High‐Throughput Screen of Natural Product Extracts in A Yeast Model of Polyglutamine Proteotoxicity

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106714/1/cbdd12259.pd

Kinetic modelling of competition and depletion of shared miRNAs by competing endogenous RNAs

Non-conding RNAs play a key role in the post-transcriptional regulation of mRNA translation and turnover in eukaryotes. miRNAs, in particular, interact with their target RNAs through protein-mediated, sequence-specific binding, giving rise to extended and highly heterogeneous miRNA-RNA interaction networks. Within such networks, competition to bind miRNAs can generate an effective positive coupling between their targets. Competing endogenous RNAs (ceRNAs) can in turn regulate each other through miRNA-mediated crosstalk. Albeit potentially weak, ceRNA interactions can occur both dynamically, affecting e.g. the regulatory clock, and at stationarity, in which case ceRNA networks as a whole can be implicated in the composition of the cell's proteome. Many features of ceRNA interactions, including the conditions under which they become significant, can be unraveled by mathematical and in silico models. We review the understanding of the ceRNA effect obtained within such frameworks, focusing on the methods employed to quantify it, its role in the processing of gene expression noise, and how network topology can determine its reach.Comment: review article, 29 pages, 7 figure

Lactobacillus rhamnosus GG-supplemented formula expands butyrate-producing bacterial strains in food allergic infants.

Dietary intervention with extensively hydrolyzed casein formula supplemented with Lactobacillus rhamnosus GG (EHCF+LGG) accelerates tolerance acquisition in infants with cow's milk allergy (CMA). We examined whether this effect is attributable, at least in part, to an influence on the gut microbiota. Fecal samples from healthy controls (n=20) and from CMA infants (n=19) before and after treatment with EHCF with (n=12) and without (n=7) supplementation with LGG were compared by 16S rRNA-based operational taxonomic unit clustering and oligotyping. Differential feature selection and generalized linear model fitting revealed that the CMA infants have a diverse gut microbial community structure dominated by Lachnospiraceae (20.5±9.7%) and Ruminococcaceae (16.2±9.1%). Blautia, Roseburia and Coprococcus were significantly enriched following treatment with EHCF and LGG, but only one genus, Oscillospira, was significantly different between infants that became tolerant and those that remained allergic. However, most tolerant infants showed a significant increase in fecal butyrate levels, and those taxa that were significantly enriched in these samples, Blautia and Roseburia, exhibited specific strain-level demarcations between tolerant and allergic infants. Our data suggest that EHCF+LGG promotes tolerance in infants with CMA, in part, by influencing the strain-level bacterial community structure of the infant gut

Masked suffix priming and morpheme positional constraints

Although masked stem priming (e.g., dealer\u2013DEAL) is one of the most established effects in visual word identification (e.g., Grainger et al., 1991), it is less clear whether primes and targets sharing a suffix (e.g., kindness\u2013WILDNESS) also yield facilitation (Giraudo & Grainger, 2003; Du\uf1abeitia et al., 2008). In a new take on this issue, we show that prime nonwords facilitate lexical decisions to target words ending with the same suffix (sheeter\uac\u2013TEACHER) compared to a condition where the critical suffix was substituted by another one (sheetal\u2013TEACHER) or by an unrelated non\u2013morphological ending (sheetub\u2013 TEACHER). We also show that this effect is genuinely morphological, as no priming emerged in non\u2013complex items with the same orthographic characteristics (sportel\u2013BROTHEL vs. sportic\u2013BROTHEL vs. sportur\u2013BROTHEL). In a further experiment, we took advantage of these results to assess whether suffixes are recognized in a position\u2013specific fashion. Masked suffix priming did not emerge when the relative order of stems and suffixes was reversed in the prime nonwords\u2014ersheet did not yield any time saving in the identification of teacher as compared to either alsheet or obsheet. We take these results to show that \u2013er was not identified as a morpheme in ersheet, thus indicating that suffix identification is position specific. This conclusion is in line with data on interference effects in nonword rejection (Crepaldi, Rastle, & Davis, 2010), and strongly constrains theoretical proposals on how complex words are identified. In particular, because these findings were reported in a masked priming paradigm, they suggest that positional constraints operate early, most likely at a pre\u2013lexical level of morpho\u2013orthographic analysi

Thermal Stability of Filtered Vacuum Arc Deposited Er2O3 Coatings

Erbium oxide (Er2O3) coatings were deposited using filtered vacuum arc deposition (FVAD) and their structure and thermal stability were studied as a function of fabrication parameters. The coatings were deposited on silicon wafer and tantalum substrates with an arc current of 50 A and a deposition rate of 1.6 ± 0.4 nm/s. The arc was sustained on truncated cone Er cathodes. The influence of oxygen pressure (P= 0.40-0.93 Pa), bias voltage (Vb= -20, -40 or grounded) and substrate temperature (room temperature (RT) or 673K) on film properties was studied before and after post deposition annealing (1273K for 1 hour, at P~ 1.33 Pa). The coatings were characterized using X-ray diffraction (XRD), optical microscopy, scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and Knoop Hardness. Optical microscope images indicated that the coatings had very low macroparticle concentration on their surface. The macroparticle diameters were less than 2.5 μm. The coatings were composed of only Er2O3 without any metallic phase under all deposition parameters tested. The coatings deposited on RT substrates were XRD amorphous and had a featureless cross-section microstructure. However, the coatings deposited on 673K heated substrates had a C-Er2O3 structure with (222) preferred orientation and weak columnar microstructure. The coating hardness varied with deposition pressure and substrate bias, and reached a maximum value of 10 GPa at P = 0.4 Pa and Vb = -40 V. The post-deposition annealing caused crystallization, and the coatings hardness dropped to 4 GPa with thermal treatment. However, after post-deposition annealing, no peeling or cracking appeared at the coating surface or the interface with the substrate

Effect of promoter architecture on the cell-to-cell variability in gene expression

According to recent experimental evidence, the architecture of a promoter, defined as the number, strength and regulatory role of the operators that control the promoter, plays a major role in determining the level of cell-to-cell variability in gene expression. These quantitative experiments call for a corresponding modeling effort that addresses the question of how changes in promoter architecture affect noise in gene expression in a systematic rather than case-by-case fashion. In this article, we make such a systematic investigation, based on a simple microscopic model of gene regulation that incorporates stochastic effects. In particular, we show how operator strength and operator multiplicity affect this variability. We examine different modes of transcription factor binding to complex promoters (cooperative, independent, simultaneous) and how each of these affects the level of variability in transcription product from cell-to-cell. We propose that direct comparison between in vivo single-cell experiments and theoretical predictions for the moments of the probability distribution of mRNA number per cell can discriminate between different kinetic models of gene regulation.Comment: 35 pages, 6 figures, Submitte

GTP binding and intramolecular regulation by the ROC domain of Death Associated Protein Kinase 1

The ROCO proteins are a family of large, multidomain proteins characterised by the presence of a Ras of complex proteins (ROC) domain followed by a COR, or C-terminal of ROC, domain. It has previously been shown that the ROC domain of the human ROCO protein Leucine Rich Repeat Kinase 2 (LRRK2) controls its kinase activity. Here, the ability of the ROC domain of another human ROCO protein, Death Associated Protein Kinase 1 (DAPK1), to bind GTP and control its kinase activity has been evaluated. In contrast to LRRK2, loss of GTP binding by DAPK1 does not result in loss of kinase activity, instead acting to modulate this activity. These data highlight the ROC domain of DAPK1 as a target for modifiers of this proteins function, and casts light on the role of ROC domains as intramolecular regulators in complex proteins with implications for a broad range of human diseases

Binding Modes of Peptidomimetics Designed to Inhibit STAT3

STAT3 is a transcription factor that has been found to be constitutively activated in a number of human cancers. Dimerization of STAT3 via its SH2 domain and the subsequent translocation of the dimer to the nucleus leads to transcription of anti-apoptotic genes. Prevention of the dimerization is thus an attractive strategy for inhibiting the activity of STAT3. Phosphotyrosine-based peptidomimetic inhibitors, which mimic pTyr-Xaa-Yaa-Gln motif and have strong to weak binding affinities, have been previously investigated. It is well-known that structures of protein-inhibitor complexes are important for understanding the binding interactions and designing stronger inhibitors. Experimental structures of inhibitors bound to the SH2 domain of STAT3 are, however, unavailable. In this paper we describe a computational study that combined molecular docking and molecular dynamics to model structures of 12 peptidomimetic inhibitors bound to the SH2 domain of STAT3. A detailed analysis of the modeled structures was performed to evaluate the characteristics of the binding interactions. We also estimated the binding affinities of the inhibitors by combining MMPB/GBSA-based energies and entropic cost of binding. The estimated affinities correlate strongly with the experimentally obtained affinities. Modeling results show binding modes that are consistent with limited previous modeling studies on binding interactions involving the SH2 domain and phosphotyrosine(pTyr)-based inhibitors. We also discovered a stable novel binding mode that involves deformation of two loops of the SH2 domain that subsequently bury the C-terminal end of one of the stronger inhibitors. The novel binding mode could prove useful for developing more potent inhibitors aimed at preventing dimerization of cancer target protein STAT3

Optical Control of Metabotropic Glutamate Receptors

G-protein coupled receptors (GPCRs), the largest family of membrane signaling proteins, respond to neurotransmitters, hormones and small environmental molecules. The neuronal function of many GPCRs has been difficult to resolve because of an inability to gate them with subtype-specificity, spatial precision, speed and reversibility. To address this, we developed an approach for opto-chemical engineering native GPCRs. We applied this to the metabotropic glutamate receptors (mGluRs) to generate light-agonized and light-antagonized “LimGluRs”. The light-agonized “LimGluR2”, on which we focused, is fast, bistable, and supports multiple rounds of on/off switching. Light gates two of the primary neuronal functions of mGluR2: suppression of excitability and inhibition of neurotransmitter release. The light-antagonized “LimGluR2block” can be used to manipulate negative feedback of synaptically released glutamate on transmitter release. We generalize the optical control to two additional family members: mGluR3 and 6. The system works in rodent brain slice and in zebrafish in vivo, where we find that mGluR2 modulates the threshold for escape behavior. These light-gated mGluRs pave the way for determining the roles of mGluRs in synaptic plasticity, memory and disease