Summary recommendations on "Analytical methods for substances in the Watch List under the Water Framework Directive".

The Watch List (WL) is a monitoring program under the European Water Framework Directive (WFD) to obtain high-quality Union-wide monitoring data on potential water pollutants for which scarce monitoring data or data of insufficient quality are available. The main purpose of the WL data collection is to determine if the substances pose a risk to the aquatic environment at EU level and subsequently to decide whether a threshold, the Environmental Quality Standards (EQS) should be set for them and, potentially to be listed as priority substance in the WFD. The first WL was established in 2015 and contained 10 individual or groups of substances while the 4th WL was launched in 2022. The results of monitoring the substances of the first WL showed that some countries had difficulties to reach an analytical Limit of Quantification (LOQ) below or equal to the Predicted No-Effect Concentrations (PNEC) or EQS. The Joint Research Centre (JRC) of the European Commission (EC) organised a series of workshops to support the EU Member States (MS) and their activities under the WFD. Sharing the knowledge among the Member States on the analytical methods is important to deliver good data quality. The outcome and the discussion engaged with the experts are described in this paper, and in addition a literature review of the most important publications on the analysis of 17-alpha-ethinylestradiol (EE2), amoxicillin, ciprofloxacin, metaflumizone, fipronil, metformin, and guanylurea from the last years is presented.

A rapid screening method for testing the efficiency of masks in breaking down aerosols.

The highest risk of novel coronavirus SARS-CoV-2 to be spread through human-to-human transmission has boosted the use of personal protective equipment at worldwide level. In Europe, the medical face masks must be tested to certify the essential requirements in agreement with European Standard EN 14683:2019, and face masks for industrial use in agreement with European Standard EN 149:2009. Due to the need of large quantitative of medical and non-medical face masks in coronavirus outbreak, several Italian industries are working for shift a portion of their manufacturing capacity for producing medical and non-medical face mask. For screening evaluation of the effectiveness of personal protective equipment produced by reconverted industries, ARPA Lazio and the Department of Chemical Science and Technologies of Tor Vergata University have set-up an analytical system able to simulate the respiratory action and to measure the percentage of particles that pass through the face masks using optical particle counter (based on the EN 16890: 2017 that uses the same light scattering principle to evaluate the filter filtration efficiency). This set-up was challenged using face masks produced by reconverted industries and the data were compared with ones obtained using medical face mask.

Numbers around Descartes: A preregistered study on the three-dimensional SNARC effect.

The Spatial-Numerical Association of Response Codes (SNARC) effect suggests that numbers are represented along a horizontal left-to-right oriented, mental number line, with small numbers on the left and large numbers on the right. Much less evidence exists for vertical (down-to-up) and sagittal (near-to-far) SNARC effects. This might be due to the employment of different experimental paradigms among studies and to the, sometimes, inexact definition of the vertical and sagittal axes. We investigated for the first time the SNARC effect along the horizontal, vertical, and sagittal axes, by means of a classic SNARC task. Our results suggest the presence of three equally-strong SNARC effects. Our findings can be considered as evidence in favor of a three-dimensional, mental representation of numbers, in the form of a mental number space, defined by Cartesian coordinates.

A MALDI-TOF MS Approach for Mammalian, Human, and Formula Milks' Profiling.

Human milk composition is dynamic, and substitute formulae are intended to mimic its protein content. The purpose of this study was to investigate the potentiality of matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS), followed by multivariate data analyses as a tool to analyze the peptide profiles of mammalian, human, and formula milks. Breast milk samples from women at different lactation stages (2 (n = 5), 30 (n = 6), 60 (n = 5), and 90 (n = 4) days postpartum), and milk from donkeys (n = 4), cows (n = 4), buffaloes (n = 7), goats (n = 4), ewes (n = 5), and camels (n = 2) were collected. Different brands (n = 4) of infant formulae were also analyzed. Protein content (<30 kDa) was analyzed by MS, and data were exported for statistical elaborations. The mass spectra for each milk closely clustered together, whereas different milk samples resulted in well-separated mass spectra. Human samples formed a cluster in which colostrum constituted a well-defined subcluster. None of the milk formulae correlated with animal or human milk, although they were specifically characterized and correlated well with each other. These findings propose MALDI-TOF MS milk profiling as an analytical tool to discriminate, in a blinded way, different milk types. As each formula has a distinct specificity, shifting a baby from one to another formula implies a specific proteomic exposure. These profiles may assist in milk proteomics for easiness of use and minimization of costs, suggesting that the MALDI-TOF MS pipelines may be useful for not only milk adulteration assessments but also for the characterization of banked milk specimens in pediatric clinical settings.

Phenotypic typing and epidemiological survey of antifungal resistance of Candida species detected in clinical samples of Italian patients in a 17 months' period.

INTRODUCTION: Yeast pathogens are emerging agents of nosocomial as well as community-acquired infections and their rapid and accurate identification is crucial for a better management of high-risk patients and for an adequate treatment. METHODS: We performed a retrospective review of 156 yeast isolates collected during a 17 months' period of regular clinical practice at the Microbiology Department of San Camillo Hospital in Treviso, Italy and analyzed by the traditional culture-based method combined with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). RESULTS: Out of all the samples collected MALDI-TOF MS was able to characterize with a MT score ≥1.7 (accurate result at species level) 12 different yeast and yeast-like species from 140 samples: Candida albicans (63.7%), Candida glabrata (13.6%), Saccharomyces cerevisiae (6.5%), Candida parapsilosis (5.7%), Candida tropicalis (2.1%), Candida pararugosa (2.1%), Candida guilliermondii (2.1%), Candida kefyr (1.4%), Candida lusitaniae (0.7%), Candida palmioleophila (0.7%), Geotrichum silvicola (0.7%), Rhodotorula mucilaginosa (0.7%). Susceptibility testing toward seven common antifungal agents showed a characteristic MIC distribution of C. albicans isolates for echinocandins: particularly we noticed that 72% and 46% of C. albicans showed an MIC value close to clinical breakpoint as defined by EUCAST, respectively for anidulafungin and micafungin. CONCLUSION: Accurate identification of microorganisms and the study of their antifungal susceptibility allow to understand the epidemiology of a particular area, permitting the choice of the most appropriate early antifungal treatment.

"Omic" investigations of protozoa and worms for a deeper understanding of the human gut "parasitome".

The human gut has been continuously exposed to a broad spectrum of intestinal organisms, including viruses, bacteria, fungi, and parasites (protozoa and worms), over millions of years of coevolution, and plays a central role in human health. The modern lifestyles of Western countries, such as the adoption of highly hygienic habits, the extensive use of antimicrobial drugs, and increasing globalisation, have dramatically altered the composition of the gut milieu, especially in terms of its eukaryotic "citizens." In the past few decades, numerous studies have highlighted the composition and role of human intestinal bacteria in physiological and pathological conditions, while few investigations exist on gut parasites and particularly on their coexistence and interaction with the intestinal microbiota. Studies of the gut "parasitome" through "omic" technologies, such as (meta)genomics, transcriptomics, proteomics, and metabolomics, are herein reviewed to better understand their role in the relationships between intestinal parasites, host, and resident prokaryotes, whether pathogens or commensals. Systems biology-based profiles of the gut "parasitome" under physiological and severe disease conditions can indeed contribute to the control of infectious diseases and offer a new perspective of omics-assisted tropical medicine.

Food labeling issues in patients with severe food allergies: solving a hamlet-like doubt.

PURPOSE OF REVIEW: We review the laws on labeling in the international community, the difficulties they pose to the food manufacturers to prepare the food labels and the methodologies to determine the concentration of potential allergens in foods. RECENT FINDINGS: European Food Safety Authority and International Life Sciences Institute Europe are evaluating strategies to identify the threshold level of allergen that can trigger a reaction in individuals. The most used techniques to detect the presence of protein in food are Enzyme-linked immunosorbent assay, polymerase chain reaction and real time polymerase chain reaction. Researchers are now trying to apply proteomics to estimate the amount of protein within the food.In order to protect the health of consumers, the Codex Alimentarius Commission updates constantly the list of allergens. In response to these regulations, some industries have also added some precautionary allergen labeling (PAL). It was generally agreed that PAL statements needed to be visible, simple, and safe. It was suggested that PAL be standardized, an action that would occur if the 'Voluntary Incidental Trace Allergen Labelling' process was made mandatory. SUMMARY: So far, no laboratory technique is able to reassure the consumers about the composition of foods found on the packaging. International authorities produced increasingly stringent laws, but more is still to do.

Sensitization pattern to inhalant and food allergens in symptomatic children at first evaluation.

BACKGROUND: Data on specific IgE sensitization prevalence in children with allergy-like symptoms seen in the primary care setting are rare. Early diagnosis of allergic diseases is important to prevent clinical manifestations, exacerbations or expansion of allergic diseases to other organ systems. The present study aims to assess the usefulness of early serological diagnosis in children with common allergic symptoms. METHODS: 532 children (<15 years of age), with at least one of ten allergy-like symptoms, from 21 primary care centers in two geographic areas of Italy and Spain were included in the study. Patients were tested with, either Phadiatop® Infant (0-5 years of age) or Phadiatop® and food mix (fx5e) (>5 years of age) to discriminate atopic from non-atopic subjects. A blood sample of atopic subjects was taken for additional 6-26 specific IgE antibody determinations from a predefined panel using the ImmunoCAP® System. RESULTS: 267 children (50.2 %) were positive in the initial test and were classified as atopic. 14 % were mono-sensitized, 37 % were sensitized to 2-3 allergens and 49 % to more than 3 allergens. The average number of symptoms in the atopic group was 3.3 vs 2.8 in the non-atopic group. The prevalence of sensitization to single allergens was highest for grass and ragweed pollen and house-dust mites (19-28 %). Sensitization to tree allergens was highest for olive tree (16.5 %). Cow's milk and egg white were the most sensitizing foods (~15 %). Food allergen sensitization predominated in younger children (OR = 2.8) whereas the inverse occurred with inhalant allergens (OR = 2.5 to 5.6). A significant positive correlation between patient age and the number of sensitizations was found. CONCLUSIONS: Specific IgE sensitization in children with allergy-like symptoms is common. Multiple sensitization is predominating. Number of clinical symptoms was higher in the atopic group compared to the non-atopic without a correlation with the number of positive allergens. Age seems to play a crucial role in the development of sensitization with a significant positive correlation between patient age and the number of sensitizations.

A Simple and Effective Mass Spectrometric Approach to Identify the Adulteration of the Mediterranean Diet Component Extra-Virgin Olive Oil with Corn Oil.

Extra virgin olive oil (EVOO) with its nutraceutical characteristics substantially contributes as a major nutrient to the health benefit of the Mediterranean diet. Unfortunately, the adulteration of EVOO with less expensive oils (e.g., peanut and corn oils), has become one of the biggest source of agricultural fraud in the European Union, with important health implications for consumers, mainly due to the introduction of seed oil-derived allergens causing, especially in children, severe food allergy phenomena. In this regard, revealing adulterations of EVOO is of fundamental importance for health care and prevention reasons, especially in children. To this aim, effective analytical methods to assess EVOO purity are necessary. Here, we propose a simple, rapid, robust and very sensitive method for non-specialized mass spectrometric laboratory, based on the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) coupled to unsupervised hierarchical clustering (UHC), principal component (PCA) and Pearson's correlation analyses, to reveal corn oil (CO) adulterations in EVOO at very low levels (down to 0.5%).

Proteomic applications in food allergy: food allergenomics.

PURPOSE OF REVIEW: To familiarize the reader with the recent developments in the identification of food protein allergens by proteomics mass spectrometry-based methods, named allergenomics. RECENT FINDINGS: The proteomic analysis of food protein allergens has became a hot topic in the food safety field in recent years. Indeed, food allergies represent a current and relevant problem in clinical medicine. Several food allergenomics studies have recently been performed, aiming at better understanding the cause of sensitization to cow's milk in breastfed infants and at assessing both the safety of food (e.g. transgenic) and in particular the allergenic properties of processed fish and seafood. SUMMARY: Food protein allergen characterization and quantification, together with the immunoglobulin E epitope mapping, will contribute to the diagnosis/prognosis of food allergy and will lead to a better safety assessment of foods (e.g. novel transgenic foods).

Farm animal serum proteomics and impact on human health.

Due to the incompleteness of animal genome sequencing, the analysis and characterization of serum proteomes of most farm animals are still in their infancy, compared to the already well-documented human serum proteome. This review focuses on the implications of the farm animal serum proteomics in order to identify novel biomarkers for animal welfare, early diagnosis, prognosis and monitoring of infectious disease treatment, and develop new vaccines, aiming at determining the reciprocal benefits for humans and animals.

A sensitive and effective proteomic approach to identify she-donkey's and goat's milk adulterations by MALDI-TOF MS fingerprinting.

She-donkey's milk (DM) and goat's milk (GM) are commonly used in newborn and infant feeding because they are less allergenic than other milk types. It is, therefore, mandatory to avoid adulteration and contamination by other milk allergens, developing fast and efficient analytical methods to assess the authenticity of these precious nutrients. In this experimental work, a sensitive and robust matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling was designed to assess the genuineness of DM and GM milks. This workflow allows the identification of DM and GM adulteration at levels of 0.5%, thus, representing a sensitive tool for milk adulteration analysis, if compared with other laborious and time-consuming analytical procedures.

The possible implication of the S250C variant of the autoimmune regulator protein in a patient with autoimmunity and immunodeficiency: in silico analysis suggests a molecular pathogenic mechanism for the variant.

Autoimmunity can develop from an often undetermined interplay of genetic and environmental factors. Rare forms of autoimmune conditions may also result from single gene mutations as for autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, an autosomal recessive disease associated with mutated forms of the autoimmune regulator gene. It was proposed that genetic variability in the autoimmune regulator locus, in particular heterozygous loss-of-function mutations, might favor the development of organ-specific autoimmunity by affecting the presentation of self-antigens in the thymus. Indeed, heterozygous mutations of the autoimmune regulator gene were reported in patients with organ-specific autoimmunity. Also, in primary immunodeficiencies, a breakdown in central/peripheral tolerance frequently produces association with autoimmunity. The causative link may involve a common genetic background and several gene defects have been identified as putative culprits. We report a unique patient, a 14 year old male from Lazio region, affected by common variable immunodeficiency associated with autoimmune manifestations (alopecia, onychodystrophy) and heterozygote for the S250C variant located in the SAND domain of the autoimmune regulator gene protein. To our knowledge this is the first report of the S250C variant in a patient bearing this unusual combination of autoimmunity and immunodeficiency. To obtain insights into the possible molecular effects of the S250C variant, we have carried out an in silico analysis of the SAND domain structure of the autoimmune regulator protein. In particular, homology modeling has allowed us to observe that the cysteine introduced by the S250C variant is surrounded by cationic residues, and by means of molecular dynamics simulations together with pKa calculations, we have shown that these residues remain stably proximal to cysteine-250 lowering its pKa and thus conferring high chemical reactivity to the mutated residue. We propose that the enhanced reactivity of cysteine-250, which is likely to impair the protein function but probably insufficient to produce alone a phenotype as a heterozygous S250C variant due to compensation mechanisms, might become manifest when combined with other genetic/environmental factors. These results can provide the rationale for the patient's unusual phenotype, shedding new light into the pathogenesis of the clinical association of autoimmunity and immunodeficiency.

The Role of Mass Spectrometry in the "Omics" Era.

Mass spectrometry (MS) is one of the key analytical technology on which the emerging ''-omics'' approaches are based. It may provide detection and quantization of thousands of proteins and biologically active metabolites from a tissue, body fluid or cell culture working in a ''global'' or ''targeted'' manner, down to ultra-trace levels. It can be expected that the high performance of MS technology, coupled to routine data handling, will soon bring fruit in the request for a better understanding of human diseases, leading to new molecular biomarkers, hence affecting drug targets and therapies. In this review, we focus on the main advances in the MS technologies, influencing genomics, transcriptomics, proteomics, lipidomics and metabolomics fields, up to the most recent MS applications to meta-omic studies.

Proteomic analysis of detergent-resistant membrane microdomains in trophozoite blood stage of the human malaria parasite Plasmodium falciparum.

Intracellular pathogens contribute to a significant proportion of infectious diseases worldwide. The successful strategy of evading the immune system by hiding inside host cells is common to all the microorganism classes, which exploit membrane microdomains, enriched in cholesterol and sphingolipids, to invade and colonize the host cell. These assemblies, with distinct biochemical properties, can be isolated by means of flotation in sucrose density gradient centrifugation because they are insoluble in nonionic detergents at low temperature. We analyzed the protein and lipid contents of detergent-resistant membranes from erythrocytes infected by Plasmodium falciparum, the most deadly human malaria parasite. Proteins associated with membrane microdomains of trophic parasite blood stages (trophozoites) include an abundance of chaperones, molecules involved in vesicular trafficking, and enzymes implicated in host hemoglobin degradation. About 60% of the identified proteins contain a predicted localization signal suggesting a role of membrane microdomains in protein sorting/trafficking. To validate our proteomic data, we raised antibodies against six Plasmodium proteins not characterized previously. All the selected candidates were recovered in floating low-density fractions after density gradient centrifugation. The analyzed proteins localized either to internal organelles, such as the mitochondrion and the endoplasmic reticulum, or to exported membrane structures, the parasitophorous vacuole membrane and Maurer's clefts, implicated in targeting parasite proteins to the host erythrocyte cytosol or surface. The relative abundance of cholesterol and phospholipid species varies in gradient fractions containing detergent-resistant membranes, suggesting heterogeneity in the lipid composition of the isolated microdomain population. This study is the first report showing the presence of cholesterol-rich microdomains with distinct properties and subcellular localization in trophic stages of Plasmodium falciparum.

Reproducibility of combinatorial peptide ligand libraries for proteome capture evaluated by selected reaction monitoring.

UNLABELLED: Systems biology studies require the capability to quantify with high precision proteins spanning a broad range of abundances across multiple samples. However, the broad range of protein expression in cells often precludes the detection of low-abundance proteins. Different sample processing techniques can be applied to increase proteome coverage. Among these, combinatorial (hexa)peptide ligand libraries (CPLLs) bound to solid matrices have been used to specifically capture and detect low-abundance proteins in complex samples. To assess whether CPLL capture can be applied in systems biology studies involving the precise quantitation of proteins across a multitude of samples, we evaluated its performance across the whole range of protein abundances in Saccharomyces cerevisiae. We used selected reaction monitoring assays for a set of target proteins covering a broad abundance range to quantitatively evaluate the precision of the approach and its capability to detect low-abundance proteins. Replicated CPLL-isolates showed an average variability of ~10% in the amount of the isolated proteins. The high reproducibility of the technique was not dependent on the abundance of the protein or the amount of beads used for the capture. However, the protein-to-bead ratio affected the enrichment of specific proteins. We did not observe a normalization effect of CPLL beads on protein abundances. However, CPLLs enriched for and depleted specific sets of proteins and thus changed the abundances of proteins from a whole proteome extract. This allowed the identification of ~400 proteins otherwise undetected in an untreated sample, under the experimental conditions used. CPLL capture is thus a useful tool to increase protein identifications in proteomic experiments, but it should be coupled to the analysis of untreated samples, to maximize proteome coverage. Our data also confirms that CPLL capture is reproducible and can be confidently used in quantitative proteomic experiments. SIGNIFICANCE: Combinatorial hexapeptide ligand libraries (CPLLs) bound to solid matrices have been proposed to specifically capture and detect low-abundance proteins in complex samples. To assess whether the CPLL capture can be confidently applied in systems biology studies involving the precise quantitation of proteins across a broad range of abundances and a multitude of samples, we evaluated its reproducibility and performance features. Using selected reaction monitoring assays for proteins covering the whole range of abundances we show that the technique is reproducible and compatible with quantitative proteomic studies. However, the protein-to-bead ratio affects the enrichment of specific proteins and CPLLs depleted specific sets of proteins from a whole proteome extract. Our results suggest that CPLL-based analyses should be coupled to the analysis of untreated samples, to maximize proteome coverage. Overall, our data confirms the applicability of CPLLs in systems biology research and guides the correct use of this technique.

Human serum proteome analysis: new source of markers in metabolic disorders.

The prevalence of metabolic disorders (MDs), especially diabetes, is rapidly increasing worldwide, leading to an increasing risk of cardiovascular and other socially relevant complications. To boost MD biomarker discovery, advanced proteomics can harmonize metabolomics. Indeed, the rapid development of mass spectrometry (MS) has designated proteomics as an emerging platform to interrogate the plasma/serum proteome for the discovery of next-generation biomarkers exploitable for risk assessment, early detection and prognosis of MDs. Preanalytical plasma/serum treatment, such as combinatorial peptide ligand libraries with nano-liquid chromatography coupled with tandem MS or selected reaction monitoring coupled to triple-quadrupole time-of-flight instruments, are proven clinical laboratory techniques for quantitative analyses. New strategies, such as SWATH™ MS, which allows us to systematically characterize and quantify query sample sets of 'any protein of interest' in complex biological samples, may dramatically improve next-generation MD biomarkers, especially considering the plethora of candidates coming from the 'bioreactor' gut microbiota affecting MD onset and progression.

Assessment of the floral origin of honey via proteomic tools.

The honey from chestnut, acacia, sunflower, eucalyptus and orange was analysed for its proteome content, in order to see if any plant proteins present would allow the proteo-typing of these different varieties. Since the total protein content turned out to be minute, 200g of each honey type were diluted to 1L and then added with ProteoMiner to enhance the visibility of the proteinaceous material. All bands visible in the SDS-PAGE profile of each type of honey were eluted, digested and identified by mass spectrometry in a LTQ-XL instrument. It turned out that all proteins identified (except one, the enzyme glyceraldehyde-3-phosphate dehydrogenase from Mesembryanthemum crystallinum) were not of plant origin but belonged to the Apis mellifera proteome. Among the total proteins identified (eight, but only seven as basic constituents of all types of honey) five belonged to the family of major royal jelly proteins 1-5, and were also the most abundant ones in any type of honey, together with α-glucosidase and defensin-1. It thus appears that honey has a proteome resembling the royal jelly proteome (but with considerably fewer species), except that its protein concentration is lower by three to four orders of magnitude as compared to royal jelly. Attempts at identifying additional plant (pollen, nectar) proteins via peptidome analysis were unsuccessful.

Plasma proteomics for biomarker discovery: a study in blue.

The performance of Cibacron Blue dye (HiTrapBlue or Affigel Blue) in depleting albumin from plasma, as a pre-treatment for biomarker searching in the low-abundance proteome, is here assessed. It is shown that (i) co-depletion of non-albumin species is an ever-present hazard; (ii) the only proper eluant able to release quantitatively the proteins bound to the dye is boiling 4% SDS-25 mM DTT, an ion shock (2 M NaCl) being quite ineffective in releasing the low-abundance species tightly bound to the dye moiety; (iii) the mechanism of dye-protein interaction, after an initial ion-ion docking, is a robust hydrophobic interaction, which progressively augments at lower and lower pH values; (iv) at pH 2.2 in the presence of 0.1% TFA, the blue resin behaves, for all practical purposes, just as a reverse-phase chromatography column, since all residual proteins present in plasma are completely harvested. However Cibacron Blue technology should not necessarily be discarded: As long as also the plasma fraction adsorbed is properly released and analyzed, together with the flow through, one should be able to perform a viable analysis of the low-abundance proteome.

Horam nonam exclamavit: sitio. The trace proteome of your daily vinegar.

The trace proteome of white-wine vinegar has been identified via capture with home-made combinatorial peptide ligand libraries under conditions mimicking reverse-phase capture, i.e. at pH 2.2 in presence of 0.1% trifluoroacetic acid. A total of 27 unique gene products have been identified, of which 10 specific of the database Vitis vinifera, 13 found in the general database Uniprot_viridiplantae and 4 in Swiss Prot_all entries. The most abundant species detected, on the basis of spectral counts, appears to be the whole genome shotgun sequence of line PN40024, scaffold_22 (a protein of the glycosyl hydrolase family). Curiously, up to the present, no information had been available on vinegar proteome.