Inhibition of human LDL lipid peroxidation by phenol-rich beverages and their impact on plasma total antioxidant capacity in humans

Mounting evidence shows that phenol-rich beverages exert strong antioxidant activity. However, in vivo evidence has produced conflicting results. In the present study, we studied the impact of the ingestion of 300 mL of black and green tea, alcohol-free red wine, alcohol-free white wine, or water on plasma total antioxidant capacity in five healthy volunteers. Red wine has the highest content of phenolics (3.63 ± 0.48 g QE/L), followed by green tea (2.82 ± 0.07 g QE/L), black tea (1.37 ± 0.15 g QE/L), and white wine (0.31 ± 0.01 g QE/L). Plasma total antioxidant capacity values of subjects who drank green tea rose at 30 min (P < 0.05). After black tea and red wine ingestion, the peaks were at 50 min (P < 0.05 and P < 0.01, respectively). No changes were observed in the control and white wine groups. Red wine and green tea were the most efficient in protecting low density lipoprotein from oxidation driven by peroxyl and ferril radicals, respectively. Phenol-rich beverages are a natural source of antioxidants; however, the phenolic content alone cannot be considered an index of their in vivo antioxidant activity.http://www.sciencedirect.com/science/article/B6T8P-420SK92-B/1/657ecacc07210bc6cfa30dc5753d761

Neuropathic pain is associated with depressive behaviour and induces neuroplasticity in the amygdala of the rat

Chronic pain is associated with the development of affective disorders but the underlying mechanisms are not fully understood. Changes in brain centres implicated in both emotional and pain processing are likely to be critical in the interplay of pain control and affective emotional behaviour. In the present study, we assessed emotional behaviour and performed a structural analysis of the amygdala (AMY) in neuropathic rats after two months of hyperalgesia and allodynia, induced by the spared nerve injury model (SNI). When compared with Sham-controls, SNI animals displayed signs of depressive-like behaviour. In addition, we found an increased amygdalar volume in SNI rats. No alterations were found in the dendritic arborizations of AMY neurons but, surprisingly, the amygdalar hypertrophy was associated with an increased cell proliferation [bromodeoxyuridine (BrdU)-positive cells] in the central (CeA) and basolateral (BLA) amygdaloid nuclei. The phenotypic analysis of the newly-acquired cells revealed that they co-label for neuronal markers (BrdU + NeuN and BrdU + Calbindin), but not for differentiated glial cells (BrdU + glial fibrillary acidic protein). We demonstrate that neuropathic pain promotes generation of new neurons in the AMY. Given the established role of the AMY in emotional behaviour, we propose that these neuroplastic changes might contribute for the development of depressive-like symptoms that are usually present in prolonged pain syndromes in humans.Fundação para a Ciência e Tecnologia (FCT) Project no. POCTI/NSE/46399/2002FEDERFundação Calouste Gulbenkian Project no. 7455

Bioprocessing of recombinant proteins using alternative purification platforms

The advent of biopharmaceuticals in modern medicine brought enormous benefits to diverse human diseases and improved the well-being of many people worldwide. Since the FDA approval of humulin (recombinant insulin) in 19821, remarkable advances in the treatment of chronic diseases have been achieved with biopharmaceutical-based therapies2. This sector represents 40% of ca. 6000 products currently in clinical development2, and is dominated by therapeutic proteins with over 200 protein drugs in the market1. Along the years, many improvements in the biopharmaceuticals upstream stage resulted in high titers of the desired product, and shifted the bioprocess bottleneck to the downstream processing, which is currently dominated by chromatography, accounting with more than 70% of total downstream costs3. Aiming at finding new cost-effective, efficient and sustainable technologies for proteins purification, novel polymer-polymer aqueous biphasic systems (ABS) with ionic liquids (ILs) as adjuvants are investigated as alternative purification platforms for the downstream of interferon alfa 2b (IFNα2b) from Escherichia coli BL21 cultures. Initial experiments showed that the production of IFNα2b is higher using the SOB culture medium and the western-blot analysis revealed that it is present in the inclusion body fraction. This fraction was washed, solubilized using a specific buffer and, finally dialyzed. After analyzing the stability of the target protein in several phase-forming components, the ternary phase diagrams of ABS composed by polyethylene glycol (PEG), polypropylene glycol (PPG) with ILs as adjuvants were determined at 25 °C, as well as the corresponding tie-lines, tie-line lengths and critical points. Chloride-based ILs combined with cholinium, imidazolium, pyrrolidinium, piperidinium, tetralkylammonium and tetralkylphosphonium cations were investigated. In summary, this study reports effective IFNα2b purification platforms from E. coli based on polymer-polymer-ABS, being highlighted the beneficial role of ILs in the downstream processing of proteins, either as adjuvants in ABS or by exploring new ILs features in protein stabilization.publishe

PRIMARY CUTANEOUS INFECTION WITH SCEDOSPORIUM APIOSPERMUM IN AN IMMUNOCOMPROMISED PATIENT SUCCESSFULLY TREATED WITH VORICONAZOLE

Durante as últimas décadas, a incidência de infecções fúngicas oportunistas tem vindo a aumentar, nomeadamente no contexto de imunossupressão. O Scedosporium apiospermum é um fungo filamentoso ubiquitário no solo, vegetação em decomposição, esgotos e em águas poluídas. Pode originar infecções em doentes imunocompetentes após trauma e infecções graves e, potencialmente fatais em doentes imunocomprometidos. Descrevemos um caso de uma infecção por S. apiospermum no dorso do pé esquerdo de um doente sob corticoterapia sistémica prolongada em combinação com metotrexato devido ao diagnóstico de arterite de células gigantes. O tratamento destas infecções pode ser um desafio devido à inerente resistência a muitos dos antifúngicos sistémico disponíveis, incluindo a anfotericina B. No caso descrito, foi realizada terapêutica com voriconazol, com resolução completa das lesões.During the last decades, the incidence of opportunistic fungal infections has been increasing, namely in the context of immunosuppression. The Scedosporium apiospermum is a ubiquitous filamentous fungus in soil, decaying vegetation, sewers and polluted waters. Can cause infections in immunocompetent patients after trauma and severe and potentially fatal infections in immunocompromised patients. The authors describe a case of an infection by S. apiospermum on the dorsum of the left foot of a patient under prolonged systemic corticosteroid therapy combined with methotrexate due to the diagnosis of giant cell arteritis. Treatment of these infections can be a challenge due to the inherent resistance to many of the available systemic antifungal agents, including amphotericin B. In the case described, therapy with voriconazole was performed, with complete resolution of the lesions

Aqueous two-phase systems comprising ionic liquids for the extraction of recombinant proteins

The progress made on the use of new biopharmaceuticals has a high influence on the global health, and since the approval of humulin (recombinant insulin) by FDA, new protein-based pharmaceuticals have been introduced in the market such as the interferon alfa 2b (IFNα2b), which has been used in in the treatment of oncological diseases for more than 30 years. Nowadays, the (bio)pharmaceutical industry is committed to improve the quality of their products while endeavoring a reduction of its production costs. Currently, the downstream processing of biopharmaceuticals is dominated by chromatography and, despite being highly effective, is responsible for their current high-cost [1]. Accordingly, there is a crucial need to develop effective alternative purification platforms. In this way, aqueous two-phase systems (ATPS) emerge. Due to their high-water content, ATPS may be considered a biocompatible technique for the purification of (bio)molecules. Additionally, the use of ionic liquids (ILs) in low concentrations as adjuvants in ATPS affords an enhanced performance in what concerns to (bio)molecules selectivity. In this work it is shown the use of polymer-polymer ATPS comprising ILs as adjuvants to purify recombinant proteins. Several variables were studied in the extraction of the target proteins, namely pH, polymer molecular weight, and the effect of different families of ILs and their concentration. Overall, imidazolium-based ILs seem responsible for an improved ATPS performance towards a higher purity, and is additionally shown that electrostatic interactions between the negatively charged protein and ILs cations may account for this behavior.publishe

Downstream processing of recombinant proteins using alternative purification platforms based on ionic liquids

Remarkable advances in the treatment of chronic diseases have been achieved with biopharmaceutical-based therapies, a sector that is currently dominated by therapeutic proteins with over 200 protein drugs in the market1. Improvements in the biopharmaceuticals upstream stage have resulted in high yields of the desired product, and shifted the bioprocess bottleneck to the downstream processing, which is currently dominated by chromatography, accounting with more than 70% of total downstream costs2. Aiming at finding new cost-effective, efficient and sustainable technologies for proteins purification, novel polymer-polymer aqueous biphasic systems (ABS) using ionic liquids (ILs) as adjuvants were investigated in the downstream of interferon alfa 2b (IFNα2b) from Escherichia coli BL21 cultures. The phase diagrams of ABS composed by polyethylene glycol (PEG) and polypropylene glycol (PPG), with ILs as adjuvants, were determined at 25 °C. Chloride-based ILs combined with cholinium, imidazolium, pyrrolidinium, piperidinium, tetralkylammonium and tetralkylphosphonium cations were investigated. The use of ILs allows the selective removal of the protein impurities present in the culture medium. In summary, the application of ILs allowed to develop effective IFNα2b purification platforms from E. coli based on polymer-polymer-ABS, having significant benefits in terms of economic, biocompatible and environmental issues. References: 1Walsh G, Nat Biotechnol, 2018, 36, 1136-1145; 2Azevedo AM, Rosa PAJ, Ferreira IF, et al. Trends Biotechnol, 2009, 27, 240-247.publishe

Improving the purification of recombinant proteins by the use of ionic liquids as adjuvants in aqueous two-phase systems

The development of new drugs has a high impact on the global health, and since the approval of humulin (recombinant insulin) by FDA, several protein-based biopharmaceuticals have been introduced in the market. These products are usually attained through biotechnological processes using engineered biological sources and have led to remarkable advances in the treatment of distinct human diseases, as for example the case of interferon alfa 2b (IFNα2b) which has been used in in the treatment of oncological diseases for more than 30 years [1]. Currently, the (bio)pharmaceutical industry is committed to improve the quality of their products while endeavouring a reduction of its production costs, which is intimately related to the necessity of improving their downstream processes, since this field is mainly dominated by chromatographic techniques [2] and are responsible for their current high-cost. Therefore, envisaging to develop new, cost-effective, efficient and sustainable techniques for proteins purification, polymer-polymer aqueous two-phase systems (ATPS) with ionic liquids (ILs) as adjuvants are herein proposed for the extraction and purification of IFNα2b from Escherichia coli BL21 cultures. The recombinant protein was produced in the form of inclusion bodies, followed by a dialysis step in order to solubilize the target biomolecule in a specific buffer, with the goal of maintaining the biomolecule stability. Afterwards, several polymers where studied as phase-forming components of ATPS, namely polyethylene glycol (PEG), polypropylene glycol (PPG), as well as Dextran. Furthermore, several ILs belonging to the cholinium, imidazolium, tetralkylammonium and tetralkylphosphonium families, were tested as co-adjuvants for ATPS formation in order to extract and purify of IFNα2b.publishe

Diphenyl diselenide, a simple glutathione peroxidase mimetic, inhibits human LDL oxidation in vitro

Oxidative modification of low-density lipoprotein (LDL) represents an important factor in atherogenesis. In the present study, we have investigated the antioxidant capability of diphenyl diselenide (PhSe)2, a simple organoseleno compound, against copper (Cu2+) and peroxyl radical-induced human LDL oxidation in vitro. In initial studies using human serum, (PhSe)2 caused a dose-dependent inhibition of Cu2+-induced lipid peroxidation, which was correlated to thiol consumption. (PhSe)2 increased lipid peroxidation lag phase and decreased lipid peroxidation rate in isolated human LDL, evaluated by measuring both conjugated diene (CD) and thiobarbituric acid reactive substances (TBARS) levels. Consistent with these observations, (PhSe)2 showed a marked inhibitory effect on 2,2-azobis(2-amidinopropane dihydrochloride) (AAPH)-induced oxidation of LDL or parinaric acid (PnA) incorporated into LDL. (PhSe)2 also displayed a dose-dependent protective effect against Cu2+-induced lipid peroxidation in rat aortic slices. Interestingly, besides the antioxidant effects of (PhSe)2 toward the lipid moieties of LDL, which was related to its thiol-peroxidase activity, protein moieties from human isolated LDL were also protected against Cu2+-induced oxidation. The results presented herein are the first to show that (i) (PhSe)2 inhibits lipid peroxidation in human isolated LDL in vitro, (ii) this phenomenon is related to its thiol-peroxidase activity, and (iii) this chalcogen also prevents the oxidation of protein moieties of human LDL. Taken together, such data render (PhSe)2 a promising molecule for pharmacological studies with respect to the atherogenic process.http://www.sciencedirect.com/science/article/B6T12-4S21TS2-1/1/38250cf8bae4a4195ccd8905cf5c2a8

Studies on the mechanism of action of anti-tumor bis(aminophenolate) ruthenium(III) complexes

Two recently published Ru(III) comp lexes bearing ( N 2 O 2 ) tetradentate bis(aminophenolate) ligands, 20 formulated as [Ru(III)( s alan )(PPh 3 )Cl] ( where s alan is the tetradentate ligand 6,6' - (1S,2S) - 21 cyclohexane - 1,2 - diylbis(azanediyl)bis(met hylene)bis(3 - methoxyphenol) in c omplex 1 , or 2,2' - (1S,2S) - 22 cyclohexane - 1,2 - diylbis(azanediyl)bis(meth ylene)bis(4 - methoxyphenol) in c omplex 2 , and PPh 3 is 23 triphenylphosphane ) were studied herein to outline their antitumor mode of action. Previously, it was 24 found that they were very active against human ovar ian and bre a st adenocarcinoma cells (on a 72 h 25 challenge ) . In this work, the human cisplatin - sensitive o varian adeno carcinoma line A2780 was used 26 as the cell model for further studies a t a 24 h challenge . B oth complexes are active , their cytotoxicity 27 being c omparable to that of c isplatin in the same conditions. 28 A s a possible target in the cell for their action, the int eraction of 1 and 2 with DNA was assessed 29 through displacement of well - established DNA fluorescent probes ( ethidium bromide , EB, and 4',6 - 30 d iamidino - 2 - p henylindole, DAPI ) through s teady - state and time - resolved fluorescence spectroscopy . 31 The complete emission spectra were analysed globally for the binary DNA ‒ p robe and ternary DNA ‒ 32 p robe ‒ Ru(III) complex systems . B oth Ru(III) complexes can displace EB and bind to DNA with 33 similar and moderate strong affinity with conditional stability constants of log K’ = ( 5.05 ± 0.01 ) for 1 34 and log K’= ( 4.79 ± 0.01 ) for 2 . The analysis of time - domain fluorescence intensity decays confirmed 35 both qualitatively and quantitatively the model used to describe the binding and competition processes. 36 Cell studies indicated that apoptosis is t he major mechanism of cell death for both complexes , with 2 37 (the mo re active complex ) promoting that process more efficiently than 1 . Transmission electron 38 micro graphs revealed clear a lteration s on intracellular organization consistent with the induction of 39 programmed cell death processes

The antibacterial, anti-biofilm, anti-inflammatory and virulence inhibition properties of Portuguese honeys

In Portugal, beekeeping activity has a significant weight among livestock production. The antimicrobial activities of Portuguese honeys have been reported, but the anti-biofilm formation and anti-virulence abilities have not been investigated. The main goal of this work was to study the impact of three monofloral honeys (citrus, lavender and strawberry tree) honeys on adherence of Staphylococcus aureus, methicillin-resistant S. aureus (MRSA) and Pseudomonas aeruginosa, as well as the influence of the same honeys on virulence using Galleria mellonella as a model. In addition, the general physico-chemical characterization of these honeys and the microbial quality were also performed. The anti-inflammatory activity was also estimated by analyzing the activity of the enzymes hyaluronidase and lipoxygenase. The tested honeys complied with European legislation and no microbial contamination was observed. Of all the honeys at 12.5 and 25%, w/v the citrus honey caused the highest inhibitory activity against P. aeruginosa. Strawberry tree honey at 25% w/v was able to significantly inhibit the MRSA strains. Anti-biofilm formation and anti-inflammatory activities were observed. The different honeys impaired the virulence of S. aureus and MRSA strains. The Portuguese honeys were capable of combating the tested bacterial pathogens not only by inhibiting their growth but also by affecting important pathogenicity properties, such as adherence and virulence