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.

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.

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%).

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.

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.

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.

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.

"Proteomineering" serum biomarkers. A study in scarlet.

The performance of sera pre-treatment for biomarker searching via combinatorial peptide ligand libraries (CPLL) has recently been challenged (Proteomics 2010, 10, 1416-1425) and stated to allow discovery of only medium to high-abundance proteins. We have thus investigated four elution protocols, as published in recent reports: (i) in 4 M urea+1% CHAPS; (ii) in 4 M urea+1% CHAPS+5% acetic acid; (iii) in 8 M urea+2% CHAPS+5% acetic acid; (iv) in boiling 4% SDS+25 mM DTT. One milliliter of serum, in all cases, was captured with 50 µL of CPLL beads, which were then eluted with the four eluants described above. In the first three cases, after the first elution, the beads were re-eluted with cocktail (iv), known to offer maximal release of proteins adsorbed by the CPLL ligands. Eluant (i) released only ca. 20% of the species adsorbed, eluant (ii) ca. 60%, eluant (iii) ca. 80%. Thus, the poor performance of the CPLL methodology, as reported in (i) is not due to any fault of the capture technique, but simply to the adoption of a very poor elution protocol. Even those using eluants (ii) and (iii) should know that a substantial fraction of the captured species still remains bound to the beads and is thus not available to biomarker discovery. Once more, eluant (iv) is recognized as the only one able to offer optimal recovery from the CPLL baits.

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.

Biochemical characterization of MLC1 protein in astrocytes and its association with the dystrophin-glycoprotein complex.

MLC1 gene mutations have been associated with megalencephalic leukoencephalopathy with subcortical cysts (MLC), a rare neurologic disorder in children. The MLC1 gene encodes a membrane protein (MLC1) with unknown function which is mainly expressed in astrocytes. Using a newly developed anti-human MLC1 polyclonal antibody, we have investigated the biochemical properties and localization of MLC1 in cultured astrocytes and brain tissue and searched for evidence of a relationship between MLC1 and proteins of the dystrophin-glycoprotein complex (DGC). Cultured astrocytes express two MLC1 components showing different solubilisation properties and subcellular distribution. Most importantly, we show that the membrane-associated component of MLC1 (60-64 kDa) localizes in astrocytic lipid rafts together with dystroglycan, syntrophin and caveolin-1, and co-fractionates with the DGC in whole rat brain tissue. In the human brain, MLC1 protein is expressed in astrocyte processes and ependymal cells, where it colocalizes with dystroglycan and syntrophin. These data indicate that the DGC may be involved in the organization and function of the MLC1 protein in astrocyte membranes.

Mass spectrometric identification of hemoglobin modifications induced by nitrosobenzene.

Aniline and nitrobenzene (NB) are widely used industrial chemicals. Early effects of aniline toxicity include methemoglobin formation and damage to erythrocytes (Jenkins, F.P., 1972. The no-effect dose of anilne in human subjects and a comparison of aniline toxicity in man and rat. Food Cosmet. Toxicol. 10, 671-679; Bus, J.S., Popp, J.A., 1987. Perspectives on the mechanism of action of the splenic toxicity of aniline and structurally-related. Food Chem. Toxicol. 25, 619-627). In this report, we describe an analytical method, based on LC techniques and mass spectrometry, which could help in monitoring the exposure to aniline and NB. In particular, we describe and characterize the formation of specific adducts during an in vitro reaction of nitrosobenzene (NOB), the main metabolite of aniline and NB, and human hemoglobin.

Plasmodium lipid rafts contain proteins implicated in vesicular trafficking and signalling as well as members of the PIR superfamily, potentially implicated in host immune system interactions.

Plasmodium parasites, the causal agents of malaria, dramatically modify the infected erythrocyte by exporting parasite proteins into one or multiple erythrocyte compartments, the cytoplasm and the plasma membrane or beyond. Despite advances in defining signals and specific cellular compartments implicated in protein trafficking in Plasmodium-infected erythrocytes, the contribution of lipid-mediated sorting to this cellular process has been poorly investigated. In this study, we examined the proteome of cholesterol-rich membrane microdomains or lipid rafts, purified from erythrocytes infected by the rodent parasite Plasmodium berghei. Besides structural proteins associated with invasive forms, we detected chaperones, proteins implicated in vesicular trafficking, membrane fusion events and signalling. Interestingly, the raft proteome of mixed P. berghei blood stages included proteins encoded by members of a large family (bir) of putative variant antigens potentially implicated in host immune system interactions and targeted to the surface of the host erythrocytes. The generation of transgenic parasites expressing BIR/GFP fusions confirmed the dynamic association of members of this protein family with membrane microdomains. Our results indicated that lipid rafts in Plasmodium-infected erythrocytes might constitute a route to sort and fold parasite proteins directed to various host cell compartments including the cell surface.

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.

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.

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.

"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.

Functional genomics, new tools in malaria research.

The mosquito-transmitted unicellular parasite Plasmodium falciparum, the agent of malaria disease, still causes more than one million deaths every year in the tropical and subtropical areas of the world. New intervention strategies are needed to contrast the insurgence of resistance to effective drugs and insecticides. The complete annotated genomes of the human parasite P. falciparum and the rodent model P. yoelii is now available thus providing a prediction of their possible gene products. This makes feasible the application of functional genomics to malaria research with the final goal of providing a complete survey of Plasmodium life cycle. Genome-wide approaches to the study of transcriptome or proteome were successfully applied to malaria parasite with the promise for new drug and vaccine candidates in the next future.

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).

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.