Biology subjects

Kobayashi, T.

Publications and source records attributed to Kobayashi, T..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

Reduced glycolysis links resting zone chondrocyte proliferation in the growth plate

A gain-of-function mutation of the chondrocyte-specific microRNA, miR-140-5p, encoded by the MIR140 gene, causes spondyloepiphyseal dysplasia, Nishimura type (SEDN, also known as SED, MIR140 type; MIM, 611894). We reported that a mouse model for SEDN showed a unique growth plate phenotype that is characterized by an expansion of the resting zone of the growth plate and an increase in resting chondrocytes, of which the mechanism of regulation is poorly understood. We found that the miR-140 mutant chondrocytes showed a significant reduction of Hif1a, the master transcription factor that regulates energy metabolism in response to hypoxia. Based on this finding, we hypothesized that energy metabolism plays a regulatory role in resting chondrocyte proliferation and growth plate development. In this study, we show that suppression of glycolysis via LDH ablation causes an expansion of the resting zone and skeletal developmental defects. We have also found that reduced glycolysis results in reduced histone acetylation in the miR-140 mutant as well as LDH-deficient chondrocytes likely due to the reduction in acetyl-CoA generated from mitochondria-derived citrate. Reduction in acetyl-CoA conversion from citrate by deleting Acly caused an expansion of the resting zone and a similar gross phenotype to LDH-deficient bones without inducing energy deficiency, suggesting that the reduced acetyl-CoA, but not the ATP synthesis deficit, is responsible for the increase in resting zone chondrocytes. Comparison of the transcriptome between LDH-deficient and Acly-deficient chondrocytes also showed overlapping changes including upregulation in Fgfr3. We also confirmed that overexpression of an activation mutation of Ffgr3 causes an expansion of resting zone chondrocytes. These data demonstrate the association between reduced glycolysis and an expansion of the resting zone and suggest that it is caused by acetyl-CoA deficiency, but not energy deficiency, possibly through epigenetic upregulation of FGFR3 signaling.

developmental biology↗

bioRxiv2023

Identification of a novel enhancer essential for Satb1 expression in TH2 cells and activated ILC2s.

The genome organizer, special AT-rich binding protein-1 (SATB1) functions to globally regulate gene networks during primary T cells development and plays a pivotal role in lineage-specification in CD4+ helper-, CD8+ cytotoxic- and FOXP3+ regulatory-T cell subsets. However, it remains unclear how Satb1 gene expression is controlled, particularly in effector T cell function. Here, by using a novel reporter mouse strain expressing SATB1-Venus and genome editing, we have identified a cis-regulatory enhancer, essential for maintaining Satb1 expression specifically in TH2 cells. This enhancer is occupied by STAT6 and interacts with Satb1 promoters through chromatin looping in TH2 cells. Reduction of Satb1 expression, by the lack of this enhancer, resulted in elevated IL-5 expression in TH2 cells. In addition, we found that Satb1 is induced in activated group 2 innate lymphoid cells (ILC2s) through this enhancer. Collectively, these results provide novel insights into how Satb1 expression is regulated in TH2 cells and ILC2s during type 2 immune responses.

immunology↗

bioRxiv2022

Limited roles of Piezo mechanosensing channels in articular cartilage development and osteoarthritis progression

Osteoarthritis (OA) is a prevalent disease characterized by degeneration of the joint and pain. Mechanical stress plays a central role in OA development. It is hypothesized that cells in the OA joints produce OA-promoting molecules upon mechanical stress, and therefore, the mechanosensing systems are a theoretical target for OA treatment. Piezo mechanosensing channels mediate high-level mechanical stress in chondrocytes and have been suggested to play an important role during OA progression. To test this hypothesis, we ablated Piezo1 and Piezo2 in joint tissues using Gdf5-Cre transgenic mice [Piezo1 and 2 doubly conditional knockout (cKO) mice, cKO mice]. cKO mice showed normal development of knee joints. Both control and cKO mice developed modest to severe OA 12 weeks after the induction of OA, although some cKO mice showed milder OA. We did not find significant differences in pain in mice or gene expression after fluid flow stress in primary cells between control and cKO. Our data demonstrate the limited role of Piezo channels in joint development and OA progression. SummaryO_ST_ABSObjectiveC_ST_ABSTo investigate the role of Piezo1 and Piezo 2 in surgically induced osteoarthritis (OA) in mice. DesignMale conditional knockout (cKO) mice missing Piezo1 and Piezo2 in the joint via Gdf5-Cre transgenic mice were induced post-traumatic osteoarthritis (OA) by destabilization of the medial meniscus (DMM) of the right knee joint at 12 weeks of age. The severity of OA was assessed at 24 weeks of age using a modified Osteoarthritis Research Society International (OARSI) scoring system. OA-associated pain was evaluated by static weight bearing analysis at 4, 8, and 12 weeks post-operation. Additionally, articular chondrocytes isolated from cKO mice were exposed to fluid flow shear stress (FFSS) to evaluate the expression of OA-associated genes. ResultsMice with conditional deletion of Piezo1 and Piezo 2 showed normal joint development with no overt histological changes in the knee joint at 12 weeks and 24 weeks. DMM surgery induced moderate to severe OA in both control and cKO mice, although a few cKO mice showed milder OA. Pain assessment by static weight-bearing analysis suggested Piezo ablation in the joint has no beneficial effects on pain. FFSS increased the expression of OA-related genes both in control and cKO mice to similar extents. ConclusionPiezo1 and Piezo2 are not essential for normal joint development. Genetic ablation of Piezo channels did not confer evident protective effects on OA progression in mice. In vitro data suggests that different mechanotransducers other than Piezo channels mediate FFSS in mechanical stress-induced gene expression.

pathology↗

bioRxiv2022

Looking for a sign forecasting failure in actions: reaching errors triggered by a slowdown of movement and specific brain activity in preceding trials

Even experts can sometimes fail while performing fully learned movements. Do such failures suddenly arise, or are there any forecasting signs? It has been reported that the kinematics of the early phase of movements can predict the failure, and brain activity patterns specific to failures are observed just before the movement onset. The presence of abnormal brain activity patterns long before (> 30 s) a failure in a cognitive task leads us to question if signs of a failure in action could exist in trials preceding the failure. Here, we examined this question using a reaching movement adaptation paradigm conventionally used to test motor learning dynamics. Firstly, the presence of a behavioral sign that preceded failures was observed: the peak velocity of the reaching movement significantly decreased in the preceding two trials. Secondly, specific theta and alpha band activity of EEG were observed in the failure trials and the trials preceding the failure. These results suggest that a failure in actions does not occur suddenly, and some signs preceding failures can be observed in the prior trials. Our approach may pave the way to investigate how we prevent failures and improve motor performance.

neuroscience↗

bioRxiv2022

Increased mitochondrial Ca2+ contributes to health decline with age and Duchene muscular dystrophy in C. elegans.

Sarcopenia is a geriatric syndrome characterized by an age-related decline in skeletal muscle mass and strength. Here, we show that suppression of mitochondrial calcium uniporter (MCU)-mediated Ca2+ influx into mitochondria in the body wall muscles of the nematode Caenorhabditis elegans improved the sarcopenic phenotypes, blunting movement and mitochondrial structural and functional decline with age. We found that normally aged muscle cells exhibited elevated resting mitochondrial Ca2+ levels and increased mitophagy to eliminate damaged mitochondria. Similar to aging muscle, we found that suppressing MCU function in muscular dystrophy improved movement via reducing elevated resting mitochondrial Ca2+ levels. Taken together, our results reveal that elevated resting mitochondrial Ca2+ levels contribute to muscle decline with age and in muscular dystrophy. Further, modulation of MCU activity may act as a potent pharmacological target in various conditions involving muscle loss.

cell biology↗

bioRxiv2022

Origin and segregation of the human germline

Human germline-soma segregation occurs during weeks 2-3 in gastrulating embryos. While direct studies are hindered, here we investigate the dynamics of human primordial germ cell (PGCs) specification using in vitro models with temporally resolved single-cell transcriptomics and in-depth characterisation to in vivo datasets from human and non-human primates, including a 3D marmoset reference atlas. We elucidate the molecular signature for the transient gain of competence for germ cell fate during peri-implantation epiblast development. Further, we show that both the PGCs and amnion arise from transcriptionally similar TFAP2A positive progenitors at the posterior end of the embryo. Notably, genetic loss of function experiments show that TFAP2A is crucial for initiating the PGC fate without detectably affecting the amnion, and its subsequently replaced by TFAP2C as an essential component of the genetic network for PGC fate. Accordingly, amniotic cells continue to emerge from the progenitors in the posterior epiblast, but importantly, this is also a source of nascent PGCs.

developmental biology↗

bioRxiv2022

High-throughput platform for depolarization-induced Ca2+ release for skeletal muscle disease mutation validation and drug discovery

In skeletal muscle excitation-contraction (E-C) coupling, depolarization of the plasma membrane triggers Ca2+ release from the sarcoplasmic reticulum (SR), referred to as depolarization-induced Ca2+ release (DICR). DICR occurs via the type 1 ryanodine receptor (RyR1), which physically interacts with the dihydropyridine receptor Cav1.1 subunit in specific machinery formed with additional essential components including {beta}1a, Stac3 adaptor protein and junctophilins. Exome sequencing has accelerated the discovery of many novel mutations in genes encoding DICR machinery in various skeletal muscle diseases. However, functional validation is time-consuming because it must be performed in a skeletal muscle environment. In this study, we established a platform of the reconstituted DICR in HEK293 cells. The essential components were effectively transduced into HEK293 cells expressing RyR1 using baculovirus vectors, and Ca2+ release was quantitatively measured with R-CEPIA1er, a fluorescent ER Ca2+ indicator, without contaminant of extracellular Ca2+ influx. In these cells, [K+]-dependent Ca2+ release was triggered by chemical depolarization with the aid of inward rectifying potassium channel, indicating a successful reconstitution of DICR. Using the platform, we evaluated several Cav1.1 mutations that are implicated in malignant hyperthermia and myopathy. We also tested several RyR1 inhibitors; whereas dantrolene and Cpd1 inhibited DICR, procaine had no effect. Furthermore, twitch potentiators such as perchlorate and thiocyanate shifted the voltage dependence of DICR to more negative potentials without affecting Ca2+-induced Ca2+ release. These results well reproduced the findings with the muscle fibers and the cultured myotubes. Since the procedure is simple and reproducible, the reconstituted DICR platform will be highly useful for validation of mutations and drug discovery for skeletal muscle diseases. SummaryMutations in essential components for depolarization-induced Ca2+ release (DICR) are implicated into various skeletal muscle diseases. Murayama et al. establish a reconstituted DICR platform in nonmuscle cells for evaluation of disease-causing mutations and drug discovery.

physiology↗

bioRxiv2022

Characterization of a KDM5 Small Molecule Inhibitor with Antiviral Activity against Hepatitis B Virus

Chronic hepatitis B (CHB) is a global health care challenge and a major cause of liver disease. To find new therapeutic avenues with a potential to functionally cure chronic Hepatitis B virus (HBV) infection, we performed a focused screen of epigenetic modifiers to identify replication inhibitors. From this work we identified isonicotinic acid inhibitors of the histone lysine demethylase 5 (KDM5) with potent anti-HBV activity. To enhance the cellular permeability and liver accumulation of the most potent KDM5 inhibitor identified (GS-080) an ester prodrug was developed (GS-5801) that resulted in improved bioavailability and liver exposure as well as an increased H3K4me3:H3 ratio on chromatin. GS-5801 treatment of HBV-infected primary human hepatocytes inhibited HBV replication and antigen levels. Evaluation of GS-5801 antiviral activity in a humanized mouse model of HBV infection, however, did not result in antiviral efficacy, despite achieving pharmacodynamic levels of H3K4me3:H3 predicted to be efficacious from the in vitro model. Here we discuss potential reasons for the disconnect between in vitro and in vivo efficacy, which highlight the translational difficulties of epigenetic targets for viral diseases.

pharmacology and toxicology↗

bioRxiv2022

Lymphoma cells are circulating in blood of Enzootic Bovine Leukosis and clonality value of virus-infected cells is a useful information for the diagnostic test

Bovine leukemia virus (BLV), a retrovirus, causes Enzootic Bovine Leukosis (EBL) in cattle following a latent infection period. The BLV infection results in polyclonal expansion of infected B-lymphocytes and [~]5% of infected cattle develop monoclonal leukosis. Since the clonal expansion of virus-infected cell is a key in the pathogenesis of EBL, assessing the clonality of malignant cells is crucial for both understanding viral pathogenesis, which might be useful for EBL diagnosis. For the investigation of clonality of BLV-infected cells in non-EBL and EBL cattle, two methods were used to evaluate the status of EBL; BLV-DNA-capture-seq method with high sensitivity and specificity and simple and cost-effective Rapid Amplification of Integration Site for BLV (BLV-RAIS) method. We found that the RAIS method efficiently detect expanded clone in EBL tissue sample as BLV-DNA-capture-seq method. Taking advantage of high frequency of BLV-infected cells in blood, we simplified RAIS method and showed that similar to BLV-DNA-capture-seq, this method could reliably provide quantitative value about clonal abundance of BLV-infected cells. Next, we aimed to establish a diagnostic blood test for EBL by using the clonality information. First, we compared clonality of BLV-infected cells in blood with that in tumor tissue in EBL cattle. There was a remarkably similar clonality between blood and tissue in each animal. Furthermore, BLV integration site information clearly showed that the same clone was the most expanded in both blood and tumor tissue, indicating that tumor cells were circulating in blood in the disease cattle. We also analyzed tumor tissue at two independent anatomical regions and found the same clones was most expanded in both regions, supporting the idea that tumor cells are systemically circulating in the diseased cattle. Finally, we compared clonality value between non-EBL and EBL cattle by using BLV-RAIS method and found that there was clear difference between non-EBL and EBL. More importantly, we found that clonality value was low in asymptomatic phase but high in EBL phase in the longitudinal cohort study. These findings have demonstrated that BLV integration site and clonality value are is a useful information to establish diagnostic blood test for EBL. That would contribute to reduction of economic damage caused by EBL and improvement of productivity in cattle industry.

zoology↗

bioRxiv2022

Spt4 drives cellular senescence by activating non-coding RNA transcription in ribosomal RNA gene clusters

Genome instability can drive aging in many organisms. The ribosomal RNA gene (rDNA) cluster is one of the most unstable regions in the genome. Replicative lifespan in budding yeast is correlated to rDNA stability, suggesting that the rDNA locus produces an aging signal. To understand the underlying mechanism, we looked for yeast mutants with more stable rDNA and longer lifespan than wild-type cells. We reveal that absence of a transcription elongation factor, Spt4, resulted in an increased rDNA stability, a reduced activity of the regulatory E-pro promoter in the rDNA, and extended replicative lifespan in a SIR2-dependent manner. Spt4-dependent lifespan restriction was abolished in the absence of non-coding RNA transcription at the E-pro locus. The amount of Spt4 increases and its function becomes more important as cells age. These findings suggest that Spt4 is a promising aging factor that accelerates cellular senescence through rDNA instability driven by non-coding RNA transcription

molecular biology↗

bioRxiv2022

Conservation and divergence of canonical and non-canonical imprinting in murids

BackgroundGenomic imprinting affects gene expression in a parent-of-origin manner and has a profound impact on complex traits including growth and behaviour. While the rat is widely used to model human pathophysiology, few imprinted genes have been identified in this murid. To systematically identify imprinted genes and genomic imprints in the rat, we used low input methods for genome-wide analyses of gene expression and DNA methylation to profile embryonic and extra-embryonic tissues at allele-specific resolution. ResultsWe identify 14 and 26 imprinted genes in these tissues, respectively, with 10 of these genes imprinted in both tissues. Comparative analyses with mouse revealed that orthologous imprinted gene expression and associated canonical DNA methylation imprints are conserved in the embryo proper of the Muridae family. However, only 3 paternally expressed imprinted genes are conserved in the extra-embryonic tissue of murids, all of which are associated with non-canonical H3K27me3 imprints. The discovery of 8 novel non-canonical imprinted genes unique to the rat is consistent with more rapid evolution of extra-embryonic imprinting. Meta-analysis of novel imprinted genes revealed multiple mechanisms by which species-specific imprinted expression may be established, including H3K27me3 deposition in the oocyte, the birth of ZFP57 binding motifs and the insertion of endogenous retroviral promoters. ConclusionsIn summary, we provide a comprehensive list of imprinted loci in the rat, reveal the extent of conservation of imprinted gene expression, and identify potential mechanisms responsible for the evolution of species-specific imprinting.

genomics↗

bioRxiv2022

KIF24 controls the clustering of supernumerary centrosomes in pancreatic ductal adenocarcinoma cells

Clustering of supernumerary centrosomes, potentially leading to cell survival and chromosomal instability, is frequently observed in cancers. However, the molecular mechanisms by which centrosome clustering is controlled in cancer cells remain largely unknown. A centrosomal kinesin, KIF24, was previously shown to restrain the assembly of primary cilia in mammalian cells. Here, we revealed that KIF24 depletion suppresses multipolar spindle formation by clustering centrosomes in pancreatic ductal adenocarcinoma (PDAC) cells harboring supernumerary centrosomes. KIF24 depletion also induced hyperproliferation and improved the mitotic progression in PDAC cells. On the other hand, disruption of primary cilia failed to affect the proliferation and spindle formation in KIF24-depleted cells. These results represent a novel role of KIF24 in suppressing centrosome clustering independent of primary ciliation in centrosome-amplified PDAC cells.

cell biology↗

bioRxiv2022

Atypical small GTPase RABL3 interacts with RAB11 to regulate early ciliogenesis in human cells

Primary cilia are near-ubiquitously assembled on cells in the human body and are broadly associated with genetic diseases and cancers. In the early stage of ciliogenesis, the ciliary vesicle (CV) is formed on the mother centriole, which nucleates the primary cilium. However, the regulatory mechanisms underlying CV formation have not yet been fully elucidated. Here, we found that the atypical small GTPase RAB-Like 3 (RABL3) is necessary to assemble primary cilia in human cells. RABL3 directly interacts with RAB11, which is involved in CV formation. RABL3 localizes around the centrosome during early ciliogenesis, reminiscent of RAB11 dynamics. Furthermore, RABL3 positively controls the CV formation like RAB11. These findings suggest that RABL3 plays an important role, in cooperation with RAB11, in CV formation during early ciliogenesis.

cell biology↗

bioRxiv2022

A Bayesian Hierarchical Model for Signal Extraction from Protein Microarrays

SO_SCPLOWUMMARYC_SCPLOWProtein microarrays are a promising technology that measure protein levels in serum or plasma samples. Due to the high technical variability of these assays and high variation in protein levels across serum samples in any population, directly answering biological questions of interest using protein microarray measurements is challenging. Using within-array ranks of protein levels for analysis can mitigate the impact of between-sample variation on downstream analysis. Although ranks are sensitive to pre-processing steps, ranking methods that accommodate uncertainty provide robust and loss-function optimal ranks. Such ranking methods require Bayesian modeling that produces full posterior distributions for parameters of interest. Bayesian models that produce such outputs have been developed for other assays, for example DNA microarrays, but those modeling assumptions are not appropriate for protein microarrays. We develop and evaluate a Bayesian model to extract a full posterior distribution of normalized fluorescent signals and associated ranks for protein microarrays, and show that it fits well to data from two studies that use protein microrarrays from different manufacturing processes. We validate the model via simulation and demonstrate the downstream impact of using estimates from this model to obtain optimal ranks.

bioinformatics↗

bioRxiv2022

A Random Forest Classifier Uses Antibody Responses to Plasmodium Antigens to Reveal Candidate Biomarkers of the Intensity and Timing of Past Exposure to Plasmodium falciparum

1Important goals of malaria surveillance efforts include accurately quantifying the burden of malaria over time, which can be useful to target and evaluate interventions. The majority of malaria surveillance methods capture active or recent infections which poses several challenges to achieving malaria surveillance goals. In high transmission settings, asymptomatic infections are common and therefore accurate measurement of malaria burden often demands active surveillance; in low transmission regions where infections are rare accurate surveillance requires sampling a large subset of the population; and in any context monitoring malaria burden over time necessitates serial sampling. Antibody responses to Plasmodium falciparum parasites persist after infection and therefore measuring antibodies has the potential to overcome several of the current difficulties associated with malaria surveillance. However, identifying which antibody responses are markers of the timing and intensity of past exposure to P. falciparum is challenging, particularly among adults who tend to be re-exposed multiple times over the course of their lifetime and therefore have similarly high antibody responses to many P. falciparum antigens. A previous analysis of 479 serum samples from individuals in three regions in southern Africa with different historical levels of P. falciparum malaria transmission (high, intermediate, and low) revealed regional differences in antibody responses to P. falciparum antigens among children under 5 years of age. Using a novel bioinformatic pipeline optimized for protein microarrays that minimizes between-sample technical variation, we used antibody responses to P. falciparum and P. vivax antigens as predictors in random forest models to classify adult samples into these three regions of differing historical malaria transmission with high accuracy. Many of the antigens that were most important for classification in these models do not overlap with previously published results and are therefore novel candidate markers for the timing and intensity of past exposure to P. falciparum. Measuring antibody responses to these antigens could potentially lead to improved malaria serosurveillance that captures the timing and intensity of past exposure.

immunology↗

bioRxiv2022

Mechanistic insights into intramembrane proteolysis by E. coli site-2 protease homolog RseP

Site-2 proteases are a conserved family of intramembrane proteases that cleave transmembrane substrates to regulate signal transduction and maintain proteostasis. Here, we elucidated crystal structures of inhibitor-bound forms of bacterial site-2 proteases including E. coli RseP. Our observations are consistent with a rearrangement of the RseP domains surrounding the active center to expose the substrate-binding site where a conserved electrostatic linkage between the transmembrane and membrane-associated domains mediates the conformational changes, suggesting that RseP has a gating mechanism to regulate substrate entry. Mutational analysis also supports that the substrate transmembrane helix is unwound by strand addition to the intramembrane {beta} sheet and is clamped at the active center for efficient cleavage. Furthermore, this substrate accommodation mechanism appears to be common across distinct intramembrane proteases.

biochemistry↗

bioRxiv2021

Microplastic pollution of commercial fishes from coastal and offshore waters, Japan

Microplastics (MPs) pollution is a worldwide issue in the marine environment. There is growing concern of consuming MPs through fish, yet the current contamination status of fish collected from the deep sea surrounding Japan remains limited. We present baseline data on MPs in commercially important fishes from the coastal and offshore waters near Kyushu, Japan (East China Sea). We examined the MPs in the digestive tracts of two pelagic (n = 150 in total) and five demersal species (n = 235 in total). The fish were caught by pole and line, and bottom trawl at different geographical positions. The MPs in pelagic fish (39.1 %) were higher than demersal fish (10.3 %) and of larger sizes. The MPs correlated with habitat depth and type. There was species variation in the shape and polymer composition of MPs. These results increase our understanding of the heterogeneous uptake of MPs by fishes.

ecology↗

bioRxiv2021

Reversing the miRNA -5p/-3p stoichiometry reveals physiological roles and targets of miR-140 miRNAs

The chondrocyte specific miR-140 miRNAs are necessary for normal endochondral bone growth in mice. miR-140 deficiency causes dwarfism and craniofacial deformity. However, the physiologically important targets of miR-140 miRNAs are still unclear. The miR-140 gene (Mir140) encodes three chondrocyte-specific microRNAs, miR-140-5p, derived from the 5 strand of primary miR-140, and miR140-3p.1 and -3p.2, derived from the 3 strand of primary miR-140. miR-140-3p miRNAs are ten times more abundant than miR-140-5p likely due to the non-preferential loading of miR-140-5p to Argonaute proteins. To differentiate the role of miR-140-5p and -3p miRNAs in endochondral bone development, two distinct mouse models, miR140-C>T, in which the first nucleotide of miR-140-5p was altered from cytosine to uridine, and miR140-CG, where the first two nucleotides of miR-140-3p were changed to cytosine and guanine, were created. These changes are expected to alter Argonaute protein loading preference of -5p and -3p to increase -5p loading and decrease -3p loading without changing the function of miR140-5p. These models presented a mild delay in epiphyseal development with delayed chondrocyte maturation. Using RNA-sequencing analysis of the two models, direct targets of miR140-5p, including Wnt11, were identified. Disruption of the predicted miR140-5p binding site in the 3 untranslated region of Wnt11 was shown to increase Wnt11 mRNA expression and caused a modest acceleration of epiphyseal development. These results show that the relative abundance of miRNA-5p and -3p can be altered by changing the first nucleotide of miRNAs in vivo, and this method can be useful to identify physiologically important miRNA targets.

genetics↗