Perfusion based microfluidic system for pharmacological profiling of neuronal networks

This work presents the integration of a semi-automated microfluidic platform that utilizes calcium imaging to enable the pharmacological characterization of functionally connected, but environmentally isolated neuronal networks. This approach allows, for the first time, to assess the cause-effect relationship of neuronal communication following drug application, thus allowing the pharmacological characterisation of novel drugs proposed to influence communication between neuronal networks

A 340/380 nm light emitting diode illuminator for Fura-2 AM ratiometric Ca2+ imaging of live cells with better than 5 nM precision

We report the first demonstration of a fast wavelength-switchable 340/380 nm light emitting diode (LED) illuminator for Fura-2 ratiometric Ca2+ imaging of live cells. The LEDs closely match the excitation peaks of bound and free Fura-2 and enables the precise detection of cytosolic Ca2+ concentrations, which is only limited by the Ca2+ response of Fura-2. Using this illuminator, we have shown that Fura-2 acetoxymethyl ester (AM) concentrations as low as 250 nM can be used to detect induced Ca2+ events in tsA-201 cells and while utilizing the 150 µs switching speeds available, it was possible to image spontaneous Ca2+ transients in hippocampal neurons at a rate of 24.39 Hz that were blunted or absent at typical 0.5 Hz acquisition rates. Overall, the sensitivity and acquisition speeds available using this LED illuminator significantly improves the temporal resolution that can be obtained in comparison to current systems and supports optical imaging of fast Ca2+ events using Fura-2

Normal Cones and Thompson Metric

The aim of this paper is to study the basic properties of the Thompson metric $d_T$ in the general case of a real linear space $X$ ordered by a cone $K$. We show that $d_T$ has monotonicity properties which make it compatible with the linear structure. We also prove several convexity properties of $d_T$ and some results concerning the topology of $d_T$, including a brief study of the $d_T$-convergence of monotone sequences. It is shown most of the results are true without any assumption of an Archimedean-type property for $K$. One considers various completeness properties and one studies the relations between them. Since $d_T$ is defined in the context of a generic ordered linear space, with no need of an underlying topological structure, one expects to express its completeness in terms of properties of the ordering, with respect to the linear structure. This is done in this paper and, to the best of our knowledge, this has not been done yet. The Thompson metric $d_T$ and order-unit (semi)norms $|\cdot|_u$ are strongly related and share important properties, as both are defined in terms of the ordered linear structure. Although $d_T$ and $|\cdot|_u$ are only topological (and not metrical) equivalent on $K_u$, we prove that the completeness is a common feature. One proves the completeness of the Thompson metric on a sequentially complete normal cone in a locally convex space. At the end of the paper, it is shown that, in the case of a Banach space, the normality of the cone is also necessary for the completeness of the Thompson metric.Comment: 36 page

Asymptotic behaviour of random tridiagonal Markov chains in biological applications

Discrete-time discrete-state random Markov chains with a tridiagonal generator are shown to have a random attractor consisting of singleton subsets, essentially a random path, in the simplex of probability vectors. The proof uses the Hilbert projection metric and the fact that the linear cocycle generated by the Markov chain is a uniformly contractive mapping of the positive cone into itself. The proof does not involve probabilistic properties of the sample path and is thus equally valid in the nonautonomous deterministic context of Markov chains with, say, periodically varying transitions probabilities, in which case the attractor is a periodic path.Comment: 13 pages, 22 bibliography references, submitted to DCDS-B, added references and minor correction

Compartmentalisation and localisation of the translation initiation factor (eIF) 4F complex in normally growing fibroblasts

Previous observations of association of mRNAs and ribosomes with subcellular structures highlight the importance of localised translation. However, little is known regarding associations between eukaryotic translation initiation factors and cellular structures within the cytoplasm of normally growing cells. We have used detergent-based cellular fractionation coupled with immunofluorescence microscopy to investigate the subcellular localisation in NIH3T3 fibroblasts of the initiation factors involved in recruitment of mRNA for translation, focussing on eIF4E, the mRNA cap-binding protein, the scaffold protein eIF4GI and poly(A) binding protein (PABP). We find that these proteins exist mainly in a soluble cytosolic pool, with only a subfraction tightly associated with cellular structures. However, this "associated" fraction was enriched in active "eIF4F" complexes (eIF4E.eIF4G.eIF4A.PABP). Immunofluorescence analysis reveals both a diffuse and a perinuclear distribution of eIF4G, with the perinuclear staining pattern similar to that of the endoplasmic reticulum. eIF4E also shows both a diffuse staining pattern and a tighter perinuclear stain, partly coincident with vimentin intermediate filaments. All three proteins localise to the lamellipodia of migrating cells in close proximity to ribosomes, microtubules, microfilaments and focal adhesions, with eIF4G and eIF4E at the periphery showing a similar staining pattern to the focal adhesion protein vinculin

Using climate information to support crop breeding decisions and adaptation in agriculture

Population growth in the next few decades will increase the need for food production, while the yields of major food crops could be impacted by the changing climate and changing threats from pests and pathogens. Crop breeding, both through conventional techniques, and GM assisted breeding could help meet these challenges, if adequately supported by appropriate information on the future climate. We highlight some of the major challenges for crop breeders and growers in the coming decades, and describe the main characteristics of crop breeding techniques and other adaptation options for agriculture. We review recent uses of climate information to support crop breeding decisions and make recommendations for how this might be improved. We conclude that there is significant potential for breeders to work more closely with climate scientists and crop modellers in order to address the challenges of climate change. It is not yet clear how climate information can best be used. Fruitful areas of investigation include: provision of climate information to identify key target breeding traits and develop improved success criteria (e.g. for heat/drought stress); identification of those conditions under which multiple stress factors (for example, heat stress, mid-season drought stress, flowering drought stress, terminal drought stress) are important in breeding programmes; use of climate information to inform selection of trial sites; identification of the range of environments and locations under which crop trials should be performed (likely to be a wider range of environments than done at present); identification of appropriate duration of trials (likely to be longer than current trials, due to the importance of capturing extreme events); and definition of appropriate methods for incorporating climate information into crop breeding programmes, depending on the specific needs of the breeding programme and the strengths and weaknesses of available approaches. Better knowledge is needed on climate-related thresholds important to crop breeders, for example on the frequency and severity of extreme climate events relevant to the product profile, or to help provide tailored climate analyses (particularly for extreme events). The uncertainties inherent in climate and impact projections provide a particular challenge for translating climate science into actionable outcomes for agriculture. Further work is needed to explore relevant social and economic assumptions such as the level and distribution of real incomes, changing consumption patterns, health impacts, impacts on markets and trade, and the impact of legislation relating to conservation, the environment and climate change

Interaction of eukaryotic translation initiation factor 4G with the nuclear cap-binding complex provides a link between nuclear and cytoplasmic functions of the m7 guanosine cap

In eukaryotes the majority of mRNAs have an m7G cap that is added cotranscriptionally and that plays an important role in many aspects of mRNA metabolism. The nuclear cap-binding complex (CBC; consisting of CBP20 and CBP80) mediates the stimulatory functions of the cap in pre-mRNA splicing, 3' end formation, and U snRNA export. As little is known about how nuclear CBC mediates the effects of the cap in higher eukaryotes, we have characterized proteins that interact with CBC in HeLa cell nuclear extracts as potential mediators of its function. Using cross-linking and coimmunoprecipitation, we show that eukaryotic translation initiation factor 4G (eIF4G), in addition to its function in the cytoplasm, is a nuclear CBC-interacting protein. We demonstrate that eIF4G interacts with CBC in vitro and that, in addition to its cytoplasmic localization, there is a significant nuclear pool of eIF4G in mammalian cells in vivo. Immunoprecipitation experiments suggest that, in contrast to the cytoplasmic pool, much of the nuclear eIF4G is not associated with eIF4E (translation cap binding protein of eIF4F) but is associated with CBC. While eIF4G stably associates with spliceosomes in vitro and shows close association with spliceosomal snRNPs and splicing factors in vivo, depletion studies show that it does not participate directly in the splicing reaction. Taken together the data indicate that nuclear eIF4G may be recruited to pre-mRNAs via its interaction with CBC and accompanies the mRNA to the cytoplasm, facilitating the switching of CBC for eIF4F. This may provide a mechanism to couple nuclear and cytoplasmic functions of the mRNA cap structure

Mitogen-activated protein kinase phosphatase-2 deletion impairs synaptic plasticity and hippocampal-dependent memory

Mitogen-activated protein kinases (MAPKs) regulate brain function and their dysfunction is implicated in a number of brain disorders, including Alzheimer’s disease. Thus there is great interest in understanding the signalling systems that control MAPK function. One family of proteins that contribute to this process, the mitogen-activated protein kinase phosphatases (MKPs), directly inactivate MAPKs through dephosphorylation. Recent studies have identified novel functions of MKPs in development, the immune system and cancer. However, a significant gap in our knowledge remains in relation to their role in brain functioning. Here, using transgenic mice where the Dusp4 gene encoding MKP-2 has been knocked out (MKP-2-/- mice), we show that long-term potentiation (LTP) is impaired in MKP-2-/- mice compared to MKP-2+/+ controls whereas neuronal excitability, evoked synaptic transmission and paired-pulse facilitation remain unaltered. Furthermore, spontaneous excitatory postsynaptic currents (sEPSC) frequency was increased in acute slices and primary hippocampal cultures prepared from MKP-2-/- mice with no effect on EPSC amplitude observed. An increase in synapse number was evident in primary hippocampal cultures which may account for the increase in spontaneous EPSC frequency. In addition no change in ERK activity was detected in both brain tissue and primary hippocampal cultures, suggesting that the effects of MKP-2 deletion were MAPK independent. Consistent with these alterations in hippocampal function, MKP-2-/- mice show deficits in spatial reference and working memory when investigated using the Morris water maze. These data show that MKP-2 plays a role in regulating hippocampal function and that this effect may be independent of MAPK signalling

Old case, new leads: miRNA links Kaposi’s sarcoma-associated herpesvirus with sepsis

No abstract available