Biology subjects

Hayashi, K.

Publications and source records attributed to Hayashi, K..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Human fibrosarcoma cells selected for very-high doxorubicin resistance, acquire trabectedin and eribulin cross-resistance, remain sensitive to recombinant methioninase, and have increased c-MYC expression

Doxorubicin is first-line chemotherapy for soft tissue sarcoma; however, the development of drug resistance limits its efficacy. The purpose of the present study was to select very-high doxorubicin-resistant (VHDR) HT1080 fibrosarcoma cells, determine cross-resistance to second-line chemotherapy drugs, determine maintenance of sensitivity to recombinant methioninase (r-METase) alone and in combination with doxorubicin, and measure the level of c-MYC expression. VHDR-HT1080 cells were generated by cultivating HT-1080 cells in a series of step-wise progressively higher concentrations of doxorubicin, ranging from 8 nM to 15 {micro}M, an 1875-fold increase, over a five-month period. The WST-8 reagent was used to assess cell viability. Four groups of in vitro drug-sensitivity tests were conducted, which involved both parental HT1080 and VHDR-HT1080 cells: 1) doxorubicin alone; 2) rMETase alone; 3) a combination of doxorubicin and rMETase; and 4) untreated control. The cross-resistance of VHDR-HT1080 cells to eribulin, trabectedin, gemcitabine and docetaxel was determined. The c-MYC levels in HT1080 and VHDR-HT1080 cells were measured using Western blotting. Doxorubicin had an IC50 of 3.3 {micro}M against HT1080 cells and 38.2 {micro}M against VHDR-HT1080 cells an 11.6-fold increase. The rMETase IC50 value for HT1080 was 0.75 U/ml and 0.59 U/ml for VHDR-HT1080. rMETase sensitized VHDR-HT1080 cells to doxorubicin. VHDR-HT1080 cells were cross-resistant to trabectedin 8.9-fold and cross-resistant to eribulin 1.87-fold compared to parental HT1080 cells. c-MYC expression was 8.4 times higher in VHDR-HT1080 cells compared to HT-1080 cells. The present results suggest rMETase may be used as a future clinical strategy to overcome super-doxorubicin resistance in soft tissue sarcoma.

cancer biology↗

bioRxiv2025

Heat-mediated manipulation of gene expression by IR-LEGO in the developing genitalia in Drosophila

Manipulating gene expression in a tissue-specific and temporally controlled manner is essential for understanding the function of the focal genes. Still, in many cases, the limited availability of specific promotors to drive ectopic manipulation remains a restricting factor in developing organs, even in Drosophila. Developing external genitalia is one such organ with a complex anatomical structure shaped by a joint regulatory network of many transcription factors. To overcome the restriction, we employed the infrared laser-evoked gene operator system (IR-LEGO), in which infrared laser (1,480 nm) irradiation induces gene expression under the control of a heat shock promoter. Pupal genital structures were irradiated at approximately 24 or 48 hours after puparium formation. We tested a range of laser power and depth to the target structure by a reporter assay using green fluorescent protein, which was induced under the control of the heat shock protein 70 promoter (hs-GAL4). In previous studies, the IR-LEGO has been used as a tool to induce ectopic transgene expression. In this study, we attempted to knock down genes such as yellow (y) and odd-paired (opa) ectopically by RNAi using the GAL4/UAS system. The results demonstrated that this technique has a high potential in manipulating transcript abundance levels in small groups of cells in specific genital structures to unravel novel functions of genes involved in the morphogenesis of species-specific and rapidly evolving anatomical structures. Article summaryManipulating gene expression is essential for understanding the function of the genes at specific regions of the body. In particular, knowing how different genes contribute to shaping complex anatomical structures, such as insect external genitalia, is an intriguing question. While manipulating gene expression precisely at a specific time and space remains challenging, we show that we can knock down a gene in a specific group of cells in the developing genitalia of Drosophila pupae, using infrared laser irradiation. This is a new approach to investigating how activity of genes shapes the rapidly evolving male genitalia in insects.

genetics↗

bioRxiv2025

Transgenerational effects of perinatal cannabis exposure on female reproductive parameters in mice

The use of cannabis during pregnancy and nursing is a growing public health concern, and the multigenerational impacts of perinatal cannabis exposure remain largely unknown. To address this knowledge gap, we sought to examine the long-term consequences of perinatal cannabis use on reproductive function and how it might impact subsequent generations. Pregnant female mice were exposed to control vehicle or cannabis extract [25, 100, or 200 mg/ml {Delta}9-tetrahydrocannabinol (THC) in the cannabis extract] from gestational day 1 to postnatal day 21 (twice/day), encompassing the duration of pregnancy through weaning. Based on plasma THC concentrations in F0 females, we chose 100 and 200 mg/ml THC in the cannabis extract for subsequent studies. The selected doses and exposure conditions did not disrupt pregnancy or nursing in F0 females. Pregnancy and neonatal outcomes, including gestational length, litter size, and sexual ratio, were not affected by cannabis exposure. However, cannabis-exposed neonatal F1 pups were smaller. Cannabis exposure delayed vaginal opening as a sign of puberty onset and disrupted estrous cyclicity in F1 females. However, its effects were minor in F2 and F3 females. F1-F3 females showed no abnormal ovarian and uterine histology or plasma estradiol-17{beta} levels and could produce normal offspring without pregnancy issues. These results suggest that the hypothalamus and pituitary are likely perturbed by perinatal cannabis exposure, and the early hypothalamus-pituitary-ovarian axis is disrupted in F1 females. However, they are not sufficient to compromise adult reproductive function. The present results indicate limited transgenerational effects of perinatal cannabis exposure on female reproductive parameters.

pharmacology and toxicology↗

bioRxiv2025

Multiple lesion inductions intensify central sensitization driven by neuroinflammation in a mouse model of endometriosis.

IntroductionEndometriosis is an inflammatory disease associated with chronic pelvic pain (CPP). Growing evidence indicates that endometriotic lesions are not the sole source of pain. Instead, central nervous system (CNS) dysfunction created by prolonged peripheral and central sensitization plays a role in developing endometriosis-associated CPP. This study investigated how CPP is established using a multiple lesion induction mouse model of endometriosis, as repeated retrograde menstruation is considered underlying endometriosis pathogenesis. MethodsWe generated endometriosis-like lesions by injecting endometrial tissue fragments into the peritoneal cavity in mice. The mice received a single (1x) or multiple inductions (6x) to simulate recurrent retrograde menstruation. Lesion development, hyperalgesia by behavioral testing, signs of peripheral sensitization, chronic inflammation, and neuroinflammation were examined with lesions, peritoneal fluids, dorsal root ganglia (DRG), spinal codes, and brain. ResultsMultiple lesion inductions increased lesion numbers and elevated abdominal and hind paw hypersensitivity compared to single induction mice. Elevated persistent glial cell activation across several brain regions and/or spinal cords was found in the multiple induction mice. Specifically, IBA1+ microglial soma size was increased in the hippocampus and thalamus. IBA1+ cells were abundant in the cortex, hippocampus, thalamus, and hypothalamus of the multiple induction mice. GFAP+ astrocytes were mainly elevated in the hippocampus. Elevated TRPV1, SP, and CGRP expressions in the DRG were persistent in the multiple induction mice. Furthermore, multiple inductions induced the severe disappearance of TIM4hi MHCIIlo residential macrophages and the influx of increased proinflammatory TIM4lo MHCIIhi macrophages in the peritoneal cavity. The single and multiple inductions elevated secreted TNF, IL-1{beta}, and IL-6 levels in the peritoneal cavity at 2 weeks. Elevated cytokine levels returned to the pre-induction levels in the single induction mice at 6 weeks; however, they remained elevated in the multiple induction mice. ConclusionsOur results indicate that the repeatedly occurring lesion inductions (=mimic retrograde menstruation) can be a peripheral stimulus that induces nociceptive pain and creates composite chronic inflammatory stimuli to cause neuroinflammation and sensitize the CNS. The circuits of neuroplasticity and stimulation of peripheral organs via a feedback loop of neuroinflammation may mediate widespread endometriosis-associated CPP.

molecular biology↗

bioRxiv2025

Influence of solvent, sex, and age on pharmacokinetic and acute behavioral effects of vaporized cannabis extract in mice

The legalization of cannabis in several states across the US has increased the need to better understand its effects on the body, brain, and behavior, particularly in different populations. Rodent models are particularly valuable in this respect because they provide precise control over external variables. Previous rodent studies have found age and sex differences in response to injected {Delta}9-tetrahydrocannabinol (THC), the major psychoactive component of cannabis. However, this route of administration does not mimic the most common way humans consume cannabis, i.e. through inhalation. Here, we sought to address this gap by investigating age and sex differences in pharmacokinetics and the acute behavioral effects of vaporized cannabis extract in mice. Adolescent (postnatal day [P] 35-50) and adult ([≥] P70) mice of both sexes received noncontingent exposure to 0 mg/ml, 150 mg/ml, or 300 mg/ml vaporized cannabis extract diluted in either 80% propylene glycol/20% vegetable glycerol (PG/VG) or 100% polyethylene glycol 400 (PEG). Immediately after exposure, body temperature, hot plate withdrawal latency, and locomotion were assessed. Blood was collected at 0, 30, and 60 min after vapor exposure, and plasma THC and its metabolites were analyzed. Measured THC levels were higher in both the plasma of vapor-exposed mice and the cannabis extract solutions themselves when PEG was the solvent compared to PG/VG. Vaporized cannabis (dissolved in PEG) at the highest dose tested induced hypothermic, antinociceptive, and locomotor-suppressing effects in all groups of mice. We found a dose-dependent age difference in locomotion, indicating that adolescents were less sensitive to the locomotor-suppressing effects of vaporized cannabis, which may be related to the plasma THC levels achieved. Although we found no significant sex differences in the acute behavioral effects of vaporized cannabis, there were significant sex differences in plasma THC metabolites indicating that female mice may metabolize vaporized cannabis more slowly than male mice. Taken together, the current findings add to a growing number of studies implementing vaporized cannabinoid delivery approaches by revealing PEG as the superior solvent for studies involving cannabis extract.

animal behavior and cognition↗

bioRxiv2024

Environmentally-relevant doses of bisphenol A and S exposure in utero disrupt germ cell programming across generations resolved by single nucleus multi-omics

BackgroundExposure to endocrine-disrupting chemicals (EDCs), such as bisphenol A (BPA), disrupts reproduction across generations. Germ cell epigenetic alterations are proposed to bridge transgenerational reproductive defects resulting from EDCs. Previously, we have shown that prenatal exposure to environmentally relevant doses of BPA or its substitute, BPS, caused transgenerationally maintained reproductive impairments associated with neonatal spermatogonial epigenetic changes in male mice. While epigenetic alterations in germ cells can lead to transgenerational phenotypic variations, the mechanisms sustaining these changes across generations remain unclear. ObjectivesThis study aimed to systematically elucidate the mechanism of transgenerational inherence by prenatal BPA and BPS exposure in the murine germline from F1 to F3 generations at both transcriptomic and epigenetic levels. MethodsBPA or BPS with doses of 0 (vehicle control), 0.5, 50, or 1000 {micro}g/kg/b.w./day was orally administered to pregnant CD-1 females (F0) from gestational day 7 to birth. Sperm counts and motility were examined in F1, F2, and F3 adult males. THY1+ germ cells on postnatal day 6 from F1, F2, and F3 males at a dose of 50 {micro}g/kg/b.w./day were used for analysis by single-nucleus (sn) multi-omics (paired snRNA-seq and snATAC-seq on the same nucleus). ResultsPrenatal exposure to BPA and BPS with 0.5, 50, and 1000 {micro}g/kg/b.w./day reduced sperm counts in mice across F1 to F3 generations. In the F1 neonatal germ cells, ancestral BPA or BPS exposure with 50 {micro}g/kg/b.w./day resulted in increased differentially expressed genes (DEGs) associated with spermatogonial differentiation. It also disrupted the balance between maintaining the undifferentiated and differentiating spermatogonial populations. Differentially accessible peaks (DAPs) by snATAC-seq were primarily located in the promoter regions, with elevated activity of key transcription factors, including SP1, SP4, and DMRT1. Throughout F1-F3 generations, biological processes related to mitosis/meiosis and metabolic pathways were substantially up-regulated in BPA-or BPS-exposed groups. While the quantities of DEGs and DAPs were similar in F1 and F2 spermatogonia, with both showing a significant reduction in F3. Notably, approximately 80% of DAPs in F1 and F2 spermatogonia overlapped with histone post-translational modifications linked to transcription activation, such as H3K4me1/2/3 and H3K27ac. Although BPA exerted more potent effects on gene expression in F1 spermatogonia, BPS induced longer-lasting effects on spermatogonial differentiation across F1 to F3 males. Interestingly, DMRT1 motif activity was persistently elevated across all three generations following ancestral BPA or BPS exposure. DiscussionOur work provides the first systematic analyses for understanding the transgenerational dynamics of gene expression and chromatin landscape following prenatal exposure to BPA or BPS in neonatal spermatogonia. These results suggest that prenatal exposure to environmentally relevant doses of BPA or BPS alters chromatin accessibility and transcription factor motif activities, consequently contributing to disrupted transcriptional levels in neonatal germ cells, and some are sustained to F3 generations, ultimately leading to the reduction of sperm counts in adults.

pharmacology and toxicology↗

bioRxiv2024

Split RNA switch: Programmable and precise control of gene expression by ensemble of pre- and post-translational regulation

Regulating gene expression in response to biomolecules is a powerful strategy for monitoring intracellular environments and controlling cellular programs. RNA switch is a synthetic mRNA-based technology that controls gene expression at the translational level in response to cellular RNA and protein molecules, thus enabling cell type-specific gene regulation and showing promise for gene therapy, regenerative medicine, and cell therapy. However, single RNA switches often lack the specificity required for practical applications due to low ON/OFF ratios and difficulty in finding distinct and single biomolecule targets. To address these issues, we developed "split RNA switches" that integrate outputs from multiple RNA switches by exploiting protein splicing, a post-translational modification mechanism. We demonstrated that split RNA switches significantly improve the ON/OFF ratio of microRNA (miRNA)-responsive ON switch systems by canceling undesirable leaky OFF level. We achieved efficient and robust target cell purification based on endogenous miRNA profiles, which was impossible with an ON switch alone. Additionally, we constructed multi-output and multi-input RNA-based synthetic circuits using split RNA switches to enable the detection of multiple miRNAs for precise gene control with logical operations. Split RNA switches illustrate a novel application of protein splicing and demonstrate the potential of post-translational processing as a comprehensive solution for advancing translational control technologies toward widespread mRNA therapies.

synthetic biology↗

bioRxiv2024

Isotonic and minimally invasive optical clearing media for live cell imaging ex vivo and in vivo

Tissue clearing has been widely used for fluorescence imaging of fixed tissues, but not for live tissues due to its toxicity. Here we develop minimally invasive optical clearing media for fluorescence imaging of live mammalian tissues. Light scattering is minimized by adding spherical polymers with low osmolarity to the extracellular medium. A clearing medium containing bovine serum albumin (SeeDB-Live) is minimally invasive to live cells, allowing for structural and functional imaging of live tissues, such as spheroids, organoids, acute brain slices, and the mouse brain in vivo. SeeDB-Live minimally affects the electrophysiological properties and sensory responses of neurons. We demonstrate its utility for widefield imaging of subcellular voltage dynamics, such as backpropagating action potentials, in acute brain slices. We also utilize SeeDB-Live for widefield voltage imaging of dozens of dendrites in vivo, demonstrating population dynamics. Thus, SeeDB-Live expands the scale and modalities of fluorescence imaging of live mammalian tissues.

neuroscience↗

bioRxiv2024

Disruption of ultrasonic vocalization with systemic administration of the non-competitive N-methyl-d-aspartate receptor antagonist MK-801 in adult male mice

Objective: Social behavior and communication, including ultrasonic vocalizations (USVs), are important for survival and reproductive success across species. N-methyl-D-aspartate (NMDA) receptors contribute to the neural mechanisms underlying social behavior, and pharmacological modulation of these receptors can alter both social behavior and USVs in rodents. However, the effects of NMDA receptor blockade on dyadic USVs and social approach during male-female interactions in mice remain incompletely understood. We therefore investigated the effects of systemic administration of MK-801, a noncompetitive NMDA receptor antagonist, on dyadic vocal activity, social approach, locomotor behavior, and excretory outcomes in adult male mice. Methods: Sexually experienced male mice were tested in male-female interaction and same-sex social-preference tasks. Using VocalMat, a machine-learning-based tool, we classified the USVs recorded during male-female interactions into 11 call categories and examined their temporal relationship to the proximity of the male subject to the female stimulus. Results: Administration of MK-801 to the male dose-dependently reduced the total number of USVs recorded from the male-female dyad, with a marked reduction at the highest dose, while leaving the treated male's proximity to the female stimulus unchanged. In the same-sex social-preference task, MK-801 reduced the time spent near the social stimulus at the highest dose, although preference for the social stimulus over the non-social object remained significant under all treatment conditions. MK-801 also increased locomotor activity and reduced fecal output and estimated urine output in the open-field task. These findings demonstrate that systemic NMDA receptor blockade differentially affects dyadic vocal communication, social approach, locomotor activity, and excretory outcomes. Conclusion: Systemic MK-801 administration to the male reduced the number of USVs recorded during male-female interactions and produced distinct effects on social approach, locomotor activity, and excretory outcomes. These findings indicate that NMDA receptor blockade differentially affects multiple behavioral domains, although the mechanisms underlying the reduction in USVs remain to be determined.

pharmacology and toxicology↗

bioRxiv2024

Integrative Transcriptomics Reveals Layer 1 Astrocytes Altered in Schizophrenia

Schizophrenia is one of the most prevalent psychiatric disorders with unclear pathophysiology despite a century-long history of intense research. Schizophrenia affects multiple networks across different brain regions. The anterior cingulate cortex (ACC) is the region that connects the limbic system to cognitive areas such as the prefrontal cortex and represents a pivotal region for the etiology of schizophrenia; however, the molecular pathology, considering its cellular and anatomical complexity, is not well understood. Here, we performed an integrative analysis of spatial and single-nucleus transcriptomics of the postmortem ACC of people with schizophrenia, together with a thorough histological analysis. The data revealed major transcriptomics signatures altered in schizophrenia, pointing at the dysregulation of glial cells, primarily in astrocytes. We further discovered a decrease in the cellular density and abundance of processes of interlaminar astrocytes, a subpopulation of astrocytes specific to primates that localize in the layer 1 and influence the superficial cortical microenvironment across layer 1 and layers 2/3 of the cortex. Our study suggests that aberrant changes in interlaminar astrocytes could explain the cell-to-cell circuit alterations found in schizophrenia and represent novel therapeutic targets to ameliorate schizophrenia-associated dysfunction.

neuroscience↗

bioRxiv2024

Linear models replicate the energy landscape and dynamics of resting-state brain activity

The spatiotemporal dynamics of resting-state brain activity can be characterized by switching between multiple brain states, and numerous techniques have been developed to extract such dynamic features from resting-state functional magnetic resonance imaging (fMRI) data. However, many of these techniques are based on momentary temporal correlation and co-activation patterns and merely reflect stationary, linear features of the data, suggesting that the non-stationary dynamic features extracted by these techniques may be misinterpreted. To examine whether such misinterpretations occur when using techniques that are not based on momentary temporal correlation or co-activation patterns, we addressed Energy Landscape Analysis (ELA), a statistical physics-inspired method that was designed to extract multiple brain states and dynamics of resting-state fMRI data. We found that the ELA-derived features were almost identical for real data and surrogate data suggesting that the features derived from ELA were accounted for by stationary and linear properties of the real data rather than non-stationary or non-linear properties. To confirm that surrogate data were distinct from the real data, we replicated a previous finding that some topological properties of resting-state fMRI data differed between the real and surrogate data. Overall, the present finding that the energy landscape and activity dynamics of resting-state fMRI data were well captured by stationary and linear surrogate data supports the notion that linear models sufficiently describe the dynamics of resting-state brain activity.

neuroscience↗

bioRxiv2024

Sexual dimorphism in the tardigrade Paramacrobiotus metropolitanus transcriptome

BackgroundIn gonochoristic animals, the sex determination pathway induces different morphological and behavioral features that can be observed between sexes, a condition known as sexual dimorphism. While many components of this sex differentiation cascade shows high levels of diversity, factors such as the Doublesex-Mab-3-related transcription factor (DMRT) are highly conserved throughout animals. Species of the phylum Tardigrada exhibits remarkable diversity in morphology and behavior between sexes, suggesting a pathway regulating such dimorphism. Despite the wealth of genomic and zoological knowledge accumulated in recent studies, the sexual differences in tardigrades genomes have not been identified. In this study, we focused on the gonochoristic species Paramacrobiotus metropolitanus and employed omics analyses to unravel the molecular basis of sexual dimorphism. ResultsTranscriptome analysis between sex identified numerous differentially expressed genes, of which approximately 2,000 male-biased genes were focused on 29 non-male-specific genomic loci. From these regions, we identified two Macrobiotidae family specific DMRT paralogs, which were significantly upregulated in males and lacked sex specific splicing variants. Furthermore, phylogenetic analysis indicated all tardigrade genomes lacks the doublesex ortholog, suggesting doublesex emerged after the divergence of Tardigrada. In contrast to sex-specific expression, no evidence of genomic difference between the sexes were found. We also identified several anhydrobiosis genes exhibiting sex-biased expression, possibly suggesting a mechanism for protection of sex specific tissues against extreme stress. ConclusionsThis study provides a comprehensive analysis for analyzing the genetic differences between sexes in tardigrades. The existence of male-biased, but not male-specific, genomic loci and identification of the family specific male-biased DMRT subfamily would provide the foundation for understanding the sex determination cascade. In addition, sex-biased expression of several tardigrade-specific genes which are involved their stress tolerance suggests a potential role in protecting sex-specific tissue and gametes.

zoology↗

bioRxiv2023

Neural correlates of category learning in monkey inferior temporal cortex

We trained two monkeys implanted with multi-electrode arrays to categorize natural images of cats and dogs, in order to observe changes in neural activity related to category learning. We recorded neural activity from area TE, which is required for normal learning of visual categories based on perceptual similarity. Neural activity during a passive viewing task was compared pre- and post-training. After the category training, the accuracy of abstract category decoding improved. Specifically, the proportion of single units with category selectivity increased, and units sustained their category-specific responses for longer. Visual category learning thus appears to enhance category separability in area TE by driving changes in the stimulus selectivity of individual neurons and by recruiting more units to the active network.

neuroscience↗

bioRxiv2023

High-fat diets promote peritoneal inflammation and augment endometriosis-associated abdominal hyperalgesia

Immune dysfunction is one of the central components in the development and progression of endometriosis by establishing a chronic inflammatory environment. Western-style high-fat diets (HFD) have been linked to greater systemic inflammation to cause metabolic and chronic inflammatory diseases, and are also considered an environmental risk factor for gynecologic diseases. Here, we aimed to examine how HFD alter an inflammatory environment in endometriosis and discern their contribution to endometriotic-associated hyperalgesia. Our results showed that HFD-induced obesity enhanced abdominal mechanical allodynia that was induced by endometriotic lesions. Peritoneal inflammatory macrophages and cytokine levels increased by lesion induction were elevated by chronic exposure to HFD. Pain-related mediators in the dorsal root ganglia were further stimulated after lesion induction under the HFD condition. Although HFD did not affect inflammatory macrophages in the peritoneal cavity without lesion induction, the diversity and composition of the gut microbiota were clearly altered by HFD as a sign of low-grade systemic inflammation. Thus, HFD alone might not establish a local inflammatory environment in the pelvic cavity, but it can contribute to further enhancing chronic inflammation, leading to the exacerbation of endometriosis-associated abdominal hyperalgesia following the establishment and progression of the disease.

physiology↗

bioRxiv2023

Brain-derived neurotrophic factor contributes to activity-induced muscle pain in male but not female mice

Activity-induced muscle pain increases release of interleukin-1{beta} (IL-1{beta}) in muscle macrophages and the development of pain is prevented by blockade of IL-1{beta}. Brain derived neurotrophic factor (BDNF) is released from sensory neurons in response to IL-1{beta} and mediates both inflammatory and neuropathic pain. Thus, we hypothesized that metabolites released during fatiguing muscle contractions activate macrophages to release IL-1{beta}, which subsequently activate sensory neurons to secrete BDNF. To test this hypothesis, we used an animal model of activity-induced pain induced by repeated intramuscular acidic saline injections combined with fatiguing muscle contractions. Intrathecal or intramuscular injection of inhibitors of BDNF-Tropomyosin receptor kinase B (TrkB) signaling, ANA-12 or TrkB-Fc, reduced the decrease in muscle withdrawal thresholds in male, but not in female, mice when given before or 24hr after, but not 1 week after induction of the model. BDNF messenger ribonucleic acid (mRNA) was significantly increased in L4-L6 dorsal root ganglion (DRG), but not the spinal dorsal horn or gastrocnemius muscle, 24hr after induction of the model in either male or female mice. No changes in TrkB mRNA or p75 neurotrophin receptor mRNA were observed. BDNF protein expression via immunohistochemistry was significantly increased in L4-L6 spinal dorsal horn and retrogradely labelled muscle afferent DRG neurons, at 24hr after induction of the model in both sexes. In cultured DRG, fatigue metabolites combined with IL-1{beta} significantly increased BDNF expression in both sexes. In summary, fatigue metabolites release, combined with IL-1{beta}, BDNF from primary DRG neurons and contribute to activity-induced muscle pain only in males, while there were no sex differences in the changes in expression observed in BDNF.

neuroscience↗

bioRxiv2023

Engineered Active Zymogen of Microbial Transglutaminase

Microbial transglutaminase (MTG) has shown to be a powerful biocatalytic glue for site-specific crosslinking of a range of biomolecules and synthetic molecules, those handled with an MTG-reactive moiety. The preparation of active recombinant MTG requires the posttranslational proteolytic digestion of propeptide working as an intramolecular chaperon to assist the correct folding of MTG zymogen (MTGz) in the biosynthesis. Herein, we propose an engineered active zymogen of MTG (EzMTG) that is expressed as soluble form in the host E. coli cytosol and exhibits the cross-linking activity without limited proteolysis. Based on the 3D structure of MTGz and serendipitous findings, saturated mutagenesis of K10 or Y12 in propeptide domain leads to generate several active MTGz mutants. In particular, K10D/Y12G mutant exhibited the catalytic activity comparable with a mature form. However, the expression level was low possibly due to the reduction of chaperone activity and/or the promiscuous substrate specificity of MTG, which is potentially harmful to the host cells. By contrast, soluble K10R/Y12A mutant is expressed in the cytosol of host E. coli and exhibited unique substrate-dependent reactivity toward peptidyl substrates. The quantitative analysis of the binding affinity of mutated propeptide to the active site suggested the trade-off relationship of EzMTGs between the binding affinity and the catalytic activity. Our proof-of-concept study provides insights into the design of a new biocatalyst by using the zymogen as a scaffold and will convey a potential route to the high-throughput screening of MTG mutants for bioconjugation applications.

biochemistry↗

bioRxiv2023

Spatio-temporal Coordination of Active Deformation Forces and Wnt / Hippo-Yap Signaling in Hydra Regeneration

Ample evidence suggests that Wnt signaling and tissue deformation are key determinants for pattern formation in animals. The coordination of these biochemical and biomechanical spatio-temporal asymmetries is often unknown or controversial. We investigated this relationship by studying regeneration in the freshwater polyp Hydra. In both reaggregates of dissociated cells and tissue regenerates, we found significant tissue contraction waves and upregulation of Wnt signaling. Applying a simple mechanical model to the mode analysis of the active deformations, we quantitatively defined the phase reversal of size change and axial deformation in those oscillations as the time point of "biomechanical" symmetry breaking. Moreover, overexpression and inhibition of canonical Wnt signaling modulated the timing of this biomechanical symmetry breaking. A direct comparison with the RNAseq data indicates that the biomechanical symmetry breaking occurs only after the upregulation of canonical Wnt signaling. Further data suggest that biochemical signaling and biomechanical active deformation synergistically stabilize the body axis and hence the following head structure formation by Hippo-Yap signaling. The symmetry breaking mechanism identified here in Hydra most likely represents a patterning module that is evolutionary conserved from early metazoan to bilaterian animals.

biophysics↗

bioRxiv2023

Number of kinesins engaged in axonal cargo transport: A novel biomarker for neurological disorders

Kinesin motor proteins play crucial roles in anterograde transport of cargo vesicles in neurons, moving them along axons from the cell body towards the synaptic region. Not only the transport force and velocity of single motor protein, but also the number of kinesin molecules involved in transporting a specific cargo, is pivotal for synapse formation. This collective transport by multiple kinesins ensures stable and efficient cargo transport in neurons. Abnormal increases or decreases in the number of engaged kinesin molecules per cargo could potentially act as biomarkers for neurodegenerative diseases such as Alzheimers, Parkinsons, amyotrophic lateral sclerosis (ALS), spastic paraplegia, polydactyly syndrome, and virus transport disorders. We review here a model constructed using physical measurements to quantify the number of kinesin molecules associated with their cargo, which could shed light on the molecular mechanisms of neurodegenerative diseases related to axonal transport.

biophysics↗