Biology subjects

Lee, A. C.

Publications and source records attributed to Lee, A. C..

11 recordsLinked to original sources

bioRxiv2026

twist1a and twist1b play overlapping roles during zebrafish pectoral fin development

Background: In humans, haploinsufficiency of TWIST1 leads to Saethre-Chotzen Syndrome, characterized by limb abnormalities and craniofacial defects. In mouse, Twist1 promotes limb bud outgrowth via maintenance of the apical ectodermal ridge (AER), in addition to its craniofacial roles. In zebrafish, two twist1 paralogs, twist1a and twist1b, are known to influence cranial neural crest and cranial suture development but have not yet been implicated in limb formation. Results: We find that twist1a and twist1b act redundantly during zebrafish pectoral fin development. While twist1b mutants have normal fins, twist1a mutants have small fins, and twist1a;twist1b double mutants lack fins. These defects appear to originate with impaired establishment of the pectoral fin field, followed by failure of AER formation, likely due to reduced fgf10a expression in the fin mesenchyme. Anterior-posterior patterning of the fin bud is relatively normal in twist1a mutants and twist1a;twist1b double mutants, albeit with an anterior extension of shha expression in some embryos. Conclusions: Together, our results show that twist1a and twist1b play crucial and overlapping roles in zebrafish pectoral fin development, echoing roles played by Twist1 in the mouse limb bud and highlighting an early impact of twist1 genes on the initial formation of the zebrafish forelimb field.

developmental biology↗

bioRxiv2026

Balancing spatial resolution and proteome depth in LC-MS based spatialproteomics

Spatial proteomics aims to resolve protein composition within intact tissues, yet extraction-based liquid chromatography-mass spectrometry (LC-MS) workflows face an inherent trade-off: smaller sampling units increase spatial specificity, whereas larger sampling units provide greater proteome depth and robustness. As analytical sensitivity improves, sampling-unit size therefore becomes a key experimental design parameter. Current extraction-based LC-MS workflows typically rely on laser capture microdissection (LCM), where sample recovery and scalability can become limiting at low input. Spatially resolved laser-activated cell sorting (SLACS) offers an alternative tissue-isolation strategy based on single-pulse near-infrared laser activation. Here, we use SLACS to systematically examine the resolution-sensitivity trade-off across sampling units ranging from single-cell-equivalent to larger low-input tissue regions. Few-cell sampling retained substantial proteomic information relative to larger regions while increasing spatial specificity. Applied to the mouse somatosensory cortex, SLACS generated deep, layer-resolved proteomic profiles from regions corresponding to approximately 60 cells and preserved major layer-specific molecular patterns at inputs as low as approximately 6 cells. These results highlight sampling-unit size as an important experimental design parameter in extraction-based spatial proteomics and support few-cell sampling as a practical compromise between spatial specificity, proteome depth and robustness.

biochemistry↗

bioRxiv2026

Cryptic disease-prone states in human mesocortical assembloids revealed by multimodal profiling

Neurodegenerative diseases are characterized by synaptic failure, aberrant protein accumulation, and neuroglial dysfunction that emerge long before clinical onset. Although brain assembloids, which recapitulate interregional circuit connectivity beyond the scope of single organoids, significantly advance the modeling of circuit pathophysiology, they are predominantly evaluated through single-modality approaches that cannot resolve the functional and molecular heterogeneity underlying differential disease susceptibility. Here we show that morphologically identical human iPSC-derived dorso forebrain-midbrain mesocortical assembloids (MCAs) spontaneously bifurcate into disease-prone and non-prone states under identical culture conditions, revealing that MCA heterogeneity reflects intrinsic neurodegeneration susceptibility rather than stochastic culture variability. Using integrated electrophysiological, molecular, and spatial profiling, we find that disease-prone MCAs exhibit a temporally ordered molecular cascade in which neurofilament light chain elevation precedes tau dysregulation, mirroring the sequential biomarker trajectories observed in pre-symptomatic human neurodegeneration. Disease-prone MCAs further display selective cortical hyperexcitability and aberrant brainwave-like oscillatory dynamics that remain undetectable by any single modality. Spatially, a discrete junction-like neuronal population at the midbrain-forebrain interface shows transcriptional priming for synaptic overactivation alongside impaired astrocytic glutamate clearance, defining a spatially confined neuron-glial uncoupling as a candidate early origin of the disease-prone state. These findings reframe MCA heterogeneity as a biological window into pre-symptomatic neurodegeneration, with broad implications for disease modeling, risk stratification, and therapeutic discovery.

neuroscience↗

bioRxiv2026

Chemogenetic inhibition of the noradrenergic locus coeruleus promotes the development of risk-taking decisional strategies and selectively enhances motor impulsivity in females

The locus coeruleus noradrenaline (LC-NA) system is a key regulator of arousal, attention, and reward learning. Noradrenaline plays a critical role in impulse control, and recent evidence indicates the importance of noradrenaline signaling in cost-benefit decision making once choice strategies are established. However, whether the LC causally shapes the acquisition of decision strategies, and how this contribution may differ across sexes, remains unclear. We addressed these questions by chemogenetically inhibiting catecholaminergic neurons within the LC of adult tyrosine-hydroxylase Cre (TH::Cre) rats (n=69; 35 females) throughout acquisition of the cued rat gambling task (crGT), a probabilistic decision making paradigm that incorporates salient audiovisual reward-paired cues and simultaneously measures motor impulsivity. LC inhibition accelerated the development of risky choice strategies early in training in both males and females, reflected by impaired adoption of the most advantageous option and increased preference for risky options. Trial-by-trial analyses reveal that LC inhibition promoted switches in choice strategy following safe wins, while reducing switches away from risky options after both wins and losses. LC inhibition therefore seemed to encourage the repetition of actions that resulted in more uncertain outcomes. LC inhibition also selectively enhanced motor impulsivity in females, particularly early in training. These results provide causal evidence that the LC system guides the formation of optimal decisional strategies, while exerting sex-specific control over impulsive action. Abbreviated summaryChernoff et al. demonstrate that noradrenergic locus coeruleus (LC) neurons are required to develop optimal decisional strategies, as their inhibition hastens the adoption of risk-preferring choice tendencies. This may be underpinned by reduced behavioural sensitivity to likely outcomes. They also show that the LC regulates impulse control, specifically in females.

neuroscience↗

bioRxiv2025

Aberrant recursive splicing in a human disease locus

Recursive splice sites are rare motifs postulated to facilitate splicing across massive introns and shape isoform diversity, especially for long, brain-expressed genes. The necessity of this unique mechanism remains unsubstantiated, as does the role of recursive splicing (RS) in human disease. From analyses of rare copy number variants (CNVs) from almost one million individuals, we previously identified large, heterozygous deletions eliminating an RS site (RS1) in the first intron of CADM2 that conferred substantial risk for attention deficit hyperactivity disorder (ADHD) and other neurobehavioral traits. CADM2 encodes a neuronally expressed cell adhesion molecule that has repeatedly been associated with ADHD and numerous similar traits. To explore the molecular impact of RS ablation in CADM2, we used CRISPR to model patient deletions and to target a smaller region ([~]500 base pairs) containing RS1 in both human induced neurons (iNs) and rats. Transcriptome analyses in unedited iNs provided a catalog of CADM2 transcripts, including novel transcripts that retained RS exons. Intriguingly, ablating RS1 altered the gradient of RNA abundance across the first intron of CADM2, decreased the level of CADM2 expression, and impacted transcript usage. Decreased CADM2 expression was reflected in reduced exon usage downstream of the RS1 site and global alteration to genes involved in neuronal processes including synapse and axon development. Given the scale of our analyses and the widespread association of CADM2 with neurobehavioral traits, we sought to validate these findings using in vivo models and found that rodent models harboring Cadm2 RS1 deletions exhibited significant changes in relevant behaviors and functional brain connectivity. In summary, our analyses demonstrate a functional role for RS as a noncoding regulatory mechanism in a gene associated with a spectrum of neuropsychiatric and behavioral traits. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/666599v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@171156borg.highwire.dtl.DTLVardef@13553baorg.highwire.dtl.DTLVardef@bee7forg.highwire.dtl.DTLVardef@156f19e_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

genomics↗

bioRxiv2022

Rodent perirhinal cortex, but not ventral hippocampus, inhibition induces approach bias under object-based approach-avoidance conflict

Neural models of approach-avoidance (AA) conflict behavior and its dysfunction have focused traditionally on the hippocampus, with the assumption that this medial temporal lobe (MTL) structure plays a ubiquitous role in arbitrating AA conflict. We challenge this perspective by using three different AA behavioural tasks in conjunction with optogenetics, to demonstrate that a neighbouring region in rats, perirhinal cortex, is also critically involved but only when conflicting motivational values are associated with objects and not contextual information. The ventral hippocampus, in contrast, was found not to be essential for object-associated AA conflict, suggesting its preferential involvement in context-associated conflict. We propose that stimulus type can impact MTL involvement during AA conflict and that a more nuanced understanding of MTL contributions to impaired AA behaviour (e.g., anxiety) is required. These findings serve to expand upon the established functions of the perirhinal cortex while concurrently presenting innovative behavioural paradigms that permit the assessment of different facets of AA conflict behaviour.

neuroscience↗

bioRxiv2022

Whole genome sequencing of hematologically stained cells catapulted from Cell smears

Analyzing archived peripheral blood smears is a potential route towards gaining cell morphology and genome information of blood cell types from various diseases. Yet, acquiring whole genome information from morphologically targeted cells was difficult, especially for rare cell types. The main causes for such difficulty were the inevitable usage of cell stains leading to whole genome amplification inhibition, and insufficient cell isolation performance of previously introduced laser microdissection (LMD) techniques. Here, we introduce a new laser-based cell isolation technique and a whole genome amplification (WGA) protocol optimized for whole genome analysis from minute input of hematologically stained cells. We were able to perform whole genome copy number profiling and SNP analysis from as little as 5 cells.

genomics↗

bioRxiv2020

Tobacco, but not nicotine and flavor-less electronic cigarettes, induces ACE2 and immune dysregulation

COVID-19, caused by the virus SARS-CoV-2, has infected millions worldwide. This pandemic overlaps with the ongoing epidemics of cigarette smoking and electronic cigarette (e-cig) vaping, with over 1 billion smokers and vapers worldwide. However, there is scarce data relating COVID-19 risks and outcome with cigarette or e-cig use. In this study, we mined 3 independent RNA expression datasets from smokers and vapers to understand the potential relationship between vaping/smoking and the dysregulation of key genes and pathways related to COVID-19. We found that smoking, but not vaping, upregulates ACE2, the cellular receptor that SARS-CoV-2 requires for infection. Both smoking and use of nicotine and flavor-containing e-cig led to upregulations of pro-inflammatory cytokine production and expression of genes related to inflammasomes. Vaping flavor-less and nicotine-less e-cig, however, did not lead to significant cytokine dysregulation and inflammasome activation. Release of inflammasome products, such as IL-1B, and cytokine storms are hallmarks of COVID-19 infection, especially in severe cases. Therefore, our findings demonstrated that smoking or vaping, specifically use of flavored or nicotine-containing e-cigs, may critically exacerbate COVID-19-related inflammation or increase susceptibility to the disease. Further scientific and public health investigations should be undertaken to address these concerning links between COVID-19 and e-cig/smoking.

immunology↗

bioRxiv2020

DNA micro-disk for the management of DNA-based data storage with index and write-once-read-many (WORM) memory features

DNA-based data storage has attracted attention because of its higher physical density of the data and longer retention time than those of conventional digital data storage1-7. However, previous DNA-based data storage lacked index features and the data quality of storage after a single access is not preserved, obstructing its industrial use. Here, we propose DNA micro-disks, quick response (QR)-coded micro-sized disks that harbour data-encoded DNA molecules for the efficient management of DNA-based data storage. We demonstrate the two major features that previous DNA-based data storage studies could not achieve. One feature is accessing data items efficiently by indexing the data-encoded DNA library. Another is achieving write-once-read-many (WORM) memory through the immobilization of DNA molecules on the disk and their enrichment through in situ DNA production. Through these features, the reliability of DNA-based data storage was increased by allowing multiple accession of data-encoded DNA without data loss.

bioengineering↗

bioRxiv2018

High-throughput retrieval of physical DNA for NGS-identifiable clones in phage display library

In antibody discovery, in-depth analysis of an antibody library and high-throughput retrieval of clones in the library are crucial to identifying and exploiting rare clones with different properties. However, existing methods have several technical limitations such as low process throughput from laborious cloning process and waste of the phenotypic screening capacity from unnecessary repetitive tests on the dominant clones. To overcome the limitations, we developed a new high-throughput platform for the identification and retrieval of clones in the library, TrueRepertoire. TrueRepertoire provides highly accurate sequences of the clones with linkage information between heavy and light chains of the antibody fragment. Additionally, the physical DNA of clones can be retrieved in high throughput based on the sequence information. We validated the high accuracy of the sequences and demonstrated that there is no platform-specific bias. Moreover, the applicability of TrueRepertoire was demonstrated by a phage-displayed single-chain variable fragment (scFv) library targeting human hepatocyte growth factor (hHGF) protein.

bioengineering↗

bioRxiv2018

Constructing and visualizing cancer genomic maps in 3D spatial context by phenotype-based high-throughput laser-aided isolation and sequencing (PHLI-seq)

A spatially resolved analysis of the heterogeneous cancer genome, in which the data are connected to the three-dimensional space of a tumour, is crucial to understand cancer biology and the clinical impact of cancer heterogeneity on patients. However, despite recent progress in spatially resolved transcriptomics, spatial mapping of genomic data in a high-throughput and high-resolution manner has been challenging due to current technical limitations. Here, we describe a novel approach, phenotype-based high-throughput laser-aided isolation and sequencing (PHLI-seq), which enables high-throughput isolation of a single-cell or a small number of cells and their genome-wide sequence analysis to construct genomic maps within cancer tissue in relation to the phenotypes of the cells. By applying PHLI-seq, we reveal the heterogeneity of breast cancer tissues at a high resolution and map the genomic landscape of the cells to their corresponding spatial locations and phenotypes in the tumour mass. Additionally, with different staining modalities, the genotypes of the cells can be connected to corresponding phenotypic information of the tissue. Together with the spatially resolved genomic analysis, we can infer the histories of heterogeneous cancer cells in two or three dimensions, providing significant insight into cancer biology and precision medicine.

genomics↗