Biology subjects

Eguchi, A.

Publications and source records attributed to Eguchi, A..

9 recordsLinked to original sources

bioRxiv2026

AAV and lentiviral transduction in Duchenne muscular dystrophy cardiomyocytes activate cell stress responses

Duchenne muscular dystrophy (DMD) is an X-linked muscle wasting disorder marked by lack of dystrophin expression. Symptoms include loss of ambulation, respiratory problems, and cardiac complications with heart failure being the leading cause of death. Dystrophin transduces force from the actin cytoskeleton to the extracellular matrix to protect cells during muscle contraction. Restoration of dystrophin expression by gene transfer holds promise in addressing the root cause of disease. We compared the changes to transcriptional profiles after gene transfer by adeno-associated virus or lentivirus to examine whether viral treatment alone impacts cell homeostasis. We delivered GFP to cardiomyocytes differentiated from induced pluripotent stem cells (iPSCs) with DMD mutations. Global transcriptional profiling revealed a downregulation of metabolic genes after lentiviral transduction compared to untreated controls. In both AAV and lentivirus-treated DMD iPSC-cardiomyocytes, we observed an activation of the p53 DNA damage response in addition to a downregulation of cell cycle genes, suggesting stress-induced G2/M checkpoint arrest following viral delivery. These findings demonstrate that gene therapy mediated by viral vectors activates cell stress pathways. Interventions to mitigate these stress responses may be necessary for safe and effective gene transfer in diseased cells.

molecular biology↗

bioRxiv2026

Decoupling Fabrication from Encoding: DNA-Addressable Template Microparticles for Large, User-Defined Optical Barcode Libraries

Optical barcodes for pooled high-throughput screening must support large libraries while remaining decodable in a single imaging step. Existing approaches often trade design control for manufacturability: deterministic barcodes often require per-code redesign of particle fabrication, whereas stochastic combinatorial barcodes are difficult to generate as predefined batches. Here we introduce a chemically programmable barcoding architecture that decouples particle fabrication from barcode assignment. Using a contact-free multilaminar flow lithography platform with all-around three-dimensional sheathing, we continuously fabricate a universal hydrogel scaffold containing five spatially segregated DNA-addressable domains at rates >106 particles/h. Chosen barcode identities are subsequently written on demand onto the same template batch by domain-selective DNA hybridization. Single-domain measurements resolved 64 candidate optical states, indicating an experimentally informed theoretical upper bound of 645 {approx} 1.1 x 109 barcodes. We further implemented a predefined 59,049-code library by split-pool labeling, achieving an 88% recovery of decoded beads at a stringent posterior threshold (>0.95). After 11 days, >7,800 beads were correctly re-identified at >0.95 accuracy in matched fields of view. This strategy provides a highly scalable, chemically programmable route to build large, user-defined optical barcode libraries with single-image optical readout and longitudinal traceability.

bioengineering↗

bioRxiv2024

Phosphoproteomic investigation of targets of protein phosphatases in EGFR signaling

Receptor tyrosine kinases (RTKs) initiate cellular signaling pathways, which are regulated through a delicate balance of phosphorylation and dephosphorylation events. While many studies of RTKs have focused on downstream-activated kinases catalyzing the site-specific phosphorylation, few studies have focused on the phosphatases carrying out the dephosphorylation. In this study, we analyzed six protein phosphatase networks using chemical inhibitors in context of epidermal growth factor receptor (EGFR) signaling by mass spectrometry-based phosphoproteomics. Specifically, we focused on protein phosphatase 2C (PP2C), involved in attenuating p38-dependent signaling pathways in various cellular responses, and confirmed its effect in regulating p38 activity in EGFR signaling. Furthermore, utilizing a p38 inhibitor, we classified phosphosites whose phosphorylation status depends on PP2C inhibition into p38-dependent and p38-independent sites. This study provides a large-scale dataset of phosphatase-regulation of EGF-responsive phosphorylation sites, which serves as a useful resource to deepen our understanding of EGFR signaling.

cell biology↗

bioRxiv2023

Spatiotemporal Proteomics Deciphers Functional Selectivity of EGFR ligands

The epidermal growth factor receptor (EGFR) induces different signaling outputs depending on ligand identity and biological context. This phenomenon is known as functional selectivity, but the underlying molecular mechanisms remain elusive. Here, we investigated this on a global scale and time-resolved by high-throughput multilayered proteomics integrating dynamic changes in the EGFR interaction network by proximity biotinylation using EGFR- TurboID, phosphoproteome, and proteome in response to stimulation with the six highest-affinity EGFR ligands. We obtained comprehensive temporal profiles of protein recruitment and phosphosite changes pinpointing signaling proteins differentially regulated by the six ligands with key impact on EGFR endocytic fate, e.g. degradation or recycling. Specifically, the Epsin family protein, Clint1 was identified to control the endocytic trafficking of EGFR towards degradation. Moreover, we characterized the protein interaction selectivity of EGFR C-terminally phosphorylated tyrosine residues using a panel of tyrosine mutated constructs showing STAT5 specificity for EGFR Y1173. These data provide a comprehensive resource deciphering functional selectivity of EGFR signaling to support discovery of novel drug targets.

molecular biology↗

bioRxiv2023

One-Tip enables comprehensive proteome coverage in minimal cells and single zygotes

We present One-Tip, a lossless proteomics methodology that seamlessly combines swift, one-pot sample preparation with narrow-window data-independent acquisition mass spectrometric analysis. With simplest sample processing, One-Tip reproducibly identifies > 9,000 proteins from [~]1000 cells and [~] 6,000 proteins in a single mouse zygote with a throughput of 40 samples-per-day. This easy-to-use method expands capabilities of proteomics research by enabling greater depth, scalability and throughput covering low to high input samples.

biochemistry↗

bioRxiv2023

Subchronic Toxicity Assessment of Perfluoroalkyl Substances by Mixed Exposure of Nine Substances at Daily Intake Relevant Concentration

Per- and poly-fluoroalkyl substances (PFAS) have been utilized extensively for various applications owing to their distinctive chemical properties. They exhibit high persistence in the environment and accumulate within the human body, necessitating toxicity assessments. However, the consequences of prolonged, low-level exposure to PFAS and concurrent exposure to multiple PFAS have not been explored. In this study, male C57BL/6J mice (aged 8 weeks) were exposed to a composite of nine PFAS, which include long-chain PFAS (e.g. perfluorooctanoic acid and perfluorooctanesulfonic acid) and short-chain PFAS (e.g. perfluorobutanoic acid and perfluorobutanesulfonic acid) at concentrations equivalent to the estimated daily human intake in the composition reported (1 {micro}g/L [sum of the nine compounds], the maximum reported exposure concentration) via drinking water. Histological examination revealed vacuolization of hepatocytes and irregular arrangement of hepatocyte cords, suggesting that exposure to low levels of the PFAS mixture causes morphological changes in liver tissues. Transcriptome analysis revealed that PFAS exposure mainly altered a group of genes related to metabolism and chemical carcinogenesis. Machine learning analysis of the liver metabolome showed a typical concentration-independent alteration upon PFAS exposure, and in addition to known substances such as glutathione, a compound with unknown biological function; 2,5-dihydro-2,4-dimethyloxazole was found. This study demonstrates that daily exposure to PFAS leads to morphological changes in liver tissues and alters the expression of metabolism-and cancer-related genes as well as phospholipid metabolism. Future studies are required to evaluate the chronic toxicity of prolonged, low-level exposure to PFAS mixtures and to investigate the health effects of PFAS.

pharmacology and toxicology↗

bioRxiv2022

TRF2 rescues pathogenic phenotypes in Duchenne muscular dystrophy cardiomyocytes derived from human iPSCs

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease caused by the lack of dystrophin. Heart failure, driven by cardiomyocyte death, fibrosis, and the development of dilated cardiomyopathy, is the leading cause of death in DMD patients. Current treatments decrease the mechanical load on the heart, but do not address the root cause of dilated cardiomyopathy: cardiomyocyte death. Previously, we showed that telomere shortening is a hallmark of cardiomyocytes in DMD patient cardiac tissues and in a mouse model of DMD we generated that manifests dilated cardiomyopathy. We also found that this telomere shortening is recapitulated in DMD cardiomyocytes differentiated from patient-derived induced pluripotent stem cells (iPSCs). Here we further characterize that DMD cardiomyocytes exhibit reduced cell size, nuclear size, and sarcomere density compared to healthy isogenic controls. The telomere-binding protein, TRF2, is a core component of the shelterin complex, which protects chromosome ends and is expressed at lower levels in DMD cardiomyocytes compared to controls. We investigated whether preservation of telomere length could be achieved with upregulation of TRF2. We found that TRF2 upregulation in DMD cardiomyocytes increased telomere lengths, cell size, nuclear size, sarcomere density, and cell survival. These data suggest TRF2 gene therapy has the potential to delay the onset of dilated cardiomyopathy.

cell biology↗

bioRxiv2020

Single position substitution of hairpin pyrrole-imidazole polyamides imparts distinct DNA-binding profiles across the human genome

Regulating desired loci in the genome with sequence-specific DNA-binding molecules is a major goal for the development of precision medicine. Pyrrole-imidazole (Py-Im) polyamides are synthetic molecules that can be rationally designed to target specific DNA sequences to both disrupt and recruit transcriptional machinery. While in vitro binding has been extensively studied, in vivo effects are often difficult to predict using current models of DNA binding. Determining the impact of genomic architecture and the local chromatin landscape on polyamide-DNA sequence specificity remains an unresolved question that impedes their effective deployment in vivo. In this report we identified polyamide-DNA interaction sites across the entire genome, by covalently crosslinking and capturing these events in the nuclei of human LNCaP cells. This method, termed COSMIC-seq, confirms the ability of hairpin-polyamides, with similar architectures but differing at a single ring position, to retain in vitro specificities and display distinct genome-wide binding profiles. These results underpin the development of Py-Im polyamides as DNA-targeting molecules that mediate their regulatory or remedial functions at desired genomic loci.

biochemistry↗

bioRxiv2020

The TAT Protein Transduction Domain as an Intra-articular Drug-Delivery Technology

ObjectiveIntra-articular drug delivery holds great promise for the treatment of joint diseases such as osteoarthritis. The objective of this study was to evaluate the TAT peptide transduction domain (TAT-PTD) as a potential intra-articular drug delivery technology for synovial joints. DesignExperiments examined the ability of TAT conjugates to associate with primary chondrocytes and alter cellular function both in vitro and in vivo. Further experiments examined the ability of the TAT-PTD to bind to human osteoarthritic cartilage. ResultsThe results show that the TAT-PTD associates with chondrocytes, is capable of delivering siRNA for chondrocyte gene knockdown, and that the recombinant enzyme TAT-Cre is capable of inducing in vivo genetic recombination within the knee joint in a reporter mouse model. Lastly, binding studies show that osteoarthritic cartilage preferentially uptakes the TAT-PTD from solution. ConclusionsThe results suggest that the TAT-PTD is a promising delivery strategy for intra-articular therapeutics.

bioengineering↗