Biology subjects

Fujii, T.

Publications and source records attributed to Fujii, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Microbead-Based Heterodimer Assay for Amplification-Free Detection of Single-Stranded DNA

Direct detection of rare nucleic-acid targets without molecular amplification requires specific recognition events to be made observable while remaining distinguishable from nonspecific interactions. Here, we introduce a microbead-based heterodimer assay (MBHA) that converts two-site recognition of single-stranded DNA into spatially resolved, two-color bead-pairing events for direct nucleic acid detection. Two spectrally distinct microbead populations are functionalized with partially double-stranded DNA probes exposing target-binding domains complementary to separate regions of the target. Target-mediated bridging produces heterotypic bead pairs that are classified by fluorescence identity and an interparticle-distance criterion, while residual background is evaluated relative to blank measurements. Under the tested conditions, 166 fM was the lowest target concentration exceeding the blank-derived detection threshold. A sequence-scrambled synthetic non-target DNA remained below threshold across the tested concentration range, and detection at 166 fM was retained in the presence of 1 uM non-target DNA, corresponding to an approximately six-million-fold molar excess. Probe redesign further extended the assay to a second 60-nucleotide synthetic DNA target derived from the SARS-CoV-2 ORF1ab sequence. This proof-of-concept study establishes heterodimer counting as an amplification-free measurement strategy in which rare molecular-recognition events are made directly observable and individually classifiable without increasing target copy number.

biophysics↗

bioRxiv2026

Prior Bombyx mori latent virus exposure enhances subsequent BmNPV infection and suppresses antiviral responses in the silkworm, Bombyx mori

Bombyx mori latent virus (BmLV) is a persistent RNA virus frequently detected in silkworm-derived cell lines and has been reported to interact with B. mori nucleopolyhedrovirus (BmNPV). However, whether previous BmLV exposure alters subsequent BmNPV infection in silkworm larvae remains unclear. Here, we examined how BmLV exposure history affects subsequent BmNPV infection in cultured cells and fifth-instar B. mori larvae. Persistent BmLV infection increased BmNPV-mediated reporter protein expression and BmNPV genome abundance in cultured cells, whereas short-term BmLV exposure or simultaneous BmLV and BmNPV coinfection did not reproduce the increases observed in persistently BmLV-positive cultures. In silkworm larvae (n22 strain), 24 h BmLV pre-exposure before BmNPV challenge increased BmNPV-mediated reporter protein expression and viral genome accumulation compared with BmNPV infection alone. BmLV antigenome RNA was detected in multiple larval tissues after BmLV exposure, indicating BmLV replication during the pre-exposure period. Notably, this prior BmLV exposure was associated with altered antiviral responses upon subsequent BmNPV infection. Specifically, BmLV-pre-exposed larvae showed reduced induction of Dicer-2, R2D2, and stimulator of interferon genes (STING) in the fat body. These findings indicate that prior BmLV exposure enhances subsequent BmNPV infection and is associated with attenuated induction of host antiviral genes. This study expands our understanding of how sequential viral infections shape host-virus interactions and suggests a novel approach for increasing recombinant protein production in baculovirus-based expression systems.

microbiology↗

bioRxiv2026

DVT-primed neutrophils reshape the brain microenvironment to promote brain metastasis

Brain metastasis (BrM) is a devastating complication of triple-negative breast cancer (TNBC), yet how host macroenvironmental conditions influence brain metastatic susceptibility remains poorly understood. Here, we investigated the impact of deep vein thrombosis (DVT), a common complication in patients with cancer, on TNBC brain metastasis. Analysis of a large clinical cohort identified DVT as an independent risk factor for BrM in patients with metastatic breast cancer. Using two syngeneic TNBC models, we demonstrated that DVT selectively enhanced brain metastasis without significantly affecting primary tumor growth or lung metastasis. Mechanistically, DVT increased neutrophil accumulation in the brain metastatic microenvironment, and neutrophil depletion completely abrogated the metastasis-promoting effect of DVT. Single-cell RNA sequencing of CD45+ cells in peripheral blood revealed that DVT reprogrammed circulating neutrophils toward migratory and inflammatory transcriptomic profiles characterized by neutrophil extracellular trap (NET) formation. DVT increased CXCR2 expression on circulating neutrophils and enhanced neutrophil recruitment in brain. Moreover, DVT significantly increased circulating NETs. Collectively, our findings identify DVT as a systemic driver of TNBC brain metastasis and reveal that DVT primes circulating neutrophils toward enhanced vascular recruitment and NET formation, providing a mechanistic link between cancer-associated thrombosis and brain metastatic susceptibility.

molecular biology↗

bioRxiv2026

SOX2 terminates trophectoderm competence in inner cell mass by closing trophectoderm enhancers

During embryonic development, cellular competence to respond to differentiation signals changes dynamically. Although the mechanisms underlying competence acquisition have been extensively studied, those underlying competence loss remain unclear. In preimplantation mouse embryos, Hippo signaling shifts from regulating trophectoderm (TE) fate specification to promoting epiblast maturation. During the blastocyst stage, inner cell mass (ICM) cells lose TE competence in response to the Hippo signaling effector TEAD-YAP. Here, we show that the pioneer factor SOX2 terminates TE competence in the ICM. SOX2 binding to the TEAD-YAP-dependent TE enhancer (TEE) of the TE regulator Gata3 induces chromatin closure, suppressing TEE responsiveness to TEAD-YAP activity. This function of SOX2 requires its interaction with the corepressor TLE4 and histone deacetylase. Similar SOX2-dependent chromatin closure also occurs around other TE genes, including the TE enhancer of another TE regulator, Cdx2. Thus, SOX2 terminates TE competence in ICM cells by closing Hippo signaling-responsive enhancers.

developmental biology↗

bioRxiv2026

Dysregulation of the p16-CDK6 axis characterizes HPV-unrelated p16-positive oropharyngeal carcinoma

Human papillomavirus (HPV)-related and HPV-unrelated oropharyngeal squamous cell carcinomas (OPCs) are distinct entities with different clinical outcomes. While p16 immunohistochemistry (IHC) is widely used as a surrogate marker for HPV-driven OPC, a subset of HPV-unrelated OPCs also overexpress p16, and the biological basis of this discordance remains unclear. Here, we performed integrated clinicopathological, transcriptomic, genomic, and functional analyses of OPCs and demonstrated that dysregulation of the p16-CDK6 axis characterizes HPV-unrelated p16-positive OPCs. Although these tumors closely resembled HPV-unrelated p16-negative OPCs in their clinicopathological and transcriptomic characteristics, they exhibited a more favorable prognosis. CDK6 was recurrently upregulated in HPV-unrelated OPC regardless of p16 status and was already detectable in high-grade dysplastic leukoplakia, suggesting that CDK6 activation is an early event in HPV-unrelated tumorigenesis. In experimental models, CDK6 overexpression induced compensatory p16 upregulation, creating selective pressure for subsequent CDKN2A inactivation. Consistent with this model, homozygous CDKN2A loss predominated in p16-negative tumors. We further identified CDKN2A frameshift mutations generating p14ARF-p16 chimeric proteins that retain p16 immunoreactivity despite functional loss of wild-type p16, revealing a previously unrecognized diagnostic pitfall of p16 IHC. These findings provide a biological framework for p16 overexpression in HPV-unrelated OPC and suggest that assessment of the p16-CDK6 axis may refine molecular classification and risk stratification beyond p16 IHC alone.

cancer biology↗

bioRxiv2026

The Il1r2-CreERT2 knock-in mouse enables inducible labeling of slow-cycling epidermal basal cells and genetic ablation of the IL-1 decoy receptor

The interfollicular epidermis is maintained by spatially organized basal cell populations with distinct molecular signatures and division kinetics; however, the markers that define these populations remain poorly defined. In this study, we identified Il1r2, which encodes the IL-1 decoy receptor IL-1R2, as a marker of the slow-cycling basal population in the epidermis. Single-cell RNA-seq analysis of epidermal and hair follicle basal populations in the murine tail skin revealed that Il1r2 is preferentially expressed in the slow-cycling epidermal basal population, and immunofluorescence staining confirmed its protein localization in tissues. To enable fate mapping of this population, we generated an Il1r2-CreERT2 knock-in mouse line using a CRISPR-Cas9-based PITCh method. Tamoxifen induction in Il1r2-CreERT2/Rosa-tdTomato mice exhibited selective labeling of basal cells localized to the slow-cycling interscale region of the tail epidermis. Because the CreERT2 cassette was inserted into the Il1r2 coding sequence, homozygous Il1r2-CreERT2 knock-in mice can also serve as an Il1r2 knockout model through targeted gene ablation. Thus, the Il1r2-CreERT2 mouse line provides a dual genetic tool for lineage tracing of the slow-cycling epidermal basal population and for functional modulation of IL-1 signaling in vivo.

developmental biology↗

bioRxiv2026

Phytochemical Modulation of Astrocyte A1/A2 Polarization and Hepcidin-Associated Iron Dysregulation in LPS-Driven Neuroinflammation

Background/ObjectivesNeuroinflammation-driven iron dysregulation and neurotoxic astrocyte polarization are increasingly recognized as interconnected pathological mechanisms in neurodegenerative diseases. Systemic inflammation triggered by strenuous exercise or infection can engage the central nervous system and astrocytic inflammatory responses and perturb iron homeostasis; however, targeted nutritional strategies to counteract these processes remain limited. Inflamate(R) is a multi-component botanical supplement comprising boswellic acids, astilbin, xanthohumol, and cinnamaldehyde, each with documented anti-inflammatory properties. However, whether this combined formulation can modulate the inflammatory-iron metabolic axis and astrocyte phenotypic polarization remains unexplored. This study aimed to investigate the effects of Inflamate(R) on LPS-induced pro-inflammatory gene expression, iron metabolism-related gene regulation, and A1/A2 astrocyte phenotypic polarization in mouse astrocytes. MethodsMouse astrocytes (AWT) were pre-treated with Inflamate(R) (0.0375 g/mL) or DMSO vehicle for 24 h, followed by lipopolysaccharide (LPS; 1 g/mL) stimulation for an additional 24 h. The non-cytotoxic working concentration was determined by morphological assessment, CCK-8 cell viability, and LDH cytotoxicity assays. Expression of 14 target genes spanning pro-inflammatory mediators (NOS2, IL6, C3, COX2, PLA2g15, SOCS3), iron metabolism regulators (FTH1, Hepcidin, TFRC, SLC40A1, RGMa, RGMb), and astrocyte polarization markers (S100A10, GFAP) was quantified by qRT-PCR. ResultsUnder normal culture conditions, Inflamate(R) did not significantly alter the expression of any target gene except S100A10, confirming the absence of baseline cytotoxicity or transcriptional homeostatic perturbation. Upon LPS stimulation, Inflamate(R) selectively suppressed NOS2 (approximately 64% reduction, p < 0.0001), IL6 (approximately 37% reduction, p < 0.0001), and C3 (approximately 47% reduction, p < 0.0001), while COX2, PLA2g15, and SOCS3 remained unaffected. Concurrently, Inflamate(R) significantly reduced LPS-induced Hepcidin expression to approximately 17% of the control level (p < 0.05) and attenuated FTH1 upregulation (p < 0.01), without altering the expression of iron transporters (TFRC, SLC40A1) or BMP-SMAD pathway components (RGMa, RGMb). Furthermore, Inflamate(R) upregulated the neuroprotective A2 marker S100A10 under both basal (p < 0.05) and LPS-stimulated conditions (p < 0.01), while the general reactivity marker GFAP remained unchanged. ConclusionsInflamate(R) exerts a selective, multi-target modulatory effect at the transcriptional level in LPS-stimulated astrocytes, encompassing suppression of the iNOS-NO and IL-6 signaling axes, attenuation of inflammation-driven hepcidin-ferritin iron dysregulation via the IL-6-STAT3 pathway, and promotion of a phenotypic shift from neurotoxic A1 toward neuroprotective A2 astrocyte polarization. Given that the IL-6-JAK-STAT3-hepcidin axis is also activated during exercise-induced systemic inflammation, these findings suggest that Inflamate(R) may represent a targeted nutritional strategy for preserving CNS iron homeostasis and supporting neuroprotective astrocyte function in both neurodegenerative and exercise-related neuroinflammatory contexts. Further validation in in vivo neurodegenerative and exercise models, including protein-level analyses, is warranted to confirm these transcriptional findings.

neuroscience↗

bioRxiv2026

Minute-scale coupling of chromatin marks and transcriptional bursts

Histone modifications are often described as stable epigenetic marks that contribute to maintaining gene-expression programs during development and environmental responses 1-5. However, transcription of many genes is intermittent, switching between transcriptionally active and inactive episodes within minutes 6-10. Whether chromatin marks around individual genes change on these rapid timescales remains unclear. Here we show that local chromatin modification signals around endogenous genes in mouse embryonic stem cells fluctuate reversibly with transcriptional state, using live imaging of individual genes together with fluorescent probes that report histone modifications11-16. Activation-associated acetylation and methylation marks increased in association with transcriptional activation and decreased with inactivation, whereas a Polycomb-associated repressive mark behaved oppositely. Transcriptional coactivators and both histone acetyltransferase and deacetylase complexes were enriched during transcriptionally active state, consistent with opposing enzymatic activities shaping local acetylation levels 17,18. Inhibiting histone deacetylases altered the durations of active and inactive events, supporting a role for deacetylation in regulating transcriptional state transitions. Thus, histone modifications undergo reversible, minute-scale changes coupled to transcriptional activity. This framework helps explain how stochastic transcriptional bursts can occur with stable gene regulation over longer timescales.

molecular biology↗

bioRxiv2025

Efficient gene disruption with CRISPR-Cas3 in human T cells

The CRISPR-Cas9 system has been widely adopted as a genome editing tool due to its high efficiency and versatility, contributing to the development of various therapeutic strategies. However, its clinical application remains limited by safety concerns, including off-target effects and large-scale chromosomal rearrangements such as translocations and inversions. Recently, the CRISPR-Cas3 system, a Class 1 CRISPR effector complex with unidirectional DNA degradation activity, has gained attention as a potential alternative, offering reduced off-target activity. In this study, we applied the CRISPR-Cas3 system to human T cells and successfully disrupted two clinically relevant genes, T cell receptor alpha constant (TRAC) and beta-2 microglobulin (B2M). These gene deletions were associated with a reduction in both graft-versus-host disease (GVHD) risk and host immune rejection. Importantly, no off-target mutations were detected in CRISPR-Cas3-edited cells, in contrast to the off-target effects observed with CRISPR-Cas9. Furthermore, CAR-T cells generated by deleting TRAC or B2M using CRISPR-Cas3 maintained their antigen-specific cytotoxicity against tumor cells, while exhibiting reduced alloreactivity. These results suggest that CRISPR-Cas3 provides a safer and promising platform for genome editing in T cell engineering, with potential applications in the development of next-generation allogeneic T cell therapies.

cancer biology↗

bioRxiv2025

In vivo efficacy of fidaxomicin against rpoB mutant Clostridioides difficile infection.

ObjectivesClostridioides difficile infection (CDI) is a well-known healthcare-associated diarrheal disease. Fidaxomicin, a key antibiotic used to treat CDI, targets rpoB. However, some clinical isolates have mutations in rpoB, which reduces their susceptibility to this antibiotic. In this study, the effects of rpoB mutations on the virulence of C. difficile and efficacy of fidaxomicin against CDI were evaluated in vivo. MethodsAn rpoB mutant strain (C. difficile G1073R-2024) with reduced fidaxomicin susceptibility was generated through spontaneous induction in a murine CDI model from the parental strain C. difficile VPI 10463. The virulence and therapeutic responses of the mutant strain were compared with those of the parental strain using a CDI model, including survival rate, body weight changes, clinical scores, and bacterial loads in feces. ResultsC. difficile G1073R-2024 had an amino acid alteration in Gln1073Arg and the minimum inhibitory concentration of fidaxomicin was >64 g/mL. In vivo virulence was not significantly different between strains. Fidaxomicin treatment resulted in 100% survival rates and a comparable reduction in the bacterial load for both strains. ConclusionsFidaxomicin was effective against CDI caused by the rpoB mutant strain. The emergence of such mutations highlights the need for ongoing surveillance of drug resistance trends in clinical settings.

microbiology↗

bioRxiv2024

Nemo knows: clownfishes differentiate cryptic host species across fine and broad geographic scales and reveal a potential adaptive radiation in the clownfish-hosting sea anemones.

The symbiosis between clownfishes (or anemonefishes) and their host sea anemones ranks among the most recognizable animal interactions on the planet. Found on coral reef habitats across the Indian and Pacific Oceans, 28 recognized species of clownfishes adaptively radiated from a common ancestor to live obligately with only 10 nominal species of host sea anemones. Are the host sea anemones truly less diverse than clownfishes? Did the symbiosis with clownfishes trigger a reciprocal adaptive radiation in sea anemones, or minimally, a co-evolutionary response to the mutualism? To address these questions, we combined fine- and broad-scale biogeographic sampling with multiple independent genomic datasets for the bubble-tip sea anemone, Entacmaea quadricolor--the most common clownfish host anemone throughout the Indo-West Pacific. Fine-scale sampling and restriction site associated DNA sequencing (RADseq) throughout the Japanese Archipelago revealed three highly divergent cryptic species: two of which co-occur throughout the Ryukyu Islands and can be differentiated by the clownfish species they host. Remarkably, broader biogeographic sampling and bait-capture sequencing reveals that this pattern is not simply the result of local ecological processes unique to Japan, but part of a deeper evolutionary signal where some species of E. quadricolor serve as host to the generalist clownfish species Amphiprion clarkii and others serve as host to the specialist clownfish A. frenatus. In total, we delimit at least five cryptic species in E. quadricolor that have diversified within the last five million years. The rapid diversification of E. quadricolor combined with functional ecological and phenotypic differentiation supports the hypothesis that this may represent an adaptive radiation in response to mutualism with clownfishes. Our data indicate that clownfishes are not merely settling in locally available hosts but recruiting to specialized host lineages with which they have co-evolved. These findings have important implications for understanding how the clownfish-sea anemone symbiosis has evolved and will shape future research agendas on this iconic model system.

evolutionary biology↗

bioRxiv2024

Intervening to preserve function in ischemic cardiomyopathy with a porous hydrogel and extracellular matrix composite in a rat myocardial infarction model

A variety of hydrogels have been developed for intramyocardial injection therapy after myocardial infarction. Some of these biomaterials have incorporated bioactive substances that promote local tissue regeneration and integration, while others have emphasized the mechanical role of the injectate in providing functional benefit. In this study, we incorporated two bioactive features, porosity, and extracellular matrix derived hydrogel (ECM), into a mechanically optimized, thermoresponsive, degradable hydrogel (poly(N-isopropylacrylamide-co-N-vinylpyrrolidone-co-MAPLA) copolymer) and evaluated whether injection of this biomaterial could abrogate the remodeling process in a rat ischemic cardiomyopathy model. After myocardial infarction by coronary artery ligation, rats were randomly divided into four groups: group NP (non-porous hydrogel) without either ECM or porosity, group PM (porous hydrogel) from the same synthetic copolymer with mannitol beads as porogens, and group PME with porosity and ECM digest added to the synthetic copolymer. A group with PBS injection alone served as a control. Intramyocardial injections were made 3 days after myocardial infarction. Serial echocardiography was assessed over time, and histological assessments were performed eight weeks after infarction. Results demonstrated improved echocardiographic function and neovascularization in the PME group compared to the other hydrogels and PBS injection. The PME group also demonstrated significant improvement in LV geometry and macrophage polarization (towards M2) compared to the PBS group, whereas these differences were not observed in the NP or PM groups versus the control. These results demonstrate that further functional improvement may be achieved in hydrogel injection therapy for ischemic cardiomyopathy by incorporating porosity and ECM digest, representing a combination of mechanical and biological effects.

bioengineering↗

bioRxiv2024

Identification of Large Japanese field mouse Apodemus speciosus food plant resources in an industrial green space using DNA metabarcoding

DNA metabarcoding was employed to identify the food plant resources of the Large Japanese field mouse Apodemus speciosus, inhabiting an artificial green space on reclaimed land on the Chita Peninsula in Aichi Prefecture, Central Japan, from 2012 to 2014. DNA metabarcoding was performed using high-throughput sequencing of partial rbcL sequences extracted from feces samples collected in the study area. The obtained sequences, which were analyzed using a constructed local database, revealed that a total of 72 plant taxa were utilized as food plant resources by A. speciosus. Of these plant taxa, 43 could be assigned to species (59.7%), 16 to genus (22.2%), and 13 to family (18.1%). Of the 72 plant taxa identified in this study, the dominant families throughout all collection periods were Lauraceae (81.0% of 100 fecal samples), followed by Fagaceae (70.0%), Rosaceae (68.0%), and Oleaceae (48.0%). Fifty of the 72 plant taxa identified as food plant resources were woody plants. An analysis employing rarefaction techniques for each season in the study site indicated comprehensive coverage of the food plant resources, ranging from 86.4% in winter to 93.6% in spring. Further, 96.5% of the food plant taxa were found throughout the study period. The findings showed that DNA metabarcoding using a local database constructed from the National Center for Biotechnology Information (NCBI) database and field surveys was effective for identifying the dominant food plants in the diet of A. speciosus. The results of this study provided basic information that can be applied to formulation and implementation of management and conservation strategies for local wildlife.

molecular biology↗

bioRxiv2024

Changes in plant physiology during cultivation periods influence the hydrogen isotope ratio of the water-insect relationship

O_LIThe hydrogen stable isotope ratios ({delta}2H) in tissues of terrestrial insects are widely applied to estimate natal origins in field populations. The hydrogen isotopes of insect tissues incorporate those of environmental waters through the insects metabolic processes. Water sources and abiotic environmental factors reflect changes in plant physiology, as indicated by the {delta}2H values of plants. However, the influence of plant physiology on the assimilation of hydrogen in insect tissues derived from water through feeding diets is still unknown. C_LIO_LIWe experimentally examined the influence of water on the {delta}2H values of maize leaf and of forewings of Mythimna separata (Lepidoptera: Noctuidae) and Spodoptera frugiperda (Noctuidae). We prepared five specific water samples for cultivating maize, aiming to replicate the gradient of {delta}2H values observed in environmental waters across the Japanese archipelago. C_LIO_LIThe mean-percentage contribution of water to hydrogen in maize leaves was 17.6% (July-August) and 25.1% (September-October). Linear analyses indicated that 17.4% and 32.7% of hydrogen in M. separata and S. frugiperda forewings were derived from water through the consumption of maize leaves. The slope values of linear regression between the insect forewings and the maize leaves supplied in the final instar were closest to 1.0. These results indicated that the {delta}2H values of maize leaves and insect forewings reflected those of water resources. C_LIO_LIThis study presented the seasonal changes in climate conditions that affected the {delta}2H values of host plants and insect tissues. Changes in plant physiology with seasonal variations may influence the interpretation of the linear relationship between water and insect tissues on the estimation of natal origins. C_LI

ecology↗

bioRxiv2024

High-throughput screening for Cushing's disease: therapeutic potential of thiostrepton via cell cycle regulation

Cushings disease is a life-threatening disorder caused by autonomous secretion of adrenocorticotropic hormone (ACTH) from pituitary neuroendocrine tumors (PitNETs). Few drugs are indicated for inoperative Cushings disease, in particular that due to aggressive PitNETs. To explore agents that regulate ACTH-secreting PitNETs, we conducted high-throughput screening (HTS) using AtT-20, a murine pituitary tumor cell line characterized by ACTH secretion. For the HTS, we constructed a live cell- based ACTH reporter assay for high-throughput evaluation of ACTH changes. This assay was based on HEK293T cells overexpressing components of the ACTH receptor and a fluorescent cAMP biosensor, with high-throughput acquisition of fluorescence images at the single-cell level. Of 2480 screened bioactive compounds, over 50% inhibition of ACTH secreted from AtT-20 cells was seen with 84 compounds at 10 M, and 20 compounds at 1 M. Among these hit compounds, we focused on thiostrepton (TS) and determined its antitumor effects in both in vitro and in vivo xenograft models of Cushings disease. Transcriptome and flow cytometry analyses revealed that TS administration induced AtT-20 cell cycle arrest at the G2/M phase, which was mediated by FOXM1-independent mechanisms including downregulation of cyclins. Simultaneous TS administration with a CDK 4/6 inhibitor that affected the cell cycle at the G0/1 phase showed cooperative antitumor effects. Thus, TS is a promising therapeutic agent for Cushings disease. Our list of hit compounds and new mechanistic insights into TS effects serve as a valuable foundation for future research.

cell biology↗

bioRxiv2023

Transcription-coupled changes in higher-order genomic structure and transcription hub viscosity prolong enhancer-promoter connectivity

The orchestration of our genes heavily relies on a coordinated communication between enhancers and promoters, yet how this dynamic interplay remains elusive while transcription is active. Here, we investigated enhancer-promoter (E-P) interactions and relative to transcriptional bursting in mouse embryonic stem cells using sequential DNA/RNA/immunofluorescence (IF)-FISH analyses and computational simulations. Our data reveal that the active state of specific genes is characterized by higher-order genomic structures and local condensates of transcriptional regulatory factors, leading to an elevation in local viscosity that highly stabilizes the duration of E-P interactions. Our study underscores the pivotal role of viscosity in transcriptional dynamics and paves the way for a more nuanced understanding of gene-specific regulatory mechanisms.

molecular biology↗

bioRxiv2023

Transcriptomic Landscape of Hyperthyroidism in Mice Overexpressing Thyroid Stimulating Hormone

Hyperthyroidism is a condition with excessive thyroid hormone secretion. Activation of thyroid stimulating hormone receptor (TSHR) fundamentally leads to hyperthyroidism. The details of TSHR signaling remain to be elucidated. We conducted transcriptome analyses for hyperthyroid mice that we generated by overexpressing TSH. TSH overexpression via hydrodynamic gene delivery with pLIVE-TSHB and pLIVE-CGA vectors consistently caused hyperthyroidism and goiters for at least 4 weeks in C57BL/6J mice. RNA sequencing analysis of their thyroid glands revealed that thiamazole slightly changed the thyroid transcriptome, which reinforces a conventional theory that thiamazole decreases thyroid hormone secretion via inhibition of thyroid peroxidase activity. Meanwhile, TSH overexpression drastically changed the thyroid transcriptome. In particular, enrichment analyses identified the cell cycle, phosphatidylinositol-3 kinase/Akt pathway, and Ras-related protein 1 pathway as possibly associated with goiter development. Regarding the role of TSHR signaling in hyperthyroidism, it is noteworthy that Slc26a4 was exclusively upregulated among genes crucial to thyroid hormone secretion at both 1 and 4 weeks after hydrodynamic gene delivery. To verify the relationship between this upregulation and hyperthyroidism, we overexpressed TSH in Slc26a4 knockout mice. TSH overexpression caused hyperthyroidism in Slc26a4 knockout mice, equivalent to that in control mice. To summarize, we analyzed hyperthyroid mice generated by TSH overexpression. We did not observe significant changes in known genes and pathways involved in thyroid hormone secretion. Thus, our datasets might include candidate genes that have not yet been identified as regulators of thyroid function. Our transcriptome datasets regarding hyperthyroidism can contribute to future research on TSHR signaling.

physiology↗

bioRxiv2023

Precise immunofluorescence canceling for highly multiplexed imaging capturing specific cell state

Cell states are regulated by the response of signaling pathways to receptor ligand-binding and inter-cellular interactions. High-resolution imaging has been attempted to explore the dynamics of these processes and, recently, multiplexed imaging has profiled cell states by achieving a comprehensive acquisition of spatial protein information from cells. However, the specificity of antibodies is still compromised when visualizing activated signals. Here, we developed Precise Emission Canceling Antibodies (PECAbs) that have cleavable fluorescent labeling. PECAbs enable high-specificity sequential imaging using hundreds of antibodies, allowing for reconstruction of the spatiotemporal dynamics of signaling pathways. Additionally, combining this approach with seq-smFISH can effectively classify cells and identify their signal activation states in human tissue. Overall, the PECAb system can serve as a comprehensive platform for analyzing complex cell processes.

cell biology↗