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Shedding Light on Chandelier Cell Development, Connectivity, and Contribution to Neural Disorders
No full textChandelier cells (ChCs) are a unique type of GABAergic interneuron that selectively innervate the axon initial segment (AIS) of excitatory pyramidal neurons; the subcellular domain where action potentials are initiated. The proper genesis and maturation of ChCs is critical for regulating neural ensemble firing in the neocortex throughout development and adulthood. Recently, genetic and molecular studies have shed new light on the complex innerworkings of ChCs in health and disease. This review presents an overview of recent studies on the developmental origins, migratory properties, and morphology of ChCs. In addition, attention is given to newly identified molecules regulating ChC morphogenesis and connectivity as well as recent work linking ChC dysfunction to neural disorders, including schizophrenia, epilepsy, and autism spectrum disorder (ASD)
A fluorogenic cyclic peptide for imaging and quantification of drug-induced apoptosis
No full textProgrammed cell death or apoptosis is a central biological process that is dysregulated in many diseases, including inflammatory conditions and cancer. The detection and quantification of apoptotic cells in vivo is hampered by the need for fixatives or washing steps for non-fluorogenic reagents, and by the low levels of free calcium in diseased tissues that restrict the use of annexins. In this manuscript, we report the rational design of a highly stable fluorogenic peptide (termed Apo-15) that selectively stains apoptotic cells in vitro and in vivo in a calcium-independent manner and under wash-free conditions. Furthermore, using a combination of chemical and biophysical methods, we identify phosphatidylserine as a molecular target of Apo-15. We demonstrate that Apo-15 can be used for the quantification and imaging of drug-induced apoptosis in preclinical mouse models, thus creating opportunities for assessing the in vivo efficacy of anti-inflammatory and anti-cancer therapeutics
Mechanisms of Phosphorylation-Mediated Regulation of the RNAi Effector Protein Argonaute 2
No full textDisruption of splicing-regulatory elements using CRISPR/Cas9 to rescue spinal muscular atrophy in human iPSCs and mice
Get PDFWe here report a genome-editing strategy to correct spinal muscular atrophy (SMA). Rather than directly targeting the pathogenic exonic mutations, our strategy employed Cas9 and guide-sgRNA for the targeted disruption of intronic splicing-regulatory elements. We disrupted intronic splicing silencers (ISSs, including ISS-N1 and ISS + 100) of survival motor neuron (SMN) 2, a key modifier gene of SMA, to enhance exon 7 inclusion and full-length SMN expression in SMA iPSCs. Survival of splicing-corrected iPSC-derived motor neurons was rescued with SMN restoration. Furthermore, co-injection of Cas9 mRNA from Streptococcus pyogenes (SpCas9) or Cas9 from Staphylococcus aureus (SaCas9) alongside their corresponding sgRNAs targeting ISS-N1 into zygotes rescued 56% and 100% of severe SMA transgenic mice (Smn , SMN2 ). The median survival of the resulting mice was extended to >400 days. Collectively, our study provides proof-of-principle for a new strategy to therapeutically intervene in SMA and other RNA-splicing-related diseases. -/- tg/
MAVE-NN: learning genotype-phenotype maps from multiplex assays of variant effect
Get PDFMultiplex assays of variant effect (MAVEs), which include massively parallel reporter assays (MPRAs) and deep mutational scanning (DMS) experiments, are being rapidly adopted in many areas of biology. However, inferring quantitative models of genotype-phenotype (G-P) maps from MAVE data remains challenging, and different inference approaches have been advocated in different MAVE contexts. Here we introduce a conceptually unified approach to the problem of learning G-P maps from MAVE data. Our strategy is grounded in concepts from information theory, and is based on the view of G-P maps as a form of information compression. We also introduce MAVE-NN, a Python package that implements this approach using a neural network backend. The capabilities and advantages of MAVE-NN are then demonstrated on three diverse DMS and MPRA datasets. MAVE-NN thus fills a major need in the computational analysis of MAVE data. Installation instructions, tutorials, and documentation are provided at https://mavenn.readthedocs.io
Publisher Correction: Structure and assembly of calcium homeostasis modulator proteins.
No full textAn amendment to this paper has been published and can be accessed via a link at the top of the paper
CXCR4 inhibition in human pancreatic and colorectal cancers induces an integrated immune response
No full textInhibition of the chemokine receptor CXCR4 in combination with blockade of the PD-1/PD-L1 T cell checkpoint induces T cell infiltration and anticancer responses in murine and human pancreatic cancer. Here we elucidate the mechanism by which CXCR4 inhibition affects the tumor immune microenvironment. In human immune cell-based chemotaxis assays, we find that CXCL12-stimulated CXCR4 inhibits the directed migration mediated by CXCR1, CXCR3, CXCR5, CXCR6, and CCR2, respectively, chemokine receptors expressed by all of the immune cell types that participate in an integrated immune response. Inhibiting CXCR4 in an experimental cancer medicine study by 1-wk continuous infusion of the small-molecule inhibitor AMD3100 (plerixafor) induces an integrated immune response that is detected by transcriptional analysis of paired biopsies of metastases from patients with microsatellite stable colorectal and pancreatic cancer. This integrated immune response occurs in three other examples of immune-mediated damage to noninfected tissues: Rejecting renal allografts, melanomas clinically responding to anti-PD1 antibody therapy, and microsatellite instable colorectal cancers. Thus, signaling by CXCR4 causes immune suppression in human pancreatic ductal adenocarcinoma and colorectal cancer by impairing the function of the chemokine receptors that mediate the intratumoral accumulation of immune cells
Loss of Small-RNA-Directed DNA Methylation in the Plant Cell Cycle Promotes Germline Reprogramming and Somaclonal Variation
No full text5-methyl cytosine is widespread in plant genomes in both CG and non-CG contexts. During replication, hemi-methylation on parental DNA strands guides symmetric CG methylation on nascent strands, but non-CG methylation requires modified histones and small RNA guides. Here, we used immortalized Arabidopsis cell suspensions to sort replicating nuclei and determine genome-wide cytosine methylation dynamics during the plant cell cycle. We find that symmetric mCG and mCHG are selectively retained in actively dividing cells in culture, whereas mCHH is depleted. mCG becomes transiently asymmetric during S phase but is rapidly restored in G2, whereas mCHG remains asymmetric throughout the cell cycle. Hundreds of loci gain ectopic CHG methylation, as well as 24-nt small interfering RNAs (siRNAs) and histone H3 lysine dimethylation (H3K9me2), without gaining CHH methylation. This suggests that spontaneous epialleles that arise in plant cell cultures are stably maintained by siRNA and H3K9me2 independent of the canonical RNA-directed DNA methylation (RdDM) pathway. In contrast, loci that fail to produce siRNA may be targeted for demethylation when the cell cycle arrests. Comparative analysis with methylomes of various tissues and cell types suggests that loss of small-RNA-directed non-CG methylation during DNA replication promotes germline reprogramming and epigenetic variation in plants propagated as clones
Highly accurate long-read HiFi sequencing data for five complex genomes
No full textThe PacBio(®) HiFi sequencing method yields highly accurate long-read sequencing datasets with read lengths averaging 10-25 kb and accuracies greater than 99.5%. These accurate long reads can be used to improve results for complex applications such as single nucleotide and structural variant detection, genome assembly, assembly of difficult polyploid or highly repetitive genomes, and assembly of metagenomes. Currently, there is a need for sample data sets to both evaluate the benefits of these long accurate reads as well as for development of bioinformatic tools including genome assemblers, variant callers, and haplotyping algorithms. We present deep coverage HiFi datasets for five complex samples including the two inbred model genomes Mus musculus and Zea mays, as well as two complex genomes, octoploid Fragaria × ananassa and the diploid anuran Rana muscosa. Additionally, we release sequence data from a mock metagenome community. The datasets reported here can be used without restriction to develop new algorithms and explore complex genome structure and evolution. Data were generated on the PacBio Sequel II System