Author Correction: Tertiary lymphoid structures improve immunotherapy and survival in melanoma.

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

Tertiary lymphoid structures improve immunotherapy and survival in melanoma.

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.

Synthesis, Biological Evaluation and Computational Studies of New Hydrazide Derivatives Containing 1,3,4-Oxadiazole as Antitubercular Agents.

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.

Evaluation of Microbial Growth in Hospital Textiles Through Challenge Test.

INTRODUCTION: 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. METHODS: 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. RESULTS: 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 104 CFU/cm2 and a final concentration of 1.5 × 105 CFU/cm2 at 22 °C and 1 × 105 CFU/cm2 at 37 °C 72 h after inoculum. CONCLUSION: The 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.

Potential issues of well water in domestic use in a retrospective study from 2014 to 2018.

Well water requires chemical, physical-chemical, and microbiological analysis to ensure that the water used for irrigation or for human consumption presents no specific risks. The aim of the present work was to determine the microbiological risk of samples taken from wells in south-central Sardinia. In this survey, 55 water samples were taken from private wells in the period from 2014 to 2018. Quality was assessed through the detection of the parameters required by national law. The analysis of the data showed that only 36.4% of the samples (20 wells) complied with legal limits for water for human consumption. The most isolated species was P. aeruginosa, present in 29.1%; its high concentration and recorded Coliforms suggest that the disinfection of the wells was insufficient or not performed properly if implemented. Such data suggest the need to pay more attention to the waters used not only for human consumption but also for irrigation, since the cultivated vegetables could become a vehicle for potentially pathogenic microorganisms. National legislation sets contamination limits only for water intended for human consumption and not for irrigation water. However, based on these considerations, it appears necessary to develop reference standards for irrigation water.

Design, synthesis and antimycobacterial activity of benzoxazinone derivatives and open-ring analogues: Preliminary data and computational analysis.

This study examines in depth benzoxazine nucleus for antimycobacterial property. We synthesized some benzoxazin-2-one and benzoxazin-3-one derivatives, which were tested for activity against a panel of Mycobacterium tuberculosis (Mtb) strains, including H37Ra, H37Rv and some resistant strains. Several compounds displayed a high antimycobacterial activity and the three isoniazid analogue derivatives 8a-c exhibited a MIC range of 0.125-0.250 µg/mL (0.37-0.75 µM) against strain H37Ra, therefore lower than the isoniazid reference drug. Two benzoxazin-2-one derivatives, 1c and 5j, together with isoniazid-analogue compound 8a, also revealed low MIC values against resistant strains and proved highly selective for mycobacterial cells, compared to mammalian Vero cells. To predict whether molecule 8a is able to interact with the active site of InhA, we docked it into the crystal structure; indeed, during the molecular dynamic simulation the compound never left the protein pocket. The more active compounds were predicted for ADME properties and all proved to be potentially orally active in humans.

Design, synthesis, SAR and biological investigation of 3-(carboxymethyl)rhodanine and aminothiazole inhibitors of Mycobacterium tuberculosis Zmp1.

Sixteen 3-(carboxymethyl)rhodanines, and twelve aminothiazoles as rhodanine-mimetics were designed, synthesized and tested as inhibitors of the Zmp1 enzyme from Mycobacterium tuberculosis (Mtb). Almost all rhodanines (5a-d, 5f-n, and 7a-b) exhibited Zmp1 inhibition with IC50 values in the range 1.3-43.9 µM, whereas only aminothiazoles 12b and 12d proved active with IC50 values of 41.3 and 35.7 µM, respectively. Structure-activity relationships (SAR) were coupled with molecular modeling studies to highlight structural determinants for Zmp1 inhibition. Moreover, rhodanines 5a and 5c induced 23.4 and 53.8% of Mtb growth inhibition in THP-1 infected cells, respectively, at the non-toxic concentration of 10 µg/ml. This work represents a step forward in targeting Zmp1 by small molecules.

Annexin A2 antibodies but not inhibitors of the annexin A2 heterotetramer impair productive HIV-1 infection of macrophages in vitro.

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.

Exploring the thiazole scaffold for the identification of new agents for the treatment of fluconazole resistant Candida.

Cyclohexyliden- and 2-methylcyclohexyliden-hydrazo-4-arylthiazoles were synthesized and tested as antifungal agents. All compounds exhibited minimal inhibitory concentration (MIC) values comparable with those of fluconazole (FLC). Moreover, some compounds showed fungicidal activity at low concentration. Worth noting five out of nine compounds were active towards Candida albicans 25 FLC resistant isolated from clinical specimens. The cellular toxicity was evaluated and none of the compounds is toxic at the MIC. On the basis of our data we can conclude that these derivatives are promising agents for the treatment of resistant C. albicans.

Antimicrobial Activity of Grapefruit Seed Extract on Edible Mushrooms Contaminations: Efficacy in Preventing Pseudomonas spp. in Pleurotus eryngii.

Pleurotus eryngii is an edible mushroom that suffers significant losses due to fungal contamination and bacteriosis. The Pseudomonadaceae family represents one of the most frequent etiologic agents. Grapefruit seed extract (GSE) is a plant extract that contains different bioactive components, such as naringin, and exhibits a strong antibacterial and antioxidant activity. Over the last decade, GSE use as an alternative to chemical treatments in the food sector has been tested. However, to our knowledge, its application on mushroom crops has never been investigated. This study focuses on evaluating GSE efficacy in preventing P. eryngii yellowing. GSE antibiotic activity, inhibitory and bactericidal concentrations, and antibiofilm activity against several microorganisms were tested with the Kirby-Bauer disk diffusion assay, the broth microdilution susceptibility test, and the Crystal violet assay, respectively. In vitro, the extract exhibited antimicrobial and antibiofilm activity against Staphylococcus aureus 6538 and MRSA (wild type), Escherichia coli ATCC 8739, and Pseudomonas spp. (Pseudomonas aeruginosa 9027, P. fluorescens (wild type)). GSE application in vivo, in pre- and post-sprouting stages, effectively prevented bacterial infections and subsequent degradation in the mushroom crops: none of the P. eryngii treated manifested bacteriosis. Our findings support the use of GSE as an eco-friendly and sustainable alternative to chemical treatments for protecting P. eryngii crops from bacterial contamination, consequently ensuring food safety and preventing financial losses due to spoilage. Furthermore, GSE's potential health benefits due to its content in naringin and other bioactive components present new possibilities for its use as a nutraceutical in food fortification and supplementation.

[Evaluation of microbial contamination of linens in industrial laundry processes]. / Valutazione della biocontaminazione nel processo produttivo delle lavanderie industriali.

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.

Legionella spp. Monitoring in the Water Supply Systems of Accommodation Facilities in Sardinia, Italy: A Two-Year Retrospective Analysis.

Travel-associated Legionnaires' disease is a significant public health concern worldwide. A high number of cases are reported every year among travellers who stay at guest houses, hotels, and spas. Indeed, hot water systems, showers, and air-conditioning systems can be contaminated by Legionella, which grows at 25-42 °C. Studies have shown that in Sardinia, especially during the summer months, the water circulation in the hotels' pipes is exposed to extremely high temperatures. As a result, this study was conducted to assess the colonization of hotel water systems by Legionella in Sardinia, concerning a recent EU directive 2020/2184 for drinking water with a limit of 1000 CFU /L. Methods. A total of 112 accommodation facilities were analyzed, of which 61.3% were found to be colonized with Legionella, and out of a total of 807 samples, 32.5% were positive for Legionella presence. The results showed a higher number of positive samples in the summer season. This was also associated with the higher concentration presence of >1000 CFU/L in the samples. Consequently, this study confirms that local hotel operators should improve their water safety and prevention plans, especially in spring and summer.

DNA promoter hypermethylation of melanocyte lineage genes determines melanoma phenotype.

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.

Evaluation of growth potential and growth dynamics of Listeria monocytogenes on ready-to-eat fresh fruit.

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 Role of PTEN Loss in Immune Escape, Melanoma Prognosis and Therapy Response.

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.

Tumor genetic heterogeneity analysis of chronic sun-damaged melanoma.

Chronic sun-damaged (CSD) melanoma represents 10%-20% of cutaneous melanomas and is characterized by infrequent BRAF V600E mutations and high mutational load. However, the order of genetic events or the extent of intra-tumor heterogeneity (ITH) in CSDhigh melanoma is still unknown. Ultra-deep targeted sequencing of 40 cancer-associated genes was performed in 72 in situ or invasive CMM, including 23 CSDhigh cases. In addition, we performed whole exome and RNA sequencing on multiple regions of primary tumor and multiple in-transit metastases from one CSDhigh melanoma patient. We found no significant difference in mutation frequency in melanoma-related genes or in mutational load between in situ and invasive CSDhigh lesions, while this difference was observed in CSDlow lesions. In addition, increased frequency of BRAF V600K, NF1, and TP53 mutations (p < .01, Fisher's exact test) was found in CSDhigh melanomas. Sequencing of multiple specimens from one CSDhigh patient revealed strikingly limited ITH with >95% shared mutations. Our results provide evidence that CSDhigh and CSDlow melanomas are distinct molecular entities that progress via different genetic routes.

Imidazole and 1,2,4-Triazole-based Derivatives Gifted with Antitubercular Activity: Cytotoxicity and Computational Assessment.

BACKGROUND: Mycobacterium Tuberculosis (Mtb) is the causative pathogen of Tuberculosis (TB) and outbreaks are more common among immunosuppressed persons infected with HIV. The current treatment regimens are lengthy and toxic, yet the therapy has remained unchanged for many decades, so there is a need to find new structures with selective mechanism of action. Moreover, the increased incidence of severe disseminated infections produced by undiagnosed Multidrug-resistant (MDR), worsen clinical treatment and contribute the spread of the disease. OBJECTIVE: The aim of our study was to evaluate the potential of imidazole and triazole moieties for antimycobacterial activity, by synthesizing some 1-(1-(aryl)-2-(2,6-dichlorophenyl)hydrazono)ethyl- 1H-imidazole and 1H-1,2,4-triazole derivatives 2a-l. METHODS: The title compounds were obtained via classical organic synthesis. The antimicrobial activity was evaluated using the method of microdilution and the cytotoxicity assay was performed by MTT method. RESULTS: The results indicated that the presence of both the imidazole ring and that of the 2,6- dichlorosubstituted phenyl moiety, is more relevant for inhibitory activity against Mtb than the triazole nucleus and the unsubstituted phenyl ring. Among the series, (E)-1-(2-(5-chlorothiophen-2-yl)-2-(2- (2,6-dichlorophenyl)hydrazono)ethyl)-1H-imidazole derivative 2f and (Z)-1-(2-([1,1'-biphenyl]-4-yl)- 2-(2-(2,6-dichlorophenyl)hydrazono)ethyl]-1H-imidazole derivatives 2e exhibited a promising antimycobacterial property and the latter also displayed a safe cytotoxic profile. CONCLUSION: The synthesized compounds were studied for their antitubercular activity. Among the series, the compounds 2e and 2f appeared to be the most promising agents and, according to the docking assessment, the compounds could be CYP51 inhibitors. These evidences could be useful for the future development of new antimycobacterial derivatives targeting CYP51 with more specificity for the mycobacterial cell enzyme.

The X-Linked DDX3X RNA Helicase Dictates Translation Reprogramming and Metastasis in Melanoma.

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.

Naturally occurring Diels-Alder-type adducts from Morus nigra as potent inhibitors of Mycobacterium tuberculosis protein tyrosine phosphatase B.

Mycobacterium tuberculosis (Mtb) protein tyrosine phosphatases A and B (PtpA and PtpB) have been recognized as potential molecular targets for the development of new therapeutic strategies against tuberculosis (TB). In this context, we have recently reported that the naturally occurring Diels-Alder-type adduct Kuwanol E is an inhibitor of PtpB (Ki = 1.6 ± 0.1 µM). Here, we describe additional Diels-Alder-type adducts isolated from Morus nigra roots bark that inhibit PtpB at sub-micromolar concentrations. The two most potent compounds, namely Kuwanon G and Kuwanon H, showed Ki values of 0.39 ± 0.27 and 0.20 ± 0.01 µM, respectively, and interacted with the active site of the enzyme as suggested by kinetics and mass spectrometry studies. Molecular docking coupled with intrinsic fluorescence analysis and isothermal titration calorimetry (ITC) further characterized the interaction of these promising PtpB inhibitors. Notably, in an Mtb survival assay inside macrophages, Kuwanon G showed inhibition of Mtb growth by 61.3%. All these results point to the common Diels-Alder-type adduct scaffold, and highlight its relevance for the development of PtpB inhibitors as candidate therapeutics for TB.

Antiherpevirus activity of Artemisia arborescens essential oil and inhibition of lateral diffusion in Vero cells.

BACKGROUND: New prophylactic and therapeutic tools are needed for the treatment of herpes simplex virus infections. Several essential oils have shown to possess antiviral activity in vitro against a wide spectrum of viruses. AIM: The present study was assess to investigate the activities of the essential oil obtained from leaves of Artemisia arborescens against HSV-1 and HSV-2 METHODS: The cytotoxicity in Vero cells was evaluated by the MTT reduction method. The IC50 values were determined by plaque reduction assay. In order to characterize the mechanism of action, yield reduction assay, inhibition of plaque development assay, attachment assay, penetration assay and post-attachment virus neutralization assay were also performed. RESULTS: The IC50 values, determined by plaque reduction assay, were 2.4 and 4.1 microg/ml for HSV-1 and HSV-2, respectively, while the cytotoxicity assay against Vero cells, as determined by the MTT reduction method, showed a CC50 value of 132 mug/ml, indicating a CC50/IC50 ratio of 55 for HSV-1 and 32.2 for HSV-2. The antiviral activity of A. arborescens essential oil is principally due to direct virucidal effects. A poor activity determined by yield reduction assay was observed against HSV-1 at higher concentrations when added to cultures of infected cells. No inhibition was observed by attachment assay, penetration assay and post-attachment virus neutralization assay. Furthermore, inhibition of plaque development assay showed that A. arborescens essential oil inhibits the lateral diffusion of both HSV-1 and HSV-2. CONCLUSION: This study demonstrates the antiviral activity of the essential oil in toto obtained from A. arborescens against HSV-1 and HSV-2. The mode of action of the essential oil as antiherpesvirus agent seems to be particularly interesting in consideration of its ability to inactivate the virus and to inhibit the cell-to-cell virus diffusion.