Biology subjects

Sato, T.

Publications and source records attributed to Sato, T..

At least 91 records · Page 5Linked to original sources

bioRxiv2023

Effects of focused ultrasound in a "clean" mouse model of ultrasonic neuromodulation

Recent studies on ultrasonic neuromodulation (UNM) in rodents have shown that focused ultrasound (FUS) can activate peripheral auditory pathways, leading to off-target and brain-wide excitation, which obscures the direct activation of the target area by FUS. To address this issue, we developed a new mouse model, the double transgenic Pou4f3+/DTR x Thy1-GCaMP6s, which allows for inducible deafening using diphtheria toxin and minimizes off-target effects of UNM while allowing effects on neural activity to be visualized with fluorescent calcium imaging. Using this model, we found that the auditory confounds caused by FUS can be significantly reduced or eliminated within a certain pressure range. At higher pressures, FUS can result in focal fluorescence dips at the target, elicit non-auditory sensory confounds, and damage tissue, leading to spreading depolarization. Under the acoustic conditions we tested, we did not observe direct calcium responses in the mouse cortex. Our findings provide a cleaner animal model for UNM and sonogenetics research, establish a parameter range within which off-target effects are confidently avoided, and reveal the non-auditory side effects of higher-pressure stimulation.

neuroscience↗

bioRxiv2023

UBL3 interaction with α-synuclein is downregulated by silencing MGST3

Ubiquitin-like 3 (UBL3) is a membrane-anchored protein that has been discovered to function as a protein post-translational modifier, helping to sort proteins into small extracellular vesicles (sEVs). Aggregations of alpha-synuclein (-syn) are associated with the pathology of neurodegenerative diseases such as Parkinsons disease (PD). The aggregation and toxicity of -syn can be influenced by its interactions with specific proteins. Recently, the interactions between UBL3 and -syn was uncovered. It is believed to play a role in eliminating excess -syn from neurons, or in the spread of -syn pathology in the brain associated with neurodegenerative diseases. However, the regulator that can mediate the interaction between UBL3 and -syn remains unclear. In this study, we employed the split gaussian luciferase complementation assay and RNA interference (RNAi) technology to discover that QSOX2, HTATIP2, UBE3C, MGST3, NSF, HECTD1, SAE1 and ATG3 are involved in downregulating the interaction between UBL3 and -syn. Among these proteins, silencing MGST3 had the most significant impact on the UBL3--syn interaction (with a fold change log2 of less than - 1). MGST3 is a part of the antioxidant system, and silencing MGST3 is believed to contribute to oxidative stress. We used hydrogen peroxide (H2O2) to induce oxidative stress and observed its effect on the UBL3--syn interaction. Our finding showed that an 800M concentration of H2O2 could also downregulate this interaction. However, the effect of oxidative stress caused by 800M H2O2 on the UBL3--syn interaction did not be enhanced by silencing MGST3. In conclusion, the interaction between UBL3 and -syn is downregulated by silencing MGST3.

molecular biology↗

bioRxiv2023

The plasma degradome reflects later development of NASH fibrosis after liver transplant

Although liver transplantation (LT) is an effective therapy for cirrhosis, the risk of post-LT NASH is alarmingly high and is associated with accelerated progression to fibrosis/cirrhosis, cardiovascular disease, and decreased survival. Lack of risk stratification strategies hamper liver undergoes significant remodeling during inflammatory injury. During such remodeling, degraded peptide fragments (i.e., degradome) of the ECM and other proteins increase in plasma, making it a useful diagnostic/prognostic tool in chronic liver disease. To investigate whether inflammatory liver injury caused by post-LT NASH would yield a unique degradome profile, predictive of severe post-LT NASH fibrosis, we performed a retrospective analysis of 22 biobanked samples from the Starzl Transplantation Institute (12 with post-LT NASH after 5 years and 10 without). Total plasma peptides were isolated and analyzed by 1D-LC-MS/MS analysis using a Proxeon EASY-nLC 1000 UHPLC and nanoelectrospray ionization into an Orbitrap Elite mass spectrometer. Qualitative and quantitative peptide features data were developed from MSn datasets using PEAKS Studio X (v10). LC-MS/MS yielded [~]2700 identifiable peptide features based on the results from Peaks Studio analysis. Several peptides were significantly altered in patients that later developed fibrosis and heatmap analysis of the top 25 most significantly-changed peptides, most of which were ECM-derived, clustered the 2 patient groups well. Supervised modeling of the dataset indicated that a fraction of the total peptide signal ([~]15%) could explain the differences between the groups, indicating a strong potential for representative biomarker selection. A similar degradome profile was observed when the plasma degradome patterns were compared being obesity sensitive (C57Bl6/J) and insensitive (AJ) mouse strains. Both The plasma degradome profile of post-LT patients yields stark difference based on later development of post-LT NASH fibrosis. This approach could yield new "fingerprints" that can serve as minimally-invasive biomarkers of negative outcomes post-LT.

pathology↗

bioRxiv2023

SIRT2 inhibition protects against cardiac hypertrophy and heart failure

Sirtuins (SIRT) exhibit deacetylation or ADP-ribosyltransferase activity and regulate a wide range of cellular processes in the nucleus, mitochondria and cytoplasm. The role of the only sirtuin that resides in the cytoplasm, SIRT2, in the development of heart failure (HF) and cardiac hypertrophy is not known. In this paper, we show that the hearts of mice with deletion of Sirt2 (Sirt2-/-) display improved cardiac function after ischemia-reperfusion (I/R) and pressure overload (PO), suggesting that SIRT2 exerts maladaptive effects in the heart in response to stress. Similar results were obtained in mice with cardiomyocyte-specific Sirt2 deletion. Mechanistic studies suggest that SIRT2 modulates cellular levels and activity of nuclear factor (erythroid-derived 2)-like 2 (NRF2), which results in reduced expression of antioxidant proteins. Deletion of Nrf2 in the hearts of Sirt2-/- mice reversed protection after PO. Finally, treatment of mouse hearts with a specific SIRT2 inhibitors reduces cardiac size and attenuates cardiac hypertrophy in response to PO. These data indicate that SIRT2 has detrimental effects in the heart and plays a role in the progression of HF and cardiac hypertrophy, which makes this protein a unique member of the SIRT family. Additionally, our studies provide a novel approach for treatment of cardiac hypertrophy by targeting SIRT2 pharmacologically, providing a novel avenue for the treatment of this disorder.

molecular biology↗

bioRxiv2022

Cerebrospinal fluid-contacting neuron tracing reveals structural and functional connectivity for locomotion in the mouse spinal cord

Cerebrospinal fluid-contacting neurons (CSF-cNs) are enigmatic mechano- or chemosensory cells lying along the central canal of the spinal cord. Recent studies in zebrafish larvae and lampreys have shown that CSF-cNs control postures and movements via spinal connections. However, the structures, connectivity, and functions in mammals remain largely unknown. Here we developed a method to genetically target mouse CSF-cNs that highlighted structural connections and functions. We first found that intracerebroventricular injection of adeno-associated virus with a neuron-specific promoter and Pkd2l1-Cre mice specifically labeled CSF-cNs. Single-cell labeling of 71 CSF-cNs revealed rostral axon extensions of over 1800 m in unmyelinated bundles in the ventral funiculus and terminated on CSF-cNs to form a recurrent circuitry, which was further determined by serial electron microscopy and electrophysiology. CSF-cNs were also found to connect with axial motor neurons and premotor interneurons around the central canal and within the axon bundles. Chemogenetic CSF-cNs inactivation reduced speed and step frequency during treadmill locomotion. Our data revealed the basic structures and connections of mouse CSF-cNs to control spinal motor circuits for proper locomotion. The versatile methods developed in this study will contribute to further understanding of CSF-cNs functions in mammals.

neuroscience↗

bioRxiv2022

Metagenomic thermometer

Various microorganisms exist in environments, and each of which has an optimal growth temperature (OGT). The relationship between genomic information and OGT of each species has long been studied, and one such study revealed that OGT of prokaryotes can be accurately predicted based on the fraction of seven amino acids (IVYWREL) among all encoded amino-acid sequences in its genome. Extending this discovery, the fraction of IVYWREL among all amino acids encoded in the metagenomic DNA in an environment may reflect the environmental temperature to which the microorganisms have adapted. In the present study, we developed a Metagenomic Thermometer as a means of predicting environmental temperature based on metagenomic sequences. Analysis of 41 metagenomic samples obtained from hot springs of varying temperature revealed that the Metagenomic Thermometer could indeed predict environmental temperature with high accuracy (R2 = 0.75). Although the taxonomic composition of microbial community was substantially affected by pH, the performance of the Metagenomic Thermometer was refractory to changes in community composition. The Metagenomic Thermometer was also applied to 616 human gut metagenomic samples, yielding a reasonably accurate value (i.e., 36.22 {+/-} 1.98{degrees}C) for human body temperature. Our results suggest that environmental temperature can be predicted based on metagenomic sequence data.

bioinformatics↗

bioRxiv2022

A fungal secondary metabolism gene cluster enables mutualist-pathogen transition in root endophyte Colletotrichum tofieldiae

Plant-associated fungi show diverse lifestyles from pathogenic to mutualistic to the host; however, the principles and mechanisms through which they shift the lifestyles require elucidation. The root fungus Colletotrichum tofieldiae (Ct) promotes Arabidopsis thaliana growth under phosphate limiting conditions. We reveal a Ct strain, designated Ct3, that severely inhibits plant growth. Ct3 pathogenesis occurs through activation of host abscisic acid (ABA) pathways via a fungal secondary metabolism gene cluster related to sesquiterpene ABA and botrydial (BOT) biosynthesis. ABA-BOT cluster activation during root infection suppresses host nutrient uptake-related genes and changes the mineral contents, suggesting its role in manipulating host nutrition states. Conversely, disruption or environmental suppression of the cluster renders Ct3 beneficial for plant growth, in a manner dependent on host phosphate starvation response regulators. Our findings indicate that a fungal metabolism cluster provides a means by which infectious fungi modulate lifestyles along the parasitic-mutualistic continuum in fluctuating environments.

microbiology↗

bioRxiv2022

Cost-efficient whole genome-sequencing using novel mostly natural sequencing-by-synthesis chemistry and open fluidics platform

We introduce a massively parallel novel sequencing platform that combines an open flow cell design on a circular wafer with a large surface area and mostly natural nucleotides that allow optical end-point detection without reversible terminators. This platform enables sequencing billions of reads with longer read length ([~]300bp) and fast runs times (<20hrs) with high base accuracy (Q30 > 85%), at a low cost of $1/Gb. We establish system performance by whole-genome sequencing of the Genome-In-A-Bottle reference samples HG001-7, demonstrating high accuracy for SNPs (99.6%) and Indels in homopolymers up to length 10 (96.4%) across the vast majority (>98%) of the defined high-confidence regions of these samples. We demonstrate scalability of the whole-genome sequencing workflow by sequencing an additional 224 selected samples from the 1000 Genomes project achieving high concordance with reference data.

genomics↗

bioRxiv2022

Transcriptional circuitry of NKX2-1 and SOX1 defines an unrecognized lineage subtype of small cell lung cancer

RationaleThe current molecular classification of small cell lung cancer (SCLC) based on expression of four lineage transcription factors still leaves its major subtype SCLC-A as a heterogeneous group, necessitating more precise characterization of lineage subclasses. ObjectivesTo refine the current SCLC classification with epigenomic profiles and to identify features of the re-defined SCLC subtypes. MethodsWe performed unsupervised clustering of epigenomic profiles on 25 SCLC cell lines. Functional significance was evaluated by cell growth, apoptosis and xenograft using CRISPR-Cas9-mediated deletion. The specific cistromic profiles by ChIP-seq and its functional transcriptional partners using co-immunoprecipitation followed by mass spectrometry were determined. Rb1fl/flTrp53fl/fl and Rb1fl/flNkx2-1fl/fl mouse models were engineered to explore the function of Nkx2-1 in tumor initiation and differentiation. H3K27ac profiles were analyzed to reveal 6 human SCLC specimen and 20 mice tumors epigenomic landscapes. Measurements and Main ResultsWe identified an epigenomic subclusters of the major SCLC-A subtype, named SCLC-A and SCLC-A{sigma}. SCLC-A was characterized by the presence of a super-enhancer at the NKX2-1 locus, which was observed in human SCLC specimens and a murine SCLC model. We found NKX2-1, a dual lung and neural lineage factor, is uniquely relevant in SCLC-A. We further found maintenance of this neural identity in SCLC-A is mediated by collaborative transcriptional activity with another neuronal transcriptional factor SOX1. ConclusionsWe comprehensively describe an additional epigenomic heterogeneity of the major SCLC-A subtype, and define SCLC-A subtype by the core regulatory circuitry representing NKX2-1 and SOX1 super-enhancers and their functional collaborations to maintain neuronal linage state.

molecular biology↗

bioRxiv2022

Interrogating Metabolic Interactions Between Skeletal Muscle and Liver Circadian Clocks In Vivo

Expressed throughout the body, the circadian clock system achieves daily metabolic homeostasis at every level of physiology, with clock disruption associated with metabolic disease (1, 2). Molecular clocks present in the brain, liver, adipose, pancreas and skeletal muscle each contribute to glucose homeostasis (3). However, it is unclear; 1) which organ clocks provide the most essential contributions, and 2) if these contributions depend on inter-organ communication. We recently showed that the liver clock alone is insufficient for most aspects of daily liver glucose handling and requires connections with other clocks (4). Considering the pathways that link glucose metabolism between liver and skeletal muscle, we sought to test whether a clock connection along this axis is important. Using our previous published methodology for tissue-specific rescue of Bmal1 in vivo (4, 5), we now show that in the absence of feeding-fasting cycles, liver and muscle clocks are not sufficient for systemic glucose metabolism, nor do they form a functional connection influencing local glucose handling or daily transcriptional rhythms in each tissue. However, the introduction of a daily feeding-fasting rhythm enables a synergistic state between liver and muscle clocks that leads to restoration of systemic glucose tolerance. These findings reveal limited autonomous capabilities of liver and muscle clocks and highlight the need for inter-organ clock communication for glucose homeostasis which involves at least two peripheral metabolic organs.

cell biology↗

bioRxiv2022

Fibrosis resolution involves more than collagen; new potential players of fibrosis recovery in mouse liver

Although most work has focused on resolution of collagen ECM, fibrosis resolution involves changes to several ECM proteins. The purpose of the current study was two-fold: 1) to examine the role of MMP12 and elastin; and 2) to investigate the changes in degraded proteins in plasma (i.e., the "degradome") in a preclinical model of fibrosis resolution. Fibrosis was induced by 4 weeks carbon tetrachloride (CCl4) exposure, and recovery was monitored for an additional 4 weeks. Some mice were treated with daily MMP12 inhibitor (MMP408) during the resolution phase. Liver injury and fibrosis was monitored by clinical chemistry, histology and gene expression. The release of degraded ECM peptides in the plasma was analyzed using by 1D-LC-MS/MS, coupled with PEAKS Studio (v10) peptide identification. Hepatic fibrosis and liver injury rapidly resolved in this mouse model. However, some collagen fibrils were still present 28d after cessation of CCl4. Despite this persistent collagen presence, expression of canonical markers of fibrosis were also normalized. The inhibition of MMP12 dramatically delayed fibrosis resolution under these conditions. LC-MS/MS analysis identified that several proteins were being degraded even at late stages of fibrosis resolution. Calpains 1/2 were identified as potential new proteases involved in fibrosis resolution. CONCLUSION. The results of this study indicate that remodeling of the liver during recovery from fibrosis is a complex and highly coordinated process that extends well beyond the degradation of the collagenous scar. These results also indicate that analysis of the plasma degradome may yield new insight into the mechanisms of fibrosis recovery, and by extension, new "theragnostic" targets. Lastly, a novel potential role for calpain activation in the degradation and turnover of proteins was identified.

pathology↗

bioRxiv2022

Discovery of S-217622, a Non-Covalent Oral SARS-CoV-2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths and threatens public health and safety. Despite the rapid global spread of COVID-19 vaccines, effective oral antiviral drugs are urgently needed. Here, we describe the discovery of S-217622, the first oral non-covalent, non-peptidic SARS-CoV-2 3CL protease inhibitor clinical candidate. S-217622 was discovered via virtual screening followed by biological screening of an in-house compound library, and optimization of the hit compound using a structure-based drug-design strategy. S-217622 exhibited antiviral activity in vitro against current outbreaking SARS-CoV-2 variants and showed favorable pharmacokinetic profiles in vivo for once-daily oral dosing. Furthermore, S-217622 dose-dependently inhibited intrapulmonary replication of SARS-CoV-2 in mice, indicating that this novel non-covalent inhibitor could be a potential oral agent for treating COVID-19.

biochemistry↗

bioRxiv2022

The central clock suffices to drive the majority of circulatory metabolic rhythms

Life on Earth anticipates recurring 24-h environmental cycles via genetically-encoded molecular clocks active in all mammalian organs. Communication between these clocks is believed to control circadian homeostasis. Metabolism can be considered a form of inter- tissue communication language that results in temporal coordination of systemic metabolism between tissues. Here we characterize the extent to which clocks in different organs employ this means of communication, an area which remains largely unexplored. For this, we analysed the metabolome of serum from mice with tissue-specific expression of the clock gene Bmal1. Notably, having functional hepatic and muscle clocks can only drive a minority (13%) of the oscillating metabolites in circulation. Conversely, limiting Bmal1 expression to Syt10- expressing neurons (which are enriched in the suprachiasmatic nucleus [SCN], the master pacemaker that regulates circadian rhythms) restores rhythms to 57% of circulatory metabolites and 28% of liver transcripts, and rescues glucose intolerance. Importantly, these parameters were also restored in clock-less mice upon rhythmic feeding, indicating that the central clock mainly regulates metabolic rhythms via behavior. These findings explicate the circadian communication between tissues and highlight the importance of the central clock in governing those signals.

physiology↗

bioRxiv2021

De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals

Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo-grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control.

molecular biology↗

bioRxiv2021

Global inflammatory response in in vitro organ cultured testes using single-cell RNA-sequencing

In vitro functional sperm production is important for understanding spermatogenesis and for the treatment of male infertility. Here, we describe similarities and differences between testis tissues in vivo and in vitro and clarify abnormalities in the early stage of in vitro spermatogenesis at single-cell resolution. While in vitro spermatogenesis progressed similarly to in vivo spermatogenesis until the early pachytene spermatocyte stage, a noticeable acute inflammatory response occurred in immune cells and non-immune testicular somatic cells immediately after cultivation. Inhibitor treatment revealed that NLRP3 inflammasome signaling is key to the inflammation. We observed damaged/dead germ cell accumulation in cultured testis, which may be due to dysfunctional phagocytosis by Sertoli cells. Our data revealed abnormal testicular milieu of in vitro cultured testes caused by tissue-wide sterile inflammation, in which the danger-associated molecular pattern-NLRP3 inflammasome axis may be a key element.

molecular biology↗

bioRxiv2021

Partial prevention of glucocorticoid-induced osteocyte deterioration with osteocrin gene therapy

Glucocorticoid (GC)-induced osteoporosis and subsequent bone fragility are preceded by death and dysfunction at the cellular level. In particular, short-term glucocorticoid excess suppresses osteocyte remodeling of the surrounding bone mineral, causes apoptosis of osteoblasts and osteocytes, and disrupts homeostatic bone remodeling. Preventing apoptosis and preserving osteocyte morphology and function could be effective means of preventing bone loss during glucocorticoid excess. We hypothesized that osteocrin, which preserves osteocyte viability and morphology in other models where osteocyte defects exist, could prevent osteocyte death and dysfunction in a GC excess model. We used a liver-targeted adeno-associated virus (AAV8) to induce osteocrin overexpression in mice one week prior to implantation with prednisolone or placebo pellets. After 28 days, tissues were collected for micro-CT and histological analysis. GC excess caused the expected reduction in cortical bone thickness and osteocyte canalicular length in control AAV8-treated mice, and these effects were blunted in mice overexpressing osteocrin. However, GC-induced changes in cortical porosity, trabecular bone mass, and gene expression were not prevented by osteocrin. While the mechanism of osteocrins effects on osteocyte morphology warrants further investigation, this study does not support a role for this model of osteocrin supplementation to combat the full skeletal effects of GC excess.

physiology↗

bioRxiv2021

Stochastic variational variable selection for high-dimensional microbiome data

BackgroundThe rapid and accurate identification of a minimal-size core set of representative microbial species plays an important role in the clustering of microbial community data and interpretation of clustering results. However, the huge dimensionality of microbial metagenomics datasets is a major challenge for the existing methods such as Dirichlet multinomial mixture (DMM) models. In the framework of the existing methods, the computational burden of identifying a small number of representative species from a large number of observed species remains a challenge. ResultsWe proposed a novel framework to improve the performance of the widely used DMM approach by combining three ideas: (i) we extended the finite DMM model to an infinite case by considering Dirichlet process mixtures and estimating the number of clusters as a random variables; (ii) we proposed an indicator variable to identify representative operational taxonomic units that substantially contribute to the differentiation among clusters; and (iii) to address the computational burden of high-dimensional microbiome data, we proposed a stochastic variational inference, which approximates the posterior distribution using a controllable distribution called variational distribution, and stochastic optimization algorithms for fast computation. Using the proposed method, stochastic variational variable selection (SVVS), we analyzed the root microbiome data collected in our soybean field experiment, the human gut microbiome data from three published datasets of large-scale case-control studies and the healthy human microbiome data from the Human Microbiome Project. ConclusionsSVVS demonstrated a better performance and significantly faster computation than those of the existing methods in all cases of testing datasets. In particular, SVVS is the only method that can analyze massive high-dimensional microbial data with more than 50,000 microbial species and 1,000 samples. Furthermore, recent microbiome studies have suggested that selection of the microbial species used as a core set is important.

bioinformatics↗

bioRxiv2021

Phosphorus is a critical factor of the in vitro monoxenic culture method for awide range of arbuscular mycorrhizal fungi culture collections

Establishing an effective way to propagate a wide range of arbuscular mycorrhizal (AM) fungi species is desirable for mycorrhizal research and agricultural applications. Although the success of mycorrhizal formation is required for spore production of AM fungi, the critical factors for its construction in the in vitro monoxenic culture protocol remain to be identified. In this study, we evaluated the growth of hairy roots from carrot, flax, and chicory, and investigated the effects of the phosphorus (P) concentration in the mother plate, as well as the levels of P, sucrose, and macronutrients in a cocultivation plate with a hairy root, amount of medium of the cocultivation plate, and location of spore inoculation, by utilizing the Bayesian information criterion model selection with greater than 800 units of data. We found that the flax hairy root was suitable for in vitro monoxenic culture, and that the concentration of P in the cocultivation plate was a critical factor for mycorrhizal formation. We showed that an extremely low concentration of P (3 M) significantly improved mycorrhizal formation for AM fungi belonging to the Glomerales order, while a high concentration of P (30 M) was suitable for Diversisporales fungi. Therefore, we anticipate that the refining the P concentration will contribute to future culture collections of a wide range of AM fungi.

microbiology↗