Transcriptional signals of transformation in human cancer.

BACKGROUND: As normal cells transform into cancers, their cell state changes, which may drive cancer cells into a stem-like or more primordial, foetal, or embryonic cell state. The transcriptomic profile of this final state may encode information about cancer's origin and how cancers relate to their normal cell counterparts. METHODS: Here, we used single-cell atlases to study cancer transformation in transcriptional terms. We utilised bulk transcriptomes across a wide spectrum of adult and childhood cancers, using a previously established method to interrogate their relationship to normal cell states. We extend and validate these findings using single-cell cancer transcriptomes and organ-specific atlases of colorectal and liver cancer. RESULTS: Our bulk transcriptomic data reveals that adult cancers rarely return to an embryonic state, but that a foetal state is a near-universal feature of childhood cancers. This finding was confirmed with single-cell cancer transcriptomes. CONCLUSIONS: Our findings provide a nuanced picture of transformation in human cancer, indicating cancer-specific rather than universal patterns of transformation pervade adult epithelial cancers.

Yolk sac cell atlas reveals multiorgan functions during human early development.

The extraembryonic yolk sac (YS) ensures delivery of nutritional support and oxygen to the developing embryo but remains ill-defined in humans. We therefore assembled a comprehensive multiomic reference of the human YS from 3 to 8 postconception weeks by integrating single-cell protein and gene expression data. Beyond its recognized role as a site of hematopoiesis, we highlight roles in metabolism, coagulation, vascular development, and hematopoietic regulation. We reconstructed the emergence and decline of YS hematopoietic stem and progenitor cells from hemogenic endothelium and revealed a YS-specific accelerated route to macrophage production that seeds developing organs. The multiorgan functions of the YS are superseded as intraembryonic organs develop, effecting a multifaceted relay of vital functions as pregnancy proceeds.

Myeloid cell interferon secretion restricts Zika flavivirus infection of developing and malignant human neural progenitor cells.

Zika virus (ZIKV) can infect human developing brain (HDB) progenitors resulting in epidemic microcephaly, whereas analogous cellular tropism offers treatment potential for the adult brain cancer, glioblastoma (GBM). We compared productive ZIKV infection in HDB and GBM primary tissue explants that both contain SOX2+ neural progenitors. Strikingly, although the HDB proved uniformly vulnerable to ZIKV infection, GBM was more refractory, and this correlated with an innate immune expression signature. Indeed, GBM-derived CD11b+ microglia/macrophages were necessary and sufficient to protect progenitors against ZIKV infection in a non-cell autonomous manner. Using SOX2+ GBM cell lines, we found that CD11b+-conditioned medium containing type 1 interferon beta (IFNß) promoted progenitor resistance to ZIKV, whereas inhibition of JAK1/2 signaling restored productive infection. Additionally, CD11b+ conditioned medium, and IFNß treatment rendered HDB progenitor lines and explants refractory to ZIKV. These findings provide insight into neuroprotection for HDB progenitors as well as enhanced GBM oncolytic therapies.

Single-cell roadmap of human gonadal development.

Gonadal development is a complex process that involves sex determination followed by divergent maturation into either testes or ovaries1. Historically, limited tissue accessibility, a lack of reliable in vitro models and critical differences between humans and mice have hampered our knowledge of human gonadogenesis, despite its importance in gonadal conditions and infertility. Here, we generated a comprehensive map of first- and second-trimester human gonads using a combination of single-cell and spatial transcriptomics, chromatin accessibility assays and fluorescent microscopy. We extracted human-specific regulatory programmes that control the development of germline and somatic cell lineages by profiling equivalent developmental stages in mice. In both species, we define the somatic cell states present at the time of sex specification, including the bipotent early supporting population that, in males, upregulates the testis-determining factor SRY and sPAX8s, a gonadal lineage located at the gonadal-mesonephric interface. In females, we resolve the cellular and molecular events that give rise to the first and second waves of granulosa cells that compartmentalize the developing ovary to modulate germ cell differentiation. In males, we identify human SIGLEC15+ and TREM2+ fetal testicular macrophages, which signal to somatic cells outside and inside the developing testis cords, respectively. This study provides a comprehensive spatiotemporal map of human and mouse gonadal differentiation, which can guide in vitro gonadogenesis.

Cationic Geminoid Peptide Amphiphiles Inhibit DENV2 Protease, Furin, and Viral Replication.

Dengue is an important arboviral infectious disease for which there is currently no specific cure. We report gemini-like (geminoid) alkylated amphiphilic peptides containing lysines in combination with glycines or alanines (C15H31C(O)-Lys-(Gly or Ala)nLys-NHC16H33, shorthand notation C16-KXnK-C16 with X = A or G, and n = 0-2). The representatives with 1 or 2 Ala inhibit dengue protease and human furin, two serine proteases involved in dengue virus infection that have peptides with cationic amino acids as their preferred substrates, with IC50 values in the lower µM range. The geminoid C16-KAK-C16 combined inhibition of DENV2 protease (IC50 2.3 µM) with efficacy against replication of wildtype DENV2 in LLC-MK2 cells (EC50 4.1 µM) and an absence of toxicity. We conclude that the lysine-based geminoids have activity against dengue virus infection, which is based on their inhibition of the proteases involved in viral replication and are therefore promising leads to further developing antiviral therapeutics, not limited to dengue.

Single-cell Atlas of common variable immunodeficiency shows germinal center-associated epigenetic dysregulation in B-cell responses.

Common variable immunodeficiency (CVID), the most prevalent symptomatic primary immunodeficiency, displays impaired terminal B-cell differentiation and defective antibody responses. Incomplete genetic penetrance and ample phenotypic expressivity in CVID suggest the participation of additional pathogenic mechanisms. Monozygotic (MZ) twins discordant for CVID are uniquely valuable for studying the contribution of epigenetics to the disease. Here, we generate a single-cell epigenomics and transcriptomics census of naïve-to-memory B cell differentiation in a CVID-discordant MZ twin pair. Our analysis identifies DNA methylation, chromatin accessibility and transcriptional defects in memory B-cells mirroring defective cell-cell communication upon activation. These findings are validated in a cohort of CVID patients and healthy donors. Our findings provide a comprehensive multi-omics map of alterations in naïve-to-memory B-cell transition in CVID and indicate links between the epigenome and immune cell cross-talk. Our resource, publicly available at the Human Cell Atlas, gives insight into future diagnosis and treatments of CVID patients.

Mapping the temporal and spatial dynamics of the human endometrium in vivo and in vitro.

The endometrium, the mucosal lining of the uterus, undergoes dynamic changes throughout the menstrual cycle in response to ovarian hormones. We have generated dense single-cell and spatial reference maps of the human uterus and three-dimensional endometrial organoid cultures. We dissect the signaling pathways that determine cell fate of the epithelial lineages in the lumenal and glandular microenvironments. Our benchmark of the endometrial organoids reveals the pathways and cell states regulating differentiation of the secretory and ciliated lineages both in vivo and in vitro. In vitro downregulation of WNT or NOTCH pathways increases the differentiation efficiency along the secretory and ciliated lineages, respectively. We utilize our cellular maps to deconvolute bulk data from endometrial cancers and endometriotic lesions, illuminating the cell types dominating in each of these disorders. These mechanistic insights provide a platform for future development of treatments for common conditions including endometriosis and endometrial carcinoma.

Cells of the human intestinal tract mapped across space and time.

The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. Here, to comprehensively map cell lineages, we use single-cell RNA sequencing and antigen receptor analysis of almost half a million cells from up to 5 anatomical regions in the developing and up to 11 distinct anatomical regions in the healthy paediatric and adult human gut. This reveals the existence of transcriptionally distinct BEST4 epithelial cells throughout the human intestinal tract. Furthermore, we implicate IgG sensing as a function of intestinal tuft cells. We describe neural cell populations in the developing enteric nervous system, and predict cell-type-specific expression of genes associated with Hirschsprung's disease. Finally, using a systems approach, we identify key cell players that drive the formation of secondary lymphoid tissue in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. This catalogue of intestinal cells will provide new insights into cellular programs in development, homeostasis and disease.

Flexible comparison of batch correction methods for single-cell RNA-seq using BatchBench.

As the cost of single-cell RNA-seq experiments has decreased, an increasing number of datasets are now available. Combining newly generated and publicly accessible datasets is challenging due to non-biological signals, commonly known as batch effects. Although there are several computational methods available that can remove batch effects, evaluating which method performs best is not straightforward. Here, we present BatchBench (https://github.com/cellgeni/batchbench), a modular and flexible pipeline for comparing batch correction methods for single-cell RNA-seq data. We apply BatchBench to eight methods, highlighting their methodological differences and assess their performance and computational requirements through a compendium of well-studied datasets. This systematic comparison guides users in the choice of batch correction tool, and the pipeline makes it easy to evaluate other datasets.

Comparison of visualization tools for single-cell RNAseq data.

In the last decade, single cell RNAseq (scRNAseq) datasets have grown in size from a single cell to millions of cells. Due to its high dimensionality, it is not always feasible to visualize scRNAseq data and share it in a scientific report or an article publication format. Recently, many interactive analysis and visualization tools have been developed to address this issue and facilitate knowledge transfer in the scientific community. In this study, we review several of the currently available scRNAseq visualization tools and benchmark the subset that allows to visualize the data on the web and share it with others. We consider the memory and time required to prepare datasets for sharing as the number of cells increases, and additionally review the user experience and features available in the web interface. To address the problem of format compatibility we have also developed a user-friendly R package, sceasy, which allows users to convert their own scRNAseq datasets into a specific data format for visualization.

Many si/shRNAs can kill cancer cells by targeting multiple survival genes through an off-target mechanism.

Over 80% of multiple-tested siRNAs and shRNAs targeting CD95 or CD95 ligand (CD95L) induce a form of cell death characterized by simultaneous activation of multiple cell death pathways preferentially killing transformed and cancer stem cells. We now show these si/shRNAs kill cancer cells through canonical RNAi by targeting the 3'UTR of critical survival genes in a unique form of off-target effect we call DISE (death induced by survival gene elimination). Drosha and Dicer-deficient cells, devoid of most miRNAs, are hypersensitive to DISE, suggesting cellular miRNAs protect cells from this form of cell death. By testing 4666 shRNAs derived from the CD95 and CD95L mRNA sequences and an unrelated control gene, Venus, we have identified many toxic sequences - most of them located in the open reading frame of CD95L. We propose that specific toxic RNAi-active sequences present in the genome can kill cancer cells.

Mirnovo: genome-free prediction of microRNAs from small RNA sequencing data and single-cells using decision forests.

The discovery of microRNAs (miRNAs) remains an important problem, particularly given the growth of high-throughput sequencing, cell sorting and single cell biology. While a large number of miRNAs have already been annotated, there may well be large numbers of miRNAs that are expressed in very particular cell types and remain elusive. Sequencing allows us to quickly and accurately identify the expression of known miRNAs from small RNA-Seq data. The biogenesis of miRNAs leads to very specific characteristics observed in their sequences. In brief, miRNAs usually have a well-defined 5' end and a more flexible 3' end with the possibility of 3' tailing events, such as uridylation. Previous approaches to the prediction of novel miRNAs usually involve the analysis of structural features of miRNA precursor hairpin sequences obtained from genome sequence. We surmised that it may be possible to identify miRNAs by using these biogenesis features observed directly from sequenced reads, solely or in addition to structural analysis from genome data. To this end, we have developed mirnovo, a machine learning based algorithm, which is able to identify known and novel miRNAs in animals and plants directly from small RNA-Seq data, with or without a reference genome. This method performs comparably to existing tools, however is simpler to use with reduced run time. Its performance and accuracy has been tested on multiple datasets, including species with poorly assembled genomes, RNaseIII (Drosha and/or Dicer) deficient samples and single cells (at both embryonic and adult stage).

Transposon-driven transcription is a conserved feature of vertebrate spermatogenesis and transcript evolution.

Spermatogenesis is associated with major and unique changes to chromosomes and chromatin. Here, we sought to understand the impact of these changes on spermatogenic transcriptomes. We show that long terminal repeats (LTRs) of specific mouse endogenous retroviruses (ERVs) drive the expression of many long non-coding transcripts (lncRNA). This process occurs post-mitotically predominantly in spermatocytes and round spermatids. We demonstrate that this transposon-driven lncRNA expression is a conserved feature of vertebrate spermatogenesis. We propose that transposon promoters are a mechanism by which the genome can explore novel transcriptional substrates, increasing evolutionary plasticity and allowing for the genesis of novel coding and non-coding genes. Accordingly, we show that a small fraction of these novel ERV-driven transcripts encode short open reading frames that produce detectable peptides. Finally, we find that distinct ERV elements from the same subfamilies act as differentially activated promoters in a tissue-specific context. In summary, we demonstrate that LTRs can act as tissue-specific promoters and contribute to post-mitotic spermatogenic transcriptome diversity.

MiR-277/4989 regulate transcriptional landscape during juvenile to adult transition in the parasitic helminth Schistosoma mansoni.

Schistosomes are parasitic helminths that cause schistosomiasis, a disease affecting circa 200 million people, primarily in underprivileged regions of the world. Schistosoma mansoni is the most experimentally tractable schistosome species due to its ease of propagation in the laboratory and the high quality of its genome assembly and annotation. Although there is growing interest in microRNAs (miRNAs) in trematodes, little is known about the role these molecules play in the context of developmental processes. We use the completely unaware "miRNA-blind" bioinformatics tool Sylamer to analyse the 3'-UTRs of transcripts differentially expressed between the juvenile and adult stages. We show that the miR-277/4989 family target sequence is the only one significantly enriched in the transition from juvenile to adult worms. Further, we describe a novel miRNA, sma-miR-4989 showing that its proximal genomic location to sma-miR-277 suggests that they form a miRNA cluster, and we propose hairpin folds for both miRNAs compatible with the miRNA pathway. In addition, we found that expression of sma-miR-277/4989 miRNAs are up-regulated in adults while their predicted targets are characterised by significant down-regulation in paired adult worms but remain largely undisturbed in immature "virgin" females. Finally, we show that sma-miR-4989 is expressed in tegumental cells located proximal to the oesophagus gland and also distributed throughout the male worms' body. Our results indicate that sma-miR-277/4989 might play a dominant role in post-transcriptional regulation during development of juvenile worms and suggest an important role in the sexual development of female schistosomes.

Large-scale analysis of microRNA expression, epi-transcriptomic features and biogenesis.

MicroRNAs are important genetic regulators in both animals and plants. They have a range of functions spanning development, differentiation, growth, metabolism and disease. The advent of next-generation sequencing technologies has made it a relatively straightforward task to detect these molecules and their relative expression via sequencing. There are a large number of published studies with deposited datasets. However, there are currently few resources that capitalize on these data to better understand the features, distribution and biogenesis of miRNAs. Herein, we focus on Human and Mouse for which the majority of data are available. We reanalyse sequencing data from 461 samples into a coordinated catalog of microRNA expression. We use this to perform large-scale analyses of miRNA function and biogenesis. These analyses include global expression comparison, co-expression of miRNA clusters and the prediction of miRNA strand-specificity and underlying constraints. Additionally, we report for the first time a global analysis of miRNA epi-transcriptomic modifications and assess their prevalence across tissues, samples and families. Finally, we report a list of potentially mis-annotated miRNAs in miRBase based on their aggregated modification profiles. The results have been collated into a comprehensive online repository of miRNA expression and features such as modifications and RNA editing events, which is available at: http://wwwdev.ebi.ac.uk/enright-dev/miratlas. We believe these findings will further contribute to our understanding of miRNA function in animals and benefit the miRNA community in general.

Exploring regulatory networks of miR-96 in the developing inner ear.

Mutations in the microRNA Mir96 cause deafness in mice and humans. In the diminuendo mouse, which carries a single base pair change in the seed region of miR-96, the sensory hair cells crucial for hearing fail to develop fully and retain immature characteristics, suggesting that miR-96 is important for coordinating hair cell maturation. Our previous transcriptional analyses show that many genes are misregulated in the diminuendo inner ear and we report here further misregulated genes. We have chosen three complementary approaches to explore potential networks controlled by miR-96 using these transcriptional data. Firstly, we used regulatory interactions manually curated from the literature to construct a regulatory network incorporating our transcriptional data. Secondly, we built a protein-protein interaction network using the InnateDB database. Thirdly, gene set enrichment analysis was used to identify gene sets in which the misregulated genes are enriched. We have identified several candidates for mediating some of the expression changes caused by the diminuendo mutation, including Fos, Myc, Trp53 and Nr3c1, and confirmed our prediction that Fos is downregulated in diminuendo homozygotes. Understanding the pathways regulated by miR-96 could lead to potential therapeutic targets for treating hearing loss due to perturbation of any component of the network.

Processive catalysis.

Nature's enzymes are an ongoing source of inspiration for scientists. The complex processes behind their selectivity and efficiency is slowly being unraveled, and these findings have spawned many biomimetic catalysts. However, nearly all focus on the conversion of small molecular substrates. Nature itself is replete with inventive catalytic systems which modify, replicate, or decompose entire polymers, often in a processive fashion. Such processivity can, for example, enhance the rate of catalysis by clamping to the polymer substrate, which imparts a large effective molarity. Reviewed herein are the various strategies for processivity in nature's arsenal and their properties. An overview of what has been achieved by chemists aiming to mimic one of nature's greatest tricks is also included.

Structure-delivery relationships of lysine-based gemini surfactants and their lipoplexes.

The synthesis and properties of gemini surfactants of the type (R(1)(CO)-Lys(H)-NH)2(CH2)n are reported. For a spacer length of n = 6, the hydrophobic acyl tail was varied in length (R(1) = C8, C10, C12, C14, C16, and C18) and, for R(1) = C18, the degree of unsaturation. For R(1)(CO) = oleoyl (C18:1 Z) the spacer length (n = 2-8) and the stereochemistry of the lysine building block were varied; a 'half-gemini' derivative with a single oleoyl tail and head group was also prepared. The potential of the gemini surfactants to transfer polynucleotides across a cell membrane was investigated by transfection of HeLa cells with beta-galactosidase, both in the presence and absence of the helper lipid DOPE. Oleoyl was found to be by far the best hydrophobic tail for this biological activity, whereas the effect of the lysine stereochemistry was less pronounced. The effect of an optimum spacer length (n = 6) was observed only in the absence of helper lipid. The most active surfactant, i.e. the one with oleoyl chains and n = 6, formed liposomes with sizes in the range of 60-350 nm, and its lipoplex underwent a transition from a lamellar to a hexagonal morphology upon lowering the pH from 7 to 3.

A reagent-based dynamic trigger for cell adhesion, shape change, or cocultures.

The described protocol is a simple and easily implemented method for making dynamic micropatterns for cell culture. It is based on the use of a surface coating material (azido-PLL-g-PEG (APP)) that initially repels cells, but which can be made strongly adherent by addition of a small functional peptide (BCN-RGD) to the cell culture medium. The method can be applied to trigger the adhesion, migration, or shape change of single cells or of populations of cells, and it can be used to create patterned cocultures. The entire process can be subdivided into three main parts. The first part describes the creation of patterned APP substrates. The second part describes cell seeding and "click" triggering of cell adhesion; the final part describes variations that allow the overlay of multiple patterns or the creation of patterned cocultures. The APP coating of substrates and the triggering of adhesion only involves treating the surface with aqueous stock solutions, allowing any biology lab to adopt this technique.