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BACKGROUND & AIMS: Sirtuin 5, encoded by the SIRT5 gene, is a NAD+-dependent deacylase that modulates mitochondrial metabolic processes through post-translational modifications. In this study, we aimed to examine the impact of the SIRT5 rs12216101 T>G non-coding single nucleotide polymorphism on disease severity in patients with non-alcoholic fatty liver disease (NAFLD). METHODS: The rs12216101 variant was genotyped in 2,606 consecutive European patients with biopsy-proven NAFLD. Transcriptomic analysis, expression of mitochondrial complexes and oxidative stress levels were measured in liver samples from a subset of bariatric patients. Effects of SIRT5 pharmacological inhibition were evaluated in HepG2 cells exposed to excess free fatty acids. Mitochondrial energetics in vitro were investigated by high-performance liquid chromatography. RESULTS: In the whole cohort, the frequency distribution of SIRT5 rs12216101 TT, TG and GG genotypes was 47.0%, 42.3% and 10.7%, respectively. At multivariate logistic regression analysis adjusted for sex, age >50 years, diabetes, and PNPLA3 rs738409 status, the SIRT5 rs12216101 T>G variant was associated with the presence of non-alcoholic steatohepatitis (odds ratio 1.20, 95% CI 1.03-1.40) and F2-F4 fibrosis (odds ratio 1.18; 95% CI 1.00-1.37). Transcriptomic analysis showed that the SIRT5 rs12216101 T>G variant was associated with upregulation of transcripts involved in mitochondrial metabolic pathways, including the oxidative phosphorylation system. In patients carrying the G allele, western blot analysis confirmed an upregulation of oxidative phosphorylation complexes III, IV, V and consistently higher levels of reactive oxygen species, reactive nitrogen species and malondialdehyde, and lower ATP levels. Administration of a pharmacological SIRT5 inhibitor preserved mitochondrial energetic homeostasis in HepG2 cells, as evidenced by restored ATP/ADP, NAD+/NADH, NADP+/NADPH ratios and glutathione levels. CONCLUSIONS: The SIRT5 rs12216101 T>G variant, heightening SIRT5 activity, is associated with liver damage, mitochondrial dysfunction, and oxidative stress in patients with NAFLD. IMPACT AND IMPLICATIONS: In this study we discovered that the SIRT5 rs12216101 T>G variant is associated with higher disease severity in patients with non-alcoholic fatty liver disease (NAFLD). This risk variant leads to a SIRT5 gain-of-function, enhancing mitochondrial oxidative phosphorylation and thus leading to oxidative stress. SIRT5 may represent a novel disease modulator in NAFLD.
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Doenças Mitocondriais , Hepatopatia Gordurosa não Alcoólica , Sirtuínas , Humanos , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/complicações , Genótipo , Polimorfismo de Nucleotídeo Único , Fígado , Doenças Mitocondriais/complicações , Trifosfato de Adenosina , Predisposição Genética para Doença , Sirtuínas/genéticaRESUMO
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.
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Glioblastoma , Animais , Humanos , Camundongos , Aquaporina 4 , Astrócitos , Biomarcadores , Plectina , Isoformas de ProteínasRESUMO
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.
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Neural stem cell (NSC) transplantation induces recovery in animal models of central nervous system (CNS) diseases. Although the replacement of lost endogenous cells was originally proposed as the primary healing mechanism of NSC grafts, it is now clear that transplanted NSCs operate via multiple mechanisms, including the horizontal exchange of therapeutic cargoes to host cells via extracellular vesicles (EVs). EVs are membrane particles trafficking nucleic acids, proteins, metabolites and metabolic enzymes, lipids, and entire organelles. However, the function and the contribution of these cargoes to the broad therapeutic effects of NSCs are yet to be fully understood. Mitochondrial dysfunction is an established feature of several inflammatory and degenerative CNS disorders, most of which are potentially treatable with exogenous stem cell therapeutics. Herein, we investigated the hypothesis that NSCs release and traffic functional mitochondria via EVs to restore mitochondrial function in target cells. Untargeted proteomics revealed a significant enrichment of mitochondrial proteins spontaneously released by NSCs in EVs. Morphological and functional analyses confirmed the presence of ultrastructurally intact mitochondria within EVs with conserved membrane potential and respiration. We found that the transfer of these mitochondria from EVs to mtDNA-deficient L929 Rho0 cells rescued mitochondrial function and increased Rho0 cell survival. Furthermore, the incorporation of mitochondria from EVs into inflammatory mononuclear phagocytes restored normal mitochondrial dynamics and cellular metabolism and reduced the expression of pro-inflammatory markers in target cells. When transplanted in an animal model of multiple sclerosis, exogenous NSCs actively transferred mitochondria to mononuclear phagocytes and induced a significant amelioration of clinical deficits. Our data provide the first evidence that NSCs deliver functional mitochondria to target cells via EVs, paving the way for the development of novel (a)cellular approaches aimed at restoring mitochondrial dysfunction not only in multiple sclerosis, but also in degenerative neurological diseases.
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Vesículas Extracelulares/metabolismo , Mitocôndrias/metabolismo , Células-Tronco Neurais/metabolismo , Animais , Transporte Biológico , Células Cultivadas , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Neurais/ultraestruturaRESUMO
BACKGROUND AND OBJECTIVE: Neuropathic pain is a chronic condition characterized by aberrant signaling within the somatosensory system, affecting millions of people worldwide with limited treatment options. Herein, we aim at investigating the potential of a sigma-1 receptor (σ1R) antagonist in managing neuropathic pain. METHODS: A Chronic Constriction Injury (CCI) model was used to induce neuropathic pain. The potential of (+)-MR200 was evaluated following daily subcutaneous injections of the compound. Its mechanism of action was confirmed by administration of a well-known σ1R agonist, PRE084. RESULTS: (+)-MR200 demonstrated efficacy in protecting neurons from damage and alleviating pain hypersensitivity in CCI model. Our results suggest that (+)-MR200 reduced the activation of astrocytes and microglia, cells known to contribute to the neuroinflammatory process, suggesting that (+)-MR200 may not only address pain symptoms but also tackle the underlying cellular mechanism involved. Furthermore, (+)-MR200 treatment normalized levels of the gap junction (GJ)-forming protein connexin 43 (Cx43), suggesting a reduction in harmful intercellular communication that could fuel the chronicity of pain. CONCLUSIONS: This approach could offer a neuroprotective strategy for managing neuropathic pain, addressing both pain symptoms and cellular processes driving the condition. Understanding the dynamics of σ1R expression and function in neuropathic pain is crucial for clinical intervention.
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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.
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Diferenciação Celular , Melatonina , Células-Tronco Mesenquimais , Melatonina/farmacologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Humanos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Tecido Adiposo/citologia , Neurônios/citologia , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Células de Schwann/citologia , Células de Schwann/metabolismo , Células de Schwann/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Adulto , Nestina/metabolismo , Nestina/genética , Proteína Glial Fibrilar Ácida/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/citologia , Neuroglia/metabolismo , Sinapsinas/metabolismoRESUMO
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.
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Neoplasias Encefálicas , Glioblastoma , Animais , Humanos , Glioblastoma/patologia , Microglia/metabolismo , Proteína 6 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Proteína 6 de Ligação a Fator de Crescimento Semelhante à Insulina/uso terapêutico , Ácido Láctico/metabolismo , Ácido Láctico/uso terapêutico , Microambiente Tumoral , Peixe-Zebra/metabolismo , Linhagem Celular Tumoral , Proteínas Hedgehog , Neoplasias Encefálicas/patologiaRESUMO
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.
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Sobrecarga de Ferro , Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide , Humanos , Carcinoma Anaplásico da Tireoide/complicações , Sobrecarga de Ferro/complicações , Sobrecarga de Ferro/tratamento farmacológico , Sobrecarga de Ferro/metabolismo , Morte Celular , Ferro/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
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.
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Células-Tronco Adultas , Nicho de Células-Tronco , Adulto , Diferenciação Celular/fisiologia , Homeostase/fisiologia , Humanos , Células-Tronco/metabolismoRESUMO
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.
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Conexinas , Junções Comunicantes , Comunicação Celular/fisiologia , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Neuroglia/metabolismo , Transdução de Sinais/fisiologiaRESUMO
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.
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Melanoma , Neoplasias Uveais , Humanos , Ácido Láctico/metabolismo , Melanoma/metabolismo , Transdução de Sinais , Receptores Acoplados a Proteínas G/metabolismo , Neoplasias Uveais/patologia , Linhagem Celular TumoralRESUMO
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.
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Neuralgia , Receptores Opioides delta , Analgésicos Opioides/farmacologia , Conexina 43/uso terapêutico , Humanos , Hiperalgesia/tratamento farmacológico , Neuralgia/tratamento farmacológico , Receptores Opioides , Receptores Opioides mu , Medula EspinalRESUMO
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.
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Histonas , Doenças Neurodegenerativas , Humanos , Histonas/metabolismo , Doenças Neuroinflamatórias , Epigênese Genética , Epigenômica , Doenças Neurodegenerativas/genéticaRESUMO
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.
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Comunicação Celular/fisiologia , Canais Iônicos/fisiologia , Neuralgia/etiologia , Animais , Doença Crônica , Conexina 43/fisiologia , Junções Comunicantes/fisiologia , HumanosRESUMO
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.
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Conexina 43/metabolismo , Conexinas/metabolismo , Células-Tronco Mesenquimais , Adipogenia , Adulto , Diferenciação Celular , Células Cultivadas , Feminino , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Proteína beta-1 de Junções ComunicantesRESUMO
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.
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Neoplasias Encefálicas/enzimologia , Glioblastoma/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Quinases da Família src/antagonistas & inibidores , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular , Dano ao DNA , Ensaios de Seleção de Medicamentos Antitumorais , Histonas/metabolismo , Humanos , Hipóxia , Cinética , Microscopia de Fluorescência , Recidiva Local de Neoplasia/tratamento farmacológico , Prognóstico , Radiação Ionizante , Radioterapia , Raios X , Quinases da Família src/metabolismoRESUMO
An innovative miniaturized silicon-chip was developed for highly sensitive detection of pathogen genomes of both viruses and bacteria through real time PCR (qRT-PCR). The device was properly designed to enhance the optical signal and perform accurate thermal control. Results show an improvement of PCR amplification by one order of magnitude in sensitivity compared to the standard RT-PCR method. In particular for hepatitis B virus a decrease of the mean value of Ct of about 2.9 ± 0.9 compared to the standard system was observed. Similarly, for the bacteria Pseudomonas aeruginosa, Staphylococcus aureus and Acinetobacter baumannii, a decrease of the mean values of Ct of 1.8 ± 0.5, 3.1 ± 0.5 and 3.9 ± 0.9, respectively, was observed.
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Bactérias/genética , Bactérias/isolamento & purificação , Vírus da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Silício , Desenho de Equipamento , Genoma Viral/genética , Limite de DetecçãoRESUMO
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.
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Atrofia Muscular Espinal/fisiopatologia , Junção Neuromuscular/fisiopatologia , Plasticidade Neuronal , Animais , Toxina da Cólera/toxicidade , Masculino , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiopatologia , Atrofia Muscular Espinal/etiologia , Atrofia Muscular Espinal/patologia , Junção Neuromuscular/patologia , Saporinas/toxicidade , Medula Espinal/patologia , Medula Espinal/fisiopatologiaRESUMO
Protein SUMOylation is a dynamic post-translational modification which is involved in a diverse set of physiologic processes throughout the cell. Of note, SUMOylation also plays a role in the pathobiology of a myriad of cancers, one of which is glioblastoma (GBM). Accordingly, herein, we review core aspects of SUMOylation as it relates to GBM and in so doing highlight putative methods/modalities capable of therapeutically engaging the pathway for treatment of this deadly neoplasm.