Biology subjects

Ai, T.

Publications and source records attributed to Ai, T..

7 recordsLinked to original sources

bioRxiv2026

CellCage™ Technology Enables High-throughput Isolation of Defined Cell Combinations to Resolve the Determinants of CAR-T mediated Cytotoxicity

Understanding the cellular determinants of CAR-T cell-mediated cytotoxicity at the single-cell level remains a critical challenge in developing effective cellular immunotherapies. Conventional bulk co-culture assays provide only population-level measurements of cytotoxic activity and cannot resolve the functional heterogeneity of individual effector-target interactions. Here, we demonstrate the Cellanome platform, which enables the formation of CellCage Enclosures (CCEs) through spatially controlled photopolymerization, as a tool for high-resolution, high-throughput characterization of CAR-T cell-mediated cytotoxicity. By isolating defined effector-target combinations within individual CCEs and coupling encapsulation with longitudinal time-lapse imaging and automated image analysis, we resolve cytotoxic activity across thousands of individually tracked CCEs. Using anti-CD19 CAR-T effectors and NALM6-GFP targets, we show that individual CD8+ CAR-T effectors exhibit substantial functional heterogeneity, with serial killing capacity, effector-target contact dynamics, and intrinsic motility each independently correlating with cytotoxic potency. Cytotoxic efficacy scaled positively with effector number, and CD4+ T helper cells augmented CD8+-mediated killing in a dose-dependent manner, providing direct evidence for cooperative T cell behavior during tumor cell clearance. Extending the platform to a solid tumor model using AIC100 anti-ICAM-1 CAR-T cells and HeLa-GFP targets, we show that individual AIC100 effectors are insufficient to mediate effective killing, while multi-effector CCEs exhibit robust cytotoxicity scaling with effector abundance and CD4+:CD8+ composition. These data establish the Cellanome platform as a broadly applicable system for dissecting the determinants of CAR-T cell cytotoxicity at single-cell resolution, with direct translational relevance to the optimization of cellular immunotherapy.

immunology↗

bioRxiv2026

SRPK3 regulates back-splicing of pre-MYOCD through Phosphorylating SRSF10 to modulate cardiac hypertrophy

Cardiac hypertrophy represents a hallmark of pathological remodeling in heart failure, involving complex molecular networks that remain incompletely defined. While post-transcriptional regulation is increasingly recognized as a key control layer, the role of splicing kinases in this process requires further elucidation. This study identifies serine/arginine-rich protein kinase 3 (SRPK3) as a central regulator of myocardial hypertrophy through its control of circular RNA (circRNA) biogenesis. We observed marked SRPK3 upregulation in phenylephrine/angiotensin II-treated cardiomyocytes, transverse aortic constriction (TAC) rats, and human heart failure datasets. Gain- and loss-of-function studies demonstrated that SRPK3 overexpression exacerbates hypertrophic phenotypes, including increased cell surface area and upregulation of markers (ANP, BNP, Myh7). Conversely, siRNA knockdown or pharmacological inhibition with the pan-SRPK inhibitor MSC-1186 effectively attenuated these pathological features. Mechanistically, SRPK3 interacts with and phosphorylates the splicing factor SRSF10 at specific serine residues (S129, S131, and S133). This phosphorylation event inhibits the back-splicing of pre-MYOCD, resulting in reduced cardioprotective circ-MYOCD levels and a concomitant increase in linear MYOCD expression. The depletion of circ-MYOCD relieves its competitive inhibition on linear splicing, driving the transcriptional activation of pro-hypertrophic signaling pathways. Crucially, restoring circ-MYOCD expression reversed SRPK3-driven pathology, confirming its protective role. These findings establish the SRPK3-SRSF10-circ-MYOCD axis as a critical regulatory circuit in cardiac remodeling. By bridging kinase activity with splicing dynamics, this study highlights RNA processing as a vital therapeutic target. Targeting this axis via SRPK3 inhibition or circ-MYOCD delivery represents a promising dual-modality strategy for treating heart failure.

Molecular Biology↗

bioRxiv2025

The abscisic acid could reverse the cuproptosis in sepsis-associated acute kidney injury through the inhibition of ferredoxin-1

Sepsis-associated acute kidney injury (S-AKI) is a grave clinical condition with a poor prognosis. Prior studies have observed a potential correlation between copper (Cu) and S-AKI, though the underlying mechanisms remain to be fully elucidated. The present study aims to investigate the underlying mechanism between Cu and renal dysfunction under sepsis conditions. Utilizing a cecal ligation and puncture (CLP) murine model, we observed a substantial upregulation of inflammatory factors and deteriorated renal function (p<0.05), concomitant with an augmented mortality rate (p<0..05). Concurrently, the renal tissue exhibited a series of pathological manifestations, including fibrosis and cell death. Concurrently, the expression of the cuproptosis-regulating protein FDX-1 (p<0.05) was found to be elevated in CLP mice. Collectively, these data indicated the activation of cuproptosis in CLP-induced S-AKI. Intriguingly, the application of abscisic acid (ABA), a FDX-1 inhibitor, led to a substantial alleviation of mortality rate and inflammatory factor level (p<0.05). Furthermore, a significant alleviation was observed in renal pathology and biofunction (p<0.05). Collectively, these observations suggest that ABA exerts a protective effect on renal function during cuproptosis promoted S-AKI. Consequently, it can be concluded that ABA has the potential to reverse cuproptosis in S-AKI by inhibiting FDX-1.

molecular biology↗

bioRxiv2024

BCR, not TCR, repertoire diversity is associated with favorable COVID-19 prognosis

The SARS-CoV-2 pandemic has had a widespread and severe impact on society, yet there have also been instances of remarkable recovery, even in critically ill patients. In this study, we used single-cell RNA sequencing to analyze the immune responses in recovered and deceased COVID-19 patients during moderate and critical stages. The study included three unvaccinated patients from each outcome category. Although expanded T cell receptor (TCR) clones were predominantly SARS-CoV-2-specific, they represented only a small fraction of the total repertoire in all patients. In contrast, while deceased patients exhibited monoclonal B cell receptor (BCR) expansions without COVID-19 specificity, survivors demonstrated diverse and specific BCR clones. These findings suggest that neither TCR diversity nor BCR monoclonal expansions are sufficient for viral clearance and subsequent recovery. Differential gene expression analysis revealed that protein biosynthetic processes were enriched in survivors, but that potentially damaging mitochondrial ATP metabolism was activated in the deceased. This study underscores that BCR repertoire diversity, but not TCR diversity, correlates with favorable outcomes in COVID-19.

immunology↗

bioRxiv2024

Zinc finger transcription factors BnaSTOP2s regulate sulfur metabolism and confer resistance to Sclerotinia sclerotiorum in Brassica napus

Rapeseed (Brassica napus L.) has a high sulfur requirement for optimal growth, development, and pathogen resistance. In this study, we identified zinc finger transcription factors, BnaSTOP2s, that play key roles in sulfur metabolism and Sclerotinia sclerotiorum resistance. First, our results suggested that BnaSTOP2s are involved in sulfur as evidenced from extensive protein interaction screening. Knockout of BnaSTOP2s reduced the response sensitivity in both sulfur-deficient and sulfur-excessive conditions by promoting the elongation of primary roots of seedlings. Furthermore, the content of essential sulfur-containing metabolites, including glucosinolate and glutathione, were substantially down-regulated in roots and leaves of Bnastop2 mutants, which is consistent with the significantly lowered transcriptional levels of key players of GSL synthesis and transportation, BnaMYB28s and BnaGTR2s, respectively. Through comprehensive RNA-seq analysis, we revealed the substantial effect of BnaSTOP2s on sulfur metabolism from source to sink. Additionally, we observed a significant decrease while increase in leaf lesion sizes of the BnaSTOP2-OE and Bnastop2 mutants, respectively, when compared to the wild type during Sclerotinia sclerotiorum infection, suggesting the vital role of BnaSTOP2 in plant defense response. Overall, our findings highlight that BnaSTOP2s seems to be global regulators of sulfur metabolism and confer resistance to Sclerotinia sclerotiorum infection in B. napus.

plant biology↗

bioRxiv2023

Carvedilol suppresses ryanodine receptor-dependent Ca2+ bursts in human neurons bearing PSEN1 variants found in early onset Alzheimer's disease

Seizures are increasingly being recognized as the hallmark of Alzheimers disease (AD). Neuronal hyperactivity can be a consequence of neuronal damage caused by abnormal amyloid {beta} (A{beta}) depositions. However, it can also be a cell-autonomous phenomenon causing AD by A{beta}-independent mechanisms. Indeed, various studies using animal models showed that Ca2+ releases from the endoplasmic reticulum (ER) via type 1 inositol triphosphate receptors (InsP3R1s) and ryanodine receptors (RyRs). To investigate which is the main pathophysiological mechanism in human neurons, we measured Ca2+ signaling in neural cells derived from three early-onset AD patients harboring variants of Presenilin-1 (PSEN1 p.A246E, p.L286V, and p.M146L). Of these, it has been reported that PSEN1 p.A246E and p.L286V did not produce a significant amount of abnormal A{beta}. We found that all PSEN1-mutant neurons, but not wild-type, caused abnormal Ca2+-bursts in a manner dependent on the calcium channel, Ryanodine Receptor 2 (RyR2). Indeed, carvedilol, anRyR2 inhibitor, and VK-II-86, an analog of carvedilol without the {beta}-blocking effects, sufficiently eliminated the abnormal Ca2+ bursts. In contrast, Dantrolene, a RyR1 inhibitor, and Xestospongin c, an IP3R inhibitor, did not attenuate the Ca2+-bursts. The RNA-Seq data revealed that ER-stress responsive genes were increased, and mitochondrial Ca2+-transporter genes were decreased in PSEN1A246E cells compared to the WT neurons. Thus, we propose that aberrant Ca2+ signaling is a key link between human pathogenic PSEN1 variants and cell-intrinsic hyperactivity prior to deposition of abnormal A{beta}, offering prospects for the development of targeted prevention strategies for at-risk individuals. One Sentence SummaryAberrant Ca2+-signaling causes PSEN1-related early onset Alzheimers disease.

neuroscience↗

bioRxiv2018

A new highly sensitive real-time quantitative-PCR method for detection of BCR-ABL1 to monitor minimal residual disease in chronic myeloid leukemia after discontinuation of imatinib

Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL1 fusion protein, encoded by the Philadelphia chromosome, have drastically improved the outcomes for patients with chronic myeloid leukemia (CML). Although several real-time quantitative polymerase chain reaction (RQ-PCR) kits for the detection of BCR-ABL1 transcripts are commercially available, their accuracy and efficiency in laboratory practice require reevaluation. We have developed a new in-house RQ-PCR method to detect minimal residual disease (MRD) in CML cases. MRD was analyzed in 102 patients with CML from the DOMEST study, a clinical trial to study the rationale for imatinib mesylate discontinuation in Japan. The BCR-ABL1/ABL1 ratio was evaluated using the international standard (IS) ratio, where IS < 0.01% was defined as a major molecular response. At enrollment, BCR-ABL1 transcripts were undetectable in all samples using a widely-applied RQ-PCR method performed in the commercial laboratory, BML (BML Inc., Tokyo, Japan); however, the in-house method detected the BCR-ABL1 transcripts in five samples (5%) (mean IS ratio: 0.0062 {+/-} 0.0010%). After discontinuation of imatinib, BCR-ABL1 transcripts were detected using the in-house RQ-PCR in 21 patients (21%) that were not positive using the BML method. Nineteen samples were also tested using a commercially available RQ-PCR assay kit with a detection limit of IS ratio, 0.0007% (ODK-1201, Otsuka Pharmaceutical Co., Tokyo, Japan). This method detected low levels of BCR-ABL1 transcripts in 14 samples (74%), but scored negative for five samples (26%) that were positive using the in-house method. These data suggest that our new in-house RQ-PCR method is effective for monitoring MRD in CML.

cancer biology↗