Tracking leukemic T-cell transcriptional dynamics in vivo with a blood-based reporter assay.

Transcriptional dynamics of cancer cells govern cell fate decisions and are therapeutically actionable drug targets. In this study, we engineered a circulating cancer cell line that secretes a luciferase reporter to capture constitutive and circadian clock-driven transcription dynamics over the course of a day. Engineered human leukemic T cells (Jurkat) were observed to rhythmically secrete luciferase in a continuous flow cell culture system.

Quantitative assessment of LASSO probe assembly and long-read multiplexed cloning.

Background: Long Adapter Single-Stranded Oligonucleotide (LASSO) probes were developed as a novel tool for massively parallel cloning of kilobase-long genomic DNA sequences. LASSO dramatically improves the capture length limit of current DNA padlock probe technology from approximately 150 bps to several kbps. High-throughput LASSO capture involves the parallel assembly of thousands of probes.

Effects of intermittent T-cell cluster disaggregation on proliferative capacity and checkpoint marker expression.

T-cell immunotherapies are rapidly gaining grounds in clinical success. Presently, there is first-to-market knowledge on the translation of research scale methods to clinical and commercial scales. Improved understanding can lead to more consistent and efficient production, scaling, and eventual potency.

Computational Simulation of Adapter Length-Dependent LASSO Probe Capture Efficiency.

Multiplexed cloning of long DNA sequences is a valuable technique in many biotechnology applications, such as long-read genome sequencing and the creation of open reading frame (ORF) libraries. Long-adapter single-stranded oligonucleotide (LASSO) probes have shown promise as a tool to clone long DNA fragments. LASSO probes are molecular inversion probes (MIP) engineered with an adapter region of user-defined length, flanked between template-specific probe sequences.

Closed loop bioreactor system for the ex vivo expansion of human T cells.

Background Aim: Translation of therapeutic cell therapies to clinical-scale products is critical to realizing widespread success. Currently, however, there are limited tools that are accessible at the research level and readily scalable to clinical-scale needs.

Methods: We herein developed and assessed a closed loop bioreactor system in which (i) a highly gas-permeable silicone material was used to fabricate cell culture bags and (ii) dynamic flow was introduced to allow for dissociation of activated T-cell aggregates.

A simple method to measure CLOCK-BMAL1 DNA binding activity in tissue and cell extracts.

The proteins CLOCK and BMAL1 form a heterodimeric transcription factor essential to circadian rhythms in mammals.  Daily rhythms of CLOCK-BMAL1 DNA binding activity are known to oscillate with target gene expression in vivo. Here we present a highly sensitive assay that recapitulates native CLOCK-BMAL1 DNA binding rhythms from crude tissue extracts, which we call the Clock Protein-DNA Binding Assay (CPDBA).

Macromolecular Assemblies of the Mammalian Circadian Clock.

The mammalian circadian clock is built on a feedback loop in which PER and CRY proteins repress their own transcription. We found that in mouse liver nuclei all three PERs, both CRYs, and Casein Kinase-1δ (CK1δ) are present together in an ∼1.9-MDa repressor assembly that quantitatively incorporates its CLOCK-BMAL1 transcription factor target.

Core Circadian Clock Genes Regulate Leukemia Stem Cells in AML.

Leukemia stem cells (LSCs) have the capacity to self-renew and propagate disease upon serial transplantation in animal models, and elimination of this cell population is required for curative therapies. Here, we describe a series of pooled, in vivo RNAi screens to identify essential transcription factors (TFs) in a murine model of acute myeloid leukemia (AML) with genetically and phenotypically defined LSCs. These screens reveal the heterodimeric, circadian rhythm TFs Clock and Bmal1 as genes required for the growth of AML cells in vitro and in vivo.

Histone monoubiquitination by Clock-Bmal1 complex marks Per1 and Per2 genes for circadian feedback.

Circadian rhythms in mammals are driven by a feedback loop in which the transcription factor Clock-Bmal1 activates expression of Per and Cry proteins, which together form a large nuclear complex (Per complex) that represses Clock-Bmal1 activity. We found that mouse Clock-Bmal1 recruits the Ddb1-Cullin-4 ubiquitin ligase to Per (Per1 and Per2), Cry (Cry1 and Cry2) and other circadian target genes. Histone H2B monoubiquitination at Per genes was rhythmic and depended on Bmal1, Ddb1 and Cullin-4a.

GRP78(BiP) facilitates the cytosolic delivery of anthrax lethal factor (LF) in vivo and functions as an unfoldase in vitro.

Anthrax toxin is an A/B bacterial protein toxin which is composed of the enzymatically active Lethal Factor (LF) and/or Oedema Factor (EF) bound to Protective Antigen 63 (PA63) which functions as both the receptor binding and transmembrane domains. Once the toxin binds to its cell surface receptors it is internalized into the cell and traffics through Rab5- and Rab7-associated endosomal vesicles. Following acidification of the vesicle lumen, PA63 undergoes a dynamic change forming a beta-barrel that inserts into and forms a pore through the endosomal membrane.

COPI coatomer complex proteins facilitate the translocation of anthrax lethal factor across vesicular membranes in vitro.

The delivery of the diphtheria toxin catalytic domain (DTA) from acidified endosomes into the cytoplasm of eukaryotic cells requires protein-protein interactions between the toxin and a cytosolic translocation factor (CTF) complex. A conserved peptide motif, T1, within the DT transmembrane helix 1 mediates these interactions. Because the T1 motif is also present in the N-terminal segments of lethal factor (LF) and edema factor (EF) in anthrax toxin, we asked whether LF entry into the cell might also be facilitated by target cell cytosolic proteins.