Confidence Candidate Research Articles

Mass Spectrometry Offers Insight into the Role of Ser/Thr/Tyr Phosphorylation in the Mycobacteria.

Phosphorylation is a post translational modification which can rapidly regulate biochemical pathways by altering protein function, and has been associated with pathogenicity in bacteria. Once engulfed by host macrophages, pathogenic bacteria are exposed to harsh conditions and must respond rapidly in order to survive. The causative agent of TB, Mycobacterium tuberculosis, is unusual amongst the bacteria because it can survive within the host macrophage for decades in a latent state, demonstrating a remarkable capacity to successfully evade the host immune response. This ability may be mediated in part by regulatory mechanisms such as ser/thr/tyr phosphorylation. Mass spectrometry-based proteomics has afforded us the capacity to identify hundreds of phosphorylation sites in the bacterial proteome, allowing for comparative phosphoproteomic studies in the mycobacteria. There remains an urgent need to validate the reported phosphosites, and to elucidate their biological function in the context of pathogenicity. However, given the sheer number of putative phosphorylation events in the mycobacterial proteome, and the technical difficulty of assigning biological function to a phosphorylation event, it will not be trivial to do so. There are currently six published phosphoproteomic investigations of a member of mycobacteria. Here, we combine the datasets from these studies in order to identify commonly detected phosphopeptides and phosphosites in order to present high confidence candidates for further validation. By applying modern mass spectrometry-based techniques to improve our understanding of phosphorylation and other PTMs in pathogenic bacteria, we may identify candidates for therapeutic intervention.

Abstract 529: High-dimensional Genetic Analysis of Lipoprotein Composition and Size in the Mouse

Objective: Given the clinical relevance of variation in lipoproteins to heart disease we performed a systems genetics analysis of lipoprotein diversity in mice. Using a panel of inbred mice termed the Hybrid Mouse Diversity Panel (HMDP) we were able to perform genome-wide association (GWA) to identify more than 50 loci contributing to lipoprotein and apolipoprotein levels and composition. Approach and Results: To understand the genetic regulation of lipoproteins we quantitated more than 25 unique parameters of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) in a population of 100 genetically diverse strains of mice, termed the HMDP. Specifically, we measured apoproteins (ApoA1, ApoA2, ApoA4, ApoB100, ApoC2, ApoC3, ApoD, ApoE, ApoM, ApoJ), lipoprotein-associated enzymes (LCAT, LpPLA2, PON1), and sizes of VLDL/LDL ranging from small dense LDL to larger VLDL (180 to 300 angstroms). Performing GWA we were able to identify more than 50 genetic loci. Some loci contain genes with known roles in lipoprotein metabolism while others contain genes with no known role. To identify high confidence candidate genes and validate novel genes influencing lipoprotein metabolism, we performed systems genetics analysis and integrated global gene expression, metabolomic, and proteomic data from the same mice. Through this approach along with computational gene co-expression network analysis we were able to identify two loci with strong candidate genes never before linked to ApoA1 and ApoB metabolism. Validation studies in gene-targeted knockout mice with the high confidence gene associated with ApoA1 levels,Cds2 (CDP-diacylglycerol synthase), confirmed that Cds2 contributes to plasma ApoA1 levels. Conclusions: Lipoprotein protein composition and size show large diversity across inbred strains of mice. Using a systems genetics approach we were able to identify and validate a novel gene influencing plasma ApoA1 levels.

Hundreds of SNPs for the Endangered pygmy hippopotamus (Choeropsis liberiensis)

The pygmy hippo is an Endangered mammal endemic to West Africa, of which only 2,000–3,000 are left in the wild. Until now genetic resources to conduct monitoring of wild populations and to facilitate captive breeding have been lacking. In this study we used restriction-site associated DNA sequencing of five pygmy hippo samples to generate 1,619 high confidence candidate single nucleotide polymorphisms (SNPs) suitable for population genetic analysis. A subset of 10 of SNPs generated were validated via resequencing with 100 % success rate and through the use of KASPar DNA probes (Kbiosciences) with 90 % success rate. To facilitate future research we present the list of 1,619 SNPs ranked according to mean genotype confidence and mean coverage.

Executive Confidence and New CEO Selection

Are overconfident executives more likely to be promoted to CEO? Using an option-based overconfidence measure, we show that firms with overconfident executives tend to hire internally. Further, when firms hire internally, they are more likely to pick a more confident candidate. We propose, and test between two hypotheses, matching and board-failure, for favoring overconfident executives. We find support for the matching hypothesis. Specifically, firms that are candidates for a change in strategy (i.e., large and low-risk firms) tend to appoint overconfident-executives, and benefit in terms of improved corporate value and innovative efficiency.

The VMC Survey

The number and spatial distribution of confirmed quasi-stellar objects (QSOs) behind the Magellanic system is limited. This undermines their use as astrometric reference objects for different types of studies. We have searched for criteria to identify candidate QSOs using observations from the VISTA survey of the Magellanic Clouds system (VMC) that provides photometry in the YJKs bands and 12 epochs in the Ks band. The (Y-J) versus (J-Ks) diagram has been used to distinguish QSO candidates from Milky Way stars and stars of the Magellanic Clouds. Then, the slope of variation in the Ks band has been used to identify a sample of high confidence candidates. These criteria were developed based on the properties of 117 known QSOs presently observed by the VMC survey. VMC YJKs magnitudes and Ks light-curves of known QSOs behind the Magellanic system are presented. About 75% of them show a slope of variation in Ks>10^-4 mag/day and the shape of the light-curve is in general irregular and without any clear periodicity. The number of QSO candidates found in tiles including the South Ecliptic Pole and the 30 Doradus regions is 22 and 26, respectively, with a ~20% contamination by young stellar objects, planetary nebulae, stars and normal galaxies. By extrapolating the number of QSO candidates to the entire VMC survey area we expect to find about 1200 QSOs behind the LMC, 400 behind the SMC, 200 behind the Bridge and 30 behind the Stream areas, but not all will be suitable for astrometry. Further, the Ks band light-curves can help support investigations of the mechanism responsible for the variations.

Identification of Druggable Targets for Radiation Mitigation Using a Small Interfering RNA Screening Assay

Currently, there is a serious absence of pharmaceutically attractive small molecules that mitigate the lethal effects of an accidental or intentional public exposure to toxic doses of ionizing radiation. Moreover, cellular systems that emulate the radiobiologically relevant cell populations and that are suitable for high-throughput screening have not been established. Therefore, we examined two human pluripotent embryonal carcinoma cell lines for use in an unbiased phenotypic small interfering RNA (siRNA) assay to identify proteins with the potential of being drug targets for the protection of human cell populations against clinically relevant ionizing radiation doses that cause acute radiation syndrome. Of the two human cell lines tested, NCCIT cells had optimal growth characteristics in a 384 well format, exhibited radiation sensitivity (D(0) = 1.3 ± 0.1 Gy and ñ = 2.0 ± 0.6) comparable to the radiosensitivity of stem cell populations associated with human death within 30 days after total-body irradiation. Moreover, they internalized siRNA after 4 Gy irradiation enabling siRNA library screening. Therefore, we used the human NCCIT cell line for the radiation mitigation study with a siRNA library that silenced 5,520 genes known or hypothesized to be potential therapeutic targets. Exploiting computational methodologies, we identified 113 siRNAs with potential radiomitigative properties, which were further refined to 29 siRNAs with phosphoinositide-3-kinase regulatory subunit 1 (p85α) being among the highest confidence candidate gene products. Colony formation assays revealed radiation mitigation when the phosphoinositide-3-kinase inhibitor LY294002 was given after irradiation of 32D cl 3 cells (D(0) = 1.3 ± 0.1 Gy and ñ = 2.3 ± 0.3 for the vehicle control treated cells compared to D(0) = 1.2 ± 0.1 Gy and ñ = 6.0 ± 0.8 for the LY294002 treated cells, P = 0.0004). LY294002 and two other PI3K inhibitors, PI 828 and GSK 1059615, also mitigated radiation-induced apoptosis in NCCIT cells. Treatment of mice with a single intraperitoneal LY294002 dose of 30 mg/kg at 10 min, 4, or 24 h after LD(50/30) whole-body dose of irradiation (9.25 Gy) enhanced survival. This study documents that an unbiased siRNA assay can identify new genes, signaling pathways, and chemotypes as radiation mitigators and implicate the PI3K pathway in the human radiation response.

Dose‐dependent proteomic analysis of glioblastoma cancer stem cells upon treatment with γ‐secretase inhibitor

Notch signaling has been demonstrated to have a central role in glioblastoma (GBM) cancer stem cells (CSCs) and we have demonstrated recently that Notch pathway blockade by γ-secretase inhibitor (GSI) depletes GBM CSCs and prevents tumor propagation both in vitro and in vivo. In order to understand the proteome alterations involved in this transformation, a dose-dependent quantitative mass spectrometry (MS)-based proteomic study has been performed based on the global proteome profiling and a target verification phase where both Immunoassay and a multiple reaction monitoring (MRM) assay are employed. The selection of putative protein candidates for confirmation poses a challenge due to the large number of identifications from the discovery phase. A multilevel filtering strategy together with literature mining is adopted to transmit the most confident candidates along the pipeline. Our results indicate that treating GBM CSCs with GSI induces a phenotype transformation towards non-tumorigenic cells with decreased proliferation and increased differentiation, as well as elevated apoptosis. Suppressed glucose metabolism and attenuated NFR2-mediated oxidative stress response are also suggested from our data, possibly due to their crosstalk with Notch Signaling. Overall, this quantitative proteomic-based dose-dependent work complements our current understanding of the altered signaling events occurring upon the treatment of GSI in GBM CSCs.

RNAi Screening in Drosophila Cells Identifies New Modifiers of Mutant Huntingtin Aggregation

The fruitfly Drosophila melanogaster is well established as a model system in the study of human neurodegenerative diseases. Utilizing RNAi, we have carried out a high-throughput screen for modifiers of aggregate formation in Drosophila larval CNS-derived cells expressing mutant human Huntingtin exon 1 fused to EGFP with an expanded polyglutamine repeat (62Q). 7200 genes, encompassing around 50% of the Drosophila genome, were screened, resulting in the identification of 404 candidates that either suppress or enhance aggregation. These candidates were subjected to secondary screening in normal length (18Q)-expressing cells and pruned to remove dsRNAs with greater than 10 off-target effects (OTEs). De novo RNAi probes were designed and synthesized for the remaining 68 candidates. Following a tertiary round of screening, 21 high confidence candidates were analyzed in vivo for their ability to modify mutant Huntingtin-induced eye degeneration and brain aggregation. We have established useful models for the study of human HD using the fly, and through our RNAi screen, we have identified new modifiers of mutant human Huntingtin aggregation and aggregate formation in the brain. Newly identified modifiers including genes related to nuclear transport, nucleotide processes, and signaling, may be involved in polyglutamine aggregate formation and Huntington disease cascades.

Prediction of Novel Modifications by Unrestrictive Search of Tandem Mass Spectra

Post-translational modifications (PTMs) greatly increase the complexity and diversity of the proteome so that a protein can carry out a wide variety of functions. PTM prediction on proteins is one of the major challenges in proteomics research. Various approaches have been developed for an unrestrictive search of PTMs in proteins using tandem mass spectrometry (MS/MS). However, most tools usually addressed frequent modifications despite the fact that critical biological modifications may be rare. Here, we present MODmap for exploring potentially important rare and unknown modifications from MS/MS spectra. Extended sequence tag-based spectral alignment is proposed. It is highly sensitive to modified regions in an MS/MS spectrum and is tolerant of multiple modifications per peptide. We have developed an unrestrictive algorithm (MOD(i)), which rapidly searches for all known types of PTMs at once, without limiting a multitude of modified sites in a peptide. After MOD(i) produces spectral alignment results using all known types of PTMs, high-quality spectral alignments are subject to MODmap and are re-estimated. New mass offsets are reported via local alignment and MODmap determines novel modification candidates. In analyses of PTM-rich lens proteins, our methodology was demonstrated to be sensitive to rare modifications and suggested several confident novel modification candidates.

Intravenous α1-antitrypsin in a child with deficiency and severe lung disease: Fig. 1—

<h3>Abstract</h3> Planarians have become an established model system to study regeneration and stem cells, but the regulatory elements in the genome remain almost entirely undescribed. Here, by integrating epigenetic and expression data we use multiple sources of evidence to identify enhancer elements active in the adult stem cell populations that drive regeneration. We have used ChIP-seq data to identify regions with histone modifications consistent with enhancer identity and activity, and ATAC-seq data to identify accessible chromatin. Overlapping these signals allowed for the identification of a set of high confidence candidate enhancers predicted to be active in planarian adult stem cells. These enhancers are enriched for conserved transcription factor (TF) binding sites for TFs and TF families expressed in planarian adult stem cells. Foot-printing analyses provided further evidence that these potential TF binding sites are occupied in adult stem cells. We integrated these analyses to build testable hypotheses for the regulatory function of transcription factors in stem cells, both with respect to how pluripotency might be regulated, and to how lineage differentiation programs are controlled. Our work identifies active enhancers regulating adult stem cells and regenerative mechanisms.

Therapeutic Potential and Possible Risks of Pleiotropic Growth Factors in Ischemic Stroke

To the Editor: In their meta-analysis, Minnerup and collegues1 summarize work on granulocyte-colony stimulating factor (G-CSF) in rat and mouse models of focal cerebral ischemia, concluding that G-CSF based on its survival- and recovery-promoting effects in a total of 13 experimental stroke studies is a confident candidate for stroke treatment. Unlike other neuroprotectants, G-CSF appears to exhibit a particularly long time-window of opportunity, which makes this factor interesting for clinical application. …

Functional characterization in Caenorhabditis elegans of transmembrane worm-human orthologs.

BackgroundThe complete genome sequences for human and the nematode Caenorhabditis elegans offer an opportunity to learn more about human gene function through functional characterization of orthologs in the worm. Based on a previous genome-wide analysis of worm-human orthologous transmembrane proteins, we selected seventeen genes to explore experimentally in C. elegans. These genes were selected on the basis that they all have high confidence candidate human orthologs and that their function is unknown. We first analyzed their phylogeny, membrane topology and domain organization. Then gene functions were studied experimentally in the worm by using RNA interference and transcriptional gfp reporter gene fusions.ResultsThe experiments gave functional insights for twelve of the genes studied. For example, C36B1.12, the worm ortholog of three presenilin-like genes, was almost exclusively expressed in head neurons, suggesting an ancient conserved role important to neuronal function. We propose a new transmembrane topology for the presenilin-like protein family. sft-4, the worm ortholog of surfeit locus gene Surf-4, proved to be an essential gene required for development during the larval stages of the worm. R155.1, whose human ortholog is entirely uncharacterized, was implicated in body size control and other developmental processes.ConclusionsBy combining bioinformatics and C. elegans experiments on orthologs, we provide functional insights on twelve previously uncharacterized human genes.