Biology subjects

Nishimura, R.

Publications and source records attributed to Nishimura, R..

12 recordsLinked to original sources

bioRxiv2026

Hidden Structural Bias in Proteomics: Sonication-induced Selective Fragmentation of Intrinsically Disordered Regions

Sonication is a fundamental technique in proteome sample preparation, primarily used for protein solubilization and shearing of genomic DNA. Although the mechanical shearing of DNA is well-characterized, its unintended impact on protein structural integrity remains a significant "blind spot" in high-throughput analytical workflows. In this study, we systematically investigated sonication-induced protein fragmentation by combining gel-based fractionation (PEPPI-MS) with sequence-level compositional analysis and bioinformatic mapping. Our results demonstrate that sonication does not significantly alter overall proteome identification or the recovery of membrane proteins; however, it induces extensive and non-random protein fragmentation. Sonication caused an approximately three-fold increase in the abundance of >45 kDa protein-derived fragments migrating into the <40 kDa fraction, and 1,620 high-molecular-weight (MW) proteins were uniquely detected in the lower-MW fraction upon sonication, an eight-fold increase over non-sonicated controls. Peptide-level amino acid composition analysis revealed subtle but directional shifts in the sonication-derived fragments. This residue-level signature is reinforced by two orthogonal structural analyses (MobiDB peptide-level mapping and protein-level profiling using metapredict V3 software), which show that sonication-susceptible proteins harbor more than twice the disordered content of length-matched controls (median 40% vs. 18%). This study identifies a previously unrecognized "structural bias" whereby intrinsically disordered region (IDR)-rich proteins are selectively compromised during sample preparation. Because these fragments are indistinguishable from enzymatic digestion products in conventional bottom-up proteomics, the underlying structural damage is effectively masked in global quantitative datasets, potentially distorting biological interpretations related to protein size, isoforms, and stability, particularly for IDR-rich classes, such as transcription factors and signaling molecules. We propose that optimizing and standardizing sonication parameters is essential for ensuring the accuracy and reproducibility of quantitative proteomic analyses.

cell biology↗

bioRxiv2026

Rapid Intracellular Delivery of Human Heat Shock Protein 72 Prevents Memory Loss and Inhibits Neurodegeneration up to 9 Days After a Blast Injury

Traumatic brain injuries (TBIs) are increasingly prevalent among military service members and are associated with long-term neurological impairment and neurodegeneration. Heat shock protein 72 (HSP72) has demonstrated cytoprotective properties and has been shown to cross the blood brain barrier in rat models of blast injury, remaining in brain tissue for up to 12 hours. In this study, we evaluate engineered Fv-HSP72 variants for their ability to reduce neurodegeneration and preserve short-term memory following blast-induced TBI. Male Sprague-Dawley rats were assigned to 9 groups of n = 8 rats. Animals were either not exposed to blast (Sham), exposed to blast (Blast Only), blast exposed and given buffer (Vehicle), or blast exposed and treated with one of three Fv-HSP72 variants, dosed at 10 or 30mg/kg at 15m post-blast. Blast exposure was generated using an Advanced Blast Simulator (ABS) producing positive static pressure to model moderate to severe blast injury. Animals were euthanized 48 hours post injury for neurodegeneration and immunologic biomarker analysis. After selecting an effective Fv-HSP72 variant using the biomarker data, additional rats were divided into Sham, Vehicle, and Fv-HSP72 treatment groups to evaluate short-term memory function through the Novel Object Recognition (NOR) test on days 2 and 8 post-blast. Analysis of cortical and spinal cord tissues demonstrated a statistically significant reduction in expression of neurodegenerative markers of Tau phosphorylation and glial injury (GFAP) for rats receiving a single dose of our clinical candidate, RBB012-CTB. In fact, the drug drove astrogliosis toward a neuroprotective state in blast exposed rats. In the NOR assay, Fv-HSP72 treated rats showed improved recognition performance, indicating preservation of short-term memory function. With similar biomarker results obtained for a controlled cortical impact injury model published elsewhere (Chan et al. manuscript submitted), the analyses suggest Fv-HSP72 is neuroprotective following a blast injury as well. One sentence summaryThis study describes the effectiveness of a biologic agent, Fv-HSP72, in significantly preventing learning and memory loss in rats for up to 9 days after a blast injury.

neuroscience↗

bioRxiv2026

Rapid Intracellular Delivery of Human Heat Shock Protein 72 Inhibits Neurodegeneration and Oxidative Damage After a Traumatic Brain Injury

Fv-HSP72 is a rapid cell-penetrating human heat shock protein for the treatment of traumatic organ injuries. We have shown this re-engineered protein (HSP72) is capable of crossing the blood brain barrier (BBB) of rats suffering a controlled cortical impact (CCI) and remains in brain tissue for up to 12 hours; long after clearance from the cortex of uninjured rats. Peptide sequences unique to Fv-HSP72 allow for its differential detection from endogenous HSP72. Male Sprague-Dawley rats were divided into 10 groups of n=10 with those animals receiving a CCI subjected to a unilateral cortical contusion simulating a moderate to severe brain injury using an electronically controlled pneumatic impact device. Control groups were either uninjured (Sham), injured (TBI Only), or injured and given buffer (TBI+Vehicle). Rats treated with one of three Fv-HSP72 variants were dosed at 10 or 30mg/kg 15m post-impact, then sacrificed 48 hours later. Cortical tissues were extracted from the ipsilateral and contralateral hemispheres for biomarker analysis. Here we report results of our drug inhibiting neurodegeneration based on five biomarkers (NF-L, pNF-H, pTau [T181, T231, S396]). These results were statistically significant, especially for one of the Fv-HSP72 variants, when comparing differences both between treatment groups and within groups (i.e. when comparing ipsi-vs. contralateral hemispheres). Significant inhibition of oxidative stress (3-NT) and inflammatory (IL-6) biomarkers were also observed (both p<0.0001). With similar results obtained for a blast injury model being published elsewhere, the analyses suggest Fv-HSP72 is neuroprotective following a direct impact brain injury. One sentence summaryThis study describes the effectiveness of a biologic agent, Fv-HSP72, in significantly inhibiting neuronal tissue damage in the brain when administered after a direct cortical impact.

neuroscience↗

bioRxiv2025

Genotype-first approach revealed unrecognized breed-specific genetic diseases in dogs

Numerous dogs are affected by genetic diseases because of their unique breeding situation. The identification of pathogenic variants in dogs not only has veterinary benefits but also leads to the development of new human disease models. However, fewer pathogenic variants have been identified in dogs than in humans, and standard genetic approaches, such as large-scale case/control studies and family analyses, often fail to identify novel causative variants in dogs. To address this, we applied a genotype-first approach by developing a multiplex polymerase chain reaction (PCR)-based targeted sequencing method for dogs, analyzing 203 genes--most of which are related to human disease--from 6,333 dogs collected from a veterinary hospital, and characterizing pathogenic variant carriers. In total, 120 pathogenic variants in 83 genes were identified. Frequent pathogenic variants were enriched in specific dog breeds. Dogs homozygous for a pathogenic CHEK2 variant, frequently identified in French Bulldogs (5.74%, homozygous), showed a five-fold increased risk of cancer (P = 3.45 x 10-2). A pathogenic NOD2 variant was detected in half of the Italian Greyhounds. However, their medical records did not document the typical clinical symptoms observed in humans with NOD2 deficiency. Some pathogenic variants were present in three or fewer dogs, and 11.0% of these dogs exhibited symptoms potentially associated with gene defects. These rare variants could become common within a short period due to the canine breeding system to induce breed-specific genetic diseases. These findings suggest that the genotype-first approach successfully identified unrecognized breed-specific genetic diseases. Further characterization of dogs with pathogenic variants enable to define clinical diagnosis, providing an important spontaneous model for human genetic diseases.

genomics↗

bioRxiv2025

Optogenetic control of mechanotransduction based on light-induced homodimerization of talin

Integrin-based cell adhesions (IACs) serve as primary sites where piconewton-scale actomyosin-generated mechanical forces are transmitted to the extracellular matrix (ECM), generating traction forces that drive cell-ECM responses including adhesion, migration, and mechano-signaling. Talin, a large (270 kDa) cytosolic adaptor protein, is the principal force-transmission protein in integrin-based adhesions, containing multiple mechanosensitive domains and protein-protein interaction sites that orchestrate molecular events in mechanosensing. As a highly modular multi-domain protein, talin has been identified as an effective target for chemogenetic and optogenetic manipulation of integrin-based mechanotransduction. However, a key limitation of previous approaches is the reliance on heterodimerization modules to control talin function, requiring the expression of two modified talin fragments. In practice, achieving precise expression levels in such a 2-component approach can be challenging, particularly when combined with other genetic tools. Since talin naturally contains a C-terminal dimerization domain that forms part of its actin-binding site, we reasoned that the molecularly engineered talin with a C-terminal optically-controlled homodimerizer could enable single-component optogenetic control of mechanotransduction. This approach would facilitate multiplexing with other molecular perturbations or experimental techniques. Here, we describe an opto-homodimerizable talin based on the pdDronpa1.2 optogenetic module, which enables optogenetic control of talin by a single construct. We demonstrate that light-induced talin dimerization promotes talin recruitment to IACs, adhesion formation, actin retrograde flow engagement, and downstream mechanotransduction signaling. Conversely, light-induced talin monomerization rapidly disassembles focal adhesions, disrupts talin-actin linkages, and accelerates actin retrograde flow, underscoring the critical roles of talin dimerization. Furthermore, our single-construct design allows facile multiplexing of optogenetic modulation of integrin-mediated mechanotransduction with super-resolution single-molecule tracking, revealing the essential role of talin dimerization for integrin v{beta}5 engagement.

cell biology↗

bioRxiv2023

SLC26A2-mediated sulfate metabolism is essential for the tooth development

The sulfate transporter gene SLC26A2 is responsible for diastrophic dysplasia, which represents skeletal dysplasia in humans. This highlights the importance of sulfate metabolism in skeletal formation. SLC26A2-related chondrodysplasia is also known to exhibit abnormalities in craniofacial and tooth development. Although the function of SLC26A2 in mammals has been investigated using genetic mouse models, the essential role of SLC26A2 during craniofacial and tooth development has not been elucidated. In this study, we demonstrate the pivotal roles of SLC26A2-mediated sulfate metabolism during tooth development. Analysis of Slc26a2 expression reveals that it is predominantly expressed in dental tissues, including odontoblasts and ameloblasts, during tooth development. Slc26a2 knockout mice (Slc26a2-KO-{Delta}exon2) exhibit a retrognathic upper jaw, small upper incisors, and hypoplasia of upper molars. Additionally, upper incisors and molars in Slc26a2-KO-{Delta}exon2 mice display flattened odontoblasts and nuclei that lack intracellular polarity. In contrast, tooth phenotype is not remarkable in lower incisors and molars. Furthermore, the expression of odontoblast differentiation markers, Dspp and Dmp1, is significantly decreased in the upper molars of Slc26a2-deficient mice. Ex vivo organ culture of tooth germs by implantation of Slc26a2-deficient tooth germs under the kidney capsule reveals hypoplasia of the dentin matrix as well as tooth root shortening. In vitro studies using human dental pulp stem cells (hDPSCs) show that the expression levels of Dspp and Dmp1 in shSlc26a2 knockdown cells are significantly decreased compared to control cells. Collectively, our data demonstrate that SLC26A2-mediated sulfate metabolism is essential for tooth development. This study may provide insight into the mechanisms underlying tooth abnormalities in patients with recessively inherited chondrodysplasias caused by mutations in the SLC26A2 gene.

developmental biology↗

bioRxiv2022

The GPI-anchored Ly6 protein Belly roll regulates Drosophila melanogaster escape behaviors by modulating the excitability of nociceptive peptidergic interneurons

Appropriate modulation of escape behaviors in response to potentially damaging stimuli is essential for survival. Although nociceptive circuitry has been studied, it is poorly understood how genetic contexts affect the relevant escape responses. Using an unbiased genome-wide association analysis, we identified a Ly6/-neurotoxin family protein, Belly roll (Bero), which negatively regulates Drosophila nociceptive escape behavior. We show that Bero is expressed in abdominal leucokinin-producing neurons (ABLK neurons) and bero knockdown in ABLK neurons resulted in enhanced escape behavior. Furthermore, we demonstrated that ABLK neurons responded to the activation of nociceptors and initiated the behavior. Notably, bero knockdown reduced the persistent neuronal activity and increased the evoked nociceptive responses in ABLK neurons. Our findings reveal that Bero modulates an escape response by regulating distinct neuronal activities in ABLK neurons.

neuroscience↗

bioRxiv2021

Appropriate tension sensitivity of α-catenin ensures rounding morphogenesis of epithelial spheroids

The adherens junction (AJ) is an actin filament-anchoring junction. It plays a central role in epithelial morphogenesis through cadherin-based recognition and adhesion among cells. The stability and plasticity of AJs are required for the morphogenesis. An actin-binding -catenin is an essential component of the cadherin-catenin complex and functions as a tension transducer that changes its conformation and induces AJ development in response to tension. Despite much progress in understanding molecular mechanisms of tension sensitivity of -catenin, its significance on epithelial morphogenesis is still unknown. Here we show that the tension sensitivity of -catenin is essential for epithelial cells to form round spheroids through proper multicellular rearrangement. Using a novel in vitro suspension culture model, we found that epithelial cells form round spheroids even from rectangular-shaped cell masses with high aspect ratios without using high tension and that hypersensitive mutants affected this morphogenesis. Analyses of AJ formation and cellular tracking during rounding morphogenesis showed cellular rearrangement, probably through AJ remodeling. The rearrangement occurs at the cell mass level, but not single-cell level. Hypersensitive -catenin mutant-expressing cells did not show cellular rearrangement at the cell mass level, suggesting that proper AJ plasticity requires appropriate tension sensitivity of -catenin.

cell biology↗

bioRxiv2021

The cell surface hyaluronidase TMEM2 plays an essential role in mouse neural crest cell development and survival

Neural crest cells (NCCs) are a migratory population that gives rise to a diverse cell lineage, including the craniofacial complex, the peripheral nervous system, and a part of the heart. Hyaluronan (HA) is a major component of the extracellular matrix, and its tissue levels are dynamically regulated in during development. Although the synthesis of HA has been shown to exert substantial influence on embryonic morphogenesis, the functional importance of the catabolic side of HA turnover is poorly understood. Here, we demonstrate that the transmembrane hyaluronidase TMEM2 plays an essential role in NCC development and the morphogenesis of their derivatives. Wnt1-Cre-mediated Tmem2 knockout (Tmem2CKO) mice exhibit severe craniofacial and cardiovascular abnormalities. Analysis of Tmem2 expression using Tmem2 knock-in reporter mice reveals that Tmem2 is expressed at the site of NCC delamination in the neural tube and in Sox9-positive emigrating NCCs, suggesting that Tmem2 is critical for NCC development. Consistent with this possibility, linage tracing analysis reveals that the contribution of Wnt1-Cre-labeled cells to NCC derivatives is significantly reduced in a Tmem2-deficient background. Moreover, the emigration of NCCs from the neural tube is greatly reduced in Tmem2CKO mice. In vitro assays demonstrate that Tmem2 expression is essential for the ability of mouse O9-1 NCCs to form focal adhesion on and migrate into HA-containing substrates. Tmem2CKO mice also exhibit increased apoptotic cell death in NCC-derived tissues. Collectively, our data demonstrate that Tmem2 is essential for normal development of NCC-derivatives, including the craniofacial complex, and that TMEM2-mediated HA degradation allows NCCs to generate a tissue environment suitable for efficient focal adhesion assembly and migration. This study reveals the hitherto unrecognized functional importance of the catabolic side of HA metabolism in embryonic development and highlights the pivotal role of Tmem2 in the process. Author SummaryThe functional significance of hyaluronan (HA) in embryonic developmental processes has been demonstrated by studies using genetic manipulation of HA synthesis. However, the expression of HA is regulated not only by its synthesis, but also by its degradation. This issue is of particular importance due to the extremely rapid metabolic turnover of HA. Curiously, mice with mutations/ablations of known hyaluronidase molecules, such as the lysosomal hyaluronidases HYAL1 and HYAL2, exhibit little embryonic phenotypes. This suggests the existence of yet another hyaluronidase molecule that plays a key role in regulating extracellular HA balance in developing tissues. In this context, transmembrane protein 2 (TMEM2) is a novel hyaluronidase that functions on the cell surface. Here, we demonstrate that TMEM2 is expressed at the site of neural crest development and in neural crest cell (NSC)-derived craniofacial tissue, and that NCC-targeted Tmem2 conditional knockout mice develop severe craniofacial defects, which attests to a requirement for TMEM2-mediated extracellular HA degradation in neural crest development. Our in vitro and in vivo analyses on the underlying mechanisms of the phenotype demonstrate that TMEM2 is essential for generating a tissue environment suitable for efficient focal adhesion formation by NCCs. This paper reveals for the first time that the catabolic machinery for HA exerts a specific regulatory role in embryonic morphogenesis, and that its dysregulation of HA degradation leads to severe developmental defects.

developmental biology↗

bioRxiv2021

CCR4 blockade leads to clinical activity and prolongs survival in a canine model of advanced prostate cancer

Targeting regulatory T cell (Treg) infiltration is an emerging strategy for cancer immunotherapy. However, the efficacy of this strategy in advanced prostate cancer remains unclear. Here, we describe the therapeutic efficacy of this strategy in a canine model of advanced prostate cancer. We used dogs with naturally occurring prostate cancer to study the molecular mechanism underlying Treg infiltration into tumor tissues and the effect of anti-Treg treatment. We found that tumor-infiltrating Tregs were associated with poor prognosis in dogs bearing spontaneous prostate cancer. RNA sequencing and protein analyses showed that Treg infiltration was mediated by interaction between the tumor-producing chemokine, CCL17, and the receptor CCR4 expressed on Tregs. Dogs with advanced prostate cancer responded to mogamulizumab, a monoclonal antibody targeting CCR4, with improved survival and low incidence of clinically relevant adverse events. Exploratory analyses showed urinary CCL17 concentration and BRAFV595E mutation to be independently predictive of the response to mogamulizumab. Analysis of a publicly available transcriptomic dataset of human prostate cancer showed that the CCL17/CCR4 axis correlated with the Treg marker, Foxp3. In silico survival analyses showed that high expression of CCL17 was associated with poor prognosis. Immunohistochemistry confirmed that tumor-infiltrating Tregs expressed CCR4 in human patients with prostate cancer. These findings suggest that anti-Treg treatment through the blocking of CCR4 is a promising therapeutic approach for advanced prostate cancer. One Sentence SummaryTargeting regulatory T cell infiltration by CCR4 blockade induces objective responses and improves survival in a canine model of prostate cancer.

cancer biology↗

bioRxiv2019

Aberrant expression of the COX2/PGE2 axis is induced by activation of the RAF/MEK/ERK pathway in BRAFV595E canine urothelial carcinoma

Cancer-promoting inflammation is an important event in cancer development. The cyclooxygenase 2 (COX2)/prostaglandin E2 (PGE2) axis is a prominent inducer of inflammation. Canine urothelial carcinoma (cUC) uniquely overexpresses PGE2 and is often managed well with COX inhibitor monotherapy. In most cases, cUC tissue harbours homologous human BRAFV600E mutation, which causes aberrant activation of the RAF/MEK/ERK pathway in human cancer. However, mechanisms underlying aberrant PGE2 production and the importance of the BRAF mutation remain unclear. We show that activation of the RAF/MEK/ERK pathway in BRAF mutant cUC cells leads to COX2 overexpression and PGE2 production. Drug screening revealed that treatment with inhibitors of the arachidonic acid cascade (FDR<0.086), RAF/MEK/ERK pathway (FDR<0.067), and p38/JNK pathway (FDR<0.067) significantly reduced PGE2 production in cUC cells. We also validated the association between RAF/MEK/ERK pathway activation and COX2/PGE2 overexpression in BRAF mutant cUC cells using protein detection techniques. In histochemical analysis, BRAF mutant cUC tissue showed higher COX2 expression. Therefore, the driver mutation in the BRAF gene probably promotes tumour-promoting inflammation. These findings would benefit dogs suffering from cUC and can be extrapolated to human cancer. Finally, cUC can serve as a valuable model to elucidate the association between driver mutations and tumour-promoting inflammation.

cancer biology↗

bioRxiv2019

The Ficus erecta genome to identify the Ceratocystis canker resistance gene for breeding programs in common fig (F. carica)

Ficus erecta, a wild relative of common fig (F. carica), is a donor of Ceratocystis canker resistance in fig breeding programs. Interspecific hybridization followed by recurrent backcrossing is an effective method to transfer the resistance trait from wild to cultivated fig; however, this is time consuming and labor-intensive for trees, especially for gynodioecious plants such as fig. In this study, genome resources were developed for F. erecta to facilitate fig breeding programs. The genome sequence of F. erecta was determined using single-molecule real-time sequencing technology. The resultant assembly spanned 331.6 Mb with 538 contigs and an N50 length of 1.9 Mb, from which 51,806 high-confidence genes were predicted. Pseudomolecule sequences corresponding to the chromosomes of F. erecta were established with a genetic map based on single nucleotide polymorphisms from double-digest restriction-site associated DNA sequencing. Subsequent linkage analysis and whole genome resequencing identified a candidate gene for the Ceratocystis canker resistance trait. Genome-wide genotyping analysis enabled selection of female lines that possessed resistance and effective elimination of donor genome from progeny. The genome resources provided in this study will accelerate and enhance disease resistance breeding programs in fig.

genomics↗