Analysis of transient phosphorylation-dependent protein-protein interactions in living mammalian cells using split-TEV

Abstract Background Regulated protein-protein interactions (PPIs) are pivotal molecular switches that are important for the regulation of signaling processes within eukaryotic cells. Cellular signaling is altered in various disease conditions and offers interesting options for pharmacological interventions. Constitutive PPIs are usually mediated by large interaction domains. In contrast, stimulus-regulated PPIs often depend on small post-translational modifications and are thus better suited targets for drug development. However, the detection of modification-dependent PPIs with biochemical methods still remains a labour- and material-intensive task, and many pivotal PPIs that are potentially suited for pharmacological intervention most likely remain to be identified. The availability of methods to easily identify and quantify stimulus-dependent, potentially also transient interaction events, is therefore essential. The assays should be applicable to intact mammalian cells, optimally also to primary cells in culture. Results In this study, we adapted the split-TEV system to quantify phosphorylation-dependent and transient PPIs that occur at the membrane and in the cytosol of living mammalian cells. Split-TEV is based on a PPI-induced functional complementation of two inactive TEV protease fragments fused to interaction partners of choice. Genetically encoded transcription-coupled and proteolysis-only TEV reporter systems were used to convert the TEV activity into an easily quantifiable readout. We measured the phosphorylation-dependent interaction between the pro-apoptotic protein Bad and the adapter proteins 14-3-3ε and ζ in NIH-3T3 fibroblasts and in primary cultured neurons. Using split-TEV assays, we show that Bad specifically interacts with 14-3-3 isoforms when phosphorylated by protein kinase Akt-1/PKB at Ser136. We also measured the phosphorylation-dependent Bad/14-3-3 interactions mediated by endogenous and transient Akt-1 activity. We furthermore applied split-TEV assays to measure the phosphorylation-dependent interactions of Neuregulin-1-stimulated ErbB4 receptors with several adapter proteins. Conclusion Split-TEV assays are well suited to measure phosphorylation-dependent and transient PPIs that occur specifically at the membrane and in the cytosol of heterologous and primary cultured mammalian cells. Given the high sensitivity of the split-TEV system, all assays were performed in multi-plate formats and could be adapted for higher throughput to screen for pharmacologically active substances.</p

Bienestar animal aplicado a la producción bovina

La preocupación acerca del bienestar animal ha ido en aumento, particularmente por la presión del público consumidor. Se entiende por bienestar al estado del individuo en relación a sus intentos de adaptarse al ambiente en un momento determinado. Al ser un concepto con bases científicas debe ser medido objetivamente. Por otra parte, la definición tiene algunas implicancias como la de ser una característica del animal, en lugar de algo que se le pueda brindar, así como variar dentro de un rango –desde muy bueno a muy malo– siendo malo cuando existen dificultades para mantener el control de las funciones o fallas en las tentativas de adaptarse al ambiente. En la cría y producción de bovinos existe una opinión generalizada que los sistemas extensivos son siempre buenos, porque mantienen a los animales lo más cerca posible del estado natural y que los sistemas intensivos son malos porque las libertades de elección y movimientos están restringidas. No obstante, en cualquier sistema productivo, extensivo o intensivo, pueden existir fracasos en la adaptación y consecuente estrés. Actualmente algunas organizaciones internacionales se han planteado como objetivo lograr que las nuevas tecnologías mejoren el bienestar sin producir mermas en la producción. Esto se puede alcanzar mediante el reconocimiento de los comportamientos naturales del bovino, teniendo en cuenta sus señales visuales, auditivas y olfativas, bases del manejo racional de los animales. Esta práctica no solo permite mejorar la relación humano–animal disminuyendo los signos de estrés, sino que también proporciona beneficios prácticos y económicos, generando efectos psicosociales positivos para el bienestar humano.

The multispecific thyroid hormone transporter OATP1C1 mediates cell-specific sulforhodamine 101-labeling of hippocampal astrocytes

Sulforhodamine 101 (SR101) is widely used for astrocyte identification, though the labeling mechanism remains unknown and the efficacy of labeling in different brain regions is heterogeneous. By combining region-specific isolation of astrocytes followed by transcriptome analysis, two-photon excitation microscopy, and mouse genetics, we identified the thyroid hormone transporter OATP1C1 as the SR101-uptake transporter in hippocampus and cortex. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00429-013-0645-0) contains supplementary material, which is available to authorized users

Problemas de agresividad canina. Estudio preliminar en las ciudades de Corrientes y Resistencia

La agresión canina es un problema de comportamiento que supone un riesgo potencial para la sociedad. En este estudio se evaluó el comportamiento de 120 perros mediante el “Cuestionario de Evaluación e Investigación del Comportamiento de los Caninos” (C-BARQ) con el objeto de evidenciar la presencia de conductas agresivas en perros cuyos propietarios acudieron a la consulta clínica privada por causas ajenas a este problema, en las ciudades de Corrientes y Resistencia (Argentina). Del análisis descriptivo de los datos se pudo apreciar que un 55% de los perros mostró agresión hacia los propietarios. Si bien la frecuencia promedio no fue alta, la presencia de conducta agresiva implica un riesgo potencial para las personas. Además, se debe tener en cuenta que ninguno de los propietarios de los canes acudió a la clínica veterinaria para consultar por un problema asociado al comportamiento. Se observa que los dueños de los perros no consideran importantes a las señales sutiles de agresión, ignorando el peligro que podrían significar. Ello impone la necesidad de continuar realizando estudios sobre el tema y de analizar una forma efectiva de educación e información

Expression of Lineage Transcription Factors Identifies Differences in Transition States of Induced Human Oligodendrocyte Differentiation

Oligodendrocytes (OLs) are critical for myelination and are implicated in several brain disorders. Directed differentiation of human-induced OLs (iOLs) from pluripotent stem cells can be achieved by forced expression of different combinations of the transcription factors SOX10 (S), OLIG2 (O), and NKX6.2 (N). Here, we applied quantitative image analysis and single-cell transcriptomics to compare different transcription factor (TF) combinations for their efficacy towards robust OL lineage conversion. Compared with S alone, the combination of SON increases the number of iOLs and generates iOLs with a more complex morphology and higher expression levels of myelin-marker genes. RNA velocity analysis of individual cells reveals that S generates a population of oligodendrocyte-precursor cells (OPCs) that appear to be more immature than those generated by SON and to display distinct molecular properties. Our work highlights that TFs for generating iOPCs or iOLs should be chosen depending on the intended application or research question, and that SON might be beneficial to study more mature iOLs while S might be better suited to investigate iOPC biology

Sharp-1 regulates TGF-β signaling and skeletal muscle regeneration

10.1242/jcs.136648Journal of Cell Science1273599-608JNCS

Twelve experiments in restorative justice: the Jerry Lee program of randomized trials of restorative justice conferences

Objectives: We conducted and measured outcomes from the Jerry Lee Program of 12 randomized trials over two decades in Australia and the United Kingdom (UK), testing an identical method of restorative justice taught by the same trainers to hundreds of police officers and others who delivered it to 2231 offenders and 1179 victims in 1995–2004. The article provides a review of the scientific progress and policy effects of the program, as described in 75 publications and papers arising from it, including previously unpublished results of our ongoing analyses. Methods: After random assignment in four Australian tests diverting criminal or juvenile cases from prosecution to restorative justice conferences (RJCs), and eight UK tests of supplementing criminal or juvenile proceedings with RJCs, we followed intention-to-treat group differences between offenders for up to 18 years, and for victims up to 10 years. Results: We distil and modify prior research reports into 18 updated evidence-based conclusions about the effects of RJCs on both victims and offenders. Initial reductions in repeat offending among offenders assigned to RJCs (compared to controls) were found in 10 of our 12 tests. Nine of the ten successes were for crimes with personal victims who participated in the RJCs, with clear benefits in both short- and long-term measures, including less prevalence of post-traumatic stress symptoms. Moderator effects across and within experiments showed that RJCs work best for the most frequent and serious offenders for repeat offending outcomes, with other clear moderator effects for poly-drug use and offense seriousness. Conclusions: RJ conferences organized and led (most often) by specially-trained police produced substantial short-term, and some long-term, benefits for both crime victims and their offenders, across a range of offense types and stages of the criminal justice processes on two continents, but with important moderator effects. These conclusions are made possible by testing a new kind of justice on a programmatic basis that would allow prospective meta-analysis, rather than doing one experiment at a time. This finding provides evidence that funding agencies could get far more evidence for the same cost from programs of identical, but multiple, RCTs of the identical innovative methods, rather than funding one RCT at a time