Featured
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| Open AccessComprehensive sequence-to-function mapping of cofactor-dependent RNA catalysis in the glmS ribozyme
RNA sequence-structure-function relationships can be studied via assays of variant activity. The authors developed a method to systematically determine ribozyme variant kinetics, using it to map double-mutant interactions and show that cleavage rate drives sequence conservation in the glmS ribozyme.
- Johan O. L. Andreasson
- , Andrew Savinov
- & William J. Greenleaf
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Article
| Open AccessDeveloping a highly efficient hydroxytyrosol whole-cell catalyst by de-bottlenecking rate-limiting steps
Whole-cell catalyst-based hydroxytyrosol production is low. Here, the authors increase the efficiency of its production in E. coli by de-bottlenecking two enzymatic steps catalyzed by monooxygenase and tyramine oxidase using structure-based enzyme redesign or in vivo-directed evolution with the aid of a newly developed biosensor.
- Jun Yao
- , Yang He
- & Shuang-Yan Tang
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Article
| Open AccessFull-length transcript characterization of SF3B1 mutation in chronic lymphocytic leukemia reveals downregulation of retained introns
Long-read sequencing is useful in determining exon-connectivity of full-length mRNA isoforms. Here, by long-read nanopore sequencing, the authors report that intron retention is downregulated in SF3B1 mutant chronic lymphocytic leukemia cells than normal B cells.
- Alison D. Tang
- , Cameron M. Soulette
- & Angela N. Brooks
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Article
| Open AccessAmino acid levels determine metabolism and CYP450 function of hepatocytes and hepatoma cell lines
Hepatocytes grown in a dish are immature and do not metabolize compounds as a real liver would. Here, the authors supply stem cell-derived hepatocytes with amino acids at a higher concentration than nutritionally necessary, changing the metabolism of these cells, making them more mature and useful for drug screening and toxicity studies.
- Ruben Boon
- , Manoj Kumar
- & Catherine M. Verfaillie
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Article
| Open AccessGenetic screens in isogenic mammalian cell lines without single cell cloning
Isogenic pairs of cell lines are powerful tools but time-consuming to generate. Here the authors conduct genome-wide genetic interactions screens of ‘anchor’ genes with SaCas9 and SpCas9.
- Peter C. DeWeirdt
- , Annabel K. Sangree
- & John G. Doench
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Article
| Open AccessHigh-throughput identification of synthetic riboswitches by barcode-free amplicon-sequencing in human cells
Riboswitches can mediate ligand-dependent RNA cleavage and splicing to control gene expression. Here the authors present a method to functionally screen large libraries and identify functional variants.
- Benjamin Strobel
- , Maike Spöring
- & Sebastian Kreuz
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Article
| Open AccessBridging non-overlapping reads illuminates high-order epistasis between distal protein sites in a GPCR
Epistasis effects among amino acids at distal sites within binding pockets can have important impacts on protein fitness landscapes. Here the authors present BRIDGE, which matches non-overlapping sequence reads with their cognate DNA templates.
- Justin I. Yoo
- , Patrick S. Daugherty
- & Michelle A. O’Malley
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Article
| Open AccessTargeting FROUNT with disulfiram suppresses macrophage accumulation and its tumor-promoting properties
The cytoplasmic protein FROUNT can bind to chemokine receptors and enhance chemokine signalling. Here, the authors show that inhibiting FROUNT in macrophages either by knockdown of the gene or using the anti-alcoholism drug disulfiram, results in a reduction in tumour growth.
- Yuya Terashima
- , Etsuko Toda
- & Kouji Matsushima
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Article
| Open AccessChaperone mediated detection of small molecule target binding in cells
Quantitative profiling of small molecule-protein binding in cells can aid basic biochemical research and drug discovery. Here, the authors develop the Heat Shock Protein Inhibition Protein Stability Assay (HIPStA) as a high-throughput method to assess cellular target engagement and identify new drug targets.
- Kelvin F. Cho
- , Taylur P. Ma
- & Robert A. Blake
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Article
| Open AccessTargeting the tumor vasculature with engineered cystine-knot miniproteins
Cystine-knot miniprotein are small, highly stable, disulfide-rich peptides with increasing potential as drugs and tumor imaging agents. Here the authors develop cystine-knot miniproteins targeting the vascular tumor marker EDB, and use them as probes for in vivo tumor vasculature imaging.
- Bonny Gaby Lui
- , Nadja Salomon
- & Ugur Sahin
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Article
| Open AccessSynthetic chimeric nucleases function for efficient genome editing
CRISPR-Cas systems have well characterized, modular structures. Here the authors use that architecture to design a Cas12a library of 560 synthetic chimeras, with altered PAM preferences and specificities.
- R. M. Liu
- , L. L. Liang
- & R. T. Gill
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Article
| Open AccessA high-throughput screen identifies that CDK7 activates glucose consumption in lung cancer cells
Many cancer cells have increased glucose consumption compared to normal cells, a feature that can be exploited therapeutically. Here, the authors carry out a chemical screen and identify compounds that selectively blocks glucose metabolism in non-small-cell lung cancer cell lines.
- Chiara Ghezzi
- , Alicia Wong
- & Peter M. Clark
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Article
| Open AccessRapid selection and identification of functional CD8+ T cell epitopes from large peptide-coding libraries
High-throughput assays for TCR specificity are a bottleneck in understanding T cell immunity and harnessing it for medicine. Here the authors develop a functional screening method to identify T cell specificity in the natural context of peptide-MHC presentation, enabling detection of physiologically relevant T cell antigens from large libraries of peptide-coding sequences.
- Govinda Sharma
- , Craig M. Rive
- & Robert A. Holt
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Article
| Open AccessDe novo identification of essential protein domains from CRISPR-Cas9 tiling-sgRNA knockout screens
Tiling-sgRNA designs allow the in situ evaluation of protein domain functions. Here the authors present ProTiler - a computational method to predict CRISPR knockout hyper-sensitive regions, revealing previously unannotated domains.
- Wei He
- , Liang Zhang
- & Han Xu
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Article
| Open AccessMassive computational acceleration by using neural networks to emulate mechanism-based biological models
Mechanistic models provide valuable insights, but large-scale simulations are computationally expensive. Here, the authors show that it is possible to explore the dynamics of a mechanistic model over a large set of parameters by training an artificial neural network on a smaller set of simulations.
- Shangying Wang
- , Kai Fan
- & Lingchong You
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Article
| Open AccessAn in vitro model maintaining taxon-specific functional activities of the gut microbiome
The authors present an in vitro gut microbiome model that maintains the functional and compositional profiles of individual gut microbiomes, as assessed by metaproteomics. Gut microbiome responses to metformin in this model correlate with those shown in mice fed a high-fat diet.
- Leyuan Li
- , Elias Abou-Samra
- & Daniel Figeys
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Article
| Open AccessMaximizing binary interactome mapping with a minimal number of assays
Comprehensive mapping of binary protein-protein interactions requires to combine several complementary assays. Here, the authors show that complete coverage could be reached with a minimal number of assays as long as they explore various experimental conditions.
- Soon Gang Choi
- , Julien Olivet
- & Yves Jacob
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Article
| Open AccessRobust continuous in vitro culture of the Plasmodium cynomolgi erythrocytic stages
Present understanding of Plasmodium vivax biology is hampered by its inability to grow in vitro. Here, the authors developed an in vitro culture of its simian counterpart, P. cynomolgi, which shares morphological and phenotypic similarities with P. vivax, initiating a new phase in vivax research.
- Adeline C. Y. Chua
- , Jessica Jie Ying Ong
- & Pablo Bifani
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Article
| Open AccessSaturation mutagenesis of twenty disease-associated regulatory elements at single base-pair resolution
Interpreting genetic variation in the noncoding genome remains challenging, with functional effects difficult to predict. Here, the authors perform saturation mutagenesis combined with massively parallel reporter assays for 20 disease-associated regulatory elements, quantifying the effects of over 30,000 variants.
- Martin Kircher
- , Chenling Xiong
- & Nadav Ahituv
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Article
| Open AccessPooled library screening with multiplexed Cpf1 library
AsCpf1 is an alternative nuclease to Cas9 for CRISPR mediated genome engineering. Here the authors demonstrate functional genomic screens with AsCpf1 that minimize library size with no loss in gene targeting efficiency.
- Jintan Liu
- , Sanjana Srinivasan
- & Giulio Draetta
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| Open AccessPooled clone collections by multiplexed CRISPR-Cas12a-assisted gene tagging in yeast
Construction of yeast libraries is time-consuming, costly and limited to the genetic background of the chosen strain. Here the authors present CASTLING which uses CRISPR-Cas12a and oligonucleotide pools to rapidly generate pooled libraries with large insertions such as fluorescent protein tags.
- Benjamin C. Buchmuller
- , Konrad Herbst
- & Michael Knop
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Article
| Open AccessA high-throughput screening and computation platform for identifying synthetic promoters with enhanced cell-state specificity (SPECS)
Synthetic promoters can be superior to native ones but the design is challenging without knowledge of gene regulation. Here the authors develop a pipeline that allows for screening a synthetic promoter library to identify high performance promoters in potentially any given cell state of interest.
- Ming-Ru Wu
- , Lior Nissim
- & Timothy K. Lu
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Article
| Open AccessLive imaging screen reveals that TYRO3 and GAK ensure accurate spindle positioning in human cells
Correct spindle positioning is critical for cell division but the full set of proteins regulating this is unclear. Here, with a live imaging siRNA-based screen in human cells, the authors identify 16 candidates required for this process, 11 of which were previously unassociated with such a function, including TYRO3 and GAK.
- Benita Wolf
- , Coralie Busso
- & Pierre Gönczy
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| Open AccessMarrying chemistry with biology by combining on-chip solution-based combinatorial synthesis and cellular screening
High-throughput cell-based screening of compound libraries is utilised in drug development; however, a lack of compatible methods limits direct synthesis and testing. Here, the authors present a diverse chip based synthesis system which can be combined with cell screening and demonstrate the application.
- Maximilian Benz
- , Mijanur R. Molla
- & Pavel A. Levkin
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Article
| Open AccessCommunity assessment to advance computational prediction of cancer drug combinations in a pharmacogenomic screen
Resistance to first line treatment is a major hurdle in cancer treatment, that can be overcome with drug combinations. Here, the authors provide a large drug combination screen across cancer cell lines to benchmark crowdsourced methods and to computationally predict drug synergies.
- Michael P. Menden
- , Dennis Wang
- & Julio Saez-Rodriguez
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Article
| Open AccessEngineering brain activity patterns by neuromodulator polytherapy for treatment of disorders
Brain disorders are associated with network dysfunctions that are not addressed by conventional drug screens. Here, the authors use high-throughput functional imaging of brain activity in zebrafish larvae to study the effects of individual drugs on network connectivity and demonstrate an algorithm that predicts the most effective drug combinations to normalize both the activity patterns and the animal behavior.
- Mostafa Ghannad-Rezaie
- , Peter M. Eimon
- & Mehmet Fatih Yanik
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Article
| Open AccessDevelopment of human cGAS-specific small-molecule inhibitors for repression of dsDNA-triggered interferon expression
Cyclic GMP-AMP synthase (cGAS) is involved in the modulation of inflammatory responses. Here, the authors present small-molecule inhibitors of human cGAS, characterize their interaction with the protein, and show that the compounds are active in interferon-producing cells including primary human macrophages.
- Lodoe Lama
- , Carolina Adura
- & Thomas Tuschl
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Article
| Open AccessMEK inhibitors activate Wnt signalling and induce stem cell plasticity in colorectal cancer
Wnt signaling is necessary for colorectal cancer tumorigenesis and stem cell maintenance. Here, the authors identify MEK1/2 inhibitors as potent activators of Wnt/β-catenin signalling and show that clinically approved MEK inhibitors inadvertently induce stem cell plasticity in colorectal cancer
- Tianzuo Zhan
- , Giulia Ambrosi
- & Michael Boutros
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Article
| Open AccessCapturing single-cell heterogeneity via data fusion improves image-based profiling
A challenge with single-cell resolution methods is that cell heterogeneity should be captured while allowing for comparisons between populations. Here the authors fuse information from the dispersion profiles with the average profiles at the level of profiles’ similarity matrices for single cell imaging data.
- Mohammad H. Rohban
- , Hamdah S. Abbasi
- & Anne E. Carpenter
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Article
| Open AccessA survival selection strategy for engineering synthetic binding proteins that specifically recognize post-translationally phosphorylated proteins
Protein phosphorylation helps to control many important cellular activities. Here the authors describe a genetic selection strategy to isolate designed ankyrin repeat proteins that bind specifically to phosphomodified targets.
- Bunyarit Meksiriporn
- , Morgan B. Ludwicki
- & Matthew P. DeLisa
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| Open AccessMetabolic profiling of cancer cells reveals genome-wide crosstalk between transcriptional regulators and metabolism
Aberrant gene expression in cancer coincides with drastic changes in metabolism. Here, the authors combined metabolome, transcriptome and proteome data in 54 cancer cell lines to uncover a genome-scale network of associations between transcriptional regulators and metabolites.
- Karin Ortmayr
- , Sébastien Dubuis
- & Mattia Zampieri
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Article
| Open AccessPurification of cross-linked RNA-protein complexes by phenol-toluol extraction
RNA binding proteins are important regulators of RNA function. Here the authors describe a method for isolation of RNA-protein complexes that does not rely on a specific RNA sequence or motif, and demonstrate the approach by providing the global RNA-bound proteomes of human HEK293 cells and Salmonella Typhimurium.
- Erika C. Urdaneta
- , Carlos H. Vieira-Vieira
- & Benedikt M. Beckmann
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Article
| Open AccessThe flavonoid 4,4′-dimethoxychalcone promotes autophagy-dependent longevity across species
Although ageing is the most important risk factor for chronic ailments, effective interventions remain rare. Here, the authors identify the flavonoid 4,4’-dimethoxychalcone and demonstrate that it extends lifespan and promotes health in multiple organisms by inducing autophagy.
- Didac Carmona-Gutierrez
- , Andreas Zimmermann
- & Frank Madeo
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Article
| Open AccessPrecise tuning of gene expression levels in mammalian cells
Analogue regulation of gene expression is important for normal function in mammals but existing genetic technologies are designed to achieve ON/OFF control. Here the authors develop synthetic microRNA silencing-mediated fine-tuners (miSFITs) to precisely control target gene expression levels.
- Yale S. Michaels
- , Mike B. Barnkob
- & Tudor A. Fulga
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| Open AccessHigh-throughput single-cell rheology in complex samples by dynamic real-time deformability cytometry
Real-time deformability cytometry (RT-DC) is used for mechanical cell phenotyping but is limited to a single snapshot per cell and can only measure elastic modulus. Here the authors introduce dynamic RT-DC which can measure elasticity and viscosity in single cells by following dynamic cell shape changes.
- Bob Fregin
- , Fabian Czerwinski
- & Oliver Otto
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Article
| Open AccessA macrophage-based screen identifies antibacterial compounds selective for intracellular Salmonella Typhimurium
Salmonella Typhimurium replicates within host phagocytes. Here, the authors show that a known psychoactive drug (metergoline) inhibits growth of the pathogen in macrophages, disrupts the proton motive force at the bacterial cytoplasmic membrane, and extends animal survival during systemic infection.
- Michael J. Ellis
- , Caressa N. Tsai
- & Eric D. Brown
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| Open AccessIndustrial scale high-throughput screening delivers multiple fast acting macrofilaricides
Parasitic nematodes causing onchocerciasis and lymphatic filariasis rely on a bacterial endosymbiont, Wolbachia, which is a validated therapeutic target. Here, Clare et al. perform a high-throughput screen of 1.3 million compounds and identify 5 chemotypes with faster kill rates than existing anti-Wolbachia drugs.
- Rachel H. Clare
- , Catherine Bardelle
- & Stephen A. Ward
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Article
| Open AccessA synthetic microbial biosensor for high-throughput screening of lactam biocatalysts
Efficient biosynthesis of lactams is still undesirable due to lacking of suitable enzyme. Here, the authors develop a sensitive transcription factor-based biosensor for high-throughput screening of marine metagenome and find a cyclase that can cyclize ω-amino fatty acids to lactam.
- Soo-Jin Yeom
- , Moonjeong Kim
- & Seung-Goo Lee
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Article
| Open AccessLine excitation array detection fluorescence microscopy at 0.8 million frames per second
Demanding biological imaging applications require extremely fast detection methods. Here, the authors demonstrate Line Excitation Array Detection, their developed technique for performing fluorescence microscopy in 3D with 0.8 million frames per second, by applying it to flow cytometry and neuron detection.
- Chris Martin
- , Tianqi Li
- & Adela Ben-Yakar
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Article
| Open AccessExpression-based drug screening of neural progenitor cells from individuals with schizophrenia
Unbiased large scale screening of small molecules for drug discovery in psychiatric disease is technically challenging and financially costly. Here, Readhead and colleagues integrate in silico and in vitro approaches to design and conduct transcriptomic drug screening in schizophrenia patient-derived neural cells, in order to survey novel pathologies and points of intervention.
- Benjamin Readhead
- , Brigham J. Hartley
- & Kristen J. Brennand
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Article
| Open AccessDRUG-seq for miniaturized high-throughput transcriptome profiling in drug discovery
RNA-seq is a powerful tool to investigate how drugs affect the transcriptome but library construction can be costly. Here the authors introduce DRUG-seq, an automated platform for high-throughput transcriptome profiling.
- Chaoyang Ye
- , Daniel J. Ho
- & Ajamete Kaykas
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Article
| Open AccessMapping protein selectivity landscapes using multi-target selective screening and next-generation sequencing of combinatorial libraries
Characterizing the binding selectivity landscape of interacting proteins is crucial in protein engineering. Here the authors use multi-target selective library screening and in silico next-generation sequencing to map the binding landscape of proteins and produce improved proteases inhibitors.
- Si Naftaly
- , Itay Cohen
- & Niv Papo
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Article
| Open Accessrec-YnH enables simultaneous many-by-many detection of direct protein–protein and protein–RNA interactions
Current methods to unveil protein–protein and protein–RNA interactions often involve long, costly procedures. Here the authors present a rapid yeast 2- and 3-hybrid screening pipeline to detect interactions within protein libraries or between protein libraries and RNA fragment pools.
- Jae-Seong Yang
- , Mireia Garriga-Canut
- & Sebastian P. Maurer
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Article
| Open AccessGraphDDP: a graph-embedding approach to detect differentiation pathways in single-cell-data using prior class knowledge
Inference and representation of differentiation trajectories from single cell RNA-seq data remains a challenge. Here, the authors offer a visualization approach that captures both continuous differentiation trajectories and discrete clusters representing metastable states along the trajectories.
- Fabrizio Costa
- , Dominic Grün
- & Rolf Backofen
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Article
| Open AccessUtilization of rare codon-rich markers for screening amino acid overproducers
Current toxic analogues-based amino acid overproducer screening method cannot provide accuracy, sensitivity and high throughput simultaneously. Here, the authors use rare codon rich marker for the selection of overproducers of multiple amino acids from random mutation libraries of E. coli and C. glutamicum.
- Bo Zheng
- , Xiaoyan Ma
- & Yi-Xin Huo
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Article
| Open AccessEngineered bidirectional promoters enable rapid multi-gene co-expression optimization
Classic monodirectional promoters are of limited use for multiple gene co-expression. Here the authors generate a library of 168 bidirectional promoters for the yeast K. phaffii (syn. P. pastoris) with diverse expression profiles to optimize metabolic pathway design.
- Thomas Vogl
- , Thomas Kickenweiz
- & Anton Glieder
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Article
| Open AccessDecoding a cancer-relevant splicing decision in the RON proto-oncogene using high-throughput mutagenesis
Alternative splicing is a critical step in eukaryotic gene expression but its molecular rules are not fully understood. Here, the authors develop a high-throughput mutagenesis approach to comprehensively characterise determinants of alternative splicing for the RON proto-oncogene.
- Simon Braun
- , Mihaela Enculescu
- & Kathi Zarnack
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Article
| Open AccessDirected evolution of CRISPR-Cas9 to increase its specificity
Undesired off-target effects can hamper the use of CRISPR-Cas9 in therapeutic applications. Here the authors use a directed evolution approach to develop Sniper-Cas9 which combines high specificity with no loss of on-target activity.
- Jungjoon K. Lee
- , Euihwan Jeong
- & Jin-Soo Kim
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Article
| Open AccessDiscovery of a drug candidate for GLIS3-associated diabetes
GLIS3 mutations are associated with type 1, type 2, and neonatal diabetes. Here, the authors generate mono-hormonal glucose-responding pancreatic β-like cells in vitro and through a screen identify a drug that rescues pancreatic β-like cell death in GLIS3 mutants by inhibiting the abnormally activated TGFβ pathway.
- Sadaf Amin
- , Brandoch Cook
- & Shuibing Chen