Plectin plays a role in the migration and volume regulation of astrocytes: a potential biomarker of glioblastoma.

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.

Skeletal muscle of young females under resistance exercise exhibits a unique innate immune cell infiltration profile compared to males and elderly individuals.

Muscle damage resulting from physical activities such as exercise triggers an immune response crucial for tissue repair and recovery. This study investigates the immune cell profiles in muscle biopsies of individuals engaged in resistance exercise (RE) and explores the impact of age and sex on the immune response following exercise-induced muscle damage. Microarray datasets from muscle biopsies of young and old subjects were analyzed, focusing on the gene expression patterns associated with immune cell activation. Genes were compared with immune cell signatures to reveal the cellular landscape during exercise. Results show that the most significant modulated gene after RE was Folliculin Interacting Protein 2 (FNIP2) a crucial regulator in cellular homeostasis. Moreover, the transcriptome was stratified based on the expression of FNIP2 and the 203 genes common to the groups obtained based on sex and age. Gene ontology analysis highlighted the FLCN-FNIP1-FNIP2 complex, which exerts as a negative feedback loop to Pi3k-Akt-mTORC1 pathway. Furthermore, we highlighted that the young females exhibit a distinct innate immune cell activation signature compared to males after a RE session. Specifically, young females demonstrate a notable overlap with dendritic cells (DCs), M1 macrophages, M2 macrophages, and neutrophils, while young males overlap with M1 macrophages, M2 macrophages, and motor neurons. Interestingly, in elderly subjects, both sexes display M1 macrophage activation signatures. Comparison of young and elderly signatures reveals an increased M1 macrophage percentage in young subjects. Additionally, common genes were identified in both sexes across different age groups, elucidating biological functions related to cell remodeling and immune activation. This study underscores the intricate interplay between sex, age, and the immune response in muscle tissue following RE, offering potential directions for future research. Nevertheless, there is a need for further studies to delve deeper and confirm the dynamics of immune cells in response to exercise-induced muscle damage.

Melatonin Enhances Neural Differentiation of Adipose-Derived Mesenchymal Stem Cells.

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.

Lactate modulates microglia polarization via IGFBP6 expression and remodels tumor microenvironment in glioblastoma.

Lactic acidosis has been reported in solid tumor microenvironment (TME) including glioblastoma (GBM). In TME, several signaling molecules, growth factors and metabolites have been identified to induce resistance to chemotherapy and to sustain immune escape. In the early phases of the disease, microglia infiltrates TME, contributing to tumorigenesis rather than counteracting its growth. Insulin-like Growth Factor Binding Protein 6 (IGFBP6) is expressed during tumor development, and it is involved in migration, immune-escape and inflammation, thus providing an attractive target for GBM therapy. Here, we aimed at investigating the crosstalk between lactate metabolism and IGFBP6 in TME and GBM progression. Our results show that microglia exposed to lactate or IGFBP6 significantly increased the Monocarboxylate transporter 1 (MCT1) expression together with genes involved in mitochondrial metabolism. We, also, observed an increase in the M2 markers and a reduction of inducible nitric oxide synthase (iNOS) levels, suggesting a role of lactate/IGFBP6 metabolism in immune-escape activation. GBM cells exposed to lactate also showed increased levels of IGFBP6 and vice-versa. Such a phenomenon was coupled with a IGFBP6-mediated sonic hedgehog (SHH) ignaling increase. We, finally, tested our hypothesis in a GBM zebrafish animal model, where we observed an increase in microglia cells and igfbp6 gene expression after lactate exposure. Our results were confirmed by the analysis of human transcriptomes datasets and immunohistochemical assay from human GBM biopsies, suggesting the existence of a lactate/IGFBP6 crosstalk in microglial cells, so that IGFBP6 expression is regulated by lactate production in GBM cells and in turn modulates microglia polarization.

Anaplastic thyroid cancer cells reduce CD71 levels to increase iron overload tolerance.

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.

Adult stem cell niches for tissue homeostasis.

Adult stem cells are fundamental to maintain tissue homeostasis, growth, and regeneration. They reside in specialized environments called niches. Following activating signals, they proliferate and differentiate into functional cells that are able to preserve tissue physiology, either to guarantee normal turnover or to counteract tissue damage caused by injury or disease. Multiple interactions occur within the niche between stem cell-intrinsic factors, supporting cells, the extracellular matrix, and signaling pathways. Altogether, these interactions govern cell fate, preserving the stem cell pool, and regulating stem cell proliferation and differentiation. Based on their response to body needs, tissues can be largely classified into three main categories: tissues that even in normal conditions are characterized by an impressive turnover to replace rapidly exhausting cells (blood, epidermis, or intestinal epithelium); tissues that normally require only a basal cell replacement, though able to efficiently respond to increased tissue needs, injury, or disease (skeletal muscle); tissues that are equipped with less powerful stem cell niches, whose repairing ability is not able to overcome severe damage (heart or nervous tissue). The purpose of this review is to describe the main characteristics of stem cell niches in these different tissues, highlighting the various components influencing stem cell activity. Although much has been done, more work is needed to further increase our knowledge of niche interactions. This would be important not only to shed light on this fundamental chapter of human physiology but also to help the development of cell-based strategies for clinical therapeutic applications, especially when other approaches fail.

Connexins Signatures of the Neurovascular Unit and Their Physio-Pathological Functions.

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.

Mu and Delta Opioid Receptor Targeting Reduces Connexin 43-Based Heterocellular Coupling during Neuropathic Pain.

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.

Lactate Rewrites the Metabolic Reprogramming of Uveal Melanoma Cells and Induces Quiescence Phenotype.

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.

The Role of Epigenetics in Neuroinflammatory-Driven Diseases.

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.

Phytochemical Analysis and Anti-Inflammatory and Anti-Osteoarthritic Bioactive Potential of Verbascum thapsus L. (Scrophulariaceae) Leaf Extract Evaluated in Two In Vitro Models of Inflammation and Osteoarthritis.

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.

Intercellular communication and ion channels in neuropathic pain chronicization.

BACKGROUND: Neuropathic pain is caused by primary lesion or dysfunction of either peripheral or central nervous system. Due to its complex pathogenesis, often related to a number of comorbidities, such as cancer, neurodegenerative and neurovascular diseases, neuropathic pain still represents an unmet clinical need, lacking long-term effective treatment and complex case-by-case approach. AIM AND METHODS: We analyzed the recent literature on the role of selective voltage-sensitive sodium, calcium and potassium permeable channels and non-selective gap junctions (GJs) and hemichannels (HCs) in establishing and maintaining chronic neuropathic conditions. We finally focussed our review on the role of extracellular microenvironment modifications induced by resident glial cells and on the recent advances in cell-to-cell and cell-to-extracellular environment communication in chronic neuropathies. CONCLUSION: In this review, we provide an update on the current knowledge of neuropathy chronicization processes with a focus on both neuronal and glial ion channels, as well as on channel-mediated intercellular communication.

Connexin expression decreases during adipogenic differentiation of human adipose-derived mesenchymal stem cells.

Adipose-derived stem cells (ASCs) represent a valuable tool for regenerative medicine being able to differentiate toward several cell lines, such as adipocytes, chondrocytes and osteocytes. During ASC adipogenic differentiation, changes in connexin (Cx) expression were evaluated in the present study. Three different Cxs were investigated: Cx43, Cx32 and Cx31.9. Cx43 is the most abundant in human tissues, Cx32 is prevalently found in nervous tissue and Cx31.9 is found at the myocardial level. Human ASCs undergoing adipogenic differentiation were isolated from raw lipoaspirate and characterized as mesenchymal stem cells. After multiple days of culture (1, 7, 14, 21 and 28 days), adipogenic differentiation was assessed by Oil Red O staining and Acetyl-CoA carboxylase (ACC) levels by western blotting. Cx expression was evaluated by western blotting at the same time points. In treated ASCs, lipidic vacuoles were detected from day 7 of treatment. Their number and size progressively increased over the entire period of observation. A parallel increase of ACC expression was also found. Lower levels of Cx expression were detected during adipogenic differentiation. Such decreases were particularly evident for Cx32, already after the first day of treatment. Cx31.9 and Cx43 also decreased, but starting from day 7. Our results suggest that ASCs may initially be equipped with a variety of Cxs, which is not surprising assuming their multipotential differentiation ability. Although some Cxs may be selectively enhanced depending on specific induction strategies toward different tissues, they seem markedly downregulated during adipogenic differentiation.

SRC Tyrosine Kinase Inhibitor and X-rays Combined Effect on Glioblastoma Cell Lines.

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.

Neuromuscular Plasticity in a Mouse Neurotoxic Model of Spinal Motoneuronal Loss.

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.

Clobetasol Modulates Adult Neural Stem Cell Growth via Canonical Hedgehog Pathway Activation.

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.

Proton Therapy and Src Family Kinase Inhibitor Combined Treatments on U87 Human Glioblastoma Multiforme Cell Line.

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.

Engineered cartilage regeneration from adipose tissue derived-mesenchymal stem cells: A morphomolecular study on osteoblast, chondrocyte and apoptosis evaluation.

The poor self-repair capacity of cartilage tissue in degenerative conditions, such as osteoarthritis (OA), has prompted the development of a variety of therapeutic approaches, such as cellular therapies and tissue engineering based on the use of mesenchymal stem cells (MSCs). The aim of this study is to demonstrate, for the first time, that the chondrocytes differentiated from rat adipose tissue derived-MSCs (AMSCs), are able to constitute a morphologically and biochemically healthy hyaline cartilage after 6 weeks of culture on a Collagen Cell Carrier (CCC) scaffold. In this study we evaluated the expression of some osteoblasts (Runt-related transcription factor 2 (RUNX2) and osteocalcin), chondrocytes (collagen I, II and lubricin) and apoptosis (caspase-3) biomarkers in undifferentiated AMSCs, differentiated AMSCs in chondrocytes cultured in monolayer and AMSCs-derived chondrocytes seeded on CCC scaffolds, by different techniques such as immunohistochemistry, ELISA, Western blot and gene expression analyses. Our results showed the increased expression of collagen II and lubricin in AMSCs-derived chondrocytes cultured on CCC scaffolds, whereas the expression of collagen I, RUNX2, osteocalcin and caspase-3 resulted decreased, when compared to the controls. In conclusion, this innovative basic study could be a possible key for future therapeutic strategies for articular cartilage restoration through the use of CCC scaffolds, to reduce the morbidity from acute cartilage injuries and degenerative joint diseases.

An Advanced, Silicon-Based Substrate for Sensitive Nucleic Acids Detection.

Surface substrate and chemical functionalization are crucial aspects for the fabrication of the sensitive biosensor based on microarray technology. In this paper, an advanced, silicon-based substrate (A-MA) allowing enhancement of optical signal for microarray application is described. The substrate consists in a multilayer of Si/Al/SiO2 layers. The optical signal enhancement is reached by a combination of the mirror effect of Al film and the SiO2 thickness around 830 nm, which is able to reach the maximum of interference for the emission wavelength of the Cy5 fluorescent label. Moreover, SiO2 layer is suitable for the immobilization of single-strand DNA through standard silane chemistry, and probe densities of about 2000 F/um² are reached. The microarray is investigated in the detection of HBV (Hepatitis B Virus) pathogen with analytical samples, resulting in a dynamic linear range of 0.05⁻0.5 nM, a sensitivity of about 18000 a.u. nM-1, and a Limit of Detection in the range of 0.031⁻0.043 Nm as a function of the capture probe sequence.