Donnelly Centre for Cellular and Biomolecular Research

PubMed

Recent Publications

Evaluation of methods to assign cell type labels to cell clusters from single-cell RNA-sequencing data.

Read Full Article on External Site Read Full Article on External Site Related Articles

Evaluation of methods to assign cell type labels to cell clusters from single-cell RNA-sequencing data.

F1000Res. 2019;8:

Authors: Diaz-Mejia JJ, Meng EC, Pico AR, MacParland SA, Ketela T, Pugh TJ, Bader GD, Morris JH

Abstract
Background: Identification of cell type subpopulations from complex cell mixtures using single-cell RNA-sequencing (scRNA-seq) data includes automated steps from normalization to cell clustering. However, assigning cell type labels to cell clusters is often conducted manually, resulting in limited documentation, low reproducibility and uncontrolled vocabularies. This is partially due to the scarcity of reference cell type signatures and because some methods support limited cell type signatures. Methods: In this study, we benchmarked five methods representing first-generation enrichment analysis (ORA), second-generation approaches (GSEA and GSVA), machine learning tools (CIBERSORT) and network-based neighbor voting (METANEIGHBOR), for the task of assigning cell type labels to cell clusters from scRNA-seq data. We used five scRNA-seq datasets: human liver, 11 Tabula Muris mouse tissues, two human peripheral blood mononuclear cell datasets, and mouse retinal neurons, for which reference cell type signatures were available. The datasets span Drop-seq, 10X Chromium and Seq-Well technologies and range in size from ~3,700 to ~68,000 cells. Results: Our results show that, in general, all five methods perform well in the task as evaluated by receiver operating characteristic curve analysis (average area under the curve (AUC) = 0.91, sd = 0.06), whereas precision-recall analyses show a wide variation depending on the method and dataset (average AUC = 0.53, sd = 0.24). We observed an influence of the number of genes in cell type signatures on performance, with smaller signatures leading more frequently to incorrect results. Conclusions: GSVA was the overall top performer and was more robust in cell type signature subsampling simulations, although different methods performed well using different datasets. METANEIGHBOR and GSVA were the fastest methods. CIBERSORT and METANEIGHBOR were more influenced than the other methods by analyses including only expected cell types. We provide an extensible framework that can be used to evaluate other methods and datasets at https://github.com/jdime/scRNAseq_cell_cluster_labeling.

PMID: 31508207 [PubMed - in process]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

Close association between vasa-positive germ plasm granules and mitochondria correlates with cytoplasmic localization of 12S and 16S mtrRNAs during zebrafish spermatogenesis.

Related Articles

Close association between vasa-positive germ plasm granules and mitochondria correlates with cytoplasmic localization of 12S and 16S mtrRNAs during zebrafish spermatogenesis.

Differentiation. 2019 Aug 30;109:34-41

Authors: Reunov A, Yakovlev K, Hu J, Reunova Y, Komkova A, Alexandrova Y, Pimenova E, Tiefenbach J, Krause H

Abstract
The phenomenon of the cytoplasmic localisation of mitochondrial ribosomal subunits (12 S mitochondrial rRNA and 16 S mitochondrial rRNA) has been discovered by scientific teams working with spermatogenic cells of mice. Previous reports showed that the release of mitochondrial substance occurs during interaction of mitochondria with the germ plasm granules (GG). To determine if the interplay between the vasa-positive GG and the mitochondria is associated with cytoplasmic localisation of mtrRNAs, we studied the spermatogenic cells of zebrafish, Danio rerio. It was revealed that in type A undifferentiated spermatogonia the GG did not contact mitochondria, and the extra-mitochondrial localisation of the mtrRNAs was not found. In type A differentiated spermatogonia, the amount of GG in contact with mitochondria increased, but the extra-mitochondrial localisation of the mtrRNAs was not found either. In type B late spermatogonia, which are pre-meiotic cells, the GG/mitochondrion complexes were typically found in contact with the nucleus. This stage was associated with the intra-mitochondrial localisation of GG-originated vasa and extra-mitochondrial localisation of 12 S mtrRNA and 16 S mtrRNA. Until the onset of meiosis, which was determined by the observation of synaptonemal complexes in zygotene-pachytene spermatocytes I, the GG/mitochondrion complexes disappeared, but both types of mtrRNAs persisted in the cytoplasm of spermatids and spermatozoa.

PMID: 31494397 [PubMed - as supplied by publisher]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

Associations between Coffee Products and Breast Cancer Risk: a Case-Control study in Hong Kong Chinese Women.

Related Articles

Associations between Coffee Products and Breast Cancer Risk: a Case-Control study in Hong Kong Chinese Women.

Sci Rep. 2019 Sep 03;9(1):12684

Authors: Lee PMY, Chan WC, Kwok CC, Wu C, Law SH, Tsang KH, Yu WC, Yeung YC, Chang LDJ, Wong CKM, Wang F, Tse LA

Abstract
Coffee contains caffeine and diterpenes that were associated with decreased breast cancer risk, but results remained inconsistent. The study purpose was to investigate the associations between coffee products and breast cancer risk among Hong Kong Chinese women. We conducted a hospital-based case-control study in three public hospitals. 2169 Chinese women aged 24-84 years old were interviewed using a standardized questionnaire with questions asking types, cups and duration on coffee drinking. We used unconditional multivariate logistic regression to calculate the adjusted odds ratio (AOR) and 95% confidence interval (95% CI) for breast cancer risk with different coffee products. 238 (20.6%) cases and 179 (17.7%) controls are habitual coffee drinkers. No association was found between overall coffee drinking and breast cancer risk. Compared to the non-habitual coffee drinkers, women who consumed instant coffee (AOR = 1.50, 95% CI = 1.10-2.03) were significantly associated with an increased breast cancer risk. Women who drank brewed coffee (AOR = 0.48, 95% CI = 0.28-0.82) were negatively associated with breast cancer risk. A positive association between instant coffee and breast cancer risk was observed, contradicted to the outcomes of drinking brewed coffee. Larger studies are warranted to ascertain the role of different types of coffee products in breast cancer risk.

PMID: 31481730 [PubMed - in process]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

The marginal cells of the Caenorhabditis elegans pharynx scavenge cholesterol and other hydrophobic small molecules.

Related Articles

The marginal cells of the Caenorhabditis elegans pharynx scavenge cholesterol and other hydrophobic small molecules.

Nat Commun. 2019 Sep 02;10(1):3938

Authors: Kamal M, Moshiri H, Magomedova L, Han D, Nguyen KCQ, Yeo M, Knox J, Bagg R, Won AM, Szlapa K, Yip CM, Cummins CL, Hall DH, Roy PJ

Abstract
The nematode Caenorhabditis elegans is a bacterivore filter feeder. Through the contraction of the worm's pharynx, a bacterial suspension is sucked into the pharynx's lumen. Excess liquid is then shunted out of the buccal cavity through ancillary channels made by surrounding marginal cells. We find that many worm-bioactive small molecules (a.k.a. wactives) accumulate inside of the marginal cells as crystals or globular spheres. Through screens for mutants that resist the lethality associated with one crystallizing wactive we identify a presumptive sphingomyelin-synthesis pathway that is necessary for crystal and sphere accumulation. We find that expression of sphingomyelin synthase 5 (SMS-5) in the marginal cells is not only sufficient for wactive accumulation but is also important for absorbing exogenous cholesterol, without which C. elegans cannot develop. We conclude that sphingomyelin-rich marginal cells act as a sink to scavenge important nutrients from filtered liquid that might otherwise be shunted back into the environment.

PMID: 31477732 [PubMed - in process]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

CRISPR screens are feasible in TP53 wild-type cells.

Related Articles

CRISPR screens are feasible in TP53 wild-type cells.

Mol Syst Biol. 2019 Aug;15(8):e8679

Authors: Brown KR, Mair B, Soste M, Moffat J

Abstract
A recent study by Haapaniemi et al (2018) reported that intact p53 signaling hampers CRISPR-based functional genomic screens. Brown et al report good performance of genome-scale screens in TP53 wild-type cells and reiterate best practices for CRISPR screening.

PMID: 31464370 [PubMed - in process]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

The Epstein-Barr Virus BMRF1 Protein Activates Transcription and Inhibits the DNA Damage Response by Binding NuRD.

Related Articles

The Epstein-Barr Virus BMRF1 Protein Activates Transcription and Inhibits the DNA Damage Response by Binding NuRD.

J Virol. 2019 Aug 28;:

Authors: Salamun SG, Sitz J, De La Cruz-Herrera CF, Yockteng-Melgar J, Marcon E, Greenblatt J, Fradet-Turcotte A, Frappier L

Abstract
The BMRF1 protein of Epstein-Barr virus (EBV) has multiple roles in viral lytic infection including serving as the DNA polymerase processivity factor, activating transcription from several EBV promoters and inhibiting the host DNA damage response to double-stranded DNA breaks (DSBs). Using affinity purification coupled to mass spectrometry, we identified the nucleosome remodeling and deacetylation (NuRD) complex as the top interactor of BMRF1. We further found that NuRD components localize with BMRF1 at viral replication compartments and that this interaction occurs through the BMRF1 C-terminal region previously shown to mediate transcriptional activation. We identified an RBBP4 binding motif within this region that can interact with both RBBP4 and MTA2 components of the NuRD complex, and showed that point mutation of this motif abrogates NuRD binding as well as the ability of BMRF1 to activate transcription from the BDLF3 and BLLF1 EBV promoters. In addition to its role in transcriptional regulation, NuRD has been shown to contribute to DSB signaling in enabling recruitment of RNF168 ubiquitin ligase and subsequent ubiquitylation at the break. We showed that BMRF1 inhibited RNF168 recruitment and ubiquitylation at DSBs and that this inhibition was at least partly relieved by loss of the NuRD interaction. The results reveal a mechanism by which BMRF1 activates transcription and inhibits DSB signaling and a novel role for NuRD in transcriptional activation in EBV.IMPORTANCE The Epstein-Barr virus (EBV) BMRF1 protein is critical for EBV infection, playing key roles in viral genome replication, activation of EBV genes and inhibition of host DNA damage responses (DDRs). Here we show that BMRF1 targets the cellular nucleosome remodeling and deacetylation (NuRD) complex, using a motif in the BMRF1 transcriptional activation sequence. Mutation of this motif disrupts the ability of BMRF1 to activate transcription and interfere with DDRs, showing the importance of the NuRD interaction for BMRF1 functions. BMRF1 was shown to act at the same step in the DDR as NuRD, suggesting that it interferes with NuRD function.

PMID: 31462557 [PubMed - as supplied by publisher]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

Tailored tetravalent antibodies potently and specifically activate Wnt/Frizzled pathways in cells, organoids and mice.

Related Articles

Tailored tetravalent antibodies potently and specifically activate Wnt/Frizzled pathways in cells, organoids and mice.

Elife. 2019 Aug 27;8:

Authors: Tao Y, Mis M, Blazer L, Ustav M, Steinhart Z, Chidiac R, Kubarakos E, O'Brien S, Wang X, Jarvik N, Patel N, Adams J, Moffat J, Angers S, Sidhu SS

Abstract
Secreted Wnt proteins regulate development and adult tissue homeostasis by binding and activating cell-surface Frizzled receptors and co-receptors including LRP5/6. The hydrophobicity of Wnt proteins has complicated their purification and limited their use in basic research and as therapeutics. We describe modular tetravalent antibodies that can recruit Frizzled and LRP5/6 in a manner that phenocopies the activities of Wnts both in vitro and in vivo. The modular nature of these synthetic Frizzled and LRP5/6 Agonists, called FLAgs, enables tailored engineering of specificity for one, two or multiple members of the Frizzled family. We show that FLAgs underlie differentiation of pluripotent stem cells, sustain organoid growth, and activate stem cells in vivo. Activation of Wnt signaling circuits with tailored FLAgs will enable precise delineation of functional outcomes directed by distinct receptor combinations and could provide a new class of therapeutics to unlock the promise of regenerative medicine.

PMID: 31452509 [PubMed - in process]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

Extracellular phosphorylation drives the formation of neuronal circuitry.

Related Articles

Extracellular phosphorylation drives the formation of neuronal circuitry.

Nat Chem Biol. 2019 Aug 26;:

Authors: Harada H, Farhani N, Wang XF, Sugita S, Charish J, Attisano L, Moran M, Cloutier JF, Reber M, Bremner R, Monnier PP

Abstract
Until recently, the existence of extracellular kinase activity was questioned. Many proteins of the central nervous system are targeted, but it remains unknown whether, or how, extracellular phosphorylation influences brain development. Here we show that the tyrosine kinase vertebrate lonesome kinase (VLK), which is secreted by projecting retinal ganglion cells, phosphorylates the extracellular protein repulsive guidance molecule b (RGMb) in a dorsal-ventral descending gradient. Silencing of VLK or RGMb causes aberrant axonal branching and severe axon misguidance in the chick optic tectum. Mice harboring RGMb with a point mutation in the phosphorylation site also display aberrant axonal pathfinding. Mechanistic analyses show that VLK-mediated RGMb phosphorylation modulates Wnt3a activity by regulating LRP5 protein gradients. Thus, the secretion of VLK by projecting neurons provides crucial signals for the accurate formation of nervous system circuitry. The dramatic effect of VLK on RGMb and Wnt3a signaling implies that extracellular phosphorylation likely has broad and profound effects on brain development, function and disease.

PMID: 31451763 [PubMed - as supplied by publisher]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

Decyl gallate as a possible inhibitor of N-glycosylation process in Paracoccidioides lutzii.

Related Articles

Decyl gallate as a possible inhibitor of N-glycosylation process in Paracoccidioides lutzii.

Antimicrob Agents Chemother. 2019 Aug 26;:

Authors: de Paula E Silva ACA, de Oliveira HC, Scorzoni L, Marcos CM, Dos Santos CT, Fusco-Almeida AM, Salina ACG, Medeiros AI, Almeida F, Li SC, Boone C, Mendes-Giannini MJS

Abstract
The available antifungal therapeutic arsenal is limited. The search for alternative drugs with fewer side effects and new targets remains a major challenge. Decyl gallate (G14) is a derivative of gallic acid with a range of biological activities and wide spectra antifungal activity. Previously, our group demonstrated the promising anti-Paracoccidioides activity of G14. In this work, aiming to evaluate the antifungal characteristics of G14 for P. lutzii, chemical-genetic interaction analysis was conducted on Saccharomyces cerevisiae model. N-glycosylation and/or the unfolded protein response pathways were identified as high confidence target process prediction. The over activation of unfolded protein response (UPR) signaling was confirmed using this model with green fluorescent protein (GFP) tagged IRE1/ATF6/PERK genes. In P. lutzii, this prediction was confirmed by the low activity of glycosylated enzymes (α-(1,3)-glucanase, N-Acetyl-β-D-glucosaminidase, α-(1,4)-Amylase), by hyperexpresion of genes involved with the UPR and glycosylated enzymes, and by the reduction of glycosylated proteins and chitin amount. All these components are involved in fungal cell wall integrity and are depended of N-glycosylation process. This loss was confirmed by the reduction of mitochondrial activity, the impaired budding, the enhance of wall permeability and the decrease of viability. These events lead to a reduction of the ability of fungi to adhere on lung epithelial human cell (A549) in vitro Therefore, G14 may to have an important role in balancing the inflammatory reaction caused by fungal infection, without interfering on microbicidal activity of nitric oxide. This work provides new information on the activity of G14, a potential anti-Paracoccidioides compound.

PMID: 31451502 [PubMed - as supplied by publisher]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄

Rapid Chemical Reaction Monitoring by Digital Microfluidics-NMR: Proof of Principle Towards an Automated Synthetic Discovery Platform.

Related Articles

Rapid Chemical Reaction Monitoring by Digital Microfluidics-NMR: Proof of Principle Towards an Automated Synthetic Discovery Platform.

Angew Chem Int Ed Engl. 2019 Aug 26;:

Authors: Wu B, von der Ecken S, Swyer I, Li C, Jenne A, Vincent F, Schmidig D, Kuehn T, Busse F, Stronks H, Soong R, Wheeler AR, Simpson AJ

Abstract
Microcoil Nuclear magnetic resonance (NMR) has been interfaced with digital microfluidics (DMF) and is applied to monitor organic reactions in organic solvents as a proof of concept. DMF permits droplets to be moved and mixed inside the NMR to initiate reactions while using sub-microliter volumes of reagent, opening up the potential to follow the reactions of scarce or expensive reagents. By setting up the spectrometer shims on a reagent droplet, data acquisition can be started immediately upon droplet mixing and is only limited by the rate at which NMR data can be collected, allowing the monitoring of fast reactions. Here we report a cyclohexene carbonate hydrolysis in dimethylformamide and a Knoevenagel condensation in methanol/water. This is to our knowledge the first time rapid organic reactions in organic solvents have been monitored by high field DMF-NMR. The study represents a key first step towards larger DMF-NMR arrays that could in future serve as discovery platforms, where computer controlled DMF automates mixing/titration of chemical libraries and NMR is used to study the structures formed and kinetics in real time.

PMID: 31449724 [PubMed - as supplied by publisher]



▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄ ▄