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Pooled CRISPR-Based Genetic Screens in Mammalian Cells.

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Pooled CRISPR-Based Genetic Screens in Mammalian Cells.

J Vis Exp. 2019 Sep 04;(151):

Authors: Chan K, Tong AHY, Brown KR, Mero P, Moffat J

Abstract
Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian cells, this technology represents a novel means to perform genome-wide genetic screens for functional genomics studies. Libraries of guide RNAs (sgRNA) targeting all open reading frames permit the facile generation of thousands of genetic perturbations in a single pool of cells that can be screened for specific phenotypes to implicate gene function and cellular processes in an unbiased and systematic way. CRISPR-Cas screens provide researchers with a simple, efficient, and inexpensive method to uncover the genetic blueprints for cellular phenotypes. Furthermore, differential analysis of screens performed in various cell lines and from different cancer types can identify genes that are contextually essential in tumor cells, revealing potential targets for specific anticancer therapies. Performing genome-wide screens in human cells can be daunting, as this involves the handling of tens of millions of cells and requires analysis of large sets of data. The details of these screens, such as cell line characterization, CRISPR library considerations, and understanding the limitations and capabilities of CRISPR technology during analysis, are often overlooked. Provided here is a detailed protocol for the successful performance of pooled genome-wide CRISPR-Cas9 based screens.

PMID: 31545321 [PubMed - in process]



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Discovering genetic interactions bridging pathways in genome-wide association studies.

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Discovering genetic interactions bridging pathways in genome-wide association studies.

Nat Commun. 2019 09 19;10(1):4274

Authors: Fang G, Wang W, Paunic V, Heydari H, Costanzo M, Liu X, Liu X, VanderSluis B, Oately B, Steinbach M, Van Ness B, Schadt EE, Pankratz ND, Boone C, Kumar V, Myers CL

Abstract
Genetic interactions have been reported to underlie phenotypes in a variety of systems, but the extent to which they contribute to complex disease in humans remains unclear. In principle, genome-wide association studies (GWAS) provide a platform for detecting genetic interactions, but existing methods for identifying them from GWAS data tend to focus on testing individual locus pairs, which undermines statistical power. Importantly, a global genetic network mapped for a model eukaryotic organism revealed that genetic interactions often connect genes between compensatory functional modules in a highly coherent manner. Taking advantage of this expected structure, we developed a computational approach called BridGE that identifies pathways connected by genetic interactions from GWAS data. Applying BridGE broadly, we discover significant interactions in Parkinson's disease, schizophrenia, hypertension, prostate cancer, breast cancer, and type 2 diabetes. Our novel approach provides a general framework for mapping complex genetic networks underlying human disease from genome-wide genotype data.

PMID: 31537791 [PubMed - indexed for MEDLINE]



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Rewiring of the Human Mitochondrial Interactome during Neuronal Reprogramming Reveals Regulators of the Respirasome and Neurogenesis.

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Rewiring of the Human Mitochondrial Interactome during Neuronal Reprogramming Reveals Regulators of the Respirasome and Neurogenesis.

iScience. 2019 Sep 04;19:1114-1132

Authors: Moutaoufik MT, Malty R, Amin S, Zhang Q, Phanse S, Gagarinova A, Zilocchi M, Hoell L, Minic Z, Gagarinova M, Aoki H, Stockwell J, Jessulat M, Goebels F, Broderick K, Scott NE, Vlasblom J, Musso G, Prasad B, Lamantea E, Garavaglia B, Rajput A, Murayama K, Okazaki Y, Foster LJ, Bader GD, Cayabyab FS, Babu M

Abstract
Mitochondrial protein (MP) assemblies undergo alterations during neurogenesis, a complex process vital in brain homeostasis and disease. Yet which MP assemblies remodel during differentiation remains unclear. Here, using mass spectrometry-based co-fractionation profiles and phosphoproteomics, we generated mitochondrial interaction maps of human pluripotent embryonal carcinoma stem cells and differentiated neuronal-like cells, which presented as two discrete cell populations by single-cell RNA sequencing. The resulting networks, encompassing 6,442 high-quality associations among 600 MPs, revealed widespread changes in mitochondrial interactions and site-specific phosphorylation during neuronal differentiation. By leveraging the networks, we show the orphan C20orf24 as a respirasome assembly factor whose disruption markedly reduces respiratory chain activity in patients deficient in complex IV. We also find that a heme-containing neurotrophic factor, neuron-derived neurotrophic factor [NENF], couples with Parkinson disease-related proteins to promote neurotrophic activity. Our results provide insights into the dynamic reorganization of mitochondrial networks during neuronal differentiation and highlights mechanisms for MPs in respirasome, neuronal function, and mitochondrial diseases.

PMID: 31536960 [PubMed - as supplied by publisher]



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Age- and sex-dependent effects of metformin on neural precursor cells and cognitive recovery in a model of neonatal stroke.

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Age- and sex-dependent effects of metformin on neural precursor cells and cognitive recovery in a model of neonatal stroke.

Sci Adv. 2019 Sep;5(9):eaax1912

Authors: Ruddy RM, Adams KV, Morshead CM

Abstract
Resident neural stem and progenitor cells, collectively termed neural precursor cells (NPCs), reside in a well-defined neurogenic niche in the subventricular zone (SVZ) and contribute to ongoing postnatal neurogenesis. It is well established that the NPC niche can alter the behavior of NPCs. NPC activation is a promising therapeutic strategy for brain repair. The drug metformin has been shown to activate neural stem cells, promote differentiation, and lead to functional motor recovery in a neonatal stroke model. We demonstrate that metformin-induced NPC expansion and functional recovery is sex hormone dependent. Metformin increases the size of the NPC pool in adult females, but not males, and promotes cognitive recovery in a model of brain injury in females, but not males. Our data demonstrate that metformin has age- and sex-dependent effects on NPCs that correlate with functional recovery, which has important implications for neural repair.

PMID: 31535024 [PubMed - in process]



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Injectable and degradable methacrylic acid hydrogel alters macrophage response in skeletal muscle.

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Injectable and degradable methacrylic acid hydrogel alters macrophage response in skeletal muscle.

Biomaterials. 2019 Sep 05;223:119477

Authors: Carleton MM, Sefton MV

Abstract
After severe trauma, skeletal muscle cannot repair itself leading to scar tissue formation and functional impairment. A novel approach to overcome this issue is to alter the fibrotic response in muscle using regenerative biomaterials, such as those containing methacrylic acid (MAA). In the skin, MAA-based materials have been shown to promote wound healing and new vessel formation, through endogenous mechanisms, including macrophage polarization; however, MAA has yet to be studied outside the skin. To study the innate immune response to MAA in skeletal muscle, MAA-poly(ethylene glycol) (MAA-PEG) hydrogels were synthesized with degradation rates of either 2 (fast-degrading) or 7 days (slow-degrading). When injected into the tibialis anterior muscle of mice, both slow- and fast-degrading MAA hydrogels increased the expression of Il-10, Tnfα and M2 macrophage markers (Fizz1 and Arg for slow-and fast-degrading, respectively). Moreover, the slow degrading MAA hydrogel decreased the number of pro-inflammatory MHCII+ macrophages. An unbiased t-distributed stochastic neighbor embedding (tSNE) analysis suggested the involvement of other immune cells beyond just macrophages in the effect of MAA on skeletal muscle. Overall, this study shows that MAA hydrogels bias macrophages towards a pro-regenerative phenotype.

PMID: 31521886 [PubMed - as supplied by publisher]



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

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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]



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

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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]



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

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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]



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

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

Nat Commun. 2019 09 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 - indexed for MEDLINE]



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CRISPR screens are feasible in TP53 wild-type cells.

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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]



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