Optimization of a fast and sensitive method based on matrix solid-phase dispersion-LC-ms/ms for simultaneous determination of phthalates and bisphenols in mussel samples.

Bisphenols and phthalates are wide classes of endocrine disrupting chemicals (EDCs) extensively used as additives in plastic products. In this study, a fast and reliable analytical method based on matrix solid-phase dispersion (MSPD) coupled with LC-MS/MS was developed and optimized for simultaneous determination of 8 bisphenols and 7 phthalates in raw mussel extract. The LC-MS/MS method was tested for linearity (R2), inter- and intra-day repeatability, limit of detection and quantification, both for matrix-free and matrix-matched solutions. The MSPD method was optimized in terms of ratio between sample and sorbent, and the type and quantity of the eluents in order to maximize the recoveries and to minimize matrix effects. The obtained recoveries (values between 75% and 113%), limits of detection (values between 0.048 and 0.36 µg kg-1), limits of quantification (values between 0.16 and 1.28 µg kg-1), repeatability (RSD% between 1.30% and 8.41%) and linearity (R2 > 0.998) were satisfactory and suitable for the determination of target micropollutants in food samples. In addition, the low solvent consumption and fast execution make this method ideal for routinely determinations of bisphenols and phthalates in mussels.

Perfluorinated Compounds (PFCs) in River Waters of Central Italy: Monthly Variation and Ecological Risk Assessment (ERA).

Perfluorinated compounds (PFCs) are a wide class of emerging pollutants. In this study, we applied the US EPA method 533 for the determination of 21 PFCs in river water samples. In particular, this method was used to investigate the presence of the target PFCs in six rivers in central Italy during a 4-month-long monitoring campaign. In 73% of the analyzed samples, at least some of the target PFCs were detected at concentrations higher than the limit of detection (LOD). The sum of the 21 target analytes (∑21PFCs) ranged from 4.3 to 68.5 ng L-1, with the highest concentrations measured in the month of June, probably due to a minor river streamflow occurring in the warmer summer months. Considering the individual congeners, PFBA and PFPeA, followed by PFHxA and PFOA, were the predominantly detected compounds. Short- and medium-chain PFCs (C4-C9) prevail over the long-chain PFCs (C10-C18), likely due to the increased industrial use and the higher solubility of short-chain PFCs compared to long-chain PFCs. The ecological risk assessment, conducted by using the risk quotient method, highlighted that the risk for aquatic environments associated with PFBA, PFPeA, PFBS, PFHxA and PFOA was low or negligible. Only for PFOA, there was a medium level of risk in two rivers in the month of June. With regard to PFOS, 54% of the river water samples were classified as "high risk" for the aquatic environment. The remaining 46% of the samples were classified as "medium risk."

Occurrence and congener profiles of dioxins (PCDDs), furans (PCDFs) and polychlorinated biphenyls (PCBs) in ovine and caprine milk samples collected in a very polluted site in Central Italy.

Concentrations of 7 polychlorinated dibenzo-p-dioxins (PCDDs), 10 polychlorinated dibenzofurans (PCDFs) and 18 polychlorinated biphenyls (PCBs), including 12 dioxin-like (DL-PCBs) and 6 non-dioxin-like PCBs (NDL-PCBs) were measured in 16 ovine and caprine milk samples collected in the territory of Bussi sul Tirino, central Italy, a Site of National Interest (SNI) due to its high and widespread environmental pollution. All the analyzed samples were compliant with the maximum levels fixed by Commission Regulation (EU) 1259/2011 for the content of PCDD/Fs and the sum of PCDD/Fs and DL-PCBs. In two cases, contamination levels of the sum of PCDD/Fs and DL-PCBs were higher than the action levels fixed by EU Recommendation 663/2014. The statistical analysis, performed by Principal Component Analysis (PCA), revealed that the differences in contamination profiles of the different milk samples were independent of the distance of the farms from the Bussi illegal landfill but likely related to local emission sources influencing the exposure to POPs of studied animals.

Determination of Carbonyl Compounds in Different Work Environments: Comparison between LC-UV/DAD and LC-MS/MS Detection Methods.

There were two analytical methods for the determination of 12 carbonyl compounds (CCs) by using liquid chromatography (LC) coupled with mass spectrometry (MS/MS) and diode array detector (UV/DAD) that were developed and applied to 52 samples that were collected in 10 workplaces. Linearity (0.996 < R2 < 0.999), intra-day repeatability (0.7 < RSD% < 10), and inter-day repeatability (5 < RSD% < 16) were acceptable for both techniques, but the highest sensibility of the MS/MS method allowed us to correctly quantify 98% of the samples (versus 32% by UV/DAD). The comparison of the concentrations that were obtained by quantifying the same sample with both techniques showed good agreement for acetaldehyde and formaldehyde (0.1 < % deviation < 30) but much higher for the less abundant congeners. In real samples, formaldehyde was the most abundant congener (concentrations between 2.7 and 77 µg m-3), followed by acetaldehyde (concentrations between 1.5 and 79 µg m-3) and butyraldehyde (concentrations between 0.4 and 13 µg m-3). In all the beauty salon samples, instead, the most abundant congener was acetaldehyde (concentrations between 19 and 79 µg m-3), probably associated with the use of beauty products. Principal components analysis (PCA) confirms the ubiquitous character of formaldehyde and highlights the influence of minority CCs on different workplaces.

Determination of hair cortisol in horses: comparison of immunoassay vs LC-HRMS/MS.

The measure of hair cortisol concentration (HCC) is becoming an emerging approach to monitor mid-/long-term stress in animals, so it is more and more important to develop accurate and reliable methods. In the light of this, the aim of the present study was to compare mane HCCs of 47 horses with different managements, by means of an immunoassay (ELISA) and liquid chromatography coupled to hybrid high-resolution mass spectrometry (LC-HRMS/MS). After the washing step, the ground hair was extracted with methanol. The extract was evaporated and redissolved in two different aqueous solutions, depending on the detection technique. The methods were validated according to EMA guideline for bioanalytical method validation, in the range 2-50 pg mg-1 (ELISA) and 1-100 pg mg-1 (LC-HRMS/MS). Satisfactory quantitative performances were obtained for both of the approaches, but this latter demonstrated better precision. The detected concentrations in real samples were encompassing the range 1.3-8.8 pg mg-1 and 2.0-17.9 pg mg-1 by means of LC-HRMS/MS and ELISA, respectively. Overall, HCCs measured with ELISA technique were 1.6 times higher. The overestimation of immunoassay results might be caused by cross-reactivity phenomena of laboratory reagents and other structurally similar hormones present in the mane.

Comparison of analytical approaches for identifying airborne microorganisms in a livestock facility.

An intensive study, applied to a site characterized by multiple sources of microorganisms, was aimed at understanding the best approach to study bioaerosol. Culture-based, molecular biological, and chemical methods were applied to Particulate Matter (PM) samples collected in a livestock facility, during spring and autumn seasons, in two different outdoor areas. The first one was close to a place where feed was stored and handled and the second next to an open cowshed. Qualitative analysis of bacteria was performed by sequencing techniques applied to DNA extracted from both isolated culturable bacteria and particulate matter samples. Quantification of microorganisms was achieved through three distinct approaches. Microorganism colonies were counted, after incubation at 28 °C, and expressed as colony-forming units (CFU) per m3. Chemical method consisted in the identification of individual biomarkers, and their conversion to number of microorganisms per m3, using proper conversion factors. Finally, qPCR was applied to DNA extracted from PM samples, and the results were expressed as total amount of bacteria present in the bioaerosol (UG/m3). The presence of airborne sterols was also studied to broaden the knowledge of bioaerosol components in atmosphere. Small seasonal differences and major sampling site differences occurred. Obviously, culture-dependent method identified less and different bacteria, than culture-independent approach. The chemical approach and the culture independent metagenomic method were in good agreement. As expected, CFU/m3 accounted for not more than 0.3% of bacteria calculated as the average of chemical and culture independent metagenomic methods. The complexity of the obtained results shows that the different approaches are complementary to obtain an exhaustive description of bioaresol in terms of concentration, speciation, viability, pathogenicity.

Toxic Organic Contaminants in Airborne Particles: Levels, Potential Sources and Risk Assessment.

In the last years, many studies have focused on risk assessment of exposure of workers to airborne particulate matter (PM). Several studies indicate a strong correlation between PM and adverse health outcomes, as a function of particle size. In the last years, the study of atmospheric particulate matter has focused more on particles less than 10 µm or 2.5 µm in diameter; however, recent studies identify in particles less than 0.1 µm the main responsibility for negative cardiovascular effects. The present paper deals with the determination of 66 organic compounds belonging to six different classes of persistent organic pollutants (POPs) in the ultrafine, fine and coarse fractions of PM (PM < 0.1 µm; 0.1 < PM < 2.5 µm and 2.5 < PM < 10 µm) collected in three outdoor workplaces and in an urban outdoor area. Data obtained were analyzed with principal component analysis (PCA), in order to underline possible correlation between sites and classes of pollutants and characteristic emission sources. Emission source studies are, in fact, a valuable tool for both identifying the type of emission source and estimating the strength of each contamination source, as useful indicator of environment healthiness. Moreover, both carcinogenic and non-carcinogenic risks were determined in order to estimate human health risk associated to study sites. Risk analysis was carried out evaluating the contribution of pollutant distribution in PM size fractions for all the sites. The results highlighted significant differences between the sites and specific sources of pollutants related to work activities were identified. In all the sites and for all the size fractions of PM both carcinogenic and non-carcinogenic risk values were below acceptable and safe levels of risks recommended by the regulatory agencies.

Environmental status of an Italian site highly polluted by illegal dumping of industrial wastes: The situation 15 years after the judicial intervention.

In 2008 the Italian government classified the Bussi sul Tirino area (Central Italy) as Site of National Interest destined to remediation which, unfortunately, has not yet begun. The decision followed >20 years of illegal dumping of industrial wastes, lasting from 1984 to 2005, that generated the biggest illegal toxic waste disposal site in Europe. The contamination profile of the site was mainly characterized by PCDD/Fs, PCBs, PAHs, chlorinated solvents, Hg, and Pb. Due to the health concern of the population and local authorities, an extensive monitoring and biomonitoring campaign was carried out in 2017-2018, checking the site-specific pollutants in local food (free-range hens' eggs, milk from grazing sheep and goats, wild edible mushrooms, and drinking water), environmental (air and freshwaters) and biological (human urine) matrices. A total of 314 samples were processed, obtaining 3217 analytical data that were compared with regulatory limits, when available, and values reported by international literature. The sum PCDD/Fs and DL-PCBs ranged from 0.24 to 3.6 pg TEQ g-1 fat, and from 0.46 to 8.3 pg TEQ g-1 fat, respectively in milk in eggs, in line with the maximum levels established by CE Regulations except for an egg sample. As regards PAHs, all our results were lower than the literature data, as well as for Hg and Pb. Outdoor air showed levels of chlorinated solvents ranging from

Characterization of seven sterols in five different types of cattle feedstuffs.

This paper provides a method for the quantification of sterols in different types of calf feedstuffs based on soy, sunflower, hay, calf feed and a mixture of all of them. The free fraction and the total sterolic fraction, after saponification and acidic hydrolysis of the samples, are extracted by solvent and the sterols are identified/quantified by reversed phase HPLC coupled to tandem mass spectrometry by atmospheric pressure chemical ionization. After the recovery evaluation, the method is validated in terms of linearity (coefficient of determination R2), repeatability (coefficient of variation RSD), limit of detection and quantification. In most of the cases, the most representative phytosterol is ß-sitosterol, followed by campesterol or stigmasterol and by other minor sterols such as fucosterol, and Δ-5-avenasterol. In addition, also cholesterol and ergosterol, if present, are evaluated in all the samples. As far as we know, very little information is available on the investigated feeds, which are commonly used on farms. The results of this survey were compared to other studies, if present in literature, showing good agreement. The proposed method resulted to be simple, fast and suitable for application to other sterols, feedstuffs and derived foods. The knowledge of the sterolic content and composition is getting more and more important, both in terms of comprehension of the vegetal biochemistry and as basis for sterolomic studies.

Dye Transport through Bilayers Agrees with Lipid Electropore Molecular Dynamics.

Although transport of molecules into cells via electroporation is a common biomedical procedure, its protocols are often based on trial and error. Despite a long history of theoretical effort, the underlying mechanisms of cell membrane electroporation are not sufficiently elucidated, in part, because of the number of independent fitting parameters needed to link theory to experiment. Here, we ask if the electroporation behavior of a reduced cell membrane is consistent with time-resolved, atomistic, molecular dynamics (MD) simulations of phospholipid bilayers responding to electric fields. To avoid solvent and tension effects, giant unilamellar vesicles (GUVs) were used, and transport kinetics were measured by the entry of the impermeant fluorescent dye calcein. Because the timescale of electrical pulses needed to restructure bilayers into pores is much shorter than the time resolution of current techniques for membrane transport kinetics measurements, the lifetimes of lipid bilayer electropores were measured using systematic variation of the initial MD simulation conditions, whereas GUV transport kinetics were detected in response to a nanosecond timescale variation in the applied electric pulse lifetimes and interpulse intervals. Molecular transport after GUV permeabilization induced by multiple pulses is additive for interpulse intervals as short as 50 ns but not 5-ns intervals, consistent with the 10-50-ns lifetimes of electropores in MD simulations. Although the results were mostly consistent between GUV and MD simulations, the kinetics of ultrashort, electric-field-induced permeabilization of GUVs were significantly different from published results in cells exposed to ultrashort (6 and 2 ns) electric fields, suggesting that cellular electroporation involves additional structures and processes.

High spatial resolution analysis of polybrominated diphenyl ethers (PBDEs) using transplanted lichen Evernia prunastri: A case study in central Italy.

The ability of transplanted lichen Evernia (E.) prunastri (L.) to act as a high spatial biomonitoring tool for 14 polybrominated diphenyl ethers (PBDEs) was evaluated at 23 monitoring sites in a very polluted area in central Italy. The selected area is characterized by the presence of numerous emission sources, such as waste-to-energy plant, steel plant, vehicular traffic, and domestic heating. Transplanted E. prunastri proved to be a useful tool to biomonitor PBDEs, due to its ability to bioaccumulate individual congeners in varying concentrations in relation to the strength of the emission sources present over the territory. PBDEs levels widely ranged from 132 to 24,237 ng kg-1 dry weight, according to the sources of emission located around the monitoring sites. The highest concentrations were detected at the sites close to the municipal solid waste incinerator, steel plant, and high busy roads, confirming their important role as PBDEs emissions sources.

Polychlorinated Biphenyl Profile in Polyhydroxy-alkanoates Synthetized from Urban Organic Wastes.

The microbial synthesis of polyhydroxyalkanoates (PHA) from organic wastes is a valuable process to valorize available renewable resources, such as food wastes and biological sludge. Bioplastics find many applications in various sectors, from medical field to food industry. However, persistent organic pollutants could be transferred from wastes to the final product. The present paper demonstrates that the use of municipal wastes in PHA production is safe for the environment and human health and provides a polychlorinated biphenyl (PCB) profile in both commercial and waste-based PHA samples. PCB analysis in several PHA samples showed very low concentrations of the target analytes. Commercial PHA samples showed a similar PCB level with respect to PHA samples from municipal waste/sludge and higher than PHA samples from fruit waste. For all analyzed PCBs, detected concentrations were consistently lower than the ones reported in regulatory framework or guidelines.

Evaluation of Bioaerosol Bacterial Components of a Wastewater Treatment Plant Through an Integrate Approach and In Vivo Assessment.

Wastewater carries different pathogenic and non-pathogenic microorganisms that can be dispersed in the surrounding environment. Workers who frequent sewage treatment plants can therefore be exposed to aerosols that contain a high concentration of potentially dangerous biological agents, or they can come into direct contact with contaminated material. This can lead to allergies, infections and occupational health-associated diseases. A characterization of biological risk assessment of bioaerosol exposure is necessary. The aim of this study was to evaluate the application of an interdisciplinary method that combines chemical and biological approaches for the analysis of a bioaerosol derived from a wastewater treatment plant (WWTP) situated in Italy. Sampled filters were analyzed by HPLC-MS/MS spectroscopy that searched for different chemical biomarkers of airborne microorganisms. The analytical quantification was compared to the biological cultural method that revealed an underrated microbial concentration. Furthermore, next generation sequencing analysis was used also to identify the uncultivable species that were not detected by the culture dependent-method. Moreover, the simple animal model Caenorhabditis elegans was used to evaluate the pathogenicity of two isolates-Acinetobacter iwoffii and Micrococcus luteus-that showed multidrug-resistance. This work represents a starting point for the development of a multidisciplinary approach for the validation of bioaerosol exposure on WWTP workplaces.

Rumen microbiome in dairy calves fed copper and grape-pomace dietary supplementations: Composition and predicted functional profile.

The rumen microbiome is fundamental for the productivity and health of dairy cattle and diet is known to influence the rumen microbiota composition. In this study, grape-pomace, a natural source of polyphenols, and copper sulfate were provided as feed supplementation in 15 Holstein-Friesian calves, including 5 controls. After 75 days of supplementation, genomic DNA was extracted from the rumen liquor and prepared for 16S rRNA-gene sequencing to characterize the composition of the rumen microbiota. From this, the rumen metagenome was predicted to obtain the associated gene functions and metabolic pathways in a cost-effective manner. Results showed that feed supplementations did alter the rumen microbiome of calves. Copper and grape-pomace increased the diversity of the rumen microbiota: the Shannon's and Fisher's alpha indices were significantly different across groups (p-values 0.045 and 0.039), and Bray-Curtis distances could separate grape-pomace calves from the other two groups. Differentially abundant taxa were identified: in particular, an uncultured Bacteroidales UCG-001 genus and OTUs from genus Sarcina were the most differentially abundant in pomace-supplemented calves compared to controls (p-values 0.003 and 0.0002, respectively). Enriched taxonomies such as Ruminiclostridium and Eubacterium sp., whose functions are related to degradation of the grape- pomace constituents (e.g. flavonoids or xyloglucan) have been described (p-values 0.027/0.028 and 0.040/0.022 in Pomace vs Copper and Controls, respectively). The most abundant predicted metagenomic genes belonged to the arginine and proline metabolism and the two- component (sensor/responder) regulatory system, which were increased in the supplemented groups. Interestingly, the lipopolysaccharide biosynthetic pathway was decreased in the two supplemented groups, possibly as a result of antimicrobial effects. Methanogenic taxa also responded to the feed supplementation, and methane metabolism in the rumen was the second most different pathway (up-regulated by feed supplementations) between experimental groups.

Identification and Characterization of a Stem Cell-Like Population in Bovine Milk: A Potential New Source for Regenerative Medicine in Veterinary.

Milk is a complex fluid required for development, nutrition and immunological protection to the newborn offspring. Interestingly, latest finding proved the presence of novel stem cell population in human milk with multilineage differentiation potential. Given that little is known about cellular milk content in other mammalian species such as bovine, the purpose of our study was to isolate and characterize a potential stem cell-like population in bovine milk. In detail, we first analyzed the phenotype of the isolated cells able to grow in plastic adherence and then their capability to differentiate into osteogenic, chondrogenic, and adipogenic lineages. Bovine milk stem cells (bMSCs) resulted plastic adherent and showed a heterogeneous population with epithelial and spindle-shaped cells. Successively, their immunophenotype indicated that bovine milk cells were positive for the typical epithelial markers E-cadherin, cytokeratin-14, cytokeratin-18, and smooth muscle actin. Notably, a subset (30%-40%), constantly observed in purified milk cells, showed the typical mesenchymal surface antigens CD90, CD73, and CD105. Furthermore, the same percentage of bMSCs expressing CD90, CD73, and CD105 presented the stemness markers SOX2 and OCT4 translocated in their nuclei. Finally, our data showed that bMSCs were able to differentiate into osteoblasts, chondroblasts, and adipocytes. In addition, the flow cytometry analysis revealed the presence of a subpopulation of events characterized by typical extracellular vesicles (EVs, size 0.1-1 µm), which did not contain nuclei and were positive for the same markers identified on the surface of bMSCs (CD73, CD90, and CD105), and thus might be considered milk cell-derived EVs. In conclusion, our data suggest that bovine milk is an easily available source of multipotent stem cells able to differentiate into multiple cell lineages. These features can open new possibilities for development biology and regenerative medicine in veterinary area to improving animal health.

Computing Spatiotemporal Heat Maps of Lipid Electropore Formation: A Statistical Approach.

We extend the multiscale spatiotemporal heat map strategies originally developed for interpreting molecular dynamics simulations of well-structured proteins to liquids such as lipid bilayers and solvents. Our analysis informs the experimental and theoretical investigation of electroporation, that is, the externally imposed breaching of the cell membrane under the influence of an electric field of sufficient magnitude. To understand the nanoscale architecture of electroporation, we transform time domain data of the coarse-grained interaction networks of lipids and solvents into spatial heat maps of the most relevant constituent molecules. The application takes advantage of our earlier graph-based activity functions by accounting for the contact-forming and -breaking activity of the lipids in the bilayer. Our novel analysis of lipid interaction networks under periodic boundary conditions shows that the disruption of the bilayer, as measured by the breaking activity, is associated with the externally imposed pore formation. Moreover, the breaking activity can be used for statistically ranking the importance of individual lipids and solvent molecules through a bridging between fast and slow degrees of freedom. The heat map approach highlighted a small number of important lipids and solvent molecules, which allowed us to efficiently search the trajectories for any functionally relevant mechanisms. Our algorithms are freely disseminated with the open-source package TimeScapes.

Effect of diet supplementation with Ascophyllum nodosum on cow milk composition and microbiota.

Iodine deficiency remains a major public health concern in many countries, including some European regions. This study aimed at understanding the effect of a supplement of marine alga Ascophyllum nodosum as a iodine fortifier in the cow diet, on the compositional and microbiological quality of milk. The results obtained in this work indicated that the dietary inclusion of A. nodosum exerted significant effects on cow milk composition. In particular, it increased iodine content and reduced the quantity of free amino acids without modifying the free fatty acid content. From a microbiological point of view, statistically significant differences were found in presumptive mesophilic lactobacilli, mesophilic lactococci, and Pseudomonas spp. counts. Based on a culture-independent method, milk obtained after dietary inclusion of A. nodosum harbored the highest number of Firmicutes (e.g., Lactococcus lactis) and the lowest number of Proteobacteria (e.g., Pseudomonas). In addition to changes in bacterial population, diet supplementation with A. nodosum changed the catabolic profiles of the milk community, according to Biolog Ecoplate (Biolog Inc., Hayward, CA) results. The results of this study suggest that the dietary inclusion of the marine alga A. nodosum led to an improvement of the iodine content in milk, and to a modification of its microbiota with a positive effect on milk hygiene and transformation.

A rapid high-performance liquid chromatography-tandem mass spectrometry assay for unambiguous detection of different milk species employed in cheese manufacturing.

The aim of the study was to investigate the possibility to differentiate the 4 most important species in Italian dairy industry (cow, buffalo, sheep, and goat), applying a bottom-up proteomic approach to assess the milk species involved in cheese production. Selective peptides were detected in milk to use as markers in cheese products. Trypsin-digested milk samples of cow, sheep, goat, and buffalo, analyzed by HPLC-tandem mass spectrometry provided species-specific peptides, some of them recognized by Mascot software (Matrix Science Ltd., Boston, MA) as derived from well-known species specific proteins. A multianalyte multiple reaction monitoring method, built with these specific peptides, was successfully applied to cheeses with different composition, showing high specificity in detection of species involved. Neither aging nor production method seemed to affect the response, demonstrating that chosen peptides well act as species markers for dairy products.

Solid-state magic-angle spinning NMR of outer-membrane protein G from Escherichia coli.

Uniformly 13C-,15N-labelled outer-membrane protein G (OmpG) from Escherichia coli was expressed for structural studies by solid-state magic-angle spinning (MAS) NMR. Inclusion bodies of the recombinant, labelled protein were purified under denaturing conditions and refolded in detergent. OmpG was reconstituted into lipid bilayers and several milligrams of two-dimensional crystals were obtained. Solid-state MAS NMR spectra showed signals with an apparent line width of 80-120 Hz (including homonuclear scalar couplings). Signal patterns for several amino acids, including threonines, prolines and serines were resolved and identified in 2D proton-driven spin-diffusion (PDSD) spectra.