Vulvo-vaginal epithelial tumors in mares: A preliminary investigation on epithelial-mesenchymal transition and tumor-immune microenvironment.

Vulvo-vaginal epithelial tumors are uncommon in mares, and data on the epithelial-to-mesenchymal transition (EMT) and the tumor-immune microenvironment (TIME) are still lacking. This is a study investigating the equus caballus papillomavirus type 2 (EcPV2) infection state as well as the EMT process and the tumor microenvironment in vulvo-vaginal preneoplastic/ benign (8/22) or malignant (14/22) epithelial lesions in mares. To do this, histopathological, immunohistochemical, transcriptomic, in situ hybridization, and correlation analyses were carried out. Immunohistochemistry quantification showed that cytoplasmic E-cadherin and ß-catenin expression as well as nuclear ß-catenin expression were features of malignant lesions, while benign/preneoplastic lesions were mainly characterized by membranous E-cadherin and ß-catenin expression. Despite this, there were no differences between benign and malignant equine vulvo-vaginal lesions in the expression of downstream genes involved in the canonical and noncanonical wnt/ß-catenin pathways. In addition, malignant lesions were characterized by a lower number of cells with cytoplasmic cytokeratin expression as well as a slightly higher cytoplasmic vimentin immunolabeling. The TIME of malignant lesions was characterized by more numerous CD204+ M2-polarized macrophages. Altogether, our results support the hypothesis that some actors in TIME such as CD204+ M2-polarized macrophages may favor the EMT process in equine vulvo-vaginal malignant lesions providing new insights for future investigations in the field of equine EcPV2-induced genital neoplastic lesions.

RNAseq Analysis of Livers from Pigs Treated with Testosterone and Nandrolone Esters: Selection and Field Validation of Transcriptional Biomarkers.

The use of anabolic-androgenic steroids (AASs) as growth promoters in farm animals is banned in the European Union, representing both an illicit practice and a risk for consumer health. However, these compounds are still illegally administered, often in the form of synthetic esters. This work aimed to characterize significant coding RNA perturbations related to the illicit administration of testosterone and nandrolone esters in fattening pigs. A total of 27 clinically healthy 90-day-old pigs were randomly assigned to test and control groups. Nine animals were treated with testosterone esters (Sustanon®) and other nine with nandrolone esters (Myodine®). At the end of the trial, liver samples were collected and analyzed using RNAseq, allowing the identification of 491 differentially expressed genes (DEGs). The transcriptional signature was further characterized by a smaller sub-cluster of 143 DEGs, from which a selection of 16 genes was made. The qPCR analysis confirmed that the identified cluster could still give good discrimination between untreated gilt and barrows compared to the relative testosterone-treated counterparts. A conclusive field survey on 67 liver samples collected from pigs of different breeds and weight categories confirmed, in agreement with testosterone residue profiles, the specificity of selected transcriptional biomarkers, showing their potential applications for screening purposes when AAS treatment is suspected, allowing to focus further investigations of competent authorities and confirmatory analysis where needed.

Disclosing Frauds in Herbal Food Supplements Labeling: A Simple LC-MS/MS Approach to Detect Alkaloids and Biogenic Amines.

Food supplements are a category of products perceived safe and therefore commonly used by different categories of consumers without any particular attention or precaution. However, health risks associated with the consumption of supplements containing undeclared substances cannot be excluded. A variety of analytical methods are used to control supplement quality composition, but usually, these procedures are complex and time-consuming. Here, we report the results of a simple and fast liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, to detect and quantify simultaneously different categories of active molecules, such as biogenic ammines and natural alkaloids that at high doses can produce negative health effect in consumers. Three categories of products intended for body weight loss, energy boosting, and erectile dysfunction treatment, purchased through e-commerce sites and from local supermarkets, were analyzed (n = 91). The caffeine, synephrine, agmatine sulfate, yohimbine, phenethylamine, and icariin were correctly separated and identified with good precision (RSD < 20%) and recovery (89-109%). The identification and quantification of the analytes in real samples highlighted that the 26% of the samples were not compliant with labeling, confirming that frauds are very common also in the natural supplements market. This LC-MS/MS method could be easily used to test natural supplements in order to check the correct labeling and to protect consumers from potential health risks and food frauds.

Epithelial-mesenchymal transition in an EcPV2-positive vulvar squamous cell carcinoma of a mare.

BACKGROUND: Vulvar squamous cell carcinoma (VSCC) has been recently associated with Equus caballus papillomavirus type 2 (EcPV2) infection. Still, few reports concerning this disease are present in the literature. OBJECTIVE: To describe a case of naturally occurring EcPV2-induced VSCC, by investigating tumour ability in undergoing the epithelial-to-mesenchymal transition (EMT). STUDY DESIGN: Case report. METHODS: A 13-year-old Haflinger mare was referred for a rapidly growing vulvar mass. After surgical excision, the mass was submitted to histopathology and molecular analysis. Histopathological diagnosis was consistent with a VSCC. Real-time qPCR, real-time reverse transcriptase (RT)-qPCR and RNAscope were carried out to detect EcPV2 infection and to evaluate E6/E7 oncogenes expression. To highlight the EMT, immunohistochemistry (IHC) was performed. Expression of EMT-related and innate immunity-related genes was investigated through RT-qPCR. RESULTS: Real-time qPCR, RT-qPCR and RNAscope confirmed EcPV2 DNA presence and expression of EcPV2 oncoproteins (E6 and E7) within the neoplastic vulvar lesion. IHC highlighted a cadherin switch together with the expression of the EMT-related transcription factor HIF1α. With RT-qPCR, significantly increased gene expression of EBI3 (45.0 ± 1.62, p < 0.01), CDH2 (2445.3 ± 0.39, p < 0.001), CXCL8 (288.7 ± 0.40, p < 0.001) and decreased gene expression of CDH1 (0.3 ± 0.57, p < 0.05), IL12A (0.04 ± 1.06, p < 0.01) and IL17 (0.2 ± 0.64, p < 0.05) were detected. MAIN LIMITATIONS: Lack of ability to generalise and danger of over-interpretation. CONCLUSION: The results obtained were suggestive of an EMT event occurring within the neoplastic lesion.

Identification of Illicit Conservation Treatments in Fresh Fish by Micro-Raman Spectroscopy and Chemometric Methods.

In the field of food control for fresh products, the identification of foods subjected to illicit conservation treatments to extend their shelf life is fundamental. Fresh fish products are particularly subjected to this type of fraud due to their high commercial value and the fact that they often have to be transported over a long distance, keeping their organoleptic characteristics unaltered. Treatments of this type involve, e.g., the bleaching of the meat and/or the momentary abatement of the microbial load, while the degradation process continues. It is therefore important to find rapid methods that allow the identification of illicit treatments. The study presented here was performed on 24 sea bass samples divided into four groups: 12 controls (stored on ice in the fridge for 3 or 24 h), and 12 treated with a Cafodos-like solution for 3 or 24 h. Muscle and skin samples were then characterized using micro-Raman spectroscopy. The data were pre-processed by smoothing and taking the first derivative and then PLS-DA models were built to identify short- and long- term effects on the fish's muscle and skin. All the models provided the perfect classification of the samples both in fitting and cross-validation and an analysis of the bands responsible for the effects was also reported. To the best of the authors' knowledge, this is the first time Raman spectroscopy has been applied for the identification of a Cafodos-like illicit treatment, focusing on both fish muscle and skin evaluation. The procedure could pave the way for a future application directly on the market through the use of a portable device.

Pro-Inflammatory and Cytotoxic Effects of Polystyrene Microplastics on Human and Murine Intestinal Cell Lines.

Plastic is a polymer extremely resistant to degradation that can remain for up to hundreds or thousands of years, leading to the accumulation of massive amounts of plastic waste throughout the planet's ecosystems. Due to exposure to various environmental factors, plastic breaks down into smaller particles named microplastics (1-5000 µm) and nanoplastics (<1 µm). Microplastics (MPs) are ubiquitous pollutants but, still, little is known about their effects on human and animal health. Herein, our aim is to investigate cytotoxicity, oxidative stress, inflammation and correlated gene modulation following exposure to polystyrene microplastics (PS-MPs) in HRT-18 and CMT-93 epithelial cell lines. After 6, 24 and 48 h PS-MPs treatment, cell viability (MTT) and oxidative stress (SOD) assays were performed; subsequently, expression changes and cytokines release were investigated by Real-Time PCR and Magnetic-beads panel Multiplex Assay, respectively. For each exposure time, a significantly increased cytotoxicity was observed in both cell lines, whereas SOD activity increased only in CMT-93 cells. Furthermore, Magnetic-beads Multiplex Assay revealed an increased release of IL-8 in HRT-18 cells' medium, also confirmed by gene expression analysis. Results obtained suggest the presence of a pro-inflammatory pattern induced by PS-MPs treatment that could be related to the observed increase in cytotoxicity.

Current State-of-the-Art Spectroscopic and Chromatographic Techniques Utilized in Food Authenticity and Food Traceability.

Food products are heterogeneous and complex matrices characterized by various compounds and in variable proportions [...].

Multi-Level System to Assess Toxicity in Water Distribution Plants.

The toxicity of water samples from water distribution plants needs to be investigated further. Indeed, studies on the pro-oxidant effects driven by tap water are very limited. In this study, the water quality, pro-oxidant effects, and potential health risks driven by exposure to groundwater samples from two water plants (sites A and B) located in Northwestern Italy were investigated in a multi-level system. Physicochemical parameters and the absence of pathogens, cyanotoxins, and endocrine active substances indicated a good water quality for both sites. The 25 metals analyzed were found under the limit of quantification or compliant with the maximum limits set by national legislation. Water samples were concentrated by the solid-phase extraction system in order to assess the aquatic toxicity on Epithelioma papulosum cyprini (EPC) cell line. Levels of superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, and glutathione reductase were evaluated through the Integrated Biomarkers Response (IBRv2) index. EPC cell line was found a sensible model for assessing the antioxidant responses driven by both water concentrates. A similar antioxidant response was shown by plots and IBRv2 suggesting a muted risk for the two sampling sites.

Chemometric Differentiation of Sole and Plaice Fish Fillets Using Three Near-Infrared Instruments.

Fish species substitution is one of the most common forms of fraud all over the world, as fish identification can be very challenging for both consumers and experienced inspectors in the case of fish sold as fillets. The difficulties in distinguishing among different species may generate a "grey area" in which mislabelling can occur. Thus, the development of fast and reliable tools able to detect such frauds in the field is of crucial importance. In this study, we focused on the distinction between two flatfish species largely available on the market, namely the Guinean sole (Synaptura cadenati) and European plaice (Pleuronectes platessa), which are very similar looking. Fifty fillets of each species were analysed using three near-infrared (NIR) instruments: the handheld SCiO (Consumer Physics), the portable MicroNIR (VIAVI), and the benchtop MPA (Bruker). PLS-DA classification models were built using the spectral datasets, and all three instruments provided very good results, showing high accuracy: 94.1% for the SCiO and MicroNIR portable instruments, and 90.1% for the MPA benchtop spectrometer. The good classification results of the approach combining NIR spectroscopy, and simple chemometric classification methods suggest great applicability directly in the context of real-world marketplaces, as well as in official control plans.

Transcriptional Biomarkers and Immunohistochemistry for Detection of Illicit Dexamethasone Administration in Veal Calves.

Corticosteroids such as Dexamethasone (DEX) are commonly licensed for therapy in meat animals due to their known pharmacological properties. However, their misuse aimed to achieve anabolic effects is often found by National Residues Control Plans. The setup of a complementary "biomarker based" methods to unveil such illicit practices is encouraged by current European legislation. In this study, the combined use of molecular and histological quantitative techniques was applied on formalin fixed paraffin embedded (FFPE) muscle samples to assess the effects of illicit DEX treatment on veal calves. A PCR array, including 28 transcriptional biomarkers related to DEX exposure, was combined with a histochemical analysis of muscle fiber. An analysis based on unsupervised (PCA) and supervised (PLS-DA and Kohonen's SOM) methods, was applied in order to define multivariate models able to classify animals suspected of illicit treatment by DEX. According to the conventional univariate approach, a not-significant reduction in type I fibres was recorded in the DEX-treated group, and only 12 out of 28 targeted genes maintained their expected differential expression, confirming the technical limitations of a quantitative analysis on FFPE samples. However, the multivariate models developed highlighted the possibility to establish complementary screening strategies, particularly when based on transcriptional biomarkers characterised by low expression profiles.

Validation of serum paraoxonase/arylesterase 1 (PON1) as a protein marker of illicit dexamethasone treatment in veal calves.

The illicit use of dexamethasone and other glucocorticoids for cattle fattening in livestock production has been widely described; evidence for illegal treatments can be obtained by direct or indirect detection. In our previous study, we applied two-dimensional electrophoresis (2DE) to identify plasma protein markers of dexamethasone administration in veal calves. Comparison of 2DE maps obtained from blood samples before and after treatment showed the disappearance of two protein spots identified as serum paraoxonase/arylesterase 1 precursor (PON1). In the present study, we validated PON1 as a marker by analysing a larger number of samples treated with dexamethasone for illicit use. Analysis of samples from experimental treatment with other glucocorticoids, androgens and oestrogens confirmed that their influence on PON1 could be excluded. The specificity of the PON1 protein marker was verified on expected negative field samples to exclude interfering factors. However, there is poor statistical evidence to support a significant association between the outcome of PON1 and the considered variables. The results on field samples were compared with histological examination of the thymus as a biomarker of corticosteroid treatment monitored in the Italian histological plan for the control of growth promoters in animals. Two suspect cases were identified from two Piedmont farms where other animals had tested positive at histological examination. In conclusion, the absence of PON1 in the plasma of veal calves can indirectly reveal illicit dexamethasone treatment in individual animals and so identify suspect farms for further investigation. It is effective in a period ranging from 3 to about 10 days from illicit treatment, covering a time span that goes beyond the limits of official chemical controls and preceding histological controls on the thymus of slaughtered animals. PON1 detection in plasma can be coupled with other tests to identify illegal dexamethasone use on veal calf farms.

The Promise and Challenges of Determining Recombinant Bovine Growth Hormone in Milk.

Recombinant bovine growth hormone (rbGH) is produced in large quantities and widely used in a number of countries worldwide to stimulate milk production in dairy animals. The use of this compound in animal production is strictly regulated by food safety directives in force, in particular in the European Union (EU). Although analytical strategies for the detection of rbGH in blood have been successfully reported over the past 15 years, they do not fully answer the expectations of either competent authorities or industrials that would expect measuring its occurrence directly in the milk. As a matrix of excretion but also of consumption, milk appears indeed as the matrix of choice for detecting the use of rbGH in dairy animals. It also allows large volumes to be collected without presenting an invasive character for the animal. However, rbGH detection in milk presents several challenges, mainly related to the sensitivity required for its detection in a complex biological matrix. This review article presents the specific difficulties associated with milk and provides an overview of the analytical strategies reported in the literature and whether they concern indirect or direct approaches to the detection of rbGH administration to animals, with applications either for screening or confirmation purposes.

Nandrolone and estradiol biomarkers identification in bovine urine applying a liquid chromatography high-resolution mass spectrometry metabolomics approach.

With the aim of specifically investigating patterns associated with three steroid treatments (17ß-nandrolone, 17ß-estradiol, and 17ß-nandrolone + 17ß-estradiol) in bovine, an reversed phase liquid chromatography (RPLC)-electrospray ionization (ESI)(+/-)-high-resolution mass spectrometry (HRMS) study was conducted to characterize the urinary profiles of involved animals. Although specific fingerprints with strong differences could be highlighted between urinary metabolite profiles within urine samples collected on control and treated animals, it appeared further that significant discriminations could also be observed between steroid treatments, evidencing thus specific patterns and candidate biomarkers associated to each treatment. An MS-2 structural elucidation step enabled level-1 identification of two biomarkers mainly involved in energy pathways, in relation to skeletal muscle functioning. These results make it possible to envisage a global strategy for the detection of anabolic practices involving steroids, while at the same time providing clues as to the compounds used, which would facilitate the confirmation stage to follow.

Anabolic treatments in bovines: quantification of plasma protein markers of dexamethasone administration.

The aim of this study was to profile plasma proteome responses in bulls experimentally treated with dexamethasone at anabolic dosage. Illicit use of active substances in animal husbandry remains a matter of concern in Europe. Corticosteroids are probably one of the most widespread growth promoter family illegally used in beef cattle and veal calves. Testing for corticosteroids relies on detection of drug residues or their metabolites in biological fluids or tissues. Their indirect detection by mapping altered physiological parameters may overcome limits linked to route of administration, dosage, biotransformation and elimination kinetics that can lower residual drug concentration, hampering official controls. A set of 11 proteins proposed in literature as potential markers of anabolic treatments with dexamethasone, was quantified in bovine plasma by targeted proteomics based on liquid chromatography-high resolution tandem mass spectrometry. Among investigated proteins, sex hormone-binding globulin (SHBG), histidine-rich glycoprotein (HRG) and paraoxonase-1 (PON1) were found to be biomarkers of treatment. To investigate further such biomarkers, an additional group of veal calves was experimentally treated with dexamethasone at anabolic. These animals also demonstrated a significant alteration in SHBG, HRG and PON1 concentration, suggesting that quantification of plasma markers have the potential to detect animals illegally exposed to dexamethasone.

Differentiation between Fresh and Thawed Cephalopods Using NIR Spectroscopy and Multivariate Data Analysis.

The sale of frozen-thawed fish and fish products, labeled as fresh, is currently one of the most common and insidious commercial food frauds. For this reason, the demand of reliable tools to identify the storage conditions is increasing. The present study was performed on two species, commonly sold in large-scale distribution: Cuttlefish (Sepia officinalis) and musky octopus (Eledone spp.). Fifty fresh cephalopod specimens were analyzed at refrigeration temperature (2 ± 2 °C), then frozen at -20 °C for 10 days and finally thawed and analyzed again. The performance of three near-infrared (NIR) instruments in identifying storage conditions were compared: The benchtop NIR Multi Purpose Analyzer (MPA) by Bruker, the portable MicroNIR by VIAVI and the handheld NIR SCiO by Consumer Physics. All collected spectra were processed and analyzed with chemometric methods. The SCiO data were also analyzed using the analytical tools available in the online application provided by the manufacturer to evaluate its performance. NIR spectroscopy, coupled with chemometrics, allowed discriminating between fresh and thawed samples with high accuracy: Cuttlefish between 82.3-94.1%, musky octopus between 91.2-97.1%, global model between 86.8-95.6%. Results show how food frauds could be detected directly in the marketplace, through small, ultra-fast and simplified handheld devices, whereas official control laboratories could use benchtop analytical instruments, coupled with chemometric approaches, to develop accurate and validated methods, suitable for regulatory purposes.

A novel tool to screen for treatments with clenbuterol in bovine: Identification of two hepatic markers by metabolomics investigation.

Surveillance of illegal use of growth promoters such as ß2-agonists in food producing animals rely on the detection of drug residues by LC-MS/MS. Screening strategies focusing on indirect physiological responses following administration of active compounds are promising approaches to strengthen existing targeted methods and ensure food safety. A metabolomics analysis based on LC-HRMS was carried out on liver extracts from bulls experimentally treated with clenbuterol combined with dexamethasone (n = 8) to mimic a potential anabolic practice, and control animals (n = 8). Nicotinic acid and 5'-deoxy-5'-methylthioadenosine were identified as biomarkers of treatment. Ratio values of such markers to others of the same metabolic pathways (nicotinamide or methionine) were used to develop a classification model to assign animals as treated with clenbuterol or non-treated. The classification model was tested on an external validation set comprising 74 animals either treated with different anabolic compounds (ß2-agonists, sexual steroids, corticosteroid), or non-treated, showing 100% sensitivity and specificity.

Regucalcin expression profiles in veal calf testis: validation of histological and molecular tests to detect sex steroids illicit administration.

BACKGROUND: Sex steroids administration in meat producing animals is forbidden within the EU to preserve consumers' safety, but continuous monitoring to identify resurgence of their misuse is needed. Among biomarkers related to sex steroids abuse in veal calves the regucalcin (RGN) mRNA perturbations in testis have been described in RNAlater samples. To setup novel diagnostic method, to update current tests available in National Residue Control Plans (NRCPs) and in legal dispute when illicit practices on farm animals are suspected, the reliability of RGN profiling was assessed by histological and molecular techniques. METHODS: Formalin fixed paraffin embedded (FFPE) testis samples, chosen being the most effective preservation strategy adopted by histological NRCPs and allowing easier retrospective analysis if required by legal disputes, were analyzed from veal calves treated with nandrolone, 17ß-estradiol and a cocktail of the two hormones. RGN levels were determined by quantitative Real Time PCR and Immunohistochemistry assays. Test performances were assessed and compared by multiple ROC curves. RESULTS: Both tests resulted sensitive and specific, allowing to enrich, in future field investigation, novel integrated diagnostic protocols needed to unveil sex steroid abuse. DISCUSSION: Developed RT-qPCR and IHC methods confirmed RGN as a useful and robust biomarker to detect illegal administration of sex steroid hormones in veal calves. The developed methods, successfully applied to ten years old FFPE blocks, could allow both retrospective analysis, when supplementary investigations are requested by authorities, and future implementation of current NRCPs.

The Italian strategy to fight illegal treatment with growth promoters: Results of the 2017-2019 histological monitoring plan.

The Histological Control Plan has been introduced in Italy in 2008 as an indirect monitoring tool of illicit administration of sexual hormones and corticosteroids in bovine. Analysis of 2008-2016 results permitted to draw a new plan targeting risk category. This work presents the results of the histopathological monitoring plan that was carried out from 2017 to 2019. The overall prevalence of samples suspected of treatment with corticosteroid was 11.3% [95% confidence interval (CI) 6.6-17.8] in 2017; 10.2% (95% CI 6.6-16.9) in 2018 and 8.9% (95% CI 4.6-15.4) in 2019. The overall prevalence of samples suspected of treatment with sexual hormones was 2.3 % (95% CI 0.5-6.6) in 2017; 6.2% (95% CI 2.7-11.8) in 2018 and 12.4% (95% CI 7.4-19.1) in 2019. Although not targeting and measuring specific molecules, this strategy allows to verify the trend of illicit treatments and identify farms to be submitted to further check.

LC-MS/MS analyses of bile and histological analyses of thymus as diagnostic tools to detect low dose dexamethasone illicit treatment in beef cattle at slaughterhouse.

Dexamethasone (DXM) is a synthetic adrenal corticosteroid with anti-inflammatory properties used for therapeutic purposes in a wide range of pathologies and of the most common corticosteroids used for anabolic purposes in beef cattle. It is proven that DXM induces histological changes, traceable as increasing fatty infiltration of the thymus associated with a concurrent decrease of the cortex-medulla ratio, so the histological examination of the thymus gland has been established as an indirect morphological biomarker. The aim of the present study is to compare thymus histology and DXM concentrations in biological fluids collected at slaughterhouse after 1 month of DXM treatment. Our findings demonstrate that a low dosage of DXM administered to 12 months-old-Chianina beef cattle induces severe thymic atrophy with concurrent reduction of the cortex/medulla ratio, demonstrable even when DXM residues are not found in serum and urine samples. It is worth to note that, at the slaughterhouse, DXM residues are detectable in bile samples, indicating the ability of this biological fluid to bio-concentrate the administered drug if compared to serum and urine. Therefore, bile could be candidates as new liquid matrix for the screening programs planned to contrast the illegal use of anabolic substances.

Real-time PCR assay for detecting illicit steroid administration in veal calves allows reliable biomarker profiling of formalin-fixed, paraffin-embedded (FFPE) archival tissue samples.

In the context of mRNA biomarker profiling, formalin fixed paraffin embedded (FFPE) samples represent an interesting source for retrospective analysis. However, the implementation in routine analysis of FFPE samples, following legislation demands for validated and accredited methods, often requires critical revision and optimization. In the frame of an official control program the validation study of a molecular test for detection of sex steroids administration in calves, based on quantification of progesterone-Receptor mRNA in bulbo-urethral gland samples, was performed: analyses were made on FFPE tissues sections routinary used for histological investigations and compared with RNAlater tissue preservation. To overcome the limitations of original assays several modifications were tested. Obtained results confirmed how Progesterone-Receptor assay represent a useful tool to study suspected cases of sex steroid illicit administration in veal calves, complementary to histological and/or immune histochemical investigation, overcoming the limitation of field studies, where optimal pre-analytical condition cannot always be guarantee.