AMBRA1 levels predict resistance to MAPK inhibitors in melanoma.

Intrinsic and acquired resistance to mitogen-activated protein kinase inhibitors (MAPKi) in melanoma remains a major therapeutic challenge. Here, we show that the clinical development of resistance to MAPKi is associated with reduced tumor expression of the melanoma suppressor Autophagy and Beclin 1 Regulator 1 (AMBRA1) and that lower expression levels of AMBRA1 predict a poor response to MAPKi treatment. Functional analyses show that loss of AMBRA1 induces phenotype switching and orchestrates an extracellular signal-regulated kinase (ERK)-independent resistance mechanism by activating focal adhesion kinase 1 (FAK1). In both in vitro and in vivo settings, melanomas with low AMBRA1 expression exhibit intrinsic resistance to MAPKi therapy but higher sensitivity to FAK1 inhibition. Finally, we show that the rapid development of resistance in initially MAPKi-sensitive melanomas can be attributed to preexisting subclones characterized by low AMBRA1 expression and that cotreatment with MAPKi and FAK1 inhibitors (FAKi) effectively prevents the development of resistance in these tumors. In summary, our findings underscore the value of AMBRA1 expression for predicting melanoma response to MAPKi and supporting the therapeutic efficacy of FAKi to overcome MAPKi-induced resistance.

Modulation of innate immunity related genes resulting in prophylactic antimicrobial and antiviral properties.

BACKGROUND: The innate immunity acts during the early phases of infection and its failure in response to a multilayer network of co-infections is cause of immune system dysregulation. Epidemiological SARS-CoV-2 infections data, show that Influenza Virus (FLU-A-B-C) and Respiratory Syncytial Virus (RSV) are co-habiting those respiratory traits. These viruses, especially in children (mostly affected by 'multi-system inflammatory syndrome in children' [MIS-C] and the winter pandemic FLU), in the aged population, and in 'fragile' patients are causing alteration in immune response. Then, bacterial and fungal pathogens are also co-habiting the upper respiratory traits (e.g., Staphylococcus aureus and Candida albicans), thus contributing to morbidity in those COVID-19 affected patients. METHODS: Liquid chromatography coupled with high-resolution mass spectrometry using the quadrupole orbital ion trap analyser (i.e., UHPLC-Q-Orbitrap HRMS) was adopted to measure the polyphenols content of a new nutraceutical formula (Solution-3). Viral infections with SARS-CoV-2 (EG.5), FLU-A and RSV-A viruses (as performed in BLS3 authorised laboratory) and real time RT-PCR (qPCR) assay were used to test the antiviral action of the nutraceutical formula. Dilution susceptibility tests have been used to estimate the minimum inhibitory and bactericidal concentration (MIC and MBC, respectively) of Solution-3 on a variety of microorganisms belonging to Gram positive/ negative bacteria and fungi. Transcriptomic data analyses and functional genomics (i.e., RNAseq and data mining), coupled to qPCR and ELISA assays have been used to investigate the mechanisms of action of the nutraceutical formula on those processes involved in innate immune response. RESULTS: Here, we have tested the combination of natural products containing higher amounts of polyphenols (i.e., propolis, Verbascum thapsus L., and Thymus vulgaris L.), together with the inorganic long chain polyphosphates 'polyPs' with antiviral, antibacterial, and antifungal behaviours, against SARS-CoV-2, FLU-A, RSV-A, Gram positive/ negative bacteria and fungi (i.e., Candida albicans). These components synergistically exert an immunomodulatory action by enhancing those processes involved in innate immune response (e.g., cytokines: IFNγ, TNFα, IL-10, IL-6/12; chemokines: CXCL1; antimicrobial peptides: HBD-2, LL-37; complement system: C3). CONCLUSION: The prophylactic antimicrobial success of this nutraceutical formula against SARS-CoV-2, FLU-A and RSV-A viruses, together with the common bacteria and fungi co-infections as present in human oral cavity, is expected to be valuable.

Increasing outer membrane complexity: the case of the lipopolysaccharide lipid A from marine Cellulophaga pacifica.

Gram-negative bacteria living in marine waters have evolved peculiar adaptation strategies to deal with the numerous stress conditions that characterize aquatic environments. Among the multiple mechanisms for efficient adaptation, these bacteria typically exhibit chemical modifications in the structure of the lipopolysaccharide (LPS), which is a fundamental component of their outer membrane. In particular, the glycolipid anchor to the membrane of marine bacteria LPSs, i.e. the lipid A, frequently shows unusual chemical structures, which are reflected in equally singular immunological properties with potential applications as immune adjuvants or anti-sepsis drugs. In this work, we determined the chemical structure of the lipid A from Cellulophaga pacifica KMM 3664T isolated from the Sea of Japan. This bacterium showed to produce a heterogeneous mixture of lipid A molecules that mainly display five acyl chains and carry a single phosphate and a D-mannose disaccharide on the glucosamine backbone. Furthermore, we proved that C. pacifica KMM 3664T LPS acts as a weaker activator of Toll-like receptor 4 (TLR4) compared to the prototypical enterobacterial Salmonella typhimurium LPS. Our results are relevant to the future development of novel vaccine adjuvants and immunomodulators inspired by marine LPS chemistry.

Pushing the Boundaries of Structural Heterogeneity with the Lipid A of Marine Bacteria Cellulophaga.

Marine bacteria, which are often described as chemical gold, are considered an exceptional source of new therapeutics. Considerable research interest has been given to lipopolysaccharides (LPSs), the main components of the Gram-negative outer membrane. LPS and its lipid A portion from marine bacteria are known to exhibit a tricky chemistry that has been often associated with intriguing properties such as behaving as immune adjuvants or anti-sepsis molecules. In this scenario, we report the structural determination of the lipid A from three marine bacteria within the Cellulophaga genus, which showed to produce an extremely heterogenous blend of tetra- to hexa-acylated lipid A species, mostly carrying one phosphate and one D-mannose on the glucosamine disaccharide backbone. The ability of the three LPSs in activating TLR4 signaling revealed a weaker immunopotential by C. baltica NNO 15840T and C. tyrosinoxydans EM41T , while C. algicola ACAM 630T behaved as a more potent TLR4 activator.

A novel smaller ß-defensin-derived peptide is active against multidrug-resistant bacterial strains.

Antibiotic resistance is becoming a severe obstacle in the fight against acute and chronic infectious diseases that accompany most degenerative illnesses from neoplasia to osteo-arthritis and obesity. Currently, the race is on to identify pharmaceutical molecules or combinations of molecules able to prevent or reduce the insurgence and/or progression of infectivity. Attempts to substitute antibiotics with antimicrobial peptides have, thus far, met with little success against multidrug-resistant (MDR) bacterial strains. During the last decade, we designed and studied the activity and features of human ß-defensin analogs, which are salt-resistant, and hence active also under high salt concentrations as, for instance, in cystic fibrosis. Herein, we describe the design, synthesis, and major features of a new 21 aa long molecule, peptide γ2. The latter derives from the γ-core of the ß-defensin natural molecules, a small fragment of these molecules still bearing high antibacterial activity. We found that peptide γ2, which contains only one disulphide bond, recapitulates most of the biological properties of natural human ß-defensins and can also counteract both Gram-positive and Gram-negative MDR bacterial strains and biofilm formation. Moreover, it has great stability in human serum thereby enhancing its antibacterial presence and activity without cytotoxicity in human cells. In conclusion, peptide γ2 is a promising new weapon also in the battle against intractable infectious diseases.

Celiac disease-associated Neisseria flavescens decreases mitochondrial respiration in CaCo-2 epithelial cells: Impact of Lactobacillus paracasei CBA L74 on bacterial-induced cellular imbalance.

We previously identified a Neisseria flavescens strain in the duodenum of celiac disease (CD) patients that induced immune inflammation in ex vivo duodenal mucosal explants and in CaCo-2 cells. We also found that vesicular trafficking was delayed after the CD-immunogenic P31-43 gliadin peptide-entered CaCo-2 cells and that Lactobacillus paracasei CBA L74 (L. paracasei-CBA) supernatant reduced peptide entry. In this study, we evaluated if metabolism and trafficking was altered in CD-N. flavescens-infected CaCo-2 cells and if any alteration could be mitigated by pretreating cells with L. paracasei-CBA supernatant, despite the presence of P31-43. We measured CaCo-2 bioenergetics by an extracellular flux analyser, N. flavescens and P31-43 intracellular trafficking by immunofluorescence, cellular stress by TBARS assay, and ATP by bioluminescence. We found that CD-N. flavescens colocalised more than control N. flavescens with early endocytic vesicles and more escaped autophagy thereby surviving longer in infected cells. P31-43 increased colocalisation of N. flavescens with early vesicles. Mitochondrial respiration was lower (P < .05) in CD-N. flavescens-infected cells versus not-treated CaCo-2 cells, whereas pretreatment with L. paracasei-CBA reduced CD-N. flavescens viability and improved cell bioenergetics and trafficking. In conclusion, CD-N. flavescens induces metabolic imbalance in CaCo-2 cells, and the L. paracasei-CBA probiotic could be used to correct CD-associated dysbiosis.

Campylobacter jejuni bacteremia in Italian pediatric patients with acute lymphoblastic leukemia: Report of two cases.

Infections caused by Campylobacter jejuni are rarely associated with extraintestinal complications. C. jejuni bacteremia is difficult to detect in patients with hematological malignancies undergoing chemotherapy where the choice of appropriate antibiotic treatment is extremely important. We report two cases of C. jejuni bacteremia in Italian pediatric patients affected by acute lymphoblastic leukemia (ALL). Agreeing with the most recent epidemiological data, both clinical isolates showed a typical phenotypic antimicrobial resistance patterns with combined resistance to ciprofloxacin and tetracycline. To our knowledge, this is the first report of C. jejuni isolation from the blood of ALL pediatric patients in Italy, and it provides important epidemiological information on this rare infection.

[Validation of surgical masks during COVID19 emergency: activities at the University of Napoli Federico II]. / Validazione di maschere chirurgiche nella fase di emergenza COVID19: l'esperienza dell'Università degli Studi di Napoli Federico II.

SUMMARY: During COVID-19 pandemic crisis, Italian Government has approved Law Decree no. 18 of 17 march 2020, in which art. 15 allows enterprises to produce, import and commercialize surgical masks notwithstanding the current rules of product certification. It is just required that the interested enterprises send to the Italian National Institute of Health a selfcertification in which they declare the technical characteristics of the masks and that masks are produced according to the safety requirements. In this context, a technical-scientific unit was established at the University of Napoli Federico II to provide interested enterprises with state-of-the-art consultancy, testing and measurement services, adhering to rigorous scientific protocols. Characterization tests were carried out on 163 surgical masks and/or materials for their construction and they have enabled the identification of pre-screening criteria to simplify the procedure for evaluating surgical masks using methods for assessing the filtration efficiency of particles and aerosols. Based on experimental results, it has been observed that a filtration efficiency for particles with sizes larger that 650 nm (PFE>650) exceeding 35% might guarantees a bacterial filtration efficiency (BFE) higher than 95% while BFE values higher than 98% are obtained when the PFE>650 is larger than 40%. PFE measurement is extremely simpler with respect to BFE, the latter being time-consuming and requiring specific equipment and methods for its realization. Many tested materials have shown the capability to assure high filtration efficiencies but Spundonded-Meltblown-Spunbonded (SMS), that are layers of non-woven fabric with different weights of Meltblown, can simultaneously guarantee high particle filtration efficiencies with pressure drop values (breathability) in the limits to classify the surgical masks as Type II/IIR. In fact, the fabric products analyzed so far have not been able to simultaneously guarantee adequate BFE and breathability values. On the contrary, Spunbonds of adequate weights can virtually verify both requirements and accredit themselves as possible materials for the production of surgical masks, at least of Type I. Further studies are needed to verify the possibility of producing low-cost, reusable surgical masks that could meet the criteria of circular economy.

Virulence Traits of a Serogroup C Meningococcus and Isogenic cssA Mutant, Defective in Surface-Exposed Sialic Acid, in a Murine Model of Meningitis.

In serogroup C Neisseria meningitidis, the cssA (siaA) gene codes for an UDP-N-acetylglucosamine 2-epimerase that catalyzes the conversion of UDP-N-acetyl-α-d-glucosamine into N-acetyl-d-mannosamine and UDP in the first step in sialic acid biosynthesis. This enzyme is required for the biosynthesis of the (α2→9)-linked polysialic acid capsule and for lipooligosaccharide (LOS) sialylation. In this study, we have used a reference serogroup C meningococcal strain and an isogenic cssA knockout mutant to investigate the pathogenetic role of surface-exposed sialic acids in a model of meningitis based on intracisternal inoculation of BALB/c mice. Results confirmed the key role of surface-exposed sialic acids in meningococcal pathogenesis. The 50% lethal dose (LD50) of the wild-type strain 93/4286 was about four orders of magnitude lower than that of the cssA mutant. Compared to the wild-type strain, the ability of this mutant to replicate in brain and spread systemically was severely impaired. Evaluation of brain damage evidenced a significant reduction in cerebral hemorrhages in mice infected with the mutant in comparison with the levels in those challenged with the wild-type strain. Histological analysis showed the typical features of bacterial meningitis, including inflammatory cells in the subarachnoid, perivascular, and ventricular spaces especially in animals infected with the wild type. Noticeably, 80% of mice infected with the wild-type strain presented with massive bacterial localization and accompanying inflammatory infiltrate in the corpus callosum, indicating high tropism of meningococci exposing sialic acids toward this brain structure and a specific involvement of the corpus callosum in the mouse model of meningococcal meningitis.

Genotyping of Toxoplasma gondii strain directly from human CSF samples of congenital toxoplasmosis clinical case.

This report describes a case of congenital toxoplasmosis in a newborn in Southern Italy. A pregnant mother had been admitted at the 20th week of her pregnancy on account of pharyngodynia and laterocervical lymphadenopathy. Although serological testing of the mother's serum documented a seroconversion with positive IgG and IgM anti-Toxoplasma antibodies during II trimester, the woman refused to perform prenatal diagnosis for congenital toxoplasmosis. Fetal ultrasound scan already showed mild asymmetrical triventricular hydrocephaly and cerebral calcifications. After birth, real-time PCR on cerebrospinal fluid and blood samples of the newborn showed a positive result for 529bp-repeat element DNA of T. gondii, In addition brain magnetic resonance imaging and computed tomography showed a characteristic diffuse brain tissue loss associated with hydrocephalus. For the first time molecular characterization of T. gondii isolate was performed directly from the newborn's CSF samples by using nested-PCR-RFLP of sag-2 and pk1 genes. The PCR-RLFP analysis revealed that the isolate belongs to the clonal type II, the predominant lineage causing human toxoplasmosis, as confirmed by DNA sequencing.

Metagenomics Reveals Dysbiosis and a Potentially Pathogenic N. flavescens Strain in Duodenum of Adult Celiac Patients.

OBJECTIVES: Celiac disease (CD)-associated duodenal dysbiosis has not yet been clearly defined, and the mechanisms by which CD-associated dysbiosis could concur to CD development or exacerbation are unknown. In this study, we analyzed the duodenal microbiome of CD patients. METHODS: The microbiome was evaluated in duodenal biopsy samples of 20 adult patients with active CD, 6 CD patients on a gluten-free diet, and 15 controls by DNA sequencing of 16S ribosomal RNA libraries. Bacterial species were cultured, isolated and identified by mass spectrometry. Isolated bacterial species were used to infect CaCo-2 cells, and to stimulate normal duodenal explants and cultured human and murine dendritic cells (DCs). Inflammatory markers and cytokines were evaluated by immunofluorescence and ELISA, respectively. RESULTS: Proteobacteria was the most abundant and Firmicutes and Actinobacteria the least abundant phyla in the microbiome profiles of active CD patients. Members of the Neisseria genus (Betaproteobacteria class) were significantly more abundant in active CD patients than in the other two groups (P=0.03). Neisseria flavescens (CD-Nf) was the most abundant Neisseria species in active CD duodenum. Whole-genome sequencing of CD-Nf and control-Nf showed genetic diversity of the iron acquisition systems and of some hemoglobin-related genes. CD-Nf was able to escape the lysosomal compartment in CaCo-2 cells and to induce an inflammatory response in DCs and in ex-vivo mucosal explants. CONCLUSIONS: Marked dysbiosis and an abundance of a peculiar CD-Nf strain characterize the duodenal microbiome in active CD patients thus suggesting that the CD-associated microbiota could contribute to the many inflammatory signals in this disorder.

Fitness Cost of Rifampin Resistance in Neisseria meningitidis: In Vitro Study of Mechanisms Associated with rpoB H553Y Mutation.

Rifampin chemoprophylaxis against Neisseria meningitidis infections led to the onset of rifampin resistance in clinical isolates harboring point mutations in the rpoB gene, coding for the RNA polymerase ß chain. These resistant strains are rare in medical practice, suggesting their decreased fitness in the human host. In this study, we isolated rifampin-resistant rpoB mutants from hypervirulent serogroup C strain 93/4286 and analyzed their different properties, including the ability to grow/survive in different culture media and in differentiated THP-1 human monocytes and to compete with the wild-type strain in vitro. Our results demonstrate that different rpoB mutations (H553Y, H553R, and S549F) may have different effects, ranging from low- to high-cost effects, on bacterial fitness in vitro. Moreover, we found that the S549F mutation confers temperature sensitivity, possibly explaining why it is observed very rarely in clinical isolates. Comparative high-throughput RNA sequencing analysis of bacteria grown in chemically defined medium demonstrated that the low-cost H553Y substitution resulted in global transcriptional changes that functionally mimic the stringent response. Interestingly, many virulence-associated genes, including those coding for meningococcal type IV pili, porin A, adhesins/invasins, IgA protease, two-partner secretion system HrpA/HrpB, enzymes involved in resistance to oxidative injury, lipooligosaccharide sialylation, and capsular polysaccharide biosynthesis, were downregulated in the H553Y mutant compared to their level of expression in the wild-type strain. These data might account for the reduced capacity of this mutant to grow/survive in differentiated THP-1 cells and explain the rarity of H553Y mutants among clinical isolates.

Benzoxazolone carboxamides: potent and systemically active inhibitors of intracellular acid ceramidase.

The ceramides are a family of bioactive lipid-derived messengers involved in the control of cellular senescence, inflammation, and apoptosis. Ceramide hydrolysis by acid ceramidase (AC) stops the biological activity of these substances and influences survival and function of normal and neoplastic cells. Because of its central role in the ceramide metabolism, AC may offer a novel molecular target in disorders with dysfunctional ceramide-mediated signaling. Here, a class of benzoxazolone carboxamides is identified as the first potent and systemically active inhibitors of AC. Prototype members of this class inhibit AC with low nanomolar potency by covalent binding to the catalytic cysteine. Their metabolic stability and high in vivo efficacy suggest that these compounds may be used as probes to investigate the roles of ceramide in health and disease, and that this scaffold may represent a promising starting point for the development of novel therapeutic agents.

Identification of Inquilinus limosus in cystic fibrosis: a first report in Italy.

Cystic fibrosis is a genetic disorder associated with a polymicrobial lung infection where classical pathogens and newly identified bacteria may interact. Inquilinus limosus is an a-proteobacterium recently isolated in the airways of cystic fibrosis patient. We report the first case in Italy of I.limosus isolation from the sputum sample of a cystic fibrosis patient. The patient is a 20-years-old man with cystic fibrosis, regularly attending the Regional Care Center for Cystic Fibrosis at the Federico II University Hospital of Naples. Microbiological culture methods detected a mu- coid gram negative bacillus in the patient's sputum sample. The isolate exhibited a distinct antimicrobial suscep- tibility profile with a high MIC for several drugs. The MALDI-TOF mass spectrometry analysis indicated the bac- terium isolated as I. limosus, confirmed by 16s rDNA sequence analysis. The described clinical case demonstrates how the bacterial biodiversity in the airways of cystic fibrosis patients is still underestimated. Cystic fibrosis lung represents an ecological niche suitable for growth of a wide variety of unusual bacteria not commonly associated with human diseases, such as I. limosus. Therefore further studies are needed to evaluate the epidemiology and clinical implications of I. limosus in the physiopathology of cystic fibrosis lung infection.

Vine-Winery Byproducts as Precious Resource of Natural Antimicrobials: In Vitro Antibacterial and Antibiofilm Activity of Grape Pomace Extracts against Foodborne Pathogens.

Grape pomace is the main by-product of vine-winery chains. It requires adequate treatment and disposal but is also an economically underused source of bioactive plant secondary metabolites. This study aimed to investigate the antibacterial effects of polyphenolic extracts from Aglianico (Vitis vinifera L.) grape pomace. In particular, hydroethanolic extracts obtained via an ultrasonic-assisted extraction technique were selected for antimicrobial tests. The extracts were screened for their antibacterial effects against foodborne pathogens that were both Gram-positive, in the case of Staphylococcus aureus and Bacillus cereus, and Gram-negative, in the case of Escherichia coli and Salmonella enterica subsp. enterica serovar Typhimurium, showing variable bacteriostatic and bactericidal effects. In addition, our results demonstrated that the tested grape pomace extracts can reduce the inhibitory concentration of standard antibiotics. Interestingly, selected extracts inhibited biofilm development by S. aureus and B. cereus. Overall, these new insights into the antibacterial properties of grape pomace extracts may represent a relevant step in the design of novel therapeutic tools to tackle foodborne diseases, and in the management of resistant biofilm-related infections.

Eco-Friendly Sanitization of Indoor Environments: Effectiveness of Thyme Essential Oil in Controlling Bioaerosol Levels and Disinfecting Surfaces.

Bioaerosols and pathogens in indoor workplaces and residential environments are the primary culprits of several infections. Techniques for sanitizing air and surfaces typically involve the use of UV rays or chemical sanitizers, which may release chemical residues harmful to human health. Essential oils, natural substances derived from plants, which exhibit broad antimicrobial properties, could be a viable alternative for air and surface sanitation. The objective of this study has been to investigate the efficacy of thyme essential oil (TEO) in environmental sanitation processes. In Vitro assays through agar well diffusion, disk volatilization and tube dilution methods revealed significant antimicrobial activity of TEO 100% against foodborne and environmental isolates, with both bacteriostatic/fungistatic and bactericidal/fungicidal effects. Therefore, aqueous solutions of TEO 2.5% and 5% were formulated for air sanitation through nebulization and surface disinfection via direct contact. Bioaerosol samples and surface swabs were analyzed before and after sanitation, demonstrating the efficacy of aqueous solutions of TEO in reducing mesophilic and psychrophilic bacteria and environmental fungi levels in both air and on surfaces. The obtained results prove the antimicrobial potential of aqueous solutions of TEO in improving indoor air quality and surface cleanliness, suggesting thyme essential oil as an effective and safe natural sanitizer with minimal environmental impact compared to dangerous chemical disinfectants.

Antimicrobial efficacy of Punica granatum Lythraceae peel extract against pathogens belonging to the ESKAPE group.

The improper use and abuse of antibiotics have led to an increase in multidrug-resistant (MDR) bacteria resulting in a failure of standard antibiotic therapies. To date, this phenomenon represents a leading public health threat of the 21st century which requires alternative strategies to fight infections such as the identification of new molecules active against MDR strains. In the last 20 years, natural extracts with biological activities attracted scientific interest. Following the One Health Approach, natural by-products represent a sustainable and promising alternative solution. Consistently, the aim of the present study was to evaluate the antimicrobial activity of hydro-alcoholic pomegranate peel extract (PPE) against MDR microorganisms belonging to Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. "ESKAPE" group pathogens. Through semiquantitative and quantitative methods, the PPE showed effective antimicrobial activity against Gram-positive and Gram-negative MDR bacteria. The kinetics of bactericidal action of PPE highlighted that microbial death was achieved in a time- and dose-dependent manner. High concentrations of PPE exhibited antioxidant activity, providing a protective effect on cellular systems and red blood cell membranes. Finally, we report, for the first time, a significant intracellular antibacterial property of PPE as highlighted by its bactericidal action against the staphylococcal reference strain and its bacteriostatic effect against clinical resistant strain in the HeLa cell line. In conclusion, due to its characterized content of polyphenolic compounds and antioxidant activity strength, the PPE could be considered as a therapeutic agent alone or in conjunction with standard antibiotics against challenging infections caused by ESKAPE pathogens.

Antibacterial and Antibiofilm Efficacy of Thyme (Thymus vulgaris L.) Essential Oil against Foodborne Illness Pathogens, Salmonella enterica subsp. enterica Serovar Typhimurium and Bacillus cereus.

Nowadays, the wide spread of foodborne illness and the growing concerns about the use of synthetic food additives have shifted the focus of researchers towards essential oils (EOs) as possible antimicrobials and preservatives of natural origin. Thanks to their antimicrobial properties against pathogenic and food spoilage microorganisms, EOs have shown good potential for use as alternative food additives, also to counteract biofilm-forming bacterial strains, the spread of which is considered to be among the main causes of the increase in foodborne illness outbreaks. In this context, the aim of this study has been to define the antibacterial and antibiofilm profile of thyme (Thymus vulgaris L.) essential oil (TEO) against widespread foodborne pathogens, Salmonella enterica subsp. enterica serovar Typhimurium and Bacillus cereus. TEO chemical composition was analyzed through gas chromatography-mass spectrometry (GC-MS). Preliminary in vitro antibacterial tests allowed to qualitatively verify TEO efficacy against the tested foodborne pathogens. The subsequent determination of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values allowed to quantitatively define the bacteriostatic and bactericidal effects of TEO. To evaluate the ability of essential oils to inhibit biofilm formation, a microplate assay was performed for the bacterial biofilm biomass measurement. Results suggest that TEO, rich in bioactive compounds, is able to inhibit the growth of tested foodborne bacteria. In addition, the highlighted in vitro anti-biofilm properties of TEO suggest the use of this natural agent as a promising food preservative to counteract biofilm-related infections in the food industry.

Ambra1 modulates the tumor immune microenvironment and response to PD-1 blockade in melanoma.

BACKGROUND: Loss of Ambra1 (autophagy and beclin 1 regulator 1), a multifunctional scaffold protein, promotes the formation of nevi and contributes to several phases of melanoma development. The suppressive functions of Ambra1 in melanoma are mediated by negative regulation of cell proliferation and invasion; however, evidence suggests that loss of Ambra1 may also affect the melanoma microenvironment. Here, we investigate the possible impact of Ambra1 on antitumor immunity and response to immunotherapy. METHODS: This study was performed using an Ambra1-depleted BrafV600E /Pten-/ - genetically engineered mouse (GEM) model of melanoma, as well as GEM-derived allografts of BrafV600E /Pten-/ - and BrafV600E /Pten-/ -/Cdkn2a-/ - tumors with Ambra1 knockdown. The effects of Ambra1 loss on the tumor immune microenvironment (TIME) were analyzed using NanoString technology, multiplex immunohistochemistry, and flow cytometry. Transcriptome and CIBERSORT digital cytometry analyses of murine melanoma samples and human melanoma patients (The Cancer Genome Atlas) were applied to determine the immune cell populations in null or low-expressing AMBRA1 melanoma. The contribution of Ambra1 on T-cell migration was evaluated using a cytokine array and flow cytometry. Tumor growth kinetics and overall survival analysis in BrafV600E /Pten-/ -/Cdkn2a-/ - mice with Ambra1 knockdown were evaluated prior to and after administration of a programmed cell death protein-1 (PD-1) inhibitor. RESULTS: Loss of Ambra1 was associated with altered expression of a wide range of cytokines and chemokines as well as decreased infiltration of tumors by regulatory T cells, a subpopulation of T cells with potent immune-suppressive properties. These changes in TIME composition were associated with the autophagic function of Ambra1. In the BrafV600E /Pten-/ -/Cdkn2a-/ - model inherently resistant to immune checkpoint blockade, knockdown of Ambra1 led to accelerated tumor growth and reduced overall survival, but at the same time conferred sensitivity to anti-PD-1 treatment. CONCLUSIONS: This study shows that loss of Ambra1 affects the TIME and the antitumor immune response in melanoma, highlighting new functions of Ambra1 in the regulation of melanoma biology.