Nanocomposite microbeads made of recycled polylactic acid for the magnetic solid phase extraction of xenobiotics from human urine.

Nanocomposite microbeads (average diameter = 10-100 µm) were prepared by a microemulsion-solidification method and applied to the magnetic solid-phase extraction (m-SPE) of fourteen analytes, among pesticides, drugs, and hormones, from human urine samples. The microbeads, perfectly spherical in shape to maximize the surface contact with the analytes, were composed of magnetic nanoparticles dispersed in a polylactic acid (PLA) solid bulk, decorated with multi-walled carbon nanotubes (mPLA@MWCNTs). In particular, PLA was recovered from filters of smoked electronic cigarettes after an adequate cleaning protocol. A complete morphological characterization of the microbeads was performed via Fourier-transform infrared (FTIR) spectroscopy, UV-Vis spectroscopy, thermogravimetric and differential scanning calorimetry analysis (TGA and DSC), scanning electron microscopy (SEM) and X-ray diffraction analysis (XRD). The recovery study of the m-SPE procedure showed yields ≥ 64%, with the exception of 4-chloro-2-methylphenol (57%) at the lowest spike level (3 µg L-1). The method was validated according to the main FDA guidelines for the validation of bioanalytical methods. Using liquid chromatography-tandem mass spectrometry, precision and accuracy were below 11% and 15%, respectively, and detection limits of 0.1-1.8 µg L-1. Linearity was studied in the range of interest 1-15 µg L-1 with determination coefficients greater than 0.99. In light of the obtained results, the nanocomposite microbeads have proved to be a valid and sustainable alternative to traditional sorbents, offering good analytical standards and being synthetized from recycled plastic material. One of the main objectives of the current work is to provide an innovative and optimized procedure for the recycling of a plastic waste, to obtain a regular and reliable microstructure, whose application is here presented in the field of analytical chemistry. The simplicity and greenness of the method endows the procedure with a versatile applicability in different research and industrial fields.

Recovery of cellulose acetate bioplastic from cigarette butts: realization of a sustainable sorbent for water remediation.

Cigarette butts, one of the most common forms of litter in the world, represent a source of chemical and plastic pollution releasing thousands of toxic compounds and microfibers of cellulose acetate (CA). Besides the correct waste management, the recovery of CA from cigarette filters is a way to cushion their negative effects on the environment. Thus far, recycling strategies have been limited to industrial applications, while not many solutions have designed for water remediation. This work describes a strategy to valorize this harmful waste and to reverse its environmental impact, proposing a simple and effective procedure of reclamation of CA and its reuse to prepare a composite sorbent for the treatment of polluted water. The first step entails the washing of filters with hot water (T = 90 °C) and hot ethanol (T = 58-68 °C) to remove the impurities produced during cigarette burning, as verified by means of UV and attenuated total reflection-Fourier-transform infrared (ATR-FTIR) spectroscopy, thermal gravimetric analysis (TGA), and differential scanning calorimetry (DSC). The second step involves the use of the regenerated CA to prepare porous cylinder-shaped cryogels (15 mm × 10 mm) whose sorption properties are enhanced by the combination with AC (15 % w/w). The synthesis takes advantage of the sol-gel transition of the polymer dispersion (5 % w/V) in a solution acetone/water 5 mM in NH3 (60/40, v/v). After characterization by dynamic mechanical analysis (DMA), TGA, FT-IR, and scanning electron microscopy (SEM), the adsorption capability of the physical cryogel was studied in terms of treated environmental water volume, contact time and concentration of the selected pollutants. The results have shown that the proposed strategy is a low-cost way to recycle CA from cigarette butts and that the designed sorbent is a promising material for water treatment, allowing quick removal times and yields >79.6 %.

Identification of the pyridoxal 5'-phosphate allosteric site in human pyridox(am)ine 5'-phosphate oxidase.

Adequate levels of pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6 , and its proper distribution in the body are essential for human health. The PLP recycling pathway plays a crucial role in these processes and its defects cause severe neurological diseases. The enzyme pyridox(am)ine 5'-phosphate oxidase (PNPO), whose catalytic action yields PLP, is one of the key players in this pathway. Mutations in the gene encoding PNPO are responsible for a severe form of neonatal epilepsy. Recently, PNPO has also been described as a potential target for chemotherapeutic agents. Our laboratory has highlighted the crucial role of PNPO in the regulation of PLP levels in the cell, which occurs via a feedback inhibition mechanism of the enzyme, exerted by binding of PLP at an allosteric site. Through docking analyses and site-directed mutagenesis experiments, here we identified the allosteric PLP binding site of human PNPO. This site is located in the same protein region as the allosteric site we previously identified in the Escherichia coli enzyme homologue. However, the identity and arrangement of the amino acid residues involved in PLP binding are completely different and resemble those of the active site of PLP-dependent enzymes. The identification of the PLP allosteric site of human PNPO paves the way for the rational design of enzyme inhibitors as potential anti-cancer compounds.

Fragment Merging, Growing, and Linking Identify New Trypanothione Reductase Inhibitors for Leishmaniasis.

Trypanothione reductase (TR) is a suitable target for drug discovery approaches against leishmaniasis, although the identification of potent inhibitors is still challenging. Herein, we harnessed a fragment-based drug discovery (FBDD) strategy to develop new TR inhibitors. Previous crystallographic screening identified fragments 1-3, which provided ideal starting points for a medicinal chemistry campaign. In silico investigations revealed critical hotspots in the TR binding site, guiding our structure- and ligand-based structure-actvity relationship (SAR) exploration that yielded fragment-derived compounds 4-14. A trend of improvement in Leishmania infantum TR inhibition was detected along the optimization and confirmed by the crystal structures of 9, 10, and 14 in complex with Trypanosoma brucei TR. Compound 10 showed the best TR inhibitory profile (Ki = 0.2 µM), whereas 9 was the best one in terms of in vitro and ex vivo activity. Although further fine-tuning is needed to improve selectivity, we demonstrated the potentiality of FBDD on a classic but difficult target for leishmaniasis.

A liquid chromatography-mass spectrometry method for the enantioselective multiresidue determination of nine chiral agrochemicals in urine using an enrichment procedure based on graphitized carbon black.

Many agrochemicals are chiral molecules, and most of them are marketed as racemates or diastereomeric mixtures. Stereoisomers that are not the active enantiomer have little or no pesticidal activity and can exert serious toxic effects towards non-target organisms. Thus, investigating the possible exposure to different isomers of chiral pesticides is an urgent need. The present work was aimed at developing a new enantioselective high-performance liquid chromatography-mass spectrometry method for the simultaneous determination of nine chiral pesticides in urine. Two solid-phase extraction (SPE) procedures, based on different carbon-based sorbents (graphitized carbon black (GCB) and buckypaper (BP)), were developed and compared. By using GCB, all analytes were recovered with yields ranging from 60 to 97%, while BP allowed recoveries greater than 54% for all pesticides except those with acid characteristics. Baseline separation was achieved for the enantiomers of all target agrochemicals on a Lux Cellulose-2 column within 24 min under reversed-phase mode. The developed method was then validated according to the FDA guidelines for bioanalytical methods. Besides recovery, the other evaluated parameters were precision (7-15%), limits of detection (0.26-2.21 µg/L), lower limits of quantitation (0.43-3.68 µg/L), linear dynamic range, and sensitivity. Finally, the validated method was applied to verify the occurrence of the pesticide enantiomers in urine samples from occupationally exposed workers.

An enantioselective high-performance liquid chromatography-mass spectrometry method to study the fate of quizalofop-P-ethyl in soil and selected agricultural products.

In this study, the attention was focused on quizalofop-ethyl, a chiral herbicide whose formulation has recently been marketed as quizalofop-P-ethyl, i.e. the (+)-enantiomer exhibiting herbicidal activity. To verify the real enantiomeric purity of this product as well as to study its environmental fate, the enantioselective separation of the P- and M- enantiomers of quizalofop-ethyl was achieved on Lux Cellulose-2 column (3­chloro,4-methylphenilcarbamate cellulose) under isocratic conditions in polar organic mode. Once established that the commercial formulation contains ˜ 0.6% (enantiomeric fraction) of M as an impurity, an HPLC-MS/MS method was developed, validated and applied to the analysis of soil, carrots and turnips treated with the herbicide. A simple solid-liquid extraction allowed recoveries greater than 70%; limits of detections of P and M enantiomers were below 5 ng g-1. The analyses of the real samples showed a modification of the enantiomeric fraction of quizalofop-M-ethyl between the commercial formulation (EFM = 0.63 ± 0.03%) and the analysed matrices (EFM = 7.6 ± 0.1% for carrots; EFM = 0% for the other matrices). This outcome highlighted the occurrence of an enantioselective biotic dissipation, responsible for a greater persistency of the distomer in carrots. On the other hand, since screening analyses revealed the occurrence of residues of the metabolite quizalofop-acid with the same EFs as the ester precursor, it was concluded that the hydrolytic conversion was an abiotic process.

Investigation of the Entry Pathway and Molecular Nature of σ1 Receptor Ligands.

The σ1 receptor (σ1-R) is an enigmatic endoplasmic reticulum resident transmembrane protein implicated in a variety of central nervous system disorders and whose agonists have neuroprotective activity. In spite of σ1-R's physio-pathological and pharmacological importance, two of the most important features required to fully understand σ1-R function, namely the receptor endogenous ligand(s) and the molecular mechanism of ligand access to the binding site, have not yet been unequivocally determined. In this work, we performed molecular dynamics (MD) simulations to help clarify the potential route of access of ligand(s) to the σ1-R binding site, on which discordant results had been reported in the literature. Further, we combined computational and experimental procedures (i.e., virtual screening (VS), electron density map fitting and fluorescence titration experiments) to provide indications about the nature of σ1-R endogenous ligand(s). Our MD simulations on human σ1-R suggested that ligands access the binding site through a cavity that opens on the protein surface in contact with the membrane, in agreement with previous experimental studies on σ1-R from Xenopus laevis. Additionally, steroids were found to be among the preferred σ1-R ligands predicted by VS, and 16,17-didehydroprogesterone was shown by fluorescence titration to bind human σ1-R, with significantly higher affinity than the prototypic σ1-R ligand pridopidine in the same essay. These results support the hypothesis that steroids are among the most important physiological σ1-R ligands.

Inhibition of Leishmania infantum Trypanothione Reductase by New Aminopropanone Derivatives Interacting with the NADPH Binding Site.

BACKGROUND: As a result of the paucity of treatment, Leishmaniasis continues to provoke about 60,000 deaths every year worldwide. New molecules are needed, and drug discovery research is oriented toward targeting proteins crucial for parasite survival. Among them, trypanothione reductase (TR) is of remarkable interest owing to its vital role in Leishmania species protozoan parasite life. Our previously identified compound 1 is a novel chemotype endowed with a unique mode of TR inhibition thanks to its binding to a formerly unknown but druggable site at the entrance of the NADPH binding cavity, absent in human glutathione reductase (hGR). METHODS: We designed and synthesized new 3-amino-1-arylpropan-1-one derivatives structurally related to compound 1 and evaluated their potential inhibition activity on TR from Leishmania infantum (LiTR). Cluster docking was performed to assess the binding poses of the compounds. RESULTS: The newly synthesized compounds were screened at a concentration of 100 µM in in vitro assays and all of them proved to be active with residual activity percentages lower than 75%. CONCLUSIONS: Compounds 2a and 2b were the most potent inhibitors found, suggesting that an additional aromatic ring might be promising for enzymatic inhibition. Further structure-activity relationships are needed to optimize our compounds activity.

Enantioselective high-performance liquid chromatographic separations to study occurrence and fate of chiral pesticides in soil, water, and agricultural products.

As many as 40% of all plant protection products currently used contain chiral active ingredients. Enantiomers of the same pesticide have identical physicochemical properties in an isotropic medium, but they may display different activity and toxicity because of their interaction with enzymes or other naturally occurring asymmetric molecules. This difference may also lead to variations in biotic degradation rates, making one enantiomer more persistent than the other in natural and agricultural environments. In terms of methodological aspects, this critical review describes the most used chiral stationary phases for HPLC enantioseparations of chiral pesticides, pinpointing their strengths and weaknesses. As far as their applicability is concerned, most research has been carried out by means of columns based on derivatized amylose/cellulose due to their rather universal analyte coverage. The chromatographic compatibility with sensitive detection techniques, such as mass spectrometry, has allowed the trace analysis of stereoisomers, revealing ubiquitous occurrence of some chiral pesticides in surface waters, sediments, plants, agricultural soils, roots, fruit and vegetables. The study of their distribution and degradation in various environmental compartments and agricultural soil-plant systems has highlighted the enrichment with one enantiomer over the other in certain matrices following the enantioselective dissipation catalysed by microorganisms or plant enzymes as well as the phenomenon of chiral inversion in some cases. Irrespective of the reliability of a chiral method, such investigations are often hindered by the lack of pure standards of single enantiomers, which makes it difficult to identify their stereochemical configuration and requires precise strategies of quantification. Surely, the research in this field has been grown over the last few years due to the necessity of assessing and limiting risks related to exposure to chiral pesticides, which can be considered emerging contaminants in all aspects.

Formulation matters! A spectroscopic and molecular dynamics investigation on the peptide CIGB552 as itself and in its therapeutical formulation.

Synthetic therapeutic peptides (STP) are intensively studied as new-generation drugs, characterized by high purity, biocompatibility, selectivity and stereochemical control. However, most of the studies are focussed on the bioactivity of STP without considering how the formulation actually used for therapy administration could alter the physico-chemical properties of the active principle. The aggregation properties of a 20-mer STP (Ac-His-Ala-Arg-Ile-Lys-D-Pro-Thr-Phe-Arg-Arg-D-Leu-Lys-Trp-Lys-Tyr-Lys-Gly-Lys-Phe-Trp-NH2 ), showing antitumor activity, were investigated by optical spectroscopy and atomic force microscopy imaging, as itself (CIGB552) and in its therapeutic formulation (CIGB552TF). It has found that the therapeutic formulation deeply affects the aggregation properties of the investigated peptide and the morphology of the aggregates formed on mica by deposition of CIGB552 and CIGB552TF millimolar solutions. Molecular dynamics simulations studied the first steps of CIGB552 aggregation under physiological ionic strength conditions (NaCl 150 mM), showing that peptide oligomers, from dimers to tetramers, are preferentially formed in this environment. Interestingly, cell viability assays performed on H-460 cell lines indicate a major antiproliferative activity of the peptide in its therapeutic formulation with respect to the peptide aqueous solution.