A Small Conductance Calcium-Activated K⁺ Channel in C. elegans, KCNL-2, Plays a Role in the Regulation of the Rate of Egg-Laying

In the nervous system of mice, small conductance calcium-activated potassium (SK) channels function to regulate neuronal excitability through the generation of a component of the medium afterhyperpolarization that follows action potentials. In humans, irregular action potential firing frequency underlies diseases such as ataxia, epilepsy, schizophrenia and Parkinson's disease. Due to the complexity of studying protein function in the mammalian nervous system, we sought to characterize an SK channel homologue, KCNL-2, in C. elegans, a genetically tractable system in which the lineage of individual neurons was mapped from their early developmental stages. Sequence analysis of the KCNL-2 protein reveals that the six transmembrane domains, the potassium-selective pore and the calmodulin binding domain are highly conserved with the mammalian homologues. We used widefield and confocal fluorescent imaging to show that a fusion construct of KCNL-2 with GFP in transgenic lines is expressed in the nervous system of C. elegans. We also show that a KCNL-2 null strain, kcnl-2(tm1885), demonstrates a mild egg-laying defective phenotype, a phenotype that is rescued in a KCNL-2-dependent manner. Conversely, we show that transgenic lines that overexpress KCNL-2 demonstrate a hyperactive egg-laying phenotype. In this study, we show that the vulva of transgenic hermaphrodites is highly innervated by neuronal processes and by the VC4 and VC5 neurons that express GFP-tagged KCNL-2. We propose that KCNL-2 functions in the nervous system of C. elegans to regulate the rate of egg-laying. © 2013 Chotoo et al

Evaluation of genetic isolation within an island flora reveals unusually widespread local adaptation and supports sympatric speciation

It is now recognized that speciation can proceed even when divergent natural selection is opposed by gene flow. Understanding the extent to which environmental gradients and geographical distance can limit gene flow within species can shed light on the relative roles of selection and dispersal limitation during the early stages of population divergence and speciation. On the remote Lord Howe Island (Australia), ecological speciation with gene flow is thought to have taken place in several plant genera. The aim of this study was to establish the contributions of isolation by environment (IBE) and isolation by community (IBC) to the genetic structure of 19 plant species, from a number of distantly related families, which have been subjected to similar environmental pressures over comparable time scales. We applied an individual-based, multivariate, model averaging approach to quantify IBE and IBC, while controlling for isolation by distance (IBD). Our analyses demonstrated that all species experienced some degree of ecologically driven isolation, whereas only 12 of 19 species were subjected to IBD. The prevalence of IBE within these plant species indicates that divergent selection in plants frequently produces local adaptation and supports hypotheses that ecological divergence can drive speciation in sympatry

Aptamer-based multiplexed proteomic technology for biomarker discovery

Interrogation of the human proteome in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology. We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 [mu]L of serum or plasma). Our current assay allows us to measure ~800 proteins with very low limits of detection (1 pM average), 7 logs of overall dynamic range, and 5% average coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding DNA aptamer concentration signature, which is then quantified with a DNA microarray. In essence, our assay takes advantage of the dual nature of aptamers as both folded binding entities with defined shapes and unique sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to discover unique protein signatures characteristic of various disease states. More generally, we describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine

Methods for specifying the target difference in a randomised controlled trial : the Difference ELicitation in TriAls (DELTA) systematic review

Peer reviewedPublisher PD

Caspase Inhibition Blocks Cell Death and Enhances Mitophagy but Fails to Promote T-Cell Lymphoma

Caspase-9 is a component of the apoptosome that mediates cell death following release of cytochrome c from mitochondria. Inhibition of Caspase-9 with a dominant negative construct (Casp9DN) blocks apoptosome function, promotes viability and has been implicated in carcinogenesis. Inhibition of the apoptosome in vitro impairs mitochondrial function and promotes mitophagy. To examine whether inhibition of the apoptosome would enhance mitophagy and promote oncogenesis in vivo, transgenic mice were generated that express Casp9DN in the T cell lineage. The effects of Casp9DN on thymocyte viability, mitophagy and thymic tumor formation were examined. In primary thymocytes, Casp9DN delayed dexamethasone (Dex)-induced cell death, altered mitochondrial structure, and decreased oxidant production. Transmission electron microscopy (TEM) revealed that inhibition of the apoptosome resulted in structurally abnormal mitochondria that in some cases were engulfed by double-membrane structures resembling autophagosomes. Consistent with mitochondria being engulfed by autophagosomes (mitophagy), confocal microscopy showed colocalization of LC3-GFP and mitochondria. However, Casp9DN did not significantly accelerate T-cell lymphoma alone, or in combination with Lck-Bax38/1, or with Beclin 1+/− mice, two tumor-prone strains in which altered mitochondrial function has been implicated in promoting tumor development. In addition, heterozygous disruption of Beclin 1 had no effect on T-cell lymphoma formation in Lck-Bax38/1 mice. Further studies showed that Beclin 1 levels had no effect on Casp9DN-induced loss of mitochondrial function. These results demonstrate that neither inhibition of apoptosome function nor Beclin 1 haploinsufficiency accelerate T-cell lymphoma development in mice

Pavlovian Fear Conditioning Activates a Common Pattern of Neurons in the Lateral Amygdala of Individual Brains

Understanding the physical encoding of a memory (the engram) is a fundamental question in neuroscience. Although it has been established that the lateral amygdala is a key site for encoding associative fear memory, it is currently unclear whether the spatial distribution of neurons encoding a given memory is random or stable. Here we used spatial principal components analysis to quantify the topography of activated neurons, in a select region of the lateral amygdala, from rat brains encoding a Pavlovian conditioned fear memory. Our results demonstrate a stable, spatially patterned organization of amygdala neurons are activated during the formation of a Pavlovian conditioned fear memory. We suggest that this stable neuronal assembly constitutes a spatial dimension of the engram