Kα2/Kα1 ratio, are parameters with a large application in atomic physics and related scientific and technological areas. D.

The majority of heterocycle compounds and typically common heterocycle fragments present in most pharmaceuticals currently marketed, alongside with their intrinsic versatility and unique physicochemical properties, have poised them as true cornerstones of medicinal chemistry. Apart from the already marketed drugs, there are many other being investigated for their promising activity against several malignancies. In particular, anticancer research has been capitalizing on the intrinsic versatility and dynamic core scaffold of these compounds. Nevertheless, as for any other promising anticancer drugs, heterocyclic compounds do not come without shortcomings. In this review, we provide for a concise overview of heterocyclic active compounds and families and their main applications in medicine. We shall focus on those suitable for cancer therapy while simultaneously addressing main biochemical modes of action, biological targets, structure-activity relationships as well as intrinsic limitation issues in the use of these compounds. Finally, considering the advent of nanotechnology for effective selective targeting of drugs, we shall discuss fundamental aspects and considerations on nanovectorization of such compounds that may improve pharmacokinetic/pharmacodynamic properties of heterocycles.

Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) are considered exclusive animal pathogens; however, a putative zoonotic upper limb cellulitis, a prosthetic joint infection and an infective endocarditis were described in humans. To unravel if bovine SDSD isolates are able to infect human cells, the adherence and internalization to human primary keratinocytes of two bovine SDSD strains isolated from milk collected from udder were analyzed. Bacterial adhesion assays and confocal microscopy indicate a high adherence and internalization of SDSD isolates to human cells, suggesting for the first time the ability of bovine isolates to infect human cells.