Alpha-SNAP (M105I) mutation promotes neuronal differentiation of neural stem/progenitor cells through overactivation of AMPK.

Background: The M105I point mutation in α-SNAP (Soluble N-ethylmaleimide-sensitive factor attachment protein-alpha) leads in mice to a complex phenotype known as hyh (hydrocephalus with hop gait), characterized by cortical malformation and hydrocephalus, among other neuropathological features. Studies performed by our laboratory and others support that the hyh phenotype is triggered by a primary alteration in embryonic neural stem/progenitor cells (NSPCs) that leads to a disruption of the ventricular and subventricular zones (VZ/SVZ) during the neurogenic period. Besides the canonical role of α-SNAP in SNARE-mediated intracellular membrane fusion dynamics, it also negatively modulates AMP-activated protein kinase (AMPK) activity. AMPK is a conserved metabolic sensor associated with the proliferation/differentiation balance in NSPCs. Methods: Brain samples from hyh mutant mice (hydrocephalus with hop gait) (B6C3Fe-a/a-Napahyh/J) were analyzed by light microscopy, immunofluorescence, and Western blot at different developmental stages. In addition, NSPCs derived from WT and hyh mutant mice were cultured as neurospheres for in vitro characterization and pharmacological assays. BrdU labeling was used to assess proliferative activity in situ and in vitro. Pharmacological modulation of AMPK was performed using Compound C (AMPK inhibitor) and AICAR (AMPK activator). Results: α-SNAP was preferentially expressed in the brain, showing variations in the levels of α-SNAP protein in different brain regions and developmental stages. NSPCs from hyh mice (hyh-NSPCs) displayed reduced levels of α-SNAP and increased levels of phosphorylated AMPKα (pAMPKαThr172), which were associated with a reduction in their proliferative activity and a preferential commitment with the neuronal lineage. Interestingly, pharmacological inhibition of AMPK in hyh-NSPCs increased proliferative activity and completely abolished the increased generation of neurons. Conversely, AICAR-mediated activation of AMPK in WT-NSPCs reduced proliferation and boosted neuronal differentiation. Discussion: Our findings support that α-SNAP regulates AMPK signaling in NSPCs, further modulating their neurogenic capacity. The naturally occurring M105I mutation of α-SNAP provokes an AMPK overactivation in NSPCs, thus connecting the α-SNAP/AMPK axis with the etiopathogenesis and neuropathology of the hyh phenotype.

Expression and distribution of kallikrein-related peptidases 5, 7, 8 and 10 in normal apocrine gland of canine skin / Expresión y distribución de péptidos relacionados con la calicreína 5, 7, 8 y 10 en glándula apocrina normal de piel canina

SUMMARY: Apocrine glands are sweat glands that are located in the skin of the dog. Anal sac apocrine, circunanal apocrine, and mammary glands are considered modified apocrine structures, and there are about nine possible types of neoplasms and other tumors in the apocrine glands of the dog and cat, including cysts, adenoma, carcinoma, and adenocarcinoma. Thus, it is important to provide new markers to characterize these glands to improve the histopathological diagnosis. In this article, we describe the distribution of kallikrein- related peptidases 5, 7, 8, and 10 in the normal apocrine glands of the dog's skin. These proteases have been shown to play a fundamental role in the homeostasis of the human skin barrier but have been scarcely studied in canine skin.

Las glándulas apocrinas son glándulas sudoríparas que se encuentran en la piel del perro. Las glándulas apocrinas del saco anal, apocrinas circunanales y mamarias se consideran estructuras apocrinas modificadas, y existen alrededor de nueve tipos posibles de neoplasias y otros tumores en las glándulas apocrinas del perro y el gato, incluidos quistes, adenoma, carcinoma y adenocarcinoma. Por lo tanto, es importante proporcionar nuevos marcadores para caracterizar estas glándulas para mejorar el diagnóstico histopatológico. En este artículo, describimos la distribución de las peptidasas 5, 7, 8 y 10 relacionadas con la calicreína en las glándulas apocrinas normales de la piel del perro. Se ha demostrado que estas proteasas desempeñan un papel fundamental en la homeostasis de la barrera de la piel humana, pero apenas se han estudiado en la piel canina.

TNF-α-activated eNOS signaling increases leukocyte adhesion through the S-nitrosylation pathway.

Nitric oxide (NO) is a key factor in inflammation. Endothelial nitric oxide synthase (eNOS), whose activity increases after stimulation with proinflammatory cytokines, produces NO in endothelium. NO activates two pathways: 1) soluble guanylate cyclase-protein kinase G and 2) S-nitrosylation (NO-induced modification of free-thiol cysteines in proteins). S-nitrosylation affects phosphorylation, localization, and protein interactions. NO is classically described as a negative regulator of leukocyte adhesion to endothelial cells. However, agonists activating NO production induce a fast leukocyte adhesion, which suggests that NO might positively regulate leukocyte adhesion. We tested the hypothesis that eNOS-induced NO promotes leukocyte adhesion through the S-nitrosylation pathway. We stimulated leukocyte adhesion to endothelium in vitro and in vivo using tumor necrosis factor-α (TNF-α) as proinflammatory agonist. ICAM-1 changes were evaluated by immunofluorescence, subcellular fractionation, immunoprecipitation, and fluorescence recovery after photobleaching (FRAP). Protein kinase Cζ (PKCζ) activity and S-nitrosylation were evaluated by Western blot analysis and biotin switch method, respectively. TNF-α, at short times of stimulation, activated the eNOS S-nitrosylation pathway and caused leukocyte adhesion to endothelial cells in vivo and in vitro. TNF-α-induced NO led to changes in ICAM-1 at the cell surface, which are characteristic of clustering. TNF-α-induced NO also produced S-nitrosylation and phosphorylation of PKCζ, association of PKCζ with ICAM-1, and ICAM-1 phosphorylation. The inhibition of PKCζ blocked leukocyte adhesion induced by TNF-α. Mass spectrometry analysis of purified PKCζ identified cysteine 503 as the only S-nitrosylated residue in the kinase domain of the protein. Our results reveal a new eNOS S-nitrosylation-dependent mechanism that induces leukocyte adhesion and suggests that S-nitrosylation of PKCζ may be an important regulatory step in early leukocyte adhesion in inflammation.NEW & NOTEWORTHY Contrary to the well-established inhibitory role of NO in leukocyte adhesion, we demonstrate a positive role of nitric oxide in this process. We demonstrate that NO induced by eNOS after TNF-α treatment induces early leukocyte adhesion activating the S-nitrosylation pathway. Our data suggest that PKCζ S-nitrosylation may be a key step in this process.

Antibacterial and cytotoxic evaluation of copper and zinc oxide nanoparticles as a potential disinfectant material of connections in implant provisional abutments: An in-vitro study.

OBJECTIVE: This study evaluates the antibacterial activity against mono and multispecies bacterial models and the cytotoxic effects of zinc oxide and copper nanoparticles(ZnO-NPs/Cu-NPs) in cell cultures of human gingival fibroblasts(HGFs). DESIGN: The antibacterial activities of ZnO-NPs and Cu-NPs against 4 bacteria species were tested according to their minimum inhibitory concentrations(MICs) and against mature multispecies anaerobic model by spectral confocal laser scanning microscopy. The viabilities and cytotoxic effects of ZnO-NPs and Cu-NPs to HGFs cell cultures were tested by MTT, LDH assays, production of ROS, and the activation of caspase-3. The results were analyzed using one-way ANOVA followed by Tukey tests, considering p < 0.05 as statistically significant. RESULTS: For all strains, MICs of ZnO-NPs and Cu-NPs were in the range of 78.3 µg/mL-3906 µg/mL and 125 µg/mL-625 ug/mL, respectively. In a multispecies model, a significant decrease in the total biomass volume(µ3) was observed in response to exposure to 125 µg/mL of each NPs for which there was bactericidal activity. Significant differences were found between the volumes of viable and nonviable biomass exposed to nanostructures with Cu-NPs compared to ZnO-NPs. Both NPs induced mitochondrial dose-dependent cytotoxicity, ZnO-NPs increases LDH release and intracellular ROS generation. Cu-NPs at a concentration of 50 µg/mL induced production of cleaved caspase-3, activating the apoptotic pathway early and at low doses. CONCLUSIONS: After 24 h, ZnO-NPs are biocompatible between 78-100 µg/mL and Cu-NPs below 50 µg/mL. Antibacterial activity in a monospecies model is strain dependent, and in a multispecies model was a lower doses after 10 min of exposure.

Role of NO and S-nitrosylation in the Expression of Endothelial Adhesion Proteins That Regulate Leukocyte and Tumor Cell Adhesion.

Leukocyte recruitment is one of the most important cellular responses to tissue damage. Leukocyte extravasation is exquisitely regulated by mechanisms of selective leukocyte-endothelium recognition through adhesion proteins in the endothelial cell surface that recognize specific integrins in the activated leukocytes. A similar mechanism is used by tumor cells during metastasis to extravasate and form a secondary tumor. Nitric oxide (NO) has been classically described as an anti-inflammatory molecule that inhibits leukocyte adhesion. However, the evidence available shows also a positive role of NO in leukocyte adhesion. These apparent discrepancies might be explained by the different NO concentrations reached during the inflammatory response, which are highly modulated by the expression of different nitric oxide synthases, along the inflammatory response and by changes in their subcellular locations.

Biodentine Induce Proteínas Apoptóticas Caspasa 3 y PARP-1 en Células de la Pulpa Dental Humana / Biodentine Induces Apoptotic Proteins Caspase 3 and PARP - 1 on Human Dental Pulp Cells

RESUMEN: Las patologías pulpares han sido un verdadero reto para la odontología principalmente por su tratamiento. Actualmente, existen numerosos biomateriales en el mercado que reportan tener propiedades inherentes en los tejidos dentarios. Sin embargo, diferentes estudios sobre múltiples líneas celulares expuestas a estos biomateriales demuestran resultados controversiales como biocompatiblidad y citotoxicidad celular. Biodentine, es un cemento endodóntico en base a silicatos cálcico de múltiples aplicaciones, que prestaría propiedades de biocompatibilidad como bioactividad celular, características que le permitirían incluso ser utilizado en contacto directo con la pulpa dental. El objetivo de este estudio es la evaluación in-vitro de Biodentine, sobre cultivos de células de la pulpa dental humana (CCPDH). Se prepararon discos de cemento de Biodentine™ de 2 x 6 mm, los que se expusieron a cultivos de células aisladas de la pulpa dental humana. Luego de 24, 48 y 72 horas de exposición, se realizaron ensayos de viabilidad celular utilizando el método colorimétrico MTT. También se realizaron ensayos de expresión proteica de dos proteínas involucradas en la vía de señalización de la apoptosis celular: Caspasa - 3 clivada y Poli (ADP-Ribosa) Polimerasa, PARP - 1. Existen diferencias estadísticamente significativas (p<0,05) en los ensayos de viabilidad celular entre las células expuestas a Biodentine y el grupo control, como también a medida que aumenta el tiempo de exposición (p<0,05). Por otra parte, también existen diferencias significativas (p<0,05) en la expresión de PARP- 1 en los grupos sometidos a Biodentine. Los resultados obtenidos en este estudio demuestran que Biodentine genera citotoxicidad celular en cultivos celulares de pulpa dental humana, por disminución de la viabilidad celular como por la expresión de proteínas apoptóticas. Es por esto que la utilización de este biomaterial debería ser estudiado y considerarse en cada caso clínico, especialmente como recubridor pulpar directo.

ABSTRACT: Oral pathologies have been a real challenge for dentistry, mainly due to its treatment. Currently, there are numerous biomaterials on the market that may present inherent properties in dental tissues. However, studies on multiple cell lines are based on biocompatible results such as biocompatibility and cellular cytotoxicity. Biodentine is endodontic cement based on calcium silicates of multiple applications, which would provide biocompatibility properties as cellular bioactivity, characteristics that will allow it to be used in direct contact with the dental pulp. The objective of this study is the in vitro evaluation of Biodentine, on cultures of cells of the human dental pulp (HDPC). Biodentine cement disks of 2 x 6 mm were prepared, and HDPC culture plates were introduced. After 24, 48 and 72 hours of exposure, cell viability tests were performed using the MTT colorimetric method. On the other hand, protein expression assays of two proteins involved in the signaling pathway of cell apoptosis Caspase-3 cleaved (cas-3 clv) and PARP-1 are carried out. There are statistically significant differences (p <0,05) in the cell viability tests between Biodentine and control group, as well as the exposure time increases (p <0,05). Otherwise, there are also significant differences (p <0,05) in the expression of PARP-1 in the groups, sometimes a Biodentine. The results in this study that Biodentine generates a cellular cytotoxicity in HDPC cultures, therefore, cell viability as the expression of apoptotic proteins. This is why the use of this biomaterial should be studied for each particular clinical case, especially as a direct pulp capping agent.

Interleukin-8 Secreted by Glioblastoma Cells Induces Microvascular Hyperpermeability Through NO Signaling Involving S-Nitrosylation of VE-Cadherin and p120 in Endothelial Cells.

Glioblastoma is a highly aggressive brain tumor, characterized by the formation of dysfunctional blood vessels and a permeable endothelial barrier. S-nitrosylation, a post-translational modification, has been identified as a regulator of endothelial function. In this work we explored whether S-nitrosylation induced by glioblastoma tumors regulates the endothelial function. As proof of concept, we observed that S-nitrosylation is present in the tumoral microenvironment of glioblastoma in two different animal models. Subsequently, we measured S nitrosylation and microvascular permeability in EAhy296 endothelial cells and in cremaster muscle. In vitro, conditioned medium from the human glioblastoma cell line U87 activates endothelial nitric oxide synthase, causes VE-cadherin- S-nitrosylation and induces hyperpermeability. Blocking Interleukin-8 (IL-8) in the conditioned medium inhibited S-nitrosylation of VE-cadherin and hyperpermeability. Recombinant IL-8 increased endothelial permeability by activating eNOS, S-nitrosylation of VE-cadherin and p120, internalization of VE-cadherin and disassembly of adherens junctions. In vivo, IL-8 induced S-nitrosylation of VE-cadherin and p120 and conditioned medium from U87 cells caused hyperpermeability in the mouse cremaster muscle. We conclude that eNOS signaling induced by glioma cells-secreted IL-8 regulates endothelial barrier function in the context of glioblastoma involving S-nitrosylation of VE-cadherin and p120. Our results suggest that inhibiting S-nitrosylation may be an effective way to control and/or block damage to the endothelial barrier and prevent cancer progression.

Coagulation Factor Xa Promotes Solid Tumor Growth, Experimental Metastasis and Endothelial Cell Activation.

Hypercoagulable state is linked to cancer progression; however, the precise role of the coagulation cascade is poorly described. Herein, we examined the contribution of a hypercoagulative state through the administration of intravenous Coagulation Factor Xa (FXa), on the growth of solid human tumors and the experimental metastasis of the B16F10 melanoma in mouse models. FXa increased solid tumor volume and lung, liver, kidney and lymph node metastasis of tail-vein injected B16F10 cells. Concentrating on the metastasis model, upon coadministration of the anticoagulant Dalteparin, lung metastasis was significantly reduced, and no metastasis was observed in other organs. FXa did not directly alter proliferation, migration or invasion of cancer cells in vitro. Alternatively, FXa upon endothelial cells promoted cytoskeleton contraction, disrupted membrane VE-Cadherin pattern, heightened endothelial-hyperpermeability, increased inflammatory adhesion molecules and enhanced B16F10 adhesion under flow conditions. Microarray analysis of endothelial cells treated with FXa demonstrated elevated expression of inflammatory transcripts. Accordingly, FXa treatment increased immune cell infiltration in mouse lungs, an effect reduced by dalteparin. Taken together, our results suggest that FXa increases B16F10 metastasis via endothelial cell activation and enhanced cancer cell-endothelium adhesion advocating that the coagulation system is not merely a bystander in the process of cancer metastasis.

Galectin-8 induces endothelial hyperpermeability through the eNOS pathway involving S-nitrosylation-mediated adherens junction disassembly.

The permeability of endothelial cells is regulated by the stability of the adherens junctions, which is highly sensitive to kinase-mediated phosphorylation and endothelial nitric oxide synthase (eNOS)-mediated S-nitrosylation of its protein components. Solid tumors can produce a variety of factors that stimulate these signaling pathways leading to endothelial cell hyperpermeability. This generates stromal conditions that facilitate tumoral growth and dissemination. Galectin-8 (Gal-8) is overexpressed in several carcinomas and has a variety of cellular effects that can contribute to tumor pathogenicity, including angiogenesis. Here we explored whether Gal-8 has also a role in endothelial permeability. We show that recombinant Gal-8 activates eNOS, induces S-nitrosylation of p120-catenin (p120) and dissociation of adherens junction, leading to hyperpermeability of the human endothelial cell line EAhy926. This pathway involves focal-adhesion kinase (FAK) activation downstream of eNOS as a requirement for eNOS-mediated p120 S-nitrosylation. This suggests a reciprocal, yet little understood, regulation of phosphorylation and S-nitrosylation events acting upon adherens junction permeability. In addition, glutathione S-transferase (GST)-Gal-8 pull-down experiments and function-blocking ß1-integrin antibodies point to ß1-integrins as cell surface components involved in Gal-8-induced hyperpermeability. Endogenous Gal-8 secreted from the breast cancer cell line MCF-7 has similar hyperpermeability and signaling effects. Furthermore, the mouse cremaster model system showed that Gal-8 also activates eNOS, induces S-nitrosylation of adherens junction components and is an effective hyperpermeability agent in vivo. These results add endothelial permeability regulation by S-nitrosylation as a new function of Gal-8 that can potentially contribute to the pathogenicity of tumors overexpressing this lectin.