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Hydrogen peroxide (H2O2) is an important metabolite involved in most of the redox metabolism reactions and processes of the cells. H2O2 is recognized as one of the main molecules in the sensing, modulation and signaling of redox metabolism, and it is acting as a second messenger together with hydrogen sulfide (H2S) and nitric oxide (NO). These second messengers activate in turn a cascade of downstream proteins via specific oxidations leading to a metabolic response of the cell. This metabolic response can determine proliferation, survival or death of the cell depending on which downstream pathways (homeostatic, pathological, or protective) have been activated. The cells have several sources of H2O2 and cellular systems strictly control its concentration in different subcellular compartments. This review summarizes research on the role played by H2O2 in signaling pathways of eukaryotic cells and how this signaling leads to homeostatic or pathological responses.

Glioblastoma multiforme (GBM) is a grade IV astrocytoma and the most common form of malignant brain tumor in adults. GBM remains one of the most fatal and least successfully treated solid tumors: current therapies provide a median survival of 12-15 months after diagnosis, due to the high recurrence rate. Glioma Stem Cells (GSCs) are believed to be the real driving force of tumor initiation, progression and relapse. Therefore, better therapeutic strategies GSCs-targeted are needed. Resveratrol is a polyphenolic phytoalexin found in fruits and vegetables displaying pleiotropic health benefits. Many studies have highlighted its chemo-preventive and chemotherapeutic activities in a wide range of solid tumors. In this work, we analyzed the effects of Resveratrol exposure on cell viability, proliferation and motility in seven GSC lines isolated from GBM patients. For the first time in our knowledge, we investigated Resveratrol impact on Wnt signaling pathway in GSCs, evaluating the expression of seven Wnt signaling pathway-related genes and the protein levels of c-Myc and β-catenin. Finally, we analyzed Twist1 and Snail1 protein levels, two pivotal activators of epithelial-mesenchymal transition (EMT) program. Results showed that although response to Resveratrol exposure was highly heterogeneous among GSC lines, generally it was able to inhibit cell proliferation, increase cell mortality, and strongly decrease cell motility, modulating the Wnt signaling pathway and the EMT activators. Treatment with Resveratrol may represent a new interesting therapeutic approach, in order to affect GSCs proliferation and motility, even if further investigations are needed to deeply understand the GSCs heterogeneous response.

DNAM-1 is a major NK cell activating receptor and, together with NKG2D and NCRs, by binding specific ligands, strongly contributes to mediating the killing of tumor or virus-infected cells. DNAM-1 specifically recognizes PVR and Nectin-2 ligands that are expressed on some virus-infected cells and on a broad spectrum of tumor cells of both hematological and solid malignancies. So far, while NK cells engineered for different antigen chimeric receptors (CARs) or chimeric NKG2D receptor have been extensively tested in preclinical and clinical studies, the use of DNAM-1 chimeric receptor-engineered NK cells has been proposed only in our recent proof-of-concept study and deserves further development. The aim of this perspective study is to describe the rationale for using this novel tool as a new anti-cancer immunotherapy.

Abstract BACKGROUND The lack of immune synergy with conventional chemoradiation could explain the failure of checkpoint inhibitors in current clinical trials for high-grade gliomas (HGGs). OBJECTIVE To analyze the impact of radiotherapy (RT), Temozolomide (TMZ) and antiprogrammed cell death protein 1 (αPD1) (as single or combined treatments) on the immune microenvironment of experimental HGGs. METHODS Mice harboring neurosphere /CT-2A HGGs received RT (4 Gy, single dose), TMZ (50 mg/kg, 4 doses) and αPD1 (100 μg, 3 doses) as monotherapies or combinations. The influence on survival, tumor volume, and tumor-infiltrating immune cells was analyzed. RESULTS RT increased total T cells (P = .0159) and cluster of differentiation (CD)8+ T cells (P = .0078) compared to TMZ. Lymphocyte subpopulations resulting from TMZ or αPD1 treatment were comparable with those of controls. RT reduced M2 tumor-associated macrophages/microglia (P = .0019) and monocytic myeloid derived suppressor cells (mMDSCs, P = .0003) compared to controls. The effect on mMDSC was also seen following TMZ and αPD1 treatment, although less pronounced (P = .0439 and P = .0538, respectively). Combining RT with TMZ reduced CD8+ T cells (P = .0145) compared to RT alone. Adding αPD1 partially mitigated this effect as shown by the increased CD8+ T cells/Tregs ratio, even if this result failed to reach statistical significance (P = .0973). Changing the combination sequence of RT, TMZ, and αPD1 did not alter survival nor the immune effects. CONCLUSION RT, TMZ, and αPD1 modify the immune microenvironment of HGG. The combination of RT with TMZ induces a strong immune suppression which cannot be effectively counteracted by αPD1.

The INFOGEST protocol is widely used for standardized in vitro digestion studies, but its application to cell models remains challenging due to residual enzyme activity, bile salts, and matrix effects that impair cell metabolism. This study investigated a post-digestion processing strategy to enable direct testing of INFOGEST digesta on 2D Caco-2 monolayers and intestine-on-chip systems without chemical inhibitors. Five post-digestion storage methodologies were compared: A, no storage; B, liquid nitrogen at − 80 °C; C, liquid nitrogen/freeze-drying; D, − 20 °C/freeze-drying; and E, − 20 °C. Residual enzymatic activities and effects on epithelial cell morphology and viability were evaluated, and the optimized workflow was validated in a microfluidic intestine-on-chip, including plant matrices. The condition D significantly reduced residual enzymatic activity, especially trypsin, identified as a key mediator of epithelial cell disruption. This storage method preserved Caco-2 monolayer integrity at a 25% dilution, with no cytotoxicity or cell detachment. When applied to digested Brassica oleracea and Malus domestica matrices, condition D did not compromise cell viability as evidenced by the results obtained with INFOGEST digesta in the absence of plant food matrices. In intestine-on-chip experiments, treated digesta produced stable TEER values and continuous ZO-1 localization, confirming barrier integrity. This simple freeze-then-lyophilize workflow enables a scalable and biocompatible integration of INFOGEST digestion with advanced cellular models for studying food bioaccessibility and intestinal absorption.

Hermetia illucens is a species of great interest for numerous industrial applications. A high-quality reference genome is already available for H. illucens. However, the worldwide maintenance of numerous captive populations of H. illucens, each with its own genotypic and phenotypic characteristics, made it of interest to perform a de novo genome assembly on one population of H. illucens to define a chromosome-scale genome assembly. By combining the PacBio and the Omni-C proximity ligation technologies, a new H. illucens chromosome-scale genome of 888.59 Mb, with a scaffold N50 value of 162.19 Mb, was assembled. The final chromosome-scale assembly obtained a BUSCO completeness of 89.1%. By exploiting the Omni-C proximity ligation technology, topologically associated domains and other topological features that play a key role in the regulation of gene expression were identified. Further, 65.62% of genomic sequences were masked as repeated sequences, and 32,516 genes were annotated using the MAKER pipeline. The H. illucens Lsp-2 genes that were annotated were further characterized, and the three-dimensional organization of the encoded proteins was predicted. A new chromosome-scale genome assembly of good quality for H. illucens was assembled, and the genomic annotation phase was initiated. The availability of this new chromosome-scale genome assembly enables the further characterization, both genotypically and phenotypically, of a species of interest for several biotechnological applications.

Recently, in vitro models emerge as valuable tools in biomedical research by enabling the investigation of complex physiological processes in a controlled environment, replicating some traits of interest of the biological tissues. This study focuses on the development of tubular polymeric scaffolds, made of electrospun fibers, aimed to generate three‐dimensional (3D) in vitro intestinal models resembling the lumen of the gut. Polycaprolactone (PCL) and polyacrylonitrile (PAN) are used to produce tightly arranged ultrafine fiber meshes via electrospinning in the form of continuous tubular structures, mimicking the basement membrane on which the epithelial barrier is formed. Morphological, physical, mechanical, and piezoelectric properties of the PCL and PAN tubular scaffolds are investigated. They are cultured with Caco‐2 cells using different biological coatings (i.e., collagen, gelatin, and fibrin) and their capability of promoting a compact epithelial layer is assessed. PCL and PAN scaffolds show 42% and 50% porosity, respectively, with pore diameters and size suitable to impede cell penetration, thus promoting an intestinal epithelial barrier formation. Even if both polymeric structures allow Caco‐2 cell adhesion, PAN fiber meshes best suit many requirements needed by this model, including highest mechanical strength upon expansion, porosity and piezoelectric properties, along with the lowest pore size. This study showcases the creation of tubular continuous scaffolds through electrospinning, composed of finely woven fibers of polyacrylonitrile (PAN) and polycaprolactone (PCL). These scaffolds support the formation of an intestinal epithelial layer in vitro, aiming to replicate the small intestine 3D structure. PAN emerges as the most promising material based on wettability, mechanical, piezoelectric and biological properties.

Background Checkpoint immunotherapy unleashes tumor control by T cells, but it is undermined in non-immunogenic tumors, e.g. with low MHC class I expression and low neoantigen burden, such as neuroblastoma (NB). Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an enzyme that trims peptides before loading on MHC class I molecules. Inhibition of ERAP1 results in the generation of new antigens able of inducing potent anti-tumor immune responses. Here, we identify a novel non-toxic combinatorial strategy based on genetic inhibition of ERAP1 and administration of the HDAC inhibitor (HDACi) entinostat that increase the immunogenicity of NB, making it responsive to PD-1 therapy. Methods CRISPR/Cas9-mediated gene editing was used to knockout (KO) the ERAP1 gene in 9464D NB cells derived from spontaneous tumors of TH-MYCN transgenic mice. The expression of MHC class I and PD-L1 was evaluated by flow cytometry (FC). The immunopeptidome of these cells was studied by mass spectrometry. Cocultures of splenocytes derived from 9464D bearing mice and tumor cells allowed the assessment of the effect of ERAP1 inhibition on the secretion of inflammatory cytokines and activation and migration of immune cells towards ERAP1 KO cells by FC. Tumor cell killing was evaluated by Caspase 3/7 assay and flow cytometry analysis. The effect of ERAP1 inhibition on the immune content of tumors was analyzed by FC, immunohistochemistry and multiple immunofluorescence. Results We found that inhibition of ERAP1 makes 9464D cells more susceptible to immune cell-mediated killing by increasing both the recall and activation of CD4 + and CD8 + T cells and NK cells. Treatment with entinostat induces the expression of MHC class I and PD-L1 molecules in 9464D both in vitro and in vivo. This results in pronounced changes in the immunopeptidome induced by ERAP1 inhibition, but also restrains the growth of ERAP1 KO tumors in vivo by remodelling the tumor-infiltrating T-cell compartment. Interestingly, the absence of ERAP1 in combination with entinostat and PD-1 blockade overcomes resistance to PD-1 immunotherapy and increases host survival. Conclusions These findings demonstrate that ERAP1 inhibition combined with HDACi entinostat treatment and PD-1 blockade remodels the immune landscape of a non-immunogenic tumor such as NB, making it responsive to checkpoint immunotherapy.

Background Lung cancer is still the main cause of cancer death worldwide despite the availability of targeted therapies and immune-checkpoint inhibitors combined with chemotherapy. Cancer cell heterogeneity and primary or acquired resistance mechanisms cause the elusive behaviour of this cancer and new biomarkers and active drugs are urgently needed to overcome these limitations. p65BTK, a novel isoform of the Bruton Tyrosine Kinase may represent a new actionable target in non-small cell lung cancer (NSCLC). Methods p65BTK expression was evaluated by immunohistochemistry in 382 NSCLC patients with complete clinico-pathological records including smoking habit, ALK and EGFR status, and in metastatic lymph nodes of 30 NSCLC patients. NSCLC cell lines mutated for p53 and/or a component of the RAS/MAPK pathway and primary lung cancer-derived cells from Kras/Trp53 null mice were used as a preclinical model. The effects of p65BTK inhibition by BTK Tyrosine Kinase Inhibitors (TKIs) (Ibrutinib, AVL-292, RN486) and first-generation EGFR-TKIs (Gefitinib, Erlotinib) on cell viability were evaluated by MTT. The effects of BTK-TKIs on cell growth and clonogenicity were assessed by crystal violet and colony assays, respectively. Cell toxicity assays were performed to study the effect of the combination of non-toxic concentrations of BTK-TKIs with EGFR-TKIs and standard-of-care (SOC) chemotherapy (Cisplatin, Gemcitabine, Pemetrexed). Results p65BTK was significantly over-expressed in EGFR-wild type (wt) adenocarcinomas (AdC) from non-smoker patients and its expression was also preserved at the metastatic site. p65BTK was also over-expressed in cell lines mutated for KRAS or for a component of the RAS/MAPK pathway and in tumors from Kras/Trp53 null mice. BTK-TKIs were more effective than EGFR-TKIs in decreasing cancer cell viability and significantly impaired cell proliferation and clonogenicity. Moreover, non-toxic doses of BTK-TKIs re-sensitized drug-resistant NSCLC cell lines to both target- and SOC therapy, independently from EGFR/KRAS status. Conclusions p65BTK results as an emerging actionable target in non-smoking EGFR-wt AdC, also at advanced stages of disease. Notably, these patients are not eligible for EGFR-TKIs-based therapy due to a lack of EGFR mutation. The combination of BTK-TKIs with EGFR-TKIs is cytotoxic for EGFR-wt/KRAS-mutant/p53-null tumors and BTK-TKIs re-sensitizes drug-resistant NSCLC to SOC chemotherapy. Therefore, our data suggest that adding BTK-TKIs to SOC chemotherapy and EGFR-targeted therapy may open new avenues for clinical trials in currently untreatable NSCLC.

Future long-duration human space missions will expose astronauts to chronically reduced gravitational loading, a condition associated with oxidative stress and epithelial barrier dysfunction. The intestinal epithelial barrier depends on tight junctions (TJs), yet the impact of partial gravity on TJ composition, assembly, and claudin organization remains poorly defined. Here, we show that differentiated intestinal epithelial monolayers exposed to simulated Mars gravity undergo TJ ultrastructural remodeling, characterized by loss of apical membrane “kissing points” and widening of the paracellular space, accompanied by impaired barrier function. Simulated Mars gravity also induces oxidative stress and accumulation of cytoplasmic and nuclear lipid droplets, consistent with altered membrane and lipid homeostasis. At the molecular level, simulated Mars gravity promotes selective TJ changes, with significant downregulation—but not mislocalization—of barrier-forming claudins CLDN1 and CLDN3 and the scaffolding protein ZO-1, while CLDN2, CLDN4, CLDN7, CLDN12, CLDN23, and OCLN remain unchanged. STAT3 activation, but not ERK or NF-κB signaling, may be associated with these alterations and is consistent with a stress-adaptive remodeling response to oxidative stress under simulated Mars gravity. Overall, these findings identify simulated Mars gravity as a disruptor of intestinal barrier homeostasis and highlight TJ remodeling as a target for countermeasures to preserve gut integrity during deep-space missions.