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School share tables offer opportunities for food recovery and increased access to healthy foods by allowing students to donate or consume unopened items, such as cartons of milk. However, stakeholders have concerns about temperature abuse potentially causing premature milk spoilage. While previous research showed short ambient temperature abuse of milk (under conditions representing share tables) does not meaningfully impact microbial milk quality, differences across school cafeterias (e.g., ambient temperatures, storage systems, bell schedules, refrigeration temperature) may limit the generalizability of this conclusion. To address this, the overnight refrigeration temperature and the milk's initial contamination were predicted to be the main drivers for milk spoilage. Share tables were predicted to only cause inconsequential microbial quality changes (4 spoiled milk per million served, which would be ≤2 milk cartons spoiled per school year) under short and medium bell schedules (≤125 min of total service), even without temperature control during the lunch period. Under long (221 min) and very long (266 min) bell schedules, share tables with ambient temperature storage were predicted to have higher milk spoilage (19 and 42 spoiled milk cartons per million served, respectively), and adding storage systems was predicted to reduce the decline in milk quality (12 and 24 spoiled milk cartons per million served, respectively). These results provide a resource to support science-based decision-making for the inclusion of milk in school cafeteria share tables, ultimately working to reduce food waste and address food insecurity.
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Due to the increase in the rate of male and female infertility, assisted fertilization practices are currently adopted as valid support for couples unable to get pregnant. Analytical approaches for fertility hormone dosages are constantly being developed, following the technological progress of fertilization methods that have evolved for more than a century. Indeed, the analysis of fertility hormones in serum samples is a common clinical practice to check the fertility state, but absolute quantification of these hormones is a great challenge due to biological variability and low serum concentrations. Currently, ELISA (enzyme-linked immunosorbent assay) based methods are the most used analytical techniques to quantify hormones in blood in clinical settings. The current Article discusses the development of a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) to monitor multiple fertility hormones of a protein nature in a single chromatographic run, i.e., LH (luteinizing hormone), FSH (follicle-stimulating hormone), TSH (thyroid-stimulating hormone), AMH (anti-Müllerian hormone), adiponectin, ghrelin, leptin, glucagon, and obestatin. Particular attention has been paid to the AMH hormone, whose ELISA-based quantification is known to be controversial due to the poor reproducibility between the various kits used. For AMH, the internal standard method was used for the quantitative determination to compare mass spectrometry data to the ELISA assays performed by an accredited analysis laboratory on a cohort of samples from women aged between 18 and 60 years. The ability to monitor multiple transitions by LC-MRM/MS ensured both high specificity and high selectivity, which is necessary for the quantification of protein and steroid hormones, besides improvements in data reproducibility and reduced analysis times and costs.
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To evaluate the potential differences in the propensity of ß-casein A1 (ß-CNA1) and A2 (ß-CNA2) from bovine milk to release health-relevant ß-casomorphins (BCMs), food-derived peptides were monitored over time in the blood of eight human volunteers who consumed milk containing both protein variants. Liquid chromatography coupled with high resolution tandem mass spectrometry revealed interindividual variability of milk peptidomic profiles in human blood. BCMs were not detected, whereas BCM precursors originating from both ß-CNA1 and ß-CNA2 were ascertained, with ß-CNA2-derived peptides showing a slightly greater susceptibility to proteolysis. Ten synthetic peptides mimicking circulating BCM precursors from ß-CNA1 and ß-CNA2, which were incubated ex vivo with the blood of two volunteers, showed comparable potential to generate BCMs. The formation of BCMs seemed to depend mainly on the size of the BCM precursors and less on the presence of His67 or Pro67. These findings challenge the belief that BCMs are released exclusively from ß-CNA1 and support the nutritional safety of conventional milk, informing health policies regarding milk consumption.
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Over the past few years, l-iminosugars have revealed attractive pharmacological properties for managing rare diseases including Cystic Fibrosis (CF). The iminosugar N-butyl-l-deoxynojirimycin (l-NBDNJ, ent-1), prepared by a carbohydrate-based route, was herein evaluated for its anti-inflammatory and anti-infective potential in models of CF lung disease infection. A significant decrease in the bacterial load in the airways was observed in the murine model of Pseudomonas aeruginosa chronic infection in the presence of l-NBDNJ, also accompanied by a modest reduction of inflammatory cells. Mechanistic insights into the observed activity revealed that l-NBDNJ interferes with the expression of proteins regulating cytoskeleton assembly and organization of the host cell, downregulates the main virulence factors of P. aeruginosa involved in the host response, and affects pathogen adhesion to human cells. These findings along with the observation of the absence of an in vitro bacteriostatic/bactericidal action of l-NBDNJ suggest the potential use of this glycomimetic as an antivirulence agent in the management of CF lung disease.
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We investigated the effect of growing on lactate instead of glucose in human cardiomyocyte assessing their viability, cell cycle activity, oxidative stress and metabolism by a proteomic and metabolomic approach. In previous studies performed on elite players, we found that adaptation to exercise is characterized by a chronic high plasma level of lactate. Lactate is considered not only an energy source but also a signalling molecule and is referred as "lactormone"; heart is one of the major recipients of exogenous lactate. With this in mind, we used a cardiac cell line AC16 to characterize the lactate metabolic profile and investigate the metabolic flexibility of the heart. Interestingly, our data indicated that cardiomyocytes grown on lactate (72 h) show change in several proteins and metabolites linked to cell hypertrophy and cytoskeleton remodelling. The obtained results could help to understand the effect of this metabolite on heart of high-performance athletes.
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Share tables (ST) are tables or stations in school cafeterias where students can return unopened foods and beverages, providing an opportunity to access these items at no cost. Currently, research suggests that milk is among the most wasted items in breakfast and lunch programs in the United States. Share tables present a simple solution for reducing milk waste, but research is needed to understand the microbial spoilage potential of milk in ST. To this end, uninoculated milk cartons and milk cartons inoculated with 2 to 3 log10(cfu/mL) Pseudomonas poae, a fast-growing psychrotroph, was exposed to ambient temperature during winter (mean temperature = 20.3°C) and summer (23.1°C) for 125 min, repeated over 5 d (the length of a school week). Microbial counts in the inoculated milk cartons increased linearly, exceeding the spoilage threshold of 6.0 log10(cfu/mL) after d 3 and after d 4 in the winter and summer season trials, respectively. In the winter trial, the microbial counts for uninoculated milk cartons never exceeded the lower limit of detection, 2.31 log10(cfu/mL), and in the summer trials, microbial counts never reached the spoilage threshold, indicating that initial contamination is a driving factor of microbial milk spoilage. Regardless of sharing status or seasonality, the greatest changes in counts for inoculated milk cartons occurred during overnight refrigeration, ranging from 0.56 to 1.4 log10(cfu/mL), while during the share table ranged from no observable change up to 0.29 log10(cfu/mL), emphasizing that school nutrition personnel should focus efforts on tightly controlling refrigeration temperatures and returning milk to refrigeration as soon as possible. A previously developed model for school cafeteria share tables was adapted to understand the typical residence time of milk in a simulated cafeteria with an ambient temperature share table for the summer and winter seasons over 1,000 wk. Milk was predicted to have a very short mean residence time (85 min) regardless of sharing status or season, with 99.8% of milk consumed, discarded, or donated within the first 2 d. As a result, only 3 out of 451,410 and 6 out of 451,410 simulated milks spoiled in the winter and summer seasons, respectively. The data generated here can be used to inform science-based decision-making for including milk in share tables, or applied to any system where one might have to accept short-term unrefrigerated storage of milk to meet a waste reduction or food security goal.
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OBJECTIVE: The etiopathogenesis of systemic sclerosis (SSc) is unknown. Platelet-derived growth factor receptors (PDGFRs) are overexpressed in patients with SSc. Because PDGFRα is targeted by the adeno-associated virus type 5 (AAV5), we investigated whether AAV5 forms a complex with PDGFRα exposing epitopes that may induce the immune responses to the virus-PDGFRα complex. METHODS: The binding of monomeric human PDGFRα to the AAV5 capsid was analyzed by in silico molecular docking, surface plasmon resonance (SPR), and genome editing of the PDGFRα locus. AAV5 was detected in SSc lungs by in situ hybridization, immunohistochemistry, confocal microscopy, and molecular analysis of bronchoalveolar lavage (BAL) fluid. Immune responses to AAV5 and PDGFRα were evaluated by SPR using SSc monoclonal anti-PDGFRα antibodies and immunoaffinity-purified anti-PDGFRα antibodies from sera of patients with SSc. RESULTS: AAV5 was detected in the BAL fluid of 41 of 66 patients with SSc with interstitial lung disease (62.1%) and in 17 of 66 controls (25.75%) (P < 0.001). In SSc lungs, AAV5 localized in type II pneumocytes and in interstitial cells. A molecular complex formed of spatially contiguous epitopes of the AAV5 capsid and of PDGFRα was identified and characterized. In silico molecular docking analysis and binding to the agonistic anti-PDGFRα antibodies identified spatially contiguous epitopes derived from PDGFRα and AAV5 that interacted with SSc agonistic antibodies to PDGFRα. These peptides were also able to bind total IgG isolated from patients with SSc, not from healthy controls. CONCLUSION: These data link AVV5 with the immune reactivity to endogenous antigens in SSc and provide a novel element in the pathogenesis of SSc.
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Enfermedades Pulmonares Intersticiales , Esclerodermia Sistémica , Humanos , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Epítopos , Dependovirus/metabolismo , Autoanticuerpos , Simulación del Acoplamiento Molecular , Esclerodermia Sistémica/patología , Péptidos , Pulmón/patologíaRESUMEN
The in-depth studies over the years on the defence barriers by tomato plants have shown that the Systemin peptide controls the response to a wealth of environmental stress agents. This multifaceted stress reaction seems to be related to the intrinsic disorder of its precursor protein, Prosystemin (ProSys). Since latest findings show that ProSys has biological functions besides Systemin sequence, here we wanted to assess if this precursor includes peptide motifs able to trigger stress-related pathways. Candidate peptides were identified in silico and synthesized to test their capacity to trigger defence responses in tomato plants against different biotic stressors. Our results demonstrated that ProSys harbours several repeat motifs which triggered plant immune reactions against pathogens and pest insects. Three of these peptides were detected by mass spectrometry in plants expressing ProSys, demonstrating their effective presence in vivo. These experimental data shed light on unrecognized functions of ProSys, mediated by multiple biologically active sequences which may partly account for the capacity of ProSys to induce defense responses to different stress agents.
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Péptidos , Proteínas de Plantas , Péptidos/metabolismo , Proteínas de Plantas/metabolismoRESUMEN
The valorisation and conservation of plant genetic resources (PGRs) and wild fruit PGRs are critical to ensure the maintenance of genetic and cultural heritage and to promote new perspectives on resource use. New strategies to characterize PGRs are needed, and the omics approach can provide information that is still largely unknown. The Strawberry tree (Arbutus unedo L.) is an underutilized, drought and fire-resistant species distributed in the Mediterranean area and its berries have large ethnobotanical use. Although their phenolic profile and antioxidant capacity are known, they are not well characterised, particularly from a proteomic perspective. The aim of this work is the characterisation of two ecotypes of A. unedo (Campania and Sicily) from a molecular viewpoint to valorise and encourage the preservation of this wild fruit. Samples were collected from two different geographical areas to assess whether different geographical conditions could influence the characteristics of leaves and fruits at the three stages of ripening (green, veraison, red). Proteomic analysis identified 904 proteins, of which 122 showed significance along the ripening. Some of these differentially abundant proteins, such as chalcone synthase, show a marked increase during ripening. The protein functional classes with the highest representation are involved in protein and amino acid metabolism, glycolysis and in secondary metabolism. From a proteomic perspective, there are no differences between the fruits from the two regions compared by the ripening stage. However, the pedoclimatic metabolic imprinting allowed the observation of good diversity in the metabolomic profiles between the two ecotypes, especially for anthocyanins, 4 times more abundant in the Sicilian veraisoned fruit than in the Campania one, and catechins, with double the abundance in the Campania ecotype compared to the Sicilian ecotype in the green phase, but more abundant (3x) in the Sicilian veraisoned fruit. Phenolic compounds show a 20% greater abundance in the Campania green arbutus fruit than in the Sicilian one, values that then equalise as ripening progresses. Multi-omic characterisation enhanced the knowledge on a wild fruit plant species which shows specific adaptations and responses to the environment to be considered when addressing the issue of local agrobiodiversity.
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Inflammatory response triggered by innate immunity can act to protect against microorganisms that behave as pathogens, with the aim to restore the homeostatic state between host and beneficial microbes. As a filter-feeder organism, the ascidian Ciona robusta is continuously exposed to external microbes that may be harmful under some conditions. In this work, we used transcriptional and proteomic approaches to investigate the inflammatory response induced by stimuli of bacterial (lipopolysaccharide -LPS- and diacylated lipopeptide - Pam2CSK4) and fungal (zymosan) origin, in Ciona juveniles at stage 4 of metamorphosis. We focused on receptors, co-interactors, transcription factors and cytokines belonging to the TLR and Dectin-1 pathways and on immune factors identified by homology approach (i.e. immunoglobulin (Ig) or C-type lectin domain containing molecules). While LPS did not induce a significant response in juvenile ascidians, Pam2CSK4 and zymosan exposure triggered the activation of specific inflammatory mechanisms. In particular, Pam2CSK4-induced inflammation was characterized by modulation of TLR and Dectin-1 pathway molecules, including receptors, transcription factors, and cytokines, while immune response to zymosan primarily involved C-type lectin receptors, co-interactors, Ig-containing molecules, and cytokines. A targeted proteomic analysis enabled to confirm transcriptional data, also highlighting a temporal delay between transcriptional induction and protein level changes. Finally, a protein-protein interaction network of Ciona immune molecules was rendered to provide a wide visualization and analysis platform of innate immunity. The in vivo inflammatory model described here reveals interconnections of innate immune pathways in specific responses to selected microbial stimuli. It also represents the starting point for studying ontogeny and regulation of inflammatory disorders in different physiological conditions.
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Cordados no Vertebrados , Animales , Lipopolisacáridos , Proteómica , Zimosan , Inmunidad Innata , Citocinas , Anticuerpos , Lectinas Tipo CRESUMEN
Here, an unprecedented biorefinery approach has been designed to recover high-added value bioproducts starting from the culture ofPorphyridium cruentum. This unicellular marine red alga can secrete and accumulate high-value compounds that can find applications in a wide variety of industrial fields. 300 ± 67 mg/L of exopolysaccharides were obtained from cell culture medium; phycoerythrin was efficiently extracted (40% of total extract) and isolated by single chromatography, with a purity grade that allowed the crystal structure determination at 1.60 Å; a twofold increase in ß-carotene yield was obtained from the residual biomass; the final residual biomass was found to be enriched in saturated fatty acids. Thus, for the first time, a complete exploitation ofP. cruentumculture was set up.
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The aim of this study was to characterize the salivary proteome and metabolome of highly trained female and male young basketball players, highlighting common and different traits. A total of 20 male and female basketball players (10 female and 10 male) and 20 sedentary control subjects (10 female and 10 male) were included in the study. The athletes exercised at least five times per week for 2 h per day. Saliva samples were collected mid-season, between 9:00 and 11:00 a.m. and away from sport competition. The proteome and metabolome were analyzed by using 2DE and GC-MS techniques, respectively. A computerized 2DE gel image analysis revealed 43 spots that varied in intensity among groups. Between these spots, 10 (23.2%) were differentially expressed among male athletes and controls, 22 (51.2%) between female basketball players and controls, 11 spots (25.6%) between male and female athletes, and 13 spots (30.2%) between male and female controls. Among the proteins identified were Immunoglobulin, Alpha-Amylase, and Dermcidin, which are inflammation-related proteins. In addition, several amino acids, such as glutamic acid, lysine, ornithine, glycine, tyrosine, threonine, and valine, were increased in trained athletes. In this study, we highlight that saliva is a useful biofluid to assess athlete performance and confirm that the adaptation of men and women to exercise has some common features, but also some different sex-specific behaviors, including differential amino acid utilization and expression of inflammation-related proteins, which need to be further investigated. Moreover, in the future, it will be interesting to examine the influence of sport-type on these differences.
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Antimicrobial peptides (AMPs) are a unique and diverse group of molecules endowed with a broad spectrum of antibiotics properties that are being considered as new alternative therapeutic agents. Most of these peptides are membrane-active molecules, killing bacteria by membrane disruption. However, recently an increasing number of AMPs was shown to enter bacterial cells and target intracellular processes fundamental for bacterial life. In this paper we investigated the mechanism of action of Maganin-2 (Mag-2), a well-known antimicrobial peptide isolated from the African clawed frog Xenopus laevis, by functional proteomic approaches. Several proteins belonging to E. coli macromolecular membrane complexes were identified as Mag-2 putative interactors. Among these, we focused our attention on BamA a membrane protein belonging to the BAM complex responsible for the folding and insertion of nascent ß-barrel Outer Membrane Proteins (OMPs) in the outer membrane. In silico predictions by molecular modelling, in vitro fluorescence binding and Light Scattering experiments carried out using a recombinant form of BamA confirmed the formation of a stable Mag-2/BamA complex and indicated a high affinity of the peptide for BamA. Functional implications of this interactions were investigated by two alternative and complementary approaches. The amount of outer membrane proteins OmpA and OmpF produced in E. coli following Mag-2 incubation were evaluated by both western blot analysis and quantitative tandem mass spectrometry in Multiple Reaction Monitoring scan mode. In both experiments a gradual decrease in outer membrane proteins production with time was observed as a consequence of Mag-2 treatment. These results suggested BamA as a possible good target for the rational design of new antibiotics since this protein is responsible for a crucial biological event of bacterial life and is absent in humans.
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In the last decade or so, polychlorinated biphenyls (PCBs) garnered renewed attention in the scientific community due to new evidence pointing at their continued presence in the environment and workplaces and the potential human risks related to their presence. PCBs move from the environment to humans through different routes; the dominant pathway is the ingestion of contaminated foods (fish, seafood and dairy products), followed by inhalation (both indoor and outdoor air), and, to a lesser extent, dust ingestion and dermal contact. Numerous studies reported the environmental and occupational exposure to these pollutants, deriving from building materials (flame-retardants, plasticizers, paints, caulking compounds, sealants, fluorescent light ballasts, etc.) and electrical equipment. The highest PCBs contaminations were detected in e-waste recycling sites, suggesting the need for the implementation of remediation strategies of such polluted areas to safeguard the health of workers and local populations. Furthermore, a significant correlation between PCB exposure and increased blood PCB concentrations was observed in people working in PCB-contaminated workplaces. Several epidemiological studies suggest that environmental and occupational exposure to high concentrations of PCBs is associated with different health outcomes, such as neuropsychological and neurobehavioral deficits, dementia, immune system dysfunctions, cardiovascular diseases and cancer. In addition, recent studies indicate that PCBs bioaccumulation can reduce fertility, with harmful effects on the reproductive system that can be passed to offspring. In the near future, further studies are needed to assess the real effects of PCBs exposure at low concentrations for prolonged exposure in workplaces and specific indoor environments.
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This work focused on the extraction, quantification, and characterization of bioactive compounds of Arbutus unedo L. fruits, comparing the results obtained from the different ripening states. Extractions were performed by different methods (such as maceration extraction and ultrasonic extraction) and food grade solvents (aqueous and hydroalcoholic solvents) in each of the all ripening states (four states considered, associated with four different colors, i.e., green, yellow, orange, and red). The presence of (poly)phenols was quantified and characterized, and scavenging activity was determined by the Folin-Ciocâlteu reagent and the DPPH method, respectively. The content of bioactive compounds was characterized by LC-MS/MS, such as multiple reaction monitoring (MRM) mass spectrometry. The results showed that ultrasound-assisted extraction (UAE) performed better than maceration extraction; ethanol-water mixture extracts showed a more positive effect than the use of aqueous extracts regarding the content of total phenolic compounds. Overall, the total phenolic compounds in the EtOH:H2O mixture at a ratio of 7:3 (v:v) were higher than that of the other solvents for both extraction methods. Some bioactive molecules were characterized for the first time in the extracts of A. unedo. The chemical profile of the strawberry tree extracts depended on the degree of fruit ripeness. The results suggest that A. unedo fruits may be of great interest for food and nutraceutical applications.
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223Radium dichloride image-based individual dosimetry requires an optimal acquisition and reconstruction protocol and proper image correction methods for theranostic applications. To assess this problem, radium-223 dichloride SPECT images were acquired from a Jaszczak simulator with a dual-headed gamma camera, LEHR collimator, 128 × 128 matrix, and total time of 32 minutes. A cylindrical PMMA phantom was used to calibrate the measurements performed with Jaszczak. The image quality parameters (noise coefficient, contrast, contrast-to-noise ratio and recovery coefficient) and septal penetration correction were calculated by MATLAB®. The best results for the investigated image quality parameters were obtained with an 89 keV energy window (24% wide) produced together with OSEM/MLEM reconstruction (8 subsets and 4 iterations) applying a Butterworth filter (order 10 and cutoff frequency of 0.48 cycles·cm-1). The successfully performed recovery coefficient parameter evaluation allows uptake correction for future patient dosimetry applications.
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Medicina de Precisión , Tomografía Computarizada de Emisión de Fotón Único , Humanos , Fantasmas de ImagenRESUMEN
Oxylipins are signaling molecules originated by fatty acids that modulate vascular and bronchial tone, bronchial secretion, cytokine production and immune cell activity. The unbalanced production of pro-inflammatory and pro-resolving (i.e., anti-inflammatory) oxylipins has a relevant role in the pathogenesis of pulmonary inflammation like in cystic fibrosis (CF). We analyzed by LC-MRM/MS 65 oxylipins and 4 fatty acids in resting saliva from 69 patients with CF and 50 healthy subjects (controls). The salivary levels of 48/65 oxylipins were significantly different between CF patients and controls. Among these, EpETE, DHET, 6ketoPGE1 and HDHA were significantly higher in saliva from CF patients than in controls. All these molecules display anti-inflammatory effects, i.e., releasing of bronchial and vascular tone, modulation of cytokine release. While 20-hydroxyPGF2A, PGB2, EpDPE, 9 K-12-ELA, bicyclo-PGE2, oleic acid, LTC4, linoleic acid, 15oxoEDE, 20 hydroxyPGE2 and DHK-PGD2/PGE2 (mostly associated to pro-inflammatory effects) resulted significantly lower in CF patients than in controls. Our data suggest that the salivary oxylipins profile in CF patients is addressed toward a global anti-inflammatory effect. Although these findings need be confirmed on larger populations in prospective studies, they will contribute to better understand the pathogenesis of CF chronic inflammation and to drive targeted therapies based on the modulation of oxylipins synthesis and degradation.
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Fibrosis Quística , Oxilipinas , Antiinflamatorios/metabolismo , Fibrosis Quística/metabolismo , Citocinas/metabolismo , Dinoprostona , Ácidos Grasos , Humanos , Oxilipinas/metabolismo , Estudios Prospectivos , Saliva/metabolismoRESUMEN
Monitoring fatigue and recovery during training periods contributes to identifying the best training methods to achieve sports performance. To date, little is known about sex-related differences in sports adaptations. The aim of the present study is to identify sex-related sports adaptation proteins in female basketball players and male basketball players using proteomics approach on plasma samples withdrawn from athletes during in-season training period but far from a competition. A cohort of 20 professional basketball players, 10 female (BF) and 10 male (BM), and 20 sedentary male (10 CM) and female (10 CF) as control, of comparable age and BMI, were involved in this study. Protein profiles of plasma samples obtained from BM, BF, CM, and CF were analyzed by two-dimensional electrophoresis (2-DE). Differentially expressed proteins were identified by mass spectrometry. The computational 2-DE gel image analysis pointed out 33 differentially expressed protein spots (ANOVA p-value < 0.05) and differences between male and female basketball players are more evident among the players than controls. The expression profile of 54.5% of the total proteins is affected by sports activity. Furthermore, 14 proteins are differentially expressed in basket female players in comparison with their relative controls while seven are differentially expressed in basket male players in comparison with their controls. In conclusion, we identify in female athletes a reduction in proteins related to transcription regulation, most of these modulate chronic inflammation confirming the anti-inflammatory effect of regular training in female muscle metabolism. In male and female athletes, we found a decrease in Transthyretin involved in muscle homeostasis and regeneration and Dermcidin a stress-induced myokine linked to inflammatory and it will be interesting to fully understand the role of its different isoforms in male and female skeletal muscle contraction.