Biology subjects

Yang, C.-C.

Publications and source records attributed to Yang, C.-C..

18 recordsLinked to original sources

bioRxiv2026

De Novo Regeneration of Rete Ridges during Cetacean skin wound healing

Humans are tight-skinned mammals who typically fail to regenerate large full-thickness skin wounds, instead healing with substantial scarring and concomitant loss of function. Mechanical context is a major determinant of this outcome: elevated tissue tension or stiffness promotes fibrotic repair associated with hypertrophic or keloid scarring. Accordingly, regenerative medicine research has relied on diverse animal models to understand scar development and skin regeneration. Loose-skinned mammals exhibit greater regeneration ability. Furthermore, spiny mouse skin is significantly less stiff and associated with enhanced regenerative ability. Interestingly, this skin wound stiffness can be modulated to shift healing toward more regenerative or more fibrotic trajectories. Despite of this progress, the restoration of normal skin architecture after large-full thickness injury has not been elucidated in tight-skinned mammals. Can large full-thickness wounds regenerate with minimal scarring in tight-skinned mammals? Here we show the tight-skinned mammal Frasers Dolphin regenerates de novo a complex rete ridge architecture with associated vasculature and minimal scar following large full-thickness wound healing. Counterintuitively, this skin regeneration occurs in an aqueous, high-shear stress and high-tension environment. Complete rete ridge regeneration in tight-skinned mammals has not been documented and not observed in humans except in utero. This unique ability to rebuild elaborate rete ridges under tension is an opportunity to uncover molecular, cellular, and tissue-level mechanisms that enable regenerative wound healing in a mechanical regime typically associated with fibrosis.

pathology↗

bioRxiv2025

Automation workflow for high-throughput arrayed plasmid DNA preparation and quantification.

High-throughput generation of arrayed plasmid DNA library using commercially available miniprep kits remains labor-intensive and costly. The yield and quality of plasmid preparations directly affect downstream applications, including arrayed viral library production and CRISPR library screening. Insufficient plasmid yield or DNA concentration often requires repeated preparations or additional DNA concentration steps to obtain adequate quantities. Similarly, higher variations in yield or quality across wells or plates can render an entire library unsuitable for subsequent experiments. To increase productivity and mitigate human intervention and errors, the present study established an automated workflow for high-throughput plasmid DNA preparation and quantification. The workflow was carried out by the Biomek i7 Hybrid automated workstation, synergizing a robotic liquid handler and multiple peripheral instruments to produce and measure plasmid DNA in a 96-well plate format. Bacterial competent cells were alkaline lysed and plasmid DNA was purified using magnetic beads, followed by quantification with the PicoGreen assay. The PicoGreen assay reported median and average yields of approximately 9.5 and 10 {micro}g per sample, respectively, which are equivalent to 7.6 and 8 {micro}g/mL of bacterial culture. Plasmid DNA concentrations measured by the PicoFluor fluorometer were consistently lower than those obtained using the NanoDrop UV spectrophotometer. The comparison demonstrated robust positive correlation between PicoGreen assay and NanoDrop measurements (R2 > 0.8). Among 480 plasmid DNA samples, average and median yields measured by the NanoDrop reached approximately 24 and 25 {micro}g per sample per well, corresponding to 19 and 20 {micro}g/mL of bacterial culture. Over 98% of samples exceeded the high-yield threshold of 10 {micro}g/mL of culture, with high plasmid quality validated through DNA gel electrophoresis. Collectively, this study demonstrated a robust, scalable, and cost-effective automation platform for high throughput arrayed plasmid library generation and quantification.

molecular biology↗

bioRxiv2025

Automation of high-throughput workflow for arrayed CRISPR activation library screening

CRISPR-mediated gene activation (CRISPRa) is among the most efficient and reliable strategies for mimicking sustained activation of endogenous promoters and their corresponding genes at physiological levels. By leveraging guide-RNA (gRNA) library design, CRISPRa screens can be applied on a whole-genome scale and are compatible with both arrayed and pooled formats, depending on assay requirements. Compared with conventional arrayed CRISPRa libraries that use single or dual gRNAs and often require multiple gRNA candidates per target, a recently developed CRISPRa library (termed T. gonfio) incorporates four tandem gRNAs per lentivector per target, thereby reducing library complexity and representing the smallest arrayed genome-wide CRISPRa library. To streamline genome-wide arrayed CRISPRa screening, this study developed a high-throughput automated workflow using the Biomek i7 Hybrid liquid-handling platform, integrated with multiple peripheral instruments. The workflow comprises three pipelines: lentiviral library transduction, cell library passaging, and assay processing. These pipelines together establish and maintain the transduced cell library for extended screening times. This enables assay processing at desired extended time points and improves the likelihood of identifying phenotypes that require longer time to develop, making the workflow suitable even for rapidly proliferating cell models. In a pilot arrayed screen using a T. gonfio mini-library targeting kinases and phosphatases, activation of the EPHA2 receptor promoter induced a growth reduction phenotype in the HEK293 cell model. This phenotype was recapitulated in a parallel pooled CRISPRa screen using the same mini-library and further validated in a co-culture assay.

cancer biology↗

bioRxiv2025

Automation of high-throughput arrayed lentivirus production and titration

Generation of arrayed genome-wide CRISPR libraries in a ready-to-transduce lentiviral format remains laborious, time-consuming, and costly. To address these limitations, the present study developed a fully automated lentivirus production and titration workflow using a Biomek i7 Hybrid automated workstation, integrated with multiple instruments and managed by SAMI EX software. The workflow produced and titrated viruses in 96 and 384-well plate formats, respectively. It employed reverse transfection and triplicate wells per lentivector to reduce variability and yielded an average of three viral particles in transduction unit (TU) per producing HEK293T cell. Titration was performed using U937-mCherry suspension cells, with the percentage of transduced cells converted from U937 (X%) to HEK293T (Y%) values via a linear regression equation (Y% = 4.3X% + 9.3%). The titer calculation was based on the initial seeding cell number, the converted percentage of HEK293T transduced cells, and virus input volume. The titration demonstrated strong reproducibility across LSRFortessa (BD) and Aurora (Cytek) flow cytometers (R2 = 0.9). Among 1,760 unconcentrated virus preparations, median and mean titers reached approximately 1.2 x 106 TU/mL, with over 97% of samples exceeding the high-titer threshold of 2x105 TU/mL, thus demonstrating a robust, scalable, and cost-effective automation platform for high throughput arrayed lentiviral library production and titration.

microbiology↗

bioRxiv2025

Automation of high-throughput arrayed mammalian cell line cultivation

Cell culture automation has traditionally been limited to basic tasks at low throughput, which are insufficient for passaging rapidly proliferating cell lines or for generating stable clonal lines. To address unmet needs, this study implemented a Biomek i7 Hybrid automated workstation, integrated with peripheral instruments and coordinated by SAMI EX software, to enable automated, high throughput mammalian cell culture workflows. The workflows support cell density monitoring, arrayed passaging, sample cherry-picking, plate reformatting, cell density normalization, and cryopreservation in 96-well plates. Integration with the CloneSelect imager allows rapid confluency monitoring and monoclonality assessment (<100 sec per plate). Cell passaging and density normalization require 32 minutes for one plate and 61 minutes for two plates. Workflow consistency was demonstrated across multiple cell lines and biological replicates, with wells showing comparable confluency within three standard deviations, lower coefficient of variation, and substantially narrower interquartile ranges after a single cell passage and density normalization. Four automation pipelines, including monoclonality screening, cell passaging and cherry-picking, density normalization, and cryopreservation, collectively enable clonal line establishment. Depending on scale, one to eight 384-well plates were processed in 69 to 355 minutes, yielding an average of 35 clonal lines per plate suitable for downstream genomic DNA sequence confirmation.

cell biology↗

bioRxiv2025

Phosphorylation of Claspin by elF2α kinase protects cells from heat stress

Various biological stresses can induce replication stress, potentially leading to cancer development. Replication checkpoint protects cells against the threat of DNA damages by suspending cell cycle, allowing DNA repair. Chk1 kinase is activated by various stresses and is required for replication checkpoint. An adaptor protein, Claspin, mediates Chk1 activation, but specific mechanisms involved are still unknown. We demonstrate that heat stress induces hyperphosphorylation of Claspin, resulting in Chk1 activation. Claspin phosphorylation is impaired in cells lacking two eIF2 kinases, GCN2 and HRI, which are parts of the integrated stress response (ISR) pathway. Mass spectrometry identified five heat-inducible phosphorylation sites in the C-terminal region of Claspin, and Claspin lacking these sites failed to activate checkpoint. Our data support a model that the unphosphorylated C-terminal tail of Claspin masks its CKBD (Chk1 Binding Domain)-AP (Acidic Patch) domain, preventing it from interacting with Cdc7. Phosphorylation of the tail causes it to dissociate from CKBD-AP and facilitates Cdc7 interaction, leading to increased Chk1 activation. Our findings demonstrate crosstalk between replication checkpoint and ISR pathways, and suggest potential new strategies for cancer therapy.

molecular biology↗

bioRxiv2024

A Randomized, Controlled Clinical Trial Demonstrates Improved Cognitive Function in Senior Dogs Supplemented with a Senolytic and NAD+ Precursor Combination.

Age-related decline in mobility and cognition are associated with cellular senescence and NAD+ depletion in dogs and people. A combination of a novel NAD+ precursor and senolytic, LY-D6/2 was examined in this randomized controlled trial. Seventy dogs were enrolled and allocated into placebo, low or full dose groups. Primary outcomes were change in cognitive impairment measured with the owner-reported Canine Cognitive Dysfunction Rating (CCDR) scale and change in activity measured with physical activity monitors. Fifty-nine dogs completed evaluations at the three-month primary endpoint, and 51 reached the six-month secondary endpoint. There was a significant difference in CCDR score across treatment groups from baseline to the primary endpoint (p=0.02) with the largest decrease in the full dose group. There were no significant differences between groups in changes in measured activity. However, the proportion of dogs that improved in frailty and owner-reported activity levels and happiness was higher in the full dose group than other groups. Adverse events occurred equally across groups. All groups showed improvement in cognition, frailty, and activity suggesting placebo effect and benefits of trial participation. We conclude that LY-D6/2 significantly improves owner-assessed cognitive function and may have broader effects on frailty, activity and happiness as reported by owners.

animal behavior and cognition↗

bioRxiv2024

Phosphorylation inhibits intramolecular interactions, DNA-binding and protein interactions of Claspin through disordered/ structured conformation transition

Claspin, known to be highly disordered, plays important roles in replication fork progression, initiation and cellular responses to replication stress. However, regulation of its structure and molecular interactions is not completely understood. We show here, through Proximity-Ligation-Assays, the evidence for intramolecular interaction between the N- and C-terminal segments of Claspin, which depends on the Acidic-Patch [AP] segment near its C-terminus. Interaction of Claspin with DNA and replication factors is highly stimulated in {Delta}AP mutant and by prior dephosphorylation. The wild-type Claspin inhibits the helicase activity of MCM in an AP-dependent manner. {Delta}AP and dephosphorylated Claspin exhibit resistance to trypsin digestion compared to wild-type, suggesting the presence of structural domains in the formers. We propose that Claspin is converted from disordered (closed) to structured (open) conformation at initiation, which stimulates its DNA binding and interaction with replication factors and counteracts its helicase inhibitory activity to trigger initiation of DNA replication.

molecular biology↗

bioRxiv2023

OICNet: A Neural Network for Online EEG Source Separation using Independent Component Analysis

Online source separation of EEG signals plays a crucial role in understanding and interpreting brain dynamics in real-time applications such as brain-computer interfaces (BCIs). In this paper, we propose OICNet, a novel neural network designed specifically for online EEG source separation using independent component analysis, aiming to address the challenges of real-time computational efficiency and reliable extraction of independent components from EEG data streams. The OICNet is trained on a loss function that integrates non-Gaussianity measurement and an orthogonality constraint to achieve effective decomposition of multi-channel EEG signals. We conducted comprehensive evaluation of OICNet on both task-related and task-free EEG datasets with comparison against conventional and network-based ICA counterparts. The results demonstrate that OICNet outperforms existing methods in terms of accuracy and computational efficiency. Overall, OICNet provides high-efficiency real-time EEG source separation capabilities and paves the way for advancements in deep-learning EEG processing in real-world BCI applications.

bioinformatics↗

bioRxiv2023

Probe capture enrichment sequencing of amoA genes discloses diverse ammonia-oxidizing archaeal and bacterial populations

The ammonia monooxygenase subunit A (amoA) gene has been used to investigate the phylogenetic diversity, spatial distribution, and activity of ammonia-oxidizing archaeal (AOA) and bacterial (AOB), which contribute significantly to the nitrogen cycle in various ecosystems. Amplicon sequencing of amoA is a widely used method; however, it produces inaccurate results owing to the lack of a universal primer set. Moreover, currently available primer sets suffer from amplification biases, which can lead to severe misinterpretation. Although shotgun metagenomic and metatranscriptomic analyses are alternative approaches without amplification bias, the low concentration of target genes in heterogeneous environmental DNA restricts a comprehensive analysis to a realizable sequencing depth. In this study, we developed a method for amoA enrichment sequencing using a hybridization capture technique. Using metagenomic mock community samples, our approach effectively enriched amoA genes with low compositional changes, outperforming amplification and meta-omics sequencing analyses. Following the analysis of metatranscriptomic marine samples, we predicted 80 operational taxonomic units (OTUs) assigned to either AOA or AOB, of which 30 OTUs were unidentified using simple metatranscriptomic or amoA gene amplicon sequencing. Mapped read ratios to all the detected OTUs were significantly higher for the capture samples (50.4 {+/-} 27.2%) than for non-capture samples (0.05 {+/-} 0.02%), demonstrating the high enrichment efficiency of the method. The analysis also revealed the spatial diversity of AOA ecotypes with high sensitivity and phylogenetic resolution, which are difficult to examine using conventional approaches.

microbiology↗

bioRxiv2022

Claspin-dependent and -independent Chk1 activation by a panel of biological stresses

Replication stress has been suggested to be an ultimate trigger of carcinogenesis. Oncogenic signal, such as overexpression of CyclinE, has been shown to induce replication stress. Here, we show that various biological stresses, including heat, oxidative stress, osmotic stress, LPS, hypoxia, and arsenate induce activation of Chk1, a key effector kinase for replication checkpoint. Some of these stresses indeed reduce the fork rate, inhibiting DNA replication. Analyses of Chk1 activation in the cell population with western analyses showed that Chk1 activation by these stresses is largely dependent on Claspin. On the other hand, single cell analyses with Fucci cells indicated that while Chk1 activation during S phase is dependent on Claspin, that in G1 is mostly independent of Claspin. We propose that various biological stresses activate Chk1 either directly by stalling DNA replication fork or by some other mechanism that does not involve replication inhibition. The former pathway predominantly occurs in S phase and depends on Claspin, while the latter pathway, which may occur throughout the cell cycle, is largely independent of Claspin. Our findings provide evidence for novel links between replication stress checkpoint and other biological stresses and points to the presence of unknown mechanisms of Chk1 activation in mammalian cells.

molecular biology↗

bioRxiv2022

On Correlation between Structural Properties and Viral Escape Measurements from Deep Mutational Scanning

Encouraged by recent efforts to map responses of SARS-CoV-2 mutations to various antibody treatments with deep mutational scanning, we explored the possibility of tying measurable structural contact information from the binding complexes of antibodies and their targets to experimentally determined viral escape responses. With just a single crystal structure for each binding complex, we find that the average correlation coefficient R is surprisingly high at 0.76. Our two methods for calculating contact information use binary contacts measured between all residues of two proteins. By varying the parameters to obtain binary contacts, we find that 3.6 [A] and 7 [A] are pivotal distances to toggle the binary step function when tallying the contacts for each method. The correlations are improved by short simulations ([~]25 ns), which increase average R to 0.78. With blind tests using the random forest model, we can further improve average R to 0.84. These easy-to-implement measurements can be utilized in computational screening of viral mutations that escape antibody treatments and potentially other protein-protein interaction problems.

bioinformatics↗

bioRxiv2022

Claspin is required for growth recovery from serum starvation through regulating the PI3K-PDK1-mTOR pathway

Growth recovery from serum starvation requires the activation of PI3 kinase (PI3K)-PDK1-Akt-mTOR pathways. Claspin plays multiple important roles in regulation of DNA replication as a mediator for the cellular response to replication stress, an integral replication fork factor that facilitates replication fork progression and a factor that promotes initiation by recruiting Cdc7 kinase. Here, we report a novel role of Claspin in growth recovery from serum starvation. In the absence of Claspin, cells do not proceed into S phase and eventually die. Claspin interacts with PI3K and mTOR, and is required for activation of PI3K-PDK1-mTOR and for that of mTOR downstream factors, p70S6K and 4E-BP1, but not for p38 MAPK cascade during the recovery from serum starvation. PDK1 interacts with Claspin, notably with CKBD, in a manner dependent on phosphorylation of the latter protein, and is required for interaction of mTOR with Claspin. p53 and ROS (Reactive Oxygen Species) inhibitors increased survival of Claspin-deficient cells released from serum starvation. Thus, Claspin plays a novel role as a mediator/protein platform for nutrition-induced proliferation/survival signaling by activating the mTOR pathway.

molecular biology↗

bioRxiv2020

SARS-CoV-2 ORF9c Is a Membrane-Associated Protein that Suppresses Antiviral Responses in Cells

Disrupted antiviral immune responses are associated with severe COVID-19, the disease caused by SAR-CoV-2. Here, we show that the 73-amino-acid protein encoded by ORF9c of the viral genome contains a putative transmembrane domain, interacts with membrane proteins in multiple cellular compartments, and impairs antiviral processes in a lung epithelial cell line. Proteomic, interactome, and transcriptomic analyses, combined with bioinformatic analysis, revealed that expression of only this highly unstable small viral protein impaired interferon signaling, antigen presentation, and complement signaling, while inducing IL-6 signaling. Furthermore, we showed that interfering with ORF9c degradation by either proteasome inhibition or inhibition of the ATPase VCP blunted the effects of ORF9c. Our study indicated that ORF9c enables immune evasion and coordinates cellular changes essential for the SARS-CoV-2 life cycle. One-sentence summarySARS-CoV-2 ORF9c is the first human coronavirus protein localized to membrane, suppressing antiviral response, resembling full viral infection.

bioinformatics↗

bioRxiv2020

Reciprocal transplant experiment in lakes with disparate trophic states show resolution dependent partial functional redundancy

Microbial communities are considered to be functionally redundant, but few studies have tested this hypothesis empirically. In this study, we performed an in situ reciprocal transplant experiment on the surface and bottom waters of two lakes with disparate trophic states and tracked changes in their microbial community and functional attributes for 6 weeks using high-throughput sequencing and functional approaches. The communities from both lakes were resistant to changes in composition after the reciprocal transplant, but their functions tended to become similar to the incubating lakes functional profiles. A significant linear positive relationship was observed between the microbial community and functional attributes, though with varying scales of similarity, suggesting partial functional redundancy. Furthermore, the entropy-based L-divergence measure quantified the scale of partial functional redundancy in the lakes surface and bottom waters. This study establishes and quantifies the scale of partial functional redundancy in the freshwater ecosystem through empirical investigation.

ecology↗

bioRxiv2019

Crosstalk between eIF2α and eEF2 phosphorylation pathways optimizes translational arrest in response to oxidative stress

The cellular stress response triggers a cascade of events leading to transcriptional reprogramming and a transient inhibition of global protein synthesis, which is thought to be mediated by phosphorylation of eukaryotic initiation factor-2 (eIF2). Using mouse embryonic fibroblasts (MEFs) and the fission yeast S. pombe, we report here that rapid translational arrest and cell survival in response to hydrogen peroxide-induced oxidative stress do not rely on eIF2 kinases and eIF2 phosphorylation. Rather H2O2 induces a block in elongation through phosphorylation of eukaryotic elongation factor 2 (eEF2). Kinetic and dose-response analyses uncovered crosstalk between the eIF2 and eEF2 phosphorylation pathways, indicating that, in MEFs, eEF2 phosphorylation initiates the acute shutdown in translation, which is then maintained by eIF2 phosphorylation. Our results challenge the common conception that eIF2 phosphorylation is the primary trigger of translational arrest in response to oxidative stress and point to integrated control that may facilitate the survival of cancer cells.\n\nHIGHLIGHTSO_LIOxidative stress-induced translation arrest is independent of eIF2 phosphorylation\nC_LIO_LIOxidative stress blocks translation elongation\nC_LIO_LIOxidative stress triggers eEF2 kinase activation\nC_LIO_LIeEF2K KO cells are hypersensitive to oxidative stress\nC_LI

cell biology↗

bioRxiv2019

Divergence in Thermostability of Arabidopsis Mitochondrial Nucleotide Exchange Factors Encoded by Duplicate Genes, MGE1 and MGE2

The divergence of duplicate genes links to organismic adaptation. In Arabidopsis thaliana two nuclear genes encode mitochondrial GrpEs, MGE1 and MGE2, the nucleotide exchange factors of DnaK/HSP70 chaperone. MGE1 and MGE2 are duplicate genes originated from a whole genome duplication event. They respond differentially to high temperature; MGE2 is heat-inducible and is required for Arabidopsis seedlings to tolerate prolonged heat stress, while MGE1 is constitutively expressed. Heterologous expression of MGE2 but not MGE1 restored the growth of E. coli grpE mutant cells at elevated temperatures, suggesting that MGE2 is more thermostable than MGE1. In this study, we directly compared the thermostability of the purified recombinant MGE1 and MGE2 by circular dichroism spectroscopy. The temperature midpoints of the unfolding transition (Tm) of MGE1 and MGE2 were about 38 and 46 {degrees}C, respectively, indicating that MGE2 is remarkably more stable than MGE1 at higher temperature. Domain swapping between the two homologous proteins showed that the N-terminal region, including an unstructured sequence and a long -helix domain, is the major determinant of the thermostability. Although MGE2 contains a conserved sequence derived from an exonized intron within the N-terminus unstructured region, deletion of this sequence did not substantially affect protein thermostability in vitro and complementation of E. coli and Arabidopsis heat sensitive mutants. Taken together, our results suggest that Arabidopsis MGE1 and MGE2 had diverged not only in transcriptional response but also in the thermostability of the encoded proteins, which may contribute to adaptation of plants to higher temperatures.

evolutionary biology↗

bioRxiv2018

A prospective, observational study on conversion of Clinically Isolated Syndrome to Multiple Sclerosis during 4-year period (MS NEO study) in Taiwan

ImportanceCIS to MS conversion rates vary depending on population cohorts, initial manifestations, and durations of follow-up.\n\nObjectiveTo investigate conversion rate of patients from CIS to MS and the prognostic significance of demographic and clinical variables in Taiwanese population.\n\nDesignNationwide, prospective, multi-centric, observational study from November 2008 to November 2014 with 4 years follow-up.\n\nSettingMulti-centre setting at 5 institutions in Taiwan.\n\nParticipants152 patients having single clinical event potentially suggestive of MS in last 2 years were enrolled as consecutive sample. 33 patients were lost to follow-up and 16 patients did not complete the study.103 patients completed the study.\n\nIntervention(s) (for clinical trials) or Exposure(s) (for observational studies)Natural progression from first episode of CIS to MS or NMO was observed.\n\nMain Outcome(s) and Measure(s)Variables analysed were proportion of patients converting to MS or NMO after first episode of CIS, duration between first episode of neurological event and diagnosis of MS, status of anti-AQP4 IgG and length of longest contiguous spinal cord lesion in MS patients. Association between baseline characteristics and progression to MS from CIS was analyzed using multiple logistic regression. Multivariate time dependent effect of baseline characteristics on progression to MS was plotted.\n\nResults14.5% patients with CIS converted to MS after 1.1 {+/-} 1.0 years with greater predisposition (18.8%) in those having syndromes referable to the cerebral hemispheres. Conversion rate from ON to MS was 9.7%. 90.9% patients had benign disease course. 46.7% patients had abnormal MRIs at baseline, with 0.6{+/-}0.5 contrast enhanced lesions. Below normal BMI and MRI lesion load ([≥] 4 lesions) were identified as risk indicators for the development of MS. Only 4.5% were positive for anti-AQP4 antibody in MS patients and amongst them, 80% were NMO patients as diagnosed by modern criteria.\n\nConclusions and Relevance Below normal BMI and number of demyelinating lesions ([≥]4) are significant predictors of conversion from CIS to MS. A low conversion rate to MS in Taiwanese CIS patients and majority of them having a benign course and minimal disability suggest the roles of geographic, genetic and ethnic factors.\n\nTrial RegistrationNon-trial observational study.

neuroscience↗