Royal Netherlands Academy of Arts and Sciences

Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it is difficult to amplify in fungi, often includes large introns, and can be insufficiently variable. Three subunits from the nuclear ribosomal RNA cistron were compared together with regions of three representative protein-coding genes (largest subunit of RNA polymerase II, second largest subunit of RNA polymerase II, and minichromosome maintenance protein). Although the protein-coding gene regions often had a higher percent of correct identification compared with ribosomal markers, low PCR amplification and sequencing success eliminated them as candidates for a universal fungal barcode. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter- and intraspecific variation. The nuclear ribosomal large subunit, a popular phylogenetic marker in certain groups, had superior species resolution in some taxonomic groups, such as the early diverging lineages and the ascomycete yeasts, but was otherwise slightly inferior to the ITS. The nuclear ribosomal small subunit has poor species-level resolution in fungi. ITS will be formally proposed for adoption as the primary fungal barcode marker to the Consortium for the Barcode of Life, with the possibility that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups.

Fungi play major roles in ecosystem processes, but the determinants of fungal diversity and biogeographic patterns remain poorly understood. Using DNA metabarcoding data from hundreds of globally distributed soil samples, we demonstrate that fungal richness is decoupled from plant diversity. The plant-to-fungus richness ratio declines exponentially toward the poles. Climatic factors, followed by edaphic and spatial variables, constitute the best predictors of fungal richness and community composition at the global scale. Fungi show similar latitudinal diversity gradients to other organisms, with several notable exceptions. These findings advance our understanding of global fungal diversity patterns and permit integration of fungi into a general macroecological framework.

Microbial communities play a pivotal role in the functioning of plants by influencing their physiology and development. While many members of the rhizosphere microbiome are beneficial to plant growth, also plant pathogenic microorganisms colonize the rhizosphere striving to break through the protective microbial shield and to overcome the innate plant defense mechanisms in order to cause disease. A third group of microorganisms that can be found in the rhizosphere are the true and opportunistic human pathogenic bacteria, which can be carried on or in plant tissue and may cause disease when introduced into debilitated humans. Although the importance of the rhizosphere microbiome for plant growth has been widely recognized, for the vast majority of rhizosphere microorganisms no knowledge exists. To enhance plant growth and health, it is essential to know which microorganism is present in the rhizosphere microbiome and what they are doing. Here, we review the main functions of rhizosphere microorganisms and how they impact on health and disease. We discuss the mechanisms involved in the multitrophic interactions and chemical dialogues that occur in the rhizosphere. Finally, we highlight several strategies to redirect or reshape the rhizosphere microbiome in favor of microorganisms that are beneficial to plant growth and health.

UNLABELLED: Sambamba is a high-performance robust tool and library for working with SAM, BAM and CRAM sequence alignment files; the most common file formats for aligned next generation sequencing data. Sambamba is a faster alternative to samtools that exploits multi-core processing and dramatically reduces processing time. Sambamba is being adopted at sequencing centers, not only because of its speed, but also because of additional functionality, including coverage analysis and powerful filtering capability. AVAILABILITY AND IMPLEMENTATION: Sambamba is free and open source software, available under a GPLv2 license. Sambamba can be downloaded and installed from http://www.open-bio.org/wiki/Sambamba.Sambamba v0.5.0 was released with doi:10.5281/zenodo.13200.

Pancreatic stellate cells (PSCs) differentiate into cancer-associated fibroblasts (CAFs) that produce desmoplastic stroma, thereby modulating disease progression and therapeutic response in pancreatic ductal adenocarcinoma (PDA). However, it is unknown whether CAFs uniformly carry out these tasks or if subtypes of CAFs with distinct phenotypes in PDA exist. We identified a CAF subpopulation with elevated expression of α-smooth muscle actin (αSMA) located immediately adjacent to neoplastic cells in mouse and human PDA tissue. We recapitulated this finding in co-cultures of murine PSCs and PDA organoids, and demonstrated that organoid-activated CAFs produced desmoplastic stroma. The co-cultures showed cooperative interactions and revealed another distinct subpopulation of CAFs, located more distantly from neoplastic cells, which lacked elevated αSMA expression and instead secreted IL6 and additional inflammatory mediators. These findings were corroborated in mouse and human PDA tissue, providing direct evidence for CAF heterogeneity in PDA tumor biology with implications for disease etiology and therapeutic development.

Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly ‘housekeeping’, whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research. A study from the FANTOM consortium using single-molecule cDNA sequencing of transcription start sites and their usage in human and mouse primary cells, cell lines and tissues reveals insights into the specificity and diversity of transcription patterns across different mammalian cell types. FANTOM5 (standing for functional annotation of the mammalian genome 5) is the fifth major stage of a major international collaboration that aims to dissect the transcriptional regulatory networks that define every human cell type. Two Articles in this issue of Nature present some of the project's latest results. The first paper uses the FANTOM5 panel of tissue and primary cell samples to define an atlas of active, in vivo bidirectionally transcribed enhancers across the human body. These authors show that bidirectional capped RNAs are a signature feature of active enhancers and identify more than 40,000 enhancer candidates from over 800 human cell and tissue samples. The enhancer atlas is used to compare regulatory programs between different cell types and identify disease-associated regulatory SNPs, and will be a resource for studies on cell-type-specific enhancers. In the second paper, single-molecule sequencing is used to map human and mouse transcription start sites and their usage in a panel of distinct human and mouse primary cells, cell lines and tissues to produce the most comprehensive mammalian gene expression atlas to date. The data provide a plethora of insights into open reading frames and promoters across different cell types in addition to valuable annotation of mammalian cell-type-specific transcriptomes.

Disorders of the brain can exhibit considerable epidemiological comorbidity and often share symptoms, provoking debate about their etiologic overlap. We quantified the genetic sharing of 25 brain disorders from genome-wide association studies of 265,218 patients and 784,643 control participants and assessed their relationship to 17 phenotypes from 1,191,588 individuals. Psychiatric disorders share common variant risk, whereas neurological disorders appear more distinct from one another and from the psychiatric disorders. We also identified significant sharing between disorders and a number of brain phenotypes, including cognitive measures. Further, we conducted simulations to explore how statistical power, diagnostic misclassification, and phenotypic heterogeneity affect genetic correlations. These results highlight the importance of common genetic variation as a risk factor for brain disorders and the value of heritability-based methods in understanding their etiology.

The mammalian intestine is covered by a single layer of epithelial cells that is renewed every 4-5 days. This high cell turnover makes it a very attractive and comprehensive adult organ system for the study of cell proliferation and differentiation. The intestine is composed of proliferative crypts, which contain intestinal stem cells, and villi, which contain differentiated specialized cell types. Through the recent identification of Lgr5, an intestinal stem cell marker, it is now possible to visualize stem cells and study their behavior and differentiation in a much broader context. In this review we describe the identification of intestinal stem cells. We also discuss genetic studies that have helped to elucidate those signals important for progenitor cells to differentiate into one of the specialized intestinal epithelial cell types. These studies describe a genetic hierarchy responsible for cell fate commitment in normal gut physiology. Where relevant we also mention aberrant deregulation of these molecular pathways that results in colon cancer.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause coronavirus disease 2019 (COVID-19), an influenza-like disease that is primarily thought to infect the lungs with transmission through the respiratory route. However, clinical evidence suggests that the intestine may present another viral target organ. Indeed, the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) is highly expressed on differentiated enterocytes. In human small intestinal organoids (hSIOs), enterocytes were readily infected by SARS-CoV and SARS-CoV-2, as demonstrated by confocal and electron microscopy. Enterocytes produced infectious viral particles, whereas messenger RNA expression analysis of hSIOs revealed induction of a generic viral response program. Therefore, the intestinal epithelium supports SARS-CoV-2 replication, and hSIOs serve as an experimental model for coronavirus infection and biology.

Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy.

To understand organ function, it is important to have an inventory of its cell types and of their corresponding marker genes. This is a particularly challenging task for human tissues like the pancreas, because reliable markers are limited. Hence, transcriptome-wide studies are typically done on pooled islets of Langerhans, obscuring contributions from rare cell types and of potential subpopulations. To overcome this challenge, we developed an automated platform that uses FACS, robotics, and the CEL-Seq2 protocol to obtain the transcriptomes of thousands of single pancreatic cells from deceased organ donors, allowing in silico purification of all main pancreatic cell types. We identify cell type-specific transcription factors and a subpopulation of REG3A-positive acinar cells. We also show that CD24 and TM4SF4 expression can be used to sort live alpha and beta cells with high purity. This resource will be useful for developing a deeper understanding of pancreatic biology and pathophysiology of diabetes mellitus.

Stem cells fuel tissue development, renewal, and regeneration, and these activities are controlled by the local stem cell microenvironment, the "niche." Wnt signals emanating from the niche can act as self-renewal factors for stem cells in multiple mammalian tissues. Wnt proteins are lipid-modified, which constrains them to act as short-range cellular signals. The locality of Wnt signaling dictates that stem cells exiting the Wnt signaling domain differentiate, spatially delimiting the niche in certain tissues. In some instances, stem cells may act as or generate their own niche, enabling the self-organization of patterned tissues. In this Review, we discuss the various ways by which Wnt operates in stem cell control and, in doing so, identify an integral program for tissue renewal and regeneration.

To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation.

Recent examples have highlighted how stem cells have the capability to initiate morphogenesis in vitro; that is, to generate complex structures in culture that closely parallel their in vivo counterparts. Lgr5, the receptor for the Wnt-agonistic R-spondins, marks stem cells in multiple adult organs of mice and humans. In R-spondin-based three-dimensional cultures, these Lgr5 stem cells can grow into ever-expanding epithelial organoids that retain their original organ identity. Single Lgr5 stem cells derived from the intestine can be cultured to build epithelial structures that retain hallmarks of the in vivo epithelium. Here, we review the mechanisms that support this notable example of self-organization and discuss applications of this technology for stem cell research, disease modeling (e.g., for colorectal cancer and cystic fibrosis), and regenerative medicine.

The phylum Acidobacteria is one of the most widespread and abundant on the planet, yet remarkably our knowledge of the role of these diverse organisms in the functioning of terrestrial ecosystems remains surprisingly rudimentary. This blatant knowledge gap stems to a large degree from the difficulties associated with the cultivation of these bacteria by classical means. Given the phylogenetic breadth of the Acidobacteria, which is similar to the metabolically diverse Proteobacteria, it is clear that detailed and functional descriptions of acidobacterial assemblages are necessary. Fortunately, recent advances are providing a glimpse into the ecology of members of the phylum Acidobacteria. These include novel cultivation and enrichment strategies, genomic characterization and analyses of metagenomic DNA from environmental samples. Here, we couple the data from these complementary approaches for a better understanding of their role in the environment, thereby providing some initial insights into the ecology of this important phylum. All cultured acidobacterial type species are heterotrophic, and members of subdivisions 1, 3, and 4 appear to be more versatile in carbohydrate utilization. Genomic and metagenomic data predict a number of ecologically relevant capabilities for some acidobacteria, including the ability to: use of nitrite as N source, respond to soil macro-, micro nutrients and soil acidity, express multiple active transporters, degrade gellan gum and produce exopolysaccharide (EPS). Although these predicted properties allude to a competitive life style in soil, only very few of these prediction shave been confirmed via physiological studies. The increased availability of genomic and physiological information, coupled to distribution data in field surveys and experiments, should direct future progress in unraveling the ecology of this important but still enigmatic phylum.

Abstract Pancreatic cancer is the most lethal common solid malignancy. Systemic therapies are often ineffective, and predictive biomarkers to guide treatment are urgently needed. We generated a pancreatic cancer patient–derived organoid (PDO) library that recapitulates the mutational spectrum and transcriptional subtypes of primary pancreatic cancer. New driver oncogenes were nominated and transcriptomic analyses revealed unique clusters. PDOs exhibited heterogeneous responses to standard-of-care chemotherapeutics and investigational agents. In a case study manner, we found that PDO therapeutic profiles paralleled patient outcomes and that PDOs enabled longitudinal assessment of chemosensitivity and evaluation of synchronous metastases. We derived organoid-based gene expression signatures of chemosensitivity that predicted improved responses for many patients to chemotherapy in both the adjuvant and advanced disease settings. Finally, we nominated alternative treatment strategies for chemorefractory PDOs using targeted agent therapeutic profiling. We propose that combined molecular and therapeutic profiling of PDOs may predict clinical response and enable prospective therapeutic selection. Significance: New approaches to prioritize treatment strategies are urgently needed to improve survival and quality of life for patients with pancreatic cancer. Combined genomic, transcriptomic, and therapeutic profiling of PDOs can identify molecular and functional subtypes of pancreatic cancer, predict therapeutic responses, and facilitate precision medicine for patients with pancreatic cancer. Cancer Discov; 8(9); 1112–29. ©2018 AACR. See related commentary by Collisson, p. 1062. This article is highlighted in the In This Issue feature, p. 1047

Organoids are self‐organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here, we describe a method to establish long‐term‐expanding human airway organoids from broncho‐alveolar resections or lavage material. The pseudostratified airway organoids consist of basal cells, functional multi‐ciliated cells, mucus‐producing secretory cells, and CC10‐secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoids established from lung cancer resections and metastasis biopsies retain tumor histopathology as well as cancer gene mutations and are amenable to drug screening. Respiratory syncytial virus (RSV) infection recapitulates central disease features, dramatically increases organoid cell motility via the non‐structural viral NS2 protein, and preferentially recruits neutrophils upon co‐culturing. We conclude that human airway organoids represent versatile models for the in vitro study of hereditary, malignant, and infectious pulmonary disease. To date, persistent in vitro culture of adult human lung epithelium remains elusive. In this methods resource article, culture conditions to maintain three‐dimensional pulmonary tissue long‐term are reported and applied to recapitulate related diseases. Three‐dimensional human pulmonary tissue culture allows for investigation of hereditary diseases.

Richard O'Connell and colleagues report the genomes and transcriptomes of two Colletotrichum plant fungal pathogens. C. higginsianum infects Arabidopsis thaliana, and C. graminicola infects maize (Zea mays); comparative genomics in both species lead to molecular insights into the transition from biotrophic to necrotrophic life stages. Colletotrichum species are fungal pathogens that devastate crop plants worldwide. Host infection involves the differentiation of specialized cell types that are associated with penetration, growth inside living host cells (biotrophy) and tissue destruction (necrotrophy). We report here genome and transcriptome analyses of Colletotrichum higginsianum infecting Arabidopsis thaliana and Colletotrichum graminicola infecting maize. Comparative genomics showed that both fungi have large sets of pathogenicity-related genes, but families of genes encoding secreted effectors, pectin-degrading enzymes, secondary metabolism enzymes, transporters and peptidases are expanded in C. higginsianum. Genome-wide expression profiling revealed that these genes are transcribed in successive waves that are linked to pathogenic transitions: effectors and secondary metabolism enzymes are induced before penetration and during biotrophy, whereas most hydrolases and transporters are upregulated later, at the switch to necrotrophy. Our findings show that preinvasion perception of plant-derived signals substantially reprograms fungal gene expression and indicate previously unknown functions for particular fungal cell types.

The concept that tumors are maintained by dedicated stem cells, the so-called cancer stem cell hypothesis, has attracted great interest but remains controversial. Studying mouse models, we provide direct, functional evidence for the presence of stem cell activity within primary intestinal adenomas, a precursor to intestinal cancer. By "lineage retracing" using the multicolor Cre-reporter R26R-Confetti, we demonstrate that the crypt stem cell marker Lgr5 (leucine-rich repeat-containing heterotrimeric guanine nucleotide-binding protein-coupled receptor 5) also marks a subpopulation of adenoma cells that fuel the growth of established intestinal adenomas. These Lgr5(+) cells, which represent about 5 to 10% of the cells in the adenomas, generate additional Lgr5(+) cells as well as all other adenoma cell types. The Lgr5(+) cells are intermingled with Paneth cells near the adenoma base, a pattern reminiscent of the architecture of the normal crypt niche.

Cognitive functions rely on the coordinated activity of neurons in many brain regions, but the interactions between cortical areas are not yet well understood. Here we investigated whether low-frequency (α) and high-frequency (γ) oscillations characterize different directions of information flow in monkey visual cortex. We recorded from all layers of the primary visual cortex (V1) and found that γ-waves are initiated in input layer 4 and propagate to the deep and superficial layers of cortex, whereas α-waves propagate in the opposite direction. Simultaneous recordings from V1 and downstream area V4 confirmed that γ- and α-waves propagate in the feedforward and feedback direction, respectively. Microstimulation in V1 elicited γ-oscillations in V4, whereas microstimulation in V4 elicited α-oscillations in V1, thus providing causal evidence for the opposite propagation of these rhythms. Furthermore, blocking NMDA receptors, thought to be involved in feedback processing, suppressed α while boosting γ. These results provide new insights into the relation between brain rhythms and cognition.