Biology subjects

Keung, A. J.

Publications and source records attributed to Keung, A. J..

16 recordsLinked to original sources

bioRxiv2026

Colloidal DNA nanoaggregates applied towards file-partitioning for information storage and dynamic data obfuscation

The molecular programmability of nucleic acids has facilitated the development of architected DNA/RNA nanostructures and their applications in novel materials and technologies. We report how different types of DNA and RNA nanoaggregates, bundling digital information encoded into oligo libraries, can be formed by manipulating the ionic strength of the solution. As DNA or RNA suspensions are immersed in solutions of increasing salt concentrations, we observe the onset of aggregation. Further increase in ionic strength leads to the formation of stable, reproducible, and well-defined aggregates. We show that these nanoaggregates are kinetically trapped at room temperature, stably partition DNA libraries that encode image files, and support file-specific random access by bundling DNA libraries with unique address oligos. The nanoaggregate files can be disrupted and reformed into scrambled bundles using simple external fluid shear or temperature annealing, rapidly obfuscating the data. We term these nanoaggregates nucleic acid PACKeTs: Partitioned Aggregates of Colloidal DNA/RNA through Kinetic Trapping. Overall, the results demonstrate how gaining fundamental insights into ionic colloidal aggregation enables new forms of manipulation of DNA and RNA libraries. This understanding could lead to novel functionalities including kinetically trapped data partitioning, random access, and data encryption or obfuscation.

bioengineering↗

bioRxiv2025

Glucose levels impact the morphology and cell-type composition of human cerebral organoids

Human cerebral organoids, derived from pluripotent stem cells, are powerful models for studying human brain development. The understanding of how morphogens can be used to guide patterning and differentiation has matured rapidly, however, the influence of basal media components on organoid development remains unclear. Standard organoid media frequently contain non- physiological concentrations of nutrients, including glucose, a central regulator of cellular metabolism and signaling. Here, we examine how glucose availability shapes cerebral organoid growth, morphology, and cell-type composition by comparing conventional hyperglycemic media to media with glucose levels more closely resembling physiological conditions. We find that organoids derived from multiple human pluripotent stem cell lines can grow in physiological glucose, but exhibit altered growth rates, structural features, and lineage distributions. In H9 embryonic stem cell-derived organoids, inhibition of the mTOR pathway under physiological glucose restores neurodevelopmental cell types otherwise diminished in these conditions. These findings highlight glucose as a key determinant of organoid lineage specification and cellular signaling. Importantly, however, glucose modulation does not reduce variability across organoids or cell lines, underscoring the need to better understand and control sources of heterogeneity to improve organoid models.

cell biology↗

bioRxiv2025

A Human Angelman Syndrome Class II Pluripotent Stem Cell line with Fluorescent Paternal UBE3A Reporter

IntroductionAngelman Syndrome (AS) is characterized in large part by the loss of functional UBE3A protein in mature neurons. A majority of AS etiologies is linked to deletion of the maternal copy of the UBE3A gene and epigenetic silencing of the paternal copy. A common therapeutic strategy is to unsilence the intact paternal copy thereby restoring UBE3A levels. Identifying novel therapies has been aided by a UBE3A-YFP reporter mouse model. This study presents an analogous fluorescent UBE3A reporter system in human cells. MethodsPreviously derived induced Pluripotent Stem Cells (iPSCs) with a Class II large deletion at the UBE3A locus are used in this study. mGL and eGFP are integrated downstream of the endogenous UBE3A using CRISPR/Cas9. These reporter iPSCs are differentiated into 2D and 3D neural cultures to monitor long-term neuronal maturation. Green fluorescence dynamics are analyzed by immunostaining and flow cytometry. ResultsThe reporter is successfully integrated into the genome and reports paternal UBE3A expression. Fluorescence expression gradually reduces with UBE3A silencing in neurons as they mature. Expression patterns also reflect expected responses to molecules known to reactivate paternal UBE3A. DiscussionThis human-cell-based model can be used to screen novel therapeutic candidates, facilitate tracking of UBE3A expression in time and space, and study human-specific responses. However, its ability to restore UBE3A function cannot be studied using this model. Further research in human cells is needed to engineer systems with functional UBE3A to fully capture the therapeutic capabilities of novel candidates.

cell biology↗

bioRxiv2025

A High Sensitivity Assay of UBE3A Ubiquitin Ligase Activity

UBE3A is an E3 ubiquitin ligase associated with several neurodevelopmental disorders. The development of several preclinical therapeutic approaches involving UBE3A, such as gene therapy, enzyme replacement therapy, and epigenetic reactivation, require the detection of its ubiquitin ligase activity. Prior commercial assays leveraged Western Blotting to detect shifts in substrate size due to ubiquitination, but these suffered from long assay times and have also been discontinued. Here we develop a new assay that quantifies UBE3A activity. It measures the fluorescence intensity of ubiquitinated p53 substrates with a microplate reader, eliminating the need for Western Blot antibodies and instruments, and enables detection in just 1 hour. The assay is fast, cost-effective, low noise, and uses components with long shelf lives. Importantly, it is also highly sensitive, detecting UBE3A levels as low as 1nM, similar to that observed in human and mouse cerebrospinal fluid. It also differentiates the activity of wild-type UBE3A and catalytic mutants. We also design a p53 substrate with a triple-epitope tag HIS-HA-CMYC on the N terminus, which allows for versatile detection of UBE3A activity from diverse natural and engineered sources. This new assay provides a timely solution for growing needs in preclinical validation, quality control, endpoint measurements for clinical trials, and downstream manufacturing testing and validation.

biochemistry↗

bioRxiv2024

Nanopore Decoding with Speed and Versatility for Data Storage

MotivationAs nanopore technology reaches ever higher throughput and accuracy, it becomes an increasingly viable candidate for reading out DNA data storage. Nanopore sequencing offers considerable flexibility by allowing long reads, real-time signal analysis, and the ability to read both DNA and RNA. We need flexible and efficient designs that match nanopores capabilities, but relatively few designs have been explored and many have significant inefficiency in read density, error rate, or compute time. To address these problems, we designed a new single-read per-strand decoder that achieves low byte error rates, offers high throughput, scales to long reads, and works well for both DNA and RNA molecules. We achieve these results through a novel soft decoding algorithm that can be effectively parallelized on a GPU. Our faster decoder allows us to study a wider range of system designs. ResultsWe demonstrate our approach on HEDGES, a state-of-the-art DNA-constrained convolutional code. We implement one hard decoder that runs serially and two soft decoders that run on GPUs. Our evaluation for each decoder is applied to the same population of nanopore reads collected from a synthesized library of strands. These same strands are synthesized with a T7 promoter to enable RNA transcription and decoding. Our results show that the hard decoder has a byte error rate over 25%, while the prior state of the art soft decoder can achieve error rates of 2.25%. However, that design also suffers a low throughput of 183 seconds/read. Our new Alignment Matrix Trellis soft decoder improves throughput by 257x with the trade off of a higher byte error rate of 3.52% compared to the state-of-the-art. Furthermore, we use the faster speed of our algorithm to explore more design options. We show that read densities of 0.33 bits/base can be achieved, which is 4x larger than prior MSA-based decoders. We also compare RNA to DNA, and find that RNA has 85% as many error free reads as compared to DNA. Availability and implementationSource code for our soft decoder and data used to generate figures is available publicly in the Github repository https://github.com/dna-storage/hedges-soft-decoder (10.5281/zenodo.11454877). All raw FAST5/FASTQ data is available at 10.5281/zenodo.11985454 and 10.5281/zenodo.12014515. Contackvolkel@ncsu.edu or jtuck@ncsu.edu

synthetic biology↗

bioRxiv2024

Loss of UBE3A impacts both neuronal and non-neuronal cells in human cerebral organoids

Angelman syndrome is a neurodevelopmental disorder caused by (epi)genetic lesions of maternal UBE3A. Research has focused largely on the role of UBE3A in neurons due to its imprinting in that cell type. Yet, evidence suggests there may be broader neurodevelopmental impacts of UBE3A dysregulation. Human cerebral organoids might reveal these understudied aspects of UBE3A as they recapitulate diverse cell types of the developing human brain. We performed scRNAseq on organoids to reveal the effects of UBE3A disruption on cell type-specific compositions and transcriptomic alterations. In the absence of UBE3A, progenitor proliferation and structures were disrupted while organoid composition shifted away from proliferative cell types. We observed impacts on non-neuronal cells, including choroid plexus enrichment. Furthermore, EMX1+ cortical progenitors were negatively impacted, disrupting corticogenesis, and potentially delaying excitatory neuron maturation. This work reveals novel impacts of UBE3A on understudied cell types and related neurodevelopmental processes and elucidates potential new therapeutic targets. TeaserHuman cerebral organoids exhibit compositional and transcriptomic alterations in both neuronal and non-neuronal cells in the absence of UBE3A.

developmental biology↗

bioRxiv2023

Reactive Oxygen Species Mediate Transcriptional Responses to Dopamine and Cocaine in Human Cerebral Organoids

Dopamine signaling in the adult ventral forebrain regulates behavior, stress response, and memory formation and in neurodevelopment regulates neural differentiation and cell migration. Excessive dopamine levels including due to cocaine use both in utero and in adults could lead to long-term adverse consequences. The mechanisms underlying both homeostatic and pathological changes remain unclear, partly due to the diverse cellular responses elicited by dopamine and the reliance on animal models that exhibit species- specific differences in dopamine signaling. To address these limitations, 3-D cerebral organoids have emerged as human-derived models, recapitulating salient features of human cell signaling and neurodevelopment. Organoids have demonstrated responsiveness to external stimuli, including substances of abuse, making them valuable investigative models. In this study we utilize the Xiang-Tanaka ventral forebrain organoid model and characterize their response to acute and chronic dopamine or cocaine exposure. The findings revealed a robust immune response, novel response pathways, and a potential critical role for reactive oxygen species (ROS) in the developing ventral forebrain. These results highlight the potential of cerebral organoids as in vitro human models for studying complex biological processes in the brain.

cell biology↗

bioRxiv2023

Single cell assessment of human stem cell derived mesolimbic models and their responses to substances of abuse.

The mesolimbic pathway connects ventral tegmental area dopaminergic neurons and striatal medium spiny neurons, playing a critical role in reward and stress behaviors. Exposure to substances of abuse during development and adulthood has been linked to adverse outcomes and molecular changes. The rise of human cell repositories and whole genome sequences enables human functional genomics in a dish, offering insights into human-specific responses to substances of abuse. Characterizations of in vitro models are necessary to ensure appropriate experimental designs and accurate interpretation of results. This study provides a comprehensive characterization of these models and their responses to substances of abuse, introducing new culture conditions for generating medium spiny neurons and dopaminergic neurons from human pluripotent stem cells. Single cell analysis reveals cell type-specific transcriptomic responses to dopamine, cocaine, and morphine, including compound and cell type-specific transcriptomic signatures related to neuroinflammation and alterations in signaling pathways. These findings offer a resource for future genomics studies leveraging human stem cell-derived models. TeaserGeneration and characterization of a novel mesolimbic pathway model and its response to acute dopamine, morphine, and cocaine.

cell biology↗

bioRxiv2021

Identifying challenges and opportunities for histone peptides in protein engineering platforms

Histone post-translational modifications are small chemical changes to histone protein structure that have cascading effects on diverse cellular functions. Detecting histone modifications and characterizing their binding partners are critical steps in understanding chromatin biochemistry and have been accessed using common reagents such as antibodies, recombinant assays, and FRET based systems. High throughput platforms could accelerate work in this field, and also could be used to engineer de novo histone affinity reagents; yet published studies on their use with histones have been noticeably sparse. Here we describe specific experimental conditions that affect binding specificities of post-translationally modified histones in classic protein engineering platforms and likely explain the relative difficulty with histone targets in these platforms. We also show that manipulating avidity of binding interactions may improve specificity of binding.

bioengineering↗

bioRxiv2021

REMY: A platform for the rapid interrogation of epigenome modifications on yeast

Histone proteins are decorated with a combinatorially and numerically diverse set of biochemical modifications. Here we describe a versatile and scalable platform termed Rapid interrogation of Epigenome Modifications using Yeast surface display (REMY), which enables efficient characterization of histone modifications without the need for recombinant protein production. As proof-of-concept, we first used REMY to rapidly profile the histone H3 and H4 residue writing specificities of the human histone acetyltransferase, p300. Subsequently, we used REMY to screen a large panel of commercially available anti-acetylation antibodies for their specificities, identifying many suitable and unsuitable reagents. Further, use of REMY enabled efficient mapping of the large binary crosstalk space between acetylated residues on histones H3 and H4, and uncovered previously unreported residue interdependencies affecting p300 activity. Our results show that REMY is a useful tool that can advance our understanding of chromatin biology by enabling efficient interrogation of the complexity of epigenome modifications.

synthetic biology↗

bioRxiv2021

Effects of early geometric confinement on the transcriptomic profile of human cerebral organoids

BackgroundBiophysical factors such as shape and mechanical forces are known to play crucial roles in stem cell differentiation, embryogenesis and neurodevelopment. However, the complexity and experimental challenges capturing such early stages of development, and ethical concerns associated with human embryo and fetal research, limit our understanding of how these factors affect human brain organogenesis. Human cerebral organoids (hCO) are attractive models due to their ability to model important brain regions and transcriptomics of early in vivo brain development. Furthermore, they provide three-dimensional environments that better mimic the in vivo environment. To date, they have been used to understand the effects of genetics and soluble factors on neurodevelopment. Establishing links between spatial factors and hCO development will require the development of new approaches. ResultsHere, we investigated the effects of early geometric confinements on transcriptomic changes during hCO differentiation. Using a custom and tunable agarose microwell platform we generated embryoid bodies (EB) of diverse shapes and then further differentiated those EBs to whole brain hCOs. Our results showed that the microwells did not have negative gross impacts on the ability of the hCOs to differentiate generally towards neural fates, and there were clear shape dependent effects on neural lineage specification. In particular, we observed that non-spherical shapes showed signs of altered neurodevelopmental kinetics and favored the development of medial ganglionic eminence-associated brain regions and cell types over cortical regions. ConclusionsThe findings presented here suggest a role for spatial factors in brain region specification during hCO development. Understanding these spatial patterning factors will not only improve understanding of in vivo development and differentiation, but also provide important handles with which to advance and improve control over human model systems for in vitro applications.

bioengineering↗

bioRxiv2021

Mapping the dynamic transfer functions of epigenome editing

Biological information can be encoded in the dynamics of signaling components which has been implicated in a broad range of physiological processes including stress response, oncogenesis, and stem cell differentiation. To study the complexity of information transfer across the eukaryotic promoter, we screened 119 dynamic conditions--modulating the frequency, intensity, and pulse width of light--regulating the binding of an epigenome editor to a fluorescent reporter. This system revealed highly tunable gene expression and filtering behaviors and provided the most comprehensive quantification to date of the maximum amount of information that can be reliably transferred across a promoter as [~]1.7 bits. Using a library of over 100 orthogonal epigenome editors, we further determined that chromatin state could be used to tune mutual information and expression levels, as well as completely alter the input-output transfer function of the promoter. This system unlocks the information-rich content of eukaryotic epigenome editing.

synthetic biology↗

bioRxiv2019

Dynamic DNA-based information storage

Technological leaps are often driven by key innovations that transform the underlying architectures of systems. Current DNA storage systems largely rely on polymerase chain reaction, which broadly informs how information is encoded, databases are organized, and files are accessed. Here we show that a hybrid toehold DNA structure can unlock a fundamentally different, dynamic DNA-based information storage system architecture with broad advantages. This innovation increases theoretical storage densities and capacities by eliminating non-specific DNA-DNA interactions common in PCR and increasing the encodable sequence space. It also provides a physical handle with which to implement a range of in-storage file operations. Finally, it reads files non-destructively by harnessing the natural role of transcription in accessing information from DNA. This simple but powerful toehold structure lays the foundation for an information storage architecture with versatile capabilities.

synthetic biology↗

bioRxiv2019

Human cerebral organoids capture the spatiotemporal complexity and disease dynamics of UBE3A

Human neurodevelopment and its associated diseases are complex and challenging to study. This has driven recent excitement for human cerebral organoids (hCOs) as research and screening tools. These models are steadily proving their utility; however, it remains unclear what limits they will face in recapitulating the complexities of neurodevelopment and disease. Here we show that their utility extends to key (epi)genetic and disease processes that are complex in space and time. Specifically, hCOs capture UBE3As dynamically imprinted expression and subcellular localization patterns. Furthermore, given UBE3As direct links to Angelman Syndrome and Autism Spectrum Disorder, we show that hCOs respond to candidate small molecule therapeutics. This work demonstrates that hCOs can provide important insights to focus the scope of mechanistic and therapeutic strategies including revealing difficult to access prenatal developmental time windows and cell types key to disease etiology.

bioengineering↗

bioRxiv2019

Driving the scalability of DNA-based information storage systems

The extreme density of DNA presents a compelling advantage over current storage media; however, in order to reach practical capacities, new approaches for organizing and accessing information are needed. Here we use chemical handles to selectively extract unique files from a complex database of DNA mimicking 5 TB of data and design and implement a nested file address system that increases the theoretical maximum capacity of DNA storage systems by five orders of magnitude. These advancements enable the development and future scaling of DNA-based data storage systems with reasonable modern capacities and file access capabilities.

bioengineering↗

bioRxiv2018

One-step assembly of large CRISPR arrays enables multi-functional targeting and reveals constraints on array design

CRISPR-Cas systems inherently multiplex through their CRISPR arrays--whether to confer immunity against multiple invaders or by mediating multi-target editing, regulation, imaging, and sensing. However, arrays remain difficult to generate due to their reoccurring repeat sequences. Here, we report an efficient, one-step scheme called CRATES to construct large CRISPR arrays through defined assembly junctions within the trimmed portion of array spacers. We show that the constructed arrays function with the single-effector nucleases Cas9, Cas12a, and Cas13a for multiplexed DNA/RNA cleavage and gene regulation in cell-free systems, bacteria, and yeast. We also applied CRATES to assemble composite arrays utilized by multiple Cas nucleases, where these arrays enhanced DNA targeting specificity by blocking off-target sites. Finally, array characterization revealed context-dependent loss of spacer activity and processing of unintended guide RNAs derived from Cas12a terminal repeats. CRATES thus can facilitate diverse applications requiring CRISPR multiplexing and help elucidate critical factors influencing array function.

synthetic biology↗