Biology subjects

Chen, A. T.

Publications and source records attributed to Chen, A. T..

12 recordsLinked to original sources

bioRxiv2025

Senataxin is required for optimal class switch recombination to the immunoglobulin A isotype

Senataxin (SETX) is an RNA/DNA helicase that plays a pivotal role in transcription, R-loop resolution, and the DNA damage response (DDR). Dysfunction of SETX, however, has been implicated in neurodegenerative diseases, including amyotrophic lateral sclerosis and ataxia with oculomotor apraxia type 2. Importantly, both R-loop resolution and the DDR are essential for class switch recombination (CSR) in B cells, a critical step in generating antibody diversity. Here we demonstrate that ex vivo polyclonal stimulation of SETX+/+ and SETX-/- mouse splenocytes results in equivalent amounts of IgM- and IgG-secreting B cells but significantly fewer IgA-producing B cells in SETX-/- mice. Additionally, SETX-/- mice generate significantly reduced antigen specific IgA titers following infection with influenza A virus. Sequencing of the IgA-secreting B cell repertoire revealed reduced clonal diversity in SETX-/- mice. SETX-/- mice also had increased R-loop formation within the IgA locus. Collectively, these data highlight an important role for SETX in mediating CSR to IgA. As such, a further understanding of SETXs role in the immune response is important for expanding our knowledge of both the general immunobiology relating to CSR as well as the immunological phenotypes associated with neurodegenerative diseases associate with mutations in SETX. One sentence summaryA deficiency in senataxin results in impaired class switching to IgA in vitro with decreased IgA response to infection and IgA repertoire diversity in vivo.

immunology↗

bioRxiv2023

An AAV capsid reprogrammed to bind human Transferrin Receptor mediates brain-wide gene delivery

Developing vehicles that efficiently deliver genes throughout the human central nervous system (CNS) will broaden the range of treatable genetic diseases. We engineered an AAV capsid, BI-hTFR1, that binds human Transferrin Receptor (TfR1), a protein expressed on the blood-brain barrier (BBB). BI-hTFR1 was actively transported across a human brain endothelial cell layer and, relative to AAV9, provided 40-50 times greater reporter expression in the CNS of human TFRC knock-in mice. The enhanced tropism was CNS-specific and absent in wild type mice. When used to deliver GBA1, mutations of which cause Gaucher disease and are linked to Parkinsons disease, BI-hTFR1 substantially increased brain and cerebrospinal fluid glucocerebrosidase activity compared to AAV9. These findings establish BI-hTFR1 as a promising vector for human CNS gene therapy.

neuroscience↗

bioRxiv2023

Switching circuits: Modulating NK cell response to TGF-β using novel receptors

Engineered natural killer (NK) cells offer a promising treatment strategy for multiple therapeutic areas, yet tuning cell function to diverse environmental and inhibitory contexts remains a significant challenge. An attractive strategy for functional rescue in the presence of inhibitory ligands is the use of switch receptors, surface-expressed chimeric receptors that convert an external inhibitory cue into an intracellular activation signal. Here, we discover novel engineered switch receptors in NK cells that are responsive to TGF-{beta}, a soluble inhibitory factor present in many therapeutic contexts. Through a pooled screen of an 11,131 member library, we identified multiple novel signaling endodomains and endodomain combinations that improve both the acute cytotoxicity and persistence of NK cells in the presence of TGF-{beta}, demonstrating a novel and flexible approach to switch receptor discovery.

immunology↗

bioRxiv2023

TRAFfic signals: High-throughput CAR discovery in NK cells reveals novel TRAF-binding endodomains that drive enhanced persistence and cytotoxicity

Natural killer (NK) cells are a promising alternative therapeutic platform to CAR T cells given their favorable safety profile and potent killing ability. However, CAR NK cells suffer from limited persistence in vivo, which is, in part, thought to be the consequence of limited cytokine signaling. To address this challenge, we developed an innovative high-throughput screening strategy to identify CAR endodomains that could drive enhanced persistence while maintaining potent cytotoxicity. We uncovered a family of TRAF-binding endodomains that outperform benchmarks in primary NK cells along dimensions of persistence and cytotoxicity, even in low IL-2 conditions. This work highlights the importance of cell-type-specific cell therapy engineering and unlocks a wide range of high-throughput molecular engineering avenues in NK cells.

immunology↗

bioRxiv2022

Systematic multi-trait AAV capsid engineering for efficient gene delivery

Broadening gene therapy applications requires manufacturable vectors that efficiently transduce target cells in humans and preclinical models. Conventional selections of adeno-associated virus (AAV) capsid libraries are inefficient at searching the vast sequence space for the small fraction of vectors possessing multiple traits essential for clinical translation. Here, we present Fit4Function, a generalizable machine learning (ML) approach for systematically engineering multi-trait AAV capsids. By leveraging a capsid library that evenly samples the manufacturable sequence space, reproducible screening data are generated to train accurate sequence-to-function models. Combining six models, we designed a multi-trait (liver-targeted, manufacturable) capsid library and validated 89% of library variants on all six predetermined criteria. Furthermore, the models, trained only on mouse in vivo and human in vitro Fit4Function data, accurately predicted AAV capsid variant biodistribution in macaque. Top candidates exhibited high production yields, efficient murine liver transduction, up to 1000-fold greater human hepatocyte transduction, and increased enrichment, relative to AAV9, in a screen for liver transduction in macaques. The Fit4Function strategy ultimately makes it possible to predict cross-species traits of peptide-modified AAV capsids and is a critical step toward assembling an ML atlas that predicts AAV capsid performance across dozens of traits.

synthetic biology↗

bioRxiv2022

A compound PCP scheme underlies sequential rosettes-based cell intercalation

Sequential rosettes are a type of collective cell behavior recently discovered in the C. elegans embryo to mediate directional cell migration through sequential formation and resolution of multicellular rosettes involving the migrating cell and its neighboring cells along the way. Here we show that a Planar Cell Polarity (PCP)-related polarity scheme regulates sequential rosettes, which is distinct from the known mode of how PCP regulates multicellular rosettes in the process of convergent extension. Specifically, non-muscle myosin (NMY) localization and edge contraction are perpendicular to that of Van Gogh as opposed to colocalizing with Van Gogh. Further analyses suggest a two-component polarity scheme: one being the canonical PCP pathway with MIG-1/Frizzled and VANG-1/Van Gogh localized to the vertical edges, the other being MIG-1/Frizzled and NMY-2 localized to the midline/contracting edges. Both MIG-1/Frizzled and VANG-1/Van Gogh are required for NMY-2 localization and contraction of the midline edges, but in redundancy with LAT-1/Latrophilin, an adhesion GPCR which has not been shown to regulate multicellular rosettes. Our results establish a distinct mode of PCP-mediated cell intercalation and shed light on the versatile nature of PCP pathway.

developmental biology↗

bioRxiv2022

Targeting AAV vectors to the CNS via de novo engineered capsid-receptor interactions

Viruses have evolved the ability to bind and enter cells through interactions with a wide variety of host cell macromolecules. Here, we screened for AAV capsids that bind two host cell proteins expressed on the mouse blood-brain barrier, LY6A or the related protein LY6C1. Introducing interactions with either protein target generated hundreds of capsids with dramatically enhanced central nervous system (CNS) tropisms. In contrast to the AAV-PHP.B capsid family, which interacts with LY6A and only exhibits its enhanced CNS tropism in a subset of mouse strains, the capsids that engage LY6C1 maintain their CNS tropism in BALB/cJ mice. Compared to conventional in vivo screens for CNS cell transducing capsids, a single round of protein target binding screening recovered significantly more capsids with enhanced performance that were validated in subsequent in vivo screens. Moreover, the initial screening round generated reproducible and quantitative target binding data that enabled the efficient machine learning-guided generation of more diverse targetspecific capsids. This work demonstrates that AAV capsids can be directly targeted to specific proteins to generate potent gene delivery vectors with known mechanisms of action and predictable tropisms.

synthetic biology↗

bioRxiv2022

Comprehensive characterization of the transcriptional response to COVID-19 in multiple organs reveals shared signatures across tissues

Infection by Coronavirus SARS-CoV2 is a severe and often deadly disease that has implications for the respiratory system and multiple organs across the human body. While the effects in the lung have been extensively studied, less is known about COVID-19s cellular impact across other organs. Here we contribute a single-nuclei RNA sequencing atlas comprising six human organs across 20 autopsies where we analyzed the transcriptional changes due to COVID-19 in multiple cell types. Computational cross-organ analysis for endothelial cells and macrophages identified systemic transcriptional changes in these cell types in COVID-19 samples. In addition, analysis of signaling pathways from multiple datasets showed several systemic dysregulations of signaling interaction in different cell types. Altogether, the COVID Tissue Atlas enables the investigation of both cell type-specific and cross-organ transcriptional responses to COVID-19, providing insights into the molecular networks affected by the disease and highlighting novel potential targets for therapies and drug development.

genomics↗

bioRxiv2021

Peer teaching as bioinformatics training strategy: incentives, challenges, and benefits.

As biomedical research becomes more data-intensive, bioinformatics is becoming essential to understanding biological processes, systems, and diseases. In this paper we describe the use of a series of peer teaching workshops as a strategy to respond to the bioinformatics training needs at a research-intensive institution. In addition to the data collected from the workshops, we also used personal experiences of researchers who participated as peer teachers to understand the incentives, challenges, and benefits of peer teaching. Developing communication skills such as confidence in teaching, explaining complex concepts, and better understanding of the topic emerged as primary benefits that the teachers obtained from this experience. Lack of time for teaching and the struggles of classroom management were identified as two major challenges. We suggest that peer teaching can be beneficial not only to train researchers in bioinformatics, but also as a professional development opportunity for graduate students and postdoctoral trainees.

scientific communication and education↗

bioRxiv2020

COVID-19 CG: Tracking SARS-CoV-2 mutations by locations and dates of interest

COVID-19 CG is an open resource for tracking SARS-CoV-2 single-nucleotide variations (SNVs) and lineages while filtering by location, date, gene, and mutation of interest. COVID-19 CG provides significant time, labor, and cost-saving utility to diverse projects on SARS-CoV-2 transmission, evolution, emergence, immune interactions, diagnostics, therapeutics, vaccines, and intervention tracking. Here, we describe case studies in which users can interrogate (1) SNVs in the SARS-CoV-2 Spike receptor binding domain (RBD) across different geographic regions to inform the design and testing of therapeutics, (2) SNVs that may impact the sensitivity of commonly used diagnostic primers, and (3) the recent emergence of a dominant lineage harboring an S477N RBD mutation in Australia. To accelerate COVID-19 research and public health efforts, COVID-19 CG will be continually upgraded with new features for users to quickly and reliably pinpoint mutations as the virus evolves throughout the pandemic and in response to therapeutic and public health interventions.

bioinformatics↗

bioRxiv2019

DO-MS: Data-Driven Optimization of Mass Spectrometry Methods

The performance of ultrasensitive LC-MS/MS methods, such as Single-Cell Proteomics by Mass Spectrometry (SCoPE-MS), depends on multiple interdependent parameters. This interdependence makes it challenging to specifically pinpoint bottlenecks in the LC-MS/MS methods and approaches for resolving them. For example, low signal at MS2 level can be due to poor LC separation, ionization, apex targeting, ion transfer, or ion detection. We sought to specifically diagnose such bottlenecks by interactively visualizing data from all levels of bottom-up LC-MS/MS analysis. Many search engines, such as MaxQuant, already provide such data, and we developed an open source platform for their interactive visualization and analysis: Data-driven Optimization of MS (DO-MS). We found that in many cases DO-MS not only specifically diagnosed bottlenecks but also enabled us to rationally optimize them. For example, we used DO-MS to diagnose poor sampling of the elution peak apex and to optimize it, which increased the efficiency of delivering ions for MS2 analysis by 370%. DO-MS is easy to install and use, and its GUI allows for interactive data subsetting and high-quality figure generation. The modular design of DO-MS facilitates customization and expansion. DO-MS is available for download from GitHub: github.com/SlavovLab/DO-MS

bioinformatics↗

bioRxiv2018

Ex vivo dynamics of human glioblastoma cells in a microvasculature-on-a-chip system correlates with tumor heterogeneity and subtypes

The perivascular niche (PVN) plays an essential role in brain tumor stem-like cell (BTSC) fate control, tumor invasion, and therapeutic resistance. Herein we report on the use of a microvasculature-on-a-chip system as a PVN model to evaluate the dynamics of BTSCs ex vivo from 10 glioblastoma patients. We observed that BTSCs preferentially localize in the perivascular zone. Live cell tracking revealed that the cells residing in the vicinity of microvessels had the lowest motility, while a fraction of cells on the microvessels unexpectedly possessed the highest motility and migrated over the longest distance. These results indicate that the perivascular zone is a niche for BTSCs, while the microvascular tracks are also a path for long-distance tumor cell migration and invasion. Additionally, the degree of co-localization between tumor cells and microvessels varied significantly across patients. To validate the results from our microvasculature-on-a-chip system, we used single-cell transcriptome sequencing (10 patients and 21,750 single cells in total) to identify the subtype of each tumor cell. The co-localization coefficient was found to correlate positively with proneural (stem-like) or mesenchymal (invasive) but not classical (proliferative) tumor cells. Furthermore, we found that a gene signature profile including PDGFRA correlated strongly with the \"homing\" of brain tumor cells to the PVN. Our findings demonstrated that ex vivo dynamics of human brain tumor cells in a microvasculature-on-a-chip model can recapitulate in vivo tumor cell dynamics, heterogeneity, and subtypes, representing a new route to the study of human tumor cell biology and uncover patient-specific tumor cell functions.

bioengineering↗