A highly reproducible and efficient method for retinal organoid differentiation from human pluripotent stem cells.

Human pluripotent stem cell (hPSC)-derived retinal organoids are three-dimensional cellular aggregates that differentiate and self-organize to closely mimic the spatial and temporal patterning of the developing human retina. Retinal organoid models serve as reliable tools for studying human retinogenesis, yet limitations in the efficiency and reproducibility of current retinal organoid differentiation protocols have reduced the use of these models for more high-throughput applications such as disease modeling and drug screening. To address these shortcomings, the current study aimed to standardize prior differentiation protocols to yield a highly reproducible and efficient method for generating retinal organoids. Results demonstrated that through regulation of organoid size and shape using quick reaggregation methods, retinal organoids were highly reproducible compared to more traditional methods. Additionally, the timed activation of BMP signaling within developing cells generated pure populations of retinal organoids at 100% efficiency from multiple widely used cell lines, with the default forebrain fate resulting from the inhibition of BMP signaling. Furthermore, given the ability to direct retinal or forebrain fates at complete purity, mRNA-seq analyses were then utilized to identify some of the earliest transcriptional changes that occur during the specification of these two lineages from a common progenitor. These improved methods also yielded retinal organoids with expedited differentiation timelines when compared to traditional methods. Taken together, the results of this study demonstrate the development of a highly reproducible and minimally variable method for generating retinal organoids suitable for analyzing the earliest stages of human retinal cell fate specification.

[Duration of antibiotic therapy for common respiratory infections in adults]. / Durées d'antibiothérapie des infections respiratoires courantes de l'adulte.

Antibiotic therapy is not recommended for the majority of adult outpatients with a respiratory tract infection. When antibiotic therapy is mandatory, its duration should be as short as possible in order to avoid side effects and reduce costs. This literature review summarises the recommendations for new, shorter courses of antibiotics. The recommended duration of antibiotic treatment for community-acquired pneumonia, chronic obstructive pulmonary disease exacerbation and acute otitis media is now 5 days, 6 days for pharyngitis and between 5 and 7 days for sinusitis. These recommendations do not apply to severe or complicated infections, infections with resistant agents or in immunosuppressed patients.

Une antibiothérapie n'est pas recommandée pour la majorité des patients adultes se présentant avec une infection des voies respiratoires en ambulatoire. Lorsqu'elle est indiquée, sa durée doit être aussi courte que possible afin d'éviter les effets indésirables et diminuer les coûts. Cette revue de littérature résume les recommandations des nouvelles durées raccourcies d'antibiothérapies. Désormais, la durée d'antibiothérapie recommandée pour la pneumonie acquise en communauté, l'exacerbation de BPCO et l'otite moyenne aiguë est de 5 jours, 6 pour la pharyngite et entre 5 et 7 pour les sinusites. Ces recommandations ne sont pas applicables pour les infections sévères ou compliquées, celles à agent résistant et chez les patients immunosupprimés.

Development of a three-dimensional organoid model to explore early retinal phenotypes associated with Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of Aß plaques and neurofibrillary tangles, resulting in synaptic loss and neurodegeneration. The retina is an extension of the central nervous system within the eye, sharing many structural similarities with the brain, and previous studies have observed AD-related phenotypes within the retina. Three-dimensional retinal organoids differentiated from human pluripotent stem cells (hPSCs) can effectively model some of the earliest manifestations of disease states, yet early AD-associated phenotypes have not yet been examined. Thus, the current study focused upon the differentiation of hPSCs into retinal organoids for the analysis of early AD-associated alterations. Results demonstrated the robust differentiation of retinal organoids from both familial AD and unaffected control cell lines, with familial AD retinal organoids exhibiting a significant increase in the Aß42:Aß40 ratio as well as phosphorylated Tau protein, characteristic of AD pathology. Further, transcriptional analyses demonstrated the differential expression of many genes and cellular pathways, including those associated with synaptic dysfunction. Taken together, the current study demonstrates the ability of retinal organoids to serve as a powerful model for the identification of some of the earliest retinal alterations associated with AD.

Retinal Ganglion Cells in a Dish: Current Strategies and Recommended Best Practices for Effective In Vitro Modeling of Development and Disease.

The ability to derive retinal ganglion cells (RGCs) from human pluripotent stem cells (hPSCs) provides an extraordinary opportunity to study the development of RGCs as well as cellular mechanisms underlying their degeneration in optic neuropathies. In the past several years, multiple approaches have been established that allow for the generation of RGCs from hPSCs, with these methods greatly improved in more recent studies to yield mature RGCs that more faithfully recapitulate phenotypes within the eye. Nevertheless, numerous differences still remain between hPSC-RGCs and those found within the human eye, with these differences likely explained at least in part due to the environment in which hPSC-RGCs are grown. With the ultimate goal of generating hPSC-RGCs that most closely resemble those within the retina for proper studies of retinal development, disease modeling, as well as cellular replacement, we review within this manuscript the current effective approaches for the differentiation of hPSC-RGCs, as well as how they have been applied for the investigation of RGC neurodegenerative diseases such as glaucoma. Furthermore, we provide our opinions on the characteristics of RGCs necessary for their use as effective in vitro disease models and importantly, how these current systems should be improved to more accurately reflect disease states. The establishment of characteristics in differentiated hPSC-RGCs that more effectively mimic RGCs within the retina will not only enable their use as effective models of RGC development, but will also create a better disease model for the identification of mechanisms underlying the neurodegeneration of RGCs in disease states such as glaucoma, further facilitating the development of therapeutic approaches to rescue RGCs from degeneration in disease states.

Autophagy disruption reduces mTORC1 activation leading to retinal ganglion cell neurodegeneration associated with glaucoma.

Autophagy dysfunction has been associated with several neurodegenerative diseases including glaucoma, characterized by the degeneration of retinal ganglion cells (RGCs). However, the mechanisms by which autophagy dysfunction promotes RGC damage remain unclear. Here, we hypothesized that perturbation of the autophagy pathway results in increased autophagic demand, thereby downregulating signaling through mammalian target of rapamycin complex 1 (mTORC1), a negative regulator of autophagy, contributing to the degeneration of RGCs. We identified an impairment of autophagic-lysosomal degradation and decreased mTORC1 signaling via activation of the stress sensor adenosine monophosphate-activated protein kinase (AMPK), along with subsequent neurodegeneration in RGCs differentiated from human pluripotent stem cells (hPSCs) with a glaucoma-associated variant of Optineurin (OPTN-E50K). Similarly, the microbead occlusion model of glaucoma resulting in ocular hypertension also exhibited autophagy disruption and mTORC1 downregulation. Pharmacological inhibition of mTORC1 in hPSC-derived RGCs recapitulated disease-related neurodegenerative phenotypes in otherwise healthy RGCs, while the mTOR-independent induction of autophagy reduced protein accumulation and restored neurite outgrowth in diseased OPTN-E50K RGCs. Taken together, these results highlight an important balance between autophagy and mTORC1 signaling essential for RGC homeostasis, while disruption to these pathways contributes to neurodegenerative features in glaucoma, providing a potential therapeutic target to prevent neurodegeneration.

Astrocytes modulate neurodegenerative phenotypes associated with glaucoma in OPTN(E50K) human stem cell-derived retinal ganglion cells.

Although the degeneration of retinal ganglion cells (RGCs) is a primary characteristic of glaucoma, astrocytes also contribute to their neurodegeneration in disease states. Although studies often explore cell-autonomous aspects of RGC neurodegeneration, a more comprehensive model of glaucoma should take into consideration interactions between astrocytes and RGCs. To explore this concept, RGCs and astrocytes were differentiated from human pluripotent stem cells (hPSCs) with a glaucoma-associated OPTN(E50K) mutation along with corresponding isogenic controls. Initial results indicated significant changes in OPTN(E50K) astrocytes, including evidence of autophagy dysfunction. Subsequently, co-culture experiments demonstrated that OPTN(E50K) astrocytes led to neurodegenerative properties in otherwise healthy RGCs, while healthy astrocytes rescued some neurodegenerative features in OPTN(E50K) RGCs. These results are the first to identify disease phenotypes in OPTN(E50K) astrocytes, including how their modulation of RGCs is affected. Moreover, these results support the concept that astrocytes could offer a promising target for therapeutic intervention in glaucoma.

Neurotoxic Astrocytes Directly Converted from Sporadic and Familial ALS Patient Fibroblasts Reveal Signature Diversities and miR-146a Theragnostic Potential in Specific Subtypes.

A lack of stratification methods in patients with amyotrophic lateral sclerosis (ALS) is likely implicated in therapeutic failures. Regional diversities and pathophysiological abnormalities in astrocytes from mice with SOD1 mutations (mSOD1-ALS) can now be explored in human patients using somatic cell reprogramming. Here, fibroblasts from four sporadic (sALS) and three mSOD1-ALS patients were transdifferentiated into induced astrocytes (iAstrocytes). ALS iAstrocytes were neurotoxic toward HB9-GFP mouse motor neurons (MNs) and exhibited subtype stratification through GFAP, CX43, Ki-67, miR-155 and miR-146a expression levels. Up- (two cases) and down-regulated (three cases) miR-146a values in iAstrocytes were recapitulated in their secretome, either free or as cargo in small extracellular vesicles (sEVs). We previously showed that the neuroprotective phenotype of depleted miR-146 mSOD1 cortical astrocytes was reverted by its mimic. Thus, we tested such modulation in the most miR-146a-depleted patient-iAstrocytes (one sALS and one mSOD1-ALS). The miR-146a mimic in ALS iAstrocytes counteracted their reactive/inflammatory profile and restored miR-146a levels in sEVs. A reduction in lysosomal activity and enhanced synaptic/axonal transport-related genes in NSC-34 MNs occurred after co-culture with miR-146a-modulated iAstrocytes. In summary, the regulation of miR-146a in depleted ALS astrocytes may be key in reestablishing their normal function and in restoring MN lysosomal/synaptic dynamic plasticity in disease sub-groups.

Extension of retinofugal projections in an assembled model of human pluripotent stem cell-derived organoids.

The development of the visual system involves the coordination of spatial and temporal events to specify the organization of varied cell types, including the elongation of axons from retinal ganglion cells (RGCs) to post-synaptic targets in the brain. Retinal organoids recapitulate many features of retinal development, yet have lacked downstream targets into which RGC axons extend, limiting the ability to model projections of the human visual system. To address these issues, retinal organoids were generated and organized into an in vitro assembloid model of the visual system with cortical and thalamic organoids. RGCs responded to environmental cues and extended axons deep into assembloids, modeling the projections of the visual system. In addition, RGC survival was enhanced in long-term assembloids, overcoming prior limitations of retinal organoids in which RGCs are lost. Overall, these approaches will facilitate studies of human visual system development, as well as diseases or injuries to this critical pathway.

Recovery of Depleted miR-146a in ALS Cortical Astrocytes Reverts Cell Aberrancies and Prevents Paracrine Pathogenicity on Microglia and Motor Neurons.

Reactive astrocytes in Amyotrophic Lateral Sclerosis (ALS) change their molecular expression pattern and release toxic factors that contribute to neurodegeneration and microglial activation. We and others identified a dysregulated inflammatory miRNA profile in ALS patients and in mice models suggesting that they represent potential targets for therapeutic intervention. Such cellular miRNAs are known to be released into the secretome and to be carried by small extracellular vesicles (sEVs), which may be harmful to recipient cells. Thus, ALS astrocyte secretome may disrupt cell homeostasis and impact on ALS pathogenesis. Previously, we identified a specific aberrant signature in the cortical brain of symptomatic SOD1-G93A (mSOD1) mice, as well as in astrocytes isolated from the same region of 7-day-old mSOD1 mice, with upregulated S100B/HMGB1/Cx43/vimentin and downregulated GFAP. The presence of downregulated miR-146a on both cases suggests that it can be a promising target for modulation in ALS. Here, we upregulated miR-146a with pre-miR-146a, and tested glycoursodeoxycholic acid (GUDCA) and dipeptidyl vinyl sulfone (VS) for their immunoregulatory properties. VS was more effective in restoring astrocytic miR-146a, GFAP, S100B, HMGB1, Cx43, and vimentin levels than GUDCA, which only recovered Cx43 and vimentin mRNA. The miR-146a inhibitor generated typical ALS aberrancies in wild type astrocytes that were abolished by VS. Similarly, pre-miR-146a transfection into the mSOD1 astrocytes abrogated aberrant markers and intracellular Ca2+ overload. Such treatment counteracted miR-146a depletion in sEVs and led to secretome-mediated miR-146a enhancement in NSC-34-motor neurons (MNs) and N9-microglia. Secretome from mSOD1 astrocytes increased early/late apoptosis and FGFR3 mRNA in MNs and microglia, but not when derived from pre-miR-146a or VS-treated cells. These last strategies prevented the impairment of axonal transport and synaptic dynamics by the pathological secretome, while also averted microglia activation through either secretome, or their isolated sEVs. Proteomic analysis of the target cells indicated that pre-miR-146a regulates mitochondria and inflammation via paracrine signaling. We demonstrate that replenishment of miR-146a in mSOD1 cortical astrocytes with pre-miR-146a or by VS abrogates their phenotypic aberrancies and paracrine deleterious consequences to MNs and microglia. These results propose miR-146a as a new causal and emerging therapeutic target for astrocyte pathogenic processes in ALS.

Astrocyte regional diversity in ALS includes distinct aberrant phenotypes with common and causal pathological processes.

Astrocytes are major contributors of motor neuron (MN) degeneration in amyotrophic lateral sclerosis (ALS). We investigated whether regional and cell maturation differences influence ALS astrocyte malfunction. Spinal and cortical astrocytes from SOD1G93A (mSOD1) 7-day-old mice were cultured for 5 and 13 days in vitro (DIV). Astrocyte aberrancies predominated in 13DIV cells with region specificity. 13DIV cortical mSOD1 astrocytes showed early morphological changes and a predominant reactive and inflammatory phenotype, while repressed proteins and genes were found in spinal cells. Inflammatory-associated miRNAs, e.g. miR-155/miR-21/miR-146a, were downregulated in the first and upregulated in the later ones. Interestingly, depleted miR-155/miR-21/miR-146a in small extracellular vesicles (sEVs/exosomes) was a common pathological feature. Cortical mSOD1 astrocytes induced late apoptosis and kinesin-1 downregulation in mSOD1 NSC-34 MNs, whereas spinal cells upregulated dynein, while decreased nNOS and synaptic-related genes. Both regional-distinct mSOD1 astrocytes enhanced iNOS gene expression in mSOD1 MNs. We provide information on the potential contribution of astrocytes to ALS bulbar-vs. spinal-onset pathology, local influence on neuronal dysfunction and their shared miRNA-depleted exosome trafficking. These causal and common features may have potential therapeutic implications in ALS. Future studies should clarify if astrocyte-derived sEVs are active players in ALS-related neuroinflammation and glial activation.

Toma de decisiones para el tratamiento de caries oclusales en el sistema de salud público brasileño / Decision-making for the treatment of occlusal caries in the Brazilian public health system

ABSTRACT Introduction: Caries diagnosis errors have become a public health problem. Objective: Verify the consistency in terms of decision-making between Brazilian dentists from the country's public health system and the International Caries Detection and Assessment System (ICDAS), and evaluate the influence of professional experience on the management of the decay. Methods: There are 80 dentists in the public health service of Recife city, Brazil. All dentists were invited to participate in this cross-sectional study. Forty dentists agreed to answer questions about their decision-making in relation to the treatment of occlusal caries in low, moderate and high-risk caries situations. The time of clinical experience, the use of methods for the assessment of caries risk and the lesion activity were also inquired about. The T-test was applied with a significance level of 5 por ciento to compare the correlation between the International Caries Detection and Assessment System scores regarding the mean age of participants. Results: The disagreement between the dentists and the criteria used by the International Caries Detection and Assessment System for decision-making were mainly related to the sound surfaces or lesions restricted to the tooth enamel. It was thus verified that lack of consistency for the low-risk condition reached a score of 3 (32 percent), while moderate (95 percent) and high-risk (85 percent) conditions lay at a classification code of 0. The time of professional experience was not considered to have interfered with decision-making on any scale, regardless of the risk condition of the patient (p> 0.05). Conclusions: A divergence in terms of decision-making between dentists of the public health system and the International Caries Detection and Assessment System could be observed, specifically regarding sound surfaces or surfaces with lesions restricted to the tooth enamel. Furthermore, professional experience was seen as not influencing the management of caries(AU)

RESUMEN Introducción: Los errores de diagnóstico de caries se han convertido en un problema de salud pública. Objetivo: Verificar la concordancia de la toma de decisiones entre los dentistas brasileños del sistema de salud pública del país y el Sistema Internacional de Detección y Evaluación de Caries (ICDAS), así como evaluar la influencia de la experiencia profesional en el manejo de la caries. Métodos: De los 80 dentistas que existen en servicio público de salud en la ciudad de Recife, Brasil, todos fueron invitados a participar en este estudio transversal. Cuarenta dentistas acordaron responder sobre su toma de decisión en relación con el tratamiento de la caries oclusal en situaciones de caries de bajo, moderado y alto riesgo. El tiempo de experiencia clínica, el uso de métodos para la evaluación del riesgo de caries y la actividad de la lesión también fueron cuestionados. La prueba T, para un nivel de significancia del 5 por ciento, fue aplicada para comparar la correlación entre los escores del Sistema Internacional de Detección y Evaluación de Caries en relación con el promedio de edad de los participantes. Resultados: El desacuerdo entre los dentistas y los criterios utilizados por el ICDAS para la toma de decisiones se concentró en los códigos de clasificación que representan superficies sanas o lesiones restringidas al esmalte de los dientes. Se verificó así que la falta de consistencia para la condición de bajo riesgo se concentró en la puntuación de 3 (32 por ciento), mientras que las condiciones moderada (95 por ciento) y de alto riesgo (85 por ciento) se concentraron en los códigos de clasificación de 0. El factor tiempo de experiencia profesional no interfirió en la prevalencia de concordancia de la toma de decisión para cualquier escore, independientemente de la condición de riesgo del paciente (p> 0,05). Conclusiones: Se pudo observar una divergencia en cuanto a la toma de decisiones entre los dentistas del sistema de salud pública y el Sistema Internacional de Detección y Evaluación de Caries, específicamente con respecto a superficies sanas o superficies con lesiones restringidas al esmalte de los dientes. Además, la experiencia profesional no influyó en el manejo de la caries(AU)

Cortical Neurotoxic Astrocytes with Early ALS Pathology and miR-146a Deficit Replicate Gliosis Markers of Symptomatic SOD1G93A Mouse Model.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron (MN) loss. Recent evidences highlight astrocytes as important players in MN death, but the mechanism-based neurotoxicity is still unknown. It is also unclear whether activation of astrocytes in ALS occurs differently in the cerebral cortex and spinal cord. We investigated glial and neuronal alterations in the cortex of SOD1G93A (mSOD1) mice in pre-symptomatic and symptomatic stages. We also characterized astrocytes isolated from the cortex of 7-day-old mSOD1 mice for their aberrancy and MN-induced degenerative effects. In the early stage, we identified a reduction of cell proliferation, NF-kB expression, and of vimentin and micro(miR)-146a expression, suggesting a restrained cortical inflammatory status. However, increased NF-kB expression, cell proliferation, and gene expression of HMGB1, connexin 43 and S100B were distinctive of the symptomatic stage, together with MN loss, downregulated unfold protein response, and decreased expression of synaptic proteins, together with that of miR-125b, miR-21, miR-146a, GFAP, and glutamate transporters. Astrocytes cultured for 13 days in vitro showed comparable NF-kB expression and cell proliferation increase, as well as similar microRNA and gene/protein expression profiles (decreased miR-21, miR-146a, GLT-1 and GFAP, and upregulated HMGB1, S100B and connexin-43), thus sustaining astrocytes as the major contributors of cortical homeostasis deregulation in the symptomatic stage. These reactive astrocytes reduced neurite length and synaptophysin expression in NSC-34/hSOD1WT MN-like cells, and induced mitochondria dysfunction, PSD-95 downregulation, metalloproteinase-9 activation, and late apoptosis in NSC-34/hSOD1G93A cells. Data indicate that astrocytes in mSOD1 mice model acquire early phenotypic aberrancies and highlight downregulated miR-146a as a biomarker and drug target in ALS.

Downregulated Glia Interplay and Increased miRNA-155 as Promising Markers to Track ALS at an Early Stage.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of unknown cause. Absence of specific targets and biomarkers compromise the development of new therapeutic strategies and of innovative tools to stratify patients and assess their responses to treatment. Here, we investigate changes in neuroprotective-neuroinflammatory actions in the spinal cord of SOD1 G93A mice, at presymptomatic and symptomatic stages to identify stage-specific biomarkers and potential targets. Results showed that in the presymptomatic stage, there are alterations in both astrocytes and microglia, which comprise decreased expression of GFAP and S100B and upregulation of GLT-1, as well as reduced expression of CD11b, M2-phenotype markers, and a set of inflammatory mediators. Reduced levels of Connexin-43, Pannexin-1, CCL21, and CX3CL1 further indicate the existence of a compromised intercellular communication. In contrast, in the symptomatic stage, increased markers of inflammation became evident, such as NF-κB/Nlrp3-inflammasome, Iba1, pro-inflammatory cytokines, and M1-polarizion markers, together with a decreased expression of M2-phenotypic markers. We also observed upregulation of the CX3CL1-CX3CR1 axis, Connexin-43, Pannexin-1, and of microRNAs (miR)-124, miR-125b, miR-146a and miR-21. Reduced motor neuron number and presence of reactive astrocytes with decreased GFAP, GLT-1, and GLAST further characterized this inflammatory stage. Interestingly, upregulation of miR-155 and downregulation of MFG-E8 appear as consistent biomarkers of both presymptomatic and symptomatic stages. We hypothesize that downregulated cellular interplay at the early stages may represent neuroprotective mechanisms against inflammation, SOD1 aggregation, and ALS onset. The present study identified a set of inflamma-miRNAs, NLRP3-inflammasome, HMGB1, CX3CL1-CX3CR1, Connexin-43, and Pannexin-1 as emerging candidates and promising pharmacological targets that may represent potential neuroprotective strategies in ALS therapy.

Percepção de uma equipe de enfermagem sobre a utilização do checklist cirúrgico / Perception of a nursing team about the use of surgical checklist / Percepción del equipo de enfermería sobre la utilización de lista de verificacion quirúrgica

Objetivo: Conhecer a percepção de profissionais de enfermagem que atuam em centro cirúrgico em relação à utilização do checklist cirúrgico. Método: Estudo exploratório, qualitativo. Os dados foram coletados de março a abril de 2015, por meio de entrevista gravada, com roteiro semiestruturado contendo dez perguntas, analisada sob a ótica da análise temática. Resultados: Participaram do estudo 13 profissionais de enfermagem. Os resultados foram organizados em três categorias: gerenciamento de risco em centro cirúrgico: dificuldades conceituais e na prática de trabalho; checklist de cirurgia segura e sua contribuição na prática de trabalho; e potencialidades e fragilidades na utilização do checklist de cirurgia segura. Conclusão: Profissionais de enfermagem percebem a necessidade de garantir a segurança do paciente, apontam que protocolos contribuem para a qualidade da assistência e dos serviços. O checklist é a principal ferramenta utilizada pela equipe visando à redução de danos e eventos adversos.

Objective: To understand how nursing professionals who work in a surgical center, perceive the use of a surgical checklist. Method: Exploratory, qualitative study. The data were collected from March to April 2015, through recorded interviews with a semi-structured script containing ten questions. The data were analyzed by using thematic analyses. Results: The study included 13 nursing professionals. The results were organized into three categories: risk management in a surgical center: difficulties in concept and in work practice; checklist for safe surgery and its contribution to the working practice; strengths and weaknesses on using the checklist for safe surgery. Conclusion: Nursing professionals realize the need to ensure patient safety and think that protocols contribute to the quality of care and services. The checklist is the main tool used by the health team in order to reduce adverse events and damage.

Objetivo: Conocer la percepción de los profesionales de enfermería que trabajan en la sala de operaciones para el uso de la lista de verificación quirúrgica. Método: Estudio exploratorio, cualitativo. Se recogieron datos de marzo a abril de 2015 a través de entrevista grabada, semiestructurada que contiene diez preguntas. Los datos fueron analizados según el análisis temático. Resultados: Participaron del estudio 13 profesionales de enfermería. Los resultados fueron organizados en tres categorías: gestión de riesgos en el centro quirúrgico: dificultades conceptuales y prácticas del trabajo; lista de verificación para una cirugía segura y su contribución a la práctica del trabajo; potencialidades y debilidades de la lista de verificación de cirugía segura. Conclusión: Profesionales de enfermería reconocen la necesidad de garantizar la seguridad del paciente y señalan que los protocolos contribuyen a la calidad de la atención. La lista de verificación es la principal herramienta utilizada con el fin de reducir los eventos adversos y daños.

Exploring New Inflammatory Biomarkers and Pathways during LPS-Induced M1 Polarization.

Identification of mediators triggering microglia activation and transference of noncoding microRNA (miRNA) into exosomes are critical to dissect the mechanisms underlying neurodegeneration. We used lipopolysaccharide- (LPS-) induced N9 microglia activation to explore new biomarkers/signaling pathways and to identify inflammatory miRNA (inflamma-miR) in cells and their derived exosomes. Upregulation of iNOS and MHC-II (M1-markers) and downregulation of arginase 1, FIZZ1 (M2-markers), and CX3CR1 (M0/M2 polarization) confirmed the switch of N9 LPS-treated cells into the M1 phenotype, as described for macrophages/microglia. Cells showed increased proliferation, activated TLR4/TLR2/NF-κB pathway, and enhanced phagocytosis, further corroborated by upregulated MFG-E8. We found NLRP3-inflammasome activation in these cells, probably accounting for the increased extracellular content of the cytokine HMGB1 and of the MMP-9 we have observed. We demonstrate for the first time that the inflamma-miR profiling (upregulated miR-155 and miR-146a plus downregulated miR-124) in M1 polarized N9 cells, noticed by others in activated macrophages/microglia, was replicated in their derived exosomes, likely regulating the inflammatory response of recipient cells and dissemination processes. Data show that LPS-treated N9 cells behave like M1 polarized microglia/macrophages, while providing new targets for drug discovery. In particular, the study yields novel insights into the exosomal circulating miRNA during neuroinflammation important for emerging therapeutic approaches targeting microglia activation.

Glycoursodeoxycholic acid reduces matrix metalloproteinase-9 and caspase-9 activation in a cellular model of superoxide dismutase-1 neurodegeneration.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects mainly motor neurons (MNs). NSC-34 MN-like cells carrying the G93A mutation in human superoxide dismutase-1 (hSOD1(G93A)) are a common model to study the molecular mechanisms of neurodegeneration in ALS. Although the underlying pathways of MN failure still remain elusive, increased apoptosis and oxidative stress seem to be implicated. Riluzole, the only approved drug, only slightly delays ALS progression. Ursodeoxycholic acid (UDCA), as well as its glycine (glycoursodeoxycholic acid, GUDCA) and taurine (TUDCA) conjugated species, have shown therapeutic efficacy in neurodegenerative models and diseases. Pilot studies in ALS patients indicate safety and tolerability for UDCA oral administration. We explored the mechanisms associated with superoxide dismutase-1 (SOD1) accumulation and MN degeneration in NSC-34/hSOD1(G93A) cells differentiated for 4 days in vitro (DIV). We examined GUDCA efficacy in preventing such pathological events and in restoring MN functionality by incubating cells with 50 µM GUDCA at 0 DIV and at 2 DIV, respectively. Increased cytosolic SOD1 inclusions were observed in 4 DIV NSC-34/hSOD1(G93A) cells together with decreased mitochondria viability (1.2-fold, p < 0.01), caspase-9 activation (1.8-fold, p < 0.05), and apoptosis (2.1-fold, p < 0.01). GUDCA exerted preventive effects (p < 0.05) while also reduced caspase-9 levels when added at 2 DIV (p < 0.05). ATP depletion (2-fold, p < 0.05), increased nitrites (1.6-fold, p < 0.05) and metalloproteinase-9 (MMP-9) activation (1.8-fold, p < 0.05), but no changes in MMP-2, were observed in the extracellular media of 4 DIV NSC-34/hSOD1(G93A) cells. GUDCA inhibited nitrite production (p < 0.05) while simultaneously prevented and reverted MMP-9 activation (p < 0.05), but not ATP depletion. Data highlight caspase-9 and MMP-9 activation as key pathomechanisms in ALS and GUDCA as a promising therapeutic strategy for slowing disease onset and progression.

Directing mouse embryonic neurosphere differentiation toward an enriched neuronal population.

Neural stem cells (NSC) are self-renewing multipotent cells that have emerged as a powerful tool to repair the injured brain. These cells can be cultured as neurospheres, which are floating aggregates of neural stem/progenitor cells (NSPCs). Despite their high clonal expansion capacity, it has been suggested that in neurospheres, only a small percentage of cells are capable of proliferation and that this system is not efficient in terms of neurogenic competence. Thus, our aim was to develop a neurosphere culture method with a highly proliferative stem/progenitor cell population and particularly with a prominent neurogenic potential, surpassing some of the claimed weaknesses of the neurosphere assay. In our model, mouse neurospheres were harvested from neural tissue at E15 and after only 4 days in vitro (DIV), we have achieved highly proliferative primary neurospheres (81% Sox2 and 76% Ki67 positive cells) and a rather low number of cells expressing glial and neuronal markers (∼10%). After inducing differentiation, we have attained an enriched neuronal population (45% ß-III-tubulin positive cells at 15 DIV). Using a simple methodology, we have developed a NSPC model that can provide a valuable source of neuronal precursors, thus offering a potential starting point for cell replacement therapies following CNS injury.

Cross-talk between neurons and astrocytes in response to bilirubin: adverse secondary impacts.

Previous studies using monotypic nerve cell cultures have shown that bilirubin-induced neurological dysfunction (BIND) involves apoptosis and necrosis-like cell death, following neuritic atrophy and astrocyte activation,and that glycoursodeoxycholic acid (GUDCA) has therapeutic efficacy against BIND. Cross-talk between neurons and astrocytes may protect or aggravate neurotoxicity by unconjugated bilirubin (UCB). In a previous work we have shown that bidirectional signaling during astrocyte-neuron recognition attenuates neuronal damage by UCB. Here, we investigated whether the establishment of neuron-astrocyte homeostasis prior to cell exposure to UCB was instead associated with a lower resistance of neurons to UCB toxicity, and if the pro-survival properties of GUDCA were replicated in that experimental model. We have introduced a 24 h adaptation period for neuron-glia communication prior to the 48 h treatment with UCB. In such conditions, UCB induced glial activation, which aggravated neuronal damage, comprising increased apoptosis,cell demise and neuritic atrophy, which were completely prevented in the presence of GUDCA. Neuronal multidrug resistance-associated protein 1 expression and tumor necrosis factor-a secretion, although unchanged by UCB, increased in the presence of astrocytes. The rise in S100B and nitric oxide in the co-cultures medium may have contributed to UCB neurotoxicity. Since the levels of these diffusible molecules did not change by GUDCA we may assume that they are not directly involved in its beneficial effects. Data indicate that astrocytes, in an indirect neuron-astrocyte co-culture model and after homeostatic setting regulation of the system, are critically influencing neurodegeneration by UCB, and support GUDCA for the prevention of BIND.