A single cell framework identifies functionally and molecularly distinct multipotent progenitors in adult human hematopoiesis.

Hematopoietic multipotent progenitors (MPPs) regulate blood cell production to appropriately meet the biological demands of the human body. Human MPPs remain ill-defined whereas mouse MPPs have been well characterized with distinct immunophenotypes and lineage potencies. Using multiomic single cell analyses and complementary functional assays, we identified new human MPPs and oligopotent progenitor populations within Lin-CD34+CD38dim/lo adult bone marrow with distinct biomolecular and functional properties.

Single cell multiomic analysis of T cell exhaustion in vitro.

T-cell activation is a key step in the amplification of an immune response. Over the course of an immune response, cells may be chronically stimulated, with some proportion becoming exhausted; an enormous number of molecules are involved in this process. There remain a number of questions about the process, namely: (1) what degree of heterogeneity and plasticity do T-cells exhibit during stimulation? (2) how many unique cell states define chronic stimulation? and (3) what markers discriminate activated from exhausted cells? We addressed these questions by performing single-cell multiomic analysis to simultaneously measure expression of 38 proteins and 399 genes in human T cells expanded in vitro.

AbSeq Protocol Using the Nano-Well Cartridge-Based Rhapsody Platform to Generate Protein and Transcript Expression Data on the Single-Cell Level.

By including oligonucleotide-labeled antibodies into high-throughput single-cell RNA-sequencing protocols, combined transcript and protein expression data can be acquired on the single-cell level. Here, we describe a protocol for the combined analysis of over 40 proteins and 400 genes on over 10 cells using the nano-well based Rhapsody platform. We also include a workflow for sample multiplexing, which uniquely identifies the initial source of cells (such as tissue type or donor) in the downstream analysis after upstream pooling.

Direct Selection of Fluorescence-Enhancing RNA Aptamers.

RNA aptamers that generate a strong fluorescence signal upon binding a nonfluorescent small-molecule dye offer a powerful means for the selective imaging of individual RNA species. Unfortunately, conventional in vitro discovery methods are not efficient at generating such fluorescence-enhancing aptamers, because they primarily exert selective pressure based on target affinity-a characteristic that correlates poorly with fluorescence enhancement. Thus, only a handful of fluorescence-enhancing aptamers have been reported to date.

Strategy for Generating Sequence-Defined Aptamer Reagent Sets for Detecting Protein Contaminants in Biotherapeutics.

Biologic drugs are typically manufactured in mammalian host cells, and it is critical from a drug safety and efficacy perspective to detect and remove host cell proteins (HCPs) during production. This is currently achieved with sets of polyclonal antibodies (pAbs), but these suffer from critical shortcomings because their composition is inherently undefined, and they cannot detect nonimmunogenic HCPs. In this work, we report a high-throughput screening and array-based binding characterization strategy that we employed to generate a set of aptamers that overcomes these limitations to achieve sensitive, broad-spectrum detection of HCPs from the widely used Chinese hamster ovary (CHO) cell line.

mRNA pseudouridylation affects RNA metabolism in the parasite .

RNA contains over 100 modified nucleotides that are created post-transcriptionally, among which pseudouridine (Ψ) is one of the most abundant. Although it was one of the first modifications discovered, the biological role of this modification is still not fully understood. Recently, we reported that a pseudouridine synthase (TgPUS1) is necessary for differentiation of the single-celled eukaryotic parasite from active to chronic infection.

High-Throughput Discovery of Aptamers for Sandwich Assays.

Sandwich assays are among the most powerful tools in molecular detection. These assays use "pairs" of affinity reagents so that the detection signal is generated only when both reagents bind simultaneously to different sites on the target molecule, enabling highly sensitive and specific measurements in complex samples. Thus, the capability to efficiently screen affinity reagent pairs at a high throughput is critical.

Advancements in Aptamer Discovery Technologies.

Affinity reagents that specifically bind to their target molecules are invaluable tools in nearly every field of modern biomedicine. Nucleic acid-based aptamers offer many advantages in this domain, because they are chemically synthesized, stable, and economical. Despite these compelling features, aptamers are currently not widely used in comparison to antibodies.

Virus recognition by Toll-7 activates antiviral autophagy in Drosophila.

Innate immunity is highly conserved and relies on pattern recognition receptors (PRRs) such as Toll-like receptors (identified through their homology to Drosophila Toll) for pathogen recognition. Although Drosophila Toll is vital for immune recognition and defense, roles for the other eight Drosophila Tolls in immunity have remained elusive. Here we have shown that Toll-7 is a PRR both in vitro and in adult flies; loss of Toll-7 led to increased vesicular stomatitis virus (VSV) replication and mortality.