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Anti-CRISPR AcrIIA5 Potently Inhibits All Cas9 Homologs Used for Genome Editing.

Anti-CRISPR AcrIIA5 Potently Inhibits All Cas9 Homologs Used for Genome Editing.

Cell Rep. 2019 Nov 12;29(7):1739-1746.e5

Authors: Garcia B, Lee J, Edraki A, Hidalgo-Reyes Y, Erwood S, Mir A, Trost CN, Seroussi U, Stanley SY, Cohn RD, Claycomb JM, Sontheimer EJ, Maxwell KL, Davidson AR

Abstract
CRISPR-Cas9 systems provide powerful tools for genome editing. However, optimal employment of this technology will require control of Cas9 activity so that the timing, tissue specificity, and accuracy of editing may be precisely modulated. Anti-CRISPR proteins, which are small, naturally occurring inhibitors of CRISPR-Cas systems, are well suited for this purpose. A number of anti-CRISPR proteins have been shown to potently inhibit subgroups of CRISPR-Cas9 systems, but their maximal inhibitory activity is generally restricted to specific Cas9 homologs. Since Cas9 homologs vary in important properties, differing Cas9s may be optimal for particular genome-editing applications. To facilitate the practical exploitation of multiple Cas9 homologs, here we identify one anti-CRISPR, called AcrIIA5, that potently inhibits nine diverse type II-A and type II-C Cas9 homologs, including those currently used for genome editing. We show that the activity of AcrIIA5 results in partial in vivo cleavage of a single-guide RNA (sgRNA), suggesting that its mechanism involves RNA interaction.

PMID: 31722192 [PubMed - in process]



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Cranial irradiation in juvenile mice leads to early and sustained defects in the stem and progenitor cell pools and late cognitive impairments.

Cranial irradiation in juvenile mice leads to early and sustained defects in the stem and progenitor cell pools and late cognitive impairments.

Brain Res. 2019 Nov 09;:146548

Authors: Ruddy RM, Derkach D, Dadwal P, Morshead CM

Abstract
Cranial irradiation is used in combination with other therapies as a treatment for brain tumours and is thought to contribute to long-term cognitive deficits. Several rodent models have demonstrated that these cognitive deficits may be correlated with damage to neural progenitor cells in the subventricular zone (SVZ) and dentate gyrus (DG), the two neurogenic niches of the brain. Studies in rodent models typically assess the proliferating progenitor population, but rarely investigate the effect of cranial irradiation on the neural stem cell pool. Further, few studies evaluate the effects in juveniles, an age when children typically receive this treatment. Herein, we examine the cellular and behavioural effects of juvenile cranial irradiation on stem and progenitor populations in the two neurogenic regions of the brain and assess cognitive outcomes. We found regionally distinct effects of cranial irradiation in the juvenile brain. In the SVZ, we observed a defect in the stem cell pool and a concomitant decrease in proliferating cells that were maintained for at least one week. In the DG, a similar defect in the stem cell pool and proliferating cells was observed and persisted in the stem cell population. Finally, we demonstrated that cranial irradiation resulted in late cognitive deficits. This study demonstrates that juvenile cranial irradiation leads to regionally distinct defects in the stem and progenitor populations, and late cognitive deficits, which may be important factors in determining therapeutic targets and timing of interventions following cranial irradiation.

PMID: 31715143 [PubMed - as supplied by publisher]



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A CD133-AKT-Wnt signaling axis drives glioblastoma brain tumor-initiating cells.

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A CD133-AKT-Wnt signaling axis drives glioblastoma brain tumor-initiating cells.

Oncogene. 2019 Nov 06;:

Authors: Manoranjan B, Chokshi C, Venugopal C, Subapanditha M, Savage N, Tatari N, Provias JP, Murty NK, Moffat J, Doble BW, Singh SK

Abstract
Mechanistic insight into signaling pathways downstream of surface receptors has been revolutionized with integrated cancer genomics. This has fostered current treatment modalities, namely immunotherapy, to capitalize on targeting key oncogenic signaling nodes downstream of a limited number of surface markers. Unfortunately, rudimentary mechanistic understanding of most other cell surface proteins has reduced the clinical utility of these markers. CD133 has reproducibly been shown to correlate with disease progression, recurrence, and poor overall survivorship in the malignant adult brain tumor, glioblastoma (GBM). Using several patient-derived CD133high and CD133low GBMs we describe intrinsic differences in determinants of stemness, which we owe to a CD133-AKT-Wnt signaling axis in which CD133 functions as a putative cell surface receptor for AKT-dependent Wnt activation. These findings may have implications for personalized oncology trials targeting PI3K/AKT or Wnt as both pathways may be activated independent of their canonical drivers, leading to treatment resistance and disease relapse.

PMID: 31695152 [PubMed - as supplied by publisher]



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A potent anti-SpuE antibody allosterically inhibits type III secretion system and attenuates virulence of Pseudomonas aeruginosa.

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A potent anti-SpuE antibody allosterically inhibits type III secretion system and attenuates virulence of Pseudomonas aeruginosa.

J Mol Biol. 2019 Nov 01;:

Authors: Zhang Y, Sun X, Qian Y, Yi H, Song K, Zhu H, Zonta F, Chen W, Ji Q, Miersch S, Sidhu SS, Wu D

Abstract
Multidrug-resistant Gram-negative bacteria infection is particularly severe within the designated ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species), which underscores the urgent need to explore alternative therapeutic strategies. The type III secretion system (T3SS) is considered to be a key virulence factor in many Gram-negative bacteria, and T3SS is in turn regulated by SpuE in P. aeruginosa, which is a spermidine binding protein from an ATP-binding cassette transporter family and highly conserved within ESKAPE pathogens. Here, we identified a potent anti-SpuE antagonistic antibody that allosterically inhibits the expression of T3SS and attenuates virulence of P. aeruginosa. X-ray crystallography and molecular dynamics simulations revealed that binding of antibody to SpuE induces a change in the dynamics of SpuE, which in turn may reduce spermidine uptake by P. aeruginosa. The antibody could serve as a template for developing novel biologics to target a broad spectrum of Gram-negative bacteria.

PMID: 31682834 [PubMed - as supplied by publisher]



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MaveDB: an open-source platform to distribute and interpret data from multiplexed assays of variant effect.

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MaveDB: an open-source platform to distribute and interpret data from multiplexed assays of variant effect.

Genome Biol. 2019 Nov 04;20(1):223

Authors: Esposito D, Weile J, Shendure J, Starita LM, Papenfuss AT, Roth FP, Fowler DM, Rubin AF

Abstract
Multiplex assays of variant effect (MAVEs), such as deep mutational scans and massively parallel reporter assays, test thousands of sequence variants in a single experiment. Despite the importance of MAVE data for basic and clinical research, there is no standard resource for their discovery and distribution. Here, we present MaveDB ( https://www.mavedb.org ), a public repository for large-scale measurements of sequence variant impact, designed for interoperability with applications to interpret these datasets. We also describe the first such application, MaveVis, which retrieves, visualizes, and contextualizes variant effect maps. Together, the database and applications will empower the community to mine these powerful datasets.

PMID: 31679514 [PubMed - in process]



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Highly Combinatorial Genetic Interaction Analysis Reveals a Multi-Drug Transporter Influence Network.

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Highly Combinatorial Genetic Interaction Analysis Reveals a Multi-Drug Transporter Influence Network.

Cell Syst. 2019 Oct 14;:

Authors: Celaj A, Gebbia M, Musa L, Cote AG, Snider J, Wong V, Ko M, Fong T, Bansal P, Mellor JC, Seesankar G, Nguyen M, Zhou S, Wang L, Kishore N, Stagljar I, Suzuki Y, Yachie N, Roth FP

Abstract
Many traits are complex, depending non-additively on variant combinations. Even in model systems, such as the yeast S. cerevisiae, carrying out the high-order variant-combination testing needed to dissect complex traits remains a daunting challenge. Here, we describe "X-gene" genetic analysis (XGA), a strategy for engineering and profiling highly combinatorial gene perturbations. We demonstrate XGA on yeast ABC transporters by engineering 5,353 strains, each deleted for a random subset of 16 transporters, and profiling each strain's resistance to 16 compounds. XGA yielded 85,648 genotype-to-resistance observations, revealing high-order genetic interactions for 13 of the 16 transporters studied. Neural networks yielded intuitive functional models and guided exploration of fluconazole resistance, which was influenced non-additively by five genes. Together, our results showed that highly combinatorial genetic perturbation can functionally dissect complex traits, supporting pursuit of analogous strategies in human cells and other model systems.

PMID: 31668799 [PubMed - as supplied by publisher]



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Long Noncoding RNAs and Repetitive Elements: Junk or Intimate Evolutionary Partners?

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Long Noncoding RNAs and Repetitive Elements: Junk or Intimate Evolutionary Partners?

Trends Genet. 2019 Oct 26;:

Authors: Lee H, Zhang Z, Krause HM

Abstract
Our recent ability to sequence entire genomes, along with all of their transcribed RNAs, has led to the surprising finding that only ∼1% of the human genome is used to encode proteins. This finding has led to vigorous debate over the functional importance of the transcribed but untranslated portions of the genome. Currently, scientists tend to assume coding genes are functional until proven not to be, while the opposite is true for noncoding genes. This review takes a new look at the evidence for and against widespread noncoding gene functionality. We focus in particular on long noncoding RNA (noncoding RNAs longer than 200 nucleotides) genes and their 'junk' associates, transposable elements, and satellite repeats. Taken together, the suggestion put forward is that more of this junk DNA may be functional than nonfunctional and that noncoding RNAs and transposable elements act symbiotically to drive evolution.

PMID: 31662190 [PubMed - as supplied by publisher]



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Comprehensive analysis of all evolutionary paths between two divergent PDZ domain specificities.

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Comprehensive analysis of all evolutionary paths between two divergent PDZ domain specificities.

Protein Sci. 2019 Oct 25;:

Authors: Teyra J, Ernst A, Singer A, Sicheri F, Sidhu SS

Abstract
To understand the molecular evolution of functional diversity in protein families, we comprehensively investigated the consequences of all possible mutation combinations separating two peptide-binding domains with highly divergent specificities. We analyzed the Erbin PDZ domain (Erbin-PDZ), which exhibits canonical type I specificity, and a synthetic Erbin-PDZ variant (E-14) that differs at six positions and exhibits an atypical specificity that closely resembles that of the natural Pdlim4 PDZ domain (Pdlim4-PDZ). We constructed a panel of 64 PDZ domains covering all possible transitions between Erbin-PDZ and E-14 (i.e. the panel contained variants with all possible combinations of either the Erbin-PDZ or E-14 sequence at the six differing positions). We assessed the specificity profiles of the 64 PDZ domains using a C-terminal phage-displayed peptide library containing all possible genetically encoded heptapeptides. The specificity profiles clustered into six distinct groups, showing that intermediate domains can be nodes for the evolution of divergent functions. Remarkably, three substitutions were sufficient to convert the specificity of Erbin-PDZ to that of Pdlim4-PDZ, whereas Pdlim4-PDZ contains 71 differences relative to Erbin-PDZ. X-ray crystallography revealed the structural basis for specificity transition: a single substitution in the center of the binding site, supported by contributions from auxiliary substitutions, altered the main chain conformation of the peptide ligand to resemble that of ligands bound to Pdlim4-PDZ. Our results show that a very small set of mutations can dramatically alter protein specificity, and these findings support the hypothesis whereby complex protein functions evolve by gene duplication followed by cumulative mutations.

PMID: 31654425 [PubMed - as supplied by publisher]



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Author Correction: Identification and Characterization of USP7 Targets in Cancer Cells.

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Author Correction: Identification and Characterization of USP7 Targets in Cancer Cells.

Sci Rep. 2019 Oct 25;9(1):15664

Authors: Georges A, Marcon E, Greenblatt J, Frappier L

Abstract
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

PMID: 31653879 [PubMed - in process]



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A Functional Analysis of the Drosophila Gene hindsight: Evidence for Positive Regulation of EGFR Signaling.

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A Functional Analysis of the Drosophila Gene hindsight: Evidence for Positive Regulation of EGFR Signaling.

G3 (Bethesda). 2019 Oct 24;:

Authors: Kim M, Du OY, Whitney RJ, Wilk R, Hu J, Krause HM, Kavaler J, Reed BH

Abstract
We have investigated the relationship between the function of the gene hindsight (hnt), which is the Drosophila homolog of Ras Responsive Element Binding protein-1 (RREB-1), and the EGFR signaling pathway. We report that hnt mutant embryos are defective in EGFR signaling dependent processes, namely chordotonal organ recruitment and oenocyte specification. We also show the temperature sensitive hypomorphic allele hntpebbled is enhanced by the hypomorphic MAPK allele rolled (rl1 ). We find that hnt overexpression results in ectopic DPax2 expression within the embryonic peripheral nervous system, and we show that this effect is EGFR-dependent. Finally, we show that the canonical U-shaped embryonic lethal phenotype of hnt, which is associated with premature degeneration of the extraembyonic amnioserosa and a failure in germ band retraction, is rescued by expression of several components of the EGFR signaling pathway (sSpi, Ras85DV12 , pntP1 ) as well as the caspase inhibitor p35 Based on this collection of corroborating evidence, we suggest that an overarching function of hnt involves the positive regulation of EGFR signaling.

PMID: 31649045 [PubMed - as supplied by publisher]



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