Methylarginine targeting chimeras for lysosomal degradation of intracellular proteins.

A paradigm shift in drug development is the discovery of small molecules that harness the ubiquitin-proteasomal pathway to eliminate pathogenic proteins. Here we provide a modality for targeted protein degradation in lysosomes. We exploit an endogenous lysosomal pathway whereby protein arginine methyltransferases (PRMTs) initiate substrate degradation via arginine methylation.

A high throughput bispecific antibody discovery pipeline.

Bispecific antibodies (BsAbs) represent an emerging class of immunotherapy, but inefficiency in the current discovery has limited their broad clinical availability. Here we report a high throughput, agnostic, single-cell-based functional screening pipeline, comprising molecular and cell engineering for efficient generation of BsAb library cells, followed by functional interrogation at the single-cell level to identify and sort positive clones and downstream sequence identification and functionality characterization. Using a CD19xCD3 bispecific T cell engager (BiTE) as a model, we demonstrate that our single-cell platform possesses a high throughput screening efficiency of up to one and a half million variant library cells per run and can isolate rare functional clones at a low abundance of 0.

An Optimized Enzyme-Nucleobase Pair Enables RNA Metabolic Labeling with Improved Cell-Specificity.

Current transcriptome-wide analyses have identified a growing number of regulatory RNA with expression that is characterized in a cell-type-specific manner. Herein, we describe RNA metabolic labeling with improved cell-specificity utilizing the expression of an optimized uracil phosphoribosyltransferase (UPRT) enzyme. We demonstrate improved selectivity for metabolic incorporation of a modified nucleobase (5-vinyuracil) into nascent RNA, using a battery of tests.

Repurposing a DNA-Repair Enzyme for Targeted Protein Degradation.

Herein we present the exploration of the utility of DNA demethylase enzymes for targeted protein degradation. Novel benzylguanine substrates are characterized for their ability to control protein degradation in cells. Our data demonstrate the utility of this approach to degrade fusion proteins in different localizations within living cells.

Spatial transcriptomics using combinatorial fluorescence spectral and lifetime encoding, imaging and analysis.

Multiplexed mRNA profiling in the spatial context provides new information enabling basic research and clinical applications. Unfortunately, existing spatial transcriptomics methods are limited due to either low multiplexing or complexity. Here, we introduce a spatialomics technology, termed Multi Omic Single-scan Assay with Integrated Combinatorial Analysis (MOSAICA), that integrates in situ labeling of mRNA and protein markers in cells or tissues with combinatorial fluorescence spectral and lifetime encoded probes, spectral and time-resolved fluorescence imaging, and machine learning-based decoding.

Epigenetic silencing directs expression heterogeneity of stably integrated multi-transcript unit genetic circuits.

We report that epigenetic silencing causes the loss of function of multi-transcript unit constructs that are integrated using CRISPR-Cas9. Using a modular two color reporter system flanked by selection markers, we demonstrate that expression heterogeneity does not correlate with sequence alteration but instead correlates with chromosomal accessibility. We partially reverse this epigenetic silencing via small-molecule inhibitors of methylation and histone deacetylation.

Rapid bacterial detection and antibiotic susceptibility testing in whole blood using one-step, high throughput blood digital PCR.

Sepsis due to antimicrobial resistant pathogens is a major health problem worldwide. The inability to rapidly detect and thus treat bacteria with appropriate agents in the early stages of infections leads to excess morbidity, mortality, and healthcare costs. Here we report a rapid diagnostic platform that integrates a novel one-step blood droplet digital PCR assay and a high throughput 3D particle counter system with potential to perform bacterial identification and antibiotic susceptibility profiling directly from whole blood specimens, without requiring culture and sample processing steps.

An ultrasensitive test for profiling circulating tumor DNA using integrated comprehensive droplet digital detection.

Current cancer detection systems lack the required sensitivity to reliably detect minimal residual disease (MRD) and recurrence at the earliest stages when treatment would be most effective. To address this issue, we present a novel liquid biopsy approach that utilizes an integrated comprehensive droplet digital detection (IC3D) digital PCR system which combines microfluidic droplet partitioning, fluorescent multiplex PCR chemistry, and our rapid 3D, large-volume droplet counting technology. The IC3D ddPCR assay can detect cancer-specific, ultra-rare genomic targets due to large sample input and high degree of partitioning.

The coordinating role of IQGAP1 in the regulation of local, endosome-specific actin networks.

IQGAP1 is a large, multi-domain scaffold that helps orchestrate cell signaling and cytoskeletal mechanics by controlling interactions among a spectrum of receptors, signaling intermediates, and cytoskeletal proteins. While this coordination is known to impact cell morphology, motility, cell adhesion, and vesicular traffic, among other functions, the spatiotemporal properties and regulatory mechanisms of IQGAP1 have not been fully resolved. Herein, we describe a series of super-resolution and live-cell imaging analyses that identified a role for IQGAP1 in the regulation of an actin cytoskeletal shell surrounding a novel membranous compartment that localizes selectively to the basal cortex of polarized epithelial cells (MCF-10A).

Elucidation of Exosome Migration across the Blood-Brain Barrier Model In Vitro.

The delivery of therapeutics to the central nervous system (CNS) remains a major challenge in part due to the presence of the blood-brain barrier (BBB). Recently, cell-derived vesicles, particularly exosomes, have emerged as an attractive vehicle for targeting drugs to the brain, but whether or how they cross the BBB remains unclear. Here, we investigated the interactions between exosomes and brain microvascular endothelial cells (BMECs) under conditions that mimic the healthy and inflamed BBB .

Programming in situ immunofluorescence intensities through interchangeable reactions of dynamic DNA complexes.

The regulation of antibody reporting intensities is critical to various in situ fluorescence-imaging analyses. Although such control is often necessary to visualize sparse molecular targets, the ability to tune marker intensities is also essential for highly multiplexed imaging strategies in which marker reporting levels must be tuned both to optimize dynamic detection ranges and to minimize crosstalk between different signals. Existing chemical amplification approaches generally lack such control.

Configuring robust DNA strand displacement reactions for in situ molecular analyses.

The number of distinct biomolecules that can be visualized within individual cells and tissue sections via fluorescence microscopy is limited by the spectral overlap of the fluorescent dye molecules that are coupled permanently to their targets. This issue prohibits characterization of important functional relationships between different molecular pathway components in cells. Yet, recent improved understandings of DNA strand displacement reactions now provides opportunities to create programmable labeling and detection approaches that operate through controlled transient interactions between different dynamic DNA complexes.

Theoretical investigation of the role of the RANK-RANKL-OPG system in bone remodeling.

The RANK-RANKL-OPG system is an essential signaling pathway involved in bone cell-cell communication, with ample evidence that modification of the RANK-RANKL-OPG signaling pathway has major effects on bone remodeling. The first focus of this paper is to demonstrate that a theoretical model of bone cell-cell interactions is capable of qualitatively reproducing changes in bone associated with RANK-RANKL-OPG signaling. To do this we consider either biological experiments or bone diseases related to receptor and/or ligand deficiencies, including RANKL over-expression, ablation of OPG production and/or RANK receptor modifications.

Model structure and control of bone remodeling: a theoretical study.

It is generally accepted that RANKL is highly expressed in osteoblast precursor cells while OPG is highly expressed in mature osteoblasts, but to date no functional utility to the BMU has been proposed for this particular ligand-decoy-receptor expression profile. As discovered in the mid 90s, the RANK-RANKL-OPG signaling cascade is a major signaling pathway regulating bone remodeling. In this paper we study theoretically the functional implications of particular RANKL/OPG expression profiles on bone volume.