Biology subjects

Okumura, Y.

Publications and source records attributed to Okumura, Y..

8 recordsLinked to original sources

bioRxiv2024

Shaving black fur uncovers hidden issues in p16-3MR mice

The p16-3MR mouse model has previously been reported to effectively visualize and eliminate senescent cells in vivo. However, we now report significant issues with this model. Noninvasive bioluminescence imaging without shaving the black fur detected almost no luminescence signals, but they became visible after shaving. However, these signals were very faint and attributed to the auto-luminescence of coelenterazine-h, rather than reporter gene expression. Further experiments showed no significant bioluminescent changes with aging, doxorubicin treatment, or during wound healing, contradicting earlier reports. Comparative analysis between p16-3MR mice from different sources revealed no differences, suggesting functional issues with Renilla luciferase (Rluc) and herpes simplex virus 1 thymidine kinase (HSV-TK) expression in this mouse model. Our findings underscore the necessity of using white-furred mice or ensuring proper fur shaving for accurate in vivo bioluminescence imaging. We recommend the reevaluation of previously published studies that used p16-3MR mice without proper controls to ensure data accuracy. These results provide critical insights for researchers using p16-3MR mice in senescence studies.

molecular biology↗

bioRxiv2024

Phosphodiesterase-5 inhibition inhibits epithelial ATP release and restores detrusor contractility in rats with type 2 diabetes via an increase in bladder blood flow

PurposeThe bladder dysfunction associated with type 2 diabetes mellitus (T2DM) involves urine storage and voiding disorders. We evaluated the pathologic conditions of bladder wall in a rat model of T2DM and evaluated the effects of the phosphodiesterase-5 (PDE-5) inhibitor tadalafil (TA). Materials and MethodsMale Otsuka Long-Evans Tokushima Fatty (OLETF) rats and Long-Evans Tokushima Otsuka (LETO) rats comprised T2DM and control groups. TA was orally administered for 12 weeks. The bladder blood flow and ATP released from the bladder epithelium were measured using laser speckle imaging and an organ bath bladder distention test. The expression levels of markers of hypoxia, pro-inflammatory cytokines, and growth factors in the bladder wall were measured by real-time PCR and ELISA. The contractions of bladder strips in response to KCl and carbachol were monitored in OLETF rats. ResultsThe bladder blood flow was impaired and there was greater ATP release and vesicular nucleotide transporter (VNUT) expression in the OLETF rats than in the LETO rats, but these effects were suppressed by TA administration. Furthermore, the high expression of HIF-1, 8-OHdG, IL-6, TNF-, IGF-1, and bFGF in the OLETF rats was reduced by TA administration. In the OLETF rats, the contractile responses of bladder strips to KCl and carbachol were impaired, but were restored by TA administration. ConclusionsThe impairment of bladder blood flow in rats with T2DM is associated with greater ATP release and the upregulation of VNUT, markers of hypoxia, proinflammatory cytokines, and growth factors in the bladder epithelium. PDE5 inhibition has the potential to prevent the storage and voiding dysfunction associated with T2DM.

pharmacology and toxicology↗

bioRxiv2021

All-trans retinoic acid induces GADD34 gene expression via transcriptional regulation by Six1-TLE3 and post-transcriptional regulation by p38-TTP in skeletal muscle

All-trans retinoic acid (ATRA) increases the sensitivity to unfolded protein response (UPR) in differentiating leukemic blasts. The downstream transcriptional factors of PERK, a major arm of UPR, regulates muscle differentiation. However, the role of growth arrest and DNA damage-inducible protein 34 (GADD34), one of the downstream factors of PERK, and the effects of ATRA on GADD34 expression in muscle remain unclear. In this study, we identified ATRA increased the GADD34 expression independent of the PERK signal in the gastrocnemius muscle of mice. ATRA up-regulated GADD34 expression through the transcriptional activation of it via inhibiting the interaction of homeobox Six1 and transcription co-repressor TLE3 with the MEF3-binding site on the GADD34 gene promoter in myoblasts. ATRA also inhibited the interaction of TTP, which induces mRNA degradation, with AU-rich element on GADD34 mRNA via p38 MAPK, resulting in the instability of GADD34 mRNA. Overexpressed GADD34 in myoblasts changes the type of myosin heavy chain in myotubes. These results suggest ATRA increases GADD34 expression via transcriptional and post-transcriptional regulation in myoblasts, which changes muscle fiber type in myotubes.

biochemistry↗

bioRxiv2021

Empirical estimation of marine phytoplankton assemblages in coastal and offshore areas using an in situ multi-wavelength excitation fluorometer

Phytoplankton assemblages are important for understanding the quality of primary production in marine ecosystems. Here, we describe development of a methodology for monitoring marine phytoplankton assemblages using an in situ multi-wavelength excitation fluorometer (MEX) and its application for seasonal observations in coastal and offshore areas around Japan. The MEX recorded the fluorescence excited with nine light-emitting diodes, temperature, and sensor depth. We prepared reference datasets comprising temperature, MEX fluorescence, and plant-pigment-based chemotaxonomy phytoplankton assemblages. Target MEX fluorescence was decomposed by reference MEX fluorescence using a linear inverse model for calculating coefficients after the reference data were limited by temperature, followed by reconstruction of plant-pigment-based chemotaxonomy of the target MEX fluorescence using the coefficients and the chemotaxonomy assemblages of the reference data. Sensitivity analysis indicated poor estimation of the proportion and/or chlorophyll a-based abundance of chlorophytes, haptophytes, prasinophytes, and prochlorophytes; however, limiting the estimations to five chemotaxonomic groups [diatoms, dinoflagellates, cryptophytes, cyanobacteria (cyanophytes and prochlorophytes), and other eukaryotes (chlorophytes, haptophytes, and prasinophytes)] resulted in positive correlations of both the proportion and abundances, suggesting that the five taxonomic abundances were well-estimated using the MEX. Additionally, MEX observations denoted spatial and seasonal variations of phytoplankton assemblages, with high contributions from other eukaryotes in every area and season, cyanobacteria highly during the summer in surface Kuroshio and Japan Sea waters, and diatoms in the Oyashio and Oyashio-Kuroshio transition areas and the Okhotsk Sea. Furthermore, ratios of water-column-integrated chlorophyll-based abundances to those on the surface at the chemotaxonomy group level were differed among the areas and groups. These findings suggested that phytoplankton-assemblage monitoring in the vertical direction is essential for evaluation of their current biomass, and that the MEX promotes the acquisition of these observations.

ecology↗

bioRxiv2020

Cystal structure of inhibitor-bound human MSPL/TMPRSS13 that can activate high pathogenic avian influenza

Infection of certain influenza viruses is triggered when its hemagglutinin (HA) is cleaved by host cell proteases such as proprotein convertases and type II transmembrane serine proteases (TTSP). HA with a monobasic motif is cleaved by trypsin-like proteases, including TMPRSS2 and HAT, while the multibasic motif found in high pathogenicity avian influenza HA is cleaved by furin, PC5/6, or MSPL. MSPL belongs to the TMPRSS family and preferentially cleaves [R/K]-K-K-R{downarrow} sequences. Here, we solved the crystal structure of the extracellular region of human MSPL in complex with an irreversible substrate-analog inhibitor. The structure revealed three domains clustered around the C-terminal -helix of the SPD. The inhibitor structure and its putative model show that the P1-Arg inserts into the S1 pocket, whereas the P2-Lys and P4-Arg interacts with the Asp/Glu-rich 99-loop that is unique to MSPL. Based on the structure of MSPL, we also constructed a homology model of TMPRSS2, which is essential for the activation of the SARS-CoV-2 spike protein and infection. The model may provide the structural insight for the drug development for COVID-19.

biophysics↗

bioRxiv2020

Outer membrane-deprived cyanobacteria liberate periplasmic and thylakoid luminal components that support the growth of heterotrophs

Chloroplasts originate from endosymbiosis of a cyanobacterium within a heterotrophic host cell. Establishing endosymbiosis requires the translocation across its envelope of photosynthetic products generated inside the once free-living cyanobacterium to be exploited by host metabolism. However, the nature of this translocation event is unknown. We previously found that most cyanobacterial outer membrane components were eliminated during the primitive stage of chloroplast evolution, suggesting the importance of evolutionary changes of the outer membrane. Here, we removed the outer membrane from Synechocystis sp. PCC 6803 by disrupting the physical interaction with peptidoglycan, and characterized the effects on cell function. Outer membrane-deprived cells liberated diverse substances into the environment without significantly compromising photoautotrophic growth. The amount of liberated proteins increased to ~0.35 g/L within five days of culture. Proteomic analysis showed that most liberated proteins were periplasmic and thylakoid luminal components. Connectivity between the thylakoid lumen-extracellular space was confirmed by findings that an exogenous hydrophilic oxidant was reduced by photosynthetic electron transport chain on the thylakoid membrane. Metabolomic analysis detected the release of nucleotide-related metabolites at concentrations around 1 M. The liberated materials supported the proliferation of heterotrophic bacteria. These findings show that breaching the outer membrane, without any manipulations to the cytoplasmic membrane, converts a cyanobacterium to a chloroplast-like organism that conducts photosynthesis and releases its biogenic materials. This conversion not only represents a potential explanation why the outer membrane markedly changed during the earliest stage of chloroplast evolution, but also provides the opportunity to harness cyanobacterial photosynthesis for biomanufacturing processes. SIGNIFICANCE STATEMENTAlthough it is well accepted that chloroplasts stem from endosymbiosis of a cyanobacterium within a heterotrophic host cell, the issue of how photosynthetic products generated inside a formerly free-living cyanobacterium are translocated across its envelope and exploited by host metabolism has been little addressed. Here we show that breaching the cyanobacterial outer membrane barrier converts a cyanobacterium to a chloroplast-like organism that conducts photosynthesis and releases its diverse biogenic materials into its external environment, which sustains the growth of heterotrophic organisms. This conversion represents a possible example of metabolic exploitation of cyanobacterial photosynthesis. Further, this "quasi-chloroplast" provides a potential opportunity for industrial application such as producing feedstock for biomanufacturing processes that harnesses heterotrophic bacteria.

microbiology↗

bioRxiv2019

The Prevalence and Patterns of Combined Psychotropic Polypharmacy among Acute Care Hospitals in Japan

PurposeTo evaluate the prescribing patterns of psychotropic polypharmacy for inpatients of acute care hospitals in Japan.\n\nMethodsAdministrative data on 2,639,885 patients admitted to acute care hospitals in Japan between July and December of 2008 were analyzed retrospectively. We defined psychotropic medications as antipsychotics, antidepressants, benzodiazepines, and other sedatives/hypnotics and studied their prescription patterns during the hospitalization of patients with stroke, acute cardiac infarction, cancer, and diabetes mellitus.\n\nResultsAt least one psychotropic drug was prescribed in 35.9% of all cases. Two-drug combinations of antipsychotic drugs were prescribed for stroke patients in 14,615 cases (1.4%), more than 2 in 3,132 cases (0.3%), and 22.4% of cases were prescribed 2 or more psychotropic drugs in addition to antipsychotic drugs. Amongst upper gastrointestinal cancer patients, 7.7% were prescribed a combination of 2 or more drugs, including benzodiazepines. Of the upper gastrointestinal cancer patients who were prescribed benzodiazepines, 20.3% were also prescribed 2 or more psychotropic drugs. Amongst stroke and upper gastrointestinal cancer patients, 36.6% and 35.6%, respectively, were treated with combination therapy using drugs of this class and others.\n\nConclusionThere is a pattern of polypharmacy that combines benzodiazepines and other sedatives/hypnotics with antidepressants or antipsychotic drugs, and this study provides a detailed analysis of this within acute care hospitals. Our results indicate the need for additional research into the efficacy of polypharmacy for inpatients in non-psychiatric settings.

epidemiology↗

bioRxiv2018

Parallelized analysis of spatial gene expression patterns by database integration

We developed a computational framework for automated integration of a large number of two-dimensional (2D) images with three-dimensional (3D) image datasets located in the standard 3D coordinate. We applied the framework to 2,810 para-sagittal sectioned mouse brain 2D images of in situ hybridization (ISH), archived in the BrainTx database (http://www.cdtdb.neuroinf.jp). We registered the ISH images into the mouse standard coordinate space for MR images, Waxholm space (WHS, https://www.nitrc.org/projects/incfwhsmouse) by linearly transforming them into each of a series of para-sagittal MR image slices, and identifying the best-fit slice by calculating the similarity metric value ({delta}). Transformed 2D images were compared with 3D gene expression image datasets, which were made using a microtomy-based microarray assay system, Transcriptome Tomography, and archived in the ViBrism DB (http://vibrism.neuroinf.jp): the 3D images are located in the WHS.\n\nWe first transformed ISH images of 10 regionally expressed genes and compared them to signals of corresponding 3D expression images in ViBrism DB for evaluating the integration schema: two types of data, produced with different modalities and originally located in different dimensions, were successfully compared after enhancing ISH signals against background noise. Then, for the massive transformation of BrainTx database images, we parallelized our framework, using the IPython cluster package, and implemented it on the PC cluster provided for the Brain Atlasing Hackathon activity hosted by Neuroinformatics Japan Center in Japan. We could identify the best-fit positions for all of the ISH images. All programs were made available through the GitHub repository, at the web site of neuroinformatics/bah2016_registration (https://github.com/neuroinformatics/bah2016_registration).

neuroscience↗