A versatile toolkit to produce sensitive FRET biosensors to visualize signaling in time and space

Genetically encoded, ratiometric biosensors based on fluorescence resonance energy transfer (FRET) are powerful tools to study the spatiotemporal dynamics of cell signaling. However, many biosensors lack sensitivity. We present a biosensor library that contains circularly permutated mutants for both the donor and acceptor fluorophores, which alter the orientation of the dipoles and thus better accommodate structural constraints imposed by different signaling molecules while maintaining FRET efficiency. Our strategy improved the brightness and dynamic range of preexisting RhoA and extracellular signal-regulated protein kinase (ERK) biosensors. Using the improved RhoA biosensor, we found micrometer-sized zones of RhoA activity at the tip of F-actin bundles in growth cone filopodia during neurite extension, whereas RhoA was globally activated throughout collapsing growth cones. RhoA was also activated in filopodia and protruding membranes at the leading edge of motile fibroblasts. Using the improved ERK biosensor, we simultaneously measured ERK activation dynamics in multiple cells using low-magnification microscopy and performed in vivo FRET imaging in zebrafish. Thus, we provide a construction toolkit consisting of a vector set, which enables facile generation of sensitive biosensors.

Two distinct filopodia populations at the growth cone allow to sense nanotopographical extracellular matrix cues to guide neurite outgrowth

BACKGROUND The process of neurite outgrowth is the initial step in producing the neuronal processes that wire the brain. Current models about neurite outgrowth have been derived from classic two-dimensional (2D) cell culture systems, which do not recapitulate the topographical cues that are present in the extracellular matrix (ECM) in vivo. Here, we explore how ECM nanotopography influences neurite outgrowth. METHODOLOGY/PRINCIPAL FINDINGS We show that, when the ECM protein laminin is presented on a line pattern with nanometric size features, it leads to orientation of neurite outgrowth along the line pattern. This is also coupled with a robust increase in neurite length. The sensing mechanism that allows neurite orientation occurs through a highly stereotypical growth cone behavior involving two filopodia populations. Non-aligned filopodia on the distal part of the growth cone scan the pattern in a lateral back and forth motion and are highly unstable. Filopodia at the growth cone tip align with the line substrate, are stabilized by an F-actin rich cytoskeleton and enable steady neurite extension. This stabilization event most likely occurs by integration of signals emanating from non-aligned and aligned filopodia which sense different extent of adhesion surface on the line pattern. In contrast, on the 2D substrate only unstable filopodia are observed at the growth cone, leading to frequent neurite collapse events and less efficient outgrowth. CONCLUSIONS/SIGNIFICANCE We propose that a constant crosstalk between both filopodia populations allows stochastic sensing of nanotopographical ECM cues, leading to oriented and steady neurite outgrowth. Our work provides insight in how neuronal growth cones can sense geometric ECM cues. This has not been accessible previously using routine 2D culture systems.

Performance Evaluation of the New Connecticut Leading Employment Index Using Lead Profiles and BVAR Models

Dua and Miller (1996) created leading and coincident employment indexes for the state of Connecticut, following Moore's (1981) work at the national level. The performance of the Dua-Miller indexes following the recession of the early 1990s fell short of expectations. This paper performs two tasks. First, it describes the process of revising the Connecticut Coincident and Leading Employment Indexes. Second, it analyzes the statistical properties and performance of the new indexes by comparing the lead profiles of the new and old indexes as well as their out-of-sample forecasting performance, using the Bayesian Vector Autoregressive (BVAR) method. The new indexes show improved performance in dating employment cycle chronologies. The lead profile test demonstrates that superiority in a rigorous, non-parametric statistic fashion. The mixed evidence on the BVAR forecasting experiments illustrates the truth in the Granger and Newbold (1986) caution that leading indexes properly predict cycle turning points and do not necessarily provide accurate forecasts except at turning points, a view that our results support.

Diversity of neuronal connexins in the mammalian retina

Many neurons in the mammalian retina are electrically coupled by intercellular channels or gap junctions, which are assembled from a family of proteins called connexins. Numerous studies indicate that gap junctions differ in properties such as conductance and tracer permeability. For example, A-type horizontal cell gap junctions are permeable to Lucifer Yellow, but B-type horizontal cell gap junctions are not. This suggests the two cell types express different connexins. My hypothesis is that multiple neuronal connexins are expressed in the mammalian retina in a cell type specific manner. Immunohistochemical techniques and confocal microscopy were used to localize certain connexins within well-defined neuronal circuits. The results of this study can be summarized as follows: AII amacrine cells, which receive direct input from rod bipolar cells, are well-coupled to neighboring AIIs. In addition, AII amacrine cells also form gap junctions with ON cone bipolar cells. This is a complex heterocellular network. In both rabbit and primate retina, connexin36 occurs at dendritic crossings in the AII matrix as well as between AIIs and ON cone bipolar cells. Coupling in the AII network is thought to reduce noise in the rod pathway while AII/bipolar gap junctions are required for the transmission of rod signals to ON ganglion cells. In the outer plexiform layer, connexin36 forms gap junctions between cones and between rods and cones via cone telodendria. Cone to cone coupling is thought to reduce noise and is partly color selective. Rod to cone coupling forms an alternative rod pathway thought to operate at intermediate light intensity. A-type horizontal cells in the rabbit retina are strongly coupled via massive low resistance gap junctions composed from Cx50. Coupling dramatically extends the receptive field of horizontal cells and the modulation of coupling is thought to change the strength of the feedback signal from horizontal cells to cones. Finally, there are other coupled networks, such as B-type horizontal cells and S1/S2 amacrine cells, which do not use either connexin36 or Cx50. These results confirm the hypothesis that multiple neuronal connexins are expressed in the mammalian retina and these connexins are localized to particular retinal circuits. ^

Functional architecture of mammalian horizontal cells

In the rabbit retina, there are two kinds of horizontal cells (HCs). The A-type HC is a large axonless cell which contacts cones exclusively. The B-type HC is an axon bearing cell. While the somatic dendrites of B-type HCs also contact cones, the axon expands into an elaborately branched structure, the axon terminal (AT), which contacts a large number of rods. It is difficult to label the different HCs selectively by immunochemical methods. Therefore, we developed dye injection methods to label each type of HC. Then it was possible, (1) to describe the detailed structure of the AT (2) to identify the glutamate receptors mediating cone input to A and B-type HCs and rod input to ATs and (3) to test the hypothesis that the B-type HCs are coupled via Cx57 gap junctions. ^ To obtain well filled examples of single HCs, it was necessary to block gap junction coupling to stop the spread of Neurobiotin through the network. We used dye coupling in A-type HCs to screen a series of potential gap junction antagonists. One of these compounds, meclofenamic acid (MFA), was potent, water soluble and easily reversible. This compound may be a useful tool to manipulate gap junction coupling. ^ In the presence of MFA, Neurobiotin passed down the axon of B-type HCs to reveal the detailed structure of the AT. We observed that only one AT ending entered each rod spherule invagination. This observation was confirmed by calculation and two dye injections. ^ Glutamate is the neurotransmitter used by both rods and cones. AMPA receptors were colocalized with the dendrites of A and B-type HCs at each cone pedicle. In addition, AMPA receptors were located on the AT ending at each rod spherule. Thus rod and cone input to HCs is mediated by AMPA receptors. ^ A-type and B-type HCs may express different connexins because they have different dye-coupling properties. Recently, we found that connexin50 (Cx50) is expressed by A-type HCs. B-type HCs and B-type ATs are also independently coupled. Cx57 was expressed in the OPL and double label studies showed that Cx 57 was colocalized with the AT matrix but not with the somatic dendrites of B-type HCs. ^ In summary, we have identified a useful gap junction antagonist, MFA. There is one AT ending at each rod spherule, rods inputs to ATs is mediated by AMPA receptors and coupling in the AT matrix is mediated by Cx57. This confirms that HCs with different properties use distinct connexins. The properties of ATs described in this research are consistent. The connections and properties reported here suggest that ATs functions as rod HCs and provide a negative feedback signal to rods. ^

Evaluación de la contaminación de acuíferos producida por actividades de saneamiento y re-uso de efluentes en el norte de la provincia de Mendoza

En la zona norte de la provincia de Mendoza se desarrollan actividades que pueden afectar la calidad natural del agua subterránea: disposición y re-uso de efluentes industriales para riego agrícola, utilización de fertilizantes, saneamiento in-situ, fugas de redes de alcantarillado, etc. En esta región, surcada superficialmente por los ríos Mendoza y Tunuyán, la sedimentación cuaternaria determinó la formación de dos grandes unidades hidrogeológicas: acuíferos libres (sector de conos aluviales), y acuífero freático superior y acuíferos subyacentes confinados y/o semiconfinados. El área de estudio se encuentra ubicada en esta última unidad hidrogeológica donde se ha detectado contaminación de acuíferos por nitratos. El objetivo de este trabajo es identificar el origen de la contaminación, utilizando metodología hidroquímica mediante la evaluación de diversos parámetros físicoquímicos y biológicos, y técnicas isotópicas para corroborar la procedencia del agua subterránea y el origen de los nitratos. Los resultados obtenidos muestran que la presencia de nitratos en los acuíferos semiconfinado y confinado no proviene de la influencia del acuífero libre suprayacente, afectado por el re-uso de efluentes, sino que se relaciona con el ingreso de flujo horizontal de aguas subterráneas contaminadas provenientes del área del Gran Mendoza, debido a las pérdidas en las redes de alcantarillado y obras de saneamiento in situ.

Actividad industrial y crisis del régimen de convertibilidad en el Gran La Plata : Estudio de una fábrica recuperada

En el Gran La Plata las políticas económicas implementadas durante el régimen de convertibilidad profundizaron la desindustrialización y reestructuración de los años 70 y provocaron un achicamiento general del sector industrial y una fuerte expulsión de mano de obra y precarización laboral. La recesión y crisis del régimen de convertibilidad incrementaron el deterioro de la actividad económica y el quiebre de empresas, ante lo cual trabajadores de algunas industrias de la región decidieron impedir el cierre de las mismas permaneciendo en los establecimientos para evitar el desempleo. La situación más significativa es la de la actual Cooperativa Unión Papelera Platense la que, una vez lograda la compra de la empresa luego de su recuperación, orientó su estrategia hacia su consolidación y a enfrentar las problemáticas que afectan al conjunto de las pymes industriales: sostenimiento en el mercado, dificultades de financiamiento, incremento de los costos internos

Dinámica macroeconómica, mercado de trabajo y distribución del ingreso en el período 2003-2005

El período post-devaluación 2003-2005 presenta una combinación de cambios y continuidades en la dinámica socioeconómica y territorial con respecto a la fase de recesión y crisis del Régimen de Convertibilidad. Se registra una significativa recuperación de la actividad socioeconómica en diversos sectores, ramas y empresas que no se traduce con el mismo vigor en los indicadores sociales sobre mercado de trabajo y pobreza, o incluso sin que se produzcan modificaciones sustantivas en los referidos a distribución del ingreso. En el territorio argentino estos procesos se reflejan a través de una desigual distribución de las inversiones y de la captación de los beneficios que el crecimiento económico genera. El presente artículo tiene como objetivos caracterizar este período a partir de un análisis de las principales variables socioeconómicas, presentar los principales debates en torno a los cambios y continuidades respecto del régimen de convertibilidad y dar cuenta de sus vinculaciones con la dinámica territorial.

Actividad industrial y crisis del régimen de convertibilidad en el Gran La Plata : Estudio de una fábrica recuperada

En el Gran La Plata las políticas económicas implementadas durante el régimen de convertibilidad profundizaron la desindustrialización y reestructuración de los años 70 y provocaron un achicamiento general del sector industrial y una fuerte expulsión de mano de obra y precarización laboral. La recesión y crisis del régimen de convertibilidad incrementaron el deterioro de la actividad económica y el quiebre de empresas, ante lo cual trabajadores de algunas industrias de la región decidieron impedir el cierre de las mismas permaneciendo en los establecimientos para evitar el desempleo. La situación más significativa es la de la actual Cooperativa Unión Papelera Platense la que, una vez lograda la compra de la empresa luego de su recuperación, orientó su estrategia hacia su consolidación y a enfrentar las problemáticas que afectan al conjunto de las pymes industriales: sostenimiento en el mercado, dificultades de financiamiento, incremento de los costos internos

Dinámica macroeconómica, mercado de trabajo y distribución del ingreso en el período 2003-2005

El período post-devaluación 2003-2005 presenta una combinación de cambios y continuidades en la dinámica socioeconómica y territorial con respecto a la fase de recesión y crisis del Régimen de Convertibilidad. Se registra una significativa recuperación de la actividad socioeconómica en diversos sectores, ramas y empresas que no se traduce con el mismo vigor en los indicadores sociales sobre mercado de trabajo y pobreza, o incluso sin que se produzcan modificaciones sustantivas en los referidos a distribución del ingreso. En el territorio argentino estos procesos se reflejan a través de una desigual distribución de las inversiones y de la captación de los beneficios que el crecimiento económico genera. El presente artículo tiene como objetivos caracterizar este período a partir de un análisis de las principales variables socioeconómicas, presentar los principales debates en torno a los cambios y continuidades respecto del régimen de convertibilidad y dar cuenta de sus vinculaciones con la dinámica territorial.

Dinámica macroeconómica, mercado de trabajo y distribución del ingreso en el período 2003-2005

El período post-devaluación 2003-2005 presenta una combinación de cambios y continuidades en la dinámica socioeconómica y territorial con respecto a la fase de recesión y crisis del Régimen de Convertibilidad. Se registra una significativa recuperación de la actividad socioeconómica en diversos sectores, ramas y empresas que no se traduce con el mismo vigor en los indicadores sociales sobre mercado de trabajo y pobreza, o incluso sin que se produzcan modificaciones sustantivas en los referidos a distribución del ingreso. En el territorio argentino estos procesos se reflejan a través de una desigual distribución de las inversiones y de la captación de los beneficios que el crecimiento económico genera. El presente artículo tiene como objetivos caracterizar este período a partir de un análisis de las principales variables socioeconómicas, presentar los principales debates en torno a los cambios y continuidades respecto del régimen de convertibilidad y dar cuenta de sus vinculaciones con la dinámica territorial.

Pollen and macrofossils profiles and age determination from three cores in Northeastern Kansas, U.S.A.

Two marshes near Muscotah and Arrington, Atchison County, northeastern Kansas, yielded a pollen sequence covering the last 25,000 yrs of vegetation development. The earliest pollen spectra are comparable with surface pollen spectra from southern Saskatchewan and southeastern Manitoba and might indicate a rather open vegetation but with some pine, spruce, and birch as the most important tree species, with local stands of alder and willow. This type of vegetation changed about 23,000 yrs ago to a spruce forest, which prevailed in the region until at least 15,000 yrs ago. Because of a hiatus, the vegetation changes resulting in the spread of a mixed deciduous forest and prairie, which was present in the region from 11,000 to 9,000 yrs ago, remain unknown. Prairie vegetation, with perhaps a few trees along the valleys, covered the region until about 5,000 yrs ago, when a re-expansion of deciduous trees began in the lowlands.

Comparison of accumulation rates and stable isotope ratios

Since 1979/80, glaciological studies have been carried out at Ekströmisen, Antarctica, including accumulation-stake measurements, snow-pit and shallow-firn-core studies. Snowstratigraphy, chemical properties and stable-isotope ratios (d18O) were investigated. This study focuses on three cores taken between 1982 and 1998. The 1998 core was dated using dielectric profiling, d18O profiles and stake measurements. Accumulation rates showhigh interannual and spatial variability due to the extreme wind influence. No significant trend was found for the last 50 years; during the first half of the 20th century, accumulation decreased. The high spatial and interannual variability, however, means that trends must be interpreted with care. In spite of the highly irregular accumulation distribution, stable-isotope ratios show little spatial variability. The mean annual d18O values of cores B04 and FB0198 agree fairly well for the time period 1955-82 covered by both cores. d18O values have increased during most of the 20th century; since the late 1980s a decrease is observed. This change is not related to air temperature, since mean annual air temperatures at Neumayer show no significant trend over the last two decades.