Biology subjects

Suzuki, M.

Publications and source records attributed to Suzuki, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Single-cell map of the female brain across reproductive transitions

Ovarian hormone shifts enable reproduction and are associated with substantial brain plasticity and disease risks. While imaging studies provide (micro)structural insights into brain changes across the ovarian cycle and pregnancy, the high resolution, single-cell map of the brain across reproductive transitions is missing. Here, we performed multiome (gene expression and chromatin accessibility) analysis of the mouse ventral hippocampus (vHIP) across sex, estrous cycle, and peripartum period at single cell resolution. We identify dynamic changes in vHIP cellular composition across the estrous cycle and pregnancy, including in the neural stem cells of the dentate gyrus (DG), enabling hormone-driven neurogenesis. Major gene expression changes are neuronal function-relevant, cell type-specific, and found in excitatory neurons of CA1, CA3, and DG subfields, across sex and reproductive transitions. In contrast, chromatin accessibility changes are more extensive and found across cell types, likely driven by estrogen level shifts in both within-female and between-sex comparisons. We show that chromatin remodeling during the estrous cycle primes the genome for gene expression changes during pregnancy and is also enriched for brain disease-relevant genes. Finally, we reveal a thyroid hormone transporter (Transthyretin, Ttr) gene as the major candidate gene that drives structural and behavioral changes across the estrous cycle and pregnancy. Our study provides an extensive cellular and molecular view of how reproductive transitions shape the brain and opens the possibility to target downstream targets of estrogen, including thyroid hormone signaling, as a treatment option for hormone-sensitive periods in women.

neuroscience↗

bioRxiv2025

L-Phenylalanine restriction amplifies boron neutron capture therapy efficacy through increased L-boronophenylalanine uptake and induces activating transcription factor 4 stress response in tumor cell lines

Boron neutron capture therapy (BNCT) relies on the selective uptake of boron-10 compounds by tumor cells. L-boronophenylalanine (BPA) serves as a key carrier, and enhancing its accumulation is critical for improving BNCT efficacy. In this study, we investigated the effects of L-phenylalanine (Phe) restriction on the tumor cell lines SAS, U87-MG, PANC-1, and A375, and the immortalized keratinocyte line HaCaT on modulating BPA uptake and associated cellular responses. Quantitative analysis using inductively coupled plasma atomic emission spectroscopy (ICP-AES) showed that 24 h Phe restriction increased BPA uptake in the SAS, U87-MG, and PANC-1 cell lines. Colony formation assays confirmed enhanced sensitivity to neutron irradiation in these cells. RNA sequencing indicated that Phe restriction activated the integrated stress response downstream of activating transcription factor 4 (ATF4), although this pathway was not directly linked to increased BPA uptake. The L-type amino acid transporter 1 (LAT1)/4F2 heavy chain (4F2HC) complex was identified as the exclusive transport route for BPA. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that Phe restriction altered intracellular levels of amino acids that serve as LAT1/4F2HC exchange substrates, suggesting a metabolic basis for enhanced BPA transport. Our results reveal that Phe restriction enhances BPA uptake and BNCT efficacy in a cell line-dependent manner, likely through the modulation of amino-acid metabolism. Therefore, targeted amino-acid manipulation prior to BNCT may represent a promising strategy to improve therapeutic outcomes.

cancer biology↗

bioRxiv2025

Axis-specific neuro-musculoskeletal contributions to postural stability and vibration avoidance in mice

Animals must appropriately regulate their responses to external mechanical oscillations such as whole-body (WB) vibration to ensure postural stability. However, knowledge of mouse responses to WB vibration remains limited. Here, we used high-speed video-based quantitative analyses to characterize body-part movements in awake and anesthetized mice under vertical, longitudinal, and lateral WB vibration. Awake mice exhibited lower resonance frequencies and smaller displacement amplitudes than anesthetized mice during vertical and lateral WB vibration, but not longitudinal vibration, indicating that the neuro-musculoskeletal (NMS) system contributes to postural stability in a vibration axis-dependent manner. Vibration modeling suggests that the NMS system acts as an active vibration absorber by reducing effective stiffness and dynamically adjusting effective damping, analogous to active vehicle suspensions. Axis-specific vibration control may reflect an evolutionary balance between postural stability and locomotor efficiency in quadrupeds, paralleling the design principles of rear-wheel-drive vehicles. Behavioral preference tests revealed that mice selectively avoid vertical WB vibration at specific frequencies, but neither lateral nor longitudinal WB vibration. These findings reveal an axis-specific NMS control principle analogous to vehicle design, linking postural regulation, locomotor efficiency and avoidance behavior, and advancing our understanding of the biomechanics and behavior of quadrupeds exposed to WB vibration.

biophysics↗

bioRxiv2025

Continuous Flash Suppression responses in mouse visual cortex: stimulus laterality and anesthesia effects

We investigated whether binocularly conflicting stimuli adapted from primate binocular rivalry studies could induce binocular response suppression in mouse visual cortex. We presented binocularly conflicting stimuli adapted from the paradigm of continuous flash suppression to awake and anesthetized mice and examined neuronal responses in visual areas V1 and LM. Neurons often showed a preference for the left or right eye where a monocular grating was presented, and their responses were modulated by presenting a conflicting flashing Mondrian stimulus to the other eye. The direction of this modulation depended on whether neurons preferentially responded to contralateral or ipsilateral gratings. Responses of cells with a preference for ipsilateral gratings were suppressed during binocular conflict, while contralateral-preferring cells were less suppressed or even enhanced by binocular conflict. Binocular conflict modulation continued to occur under anesthesia, but response recovery after intermittent binocular conflict only occurred in awake mice. Responses to Mondrians were modulated by binocular conflict in a similar fashion as those to grating stimuli, which suggests that mouse binocular conflict processing exhibits a distinctive dependency on stimulus laterality over other stimulus features. Finally, a canonical binocular rivalry model could be successfully fitted to our data but lacked sufficient competitive inhibition and adaptation strength to create oscillatory activity under binocular conflict.

neuroscience↗

bioRxiv2025

Genetic background and transient prenatal disruption of vitamin A signaling determine susceptibility to airway hyperresponsiveness in mice

Airway structural changes and hyperresponsiveness (AHR), hallmarks of asthma, are crucially influenced by genetic variations and adverse exposures. While intrauterine environmental perturbations leading to dysfunctional lung development have been linked to adult pulmonary disease, still little is known about the developmental events leading to these postnatal abnormalities. Here, we provide evidence of genetic background playing a key role in this process. Using A/J and C57BL/6J mice known for their distinct susceptibility to AHR, we show that A/J but not C57BL/6J develop an aberrant airway smooth muscle (SM) program and AHR in adulthood when exposed transiently to a vitamin A/retinoic acid (RA)-disrupted intrauterine environment in vivo by a maternal BMS493 administration. Single nuclei multiomics analysis identified a subpopulation of mesenchymal cells that overactivated TGF{beta} targets in response to BMS selectively in A/J, but not C57BL/6J, embryonic lungs. These cells, localized to sites of airway SM initiation, exhibited robust BMS-mediated upregulation of SMAD2/3 targets, including regulators of SM program Pdgfra and Tnc, and showed stable cell proportions despite the marked transcriptional rewiring following RA disruption. These findings identify TGF{beta}-activating mesenchymal cells as a critical niche responsive to RA signaling and reveal how genetic background determines developmental susceptibility to micronutrient perturbations with long-term impact on airway function.

developmental biology↗

bioRxiv2025

Human Interleukin-4-Dependent Facilitation of Human IgG Production in PBL-NOG-hIL-4-Tg mice

Immune-humanized mice provide valuable experimental models for evaluating immune-based therapies, yet the induction of human antigen-specific IgG production remains limited due to species-specific incompatibilities. Our previous work demonstrated that NOG-hIL-4-Tg mice, which express human interleukin-4 (IL-4), support human T and B cell maintenance and enable antigen-specific IgG production following transplantation of human peripheral blood mononuclear cells (PBMCs). In this study, we investigated how IL-4 enhances antibody responses in this model. Flow cytometry and histological analysis revealed that human B cell maintenance was associated with specific plasma hIL-4 concentration ranges at supraphysiological levels. T and B cell receptor repertoire analysis using next-generation sequencing showed that clonal diversity remained largely conserved for one month and decreased to three months post-engraftment. Immunoglobulin repertoire profiling confirmed IgG class switching in an IL-4-concentration-dependent manner. Among the IgG subclasses, IgG3 increased during the first and second months and then decreased thereafter. In contrast, IgG1 tended to increase over time; however, the proportion of IgG subclass varied among individual donors. Following immunization with two distinct peptide antigens, the mice produced enhanced levels of antigen-reactive IgG. However, many B cell clones also exhibited weak responses to unrelated third-party antigens, likely reflecting insufficient affinity maturation. Histological evaluation showed tertiary lymphoid structure (TLS)-like accumulations of B and T cells in the spleen, although fully developed germinal centers were absent. Taken together, these findings demonstrate that NOG-hIL-4-Tg mice maintain human B cells within a regulated IL-4 environment, promote early formation of splenic tertiary lymphoid structures, and support IgG production characterized by clonal expansion, class switching, and somatic hypermutation. These results confirm that human IL-4 expression supports human antibody responses in the PBL-NOG-hIL-4-Tg mouse system. (278)

immunology↗

bioRxiv2025

High-content live-cell time-lapse imaging predicts cells about to die via apoptosis

Cell death is a dynamic process that unfolds through time. Live-cell time-lapse imaging captures these dynamics in a way thats impossible for static snapshots. High-content imaging (HCI), which has been developed for static microscopy, applied to time-lapse imaging can quantify how single-cell states change through time. Here we show the ability of high-content live-cell time-lapse imaging (HCLTI) to quantify the onset and progression of one form of cell death called apoptosis. We apply the Live Cell Painting assay called ChromaLIVETM and develop an HCLTI analysis pipeline. We show that HCLTI can discern the morphology dynamics of cells undergoing apoptosis, and demonstrate that machine learning can predict apoptosis as early as 100 minutes after exposing HeLa cells to the apoptosis inducer Staurosporine. This technical advancement paves the way for future studies to better understand the dynamics of other forms of cell death. Understanding cell death dynamics is one piece of solving larger biomedical puzzles like understanding how cells resist death (e.g., therapeutic resistance of cancer cells) and how cells die too soon (e.g., neurodegeneration).

systems biology↗

bioRxiv2025

Highly Correlated Activity across Higher-Order Thalamic Nuclei in Awake and Anesthetized States

The thalamus is an egg-shaped structure deep inside the brain and is of central importance for our cognitive functions. The thalamus can be divided into first- and higher-order (HO) nuclei. Unlike first-order thalamic regions which are known to primarily relay sensory signals to the neocortex, the functions of HO thalamic nuclei remain far less clear. Although previous studies indicate HO thalamic nuclei influence cortical processing and consciousness, most studies examined single thalamic nuclei in isolation. To investigate the relation between distinct HO thalamic nuclei, we recorded local field potentials (LFP) and multi-unit activity (MUA) across multiple thalamic nuclei simultaneously in awake and anesthetized mice with sensory stimulation. The effect of voluntary locomotion was also studied in the awake state. Surprisingly, we found that both LFP and MUA were strongly correlated across HO nuclei in both states, which cannot be explained by volume conduction alone but suggests the existence of shared synaptic inputs. Furthermore, in the awake state locomotion and sensory stimuli activated many HO nuclei, including those that have not classically been regarded as sensory or motor. These findings challenge the classical view of the thalamus being the aggregate of isolated, independent nuclei and suggest that the thalamus serves as an ideal bottleneck whose coordination resulted in effective orchestration of the entire brain.

neuroscience↗

bioRxiv2025

Plasmid-mediated macrolide resistance among rapidly growing mycobacteria in Japan

ObjectivesThe spread of a transmissible plasmid carrying the 23S rRNA methylase gene erm(55), which confers inducible macrolide resistance in rapidly growing mycobacteria (RGM), has raised significant clinical concerns. The aim of this study was to investigate the prevalence of erm(55)-carrying plasmids in clinically isolated RGM strains in Japan. MethodsIn total, 607 RGM clinical isolates, representing 32 species or complexes, collected between 2019 and 2023 in Japan were examined. To detect the presence of erm(55)-carrying plasmids, we conducted PCR screening, minimum inhibitory concentration testing for clarithromycin, and whole-plasmid genome sequencing. Comparative genomic analyses were performed to characterise the plasmids. ResultsAmong the 607 RGM isolates, 0.8% (5/607) possessed the plasmid with the erm(55) gene and exhibited inducible macrolide resistance, with ratios of 100% (1/1) in Mycobacterium murale, 50% (3/6) in M. obuense, and 0.8% (1/125) in M. chelonae. The erm(55)-carrying plasmids ranged from 126,187 to 170,220 bp in size. Pairwise BLASTn comparisons of the erm(55)-carrying plasmids showed weighted percent identity values ranging from 99.5% to 99.9%, with query and subject coverage values ranging from 74.2% to 100%. All erm(55) sequences (813 bp) were identical and located within a horizontal gene transfer region. ConclusionsThis study confirmed the presence of macrolide-resistant RGMs related to the erm(55)-carrying plasmid in Japan, although the overall prevalence remains low. These findings emphasise the need to consider plasmid-mediated resistance when treating infections caused by the RGM species.

microbiology↗

bioRxiv2025

GLP1 receptor agonists attenuate systemic inflammation through microbiota mediated enrichment of Faecalibacterium prausnitzii and Roseburia spp

This study investigated the comparative anti-inflammatory effects of glucagon-like peptide-1 receptor agonists (GLP-1 RAs) and nonsteroidal anti-inflammatory drugs (NSAIDs), with a particular focus on microbiota-mediated mechanisms. A total of 120 participants were enrolled, including a GLP-1 RA group (n = 60) and an NSAID control group (n = 60). After 12 weeks of treatment, the GLP-1 RA group showed significant reductions in systemic inflammatory markers, with C-reactive protein (CRP) decreasing from 4.3 {+/-} 0.6 to 2.1 {+/-} 0.4 mg/L (p < 0.01), interleukin-6 (IL-6) decreasing from 3.6 {+/-} 0.5 to 1.8 {+/-} 0.3 pg/mL (p < 0.01), and tumor necrosis factor- (TNF-) decreasing from 18.2 {+/-} 1.5 to 12.5 {+/-} 1.2 pg/mL (p < 0.05). In contrast, the NSAID group exhibited only modest reductions in these markers without achieving normalization. Microbiome analysis revealed a significant post-treatment enrichment of Faecalibacterium prausnitzii (r = -0.46 with CRP, p < 0.001) and Roseburia spp. (r = -0.39 with CRP, p < 0.01) in the GLP-1 RA group, whereas NSAIDs showed no comparable effect. These findings establish a novel mechanistic link between incretin-based therapies and host immune homeostasis, highlighting the potential of GLP-1 RAs as systemic anti-inflammatory agents beyond their metabolic benefits.

immunology↗

bioRxiv2025

Atlas of innate immune responses to experimental cholera and IL22 treatment demonstrates protection by mucus-secreting cells

The diarrheal disease cholera remains a global threat, but there is limited knowledge of the innate immune defenses in the small intestine that protect against the causative agent, Vibrio cholerae. Here, single-cell RNA-sequencing of epithelial and immune cells mapped gene expression patterns in the infant mouse small intestine and revealed changes in response to V. cholerae infection and prophylactic treatment with an IL22 Fc-fusion protein. Infection increased the abundance of an enterocyte subtype with high expression of defense-associated functions and stimulated production of IL22, a cytokine linked to epithelial integrity, from group 3 innate lymphoid cells. Administration of IL22Fc increased production of vibriocidal Reg3{beta} from enterocytes and the abundance of secretory lineage and Muc2-producing goblet cells, which secreted mucus into the intestinal crypts, impairing V. cholerae association with the epithelium. These IL22-mediated responses limited V. cholerae intestinal colonization and protected mice from diarrhea and death. Our findings suggest enterocyte specialization in mucosal defense. SummarySingle cell studies uncovered specialization of epithelial cells in intestinal defense. The epithelial cell-targeting cytokine IL22 protected mice from cholera by promoting the genesis of mucus secreting cells.

microbiology↗

bioRxiv2025

A steady state pool of calcium-dependent actin is maintained by Homer and controls epithelial mechanosensation

Epithelial cells are inherently contractile and in homeostasis, tissue integrity is maintained by balancing the uneven contractile forces in neighboring cells at the cell-cell interface. By contrast, epithelial cells can utilize an imbalance in contractile force to communicate various information to induce tissue-wide response as in wound healing. Contractility is generated and processed at the apical junctional complex (AJC) by the dynamic behavior of the actin cytoskeleton. Calcium signaling can pattern cellular responses based on its reach and amplitude and the actin cytoskeleton is supported by its wide ranging effects on actin regulators. Calcium transients regulate various cell behaviors associated with actin remodeling, such as in damage response and developmental morphogenesis. Here we report that calcium maintains an adaptive pool of AJC-associated actin that is sensitive to tension and encoded by calcium dynamics. For this, the recently identified epithelial polarity module Homer-MUPP1/PatJ is required. Homer regulates calcium signaling in various tissue contexts through interaction with numerous components of the endoplasmic reticulum (ER) and plasma membrane (PM) calcium signal toolkit. Knockout of either Homer or MUPP1/PatJ attenuated tension-induced calcium response and severely disrupted wound healing migration, which is dependent on guidance input through AJC tension. We also show that Homer is integral to early embryonic neurodevelopment as its suppression causes failure of neural tube closure. Our findings highlight the critical role of localized calcium dynamics on AJC actin remodeling and cellular behavior, elucidating the means of tissue coordination through intercellular tension. Significance statementThis study uncovers a novel mechanism by which localized calcium dynamics at the apical junctional complex maintain an adaptive, tension sensitive pool of actin, regulated by the epithelial polarity scaffolds Homer and MUPP1/PatJ. Importantly, this mechanism operates without perturbing epithelial polarity, indicating a specific means to modulate tissue mechanics. By linking mechanical forces to localized calcium amplification, this module enables precise mechanosensation, coordinating collective behaviors such as epithelial wound healing and neural tube closure in Xenopus. These findings redefine our understanding of intercellular tension sensing in epithelial tissues and highlight the Homer-calcium signaling axis as a key driver of tissue morphogenesis and homeostasis, with far reaching implications for developmental biology, regenerative medicine, and neural tube defect pathogenesis.

cell biology↗

bioRxiv2025

Emergence of cell polarity by reciprocal interactions between Wnt and core PCP components

Planar cell polarity (PCP) is established by asymmetric localization of core PCP components in each cell. Since some Wnt proteins can induce PCP in vertebrates, it has been proposed that Wnt concentration gradients provide spatial cues for polarization. Here, however, we present evidence that Wnt11 can regulate PCP in a gradient-independent manner. In the neural plate of Xenopus embryos, endogenous Wnt11 does not form an obvious gradient in the direction of PCP, but is polarized at cell boundaries together with core PCP components. Wnt11 polarization is dependent on core PCP components, while polarization of core PCP components can also be induced by Wnt11, indicating a mutual amplification loop between Wnt11 and core PCP components in PCP formation. Furthermore, Wnt11 and core PCP components compose an intercellular loop to coordinate the direction of polarity. Thus, we propose that local and reciprocal interactions between Wnt11 and core PCP components can generate PCP.

developmental biology↗

bioRxiv2025

Dynamic polarization of heparan sulfate proteoglycans is involved in planar cell polarity and localization of endogenous Wnt11 during vertebrate neural tube formation.

Planar cell polarity (PCP) on a tissue plane is established by asymmetric polarization of core PCP components in individual cells. Recently, we found that Wnt11 organizes PCP in Xenopus embryos by accumulating locally with core PCP components to be polarized, but not by acting as a directional cue forming a global gradient. Since heparan sulfate proteoglycans (HSPGs) associate with Wnt on cell membranes, how HSPGs are involved in Wnt polarization and PCP formation is a central issue. Here, we show that HSPGs are dynamically organized in a polarized manner, as with core PCP components and Wnt11, by associating with them. While HSPGs with HS chains enriched by N-sulfation and N-deacetylation are polarized, depending on Wnt and core PCP components, NDST1, which catalyzes these modifications, is required for Wnt11 polarization and PCP formation. Thus, mutual regulation among HS chains, Wnt11, and PCP components appears to be essential for PCP formation. HighlightsO_LIndst1 is required for Wnt11 localization and proper PCP formation. C_LIO_LINDST1-modified HS chains are polarized in the neural plate during development. C_LIO_LIPolarization of NDST1-modified HS chains depends on PCP formation. C_LIO_LIPositive feedback loop results in polarized distribution of HSPGs, Wnt11, and PCP. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/669988v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@1db75adorg.highwire.dtl.DTLVardef@10aec47org.highwire.dtl.DTLVardef@1e50d5org.highwire.dtl.DTLVardef@4cd41c_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2025

Platform of single-molecule enzyme activity-based liquid biopsy for detection of pancreatic adenocarcinoma at early stages

Liquid biopsy analyzes biomolecules in biofluids to provide critical information for the early detection of diseases and the development of more effective treatment strategies. Among these biomolecules, the functional states of proteins offer particularly valuable insights due to their direct correlation with phenotypic changes. However, current approaches for assessing protein function in liquid biopsies are often limited by low detection sensitivity. Here, we present a liquid biopsy platform that analyzes single-molecule protease/peptidase activity to detect pathological alterations in enzyme function in blood samples. The platform demonstrates potential for identifying patients with early-stage (stage I-II) pancreatic ductal adenocarcinoma (PDAC).

biochemistry↗

bioRxiv2025

Benefits and costs of a hypercapsule and the mechanism of its loss in a clinical isolate of Acinetobacter baumannii

Acinetobacter baumannii is an opportunistic pathogen in which capsule production is closely linked to immune evasion and environmental persistence. Recent studies have described two seemingly contradictory phenomena--increasing prevalence of capsule-overproducing clinical isolates and frequent isolation of capsule-deficient variants. The biological significance of these phenomena remains unclear. In this study, we analyzed a clinical isolate, OCU_Ac16b, which spontaneously gives rise to two phenotypically distinct variants: the L type forming large colonies with an extremely thick capsule, and the S type forming small colonies with a substantially reduced or absent capsule. When L-type cells were cultured in test tubes under low-shaking conditions, S-type variants reproducibly emerged, constituting 45-80% of the population within 24 hours. Whole-genome sequencing revealed that this conversion is driven by distinct mutations in the capsular polysaccharide synthesis cluster, including insertion sequence (IS) insertions and a single-nucleotide deletion. PCR analysis confirmed that IS insertions in wzy (polysaccharide polymerase) occur within individual L-type colonies prior to liquid culture, providing a likely explanation for the rapid and reproducible emergence of S-type variants. Phenotypic characterization demonstrated a biological trade-off, with L-type cells exhibiting enhanced resistance to serum killing, desiccation, and certain {beta}-lactam antibiotics, whereas S-type cells showed superior surface attachment, increased biofilm formation, and a growth advantage under oxygen-limited conditions. Our findings uncover a highly reproducible, mutation-driven capsule switching mechanism that enables rapid phenotypic adaptation to changing environments. This phenotypic heterogeneity has significant implications for pathogenesis, persistence, diagnostic evaluation, and clinical management. ImportanceAcinetobacter baumannii is a clinically important opportunistic pathogen that exhibits striking phenotypic diversity. In particular, some clinical isolates produce unusually thick capsules, which are thought to contribute to immune evasion and persistence, while others lack capsule altogether. However, the biological significance of these contrasting phenotypes has remained unclear. In this study, we analyzed a clinical isolate that spontaneously gives rise to capsule-deficient variants from a hypercapsulated form. We found that the conversion is driven by spontaneous mutations in capsule biosynthesis genes that arise naturally within colonies, while the expansion of capsule-deficient cells is promoted under oxygen-limited conditions. The two variants differed in serum resistance, desiccation tolerance, growth characteristics, and antibiotic responses, revealing a trade-off between protective barriers and environmental adaptability. These findings provide new insights into how A. baumannii balances survival strategies through genetic and phenotypic heterogeneity, with important implications for diagnosis, treatment, and bacterial persistence in clinical settings.

microbiology↗

bioRxiv2025

ERSAtool: A User-Friendly R/Shiny Comprehensive Transcriptomic Analysis Interface Suitable for Education

RNA sequencing (RNA-seq) has become an essential technology for assessing gene expression profiles in biomedical research. However, the coding complexity of RNA-seq data analysis remains a significant barrier for students and researchers without extensive bioinformatics expertise. We present the Educational RNA-Seq Analysis tool (ERSAtool), a comprehensive R/Shiny interface that provides an intuitive graphical visualization of the complete RNA-seq analysis workflow. The application is built on established Bioconductor packages and upholds high standards in analyses while significantly reducing the technical expertise required to conduct sophisticated transcriptomic analyses. ERSAtool supports various input formats, such as raw count matrices and STAR alignment outputs. It generates sample information metadata through direct integration with the Gene Expression Omnibus (GEO) provided by the National Center for Biotechnology Information (NCBI). The application guides users through normalization, data visualization, differential expression analysis, and functional interpretation using Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA). All results can be compiled into comprehensive, downloadable reports that enhance reproducibility and knowledge sharing. The design includes targeted features that facilitate educational use, making it especially useful for teaching transcriptomics in undergraduate to graduate-level bioinformatics courses. By connecting command-line bioinformatics tools with accessible graphical interfaces, ERSAtool improves accessibility to advanced transcriptomic analysis capabilities, potentially accelerating discoveries across various biological fields. The source code for the ERSAtool package is available at https://github.com/SuzukiLabTAMU/ERSAtool and is released under the GNU General Public License v3.0 (GPL-3.0).

genetics↗

bioRxiv2025

A safer fluorescent in situ hybridization protocol for cryosections

Fluorescent in situ hybridization (FISH) enables highly sensitive, high-resolution detection of gene transcripts. Moreover, by employing multiple probes, this technique allows for multiplexed, simultaneous detection of distinct gene expression patterns spatiotemporally, making it a valuable spatial transcriptomics approach. Owing to these advantages, FISH techniques are rapidly being adopted across diverse areas of basic biology. However, conventional protocols often rely on volatile, toxic reagents such as formalin or methanol, posing potential health risks to researchers. Here, we present a safer protocol that replaces these chemicals with low-toxicity alternatives, without compromising the high detection sensitivity of FISH. We validated this protocol using both in situ hybridization chain reaction (HCR) and signal amplification by exchange reaction (SABER)-FISH in frozen sections of various model organisms, including mouse (Mus musculus), amphibians (Xenopus laevis and Pleurodeles waltl), and medaka (Oryzias latipes). Our results demonstrate successful multiplexed detection of morphogenetic and cell-type marker genes in these model animals using this safer protocol. The protocol has the additional advantage of requiring no proteolytic enzyme treatment, thus preserving tissue integrity. Furthermore, we show that this protocol is fully compatible with EGFP immunostaining, allowing for the simultaneous detection of mRNAs and reporter proteins in transgenic animals. This protocol retains the benefits of highly sensitive, multiplexed, and multimodal detection afforded by integrating in situ HCR and SABER-FISH with immunohistochemistry, while providing a safer option for researchers, thereby offering a valuable tool for basic biology.

molecular biology↗