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The QuEChERS approach was optimized for extracting cholesterol oxidation products in cheese, followed by LC-APCI-MS/MS analysis. Optimization of the method, including evaluations of saponification step, sample weight, and d-SPE purification resulted in good recoveries for each analyte, ensuring a reliable determination of these contaminants in cheese samples. In addition, the method was successfully validated by testing linearity of response, analytical limits (LOD and LLOQ), and precision. Sliced cheese samples wrapped in various packaging materials underwent a challenging test to simulate refrigerated storage conditions under fluorescent light, inducing photo-oxidative stress. The validated QuEChERS method revealed that only seasoned hard cow’s cheese showed an increase in the concentration of 7-ketocholesterol and its chemical precursors, 7β-hydroxycholesterol, and 7α-hydroxycholesterol, reaching levels of 0.45, 0.35, and 0.35 µg g−1, respectively. Conversely, opaque packaging and the use of a double film were found to be effective in preventing the formation of COPs in cheese samples subjected to photo-oxidative stress, such as smoked cheese and melted cheese slices (sottilette). A trade-off must be found between ensuring cheese protection and meeting the consumer’s desire to see the product.

Two different methods for single step transesterification from pig meat without fat extraction have been tested. Freeze-drying of the meat with and without anhydrous salt, followed by a base-catalyzed transmethylation (KOH/MeOH) was carried out. Both methods were compared with the standard Folch procedure of fat extraction followed by transmethylation. The methods were tested on a complete sample set of biceps femoris of pig thigh, used for the production of dry-cured ham. The set was divided in three subgroups according to total fat content. Both derivatization protocols on freeze-dried pork muscle were proven to be a valid alternative to the Folch procedure for FAME analysis. Freeze-drying method offered several advantages in comparison with the Folch procedure, including a lower solvent requirement, and process temperature, as well as considerable saving of time. In freeze-drying, the addition of an anhydrous salt (Na 2 SO 4 ) gave more friable samples which resulted in higher yields for some fatty acids, particularly evident in the case of tissues with high lipid content.

AbstractObjectiveTo evaluate pathological complete response as a surrogate endpoint for disease-free survival and overall survival in regulatory neoadjuvant trials of early stage breast cancer.DesignSystematic review and meta-analysis.Data sourcesMedline, Embase, and Scopus to 1 December 2020.Eligibility criteria for study selectionRandomised clinical trials that tested neoadjuvant chemotherapy given alone or combined with other treatments, including anti-human epidermal growth factor 2 (anti-HER2) drugs, targeted treatments, antivascular agents, bisphosphonates, and immune checkpoint inhibitors.Data extraction and synthesisTrial level associations between the surrogate endpoint pathological complete response and disease-free survival and overall survival.MethodsA weighted regression analysis was performed on log transformed treatment effect estimates (hazard ratio for disease-free survival and overall survival and relative risk for pathological complete response), and the coefficient of determination (R2) was used to quantify the association. The secondary objective was to explore heterogeneity of results in preplanned subgroups analysis, stratifying trials according treatment type in the experimental arm, definition used for pathological complete response (breast and lymph nodes v breast only), and biological features of the disease (HER2 positive or triple negative breast cancer). The surrogate threshold effect was also evaluated, indicating the minimum value of the relative risk for pathological complete response necessary to confidently predict a non-null effect on hazard ratio for disease-free survival or overall survival.Results54 randomised clinical trials comprising a total of 32 611 patients were included in the analysis. A weak association was observed between the log(relative risk) for pathological complete response and log(hazard ratio) for both disease-free survival (R2=0.14, 95% confidence interval 0.00 to 0.29) and overall survival (R2 =0.08, 0.00 to 0.22). Similar results were found across all subgroups evaluated, independently of the definition used for pathological complete response, treatment type in the experimental arm, and biological features of the disease. The surrogate threshold effect was 5.19 for disease-free survival but was not estimable for overall survival. Consistent results were confirmed in three sensitivity analyses: excluding small trials (<200 patients enrolled), excluding trials with short median follow-up (<24 months), and replacing the relative risk for pathological complete response with the absolute difference of pathological complete response rates between treatment arms.ConclusionA lack of surrogacy of pathological complete response was identified at trial level for both disease-free survival and overall survival. The findings suggest that pathological complete response should not be used as primary endpoint in regulatory neoadjuvant trials of early stage breast cancer.

Background Prevention and treatment of metastatic breast cancer (BC) is an unmet clinical need. The retinoic acid derivative fenretinide (FeR) was previously evaluated in Phase I-III clinical trials but, despite its excellent tolerability and antitumor activity in preclinical models, showed limited therapeutic efficacy due to poor bioavailability. We recently generated a new micellar formulation of FeR, Bionanofenretinide (Bio-nFeR) showing enhanced bioavailability, low toxicity, and strong antitumor efficacy on human lung cancer, colorectal cancer, and melanoma xenografts. In the present study, we tested the effect of Bio-nFeR on a preclinical model of metastatic BC. Methods We used BC cell lines for in vitro analyses of cell viability, cell cycle and migratory capacity. For in vivo studies, we used HER2/neu transgenic mice (neuT) as a model of spontaneously metastatic BC. Mice were treated orally with Bio-nFeR and at sacrifice primary and metastatic breast tumors were analyzed by histology and immunohistochemistry. Molecular pathways activated in primary tumors were analyzed by immunoblotting. Stem cell content was assessed by flow cytometry, immunoblotting and functional assays such as colony formation ex vivo and second transplantation assay in immunocompromised mice. Results Bio-nFeR inhibited the proliferation and migration of neuT BC cells in vitro and showed significant efficacy against BC onset in neuT mice. Importantly, Bio-nFeR showed the highest effectiveness against metastatic progression, counteracting both metastasis initiation and expansion. The main mechanism of Bio-nFeR action consists of promoting tumor dormancy through a combined induction of antiproliferative signals and inhibition of the mTOR pathway. Conclusion The high effectiveness of Bio-nFeR in the neuT model of mammary carcinogenesis, coupled with its low toxicity, indicates this formulation as a potential candidate for the treatment of metastatic BC and for the adjuvant therapy of BC patients at high risk of developing metastasis.

ObjectiveCancer stem cells are responsible for tumour spreading and relapse. Human epidermal growth factor receptor 2 (HER2) expression is a negative prognostic factor in colorectal cancer (CRC) and a potential target in tumours carrying the gene amplification. Our aim was to define the expression of HER2 in colorectal cancer stem cells (CR-CSCs) and its possible role as therapeutic target in CRC resistant to anti- epidermal growth factor receptor (EGFR) therapy.DesignA collection of primary sphere cell cultures obtained from 60 CRC specimens was used to generate CR-CSC mouse avatars to preclinically validate therapeutic options. We also made use of the ChIP-seq analysis for transcriptional evaluation of HER2 activation and global RNA-seq to identify the mechanisms underlying therapy resistance.ResultsHere we show that in CD44v6-positive CR-CSCs, high HER2 expression levels are associated with an activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which promotes the acetylation at the regulatory elements of the Erbb2 gene. HER2 targeting in combination with phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase kinase (MEK) inhibitors induces CR-CSC death and regression of tumour xenografts, including those carrying Kras and Pik3ca mutation. Requirement for the triple targeting is due to the presence of cancer-associated fibroblasts, which release cytokines able to confer CR-CSC resistance to PI3K/AKT inhibitors. In contrast, targeting of PI3K/AKT as monotherapy is sufficient to kill liver-disseminating CR-CSCs in a model of adjuvant therapy.ConclusionsWhile PI3K targeting kills liver-colonising CR-CSCs, the concomitant inhibition of PI3K, HER2 and MEK is required to induce regression of tumours resistant to anti-EGFR therapies. These data may provide a rationale for designing clinical trials in the adjuvant and metastatic setting.

This report of the European Food Safety Authority and the European Centre for Disease Prevention and Control presents the results of zoonoses monitoring and surveillance activities carried out in 2021 in 27 MSs, the United Kingdom (Northern Ireland) and nine non‐MSs. Key statistics on zoonoses and zoonotic agents in humans, food, animals and feed are provided and interpreted historically. In 2021, the first and second most reported zoonoses in humans were campylobacteriosis and salmonellosis, respectively. Cases of campylobacteriosis and salmonellosis increased in comparison with 2020, but decreased compared with previous years. In 2021, data collection and analysis at the EU level were still impacted by the COVID‐19 pandemic and the control measures adopted in the MSs, including partial or total lockdowns. Sixteen MSs and the United Kingdom (Northern Ireland) achieved all the established targets in poultry populations for reduction in Salmonella prevalence for the relevant serovars. Salmonella samples from carcases of various animal species and samples for Campylobacter quantification from broiler carcases were more frequently positive when performed by the competent authorities than when own‐checks were conducted. Yersiniosis was the third most reported zoonosis in humans, followed by Shiga toxin‐producing Escherichia coli (STEC) and Listeria monocytogenes infections. L. monocytogenes and West Nile virus infections were the most severe zoonotic diseases, with the most hospitalisations and highest case fatality rates. Overall, MSs reported more foodborne outbreaks and cases in 2021 than in 2020. S. Enteritidis remained the most frequently reported causative agent for foodborne outbreaks. Salmonella in ‘eggs and egg products’ and in ‘mixed foods’ were the agent/food pairs of most concern. Outbreaks linked to ‘vegetables and juices and products thereof’ rose considerably compared with previous years. This report also provides updates on brucellosis, Coxiella burnetii (Q fever), echinococcosis, rabies, toxoplasmosis, trichinellosis, tuberculosis due to Mycobacterium bovis or M. caprae, and tularaemia.

BackgroundIf Parkinson’s Disease (PD) may represent a risk factor for Coronavirus disease 2019 (COVID-19) is debated and there are few data on the direct and indirect effects of this pandemic in PD patients.ObjectiveIn the current study we evaluated the prevalence, mortality and case-fatality of COVID-19 in a PD cohort, also exploring possible risk factors. We also aimed to investigate the effect of lockdown on motor/non-motor symptoms in PD patients as well as their acceptability/accessibility to telemedicine.MethodA case-controlled survey about COVID-19 and other clinical features in PD patients living in Tuscany was conducted. In non-COVID-19 PD patients motor/non-motor symptoms subjective worsening during the lockdown as well as feasibility of telemedicine were explored.ResultsOut of 740 PD patients interviewed, 7 (0.9%) were affected by COVID-19, with 0.13% mortality and 14% case-fatality. COVID-19 PD patients presented a higher presence of hypertension (p < 0.001) and diabetes (p = 0.049) compared to non-COVID-19. In non-COVID-19 PD population (n = 733) about 70% did not experience a subjective worsening of motor symptoms or mood, anxiety or insomnia. In our population 75.2% of patients was favorable to use technology to perform scheduled visits, however facilities for telemedicine were available only for 51.2% of cases.ConclusionA higher prevalence of COVID-19 respect to prevalence in Tuscany and Italy was found in the PD population. Hypertension and diabetes, as for general population, were identified as risk factors for COVID-19 in PD. PD patients did not experience a subjective worsening of symptoms during lockdown period and they were also favorable to telemedicine, albeit we reported a reduced availability to perform it.

Peripheral neurotoxicity is a dose-limiting adverse reaction of primary frontline chemotherapeutic agents, including vincristine. Neuropathy can be so disabling that patients drop out of potentially curative therapy, negatively impacting cancer prognosis. The hallmark of vincristine neurotoxicity is axonopathy, yet its underpinning mechanisms remain uncertain. We developed a comprehensive drug discovery platform to identify neuroprotective agents against vincristine-induced neurotoxicity. Among the hits identified, SIN-1—an active metabolite of molsidomine—prevents vincristine-induced axonopathy in both motor and sensory neurons without compromising vincristine anticancer efficacy. Mechanistically, we found that SIN-1’s neuroprotective effect is mediated by activating soluble guanylyl cyclase. We modeled vincristine-induced peripheral neurotoxicity in rats to determine molsidomine therapeutic potential in vivo. Vincristine administration induced severe nerve damage and mechanical hypersensitivity that were attenuated by concomitant treatment with molsidomine. This study provides evidence of the neuroprotective properties of molsidomine and warrants further investigations of this drug as a therapy for vincristine-induced peripheral neurotoxicity.

The aim of the present study was to assess the biochemical and molecular structural characteristics of a novel alkali-thermostable GH10 xylanase (Xyl10B) identified in a termite gut microbiome by a shotgun metagenomic approach. This endoxylanase candidate was amplified, cloned, heterologously expressed in Escherichia coli and purified. The recombinant enzyme was active at a broad range of temperatures (37–60 ºC) and pH values (4–10), with optimal activity at 50 ºC and pH 9. Moreover, its activity remained at more than 80% of its maximum at 50 °C for 8 h. In addition, Xyl10B was found to be stable in the presence of salt and several ions and chemical reagents frequently used in the industry. These characteristics make this enzyme an interesting candidate for pulp and paper bleaching industries, since this process requires enzymes without cellulase activity and resistant to high temperatures and alkaline pH (thermo-alkaliphilic enzymes). The products of xylan hydrolysis by Xyl10B (short xylooligosaccharides, xylose and xylobiose) could be suitable for application as prebiotics and in the production of bioethanol.Key pointsBiochemical and molecular structural characterization of a novel GH10 xylanase (Xyl10B) from a termite gut microbiome.Xyl10B is a candidate biocatalyst in the bleaching process of pulp and the paper industries because of its inactivity on carboxymethyl cellulose.The shorter xylooligosaccharides generated from the hydrolysis of xylan would be suitable in different applications, including the food industry as prebiotics.