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Abstract Glioblastoma (GBM) represents an aggressive brain tumor, characterized by intra- and inter-tumoral heterogeneity and therapy resistance, leading to unfavourable prognosis. An increasing number of studies pays attention on the regulation of ferroptosis, an iron-dependent cell death, as a strategy to reverse drug resistance in cancer. However, the debate on whether this strategy may have important implications for the treatment of GBM is still ongoing. In the present study, we used ferric ammonium citrate and erastin to evaluate ferroptosis induction effects on two human GBM cell lines, U-251 MG, with proneural characteristics, and T98-G, with a mesenchymal profile. The response to ferroptosis induction was markedly different between cell lines, indeed T98-G cells showed an enhanced antioxidant defence, with increased glutathione levels, as compared to U-251 MG cells. Moreover, using bioinformatic approaches and analysing publicly available datasets from patients’ biopsies, we found that GBM with a mesenchymal phenotype showed an up-regulation of several genes involved in antioxidant mechanisms as compared to proneural subtype. Thus, our results suggest that GBM subtypes differently respond to ferroptosis induction, emphasizing the significance of further molecular studies on GBM to better discriminate between various tumor subtypes and progressively move towards personalized therapy.

In the era of digitalization, the biomedical sector has been affected by the spread of artificial intelligence. In recent years, the possibility of using deep and machine learning methods for clinical diagnostic and therapeutic interventions has been emerging as an essential resource for biomedical imaging. Digital pathology represents innovation in a clinical world that looks for faster and better-performing diagnostic methods, without losing the accuracy of current human-guided analyses. Indeed, artificial intelligence has played a key role in a wide variety of applications that require the analysis of a massive amount of data, including segmentation processes in medical imaging. In this context, artificial intelligence enables the improvement of image segmentation methods, moving towards the development of fully automated systems of analysis able to support pathologists in decision-making procedures. The aim of this review is to aid biologists and clinicians in discovering the most common segmentation open-source tools, including ImageJ (v. 1.54), CellProfiler (v. 4.2.5), Ilastik (v. 1.3.3) and QuPath (v. 0.4.3), along with their customized implementations. Additionally, the tools’ role in the histological imaging field is explored further, suggesting potential application workflows. In conclusion, this review encompasses an examination of the most commonly segmented tissues and their analysis through open-source deep and machine learning tools.

Abstract Background The expression of aquaporin 4 (AQP4) and intermediate filament (IF) proteins is altered in malignant glioblastoma (GBM), yet the expression of the major IF-based cytolinker, plectin (PLEC), and its contribution to GBM migration and invasiveness, are unknown. Here, we assessed the contribution of plectin in affecting the distribution of plasmalemmal AQP4 aggregates, migratory properties, and regulation of cell volume in astrocytes. Methods In human GBM, the expression of glial fibrillary acidic protein (GFAP), AQP4 and PLEC transcripts was analyzed using publicly available datasets, and the colocalization of PLEC with AQP4 and with GFAP was determined by immunohistochemistry. We performed experiments on wild-type and plectin-deficient primary and immortalized mouse astrocytes, human astrocytes and permanent cell lines (U-251 MG and T98G) derived from a human malignant GBM. The expression of plectin isoforms in mouse astrocytes was assessed by quantitative real-time PCR. Transfection, immunolabeling and confocal microscopy were used to assess plectin-induced alterations in the distribution of the cytoskeleton, the influence of plectin and its isoforms on the abundance and size of plasmalemmal AQP4 aggregates, and the presence of plectin at the plasma membrane. The release of plectin from cells was measured by ELISA. The migration and dynamics of cell volume regulation of immortalized astrocytes were assessed by the wound-healing assay and calcein labeling, respectively. Results A positive correlation was found between plectin and AQP4 at the level of gene expression and protein localization in tumorous brain samples. Deficiency of plectin led to a decrease in the abundance and size of plasmalemmal AQP4 aggregates and altered distribution and bundling of the cytoskeleton. Astrocytes predominantly expressed P1c, P1e, and P1g plectin isoforms. The predominant plectin isoform associated with plasmalemmal AQP4 aggregates was P1c, which also affected the mobility of astrocytes most prominently. In the absence of plectin, the collective migration of astrocytes was impaired and the dynamics of cytoplasmic volume changes in peripheral cell regions decreased. Plectin’s abundance on the plasma membrane surface and its release from cells were increased in the GBM cell lines. Conclusions Plectin affects cellular properties that contribute to the pathology of GBM. The observed increase in both cell surface and released plectin levels represents a potential biomarker and therapeutic target in the diagnostics and treatment of GBMs.

Abstract Background Follicular thyroid cancer (FTC) is a prevalent form of differentiated thyroid cancer, whereas anaplastic thyroid cancer (ATC) represents a rare, fast-growing, undifferentiated, and highly aggressive tumor, posing significant challenges for eradication. Ferroptosis, an iron-dependent cell death mechanism driven by the excessive production of reactive oxygen species and subsequent lipid peroxidation, emerges as a promising therapeutic strategy for cancer. It has been observed that many cancer cells exhibit sensitivity to ferroptosis, while some other histotypes appear to be resistant, by counteracting the metabolic changes and oxidative stress induced by iron overload. Methods Here we used human biopsies and in vitro approaches to analyse the effects of iron-dependent cell death. We assessed cell proliferation and viability through MTT turnover, clonogenic assays, and cytofluorimetric-assisted analysis. Lipid peroxidation assay and western blot were used to analyse molecular mechanisms underlying ferroptosis modulation. Two distinct thyroid cancer cell lines, FTC-133 (follicular) and 8505C (anaplastic), were utilized. These cell lines were exposed to ferroptosis inducers, Erastin and RSL3, while simulating an iron overload condition using ferric ammonium citrate. Results Our evidence suggests that FTC-133 cell line, exposed to iron overload, reduced their viability and showed increased ferroptosis. In contrast, the 8505C cell line seems to better tolerate ferroptosis, responding by modulating CD71, which is involved in iron internalization and seems to have a role in resistance to iron overload and consequently in maintaining cell viability. Conclusions The differential tolerance to ferroptosis observed in our study may hold clinical implications, particularly in addressing the unmet therapeutic needs associated with ATC treatment, where resistance to ferroptosis appears more pronounced compared to FTC.

Glioblastoma (GBM) is one of the most aggressive cancers, characterized by a decrease in antioxidant levels. Evidence has demonstrated that ferulic acid (FA), a natural antioxidant particularly abundant in vegetables and fruits, could be a promising candidate for GBM treatment. Since FA shows a high instability that compromises its therapeutic application, it has been encapsulated into Nanostructured Lipid Carriers (NLCs) to improve its bioavailability in the brain. It has been demonstrated that tissue transglutaminase (TG2) is a multi-functional protein implicated in many physiological and pathological processes, including cancer. TG2 is also involved in GBM correlated with metastasis formation and drug resistance. Therefore, the evaluation of TG2 expression levels and its cellular localization are important to assess the anti-cancer effect of FA against GBM cancer. Our results have demonstrated that treatment with free FA and FA-NLCs in the U87-MG cancer cell line differently modified TG2 localization and expression levels. In the cells treated with free FA, TG2 appeared expressed both in the cytosol and in the nucleus, while the treatment with FA-NLCs showed that the protein is exclusively localized in the cytosol, exerting its pro-apoptotic effect. Therefore, our data suggest that FA loaded in NLCs could represent a promising natural agent for supplementing the current anti-cancer drugs used for the treatment of GBM.

Background: Chronic myeloid leukemia is a hematological malignancy characterized by the abnormal proliferation of leukemic cells. Despite significant progress with tyrosine kinase inhibitors, such as Dasatinib, resistance remains a challenge. The aim of the present study was to investigate the potential of Selinexor, an Exportin-1 inhibitor, to improve TKI effectiveness on CML. Methods: Human CML cell lines (LAMA84 and K562) were treated with Selinexor, Dasatinib, or their combination. Apoptosis, mitochondrial membrane potential, and mitochondrial mass were assessed using flow cytometry. Real-time RT-PCR was used to evaluate the expression of genes related to mitochondrial function. Western blot and confocal microscopy examined PINK and heme oxygenase-1 (HO-1) protein levels. Results: Selinexor induced apoptosis and mitochondrial depolarization in CML cell lines, reducing cell viability. The Dasatinib/Selinexor combination further enhanced cytotoxicity, modified mitochondrial fitness, and downregulated HO-1 nuclear translocation, which has been associated with drug resistance in different models. Conclusions: In conclusion, this study suggests that Dasatinib/Selinexor could be a promising therapeutic strategy for CML, providing new insights for new targeted therapies.

Proton boron capture therapy (PBCT) in a preclinical glioblastoma model exhibits increased therapeutic effectiveness with an increased cell death and mitophagy, supporting its use as a scalable strategy to treat glioblastoma.

Adipose-derived mesenchymal stem cells (ASCs) are adult multipotent stem cells, able to differentiate toward neural elements other than cells of mesodermal lineage. The aim of this research was to test ASC neural differentiation using melatonin combined with conditioned media (CM) from glial cells. Isolated from the lipoaspirate of healthy donors, ASCs were expanded in a basal growth medium before undergoing neural differentiation procedures. For this purpose, CM obtained from olfactory ensheathing cells and from Schwann cells were used. In some samples, 1 µM of melatonin was added. After 1 and 7 days of culture, cells were studied using immunocytochemistry and flow cytometry to evaluate neural marker expression (Nestin, MAP2, Synapsin I, GFAP) under different conditions. The results confirmed that a successful neural differentiation was achieved by glial CM, whereas the addition of melatonin alone did not induce appreciable changes. When melatonin was combined with CM, ASC neural differentiation was enhanced, as demonstrated by a further improvement of neuronal marker expression, whereas glial differentiation was attenuated. A dynamic modulation was also observed, testing the expression of melatonin receptors. In conclusion, our data suggest that melatonin’s neurogenic differentiation ability can be usefully exploited to obtain neuronal-like differentiated ASCs for potential therapeutic strategies.

The aim of the present study consists of the evaluation of the biodistribution of a novel 68Ga-labeled radiopharmaceutical, [68Ga]Ga-NODAGA-Z360, injected into Balb/c nude mice through histopathological analysis on bioptic samples and radiomics analysis of positron emission tomography/computed tomography (PET/CT) images. The 68Ga-labeled radiopharmaceutical was designed to specifically bind to the cholecystokinin receptor (CCK2R). This receptor, naturally present in healthy tissues such as the stomach, is a biomarker for numerous tumors when overexpressed. In this experiment, Balb/c nude mice were xenografted with a human epidermoid carcinoma A431 cell line (A431 WT) and overexpressing CCK2R (A431 CCK2R+), while controls received a wild-type cell line. PET images were processed, segmented after atlas-based co-registration and, consequently, 112 radiomics features were extracted for each investigated organ / tissue. To confirm the histopathology at the tissue level and correlate it with the degree of PET uptake, the studies were supported by digital pathology. As a result of the analyses, the differences in radiomics features in different body districts confirmed the correct targeting of the radiopharmaceutical. In preclinical imaging, the methodology confirms the importance of a decision-support system based on artificial intelligence algorithms for the assessment of radiopharmaceutical biodistribution.

In the original publication [...]

Abstract Mesenchymal stromal cells (MSCs) within the protective microenvironment of multiple myeloma (MM) promote tumor growth, confer chemoresistance and support metabolic needs of plasma cells (PCs) even transferring mitochondria. In this scenario, heterocellular communication and dysregulation of critical signaling axes are among the major contributors to progression and treatment failure. Here, we report that myeloma MSCs have decreased reliance on mitochondrial metabolism as compared to healthy MSCs and increased tendency to deliver mitochondria to MM cells, suggesting that this intercellular exchange between PCs and stromal cells can be consider part of MSC pro-tumorigenic phenotype. Interestingly, we also showed that PCs promoted expression of connexin 43 (CX43) in MSCs leading to CXCL12 activation and stimulation of its receptor CXCR4 on MM cells favoring protumor mitochondrial transfer. Consistently, we observed that selective inhibition of CXCR4 by plerixafor resulted in a significant reduction of mitochondria trafficking. Moreover, intracellular expression of CXCR4 in myeloma PCs from BM biopsy specimens demonstrated higher CXCR4 colocalization with CD138+ cells of non-responder patients to bortezomib compared with responder patients, suggesting that CXCR4 mediated chemoresistance in MM. Taken together, our data demonstrated that CXCL12/CXCR4 axis mediates intercellular coupling thus suggesting that the myeloma niche may be exploited as a target to improve and develop therapeutic approaches.

Chronic myeloproliferative neoplasms encompass the BCR-ABL1-negative neoplasms polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). These are characterized by calreticulin (CALR), myeloproliferative leukemia virus proto-oncogene (MPL) and the tyrosine kinase Janus kinase 2 (JAK2) mutations, eventually establishing a hyperinflammatory tumor microenvironment (TME). Several reports have come to describe how constitutive activation of JAK-STAT and NFκB signaling pathways lead to uncontrolled myeloproliferation and pro-inflammatory cytokines secretion. In such a highly oxidative TME, the balance between Hematopoietic Stem Cells (HSCs) and Mesenchymal Stromal Cells (MSCs) has a crucial role in MPN development. For this reason, we sought to review the current literature concerning the interplay between HSCs and MSCs. The latter have been reported to play an outstanding role in establishing of the typical bone marrow (BM) fibrotic TME as a consequence of the upregulation of different fibrosis-associated genes including PDGF- β upon their exposure to the hyperoxidative TME characterizing MPNs. Therefore, MSCs might turn to be valuable candidates for niche-targeted targeting the synthesis of cytokines and oxidative stress in association with drugs eradicating the hematopoietic clone.

Accumulating evidence sustains glial cells as critical players during central nervous system (CNS) development, homeostasis and disease. Olfactory ensheathing cells (OECs), a type of specialized glia cells sharing properties with both Schwann cells and astrocytes, are of critical importance in physiological condition during olfactory system development, supporting its regenerative potential throughout the adult life. These characteristics prompted research in the field of cell-based therapy to test OEC grafts in damaged CNS. Neuroprotective mechanisms exerted by OEC grafts are not limited to axonal regeneration and cell differentiation. Indeed, OEC immunomodulatory properties and their phagocytic potential encourage OEC-based approaches for tissue regeneration in case of CNS injury. Herein we reviewed recent advances on the immune role of OECs, their ability to modulate CNS microenvironment via bystander effects and the potential of OECs as a cell-based strategy for tissue regeneration.

Tumor onset and its progression are strictly linked to its metabolic rewiring on the basis of the Warburg effect. In this context, fumarate emerged as a putative oncometabolite mediating cancer progression. Fumarate accumulation is usually driven by fumarate hydratase (FH) loss of function, the enzyme responsible for the reversible conversion of fumarate into malate. Fumarate accumulation acts as a double edge sword: on one hand it takes part in the metabolic rewiring of cancer cells, while on the other it also plays a crucial role in chromatin architecture reorganization. The latter is achieved by competing with a-ketoglutarate-dependent enzymes, eventually altering the cellular methylome profile, which in turn leads to its transcriptome modeling. Furthermore, in recent years, it has emerged that FH has an ability to recruit DNA double strand breaks. The accumulation of fumarate into damaged sites might also determine the DNA repair pathway in charge for the seizure of the lesion, eventually affecting the mutational state of the cells. In this work, we aimed to review the current knowledge on the role of fumarate as an oncometabolite orchestrating the cellular epigenetic landscape and DNA repair machinery.

The osteogenic and chondrogenic differentiation ability of adipose-derived mesenchymal stromal cells (ASCs) and their potential therapeutic applications in bone and cartilage defects are reported in this review. This becomes particularly important when these disorders can only be poorly treated by conventional therapeutic approaches, and tissue engineering may represent a valuable alternative. Being of mesodermal origin, ASCs can be easily induced to differentiate into chondrocyte-like and osteocyte-like elements and used to repair damaged tissues. Moreover, they can be easily harvested and used for autologous implantation. A plethora of ASC-based strategies are being developed worldwide: they include the transplantation of freshly harvested cells, in vitro expanded cells or predifferentiated cells. Moreover, improving their positive effects, ASCs can be implanted in combination with several types of scaffolds that ensure the correct cell positioning; support cell viability, proliferation and migration; and may contribute to their osteogenic or chondrogenic differentiation. Examples of these strategies are described here, showing the enormous therapeutic potential of ASCs in this field. For safety and regulatory issues, most investigations are still at the experimental stage and carried out in vitro and in animal models. Clinical applications have, however, been reported with promising results and no serious adverse effects.

Abstract Motoneuronal loss is the main feature of amyotrophic lateral sclerosis, although pathogenesis is extremely complex involving both neural and muscle cells. In order to translationally engage the sonic hedgehog pathway, which is a promising target for neural regeneration, recent studies have reported on the neuroprotective effects of clobetasol, an FDA-approved glucocorticoid, able to activate this pathway via smoothened. Herein we sought to examine functional, cellular, and metabolic effects of clobetasol in a neurotoxic mouse model of spinal motoneuronal loss. We found that clobetasol reduces muscle denervation and motor impairments in part by restoring sonic hedgehog signaling and supporting spinal plasticity. These effects were coupled with reduced pro-inflammatory microglia and reactive astrogliosis, reduced muscle atrophy, and support of mitochondrial integrity and metabolism. Our results suggest that clobetasol stimulates a series of compensatory processes and therefore represents a translational approach for intractable denervating and neurodegenerative disorders.

The tumor microenvironment (TME) plays a pivotal role in establishing malignancy, and it is associated with high glycolytic metabolism and lactate release through monocarboxylate transporters (MCTs). Several lines of evidence suggest that lactate also serves as a signaling molecule through its receptor hydroxycarboxylic acid receptor 1 (HCAR1/GPR81), thus functioning as a paracrine and autocrine signaling molecule. The aim of the present study was to investigate the role of lactate in glioblastoma (GBM) progression and metabolic reprogramming in an in vitro and in vivo model. The cell proliferation, migration, and clonogenicity were tested in vitro in three different human GBM cell lines. The expressions of MCT1, MCT4, and HCAR1 were evaluated both in vitro and in a zebrafish GBM model. The results were further validated in patient-derived GBM biopsies. Our results showed that lactate significantly increased the cell proliferation, migration, and colony formation capacity of GBM cells, both in vitro and in vivo. We also showed that lactate increased the expressions of MCT1 and HCAR1. Moreover, lactate modulated the epithelial–mesenchymal transition protein markers E-cadherin and β-catenin. Interestingly, lactate induced mitochondrial mass and the OXPHOS gene, suggesting improved mitochondrial fitness. Similar effects were observed after treatment with 3,5-dihydroxybenzoic acid, a known agonist of HCAR1. Consistently, the GBM zebrafish model exhibited an altered metabolism and increased expressions of MCT1 and HCAR1, leading to high levels of extracellular lactate and, thus, supporting tumor cell proliferation. Our data from human GBM biopsies also showed that, in high proliferative GBM biopsies, Ki67-positive cells expressed significantly higher levels of MCT1 compared to low proliferative GBM cells. In conclusion, our data suggest that lactate and its transporter and receptor play a major role in GBM proliferation and migration, thus representing a potential target for new therapeutic strategies to counteract tumor progression and recurrence.

Herein, we assess the gene expression changes activated in thyroid tumors through a computational approach, using the MapReduce algorithm. Through this predictive analysis, we identified the TfR1 gene as a critical mediator of thyroid tumor progression. Then, we investigated the effect of TfR1 gene silencing through small interfering RNA (siRNA) in the expression of extracellular signal-regulated kinase 1/2 (Erk1/2) pathway and c-Myc in human differentiated follicular and undifferentiated anaplastic thyroid cancer. The expression levels of cyclin D1, p53, and p27, proteins involved in cell cycle progression, were also evaluated. The effect of TfR1 gene silencing through siRNA on the apoptotic pathway activation was also tested. Computational prediction and in vitro studies demonstrate that TfR1 plays a key role in thyroid cancer and that its downregulation was able to inhibit the ERK pathway, reducing also c-Myc expression, which blocks the cell cycle and activates the apoptotic pathway. We demonstrate that TfR1 plays a crucial role for a rapid and transient activation of the ERK signaling pathway, which induces a deregulation of genes involved in the aberrant accumulation of intracellular free iron and in drug resistance. We also suggest that TfR1 might represent an important target for thyroid cancer therapy. Keywords: human thyroid cancer, TfR1 gene silencing, in silico studies

The central nervous system represents a complex environment in which glioblastoma adapts skillfully, unleashing a series of mechanisms suitable for its efficient development and diffusion. In particular, changes in gene expression and mutational events that fall within the domain of epigenetics interact complexly with metabolic reprogramming and stress responses enacted in the tumor microenvironment, which in turn fuel genomic instability by providing substrates for DNA modifications. The aim of this review is to analyze this complex interaction that consolidates several conditions that confer a state of immunosuppression and immunoevasion, making glioblastoma capable of escaping attack and elimination by immune cells and therefore invincible against current therapies. The progressive knowledge of the cellular mechanisms that underlie the resistance of the glioblastoma represents, in fact, the only weapon to unmask its weak points to be exploited to plan successful therapeutic strategies.

Uveal melanoma (UM), the most common primary intraocular cancer in adults, is among the tumors with poorer prognosis. Recently, the role of the oncometabolite lactate has become attractive due to its role as hydroxycarboxylic acid receptor 1 (HCAR1) activator, as an epigenetic modulator inducing lysine residues lactylation and, of course, as a glycolysis end-product, bridging the gap between glycolysis and oxidative phosphorylation. The aim of the present study was to dissect in UM cell line (92.1) the role of lactate as either a metabolite or a signaling molecule, using the known modulators of HCAR1 and of lactate transporters. Our results show that lactate (20 mM) resulted in a significant decrease in cell proliferation and migration, acting and switching cell metabolism toward oxidative phosphorylation. These results were coupled with increased euchromatin content and quiescence in UM cells. We further showed, in a clinical setting, that an increase in lactate transporters MCT4 and HCAR1 is associated with a spindle-shape histological type in UM. In conclusion, our results suggest that lactate metabolism may serve as a prognostic marker of UM progression and may be exploited as a potential therapeutic target.

Neurodegenerative disorders are characterized by the progressive loss of central and/or peripheral nervous system neurons. Within this context, neuroinflammation comes up as one of the main factors linked to neurodegeneration progression. In fact, neuroinflammation has been recognized as an outstanding factor for Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), and multiple sclerosis (MS). Interestingly, neuroinflammatory diseases are characterized by dramatic changes in the epigenetic profile, which might provide novel prognostic and therapeutic factors towards neuroinflammatory treatment. Deep changes in DNA and histone methylation, along with histone acetylation and altered non-coding RNA expression, have been reported at the onset of inflammatory diseases. The aim of this work is to review the current knowledge on this field.

Neuropathic pain is one of the most disabling forms of chronic pain and it is characterized by hyperalgesia and allodynia linked to an aberrant processing of pain transmission and to neuroinflammation. Transforming growth factor-β1 (TGF-β1) is an anti-inflammatory cytokine, which protects against neuroinflammation. It has been demonstrated that TGF-β1 and opioid receptors signalling crosstalk results in an improvement of endogenous opioid analgesia, but it is not known whether mu opioid peptide receptor (MOPr) or delta opioid peptide receptor (DOPr) agonists can positively modulate TGF-β1 pathway. In the present study, we examined the correlation between anti-allodynic effect of LP2, a dual-target MOPr/DOPr agonist, and TGF-β1 signalling in the chronic constriction injury (CCI) model. We detected a significant decrease of active TGF-β1 and of its type II receptor TGFβ-R2 levels in the spinal cord from CCI rats and a selective deficit of TGF-β1 in microglia cells both at days 11 and 21 post-ligature, as assessed by immunofluorescence analysis. LP2, when administered from the 11 days post-ligature to 21 days, was able to reduce CCI-induced mechanical allodynia by rescue of TGF-β1 and TGFβ-R2 levels. Our data suggest that the rescue of TGF-β1 signalling by dual-target MOPr/DOPr agonist LP2 could be mediated by DOPr activation in spinal microglia, thus the dual-target approach could represent a novel pharmacological approach to increase the analgesic efficacy of MOPr agonists.

Automatically recognizing negative emotions, such as anger or stress, and also positive ones, such as euphoria, can contribute to improving well-being. In real-life, emotion recognition is a difficult task since many of the technologies used for this purpose in both laboratory and clinic environments, such as electroencephalography (EEG) and electrocardiography (ECG), cannot realistically be used. Photoplethysmography (PPG) is a non-invasive technology that can be easily integrated into wearable sensors. This paper focuses on the comparison between PPG and ECG concerning their efficacy in detecting the psychophysical and affective states of the subjects. It has been confirmed that the levels of accuracy in the recognition of affective variables obtained by PPG technology are comparable to those achievable with the more traditional ECG technology. Moreover, the affective psychological condition of the participants (anxiety and mood levels) may influence the psychophysiological responses recorded during the experimental tests.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting motoneurons (MNs) with a fatal outcome. The typical degeneration of cortico-spinal, spinal, and bulbar MNs, observed in post-mortem biopsies, is associated with the activation of neuroimmune cells. GJA1, a member of the connexins (Cxs) gene family, encodes for connexin 43 (Cx43), a core gap junctions (GJs)- and hemichannels (HCs)-forming protein, involved in cell death, proliferation, and differentiation. Recently, Cx43 expression was found to play a role in ALS pathogenesis. Here, we used microarray and RNA-seq datasets from the NCBI of the spinal cord of control (NDC) and ALS patients, which were stratified according to the GJA1 gene expression. Genes that positively or negatively correlated to GJA1 expression were used to perform a genomic deconvolution analysis (GDA) using neuroimmune signatures. Expression analysis revealed a significantly higher GJA1 expression in the MNs of ALS patients as compared to NDC. Gene deconvolution analysis revealed that positively correlated genes were associated with microglia activation, whereas negatively correlated genes were associated with neuronal activation profiles. Moreover, gene ontology analysis, performed on genes characterizing either microglia or neuronal signature, indicated immune activation or neurogenesis as main biological processes. Finally, using a synthetic analysis of drugs able to revert the GJA1 transcriptomic signatures, we found a specific drug profile for ALS patients with high GJA1 expression levels, composed of amlodipine, sertraline, and prednisolone. In conclusion, our exploratory study suggests GJA1 as a new neuro-immunological gene correlated to microglial cellular profile in the spinal cord of ALS patients. Further studies are warranted to confirm these results and to evaluate the therapeutic potential of drugs able to revert typical GJA1/CX43 signature in ALS patients

Hemoglobin and iron overload is considered the major contributor to intracerebral hemorrhage (ICH)-induced brain injury. Accumulation of iron in the brain leads to microglia activation, inflammation and cell loss. Current available treatments for iron overload-mediated disorders are characterized by severe adverse effects, making such conditions an unmet clinical need. We assessed the potential of α-lipoic acid (ALA) as an iron chelator, antioxidant and anti-inflammatory agent in both in vitro and in vivo models of iron overload. ALA was found to revert iron-overload-induced toxicity in HMC3 microglia cell line, preventing cell apoptosis, reactive oxygen species generation and reducing glutathione depletion. Furthermore, ALA regulated gene expression of iron-related markers and inflammatory cytokines, such as IL-6, IL-1β and TNF. Iron toxicity also affects mitochondria fitness and biogenesis, impairments which were prevented by ALA pre-treatment in vitro. Immunocytochemistry assay showed that, although iron treatment caused inflammatory activation of microglia, ALA treatment resulted in increased ARG1 expression, suggesting it promoted an anti-inflammatory phenotype. We also assessed the effects of ALA in an in vivo zebrafish model of iron overload, showing that ALA treatment was able to reduce iron accumulation in the brain and reduced iron-mediated oxidative stress and inflammation. Our data support ALA as a novel approach for iron-overload-induced brain damage.

Central nervous system (CNS) homeostasis is closely linked to the delicate balance of the microenvironment in which different cellular components of the neurovascular unit (NVU) coexist. Intercellular communication plays a pivotal role in exchanges of signaling molecules and mediators essential for survival functions, as well as in the removal of disturbing elements that can lead to related pathologies. The specific signatures of connexins (Cxs), proteins which form either gap junctions (GJs) or hemichannels (HCs), represent the biological substrate of the pathophysiological balance. Connexin 43 (Cx43) is undoubtedly one of the most important factors in glia–neuro–vascular crosstalk. Herein, Cxs signatures of every NVU component are highlighted and their critical influence on functional processes in healthy and pathological conditions of nervous microenvironment is reviewed.

Chronic neuropathic pain emerges from either central or peripheral lesions inducing spontaneous or amplified responses to non-noxious stimuli. Despite different pharmacological approaches to treat such a chronic disease, neuropathic pain still represents an unmet clinical need, due to long-term therapeutic regimens and severe side effects that limit application of currently available drugs. A critical phenomenon involved in central sensitization is the exchange of signalling molecules and cytokines, between glia and neurons, driving the chronicization process. Herein, using a chronic constriction injury (CCI) model of neuropathic pain, we evaluated the efficacy of the mu (M-) and delta (D-) opioid receptor (-OR) targeting agent LP2 in modulating connexin-based heterocellular coupling and cytokine levels. We found that long-term efficacy of LP2 is consequent to MOR-DOR targeting resulting in the reduction of CCI-induced astrocyte-to-microglia heterocellular coupling mediated by connexin 43. We also found that single targeting of DOR reduces TNF and IL-6 levels in the chronic phase of the disease, but the peripheral and central discharge as the primary source of excitotoxic stimulation in the spinal cord requires a simultaneous MOR-DOR targeting to reduce CCI-induced neuropathic pain.

In 2021 the World Health Organization published the fifth and latest version of the Central Nervous System tumors classification, which incorporates and summarizes a long list of updates from the Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy work. Among the adult-type diffuse gliomas, glioblastoma represents most primary brain tumors in the neuro-oncology practice of adults. Despite massive efforts in the field of neuro-oncology diagnostics to ensure a proper taxonomy, the identification of glioblastoma-tumor subtypes is not accompanied by personalized therapies, and no improvements in terms of overall survival have been achieved so far, confirming the existence of open and unresolved issues. The aim of this review is to illustrate and elucidate the state of art regarding the foremost biological and molecular mechanisms that guide the beginning and the progression of this cancer, showing the salient features of tumor hallmarks in glioblastoma. Pathophysiology processes are discussed on molecular and cellular levels, highlighting the critical overlaps that are involved into the creation of a complex tumor microenvironment. The description of glioblastoma hallmarks shows how tumoral processes can be linked together, finding their involvement within distinct areas that are engaged for cancer-malignancy establishment and maintenance. The evidence presented provides the promising view that glioblastoma represents interconnected hallmarks that may led to a better understanding of tumor pathophysiology, therefore driving the development of new therapeutic strategies and approaches.

The 64Cu-labeled chelator was analyzed in vivo by positron emission tomography (PET) imaging to evaluate its biodistribution in a murine model at different acquisition times. For this purpose, nine 6-week-old female Balb/C nude strain mice underwent micro-PET imaging at three different time points after 64Cu-labeled chelator injection. Specifically, the mice were divided into group 1 (acquisition 1 h after [64Cu] chelator administration, n = 3 mice), group 2 (acquisition 4 h after [64Cu]chelator administration, n = 3 mice), and group 3 (acquisition 24 h after [64Cu] chelator administration, n = 3 mice). Successively, all PET studies were segmented by means of registration with a standard template space (3D whole-body Digimouse atlas), and 108 radiomics features were extracted from seven organs (namely, heart, bladder, stomach, liver, spleen, kidney, and lung) to investigate possible changes over time in [64Cu]chelator biodistribution. The one-way analysis of variance and post hoc Tukey Honestly Significant Difference test revealed that, while heart, stomach, spleen, kidney, and lung districts showed a very low percentage of radiomics features with significant variations (p-value < 0.05) among the three groups of mice, a large number of features (greater than 60% and 50%, respectively) that varied significantly between groups were observed in bladder and liver, indicating a different in vivo uptake of the 64Cu-labeled chelator over time. The proposed methodology may improve the method of calculating the [64Cu]chelator biodistribution and open the way towards a decision support system in the field of new radiopharmaceuticals used in preclinical imaging trials.

Iron plays a major role in multiple processes involved in cell homeostasis such as metabolism, respiration and DNA synthesis. Cancer cells exhibit pronounced iron retention as compared to healthy counterpart. This phenomenon also occurs in multiple myeloma (MM), a hematological malignancy characterized by terminally differentiated plasma cells (PCs), in which serum ferritin levels have been reported as a negative prognostic marker. The aim of current study is to evaluate the potential role of iron metabolism in promoting drug resistance in myeloma cancer cells with particular regard to the interactions between PCs and tumor-associated macrophages (TAMs) as a source of iron. Our data showed that myeloma cell lines are able to intake and accumulate iron and thus, increasing their scavenger antioxidant-related genes and mitochondrial mass. We further demonstrated that PCs pre-treated with ferric ammonium citrate (FAC) decreased bortezomib (BTZ)-induced apoptosis in vitro and successfully engrafted in zebrafish larvae treated with BTZ. Treating human macrophages with FAC, we observed a switch toward a M2-like phenotype associated with an increased expression of anti-inflammatory markers such as ARG1, suggesting the establishment of an iron-mediated immune suppressive tumor microenvironment favouring myeloma growth. Using mfap4:tomato mutant zebrafish larvae, we further confirmed the increase of PCs-monocytes interactions after FAC treatment which favour BTZ-resistance. Taken together our data support the hypothesis that targeting iron trafficking in myeloma microenvironment may represent a promising strategy to counteract a tumor-supporting milieu and drug resistance.

The purpose of this paper is to characterize the skin deterministic damage due to the effect of proton beam irradiation in mice occurred during a long-term observational experiment. This study was initially defined to evaluate the insurgence of myelopathy irradiating spinal cords with the distal part of a Spread-out Bragg peak (SOBP). To the best of our knowledge, no study has been conducted highlighting high grades of skin injury at the dose used in this paper. Nevertheless these effects occurred. In this regard, the experimental evidence of significant insurgence of skin injury induced by protons using a SOBP configuration will be shown. Skin damages were classified into six scores (from 0 to 5) according to the severity of the injuries and correlated to ED50 (i.e. the radiation dose at which 50% of animals show a specific score) at 40 days post-irradiation (d.p.i.). The effects of radiation on the overall animal wellbeing have been also monitored and the severity of radiation-induced skin injuries was observed and quantified up to 40 d.p.i.

Breast cancer is the most frequent tumor and the leading cause of cancer deaths in women. In recent years, lactate metabolism and, in particular, its receptor GPR81 have been shown to play a vital role in cancer biology. GPR81 is upregulated in breast cancer and promotes tumor growth by tumor cell-derived lactate. Therefore, the search for possible crosstalk and the involvement of new molecules capable of generating this pathology is always in continuous development. In this study, the relationship between GPR81 and IGFBP6 protein in tumor growth and oxidative stress in the human breast cancer cell line MDA-MB-231 was studied. Cells were treated with lactate or the GPR81 receptor agonist and antagonist 3,5-DHBA and 3-OBA, respectively. In addition, oxidative stress and proliferation were also evaluated in cells challenged with the recombinant IGFBP6 protein. Our data showed that lactate induced cell proliferation and wound healing of the MDA-231 breast cancer cell through the overexpression of both the lactate receptor GPR81 and IGFBP6. The increase in IGFBP6 was able, in turn, to improve the mitochondrial fitness and redox state, as suggested by the reduced levels of mitochondrial ROS production after IGFBP6 treatment, presumably mediated by the increase in the ROS detoxifying genes HMOX1, GSTK1 and NQO1. In conclusion, our data highlight a novel axis between GPR81 and IGFBP6 in MDA-231 cells able to modulate lactate metabolism and oxidative stress. This complex signaling may represent a new therapeutic target for breast cancer.

Abstract Background Our previous study demonstrates that Citrus-limon derived nanovesicles are able to decrease colon cancer cell viability, and that this effect is associated with the downregulation of the intracellular phospholipase DDHD domain-containing protein 1 (DDHD1). While few studies are currently available on the contribution of DDHD1 in neurological disorders, there is no information on its role in cancer. This study investigates the role of DDHD1 in colon cancer. Methods DDHD1 siRNAs and an overexpression vector were transfected into colorectal cancer and normal cells to downregulate or upregulate DDHD1 expression. In vitro and in vivo assays were performed to investigate the functional role of DDHD1 in colorectal cancer cell growth. Quantitative proteomics using SWATH-MS was performed to determinate the molecular effects induced by DDHD1 silencing in colorectal cancer cells. Results The results indicate that DDHD1 supports colon cancer cell proliferation and survival, since its downregulation reduces in vitro colon cancer cell viability and increases apoptosis rate, without affecting normal cells. On the contrary, in vivo studies demonstrate that the xenograft tumors, derived from DDHD1-overexpressing cells, have a higher proliferation rate compared to control animals. Additionally, we found that functional categories, significantly affected by DDHD1 silencing, were specifically related to cancer phenotype and for the first time associated to DDHD1 activity. Conclusions In conclusion, this study provides the first evidence confirming the role of DDHD1 in cancer, providing a possibility to define a new target to design more effective therapies for colon cancer patients.

Pediatric small round blue cell tumors (SRBCTs) are a heterogeneous group of neoplasms with overlapping morphological appearance. Accordingly, their diagnosis is one of the most difficult in the field of surgical pathology. The most common tumors include rhabdomyosarcoma, Ewing’s sarcoma, neuroblastoma, lymphoblastic lymphoma and Wilms’ tumor (the blastemal component). Over time their diagnosis has become more difficult due to the increasing use of small biopsies. However, the advent of immunohistochemistry has improved the quality of diagnosis in most cases by the application of an adequate panel of immunomarkers. Recently, WT1 and Cyclin D1 have been shown to be useful in the differential diagnosis of SRBCTs on surgically-resected specimens, showing a diffuse cytoplasmic positivity of the former in all RMSs and a diffuse nuclear staining of the latter in both EWS and NB. The aim of the present study was to investigate the expression of WT1 and Cyclin D1 on small biopsies from a series of 105 pediatric SRBCTs to evaluate their diagnostic utility. Both immunomarkers were differentially expressed, with a diffuse and strong cytoplasmic staining for WT1 limited to all cases of RMS, and a diffuse nuclear staining for cyclin D1 restricted to all cases of EWS and NB. Notably, the expression of WT1 and cyclin D1 was also retained in those cases in which the conventional tumor markers (myogenin, desmin and MyoD1 for RMS; CD99 for EWS; NB84 for NB) were focally expressed or more rarely absent. The present study shows that WT1 and Cyclin D1 are helpful immunomarkers exploitable in the differential diagnosis of pediatric SRBCTs on small biopsies, suggesting their applicability in routine practice.

Osteoarthritis (OA) is a complex disease, source of pain and disability that affects millions of people worldwide. OA etiology is complex, multifactorial and joint-specific, with genetic, biological and biomechanical components. Recently, several studies have suggested a potential adjuvant role for natural extracts on OA progression, in terms of moderating chondrocyte inflammation and following cartilage injury, thus resulting in an overall improvement of joint pain. In this study, we first analyzed the phenylethanoid glycosides profile and the total amount of polyphenols present in a leaf aqueous extract of Verbascum thapsus L. We then investigated the anti-inflammatory and anti-osteoarthritic bioactive potential of the extract in murine monocyte/macrophage-like cells (RAW 264.7) and in human chondrocyte cells (HC), by gene expression analysis of specifics inflammatory cytokines, pro-inflammatory enzymes and metalloproteases. Six phenylethanoid glycosides were identified and the total phenolic content was 124.0 ± 0.7 mg gallic acid equivalent (GAE)/g of extract. The biological investigation showed that the extract is able to significantly decrease most of the cellular inflammatory markers, compared to both control cells and cells treated with Harpagophytum procumbens (Burch.) DC. ex Meisn, used as a positive control. Verbascum thapsus leaf aqueous extract has the potential to moderate the inflammatory response, representing an innovative possible approach for the inflammatory joint disease treatment.

Glioblastoma (GBM) represents the most common primary brain tumor within the adult population. Current therapeutic options are still limited by high rate of recurrences and signalling axes that promote GBM aggressiveness. The contribution of gap junctions (GJs) to tumor growth and progression has been proven by experimental evidence. Concomitantly, tumor microenvironment has received increasing interest as a critical process in dysregulation and homeostatic escape, finding a close link between molecular mechanisms involved in connexin 43 (CX43)-based intercellular communication and tumorigenesis. Moreover, evidence has come to suggest a crucial role of sonic hedgehog (SHH) signalling pathway in GBM proliferation, cell fate and differentiation. Herein, we used two human GBM cell lines, modulating SHH signalling and CX43-based intercellular communication in in vitro models using proliferation and migration assays. Our evidence suggests that modulation of the SHH effector smoothened (SMO), by using a known agonist (i.e., purmorphamine) and a known antagonist (i.e., cyclopamine), affects the CX43 expression levels and therefore the related functions. Moreover, SMO activation also increased cell proliferation and migration. Importantly, inhibition of CX43 channels was able to prevent SMO-induced effects. SHH pathway and CX43 interplay acts inducing tumorigenic program and supporting cell migration, likely representing druggable targets to develop new therapeutic strategies for GBM.

Background: Deep angiomyxoma (DAM) is currently included in the category of “specific stromal tumors of the lower female genital tract”, along with angiomyofibroblastoma, cellular angiofibroma and myofibroblastoma. Given the high rate of local recurrences, it is crucial to recognize DAM from other tumors that possess indolent behaviour. In the present paper, we analyzed the morphological and immunohistochemical features of 42 surgically-resected vulvo-vaginal DAMs (36 primary and 6 recurrent lesions) in order to widen the morphological spectrum of this uncommon tumor. Methods: A series of 36 cases of surgically-resected primary vulvo-vaginal DAMs were retrospectively collected. Locally recurrent tumors were also available for six of these cases. Results: Out of the primary tumors, 25 out of 36 exhibited the classic-type morphology of DAM. In the remaining cases (11/36 cases), the following uncommon features, which sometimes coexist with one another, were observed: (i) alternating myxoid and collagenized/fibrous areas; (ii) hypercellular areas; (iii) neurofibroma-like appearance; (iv) perivascular hyalinization; (v) microcystic/reticular stromal changes; (vi) “microvascular growth pattern”; (vii) perivascular cuffing; (viii) nodular leiomyomatous differentiation; (ix) hypocellular and fibro-sclerotic stroma. Among the six locally recurrent tumors the following features were observed: (i) classic-type morphology; (ii) hypocellular fibro-sclerotic stroma; (iii) extensive perivascular hyalinization, lumen obliteration and formation of confluent nodular sclerotic masses; (iv) hypercellularity. Immunohistochemically, the neoplastic cells of classic-type DAM in both primary and recurrent tumors were diffusely stained with desmin, suggesting a myofibroblastic nature; in contrast, the neoplastic cells showing elongated fibroblastic-like morphology and set in collagenized/fibrosclerotic stroma in both primary and recurrent tumors were negative or only focally stained with desmin, which is consistent with a fibroblastic profile. Conclusion: Although diagnosis of DAM is usually straightforward if typical morphology is encountered, diagnostic problems may arise when a pathologist is dealing with unusual morphological features, especially hypercellularity, extensive collagenous/fibrosclerotic stroma or neurofibroma-like appearance.

Abstract Traumatic injury or surgical excision of diseased bone tissue usually require the reconstruction of large bone defects unable to heal spontaneously, especially in older individuals. This is a big challenge requiring the development of biomaterials mimicking the bone structure and capable of inducing the right commitment of cells seeded within the scaffold. In particular, given their properties and large availability, the human adipose-derived stem cells are considered as the better candidate for autologous cell transplantation. In order to evaluate the regenerative potential of these cells along with an osteoinductive biomaterial, we have used collagen/hydroxyapatite scaffolds to test ectopic bone formation after subcutaneous implantation in mice. The process was analysed both in vivo, by Fluorescent Molecular Tomography (FMT), and ex vivo, to evaluate the formation of bone and vascular structures. The results have shown that the biomaterial could itself be able of promoting differentiation of host cells and bone formation, probably by means of its intrinsic chemical and structural properties, namely the microenvironment. However, when charged with human mesenchymal stem cells, the ectopic bone formation within the scaffold was increased. We believe that these results represent an important advancement in the field of bone physiology, as well as in regenerative medicine.

Abstract A fundamental task in cancer research aims at the identification of new pharmacological therapies that can affect tumor growth. Differentiation therapy might exploit this function not only for hematological diseases, such as acute promyelocytic leukemia (APML) but also for epithelial tumors, including lung cancer. Here we show that Retinoic Acid (RA) arrests in vitro and in vivo the growth of Tyrosine Kinase Inhibitors (TKI) resistant Non Small Cell Lung Cancer (NSCLC). In particular, we found that RA induces G0/G1 cell cycle arrest in TKI resistant NSCLC cells and activates terminal differentiation programs by modulating the expression of GATA6, a key transcription factor involved in the physiological differentiation of the distal lung. In addition, our results demonstrate that RA inhibits EGFR and Wnt signaling activation, two pathways involved in NSCLC progression. Furthermore, we uncovered a novel mechanism in NSCLC that shows how RA exerts its function; we found that RA-mediated GATA6 activation is necessary for EGFR and Wnt inhibition, thus leading to 1) increased differentiation and 2) loss of proliferation. All together, these findings prove that differentiation therapy might be feasible in TKI resistant NSCLCs, and shed light on new targets to define new pharmacological therapies.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by hematopoietic stem-cell-derived clonal proliferation, leading to bone marrow (BM) fibrosis. Hematopoiesis alterations are closely associated with modifications of the BM microenvironment, characterized by defective interactions between vascular and endosteal niches. As such, neoangiogenesis, megakaryocytes hyperplasia and extensive bone marrow fibrosis, followed by osteosclerosis and bone damage, are the most relevant consequences of PMF. Moreover, bone tissue deposition, together with progressive fibrosis, represents crucial mechanisms of disabilities in patients. Although the underlying mechanisms of bone damage observed in PMF are still unclear, the involvement of cytokines, growth factors and bone marrow microenvironment resident cells have been linked to disease progression. Herein, we focused on the role of megakaryocytes and their alterations, associated with cytokines and chemokines release, in modulating functions of most of the bone marrow cell populations and in creating a complex network where impaired signaling strongly contributes to progression and disabilities.

The management of chondral defects represents a big challenge because of the limited self-healing capacity of cartilage. Many approaches in this field obtained partial satisfactory results. Cartilage tissue engineering, combining innovative scaffolds and stem cells from different sources, emerges as a promising strategy for cartilage regeneration. The aim of this study was to evaluate the capability of a cell-free collagen I-based scaffold to promote cartilaginous repair after orthotopic implantation in vivo. Articular cartilage lesions (ACL) were created at the femoropatellar groove in rat knees and cell free collagen I-based scaffolds (S) were then implanted into right knee defect for the ACL-S group. No scaffold was implanted for the ACL group. At 4-, 8- and 16-weeks post-transplantation, degrees of cartilage repair were evaluated by morphological, histochemical and gene expression analyses. Histological analysis shows the formation of fibrous tissue, at 4-weeks replaced by a tissue resembling the calcified one at 16-weeks in the ACL group. In the ACL-S group, progressive replacement of the scaffold with the newly formed cartilage-like tissue is shown, as confirmed by Alcian Blue staining. Immunohistochemical and quantitative real-time PCR (qRT-PCR) analyses display the expression of typical cartilage markers, such as collagen type I and II (ColI and ColII), Aggrecan and Sox9. The results of this study display that the collagen I-based scaffold is highly biocompatible and able to recruit host cells from the surrounding joint tissues to promote cartilaginous repair of articular defects, suggesting its use as a potential approach for cartilage tissue regeneration.

Glioblastoma (GBM) is one of the most lethal types of tumor due to its high recurrence level in spite of aggressive treatment regimens involving surgery, radiotherapy and chemotherapy. Hypoxia is a feature of GBM, involved in radioresistance, and is known to be at the origin of treatment failure. The aim of this work was to assess the therapeutic potential of a new targeted c-SRC inhibitor molecule, named Si306, in combination with X-rays on the human glioblastoma cell lines, comparing normoxia and hypoxia conditions. For this purpose, the dose modifying factor and oxygen enhancement ratio were calculated to evaluate the Si306 radiosensitizing effect. DNA damage and the repair capability were also studied from the kinetic of γ-H2AX immunodetection. Furthermore, motility processes being supposed to be triggered by hypoxia and irradiation, the role of c-SRC inhibition was also analyzed to evaluate the migration blockage by wound healing assay. Our results showed that inhibition of the c-SRC protein enhances the radiotherapy efficacy both in normoxic and hypoxic conditions. These data open new opportunities for GBM treatment combining radiotherapy with molecularly targeted drugs to overcome radioresistance.

The proteasome inhibitor bortezomib (BTZ) has emerged as an effective drug for the treatment of multiple myeloma even though many patients relapse from BTZ therapy. The present study investigated the metabolic pathways underlying the acquisition of bortezomib resistance in multiple myeloma. We used two different clones of multiple myeloma cell lines exhibiting different sensitivities to BTZ (U266 and U266-R) and compared them in terms of metabolic profile, mitochondrial fitness and redox balance homeostasis capacity. Our results showed that the BTZ-resistant clone (U266-R) presented increased glycosylated UDP-derivatives when compared to BTZ-sensitive cells (U266), thus also suggesting higher activities of the hexosamine biosynthetic pathway (HBP), regulating not only protein O- and N-glycosylation but also mitochondrial functions. Notably, U266-R displayed increased mitochondrial biogenesis and mitochondrial dynamics associated with stronger antioxidant defenses. Furthermore, U266-R maintained a significantly higher concentration of substrates for protein glycosylation when compared to U266, particularly for UDP-GlcNac, thus further suggesting the importance of glycosylation in the BTZ pharmacological response. Moreover, BTZ-treated U266-R showed significantly higher ATP/ADP ratios and levels of ECP and also exhibited increased mitochondrial fitness and antioxidant response. In conclusions, our findings suggest that the HBP may play a major role in mitochondrial fitness, driving BTZ resistance in multiple myeloma and thus representing a possible target for new drug development for BTZ-resistant patients.

The expression of neuronal and glial connexins (Cxs) has been evaluated in adipose-derived mesenchymal stem cells (ASCs) whose neural differentiation was promoted by a conditioned medium (CM) obtained from cultures of olfactory ensheathing cells (OECs) or Schwann cells (SCs). By immunocytochemistry and flow cytometer analysis it was found that Cx43 was already considerably expressed in naïve ASCs and further increased after 24 h and 7 days from CM exposition. Cx32 and Cx36 were significantly improved in conditioned cultures compared to control ASCs, whereas a decreased expression was noticed in the absence of CM treatments. Cx47 was virtually absent in any conditions. Altogether, high basal levels and induced increases of Cx43 expression suggest a potential attitude of ASCs toward an astrocyte differentiation, whereas the lack of Cx47 would indicate a poor propensity of ASCs to become oligodendrocytes. CM-evoked Cx32 and Cx36 increases showed that a neuronal- or a SC-like differentiation can be promoted by using this strategy. Results further confirm that environmental cues can favor an ASC neural differentiation, either as neuronal or glial elements. Of note, the use of glial products present in CM rather than the addition of chemical agents to achieve such differentiation would resemble “more physiological” conditions of differentiation. As a conclusion, the overexpression of typical neural Cxs would indicate the potential capability of neural-like ASCs to interact with neighboring neural cells and microenvironment.

Cell-to-cell interaction and cell-to-extracellular environment communication are emerging as new therapeutic targets in neurodegenerative disorders. Dynamic expression of connexins leads to distinctive hemichannels and gap junctions, characterized by cell-specific conduction, exchange of stimuli or metabolites, and particular channel functions. Herein, we briefly reviewed classical physiological traits and functions of connexins, hemichannels, and gap junctions, in order to discuss the controversial role of these proteins and their mediated interactions during neuroprotection, with a particular focus on Cx43-based channels. We pointed out the contribution of connexins in neural cells populations during neurodegenerative processes to explore potential neuroprotective therapeutic applications.

Harpagophytum procumbens (H. procumbens), also known as Devil’s Claw, has been used to treat a wide range of pathological conditions, including pain, arthritis and inflammation. Inflammatory mediators, released at the site of injury, can sensitize nociceptive terminals and are responsible for allodynia and hyperalgesia. Carbon monoxide (CO), produced in a reaction catalyzed by the enzyme heme oxygenase (HO), may play a role in nociceptive processing and has also been recognized to act as a neurotransmitter or neuromodulator in the nervous system. This study was designed to investigate whether the HO/CO pathway is involved in the analgesic response of H. procumbens in carrageenan-induced hyperalgesia in rats. Mechanical allodynia and thermal hyperalgesia were evaluated by using von Frey filaments and the plantar test, respectively. The results of our experiments showed that pretreatment with the HO inhibitor ZnPP IX significantly decreased the antihyperalgesic effect produced by H. procumbens (800 mg/kg, i.p.) in carrageenan-injected rats. Consistently, the pretreatment with hemin, a HO-1 substrate, or CORM-3, a CO releasing molecule, before a low dose of H. procumbens (300 mg/kg, i.p.) induced a clear antiallodynic response in carrageenan injected rats. These results suggest the involvement of HO-1/CO system in the antiallodynic and antihyperalgesic effect of H. procumbens in carrageenan-induced inflammatory pain.

Injured articular cartilage has a limited innate regenerative capacity, due to the avascular nature and low cellularity of the tissue itself. Although several approaches have been proposed to repair the joint cartilage, none of them has proven to be effective. The absence of suitable therapeutic options has encouraged tissue-engineering approaches combining specific cell types and biomaterials. In the present work, we have evaluated the potential of a cell-free Collagen I-based scaffold to promote the augmentation of cartilage-like phenotype after subcutaneous implantation in the mouse. Forty female mice were grafted subcutaneously with scaffolds, while four additional mice without scaffold were used as negative controls. The effects of scaffold were evaluated at 1, 2, 4, 8, or 16 weeks after implantation. Immunohistochemical analysis shows the expression of typical cartilage markers, including type-II Collagen, Aggrecan, Matrilin-1 and Sox 9. These data are also confirmed by qRT-PCR that further show that both COL2A1 and COL1A1 increase over time, but the first one increases more rapidly, thus suggesting a typical cartilage-like address. Histological analysis shows the presence of some pericellular lacunae, after 8 and 16 weeks. Results suggest that this scaffold (i) is biocompatible in vivo, (ii) is able to recruit host cells (iii) induce chondrogenic differentiation of host cells. Such evidences suggest that this cell-free scaffold is promising and represents a potential approach for cartilage regeneration.

Glioblastoma Multiforme (GBM) is the most common of malignant gliomas in adults with an exiguous life expectancy. Standard treatments are not curative and the resistance to both chemotherapy and conventional radiotherapy (RT) plans is the main cause of GBM care failures. Proton therapy (PT) shows a ballistic precision and a higher dose conformity than conventional RT. In this study we investigated the radiosensitive effects of a new targeted compound, SRC inhibitor, named Si306, in combination with PT on the U87 glioblastoma cell line. Clonogenic survival assay, dose modifying factor calculation and linear-quadratic model were performed to evaluate radiosensitizing effects mediated by combination of the Si306 with PT. Gene expression profiling by microarray was also conducted after PT treatments alone or combined, to identify gene signatures as biomarkers of response to treatments. Our results indicate that the Si306 compound exhibits a radiosensitizing action on the U87 cells causing a synergic cytotoxic effect with PT. In addition, microarray data confirm the SRC role as the main Si306 target and highlights new genes modulated by the combined action of Si306 and PT. We suggest, the Si306 as a new candidate to treat GBM in combination with PT, overcoming resistance to conventional treatments.

Sonic hedgehog (Shh) signaling is a key pathway within the central nervous system (CNS), during both development and adulthood, and its activation via the 7-transmembrane protein Smoothened (Smo) may promote neuroprotection and restoration during neurodegenerative disorders. Shh signaling may also be activated by selected glucocorticoids such as clobetasol, fluocinonide and fluticasone, which therefore act as Smo agonists and hold potential utility for regenerative medicine. However, despite its potential role in neurodegenerative diseases, the impact of Smo-modulation induced by these glucocorticoids on adult neural stem cells (NSCs) and the underlying signaling mechanisms are not yet fully elucidated. The aim of the present study was to evaluate the effects of Smo agonists (i.e., purmorphamine) and antagonists (i.e., cyclopamine) as well as of glucocorticoids (i.e., clobetasol, fluocinonide and fluticasone) on NSCs in terms of proliferation and clonal expansion. Purmorphamine treatment significantly increased NSC proliferation and clonal expansion via GLI-Kruppel family member 1 (Gli1) nuclear translocation and such effects were prevented by cyclopamine co-treatment. Clobetasol treatment exhibited an equivalent pharmacological effect. Moreover, cellular thermal shift assay suggested that clobetasol induces the canonical Smo-dependent activation of Shh signaling, as confirmed by Gli1 nuclear translocation and also by cyclopamine co-treatment, which abolished these effects. Finally, fluocinonide and fluticasone as well as control glucocorticoids (i.e., prednisone, corticosterone and dexamethasone) showed no significant effects on NSCs proliferation and clonal expansion. In conclusion, our data suggest that Shh may represent a druggable target system to drive neuroprotection and promote restorative therapies.

Despite the relevant research efforts, the causes of amyotrophic lateral sclerosis (ALS) are still unknown and no effective cure is available. Many authors suggest that ALS is a multi-system disease caused by a network failure instead of a cell-autonomous pathology restricted to motoneurons. Although motoneuronal loss is the critical hallmark of ALS given their specific vulnerability, other cell populations, including muscle and glial cells, are involved in disease onset and progression, but unraveling their specific role and crosstalk requires further investigation. In particular, little is known about the plastic changes of the degenerating motor system. These spontaneous compensatory processes are unable to halt the disease progression, but their elucidation and possible use as a therapeutic target represents an important aim of ALS research. Genetic animal models of disease represent useful tools to validate proven hypotheses or to test potential therapies, and the conception of novel hypotheses about ALS causes or the study of pathogenic mechanisms may be advantaged by the use of relatively simple in vivo models recapitulating specific aspects of the disease, thus avoiding the inclusion of too many confounding factors in an experimental setting. Here, we used a neurotoxic model of spinal motoneuron depletion induced by injection of cholera toxin-B saporin in the gastrocnemius muscle to investigate the possible occurrence of compensatory changes in both the muscle and spinal cord. The results showed that, following the lesion, the skeletal muscle became atrophic and displayed electromyographic activity similar to that observed in ALS patients. Moreover, the changes in muscle fiber morphology were different from that observed in ALS models, thus suggesting that some muscular effects of disease may be primary effects instead of being simply caused by denervation. Notably, we found plastic changes in the surviving motoneurons that can produce a functional restoration probably similar to the compensatory changes occurring in disease. These changes could be at least partially driven by glutamatergic signaling, and astrocytes contacting the surviving motoneurons may support this process.