Applications of single-cell RNA sequencing in drug discovery and development.

Single-cell technologies, particularly single-cell RNA sequencing (scRNA-seq) methods, together with associated computational tools and the growing availability of public data resources, are transforming drug discovery and development. New opportunities are emerging in target identification owing to improved disease understanding through cell subtyping, and highly multiplexed functional genomics screens incorporating scRNA-seq are enhancing target credentialling and prioritization. ScRNA-seq is also aiding the selection of relevant preclinical disease models and providing new insights into drug mechanisms of action. In clinical development, scRNA-seq can inform decision-making via improved biomarker identification for patient stratification and more precise monitoring of drug response and disease progression. Here, we illustrate how scRNA-seq methods are being applied in key steps in drug discovery and development, and discuss ongoing challenges for their implementation in the pharmaceutical industry.

Correction: Ten simple rules for leveraging virtual interaction to build higher-level learning into bioinformatics short courses.

[This corrects the article DOI: 10.1371/journal.pcbi.1010220.].

Galaxy Training: A powerful framework for teaching!

There is an ongoing explosion of scientific datasets being generated, brought on by recent technological advances in many areas of the natural sciences. As a result, the life sciences have become increasingly computational in nature, and bioinformatics has taken on a central role in research studies. However, basic computational skills, data analysis, and stewardship are still rarely taught in life science educational programs, resulting in a skills gap in many of the researchers tasked with analysing these big datasets. In order to address this skills gap and empower researchers to perform their own data analyses, the Galaxy Training Network (GTN) has previously developed the Galaxy Training Platform (https://training.galaxyproject.org), an open access, community-driven framework for the collection of FAIR (Findable, Accessible, Interoperable, Reusable) training materials for data analysis utilizing the user-friendly Galaxy framework as its primary data analysis platform. Since its inception, this training platform has thrived, with the number of tutorials and contributors growing rapidly, and the range of topics extending beyond life sciences to include topics such as climatology, cheminformatics, and machine learning. While initially aimed at supporting researchers directly, the GTN framework has proven to be an invaluable resource for educators as well. We have focused our efforts in recent years on adding increased support for this growing community of instructors. New features have been added to facilitate the use of the materials in a classroom setting, simplifying the contribution flow for new materials, and have added a set of train-the-trainer lessons. Here, we present the latest developments in the GTN project, aimed at facilitating the use of the Galaxy Training materials by educators, and its usage in different learning environments.

Training Infrastructure as a Service.

BACKGROUND: Hands-on training, whether in bioinformatics or other domains, often requires significant technical resources and knowledge to set up and run. Instructors must have access to powerful compute infrastructure that can support resource-intensive jobs running efficiently. Often this is achieved using a private server where there is no contention for the queue. However, this places a significant prerequisite knowledge or labor barrier for instructors, who must spend time coordinating deployment and management of compute resources. Furthermore, with the increase of virtual and hybrid teaching, where learners are located in separate physical locations, it is difficult to track student progress as efficiently as during in-person courses. FINDINGS: Originally developed by Galaxy Europe and the Gallantries project, together with the Galaxy community, we have created Training Infrastructure-as-a-Service (TIaaS), aimed at providing user-friendly training infrastructure to the global training community. TIaaS provides dedicated training resources for Galaxy-based courses and events. Event organizers register their course, after which trainees are transparently placed in a private queue on the compute infrastructure, which ensures jobs complete quickly, even when the main queue is experiencing high wait times. A built-in dashboard allows instructors to monitor student progress. CONCLUSIONS: TIaaS provides a significant improvement for instructors and learners, as well as infrastructure administrators. The instructor dashboard makes remote events not only possible but also easy. Students experience continuity of learning, as all training happens on Galaxy, which they can continue to use after the event. In the past 60 months, 504 training events with over 24,000 learners have used this infrastructure for Galaxy training.

Deciphering Tumour Microenvironment of Liver Cancer through Deconvolution of Bulk RNA-Seq Data with Single-Cell Atlas.

Liver cancers give rise to a heavy burden on healthcare worldwide. Understanding the tumour microenvironment (TME) underpins the development of precision therapy. Single-cell RNA sequencing (scRNA-seq) technology has generated high-quality cell atlases of the TME, but its wider application faces enormous costs for various clinical circumstances. Fortunately, a variety of deconvolution algorithms can instead repurpose bulk RNA-seq data, alleviating the need for generating scRNA-seq datasets. In this study, we reviewed major public omics databases for relevance in this study and utilised eight RNA-seqs and one microarray dataset from clinical studies. To decipher the TME of liver cancer, we estimated the fractions of liver cell components by deconvoluting the samples with Cibersortx using three reference scRNA-seq atlases. We also confirmed that Cibersortx can accurately deconvolute cell types/subtypes of interest. Compared with non-tumorous liver, liver cancers showed multiple decreased cell types forming normal liver microarchitecture, as well as elevated cell types involved in fibrogenesis, abnormal angiogenesis, and disturbed immune responses. Survival analysis shows that the fractions of five cell types/subtypes significantly correlated with patient outcomes, indicating potential therapeutic targets. Therefore, deconvolution of bulk RNA-seq data with scRNA-seq atlas references can be a useful tool to help understand the TME.

Single-Cell Analysis Identifies Thymic Maturation Delay in Growth-Restricted Neonatal Mice.

Fetal growth restriction (FGR) causes a wide variety of defects in the neonate which can lead to increased risk of heart disease, diabetes, anxiety and other disorders later in life. However, the effect of FGR on the immune system, is poorly understood. We used a well-characterized mouse model of FGR in which placental Igf-2 production is lost due to deletion of the placental specific Igf-2 P0 promotor. The thymi in such animals were reduced in mass with a ~70% reduction in cellularity. We used single cell RNA sequencing (Drop-Seq) to analyze 7,264 thymus cells collected at postnatal day 6. We identified considerable heterogeneity among the Cd8/Cd4 double positive cells with one subcluster showing marked upregulation of transcripts encoding a sub-set of proteins that contribute to the surface of the ribosome. The cells from the FGR animals were underrepresented in this cluster. Furthermore, the distribution of cells from the FGR animals was skewed with a higher proportion of immature double negative cells and fewer mature T-cells. Cell cycle regulator transcripts also varied across clusters. The T-cell deficit in FGR mice persisted into adulthood, even when body and organ weights approached normal levels due to catch-up growth. This finding complements the altered immunity found in growth restricted human infants. This reduction in T-cellularity may have implications for adult immunity, adding to the list of adult conditions in which the in utero environment is a contributory factor.

Efficacy and safety of high-dose chemotherapy with autologous stem cell transplantation in senior versus younger adults with newly diagnosed multiple myeloma.

We retrospectively studied 340 fit patients with multiple myeloma (MM) who underwent autologous stem cell transplantation (ASCT). We hypothesized that progression-free survival (PFS) of older patients was non-inferior to that of younger patients after ASCT. Our null hypothesis was that the PFS hazard ratio (HR) for a 5-year increase in age was ≥1.05; the alternative (non-inferiority) hypothesis was that the HR was ≤1. The observed HR was 0.94 (95% confidence interval [CI] 0.86-1.03); since the CI upper bound was <1.05, we reject the null hypothesis and conclude that PFS in older patients was at least as good as in younger patients. We cannot reject an analogous null hypothesis for overall survival (HR 1.06 [95% CI 0.94-1.19]), since the CI upper bound >1.05. Toxicity was similar across ages and transplant-related mortality was minimal. 28% of subjects <65 versus 45% of those ≥65 received maintenance therapy. In summary, ASCT prolongs PFS equally well in older vs. younger adults. Although we cannot exclude maintenance as a confounder, these data support ASCT for fit seniors with MM.

Mll-AF4 Confers Enhanced Self-Renewal and Lymphoid Potential during a Restricted Window in Development.

MLL-AF4+ infant B cell acute lymphoblastic leukemia is characterized by an early onset and dismal survival. It initiates before birth, and very little is known about the early stages of the disease's development. Using a conditional Mll-AF4-expressing mouse model in which fusion expression is targeted to the earliest definitive hematopoietic cells generated in the mouse embryo, we demonstrate that Mll-AF4 imparts enhanced B lymphoid potential and increases repopulation and self-renewal capacity during a putative pre-leukemic state. This occurs between embryonic days 12 and 14 and manifests itself most strongly in the lymphoid-primed multipotent progenitor population, thus pointing to a window of opportunity and a potential cell of origin. However, this state alone is insufficient to generate disease, with the mice succumbing to B cell lymphomas only after a long latency. Future analysis of the molecular details of this pre-leukemic state will shed light on additional events required for progression to acute leukemia.

Analysis of Jak2 signaling reveals resistance of mouse embryonic hematopoietic stem cells to myeloproliferative disease mutation.

The regulation of hematopoietic stem cell (HSC) emergence during development provides important information about the basic mechanisms of blood stem cell generation, expansion, and migration. We set out to investigate the role that cytokine signaling pathways play in these early processes and show here that the 2 cytokines interleukin 3 and thrombopoietin have the ability to expand hematopoietic stem and progenitor numbers by regulating their survival and proliferation. For this, they differentially use the Janus kinase (Jak2) and phosphatidylinositol 3-kinase (Pi3k) signaling pathways, with Jak2 mainly relaying the proproliferation signaling, whereas Pi3k mediates the survival signal. Furthermore, using Jak2-deficient embryos, we demonstrate that Jak2 is crucially required for the function of the first HSCs, whereas progenitors are less dependent on Jak2. The JAK2V617F mutation, which renders JAK2 constitutively active and has been linked to myeloproliferative neoplasms, was recently shown to compromise adult HSC function, negatively affecting their repopulation and self-renewal ability, partly through the accumulation of JAK2V617F-induced DNA damage. We report here that nascent HSCs are resistant to the JAK2V617F mutation and show no decrease in repopulation or self-renewal and no increase in DNA damage, even in the presence of 2 mutant copies. More importantly, this unique property of embryonic HSCs is stably maintained through ≥1 round of successive transplantations. In summary, our dissection of cytokine signaling in embryonic HSCs has uncovered unique properties of these cells that are of clinical importance.

Cyclophosphamide-based hematopoietic stem cell mobilization before autologous stem cell transplantation in newly diagnosed multiple myeloma.

High-dose cyclophosphamide (Cy) is frequently employed for peripheral blood mobilization of hematopoietic stem cells before high-dose chemotherapy with autologous stem cell transplantation (ASCT) in multiple myeloma (MM). The benefit of mobilization with Cy over filgrastim (granulocyte colony-stimulating factor; G-CSF) alone is unclear. Between 2000 and 2008, 167 patients with newly diagnosed MM underwent single ASCT after melphalan conditioning at our institution. Seventy-three patients were mobilized with G-CSF alone, and 94 patients with Cy plus G-CSF (Cy+G-CSF). We retrospectively analyzed Cy's impact on both toxicity and efficacy. Mobilization efficiency was augmented by Cy; a mean total of 12 versus 5.8 × 10(6) CD34+ cells/kg were collected from patients mobilized with Cy+G-CSF versus G-CSF, respectively, (P < 0.01), over a mean of 1.6 versus 2.2 days of peripheral blood apheresis (p = 0.001). Mobilization-related toxicity was also, however, augmented by Cy; 14% of Cy+G-CSF patients were hospitalized because of complications versus none receiving G-CSF (P < 0.0001). Toxicity, including death, related to ASCT was similar between cohorts. Regarding long-term outcomes, multivariate analysis revealed no difference for Cy+G-CSF versus G-CSF (hazard ratio 0.8 for event-free survival [95% confidence interval {CI} 0.57-1.25] and 0.96 for overall survival [95% CI 0.61-1.54]). In summary, we show that mobilization with Cy increases toxicity without positively impacting long-term outcomes in MM. Our findings place into question Cy's benefit as a routine component of stem cell mobilization regimens in MM. Randomized trials are needed to elucidate the risks and benefits of Cy more definitively.

Measurement of heterogeneous reaction rates: three strategies for controlling mass transport and their application to indium-mediated allylations.

We describe three new strategies for determining heterogeneous reaction rates using photomicroscopy to measure the rate of retreat of metal surfaces: (i) spheres in a stirred solution, (ii) microscopic powder in an unstirred solution, and (iii) spheres on a rotating shaft. The strategies are applied to indium-mediated allylation (IMA), which is a powerful tool for synthetic chemists because of its stereoselectivity, broad applicability, and high yields. The rate-limiting step of IMA, reaction of allyl halides at indium metal surfaces, is shown to be fast, with a minimum value of the heterogeneous rate constant of 1 × 10(-2) cm/s, an order of magnitude faster than the previously determined minimum value. The strategies described here can be applied to any reaction in which the surface is retreating or advancing, thereby broadening the applicability of photomicroscopy to measuring heterogeneous reaction kinetics.