Biology subjects

Lin, S.-J.

Publications and source records attributed to Lin, S.-J..

10 recordsLinked to original sources

bioRxiv2026

MCseg: AI agent-guided workflow search for no-code cell segmentation and transcript attribution in spatial transcriptomics

Cell-level analysis of high-resolution spatial transcriptomics depends on accurate segmentation and transcript assignment, yet current workflows often trade transcript capture for boundary purity and can require substantial image-analysis expertise. We developed MCseg, a downloadable no-code platform whose fixed segmentation engine was derived by an AI-agent-guided search in which an AI agent iteratively proposed and evaluated combinations of image-processing and segmentation operations against Xenium-derived cell boundaries. In a lung adenocarcinoma development set, fixed-parameter MCseg increased mean panoptic quality from 0.432 to 0.472 relative to an Optuna-tuned two-diameter Cellpose baseline, while a reference-guided calibration analysis reached 0.554. In an independent expert-annotated colorectal cancer region, MCseg showed higher lineage recall and micro-F1 than the StarDist-based ENACT workflow among cells covered by both methods. Across 15 colorectal cancer regions, MCseg increased neighborhood expression discordance and reduced lineage-exclusive co-expression relative to Space Ranger at similar UMI density. The fixed workflow also transferred to fresh-frozen breast cancer without tissue-specific architecture search, illustrating an agent-guided route to reproducible, locally deployable cell-level spatial transcriptomic analysis.

bioinformatics↗

bioRxiv2026

High-resolution volumetric intravital imaging reveals asymmetric serotonin-dependent post-shock activity in the Drosophila brain

Volumetric intravital imaging of adult Drosophila brains remains challenging due to optical scattering and speed limitations of conventional microscopy. Here, we present V-shape Bessel-beam light-sheet microscopy (vSPIM), an upright, high-speed platform achieving subcellular resolution across large, opaque volumes in vivo. Through real-time calcium imaging in adult Drosophila, vSPIM uncovered two unprecedented phenomena: first, a complex ensemble of approximately 150 olfactory projection neuron boutons (more than 400 per hemisphere) resolved 18 distinct odor valence coding patterns within the dense mushroom body calyx, aligning with connectomic architecture. Second, vSPIM revealed a previously uncharacterized asymmetric, serotonin-dependent post-shock firing (PsF) response. This PsF activity emerged within the mushroom body and dopaminergic neurons only after repetitive aversive stimulation, distinct from canonical dopamine-driven reinforcement signals. Finally, we demonstrated the versatility of vSPIM by tracking corneal endothelial dynamics during mouse wound healing. Overall, vSPIM establishes a scalable framework for intravital imaging, bridging the gap between optical innovation and circuit physiology in adult tissues.

neuroscience↗

bioRxiv2026

Microglial pruning of extinction-ensemble synapses preserves fear memory

Fear extinction suppresses learned fear without erasing the original memory, yet how competing fear and extinction ensembles are selectively updated remains unknown. Here, we show that microglia preserve fear memory by selectively editing extinction-ensemble synapses during retrieval. In medial prefrontal cortex, microglial processes, but not somata, expand, ramify, and tighten their engagement with extinction-ensemble dendrites and spines, where they preferentially engulf excitatory postsynaptic material and bias spine remodeling toward elimination. This selectivity is instructed by local find-me, eat-me, and dont-eat-me cues: purinergic signaling recruits microglial processes, phosphatidylserine exposure licenses engulfment of extinction-ensemble synapses, and CD47-SIRP protects fear ensembles from removal. Weakening microglial recruitment or engulfment, or removing this protection, accelerates extinction without impairing fear acquisition. These findings identify microglial processes as active gatekeepers of ensemble competition and reveal a neuroimmune mechanism that preserves fear memory by limiting extinction. In BriefDuring memory updating, microglial processes selectively engage extinction-ensemble synapses rather than globally pruning active circuits. Local recruitment, engulfment, and protection cues determine this choice, allowing microglia to preserve fear memory by limiting extinction. HighlightsO_LIExtinction retrieval selectively recruits microglial processes to extinction ensembles. C_LIO_LIMicroglia preferentially engulf excitatory postsynaptic material from extinction ensembles. C_LIO_LILocal "find-me," "eat-me," and "dont-eat-me" cues determine synapse selection. C_LIO_LIDisrupting microglial pruning facilitates extinction without impairing fear learning. C_LI

neuroscience↗

bioRxiv2025

Dephosphorylation of YES kinase-mediated co-chaperone DNAJB6b phosphorylation attenuates tau protein aggregation

Alzheimers disease (AD) is a neurodegenerative disorder characterized by a gradual deterioration of memory. Here, we examine the biological consequences of phosphorylation-mediated chaperone activity in AD-associated tau aggregates. Increased phosphorylation of DNAJB6b at Y53 is observed in the brain lysates of AD patients. Our research found that an activated Src family kinase, YES, phosphorylates Y53 within the J-domain of DNAJB6b and enhances the binding between DNAJB6b and Hsp70. The strengthened association between DNAJB6b and Hsp70 may lead to the accumulation of tau aggregates. These findings suggest that the up-regulation of YES kinase modifies DNAJB6b, and that the resulting alteration of DNAJB6b-dependent tau disaggregation may contribute to an increased risk of developing AD. Additionally, phosphor-DNAJB6b at Y53 may serve as a biomarker for the prognosis and diagnosis of AD.

neuroscience↗

bioRxiv2025

TadA-Derived Cytosine Base Editor for Precise Genome Editing in Zebrafish

CRISPR base editors are crucial for precise genome manipulation. Existing APOBEC-based cytosine base editors (CBEs), while powerful, exhibit indels and sequence context limitations, where TC-context preferences restrict effective editing of CC and GC motifs. To address these challenges, we evaluated various tRNA adenine deaminase (TadA)-derived CBEs, ultimately engineering zTadCBE that demonstrates high editing efficiency, minimized off-target effects, and an expanded targeting range. Our approach integrates beneficial mutations from TadA-based adenine base editors (ABEs) with SpRYCas9n-enhanced protospacer-adjacent motif (PAM) compatibility. Additionally, we engineered expanded-window zTadCBE variants, zTadCBE-ex1 and zTadCBE-ex2, to target wider nucleotide ranges, further increasing the versatility of this tool. To demonstrate the utility of zTadCBE variants in the functional assessment of genetic mutants, we generated a model for CDH23-associated hearing loss to validate the pathogenicity of a patient-specific variant in zebrafish. Furthermore, we induced a premature stop codon in the mediator complex gene med12 to inactivate its function using a CRISPR-STOP strategy and recapitulated patient-specific phenotypes in the founding (F0) generation. zTadCBE variants thus offer a robust set of CBEs for precise and efficient C-to-T editing in zebrafish, promising to advance the rapid functional assessment of genetic variants in vivo.

genetics↗

bioRxiv2024

Naa80 is required for actin N-terminal acetylation and normal hearing in zebrafish

Actin is a key component of the cytoskeleton of eukaryotic cells and is involved in numerous cellular functions. In animal cells, actins are uniquely N-terminally processed by a dedicated enzyme machinery to generate their mature acidic and acetylated forms. The final step of this maturation process involves N-terminal acetylation, a reaction catalyzed by NAA80 in humans. In human cell lines, N-terminal acetylation of actin plays a crucial role in maintaining normal cytoskeletal dynamics and cell motility. The physiological impact of actin N-terminal acetylation remains to be defined. Here, we developed a zebrafish naa80 knockout model and established that zNaa80 acetylates both muscle and non-muscle actins in vivo. Our in vitro investigation of purified zNaa80 unveiled a clear preference for acetylating N-termini derived from actins. Interestingly, zebrafish lacking actin N-terminal acetylation were viable and exhibited normal development, morphology and behaviour. In contrast, human individuals carrying pathogenic actin variants may present with hypotonia and hearing impairment. While zebrafish depleted for naa80 did not display any obvious muscle defects or abnormal muscle tissue, we found that they have abnormal inner ear development such as small otoliths and impaired response to sound stimuli. In sum, we have defined that zebrafish Naa80 N-terminally acetylates actins in vitro and in vivo and that actin N-terminal acetylation is essential for normal hearing in vivo.

biochemistry↗

bioRxiv2024

Expansion microscopy for super-resolution imaging of collagen-abundant tissues

Expansion microscopy (ExM) is popular for three-dimensional ultrastructural imaging of cultured cells and tissue slices at nanoscale resolution with conventional microscopes via physical expansion of biological tissues. However, the application of this technology to collagen-abundant thick tissues is challenging. We demonstrate a new method, collagen expansion microscopy (ColExM), optimized for expanding tissues containing more than 70% collagen. ColExM succeeded in 4.5-fold linear expansion with minimal structural distortion of corneal and skin tissues. It was also compatible with immunostaining, allowing super-resolution visualization of three-dimensional neural structures innervating hair follicles and corneas. With ColExM, we succeeded in identifying individual mitochondria and previously unrecognized dendritic spine-like structures of corneal nerves. ColExM also enabled fine mapping of structural rearrangement of tight junctions and actin cytoskeletons. Therefore, this method can facilitate the exploration of three-dimensional nanoscale structures in collagen-rich tissues.

bioengineering↗

bioRxiv2022

Prostaglandin E2 prevents radiotherapy-induced alopecia by attenuating transit amplifying cell apoptosis through promoting G1 arrest

Growing hair follicles (HFs) harbor actively dividing transit amplifying cells (TACs), rendering them highly sensitive to radiotherapy (RT). Clinically, there is still a lack of effective treatment for radiotherapy-induced alopecia (RIA). We aimed to dissect the effect and mechanism of local prostaglandin E2 (PGE2) pretreatment in RIA prevention. We found that PGE2 pretreatment reduced RIA by preventing premature termination of anagen through enhancing HF self-repair. Mechanistically, PGE2 did not activate HF stem cells, but preserved more TACs for regenerative attempts. Pretreatment of PGE2 lessened radiosensitivity of TACs by transiently arresting them in the G1 phase, thereby reducing TAC apoptosis and mitigating HF dystrophy. The preservation of more TACs accelerated HF self-repair and bypassed RT-induced premature catagen entry. Promoting G1 arrest by systemic administration of palbociclib isethionate (PD0332991), a CDK4/6 inhibitor, offered a similar protective effect against RT. Therefore, PGE2 protects HF TACs from RT by transiently inducing G1 arrest, and the regeneration of HF structures lost from RT is accelerated to resume anagen growth, thus bypassing the long downtime of hair loss. PGE2 has the potential to be repurposed as a preventive treatment for RIA.

cell biology↗

bioRxiv2020

Biallelic mutation of CLRN2 causes non-syndromic hearing loss in humans

Deafness, the most frequent sensory deficit in humans, is extremely heterogenous with hundreds of genes probably involved. Clinical and genetic analyses of an extended consanguineous family with pre-lingual, moderate-to-profound autosomal recessive sensorineural hearing loss, allowed us to identify CLRN2, encoding a tetraspan protein as a new deafness gene. Homozygosity mapping followed by exome sequencing identified a 15.2 Mb locus on chromosome 4p15.32p15.1 containing a missense pathogenic variant in CLRN2 (c.494C>A, NM_001079827.2) segregating with the disease. Using in vitro RNA splicing analysis, we show that the CLRN2 c.494C>A mutation leads to two events: 1) the substitution of a highly conserved threonine (uncharged amino acid) to lysine (charged amino acid) at position 165, p.(Thr165Lys), and 2) aberrant splicing, with the retention of intron 2 resulting in a stop codon after 26 additional amino acids, p.(Gly146Lysfs*26). Expression studies and phenotyping of newly produced zebrafish and mouse models deficient for clarin 2 further confirm that clarin 2, expressed in the inner ear hair cells, is essential for normal organization and maintenance of the auditory hair bundles, and for hearing function. Together, our findings identify CLRN2 as a new deafness gene, which will impact future diagnosis and treatment for deaf patients.

genetics↗

bioRxiv2019

N-terminal protein acetylation by NatB modulates the levels of Nmnats, the NAD+ biosynthetic enzymes in Saccharomyces cerevisiae

NAD+ is an essential metabolite participating in cellular biochemical processes and signaling. The regulation and interconnection among multiple NAD+ biosynthesis pathways are not completely understood. We previously identified the N-terminal (Nt) protein acetyltransferase complex NatB as a NAD+ homeostasis factor. Cells lacking NatB show an approximate 50% reduction in the NAD+ level and aberrant metabolism of NAD+ precursors, which are associated with a decrease of nicotinamide mononucleotide adenylyltransferases (Nmnat) protein levels. Here we show this decrease in NAD+ and Nmnat protein levels is specifically due to the absence of Nt-acetylation of Nmnat (Nma1 and Nma2) proteins, and not other NatB substrates. Nt-acetylation is a critical regulator of protein degradation by the N-end rule pathways, indicating absence of Nt-acetylation may alter Nmnat protein stability. Interestingly, the rate of protein turnover (t1/2) of non-Nt-acetylated Nmnats does not significantly differ from Nt-acetylated Nmnats, suggesting reduced Nmnat levels in NmatB mutants are not due to increased post-translational degradation of non-Nt-acetylated Nmnats. In line with these observations, deletion or depletion of N-rule pathway ubiquitin E3 ligases in NatB mutants is not sufficient to restore NAD+ levels. Moreover, the status of Nt-acetylation does not alter the rate of translation initiation of Nmnats. Collectively our studies suggest absence of Nt-acetylation may increase co-translational degradation of nascent Nmnat polypeptides, which results in reduced Nmnat levels in NatB mutants. Nmnat activities are essential for all routes of NAD+ biosynthesis. Understanding the regulation of Nmnat protein homeostasis will facilitate our understanding of the molecular basis and regulation of NAD+ metabolism.

biochemistry↗