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Food sector operators are responsible for monitoring the production cycle in order to offer a safe product from a sanitary point of view for marketing and consumption. The main goal of the work was to evaluate the degree of contamination of various surfaces in different production companies and therefore the effectiveness of the sanitization procedures performed at critical points in the aforesaid process (CCPs). As a result of this initial analysis, suggestions are then made on how to strengthen the prevention and reduce the risk of microbial contamination of the entire workflow and also on how to prevent foodborne infectious diseases and drug resistance. The analytical investigation activity was carried out in our province in the period between 2011–2020 and involved commercial catering environments, large-scale distribution, small retail centers and small producers. Significant differences emerged from the results regarding the contamination of surfaces in the operational situations considered (contamination with Enterobacteriaceae spp. was more frequent in retail centers than in the other food sectors (16.3% vs 33.0%; p

To extend our screening for novel antimycobacterial molecules, we have designed, synthesized, and biologically evaluated a library of 14 new hydrazide derivatives containing 1,3,4-oxadiazole core. A variety of mycobacterial strains, including some drug-resistant strains, were tested for antimycobacterial activity. Among the compounds tested, five showed high antimycobacterial activity (MIC values of 8 μg/mL) against M. tuberculosis H37Ra attenuated strain, and two derivatives were effective (MIC of 4 µg/mL) against pyrazinamide-resistant strains. Furthermore, the novel compounds were tested against the fungal C. albicans strain, showing no antimycotic activity, and thus demonstrating a good selectivity profile. Notably, they also exhibited low cytotoxicity against human SH-SY5Y cells. The molecular modeling carried out suggested a plausible mechanism of action towards the active site of the InhA enzyme, which confirmed our hypothesis. In conclusion, the active compounds were predicted in silico for ADME properties, and all proved to be potentially orally absorbed in humans.

Cellular stress contributes to the capacity of melanoma cells to undergo phenotype switching into highly migratory and drug-tolerant dedifferentiated states. Such dedifferentiated melanoma cell states are marked by loss of melanocyte-specific gene expression and increase of mesenchymal markers. Two crucial transcription factors, microphthalmia-associated transcription factor (MITF) and SRY-box transcription factor 10 (SOX10), important in melanoma development and progression, have been implicated in this process. In this study we describe that loss of MITF is associated with a distinct transcriptional program, MITF promoter hypermethylation, and poor patient survival in metastatic melanoma. From a comprehensive collection of melanoma cell lines, we observed that MITF-methylated cultures were subdivided in 2 distinct subtypes. Examining mRNA levels of neural crest-associated genes, we found that 1 subtype had lost the expression of several lineage genes, including SOX10. Intriguingly, SOX10 loss was associated with SOX10 gene promoter hypermethylation and distinct phenotypic and metastatic properties. Depletion of SOX10 in MITF-methylated melanoma cells using CRISPR/Cas9 supported these findings. In conclusion, this study describes the significance of melanoma state and the underlying functional properties explaining the aggressiveness of such states.

The consumption of fresh or RTE fruits is increasing every year and Listeria monocytogenes has been identified on raw or minimally processed fruits. A food product can become contaminated with L. monocytogenes anywhere along the pathway of food production during planting, harvesting, packaging, distribution and serving. The aim of this work was to assess the microbiological risks associated with consumption of ready- to- eat fruit such as melon, pineapple, coconut and fruit salad. The presence of Escherichia coli, Salmonella spp. and L. monocytogenes was also evaluated. Microbiological challenge tests were carried out for the evaluation of the L. monocytogenes growth potential in RTE fruit stored at 4 and 8°C. E. coli counts resulted under the detection limit of 10 CFU g-1, Salmonella and L. monocytogenes were not detected (absence in 25g). The growth potential values in coconut and melon (δ>0.5) showed the growth capacity of Listeria at the temperatures considered. A low initial load, also derived from good hygiene practices, and correct storage temperatures are essential to reduce bacterial growth in RTE fruit. The challenge test showed how each type of RTE fruit has a different commercial life based on its specific growth potential and that food should be stored at temperatures not higher than 4°C for a short period.

The consumption of fresh or RTE fruits is increasing every year and Listeria monocytogenes has been identified on raw or minimally processed fruits. A food product can become contaminated with L. monocytogenes anywhere along the pathway of food production during planting, harvesting, packaging, distribution and serving. The aim of this work was to assess the microbiological risks associated with consumption of ready- to- eat fruit such as melon, pineapple, coconut and fruit salad. The presence of Escherichia coli, Salmonella spp. and L. monocytogenes was also evaluated. Microbiological challenge tests were carried out for the evaluation of the L. monocytogenes growth potential in RTE fruit stored at 4 and 8°C. E. coli counts resulted under the detection limit of 10 CFU g-1, Salmonella and L. monocytogenes were not detected (absence in 25g). The growth potential values in coconut and melon (δ>0.5) showed the growth capacity of Listeria at the temperatures considered. A low initial load, also derived from good hygiene practices, and correct storage temperatures are essential to reduce bacterial growth in RTE fruit. The challenge test showed how each type of RTE fruit has a different commercial life based on its specific growth potential and that food should be stored at temperatures not higher than 4°C for a short period.

Ensuring the microbiological quality of textiles is an important requirement for health care facilities. The present study examines the way transport times and temperatures influence microbial growth in textiles. Therefore, the effectiveness of washing and disinfection processes has also been studied.Microbial Challenge Tests were set up through the artificial contamination of different dry and wet textiles which were stored at different temperatures. The bacterial concentration was evaluated in well-defined time phases aimed at simulating the time it took for the textiles to be transported from the hospital facilities to the reconditioning unit. Three times were therefore considered from T = 0 inoculation moment to T = 72 h post inoculation. At the end of each time, the increase in bacterial concentration was assessed by means of microbiological cultures, using selective media for the enumeration of each type of inoculated microorganism.In all the contaminated textiles the bacterial concentration remained unchanged at a temperature of 4 °C, while at 22 °C and 37 °C there was a significant increase (p 0.05) starting from 8 h of storage. In these textiles, the microorganism that showed the greatest growth capacity was P. aeruginosa with average initial concentration values of 10The data highlights the fact that the degree of contamination in textiles does not undergo an increase when transport takes place at a controlled temperature. Refrigerated transport of hospital textiles is thus a desirable preventive measure to keep microbiological risk under control.

Checkpoint blockade therapies that reactivate tumour-associated T cells can induce durable tumour control and result in the long-term survival of patients with advanced cancers1. Current predictive biomarkers for therapy response include high levels of intratumour immunological activity, a high tumour mutational burden and specific characteristics of the gut microbiota2,3. Although the role of T cells in antitumour responses has thoroughly been studied, other immune cells remain insufficiently explored. Here we use clinical samples of metastatic melanomas to investigate the role of B cells in antitumour responses, and find that the co-occurrence of tumour-associated CD8+ T cells and CD20+ B cells is associated with improved survival, independently of other clinical variables. Immunofluorescence staining of CXCR5 and CXCL13 in combination with CD20 reveals the formation of tertiary lymphoid structures in these CD8+CD20+ tumours. We derived a gene signature associated with tertiary lymphoid structures, which predicted clinical outcomes in cohorts of patients treated with immune checkpoint blockade. Furthermore, B-cell-rich tumours were accompanied by increased levels of TCF7+ naive and/or memory T cells. This was corroborated by digital spatial-profiling data, in which T cells in tumours without tertiary lymphoid structures had a dysfunctional molecular phenotype. Our results indicate that tertiary lymphoid structures have a key role in the immune microenvironment in melanoma, by conferring distinct T cell phenotypes. Therapeutic strategies to induce the formation of tertiary lymphoid structures should be explored to improve responses to cancer immunotherapy.

Checkpoint blockade therapies have changed the clinical management of metastatic melanoma patients considerably, showing survival benefits. Despite the clinical success, not all patients respond to treatment or they develop resistance. Although there are several treatment predictive biomarkers, understanding therapy resistance and the mechanisms of tumor immune evasion is crucial to increase the frequency of patients benefiting from treatment. The PTEN gene is thought to promote immune evasion and is frequently mutated in cancer and melanoma. Another feature of melanoma tumors that may affect the capacity of escaping T-cell recognition is melanoma cell dedifferentiation characterized by decreased expression of the microphtalmia-associated transcription factor (MITF) gene. In this study, we have explored the role of PTEN in prognosis, therapy response, and immune escape in the context of MITF expression using immunostaining and genomic data from a large cohort of metastatic melanoma. We confirmed in our cohort that PTEN alterations promote immune evasion highlighted by decreased frequency of T-cell infiltration in such tumors, resulting in a worse patient survival. More importantly, our results suggest that dedifferentiated PTEN negative melanoma tumors have poor patient outcome, no T-cell infiltration, and transcriptional properties rendering them resistant to targeted- and immuno-therapy.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Summary: The X-linked DDX3X gene encodes an ATP-dependent DEAD-box RNA helicase frequently altered in various human cancers, including melanomas. Despite its important roles in translation and splicing, how DDX3X dysfunction specifically rewires gene expression in melanoma remains completely unknown. Here, we uncover a DDX3X-driven post-transcriptional program that dictates melanoma phenotype and poor disease prognosis. Through an unbiased analysis of translating ribosomes, we identified the microphthalmia-associated transcription factor, MITF, as a key DDX3X translational target that directs a proliferative-to-metastatic phenotypic switch in melanoma cells. Mechanistically, DDX3X controls MITF mRNA translation via an internal ribosome entry site (IRES) embedded within the 5′ UTR. Through this exquisite translation-based regulatory mechanism, DDX3X steers MITF protein levels dictating melanoma metastatic potential in vivo and response to targeted therapy. Together, these findings unravel a post-transcriptional layer of gene regulation that may provide a unique therapeutic vulnerability in aggressive male melanomas. : Here, Phung et al. show that the X-linked gene DDX3X encoding an RNA helicase is frequently mutated in male melanoma and directs a post-transcriptional program that impacts clinical outcome and therapy response. These findings provide insights into the underlying mechanisms driving metastatic potential and resistance of aggressive melanomas. Keywords: DDX3X, melanocyte, melanoma, MITF, IRES, translation control, migration, metastasis, therapy, resistance

Ricotta Salata is a traditional ripened and salted whey cheese made in Sardinia (Italy) from sheep's milk. This product is catalogued as ready-to-eat food (RTE) since it is not submitted to any further treatment before consumption. Thus, foodborne pathogens, such as Listeria monocytogenes, can represent a health risk for consumers. In September 2012, the FDA ordered the recall of several batches of Ricotta Salata imported from Italy linked to 22 cases of Listeriosis in the United States. This study was aimed at evaluating the presence and virulence properties of L. monocytogenes in 87 samples of Ricotta Salata produced in Sardinia. The ability of this product to support its growth under foreseen packing and storing conditions was also evaluated in 252 samples. Of the 87 samples 17.2% were positive for the presence of L. monocytogenes with an average concentration of 2.2 log10 cfu/g. All virulence-associated genes (prfA, rrn, hlyA, actA, inlA, inlB, iap, plcA, and plcB) were detected in only one isolated strain. The Ricotta Salata samples were artificially inoculated and growth potential (δ) was assessed over a period of 3 mo. The value of the growth potential was always0.5 log10 cfu/g under foreseen packing and storing conditions. This study indicates that Ricotta Salata supports the L. monocytogenes growth to levels that may present a serious risk to public health, even while stored at refrigeration temperatures.

In the Mediterranean area, salted and dried roe from the gray Mugil cephalus "bottarga" represent a speciality food with great commercial value. Bottarga is currently produced by a traditional handmade process and, the risk of human bacterial contamination during its manufacturing is still unknown; in this perspective the foodborne pathogen Staphylococcus aureus could potentially contaminate this product due to poor sanitation or bad handling during processing. The aim of this work is: to evaluate the contamination level of foodborne pathogens at different product manufacturing stages and, in addition, to describe a fast and realizable method for the rapid detection of S. aureus in bottarga samples in the field. A cultural procedure was initially used to investigate the occurrence of S. aureus and the other main foodborne pathogens in bottarga samples at the different manufacturing stages (from roe to final product). In addition, a molecular approach was used to rapidly determine the presence of total bacteria, S. aureus, and its potential toxigenicity. Of the 194 specimens analyzed, we identified: Clostridium perfringens, Enterococcus spp. and Enterobacteriaceae. However, some samples resulted as being contaminated with S. aureus (4% in roe and 8.7% in the final product). During the bottarga manufacturing process, we observed an increase in pathogen levels (from 10(2) to 10(5) CFU/g) in contaminated samples, and entA and entB genotypes were identified. Reconstruction experiments suggest that the fresh roe and the bottarga (not completely dried) could represent a risk for the contamination and growth of pathogen bacteria.

Laundering linens and protecting them from microbiological recontamination are critical issues for the hotel and food industries and especially for hospitals. This study was performed to evaluate a sample of industrial laundries in Sardinia (Italy), to assess their compliance with national hygienic and sanitary regulations, along the complete laundering process. Study results indicate that industrial laundering processes are effective and that better awareness of staff who handle laundered textiles is required to reduce the risk of recontamination.

We investigated the usefulness of a colorimetric method based on the reduction of a tetrazolium salt (XTT) for the susceptibility testing of clinical isolates of Mycobacterium tuberculosis to isoniazid, rifampin, rifabutin, ethambutol hydrochloride, ethionamide and streptomycin. The isolates and the ATCC reference strains reported as susceptible according to the agar dilution method approved by the National Committee for Clinical Laboratory Standards were found to be susceptible by the XTT colorimetric assay after times of incubation ranging between three days for rifampin and rifabutin to eight days for isoniazid. In comparison with other colorimetric methods reviewed in this article, the proposed assay is suitable for determining the susceptibility or resistance to most antituberculous drugs and, as a consequence of the water-solubility of the formazan yielded by reduction of XTT, additional steps such as the addition of extraction buffer and further incubation before the spectrophotometric analysis are not needed. The XTT reduction assay is an inexpensive, rapid and reliable screening method for the detection of susceptible, resistant and multidrug-resistant strains of M. tuberculosis and is an alternative to the costly performance of molecular or radiometric methods.

© 2016 The Author(s). During sexual transmission of human immunodeficiency virus (HIV), macrophages are initial targets for HIV infection. Secretory leukocyte protease inhibitor (SLPI) has been shown to protect against HIV infection of macrophages through interactions with annexin A2 (A2), which is found on the macrophage cell surface as a heterotetramer (A2t) consisting of A2 and S100A10. Therefore, we investigated potential protein-protein interactions between A2 and HIV-1 gp120 through a series of co-immunoprecipitation assays and a single molecule pulldown (SiMPull) technique. Additionally, inhibitors of A2t (A2ti) that target the interaction between A2 and S100A10 were tested for their ability to impair productive HIV-1 infection of macrophages. Our data suggest that interactions between HIV-1 gp120 and A2 exist, though this interaction may be indirect. Furthermore, an anti-A2 antibody impaired HIV-1 particle production in macrophages in vitro, whereas A2ti did not indicating that annexin A2 may promote HIV-1 infection of macrophages in its monomeric rather than tetrameric form.