Biology subjects

Ishii, K.

Publications and source records attributed to Ishii, K..

33 records · Page 2Linked to original sources

bioRxiv2023

Fluctuation of lysosomal protein degradation in neural stem cells of postnatal mouse brain

Lysosomes are intracellular organelles responsible for degrading diverse macromolecules delivered from several pathways, such as the endo-lysosomal and autophagic pathways. Recent reports have suggested that lysosomes are essential in regulating neural stem cells in developing, adult, and aged brains. However, the activity of these lysosomes has not yet been monitored in these brain tissues. Here, we report a new probe to measure lysosomal protein degradation in brain tissue by immunostaining. Our results demonstrate the fluctuation of lysosomal protein degradation in neural stem cells depending on age and brain disorder. Neural stem cells increase lysosomal activity during hippocampal development in the dentate gyrus, but aging and aging-related disease reduces their activity. In addition, physical exercise increases lysosomal activity in neural stem cells and astrocytes. We hypothesize three different stages of lysosomal activity: the increase in development, the stable state for the adult stage, and the reduction by damages with age or disease.

neuroscience↗

bioRxiv2022

Oncolytic adenovirus serotype 35 mediated tumor growth suppression via efficient activation and tumor infiltration of natural killer cells

BackgroundOncolytic adenoviruses (OAds) mediate superior antitumor effects both by inducing direct oncolysis and activating antitumor immunity. Previously, we developed a novel OAd fully composed of human adenovirus serotype 35 (OAd35). OAd35 efficiently killed a variety of human tumor cells; however, OAd35-mediated activation of antitumor immunity remains to be evaluated. In this study, we examined whether OAd35-induced activation of immune cells contributes to the antitumor effects of OAd35. MethodsTumor infiltration and activation of immune cells following intratumoral administration of OAd35 in tumor-bearing immune-competent and nude mice were analyzed. The involvement of natural killer (NK) cells in the tumor growth-suppression effects of OAd35 was evaluated in NK cell-depleted mice. The key signals for the OAd35-mediated tumor infiltration of NK cells were examined in interferon (IFN) alpha and beta receptor subunit 1 (IFNAR1) knockout and toll-like receptor 9 (TLR9) knockout mice. ResultsOAd35 efficiently induced tumor infiltration of activated NK cells. NK cell depletion apparently hindered the OAd35-mediated tumor growth suppression. In IFNAR1 knockout mice, OAd35-induced tumor infiltration of activated NK cells was significantly attenuated. OAd35 did not induce tumor infiltration of NK cells in TLR9 knockout mice, although OAd35 significantly activated NK cells and showed tumor growth suppression in TLR9 knockout mice. ConclusionsOAd35 significantly promoted activation and tumor infiltration of NK cells, leading to OAd35-mediated efficient tumor growth suppression. The type-I IFN signal was crucial for the OAd35-mediated tumor infiltration and activation of NK cells. The TLR9 signal was highly related to tumor infiltration of NK cells, but not NK cell activation and antitumor effects of OAd35. These findings suggest that OAd35 becomes a promising cancer immunotherapy agent via its enhancement of the antitumor activities of NK cells.

cancer biology↗

bioRxiv2022

Drosophila epidermal cells are intrinsically mechanosensitive and drive nociceptive behavioral outputs

Somatosensory neurons (SSNs) that detect and transduce mechanical, thermal, and chemical stimuli densely innervate an animals skin. However, although epidermal cells provide the first point of contact for sensory stimuli. our understanding of roles that epidermal cells play in SSN function, particularly nociception, remains limited. Here, we show that stimulating Drosophila epidermal cells elicits activation of SSNs including nociceptors and triggers a variety of behavior outputs, including avoidance and escape. Further, we find that epidermal cells are intrinsically mechanosensitive and that epidermal mechanically evoked calcium responses require the store-operated calcium channel Orai. Epidermal cell stimulation augments larval responses to acute nociceptive stimuli and promotes prolonged hypersensitivity to subsequent mechanical stimuli. Hence, epidermal cells are key determinants of nociceptive sensitivity and sensitization, acting as primary sensors of noxious stimuli that tune nociceptor output and drive protective behaviors.

neuroscience↗

bioRxiv2022

Single Photon smFRET. III. Application to Pulsed Illumination

Forster resonance energy transfer (FRET) using pulsed illumination has been pivotal in leveraging lifetime information in FRET analysis. However, there remain major challenges in quantitative single photon, single molecule FRET (smFRET) data analysis under pulsed illumination including: 1) simultaneously deducing kinetics and number of system states; 2) providing uncertainties over estimates, particularly uncertainty over the number of system states; 3) taking into account detector noise sources such as crosstalk, and the instrument response function contributing to uncertainty; in addition to 4) other experimental noise sources such as background. Here, we implement the Bayesian nonparametric framework described in the first companion manuscript that addresses all aforementioned issues in smFRET data analysis specialized for the case of pulsed illumination. Furthermore, we apply our method to both synthetic as well as experimental data acquired using Holliday junctions. Why It MattersIn the first companion manuscript of this series, we developed new methods to analyze noisy smFRET data. These methods eliminate the requirement of a priori specifying the dimensionality of the physical model describing a molecular complexs kinetics. Here, we apply these methods to experimentally obtained datasets with samples illuminated by laser pulses at regular time intervals. In particular, we study conformational dynamics of Holliday junctions.

biophysics↗

bioRxiv2022

An attenuated vaccinia vaccine encoding the SARS-CoV-2 spike protein elicits broad and durable immune responses, and protects cynomolgus macaques and human ACE2 transgenic mice from SARS-CoV-2 and its variants

As long as the coronavirus disease 2019 (COVID-19) pandemic continues, new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with altered antigenicity will emerge. The development of vaccines that elicit robust, broad, and durable protection against SARS-CoV-2 variants is urgently needed. We have developed a vaccine (rDIs-S) consisting of the attenuated vaccinia virus DIs strain platform carrying the SARS-CoV-2 S gene. rDIs-S induced neutralizing antibody and T-lymphocyte responses in cynomolgus macaques and human angiotensin converting enzyme 2 (hACE2) transgenic mice, and showed broad protection against SARS-CoV-2 isolates ranging from the early-pandemic strain (WK-521) to the recent Omicron BA. 1 variant (TY38-839). Using a tandem mass tag (TMT) -based quantitative proteomic analysis of lung homogenates from hACE2 transgenic mice, we found that, among mice subjected to challenge infection with WK-521, vaccination with rDIs-S prevented protein expression related to the severe pathogenic effects of SARS-CoV-2 infection (tissue destruction, inflammation, coagulation, fibrosis, and angiogenesis) and restored protein expression related to immune responses (antigen presentation and cellular response to stress). Furthermore, long-term studies in mice showed that rDIs-S maintains S protein-specific antibody titers for at least 6 months after a 1st vaccination. Thus, rDIs-S appears to provide broad and durable protective immunity against SARS-CoV-2, including current and possibly future variants.

microbiology↗

bioRxiv2022

Male-killing-associated bacteriophage WO identified from comparisons of Wolbachia endosymbionts of Homona magnanima

The origin and mechanism of male-killing, an advantageous strategy employed by maternally transmitted symbionts such as Wolbachia, remain unclear. We compared genomes of four Wolbachia strains derived from Homona magnanima, a male-killing strain wHm-t (1.5 Mb), and three non-male-killing strains, wHm-a (1.1 Mb), wHm-b (1.3 Mb), and wHm-c (1.4 Mb). A wHm-t-specific 76-kbp prophage region harboured two tandemly arrayed WO-mediated killing (wmk) gene homologs (wmk-1/wmk-2 and wmk-3/wmk-4). Of these, wmk-1 or wmk-3 killed almost all Drosophila melanogaster individuals when transgenically overexpressed. Dual expression of wmk-3 and wmk-4 killed all males and rescued females. We propose a novel hypothesis wherein horizontally transmitted proto-Wolbachia with a single wmk killed both sexes, and tandem duplication of wmk allowed an evolutionary transition to a vertically transmitted symbiont, causing male-killing. Our study highlights the bacteriophage as a critical driver of the evolution of male-killing and argues for a conserved male-killing mechanism in diverse insects.

evolutionary biology↗

bioRxiv2022

An attempt of using public ambient temperature data in swine genetic evaluation for litter size traits at birth in Japan

To obtain the fundamental information on using ambient temperature information in developing the model for routine swine genetic evaluation in Japan, we analyzed total number born (TNB), number born alive (NBA), and number stillborn (NSB) collected at a Japanese farm, together with off-farm ambient temperature measured at a nearest Automated Meteorological Data Acquisition System station. Five repeatability animal models were exploited, considering the effects of farrowing season (model 1), farrowing month (model 2), quadratic regressions of daily maximum ambient temperature of farrowing day (model 3), season and temperature (model 4), or month and temperature (model 5). Patterns of the effects of daily maximum temperature of farrowing day estimated using model 3 was similar to those of farrowing season by model 1 and those of farrowing month by model 2. Adding the effect of daily maximum temperature of farrowing day (models 4 and 5) could explain phenotypic variability greater than only considering either of farrowing season and month (models 1 and 2). Estimated heritability was stable among the models and the rank correlation of predicted breeding values between models was >0.98 for all traits. The results indicate the possibility that using public ambient temperature can capture a large part of the phenotypic variability in litter size traits at birth caused by the seasonality in Japan and do not harm, at least, the performance of genetic evaluation. This study could support the availability of public meteorological data in flexible developing operational models for future swine genetic evaluation in Japan.

genetics↗

bioRxiv2020

Comprehensive characterization of migration profiles of murine cerebral cortical neurons during development using FlashTag labeling

In mammalian cerebral neocortex, different regions have different cytoarchitecture, neuronal birthdates and functions. In most regions, neuronal migratory profiles have been speculated similar to each other based on observations using thymidine analogues. Few reports investigated regional migratory differences from mitosis at the ventricular surface. Here, in mice, we applied FlashTag technology, in which dyes are injected intraventricularly, to describe migratory profiles. We revealed a mediolateral regional difference in migratory profiles of neurons that is dependent on the developmental stages, e.g., neurons labeled at E12.5-15.5 reached their destination earlier dorsomedially than dorsolaterally even where there were underlying ventricular surfaces, reflecting sojourning below the subplate. This difference was hardly recapitulated by thymidine analogues, which visualize neurogenic gradient, suggesting biological significance different from neurogenic gradient. These observations advance understanding of cortical development, portraying strength of FlashTag in studying migration, and are thus a resource for studies of normal and abnormal neurodevelopment.

neuroscience↗

bioRxiv2020

A Continuous Time Representation of smFRET for the Extraction of Rapid Kinetics

Our goal is to learn kinetic rates from single molecule FRET (smFRET) data even if these exceed the data acquisition rate. To achieve this, we develop a variant of our recently proposed hidden Markov jump process (HMJP) with which we learn transition kinetics from parallel measurements in donor and acceptor channels. Our HMJP generalizes the hidden Markov model (HMM) paradigm in two critical ways: (1) it deals with physical smFRET systems as they switch between conformational states in continuous time; (2) it estimates the transition rates between conformational states directly without having recourse to transition probabilities or assuming slow dynamics (as is necessary of the HMM). Our continuous time treatment learns transition kinetics and photon emission rates for dynamical regimes inaccessible to the HMM which treats system kinetics in discrete time. We validate the robustness of our framework on simulated data and demonstrate its performance on experimental data from FRET labeled Holliday junctions.

biophysics↗

bioRxiv2020

Direct Photon-by-photon Analysis of Time-Resolved Pulsed Excitation Data using Bayesian Nonparametrics

Lifetimes of chemical species are typically estimated, across each illuminated spot of a sample, by either fitting time correlated single photon counting (TCSPC) decay histograms or, more recently, through phasor analysis from time-resolved photon arrivals. While both methods yield lifetimes in a computationally efficient manner, the performance of both methods is limited by the choices made when fitting a TCSPC histogram. In addition, phasor analysis also requires setting the number of chemical species by hand before lifetimes can be determined. Yet the number of species itself is encoded in the photon arrival times collected for each illuminated spot and need not be set by hand a priori. Here we propose a direct photo-by-photon analysis of data drawn from pulsed excitation experiments to infer, simultaneously and self-consistently, the number of species and their associated lifetimes from as little as a few thousand photons for two species. We do so by leveraging new mathematical tools within the Bayesian nonparametric (BNP) paradigm that we have previously exploited in the analysis of single photon arrivals from single spot confocal microscopy. We benchmark our method on simulated as well as experimental data for one, two, three, and four species with data sets from both immobilized and freely diffusing molecules at the level of one illuminated spot. SUMMARYPhoton arrivals obtained from fluorescence experiments encode not only the lifetimes of chemical species but also the number of chemical species involved in the experiment. Traditional methods of analysis, such as phasor methods and methods relying on maximum likelihood or (parametric) Bayesian analysis of photon arrivals or photon arrival histograms of TCSPC data, must first ascertain the number of chemical species separately and, once specified, determine their associated lifetimes. Here we develop a method to learn the number of fluorescence species and their associated lifetimes simultaneously. We achieve this by exploiting Bayesian nonparametrics. We benchmark our approach on both simulated and experimental data for one species and mixtures of two to four species.

biophysics↗

bioRxiv2020

A neurogenetic mechanism of experience-dependent suppression of aggression

Aggression is an ethologically important social behavior1 but excessive aggression can be detrimental to animal fitness2,3. Social experiences among conspecific individuals reduce aggression in a wide range of animals4. However, the genetic and neural basis for the experience-dependent suppression of aggression remains largely unknown. Here we found that nervy (nvy), a Drosophila homolog of vertebrate myeloid translocation gene (MTG)5 involved in transcriptional regulation6-8, suppresses aggression via its action in a specific subset of neurons. Loss-of-function mutation of the nvy gene resulted in hyper-aggressiveness only in socially experienced flies, whereas overexpression of nvy suppressed spontaneous aggression in socially naive flies. The loss-of-function nvy mutant exhibited persistent aggression under various contexts in which wild-type flies transition to escape or courtship behaviors. Knockdown of nvy in octopaminergic/tyraminergic (OA/TA) neurons increased aggression, phenocopying the nvy mutation. We found that a subpopulation of OA/TA cells specifically labeled by nvy is required for the social-experience-dependent suppression of aggression. Moreover, cell-type-specific transcriptomics on nvy-expressing OA/TA neurons revealed aggression-controlling genes that are likely downstream of nvy. Our results are the first to describe the presence of a specific neuronal subpopulation in the central brain that actively suppresses aggression in a social-experience-dependent manner, illuminating the underlying genetic mechanism.

neuroscience↗

bioRxiv2020

Quantitative comparison of Drosophila behavior annotations by human observers and a machine learning algorithm

Automated quantification of behavior is increasingly prevalent in neuroscience research. Human judgments can influence machine-learning-based behavior classification at multiple steps in the process, for both supervised and unsupervised approaches. Such steps include the design of the algorithm for machine learning, the methods used for animal tracking, the choice of training images, and the benchmarking of classification outcomes. However, how these design choices contribute to the interpretation of automated behavioral classifications has not been extensively characterized. Here, we quantify the effects of experimenter choices on the outputs of automated classifiers of Drosophila social behaviors. Drosophila behaviors contain a considerable degree of variability, which was reflected in the confidence levels associated with both human and computer classifications. We found that a diversity of sex combinations and tracking features was important for robust performance of the automated classifiers. In particular, features concerning the relative position of flies contained useful information for training a machine-learning algorithm. These observations shed light on the importance of human influence on tracking algorithms, the selection of training images, and the quality of annotated sample images used to benchmark the performance of a classifier (the ‘ground truth’). Evaluation of these factors is necessary for researchers to accurately interpret behavioral data quantified by a machine-learning algorithm and to further improve automated classifications.Significance Statement Accurate quantification of animal behaviors is fundamental to neuroscience. Here, we quantitatively assess how human choices influence the performance of automated classifiers trained by a machine-learning algorithm. We found that human decisions about the computational tracking method, the training images, and the images used for performance evaluation impact both the classifier outputs and how human observers interpret the results. These factors are sometimes overlooked but are critical, especially because animal behavior is itself inherently variable. Automated quantification of animal behavior is becoming increasingly prevalent: our results provide a model for bridging the gap between traditional human annotations and computer-based annotations. Systematic assessment of human choices is important for developing behavior classifiers that perform robustly in a variety of experimental conditions.Competing Interest StatementThe authors have declared no competing interest.View Full Text

animal behavior and cognition↗

bioRxiv2019

Structural basis for ligand-induced inactivation of protein tyrosine receptor type Z (PTPRZ): Physiological relevance of head-to-toe RPTP dimerization

Protein tyrosine phosphatase receptor type Z (PTPRZ) has two receptor isoforms (PTPRZ-A and -B) containing tandem PTP-D1 and -D2 domains intracellularly, with only D1 being active. Pleiotrophin (PTN) binding to the extracellular region of PTPRZ leads to the inactivation of PTPase, thereby inducing oligodendrocyte precursor cell (OPC) differentiation and myelination in the CNS. However, the mechanisms responsible for the ligand-induced inactivation of PTPRZ remain unclear. We herein revealed that the crystal structure of the intracellular region of PTPRZ (PTPRZ-ICR) showed the \"head-to-toe\"-type dimer conformation, with D2 masking the catalytic site of D1. Mass spectrometry (MS) revealed that PTPRZ-ICR proteins remained in monomer-dimer equilibrium in aqueous solution, and a substrate-derived inhibitory peptide or competitive inhibitor (SCB4380) specifically bound to the monomer form in a 1:1 stoichiometric ratio, supporting the \"head-to-toe dimerization\" model for inactivation. A D2 deletion ({Delta}D2) or dimer interface mutation (DDKK) disrupted dimer formation, while the binding of SCB4380 was maintained. Similar to wild-type PTPRZ-B, monomer-biased PTPRZ-B-{Delta}D2 and PTPRZ-B-DDKK mutants efficiently dephosphorylated p190RhoGAP at Tyr-1105 when co-expressed in BHK-21 cells. The catalytic activities of these mutants were not suppressed by a treatment with PTN, but were inhibited by the cell-permeable PTPase inhibitor NAZ2329. The PTN treatment did not enhance OPC differentiation in primary cultured glial cells prepared from {Delta}D2 or catalytically-inactive CS mutant knock-in mice. Our results indicate that PTN-induced PTPRZ inactivation is attained by dimer formation of the intracellular tandem PTP domains in the head-to-toe configuration, which is physiologically relevant to the control of OPC differentiation in vivo.

biochemistry↗

bioRxiv2019

An asexual flower of Silene latifolia and Microbotryum lychnidis-dioicae promoting its sexual-organ development

Silene latifolia is a dioecious flowering plant with sex chromosomes in the family Caryophyllaceae. Development of a gynoecium and stamens are suppressed in the male and female flowers of S. latifolia, respectively. Microbtryum lychnidis-dioicae promotes stamen development when it infects the female flower. If suppression of the stamen and gynoecium development is regulated by the same mechanism, suppression of gynoecium and stamen development is released simultaneously with the infection by M. lychnidis-dioicae. To assess this hypothesis, an asexual mutant, without gynoecium or stamen, was infected with M. lychnidis-dioicae. A filament of the stamen in the infected asexual mutant was elongated at stages 11 and 12 of the flower bud development as well as the male, but the gynoecium did not form. Instead of the gynoecium, a filamentous structure was suppressed as in the male flower. Developmental suppression of the stamen was released by M. lychnidis-dioicae, but that of gynoecium development was not released. It is thought, therefore, that the suppression of gynoecium development was not released by the infection of M. lychnidis-dioicae. M. lychnidis-dioicae would have a function similar to SPF since the elongation of the stamen that is not observed in the healthy asexual mutant was observed after stage 8 of flower bud development. Such an infection experiment also that the Y chromosome of the asexual mutant has genes related to the differentiation of archesporial cells, but none related to maturation of the tapetal cells.

plant biology↗

bioRxiv2018

Influence of switching rule on motor learning

Humans and animals can flexibly switch rules to generate appropriate motor commands; for example, actions can be flexibly produced toward a sensory stimulus (e.g., pro-saccade or pro-reaching) or away from a sensory stimulus (e.g., anti-saccade or anti-reaching). Distinct neural activities are related to pro- and anti-movement actions; however, the effects of switching rules on motor learning are unclear. Here, we study the effect of switching rules on motor learning using pro- and anti-arm-reaching movements and a visuomotor rotation task. Although previous results support the perfect availability of learning effects under the same required movements, we show that the learning effects trained in pro-reaching movements are partially rather than perfectly available in anti-reaching movements even under the same required movement direction between those two conditions. The partial transfer is independent of the difference in the visual cue, the cognitive demand, and the actual movement direction between the pro- and anti-reaching movements. We further demonstrate that the availability of learning effects trained with pro-reaching movements is partial not only in anti-reaching movements but in reaching movements with other rules and the availability of learning effects trained with anti-reaching movements is also partial in pro-reaching movements. We thus conclude that the switching rule causes the availability of learning effects to be partial rather than perfect even under same planned movements.\n\nNew & Noteworthy\n\nMost motor learning experiments supported the involvement of planned movement directions in motor learning; the learning effects trained in a movement direction can be available at movement directions close to the trained one. Here, we show that the availability of motor learning effects is partial rather than perfect even under the same planned movements when rule is switched, which indicates that sports training and rehabilitation should include various situations under the same required motions.

neuroscience↗