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Abstract Background Melanoma progression is based on a close interaction between cancer cells and immune cells in the tumor microenvironment (TME). Thus, a better understanding of the mechanisms controlling TME dynamics and composition will help improve the management of this dismal disease. Work from our and other groups has reported the requirement of an active Hedgehog-GLI (HH-GLI) signaling for melanoma growth and stemness. However, the role of the downstream GLI1 transcription factor in melanoma TME remains largely unexplored. Methods The immune-modulatory activity of GLI1 was evaluated in a syngeneic B16F10 melanoma mouse model assessing immune populations by flow cytometry. Murine polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) were differentiated from bone marrow cells and their immunosuppressive ability was assessed by inhibition of T cells. Conditioned media (CM) from GLI1-overexpressing mouse melanoma cells was used to culture PMN-MDSCs, and the effects of CM were evaluated by Transwell invasion assay and T cell inhibition. Cytokine array analysis, qPCR and chromatin immunoprecipitation were performed to explore the regulation of CX3CL1 expression by GLI1. Human monocyte-derived dendritic cells (moDCs) were cultured in CM from GLI1-silenced patient-derived melanoma cells to assess their activation and recruitment. Blocking antibodies anti-CX3CL1, anti-CCL7 and anti-CXCL8 were used for in vitro functional assays. Results Melanoma cell-intrinsic activation of GLI1 promotes changes in the infiltration of immune cells, leading to accumulation of immunosuppressive PMN-MDSCs and regulatory T cells, and to decreased infiltration of dendric cells (DCs), CD8 + and CD4 + T cells in the TME. In addition, we show that ectopic expression of GLI1 in melanoma cells enables PMN-MDSC expansion and recruitment, and increases their ability to inhibit T cells. The chemokine CX3CL1, a direct transcriptional target of GLI1, contributes to PMN-MDSC expansion and recruitment. Finally, silencing of GLI1 in patient-derived melanoma cells promotes the activation of human monocyte-derived dendritic cells (moDCs), increasing cytoskeleton remodeling and invasion ability. This phenotype is partially prevented by blocking the chemokine CCL7, but not CXCL8. Conclusion Our findings highlight the relevance of tumor-derived GLI1 in promoting an immune-suppressive TME, which allows melanoma cells to evade the immune system, and pave the way for the design of new combination treatments targeting GLI1.

Abstract Background Glioblastoma multiforme (GBM) is a highly aggressive primary brain tumor, that is refractory to standard treatment and to immunotherapy with immune-checkpoint inhibitors (ICI). Noteworthy, melanoma brain metastases (MM-BM), that share the same niche as GBM, frequently respond to current ICI therapies. Epigenetic modifications regulate GBM cellular proliferation, invasion, and prognosis and may negatively regulate the cross-talk between malignant cells and immune cells in the tumor milieu, likely contributing to limit the efficacy of ICI therapy of GBM. Thus, manipulating the tumor epigenome can be considered a therapeutic opportunity in GBM. Methods Microarray transcriptional and methylation profiles, followed by gene set enrichment and IPA analyses, were performed to study the differences in the constitutive expression profiles of GBM vs MM-BM cells, compared to the extracranial MM cells and to investigate the modulatory effects of the DNA hypomethylating agent (DHA) guadecitabine among the different tumor cells. The prognostic relevance of DHA-modulated genes was tested by Cox analysis in a TCGA GBM patients’ cohort. Results The most striking differences between GBM and MM-BM cells were found to be the enrichment of biological processes associated with tumor growth, invasion, and extravasation with the inhibition of MHC class II antigen processing/presentation in GBM cells. Treatment with guadecitabine reduced these biological differences, shaping GBM cells towards a more immunogenic phenotype. Indeed, in GBM cells, promoter hypomethylation by guadecitabine led to the up-regulation of genes mainly associated with activation, proliferation, and migration of T and B cells and with MHC class II antigen processing/presentation. Among DHA-modulated genes in GBM, 7.6% showed a significant prognostic relevance. Moreover, a large set of immune-related upstream-regulators (URs) were commonly modulated by DHA in GBM, MM-BM, and MM cells: DHA-activated URs enriched for biological processes mainly involved in the regulation of cytokines and chemokines production, inflammatory response, and in Type I/II/III IFN-mediated signaling; conversely, DHA-inhibited URs were involved in metabolic and proliferative pathways. Conclusions Epigenetic remodeling by guadecitabine represents a promising strategy to increase the efficacy of cancer immunotherapy of GBM, supporting the rationale to develop new epigenetic-based immunotherapeutic approaches for the treatment of this still highly deadly disease.

During the production of salmonids in aquaculture, it is common to observe growth-stunted individuals. The cause for the so-called “loser fish syndrome” is unclear, which needs further investigation. Here, we present and compare computer vision systems for the automatic detection and classification of loser fish in Atlantic salmon images taken in sea cages. We evaluated two end-to-end approaches (combined detection and classification) based on YoloV5 and YoloV7, and a two-stage approach based on transfer learning for detection and an ensemble of classifiers (e.g., linear perception, Adaline, C-support vector, K-nearest neighbours, and multi-layer perceptron) for classification. To our knowledge, the use of an ensemble of classifiers, considering consolidated classifiers proposed in the literature, has not been applied to this problem before. Classification entailed the assigning of every fish to a healthy and a loser class. The results of the automatic classification were compared to the reliability of human classification. The best-performing computer vision approach was based on YoloV7, which reached a precision score of 86.30%, a recall score of 71.75%, and an F1 score of 78.35%. YoloV5 presented a precision of 79.7%, while the two-stage approach reached a precision of 66.05%. Human classification had a substantial agreement strength (Fleiss’ Kappa score of 0.68), highlighting that evaluation by a human is subjective. Our proposed automatic detection and classification system will enable farmers and researchers to follow the abundance of losers throughout the production period. We provide our dataset of annotated salmon images for further research.

The emerging field of radiomics aims to extract quantitative information from medical images. This advanced analysis technique seeks to identify specific biomarkers that can improve patient categorization and care. In oncology, clinicians collaborate with experts in image processing to design new predictive models based on artificial intelligence. This article demonstrates how technicizing diagnostic and prognostic tools has led both to a convergence of interests in this new field – that allows for an accumulation of scientific capital – and to tensions affecting, among other things, the criteria involved in the validation of technologies. In particular, the performance metrics used by researchers do not allow clinicians to measure their clinical usefulness, which is judged by the context of use. Therefore, various standards are applied to evaluate these new imaging biomarkers and their success depends on the articulation between medical and computational knowledge.

IntroductionAbout 50% of cutaneous melanoma (CM) patients present activating BRAF mutations that can be effectively targeted by BRAF inhibitors (BRAFi). However, 20% of CM patients exhibit intrinsic drug resistance to BRAFi, while most of the others develop adaptive resistance over time. The mechanisms involved in BRAFi resistance are disparate and globally seem to rewire the cellular signaling profile by up-regulating different receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR). RTKs inhibitors have not clearly demonstrated anti-tumor activity in BRAFi resistant models. To overcome this issue, we wondered whether the shared up-regulated RTK phenotype associated with BRAFi resistance could be exploited by using immune weapons as the antibody-dependent cell cytotoxicity (ADCC)-mediated effect of anti-RTKs antibodies, and kill tumor cells independently from the mechanistic roots.Methods and resultsBy using an in vitro model of BRAFi resistance, we detected increased membrane expression of EGFR, both at mRNA and protein level in 4 out of 9 BRAFi-resistant (VR) CM cultures as compared to their parental sensitive cells. Increased EGFR phosphorylation and AKT activation were observed in the VR CM cultures. EGFR signaling appeared dispensable for maintaining resistance, since small molecule-, antibody- and CRISPR-targeting of EGFR did not restore sensitivity of VR cells to BRAFi. Importantly, immune-targeting of EGFR by the anti-EGFR antibody cetuximab efficiently and specifically killed EGFR-expressing VR CM cells, both in vitro and in humanized mouse models in vivo, triggering ADCC by healthy donors’ and patients’ peripheral blood cells. ConclusionOur data demonstrate the efficacy of immune targeting of RTKs expressed by CM relapsing on BRAFi, providing the proof-of-concept supporting the assessment of anti-RTK antibodies in combination therapies in this setting. This strategy might be expected to concomitantly trigger the crosstalk of adaptive immune response leading to a complementing T cell immune rejection of tumors.

Abstract SARS-CoV-2 has evolved several strategies to overcome host cell defenses by inducing cell injury to favour its replication. Many viruses have been reported to modulate the intracellular redox balance, affecting the Nuclear factor erythroid 2-Related Factor 2 (NRF2) signaling pathway. Although antioxidant modulation by SARS-CoV-2 infection has already been described, the viral factors involved in modulating the NRF2 pathway are still elusive. Given the antagonistic activity of ORF6 on several cellular pathways, we investigated the role of the viral protein towards NRF2-mediated antioxidant response. The ectopic expression of the wt-ORF6 protein negatively impacts redox cell homeostasis, leading to an increase in ROS production, along with a decrease in NRF2 protein and its downstream controlled genes. Moreover, when investigating the Δ61 mutant, previously described as an inactive nucleopore proteins binding mutant, we prove that the oxidative stress induced by ORF6 is substantially related to its C-terminal domain, speculating that ORF6 mechanism of action is associated with the inhibition of nuclear mRNA export processes. In addition, activation by phosphorylation of the serine residue at position 40 of NRF2 is increased in the cytoplasm of wt-ORF6-expressing cells, supporting the presence of an altered redox state, although NRF2 nuclear translocation is hindered by the viral protein to fully antagonize the cell response. Furthermore, wt-ORF6 leads to phosphorylation of a stress-activated serine/threonine protein kinase, p38 MAPK, suggesting a role of the viral protein in regulating p38 activation. These findings strengthen the important role of oxidative stress in the pathogenesis of SARS-CoV-2 and identify ORF6 as an important viral accessory protein hypothetically involved in modulating the antioxidant response during viral infection.

Abstract Association with hypomethylating agents is a promising strategy to improve the efficacy of immune checkpoint inhibitors-based therapy. The NIBIT-M4 was a phase Ib, dose-escalation trial in patients with advanced melanoma of the hypomethylating agent guadecitabine combined with the anti-CTLA-4 antibody ipilimumab that followed a traditional 3 + 3 design (NCT02608437). Patients received guadecitabine 30, 45 or 60 mg/m2/day subcutaneously on days 1 to 5 every 3 weeks starting on week 0 for a total of four cycles, and ipilimumab 3 mg/kg intravenously starting on day 1 of week 1 every 3 weeks for a total of four cycles. Primary outcomes of safety, tolerability, and maximum tolerated dose of treatment were previously reported. Here we report the 5-year clinical outcome for the secondary endpoints of overall survival, progression free survival, and duration of response, and an exploratory integrated multi-omics analysis on pre- and on-treatment tumor biopsies. With a minimum follow-up of 45 months, the 5-year overall survival rate was 28.9% and the median duration of response was 20.6 months. Re-expression of immuno-modulatory endogenous retroviruses and of other repetitive elements, and a mechanistic signature of guadecitabine are associated with response. Integration of a genetic immunoediting index with an adaptive immunity signature stratifies patients/lesions into four distinct subsets and discriminates 5-year overall survival and progression free survival. These results suggest that coupling genetic immunoediting with activation of adaptive immunity is a relevant requisite for achieving long term clinical benefit by epigenetic immunomodulation in advanced melanoma patients.

The combined approaches between ex situ and in situ conservation are of great importance for threatened species in urgent need of protection. This study aims to develop concrete actions to preserve the relic of 30 adult trees of the Sicilian fir (Abies nebrodensis) from extinction using long-term germplasm conservation in liquid nitrogen (LN, −196 °C). Pollen grains were collected, and their moisture content (MC) was measured. Then, viability (2,3,5-tryphenyl tetrazolium chloride, TTC), in vitro germinability, and enzymatic antioxidant activity (ascorbate peroxidase, APX; catalase, CAT) were evaluated before and after cryopreservation. Seeds collected from mature cones underwent X-ray analysis, and only full seeds were used to excise the zygotic embryos (ZEs) for cryopreservation. The MC percentage of ZEs was determined, and then they were plunged in LN with (+PVS2) or without (−PVS2) Plant Vitrification Solution 2; untreated ZEs were used as a control. Viability (TTC test) and in vitro germination were assessed for all ZEs (+PVS2, −PVS2, and control). Embryogenic callus (EC) lines obtained from mature ZEs were cryopreserved applying the ‘encapsulation-dehydration’ technique. This study has allowed, after optimizing cryopreservation protocols for pollen, ZEs, and EC of A. nebrodensis, to establish the first cryobank of this endangered species in Polizzi Generosa (Palermo, Italy), inside the ‘Madonie Regional Park’. The strategy developed for Sicilian fir conservation will pave the way for similar initiatives for other critically endangered conifer species.

Non-structural protein 1 (Nsp1) represents one of the most crucial SARS-CoV-2 virulence factors by inhibiting the translation of host mRNAs and promoting their degradation. We selected naturally occurring virus lineages with specific Nsp1 deletions located at both the N- and C-terminus of the protein. Our data provide new insights into how Nsp1 coordinates these functions on host and viral mRNA recognition. Residues 82–85 in the N-terminal part of Nsp1 likely play a role in docking the 40S mRNA entry channel, preserving the inhibition of host gene expression without affecting cellular mRNA decay. Furthermore, this domain prevents viral mRNAs containing the 5′-leader sequence to escape translational repression. These findings support the presence of distinct domains within the Nsp1 protein that differentially modulate mRNA recognition, translation and turnover. These insights have implications for the development of drugs targeting viral proteins and provides new evidences of how specific mutations in SARS-CoV-2 Nsp1 could attenuate the virus.

Aberrant activation of Hedgehog (HH) signaling in cancer is the result of genetic alterations of upstream pathway components (canonical) or other oncogenic mechanisms (noncanonical), that ultimately concur to activate the zinc-finger transcription factors GLI1 and GLI2. Therefore, inhibition of GLI activity is a good therapeutic option to suppress both canonical and noncanonical activation of the HH pathway. However, only a few GLI inhibitors are available, and none of them have the profile required for clinical development due to poor metabolic stability and aqueous solubility, and high hydrophobicity. Two promising quinoline inhibitors of GLI were selected by virtual screening and subjected to hit-to-lead optimization, thus leading to the identification of the 4-methoxy-8-hydroxyquinoline derivative JC19. This molecule impaired GLI1 and GLI2 activities in several cellular models interfering with the binding of GLI1 and GLI2 to DNA. JC19 suppressed cancer cell proliferation by enhancing apoptosis, inducing a strong anti-tumor response in several cancer cell lines in vitro. Specificity towards GLI1 and GLI2 was demonstrated by lower activity of JC19 in GLI1- or GLI2-depleted cancer cells. JC19 showed excellent metabolic stability and high passive permeability. Notably, JC19 inhibited GLI1-dependent melanoma xenograft growth in vivo, with no evidence of toxic effects in mice. These results highlight the potential of JC19 as a novel anti-cancer agent targeting GLI1 and GLI2.

Abstract Background Improvement of efficacy of immune checkpoint blockade (ICB) remains a major clinical goal. Association of ICB with immunomodulatory epigenetic drugs is an option. However, epigenetic inhibitors show a heterogeneous landscape of activities. Analysis of transcriptional programs induced in neoplastic cells by distinct classes of epigenetic drugs may foster identification of the most promising agents. Methods Melanoma cell lines, characterized for mutational and differentiation profile, were treated with inhibitors of DNA methyltransferases (guadecitabine), histone deacetylases (givinostat), BET proteins (JQ1 and OTX-015), and enhancer of zeste homolog 2 (GSK126). Modulatory effects of epigenetic drugs were evaluated at the gene and protein levels. Master molecules explaining changes in gene expression were identified by Upstream Regulator (UR) analysis. Gene set enrichment and IPA were used respectively to test modulation of guadecitabine-specific gene and UR signatures in baseline and on-treatment tumor biopsies from melanoma patients in the Phase Ib NIBIT-M4 Guadecitabine + Ipilimumab Trial. Prognostic significance of drug-specific immune-related genes was tested with Timer 2.0 in TCGA tumor datasets. Results Epigenetic drugs induced different profiles of gene expression in melanoma cell lines. Immune-related genes were frequently upregulated by guadecitabine, irrespective of the mutational and differentiation profiles of the melanoma cell lines, to a lesser extent by givinostat, but mostly downregulated by JQ1 and OTX-015. GSK126 was the least active drug. Quantitative western blot analysis confirmed drug-specific modulatory profiles. Most of the guadecitabine-specific signature genes were upregulated in on-treatment NIBIT-M4 tumor biopsies, but not in on-treatment lesions of patients treated only with ipilimumab. A guadecitabine-specific UR signature, containing activated molecules of the TLR, NF-kB, and IFN innate immunity pathways, was induced in drug-treated melanoma, mesothelioma and hepatocarcinoma cell lines and in a human melanoma xenograft model. Activation of guadecitabine-specific UR signature molecules in on-treatment tumor biopsies discriminated responding from non-responding NIBIT-M4 patients. Sixty-five % of the immune-related genes upregulated by guadecitabine were prognostically significant and conferred a reduced risk in the TCGA cutaneous melanoma dataset. Conclusions The DNMT inhibitor guadecitabine emerged as the most promising immunomodulatory agent among those tested, supporting the rationale for usage of this class of epigenetic drugs in combinatorial immunotherapy approaches.

Abstract Background Melanoma is the deadliest form of skin cancer and metastatic disease is associated with a significant survival rate drop. There is an urgent need for consistent tumor biomarkers to scale precision medicine and reduce cancer mortality. Here, we aimed to identify a melanoma-specific circulating microRNA signature and assess its value as a diagnostic tool. Methods The study consisted of a discovery phase and two validation phases. Circulating plasma extracellular vesicles (pEV) associated microRNA profiles were obtained from a discovery cohort of metastatic melanoma patients and normal subjects as controls. A pEV-microRNA signature was obtained using a LASSO penalized logistic regression model. The pEV-microRNA signature was subsequently validated both in a publicly available dataset and in an independent internal cohort. Results We identified and validated in three independent cohorts a panel of melanoma-specific circulating microRNAs that showed high accuracy in differentiating melanoma patients from healthy subjects with an area under the curve (AUC) of 1.00, 0.94 and 0.75 respectively. Investigation of the function of the pEV-microRNA signature evidenced their possible immune suppressive role in melanoma patients. Conclusions We demonstrate that a blood test based on circulating microRNAs can non-invasively detect melanoma, offering a novel diagnostic tool for improving standard care. Moreover, we revealed an immune suppressive role for melanoma pEV-microRNAs.

Background Chemoimmunotherapy represents the standard of care for patients with advanced non-small cell lung cancer (NSCLC) and programmed death-ligand 1 (PD-L1)

Exposing fruits and vegetables to UVB radiation post-harvest is a technique used to modify secondary metabolites and prolong their shelf life. The aim of the present study was to evaluate the effects of UVB irradiation on the chemical and physical characteristics of fig cv. Dottato fruits. The UVB irradiation was 2.26 Wm−2. Two exposure times were carried out: 10 and 60 min resulting in a UVB dose of 1.4 and 8.1 kJm−2, respectively. In the control, the UVB was eliminated by a polyester film (control −UVB). After treatment, the fig fruits were stored and analyzed at different times until decay. Quality parameters (decay, weight loss, color, chlorophyll, and firmness) and physicochemical parameters (soluble solids content, pH parameters, and titratable acidity) were positively influenced by irradiation. Total and individual sugars increased gradually during the storage period in both the skin and the flesh, with glucose being higher after 10 days in the UVB treated samples. Total carotenoid content increased gradually during the storage period, with a marked increase in the +UVB fruit. The content of total and individual polyphenols was positively influenced by UVB treatment, with the UVB treated samples showing the highest values at both 7 and 10 days. The study showed an increase in by-products in both the skin and the flesh. This research confirms the effectiveness of UVB radiation in improving the nutritional qualities and shelf life of Ficus carica fruits.

Toscana virus (TOSV) is an arthropodborne virus, belonging to the genus Phlebovirus, that was first isolated in Monte Argentario in 1971 from the sand flies Phlebotomus perniciosus and P. perfiliewi [...]

We report detecting infectious Toscana virus in the seminal fluid of a 25-year-old man from Italy returning from Elba Island. The presence of infectious virus in human semen adds Toscana virus to the long list of viruses detected in this genital fluid and indicates a potential for sexual transmission.

Aim: To compare the effectiveness of different basal insulins (BI) prescribed as an add‐on to or switch from glucagon‐like peptide‐1 receptor agonist (GLP‐1 RA) therapy. Materials and Methods: Retrospective, real‐world data from electronic medical records of 32 Italian diabetes clinics were used, after propensity score adjustment, to compare effectiveness after 6 months of treatment with second‐ versus first‐generation BI (2BI vs. 1BI) or glargine 300 U/ml versus degludec 100 U/ml (Gla‐300 vs. Deg‐100), when added to (ADD‐ON) or in substitution of (SWITCH) GLP‐1 RA. Only comparisons, including a minimum of 100 patients per group, were performed to ensure adequate robustness of the analyses. Results: In the ADD‐ON cohort (N = 700), greater benefits of 2BI versus 1BI were found in glycated haemoglobin {HbA1c; estimated mean difference: −0.32% [95% confidence interval (CI) −0.62; −0.02]; p =.04} and fasting blood glucose [FBG; −20.73 mg/dl (95% CI −35.62; −5.84); p =.007]. In the SWITCH cohort (N = 2097), greater benefits of 2BI versus 1BI were found in HbA1c [−0.22% (95% CI −0.42; −0.02); p =.03], FBG [−10.15 mg/dl (95% CI −19.04; −1.26); p =.03], and body weight [−0.67 kg (95% CI −1.30; −0.04); p =.04]. In the SWITCH cohort starting 2BI (N = 688), marked differences in favour of Gla‐300 versus Deg‐100 were documented in HbA1c [−0.89% (95% CI −1.26; −0.52); p <.001] and FBG [−17.89 mg/dl (95% CI −32.45; −3.33); p =.02]. Using propensity score matching as a sensitivity analysis, the benefit on HbA1c was confirmed [−0.55% (95% CI –1.02; −0.08); p =.02]. BI titration was suboptimal in all examined cohorts. Conclusions: 2BI are a valuable option to intensify GLP‐1 RA therapy. Switching to Gla‐300 versus Deg‐100 was associated with greater HbA1c improvement. [ABSTRACT FROM AUTHOR]

The severe acute respiratory syndrome coronavirus 2 variant JN.1 recently emerged as the dominant variant despite having only one amino acid change on the spike (S) protein receptor binding domain (RBD) compared with the ancestral BA.2.86, which never represented more than 5% of global variants. To define at the molecular level the JN.1 ability to spread globally, we interrogated a panel of 899 neutralizing human monoclonal antibodies. Our data show that the single leucine-455–to–serine mutation in the JN.1 spike protein RBD unleashed the global spread of JN.1, likely occurring by elimination of more than 70% of the neutralizing antibodies mediated by IGHV3-53/3-66 germlines. However, the resilience of class 3 antibodies with low neutralization potency but strong Fc functions may explain the absence of JN.1 severe disease. Editor's summary: New variants of SARS-CoV-2 continue to emerge and enable viral immune evasion. Using a panel of 899 human monoclonal neutralizing antibodies (nAbs), Paciello et al. examined antibody neutralization activity and effector functions against the recently dominant JN.1 variant. Compared with the ancestral BA.2.86 strain, around two-thirds of nAbs lost neutralization activity against JN.1, including most derived from the immunoglobulin heavy chain V gene (IGHV) 3-53 and 3-66 germlines. The remaining nAbs were only detected in individuals with a history of multiple vaccinations and Omicron breakthrough infections. Among nAbs that could bind the JN.1 spike protein, Fc functions were largely retained and primarily driven by class 3 antibodies that bind outside of the ACE2 binding region, possibly explaining the absence of JN.1 severe disease despite strong evasion of class 1/2 nAbs. —Claire Olingy [ABSTRACT FROM AUTHOR]