Determination of Mycotoxins in Plant-Based Meat Alternatives (PBMAs) and Ingredients after Microwave Cooking.

In this study, we investigate the role of microwave cooking in reducing mycotoxin contamination in plant-based food matrices, with a focus on veggie burgers (purchased and home-made) and their ingredients (soybean, potatoes, zucchini, carrots). Two different conditions were studied (Max-Min) that were 800 W for 60 s and 800 W for 90 s, respectively. The degradation patterns of aflatoxins (AFB1, AFB2, AFG1, AFG2), fumonisins (FB1, FB2, FB3), trichothecenes (T2, HT2, ZEA), and ochratoxin A (OTA) were studied. The extraction procedures were conducted with the QuEChERS extraction, and the analyses were conducted with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Principal component analysis (PCA) showed that degradation under microwave cooking varies considerably across different food matrices and cooking conditions. This study provides valuable insights into the degradation of mycotoxins during microwave cooking and underscores the need for more research in this area to ensure food safety.

Identification of Contracaecum rudolphii (Nematoda: Anisakidae) in Great Cormorants Phalacrocorax carbo sinensis (Blumenbach, 1978) from Southern Italy.

In this study, four dead great cormorant Phalacrocorax carbo sinensis (Blumenbach, 1978) specimens, collected from the coasts and lakes of Southern Italy, were examined by necropsy for the detection of Contraceacum sp. The adults and larvae found were subjected to morphological analysis and molecular identification by PCR-RFLP. A total of 181 Contracaecum specimens were detected in all of the four great cormorants examined (prevalence = 100%), showing an intensity of infestation between nine and ninety-two. A co-infestation by adult and larval forms of Contracaecum rudolphii was found only in one of the great cormorants examined. Following molecular investigations, 48 specimens of C. rudolphii A and 38 specimens of C. rudolphii B were detected, revealing co-infestation solely for the great cormorant from Leporano Bay (Southern Italy). Our results showed an opposite ratio between C. rudolphii A and C. rudolphii B in Pantelleria and in Salso Lake (Southern Italy) compared to what was reported in the literature, probably due to migratory stopovers and the ecology of the infested fish species, confirming the role of Contracaecum nematodes as ecological tags of their hosts.

QuEChERS LC-MS/MS Screening Method for Mycotoxin Detection in Cereal Products and Spices.

We developed and validated a screening method for mycotoxin analysis in cereal products and spices. Ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) was used for the analysis. Dispersive solid-phase extractions (d-SPEs) were used for the extraction of samples. Ochratoxin A (OTA), zearalenone (ZEA), aflatoxins (AFLA; AFB1, AFB2, AFG1, AFG2), deoxynivalenol (DON), fumonisin (FUMO; FB1, FB2, FB3), T2, and HT2 were validated in maize. AFLA and DON were validated in black pepper. The method satisfies the requirements of Commission Regulation (EC) no. 401/2006 and (EC) no. 1881/2006. The screening target concentration (STC) was under maximum permitted levels (MLs) for all mycotoxins validated. The method's performance was assessed by two different proficiencies and tested with 100 real samples.

Seasonal trend of Anisakidae infestation in South Mediterranean bluefish.

A total of 1104 fish samples from markets of Sicily were analysed for the detection and species identification of Anisakidae nematodes. The preliminary analysis of the fish samples showed the presence of 2459 larvae. All the fish species revealed different prevalence of infestation, with a maximum of 100% for Lepidopus caudatus and a minimum of 4.5% in Sardina pilchardus. The 80% of the larvae examined by PCR-RFLP analysis belonged to Anisakis pegreffii species. The seasonal infestation trend of Anisakis was evaluated in all the fish sample examined. The results of the seasonal infestation trend showed a marked connection with the ecological aspects of the fish species examined. As far as we know, this work report for the first time important ecological aspects of Lepidopus caudatus specimens of South Mediterranean. This work could be useful to plan a seasonal fishing strategy aimed at reducing the health risks related to Anisakis.

Aflatoxin M1 in cow, sheep, and donkey milk produced in Sicily, Southern Italy.

Samples (n = 485) of raw (n = 394) or heat-treated (n = 91) milk of three different species (cow, n = 170; sheep, n = 133; donkey, n = 84), collected 2013-2016 in Western Sicily (Southern Italy), were analyzed for aflatoxin M1 (AFM1) by enzyme-linked immunosorbent assay (ELISA). Positive ELISA results were further analyzed by HPLC with fluorescence detection. Both methods had a detection limit for AFM1 in milk of 7 ng kg-1. ELISA yielded 12.9 and 5% positives in cows and sheep milk, respectively, all samples of donkey milk were negative. Levels of AFM1 were in most cases at 0.007-< 0.05 µg kg-1, only two samples (sheep milk) slightly exceeded the European Union maximum level of 0.05 µg kg-1. Only 6% of the samples were positive for AFM1 in a concentration range of 0.008-0.15 µg kg-1. Only milk samples collected directly from farms were positive. Overall, the levels were much lower than previously reported for Southern Italy cow and sheep milk samples purchased in retail stores. The results of this work indicate a continuous improvement of the feeding techniques on dairy farms of Southern Italy, which is essential to ensure consumers' protection.

Presence of Anisakis pegreffii in farmed sea bass (Dicentrarchus labrax L.) commercialized in Southern Italy: A first report.

We examined 151 European sea bass (Dicentrarchus labrax L.) samples from farms and fish markets of Sicily (Southern Italy) for Anisakidae larvae detection. All the samples were examined by visual inspection and modified chloro-peptic digestion. Two nematode larvae were found in the viscera of only one European sea bass sample from a farm located in Greece (FAO 37.3), giving a total prevalence of infestation of 0.7%. No other parasites were found after chloro-peptic digestion of the samples. The larvae were morphologically ascribed, at genus level, to morphotypes I and molecularly identified as Anisakis pegreffii. To the best if our knowledge, this is the first report on the presence of anisakid parasites in farmed European sea bass of Mediterranean Sea. Our findings suggest that the risk of exposure to Anisakidae nematodes in farmed European sea bass remains very low. However, further data on Mediterranean farms are needed to have a detailed risk analysis.

Molecular Identification of Hysterothylacium spp. In Fishes From the Southern Mediterranean Sea (Southern Italy).

Samples obtained from 11 teleost fish species collected in waters off of the Mediterranean coasts of Sicily, Italy, were examined for the presence of Hysterothylacium spp. larvae. In total, 3,017 fish samples were examined, and the larvae recovered were used in subsequent phylogenetic studies. Fifty-eight raphidascaridid parasitic nematodes were found in the examined fish, with prevalence values ranging from 0.2% in anchovies to 60% in forkbeard samples. Twenty-seven parasites were identified as Hysterothylacium fabri and Hysterothylacium aduncum by sequencing the following regions: the nuclear ribosomal internal transcribed spacer ( ITS) and the mitochondrial cytochrome c oxidase subunit II ( COXII). Two other larvae collected from Lophius piscatorius had very low identity scores, with ITS and COXII sequences reported in GenBank and high genetic distances (AY603539). Pairwise comparisons between the ITS region of the H. fabri isolated from fishes from Sicilian coastal waters and those isolated from the Mediterranean Sea, Turkey (KC852206), revealed genetic differences ranging from 0.015 to 0.018. Our H. aduncum samples had very low genetic differences to H. aduncum from the Adriatic Sea (KP979763, 0.00-0.003). The concatenated phylogenetic examination of the ITS- COXII sequences by using maximum likelihood analyses indicated 3 distinct clades supported by high bootstrap values. Further molecular identification and detailed morphological analyses are needed to clarify these results and confirm the diversity and relationships within Hysterothylacium.

Mercury accumulation in Mediterranean Fish and Cephalopods Species of Sicilian coasts: correlation between pollution and the presence of Anisakis parasites.

The aim of this study was to investigate mercury accumulation in some species, caught in Mediterranean Sea, in the period between May and December 2015, and to compare it to the presence of Anisakis parasites. The samples were examined by direct mercury analyzer (DMA-80) for their Hg levels. The metal concentration was compared to the presence or the absence of Anisakis parasites. Significant differences in Hg concentration in analysed samples were observed. The low-infested fishes contained 1-6 larvae of parasites whereas the high-infested one had 7-83 larvae.

Survey on the presence of A. simplex s.s. and A. pegreffii hybrid forms in Central-Western Mediterranean Sea.

In this study, 1029 fish and cephalopod samples came from Central-Western Mediterranean (FAO 37.1.1 and FAO 37.1.3) were analysed for Anisakidae larvae research with the aim to identify possible hybridisations between Anisakis pegreffii and Anisakis simplex s.s. species. A total of 1765 larvae were detected, with prevalence values between 8.1% and 100%. The morphologic analysis revealed characters attributable to morphotype I of Anisakis in 98.5% of the examined larvae, while 1.5% belonged to the morphotype II. PCR-based Restriction Fragment Length Polymorphism (PCR-RFLP) analysis of the entire ITS region (ITS1, 5.8S and ITS2) of nuclear ribosomal DNA (rDNA) was performed with HinfI and HhaI restriction enzymes. The majority of the larvae examined by PCR-RFLP were identified as A. pegreffii (71%), with a prevalence on horse mackerel from FAO 37.1.3, while 10% were identified as A. simplex s.s., 2% as A. physeteris and 17% as A. pegreffii×A. simplex s.s. hybrid genotype. The sequence analysis confirmed the hybridisation in the 85% of the larvae recognised as hybrid forms by PCR- RFLP, suggesting this form as the product of natural interspecific recombination due to the presence of sympatry areas. The presence of hybrid forms were mostly found in fish samples from FAO subzone 37.1.1. This is the first report of A. pegreffii x A. simplex s.s. hybrid genotype in fishes caught off the coasts of Sicily (Southern Italy). Finally, this study provided substantial information about the geographical distribution of Anisakidae family in Central-Western Mediterranean Sea.

Validation of the TrichinEasy® digestion system for the detection of Anisakidae larvae in fish products.

Anisakis and other parasites belonging to the Anisakidae family are organisms of interest for human health, because of their high zoonotic potential. Parasites belonging to this family can cause Anisakiasis, a parasitological disease caused by the ingestion of raw, infested fish products. Furthermore, evidence from the EFSA (European Food Safety Authority; EFSA 2010) has highlighted the allergological potential of nematodes belonging to the Anisakis genre. The detection and identification of Anisakidae larvae in fish products requires an initial visual inspection of the fish sample, as well as other techniques such as candling, UV illumination and artificial digestion. The digestion method consists of the simulation of digestive mechanics, which is made possible by the utilization of HCl and pepsin, according to EC Regulation 2075/2005. In this study, a new Anisakidae larvae detection method using a mechanical digestion system called Trichineasy® was developed. A total of 142 fish samples, belonging to 14 different species, were examined to validate the method. A reaction mixture with 100 g of sample, 10 g of pepsin (1:10000 NF) and 50 ml of 10% HCl at 36 ± 1°C for 20 minutes was evaluated to be the best condition for the digestion of fish samples. These parameters have also allowed the detection of viable larvae after digestion. The results confirm this instrumentation as a valuable and safe tool for the detection of Anisakidae larvae in fishery products.

Relationship between electrocardiographic findings and Cardiac Magnetic Resonance phenotypes in patients with Hypertrophic Cardiomyopathy.

BACKGROUND: Q waves and negative T waves are common electrocardiographic (ECG) abnormalities in patients with Hypertrophic Cardiomyopathy (HCM). Several studies correlated ECG findings with presence and extent of fibrosis and hypertrophy; however, their significance remains incompletely clarified. Our study aimed to explain the mechanism behind Q and negative T waves by comparing their positions on a 12-lead ECG with phenotypes observed at Late Gadolinium Enhancement (LGE) Cardiac Magnetic Resonance (CMR). METHODS: 12-lead ECG and LGE-CMR were performed in 88 consecutive patients with HCM (42 SD 16 years, 65 males). Using Delta Thickness ratio (DT ratio), and "global" and "parietal" LGE at CMR, the extent and distribution of myocardial hypertrophy and fibrosis were studied in correlation with ECG abnormalities. RESULTS: Q waves in different leads were not associated with "parietal" LGE score. Lateral Q waves correlated with an increased DT ratio Inferior Septum/Lateral wall (p = 0.01). A similar correlation between inferior Q waves and an increased DT Ratio Anterior wall/Inferior wall was of borderline statistical significance (p = 0.06). As expected, ECG signs of LV hypertrophy related to a raised Left Ventricular Mass Index (LVMI) (p < 0.0001) and mean wall thickness (p = 0.01). Depolarization disturbances, including negative T waves in lateral (p = 0.044) and anterior (p = 0.031) leads correlated with "parietal" LGE scores while QT dispersion (p = 0.0001) was associated with "global" LGE score. CONCLUSION: In HCM patients, Q waves are generated by asymmetric hypertrophy rather than by myocardial fibrosis, while negative T waves result from local LGE distribution at CMR.

Anisakid Nematodes as Possible Markers to Trace Fish Products.

In this work a total of 949 fish samples were analysed for the identification of nematode larvae belonging to the Anisakidae family. Biomolecular application for the identification of Anisakidae larvae can be an optimal instrument for the traceability of fish products, described on the Reg. EC 178/2002. Results confirm a correlation between geographical distribution of fishes and presence of specific Anisakid larvae. FAO 37 zone (Mediterranean sea) showed a prevailing distribution of Anisakis pegreffii and a minimal presence of A. simplex s.s. in hybrid form with Anisakis pegreffii. FAO 27 zone showed a prevailing distribution of A. simplex s.s. in fish like Brosme (Brosme brosme) and infestation prevalence of Pseudoterranova krabbei and P. decipiens s.s. in Gadus morhua. Obtained results validate the hypothesis that molecular biology methods for identifying Anisakidae larvae are effective traceability markers of fish products.

Atrial natriuretic peptide presence in parotid gland of human fetus at 13th week of development and in adult man.

Our immunohistochemical study shows that atrial natriuretic peptide (ANP) is present in both human fetal and adult parotid gland. In the fetuses ANP is localized in cell clusters, while in adult parotid gland, the ANP is seen only in the wall of intra- and extralobular ducts. The results suggest that ANP might play a role in the differentiation of the parotid gland similarly as in brain and that during fetal growth ANP may play a role in the regulating the secretion of primitive salivary fluid, even if acini are still not developed.

Expression of MMP-2 and MMP-9 in odontogenic myxoma in a child: report of a clinical case.

BACKGROUND: Odontogenic myxoma (OM) is a benign, locally invasive, non-metastasizing neoplasm of the jaw bones. Despite the benign nature of these lesions, there is a high rate of recurrence and the current recommended therapy, depending on the size and behaviour of the lesion, can vary from curettage with peripheral ostectomy, segmental resection up to radical resections for more aggressive lesions. OM is a rare tumour which occurs predominantly in the third decade of life and it is rare in children. Matrix metalloproteinases (MMPs) are a family of extracellular endopeptidases responsible for the degradation and remodelling of extracellular matrix, they are known to be involved in the progression and invasiveness of many types of tumour. MMPs have been studied in OM because of their well-known role in extracellular matrix degradation, tumour invasion and recurrence. CLINICAL CASE REPORT: We report a case of OM in a 6-year-old boy. A conservative excision was accomplished. The mass was excised without affecting the mandibular bone and the inferior alveolar nerve. Curettage and removal of the first right inferior molar were performed. After 6-month follow-up, no evidence of recurrence was found. EXPERIMENTAL DATA: We investigated the expression of MMP-2 and MMP-9 in this case of OM in a child. RT-PCR showed the expression of both MMP-2 and MMP-9 mRNAs. Immunohistochemistry showed a weak MMP-2 protein expression while MMP-9 protein was not detected. CONCLUSION: In this case of OM in a child, we report lack of recurrence after excision associated with low MMP-2 protein expression and absence of MMP-9. We believe it is worthy to deeply investigate the relationship between MMPs expression and OM behaviour with the aim to use MMPs as prognostic and/or therapeutic markers in OM.

Human umbilical cord expresses several vasoactive peptides involved in the local regulation of vascular tone: protein and gene expression of Orphanin, Oxytocin, ANP, eNOS and iNOS.

Full-term human umbilical cord contains three blood vessels: two arteries coiled around a vein and surrounded by Wharton's jelly, a mucous tissue with few mesenchymal stromal cells and abundant extracellular matrix. Umbilical vessels lack innervations, thus endothelial cells must play a role in the control of blood flow. The aim of this study was to investigate in human umbilical cord the expression of five peptides that could be involved in the regulation of vascular tone: Orphanin FQ, Oxytocin, Atrial Natriuretic Peptide (ANP), endothelial Nitric Oxide Synthase (eNOS) and inducible Nitric Oxide Synthase (iNOS). The expression of these molecules in full-term human umbilical cord was investigated through immunohistochemistry and RT-PCR. Immunoreactivity for Orphanin FQ was detected in Wharton's jelly, vessel musculature and endothelium; Oxytocin, ANP and eNOS were expressed by the umbilical epithelium, Wharton's jelly and endothelium, whereas iNOS only by endothelial cells. RT-PCR analysis showed transcriptional expression of Oxytocin, ANP and eNOS mRNAs. The presence of Orphanin, Oxytocin, ANP, eNOS and iNOS proteins was identified in the human umbilical cord. mRNA expression for Oxytocin, ANP and eNOS suggest that these molecules are synthesized by umbilical cord cells themselves. The expression of these vasoactive molecules could be part of a general mechanism locally regulating vascular tone.

Immunohistochemical and transcriptional expression of matrix metalloproteinases in full-term human umbilical cord and human umbilical vein endothelial cells.

Matrix metalloproteinases (MMPs) are extracellular zinc-dependent endopeptidases involved in the degradation and remodelling of extracellular matrix in physiological and pathological processes. MMPs also have a role on cell proliferation, migration, differentiation, angiogenesis and apoptosis. Umbilical cord is a special organ subjected to many changes during pre-natal life and whose cells can maintain a certain degree of plasticity also in post-natal period; for example recently they have been used as a source of stem cells. In this work we investigated the expression of MMPs in human umbilical cord and human umbilical vein endothelial cells (HUVEC) though immunohistochemistry, RT-PCR and gelatin zymography. MMP-2 protein is expressed in the amniotic epithelium of human umbilical cord and in few sub-epithelial fibroblasts, while MMP-3 and MMP-10 only in the umbilical epithelium. MMP-8, MMP-9 and MMP-13 immunoreactivity is localised in the epithelium and in Wharton's jelly mesenchymal cells. Immunocytochemistry also revealed protein expression for MMP-2, 3, 8, 9 and 10 in cultured HUVEC. In agreement with immunohistochemical data, RT-PCR analysis performed on samples of whole umbilical cord confirmed the transcriptional expression for the genes encoding all the six matrix metalloproteinases investigated, while in HUVEC only the expression of MMP-2, 3, 9, 10 and 13 mRNAs was detected. Gelatin zymography showed a clear MMP-2 and MMP-9 enzymatic activity in the conditioned medium of HUVEC at different culture passages, suggesting that HUVEC secrete gelatinases, that afterwards undergo extracellular activation, and this ability is not affected by passage number.

Expression of cyclooxygenase-1 and cyclooxygenase-2 in normal and pathological human oral mucosa.

Cyclooxigenase (COX) is the rate-limiting enzyme for the conversion of arachidonic acid (AA) to prostaglandins (PGs). Two isoforms of COX have been identified: COX-1 is constitutively expressed in many cells and is involved in cell homeostasis, angiogenesis and cell-cell signalling; COX-2 is not expressed in normal condition however it is strongly expressed in inflammation. The oral cavity is constantly exposed to physical and chemical trauma that could lead to mucosal reactions such as hyperplasia, dysplasia and cancer. Early diagnosis is the most important issue to address for a positive outcome of oral cancer; therefore it would be useful to identify molecular markers whose expression is associated with the various stages of oral cancer progression. Since COX enzyme has been involved, with different mechanisms, in the development and progression of malignancies we decided to investigate the expression and localization of COX-1 and COX-2 in normal human oral mucosa and three different pathologies (hyperplasia, dysplasia and carcinoma) by immunohistochemistry and RT-PCR. COX-1 mRNA and protein have been detected already in normal oral mucosa and their expression progressively increases from normal samples towards hyperplasia, dysplasia and finally carcinoma. On the contrary, COX-2 is not expressed in the normal tissue, starts to be expressed in hyperplasia, reaches the maximum activation in dysplasia and then starts to be downregulated in carcinoma.

MMP-2, MMP-9, and iNOS expression in human dental pulp subjected to orthodontic traction.

OBJECTIVE: To test the hypothesis that some metalloproteinases (MMP-2, MMP-9) and inducible nitric oxide synthetase (iNOS) enzymes in dental pulp samples do not vary when subjected to orthodontic treatment. MATERIALS AND METHODS: Human dental pulps were taken from male and female patients (N=10; age 10-14 years). A straight wire technique was used with nickel-titanium or steel archwires. The increase of pressure applied on teeth was gradual. Five patients were subjected to premolar extractions after 14 months of treatment and one after 24 months. Samples were Bouin-fixed, paraffin-embedded, and afterwards processed for immunohistochemistry using anti-MMP-2, anti-MMP-9, and anti-iNOS antibodies. RESULTS: A reduction of MMP-2, MMP-9, and iNOS expression occurred in treated samples. This became more evident with increased treatment time. CONCLUSION: The hypothesis is rejected. The reduction of expression of those proteins revealed a time-dependent relationship.

[The guiacum barrels between science and esoterism: Tommaso Campailla genius loci]. / Le botti di gujaco tra scienza ed esoterismo: Tommaso Campailla, genius loci.

Tommaso Campailla, as a typical man of his time, had a good humanistic and scientific education. He was born in Modica in 1668 [corrected] from an aristocratic family. When he was sixteen years old he moved to Catania to study law, but in a short time he came back to his native town where he preferred to study as an autodidact. He got married and had children and was elected senator seven times. In spite of his particular look, he was appreciated for his vast culture and his great sensibility. Giovanna Finocchiaro Chimirri noticed his very kind soul and called him: "the christian and Italian Lucretio". In addition, he was a friend of the Arcadian poet Girolama Lorefice Grimaldi. Campailla was a very good medical doctor, although he was an autodidact, and he was very able to inculcate love for medical studies in many students. He fought syphilis rheumatism in a "modern" way, using the "guaiacum barrels" or "vapour stovens" that he had invented. He died in 1740 in Modica.

Three-dimensional geometrical models of the liver.

In this work we used a virtual approach to study the human liver by three-dimensional geometrical models. We built the models through computer aided geometric modelling techniques starting from pictures taken during both real dissections and diagnostic medical imaging. The results show in a complete modular synthesis and with a schematic iconology the structural organization of this organ in a logic sequence of layers and topographic and spatial relationship among its components. This approach represents an amazing support to clinical anatomy for teaching and research.