Directing in Vitro Selection towards G-quadruplex-forming Aptamers to Inhibit HMGB1 Pathological Activity

In the search for novel, effective inhibitors of High-Mobility Group Box1 (HMGB1)—a protein involved in various inflammatory and autoimmune diseases as well as in cancer—we herein discovered a set of anti-HMGB1 G-quadruplex(G4)-forming aptamers by using an in vitro selection procedure applied to a doped library of guanine-rich oligonucleotides. The selected DNA sequences were then studied in a pseudo-physiological buffer mimicking the extracellular medium, where HMGB1 exerts its pathological activity, using spectroscopic, electrophoretic, and chromatographic techniques. All the oligonucleotides proved to fold into monomeric G4s and in some cases also dimeric species, stable at physiological temperature. Remarkably, the protein preferentially recognized the sequences forming dimeric parallel G4 structures, as evidenced by a properly designed chemiluminescent binding assay which also highlighted a good selectivity of these aptamers for HMGB1. Moreover, all aptamers showed anti-HMGB1 activity, inhibiting protein-induced cell migration. The acquired data allowed identifying L12 as the best anti-HMGB1 aptamer, featured by high thermal and enzymatic stability, no toxicity at least up to 5 μM concentration on healthy cells, along with potent anti-HMGB1 activity (IC₅₀ ca. 28 nM) and good binding affinity for the protein, thus indicating it as a very promising lead candidate for in vivo studies.     

Bioactive and Nutritional Potential of Medicinal and Aromatic Plant (MAP) Seasoning Mixtures

Medicinal and aromatic plants (MAPs), worldwide appreciated and used as condiments, dyes, and preservatives, possess several biological properties that justify their continuous application in the food industry. In the present study, the nutritional and chemical profiles, as well as the bioactive properties of four combinations of condiments, sold for seasoning poultry, meat, fish, and salads, were evaluated. Twenty-five phenolic compounds (HPLC-DAD-ESI/MS) were identified, with apigenin-O-malonyl-pentoside-hexoside as the major compound detected in all extracts. Oxalic and citric acids were identified in all mixtures (UFLC-PDA), as well as all the four tocopherol isoforms (HPLC-fluorescence). Regarding bioactivities, the mixtures for meat and salads (TBARS) and meat and poultry (OxHLIA) stood out for their antioxidant potential, whereas for the anti-inflammatory and antitumor properties, the mixtures revealing the greatest results were those for poultry and salad, respectively. In terms of antimicrobial activity, all the mixtures revealed the capacity to inhibit the growth of some bacterial strains. In brief, condiment mixtures showed to be a good source of bioactive compounds, as they confer health benefits, validating the importance of their inclusion in the human diet as a good dietary practice.     

Valorization of Amaranth (Amaranthus cruentus) Grain Extracts for the Development of Alginate-Based Active Films

This research work investigates the development of alginate-based films incorporating phenolic compounds extracted from Amaranthus cruentus grain using different solvents. Alginate, glycerol, and amaranth grain phenolic compounds at various concentrations were used to produce the films. An experimental Central Composite Rotatable Design (CCRD) was used to evaluate the effect of these variables on different film’s properties, i.e., water vapor permeability, hydrophobicity, moisture content, solubility, thermal, mechanical, and optical properties. This study demonstrated that high phenolic compound content and antioxidant capacity were obtained from amaranth grain using ethanol as the extraction solvent. Alginate films incorporating amaranth phenolic compounds were successfully manufactured, and this study can be used to tailor the formulation of alginate films containing amaranth phenolic compounds, depending on their final food application. For example, less flexible but more resistant and water-soluble films can be produced by increasing the alginate concentration, which was confirmed by a Principal Component Analysis (PCA) and Partial Least Squares (PLS) analysis. This study showed that active alginate films with amaranth phenolic compounds can be tailored to be used as food packaging material with potential antioxidant activity.     

Approximations and Randomization to Boost CSP Techniques

In recent years we have seen an increasing interest in combining constraint satisfaction problem (CSP) formulations and linear programming (LP) based techniques for solving hard computational problems. While considerable progress has been made in the integration of these techniques for solving problems that exhibit a mixture of linear and combinatorial constraints, it has been surprisingly difficult to successfully integrate LP-based and CSP-based methods in a purely combinatorial setting. Our approach draws on recent results on approximation algorithms based on LP relaxations and randomized rounding techniques, with theoretical guarantees, as well on results that provide evidence that the runtime distributions of combinatorial search methods are often heavy-tailed. We propose a complete randomized backtrack search method for combinatorial problems that tightly couples CSP propagation techniques with randomized LP-based approximations. We present experimental results that show that our hybrid CSP/LP backtrack search method outperforms the pure CSP and pure LP strategies on instances of a hard combinatorial problem.     

Negative inotropism of terpenes on guinea pig left atrium: structure-activity relationships

The aim of this work was to evaluate the pharmacological effect of seven structurally related terpenes on the contractility of cardiac muscle. The effect of terpenes was studied on isolated electrically driven guinea pig left atrium. From concentration–response curves for inotropic effect were determined the EC₅₀ and relative potency of such terpenes. Our results revealed that all terpenes, except phytol, showed ability to reduce the contractile response of guinea pig left atrium. Further, relative potency was directly related to the number of isoprene units and to the lipophilicity of the compounds. For example, sesquiterpenes farnesol and nerolidol showed higher relative potency when compared with the monoterpenes citronellol, geraniol and nerol. We can conclude that most of the evaluated terpenes showed a promising negative inotropism on the atrial muscle. Future studies are necessary to investigate their action mechanism.     

Analysis of the relationships between proficiency testing,validation of methods and estimation of measurement uncertainty: a qualitative study with experts

Proficiency testing (PT) is an important part in the process ensuring quality of results. Validation of methods and estimation of measurement uncertainty are also vital elements in a laboratory qualification process. The main objective of this paper is to identify the relationships between PT, validation of methods and calculation of measurement uncertainty through the drawing up of a Mind Map. The method proposed used the Web-based Delphi method to conduct a qualitative research with experts of the area (laboratory assessors and accreditation body members). Experts from five accreditation bodies (Brazil, USA, France, Portugal and China) took part in the research. The study led to the drawing up of a Mind Map showing the connections of cause and effect between the factors under study. It emerged that PT effectively contributes to the reliability of results, since it is directly related to the validation of methods and to measurement uncertainty, so as to increase laboratory’s competitiveness.     

Dispersive liquid–liquid microextraction of quinolones in porcine blood: Optimization of extraction procedure and CE separation using experimental design

A dispersive liquid–liquid microextraction procedure was developed to extract nine fluoroquinolones in porcine blood, six of which were quantified using a univariate calibration method. Extraction parameters including type and volume of extraction and dispersive solvent and pH, were optimized using a full factorial and a central composite designs. The optimum extraction parameters were a mixture of 250 μL dichloromethane (extract solvent) and 1250 μL ACN (dispersive solvent) in 500 μL of porcine blood reached to pH 6.80. After shaking and centrifugation, the upper phase was transferred in a glass tube and evaporated under N₂ steam. The residue was resuspended into 50 μL of water–ACN (70:30, v/v) and determined by CE method with DAD, under optimum separation conditions. Consequently, a tenfold enrichment factor can potentially be reached with the pretreatment, taking into account the relationship between initial sample volume and final extract volume. Optimum separation conditions were as follows: BGE solution containing equal amounts of sodium borate (Na₂B₄O₇) and di‐sodium hydrogen phosphate (Na₂HPO₄) with a final concentration of 23 mmol/L containing 0.2% of poly (diallyldimethylammonium chloride) and adjusted to pH 7.80. Separation was performed applying a negative potential of 25 kV, the cartridge was maintained at 25.0°C and the electropherograms were recorded at 275 nm during 4 min. The hydrodynamic injection was performed in the cathode by applying a pressure of 50 mbar for 10 s.     

Chemiluminescence and Bioluminescence as an Excitation Source in the Photodynamic Therapy of Cancer: A Critical Review

Photodynamic therapy (PDT) of cancer is known for its limited number of side effects, and requires light, oxygen and photosensitizer. However, PDT is limited by poor penetration of light into deeply localized tissues, and the use of external light sources is required. Thus, researchers have been studying ways to improve the effectiveness of this phototherapy and expand it for the treatment of the deepest cancers, by using chemiluminescent or bioluminescent formulations to excite the photosensitizer by intracellular generation of light. The aim of this Minireview is to give a précis of the most important general chemi‐/bioluminescence mechanisms and to analyze several studies that apply them for PDT. These studies have demonstrated the potential of utilizing chemi‐/bioluminescence as excitation source in the PDT of cancer, besides combining new approaches to overcome the limitations of this mode of treatment.