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Acute myeloid leukaemia (AML) is a rapidly fatal blood cancer that is characterised by the accumulation of immature myeloid cells in the blood and bone marrow as a result of blocked differentiation. Methods which identify master transcriptional regulators of AML subtype-specific leukaemia cell states and their combinations could be critical for discovering novel differentiation-inducing therapies. In this proof-of-concept study, we demonstrate a novel utility of the Mogrify ® algorithm in identifying combinations of transcription factors (TFs) and drugs, which recapitulate granulocytic differentiation of the NB4 acute promyelocytic leukaemia (APL) cell line, using two different approaches. In the first approach, Connectivity Map (CMAP) analysis of these TFs and their target networks outperformed standard approaches, retrieving ATRA as the top hit. We identify dimaprit and mebendazole as a drug combination which induces myeloid differentiation. In the second approach, we show that genetic manipulation of specific Mogrify ®-identified TFs (MYC and IRF1) leads to co-operative induction of APL differentiation, as does pharmacological targeting of these TFs using currently available compounds. We also show that loss of IRF1 blunts ATRA-mediated differentiation, and that MYC represses IRF1 expression through recruitment of PML-RARα, the driver fusion oncoprotein in APL, to the IRF1 promoter. Finally, we demonstrate that these drug combinations can also induce differentiation of primary patient-derived APL cells, and highlight the potential of targeting MYC and IRF1 in high-risk APL. Thus, these results suggest that Mogrify ® could be used for drug discovery or repositioning in leukaemia differentiation therapy for other subtypes of leukaemia or cancers.

Motivation The creation and analysis of gene regulatory networks have been the focus of bioinformatics research and underpins much of what is known about gene regulation. However, as a result of a bias in the availability of data types that are collected, the vast majority of gene regulatory network resources and tools have focused on either transcriptional regulation or protein–protein interactions. This has left other areas of regulation, for instance, translational regulation, vastly underrepresented despite them having been shown to play a critical role in both health and disease. Results In order to address this, we have developed CLIPreg, a package that integrates RNA, Ribo and CLIP- sequencing data in order to construct translational regulatory networks coordinated by RNA-binding proteins and micro-RNAs. This is the first tool of its type to be created, allowing for detailed investigation into a previously unseen layer of regulation. Availability and implementation CLIPreg is available at https://github.com/SGDDNB/CLIPreg. Supplementary information Supplementary data are available at Bioinformatics online.

BackgroundThe protein kinase PKN2 is required for embryonic development, and PKN2 knockout mice die as a result of failure in expansion of mesoderm tissues, cardiac development and neural tube closure. In the adult, cardiomyocyte PKN2 and PKN1 (in combination) are required for cardiac adaptation to pressure-overload. The role of PKN2 in contractile cardiomyocytes during development and its role in the adult heart remain to be fully established.MethodsWe used mice with cardiomyocyte-directed knockout of PKN2 or global PKN2 haploinsufficiency. Cardiac function and dimensions were assessed with high resolution episcopic microscopy, MRI, micro-CT and echocardiography. Biochemical and histological changes were assessed.ResultsCardiomyocyte-directed PKN2 knockout embryos displayed striking abnormalities in the compact myocardium, with frequent myocardial clefts and diverticula, ventricular septal defects and abnormal heart shape. The sub-Mendelian homozygous knockout survivors developed cardiac failure. RNASeq data showed upregulation of PKN2 in patients with dilated cardiomyopathy, suggesting an involvement in adult heart disease. Given the rarity of homozygous survivors with cardiomyocyte-specific deletion of PKN2, this was explored using mice with constitutive heterozygous PKN2 knockout. Cardiac hypertrophy resulting from hypertension induced by angiotensin II was reduced in haploinsufficient PKN2 mice relative to wild-type littermates, with suppression of cardiomyocyte hypertrophy and cardiac fibrosis.ConclusionsCardiomyocyte PKN2 is essential for heart development and formation of compact myocardium, and is also required for cardiac hypertrophy in hypertension. Thus, PKN signalling may offer therapeutic options for managing congenital and adult heart diseases.

Mito-SEPs are small open reading frame-encoded peptides that localize to the mitochondria to regulate metabolism. Motivated by an intriguing negative association between mito-SEPs and inflammation, here we screen for mito-SEPs that modify inflammatory outcomes and report a mito-SEP named “Modulator of cytochrome C oxidase during Inflammation” (MOCCI) that is upregulated during inflammation and infection to promote host-protective resolution. MOCCI, a paralog of the NDUFA4 subunit of cytochrome C oxidase (Complex IV), replaces NDUFA4 in Complex IV during inflammation to lower mitochondrial membrane potential and reduce ROS production, leading to cyto-protection and dampened immune response. The MOCCI transcript also generates miR-147b, which targets the NDUFA4 mRNA with similar immune dampening effects as MOCCI, but simultaneously enhances RIG-I/MDA-5-mediated viral immunity. Our work uncovers a dual-component pleiotropic regulation of host inflammation and immunity by MOCCI (C15ORF48) for safeguarding the host during infection and inflammation., Mito-SEPs are small peptides that can modulate oxidative metabolism in mitochondria. Here the authors show that C15ORF48 encodes a mito-SEP, MOCCI, capable of altering mitochondria respiration to suppress inflammation, while C15ORF48 3’ untranslated region also contains a miRNA, miR-147b, that synergizes with MOCCI to modulate host anti-viral responses.

Human pluripotent and trophoblast stem cells have been essential alternatives to blastocysts for understanding early human development1–4. However, these simple culture systems lack the complexity to adequately model the spatiotemporal cellular and molecular dynamics that occur during early embryonic development. Here we describe the reprogramming of fibroblasts into in vitro three-dimensional models of the human blastocyst, termed iBlastoids. Characterization of iBlastoids shows that they model the overall architecture of blastocysts, presenting an inner cell mass-like structure, with epiblast- and primitive endoderm-like cells, a blastocoel-like cavity and a trophectoderm-like outer layer of cells. Single-cell transcriptomics further confirmed the presence of epiblast-, primitive endoderm-, and trophectoderm-like cells. Moreover, iBlastoids can give rise to pluripotent and trophoblast stem cells and are capable of modelling, in vitro, several aspects of the early stage of implantation. In summary, we have developed a scalable and tractable system to model human blastocyst biology; we envision that this will facilitate the study of early human development and the effects of gene mutations and toxins during early embryogenesis, as well as aiding in the development of new therapies associated with in vitro fertilization. Human fibroblasts are reprogrammed to generate blastocyst-like structures called iBlastoids, which recapitulate aspects of embryo implantation.

The combination of single-cell RNA sequencing with CRISPR inhibition/activation provides a high-throughput approach to simultaneously study the effects of hundreds if not thousands of gene perturbations in a single experiment. One recent development in CRISPR-based single-cell techniques introduces a feature barcoding technology that allows for the simultaneous capture of mRNA and guide RNA (gRNA) from the same cell. This is achieved by introducing a capture sequence, whose complement can be incorporated into each gRNA and that can be used to amplify these features prior to sequencing. However, because the technology is in its infancy, there is little information available on how such experimental parameters can be optimized. To overcome this, we varied the capture sequence, capture sequence position, and gRNA backbone to identify an optimal gRNA scaffold for CRISPR activation gene perturbation studies. We provide a report on our screening approach along with our observations and recommendations for future use.

The reprogramming of human somatic cells to primed or naive induced pluripotent stem cells recapitulates the stages of early embryonic development 1–6. The molecular mechanism that underpins these reprogramming processes remains largely unexplored, which impedes our understanding and limits rational improvements to reprogramming protocols. Here, to address these issues, we reconstruct molecular reprogramming trajectories of human dermal fibroblasts using single-cell transcriptomics. This revealed that reprogramming into primed and naive pluripotency follows diverging and distinct trajectories. Moreover, genome-wide analyses of accessible chromatin showed key changes in the regulatory elements of core pluripotency genes, and orchestrated global changes in chromatin accessibility over time. Integrated analysis of these datasets revealed a role for transcription factors associated with the trophectoderm lineage, and the existence of a subpopulation of cells that enter a trophectoderm-like state during reprogramming. Furthermore, this trophectoderm-like state could be captured, which enabled the derivation of induced trophoblast stem cells. Induced trophoblast stem cells are molecularly and functionally similar to trophoblast stem cells derived from human blastocysts or first-trimester placentas 7. Our results provide a high-resolution roadmap for the transcription-factor-mediated reprogramming of human somatic cells, indicate a role for the trophectoderm-lineage-specific regulatory program during this process, and facilitate the direct reprogramming of somatic cells into induced trophoblast stem cells.

Hyperexcitable neurons in brain organoids Individuals with Angelman syndrome experience intellectual disability and seizures throughout their lives. In this condition, ubiquitin-mediated degradation of a key potassium channel is disrupted, allowing for the neuronal excitability and network synchronization that leads to seizure. Sun et al. used brain organoid technology to study what happens in human neurons with a mutation in a ubiquitin ligase that is implicated in Angelman syndrome. In these in vitro models and in a mouse model of Angelman syndrome, antagonists for the potassium channel normalized neuronal excitability. Science , this issue p. 1486

Synthetic biology has the potential to transform cell- and gene-based therapies for a variety of diseases. Sophisticated tools are now available for both eukaryotic and prokaryotic cells to engineer cells to selectively achieve therapeutic effects in response to one or more disease-related signals, thus sparing healthy tissue from potentially cytotoxic effects. This report summarizes the Keystone eSymposium "Synthetic Biology: At the Crossroads of Genetic Engineering and Human Therapeutics," which took place on May 3 and 4, 2021. Given that several therapies engineered using synthetic biology have entered clinical trials, there was a clear need for a synthetic biology symposium that emphasizes the therapeutic applications of synthetic biology as opposed to the technical aspects. Presenters discussed the use of synthetic biology to improve T cell, gene, and viral therapies, to engineer probiotics, and to expand upon existing modalities and functions of cell-based therapies.

The extracellular signal-regulated kinase 1/2 (ERK1/2) cascade promotes cardiomyocyte hypertrophy and is cardioprotective, with the three RAF kinases forming a node for signal integration. Our aims were to determine if BRAF is relevant for human heart failure, if BRAF promotes cardiomyocyte hypertrophy, and if Type 1 RAF inhibitors developed for cancer (that paradoxically activate ERK1/2 at low concentrations: the “RAF paradox”) may have the same effect. BRAF was upregulated in heart samples from patients with heart failure compared with normal controls. We assessed the effects of activated BRAF in the heart using mice with tamoxifen-activated Cre for cardiomyocyte-specific knock-in of the activating V600E mutation into the endogenous gene. We used echocardiography to measure cardiac dimensions/function. Cardiomyocyte BRAFV600E induced cardiac hypertrophy within 10 d, resulting in increased ejection fraction and fractional shortening over 6 weeks. This was associated with increased cardiomyocyte size without significant fibrosis, consistent with compensated hypertrophy. The experimental Type 1 RAF inhibitor, SB590885, and/or encorafenib (a RAF inhibitor used clinically) increased ERK1/2 phosphorylation in cardiomyocytes, and promoted hypertrophy, consistent with a “RAF paradox” effect. Both promoted cardiac hypertrophy in mouse hearts in vivo, with increased cardiomyocyte size and no overt fibrosis. In conclusion, BRAF potentially plays an important role in human failing hearts, activation of BRAF is sufficient to induce hypertrophy, and Type 1 RAF inhibitors promote hypertrophy via the “RAF paradox”. Cardiac hypertrophy resulting from these interventions was not associated with pathological features, suggesting that Type 1 RAF inhibitors may be useful to boost cardiomyocyte function.

The impact of aging on intestinal stem cells (ISCs) has not been fully elucidated. In this study, we identified widespread epigenetic and transcriptional alterations in old ISCs. Using a reprogramming algorithm, we identified a set of key transcription factors (Egr1, Irf1, FosB) that drives molecular and functional differences between old and young states. Overall, by dissecting the molecular signature of aged ISCs, our study identified transcription factors that enhance the regenerative capacity of ISCs.

The role of microglia cells in Alzheimer’s disease (AD) is well recognized, however their molecular and functional diversity remain unclear. Here, we isolated amyloid plaque-containing (using labelling with methoxy-XO4, XO4+) and non-containing (XO4−) microglia from an AD mouse model. Transcriptomics analysis identified different transcriptional trajectories in ageing and AD mice. XO4+ microglial transcriptomes demonstrated dysregulated expression of genes associated with late onset AD. We further showed that the transcriptional program associated with XO4+ microglia from mice is present in a subset of human microglia isolated from brains of individuals with AD. XO4− microglia displayed transcriptional signatures associated with accelerated ageing and contained more intracellular post-synaptic material than XO4+ microglia, despite reduced active synaptosome phagocytosis. We identified HIF1α as potentially regulating synaptosome phagocytosis in vitro using primary human microglia, and BV2 mouse microglial cells. Together, these findings provide insight into molecular mechanisms underpinning the functional diversity of microglia in AD., Microglia associated with Aβ plaques may have a distinct transcriptional signature compared to those in plaque-free areas of the brain in Alzheimer’s disease (AD) models. Here the authors show that amyloid plaque phagocytosis is associated with a specific microglia transcriptional signature in a mouse model of AD.

Correction to: Nature Communications https://doi.org/10.1038/s41467-019-10717-9, published online 27 May 2020.The original version of this Article omitted from the Genome Aggregation Database consortium the member Marquis P. Vawter, from the Department of Psychiatry & Human Behavior, University of California Irvine, Irvine, CA, USA. Additionally, the following was added to the Author Contributions: ‘All authors listed under The Genome Aggregation Database Consortium contributed to the generation of the primary data incorporated into the gnomAD resource’. This has been corrected in both the PDF and HTML versions of the Article.

Motivation As the generation of complex single-cell RNA sequencing datasets becomes more commonplace it is the responsibility of researchers to provide access to these data in a way that can be easily explored and shared. Whilst it is often the case that data is deposited for future bioinformatic analysis many studies do not release their data in a way that is easy to explore by non-computational researchers. Results In order to help address this we have developed ShinyCell, an R package that converts single-cell RNA sequencing datasets into explorable and shareable interactive interfaces. These interfaces can be easily customized in order to maximize their usability and can be easily uploaded to online platforms to facilitate wider access to published data. Availability and implementation ShinyCell is available at https://github.com/SGDDNB/ShinyCell and https://figshare.com/projects/ShinyCell/100439. Supplementary information Supplementary data are available at Bioinformatics online.