The Pediatric Cell Atlas:Defining the Growth Phase of Human Development at Single-Cell Resolution

Single-cell gene expression analyses of mammalian tissues have uncovered profound stage-specific molecular regulatory phenomena that have changed the understanding of unique cell types and signaling pathways critical for lineage determination, morphogenesis, and growth. We discuss here the case for a Pediatric Cell Atlas as part of the Human Cell Atlas consortium to provide single-cell profiles and spatial characterization of gene expression across human tissues and organs. Such data will complement adult and developmentally focused HCA projects to provide a rich cytogenomic framework for understanding not only pediatric health and disease but also environmental and genetic impacts across the human lifespan

Quantitative Phosphoproteomics of CXCL12 (SDF-1) Signaling

CXCL12 (SDF-1) is a chemokine that binds to and signals through the seven transmembrane receptor CXCR4. The CXCL12/CXCR4 signaling axis has been implicated in both cancer metastases and human immunodeficiency virus type 1 (HIV-1) infection and a more complete understanding of CXCL12/CXCR4 signaling pathways may support efforts to develop therapeutics for these diseases. Mass spectrometry-based phosphoproteomics has emerged as an important tool in studying signaling networks in an unbiased fashion. We employed stable isotope labeling with amino acids in cell culture (SILAC) quantitative phosphoproteomics to examine the CXCL12/CXCR4 signaling axis in the human lymphoblastic CEM cell line. We quantified 4,074 unique SILAC pairs from 1,673 proteins and 89 phosphopeptides were deemed CXCL12-responsive in biological replicates. Several well established CXCL12-responsive phosphosites such as AKT (pS473) and ERK2 (pY204) were confirmed in our study. We also validated two novel CXCL12-responsive phosphosites, stathmin (pS16) and AKT1S1 (pT246) by Western blot. Pathway analysis and comparisons with other phosphoproteomic datasets revealed that genes from CXCL12-responsive phosphosites are enriched for cellular pathways such as T cell activation, epidermal growth factor and mammalian target of rapamycin (mTOR) signaling, pathways which have previously been linked to CXCL12/CXCR4 signaling. Several of the novel CXCL12-responsive phosphoproteins from our study have also been implicated with cellular migration and HIV-1 infection, thus providing an attractive list of potential targets for the development of cancer metastasis and HIV-1 therapeutics and for furthering our understanding of chemokine signaling regulation by reversible phosphorylation

25th annual computational neuroscience meeting: CNS-2016

The same neuron may play different functional roles in the neural circuits to which it belongs. For example, neurons in the Tritonia pedal ganglia may participate in variable phases of the swim motor rhythms [1]. While such neuronal functional variability is likely to play a major role the delivery of the functionality of neural systems, it is difficult to study it in most nervous systems. We work on the pyloric rhythm network of the crustacean stomatogastric ganglion (STG) [2]. Typically network models of the STG treat neurons of the same functional type as a single model neuron (e.g. PD neurons), assuming the same conductance parameters for these neurons and implying their synchronous firing [3, 4]. However, simultaneous recording of PD neurons shows differences between the timings of spikes of these neurons. This may indicate functional variability of these neurons. Here we modelled separately the two PD neurons of the STG in a multi-neuron model of the pyloric network. Our neuron models comply with known correlations between conductance parameters of ionic currents. Our results reproduce the experimental finding of increasing spike time distance between spikes originating from the two model PD neurons during their synchronised burst phase. The PD neuron with the larger calcium conductance generates its spikes before the other PD neuron. Larger potassium conductance values in the follower neuron imply longer delays between spikes, see Fig. 17.Neuromodulators change the conductance parameters of neurons and maintain the ratios of these parameters [5]. Our results show that such changes may shift the individual contribution of two PD neurons to the PD-phase of the pyloric rhythm altering their functionality within this rhythm. Our work paves the way towards an accessible experimental and computational framework for the analysis of the mechanisms and impact of functional variability of neurons within the neural circuits to which they belong

CARBON-13 NUCLEAR MAGNETIC RESONANCE STUDIES OF CARDIAC METABOLISM (MATHEMATICAL MODELING, NMR)

The last decade has witnessed the increasing use of Nuclear Magnetic Resonance (NMR) techniques for following the metabolic fate of compounds specifically labeled with (\u2713)C. The goals of the present study are: (1) to develop reliable quantitative procedures for measuring the (\u2713)C enrichment of specific carbon sites in compounds enriched by the metabolism of (\u2713)C-labeled substrates in rat heart, and (2) to use these quantitative measurements of fractional (\u2713)C enrichment within the context of a mathematical flux model describing the carbon flow through the TCA cycle and ancillary pathways, as a means for obtaining unknown flux parameters. Rat hearts have been perfused in vitro with various combinations of glucose, acetate, pyruvate, and propionate to achieve steady state flux conditions, followed by perfusion with the same substrates labeled with (\u2713)C in specific carbon sites. The hearts were frozen at different times after addition of (\u2713)C-labeled substrates and neutralized perchloric acid extracts were used to obtain high resolution proton-decoupled (\u2713)C NMR spectra at 90.55 MHz. The fractional (\u2713)C enrichment (F.E.) of individual carbon sites in different metabolites was calculated from the area of the resolved resonances after correction for saturation and nuclear Overhauser effects. These F.E. measurements by (\u2713)C NMR were validated by the analysis of (\u2713)C-(\u271)H scalar coupling patterns observed in (\u271)H NMR spectra of the extracted metabolites. A mathematical flux model of the TCA cycle and ancillary transamination reactions was constructed to describe the formation of all the isotopic isomers of the TCA cycle and associated metabolites arising from metabolism of a variety of (\u2713)C-labeled substrates. The F.E. kinetics were used with the FACSIMILE program to solve for unknown flux parameters by means of non-linear least sqaures analysis of the more than 200 simultaneous differential equations used to describe the reactions. An alternative to using the F.E. kinetics was explored by calculation of the steady state labeling patterns in the TCA cycle using equations derived from the input fluxes (unknown) and input F.E.\u27s (measured) of the starting substrates while systematically varying the input fluxes using the oxygen consumption conservation equation. The results obtained from perfusion of hearts with glucose plus either 2-(\u2713)C acetate or 3-(\u2713)C pyruvate are similar to those obtained by previous investigators using (\u2714)C-labeled substrates. (Abstract shortened with permission of author.

Use of Non-Natural Amino Acids for the Design and Synthesis of a Selective, Cell-Permeable MALT1 Activity-Based Probe

Constitutive proteolytic activity of MALT1 is associated with highly aggressive B-cell lymphomas. Chemical tools that detect active MALT1 have been reported, but suffer from poor cell permeability and/or cross-reactivity with the cysteine protease cathepsin B. Here, we report that the non-natural amino acid pipecolinic acid in the P2 position of substrates and chemical probes leads to improved selectivity toward MALT1 and results in cell-permeable fluorescent probes.status: publishe