ABSTRACT
The first event of cellular differentiation consists of the segregation of the trophectoderm and the inner cell mass. Studies in mice suggest that cell contractility and the formation of an apical domain play important roles in this event; however, this remains unknown in the bovine. We tested the hypothesis that blocking apical domain formation would halt subsequent trophectoderm differentiation in bovine embryos. We first assessed the formation of an apical domain by the presence of Par-6 Family Cell Polarity Regulator Beta (PARD6B) and Ezrin (EZR), which appeared after the 8-cell stage. We inhibited apical domain formation by blocking cell contractility with 25µM (-)-blebbistatin. Treatment from 90 to 186h after insemination did not reduce blastocyst development compared with the untreated control group or the group treated with inactive (+)-blebbistatin. Immunofluorescence staining after blebbistatin treatment revealed the absence of EZR and the trophectoderm marker Caudal Type Homeobox 2 (CDX2). Following blebbistatin treatment, Yes1 Associated Transcriptional Regulator (YAP), which is involved in the Hippo signalling pathway, exhibited cytoplasmic staining instead of nuclear localisation. Despite changes in protein expression and localisation, no difference in trophectoderm or total cell numbers was observed. In conclusion, inhibition of cell contractility inhibited apical domain formation without impairing blastocyst formation, suggesting that a different biological mechanism is involved in trophectoderm and inner cell mass differentiation in bovine embryos.
ABSTRACT
OBJECTIVES: The aims of this study were (1) to identify attributes for patient safety at a primary healthcare level and (2) to analyze conceptions of patients, professionals, and managers about how these attributes are being addressed. STUDY DESIGN: This was a qualitative study. METHODS: Participants were recruited from three primary care settings in Brazil. A total of 37 subjects (four physicians, three nurses, three dentists, three managers, five community assistants, and 19 patients) participated on interviews about their perceptions of safety attributes at the primary care settings involved in the study. Some of these participants attended a focus group meeting. A thematic categorical analysis was carried out to interpret the interviews. RESULTS: The main attributes for patient safety were valued by the participants. However, barriers such as discontinuity of care, interruptions during consultations, breakdowns in the communication, and ineffective teamwork were reported as frequent sources of patient safety issues. Reports of patients left unattended for excessive time because of the lack of accurate information and disruptions that took up to 35 min show that there is still a long way to go for primary care to be safe and effective in the study settings. CONCLUSIONS: It is necessary that the strategies meet the patient safety needs more effectively and efficiently. Further research is needed to understand the complex nature of the problems that affect patient safety in these settings so that appropriate decisions can be made.
Subject(s)
Needs Assessment , Patient Safety , Primary Health Care/organization & administration , Administrative Personnel/psychology , Administrative Personnel/statistics & numerical data , Adult , Brazil , Communication , Female , Health Personnel/psychology , Health Personnel/statistics & numerical data , Humans , Interprofessional Relations , Male , Middle Aged , Qualitative Research , Young AdultABSTRACT
El tejido adiposo obtenido mediante liposucción es una fuente idónea para aislar células con potencial terapéutico, las denominadas células de la fracción estromal vascular (FEV), que incluyen células madre mesenquimales. Estas células se han convertido en una de las principales herramientas de terapia celular autóloga para diversas aplicaciones médicas, y en los últimos años se han ido desarrollando diversas tecnologías para su aislamiento y uso clínico. En este trabajo presentamos un nuevo método rápido, sencillo y eficiente para el aislamiento de células de la FEV mediante un dispositivo médico cerrado que permite recoger y procesar lipoaspirados humanos en el mismo procedimiento quirúrgico de manera coste-efectiva. Además describimos los métodos llevados a cabo para cuantificar la calidad, seguridad y eficacia del inóculo celular obtenido (AU)
The adipose tissue obtained by liposuction is an abundant source to isolate regenerative cells, the stromal vascular fractions cells (SVF), including adipose mesenchymal stem cells. These cells have became an excellent tool for autologous cell therapy in different clinical applications, and several platforms for adipose tissue processing have been developed for SVF isolation. In this study we describe a new medical device for SVF isolation from human lipoaspirates, using a fast, efficient and user-friendly procedure in the same surgical act in a cost-effective way. Additionally, we present different methods to guarantee the quality, safety and potency of the cell inoculum obtained (AU)
Subject(s)
Humans , Lipectomy/methods , Stromal Cells/ultrastructure , Adipose Tissue/ultrastructure , Stem Cells/ultrastructure , Transplantation, Autologous/methods , Endotoxins/isolation & purificationABSTRACT
AIMS: This study has investigated how global brain ischaemia/reperfusion (I/R) modifies levels of mRNAs encoding γ-aminobutyric acid type A (GABA(A)) receptor α1, ß2 and γ2 subunits and glutamic acid decarboxylase 65 (GAD65) in an age- and structure-dependent manner. Gene expression in response to treatment with the anti-inflammatory agent meloxicam was also investigated. METHODS: Global ischaemia was induced in 3- and 18-month-old male Sprague-Dawley rats. CA1, CA3, and dentate gyrus (DG) hippocampal areas, cerebral cortex (CC) and caudate putamen (C-Pu) from sham-operated and I/R-injured animals were excised 48 h after the insult and prepared for quantitative polymerase chain reaction assays. Following I/R, meloxicam treatment was also carried out on young animals. RESULTS: Data revealed significant decreases in the levels of all GABA(A) receptor subunit transcripts in the hippocampus of both young and older injured animals compared with sham-operated ones. In contrast, there was either an increase or no change in GAD65 mRNA levels. GABA(A) receptor subunit transcript decreases were also observed in the CC and C-Pu in young injured animals but not in the CC of the older injured ones; interestingly, significant increases were observed in the C-Pu of older injured animals compared with controls. Meloxicam treatment following the insult resulted in a diminution of the previously described I/R response. CONCLUSIONS: The data indicate that I/R results in the modification of the levels of several gene transcripts involved in GABAergic signalling in both the pre- and postsynaptic components, of this neurotransmitter system, in an age- and structure-dependent manner.
Subject(s)
Aging/physiology , Hippocampus/metabolism , Ischemic Attack, Transient/genetics , RNA, Messenger/genetics , Receptors, GABA-A/genetics , Age Factors , Animals , Gene Expression/genetics , Hippocampus/pathology , Male , Meloxicam , Protein Subunits/genetics , Protein Subunits/metabolism , Rats , Rats, Sprague-Dawley , Receptors, GABA-A/metabolism , Thiazines/pharmacology , Thiazoles/pharmacologyABSTRACT
AIM: Stroke prevalence increases with age, while alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) and inflammation have been related to ischaemia-induced damage. This study shows how age and treatment with an anti-inflammatory agent (meloxicam) modify the levels of AMPAR subunits GluR1 and GluR2, as well as the mRNA levels of the GluR2-editing enzyme, ADAR2, in a global brain ischaemia/reperfusion (I/R) model. METHODS: Two days after global ischaemia CA1, CA3, dentate gyrus and cerebral cortex were obtained from sham-operated and I/R-injured 3- and 18-month-old Sprague-Dawley rats. Real time polymerase chain reaction, Western blotting and immunohistochemical assays were performed. Meloxicam treatment was assayed on young animals. RESULTS: Data showed that age attenuates the downregulation induced by I/R in the AMPAR subunits GluR1 and GluR2 and modifies the GluR1/GluR2 mRNA level ratio in a structure-dependent way. The study of the ADAR2 mRNA levels showed more downregulation in older animals than young ones. Meloxicam treatment prevented the transcriptional arrest induced by I/R. CONCLUSION: Our data suggest that changes in the AMPAR isoforms could be associated with ageing in the different structures studied. Although GluR2 editing seems to be involved in age-dependent vulnerability to ischaemia supporting the 'GluR2 hypothesis', this alone does not explain the differential vulnerability in the different brain regions. Finally, inflammation could play a role in protection from I/R-induced injury.
Subject(s)
Aging/physiology , Receptors, AMPA/metabolism , Reperfusion Injury , Thiazines/pharmacology , Thiazoles/pharmacology , Adenosine Deaminase/genetics , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Biomarkers/metabolism , CD11b Antigen/metabolism , Down-Regulation/drug effects , Down-Regulation/physiology , Gene Expression/drug effects , Male , Meloxicam , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptors, AMPA/genetics , Reperfusion Injury/drug therapy , Reperfusion Injury/immunology , Reperfusion Injury/metabolism , Stroke/drug therapy , Stroke/immunology , Stroke/metabolismABSTRACT
Glutamatergic N-methyl-d-aspartate NMDA receptors (NMDAR) are considered to play a key role in ischemia-induced damage. Long-term (hours) changes in their expression upon ischemia have been shown. Here we report short-term changes in the mRNA levels of the major hippocampal NMDAR subunits (NR1, NR2A and NR2B), as well as c-fos, in an ex vivo ischemia model using hippocampal slices. This effect can be observed also in a calcium free incubation solution. Striking early decreases in the NMDAR subunit mRNA levels were observed after 30 min of oxygen and glucose deprivation (OGD) as well as a partial recovery when the tissues were returned to the balanced salt solution (reperfusion-like period) for 3 h. Since OGD-induced damage has been reported to be a consequence of the increase in OGD-related glutamate release, we also analyzed NMDAR mRNA levels following increased glutamate levels in hippocampal sections in which no significant effects on NMDAR subunit mRNA levels were detected. Furthermore, we describe that the presence of MK-801 (a selective NMDAR antagonist), CNQX (a selective AMPA/kainate receptor antagonist) or their combined action in the incubation solution is able to induce a significant decrease in NMDAR expression but in these conditions the OGD does not induce further decreases in mRNA levels. We suggest that the mechanisms triggered during OGD to downregulate mRNA levels of NMDAR subunits could be the same than those induced by glutamate receptor antagonists.