Genome-wide expression profiling is a widely used approach for characterizing heterogeneous populations of cells, tissues, biopsies, or other biological specimen. The exploratory analysis of such ...data typically relies on generic unsupervised methods, e.g. principal component analysis (PCA) or hierarchical clustering. However, generic methods fail to exploit prior knowledge about the molecular functions of genes. Here, I introduce GO-PCA, an unsupervised method that combines PCA with nonparametric GO enrichment analysis, in order to systematically search for sets of genes that are both strongly correlated and closely functionally related. These gene sets are then used to automatically generate expression signatures with functional labels, which collectively aim to provide a readily interpretable representation of biologically relevant similarities and differences. The robustness of the results obtained can be assessed by bootstrapping.
I first applied GO-PCA to datasets containing diverse hematopoietic cell types from human and mouse, respectively. In both cases, GO-PCA generated a small number of signatures that represented the majority of lineages present, and whose labels reflected their respective biological characteristics. I then applied GO-PCA to human glioblastoma (GBM) data, and recovered signatures associated with four out of five previously defined GBM subtypes. My results demonstrate that GO-PCA is a powerful and versatile exploratory method that reduces an expression matrix containing thousands of genes to a much smaller set of interpretable signatures. In this way, GO-PCA aims to facilitate hypothesis generation, design of further analyses, and functional comparisons across datasets.
High-throughput sequencing has allowed for unprecedented detail in gene expression analyses, yet its efficient application to single cells is challenged by the small starting amounts of RNA. We have ...developed CEL-Seq, a method for overcoming this limitation by barcoding and pooling samples before linearly amplifying mRNA with the use of one round of in vitro transcription. We show that CEL-Seq gives more reproducible, linear, and sensitive results than a PCR-based amplification method. We demonstrate the power of this method by studying early C. elegans embryonic development at single-cell resolution. Differential distribution of transcripts between sister cells is seen as early as the two-cell stage embryo, and zygotic expression in the somatic cell lineages is enriched for transcription factors. The robust transcriptome quantifications enabled by CEL-Seq will be useful for transcriptomic analyses of complex tissues containing populations of diverse cell types.
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► We present CEL-Seq, a single-cell transcriptomics method using in vitro transcription ► We show that CEL-Seq is linear, sensitive, and reproducible ► CEL-Seq is highly multiplexed, allowing for the parallel examination of hundreds of cells ► We describe early C. elegans embryology at the single-cell transcriptomic level
Yanai and colleagues have developed CEL-Seq, an RNA-Seq method for assaying the transcriptome at the single-cell level. CEL-Seq works by barcoding and pooling dozens of samples before linearly amplifying mRNA using one round of in vitro transcription. We show that CEL-Seq gives highly reproducible, linear, and sensitive results. We demonstrate the power of this method by studying early C. elegans embryonic development at single-cell resolution.
Single-cell RNA sequencing (scRNA-seq) enables the systematic identification of cell populations in a tissue, but characterizing their spatial organization remains challenging. We combine a ...microarray-based spatial transcriptomics method that reveals spatial patterns of gene expression using an array of spots, each capturing the transcriptomes of multiple adjacent cells, with scRNA-Seq generated from the same sample. To annotate the precise cellular composition of distinct tissue regions, we introduce a method for multimodal intersection analysis. Applying multimodal intersection analysis to primary pancreatic tumors, we find that subpopulations of ductal cells, macrophages, dendritic cells and cancer cells have spatially restricted enrichments, as well as distinct coenrichments with other cell types. Furthermore, we identify colocalization of inflammatory fibroblasts and cancer cells expressing a stress-response gene module. Our approach for mapping the architecture of scRNA-seq-defined subpopulations can be applied to reveal the interactions inherent to complex tissues.
Although primary graft dysfunction (PGD) is fairly common early after cardiac transplant, standardized schemes for diagnosis and treatment remain contentious. Most major cardiac transplant centers ...use different definitions and parameters of cardiac function. Thus, there is difficulty comparing published reports and no agreed protocol for management. A consensus conference was organized to better define, diagnose, and manage PGD. There were 71 participants (transplant cardiologists, surgeons, immunologists and pathologists), with vast clinical and published experience in PGD, representing 42 heart transplant centers worldwide. State-of-the-art PGD presentations occurred with subsequent breakout sessions planned in an attempt to reach consensus on various issues. Graft dysfunction will be classified into primary graft dysfunction (PGD) or secondary graft dysfunction where there is a discernible cause such as hyperacute rejection, pulmonary hypertension, or surgical complications. PGD must be diagnosed within 24 hours of completion of surgery. PGD is divided into PGD-left ventricle and PGD-right ventricle. PGD-left ventricle is categorized into mild, moderate, or severe grades depending on the level of cardiac function and the extent of inotrope and mechanical support required. Agreed risk factors for PGD include donor, recipient, and surgical procedural factors. Recommended management involves minimization of risk factors, gradual increase of inotropes, and use of mechanical circulatory support as needed. Retransplantation may be indicated if risk factors are minimal. With a standardized definition of PGD, there will be more consistent recognition of this phenomenon and treatment modalities will be more comparable. This should lead to better understanding of PGD and prevention/minimization of its adverse outcomes.
Inferring phylogenetic relationships of polyploid species and their diploid ancestors (leading to reticulate phylogenies in the case of an allopolyploid origin) based on multi-locus sequence data is ...complicated by the unknown assignment of alleles found in polyploids to diploid subgenomes. A parsimony-based approach to this problem has been proposed by Oberprieler et al. (Methods Ecol Evol 8:835-849, 2017), however, its implementation is of limited practical value. In addition to previously identified shortcomings, it has been found that in some cases, the obtained results barely satisfy the applied criterion. To be of better use to other researchers, a reimplementation with methodological refinement appears to be indispensable.
We present the AllCoPol package, which provides a heuristic method for assigning alleles from polyploids to diploid subgenomes based on the Minimizing Deep Coalescences (MDC) criterion in multi-locus sequence datasets. An additional consensus approach further allows to assess the confidence of phylogenetic reconstructions. Simulations of tetra- and hexaploids show that under simplifying assumptions such as completely disomic inheritance, the topological errors of reconstructed phylogenies are similar to those of MDC species trees based on the true allele partition.
AllCoPol is a Python package for phylogenetic reconstructions of polyploids offering enhanced functionality as well as improved usability. The included methods are supplied as command line tools without the need for prior programming knowledge.
Mutations in the ClC‐7/Ostm1 ion transporter lead to osteopetrosis and lysosomal storage disease. Its lysosomal localization hitherto precluded detailed functional characterization. Using a mutated ...ClC‐7 that reaches the plasma membrane, we now show that both the aminoterminus and transmembrane span of the Ostm1 β‐subunit are required for ClC‐7 Cl−/H+‐exchange, whereas the Ostm1 transmembrane domain suffices for its ClC‐7‐dependent trafficking to lysosomes. ClC‐7/Ostm1 currents were strongly outwardly rectifying owing to slow gating of ion exchange, which itself displays an intrinsically almost linear voltage dependence. Reversal potentials of tail currents revealed a 2Cl−/1H+‐exchange stoichiometry. Several disease‐causing CLCN7 mutations accelerated gating. Such mutations cluster to the second cytosolic cystathionine‐β‐synthase domain and potential contact sites at the transmembrane segment. Our work suggests that gating underlies the rectification of all endosomal/lysosomal CLCs and extends the concept of voltage gating beyond channels to ion exchangers.
ClC‐7/Ostm1 is a disease‐relevant lysosomal ion transporter. This study characterizes the functional properties of ClC‐7/Ostm1 and disease‐relevant mutants for the first time and extends the concept of voltage gating to ion exchangers.
The testis expresses the largest number of genes of any mammalian organ, a finding that has long puzzled molecular biologists. Our single-cell transcriptomic data of human and mouse spermatogenesis ...provide evidence that this widespread transcription maintains DNA sequence integrity in the male germline by correcting DNA damage through a mechanism we term transcriptional scanning. We find that genes expressed during spermatogenesis display lower mutation rates on the transcribed strand and have low diversity in the population. Moreover, this effect is fine-tuned by the level of gene expression during spermatogenesis. The unexpressed genes, which in our model do not benefit from transcriptional scanning, diverge faster over evolutionary timescales and are enriched for sensory and immune-defense functions. Collectively, we propose that transcriptional scanning shapes germline mutation signatures and modulates mutation rates in a gene-specific manner, maintaining DNA sequence integrity for the bulk of genes but allowing for faster evolution in a specific subset.
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•Genes expressed in the testis have reduced germline mutation rates•Germline mutational signature is tuned by spermatogenesis-gene expression levels•Genes not expressed during spermatogenesis are enriched for fast-evolving functions•A germline mutational signature generated by TCR follows a “3′-pyrimidine rule”
The male germline cells in human and mice balance the protection of genomic integrity and the evolutionary benefit of genetic mutations through transcriptional scanning, a mechanism that preferentially couples efficient DNA damage repair with high transcription activity.
Incomplete nitrification in biological filters during drinking water treatment is problematic, as it compromises drinking water quality. Nitrification problems can be caused by a lack of nutrients ...for the nitrifying microorganisms. Since copper is an important element in one of the essential enzymes in nitrification, we investigated the effect of copper dosing on nitrification in different biological rapid sand filters treating groundwater. A lab-scale column assay with filter material from a water works demonstrated that addition of a trace metal mixture, including copper, increased ammonium removal compared to a control without addition. Subsequently, another water works was investigated in full-scale, where copper influent concentrations were below 0.05 μg Cu L−1 and nitrification was incomplete. Copper dosing of less than 5 μg Cu L−1 to a full-scale filter stimulated ammonium removal within one day, and doubled the filter's removal from 0.22 to 0.46 g NH4-N m−3 filter material h−1 within 20 days. The location of ammonium and nitrite oxidation shifted upwards in the filter, with an almost 14-fold increase in ammonium removal rate in the filter's top 10 cm, within 57 days of dosing. To study the persistence of the stimulation, copper was dosed to another filter at the water works for 42 days. After dosing was stopped, nitrification remained complete for at least 238 days. Filter effluent concentrations of up to 1.3 μg Cu L−1 confirmed that copper fully penetrated the filters, and determination of copper content on filter media revealed a buildup of copper during dosing. The amount of copper stored on filter material gradually decreased after dosing stopped; however at a slower rate than it accumulated. Continuous detection of copper in the filter effluent confirmed a release of copper to the bulk phase. Overall, copper dosing to poorly performing biological rapid sand filters increased ammonium removal rates significantly, achieving effluent concentrations of below 0.01 mg NH4-N L−1, and had a long-term effect on nitrification performance.
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•Incomplete nitrification in groundwater biofilters was overcome by copper dosing.•Dosing at less than 5 μg Cu L−1 stimulated nitrification in filters within 1 day.•Copper dosing shifted ammonium and nitrite oxidation upwards in the filters.•Ammonium removal of the top 10 cm of a full-scale filter increased almost 14-fold.•After 42 days of dosing, nitrification remained complete for at least 238 days.
Arabidopsis thaliana is a host for the sugar beet cyst nematode Heterodera schachtii. Juvenile nematodes invade the roots and induce the development of a syncytium, which functions as a feeding site ...for the nematode. Here, we report on the transcriptome of syncytia induced in the roots of Arabidopsis. Microaspiration was employed to harvest pure syncytium material, which was then used to prepare RNA for hybridization to Affymetrix GeneChips. Initial data analysis showed that the gene expression in syncytia at 5 and 15 days post-infection did not differ greatly, and so both time points were compared together with control roots. Out of a total of 21 138 genes, 18.4% (3893) had a higher expression level and 15.8% (3338) had a lower expression level in syncytia, as compared with control roots, using a multiple-testing corrected false discovery rate of below 5%. A gene ontology (GO) analysis of up- and downregulated genes showed that categories related to high metabolic activity were preferentially upregulated. A principal component analysis was applied to compare the transcriptome of syncytia with the transcriptome of different Arabidopsis organs (obtained by the AtGenExpress project), and with specific root tissues. This analysis revealed that syncytia are transcriptionally clearly different from roots (and all other organs), as well as from other root tissues.
Single-cell and spatial technologies that profile gene expression across a whole tissue are revolutionizing the resolution of molecular states in clinical samples. Current commercially available ...technologies provide whole transcriptome single-cell, whole transcriptome spatial, or targeted in situ gene expression analysis. Here, we combine these technologies to explore tissue heterogeneity in large, FFPE human breast cancer sections. This integrative approach allowed us to explore molecular differences that exist between distinct tumor regions and to identify biomarkers involved in the progression towards invasive carcinoma. Further, we study cell neighborhoods and identify rare boundary cells that sit at the critical myoepithelial border confining the spread of malignant cells. Here, we demonstrate that each technology alone provides information about molecular signatures relevant to understanding cancer heterogeneity; however, it is the integration of these technologies that leads to deeper insights, ushering in discoveries that will progress oncology research and the development of diagnostics and therapeutics.
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