Psd1 binding affinity toward fungal membrane components as assessed by SPR: The role of glucosylceramide in fungal recognition and entry.

Psd1 is a plant defensin that has antifungal activity against several pathogenic and nonpathogenic fungi. Previous analysis of Psd1 chemical shift perturbations by nuclear magnetic resonance (NMR) spectroscopy demonstrated that this defensin interacts with phospholipids and the sphingolipid glucosylceramide isolated from Fusarium solani (GlcCer(Fusarium solani)). In this study, these interactions were evaluated by real-time surface plasmon resonance (SPR) analysis. The data obtained demonstrated that Psd1 could bind more strongly to small unilamellar vesicles (SUV)-containing GlcCer(Fusarium solani) than to SUV that was composed of phosphatidylcholine (PC) alone or was enriched with GlcCer that had been isolated from soybeans. An increase in the SPR response after cholesterol or ergosterol incorporation in PC-SUV was detected; however, SUV composed of PC:Erg (7:3; molar:molar) became unstable in the presence of Psd1, suggesting membrane destabilization. We also observed a lack of Psd1 internalization in Candida albicans strains that were deficient in the glucosyl ceramide synthase gene. Together, these data indicate that GlcCer is essential for Psd1 anchoring in the fungal plasma membrane as well as internalization.

Glioblastoma: therapeutic challenges, what lies ahead.

Glioblastoma (GBM) is one of the most aggressive human cancers. Despite current advances in multimodality therapies, such as surgery, radiotherapy and chemotherapy, the outcome for patients with high grade glioma remains fatal. The knowledge of how glioma cells develop and depend on the tumor environment might open opportunities for new therapies. There is now a growing awareness that the main limitations in understanding and successfully treating GBM might be bypassed by the identification of a distinct cell type that has defining properties of somatic stem cells, as well as cancer-initiating capacity - brain tumor stem cells, which could represent a therapeutic target. In addition, experimental studies have demonstrated that the combination of antiangiogenic therapy, based on the disruption of tumor blood vessels, with conventional chemotherapy generates encouraging results. Emerging reports have also shown that microglial cells can be used as therapeutic vectors to transport genes and/or substances to the tumor site, which opens up new perspectives for the development of GBM therapies targeting microglial cells. Finally, recent studies have shown that natural toxins can be conjugated to drugs that bind to overexpressed receptors in cancer cells, generating targeted-toxins to selectively kill cancer cells. These targeted-toxins are highly effective against radiation- and chemotherapy-resistant cancer cells, making them good candidates for clinical trials in GBM patients. In this review, we discuss recent studies that reveal new possibilities of GBM treatment taking into account cancer stem cells, angiogenesis, microglial cells and drug delivery in the development of new targeted-therapies.

Tenascin-C in the extracellular matrix promotes the selection of highly proliferative and tubulogenesis-defective endothelial cells.

The extracellular matrix (ECM) contains important cues for tissue homeostasis and morphogenesis. The matricellular protein tenascin-C (TN-C) is overexpressed in remodeling tissues and cancer. In the present work, we studied the effect of different ECM-which exhibited a significant diversity in their TN-C content-in endothelial survival, proliferation and tubulogenic differentiation: autologous (endothelial) ECM devoid of TN-C, but bearing large amounts of FN; fibroblast ECM, bearing both high TN-C and FN contents; and finally, glioma-derived matrices, usually poor in FN, but very rich in TN-C. HUVECs initially adhered to the immobilized matrix produced by U373 MG glioma cells, but significantly detached and died by anoikis (50 to 80%) after 24h, as compared with cells incubated with endothelial and fibroblast matrices. Surviving endothelial cells (20 to 50%) became up to 6-fold more proliferative and formed 74-97% less tube-like structures in vitro than cells grown on non-tumoral matrices. An antibody against the EGF-like repeats of tenascin-C (TN-C) partially rescued cells from the tubulogenic defect, indicating that this molecule is responsible for the selection of highly proliferative and tubulogenic defective endothelial cells. Interestingly, by using defined substrata, in conditions that mimic glioma and normal cell ECM composition, we observed that fibronectin (FN) modulates the TN-C-induced selection of endothelial cells. Our data show that TN-C is able to modulate endothelial branching morphogenesis in vitro and, since it is prevalent in matrices of injured and tumor tissues, also suggest a role for this protein in vascular morphogenesis, in these physiological contexts.

Osteoarthritic Synovial Fluid and TGF-ß1 Induce Interleukin-18 in Articular Chondrocytes.

OBJECTIVE: Synovial fluid (SF) plays an important role in the maintenance of articular cartilage. SF is a dynamic reservoir of proteins derived from cartilage and synovial tissue; thus, its composition may serve as a biomarker that reflects the health and pathophysiological condition of the joint. The purpose of the current study was to evaluate the osteoarthritic synovial fluid (OASF) and transforming growth factor-ß1 (TGF-ß1) activity in articular chondrocytes catabolic and inflammatory responses. DESIGN: Chondrocytes were seeded at passage 2 and cultured for 72 hours under different conditions. Human chondrocytes were subjected to OASF while rat chondrocytes were subjected to either healthy synovial fluid (rSF) or TGF-ß1 and then assigned for cell viability analysis. In addition, the effects of OASF and TGF-ß1 on chondrocytes metalloprotease (MMP)-3 and MMP-13 and interleukin-18 (IL-18) expression were evaluated by immunocytochemistry, ELISA, and reverse transcriptase-polymerase chain reaction. RESULTS: SF from osteoarthritic patients significantly induced MMP-3, MMP-13, and IL-18 receptor expression in chondrocytes. To put in evidence the inflammatory activity of OASF, healthy chondrocytes from rat were cultured with TGF-ß1. In the presence of TGF-ß1 these cells started to express MMP-3, MMP-13, and IL-18 genes and attached to each other forming a chondrocyte aggregated structure. Healthy SF was able to maintain a typical monolayer of rounded chondrocytes with no inflammatory response. CONCLUSION: In summary, these observations demonstrated that TGF-ß1, one of the components of OASF, has a dual effect, acting in chondrocyte maintenance and also inducing inflammatory and catabolic properties of these cells.

Perspectivas de graduandos em saúde sobre a temática minorias sexuais e de gênero na formação / Perspectives of undergraduate health students on the theme of sexual and gender minorities in training / Perspectivas de estudiantes de licenciatura en salud sobre el tema de las minorías sexuales y de género en formación

Objetivo: Caracterizar as perspectivas de graduandos da área de saúde sobre a temática minorias sexuais e de gênero na formação profissional. Métodos: Trata-se de um estudo descritivo com análise secundária dos dados qualitativos de 262 estudantes de graduação em saúde de duas Instituições de Ensino Superior no Estado de São Paulo (Brasil). Resultados: A maioria era solteiro (66%), do sexo feminino (83,4%), identidade de gênero feminina (81,2%) e heterosexual (90,6%). A maioria dos estudantes referiu não ter sofrido violência motivada pela identidade de gênero ou orientação sexual (95,4%), já ter tido essa temática durante a formação (61,7%), estar preparado profissionalmente frente a isso (88,4%) e para cuidar dessa população (77,5%). Dos discursos analisados frente à pergunta "Como você acha que a sua formação acadêmica poderia contribuir para lidar com as minorias sexuais?" emergiram duas categorias centrais: "saber lidar com as minorias sexuais e de gênero" e "tornar-se um profissional de saúde aberto à diversidade humana". Conclusão: Evidenciam-se áreas potenciais para a construção de competências sensíveis às minorias sexuais desde a graduação em saúde. (AU)

Objective: To characterize the perspectives of undergraduate students in the health field on the theme of sexual and gender minorities in professional training. Methods: This is a descriptive study with secondary analysis of the qualitative data of 262 undergraduate health students from two Higher Education Institutions in the State of São Paulo (Brazil). Results: Most was single (66%), female (83.4%), female gender identity (81.2%) and heterosexual (90.6%). Most students reported not having suffered violence motivated by gender identity or sexual orientation (95.4%), having already had this theme during training (61.7%), being professionally prepared for it (88.4%) and to care for the population (77.5%). From the speeches analyzed before the question "How do you think your academic training could contribute to dealing with sexual minorities?" two central categories emerged: "knowing how to deal with sexual and gender minorities" and "becoming a health professional open to human diversity". Conclusion: Potential areas for the construction of skills sensitive to the sexual minorities are evident since graduation in health. (AU)

Objetivo: Caracterizar las perspectivas de los estudiantes de pregrado en el campo de la salud sobre el tema de las minorías sexuales y de género en la formación profesional. Métodos: Se trata de un estudio descriptivo con análisis secundario de los datos cualitativos de 262 estudiantes del área de salud de dos Instituciones de Educación Superior en el Estado de São Paulo (Brasil). Resultados: La mayoría era soltero (66%), mujer (83,4%), identidad de género femenina (81,2%) y heterosexual (90,6%). La mayoría de los estudiantes refirió no haber sufrido violencia motivada por identidad de género u orientación sexual (95,4%), haber tenido ya esta temática durante la formación (61,7%), estar preparados profesionalmente para ello (88,4%) y atender a la población (77,5%). De los discursos analizados antes de la pregunta "¿Cómo crees que tu formación académica podría contribuir al trato con las minorías sexuales?" Surgieron dos categorías centrales: "saber lidiar con las minorías sexuales y de género" y "convertirse en un profesional de la salud abierto a la diversidad humana". Conclusión: Las áreas potenciales para la construcción de habilidades sensibles a las minorías sexuales son evidentes desde la graduación en salud. (AU)

Connective tissue growth factor (CTGF/CCN2) is negatively regulated during neuron-glioblastoma interaction.

Connective-tissue growth factor (CTGF/CCN2) is a matricellular-secreted protein involved in complex processes such as wound healing, angiogenesis, fibrosis and metastasis, in the regulation of cell proliferation, migration and extracellular matrix remodeling. Glioblastoma (GBM) is the major malignant primary brain tumor and its adaptation to the central nervous system microenvironment requires the production and remodeling of the extracellular matrix. Previously, we published an in vitro approach to test if neurons can influence the expression of the GBM extracellular matrix. We demonstrated that neurons remodeled glioma cell laminin. The present study shows that neurons are also able to modulate CTGF expression in GBM. CTGF immnoreactivity and mRNA levels in GBM cells are dramatically decreased when these cells are co-cultured with neonatal neurons. As proof of particular neuron effects, neonatal neurons co-cultured onto GBM cells also inhibit the reporter luciferase activity under control of the CTGF promoter, suggesting inhibition at the transcription level. This inhibition seems to be contact-mediated, since conditioned media from embryonic or neonatal neurons do not affect CTGF expression in GBM cells. Furthermore, the inhibition of CTGF expression in GBM/neuronal co-cultures seems to affect the two main signaling pathways related to CTGF. We observed inhibition of TGFß luciferase reporter assay; however phopho-SMAD2 levels did not change in these co-cultures. In addition levels of phospho-p44/42 MAPK were decreased in co-cultured GBM cells. Finally, in transwell migration assay, CTGF siRNA transfected GBM cells or GBM cells co-cultured with neurons showed a decrease in the migration rate compared to controls. Previous data regarding laminin and these results demonstrating that CTGF is down-regulated in GBM cells co-cultured with neonatal neurons points out an interesting view in the understanding of the tumor and cerebral microenvironment interactions and could open up new strategies as well as suggest a new target in GBM control.

Effect of thyroid hormone T3 on myosin-Va expression in the central nervous system.

Thyroid hormones (THs) are essential for brain development, where they regulate gliogenesis, myelination, cell proliferation and protein synthesis. Hypothyroidism severely affects neuronal growth and establishment of synaptic connections. Triiodothyronine (T3), the biologically active form of TH, has a central function in these activities. So, Myosin-Va (Myo-Va), a molecular motor protein involved in vesicle and RNA transport, is a good candidate as a target for T3 regulation. Here, we analyzed Myo-Va expression in euthyroid and hypothyroid adult rat brains and synaptosomes. We observed a reduction of Myo-Va expression in cultured neural cells from newborn hypothyroid rat brain, while immunocytochemical experiments showed a punctate distribution of this protein in the cytoplasm of cells. Particularly, Myo-Va co-localized with microtubules in neurites, especially in their varicosities. Myo-Va immunostaining was stronger in astrocytes and neurons of controls when compared with hypothyroid brains. In addition, supplementation of astrocyte cultures with T3 led to increased expression of Myo-Va in cells from both euthyroid and hypothyroid animals, suggesting that T3 modulates Myo-Va expression in neural cells both in vivo and in vitro. We have further analyzed Myo-Va expression in U373 cells, a human glioblastoma line, and found the same punctate cytoplasmic protein localization. As in normal neural cells, this expression was also increased by T3, suggesting that the modulatory mechanism exerted by T3 over Myo-Va remains active on astrocyte tumor cells. These data, coupled with the observation that Myo-Va is severely affected in hypothyroidism, support the hypothesis that T3 activity regulates neural motor protein expression, taking Myo-Va as a model. As a consequence, reduced T3 activity could supposedly affect axonal transport and synaptic function, and could therefore explain disturbances seen in the hypothyroid brain.

Antifungal Pisum sativum defensin 1 interacts with Neurospora crassa cyclin F related to the cell cycle.

Plant defensins, components of the plant innate immune system, are cationic cysteine-rich antifungal peptides. Evidence from the literature [Thevissen, K., et al. (2003) Peptides 24, 1705-1712] has demonstrated that patches of fungi membrane containing mannosyldiinositolphosphorylceramide and glucosylceramides are selective binding sites for the plant defensins isolated from Dahlia merckii and Raphanus sativus, respectively. Whether plant defensins interact directly or indirectly with fungus intracellular targets is unknown. To identify physical protein-protein interactions, a GAL4-based yeast two-hybrid system was performed using the antifungal plant peptide Pisum sativum defensin 1 (Psd1) as the bait. Target proteins were screened within a Neurospora crassa cDNA library. Nine out of 11 two-hybrid candidates were nuclear proteins. One clone, detected with high frequency per screening, presented sequence similarity to a cyclin-like protein, with F-box and WD-repeat domains, related to the cell cycle control. GST pull-down assay corroborated in vitro this two-hybrid interaction. Fluorescence microscopy analysis of FITC-conjugated Psd1 and DAPI-stained fungal nuclei showed in vivo the colocalization of the plant peptide Psd1 and the nucleus. Analysis of the DNA content of N. crassa conidia using flow cytometry suggested that Psd1 directed cell cycle impairment and caused conidia to undergo endoreduplication. The developing retina of neonatal rats was used as a model to observe the interkinetic nuclear migration during proliferation of an organized tissue from the S toward the M phase of the cell cycle in the presence of Psd1. The results demonstrated that the plant defensin Psd1 regulates interkinetic nuclear migration in retinal neuroblasts.

Escolha de veias periféricas para terapia intravenosa em recém-nascidos pela equipe de enfermagem / Choice made by the nursing team, of peripheral veins for intravenous terapy in newborns

A implementação da terapia intravenosa no recém-nascido é complexa e desafiadora. Objetivou-se identificar as veias periféricas de escolha, para a punção venosa, no recém-nascido e descrever os critérios adotados pela equipe de enfermagem na escolha desses vasos. O cenário foi uma Unidade de Terapia Intensiva Neonatal do Rio de Janeiro, Brasil. Foram entrevistados dez profissionais de enfermagem de maio a junho de 2007. A análise temática originou as categorias: “Escolha da veia periférica em RN: prioridades da equipe de enfermagem” e “Critérios adotados e dificuldades enfrentadas na escolha das veias para punção venosa periférica”. As veias do arco dorsal da mão foram as de primeira escolha e as epicranianas as de última escolha. Os critérios de escolha foram visualização, facilidade, durabilidade e fármaco infundido. As dificuldades enfrentadas para a realização terapia intravenosa no recém-nascido foram o desgaste da rede venosa e a dor advinda das múltiplas punções.

Avaliação da ocorrência de flebite, infiltração e extravasamento em neonatossubmetidos à terapia intravenosa / Assessment of phlebitis, infiltration and extravasation events in neonates submitted to intravenous therapy / Avaliacion de la ocurrencia de flebitis, infiltración y extravasamiento en neonatos submetidos a terapia intravenosa

O objetivo deste estudo foi descrever a ocorrência de flebite, infiltração e extravasamento em recém-nascidos internados na unidade de terapia intensiva neonatal de uma maternidade pública do Rio de Janeiro. Trata-se de um estudo quantitativo descritivo com 36 recém-nascidos em uso de terapia intravenosa e com indicação de remoção do dispositivo intravenoso periférico. Foram avaliados 50 sítios de punção imediatamente após a remoção de cateteres periféricos originando uma média de 1,40 punções venosas por neonato. As complicações foram responsáveis por 48% da remoção dos cateteres, antes da alta do tratamento, com predomínio de infiltração (79,2%), seguida por flebite (16,7%) e extravasamento (4,2%). A fim de se evitar os agravos e promover a segurança dos recém-nascidos submetidos à terapia intravenosa, a equipe de enfermagem deve avaliar periodicamente o acesso venoso periférico e obter conhecimento acerca das intervenções necessárias quando detectados sinais de complicações.

This study aimed to describe phlebitis, infiltration and extravasation events in newborn infants hospitalized at the neonatal intensive care unit of a public maternity in Rio de Janeiro, Brazil. A quantitative and descriptive study was carried out, involving 36 newborns under intravenous therapy and indicated for the removal of the peripheral intravenous device. Fifty puncture sites were assessed immediately after the peripheral catheter removal, resulting in an average 1.40 punctures per infant. Complications were responsible for 48% of catheter removals before discharge from treatment, predominantly infiltration (79.2%), followed by phlebitis (16.7%) and extravasation (4.2%). To avoid aggravations and enhance the security of newborns submitted to intravenous therapy, the nursing team should periodically assess the peripheral venous access and gain knowledge on interventions needed when signs of complications are detected.

El objetivo de este estudio fue describir la ocurrencia de flebitis, infiltración y extravasamiento en recién nacidos internados en la unidad de terapia intensiva neonatal de una maternidad pública de Rio de Janeiro. Se trata de un estudio cuantitativo descriptivo con 36 recién nacidos en uso de terapia intravenosa y con indicación de remoción del dispositivo intravenoso periférico. Fueron evaluados 50 sitios de punción inmediatamente después de la remoción de catéteres periféricos originando una media de 1,40 punciones venosas por neonato. Las complicaciones fueron responsables por 48% de las remociones de los catéteres, antes del alta del tratamiento, con predominio de infiltraciones (79,2%), seguida por flebitis (16,7%) e extravasamiento (4,2%). Con el fin de evitar los lesiones y promover la seguridad de los recién nacidos sometidos a terapia intravenosa, el equipo de enfermería debe evaluar periódicamente el acceso venoso periférico y obtener conocimiento acerca de las intervenciones necesarias cuando detectadas señales de complicaciones.

Interactive properties of human glioblastoma cells with brain neurons in culture and neuronal modulation of glial laminin organization.

The harmonious development of the central nervous system depends on the interactions of the neuronal and glial cells. Extracellular matrix elements play important roles in these interactions, especially laminin produced by astrocytes, which has been shown to be a good substrate for neuron growth and axonal guidance. Glioblastomas are the most common subtypes of primary brain tumors and may be astrocytes in origin. As normal laminin-producing glial cells are the preferential substrate for neurons, and glial tumors have been shown to produce laminin, we questioned whether glioblastoma retained the same normal glial-neuron interactive properties with respect to neuronal growth and differentiation. Then, rat neurons were co-cultured onto rat normal astrocytes or onto three human glioblastoma cell lines obtained from neurosurgery. The co-culture confirmed that human glioblastoma cells as well as astrocytes maintained the ability to support neuritogenesis, but non-neural normal or tumoral cells failed to do so. However, glioblastoma cells did not distinguish embryonic from post-natal neurons in relation to neurite pattern in the co-cultures, as normal astrocytes did. Further, the laminin organization on both normal and tumoral glial cells was altered from a filamentous arrangement to a mixed punctuate/filamentous pattern when in co-culture with neurons. Together, these results suggest that glioblastoma cells could identify neuronal cells as partners, to support their growth and induce complex neurites, but they lost the normal glia property to distinguish neuronal age. In addition, our results show for the first time that neurons modulate the organization of astrocytes and glioblastoma laminin on the extracellular matrix.