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Propofol is a widely used anesthetic. Although considered safe, propofol-related deaths occur, as it is sometimes abused recreationally or used to commit suicide. A simple, rapid, and reliable method for its analysis in various biological samples is needed. Sample clean-up is a critical step in the analysis, both in terms of time and cost, indeed. Dispersive liquid-liquid microextraction (DLLME) is a simple and fast extraction based on ternary solvent mixtures that uses small volumes of solvent and sample. A DLLME extraction followed by gas chromatography-mass spectrometry (GC-MS) analysis was developed and validated for the analysis of propofol in blood, urine, and hair. The same extraction mixture of 2.5:1 methanol/chloroform was used for the different biological samples. Validation for linearity, LOD, LOQ, precision, accuracy, and recovery gave satisfactory results for the three types of biological samples included in the study, with limits of quantification of 1 μg/mL for urine, 0.2 μg/mL for blood, and 0.1 ng/mg for hair. The DLLME procedure for purification involves a small amount of solvent, thus reducing the cost and the environmental impact. In addition, a high enrichment factor is obtained, and the time for analysis is short. The method was applied to authentic post-mortem samples for the determination of propofol in blood, urine, and hair. Also, segmental hair analysis was performed to assess chronic propofol abuse. The developed method proved to be rapid, simple, and cost-effective for blood, urine, and hair extract clean-up for the determination of propofol by GC/MS., (© 2025 The Author(s). Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Ethyl glucuronide (EtG) in hair is a reliable biomarker of alcohol consumption habits. Due to its small concentration incorporated into hair, analytical methods sensitive enough to reliably quantify EtG in this matrix are required. Sample preparation is critical in hair analysis, especially for EtG, for which extraction efficiency and matrix effect can strongly influence the results; furthermore, miniaturized methods are sought, to reduce solvent use and times of sample preparation. A micro extraction by packed sorbent (MEPS) procedure coupled to a high-performance liquid chromatography-tandem mass spectrometry method was developed and validated for quantitation of EtG in human hair samples. Fifty milligrams of hair samples were cut into snippets and extracted in water. The cleanup of the extract was carried out by using a MEPS syringe packed with anion exchange sorbent (SAX); all parameters for conditioning, washing, loading and eluting steps were optimized and the eluted aqueous volume was directly injected in the LC-MS/MS system operating in the negative ionization mode. The method was fully validated assessing LOD, LOQ, calibration curve, repeatability, accuracy, matrix effect and carryover. The method was subsequently applied to QCs and authentic hair samples. The developed MEPS method is quick and effective, with low solvent purchase and discard costs, allowing the differentiation between social drinkers and chronic excessive alcohol consumers, according to the cut-offs established by the Society of Hair Testing (SoHT)., (© 2024 The Author(s). Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Hair is the matrix of choice for investigating a subject's drug history over time, usually with specific forensic applications (license renewal, workplace drug testing, toxicological evaluation), and it is generally considered difficult to be tampered with. Nevertheless, some treatments promising to lower drug concentrations in hair are described online as how to "pass" a drug test. We selected three of these practices, claiming to be effective in decreasing drug concentrations-Treatment 1: (A) baking soda, (B) salicylic acid, (C) bleach; Treatment 2: (A) bleaching and (B) dyeing; Treatment 3: (A) white vinegar, (B) salicylic acid moisturizer, (C) liquid cleanser, and (D) dyeing. Quantitative results were compared with those of untreated hair strands, used as reference. We evaluated the efficacy of the treatment on drugs of abuse and benzodiazepines. Treatment 1 proved to be the most effective, since drug concentrations in treated hair were significantly lower than in untreated ones, although methadone and tetrahydrocannabinol (THC) seemed to be less affected than cocaine and 6-monoacetylmorphine (MAM). The mean percentage values of treatment-induced decrease were up to 90% for cocaine, 81% for benzoylecgonine, 77% for morphine, 89% for MAM, 37% for methadone, 67% for ketamine, 80% for MDMA, 76% for methamphetamine, and 60% for THC, compared with the reference samples. There was no noticeable damage or discoloration of the keratin matrix, making it difficult for the technicians to determine if there was a treatment. This could be an issue for the application of cutoffs or when low concentrations of drugs are incorporated into the keratinic matrix., (© 2023 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Green chemistry is a useful tool for producing valuable chemicals from biomass. However, extracted compounds need to be tested for safety and efficacy before their use in humans. Here we investigate the chemical composition and biological effects of a leaves Pistacia lentiscus L . supercritical carbon dioxide (SCCO 2 ) extract. Terpenes represented the main extract fraction, with Germacrene D (11.18%), delta-cadinene (10.54%), and alpha-pinene (8.7%) the most abundant molecules. Challenged with endothelial cells (ECs), increasing extract concentrations failed to affect cell proliferation or promote cell toxicity. ROS assessment in unstressed and H 2 O 2 -treated ECs revealed an extract dose-dependent antioxidant activity. Exposition of H 2 O 2 -treated ECs to increasing extract concentrations dose-dependently counteracted H 2 O 2 -induced cell impairments. The extract significantly counteracted fetal calf serum-induced ECs migration. For the first time, we report that a SCCO 2 extract obtained from PL leaves is safe on ECs and may be a useful source of valuable compounds with vasculoprotective properties.

The present study focuses on the association between road accidents and the presence of drugs of abuse markers in the biological fluids of the drivers. Biological fluids collected from 1236 drivers involved in road accidents (54 fatal and 1182 non-fatal crashes) in the Rome area were analyzed for alcohol and psychotropic drugs, as required by judicial authorities. The substance most frequently detected was alcohol (in 19% of non-fatal and 32% of fatal crashes), followed by cannabinoids (12% of non-fatal crashes) and cocaine (9% of non-fatal and 20% of fatal crashes). The results obtained for cocaine and cannabinoids in blood and urine were compared. We observed the absence or low concentrations of the active drug in blood (cocaine was often below 5 ng/ml and THC below 1 ng/ml), whereas urinary concentrations of metabolites were generally high (benzoylecgonine 250-above 5000 ng/ml, THCCOOH 15-270 ng/ml). The risk of being involved in a road accident if cocaine or cannabis markers were present in the urine specimens was evaluated compared to a control population. The odds ratios calculated, being 8.13 for cannabis and 5.32 for cocaine, suggest a strong association between the presence of these drugs in the urine of drivers and traffic accidents, regardless of their presence in blood samples. The present data suggest that the chance of being involved in a road accident is higher than in the control population even if the subject is no longer "under the influence" of cannabis or cocaine at the time of the accident., (© 2022 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

The sustainable development of modern aquaculture must rely on a significant reduction of the fish meal (FM) used in aquafeed formulations. However, FM substitution with alternative ingredients in diets for carnivorous fish species often showed reduced nutrient absorption, significantly perturbed metabolisms, and histological changes at both hepatic and intestinal levels. In the present study, rainbow trout ( Oncorhynchus mykiss ) were fed three different experimental aquafeeds. A control diet with higher FM content (27.3%) than two test formulations in which FM was substituted with two more sustainable and promising alternatives: insect meal ( Hermetia illucens larvae = 10.1%, FM = 11.6%) and poultry by-products meal (PBM = 14.8%; FM = 11.7%). Combined metabolomics and proteomics analyses of fish liver, together with histological examination of liver and intestine demonstrated that a well-balanced formulation of nutrients in the three diets allowed high metabolic compatibility of either substitution, paving the way for a deeper understanding of the impact of novel raw materials for the fish feed industry. Results show that the main metabolic pathways of nutrient absorption and catabolism were essentially unaltered by alternative feed ingredients, and also histological alterations were negligible. It is demonstrated that the substitution of FM with sustainable alternatives does not have a negative impact on fish metabolism, as long as the nutritional requirements of rainbow trout are fulfilled., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Palomba, Melis, Biosa, Braca, Pisanu, Ghisaura, Caimi, Biasato, Oddon, Gasco, Terova, Moroni, Antonini, Pagnozzi and Anedda.)

Cystic echinococcosis (CE) diagnosis by means of serological assays is hampered by the presence of parasites closely related to Echinococcus granulosus sensu lato (s.l.), responsible of the zoonotic disease and with which share cross-reacting antigens. Thus, improvements on the characterization of Echinococcus specific antigens expressed in the larval stage are required, in order to provide useful information for the development of immunological assays for the serodiagnosis of CE in sheep. Here, the proteome of the hydatid cyst fluids (HFs) of Echinococcus granulosus (hydatid fluid, EgHF) and other ovine parasites cyst fluids (CFs), Taenia hydatigena (ThCF) and Taenia multiceps (TmCF) were analyzed by a shotgun proteomic approach. Parasite and host protein profiles in the three types of cyst fluids were characterized and compared. Among the identified proteins, differential parasitic markers with serodiagnostic potential, due to their well-known immunoreactivity in human, included Ag5, AgB proteins, 8-kDa glycoproteins, hydatid disease diagnostic antigen P29 and major egg antigen P40. In particular, seven proteoforms of AgB and 8-kDa glycoprotein resulted to be the most promising diagnostic biomarkers, as they might predict CE in ovine and discriminate between different types of parasites., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The emerging market of new psychoactive substances (NPSs) is a global-scale phenomenon, and their identification in biological samples is challenging because of the lack of information about their metabolism and pharmacokinetic. In this study, we performed in silico metabolic pathway prediction and in vivo metabolism experiments, in order to identify the main metabolites of mephtetramine (MTTA), an NPS found in seizures since 2013. MetaSite™ software was used for in silico metabolism predictions and subsequently the presence of metabolites in the blood, urine, and hair of mice after MTTA administration was verified. The biological samples were analyzed by liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) using a benchtop Orbitrap instrument. This confirmed the concordance between software prediction and experimental results in biological samples. The metabolites were identified by their accurate masses and fragmentation patterns. LC-HRMS analysis identified the dehydrogenated and demethylated-dehydrogenated metabolites, together with unmodified MTTA in the blood samples. Besides unmodified MTTA, 10 main metabolites were detected in urine. In hair samples, only demethyl MTTA was detected along with MTTA. The combination of Metasite™ prediction and in vivo experiment was a powerful tool for studying MTTA metabolism. This approach enabled the development of the analytical method for the detection of MTTA and its main metabolites in biological samples. The development of analytical methods for the identification of new drugs and their main metabolites is extremely useful for the detection of NPS in biological specimens. Indeed, high throughput methods are precious to uncover the actual extent of use of NPS and their toxicity., (© 2021 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

The constant increase of new psychoactive substances, often available on the illicit drug market as 'research chemicals', poses a concern for public health and a significant analytical and legislative challenge. Β-keto-arylcyclohexamines represent a class of dissociative anesthetics recently introduced on the market of New Psychoactive Substances (NPS). There is still a lack of information about the pharmacological activity of many of such substances, usually depending on the potential chemical modifications introduced to circumvent the law. Furthermore, their intake may not be fully intentional, since consumers do not always have knowledge of the content of online purchases. The present study describes the characterization by liquid chromatography-high resolution mass spectrometry (LC-HRMS), using a benchtop Orbitrap instrument, of the novel ketamine analogues methoxpropamine, 2-fluoro-deschloroketamine and deschloroketamine, found in the post-mortem blood and hair samples from a forensic case of suicide by fall from height, and of some of their metabolites. This allowed the development of analytical methods for the determination of both the β-keto-arylcyclohexamines and the metabolites in LC-HRMS and in LC-MS/MS, providing a starting point for studying their toxicokinetics., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Context: The illegal market of counterfeit and falsified medicines and supplements containing unlabeled pharmaceuticals is expanding worldwide. They are usually referred to by the term "performance and image enhancing drugs" (PIEDs) and are mainly steroids, stimulants, hormones, and drugs for erectile dysfunction. PIEDs are easily accessible through the online or black markets. We analyzed over 400 such medicines confiscated in Italy in the period 2017-2019, to determine their composition., Methods: Confiscated products were analyzed by gas chromatography/mass spectrometry and liquid chromatography/high-resolution mass spectrometry, in order to ascertain their composition and to evaluate the correspondence between what was declared on the label and the actual content, or to identify unknown products., Results: The most commonly found substance was anabolic steroids, found in 64% of products, with 11% containing hormone modulators, 6% stimulants, 6% sexual enhancers (mainly sildenafil) and other drugs, including thyroid hormones, melanin stimulators, and vitamins. These substances were often in mixtures. The products were often mislabeled, containing contaminants in addition to the drug declared, or consisted of a drug completely different from the one reported on the label. Fifteen percent of products had a qualitative composition completely different from that declared, while 10% of products showed cross-contamination with other drugs, mainly testosterone esters, probably due to the presence of residues of other drugs in the production line. In addition, 11% of products were not labeled, so their purported composition was unknown., Discussion: PIEDs pose a threat to public health. The main risks are related to the intrinsic toxicity of the substances found, especially when taken without a therapeutic indication. Another issue is related to the mislabeling of the fake medicines, and the poor-quality standard of counterfeit product preparation, with additional risks of the presence of other toxic ingredients or microbial contamination., Conclusions: The use of counterfeit products is a public health concern, as it constitutes a high risk for consumer health. It is mainly caused by the uncontrolled use of steroids, stimulants, sexual enhancers, and other medicaments, without medical indication or supervision, with variable and unknown compositions and doses, as well as other contaminants as a result of the absence of good manufacturing practices.

We demonstrate the use of classification methods that are well-suited for forensic toxicology applications. The methods are based on penalized logistic regression, can be employed when separation occurs in a two-class classification setting, and allow for the calculation of likelihood ratios. A case study of this framework is demonstrated on alcohol biomarker data for classifying chronic alcohol drinkers. The approach can be extended to applications in the fields of analytical and forensic chemistry, where it is a common feature to have a large number of biomarkers, and allows for flexibility in model assumptions such as multivariate normality. While some penalized regression methods have been introduced previously in forensic applications, our study is meant to encourage practitioners to use these powerful methods more widely. As such, based upon our proof-of-concept studies, we also introduce an R Shiny online tool with an intuitive interface able to perform several classification methods. We anticipate that this open-source and free-of-charge application will provide a powerful and dynamic tool to infer the LR value in case of classification tasks., (Copyright © 2020 Biosa, Giurghita, Alladio, Vincenti and Neocleous.)

The quantitative determination of ethyl glucuronide (EtG) in hair samples is consistently used throughout the world to assess chronic excessive alcohol consumption. For administrative and legal purposes, the analytical results are compared with cut-off values recognised by regulatory authorities and scientific societies. However, it has been recently recognised that the analytical results depend on the hair sample pretreatment procedures, including the crumbling and extraction conditions. A systematic evaluation of the EtG extraction conditions from pulverised scalp hair was conducted by Design of Experiments (DoE) considering the extraction time, temperature, pH, and solvent composition as potential influencing factors. It was concluded that an overnight extraction at 60°C with pure water at neutral pH represents the most effective conditions to achieve high extraction yields. The absence of differential degradation of the internal standard (isotopically-labelled EtG) under such conditions was confirmed and the overall analytical method was validated according to SGWTOX and ISO17025 criteria. Twenty real hair samples with different EtG content were analysed with 3 commonly accepted procedures: (a) hair manually cut in snippets and extracted at room temperature; (b) pulverised hair extracted at room temperature; (c) hair treated with the optimised method. Average increments of EtG concentration around 69% (from a to c) and 29% (from b to c) were recorded. In light of these results, the authors urge the scientific community to undertake an inter-laboratory study with the aim of defining more in detail the optimal hair EtG detection method and verifying the corresponding cut-off level for legal enforcements., (Copyright © 2018 John Wiley & Sons, Ltd.)

The aim of this work is the evaluation of a green extraction technology to exploit winery waste byproducts. Specifically, a solid⁻liquid extraction technology (Naviglio Extractor ® ) was used to obtain polyphenolic antioxidants from the Cagnulari grape marc. The extract was then chemically characterized by spectrophotometric analysis, high-performance liquid chromatography, and mass spectrometry, revealing a total polyphenol content of 4.00 g/L ± 0.05, and the presence of anthocyanins, one of the most representative groups among the total polyphenols in grapes. To investigate potential biological activities of the extract, its ability to counteract hydrogen peroxide-induced oxidative stress and cell death was assessed in primary human endothelial cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test, used to assess potential extract cytotoxicity, failed to show any deleterious effect on cultured cells. Fluorescence measurements, attained with the intracellular reactive oxygen species (ROS) probe 2',7'-dichlorodihydrofluorescein diacetate (H₂DCF-DA), revealed a strong antioxidant potential of the marc extract on the used cells, as indicated by the inhibition of the hydrogen peroxide-induced ROS generation and the counteraction of the oxidative-induced cell death. Our results indicate the Naviglio extraction, as a green technology process, can be used to exploit wine waste to obtain antioxidants which can be used to produce enriched foods and nutraceuticals high in antioxidants.

Seven commercial products for human serum depletion/enrichment were tested and compared by shotgun proteomics. Methods were based on four different capturing agents: antibodies (Qproteome Albumin/IgG Depletion kit, ProteoPrep Immunoaffinity Albumin and IgG Depletion Kit, Top 2 Abundant Protein Depletion Spin Columns, and Top 12 Abundant Protein Depletion Spin Columns), specific ligands (Albumin/IgG Removal), mixture of antibodies and ligands (Albumin and IgG Depletion SpinTrap), and combinatorial peptide ligand libraries (ProteoMiner beads), respectively. All procedures, to a greater or lesser extent, allowed an increase of identified proteins. ProteoMiner beads provided the highest number of proteins; Albumin and IgG Depletion SpinTrap and ProteoPrep Immunoaffinity Albumin and IgG Depletion Kit resulted the most efficient in albumin removal; Top 2 and Top 12 Abundant Protein Depletion Spin Columns decreased the overall immunoglobulin levels more than other procedures, whereas specifically gamma immunoglobulins were mostly removed by Albumin and IgG Depletion SpinTrap, ProteoPrep Immunoaffinity Albumin and IgG Depletion Kit, and Top 2 Abundant Protein Depletion Spin Columns. Albumin/IgG Removal, a resin bound to a mixture of protein A and Cibacron Blue, behaved less efficiently than the other products., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We present a proteomic dataset generated from a human serum sample and the enriched/depleted fractions obtained by seven commercial products. This report is related to the research article entitled "Comparative evaluation of seven commercial products for human serum enrichment/depletion by shotgun proteomics" [1]. All samples were analyzed by LC-MS/MS, label free quantitation using the spectral counting approach, and Gene Ontology (GO) annotation. Protein relative abundances and functions were reported.

Red yeasts ascribed to the species Rhodotorula mucilaginosa are gaining increasing attention, due to their numerous biotechnological applications, spanning carotenoid production, liquid bioremediation, heavy metal biotransformation and antifungal and plant growth-promoting actions, but also for their role as opportunistic pathogens. Nevertheless, their characterization at the 'omic' level is still scarce. Here, we applied different proteomic workflows to R. mucilaginosa with the aim of assessing their potential in generating information on proteins and functions of biotechnological interest, with a particular focus on the carotenogenic pathway. After optimization of protein extraction, we tested several gel-based (including 2D-DIGE) and gel-free sample preparation techniques, followed by tandem mass spectrometry analysis. Contextually, we evaluated different bioinformatic strategies for protein identification and interpretation of the biological significance of the dataset. When 2D-DIGE analysis was applied, not all spots returned a unambiguous identification and no carotenogenic enzymes were identified, even upon the application of different database search strategies. Then, the application of shotgun proteomic workflows with varying levels of sensitivity provided a picture of the information depth that can be reached with different analytical resources, and resulted in a plethora of information on R. mucilaginosa metabolism. However, also in these cases no proteins related to the carotenogenic pathway were identified, thus indicating that further improvements in sequence databases and functional annotations are strictly needed for increasing the outcome of proteomic analysis of this and other non-conventional yeasts. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Unlabelled: The reproductive stage of Paracentrotus lividus strongly influences product quality that, in turn, impacts significantly on the market price. Large, compact and sweet gonads are preferred, and sensory attributes are positively related to the ratio of nutritive phagocytes to gametes. Gonads at advanced maturation stages, although larger, have less desirable attributes, being more watery and bitter especially in females. Therefore, the best compromise among size, texture, and taste needs to be reached. In this study, wild P. lividus were collected along coastal Sardinia, and gonads in the recovery, pre-mature, mature, and spent stages were analyzed by gel-based and by shotgun proteomics. A detailed characterization of the proteome changes occurring in gonads of both sexes along maturation was achieved, and significant modifications were seen in numerous proteins involved in nutrient accumulation in nutritive phagocytes, as well as in gamete biology and maturation. Adding to an improved understanding of the P. lividus reproductive cycle in its natural environment, the results described in this work may form the basis for defining novel protein markers and procedures for an easier sexing and staging, and for monitoring sea urchin gonad maturation cycles in aquaculture plants. Mass spectrometry data are deposited in ProteomeXchange (PXD004200)., Significance: The sensory quality of P. lividus gonads is strongly influenced by the reproductive cycle, with significant changes in flavor, texture, and size. A better knowledge of the protein profiles, patterns, and markers associated with gonad sex and maturation stage can have useful implications for understanding and monitoring these changes. One of these is the ability to identify protein profiles specifically associated with a given stage and, in perspective, to identify maturation and sex markers. The comprehensive proteomic evaluation achieved in this work was made possible by the application of combined gel-based and shotgun approaches. As a result, this study generated the largest proteomic dataset available in the literature for P. lividus, as well as a general picture of protein abundance changes occurring along maturation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We report the proteomic dataset of gonads from wild Paracentrotus lividus related to the research article entitled "Proteomic changes occurring along gonad maturation in the edible sea urchin Paracentrotus lividus" [1]. Gonads of three individuals per sex in the recovery, pre-mature, mature, and spent stages were analyzed using a shotgun proteomics approach based on filter-aided sample preparation followed by tandem mass spectrometry, protein identification carried out using Sequest-HT as the search engine within the Proteome Discoverer informatics platform, and label-free differential analysis. The dataset has been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD004200.

Background: Clinical diagnosis and follow up of cystic echinococcosis (CE) are based on imaging complemented by serology. Several immunodiagnostic tests are commercially available, but the development of new tools is still needed to overcome the lack of standardization of the target antigen, generally consisting of a crude extract of Echinococcus granulosus hydatid cyst fluid. In a previous work, we described a chromatographic method for the preparation of a highly enriched Antigen 5 fraction from hydatid cyst fluid. The high reactivity of patient sera against this preparation prompted us to evaluate further this antigen for the serodiagnosis of CE on a larger cohort of samples., Methodology/principal Findings: A total of 327 sera from CE patients with heterogeneous conditions for cyst stage, cyst number, organ localization, drug therapy, and surgical intervention, together with 253 sera from healthy controls, were first analyzed by an ELISA based on the Ag5 preparation in two different experimental setups and, in parallel, by a commercial ELISA routinely used in clinical laboratories for CE serodiagnosis. The Ag5 ELISAs revealed different sensitivity (88.3% vs 95.3%) without significant differences in specificity (94.1% vs 92.5%), for the two setups, respectively. Moreover, possible relationships between the Ag5 ELISA absorbance results and clinical variables were investigated. Chi squared test, bivariate logistic regression and multiple regression analyses highlighted differences in the serology reactivity according to pharmacological treatment, cyst activity, and cyst number., Conclusions/significance: The two Ag5 ELISAs revealed different performances depending on the setup. The good diagnostic sensitivity and the high reliability of the Ag5 preparation method make this antigen a promising candidate for the serodiagnosis of CE. Further studies will be needed to evaluate the ability of our test to provide useful information on specific CE clinical traits.

Background: The zootechnical performance of three different commercial feeds and their impact on liver and serum proteins of gilthead sea bream (Sparus aurata, L.) were assessed in a 12 week feeding trial. The three feeds, named A, B, and C, were subjected to lipid and protein characterization by gas chromatography (GC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively., Results: Feed B was higher in fish-derived lipids and proteins, while feeds C and A were higher in vegetable components, although the largest proportion of feed C proteins was represented by pig hemoglobin. According to biometric measurements, the feeds had significantly different impacts on fish growth, producing a higher average weight gain and a lower liver somatic index in feed B over feeds A and C, respectively. 2D DIGE/MS analysis of liver tissue and Ingenuity pathways analysis (IPA) highlighted differential changes in proteins involved in key metabolic pathways of liver, spanning carbohydrate, lipid, protein, and oxidative metabolism. In addition, serum proteomics revealed interesting changes in apolipoproteins, transferrin, warm temperature acclimation-related 65 kDa protein (Wap65), fibrinogen, F-type lectin, and alpha-1-antitrypsin., Conclusions: This study highlights the contribution of proteomics for understanding and improving the metabolic compatibility of feeds for marine aquaculture, and opens new perspectives for its monitoring with serological tests.

Background: Currently, the serodiagnosis of cystic echinococcosis relies mostly on crude Echinococcus granulosus hydatid cyst fluid as the antigen. Consequently, available immunodiagnostic tests lack standardization of the target antigen and, in turn, this is reflected on poor sensitivity and specificity of the serological diagnosis., Methodology/principal Findings: Here, a chromatographic method enabling the generation of highly enriched Antigen 5 (Ag5) is described. The procedure is very easy, efficient and reproducible, since different hydatid cyst fluid (HCF) sources produced very similar chromatograms, notwithstanding the clearly evident and extreme heterogeneity of the starting material. In addition, the performance of the antigen preparation in immunological assays was preliminarily assessed by western immunoblotting and ELISA on a limited panel of cystic echinococcosis patients and healthy controls. Following western immunoblotting and ELISA experiments, a high reactivity of patient sera was seen, with unambiguous and highly specific results., Conclusions/significance: The methods and results reported open interesting perspectives for the development of sensitive diagnostic tools to enable the timely and unambiguous detection of cystic echinococcosis antibodies in patient sera.

Metaproteomics enables the investigation of the protein repertoire expressed by complex microbial communities. However, to unleash its full potential, refinements in bioinformatic approaches for data analysis are still needed. In this context, sequence databases selection represents a major challenge. This work assessed the impact of different databases in metaproteomic investigations by using a mock microbial mixture including nine diverse bacterial and eukaryotic species, which was subjected to shotgun metaproteomic analysis. Then, both the microbial mixture and the single microorganisms were subjected to next generation sequencing to obtain experimental metagenomic- and genomic-derived databases, which were used along with public databases (namely, NCBI, UniProtKB/SwissProt and UniProtKB/TrEMBL, parsed at different taxonomic levels) to analyze the metaproteomic dataset. First, a quantitative comparison in terms of number and overlap of peptide identifications was carried out among all databases. As a result, only 35% of peptides were common to all database classes; moreover, genus/species-specific databases provided up to 17% more identifications compared to databases with generic taxonomy, while the metagenomic database enabled a slight increment in respect to public databases. Then, database behavior in terms of false discovery rate and peptide degeneracy was critically evaluated. Public databases with generic taxonomy exhibited a markedly different trend compared to the counterparts. Finally, the reliability of taxonomic attribution according to the lowest common ancestor approach (using MEGAN and Unipept software) was assessed. The level of misassignments varied among the different databases, and specific thresholds based on the number of taxon-specific peptides were established to minimize false positives. This study confirms that database selection has a significant impact in metaproteomics, and provides critical indications for improving depth and reliability of metaproteomic results. Specifically, the use of iterative searches and of suitable filters for taxonomic assignments is proposed with the aim of increasing coverage and trustworthiness of metaproteomic data.

This work presents a comparative evaluation of several detergent-based sample preparation workflows for the MS-based analysis of bacterial proteomes, performed using the model organism Escherichia coli. Initially, RapiGest- and SDS-based buffers were compared for their protein extraction efficiency and quality of the MS data generated. As a result, SDS performed best in terms of total protein yields and overall number of MS identifications, mainly due to a higher efficiency in extracting high molecular weight (MW) and membrane proteins, while RapiGest led to an enrichment in periplasmic and fimbrial proteins. Then, SDS extracts underwent five different MS sample preparation workflows, including: detergent removal by spin columns followed by in-solution digestion (SC), protein precipitation followed by in-solution digestion in ammonium bicarbonate or urea buffer, filter-aided sample preparation (FASP), and 1DE separation followed by in-gel digestion. On the whole, about 1000 proteins were identified upon LC-MS/MS analysis of all preparations (>1100 with the SC workflow), with FASP producing more identified peptides and a higher mean sequence coverage. Each protocol exhibited specific behaviors in terms of MW, hydrophobicity, and subcellular localization distribution of the identified proteins; a comparative assessment of the different outputs is presented., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: In the recent past, the potential suitability of fixed samples to 2D-DIGE studies has been demonstrated on model tissues, but not on "real-world" archival tissues. Therefore, this study was aimed to assess the quality of the results delivered by 2D-DIGE on samples retrieved from hospital tissue repositories., Experimental Design: Diseased and normal tissue samples (namely, human gastric adenocarcinoma and normal gastric tissue, human lung neuroendocrine tumors, canine mammary tubulo-papillary carcinoma and normal mammary tissue, sheep liver with cloudy swelling degeneration and normal liver tissue) were retrieved from human and veterinary biorepositories and subjected to full-length protein extraction, cyanine labeling, 2D-DIGE separation, image analysis, MS analysis, and protein identification., Results: Archival samples could be successfully subjected to 2D-DIGE, providing maps of satisfactory resolution, although with varying pattern complexity (possibly influenced by preanalytical variables). Moreover, differentially expressed protein identities were consistent with the disease biology., Conclusions and Clinical Relevance: 2D-DIGE can support biomarker discovery and validation studies on large sample cohorts. In fact, although some information complexity is lost when compared to fresh-frozen tissues, their vast availability and the associated patient information can considerably boost studies suffering limited sample availability or involving long-distance exchange of samples., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Bouin's solution has been used for over a century as a common fixative in several pathology laboratories worldwide. Therefore, a considerable number of Bouin-fixed paraffin-embedded (BFPE) tumor samples of various origin are available in hospital repositories as a powerful information mine for clinical investigations. To date, however, such archived tissues have not been subjected to a systematic study aimed to evaluate their potential use in proteomics. In this report, we investigated whether archival BFPE tissue specimens could be exploited for proteomic studies, upon application of protein extraction and proteomic analysis methods previously optimized for formalin-fixed samples. As a result, gastric BFPE protein extracts exhibited poor suitability for 2D-PAGE analysis, whereas over 300 unique proteins could be successfully detected when extracts were subjected to SDS-PAGE followed by LC-MS/MS (GeLC-MS/MS). Among these, several known markers for gastric cancer and normal gastric functionality were identified, indicative of biological and clinical significance of proteomic data mined from BFPE tissues. A quantitative and qualitative comparison of FFPE and BFPE tissue proteomes was also performed, and results are reported. In conclusion, we demonstrated that BFPE specimens can be analyzed by means of a proteomic approach such as GeLC-MS/MS. Although considerable molecular biases and technical constraints exist, BFPE tissue archives can be fruitfully exploited for gathering proteomic data from particularly precious samples., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Characterisation of blood serum peptides can provide valuable information on physiological and pathological processes. However, the analysis of raw serum samples by MS results in the identification of a limited number of peptides. In order to improve sensitivity, many peptide enrichment methods have been proposed during the last ten years. Here, we present a comparison of fractionation methods aimed to simplify analysis of small proteins and peptides in blood serum, one of the most promising sources of putative biomarkers. Specifically, three methods based on ultrafiltration, differential precipitation, and peptide ligand libraries (ProteoMiner) were evaluated for the enrichment of peptides and low molecular weight proteins, as demonstrated by Tricine SDS-PAGE and subsequent LC-MS/MS (GeLC-MS/MS). As a result, differential solubilisation (DS) allowed the identification of the highest number of peptides. Moreover, the DS method enabled also the quantitative comparison of samples, producing fundamental information in biomarker discovery approaches., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Milk fat globules (MFGs) are vesicles released in milk as fat droplets surrounded by the endoplasmic reticulum and apical cell membranes. During formation and apocrine secretion by lactocytes, various amounts of cytoplasmic crescents remain trapped within the released vesicle, making MFGs a natural sampling mechanism of the lactating cell contents. With the aim of investigating the events occurring in the mammary epithelium during bacterial infection, the MFG proteome was characterized by two-dimensional difference gel electrophoresis (2-D DIGE), SDS-PAGE followed by shotgun liquid chromatography-tandem mass spectrometry (GeLC-MS/MS), label-free quantification by the normalized spectral abundance factor (NSAF) approach, Western blotting, and pathway analysis, using sheep naturally infected by Mycoplasma agalactiae. A number of protein classes were found to increase in MFGs upon infection, including proteins involved in inflammation and host defense, cortical cytoskeleton proteins, heat shock proteins, and proteins related to oxidative stress. Conversely, a strikingly lower abundance was observed for proteins devoted to MFG metabolism and secretion. To our knowledge, this is the first report describing proteomic changes occurring in MFGs during sheep infectious mastitis. The results presented here offer new insights into the in vivo response of mammary epithelial cells to bacterial infection and open the way to the discovery of protein biomarkers for monitoring clinical and subclinical mastitis.

Storage conditions are known to be important for postmortem deterioration of fish muscle, and temperature is one of the factors with the strongest impact on this process. In order to shed light on the influence of temperature on the status of sea bass (Dicentrarchus labrax) muscle proteins during postmortem storage, a 2-D DIGE and mass spectrometry study was performed on fish kept at either 1 or 18°C for 5 days. As expected, the greatest alterations in sea bass filet protein composition were observed upon postmortem storage at 18°C, with distinct changes appearing in the 2-D protein profile after 5 days of storage at this temperature. In particular, degradation of the myofibrillar protein myosin heavy chain and of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, among the most abundant muscle proteins, could be clearly observed upon storage at higher temperatures. Although to a lesser extent, however, several proteins were observed to vary in abundance also upon storage for 5 days at 1°C. In particular, one of the most interesting observations was the rapid and significant decrease in the abundance of nucleoside diphosphate kinase B and phosphoglycerate mutase 2, which was observed also at low storage temperatures and appeared to be temperature-independent. The results of this study offer new knowledge on changes occurring in sea bass muscle proteins during postmortem storage at different temperatures and provide indications on protein degradation trends that might be useful for monitoring freshness of fish and quality of storage conditions., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Formalin-fixed, paraffin-embedded (FFPE) tissue banks represent an invaluable resource for biomarker discovery. Recently, the combination of full-length protein extraction, GeLC-MS/MS analysis, and spectral counting quantification has been successfully applied to mine proteomic information from these tissues. However, several sources of variability affect these samples; among these, the duration of the fixation process is one of the most important and most easily controllable ones. To assess its influence on quality of GeLC-MS/MS data, the impact of fixation time on efficiency of full-length protein extraction efficiency and on quality of label-free quantitative data was evaluated. As a result, although proteins were successfully extracted from FFPE liver samples fixed for up to eight days, fixation time appeared to negatively influence both protein extraction yield and GeLC-MS/MS quantitative proteomic data. Particularly, MS identification efficiency decreased with increasing fixation times. Moreover, amino acid modifications putatively induced by formaldehyde were detected and characterized. These results demonstrate that proteomic information can be achieved also from tissue samples fixed for relatively long times, but suggest that variations in fixation time need to be carefully taken into account when performing proteomic biomarker discovery studies on fixed tissue archives., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The propagation of traveling chemical waves in the excitable Belousov-Zhabotinsky (BZ) system when performed in the presence of 1,2-dipalmitoyl-sn-glycero-3-phosphatidyl choline (DPPC) bilayers responds sensitively to the phospholipid content. The characteristic features of wave propagation, such as spiral pitch, rotation period, and size of the spiral core region, show two regions of different behavior, one below and the other above a DPPC content of 12.5% (w/w) thus suggesting a transition in the organization of the lipid domains at a DPPC content of ∼12.5% (w/w). This transition is supported by small-angle X-ray scattering data, which show pronounced changes in the coherence lengths of the lyotropic smectic domains. Thus, the dynamics of the chemical system occurring at a macroscopic length scale reflects the organization of the water/lipid domains which extend over mesoscopic lengths. These findings indicate that in the BZ/DPPC system, there is an interaction between processes that occurs at length scales differing by as much as 3 orders of magnitude.