Biology subjects

Sato, T.

Publications and source records attributed to Sato, T..

At least 109 records · Page 6Linked to original sources

bioRxiv2021

Aging is associated with increased brain iron through brain-derived hepcidin expression

Iron is an essential molecule for biological processes, but its accumulation can lead to oxidative stress and cellular death. Due to its oxidative effects, iron accumulation is implicated in the process of aging and neurodegenerative diseases. However, the mechanism for this increase in iron with aging, and whether this increase is localized to specific cellular compartment(s), are not known. Here, we measured the levels of iron in different tissues of aged mice, and demonstrate that while cytosolic non-heme iron is increased in the liver and muscle tissue, only the aged brain exhibits an increase in both the cytosolic and mitochondrial non-heme iron. This increase in brain iron is associated with elevated levels of local hepcidin mRNA and protein in the brain. We also demonstrate that the increase in hepcidin is associated with increased ubiquitination and reduced levels of the only iron exporter, feroportin-1 (FPN1). Overall, our studies provide a potential mechanism for iron accumulation in the brain through increased local expression of hepcidin, and subsequent iron accumulation due to decreased iron export. Additionally, our data support that aging is associated with mitochondrial and cytosolic iron accumulation only in the brain and not in other tissues.

cell biology↗

bioRxiv2021

Machine-learning-guided library design cycle for directed evolution of enzymes: the effects of training data composition on sequence space exploration

Machine learning (ML) is becoming an attractive tool in mutagenesis-based protein engineering because of its ability to design a variant library containing proteins with a desired function. However, it remains unclear how ML guides directed evolution in sequence space depending on the composition of training data. Here, we present a ML-guided directed evolution study of an enzyme to investigate the effects of a known "highly positive" variant (i.e., variant known to have high enzyme activity) in training data. We performed two separate series of ML-guided directed evolution of Sortase A with and without a known highly positive variant called 5M in training data. In each series, two rounds of ML were conducted: variants predicted by the first round were experimentally evaluated, and used as additional training data for the second-round prediction. The improvements in enzyme activity were comparable between the two series, both achieving enzyme activity 2.2-2.5 times higher than 5M. Intriguingly, the sequences of the improved variants were largely different between the two series, indicating that ML guided the directed evolution to the distinct regions of sequence space depending on the presence/absence of the highly positive variant in the training data. This suggests that the sequence diversity of improved variants can be expanded not only by conventional ML using the whole training data, but also by ML using a subset of the training data even when it lacks highly positive variants. In summary, this study demonstrates the importance of regulating the composition of training data in ML-guided directed evolution.

bioengineering↗

bioRxiv2021

Internal and external features of wild birds after collisions without apparent trauma in Japan

Wild birds often require rehabilitation after collisions, even with no apparent injury. Information about aftermath of collisions is still scares. Here, we investigated external characteristics and clinical features of the internal organs of wild birds that experienced collision and compared them with birds admitted to rehabilitation center for other reasons. Necropsy was performed on 55 bird carcasses from Passeriformes and Coraciiform. Five external characteristics were recorded before necropsy including; cause of admission, keel score, life stage, fat score, and number of days before death. The median survival time was calculated by Kaplan-Meier estimates. Data on external and internal features were compared using univariate and multivariate multinomial regressions. There was no significant difference in the median survival time among the causes of admission: 1 day for collision, 2 days for trauma, and 2 days for malnutrition. Kidney discoloration was more significantly associated with collision than with other trauma (p = 0.01). Although no apparent kidney abnormality (including enlargement) were observed, anterior lobe was significantly larger than posterior lobe with collision compared with malnutrition (p = 0.045). Birds that experienced collision exhibited a higher fat score than malnourished birds (p = 0.03). Our results suggested that wild birds with abundant fat were more likely to be admitted due to collision. The gross characteristics of collision included kidney discoloration and anterior lobe extension, which was possibly due to rupture of renal blood vessels by blunt external force. From these findings, it was considered that collision caused major axis of anterior lobe significantly larger than posterior lobe, even though no abnormal finding in renal size such as hypertrophy was recognized. This was the first study to evaluate the cause of admission, necropsy results, and external characteristics in wild birds admitted to rehabilitation centers. Absolute cage rest should be adhered to restore renal function for those birds admitted due to collision, and handling and treatment should be minimized to avoid excess movement of the birds.

zoology↗

bioRxiv2021

Threonine phosphorylation regulates the molecular assembly and signaling of EGFR in cooperation with membrane lipids

The cytoplasmic domain of the receptor tyrosine kinases (RTKs) plays roles as a phosphorylation enzyme and a protein scaffold but the allocation of these two functions is not fully understood. We here analyzed assembly of the transmembrane (TM)-juxtamembrane (JM) region of EGFR, one of the best studied species of RTKs, by combining single-pair FRET imaging and a nanodisc technique. The JM domain of EGFR contains a threonine residue (Thr654) that is phosphorylated after ligand association. We observed that the TM-JM peptides of EGFR form anionic lipid-induced dimers and cholesterol-induced oligomers. The two forms involve distinct molecular interactions, with a bias towards oligomer formation upon threonine phosphorylation. We further analyzed the functions and oligomerization of whole EGFR molecules, with or without a substitution of Thr654 to alanine, in living cells. The results suggested an autoregulatory mechanism in which Thr654 phosphorylation causes a switch of the major function of EGFR from kinase activation dimers to scaffolding oligomers.

biophysics↗

bioRxiv2021

Reduction of susceptibility to azoles and 5-fluorocytosine and growth acceleration in Candida albicans by glucose in urine

Candida species are causal pathogens for urinary tract infections, vulvovaginitis, and balanitis. Diabetes mellitus is a risk factor for Candida infection. To investigate the potential effects of glucosuria on Candida spp. (C. albicans, C. krusei, and C. glabrata), we investigated the influence of their growth and antifungal susceptibilities by glucose in urine. Candida spp. exhibited greater growth in urine with glucose (300 and 3,000 mg/dL) than in plain urine taken from healthy volunteers. After 24 h incubation, the viable cell number was more than 10-fold higher in the urine added 3,000 mg/dL glucose than in plain urine. In antifungal susceptibility, more than 80% of C. albicans clinical isolates increased minimum inhibitory concentrations of azoles (fluconazole, itraconazole, voriconazole, and miconazole) and 5-fluorocytosine with the addition of glucose exceeding their breakpoints. This phenomenon was not observed in clinical isolates of C. krusei and C. glabrata. We observed the growth in the urine to which 3,000 mg/dL glucose was added even in the presence of a 128-fold higher minimum inhibitory concentration of fluconazole. In most of the C. albicans clinical isolates, the mRNA expression of the azole resistance genes ERG11, CDR1, CDR2, and MDR1 increased in glucose-added urine compared with plain urine. In conclusion, the growth of C. albicans is accelerated and azoles and 5-fluorocytosine become ineffective as a result of a high concentration of glucose in urine. These observations provide valuable information about the clinical course and therapeutic effects of azoles against C. albicans infections in patients with diabetes mellitus and hyperglucosuria. IMPORTANCEDiabetes mellitus is a chronic metabolic disease characterized by hyperglycemia and glucosuria, with a high risk of Candida infection. The current study demonstrated the acceleration of Candida growth and ineffectiveness of azoles and 5-fluorocytosine against C. albicans in urine in the presence of glucose. These observations provide novel and valuable information about the clinical course and antifungal treatment of Candida spp. in urinary tract and genital infections of diabetes mellitus patients. For the treatment of urinary tract infections caused by Candida spp., the guidelines do not mention glucosuria. Thus, this study suggests the necessity to conduct clinical evaluations for glucosuria in patients with diabetes mellitus who have urinary tract and genital infections with Candida spp.

microbiology↗

bioRxiv2021

Simple amplicon sequencing library preparation for plant root microbial community profiling

Microbiota are a major component of agroecosystems. Root microbiota, which inhabit the inside and surface of plant roots, play a significant role in plant growth and health. As next-generation sequencing technology allows the capture of microbial profiles without culturing the microbes, profiling of plant microbiota has become a staple tool in plant science and agriculture. Here, we have developed a novel high-throughput method based on a two-step PCR amplification protocol, involving DNA extraction using magnetic beads and PCR purification using exonuclease, for 16S rRNA gene amplicon sequencing of plant root microbiota. This method reduces sample handling and captures microbial diversity comparable to that obtained by the standard method. We found that using a buffer with magnetic beads enabled efficient extraction of microbial DNA directly from plant roots. In addition, we demonstrated that purification using exonuclease before the second PCR step enabled the capture of higher degrees of microbial diversity, thus allowing for the detection of minor bacteria compared with the purification using magnetic beads in this step. Our method offers a simple and high-throughput solution for maintaining the quality of plant root microbial community profiling.

plant biology↗

bioRxiv2021

Nasal delivery of single-domain antibodies improve symptoms of SARS-CoV-2 infection in an animal model

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the disease COVID-19 can lead to serious symptoms, such as severe pneumonia, in the elderly and those with underlying medical conditions. While vaccines are now available, they do not work for everyone and therapeutic drugs are still needed particularly for treating life-threatening conditions. Here, we showed nasal delivery of a new, unmodified camelid single-domain antibody (VHH), termed K-874A, effectively inhibited SARS-CoV-2 titers in infected lungs of Syrian hamsters without causing weight loss and cytokine induction. In vitro studies demonstrated that K-874A neutralized SARS-CoV-2 in both VeroE6/TMPRSS2 and human lung-derived alveolar organoid cells. Unlike other drug candidates, K-874A blocks viral membrane fusion rather than viral attachment. Cryo-electron microscopy revealed K-874A bound between the receptor binding domain and N-terminal domain of the virus S protein. Further, infected cells treated with K-874A produced fewer virus progeny that were less infective. We propose that direct administration of K-874A to the lung via a nebulizer could be a new treatment for preventing the reinfection of amplified virus in COVID-19 patients. Author summaryVaccines for COVID-19 are now available but therapeutic drugs are still needed to treat life-threatening cases and those who cannot be vaccinated. We discovered a new heavy-chain single-domain antibody that can effectively neutralize the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes COVID-19. Unlike other drug candidates, which prevent the virus from attaching to the receptor on the host cell, this new antibody acts by blocking the virus membrane from fusing with the host cell membrane. We studied the behavior of the new antibody in vitro using VeroE6/TMPRSS2 cells and human lung organoids. When delivered through the nose to infected Syrian hamsters, we found that this antibody could prevent the typical symptoms caused by SARS-CoV-2. Our results are significant because delivering simple drugs directly to infected lungs using a nebulizer could increase the potency of the drugs while reducing the risk of immune reaction that could occur if the drugs escape or are delivered through the blood stream.

microbiology↗

bioRxiv2021

Comparative analyses of gene expression in common marmoset and human pluripotent stem cells (PSCs) identify factors enhancing homologous recombination efficiency in the HPRT locus of human PSCs

A previous study assessing the efficiency of the genome editing technology CRISPR-Cas9 for knock-in gene targeting in common marmoset (marmoset; Callithrix jacchus) embryonic stem cells (ESCs) unexpectedly identified innately enhanced homologous recombination (HR) activity in marmoset ESCs (cmESCs). Here, we compared gene expression in marmoset and human pluripotent stem cells (PSCs) using transcriptomic and quantitative PCR (qPCR) analyses and found that five HR-related genes (BRCA1, BRCA2, RAD51C, RAD51D and RAD51) were upregulated in marmoset cells. Four of these upregulated genes enhanced HR efficiency with CRISPR-Cas9 in human pluripotent stem cells. Thus, the present study provides a novel insight into species-specific mechanisms for the choice of DNA repair pathways.

cell biology↗

bioRxiv2021

Identification, crystallization and epitope determination of public TCR shared and expanded in COVID-19 patients

T cells play pivotal roles in protective immunity against SARS-CoV-2 infection. Follicular helper T (Tfh) cells mediate the production of antigen-specific antibodies; however, T cell receptor (TCR) clonotypes used by SARS-CoV-2-specific Tfh cells have not been well characterized. Here, we first identified and crystallized public TCR of Tfh clonotypes that are shared and expanded in unhospitalized COVID-19-recovered patients. These clonotypes preferentially recognized SARS-CoV-2 spike (S) protein epitopes which are conserved among emerging SARS-CoV-2 variants. These clonotypes did not react with S proteins derived from common cold human coronaviruses, but cross-reacted with symbiotic bacteria, which might confer the publicity. Among SARS-CoV-2 S epitopes, S864-882, presented by frequent HLA-DR alleles, could activate multiple public Tfh clonotypes in COVID-19-recovered patients. Furthermore, S864-882-loaded HLA tetramer preferentially bound to CD4+ T cells expressing CXCR5. In this study, we identified and crystallized public TCR for SARS-CoV-2 that may contribute to the prevention of COVID-19 aggravation.

immunology↗

bioRxiv2021

Cluster Analysis of SARS-CoV-2 Gene using Deep Learning Autoencoder: Gene Profiling for Mutations and Transitions

We report on a method for analyzing the variant of coronavirus genes using autoencoder. Since coronaviruses have mutated rapidly and generated a large number of genotypes, an appropriate method for understanding the entire population is required. The method using autoencoder meets this requirement and is suitable for understanding how and when the variants emarge and disappear. For the over 30,000 SARS-CoV-2 ORF1ab gene sequences sampled globally from December 2019 to February 2021, we were able to represent a summary of their characteristics in a 3D plot and show the expansion, decline, and transformation of the virus types over time and by region. Based on ORF1ab genes, the SARS-CoV-2 viruses were classified into five major types (A, B, C, D, and E in the order of appearance): the virus type that originated in China at the end of 2019 (type A) practically disappeared in June 2020; two virus types (types B and C) have emerged in the United States and Europe since February 2020, and type B has become a global phenomenon. Type C is only prevalent in the U.S. and is suspected to be associated with high mortality, but this type also disappeared at the end of June. Type D is only found in Australia. Currently, the epidemic is dominated by types B and E.

microbiology↗

bioRxiv2021

Scx-positive tendon cells are required for correct muscle patterning

The elaborate movement of the vertebrate body is supported by the precise connection of muscle, tendon and bone. Each of the >600 distinct skeletal muscles in the human body has unique attachment sites; however, the mechanism through which muscles are reproducibly attached to designated partner tendons during embryonic development is incompletely understood. We herein show that Screlaxis-positive tendon cells have an essential role in correct muscle attachment in mouse embryos. Specific ablation of Screlaxis-positive cells resulted in dislocation of muscle attachment sites and abnormal muscle bundle morphology. Step-by-step observation of myogenic cell lineage revealed that post-fusion myofibers, but not migrating myoblasts, require tendon cells for their morphology. Furthermore, muscles could change their attachment site, even after the formation of the insertion. Our study demonstrated an essential role of tendon cells in the reproducibility and plasticity of skeletal muscle patterning, in turn revealing a novel tissue-tissue interaction in musculoskeletal morphogenesis. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

developmental biology↗

bioRxiv2020

Augmenter of Liver Regeneration Regulates Cellular Iron by Modulating Mitochondrial Transport of ATP-Binding Cassette B8

Chronic loss of Augmenter of Liver Regeneration (ALR) results in mitochondrial myopathy with cataracts, however, the mechanism for this disorder remains unclear. Here, we demonstrate that loss of ALR, a principal component of the MIA40/ALR protein import pathway, results in impaired cytosolic Fe/S cluster biogenesis in mammalian cells. Mechanistically, MIA40/ALR facilitates the mitochondrial import of ATP binding cassette (ABC)-B8, an inner mitochondrial membrane protein required for cytoplasmic Fe/S cluster maturation, through physical interaction with ABCB8. Downregulation of ALR impairs mitochondrial ABCB8 import, reduces cytoplasmic Fe/S cluster maturation, and increases cellular iron through the iron regulatory protein-iron response element system. Our finding provides a mechanistic link between MIA40/ALR import machinery and cytosolic Fe/S cluster maturation through the mitochondrial import of ABCB8, and offers a potential explanation for the pathology seen in patients with ALR mutations.

cell biology↗

bioRxiv2020

Direct Observation of the Mechanism of Antibiotic Resistance by Mix-and-Inject at the European XFEL

In this study, we follow the diffusion and buildup of occupancy of the substrate ceftriaxone in M. tuberculosis {beta}-lactamase BlaC microcrystals by structural analysis of the enzyme substrate complex at single millisecond time resolution. We also show the binding and the reaction of an inhibitor, sulbactam, on a slower millisecond time scale. We use the mix-and-inject technique to initiate these reactions by diffusion, and determine the resulting structures by serial crystallography using ultrafast, intense X-ray pulses from the European XFEL (EuXFEL) arriving at MHz repetition rates. Here, we show how to use the EuXFEL pulse structure to dramatically increase the size of the data set and thereby the quality and time resolution of "molecular movies" which unravel ligand binding and enzymatically catalyzed reactions. This shows the great potential for the EuXFEL as a tool for biomedically relevant research, particularly, as shown here, for investigating bacterial antibiotic resistance. One Sentence SummaryDirect observation of fast ligand binding in a biomedically relevant enzyme at near atomic resolution with MHz X-ray pulses at the European XFEL.

biophysics↗

bioRxiv2020

Consolidation and maintenance of Drosophila long-term memory require LIM homeodomain protein Apterous in distinct brain neurons

The LIM-homeodomain (LIM-HD) transcription factor Apterous (Ap) and its cofactor Chip (Chi) form a complex that regulates various developmental events in Drosophila. Although Ap continues to be expressed in the adult brain, the functions of the centrally expressed Ap remain incompletely understood. Here, we show that Ap and Chi in the Drosophila memory center, the mushroom bodies (MBs), are indispensable for long-term memory (LTM) maintenance, whereas Ap in a subset of clock neurons [large ventral-lateral neurons (l-LNvs)] plays a crucial role in memory consolidation in a Chi-independent manner. Ex vivo imaging revealed that Ap, but not Chi, in l-LNvs is essential for the appropriate Cl- responses to GABA. Furthermore, knockdown of GABAA receptor in l-LNvs compensated for the impairment of memory consolidation in ap null mutant flies. Our results indicate that Drosophila Ap functions differently in l-LNvs and MBs, and it contributes to the consolidation and maintenance of LTM.

neuroscience↗

bioRxiv2020

Phosphorylation of luminal region of the SUN-domain protein Mps3 promotes nuclear envelope localization during meiosis

During meiosis, protein ensembles in the nuclear envelope (NE) containing SUN- and KASH-domain proteins, called linker nucleocytoskeleton and cytoskeleton (LINC) complex, promote the chromosome motion. Yeast SUN-domain protein, Mps3, forms multiple meiosis-specific ensembles on NE, which show dynamic localisation for chromosome motion; however, the mechanism by which these Mps3 ensembles are formed during meiosis remains largely unknown. Here, we showed that the cyclin-dependent protein kinase (CDK) and Dbf4-dependent Cdc7 protein kinase (DDK) regulate meiosis-specific dynamics of Mps3 on NE, particularly by mediating the resolution of Mps3 clusters and telomere clustering. We also found that the luminal region of Mps3 juxtaposed to the inner nuclear membrane is required for meiosis-specific localisation of Mps3 on NE. Negative charges introduced by meiosis-specific phosphorylation in the luminal region of Mps3 alter its interaction with negatively charged lipids by electric repulsion in reconstituted liposomes. Phospho-mimetic substitution in the luminal region suppresses the localisation of Mps3 via the inactivation of CDK or DDK. Our study revealed multi-layered phosphorylation-dependent regulation of the localisation of Mps3 on NE for meiotic chromosome motion and NE remodelling.

molecular biology↗

bioRxiv2020

Bimolecule detection for Extracellular Vesicle Screening

Extracellular vesicle (EV) has been investigated for use in clinical testing in recent years. Specific EV surface proteins provide distinguishing characteristics, but are insufficient for more detailed classification of EVs. Here, we suggest a novel "Bimolecular surface antigen expressed in EV" (BiEV) as a potential indicator for more efficient EV screening. A BiEV can be identified using a previously developed method, enzyme-mediated activation of radical sources, to label the components proximal (within 20 nm) to a given molecule. We examined the screening of cancer cell-secreted EV (cEV) included in serum EVs from a model mouse for lung cancer. The cEV-specific BiEVs were first identified by through a comparison of serum EVs from wild-type and lung cancer mice, showing that the CHL1-SLC4A1 bimolecule was a significant candidate for cEV-specific BiEV. Enzyme-linked immunosorbent assay quantification of CHL1-SLC4A1 BiEV appeared to suggest a potential for cancer screening of these mice. Using the same protocols, we found that CHL1-caspase 14 BiEV was significantly elevated in lung cancer patients compared with healthy persons. A BiEV strategy may be able to make a contribution to more effective EV screening, resulting in novel biological and clinical applications.

biochemistry↗

bioRxiv2020

Expression of Ace2, Tmprss2, and Furin in mouse ear tissue

ObjectivesIntracellular entry of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) depends on the interaction between its spike protein to a cellular receptor named angiotensin-converting enzyme 2 (ACE2) and depends on Furin-mediated spike 23 protein cleavage and spike protein priming by host cell proteases including 24 transmembrane protease serine 2 (TMPRSS2). Tmprss1, Tmprss3, and Tmprss5 are expressed in the spiral ganglion neurons and the organ of Corti in the inner ear; however, Ace2, Tmprss2, and Furin expression profiles in the middle ear remain unclear. Therefore, this study aimed to analyze Ace2, Tmprss2, and Furin expression in the middle and inner ear of mice. Study DesignAnimal research. SettingDepartment of Otolaryngology and Head and Neck Surgery, University of Tokyo. MethodsWe performed immunohistochemical analysis to examine the distribution of Ace2, Tmprss2, and Furin in the eustachian tube, middle ear space, and cochlea of mice. ResultsAce2 was expressed in the cytoplasm in the middle ear epithelium, eustachian tube epithelium, stria vascularis, and spiral ganglion. Tmprss2 and Furin were widely expressed in the middle ear spaces and the cochlea. ConclusionCo-expression of Ace2, Tmprss2, and Furin in the middle ear indicates that the middle ear is susceptible to SARS-CoV-2 infections, thus warranting the use of personal protective equipment during mastoidectomy for coronavirus disease (COVID-19) patients.

microbiology↗

bioRxiv2020

TGN/EE SNARE protein SYP61 is ubiquitinated and required for carbon/nitrogen-nutrient responses in Arabidopsis

Ubiquitination is a post-translational modification with reversible attachment of the small protein ubiquitin, which is involved in numerous cellular processes including membrane trafficking. For example, ubiquitination of cargo proteins is known to regulate their subcellular dynamics, and plays important roles in plant growth and stress adaptation. However, the regulatory mechanism of the trafficking machinery components remains elusive. Here, we report Arabidopsis trans-Golgi network/early endosome (TGN/EE) localized soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein SYP61 as a novel ubiquitination target of a membrane localized ubiquitin ligase ATL31. SYP61 is a key component of membrane trafficking in Arabidopsis. SYP61 was ubiquitinated with K63-linked chain by ATL31 in vitro and in plants. The knockdown mutants of SYP61 were hypersensitive to the disrupted carbon (C)/nitrogen (N)-nutrient stress, suggesting its critical role in plant homeostasis in response to nutrients. We also found the ubiquitination status of SYP61 is affected by C/N-nutrient availability. These results provided possibility that ubiquitination of SNARE protein has important role in plant physiology.

plant biology↗