Cell identity is governed by the complex regulation of gene expression, represented as gene-regulatory networks1. Here we use gene-regulatory networks inferred from single-cell multi-omics data to perform in silico transcription factor perturbations, simulating the consequent changes in cell identity using only unperturbed wild-type data. We apply this machine-learning-based approach, CellOracle, to well-established paradigms—mouse and human haematopoiesis, and zebrafish embryogenesis—and we correctly model reported changes in phenotype that occur as a result of transcription factor perturbation. Through systematic in silico transcription factor perturbation in the developing zebrafish, we simulate and experimentally validate a previously unreported phenotype that results from the loss of noto, an established notochord regulator. Furthermore, we identify an axial mesoderm regulator, lhx1a. Together, these results show that CellOracle can be used to analyse the regulation of cell identity by transcription factors, and can provide mechanistic insights into development and differentiation.

We present barcoded oligonucleotides ligated on RNA amplified for multiplexed and parallel insitu analyses (BOLORAMIS), a reverse transcription-free method for spatially-resolved, targeted, in situ RNA identification of single or multiple targets. BOLORAMIS was demonstrated on a range of cell types and human cerebral organoids. Singleplex experiments to detect coding and non-coding RNAs in human iPSCs showed a stem-cell signature pattern. Specificity of BOLORAMIS was found to be 92% as illustrated by a clear distinction between human and mouse housekeeping genes in a co-culture system, as well as by recapitulation of subcellular localization of lncRNA MALAT1. Sensitivity of BOLORAMIS was quantified by comparing with single molecule FISH experiments and found to be 11%, 12% and 35% for GAPDH, TFRC and POLR2A, respectively. To demonstrate BOLORAMIS for multiplexed gene analysis, we targeted 96 mRNAs within a co-culture of iNGN neurons and HMC3 human microglial cells. We used fluorescence in situ sequencing to detect error-robust 8-base barcodes associated with each of these genes. We then used this data to uncover the spatial relationship among cells and transcripts by performing single-cell clustering and gene–gene proximity analyses. We anticipate the BOLORAMIS technology for in situ RNA detection to find applications in basic and translational research.

Since excessive amounts of catecholamines are known to produce arrhythmias and increase the plasma level of aminochrome, an oxidation product of catecholamines, we tested the hypothesis that antioxidants may reduce the formation of aminochrome and prevent the catecholamine-induced arrhythmias. For this purpose, Sprague-Dawley rats were pretreated orally, with vitamin A or vitamin C for 21 days, and their effects on ventricular arrhythmias induced by a bolus dose or cumulative doses of intravenous epinephrine were examined. Electrocardiogram recording of these animals revealed that pretreatment with either of these vitamins increased the time of onset and decreased the duration of the epinephrine-induced ventricular arrhythmias. Ventricular fibrillations due to high doses of epinephrine were also prevented by the antioxidant pretreatment. Although pretreatment with either vitamin A or vitamin C did not affect the basal malondialdehyde level in control animals, the increase in malondialdehyde level caused by epinephrine administration was significantly reduced by these agents. The elevated level of plasma aminochrome due to epinephrine was also decreased by vitamins A and C treatments. The results indicate that antioxidant may prevent catecholamine-induced arrhythmias by reducing the formation of aminochrome and thus may provide a new strategy for the management of stress-related heart disease.

We present Barcoded Oligonucleotides Ligated On RNA Amplified for Multiplexed and parallelIn-Situ analysis (BOLORAMIS), a reverse-transcription (RT)-free method for spatially-resolved, targeted, in-situ RNA identification of single or multiple targets. For this proof of concept, we have profiled 154 distinct coding and small non-coding transcripts ranging in sizes 18 nucleotides in length and upwards, from over 200, 000 individual human induced pluripotent stem cells (iPSC) and demonstrated compatibility with multiplexed detection, enabled by fluorescent in-situ sequencing. We use BOLORAMIS data to identify differences in spatial localization and cell-tocell expression heterogeneity. Our results demonstrate BOLORAMIS to be a generalizable toolset for targeted, in-situ detection of coding and small non-coding RNA for single or multiplexed applications. Manuscript:Single-cell transcriptomics is an exponentially evolving field, with recent developments in multiplexed in-situ technologies paving the way for spatial imaging of the genome and transcriptome at an unprecedented resolution 1 . We proposed Fluorescent In Situ Sequencing (FISSEQ) in 2003, and in 2014 demonstrated the generation and sequencing of highly multiplexed, spatially resolved in-situ RNA libraries in cells and tissues 2,3 . While FISSEQ's novelty lay in enabling unbiased discovery, it is primarily limited by poor detection sensitivity, and unsuitability for targeted in-situ transcriptomics (<0.005% compared to single-molecule (sm) FISH) 4,5 . Padlock probes have been demonstrated for in-situ sequencing for multiplexed transcriptomics, with singlebase resolution on a small number of transcripts 6 . However in both methods, detection efficiency is a function of RT efficiency, and subject to noise resulting from variable priming efficiency and 3 | P a g e random priming induced bias 7 . LNA modified primers have been demonstrated to increase RT efficiency with padlock probes (~30%), but require careful calibration and can be costprohibitively expensive for genome-wide applications (~2 order higher cost than unmodified primers) 5,6,8 . smFISH based multiplexed transcriptome imaging methods overcome the limitations of RT by directly hybridizing a plurality of oligo-paint like encoded DNA probes directly on target RNA, and subsequently reading out target locations using a two-stage hybridization scheme 9-14 . While smFISH based methods offer the highest in-situ RNA detection efficiency, it is best suited for large transcripts (>1500nt) and can yield lower signal than RCA based methods (~50-200 vs ~800-1000 fluorophores/transcript, respectively). As a result, a vast segment of biologically interesting RNA species, (including short-non coding RNA) remain vastly inaccessible to Multiplexed In situ (MIS) methods 1,15 . Consequently, there is a strong demand for robust MIS RNA detection methods that can overcome some of the limitations of each method (sensitivity, cost barrier and transcript-size limitation), while retaining the desirable features of these ...