A rapid magnetic solid phase extraction method followed by liquid chromatography-tandem mass spectrometry analysis for the determination of mycotoxins in cereals

Mycotoxins can contaminate various food commodities, including cereals. Moreover, mycotoxins of different classes can co-contaminate food, increasing human health risk. Several analytical methods have been published in the literature dealing with mycotoxins determination in cereals. Nevertheless, in the present work, the aim was to propose an easy and effective system for the extraction of six of the main mycotoxins from corn meal and durum wheat flour, i.e., the main four aflatoxins, ochratoxin A, and the mycoestrogen zearalenone. The developed method exploited magnetic solid phase extraction (SPE), a technique that is attracting an increasing interest as an alternative to classical SPE. Therefore, the use of magnetic graphitized carbon black as a suitable extracting material was tested. The same magnetic material proved to be effective in the extraction of mycoestrogens from milk, but has never been applied to complex matrices as cereals. Ultra high-performance liquid chromatography tandem mass spectrometry was used for detection. Recoveries were > 60% in both cereals, even if the matrix effects were not negligible. The limits of quantification of the method results were comparable to those obtained by other two magnetic SPE-based methods applied to cereals, which were limited to one or two mycotoxins, whereas in this work the investigated mycotoxins belonged to three different chemical classes

Oncostatin M is overexpressed in NASH-related hepatocellular carcinoma and promotes cancer cell invasiveness and angiogenesis

: Oncostatin M (OSM) is a pleiotropic cytokine of the interleukin (IL)-6 family that contributes to the progression of chronic liver disease. Here we investigated the role of OSM in the development and progression of hepatocellular carcinoma (HCC) in NAFLD/NASH. The role of OSM was investigated in: a) selected cohorts of NAFLD/NASH HCC patients; b) liver cancer cells exposed to human recombinant OSM or stably transfected to overexpress human OSM; c) murine HCC xenografts; d) a murine NASH-related model of hepatic carcinogenesis. OSM was found to be selectively overexpressed in HCC cells of NAFLD/NASH patients, depending on tumor grade. OSM serum levels, barely detectable in patients with simple steatosis or NASH, were increased in patients with cirrhosis, and more evident in those carrying HCC. In this latter group, OSM serum levels were significantly higher in the subjects with intermediate/advanced HCCs and correlated with poor survival. Cell culture experiments indicated that OSM upregulation in hepatic cancer cells contributes to HCC progression by inducing epithelial-to-mesenchymal transition and increased invasiveness of cancer cells as well as by inducing angiogenesis, which is of critical relevance. In murine xenografts, OSM overexpression was associated with slower tumor growth, but an increased rate of lung metastases. Overexpression of OSM and its positive correlation with the angiogenic switch were also confirmed in a murine model of NAFLD/NASH-related hepatocarcinogenesis. Consistent with this, analysis of liver specimens from human NASH-related HCCs with vascular invasion showed that OSM was expressed by liver cancer cells invading hepatic vessels. In conclusion, OSM up-regulation appears to be a specific feature of HCC arising on a NAFLD/NASH background, and it correlates with clinical parameters and disease outcome. Our data highlight a novel pro-carcinogenic contribution for OSM in NAFLD/NASH, suggesting a role of this factor as a prognostic marker and a putative potential target for therapy. This article is protected by copyright. All rights reserved

A novel BRCA1 splicing variant detected in an early onset triple-negative breast cancer patient additionally carrying a pathogenic variant in ATM: A case report

The widespread adoption of gene panel testing for cancer predisposition is leading to the identification of an increasing number of individuals with clinically relevant allelic variants in two or more genes. The potential combined effect of these variants on cancer risks is mostly unknown, posing a serious problem for genetic counseling in these individuals and their relatives, in whom the variants may segregate singly or in combination. We report a female patient who developed triple-negative high grade carcinoma in the right breast at the age of 36 years. The patient underwent bilateral mastectomy followed by combined immunotherapy and chemotherapy (IMpassion030 clinical trial). Two years later she developed a skin recurrence on the right anterior chest wall. Despite intensive treatment, the patient died at 40-year-old due to disease progression. Gene panel testing of patient’s DNA revealed the presence of a protein truncating variant in ATM [c.1672G>T; p.(Gly558Ter)] and of a not previously reported variant in the BRCA1 exon 22 donor splice site [c.5406+6T>C], whose clinical significance was unknown. The analysis of patient’s RNA revealed the up-regulation of two alternative BRCA1 mRNA isoforms derived from skipping of exon 22 and of exons 22-23. The corresponding predicted protein products, p.(Asp1778GlyfsTer27) and p.(Asp1778_His1822del) are both expected to affect the BRCA1 C Terminus (BRCT) domain. The two variants were observed to co-occur also in the proband’s brother who, in addition, was heterozygous for a common variant (c.4837A>G) mapped to BRCA1 exon 16. This allowed to ascertain, by transcript-specific amplification, the lack of functional mRNA isoforms expressed by the c.5406+6T>C allele and provided evidence to classify the BRCA1 variant as pathogenic, according to the guidelines of the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium. To our knowledge, excluding two cases detected following the screening of population specific recurrent variants, only one ATM/BRCA1 double heterozygote has been reported in the literature, being the case here described the one with the youngest age at cancer onset. The systematic collection of cases with pathogenic variants in more than one cancer predisposition gene is needed to verify if they deserve ad hoc counseling and clinical management

Hepatocyte-specific deletion of HIF2α prevents NASH-related liver carcinogenesis by decreasing cancer cell proliferation

Background & aims: Hypoxia and hypoxia-inducible factors (HIFs) are involved in chronic liver disease progression. We previously showed that hepatocyte HIF-2\u3b1 activation contributed significantly to nonalcoholic fatty liver disease progression in experimental animals and human patients. In this study, using an appropriate genetic murine model, we mechanistically investigated the involvement of hepatocyte HIF-2\u3b1 in experimental nonalcoholic steatohepatitis (NASH)-related carcinogenesis. Methods: The role of HIF-2\u3b1 was investigated by morphologic, cellular, and molecular biology approaches in the following: (1) mice carrying hepatocyte-specific deletion of HIF-2\u3b1 (HIF-2\u3b1-/- mice) undergoing a NASH-related protocol of hepatocarcinogenesis; (2) HepG2 cells stably transfected to overexpress HIF-2\u3b1; and (3) liver specimens from NASH patients with hepatocellular carcinoma. Results: Mice carrying hepatocyte-specific deletion of HIF-2\u3b1 (hHIF-2\u3b1-/-) showed a significant decrease in the volume and number of liver tumors compared with wild-type littermates. These effects did not involve HIF-1\u3b1 changes and were associated with a decrease of cell proliferation markers proliferating cell nuclear antigen and Ki67. In both human and rodent nonalcoholic fatty liver disease-related tumors, HIF-2\u3b1 levels were strictly associated with hepatocyte production of SerpinB3, a mediator previously shown to stimulate liver cancer cell proliferation through the Hippo/Yes-associated protein (YAP)/c-Myc pathway. Consistently, we observed positive correlations between the transcripts of HIF-2\u3b1, YAP, and c-Myc in individual hepatocellular carcinoma tumor masses, while HIF-2\u3b1 deletion down-modulated c-Myc and YAP expression without affecting extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase, and AKT-dependent signaling. In\ua0vitro data confirmed that HIF-2\u3b1 overexpression induced HepG2 cell proliferation through YAP-mediated mechanisms. Conclusions: These results indicate that the activation of HIF-2\u3b1 in hepatocytes has a critical role in liver carcinogenesis during NASH progression, suggesting that HIF-2\u3b1-blocking agents may serve as novel putative therapeutic tools

The BRCA2 c.68-7T > A variant is not pathogenic: A model for clinical calibration of spliceogenicity.

Although the spliceogenic nature of the BRCA2 c.68-7T>A variant has been demonstrated, its association with cancer risk remains ontroversial. In this study, we accurately quantified by real-time PCR and digital PCR the BRCA2 isoforms retaining or missing exon 3. In addition, the combined odds ratio for causality of the variant was estimated using genetic and clinical data, and its associated cancer risk was estimated by case-control analysis in 83,636 individuals. Co-occurrence in trans with pathogenic BRCA2 variants was assessed in 5,382 families. Exon 3 exclusion rate was 4.5-fold higher in variant carriers (13%) than controls (3%), indicating an exclusion rate for the c.68-7T>A allele of approximately 20%. The posterior probability of pathogenicity was 7.44 x 10-115. There was neither evidence for increased risk of breast cancer (OR 1.03; 95% CI 0.86-1.24), nor for a deleterious effect of the variant when co-occurring with pathogenic variants. Our data provide for the first time robust evidence of the non-pathogenicity of the BRCA2 c.68-7T>A. Genetic and quantitative transcript analyses together inform the threshold for the ratio between functional and altered BRCA2 isoforms compatible with normal cell function. These findings might be exploited to assess the relevance for cancer risk of other BRCA2 spliceogenic variants

Acute Delta Hepatitis in Italy spanning three decades (1991–2019): Evidence for the effectiveness of the hepatitis B vaccination campaign

Updated incidence data of acute Delta virus hepatitis (HDV) are lacking worldwide. Our aim was to evaluate incidence of and risk factors for acute HDV in Italy after the introduction of the compulsory vaccination against hepatitis B virus (HBV) in 1991. Data were obtained from the National Surveillance System of acute viral hepatitis (SEIEVA). Independent predictors of HDV were assessed by logistic-regression analysis. The incidence of acute HDV per 1-million population declined from 3.2 cases in 1987 to 0.04 in 2019, parallel to that of acute HBV per 100,000 from 10.0 to 0.39 cases during the same period. The median age of cases increased from 27 years in the decade 1991-1999 to 44 years in the decade 2010-2019 (p < .001). Over the same period, the male/female ratio decreased from 3.8 to 2.1, the proportion of coinfections increased from 55% to 75% (p = .003) and that of HBsAg positive acute hepatitis tested for by IgM anti-HDV linearly decreased from 50.1% to 34.1% (p < .001). People born abroad accounted for 24.6% of cases in 2004-2010 and 32.1% in 2011-2019. In the period 2010-2019, risky sexual behaviour (O.R. 4.2; 95%CI: 1.4-12.8) was the sole independent predictor of acute HDV; conversely intravenous drug use was no longer associated (O.R. 1.25; 95%CI: 0.15-10.22) with this. In conclusion, HBV vaccination was an effective measure to control acute HDV. Intravenous drug use is no longer an efficient mode of HDV spread. Testing for IgM-anti HDV is a grey area requiring alert. Acute HDV in foreigners should be monitored in the years to come

Determination of Aflatoxins and Ochratoxin A in Olive Oil

Scientific data on mycotoxin contamination in olive oil have shown that the presence of ochratoxin A (OTA) is more diffuse and more appreciable than that of aflatoxins (AFs). Nevertheless, the contamination levels found in olive oil do not seem to represent any considerable risk to public health. In fact, in the three countries with the largest olive oil consumption, Greece, Italy, and Spain, this ranges from 11-16 kg per capita per year, whereas in the other European and North African countries it is less than 5 kg per capita per year. In the same regions, the total consumption of cereals (i.e., the crops most affected by mycotoxin contamination) ranges from 100 to 250 kg per capita per year. Consequently, it is evident that the intake of AFs and OTA via contaminated olive oil is negligible compared to the intake via other contaminated widespread foods such as wheat and maize. However, AFs and OTA have a high toxicity and carcinogenic effect, and their simultaneous presence on food could lead to adverse additive or synergic effects. Therefore, as for other agricultural commodities, it is fundamental to prevent molds and then mycotoxin contamination in olives by ensuring optimal cultivation practices and storage conditions. © 2010 Copyright © 2010 Elsevier Inc. All rights reserved

HPLC-CHIP coupled to a triple quadrupole mass spectrometer for carbonic anhydrase II quantification in human serum

A method for carbonic anhydrase II (CA II) absolute quantification in human serum is presented. This method is based on high-performance liquid chromatography (HPLC)-Chip microfluidic device incorporating a nanoelectrospray source interfaced to a triple quadrupole mass spectrometer. The fraction containing CA II was isolated by preparative reversed-phase HPLC, and peptides obtained from the tryptic digest of the protein mixture were separated by the HPLC-Chip system. The multiple-reaction monitoring acquisition mode of a selected suitable CA II peptide and peptide internal standard allowed the selective and sensitive determination of a CA II. Absolute recovery of the method was 52 +/- 12%, while analytical recovery was 81 +/- 10%. For the eight samples analyzed, the matrix effect was found to be only -14 +/- 6%. A comparison among three regression lines type which were obtained by external calibration, matrix-matched calibration, and standard addition method, respectively, demonstrated that the first one is adequate in obtaining good accuracy and precision. Method quantification limit for CA II in serum was estimated to be 2 fmol/mL. CA II mean concentration in sera from eight healthy subjects was found to be 56 pmol/mL (relative standard deviation 24%)