Quality-based model for Life Sciences research guidelines

Nowadays, the requirement to define adequate standards and to identify and validate general guidelines for scientific activity is becoming increasingly apparent also in non-regulated scientific research. Guidelines are fundamental tools to provide valid indications for proper conduct in a research laboratory, the correct use of equipment and procedures, as well as for aligning and standardizing the procedures used in different scientific contexts. The identification, dissemination and application of common guidelines can improve significantly the reproducibility of the scientific results and the exchange of materials and data in the context of scientific consortia, comprising also industrial partners and meta-analysis projects. A Quality and Project Management OpenLab research network was formed in 2012 to develop and apply Total Quality Management models to Life Sciences research laboratories. One of the main tasks of the network has been the definition of a model for the drafting of guidelines, firmly based on quality principles and documentation management. The outcome is an operational flow describing all the phases of the process, which has been validated by four different drafting groups through the production of 13 guidelines ranging from research activity to equipment and facility management. Different institutes of the National Research Council are currently following these guidelines; some of them have also been used to define the procedures included in a certified Quality Management System for a research laboratory. Our experience shows that the model for guidelines we have developed makes drafting guidelines easier and more immediate, and significantly, it is applicable to different scientific contexts and disciplines, including both non-regulated research and technology transfer-oriented research, and also the Quality Management System of a scientific laboratory.     

Copper Catalysis in Living Systems and In Situ Drug Synthesis

The copper‐catalyzed azide–alkyne cycloaddition (CuAAC) reaction has proven to be a pivotal advance in chemical ligation strategies with applications ranging from polymer fabrication to bioconjugation. However, application in vivo has been limited by the inherent toxicity of the copper catalyst. Herein, we report the application of heterogeneous copper catalysts in azide–alkyne cycloaddition processes in biological systems ranging from cells to zebrafish, with reactions spanning from fluorophore activation to the first reported in situ generation of a triazole‐containing anticancer agent from two benign components, opening up many new avenues of exploration for CuAAC chemistry.     

Heteroditopic ligand accommodating a fused phenanthroline and a schiff base cavity as molecular spacer in the study of electron and energy transfer

The synthesis and characterisation of the heteroditopic ligand N,N'-bis(3,5-di-tert-butylsalicylidene)-5,6-(1,10-phenanthroline)diamine (DPSalH(2)) bearing a phenanthroline and a bis(salicylidene)diimine cavity are reported. This versatile ligand combines two of the most widely used ligands in coordination chemistry. Sequential metallation of the phenanthroline end with Ru(II) and the salophenic cavity with Cu(II) is described. Electrochemical behaviour of the supramolecular complexes [Ru(bpy)(2)(DPSalH(2))](2+) and [Ru(bpy)(2)(DPSalCu)](2+) are analysed in connection with UV/Vis and EPR spectroscopy. The data for the one-electron-reduced species and the singly oxidised species of the binuclear Ru(II)-Cu(II) complex confirmed the formation of metalloradical complexes. Density functional calculations on the free ligand and the copper-only complex indicate in both cases that the HOMOs and LUMOs are developed on the Schiff base cavity with minor contributions on the bipyridine end. These findings support a bichromophoric character for our ruthenium complexes in the ground state, a necessary condition in the design of supramolecular systems for the study of electron transfer. Photophysical studies indicate fast quenching of the triplet excited state in both complexes, which suggests strong intercomponent excited-state interactions. Evidence is presented that this quenching is due to intramolecular electron transfer, at least in the case of [Ru(bpy)(2)(DPSalH(2))](2+), for which a charge-separated state with a remarkable lifetime of about 30 mus was observed.     

LIC-KOR promoted formation of conjugated dienes as useful building blocks for palladium-catalyzed syntheses

It is demonstrated that α,β-unsaturated acetals can be considered a synthetic tool for transforming carbonyl derivatives into cheap and easily accessible starting materials for the construction of various and more complex structures. The lithium-potassium mixed superbase LIC-KOR induces a conjugate elimination reaction that converts α,β-unsaturated acetals into 1E-1-alkoxybuta-1,3-dienes. These derivatives can be readily metalated in situ and functionalized by reaction with electrophiles. The results can be grouped in two sections: (1) the palladium-catalyzed cross-coupling reaction between alkoxydienylboronates and tetralone- or isochromanone-derived vinyl triflates; (2) the regio- and stereoselective cross coupling reaction with aryl derivatives in the presence of a palladium catalyst (Heck conditions).     

Electron Transfer Fluorescence Quenching of Blepharisma japonicum Photoreceptor Pigments

Abstract— The hypericin analogs blepharismin (BP), oxyblepharismin (OxyBP) and stentorin (ST), the photosensing chromophores responsible for photomotile reactions in the ciliates Blepharisma japonicum (red and blue cells) and Stentor coeruleus, represent a new class of photoreceptor pigments whose chemical structures have recently been determined. In the case of ST it has been shown that the first excited singlet state can be deactivated by donation of an electron to an appropriate acceptor molecule (e.g. a quinone molecule). This charge transfer can be considered a possible mechanism for the primary photoprocess for the photomotile responses in S. coeruleus. To determine whether an electron transfer process also occurs in the deactivation of excited blepharismin, we studied the fluorescence quenching of OxyBP in dimethyl-sulfoxide (DMSO) and in ethanol using electron acceptors with different reduction potentials. Under our experimental conditions ground state and excited state complexes (like fluorescent exciplexes) are not formed between the fluorophore and the quenchers. In DMSO the bimolecular quenching constant values (k_q) calculated on the basis of the best fitting procedures clearly show that the quenching efficiency decreases with the quencher negative reduction potential, E⁰. The k_q (M^-1 s^-1) and E⁰ (V) values are, respectively, 7.8 times 10⁹ and -0.134 for 1,4-benzoquinone, 8.9 times 10⁹ and -0.309 for 1,4-naphthoquinone, 2.4 times 10⁹ and -0.8 for nitrobenzene, 0.009 times 10⁹ and -1.022 for azobenzene and 0 and -1.448 for benzophenone. These findings point to the conclusion that upon formation of the encounter complex between OxyBP and the quencher, an electron is released from excited OxyBP to the quencher, similar to what happens in ST. It is suggested that in the pigment granules such a light-induced charge transfer from excited blepharismin to a suitable electron acceptor triggers sensory transduction processes in B. japonicum.     

Model proficiency testing scheme for serological diagnosis of Brucellosis: interlaboratory study

A model interlaboratory testing scheme was developed by the Italian National Reference Laboratory for Brucellosis. This scheme was planned for both qualitative (Rose Bengal Plate Test; RBPT) and quantitative (Complement Fixation Test; CFT) serological tests and involved a total of 42 laboratories. In the preparation of this scheme, reference was made to general protocols and guidelines and to methods reported in the literature, which were applicable to analytical chemistry laboratories. Six field sera from naturally infected animals, one positive serum at a titer below the European Union (EU) positivity threshold, and 5 sera positive at titers between 20 and 851 International Units of Complement Fixation Test (IUCFT)/mL plus one negative serum were used to produce a panel of test sera. To evaluate laboratory performances in the quantitative test for each tested sample examined, z-scores based on robust summary statistics (the median and normalized interquartile range) were used. To evaluate overall laboratory performance, 2 types of combined z-scores were used: Rescaled Sum of Scores and Sum of Squared Scores. In the case of the qualitative test (RBPT), results were analyzed by a Bayesian approach. A Beta distribution, based on the result of each laboratory, was calculated and used to estimate the probability of each laboratory giving a correct result and its uncertainty.     

The Antimicrobial Activity of Origanum vulgare L. Correlated with the Gastrointestinal Perturbation in Patients with Metabolic Syndrome

(1) The metabolic syndrome (MS) promotes acute and chronic infections, due to the pro-inflammatory condition given by TNFα and IL6 or by affecting the microbiota. MS is also correlated with insulin resistance, causing inflammation and infections throughout the organism. (2) The purpose of this study was to track the effect of using the essential oil of Origanum vulgare L. (EOO) as an antibacterial treatment, compared to allopathic treatment with antibiotics in MS patients. A group of 106 people with MS was divided into four subgroups: L1—staphylococcal infection group, L2—Escherichia coli infection group, L3—streptococcal infection group with EOO treatment, and CG—control group without infection or treatment. (3) EOO is responsible for the antibacterial effect, and reduced minor uncomplicated infections. After a 10-day treatment, intestinal side effects were absent, improving the phase angle. (4) The results suggest that EOO may exhibit an antibacterial effect, similar to the antibiotic treatment, without promoting MS-specific dysbiosis, and it also improves the phase angle in patients, which is used as an index of health and cellular function.     

Rapid microwave assisted esterification method for the analysis of poly-3-hydroxybutyrate in Alcaligenes latus by gas chromatography

In the present study, we used microwave energy instead of conventional heating to transform poly-3-hydroxybutyrate (PHB) into methyl 3-hydroxybutyrate (Me-3HB) in acidified methanol (H2SO4, 10%, v/v) mixture in less than 4 min at 10% microwave power. The microwave assisted method was then applied to analyze PHB produced by Alcaligenes latus. The PHB content in the biomass determined using microwave heating was comparable to the amount found by conventional heating. Moreover, the new esterification method was at least 50 times faster than the conventional method, affording a significant saving of time and energy.     

Effects of Vanadyl Complexes with Acetylacetonate Derivatives on Non-Tumor and Tumor Cell Lines

Vanadium has a good therapeutic potential, as several biological effects, but few side effects, have been demonstrated. Evidence suggests that vanadium compounds could represent a new class of non-platinum, metal antitumor agents. In the present study, we aimed to characterize the antiproliferative activities of fluorescent vanadyl complexes with acetylacetonate derivates bearing asymmetric substitutions on the β-dicarbonyl moiety on different cell lines. The effects of fluorescent vanadyl complexes on proliferation and cell cycle modulation in different cell lines were detected by ATP content using the CellTiter-Glo Luminescent Assay and flow cytometry, respectively. Western blotting was performed to assess the modulation of mitogen-activated protein kinases (MAPKs) and relevant proteins. Confocal microscopy revealed that complexes were mainly localized in the cytoplasm, with a diffuse distribution, as in podocyte or a more aggregate conformation, as in the other cell lines. The effects of complexes on cell cycle were studied by cytofluorimetry and Western blot analysis, suggesting that the inhibition of proliferation could be correlated with a block in the G2/M phase of cell cycle and an increase in cdc2 phosphorylation. Complexes modulated mitogen-activated protein kinases (MAPKs) activation in a cell-dependent manner, but MAPK modulation can only partly explain the antiproliferative activity of these complexes. All together our results demonstrate that antiproliferative effects mediated by these compounds are cell type-dependent and involve the cdc2 and MAPKs pathway.     

Topical Dosage Formulation of Lyophilized Philadelphus coronarius L. Leaf and Flower: Antimicrobial,Antioxidant and Anti-Inflammatory Assessment of the Plant

Philadelphus coronarius is a versatile plant and its use in folk medicine has a long tradition; however, scientifically, the medical utilization of the herb is a less explored research field. The aim of our study was to identify and determine the quantity of the bioactive compounds of both the leaf and the flower and prepare a lyophilized product of them, from which medical ointments were formulated, since the topical application of P. coronarius has also not been studied. In vitro drug release, texture analysis and biocompatibility experiments were carried out, as well as the investigation of microbiological, antioxidant and anti-inflammatory properties. According to our results the composition and the selected excipients of the ointments have a great impact on the drug release, texture and bioavailability of the preparation. During the microbiological testing, the P. coronarius leaf was effective against Escherichia coli and Staphylococcus aureus, but it did not significantly decrease IL-4 production when it was tested on HaCaT cells. P. coronarius is a promising herb, and its topical application in antimicrobial therapy can be a useful addition to modern medical therapy.