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Shih, J.

Publications and source records attributed to Shih, J..

13 recordsLinked to original sources
bioRxiv2026

A Deep Quantitative Proteome Turnover Platform for Human iPSC-derived Neurons

Quantitative evaluation of protein turnover in human neurons is crucial for understanding neuron homeostasis and guiding drug development for neurological diseases. However, measuring protein turnover in postmitotic neurons remains challenging due to the high dynamic range of protein half-lives and limited proteome coverage in SILAC (Stable Isotope Labeling by Amino acids in Cell culture) experiments. Despite broad applications of dynamic SILAC proteomics to measure protein turnover in rodent tissues and primary neurons, few studies have measured protein half-lives in human neurons with limited proteome coverage. Here, we established a comprehensive platform to quantify protein half-lives in human induced pluripotent stem cell (iPSC)-derived neurons. By integrating optimized dynamic SILAC labeling in human neuron cultures, extensive peptide fractionation, optimized data-dependent and data-independent LC-MS/MS acquisition methods, and a streamlined computational pipeline, we achieved deep and accurate measurement of 10,792 protein half-lives from 162,854 unique peptides. We then compared the protein turnover and abundances in iPSC-derived glutamatergic cortical neurons and spinal motor neurons, revealing globally conserved proteome dynamics alongside subtype-specific differences consistent with specialized neuronal functions. To enable broad community access, we created NeuronProfile (www.neuronprofile.com), an interactive web platform for exploring protein turnover, abundance, and subcellular location in human neurons. Together, this work provides a comprehensive analytical platform to assess human neuronal proteostasis and a foundational resource for neurological disease research and therapeutic development.

neuroscience↗
bioRxiv2025

StrainFacts accurately quantifies both endogenous and live biotherapeutic product strain abundances in simulated and clinical vaginal microbiota samples

Live biotherapeutic products (LBPs) deliver microbial strains to modulate the host microbiota in order to promote health or treat and prevent disease. Since endogenous strains are already present, accurately evaluating LBP efficacy and mechanism of action requires distinguishing administered from endogenous strains. Although computational tools exist for inferring strains from short-read metagenomic data, few have been rigorously tested in the context of LBP treatment. Here, we assess the ability of StrainFacts, a computational tool for inferring strains from short-read metagenomic data, to estimate strain abundances and genotypes of endogenous and administered strains. We performed a simulation study of a single-strain LBP trial, modeling serial samples across a range of administered strain abundance, co-occurring endogenous strains, and sequencing depths. We found that StrainFacts accurately estimated both LBP and endogenous strain abundances and genotypes within simulated samples. We further validated methods using human vaginal microbiota samples spiked with CTV-05, the Lactobacillus crispatus strain contained in the LBP LACTIN-V, which has been shown to reduce recurrent bacterial vaginosis. Our findings demonstrate that StrainFacts can robustly assess LBP and endogenous strain colonization, abundance, and dynamics in simulated and experimental microbiota samples, supporting its utility as an analysis tool for vaginal LBP therapeutic trial data.

bioinformatics↗
bioRxiv2024

Cortical norepinephrine-astrocyte signaling critically mediates learned behavior

Updating behavior based on feedback from the environment is a crucial means by which organisms learn and develop optimal behavioral strategies1-3. Norepinephrine (NE) release from the locus coeruleus (LC) has been shown to mediate learned behaviors4-6 such that in a task with graded stimulus uncertainty and performance, a high level of NE released after an unexpected outcome causes adaptations in subsequent behavior7. Yet, how the transient activity of LC-NE neurons, lasting tens of milliseconds, alters neuronal activity and influences behavior several seconds later is unclear. Here, we show that NE released after an unexpected outcome acts directly on cortical astrocytes via 1 adrenergic (Adra1a) receptors to elicit sustained increases in intracellular calcium. Chemogenetic blockade of astrocytic calcium dynamics prevents trial-to-trial behavioral adaptation. NE stimulation of astrocytes elicits ATP release, and imaging ATP levels in the cortex reveals an increase in extracellular ATP in response to an unexpected outcome. Blocking ATP-driven signaling to neuronal adenosine A1 receptors also prevents post-reinforcement behavioral adaptation. Finally, high density neuronal recordings in prefrontal cortex reveal that a surprising outcome alters the neuronal representation of the stimulus on the subsequent trial without sustained changes in cortical activity; blocking either astrocyte calcium dynamics or A1 receptors occludes these post-reinforcement changes in single-neuron and population neuronal encoding of task variables underlying behavioral changes. Together, these data demonstrate that astrocytes play an essential role in norepinephrine-driven learned behavior: they have prolonged calcium responses to transient norepinephrine release and convey task-relevant reinforcement information across behavioral intervals, enabling selective updating of neuronal task representations to support adaptive behavior.

neuroscience↗
bioRxiv2024

The noncoding circular RNA circHomer1 regulates developmental experience-dependent plasticity in mouse visual cortex

Circular RNAs (circRNAs) are a class of closed-loop, single stranded RNAs whose expression is particularly enriched in the brain. Despite this enrichment and evidence that the expression of circRNAs are altered by synaptic development and in response to synaptic plasticity in vitro, the regulation by and function of the majority of circRNAs in experience-dependent plasticity in vivo remain unexplored. Here, we employed transcriptome-wide analysis comparing differential expression of both mRNAs and circRNAs in juvenile mouse primary visual cortex (V1) following monocular deprivation (MD), a model of experience-dependent developmental plasticity. Among the differentially expressed mRNAs and circRNAs following 3-day MD, the circular and the activity-dependent mRNA forms of the Homer1 gene, circHomer1 and Homer1a respectively, were of interest as their expression changed in opposite directions: circHomer1 expression increased while the expression of Homer1a decreased following 3-day MD. Knockdown of circHomer1 delayed the depression of closed-eye responses normally observed after 3-day MD. circHomer1-knockdown also led to a reduction in average dendritic spine size prior to MD but critically there was no further reduction after 3-day MD, consistent with impaired structural plasticity. circHomer1-knockdown also prevented the reduction of surface AMPA receptors after 3-day MD. Synapse-localized puncta of the AMPA receptor endocytic protein Arc increased in volume after MD but were smaller in circHomer1-knockdown neurons, suggesting that circHomer1 knockdown impairs experience-dependent AMPA receptor endocytosis. Thus, the expression of multiple circRNAs are regulated by experience-dependent developmental plasticity, and our findings highlight the essential role of circHomer1 in V1 synaptic development and experience-dependent plasticity. Significance StatementCircular RNAs (circRNAs) are a class of closed-loop RNAs formed through back-splicing of exon and/or intron junctions. Initially considered as byproducts of aberrant RNA splicing with limited function, recent studies have implicated circRNAs in various neurological disorders. Despite their abundant expression in the brain, the role of circRNAs in experience-dependent plasticity remains poorly understood. We conducted an in vivo transcriptome analysis of circRNAs whose expression was regulated by experience-dependent plasticity in the mouse visual cortex and highlight circHomer1, a circRNA derived from the Homer1 gene, as a circRNA critical for synaptic development and experience-dependent plasticity in vivo.

neuroscience↗
bioRxiv2024

CRISPR PERSIST-On enables heritable and fine-tunable human gene activation

Current technologies for upregulation of endogenous genes use targeted artificial transcriptional activators but stable gene activation requires persistent expression of these synthetic factors. Although general "hit-and-run" strategies exist for inducing long-term silencing of endogenous genes using targeted artificial transcriptional repressors, to our knowledge no equivalent approach for gene activation has been described to date. Here we show stable gene activation can be achieved by harnessing endogenous transcription factors (EndoTFs) that are normally expressed in human cells. Specifically, EndoTFs can be recruited to activate endogenous human genes of interest by using CRISPR-based gene editing to introduce EndoTF DNA binding motifs into a target gene promoter. This Precision Editing of Regulatory Sequences to Induce Stable Transcription-On (PERSIST-On) approach results in stable long-term gene activation, which we show is durable for at least five months. Using a high-throughput CRISPR prime editing pooled screening method, we also show that the magnitude of gene activation can be finely tuned either by using binding sites for different EndoTF or by introducing specific mutations within such sites. Our results delineate a generalizable framework for using PERSIST-On to induce heritable and fine-tunable gene activation in a hit-and-run fashion, thereby enabling a wide range of research and therapeutic applications that require long-term upregulation of a target gene.

molecular biology↗
bioRxiv2024

MOSAIC enables in situ saturation mutagenesis of genes and CRISPR prime editing guide RNA optimization in human cells

CRISPR prime editing offers unprecedented versatility and precision for the installation of genetic edits in situ. Here we describe the development and characterization of the Multiplexing Of Site-specific Alterations for In situ Characterization (MOSAIC) method, which leverages a non-viral PCR-based prime editing method to enable rapid installation of thousands of defined edits in pooled fashion. We show that MOSAIC can be applied to perform in situ saturation mutagenesis screens of: (1) the BCR-ABL1 fusion gene, successfully identifying known and potentially new imatinib drug-resistance variants; and (2) the IRF1 untranslated region (UTR), re-confirming non-coding regulatory elements involved in transcriptional initiation. Furthermore, we deployed MOSAIC to enable high-throughput, pooled screening of hundreds of systematically designed prime editing guide RNA (pegRNA) constructs for a large series of different genomic loci. This rapid screening of >18,000 pegRNA designs identified optimized pegRNAs for 89 different genomic target modifications and revealed the lack of simple predictive rules for pegRNA design, reinforcing the need for experimental optimization now greatly simplified and enabled by MOSAIC. We envision that MOSAIC will accelerate and facilitate the application of CRISPR prime editing for a wide range of high-throughput screens in human and other cell systems.

molecular biology↗
bioRxiv2022

Astrocyte glutamate transport is modulated by motor learning and regulates neuronal correlations and movement encoding by motor cortex neurons

While motor cortex is crucial for learning precise and reliable movements, whether and how astrocytes contribute to its plasticity and function during motor learning is unknown. Here we report that primary motor cortex (M1) astrocytes in mice show in vivo plasticity during learning of a lever push task, as revealed by transcriptomic and functional modifications. In particular, we observe changes in expression of glutamate transporter genes and increased coincidence of intracellular calcium events. Astrocyte-specific manipulations of M1 are sufficient to alter motor learning and execution, and neuronal population coding, in the same task. Mice expressing decreased levels of the astrocyte glutamate transporter GLT1 show impaired and variable movement trajectories. Mice with increased astrocyte Gq signaling show decreased performance rates, delayed response times and impaired trajectories, along with abnormally high levels of GLT1. In both groups of mice, M1 neurons have altered inter-neuronal correlations and impaired population representations of task parameters, including response time and movement trajectories. Thus, astrocytes have a specific role in coordinating M1 neuronal activity during motor learning, and control learned movement execution and dexterity through mechanisms that importantly include fine regulation of glutamate transport.

neuroscience↗
bioRxiv2021

solPredict: Antibody apparent solubility prediction from sequence by transfer learning

There is growing interest in developing therapeutic mAbs for the route of subcutaneous administration for several reasons, including patient convenience and compliance. This requires identifying mAbs with superior solubility that are amenable for high-concentration formulation development. However, early selection of developable antibodies with optimal high-concentration attributes remains challenging. Since experimental screening is often material and labor intensive, there is significant interest in developing robust in silico tools capable of screening thousands of molecules based on sequence information alone. In this paper, we present a strategy applying protein language modeling, named solPredict, to predict the apparent solubility of mAbs in histidine (pH 6.0) buffer condition. solPredict inputs embeddings extracted from pretrained protein language model from single sequences into a shallow neutral network. A dataset of 220 diverse, in-house mAbs, with extrapolated protein solubility data obtained from PEG-induced precipitation method, were used for model training and hyperparameter tuning through five-fold cross validation. An independent test set of 40 mAbs were used for model evaluation. solPredict achieves high correlation with experimental data (Spearman correlation coefficient = 0.86, Pearson correlation coefficient = 0.84, R2 = 0.69, and RMSE = 4.40). The output from solPredict directly corresponds to experimental solubility measurements (PEG %) and enables quantitative interpretation of results. This approach eliminates the need of 3D structure modeling of mAbs, descriptor computation, and expert-crafted input features. The minimal computational expense of solPredict enables rapid, large-scale, and high-throughput screening of mAbs during early antibody discovery.

bioinformatics↗
bioRxiv2021

Fragile X syndrome patient-derived neurons developing in the mouse brain show FMR1 -dependent phenotypes

Abnormal neuronal development in Fragile X syndrome (FXS) is poorly understood. Data on FXS patients remain scarce and FXS animal models have failed to yield successful therapies. In vitro models do not fully recapitulate the morphology and function of human neurons. Here, we co-injected neural precursor cells (NPCs) from FXS patient-derived and corrected isogenic control induced pluripotent stem cells into the brain of neonatal immune-deprived mice. The transplanted cells populated the brain and a proportion differentiated into neurons and glial cells. Single-cell RNA sequencing of transplanted cells revealed upregulated excitatory synaptic transmission and neuronal differentiation pathways in FXS neurons. Immunofluorescence analyses showed accelerated maturation of FXS neurons after an initial delay. Additionally, increased percentages of Arc- and Egr1-positive FXS neurons and wider dendritic protrusions of mature FXS striatal medium spiny neurons pointed to an increase in synaptic activity and synaptic strength as compared to control. This transplantation approach provides new insights into the alterations of neuronal development in FXS by facilitating physiological development of cells in a 3D context, and could be used to test new therapeutic compounds correcting neuronal development defects in FXS.

neuroscience↗
bioRxiv2020

Membrane Interactions of α-Synuclein Revealed by Multiscale Molecular Dynamics Simulations, Markov State Models, and NMR

-Synuclein is a presynaptic protein that binds to cell membranes and is linked to Parkinsons disease (PD). Whilst the normal function of remains -synuclein remains uncertain, it is thought that oligomerization of the protein on the cell membrane contributes to cell damage. Knowledge of how -synuclein binds to lipid bilayers is therefore of great interest as a likely first step in the molecular pathophysiology of PD, and may provide insight of the phenotype of PD-promoting mutations. We use coarse-grained and atomistic simulations in conjunction with NMR and cross-linking mass spectrometry studies of -synuclein bound to anionic lipid bilayers to reveal a break in the helical structure of the NAC region, which may give rise to subsequent oligomer formation. Coarse-grained simulations of -synuclein show that the interhelical region leads recognition and binding to both POPG and mixed composition bilayers and identifies important protein-lipid contacts, including those in the region between the two helices in the folded structure. We extend these simulations with all-atom simulations of the initial binding event to reveal details of the time-progression of lipid binding. We present secondary structure analysis that reveals points of possible {beta}-strand formation in the structure, and investigate intramolecular contacts with simulations and mass-spectrometry crosslinking. Additionally we show how Markov state models can be used to investigate possible conformational changes of membrane bound -synuclein in the NAC region, and we extract representative structures. These structural insights will aid the design and development of novel therapeutic approaches.

biophysics↗
bioRxiv2019

Simultaneous and precise generation of Zebra3 and Wsl5 mutations in rice using CRISPR/Cas9-mediated adenine base editors

The CRISPR/Cas9-mediated base editing technology can efficiently generate point mutations in the genome without introducing double-strand break (DSB) or supplying a DNA donor template for homology-dependent repair (HDR). In this study, adenine base editors (ABEs) were used for rapid generation of precise point mutations in two distinct genes, OsWsl5, and OsZebra3 (Z3), in rice protoplasts and regenerated plants. The precisely engineered point mutations were stably inherited to subsequent generations. These single nucleotide alterations resulted in single amino acid changes and associated wsl5 and z3 phenotypes as evidenced by white stripe leaf and light green/dark green leaf pattern, respectively. Through selfing and segregation, transgene-free, base edited wsl5 and z3 mutants were readily obtained in a short period of time. We noticed a novel mutation (V540A) in Z3 locus could mimic the phenotype of Z3 mutation (S542P). Furthermore, we observed unexpected non-A/G or T/C mutations in the ABE editing window in a few of the edited plants. The ABE vectors and the method from in this study could be used to simultaneously generate point mutations in multiple genes in a single transformation and serve as a useful base editing tool for crop improvement as well as basic studies in plant biology.\n\nHighlightsAdenine base editors were adapted for plant base editing that can generate precise and heritable point mutations in rice genome without indel formation. The base editing approach allows rapid generation of transgene-free rice mutants with expected phenotypic changes.

plant biology↗
bioRxiv2017

Patient-derived xenografts undergo murine-specific tumor evolution

Patient-derived xenografts (PDXs) have become a prominent model for studying human cancer in vivo. The underlying assumption is that PDXs faithfully represent the genomic features of primary tumors, retaining their molecular characteristics throughout propagation. However, the genomic stability of PDXs during passaging has not yet been evaluated systematically. Here we monitored the dynamics of copy number alterations (CNAs) in 1,110 PDX samples across 24 cancer types. We found that new CNAs accumulated quickly, such that within four passages an average of 12% of the genome was affected by newly acquired CNAs. Selection for preexisting minor clones was a major contributor to these changes, leading to both gains and losses of CNAs. The rate of CNA acquisition in PDX models was correlated with the extent of both aneuploidy and genetic heterogeneity observed in primary tumors of the same tissue. However, the specific CNAs acquired during PDX passaging differed from those acquired during tumor evolution in patients, suggesting that PDX tumors are subjected to distinct selection pressures compared to those that exist in human hosts. Specifically, several recurrent CNAs observed in primary tumors gradually disappeared in PDXs, indicating that events undergoing positive selection in humans can become dispensable during propagation in mice. Finally, we found that the genomic stability of PDX models also affected their responses to chemotherapy and targeted drugs. Our findings thus highlight the need to couple the timing of PDX molecular characterization to that of drug testing experiments. These results suggest that while PDX models are powerful tools, they should be used with caution.

cancer biology↗