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AlphaFold and AlphaFold-Multimer have become two essential tools for the modeling of unknown structures of proteins and protein complexes. In this work, we extensively benchmarked the quality of chemokine-chemokine receptor structures generated by AlphaFold-Multimer against experimentally determined structures. Our analysis considered both the global quality of the model, as well as key structural features for chemokine recognition. To study the effects of template and multiple sequence alignment parameters on the results, a new prediction pipeline called LIT-AlphaFold (https://github.com/LIT-CCM-lab/LIT-AlphaFold) was developed, allowing extensive input customization. AlphaFold-Multimer correctly predicted differences in chemokine binding orientation and accurately reproduced the unique binding orientation of the CXCL12-ACKR3 complex. Further, the predictions of the full receptor N-terminus provided insights into a putative chemokine recognition site 0.5. The accuracy of chemokine N-terminus binding mode prediction varied between complexes, but the confidence score permitted the distinguishing of residues that were very likely well positioned. Finally, we generated a high-confidence model of the unsolved CXCL12-CXCR4 complex, which agreed with experimental mutagenesis and cross-linking data.
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Benchmarking , Quimiocinas , Modelos Moleculares , Conformação Proteica , Quimiocinas/metabolismo , Quimiocinas/química , Receptores de Quimiocinas/metabolismo , Receptores de Quimiocinas/química , Ligação Proteica , Humanos , Sequência de AminoácidosRESUMO
The monitoring of contaminants represents a priority to preserve the integrity of marine ecosystems, as well as to plan and to manage restoration activities in order to protect environmental and human health. In the present study, a 6-months active biomonitoring was performed to explore the levels of eighteen trace and toxic elements, including heavy metals (TEs; i.e. Al, As, Ca, Cd, Cr, Cu, Fe, Hg, K, Mg, Mn, Na, Ni, P, Pb, Sr, Ti, and Zn), accumulated in soft tissues of blue mussel (Mytilus edulis Linnaeus, 1758) individuals transplanted at different depths (5- and 15-m depth) in five locations within the Flekkefjord fjord (Southern Norway). As this area suffered a long-lasting contamination due to both organic and inorganic contaminants, a series of restoration activities were activated to tackle and to prevent potential risks for ecosystem and local population. Our results demonstrated that the levels of TEs accumulated in edible tissues of transplanted mussels in the Flekkefjord fjord were generally low before the beginning of the restoration activities. However, location- and time-specific differences in the accumulation of TEs were noted after the implementation of such activities. Interestingly, the levels of Fe and Mn significantly increased after the beginning of the restoration activities, likely because the release of these TEs from the slag used in such operations and/or resuspension of contaminated sediments. However, assuming that native mussels can accumulate the same TEs at levels measured in transplanted individuals, our results suggest a substantial safety for human consumption of native mussels from the Flekkefjord fjord, regardless of restoration activities.
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Metais Pesados , Mytilus edulis , Oligoelementos , Poluentes Químicos da Água , Animais , Ecossistema , Monitoramento Ambiental/métodos , Estuários , Humanos , Metais Pesados/análise , Oligoelementos/análise , Poluentes Químicos da Água/análiseRESUMO
BACKGROUND: Fish consumption is increasing nowadays both because of its positive role for health due to the abundant presence of unsaturated fatty acids and for its use in many new food preparations (e.g. raw fillet used for uncooked sushi and sashimi dishes). The growing food industry and increased demand for the long-term storage and preservation of food have created the need to develop methods that can easily track and preserve food freshness and safety throughout shelf-life (production, storage, shipment, and consumption). While E-nose technologies have already been used and tested for these purposes, scarce information is available in the literature on the feasibility of using other food devices to detect changes in perishable food like fish during shelf-life in order to predict and correctly manage all food storage phases. The aim of the present study was to investigate the potential of Food Sniffer® portable devices to define the quality and safety of salmon fillet and burger (Salmo salar) packaged in modified atmosphere at two refrigerated conditions (4 and 8 °C). RESULTS: An increase in biogenic amines and volatile compounds especially ketones and alcohols were observed, with large amounts at final storage times of 8 °C temperature. CONCLUSION: The Food Sniffer® application was able to anticipate unacceptability conditions of salmon samples also correlated with chemical and microbiological parameters. This could represent a valid support for food industry and retail to manage perishable food commodities preventing possible food risk as well. © 2020 Society of Chemical Industry.
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Análise de Alimentos/métodos , Alimentos Marinhos/análise , Animais , Bactérias/crescimento & desenvolvimento , Aminas Biogênicas/análise , Análise de Alimentos/instrumentação , Embalagem de Alimentos , Inocuidade dos Alimentos , Armazenamento de Alimentos , Controle de Qualidade , Salmão/microbiologia , Alimentos Marinhos/microbiologiaRESUMO
The ability of tandem mass spectrometry to determine the primary structure of proteolytic peptides can be exploited to trace back the organisms from which the corresponding proteins were extracted. This information can be important when food products, such as protein powders, can be supplemented with lower-quality starting materials. In order to dissect the origin of proteinaceous material composing a given unknown mixture, a two-step database search strategy for bottom-up nanoscale liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) data was implemented. A single nanoLC-MS/MS analysis was sufficient not only to determine the qualitative composition of the mixtures under examination, but also to assess the relative percent composition of the various proteomes, if dedicated calibration curves were previously generated. The approach of two-step database search for qualitative analysis and proteome total ion current (pTIC) calculation for quantitative analysis was applied to several binary and ternary mixtures which mimic the composition of milk replacers typically used in calf feeding.
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Proteínas do Leite/análise , Leite/química , Proteoma/análise , Animais , Bovinos , Cromatografia Líquida , Espectrometria de Massas em TandemRESUMO
The aim of this work was to determine the microorganisms present and to investigate their metabolites that cause spoilage of many goose sausages produced in Friuli, a northeast region of Italy. The defect was observed by sensorial analysis using the "needle probing" technique; the spoiled sausages were unsafe and not marketable. Despite the addition of starter, the microorganisms, particularly enterococci and Enterobacteriaceae, grew during ripening and produced a large amount of biogenic amines; therefore, these sausages represented a risk to consumers. The production of those compounds was confirmed in vitro. Furthermore, a second cause of spoilage was attributed to moulds that grew during ripening; the fungi grew between the meat and casing, producing a large amount of total volatile nitrogen, and consequently an ammonia smell was present either in the ripening area or in the sausages. This is the first description of this type of defect in goose sausages.
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Aminas Biogênicas/metabolismo , Enterobacteriaceae/metabolismo , Enterococcus/metabolismo , Microbiologia de Alimentos , Fungos/metabolismo , Produtos da Carne/microbiologia , Animais , Aminas Biogênicas/análise , Cadaverina/metabolismo , Contagem de Colônia Microbiana , Enterobacteriaceae/crescimento & desenvolvimento , Enterococcus/crescimento & desenvolvimento , Fermentação , Inocuidade dos Alimentos , Fungos/crescimento & desenvolvimento , Gansos , Histamina/metabolismo , Itália , Nitrogênio/metabolismoRESUMO
Nowadays food wrapping assures attractive presentation and simplifies self-service shopping. Polyvinylchloride (PVC)- and polyethylene (PE)-based cling-films are widely used worldwide for wrapping cheeses. For this purpose, films used in retail possess suitable technical properties such as clinginess and unrolling capacity, that are achieved by using specific plasticizers during their manufacturing process. In the present study, the main VOCs of three cling-films (either PVC-based or PE-based) for retail use were characterized by means of Solid-Phase Micro-Extraction and GC/MS. In addition, the effects of cling film type and contact time on the migration of VOCs from the films to four different PDO Italian cheeses during cold storage under light or dark were also investigated. Among the VOCs isolated from cling-films, PVC released 2-ethylhexanol and triacetin. These compounds can likely be considered as a "non-intentionally added substance". These same compounds were also detected in cheeses wrapped in PVC films with the highest concentration found after 20 days storage. The PE cling-film was shown to possess a simpler VOC profile, lacking some molecules peculiar to PVC films. The same conclusions can be drawn for cheeses wrapped in the PE cling-film. Other VOCs found in wrapped cheeses were likely to have been released either by direct transfer from the materials used for the manufacture of cling-films or from contamination of the films. Overall, HS-SPME is shown to be a rapid and solvent free technique to screen the VOCs profile of cling-films, and to detect VOCs migration from cling-films to cheese under real retail storage conditions.
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Derivados de Benzeno/análise , Queijo/análise , Embalagem de Alimentos , Hexanóis/análise , Triacetina/análise , Xilenos/análise , Contaminação de Alimentos/análise , Cromatografia Gasosa-Espectrometria de Massas , Compostos Orgânicos Voláteis/análiseRESUMO
BACKGROUND: According to European Union legislation, prednisolone, a steroid that belongs to the glucocorticosteroid group, is banned as a growth promoter in cattle husbandry and therefore should not be present in bovine feedstuffs. As our preliminary investigations detected prednisolone in this matrix, we performed a study on different commercially available complementary feedstuffs, stored at the farm and/or in the laboratory, in order to verify whether its presence was due to neo-formation during storage. RESULTS: Prednisolone was detected in almost all (95%) feedstuffs collected at the farm.When the feedstuffs were stored at the laboratory, the frequency (31%) and the concentration of prednisolone-positives were lower. This difference, which is likely due to different environmental conditions, implies the possibility of its neo-formation. CONCLUSION: Our data indicate that the neo-formation of prednisolone can occur in feedstuff, and that the frequency and concentration could be related to the storage conditions. The individuation of an objective parameter that is useful for the identification of the compliance of feed is therefore essential.
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Ração Animal/análise , Contaminação de Alimentos/análise , Armazenamento de Alimentos , Prednisolona/análise , Criação de Animais Domésticos , Animais , Bovinos , Dieta , União Europeia , Prednisolona/metabolismoRESUMO
Meat is a highly nutritious food but there is a lot of significant evidence of negative health outcomes related to its excessive consumption, especially for processed one. Among the variety of emerging contaminants of concern for human health, a key role is played by poly- and per-fluoroalkyl substances (PFASs), which show adverse effects in humans who are exposed to them through diet. In the present study, for the first time, 70 paired batches of pre-cooked and canned bovine meat were analysed by Liquid Chromatography coupled to High Resolution Mass Spectrometry to evaluate the presence and concentration of 18 PFASs. These data were used to assess Italian consumers' health risks by performing the PFAS intake evaluation. PFBA and PFOS were detected in the precooked and canned meat samples, with PFBA mean concentration of 0.22 ± 0.36 ng g-1, and
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Infrared (IR) technology offers a promising solution for reducing microbiological loads in various food types while preserving their quality traits, such as flavour. However, research on IR's application in complex matrices is limited. Therefore, our preliminary study aimed to evaluate its effectiveness in sanitizing bovine raw milk. We assessed the bacterial count before and after IR treatment by comparing volatile organic compound profiles via headspace extraction and GC/MS analysis. Our findings showed that higher energy levels led to a greater bacterial reduction. IR85 was the most effective in reducing Coliforms and Enterobacteriaceae in non-homogenised samples, with a reduction ranging from -1.01 to >-2.99 and from -1.66 to -3.09 Log CFU/mL, respectively. IR60 and 70 showed no efficacy, while IR80 had intermediate but still satisfactory effect. IR85 notably affected volatile compounds, particularly increasing hexanal (from 0.08 to 4.21 ng g-1) and dimethyl sulphone (from 10.76 to 26.40 ng g-1), while IR80 better preserved the aroma profile. As a result, only IR80 was tested with homogenised raw milk, demonstrating significant bacterial reduction (from >2.39 to 3.06 Log CFU/mL for Coliforms and from 1.90 to >2.45 Log CFU/mL for Enterobacteriaceae) and maintaining the aroma profile quality.
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In a balanced diet, regular fish consumption provides positive outcomes for human health. On the other hand, the seafood supply chain faces a significant food safety risk due to the presence of potentially toxic elements (PTEs). In the present study, to assess the risk for Italian consumers, the concentrations of five PTEs, namely lead, chromium, cadmium, mercury, nickel, and aluminum, were determined in the three most consumed preserved fish in Italy: tuna (Thunnus albacares, Katsuwonus pelamis), mackerel (Scomber scombrus) and anchovy (Engraulis encrasicolus). Samples were collected from the national market, and the instrumental analysis was performed by inductively coupled plasma mass spectrometry (ICP-MS). The analyzed PTEs were found in all the species that were investigated. However, after considering the target hazard quotient (THQ) and the hazard index (HI), it was observed that the three fish preserves did not pose any risk of chronic toxicity for the average consumer, even at the highest concentrations detected. However, for significant consumers, mercury detected in tuna samples represented almost 90% of the tolerable weekly intake (TWI) reported by the European Food Safety Authority (EFSA), representing a matter of concern for consumers, particularly regarding developmental neurotoxicity, whose HI exceeded 111%. The acute toxicity of nickel was also considered for significant consumers at the highest concentration detected, and the margin of exposure (MOE) calculated was above 7000, much higher than the value of 30 indicated by EFSA. Due to the lack of data on non-professional carcinogenicity or human intake through foods with low cancer risk, this toxicity was not considered in the analysis of PTEs.
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BACKGROUND: The phenolic content, volatile compound fingerprint and antioxidant capacity of Waldheimia glabra (Decne.) Regel (Asteraceae), a wild plant from the Himalayan mountains used in Sherpa religious rituals and in traditional medicine, were determined for the first time to investigate its suitability as a source of natural antioxidants. RESULTS: The total phenolic content and antioxidant capacity of dried and powdered plants from two different altitudes (5000 and 5200 m above sea level) in Khumbu Valley, Nepal were estimated by the Folin-Ciocalteu and 2,2-diphenyl-1-picrylhydrazyl radical methods respectively. The volatile organic compound (VOC) fingerprints from leafing plants (in vivo and in situ VOC sampling) and from dried plant samples were evaluated by headspace solid phase microextraction. The results indicated that W. glabra was a good source of phenolics with antioxidant properties. A correlation between total phenolic content and antioxidant capacity was found (r² = 0.9111). Seychellene, a volatile compound that characterises patchouli oil, was detected for the first time in a species of the Asteraceae family. CONCLUSION: Waldheimia glabra was proved to be a good source of antioxidants, partially supporting its traditional uses. Further studies aimed at deepening knowledge on W. glabra bioactivity and its possible uses will be of interest, considering the lack of proper scientific awareness regarding these aspects.
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Antioxidantes/análise , Asteraceae/química , Fenóis/análise , Componentes Aéreos da Planta/química , Compostos Orgânicos Voláteis/análise , Altitude , Antioxidantes/química , Antioxidantes/metabolismo , Asteraceae/crescimento & desenvolvimento , Asteraceae/metabolismo , Monoterpenos Bicíclicos , Monoterpenos Cicloexânicos , Cicloexenos/análise , Cicloexenos/química , Cicloexenos/metabolismo , Etnofarmacologia , Cromatografia Gasosa-Espectrometria de Massas , Monoterpenos/análise , Monoterpenos/química , Monoterpenos/metabolismo , Nepal , Óleos Voláteis/análise , Óleos Voláteis/química , Óleos Voláteis/metabolismo , Fenóis/química , Fenóis/metabolismo , Componentes Aéreos da Planta/crescimento & desenvolvimento , Componentes Aéreos da Planta/metabolismo , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Microextração em Fase Sólida , Terpenos/análise , Terpenos/química , Terpenos/metabolismo , Compostos Orgânicos Voláteis/química , Compostos Orgânicos Voláteis/metabolismoRESUMO
Agonists of the ß2 adrenergic receptor (ADRB2) are an important class of medications used for the treatment of respiratory diseases. They can be classified as short acting (SABA) or long acting (LABA), with each class playing a different role in patient management. In this work we explored both ligand-based and structure-based high-throughput approaches to classify ß2-agonists based on their duration of action. A completely in-silico prediction pipeline using an AlphaFold generated structure was used for structure-based modelling. Our analysis identified the ligands' 3D structure and lipophilicity as the most relevant features for the prediction of the duration of action. Interaction-based methods were also able to select ligands with the desired duration of action, incorporating the bias directly in the structure-based drug discovery pipeline without the need for further processing.
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Agonistas de Receptores Adrenérgicos beta 2 , Humanos , Ligantes , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Agonistas de Receptores Adrenérgicos beta 2/uso terapêuticoRESUMO
The fragment approach has emerged as a method of choice for drug design, as it allows difficult therapeutic targets to be addressed. Success lies in the choice of the screened chemical library and the biophysical screening method, and also in the quality of the selected fragment and structural information used to develop a drug-like ligand. It has recently been proposed that promiscuous compounds, i.e., those that bind to several proteins, present an advantage for the fragment approach because they are likely to give frequent hits in screening. In this study, we searched the Protein Data Bank for fragments with multiple binding modes and targeting different sites. We identified 203 fragments represented by 90 scaffolds, some of which are not or hardly present in commercial fragment libraries. By contrast to other available fragment libraries, the studied set is enriched in fragments with a marked three-dimensional character (download at 10.5281/zenodo.7554649).
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Fish plays a key role in a healthy and balanced Italian diet, but it is also subject to the bioaccumulation of different contaminants depending on the geographical or anthropogenic context from which it is derived. In recent years, the European Food Safety Authority (EFSA) has been focusing its attention on consumer toxicological risk, considering emerging contaminants such as perfluoroalkyl substances (PFASs) and potentially toxic elements (PTEs). Regarding fish, anchovies are among the five small pelagic main commercial species in the European Union and the top five fresh species consumed by households in Italy. Considering the lack of data on PFASs and PTEs in this species, our aim was to investigate the mentioned contaminants in salted and canned anchovies collected over 10 months from different fishing areas, even those far apart, to verify possible variations in bioaccumulation and to consider the risk for the consumer. According to our results, the assessed risk was very reassuring also for large consumers. The only concern, related to Ni acute toxicity, also dependent on the different consumers' sensitivity, was related to only one sample.
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Eggs play an important role in a balanced diet; however, the European Food Safety Authority (EFSA) recognizes eggs as a major source of poly and per-fluoroalkyl substances (PFASs). In this study, the presence of PFASs was analysed in eggs produced by hens from Northern Italian regions, a PFASs-contaminated area. Sixty-five samples were analysed by high-performance liquid chromatography coupled with high-resolution mass spectrometry. The greatest presence of PFASs was found in eggs from Veneto and Emilia Romagna, and the most detected PFASs were perfluorobutanoic acid (PFBA) and perfluorooctanesulfonic acid (PFOS) (mean concentrations 0.30 ± 0.15 and 0.05 ± 0.00 ng g-1). Considering the most recent updates for the sum of the main four PFASs, the highest concentration found in the analysed samples was 0.05 ng g-1, well below the maximum limit set by the European Union. The PFAS intake evaluation confirmed that egg consumption does not represent a risk for Italian consumers.
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Bee honey has different volatile organic compound profiles that depend on the botanical origin and the state of conservation and which are mainly responsible for its specific aroma. During honey storage, the profile of these molecules and other indicators, such as 5-hydroxymethylfurfural and the diastatic index, can change depending on temperature and time. This study analyzed the variations that these parameters in acacia honey stored at three different temperatures for a total period of 550 days, using gas chromatography coupled with mass spectrometry and an electronic nose equipped with 10 different sensors. The results confirm that the composition of acacia honey varies over time due to both the reduction in the concentration of volatile molecules (e.g., formic acid, a natural acaricide) and the increase in compounds resulting from heat-dependent degradations (e.g., 5-hydroxymethylfurfural). This study supports the usefulness of the electronic nose for the early detection of aromatic alterations in honey subjected to high-temperature storage.
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Gastrointestinal nematodes (GINs) is a limiting health factor for dairy goats, and the integration of the diet with fodder containing condensed tannins (CT) is becoming increasingly important to control GINs. To preliminary evaluate their potential role as part of GIN control in goat breeding, the in vitro anthelmintic efficacy of the CTs of Silvafeed BYPRO (SBP), Silvafeed Q powder (SQ), and sainfoin hay (SH) was evaluated, and the untargeted metabolomics profiling of the selected formulations was performed. CTs were extracted in water and in ethanol, their concentration was determined, and their chemical characterization was conducted using High-Performance Liquid Chromatography (HPLC) coupled to High-Resolution Mass Spectrometry (HRMS) platform. The in vitro anthelmintic activity of the extracts was evaluated using the Eggs Hatch Test (EHT) and the Larval Migration Inhibition Test (LMIT) using different extract concentrations (150, 300, 600, and 1200 µg/mL). The metabolomic profile of the ethanol extract showed a high number of flavonoids, while the water extract showed higher levels of hydrolysable tannins. The ethanol extracts were effective on both eggs hatching and larvae migration at low concentrations (150 µg/mL) for the three analyzed samples, while the water extracts showed more varied results: SH showed the greatest ovicidal efficacy (concentration 150 µg/mL, %IH = 40.9), while SBP and SQ were more effective against the larvae migration (concentration 600 µg/mL, %LMI = 69.7% and 88%), respectively. The integration of CT-rich fodder into the diet may be considered for the control of GIN infection in goats.
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We characterized the presence of plastics in different organs of the gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) from some off-shore aquaculture facilities of the Mediterranean Sea. Plastics were detected in 38% of analyzed fish. Higher contamination was observed in fish from Turkey and Greece with respect to Italy, without significant differences between the geographical areas. Plastics accumulated mostly in the gastrointestinal tract and, to a lower extent, in the muscle, which represents the edible part of fish. Based on the particle detected, a maximum amount of 0.01 plastic/g wet weight (w.w.) can occur in muscles, suggesting a low input for humans through consumption. A large portion of the particles identified was represented by man-made cellulose-based fibers. The characterization of the polymeric composition suggests that plastics taken up by fish can have land-based and pelagic origins, but plastics can be introduced also from different aquaculture practices.
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G protein-coupled receptors are involved in many biological processes, relaying the extracellular signal inside the cell. Signaling is regulated by the interactions between receptors and their ligands, it can be stimulated by agonists, or inhibited by antagonists or inverse agonists. The development of a new drug targeting a member of this family requires to take into account the pharmacological profile of the designed ligands in order to elicit the desired response. The structure-based virtual screening of chemical libraries may prioritize a specific class of ligands by combining docking results and ligand binding information provided by crystallographic structures. The performance of the method depends on the relevance of the structural data, in particular the conformation of the targeted site, the binding mode of the reference ligand, and the approach used to compare the interactions formed by the docked ligand with those formed by the reference ligand in the crystallographic structure. Here, we propose a new method based on the conformational dynamics of a single protein-ligand reference complex to improve the biased selection of ligands with specific pharmacological properties in a structure-based virtual screening exercise. Interactions patterns between a reference agonist and the receptor, here exemplified on the ß2 adrenergic receptor, were extracted from molecular dynamics simulations of the agonist/receptor complex and encoded in graphs used to train a one-class machine learning classifier. Different conditions were tested: low to high affinity agonists, varying simulation duration, considering or ignoring hydrophobic contacts, and tuning of the classifier parametrization. The best models applied to post-process raw data from retrospective virtual screening obtained by docking of test libraries effectively filtered out irrelevant poses, discarding inactive and non-agonist ligands while identifying agonists. Taken together, our results suggest that consistency of the binding mode during the simulation is a key to the success of the method.
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Mastitis is one of the major diseases of dairy cows that affects milk quality and quantity and increases the potential risk for the presence of antimicrobial residues (AR) in milk, which could lead to the development of antimicrobial resistance (AMR) among human pathogens. Even if the presence of AR in milk and milk products is low in many countries, the threat is not negligible and cannot be ignored. These problems may be investigated by applying a One Health approach, and this prevalence study aimed to estimate the risks for human health related to milk production applied to dairy herds in Lombardy. Three hundred thirty-one bulk tank milk samples were randomly collected and analyzed by CombiFoss 7 and MilkoScan 7 (milk quality, bacteria, and somatic cell count), an HPLC system coupled to a Q-Exactive Orbitrap (AR), and qPCR (contagious pathogens). The data were analyzed by a generalized linear model. The results showed a relatively high prevalence of contagious pathogens (S. aureus 28.1%; Str. agalactiae 7.3%; M. bovis 3%), which primarily affect milk nutritional components decreasing mainly milk fat content (range 1%-2.5%), but did not show them to be associated to an increase of the risk of antimicrobial residues. These latter ones were recovered only in 7/331 samples at concentrations far below official MLRs. The results support currently active surveillance programs' efficacy in reducing AR risks, which may be further improved by prioritizing them based on geographical area characteristics.