Biology subjects

Tan, Y.

Publications and source records attributed to Tan, Y..

At least 73 records · Page 4Linked to original sources

bioRxiv2024

Neuron-astrocyte Coupling in Lateral Habenula Mediates Depressive-like Behaviors

The lateral habenular (LHb) neurons and astrocytes have been strongly implicated in depression etiology but it was not clear how the two dynamically interact during depression onset. Here, using multi-brain-region calcium photometry recording in freely-moving mice, we discover that stress induces a unique, bimodal neuronal response and a most rapid astrocytic response in the LHb. LHb astrocytic calcium requires the 1A-adrenergic receptor, and depends on a recurrent neural network between the LHb and locus coeruleus (LC). Through the gliotransmitter glutamate and ATP/Adenosine, LHb astrocytes mediate the second-wave activation of local LHb neurons as well as release of norepinephrine (NE). Activation or inhibition LHb astrocytic calcium signaling facilitates or prevents stress-induced depressive-like behaviors respectively. These results identify a stress-induced positive feedback loop in the LHb-LC axis, with astrocytes being a critical signaling relay. The identification of this prominent neuron-glia interaction may shed light on stress management and depression prevention.

neuroscience↗

bioRxiv2024

Domain Specific AI Segmentation of IMPDH2 Rod/Ring Structures in Mouse Embryonic Stem Cells

BackgroundInosine monophosphate dehydrogenase 2 (IMPDH2) is an enzyme that catalyses the rate limiting step of guanine nucleotides. In mouse embryonic stem cells (ESCs) IMPDH2 is held as large multi-protein complexes known as rod-ring (RR) structures that dissociate when ESCs differentiate. Manual analysis of RR structures from confocal microscopy images, although possible, is not feasible on a large scale due to the quantity of RR structures present in each field of view. To address this analysis bottleneck, we have created a fully automatic RR image classification pipeline to segment, characterise and measure feature distributions of these structures in ESCs. ResultsWe find that this model can automatically segment images with a Dice score of over 80% for both rods and rings for in-domain images compared to expert annotation, with a slight drop to 70% for datasets out of domain. Important feature measurements derived from these segmentations show high agreement with the measurements derived from expert annotation, achieving an R2 score of over 90% for counting the number of rings and rods over the dataset. ConclusionsWe have established for the first time a quantitative baseline for RR distribution in pluripotent ESCs and have made a pipeline available for training to be applied to other models in which RR remain an open topic of study.

bioinformatics↗

bioRxiv2024

Unlearning Virus Knowledge Toward Safe and Responsible Mutation Effect Predictions

AO_SCPLOWBSTRACTC_SCPLOWPre-trained deep protein models have become essential tools in fields such as biomedical research, enzyme engineering, and therapeutics due to their ability to predict and optimize protein properties effectively. However, the diverse and broad training data used to enhance the generalizability of these models may also inadvertently introduce ethical risks and pose biosafety concerns, such as the enhancement of harmful viral properties like transmissibility or drug resistance. To address this issue, we introduce a novel approach using knowledge unlearning to selectively remove virus-related knowledge while retaining other useful capabilities. We propose a learning scheme, PROEDIT, for editing a pre-trained protein language model toward safe and responsible mutation effect prediction. Extensive validation on open benchmarks demonstrates that PROEDIT significantly reduces the models ability to enhance the properties of virus mutants without compromising its performance on non-virus proteins. As the first thorough exploration of safety issues in deep learning solutions for protein engineering, this study provides a foundational step toward ethical and responsible AI in biology.

bioinformatics↗

bioRxiv2024

High-Throughput Empirical and Virtual Screening to Discover Novel Inhibitors of Polyploid Giant Cancer Cells in Breast Cancer

Therapy resistance in breast cancer is increasingly attributed to polyploid giant cancer cells (PGCCs), which arise through whole-genome doubling and exhibit heightened resilience to standard treatments. Characterized by enlarged nuclei and increased DNA content, these cells tend to be dormant under therapeutic stress, driving disease relapse. Despite their critical role in resistance, strategies to effectively target PGCCs are limited, largely due to the lack of high-throughput methods for assessing their viability. Traditional assays lack the sensitivity needed to detect PGCC-specific elimination, prompting the development of novel approaches. To address this challenge, we developed a high-throughput single-cell morphological analysis workflow designed to differentiate compounds that selectively inhibit non-PGCCs, PGCCs, or both. Using this method, we screened a library of 2,726 FDA Phase 1-approved drugs, identifying promising anti-PGCC candidates, including proteasome inhibitors, FOXM1, CHK, and macrocyclic lactones. Notably, RNA-Seq analysis of cells treated with the macrocyclic lactone Pyronaridine revealed AXL inhibition as a potential strategy for targeting PGCCs. Although our single-cell morphological analysis pipeline is powerful, empirically testing all existing compounds is impractical and inefficient. To overcome this limitation, we trained a machine learning model to predict anti-PGCC efficacy in silico, integrating chemical fingerprints and compound descriptions from prior publications and databases. The model demonstrated a high correlation with experimental outcomes and predicted efficacious compounds in an expanded library of over 6,000 drugs. Among the top-ranked predictions, we experimentally validated two compounds as potent PGCC inhibitors. These findings underscore the synergistic potential of integrating high-throughput empirical screening with machine learning-based virtual screening to accelerate the discovery of novel therapies, particularly for targeting therapy-resistant PGCCs in breast cancer.

bioengineering↗

bioRxiv2024

Haplotype-resolved genomes provide insights into the origin and function of genome diversity in bivalves

Bivalves are famed for exhibiting vast genetic diversity of poorly understood origins and functional significance. Within bivalves, oysters are an ancient group showing remarkable genetic and phenotypic variability alongside extensive adaptability, serving as an ideal system to understand the origins and functional significance of genomic diversity. Here, we reveal the divergent genomic landscape of Crassostrea oysters, characterized by a history of genome size reduction associated with transposable elements (TEs). By constructing a haplotype-resolved genome for Kumamoto oyster C. sikamea, we demonstrate the widespread presence of haplotype divergent sequences (HDS); genomic regions present in just one haplotype. Combined with population resequencing, we define the role of genomic divergence driven by TEs in shaping oyster genetic diversity. Comparisons of haplotype-resolved genomes across four bivalve orders reveal that while extensive HDS is common, its origins may differ markedly. We show that HDS are a hotspot of genetic innovation, harboring rapidly evolving genes of various evolutionary ages, while also strongly influencing gene expression phenotypes. A widespread lack of allele-specific expression shared among oyster individuals indicates that haplotype polymorphism provides a key source of expression variation, promoting phenotypic plasticity and adaptation. These findings advance understanding on the origins of genomic diversity and its role in adaptive evolution.

genomics↗

bioRxiv2024

A single-molecule nanopore sequencing platform

Nanopore sequencing, a third-generation sequencing technology, has revolutionized the gene sequencing industry with its advantages of long reads, fast speed, real-time sequencing and analysis, and potential in detecting base modifications. This technology allows researchers to sequence longer DNA fragments in a single read, providing more comprehensive genomic information compared to previous methods. Nanopore sequencing operates on electrical signals generated by a nanopore embedded in a membrane separating two electrolyte-filled chambers. When single-stranded DNA (ssDNA) passes through the nanopore, it creates variations in the current that correspond to different DNA bases. By analyzing these current fluctuations with machine learning algorithms, the DNA sequence can be determined. In this study, we introduced several improvements to nanopore sequencing, including nanopore local chemistry sequencing, novel motor and pore proteins, chip design, and basecalling algorithms. Our new nanopore sequencing platform, CycloneSEQ, demonstrated long-duration sequencing (107 hours) on a single chip with high yield (>50 Gb). In human genomic DNA sequencing, CycloneSEQ was able to produce long reads with N50 33.6 kb and modal identity 97.0%. Preliminary findings on human whole-genome de novo assembly, variant calling, metagenomics sequencing, and single-cell RNA sequencing have further highlighted CycloneSEQs potential across different areas of genomics.

genomics↗

bioRxiv2024

Transocular detection of premotor Parkinson's disease via retinal neurovascular coupling through functional OCT angiography

Early detection of Parkinsons disease (PD) is essential for timely initiating neuroprotective interventions before significant loss of dopaminergic (DAergic) neurons, but faces challenges in accurately and noninvasively detecting subtle neuronal changes in the midbrain. Here, we propose a transocular functional imaging method termed fOCTA-rNVC, which detects premotor PD by measuring alterations in retinal neurovascular coupling (rNVC) at the capillary level using specialized functional OCT angiography (fOCTA). Our findings demonstrate that capillary rNVC is significantly attenuated and delayed due to concurrent retinal dopaminergic degeneration in premotor PD mice. Notably, this PD-related rNVC attenuation can be temporarily reversed in acute levodopa challenge. Utilizing the functional characteristics of capillary rNVC in PD, we achieved an impressive accuracy of [~]100% in detecting premotor PD mice even with only [~]14.1% loss of midbrain DAergic neurons, at which stage prompt treatment offered superior outcomes. In contrast, no significant changes were observed in retinal thickness or vasculature in the premotor PD mice. These findings suggest that fOCTA-rNVC is a promising noninvasive solution for accurately detecting premotor PD and guiding early interventions.

bioengineering↗

bioRxiv2024

PTEN neddylation aggravates CDK4/6 inhibitor resistance in breast cancer

The gradual emergence of a novel therapeutic approach lies in the restoration of tumor suppressive machinery. PTEN is a crucial negative regulator of the PI3K/Akt signaling pathway. Protein neddylation modification contributes to PTEN inactivation and fuels breast cancer progression. Here, we highlight that an elevated level of PTEN neddylation is markedly associated with resistance to palbociclib, a CDK4/6 inhibitor used in luminal subtype breast cancer patients. Mechanistically, PTEN neddylation activates the PI3K/Akt signaling pathway, and more notably, upregulates the activity of the AP-1 transcription factor. Our data showed that PTEN neddylation stabilizes JUND, a transcription factor involved in the AP-1 complex, by disrupting its interaction with the E3 ubiquitin ligase ITCH. Consequently, activated JUND leads to the release of cytokines and chemokines, which in turn may drive an inflammatory tumor microenvironment, potentially contributing to drug resistance. Then, we identified Echinacoside as a potent inhibitor of PTEN neddylation both in vivo and in vitro by disrupting its interaction with XIAP, the E3 ligase responsible for PTEN neddylation. Combination of Echinacoside effectively overcome resistance to palbociclib in breast cancer treatment. These findings highlight targeting PTEN neddylation as a promising strategy for restoring tumor suppressor activity and overcoming resistance.

cell biology↗

bioRxiv2024

STK19 is a transcription-coupled repair factor that participates in UVSSA ubiquitination and TFIIH loading.

Transcription-coupled repair (TCR) is the major pathway to remove transcription-blocking lesions. Although discovered for nearly 40 years, the mechanism and critical players of mammalian TCR remain unclear. STK19 is a factor affecting cell survival and recovery of RNA synthesis in response to DNA damage, however, whether it is a necessary component for TCR is unknown. Here we demonstrated that STK19 is essential for human TCR. Mechanistically, STK19 is recruited to damage sites through direct interaction with CSA. It can also interact with RNA polymerase II in vitro. Once recruited, STK19 plays an important role in UVSSA ubiquitination which is needed for TCR. STK19 also promotes TCR independent of UVSSA ubiquitination by stimulating TFIIH recruitment through its direct interaction with TFIIH. In summary, our results suggest that STK19 is a key factor of human TCR that links CSA, UVSSA ubiquitination and TFIIH loading, shedding light on the molecular mechanisms of TCR.

biochemistry↗

bioRxiv2024

Cohesin-mediated 3D contacts tune enhancer-promoter regulation

Enhancers are key drivers of gene regulation thought to act via 3D physical interactions with the promoters of their target genes. However, genome-wide depletions of architectural proteins such as cohesin result in only limited changes in gene expression, despite a loss of contact domains and loops. Consequently, the role of cohesin and 3D contacts in enhancer function remains debated. Here, we developed CRISPRi of regulatory elements upon degron operation (CRUDO), a novel approach to measure how changes in contact frequency impact enhancer effects on target genes by perturbing enhancers with CRISPRi and measuring gene expression in the presence or absence of cohesin. We systematically perturbed all 1,039 candidate enhancers near five cohesin-dependent genes and identified 34 enhancer-gene regulatory interactions. Of 26 regulatory interactions with sufficient statistical power to evaluate cohesin dependence, 18 show cohesin-dependent effects. A decrease in enhancer-promoter contact frequency upon removal of cohesin is frequently accompanied by a decrease in the regulatory effect of the enhancer on gene expression, consistent with a contact-based model for enhancer function. However, changes in contact frequency and regulatory effects on gene expression vary as a function of distance, with distal enhancers (e.g., >50Kb) experiencing much larger changes than proximal ones (e.g., <50Kb). Because most enhancers are located close to their target genes, these observations can explain how only a small subset of genes -- those with strong distal enhancers -- are sensitive to cohesin. Together, our results illuminate how 3D contacts, influenced by both cohesin and genomic distance, tune enhancer effects on gene expression.

molecular biology↗

bioRxiv2024

Coordination of transcription-coupled repair and repair-independent release of stalled RNA polymerase II in response to transcription-blocking lesions

Transcription-blocking lesions (TBLs) stall elongating RNA polymerase II (PolII), which then initiates transcription-coupled repair (TCR) to remove TBLs and allow transcription recovery. In the absence of TCR, eviction of lesion-stalled PolII is required for alternative pathways to address the damage, but the mechanism is unclear. This study, utilizing Protein-Associated DNA Damage Sequencing (PADD-seq), reveals that the p97-proteasome pathway can evict lesion-stalled PolII independently of repair. Both TCR and repair-independent eviction require CSA and ubiquitination. However, p97 is dispensable for TCR and PolII eviction in TCR-proficient cells, highlighting repairs prioritization over repair-independent eviction. Moreover, ubiquitination of RPB1-K1268 is important for both pathways, with USP7s deubiquitinase activity promoting TCR without abolishing repair-independent PolII release. In summary, this study elucidates the fate of lesion-stalled PolII, and may shed light on the molecular basis of genetic diseases caused by the defects of TCR genes.

molecular biology↗

bioRxiv2024

Hormonal dynamics reveal a stimulatory role for secretoneurin in zebrafish ovulation

Surge release of luteinizing hormone (Lh) from the pituitary is essential for fertility as it triggers ovulation. While secretoneurin (SN) is a phylogenetically conserved secretogranin-2 derived peptide that stimulates Lh, its role in ovulation has not been established. To directly compare periovulatory changes in the classical hormones to the emerging reproductive neuropeptides SNa and SNb, simultaneous mass spectrometry measurement of 9 peptides and 5 steroids was conducted in female zebrafish. Regression analysis indicated that levels of SNa1-34 in the brain peaked when type 3 gonadotropin-releasing hormone (Gnrh3) increased (R2=0.71) at the time of the Lh surge, 3.5 h before ovulation. In contrast, levels of the naturally occurring derivative SNa1-14 were highest at ovulation, while SNb1-31 was invariable. The bioactivities of SNa1-34 and SNa1-14 were investigated. After injection of SNa1-34 in females that had been isolated from males, 61% (11/18) ovulated within 6 h, which was like the effects of the Lh analog human chorionic gonadotropin (hCG) (72%; 13/18 females). SNa1-34 could induce ovulation by stimulating time-dependent expression of gnrh3 in the brain, chorionic gonadotropin alpha (cga), luteinizing hormone b (lhb) subunit in pituitary, and the nuclear progesterone receptor (npr) in ovaries. In contrast, SNa1-14 exhibited far fewer effects on gene expression and did not induce ovulation. Our results support the proposal that SN is a reproductive hormone. Significance statementSecretogranin-2 is a secretory granule protein that is enzymatically processed to the bioactive neuropeptide secretoneurin. It is produced in hypothalamic neurons and anterior pituitary cells, and we provide in vivo evidence that secretoneurin plays an important role to induce ovulation. Secretoneurin levels in the brain increase concomitantly with gonadotropin-releasing hormone prior to ovulation in normal zebrafish. Injection of a synthetic secretoneurin peptide increased expression of reproduction-related genes at all levels of the hypothalamic-pituitary-ovarian axis. Secretoneurin stimulates ovulation in otherwise anovulatory females. Together these data place the evolutionarily conserved secretoneurin amongst other critical neuroendocrine regulators. Secretoneurin or synthetic agonists could be used to improve breeding in fish species, or to potentially help with infertility treatments

physiology↗

bioRxiv2024

SPACEc: A Streamlined, Interactive Python Workflow for Multiplexed Image Processing and Analysis

Multiplexed imaging technologies provide insights into complex tissue architectures. However, challenges arise due to software fragmentation with cumbersome data handoffs, inefficiencies in processing large images (8 to 40 gigabytes per image), and limited spatial analysis capabilities. To efficiently analyze multiplexed imaging data, we developed SPACEc, a scalable end-to-end Python solution, that handles image extraction, cell segmentation, and data preprocessing and incorporates machine-learning-enabled, multi-scaled, spatial analysis, operated through a user-friendly and interactive interface.

bioinformatics↗

bioRxiv2024

Proteasome inhibition induces DNA methylation alteration by attenuating the synthesis of DNA methyltransferase 1 and 3B in colorectal cancer

Proteasome is an essential organelle in guarding cellular protein homeostasis. Here, we report that inhibition of proteasome leads to alterations in DNA methylation patterns in colorectal cancer (CRC) by surpressing the synthesis of DNA methyltransferases (DNMTs). We found that treating CRC cells with proteasome inhibitors results in attenuated translation of DNMT1 and DNMT3B, mediated by the inactivation of AKT and mammalian target of rapamycin (mTOR), which is dependent on the accumulation of p300, an acetyltransferase that inhibits AKT through acetylation modification. Furthermore, we demonstrated that downregulation of DNMT1 and DNMT3B confers protection against proteasome inhibitor treatment, potentially through reprogramming the transcriptome of CRC cells, highlighting the significant role of DNMTs in response to disruptions in protein homeostasis. Interestingly, the proteasome inhibitor-induced downregulation of DNMT1 and DNMT3B appears to be CRC specific, notwithstanding the underlying mechanism remains unclear. Altogether, our findings reveal an epigenetic effect of proteasome on DNA methylation in CRC through its regulation of DNA methyltransferase synthesis.

cancer biology↗

bioRxiv2024

Polyamine Depletion by D, L-alpha-difluoromethylornithine Inhibits Ewing Sarcoma Metastasis by Inducing Ferroptosis

AO_SCPLOWBSTRACTC_SCPLOWPolyamine metabolism and signaling play important roles in multiple cancers but have not previously been studied in Ewing sarcoma. Here, we show that blocking polyamine synthesis with D, L-alpha-difluoromethylornithine (DFMO) causes a G1 cell cycle arrest, dose-dependent decreases in sarcosphere formation from Ewing sarcoma cell lines growing in non-adherent conditions and a decrease in clonogenic growth in soft agar. Further, we utilized our orthotopic implantation/amputation model of Ewing sarcoma metastasis to demonstrate that DFMO slowed primary tumor growth in addition to limiting metastasis. RNA sequencing demonstrated gene expression patterns consistent with induction of ferroptosis caused by polyamine depletion. Induction of ferroptosis was validated in vitro by demonstrating that ferrostatin-1, an inhibitor of ferroptosis, allows sphere formation even in the presence of DFMO. Collectively, these results reveal a novel mechanism by which DFMO prevents metastasis - induction of ferroptosis due to polyamine depletion. Our results provide preclinical justification to test the ability of DFMO to prevent metastatic recurrence in Ewing sarcoma patients at high risk for relapse.

cancer biology↗

bioRxiv2024

Whole-genome DNA methylomes of Tree shrew brains reveal conserved and divergent roles of DNA methylation on sex chromosome regulation

The tree shrew (Tupaia belangeri) is a promising emerging model organism in biomedical studies, notably due to their evolutionary proximity to primates. To enhance our understanding of how DNA methylation is implicated in regulation of gene expression and the X chromosome inactivation (XCI) in tree shrew brains, here we present their first genome-wide, single-base-resolution methylomes integrated with transcriptomes from prefrontal cortices. We discovered both divergent and conserved features of tree shrew DNA methylation compared to that of other mammals. DNA methylation levels of promoter and gene body regions are negatively correlated with gene expression, consistent with patterns in other mammalian brains studied. Comparing DNA methylation patterns of the female and male X chromosomes, we observed a clear and significant global reduction (hypomethylation) of DNA methylation across the entire X chromosome in females. Our data suggests that the female X hypomethylation does not directly contribute to the gene silencing of the inactivated X chromosome nor does it significantly drive sex-specific gene expression of tree shrews. However, we identified a putative regulatory region in the 5 end of the X inactive specific transcript (Xist) gene, a key gene for XCI, whose pattern of differential DNA methylation strongly relate to its differential expression between male and female tree shrews. We show that differential methylation of this region is conserved across different species. Moreover, we provide evidence suggesting that the observed difference between human and tree shrew X-linked promoter methylation is associated with the difference in genomic CpG contents. Our study offers novel information on genomic DNA methylation of tree shrews, as well as insights into the evolution of X chromosome regulation in mammals.

genomics↗

bioRxiv2024

INSIHGT: Accessible multimodal systems biology with quantitative molecular phenotyping in 3D

Biological systems are complex, encompassing intertwined spatial, molecular and functional features. However, methodological constraints always limit the completeness of information that can be extracted. Here, we report the development of INSIHGT, a minimally perturbative, accessible and cost-efficient three-dimensional (3D) spatial biology method utilizing superchaotropes and host-guest chemistry. This allows highly multiplexed and multi-modal readout of tissue biomolecules in biological systems up to centimeter scales, permitting radio-histological correlation of phosphorylated alpha-synuclein pathologies in human hemi-brainstem. The homogeneous penetration permits reliable semi-quantitative signals in 3D compared to reference signals. Diverse antigens, mRNA transcripts, neurotransmitters, and post-translational and epigenetic modifications, are well-preserved and visualized. INSIHGT also allows multi-round molecular probing for high-dimensional spatial biology and compatibility with downstream traditional histology. With INSIHGT, we mapped previously undescribed podocyte-to-parietal epithelial cell microfilaments and demonstrated their geodesic clustering in mouse glomeruli, and catalogued sparsely located neurofilament-intensive inclusion bodies in the human cerebellum, and identified NPY-proximal cell types defined by spatial morpho-proteomics in mouse hypothalamus. We anticipate INSIHGT can form the foundations for 3D spatial multi-omics technology development and holistic systems biology studies.

bioengineering↗

bioRxiv2024

The novel role of Kallistatin in linking metabolic syndromes and cognitive memory deterioration by inducing amyloid-β plaques accumulation and tau protein hyperphosphorylation

Accumulation of amyloid {beta} (A{beta}) peptides and hyperphosphorylated tau proteins in the hippocampus triggers cognitive memory decline in Alzheimers disease (AD). The incidence and mortality of sporadic AD were tightly associated with diabetes and hyperlipidemia, while the exact linked molecular is uncertain. Here, we reported that serum Kallistatin concentrations were meaningfully higher in AD patients, with a higher concentration of fasting blood glucose and triglyceride. In addition, the constructed Kallistatin-transgenic (KAL-TG) mice defined its cognitive memory impairment phenotype and lower LTP in hippocampal CA1 neurons accompanied by increased A{beta} deposition and tau phosphorylation. Mechanistically, Kallistatin could directly bind to the Notch1 receptor and thereby upregulate BACE1 expression by inhibiting PPAR{gamma} signaling, resulting in A{beta} cleavage and production. Besides, Kallistatin could promote the phosphorylation of tau by activating GSK-3{beta}. Fenofibrate, a hypolipidemic drug, could alleviate cognitive memory impairment by down-regulating A{beta} and tau phosphorylation of KAL-TG mice. Collectively, our data clarified a novel mechanism for A{beta} accumulation and tau protein hyperphosphorylation regulation by Kallistatin, which might play a crucial role in linking metabolic syndromes and cognitive memory deterioration, and suggested that fenofibrate might have the potential for treating metabolism-related AD. HighlightsO_LIKallistatin-transgenic (KAL-TG) mice defined its cognitive memory impairment phenotype accompanied by increased A{beta} deposition and tau phosphorylation. C_LIO_LIKallistatin could directly bind to the Notch1 receptor and thereby upregulate BACE1 expression by inhibiting PPAR{gamma} signaling. C_LIO_LIFenofibrate could alleviate cognitive memory impairment and down-regulate the serum Kallistatin level. C_LI

neuroscience↗