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ASO-based PKM splice-switching therapy inhibits hepatocellular carcinoma growth.
No full textThe M2 pyruvate kinase (PKM2) isoform is upregulated in most cancers and plays a crucial role in regulation of the Warburg effect, which is characterized by the preference for aerobic glycolysis over oxidative phosphorylation for energy metabolism. PKM2 is an alternative-splice isoform of the PKM gene and is a potential therapeutic target. Antisense oligonucleotides (ASO) that switch PKM splicing from the cancer-associated PKM2 to the PKM1 isoform have been shown to induce apoptosis in cultured glioblastoma cells when delivered by lipofection. Here, we explore the potential of ASO-based PKM splice switching as a targeted therapy for liver cancer. A more potent lead cEt/DNA ASO induced PKM splice-switching and inhibited the growth of cultured hepatocellular carcinoma (HCC) cells. This PKM isoform switch increased pyruvate-kinase activity and altered glucose metabolism. In an orthotopic HCC xenograft mouse model, the lead ASO and a second ASO targeting a non-overlapping site inhibited tumor growth. Finally, in a genetic HCC mouse model, a surrogate mouse-specific ASO induced Pkm splice switching and inhibited tumorigenesis, without observable toxicity. These results lay the groundwork for a potential ASO-based splicing therapy for HCC
High-Throughput and Low-Cost Genotyping Method for Plant Genome Editing
No full textGenome editing technologies have revolutionized genetic studies in the life sciences community in recent years. The application of these technologies allows researchers to conveniently generate mutations in almost any gene of interest. This is very useful for species such as maize that have complex genomes and lack comprehensive mutant collections. With the improvement of genome editing tools and transformation methods, these technologies are also widely used to assist breeding research and implementation in maize. However, the detection and genotyping of genomic edits rely on low-throughput, high-cost methods, such as traditional agarose gel electrophoresis and Sanger sequencing. This article describes a method to barcode the target regions of genomic edits from many individuals by low-cost polymerase chain reaction (PCR) amplification. It also employs next-generation sequencing (NGS) to genotype the genome-edited plants at high throughput and low cost. This protocol can be used for initial screening of genomic edits as well as derived population genotyping on a small or large scale, at high efficiency and low cost. © 2021 Wiley Periodicals LLC. Basic Protocol 1: A fast genomic DNA preparation method from genome edited plants Basic Protocol 2: Barcoding the amplicons of edited regions from each individual by two rounds of PCR Basic Protocol 3: Bioinformatics analysis
Corticostriatal Plasticity Established by Initial Learning Persists after Behavioral Reversal.
Get PDFThe neural mechanisms that allow animals to adapt their previously learned associations in response to changes in the environment remain poorly understood. To probe the synaptic mechanisms that mediate such adaptive behavior, we trained mice on an auditory-motor reversal task, and tracked changes in the strength of corticostriatal synapses associated with the formation of learned associations. Using a ChR2-based electrophysiological assay in acute striatal slices, we measured the strength of these synapses after animals learned to pair auditory stimuli with specific actions. Here, we report that the pattern of synaptic strength initially established by learning remains unchanged even when the task contingencies are reversed. Our findings reveal that synaptic changes associated with the initial acquisition of this task are not erased or overwritten, and that behavioral reversal of learned associations may recruit a separate neural circuit. These results suggest a more complex role of the striatum in regulating flexible behaviors where activity of striatal neurons may vary given the behavioral contexts of specific stimulus-action associations
BARcode DEmixing through Non-negative Spatial Regression (BarDensr).
Get PDFModern spatial transcriptomics methods can target thousands of different types of RNA transcripts in a single slice of tissue. Many biological applications demand a high spatial density of transcripts relative to the imaging resolution, leading to partial mixing of transcript rolonies in many voxels; unfortunately, current analysis methods do not perform robustly in this highly-mixed setting. Here we develop a new analysis approach, BARcode DEmixing through Non-negative Spatial Regression (BarDensr): we start with a generative model of the physical process that leads to the observed image data and then apply sparse convex optimization methods to estimate the underlying (demixed) rolony densities. We apply BarDensr to simulated and real data and find that it achieves state of the art signal recovery, particularly in densely-labeled regions or data with low spatial resolution. Finally, BarDensr is fast and parallelizable. We provide open-source code as well as an implementation for the 'NeuroCAAS' cloud platform
MaTAR25: a long non-coding RNA involved in breast cancer progression
No full textWe recently reported on the role of Mammary Tumor Associated RNA 25 (MaTAR25) in mammary tumor cell proliferation, migration, and invasion. MaTAR25 interacts with transcriptional activator protein Pur-beta (Purb) to regulate its downstream targets such as Tensin1 in trans. The human ortholog of MaTAR25, LINC01271, is upregulated with human breast cancer stage and metastasis
Characterization of Gene Expression Signatures for the Identification of Cellular Heterogeneity in the Developing Mammary Gland.
Get PDFThe developing mammary gland depends on several transcription-dependent networks to define cellular identities and differentiation trajectories. Recent technological advancements that allow for single-cell profiling of gene expression have provided an initial picture into the epithelial cellular heterogeneity across the diverse stages of gland maturation. Still, a deeper dive into expanded molecular signatures would improve our understanding of the diversity of mammary epithelial and non-epithelial cellular populations across different tissue developmental stages, mouse strains and mammalian species. Here, we combined differential mammary gland fractionation approaches and transcriptional profiles obtained from FACS-isolated mammary cells to improve our definitions of mammary-resident, cellular identities at the single-cell level. Our approach yielded a series of expression signatures that illustrate the heterogeneity of mammary epithelial cells, specifically those of the luminal fate, and uncovered transcriptional changes to their lineage-defined, cellular states that are induced during gland development. Our analysis also provided molecular signatures that identified non-epithelial mammary cells, including adipocytes, fibroblasts and rare immune cells. Lastly, we extended our study to elucidate expression signatures of human, breast-resident cells, a strategy that allowed for the cross-species comparison of mammary epithelial identities. Collectively, our approach improved the existing signatures of normal mammary epithelial cells, as well as elucidated the diversity of non-epithelial cells in murine and human breast tissue. Our study provides a useful resource for future studies that use single-cell molecular profiling strategies to understand normal and malignant breast development
PHAROH lncRNA regulates Myc translation in hepatocellular carcinoma via sequestering TIAR.
Get PDFHepatocellular carcinoma, the most common type of liver malignancy, is one of the most lethal forms of cancer. We identified a long non-coding RNA, Gm19705, that is over-expressed in hepatocellular carcinoma and mouse embryonic stem cells. We named this RNA Pluripotency and Hepatocyte Associated RNA Overexpressed in HCC, or PHAROH. Depletion of PHAROH impacts cell proliferation and migration, which can be rescued by ectopic expression of PHAROH. RNA-seq analysis of PHAROH knockouts revealed that a large number of genes with decreased expression contain a Myc motif in their promoter. MYC is decreased at the protein level, but not the mRNA level. RNA-antisense pulldown identified nucleolysin TIAR, a translational repressor, to bind to a 71-nt hairpin within PHAROH, sequestration of which increases MYC translation. In summary, our data suggest that PHAROH regulates MYC translation by sequestering TIAR and as such represents a potentially exciting diagnostic or therapeutic target in hepatocellular carcinoma
Hallucination-Like Perception in Mice Provides Neural-Circuit Insights Into Dopamine Hypothesis of Psychosis
No full textOncogenic KRAS engages an RSK1/NF1 pathway to inhibit wild-type RAS signaling in pancreatic cancer.
Get PDFPancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with limited treatment options. Although activating mutations of the KRAS GTPase are the predominant dependency present in >90% of PDAC patients, targeting KRAS mutants directly has been challenging in PDAC. Similarly, strategies targeting known KRAS downstream effectors have had limited clinical success due to feedback mechanisms, alternate pathways, and dose-limiting toxicities in normal tissues. Therefore, identifying additional functionally relevant KRAS interactions in PDAC may allow for a better understanding of feedback mechanisms and unveil potential therapeutic targets. Here, we used proximity labeling to identify protein interactors of active KRAS in PDAC cells. We expressed fusions of wild-type (WT) (BirA-KRAS4B), mutant (BirA-KRAS4BG12D), and nontransforming cytosolic double mutant (BirA-KRAS4BG12D/C185S) KRAS with the BirA biotin ligase in murine PDAC cells. Mass spectrometry analysis revealed that RSK1 selectively interacts with membrane-bound KRASG12D, and we demonstrate that this interaction requires NF1 and SPRED2. We find that membrane RSK1 mediates negative feedback on WT RAS signaling and impedes the proliferation of pancreatic cancer cells upon the ablation of mutant KRAS. Our findings link NF1 to the membrane-localized functions of RSK1 and highlight a role for WT RAS signaling in promoting adaptive resistance to mutant KRAS-specific inhibitors in PDAC
Standardized and reproducible measurement of decision-making in mice.
Get PDFProgress in science requires standardized assays whose results can be readily shared, compared, and reproduced across laboratories. Reproducibility, however, has been a concern in neuroscience, particularly for measurements of mouse behavior. Here, we show that a standardized task to probe decision-making in mice produces reproducible results across multiple laboratories. We adopted a task for head-fixed mice that assays perceptual and value-based decision making, and we standardized training protocol and experimental hardware, software, and procedures. We trained 140 mice across seven laboratories in three countries, and we collected 5 million mouse choices into a publicly available database. Learning speed was variable across mice and laboratories, but once training was complete there were no significant differences in behavior across laboratories. Mice in different laboratories adopted similar reliance on visual stimuli, on past successes and failures, and on estimates of stimulus prior probability to guide their choices. These results reveal that a complex mouse behavior can be reproduced across multiple laboratories. They establish a standard for reproducible rodent behavior, and provide an unprecedented dataset and open-access tools to study decision-making in mice. More generally, they indicate a path toward achieving reproducibility in neuroscience through collaborative open-science approaches