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ATP7B variant c.1934T > G p.Met645Arg causes Wilson disease by promoting exon 6 skipping.

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ATP7B variant c.1934T > G p.Met645Arg causes Wilson disease by promoting exon 6 skipping.

NPJ Genom Med. 2020;5:16

Authors: Merico D, Spickett C, O'Hara M, Kakaradov B, Deshwar AG, Fradkin P, Gandhi S, Gao J, Grant S, Kron K, Schmitges FW, Shalev Z, Sun M, Verby M, Cahill M, Dowling JJ, Fransson J, Wienholds E, Frey BJ

Abstract
Wilson disease is a recessive genetic disorder caused by pathogenic loss-of-function variants in the ATP7B gene. It is characterized by disrupted copper homeostasis resulting in liver disease and/or neurological abnormalities. The variant NM_000053.3:c.1934T > G (Met645Arg) has been reported as compound heterozygous, and is highly prevalent among Wilson disease patients of Spanish descent. Accordingly, it is classified as pathogenic by leading molecular diagnostic centers. However, functional studies suggest that the amino acid change does not alter protein function, leading one ClinVar submitter to question its pathogenicity. Here, we used a minigene system and gene-edited HepG2 cells to demonstrate that c.1934T > G causes ~70% skipping of exon 6. Exon 6 skipping results in frameshift and stop-gain, leading to loss of ATP7B function. The elucidation of the mechanistic effect for this variant resolves any doubt about its pathogenicity and enables the development of genetic medicines for restoring correct splicing.

PMID: 32284880 [PubMed]



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Combinatorial extracellular matrix microarray identifies novel bioengineered substrates for xeno-free culture of human pluripotent stem cells.

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Combinatorial extracellular matrix microarray identifies novel bioengineered substrates for xeno-free culture of human pluripotent stem cells.

Biomaterials. 2020 Apr 03;248:120017

Authors: Ireland RG, Kibschull M, Audet J, Ezzo M, Hinz B, Lye SJ, Simmons CA

Abstract
Stem cells in their microenvironment are exposed to a plethora of biochemical signals and biophysical forces. Interrogating the role of each factor in the cell microenvironment, however, remains difficult due to the inability to study microenvironmental cues and tease apart their interactions in high throughput. To address this need, we developed an extracellular matrix (ECM) microarray screening platform capable of tightly controlling substrate stiffness and ECM protein composition to screen the effects of these cues and their interactions on cell fate. We combined this platform with a design of experiments screening strategy to identify optimal conditions that can maintain human pluripotent stem cell (hPSC) pluripotency in chemically defined, xeno-free conditions. Combinations of ECM proteins (fibronectin, vitronectin, laminin-521, and collagen IV) were deposited on polydimethylsiloxane substrates with elastic moduli ranging from ~1 to 60 kPa using a high throughput protein plotter. Through our screening approach, we identified several non-intuitive protein-protein and protein-stiffness interactions and developed three novel culture substrates. hPSCs grown on these novel culture substrates displayed higher proliferation rates and pluripotency marker expression than current gold-standard culture substrates Geltrex- and vitronectin-coated plastic. This ECM microarray and screening approach is not limited to the factors studied here and can be broadly applied to other cell types to systematically screen microenvironmental conditions to optimally guide cell phenotype.

PMID: 32283392 [PubMed - as supplied by publisher]



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The mitochondrial peptidase, neurolysin, regulates respiratory chain supercomplex formation and is necessary for AML viability.

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The mitochondrial peptidase, neurolysin, regulates respiratory chain supercomplex formation and is necessary for AML viability.

Sci Transl Med. 2020 Apr 08;12(538):

Authors: Mirali S, Botham A, Voisin V, Xu C, St-Germain J, Sharon D, Hoff FW, Qiu Y, Hurren R, Gronda M, Jitkova Y, Nachmias B, MacLean N, Wang X, Arruda A, Minden MD, Horton TM, Kornblau SM, Chan SM, Bader GD, Raught B, Schimmer AD

Abstract
Neurolysin (NLN) is a zinc metallopeptidase whose mitochondrial function is unclear. We found that NLN was overexpressed in almost half of patients with acute myeloid leukemia (AML), and inhibition of NLN was selectively cytotoxic to AML cells and stem cells while sparing normal hematopoietic cells. Mechanistically, NLN interacted with the mitochondrial respiratory chain. Genetic and chemical inhibition of NLN impaired oxidative metabolism and disrupted the formation of respiratory chain supercomplexes (RCS). Furthermore, NLN interacted with the known RCS regulator, LETM1, and inhibition of NLN disrupted LETM1 complex formation. RCS were increased in patients with AML and positively correlated with NLN expression. These findings demonstrate that inhibiting RCS formation selectively targets AML cells and stem cells and highlights the therapeutic potential of pharmacologically targeting NLN in AML.

PMID: 32269163 [PubMed - in process]



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A Genome-Wide Screen for Genes Affecting Spontaneous Direct-Repeat Recombination in Saccharomyces cerevisiae.

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A Genome-Wide Screen for Genes Affecting Spontaneous Direct-Repeat Recombination in Saccharomyces cerevisiae.

G3 (Bethesda). 2020 Apr 07;:

Authors: Novarina D, Desai R, Vaisica JA, Ou J, Bellaoui M, Brown GW, Chang M

Abstract
Homologous recombination is an important mechanism for genome integrity maintenance, and several homologous recombination genes are mutated in various cancers and cancer-prone syndromes. However, since in some cases homologous recombination can lead to mutagenic outcomes, this pathway must be tightly regulated, and mitotic hyper-recombination is a hallmark of genomic instability. We performed two screens in Saccharomyces cerevisiae for genes that, when deleted, cause hyper-recombination between direct repeats. One was performed with the classical patch and replica-plating method. The other was performed with a high-throughput replica-pinning technique that was designed to detect low-frequency events. This approach allowed us to validate the high-throughput replica-pinning methodology independently of the replicative aging context in which it was developed. Furthermore, by combining the two approaches, we were able to identify and validate 35 genes whose deletion causes elevated spontaneous direct-repeat recombination. Among these are mismatch repair genes, the Sgs1-Top3-Rmi1 complex, the RNase H2 complex, genes involved in the oxidative stress response, and a number of other DNA replication, repair and recombination genes. Since several of our hits are evolutionary conserved, and repeated elements constitute a significant fraction of mammalian genomes, our work might be relevant for understanding genome integrity maintenance in humans.

PMID: 32265288 [PubMed - as supplied by publisher]



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MaveQuest: a web resource for planning experimental tests of human variant effects.

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MaveQuest: a web resource for planning experimental tests of human variant effects.

Bioinformatics. 2020 Apr 06;:

Authors: Kuang D, Weile J, Li R, Ouellette TW, Barber JA, Roth FP

Abstract
Fully realizing the promise of personalized medicine will require rapid and accurate classification of pathogenic human variation. Multiplexed assays of variant effect (MAVEs) can experimentally test nearly all possible missense variants in selected gene targets. Planning a MAVE study involves identifying target genes with clinical impact, and identifying scalable functional assays for that target. Here we describe MaveQuest, a web-based resource enabling systematic variant effect mapping studies by identifying potential functional assays, disease phenotypes and clinical relevance for nearly all human genes.
AVAILABILITY AND IMPLEMENTATION: MaveQuest service: https://mavequest.varianteffect.org/ MaveQuest source code: https://github.com/kvnkuang/mavequest-front-end/.

PMID: 32251504 [PubMed - as supplied by publisher]



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Genetic interaction mapping and exon-resolution functional genomics with a hybrid Cas9-Cas12a platform.

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Genetic interaction mapping and exon-resolution functional genomics with a hybrid Cas9-Cas12a platform.

Nat Biotechnol. 2020 Mar 16;:

Authors: Gonatopoulos-Pournatzis T, Aregger M, Brown KR, Farhangmehr S, Braunschweig U, Ward HN, Ha KCH, Weiss A, Billmann M, Durbic T, Myers CL, Blencowe BJ, Moffat J

Abstract
Systematic mapping of genetic interactions (GIs) and interrogation of the functions of sizable genomic segments in mammalian cells represent important goals of biomedical research. To advance these goals, we present a CRISPR (clustered regularly interspaced short palindromic repeats)-based screening system for combinatorial genetic manipulation that employs coexpression of CRISPR-associated nucleases 9 and 12a (Cas9 and Cas12a) and machine-learning-optimized libraries of hybrid Cas9-Cas12a guide RNAs. This system, named Cas Hybrid for Multiplexed Editing and screening Applications (CHyMErA), outperforms genetic screens using Cas9 or Cas12a editing alone. Application of CHyMErA to the ablation of mammalian paralog gene pairs reveals extensive GIs and uncovers phenotypes normally masked by functional redundancy. Application of CHyMErA in a chemogenetic interaction screen identifies genes that impact cell growth in response to mTOR pathway inhibition. Moreover, by systematically targeting thousands of alternative splicing events, CHyMErA identifies exons underlying human cell line fitness. CHyMErA thus represents an effective screening approach for GI mapping and the functional analysis of sizable genomic regions, such as alternative exons.

PMID: 32249828 [PubMed - as supplied by publisher]



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Diagnosing COVID-19: The Disease and Tools for Detection.

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Diagnosing COVID-19: The Disease and Tools for Detection.

ACS Nano. 2020 04 28;14(4):3822-3835

Authors: Udugama B, Kadhiresan P, Kozlowski HN, Malekjahani A, Osborne M, Li VYC, Chen H, Mubareka S, Gubbay JB, Chan WCW

Abstract
COVID-19 has spread globally since its discovery in Hubei province, China in December 2019. A combination of computed tomography imaging, whole genome sequencing, and electron microscopy were initially used to screen and identify SARS-CoV-2, the viral etiology of COVID-19. The aim of this review article is to inform the audience of diagnostic and surveillance technologies for SARS-CoV-2 and their performance characteristics. We describe point-of-care diagnostics that are on the horizon and encourage academics to advance their technologies beyond conception. Developing plug-and-play diagnostics to manage the SARS-CoV-2 outbreak would be useful in preventing future epidemics.

PMID: 32223179 [PubMed - indexed for MEDLINE]



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Targeted thermal stimulation and high-content phenotyping reveal that the C. elegans escape response integrates current behavioral state and past experience.

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Targeted thermal stimulation and high-content phenotyping reveal that the C. elegans escape response integrates current behavioral state and past experience.

PLoS One. 2020;15(3):e0229399

Authors: Byrne Rodgers J, Ryu WS

Abstract
The ability to avoid harmful or potentially harmful stimuli can help an organism escape predators and injury, and certain avoidance mechanisms are conserved across the animal kingdom. However, how the need to avoid an imminent threat is balanced with current behavior and modified by past experience is not well understood. In this work we focused on rapidly increasing temperature, a signal that triggers an escape response in a variety of animals, including the nematode Caenorhabditis elegans. We have developed a noxious thermal response assay using an infrared laser that can be automatically controlled and targeted in order to investigate how C. elegans responds to noxious heat over long timescales and to repeated stimuli in various behavioral and sensory contexts. High-content phenotyping of behavior in individual animals revealed that the C. elegans escape response is multidimensional, with some features that extend for several minutes, and can be modulated by (i) stimulus amplitude; (ii) other sensory inputs, such as food context; (iii) long and short-term thermal experience; and (iv) the animal's current behavioral state.

PMID: 32218560 [PubMed - indexed for MEDLINE]



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Shifts in Ribosome Engagement Impact Key Gene Sets in Neurodevelopment and Ubiquitination in Rett Syndrome.

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Shifts in Ribosome Engagement Impact Key Gene Sets in Neurodevelopment and Ubiquitination in Rett Syndrome.

Cell Rep. 2020 Mar 24;30(12):4179-4196.e11

Authors: Rodrigues DC, Mufteev M, Weatheritt RJ, Djuric U, Ha KCH, Ross PJ, Wei W, Piekna A, Sartori MA, Byres L, Mok RSF, Zaslavsky K, Pasceri P, Diamandis P, Morris Q, Blencowe BJ, Ellis J

Abstract
Regulation of translation during human development is poorly understood, and its dysregulation is associated with Rett syndrome (RTT). To discover shifts in mRNA ribosomal engagement (RE) during human neurodevelopment, we use parallel translating ribosome affinity purification sequencing (TRAP-seq) and RNA sequencing (RNA-seq) on control and RTT human induced pluripotent stem cells, neural progenitor cells, and cortical neurons. We find that 30% of transcribed genes are translationally regulated, including key gene sets (neurodevelopment, transcription and translation factors, and glycolysis). Approximately 35% of abundant intergenic long noncoding RNAs (lncRNAs) are ribosome engaged. Neurons translate mRNAs more efficiently and have longer 3' UTRs, and RE correlates with elements for RNA-binding proteins. RTT neurons have reduced global translation and compromised mTOR signaling, and >2,100 genes are translationally dysregulated. NEDD4L E3-ubiquitin ligase is translationally impaired, ubiquitinated protein levels are reduced, and protein targets accumulate in RTT neurons. Overall, the dynamic translatome in neurodevelopment is disturbed in RTT and provides insight into altered ubiquitination that may have therapeutic implications.

PMID: 32209477 [PubMed - in process]



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A bacteriophage mimic of the bacterial nucleoid-associated protein Fis.

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A bacteriophage mimic of the bacterial nucleoid-associated protein Fis.

Biochem J. 2020 Mar 24;:

Authors: Chakraborti S, Balakrishnan D, Trotter AJ, Gittens WH, Yang AWH, Jolma A, Paterson JR, Świątek S, Plewka J, Curtis FA, Bowers LY, Pålsson LO, Hughes TR, Taube M, Kozak M, Heddle JG, Sharples GJ

Abstract
We report the identification and characterization of a bacteriophage λ-encoded protein, NinH. Sequence homology suggests similarity between NinH and Fis, a bacterial nucleoid-associated protein involved in numerous DNA topology manipulations, including chromosome condensation, transcriptional regulation and phage site-specific recombination. We find that NinH functions as a homodimer and is able to bind and bend double-stranded DNA in vitro. Furthermore, NinH shows a preference for a 15 bp signature sequence related to the degenerate consensus favored by Fis. Structural studies reinforced the proposed similarity to Fis and supported identification of residues involved in DNA binding which were demonstrated experimentally. Overexpression of NinH proved toxic and this correlated with its capacity to associate with DNA. NinH is the first example of a phage-encoded Fis-like nucleoid-associated protein that likely influences phage excision-integration reactions or bacterial gene expression.

PMID: 32207815 [PubMed - as supplied by publisher]



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