Epilepsy-associated KCNQ2 channels regulate multiple intrinsic properties of layer 2/3 pyramidal neurons

© 2017 the authors. KCNQ2 potassium channels are critical for normal brain function, as both loss-of-function and gain-of-function KCNQ2 variants can lead to various forms of neonatal epilepsy. Despite recent progress, the full spectrum of consequences as a result of KCNQ2 dysfunction in neocortical pyramidal neurons is still unknown. Here, we report that conditional ablation of Kcnq2 from mouse neocortex leads to hyperexcitability of layer 2/3 (L2/3) pyramidal neurons, exhibiting an increased input resistance and action potential frequency, as well as a reduced medium after hyperpolarization (mAHP), a conductance partly mediated by KCNQ2 channels. Importantly, we show that introducing the KCNQ2 loss-of-function variant KCNQ2I205V into L2/3 pyramidal neurons using in utero electroporation also results in a hyperexcitable phenotype similar to the conditional knock-out. KCNQ2I205V has a right-shifted conductance-to-voltage relationship, suggesting loss of KCNQ2 channel activity at subthreshold membrane potentials is sufficient to drive large changes in L2/3 pyramidal neuronal excitability even in the presence of an intact mAHP. We also found that the changes in excitability following Kcnq2 ablation are accompanied by alterations at action potential properties, including action potential amplitude in Kcnq2-null neurons. Importantly, partial inhibition of Nav1.6 channels was sufficient to counteract the hyperexcitability of Kcnq2-null neurons. Therefore, our work shows that loss of KCNQ2 channels alters the intrinsic neuronal excitability and action potential properties of L2/3 pyramidal neurons, and identifies Nav1.6 as a new potential molecular target to reduce excitability in patients with KCNQ2 encephalopathy

Adenosine Signaling through A1 Receptors Inhibits Chemosensitive Neurons in the Retrotrapezoid Nucleus.

A subset of neurons in the retrotrapezoid nucleus (RTN) function as respiratory chemoreceptors by regulating depth and frequency of breathing in response to changes in tissue CO2/H+. The activity of chemosensitive RTN neurons is also subject to modulation by CO2/H+-dependent purinergic signaling. However, mechanisms contributing to purinergic regulation of RTN chemoreceptors are not entirely clear. Recent evidence suggests adenosine inhibits RTN chemoreception in vivo by activation of A1 receptors. The goal of this study was to characterize effects of adenosine on chemosensitive RTN neurons and identify intrinsic and synaptic mechanisms underlying this response. Cell-attached recordings from RTN chemoreceptors in slices from rat or wild-type mouse pups (mixed sex) show that exposure to adenosine (1 µM) inhibits chemoreceptor activity by an A1 receptor-dependent mechanism. However, exposure to a selective A1 receptor antagonist (8-cyclopentyl-1,3-dipropylxanthine, DPCPX; 30 nM) alone did not potentiate CO2/H+-stimulated activity, suggesting activation of A1 receptors does not limit chemoreceptor activity under these reduced conditions. Whole-cell voltage-clamp from chemosensitive RTN neurons shows that exposure to adenosine activated an inward rectifying K+ conductance, and at the network level, adenosine preferentially decreased frequency of EPSCs but not IPSCs. These results show that adenosine activation of A1 receptors inhibits chemosensitive RTN neurons by direct activation of a G-protein-regulated inward-rectifier K+ (GIRK)-like conductance, and presynaptically, by suppression of excitatory synaptic input to chemoreceptors

Tissue-Specific Requirement for the GINS Complex During Zebrafish Development

Efficient and accurate DNA replication is particularly critical in stem and progenitor cells for successful proliferation and survival. The replisome, an amalgam of protein complexes, is responsible for binding potential origins of replication, unwinding the double helix, and then synthesizing complimentary strands of DNA. According to current models, the initial steps of DNA unwinding and opening are facilitated by the CMG complex, which is composed of a GINS heterotetramer that connects Cdc45 with the mini-chromosome maintenance (Mcm) helicase. In this work, we provide evidence that in the absence of GINS function DNA replication is cell autonomously impaired, and we also show that gins1 and gins2 mutants exhibit elevated levels of apoptosis restricted to actively proliferating regions of the central nervous system (CNS). Intriguingly, our results also suggest that the rapid cell cycles during early embryonic development in zebrafish may not require the function of the canonical GINS complex as neither zygotic Gins1 nor Gins2 isoforms seem to be present during these stages

Tissue-Specific Requirement for the GINS Complex During Zebrafish Development.

Efficient and accurate DNA replication is particularly critical in stem and progenitor cells for successful proliferation and survival. The replisome, an amalgam of protein complexes, is responsible for binding potential origins of replication, unwinding the double helix, and then synthesizing complimentary strands of DNA. According to current models, the initial steps of DNA unwinding and opening are facilitated by the CMG complex, which is composed of a GINS heterotetramer that connects Cdc45 with the mini-chromosome maintenance (Mcm) helicase. In this work, we provide evidence that in the absence of GINS function DNA replication is cell autonomously impaired, and we also show that gins1 and gins2 mutants exhibit elevated levels of apoptosis restricted to actively proliferating regions of the central nervous system (CNS). Intriguingly, our results also suggest that the rapid cell cycles during early embryonic development in zebrafish may not require the function of the canonical GINS complex as neither zygotic Gins1 nor Gins2 isoforms seem to be present during these stages

Production of benzylisoquinoline alkaloids in Saccharomyces cerevisiae

The benzylisoquinoline alkaloids (BIAs) are a diverse class of metabolites that exhibit a broad range of pharmacological activities and are synthesized through plant biosynthetic pathways comprised of complex enzyme activities and regulatory strategies. We have engineered yeast to produce the key intermediate reticuline and downstream BIA metabolites from a commercially available substrate. An enzyme tuning strategy was implemented that identified activity differences between variants from different plants and determined optimal expression levels. By synthesizing both stereoisomer forms of reticuline and integrating enzyme activities from three plant sources and humans, we demonstrated the synthesis of metabolites in the sanguinarine/berberine and morphinan branches. We also demonstrated that a human P450 enzyme exhibits a novel activity in the conversion of (R)-reticuline to the morphinan alkaloid salutaridine. Our engineered microbial hosts offer access to a rich group of BIA molecules and associated activities that will be further expanded through synthetic chemistry and biology approaches

Early Marine Migration Patterns of Wild Coastal Cutthroat Trout (Oncorhynchus clarki clarki), Steelhead Trout (Oncorhynchus mykiss), and Their Hybrids

Hybridization between coastal cutthroat trout (Oncorhynchus clarki clarki) and steelhead or rainbow trout (Oncorhynchus mykiss) has been documented in several streams along the North American west coast. The two species occupy similar freshwater habitats but the anadromous forms differ greatly in the duration of marine residence and migration patterns at sea. Intermediate morphological, physiological, and performance traits have been reported for hybrids but little information has been published comparing the behavior of hybrids to the pure species.This study used acoustic telemetry to record the movements of 52 cutthroat, 42 steelhead x cutthroat hybrids, and 89 steelhead smolts, all wild, that migrated from Big Beef Creek into Hood Canal (Puget Sound, Washington). Various spatial and temporal metrics were used to compare the behavior of the pure species to their hybrids. Median hybrid residence time, estuary time, and tortuosity values were intermediate compared to the pure species. The median total track distance was greater for hybrids than for either cutthroat or steelhead. At the end of each track, most steelhead (80%) were located near or north of the Hood Canal, as expected for this seaward migrating species, whereas most cutthroat (89%) were within 8 kilometers of the estuary. Most hybrids (70%) were detected leaving Hood Canal, though a substantial percentage (20%) remained near the Big Beef Creek estuary. More hybrids (7.5%) than pure cutthroat (4.5%) or steelhead (0.0%) were last detected in the southern reaches of Hood Canal.Given the similarity in freshwater ecology between the species, differences in marine ecology may play an important role in maintaining species integrity in areas of sympatry

Metal-macrofauna interactions determine microbial community structure and function in copper contaminated sediments

Peer reviewedPublisher PD

Timescales of transformational climate change adaptation in sub-Saharan African agriculture

Climate change is projected to constitute a significant threat to food security if no adaptation actions are taken. Transformation of agricultural systems, for example switching crop types or moving out of agriculture, is projected to be necessary in some cases. However, little attention has been paid to the timing of these transformations. Here, we develop a temporal uncertainty framework using the CMIP5 ensemble to assess when and where cultivation of key crops in sub-Saharan Africa becomes unviable. We report potential transformational changes for all major crops during the twenty-first century, as climates shift and areas become unsuitable. For most crops, however, transformation is limited to small pockets (<15% of area), and only for beans, maize and banana is transformation more widespread (â 1/430% area for maize and banana, 60% for beans). We envisage three overlapping adaptation phases to enable projected transformational changes: an incremental adaptation phase focused on improvements to crops and management, a preparatory phase that establishes appropriate policies and enabling environments, and a transformational adaptation phase in which farmers substitute crops, explore alternative livelihoods strategies, or relocate. To best align policies with production triggers for no-regret actions, monitoring capacities to track farming systems as well as climate are needed

Особенности развития малого предпринимательства в Украине

В этой работе рассмотрена эволюция предпринимательства Украины на всех этапах ее общественной жизни, в том числе в период экономического кризиса в годы становления независимости. Отражены основные параметры отнесения предприятий к субъектам малого предпринимательства и их роль в развитии экономики государств.У цій роботі розглянута еволюція підприємництва України на всіх етапах її суспільного життя, зокрема в період економічної кризи в роки становлення незалежності. Відображені основні параметри віднесення підприємств до суб'єктів малого підприємництва і їх роль в розвитку економіки держав

Jet energy measurement with the ATLAS detector in proton-proton collisions at root s=7 TeV

The jet energy scale and its systematic uncertainty are determined for jets measured with the ATLAS detector at the LHC in proton-proton collision data at a centre-of-mass energy of √s = 7TeV corresponding to an integrated luminosity of 38 pb-1. Jets are reconstructed with the anti-kt algorithm with distance parameters R=0. 4 or R=0. 6. Jet energy and angle corrections are determined from Monte Carlo simulations to calibrate jets with transverse momenta pT≥20 GeV and pseudorapidities {pipe}η{pipe}<4. 5. The jet energy systematic uncertainty is estimated using the single isolated hadron response measured in situ and in test-beams, exploiting the transverse momentum balance between central and forward jets in events with dijet topologies and studying systematic variations in Monte Carlo simulations. The jet energy uncertainty is less than 2. 5 % in the central calorimeter region ({pipe}η{pipe}<0. 8) for jets with 60≤pT<800 GeV, and is maximally 14 % for pT<30 GeV in the most forward region 3. 2≤{pipe}η{pipe}<4. 5. The jet energy is validated for jet transverse momenta up to 1 TeV to the level of a few percent using several in situ techniques by comparing a well-known reference such as the recoiling photon pT, the sum of the transverse momenta of tracks associated to the jet, or a system of low-pT jets recoiling against a high-pT jet. More sophisticated jet calibration schemes are presented based on calorimeter cell energy density weighting or hadronic properties of jets, aiming for an improved jet energy resolution and a reduced flavour dependence of the jet response. The systematic uncertainty of the jet energy determined from a combination of in situ techniques is consistent with the one derived from single hadron response measurements over a wide kinematic range. The nominal corrections and uncertainties are derived for isolated jets in an inclusive sample of high-pT jets. Special cases such as event topologies with close-by jets, or selections of samples with an enhanced content of jets originating from light quarks, heavy quarks or gluons are also discussed and the corresponding uncertainties are determined. © 2013 CERN for the benefit of the ATLAS collaboration