Keras/TensorFlow in Drug Design for Immunity Disorders.

Homeostasis of the host immune system is regulated by white blood cells with a variety of cell surface receptors for cytokines. Chemotactic cytokines (chemokines) activate their receptors to evoke the chemotaxis of immune cells in homeostatic migrations or inflammatory conditions towards inflamed tissue or pathogens. Dysregulation of the immune system leading to disorders such as allergies, autoimmune diseases, or cancer requires efficient, fast-acting drugs to minimize the long-term effects of chronic inflammation. Here, we performed structure-based virtual screening (SBVS) assisted by the Keras/TensorFlow neural network (NN) to find novel compound scaffolds acting on three chemokine receptors: CCR2, CCR3, and one CXC receptor, CXCR3. Keras/TensorFlow NN was used here not as a typically used binary classifier but as an efficient multi-class classifier that can discard not only inactive compounds but also low- or medium-activity compounds. Several compounds proposed by SBVS and NN were tested in 100 ns all-atom molecular dynamics simulations to confirm their binding affinity. To improve the basic binding affinity of the compounds, new chemical modifications were proposed. The modified compounds were compared with known antagonists of these three chemokine receptors. Known CXCR3 compounds were among the top predicted compounds; thus, the benefits of using Keras/TensorFlow in drug discovery have been shown in addition to structure-based approaches. Furthermore, we showed that Keras/TensorFlow NN can accurately predict the receptor subtype selectivity of compounds, for which SBVS often fails. We cross-tested chemokine receptor datasets retrieved from ChEMBL and curated datasets for cannabinoid receptors. The NN model trained on the cannabinoid receptor datasets retrieved from ChEMBL was the most accurate in the receptor subtype selectivity prediction. Among NN models trained on the chemokine receptor datasets, the CXCR3 model showed the highest accuracy in differentiating the receptor subtype for a given compound dataset.

Determination of Pesticide Residues in IV Range Artichoke (Cynara cardunculus L.) and Its Industrial Wastes.

Fourth-range products are those types of fresh fruit and vegetables that are ready for raw consumption or after cooking, and belong to organic or integrated cultivations. These products are subject to mild post-harvesting processing procedures (selection, sorting, husking, cutting, and washing), and are afterwards packaged in packets or closed food plates, with an average shelf life of 5-10 days. Artichokes are stripped of the leaves, stems and outer bracts, and the remaining heads are washed with acidifying solutions. The A LC-MS/MS analytical method was developed and validated following SANTE guidelines for the detection of 220 pesticides. This work evaluated the distribution of pesticide residues among the fraction of artichokes obtained during the industrial processing, and the residues of their wastes left on the field were also investigated. The results showed quantifiable residues of one herbicide (pendimethalin) and four fungicides (azoxystrobin, propyzamide, tebuconazole, and pyraclostrobin). Pendimethalin was found in all samples, with the higher values in leaves 0.046 ± 8.2 mg/kg and in field waste 0.30 ± 6.7 mg/kg. Azoxystrobin was the most concentrated in the outer bracts (0.18 ± 2.9 mg/kg). The outer bracts showed the highest number of residues. The industrial waste showed a significant decrease in the number of residues and their concentration.

Computational and experimental approaches to probe GPCR activation and signaling.

G protein-coupled receptors (GPCRs) regulate different physiological functions, e.g., sensation, growth, digestion, reproductivity, nervous and immune systems response, and many others. In eukaryotes, they are also responsible for intercellular communication in response to pathogens. The major primary messengers binding to these cell-surface receptors constitute small-molecule or peptide hormones and neurotransmitters, nucleotides, lipids as well as small proteins. The simplicity of the way how GPCR signaling can be regulated by their endogenous agonists prompted the usage of GPCRs as major drug targets in modern pharmacology. Drugs targeting GPCRs inhibit pathological processes at the very beginning. This enables to significantly reduce the occurrence of morphological changes caused by diseases. Until recently, X-ray crystallography was the method of the first choice to obtain high-resolution structural information about GPCRs. Following X-ray crystallography, cryo-EM gained attention in GPCR studies as a quick and low-cost alternative. FRET microscopy is also widely used for GPCRs in the analysis of protein-protein interactions (PPIs) in intact cells as well as for screening purposes. Regarding computational methods, molecular dynamics (MD) for many years has proven its usefulness in studying the GPCR activation. MODELLER and Rosetta were widely used to generate preliminary homology models of GPCRs for MD simulation systems. Apart from the conventional all-atom approach with explicitly defined solvent, also other techniques have been applied to GPCRs, e.g., MARTINI or hybrid methods involving the coarse-grained representation, less demanding regarding computational resources, and thus offering much larger simulation timescales.

Fipronil and Fipronil Sulfone Distribution in Chicken Feathers and Eggs after Oral and Dermal Exposure.

This work aimed to investigate the bio-distribution and the persistence of fipronil and its primary metabolite fipronil sulfone after oral and dermal administration by simulating natural farming conditions. Fipronil and fipronil sulfone detection and quantification were performed in different poultry matrices using an LC-MS/MS method coupled with modified QuEChERS extraction. After oral administration, fipronil was detected in feathers at each sampling time, in eggs for 28 days, and in the internal organs at the end of the experiment. After dermal administration, high levels of fipronil were detected in feathers, accounting for 195.85 ± 8.54 mg/kg, which were reduced by a third after 60 days. No traces of fipronil were detected in the eggs or internal organs. In addition, fipronil sulfone showed remarkable residues in all samples in trial 2. The data obtained confirmed that inappropriate use of unauthorized pesticides can lead to severe contamination of entire poultry farms. The contemporary presence of fipronil sulfone in feathers and eggs associated with the lack of fipronil in eggs suggests recent dermal contamination or past oral contamination. Moreover, simultaneous analysis of hens' feathers and eggs could represent a new method to improve large-scale monitoring programs and animal welfare, limiting their slaughter.

Developmental Neurotoxicity of Environmentally Relevant Pharmaceuticals and Mixtures Thereof in a Zebrafish Embryo Behavioural Test.

Humans are exposed daily to complex mixtures of chemical substances via food intake, inhalation, and dermal contact. Developmental neurotoxicity is an understudied area and entails one of the most complex areas in toxicology. Animal studies for developmental neurotoxicity (DNT) are hardly performed in the context of regular hazard studies, as they are costly and time consuming and provide only limited information as to human relevance. There is a need for a combination of in vitro and in silico tests for the assessment of chemically induced DNT in humans. The zebrafish (Danio rerio) embryo (ZFE) provides a powerful model to study DNT because it shows fast neurodevelopment with a large resemblance to the higher vertebrate, including the human system. One of the suitable readouts for DNT testing in the zebrafish is neurobehaviour (stimulus-provoked locomotion) since this provides integrated information on the functionality and status of the entire nervous system of the embryo. In the current study, environmentally relevant pharmaceuticals and their mixtures were investigated using the zebrafish light-dark transition test. Zebrafish embryos were exposed to three neuroactive compounds of concern, carbamazepine (CBZ), fluoxetine (FLX), and venlafaxine (VNX), as well as their main metabolites, carbamazepine 10,11-epoxide (CBZ 10,11E), norfluoxetine (norFLX), and desvenlafaxine (desVNX). All the studied compounds, except CBZ 10,11E, dose-dependently inhibited zebrafish locomotor activity, providing a distinct behavioural phenotype. Mixture experiments with these pharmaceuticals identified that dose addition was confirmed for all the studied binary mixtures (CBZ-FLX, CBZ-VNX, and VNX-FLX), thereby supporting the zebrafish embryo as a model for studying the cumulative effect of chemical mixtures in DNT. This study shows that pharmaceuticals and a mixture thereof affect locomotor activity in zebrafish. The test is directly applicable in environmental risk assessment; however, further studies are required to assess the relevance of these findings for developmental neurotoxicity in humans.

Heavy metal and metalloid accumulation in wild brown trout (Salmo trutta L., 1758 complex, Osteichthyes: Salmonidae) from a mountain stream in Sardinia by ICP-OES.

 This paper reports heavy metal and metalloid accumulation in wild brown trout (Salmo trutta L., 1758 complex) raised in freshwater and uncontaminated Sardinia system (Italy). Metals are widespread pollutants of aquatic systems, and their contamination can originate from anthropogenic activities such as industrial waste, agricultural and domestic environments, and geochemical release. Fish has a relevant position within the human diet; moreover, fishes can accumulate metals, making them a valuable tool as biomarkers for risk assessment studies. The concentration of 22 metals and metalloids after chemical digestion was assessed by inductively coupled plasma-optic emission spectroscopy (ICP-OES) in both the guts and the edible part (EP, muscle + skin) of brown trout. The results, expressed as µg g-1, showed different levels of accumulation in the EP and guts, following the series Cu > Zn > Ba > Al > Sr > Fe > Pb and Fe > Al > Hg > As > Mn > Cu > Ba > B > Zn > Pb, respectively. PCA analysis showed a fairly good correlation between the total lipid and SAFA content and Cd, Hg, and Pb accumulation in the gut. Non-carcinogenic risk assessment, expressed as THQ (target hazard quotient), showed values far below 1 for all metals in muscles, while high As and Hg contamination of the gut draws attention to possible health risks which should be discarded from the fish before consumption. TR (target cancer risk) values showed alarmingly high values for As and Cd when the fish were consumed entirely (gut + EP), while Pb levels were far below the safety levels.

Zoxamide accumulation and retention evaluation after nanosuspension technology application in tomato plant.

BACKGROUND: Low water solubility of pesticide requires formulations with high levels of stabilizers and organic solvents. Moreover, only 0.1% of the applied pesticides formulation reaches the target, while 99.9% spreads in the surrounding environment. Therefore, there is the need for more efficient and environmentally sustainable alternatives. RESULTS: Zoxamide (ZO) nanosuspension was prepared through a media milling technique by using the stabilizer polysorbate 80. The thin and acicular crystals obtained, showed particle size of 227 nm, polydispersion index of 0.247 and zeta potential of -28 mV. Dimensional data and morphology of ZO nanocrystals alone, on tomato leaves and berries, were confirmed by scanning electron microscopy. The reduction in size for ZO crystals obtained after the milling process increased pesticide water solubility till 39.6 mg L-1 , about 1.6 the solubility obtained with a conventional commercial formulation. Field and dip contamination trials performed on tomato plants showed the nanosuspension's ability to increase ZO deposition and accumulation versus a coarse ZO suspension and commercial formulation, respectively. CONCLUSIONS: The nanoformulation proposed, resulted in low cost and was easy to make. Moreover, the organic solvent-free composition together with a low surfactant addition assured a minor environmental impact. Finally, the increased retention and deposition of the fungicide can reduce the amounts of ZO formulation applied to tomatoes. © 2021 Society of Chemical Industry.

Simultaneous determination and dietary risk assessment of clethodim and its metabolites in different fruits and vegetables.

Clethodim is one of the most widely used herbicides in agriculture and, after field application, is metabolised to several metabolites. The potential toxicological negative effects of these compounds are poorly understood. Thus, recently, within the risk assessment framework, the European Food Safety Authority (EFSA) proposed to include the minor metabolites in the definition of clethodim residue. In this work, an easy to use and reliable UHPLC method coupled with a triple quadrupole MS/MS was developed and validated for the detection and quantification of the herbicide clethodim and related metabolites clethodim sulphone, clethodim sulphoxide, metabolites M17R and M18R in apple, grape, olive and rice. The five analytes were extracted by using a modified QuEChERS procedure, while the active ingredients were determined in multiple reaction monitoring (MRM) ion-switching mode. The proposed method showed calibration curve linearity with r2 ≥ 0.990 for all active ingredients (a.is.) both in solvent and matrix extracts. Limits of quantitation (LOQ) of the five compounds ranged from 9.44 µg/kg for M17R in olive extract to 11.01 µg/kg for clethodim in apple extract. Recoveries values ranged from 86% to 119% at two concentration levels (LOQ and 10xLOQ), while the intraday and interday precisions of the method were both below 10% in all cases. The method was successfully used for the quantification of the five a.is. in different food matrices. Furthermore, chronic dietary risk was investigated using a hazard quotients (HQ) method based on European dietary habits. The chronic dietary exposure risk quotients ranged from 1.0 × 10-5 (lower bound scenario) to 2.7 × 10-4 (upper bound scenario) which were significantly lower than 1. Data obtained indicate that the dietary exposure risks were acceptable for clethodim and its major and minor metabolites applied in apple, table grape, rice and table olive.

Influence of the Technological Process on the Biochemical Composition of Fresh Roe and Bottarga from Liza ramada and Mugil cephalus.

Bottarga is a high-priced delicacy with high nutritional value, and, in Italy, bottarga from mullets has been recognized to be a traditional food product. The flathead grey mullet Mugil cephalus and the thinlip grey mullet Liza ramada are the main cultured grey mullets in the Mediterranean Sea. In this study, fresh roe and bottarga from these two species were investigated to evaluate the influence of the technological process and the species on their biochemical composition and health advantages. The 1 h/200 g salting-out step did not increase the levels of NaCl in the bottarga, although it highly decreased the levels of some heavy metals like Cu and Al. Processing of fresh roe in bottarga led to an essential modification of the lipid fraction, following a general series of monousatturated fatty acid (MUFA)> poliunsutturated fatti acid (PUFA) > saturated fatty acid (SAFA) and an increase in both ω3 and ω6 in Liza ramada. Moreover, bottarga showed higher levels of squalene and cholesterol and an increased Essential Amino Acid/Total Amino Acid ratio (EAA/TAA) in both species. In addition to the nutritional benefits for the consumer, the process proposed in this study may represent a reliable tool for local producers to obtain a final bottarga with both a reproducible biochemical composition and organoleptic characteristics.

Effects of Industrial Processing on Pesticide Multiresidues Transfer from Raw Tomatoes to Processed Products.

Pesticides are broadly used to improve food safety, although they can lead to adverse health effects on consumers. Various food processing approaches, at the industrial or domestic level, have been found to highly reduce the amount of pesticide residues in most food materials. In this work, samples of raw tomatoes were collected directly from the field and processed at the industrial level to produce purée, triple concentrated paste, fine pulp, and diced tomatoes. A multiresidue method based on a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged e Safe) sample preparation, followed by liquid chromatography-tandem mass spectrometry analysis (LC-MS/MS) for the assessment of 116 pesticides residues, was used. The analytical method has been validated according to SANTE indications. The recovery yields ranged from 75.5% to 115.3%, repeatability (RSDr) ranged from 3.4% to 18.3%, while reproducibility (RSDwR) ranged from 5.4% to 19.8%. The limit of quantifications (LOQs) ranged from 2.35 µg kg-1 for benthiavalicarb to 6.49 µg kg-1 for allethrin. A total of 159 raw tomato samples were collected from the field. The analysis showed the presence of 46 pesticides with azoxystrobin and chlorantraniliprole the most represented. On the other hand, all industrially processed samples showed values ≤ LOD, confirming that post-harvest processes can lead to a decrease in pesticide residues from agricultural commodities.

Integrated environmental evaluation of heavy metals and metalloids bioaccumulation in invertebrates and seaweeds from different marine coastal areas of sardinia, mediterranean sea.

In this work, three gastropods Patella vulgata, Osilinus turbinata, and Tahis clavigera, one echinoderm Parancetrotus lividus, one coelenterate Anemonia sulcata, and two seaweed Padina pavonica, and Cystoseira mediterranea were collected from three different marine areas of Sardinia in the Mediterranean sea and studied for heavy metals and metalloid content and accumulation trends. Inductively coupled plasma optical emission spectrometry (ICP-OES) was used for the determination of Al, AS, B, Ba, Be, Cd, Co, Cr, Cu, Fe, Hg, Mn, Mo, Ni, Pb, Sb, Se, Sn, Sr, Te, Ti, V and Zn in the selected samples. The results showed that gastropods were capable of accumulating Al, Ba, Cu, Fe, Sr, and Zn; seaweeds can better concentrate Al, Fe, and Zn than all other species. At the same time, echinoderms and coelenterate had limited ability to store specific metals, showing a much more homogeneous distribution. PCA analysis allowed us to discriminate among the sites and the species. Cala Zafferano was the area with the higher values of accumulation of all metals in all species as expected, considering its proximity to industrial sites. The results of the analysis showed clearly that heavy metal and metalloid accumulation was different for each species studied. Therefore, for a correct environmental assessment of a given area, a comprehensive approach is strongly recommended by exploiting the different properties of both accumulation and concentration of the metals by different aquatic species.

Volatiles, color characteristics and other physico-chemical parameters of commercial Moroccan honeys.

Seven commercial Moroccan honeys were considered for chemical characterisation. Volatile fraction, total polyphenols content, antioxidant and antiradical activities were evaluated by employing different analytical methodologies. Several physical parameters such as refractive index, pH, water content, solids content and colour were measured. Volatile fraction revealed an abundant presence of cis- and trans-linalool oxide in the seven studied samples. The presence of high levels of compounds related to the Maillard reaction, like furfural and hydroxymethylfurfural, could be the result of thermal treatments used to liquefy commercial honeys or of long storage times. The CIE L*a*b*C*(ab)h°(ab) chromatic coordinates confirmed the advanced stage of the Maillard reaction, showing L* values lower than the common values found for honey of similar typologies.