Biology subjects

Suzuki, K.

Publications and source records attributed to Suzuki, K..

At least 91 records · Page 5Linked to original sources

bioRxiv2022

Reproducible and sensitive micro-tissue RNA-sequencing from formalin-fixed paraffin-embedded tissue for spatial gene expression analysis

Spatial transcriptome analysis of formalin-fixed paraffin-embedded (FFPE) tissues using RNA-sequencing (RNA-seq) provides interactive information on morphology and gene expression, which is useful for clinical applications. However, despite the advantages of long-term storage at room temperature, FFPE tissues may be severely damaged by methylene crosslinking and provide less gene information than fresh-frozen tissues. In this study, we proposed a sensitive FFPE micro-tissue RNA-seq method that combines the punching of tissue sections (diameter: 100 m) and the direct construction of RNA-seq libraries. We evaluated a method using mouse liver tissues at 2 years after fixation and embedding and detected approximately 7,000 genes in micro-punched tissue-spots (thickness: 10 m), similar to that detected with purified total RNA (2.5 ng) equivalent to the several dozen cells in the spot. We applied this method to clinical FFPE specimens of lung cancer that had been fixed and embedded 6 years prior, and found that it was possible to determine characteristic gene expression in the microenvironment containing tumor and non-tumor cells of different morphologies. This result indicates that spatial gene expression analysis of the tumor microenvironment is feasible using FFPE tissue sections stored for extensive periods in medical facilities.

molecular biology↗

bioRxiv2022

Potent and specific human monoclonal antibodies against SARS-CoV-2 Omicron variant by rapid mRNA immunization of humanized mice

The Omicron variant (B.1.1.529) of SARS-CoV-2 rapidly becomes dominant globally. Its extensive mutations confer severe efficacy reduction to most of existing antibodies or vaccines. Here, we developed RAMIHM, a highly efficient strategy to generate fully human monoclonal antibodies (mAbs), directly applied it with Omicron-mRNA immunization, and isolated three potent and specific clones against Omicron. Rapid mRNA immunization elicited strong anti-Omicron antibody response in humanized mice, along with broader anti-coronavirus activity. Customized single cell BCR sequencing mapped the clonal repertoires. Top-ranked clones collectively from peripheral blood, plasma B and memory B cell populations showed high rate of Omicron-specificity (93.3%) from RAMIHM-scBCRseq. Clone-screening identified three highly potent neutralizing antibodies that have low nanomolar affinity for Omicron RBD, and low ng/mL level IC50 in neutralization, more potent than majority of currently approved or authorized clinical RBD-targeting mAbs. These lead mAbs are fully human and ready for downstream IND-enabling and/or translational studies.

bioengineering↗

bioRxiv2022

Decomposing predictability to identify dominant causal drivers in complex ecosystems

Ecosystems are complex systems of various physical, biological, and chemical processes. Since ecosystem dynamics are composed of a mixture of different levels of stochasticity and nonlinearity, handling these data is a challenge for existing methods of time-series based causal inferences. Here we show that, by harnessing contemporary machine learning approaches, the concept of Granger causality can be effectively extended to the analysis of complex ecosystem time series and bridge the gap between dynamical and statistical approaches. The central idea is to use an ensemble of fast and highly predictive artificial neural networks to select a minimal set of variables that maximizes the prediction of a given variable. It enables decomposition of the relationship among variables through quantifying the contribution of an individual variable to the overall predictive performance. We show how our approach, EcohNet, can improve interaction network inference for a mesocosm experiment and simulated ecosystems. The application of the method to a long-term lake monitoring dataset yielded new but interpretable results on the drivers causing cyanobacteria blooms, which is a serious threat to ecological integrity and ecosystem services. Since performance of EcohNet is enhanced by its predictive capabilities, it also provides an optimized forecasting of overall components in ecosystems. EcohNet could be used to analyze complex and hybrid multivariate time series in many scientific areas not limited to ecosystems. Significance StatementEffective use of ecosystem monitoring data to resolve global environmental issues is a major challenge of the 21st century ecology. A promising solution to address this challenge is a time-series-based causal analysis which can provide insight on the mechanical links between ecosystem components. In this work, a model-free framework named EcohNet is proposed. EcohNet utilizes ensemble predictions of echo state networks, which are known to be fast, accurate, and highly relevant for a variety of dynamical systems, and can robustly predict causal networks of ecosystem components. It also can provide an optimized forecasting of overall ecosystem components, and could be used to analyze complex and hybrid multivariate time series in many scientific areas, not limited to ecosystems.

ecology↗

bioRxiv2022

SARS-CoV-2 Omicron-specific mRNA vaccine induces potent and broad antibody responses in vivo

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has high transmissibility and recently swept the globe. Due to the extensive number of mutations, this variant has high level of immune evasion, which drastically reduced the efficacy of existing antibodies and vaccines. Thus, it is important to test an Omicron-specific vaccine, evaluate its immune response against Omicron and other variants, and compare its immunogenicity as boosters with existing vaccine designed against the reference wildtype virus (WT). Here, we generated an Omicron-specific lipid nanoparticle (LNP) mRNA vaccine candidate, and tested its activity in animals, both alone and as a heterologous booster to existing WT mRNA vaccine. Our Omicron-specific LNP-mRNA vaccine elicited strong and specific antibody response in vaccination-naive mice. Mice that received two-dose WT LNP-mRNA, the one mimicking the commonly used Pfizer/Moderna mRNA vaccine, showed a >40-fold reduction in neutralization potency against Omicron variant than that against WT two weeks post second dose, which further reduced to background level >3 months post second dose. As a booster shot for two-dose WT mRNA vaccinated mice, a single dose of either a homologous booster with WT LNP-mRNA or a heterologous booster with Omicron LNP-mRNA restored the waning antibody response against Omicron, with over 40-fold increase at two weeks post injection as compared to right before booster. Interestingly, the heterologous Omicron LNP-mRNA booster elicited neutralizing titers 10-20 fold higher than the homologous WT booster against the Omicron variant, with comparable titers against the Delta variant. All three types of vaccination, including Omicron mRNA alone, WT mRNA homologous booster, and Omicron heterologous booster, elicited broad binding antibody responses against SARS-CoV-2 WA-1, Beta, and Delta variants, as well as other Betacoronavirus species such as SARS-CoV, but not Middle East respiratory syndrome coronavirus (MERS-CoV). These data provided direct proof-of-concept assessments of an Omicron-specific mRNA vaccination in vivo, both alone and as a heterologous booster to the existing widely-used WT mRNA vaccine form.

immunology↗

bioRxiv2022

Semaphorin 3G exacerbates joint inflammation through the accumulation and proliferation of macrophages in the synovium

ObjectiveMethotrexate (MTX) is an anchor drug for rheumatoid arthritis (RA) treatment; however, the exact mechanisms by which MTX improves RA activity are still debatable. This study aimed to understand the roles of molecules whose expression is affected by MTX in RA patients and find novel therapeutic targets. MethodsCD4+ T cells from 28 treatment naive RA patients before and 3 months after the initiation of MTX treatment were subjected to DNA microarray analyses. The expression of Semaphorin 3G (Sema3G), as one of the differentially-expressed genes, and its receptor, Neuropilin-2 (Nrp2), was evaluated in RA synovium and collagen-induced arthritis (CIA) synovium. CIA and collagen antibody-induced arthritis (CAIA) were induced in Sema3G-deficient (Sema3G-/-) mice and control mice, and the clinical score, histological score, and serum cytokines were assessed. The migration and proliferation of Sema3G-stimulated bone marrow-derived macrophages (BMMs) were analyzed in vitro. The effect of local Sema3G administration during CAIA on the clinical score and the quantity of infiltrating macrophages was evaluated. ResultsThe expression of Sema3G in CD4+ T cells was downregulated by MTX treatment in RA patients. Sema3G was expressed in RA but not osteoarthritis synovium, and its receptor Nrp2 was mainly expressed on activated macrophages. Sema3G deficiency ameliorated CIA and CAIA. Sema3G stimulation enhanced the migration and proliferation of BMMs. The local administration of Sema3G deteriorated CAIA and increased infiltrating macrophages. ConclusionsUpregulation of Sema3G in RA synovium is a novel mechanism to deteriorate joint inflammation through the accumulation of macrophages. Key messagesSemaphorin 3G is expressed in the inflamed synovium in human and mice. The receptor of Semaphorin 3G is mainly expressed on M1 macrophages. Semaphorin 3G deteriorates inflammatory arthritis through macrophage proliferation and migration.

pathology↗

bioRxiv2022

Genetic and genomic analysis of oxygen consumption in mice

We estimated genetic parameters of oxygen consumption (OC), OC per metabolic body weight (OCMBW), and body weight at three through eight weeks of age in divergently selected mice populations, with an animal model considering maternal genetic, common litter environmental, and cytoplasmic inheritance effects. Cytoplasmic inheritance was considered based on maternal lineage information. For OC, estimated direct heritability was moderate (0.32) and estimated maternal heritability and proportion of the variance of cytoplasmic inheritance effects to the phenotypic variance were very low (both <0.03), implying that causal genes for OC could be located on autosomes. To assess this hypothesis, we attempted to identify possible candidate causal genes by performing pool-seq using pooled DNA samples from mice in high and low OC lines and selective signature detection. We made a list of possible candidate causal genes for OC, including those relating to electron transport chain and ATP-binging proteins (Ndufa12, Sdhc, Atp10b, etc.), Prr16 encoding Largen protein, Cry1 encoding a key component of the circadian core oscillator, and so on. The results could contribute to elucidate the genetic mechanism of OC, an indicator for maintenance energy requirement and therefore feed efficiency.

genetics↗

bioRxiv2021

Revealing a hidden intermediate of rotatory catalysis with X-ray crystallography and Molecular simulations

The mechanism of rotatory catalysis in ATP-hydrolyzing molecular motors remain an unresolved puzzle in biological energy transfer. Notwith standing the wealth of available biochemical and structural information inferred from years of experiments, knowledge on how the coupling between the chemical and mechanical steps within motors enforces directional rotatory movements remains fragmentary. Even more contentious is to pinpoint the rate-limiting step of a multi-step rotation process. Here, using Vacuolar or V1-type hexameric ATPase as an exemplary rotational motor, we present a model of the complete 4-step conformational cycle involved in rotatory catalysis. First, using X-ray crystallography a new intermediate or dwell is identified, which enables the release of an inorganic phosphate (or Pi) after ATP hydrolysis. Using molecular dynamics simulations, this new dwell is placed in a sequence with three other crystal structures to derive a putative cyclic rotation path. Free-energy simulations are employed to estimate the rate of the hexameric protein transformations, and delineate allosteric effects that allow new reactant ATP entry only after hydrolysis product exit. An analysis of transfer entropy brings to light how the sidechain-level interactions transcend into larger-scale reorganizations, highlighting the role of the ubiquitous arginine-finger residues in coupling chemical and mechanical information. Inspection of all known rates encompassing the 4-step rotation mechanism implicates overcoming of the ADP interactions with V1-ATPase to be the rate-limiting step of motor action.

biophysics↗

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

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↗

bioRxiv2021

Laboratory analysis of glucose, fructose, and sucrose contents in Japanese common beverages for the exact assessment of beverage-derived sugar intake

BackgroundThe adverse health effects of sugar-sweetened beverage consumption have been studied worldwide. There are several reports on actual sugar contents in sugar-sweetened beverages. However, there is no recent report on actual sugar contents in Japanese sugar-sweetened beverages. Therefore, we attempted to analyze glucose, fructose, and sucrose contents in Japanese common beverages. MethodsGlucose, fructose, and sucrose contents in 49 beverages including 8 energy drinks, 11 sodas, 4 fruit juices, 7 probiotic drinks, 4 sports drinks, 5 coffee drinks, 6 green tea drinks, and 4 tea drinks were determined using the enzymatic methods. ResultsTow zero calorie drinks, 2 sugarless coffee drinks, and 6 green tea drinks contained no sugar. Three coffee drinks contained only sucrose. The orders of median glucose, fructose, and sucrose contents in categorized beverages containing sugars were as follows: for glucose, fruit juice > energy drink [≥] soda >> probiotic drink > black tea drink > sports drink; for fructose, probiotic drink [≥] energy drink > fruit juice > soda >> sports drink > black tea drink; and for sucrose, black tea drink > energy drink [≥] probiotic drink > fruit juice > soda > coffee drink >> sports drink. The rate of total fructose content in total sugar content in 38 sugar-containing beverages was approximately 40-60%. The total sugar content analyzed was not always equivalent to carbohydrate content indicated on the nutrition label. ConclusionsThese results indicate that actual sugar content in Japanese common beverages is necessary for the exact assessment of beverage-derived sugar intake.

biochemistry↗

bioRxiv2021

Crocosphaera as a major consumer of fixed nitrogen despite its capability of nitrogen fixation

Crocosphaera watsonii (hereafter Crocosphaera) is a key nitrogen (N) fixer in the ocean, but its ability to consume combined N sources is still unclear. Using in situ microcosm incubations with an ecological model, we show that Crocosphaera has high competitive capability both under low and moderately high combined N concentrations. In field incubations, Crocosphaera accounted for the highest consumption of ammonium and nitrate, followed by pico-eukaryotes. The model analysis shows that cells have a high ammonium uptake rate ([~]7 mol N (mol N)-1 d-1 at the maximum), which allows them to compete against pico-eukaryotes and non-diazotrophic cyanobacteria when combined N is sufficiently available. Even when combined N is depleted, their capability of nitrogen fixation allows higher growth rates compared to potential competitors. These results suggest the high fitness of Crocosphaera in combined N limiting, oligotrophic oceans, and thus heightens its potential significance in its ecosystem and in biogeochemical cycling.

microbiology↗

bioRxiv2021

Design of complicated all-α protein structures

A wide range of de novo protein structure designs have been achieved, but the complexity of naturally occurring protein structures is still far beyond these designs. To expand the diversity and complexity of de novo designed protein structures, we sought to develop a method for designing "difficult-to-describe"-helical protein structures composed of irregularly aligned -helices like globins. Backbone structure libraries consisting of a myriad of -helical structures with 5- or 6-helices were generated by combining 18 helix-loop-helix motifs and canonical -helices, and five distinct topologies were selected for de novo design. The designs were found to be monomeric with high thermal stability in solution and fold into the target topologies with atomic accuracy. This study demonstrated that complicated -helical proteins are created using typical building blocks. The method we developed would enable us to explore the universe of protein structures for designing novel functional proteins.

biophysics↗

bioRxiv2021

Discovery of the fastest myosin, its amino acid sequence, and structural features

Cytoplasmic streaming with extremely high velocity (~70 m s-1) occurs in cells of the characean algae (Chara). Because cytoplasmic streaming is caused by organelle-associated myosin XI sliding along actin filaments, it has been suggested that a myosin XI, which has a velocity of 70 m s-1, the fastest myosin measured so far, exists in Chara cells. However, the previously cloned Chara corallina myosin XI (CcXI) moved actin filaments at a velocity of around 20 m s-1, suggesting that an unknown myosin XI with a velocity of 70 m s-1 may be present in Chara. Recently, the genome sequence of Chara braunii has been published, revealing that this alga has four myosin XI genes. In the work reported in this paper, we cloned these four myosin XIs (CbXI-1, 2, 3, and 4) and measured their velocities. While the velocities of CbXI-3 and CbXI-4 were similar to that of CcXI, the velocities of CbXI-1 and CbXI-2 were estimated to be 73 and 66 m s-1, respectively, suggesting that CbXI-1 and CbXI-2 are the main contributors to cytoplasmic streaming in Chara cells and showing that CbXI-1 is the fastest myosin yet found. We also report the first atomic structure (2.8 [A] resolution) of myosin XI using X-ray crystallography. Based on this crystal structure and the recently published cryo-EM structure of acto-myosin XI at low resolution (4.3 [A]), it appears that the actin-binding region contributes to the fast movement of Chara myosin XI. Mutation experiments of actin-binding surface loop 2 support this hypothesis. Significance statementIt has been suggested for more than 50 years that the fastest myosin in the biological world, with a velocity of 70 m s-1, exists in the alga Chara because cytoplasmic streaming with a velocity of 70 m s-1 occurs in Chara cells. However, a myosin with that velocity has not yet been identified. In this work, we succeeded in cloning a myosin XI with a velocity of 73 m s-1, the fastest myosin so far measured. We also successfully crystallized myosin XI for the first time. Structural analyses and mutation experiments suggest that the central regions that define the fast movement of Chara myosin XI are the actin-binding sites.

cell biology↗

bioRxiv2021

Reconstitution of prenyltransferase activity on nanodiscs by components of the rubber synthesis machinery of the Para rubber tree and guayule

Prenyltransferases mediate the biosynthesis of various types of polyisoprene compound in living organisms. Natural rubber (NR) of the Para rubber tree (Hevea brasiliensis) is synthesized as a result of prenyltransferase activity, with the proteins HRT1, HRT2, and HRBP having been identified as candidate components of the rubber biosynthetic machinery. To clarify the contribution of these proteins to prenyltransferase activity, we established a cell-free translation system for nanodisc-based protein reconstitution and measured the enzyme activity of the protein-nanodisc complexes. Cell-free synthesis of HRT1, HRT2, and HRBP in the presence of asolectin nanodiscs revealed that all three proteins were membrane associated. A complex of HRT1 and HRBP formed as a result of co-expression of the two proteins in the presence of nanodiscs manifested marked polyisoprene synthesis activity, whereas neither HRT1, HRT2, or HRBP alone nor a complex of HRT2 and HRBP exhibited such activity. Similar analysis of guayule (Parthenium argentatum) proteins revealed that three HRT1 homologs (CPT1-3) manifested prenyltransferease activity only if co-expressed with the homolog of HRBP (CBP). Our results thus indicate that the core prenyltransferase of the rubber biosynthetic machinery of both the Para rubber tree and guayule is formed by the assembly of heterologous subunits (HRT1 and HRBP in the former species).

plant biology↗

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

Noradrenergic alpha-2A receptor activation suppresses courtship vocalization in male Japanese quail.

Male Japanese quail produce high-frequency crow vocalizations to attract females during the breeding season. The nucleus of intercollicularis (ICo) is the midbrain vocal center in birds and electrical stimulation of the ICo produces calls that include crowing. Noradrenaline plays a significant role in sexual behavior but the contribution of noradrenaline in the control of courtship vocalizations in quail has not been well established. Using dose-dependent intracerebroventricular injection of clonidine, an 2-adrenergic receptor- specific agonist, crowing vocalization was immediately suppressed. At the same time as crow suppression by clonidine there was a reduction of immediate early gene, zenk mRNA, in the ICo; no zenk mRNA expression was detected in the dorsomedial division of the nucleus. Using histochemistry, we determined that the ICo receives noradrenergic innervation and expresses 2A-adrenergic receptor mRNA. Taken together, these data suggest that noradrenaline regulates courtship vocalization in quail, possibly via the alpha2A- adrenergic receptor expressed on ICo neurons.

animal behavior and cognition↗

bioRxiv2021

Effects of domestication on neophobia: A comparison between the domesticated Bengalese finch (Lonchura striata var. domestica) and its wild ancestor, the white-rumped munia (Lonchura striata)

Bengalese finches (Lonchura striata var. domestica) have more complex song traits than their wild ancestors, white-rumped munias (Lonchura striata). Domesticated finches are likely able to allocate more resources to song development rather than allocating resources to mechanisms aimed at coping with predation, which are no longer needed under domesticated conditions. Here, we aimed to examine the effects of changes in selection pressure due to domestication on the behaviour of Bengalese finches and to contemplate the possible evolutionary mechanisms underlying these changes. To do so, we compared neophobic responses to novel-object conditions as an assessment of reactions to potential predators. We studied groups of Bengalese finches and white-rumped munias and found that Bengalese finches were more likely to eat the food provided to them under novel-object conditions. Bengalese finches had a shorter latency time to eat, and this latency time was less affected by the novel object in the case of Bengalese finches compared to white-rumped munias. Therefore, Bengalese finches have reduced neophobic responses due to domestication. The behavioural strategies of white-rumped munias appear to be more suitable for natural environments, which include unpredictable risks, whereas Bengalese finches have likely adapted their behaviour to the conditions of artificial selection.

animal behavior and cognition↗

bioRxiv2021

Decreased synthesis and variable gene transcripts of oxytocin in a domesticated avian species

The Bengalese finch was domesticated more than 250 years ago from the wild white-rumped munia. Similar to other domesticated species, Bengalese finches show a reduced fear response and have lower corticosterone levels, compared to white-rumped munias. Bengalese finches and munias also have different song types. Since oxytocin (OT) has been found to be involved in stress coping and auditory processing, we tested whether the OT sequence and brain expression pattern and content differ in wild munias and domesticated Bengalese finches. We identified intra-strain variability in the untranslated regions of the OT sequence in Bengalese finches in comparison to the munia OT. Several of these changes fall in specific transcription factor binding sites, which show either a conserved or a relaxed evolutionary trend in the avian lineage, and in vertebrates in general. Although in situ hybridization in several hypothalamic nuclei did not reveal significant differences in the number of cells expressing OT between the two strains, real-time quantitative PCR showed significantly lower OT mRNA expression in the diencephalon of the Bengalese finches relative to munias. Our study thus points to a decreased OT synthesis in the domestic strain compared with the wild strain in birds. This is an opposite pattern from that found in some domesticated mammals, suggesting that different processes of OT function might have occurred in mammals and birds under domestication.

neuroscience↗