Biology subjects

Kobayashi, A.

Publications and source records attributed to Kobayashi, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Adolescent prodromal phenotypes of psychiatric disorders: insights from a novel DISC1-perturbed monkey model

Neurodevelopmental deficits frequently lead to mild but significant adolescent social and cognitive changes, representing prodromal manifestations of adult-onset psychiatric disorders in a cross-disease manner. Although experimental medicine utilizes animal models to address the mechanisms, rodent brains lack critical structures for human higher brain functions such as the dorsolateral prefrontal cortex. To fill this translational gap, we generated monkeys with an impaired DISC1 gene locus, targeting the multifunctional protein essential for neurodevelopment and involved in transdiagnostic mental health risks. Here we analyze the F0 animals by minimizing mosaicism. At ages equivalent to human adolescence, these monkeys exhibited blunted responsiveness to social stimuli and increased internal drives, mirroring altered social behaviors in the prodromal stage in humans. Resting-state functional MRI data indicated disrupted global functional connectivity and reorganization of network topology, marked by a shift in the dominance of brain hubs from prefrontal regions to sensory areas. This primate model provides a unique platform for studying psychiatric disorders, such as schizophrenia, across the developmental trajectory.

neuroscience↗

bioRxiv2026

Augmenting Radiation Sensitivity by Targeting PAR-Dependent Replication Fork Vulnerability in IDH-Mutant Glioma

Mutations in isocitrate dehydrogenase 1 (IDH1) drive the early stages of gliomagenesis while simultaneously imposing replication stress that creates targetable vulnerabilities. Using both in vitro and in vivo models, we show that inhibition of poly(ADP-ribose) glycohydrolase (PARG) induces a poly(ADP-ribose) (PAR)-dependent augmentation of radiosensitivity in IDH1-mutant glioma cells. Metabolic repletion of NAD+ fails to rescue this effect, indicating that the vulnerability cannot be explained solely by NAD+ depletion. Instead, PARG inhibition profoundly alters replication fork progression and S-phase kinetics in IDH1-mutant cells. Mechanistically, ionizing radiation preferentially activates replication fork-associated damage response proteins DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and X-ray repair cross-complementing protein 1 (XRCC1) in IDH1-mutant cells, a response partially reversed by pharmacologic inhibition of mutant IDH1. Importantly, pharmacologic inhibition of DNA-PKcs with AZD7648 during irradiation disrupts fork-associated repair signaling and markedly enhances cytotoxicity in IDH1-mutant glioma models. Together, these findings identify a PAR-dependent replication fork vulnerability that can be therapeutically exploited to selectively enhance radiosensitivity in IDH1-mutant gliomas. Statement of significanceIDH-mutant gliomas harbor intrinsic replication stress yet lack targeted radiosensitization strategies. We identify a PAR-dependent replication fork vulnerability in which disruption amplifies radiation cytotoxicity by deregulating S-phase fork signaling. Pharmacologic DNA-PKcs inhibition exploits this dependency, providing a genotype-selective approach to enhance radiotherapy in IDH-mutant glioma.

cancer biology↗

bioRxiv2026

tRNA-derived fragments are elevated in the aging brain and may contribute to neurodegeneration

tRNA-derived fragments (tRFs) are a class of small noncoding RNAs with emerging roles in stress responses and gene regulation, yet their dynamics across tissues and brain regions during aging remain poorly understood. Here, we systematically profiled age-associated changes in tRFs using three independent mouse small RNA-seq datasets spanning multiple organs and brain regions. Nuclear-encoded tRFs were the only small RNA class showing a strong, progressive accumulation with age, a pattern that was specific to the brain and broadly distributed across brain regions. Fragment length distributions, boundary profiles, and coverage maps were consistent with amplified cleavage at conserved sites, implicating angiogenin as a primary driver. Age-associated increases in specific tRF species, including 5CysGCA, 5GluCTC, and 5GlyGCC, were validated by northern blotting and RT-qPCR. Analogous upregulation was observed in human frontal lobe tissue from frontotemporal dementia patients and in cerebrospinal fluid from traumatic brain injury patients, suggesting that tRF accumulation is further amplified under neurological stress. Together, these findings establish nuclear-encoded tRFs as a small RNA class that accumulates selectively in the aging brain, with potential roles in neurodegeneration, and as biomarkers and targets for therapeutic intervention.

neuroscience↗

bioRxiv2026

tRNA-derived fragments elevated in Alzheimer's disease facilitate Tau aggregation

Tauopathies, including Alzheimers disease (AD), are driven by pathological Tau aggregation, a process that requires co-factors. Small RNAs (sRNA) have been proposed as such co-factors, yet little is known about endogenous transcripts that promote Tau pathology. We identify stress-induced tRNA-derived RNAs or fragments (tDRs/tRFs) as the most dysregulated sRNA class in human AD brains, PS19 mice overexpressing mutant human Tau, and human neuronal tauopathy models. Notably, the highly accumulating 5GluCTC and 5GlyGCC tRFs directly bind Tau and induce its phosphorylation, oligomerization, fibril formation, and impact neurite growth. 5GluCTC is enriched in pathological Tau precipitates and co-localizes with oligomeric Tau in PS19 mouse brains. Inhibiting 5GluCTC mitigates Tau pathology. Furthermore, these tRFs are highly secreted by neurons and can be taken up by recipient cells, contributing to Tau pathology. Our findings establish 5GluCTC as a key regulator of Tau aggregation and suggest its inhibition as a promising therapeutic strategy for tauopathies. HighlightsO_LISpecific stress-induced 5tRFs strongly accumulate in Alzheimers disease brains C_LIO_LI5GluCTC binds and co-localizes with pathologic Tau in neurons and in the brain C_LIO_LI5GluCTC promotes pathologic Tau species while its inhibition reverses these effects C_LIO_LISpecific 5tRFs are horizontally transferred between neurons C_LI

neuroscience↗

bioRxiv2026

Novel estimation of memory in molecular dynamics with extended and comprehensive single-molecule tracking software: FreeTrace

Single-molecule tracking (SMT) in live cells reveals how biomolecules explore crowded intracellular environments, yet most tracking software assumes Brownian motion, an approximation that fails when anomalous diffusion dominates. This leads to biased trajectory reconstruction and loss of biophysical information, particularly for the short trajectories typical of intracellular experiments. We present FreeTrace, an SMT framework that reconstructs trajectories under fractional Brownian motion (fBm), incorporating temporal correlations directly into linking with minimal input parameters. A deep neural network estimates diffusion properties (Hurst exponent H and generalised diffusion coefficient K) for individual trajectories, while an analytical ensemble estimator accurately recovers H from trajectories as short as three frames, conditions where mean-squared displacement methods fail. Benchmarking on simulated data demonstrates superior performance across motion types and densities. Applications to chromatin-bound histones, DNA repair proteins in S.c. yeast and human cells reveal biologically meaningful diffusion subpopulations, with H values consistent with polymer models and confined motion. FreeTrace bridges theoretical anomalous diffusion models and routine biological experiments.

molecular biology↗

bioRxiv2025

Informational and Normative Influence on Conformity in Autism

This preregistered study examined whether adults with autism spectrum disorder (ASD) show reduced social conformity and whether any such reduction depends on the type of social influence. Social conformity, defined as the tendency to adjust judgments to align with those of others, is typically driven by normative (acceptance-seeking) and informational (accuracy-seeking) motives. Thirty adults with ASD and 30 matched neurotypical (NT) adults completed two tasks: a preference rating task indexing normative influence, and a dot-counting task indexing informational influence with monetary rewards. Contrary to our predictions, adults with ASD conformed as much as NT adults in the preference rating task but showed significantly reduced conformity in the dot-counting task. Exploratory analyses indicated that this reduction was driven by a distinct subgroup of nine adults with ASD who never revised their initial estimates despite informative social cues, resulting in poorer accuracy and lower rewards. When this subgroup was excluded, group differences in conformity were no longer evident. These findings suggest that, overall, adults with ASD are as susceptible as NT adults to normative influence but less responsive to informational influence, highlighting the importance of distinguishing between types of social influence and considering individual differences when characterizing social behavior in ASD.

neuroscience↗

bioRxiv2025

A guide for Single Particle Tracking: from sample preparation and image acquisition to the analysis of individual trajectories

Live-cell Single-Particle Tracking (SPT) is a powerful super resolution microscopy approach. It allows us to observe live recordings of individual molecules in living cells at high temporal and spatial resolution (100 Hz, 20 nm). Here, we discuss the implementation and application of super-resolution microscopy to quantify the mobility of single molecules in living human cells. We first describe the human cell sample preparation for SPT with an experimental time line overview, then super-resolution microscope setting and procedure to acquire SPT films for further SPT video analyses. We provide a practical guide to determine the quality of acquired SPT films in particular, number of photons and fluorescent background. Furthermore, we introduce several SPT video analysis software comparing their advantages and limitations. Expectantly, this SPT protocol will serve as a foundation for a better SPT performance.

biophysics↗

bioRxiv2024

PAX2 is Transcriptionally Silenced by a Distinct Mechanism of Epigenetic Reprogramming to Initiate Endometrial Carcinogenesis

In most cancers, including endometrial cancer, tumor suppressor genes harboring inactivating mutations have been systematically cataloged. However, locus-specific epigenetic alterations contributing to cancer initiation and progression remain only partly described, creating knowledge gaps about functionally significant tumor suppressors and underlying mechanisms associated with their inactivation. Here, we show that PAX2 is an endometrial tumor suppressor recurrently inactivated by a distinct epigenetic reprogramming event not associated with promoter hypermethylation. PAX2 is expressed throughout normal endometrial glands; however, microscopic clones with PAX2 protein loss arise spontaneously in adulthood and progress to endometrial precancers and cancers. Here we show that PAX2 protein loss occurs via transcriptional silencing in 80% of endometrial cancers. Mechanistically, transcriptomic, epigenomic, 3D genomic, and machine learning analyses showed that silencing is associated with replacement of open/active chromatin features (H3K27ac/H3K4me3) with inaccessible/repressive features (H3K27me3). The spread of the H3K27me3 signal is restrained by cohesin loops, preventing transcriptional dysregulation of neighboring genes. Functionally, PAX2 loss promoted endometrial carcinogenesis by rewiring the transcriptional landscape via global enhancer reprogramming. Genetically engineered mouse models together with organoid and human cell line studies established Pax2 as an in vivo endometrial tumor suppressor that cooperates with Pten to produce lethal cancers. Our discovery of a specific and recurring epigenetic alteration that transcriptionally silences PAX2 to initiate most endometrial cancers opens new lines of investigation into the origins of endometrial cancer with diverse implications for its diagnosis and treatment. One Sentence SummaryWe demonstrate that PAX2 is silenced by a highly recurrent epigenetic mechanism to initiate most endometrial cancers, establishing a novel mechanism of tumor suppressor inactivation.

cancer biology↗

bioRxiv2023

Mapping adipocyte interactome networks by HaloTag enrichment mass-spectrometry

Mapping protein interaction complexes in their natural state in vivo represents the holy grail of protein network analysis. Detection of protein interaction stoichiometry has been an important technical challenge, as few studies have focused this, yet this may be solved by artificial intelligence and proteomics. Here, we describe the development of HaloMS, a high-throughput HaloTag-based affinity purification-mass spectrometry assay for protein interaction discovery. The approach enables the rapid capture of newly expressed proteins, eliminating tedious conventional one-by-one assay. As a proof-of-principle, we used HaloMS to evaluate protein complex interactions of 17 regulatory proteins in human adipocytes. The adipocyte interactome network was validated using an in vitro pull-down assay and artificial intelligence-based prediction tools. The application of HaloMS to probe adipocyte differentiation facilitated the identification of previously unknown transcription factor-protein complexes, revealing proteome-wide human adipocyte transcription factor networks, and shedding light on how different pathways are integrated.

biochemistry↗

bioRxiv2023

High-throughput structure determination of an intrinsically disordered protein using cell-free protein crystallization

Intrinsically disordered proteins (IDPs) play a crucial role in various biological phenomena, dynamically changing their conformations in response to external environmental cues. To gain a deeper understanding of these proteins, it is essential to identify the determinants that fix their structures at the atomic level. Here, we developed a pipeline for rapid crystal structure analysis of IDP using a cell-free protein crystallization (CFPC) method. Through this approach, we successfully demonstrated the determination of the structure of an IDP to uncover the key determinants that stabilize its conformation. Specifically, we focused on the 11-residue fragment of c-Myc, which forms an -helix through dimerization with a binding partner protein. This fragment was strategically fused with an in-cell crystallizing protein and was expressed in a cell-free system. The resulting crystal structures of the c-Myc fragment were successfully determined at a resolution of 1.92 [A] and we confirmed that they are identical to the structures of the complex with the native binding partner protein. This indicates that the environment of the scaffold crystal can fix the structure of c-Myc. Significantly, these crystals were obtained directly from a small reaction mixture (30 L) incubated for only 72 hours. Analysis of 8 crystal structures derived from 22 mutants revealed two hydrophobic residues as the key determinants responsible for stabilizing the -helical structure. These findings underscore the power of our CFPC screening method as a valuable tool for determining the structures of challenging target proteins and elucidating the essential molecular interactions that govern their stability.

biophysics↗

bioRxiv2022

Glutamine synthetase mRNA releases sRNA from its 3'UTR to regulate carbon/nitrogen metabolic balance

Glutamine synthetase is the key enzyme of nitrogen assimilation, which is encoded in the first cistron of glnALG operon and is induced under nitrogen limiting conditions through transcriptional activation by NtrBC in Salmonella and E. coli. 2-oxoglutarate serves as the carbon skeleton of glutamate and glutamine, but how 2-oxoglutarate fluctuation is controlled in response to nitrogen availability remained unknown. We show that the glnA mRNA produces an Hfq-dependent GlnZ sRNA from its 3 sUTR through RNase E-mediated cleavage. Through a base-pairing mechanism, GlnZ primarily regulates sucA, encoding the E1o component of 2-oxoglutarate dehydrogenase. In the cells grown on glutamine as the nitrogen source, the endogenous GlnZ represses the expression of SucA to redirect the carbon flow from the TCA cycle to the nitrogen assimilation pathway. This study also clarifies that the release of GlnZ sRNA from the glnA mRNA by RNase E is essential for the post-transcriptional regulation of sucA, and thus the mRNA coordinates the two independent functions to balance the supply and demand of the fundamental metabolites.

microbiology↗

bioRxiv2022

Cell-free Protein Crystallization for Nanocrystal Structure Determination

In-cell protein crystallization (ICPC) has attracted attention as a next-generation structural biology tool because it does not require multistep purification processes and large-scale crystallization screenings. However, significant issues remain to be solved in context of obtaining various protein crystals in sufficient amounts and quality for structure determination by ICPC. Here, we report the development of cell-free protein crystallization (CFPC), a direct protein crystallization technique which uses cell-free protein synthesis. The most crucial advantages of CFPC are that the reaction scale and time can be minimized and that various reagents can be added during the reaction. We obtained high-quality nano-sized polyhedra crystals, which are produced in insect cells by infection with cytoplasmic polyhedrosis virus, at a 200 L reaction scale within 6 h. We applied this technology to structure determination of crystalline inclusion protein A (CipA) by suppressing twin crystal formation with addition of an inhibitor to the reaction solution. We succeeded in determining a 2.11 [A] resolution structure from the nanocrystals of CipA. This technology, which integrates in-cell and in vitro crystallizations significantly expands the tools available for high throughput protein structure determination, particularly in context of unstable, low-yield, or substrate-binding proteins, which are difficult to analyze by conventional methods.

biophysics↗

bioRxiv2021

Metalloproteinase-dependent and TMPRSS2-independnt cell surface entry pathway of SARS-CoV-2 requires the furin-cleavage site and the S2 domain of spike protein

The ongoing global vaccination program to prevent SARS-CoV-2 infection, the causative agent of COVID-19, has had significant success. However, recently virus variants have emerged that can evade the immunity in a host achieved through vaccination. Consequently, new therapeutic agents that can efficiently prevent infection from these new variants, and hence COVID-19 spread are urgently required. To achieve this, extensive characterization of virus-host cell interactions to identify effective therapeutic targets is warranted. Here, we report a cell surface entry pathway of SARS-CoV-2 that exists in a cell type-dependent manner is TMPRSS2-independent but sensitive to various broad-spectrum metalloproteinase inhibitors such as marimastat and prinomastat. Experiments with selective metalloproteinase inhibitors and gene-specific siRNAs revealed that a disintegrin and metalloproteinase 10 (ADAM10) is partially involved in the metalloproteinase pathway. Consistent with our finding that the pathway is unique to SARS-CoV-2 among highly pathogenic human coronaviruses, both the furin cleavage motif in the S1/S2 boundary and the S2 domain of SARS-CoV-2 spike protein are essential for metalloproteinase-dependent entry. In contrast, the two elements of SARS-CoV-2 independently contributed to TMPRSS2-dependent S2 priming. The metalloproteinase pathway is involved in SARS-CoV-2-induced syncytia formation and cytopathicity, leading us to theorize that it is also involved in the rapid spread of SARS-CoV-2 and the pathogenesis of COVID-19. Thus, targeting the metalloproteinase pathway in addition to the TMPRSS2 and endosome pathways could be an effective strategy by which to cure COVID-19 in the future. Author SummaryTo develop effective therapeutics against COVID-19, it is necessary to elucidate in detail the infection mechanism of the causative agent, SARS-CoV-2, including recently emerging variants. SARS-CoV-2 binds to the cell surface receptor ACE2 via the Spike protein, and then the Spike protein is cleaved by host proteases to enable entry. Selection of target cells by expression of these tissue-specific proteases contributes to pathogenesis. Here, we found that the metalloproteinase-mediated pathway is important for SARS-CoV-2 infection, variants included. This pathway requires both the prior cleavage of Spike into two domains and a specific sequence in the second domain S2, conditions met by SARS-CoV-2 but lacking in the related human coronavirus SARS-CoV. The contribution of several proteases, including metalloproteinases, to SARS-CoV-2 infection was cell type dependent, especially in cells derived from kidney, ovary, and endometrium, in which SARS-CoV-2 infection was metalloproteinase-dependent. In these cells, inhibition of metalloproteinases by treatment with marimastat or prinomastat, whose safety was previously confirmed in clinical trials, was important in preventing cell death. Our study provides new insights into the complex pathogenesis unique to COVID-19 and relevant to the development of effective therapies.

microbiology↗

bioRxiv2021

Manipulating Histone Acetylation Leads to Adverse Effects in Hemangiosarcoma Cells

Canine hemangiosarcoma (HSA) is a malignant tumour derived from endothelial cells. No effective treatment has yet been developed because of the lack of understanding of its pathogenesis. Histone acetylation, an epigenetic modification, is highly associated with cancer pathogenesis. Manipulating histone acetylation by histone deacetylase inhibitors (HDACi) or bromodomain and extraterminal domain inhibitors (BETi) is one approach to treat various cancers. However, the role of histone acetylation in HSA remains unknown. This study aimed to investigate how histone acetylation functions in HSA pathogenesis using two HDACi, suberanilohydroxamic acid (SAHA) and valproic acid (VPA), and one BETi, JQ1, in vitro and in vivo. Histone acetylation levels were high in cell lines and heterogeneous in clinical cases. SAHA and JQ1 induced apoptosis in HSA cell lines. SAHA and VPA treatment in HSA cell lines upregulated inflammatory-related genes, thereby attracting macrophages. This implies that SAHA and VPA can induce anti-tumour immunity. JQ1 stimulated autophagy and inhibited the cell cycle. Finally, JQ1 suppressed HSA tumour cell proliferation in vivo. These results suggest that HDACi and BETi can be alternative drugs for HSA treatment. Although further research is required, this study provides useful insights for developing new treatments for HSA.

cancer biology↗

bioRxiv2021

Local and global crosstalk among heterochromatin marks drives epigenome patterning in Arabidopsis

Transposable elements (TEs) are robustly silenced by targeting of multiple epigenetic marks, but dynamics of crosstalk among these marks remains enigmatic. In Arabidopsis, TEs are silenced by cytosine methylation in both CpG and non-CpG contexts (mCG and mCH) and histone H3 lysine 9 methylation (H3K9me). While mCH and H3K9me are mutually dependent for their maintenance, mCG and mCH/H3K9me are independently maintained. Here we show that establishment, rather than maintenance, of mCH depends on mCG, accounting for the synergistic colocalization of these silent marks in TEs. When mCG is lost, establishment of mCH is abolished in TEs. mCG also guides mCH in active genes, although genic mCH/H3K9me is removed there. Unexpectedly, the targeting efficiency of mCH depends on relative, rather than absolute, levels of mCG, suggesting underlying global negative controls. We propose that the local positive feedback in heterochromatin dynamics, together with global negative feedback, drive robust and balanced epigenome patterning.

genomics↗

bioRxiv2021

Hemangiosarcoma cells induce M2 polarization and PD-L1 expression in macrophages

Hemangiosarcoma (HSA) is a malignant tumor derived from endothelial cells. Tumor-associated macrophages are one of the major components of tumor microenvironment and crucial for cancer development. The presence and function of macrophages in HSA have not been studied because there is no syngeneic model for HSA. In this study, we evaluated two mouse HSA cell lines and one immortalized mouse endothelial cells for their usefulness as syngeneic models for canine HSA. Our results show that the ISOS-1 cell line develops tumors with similar morphology to canine HSA. ISOS-1 cells highly express KDM2B and have similar KDM2B target expression patterns with canine HSA. Moreover, we determine that in both ISOS-1 and canine HSA tumors, macrophages are present as a major constituent of the tumor microenvironment. These macrophages are positive for CD204, an M2 macrophage marker, and express PD-L1. ISOS-1-conditioned medium can induce M2 polarization and PD-L1 expression in RAW264.7 mouse macrophage cell line. These results show that ISOS-1 can be used as a syngenic model for canine HSA and suggest that macrophages play an important role in immune evasion in HSA. Using the syngeneic mouse model for canine HSA, we can further study the role of immune cells in the pathology of HSA.

cancer biology↗

bioRxiv2021

Prevalence of floating toe and its relationship with static postural stability in children: The Yamanashi adjunct study of the Japan Environment and Childrens Study (JECS-Y)

Floating toe (FT) is a frequently seen condition in which a toe is inadequately in contact with the ground. Although toes play an important role in stabilizing standing posture and walking, many aspects of the effects of FT on the body remain unclear. To our knowledge, there have been no reports about the relationship between FT and postural stability, especially in children. This study aimed to clarify the prevalence of FT and its relationship with static postural stability in children. Of the 400 children aged 8 years who participated in our cohort study, 396, who were examined for static postural stability, were included in this study. Postural stability and FT were assessed using a foot pressure plate. The sway path length of the center of pressure and the area of the ellipse defined as the size of the area marked by the center of pressure were measured as an evaluation of static postural stability. We calculated the "floating toe score (FT score: small FT score indicates insufficient ground contact of the toes)" using the image of the plantar footprint obtained at the postural stability measurement. The FT rate was very high at more than 90%, and the FT score in the eyes-closed condition was significantly higher than that in the eyes-open condition in both sexes. The FT score significantly correlated with the center of pressure path and area. Our results suggest that ground contact of the toes is not directly related to static postural stability in children, but it may function to stabilize the body when the condition becomes unstable, such as when the eyes are closed.

physiology↗

bioRxiv2020

KDM2B promotes cell viability by enhancing DNA damage response in canine hemangiosarcoma

Epigenetic regulators have been implicated in tumorigenesis of many types of cancer; however, their roles in endothelial cell cancers such as canine hemangiosarcoma (HSA) have not been studied. In this study, we found that lysine-specific demethylase 2B (Kdm2b) was highly expressed in HSA cell lines compared to normal canine endothelial cells. Silencing of Kdm2b in HSA cells resulted to increased cell death in vitro compared to the scramble control by inducing apoptosis through the inactivation of the DNA repair pathways and accumulation of DNA damage. Similarly, doxycycline-induced Kdm2b silencing in tumor xenografts resulted to decreased tumor sizes compared to the scramble control. Furthermore, Kdm2b was also highly expressed in clinical cases of HSA, and its expression levels was higher than in hemangioma, a benign counterpart of HSA. Based on these results, we hypothesized that pharmacological Kdm2b inhibition can also induce HSA cell death and can be used as an alternative treatment for HSA. We treated HSA cells with GSK-J4, a histone demethylase inhibitor, and found that GSK-J4 treatment also induced apoptosis and cell death. On top of that, GSK-J4 treatment in HSA tumor-bearing mice decreased tumor sizes without any obvious side-effects. In this study, we demonstrated that Kdm2b acts as an oncogene in HSA by enhancing DNA damage response and can be used as a biomarker to differentiate HSA from hemangioma. Moreover, we indicated that histone demethylase inhibitor GSK-J4 can be used as a therapeutic alternative to doxorubicin for HSA treatment.

cancer biology↗