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.

Direct Evidence for the Cooperative Unfolding of

ing *Corresponding author Introduction The interaction of water-soluble proteins with membranes is a common event occurring during the course of various cellular processes. Examples include the action of bacterial toxins in host cell membranes (Parker & Pattus, 1993) and the transfer of various non-polar ligands, such as retinol and fatty acids, to a target cell surface (GodovacZimmermann, 1988; Sacchettini et al., 1988). The molecular mechanism by which soluble proteins bind and insert into biological membranes is not clearly understood. In the few cases studied, it appears that a partial unfolding of the watersoluble native structure of the protein leads to the exposure of hydrophobic patches onto the protein surface, which triggers the insertion of the protein into the membrane (Merrill et al., 1990; van der Goot et al., 1991). The mechanism is complex and appears to involve both electrostatic and hydrophobic lipid-protein interactions (Heymann et al., 1996). Cytochrome c (Cyt c

The Neuroprotective Marine Compound Psammaplysene A Binds the RNA-Binding Protein HNRNPK

In previous work, we characterized the strong neuroprotective properties of the marine compound Psammaplysene A (PA) in in vitro and in vivo models of neurodegeneration. Based on its strong neuroprotective activity, the current work attempts to identify the physical target of PA to gain mechanistic insight into its molecular action. Two distinct methods, used in parallel, to purify protein-binding partners of PA led to the identification of HNRNPK as a direct target of PA. Based on surface plasmon resonance, we find that the binding of PA to HNRNPK is RNA-dependent. These findings suggest a role for HNRNPK-dependent processes in neurodegeneration/neuroprotection, and warrant further study of HNRNPK in this context

Gene network transitions in embryos depend upon interactions between a pioneer transcription factor and core histones

Gene network transitions in embryos and other fate-changing contexts involve combinations of transcription factors. A subset of fate-changing transcription factors act as pioneers; they scan and target nucleosomal DNA and initiate cooperative events that can open the local chromatin. However, a gap has remained in understanding how molecular interactions with the nucleosome contribute to the chromatin-opening phenomenon. Here we identified a short α-helical region, conserved among FOXA pioneer factors, that interacts with core histones and contributes to chromatin opening in vitro. The same domain is involved in chromatin opening in early mouse embryos for normal development. Thus, local opening of chromatin by interactions between pioneer factors and core histones promotes genetic programming.M.I. was supported by postdoctoral fellowships from Japan Society for the Promotion of Science Foundation (H26-683), Naito Foundation (RYU10000032), Astellas Foundation for Research on Metabolic Disorders (K0076) and Uehara Memorial Foundation (H24-20124). P.S. was supported by grant nos. SAF2016-75531-R (MICINN/FEDER, UE) and B2017/BMD-3724 (Comunidad de Madrid). The research was supported by NIH grant no. GM36477 to K.S.Z.Peer reviewe

The E3 ubiquitin ligase Cul4b promotes CD4+ T cell expansion by aiding the repair of damaged DNA.

The capacity for T cells to become activated and clonally expand during pathogen invasion is pivotal for protective immunity. Our understanding of how T cell receptor (TCR) signaling prepares cells for this rapid expansion remains limited. Here we provide evidence that the E3 ubiquitin ligase Cullin-4b (Cul4b) regulates this process. The abundance of total and neddylated Cul4b increased following TCR stimulation. Disruption of Cul4b resulted in impaired proliferation and survival of activated T cells. Additionally, Cul4b-deficient CD4+ T cells accumulated DNA damage. In T cells, Cul4b preferentially associated with the substrate receptor DCAF1, and Cul4b and DCAF1 were found to interact with proteins that promote the sensing or repair of damaged DNA. While Cul4b-deficient CD4+ T cells showed evidence of DNA damage sensing, downstream phosphorylation of SMC1A did not occur. These findings reveal an essential role for Cul4b in promoting the repair of damaged DNA to allow survival and expansion of activated T cells