Biology subjects

Yamada, H.

Publications and source records attributed to Yamada, H..

33 records · Page 2Linked to original sources

bioRxiv2021

A novel hamster model of SARS-CoV-2 respiratory infection using a pseudotyped virus

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a biosafety level (BSL)-3 pathogen; therefore, its research environment is strictly limited. Pseudotyped viruses that mimic SARS-CoV-2 have been widely used for in vitro evaluation because they are available in BSL-2 containment laboratories; however, in vivo application is inadequate. Therefore, animal models that can be instigated with animal BSL-2 will increase opportunities for in vivo evaluations. MethodsHamsters (6-to 10-week-old males) were intratracheally inoculated with luciferase-expressing vesicular stomatitis virus (VSV)-based SARS-CoV-2 pseudotyped virus. The lungs were harvested 24 h after inoculation, and luminescence was measured using an in vivo imaging system. ResultsLung luminescence after inoculation with the SARS-CoV-2 pseudotyped virus increased in a dose-dependent manner. VSV-G (envelope [G]) pseudotyped virus also induced luminescence; however, a 100-fold concentration was required to reach a level similar to that of the SARS-CoV-2 pseudotyped virus. ConclusionsThe SARS-CoV-2 pseudotyped virus is applicable to SARS-CoV-2 respiratory infections in a hamster model. Because of the single-round infectious virus, the model can be used to study the steps from viral binding to entry, which will be useful for future research regarding SARS-CoV-2 entry without using live SARS-CoV-2 or transgenic animals.

microbiology↗

bioRxiv2021

Microtubule assembly by soluble tau impairs vesicle endocytosis and excitatory neurotransmission via dynamin sequestration in Alzheimer's disease synapse model

Elevation of soluble wild-type (WT) tau occurs in synaptic compartments in Alzheimers disease. We addressed whether tau elevation affects synaptic transmission at the calyx of Held in brainstem slices. Whole-cell loading of WT human tau (h-tau) in presynaptic terminals at 10-20 {micro}M caused microtubule (MT) assembly and activity-dependent rundown of excitatory neurotransmission. Capacitance measurements revealed that the primary target of WT h-tau is vesicle endocytosis. Blocking MT assembly using nocodazole prevented tau-induced impairments of endocytosis and neurotransmission. Immunofluorescence imaging analyses revealed that MT assembly by WT h-tau loading was associated with an increased bound fraction of the endocytic protein dynamin. A synthetic dodecapeptide corresponding to dynamin-1-pleckstrin-homology domain inhibited MT-dynamin interaction and rescued tau-induced impairments of endocytosis and neurotransmission. We conclude that elevation of presynaptic WT tau induces de novo assembly of MTs, thereby sequestering free dynamins. As a result, endocytosis and subsequent vesicle replenishment are impaired, causing activity-dependent rundown of neurotransmission. Significance StatementWild-type human recombinant tau loaded in rodent presynaptic terminals inhibited vesicle endocytosis, thereby causing activity-dependent rundown of excitatory transmission. This endocytic block is caused by a sequestration of dynamin by excess microtubules newly assembled by tau and can be rescued by a peptide inhibiting the microtubules-dynamin interaction, or by the microtubule disassembler nocodazole. Thus, synaptic dysfunction can be induced by pathological increase of endogenous soluble tau in Alzheimer disease slice model.

neuroscience↗

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

m6A demethylase ALKBH5 promotes tumor cell proliferation by destabilizing IGF2BPs target genes and worsens the prognosis of patients with non-small cell lung cancer

The modification of N6-methyladenosine (m6A) in RNA and its eraser ALKBH5, an m6A demethylase, play important roles across various steps of human carcinogenesis. However, the involvement of ALKBH5 in non-small cell lung cancer (NSCLC) development remains to be completely elucidated. The current study revealed that the expression of ALKBH5 were increased in NSCLC and increased expression of ALKBH5 worsened the prognosis of patients with NSCLC. In vitro study revealed that ALKBH5 knockdown suppressed cell proliferation ability of PC9 and A549 cells as well as promoted G1 arrest and increased the number of apoptotic cells. Furthermore, ALKBH5 overexpression increased the cell proliferation ability of the immortalized cell lines. Microarray analysis and western blotting revealed that the expression of CDKN1A or TIMP3 were increased by ALKBH5 knockdown. These alterations were offset by a double knockdown of both ALKBH5 and one of the IGF2BPs. The decline of mRNAs was, at least partly, owing to the destabilization of these mRNAs by one of the IGF2BPs. In conclusions, the ALKBH5-IGF2BPs axis promotes cell proliferation and tumorigenicity, which in turn causes the unfavorable prognosis of NSCLC.

cancer biology↗

bioRxiv2021

Visualization of ecological successions in lakes subdivided by volcanic eruption at Akan Caldera, Hokkaido, Japan

It is not feasible to continuously observe ecological succession in lakes because of the long time-scales generally involved. Thus, the process has been inductively deduced by comparing many lakes with different succession states, or indirectly simulated by tracking studies of smaller water bodies, experiments using microcosms or mesocosms, and reconstruction of lake history by sediment analysis. However, the reality of succession processes in large lakes with slow succession is not well understood, and new approaches are needed. Theoretically, in a group of large and small lakes of similar ages and with similar initial and watershed environments, the rate of nutrient accumulation in each lake depends on the ratio of watershed area to lake size, and the lakes are predicted to evolve to different trophic levels over time. Here, we tested this hypothesis on the 10 lakes of varying sizes in Akan Caldera, Japan, which were formed thousands of years ago by fragmentation due to volcanic eruptions within the caldera. Topographic and water quality studies showed that the ratio of accumulated watershed area to lake size (area and volume), expressed logarithmically, had a positive linear regression with the total phosphorus concentration, an indicator of trophic level. The trophic levels of the lakes were diverse, including oligotrophic, mesotrophic, and eutrophic types in the traditional "lake type" classification based on total phosphorus and chlorophyll-a concentrations. Furthermore, 21 species of aquatic macrophytes were observed by a diving survey, and the plant species composition was classified into five groups corresponding to the trophic status of the lakes, indicating a conventional "hydrarch succession". The diversity of water quality and aquatic vegetation in a group of lakes with similar origins paves the way for new comparative studies of lakes, including large lakes.

ecology↗

bioRxiv2021

Dynamic prospect theory - two core economic decision theories coexist in the gambling behavior of monkeys

Research in behavioral economics and reinforcement learning has given rise to two influential theories describing human economic choice under uncertainty. The first, prospect theory, assumes that decision-makers use static mathematical functions, utility and probability weighting, to calculate the values of alternatives. The second, reinforcement learning theory, posits that dynamic mathematical functions update the values of alternatives based on experience through reward prediction error (RPE). To date, these theories have been examined in isolation without reference to one another. Therefore, it remains unclear whether RPE affects a decision-makers utility and/or probability weighting functions, or whether these functions are indeed static as in prospect theory. Here, we propose a dynamic prospect theory model that combines prospect theory and RPE, and test this combined model using choice data on gambling behavior of captive macaques. We found that under standard prospect theory, monkeys, like humans, had a concave utility function. Unlike humans, monkeys exhibited a concave, rather than inverse-S shaped, probability weighting function. Our dynamic prospect theory model revealed that probability distortions, not the utility of rewards, solely and systematically varied with RPE: after a positive RPE, the estimated probability weighting functions became more concave, suggesting more optimistic belief about receiving rewards and over-weighted subjective probabilities at all probability levels. Thus, the probability perceptions in laboratory monkeys are not static even after extensive training, and are governed by a dynamic function well captured by the algorithmic feature of reinforcement learning. This novel evidence supports combining these two major theories to capture choice behavior under uncertainty. Significance statementWe propose and test a new decision theory under uncertainty by combining pre-existing two influential theories in the neuroeconomics: prospect theory from economics and prediction error theory from reinforcement learning. Collecting a large dataset (over 60,000 gambling decisions) from laboratory monkeys enables us to test the hybrid model of these two core decision theories reliably. Our results showed over-weighted subjective probabilities at all probability levels after lucky win, indicating that positive prediction error systematically bias decision-makers more optimistically about receiving rewards. This trial-by-trial prediction-error dynamics in probability perception provides outperformed performance of the model compared to the standard static prospect theory. Thus, both static and dynamic elements coexist in monkeys risky decision-making, an evidence contradicting the assumption of prospect theory.

animal behavior and cognition↗

bioRxiv2021

Imaging-based evaluation of pathogenicity by novel DNM2 variants associated with centronuclear myopathy

Centronuclear myopathy (CNM) is characterized clinically by muscle weakness and pathologically by the presence of centralized nuclei and disarrangement of T-tubules in muscle fibers. DNM2 which encodes a large GTPase dynamin 2 have been identified as a causative gene for CNM. Nevertheless, the identification of DNM2 variants may not always lead to the definitive diagnosis as their pathogenicity is often unknown. In this study, by imaging T-tubule-like structures reconstituted in cellulo, we demonstrated that aberrant membrane remodeling by mutant dynamin 2 is tightly associated with gain-of-function features of DNM2 variants. This simple in cellulo assay provided quantitative data required for accurately evaluating pathogenicity of reported and novel DNM2 variants identified from CNM patients in our cohort. Our approaches combining the in cellulo assay with clinical information of the patients enabled to explain the course of a disease progression by pathogenesis of each variant in DNM2-associated CNM.

cell biology↗

bioRxiv2020

Association between the extent of DNA methylation at the CpG sites of HIF3A and parameters of obesity in the general Japanese population

Obesity is a major public health problem worldwide owing to the substantial increase in risk of metabolic diseases. Hypoxia-inducible factors (HIFs) regulate transcriptional responses to hypoxic stress. DNA methylation in the CpG sites of intron 1 of HIF3A is associated with body mass index in the whole blood and adipose tissue. This study investigates the correlation between DNA methylation of HIF3A and parameters of obesity, including thickness of visceral (VAT) and subcutaneous adipose tissues, in the general Japanese population. Participants (220 men and 253 women) who underwent medical examination were enrolled in this cross-sectional study. We used pyrosequencing to quantify DNA methylation (CpG sites of cg16672562, cg22891070, and cg27146050) in HIF3A. DNA methylation of HIF3A was only different in women. Multiple regression analysis showed that DNA methylation level at cg27146050 was associated with thickness of VAT in women. DNA methylation level at cg27146050 also correlated with body mass index and percentage of body fat in women after excluding smokers and non-smokers who quit smoking with the last 5 years. DNA methylation in the CpG site (cg27146050) of HIF3A correlated with parameters of obesity in Japanese women.

biochemistry↗

bioRxiv2020

Tonic firing mode of midbrain dopamine neurons continuously tracks reward values changing moment-by-moment

Appropriate actions are taken based on the values of future rewards. The phasic activity of midbrain dopamine neurons signals these values. Because reward values often change over time, even on a subsecond-by-subsecond basis, appropriate action selection requires continuous value monitoring. However, the phasic dopamine activity, which is sporadic and has a short duration, likely fails continuous monitoring. Here, we demonstrate a tonic firing mode of dopamine neurons that effectively tracks changing reward values. We recorded dopamine neuron activity in monkeys during a Pavlovian procedure in which the value of a cued reward gradually increased or decreased. Dopamine neurons tonically increased and decreased their activity as the reward value changed. This tonic activity was evoked more strongly by non-burst spikes than burst spikes producing a conventional phasic activity. Our findings suggest that dopamine neurons change their firing mode to effectively signal reward values, which could underlie action selection in changing environments.

neuroscience↗

bioRxiv2020

Evaluation of SARS-CoV-2 neutralizing antibodies using a vesicular stomatitis virus possessing SARS-CoV-2 spike protein

SARS-CoV-2 is a novel coronavirus that emerged in 2019 and is now classified in the genus Coronavirus with closely related SARS-CoV. SARS-CoV-2 is highly pathogenic in humans and is classified as a biosafety level (BSL)-3 pathogen, which makes manipulating it relatively difficult due to its infectious nature. To circumvent the need for BSL-3 laboratories, an alternative assay was developed that avoids live virus and instead uses a recombinant VSV expressing luciferase and possesses the full length or truncated spike proteins of SARS-CoV-2. Furthermore, to measure SARS-CoV-2 neutralizing antibodies under BSL2 conditions, a chemiluminescence reduction neutralization test (CRNT) for SARS-CoV-2 was developed. The neutralization values of the serum samples collected from hospitalized patients with COVID-19 or SARS-CoV-2 PCR-negative donors against the pseudotyped virus infection evaluated by the CRNT were compared with antibody titers determined from an immunofluorescence assay (IFA). The CRNT, which used whole blood collected from hospitalized patients with COVID-19, was also examined. As a result, the inhibition of pseudotyped virus infection was specifically observed in both serum and whole blood and was also correlated with the results of the IFA. In conclusion, the CRNT for COVID-19 is a convenient assay system that can be performed in a BSL-2 laboratory with high specificity and sensitivity for evaluating the occurrence of neutralizing antibodies against SARS-CoV-2.

microbiology↗

bioRxiv2020

Tonically active neurons in the monkey dorsal striatum signal outcome feedback during trial-and-error search behavior

An animals choice behavior is shaped by the outcome feedback from selected actions in a trial-and-error approach. Tonically active neurons (TANs), presumed cholinergic interneurons in the striatum, are thought to be involved in the learning and performance of reward-directed behaviors, but it remains unclear how TANs are involved in shaping reward-directed choice behaviors based on the outcome feedback. To this end, we recorded activity of TANs from the dorsal striatum of two macaque monkeys (Macaca fuscata; 1 male, 1 female) while they performed a multi-step choice task to obtain multiple rewards. In this task, the monkeys first searched for a rewarding target from among three alternatives in a trial-and-error manner and then earned additional rewards by repeatedly choosing the rewarded target. We found that a considerable proportion of TANs selectively responded to either the reward or the no-reward outcome feedback during the trial-and-error search, but these feedback responses were not observed during repeat trials. Moreover, the feedback responses of TANs were similarly observed in any search trials, without distinctions regarding the predicted probability of rewards and the location of chosen targets. Unambiguously, TANs detected reward and no-reward feedback specifically when the monkeys performed trial-and-error searches, in which the monkeys were learning the value of the targets and adjusting their subsequent choice behavior based on the reward and no-reward feedback. These results suggest that striatal cholinergic interneurons signal outcome feedback specifically during search behavior, in circumstances where the choice outcomes cannot be predicted with certainty by the animals. HighlightsO_LITANs signal reward and no-reward outcome feedback when monkeys made search behaviors C_LIO_LITANs respond regardless of predicted reward probability or chosen target location C_LIO_LITANs may signal feedback outcomes when rewards cannot be predicted with certainty C_LI

neuroscience↗

bioRxiv2020

Neural population dynamics underlying expected value computation

Computation of expected values, i.e., probability times magnitude, seems to be a dynamic integrative process performed in the brain for efficient economic behavior. However, neural dynamics underlying this computation remain largely unknown. We examined (1) whether four core reward-related regions detect and integrate the probability and magnitude cued by numerical symbols and (2) whether these regions have distinct dynamics in the integrative process. Extractions of mechanistic structure of neural population signal demonstrated that expected-value signals simultaneously arose in central part of orbitofrontal cortex (cOFC, area 13m) and ventral striatum (VS). These expected-value signals were incredibly stable in contrast to weak and/or fluctuated signals in dorsal striatum and medial OFC. Notably, temporal dynamics of these stable expected-value signals were unambiguously distinct: sharp and gradual signal evolutions in cOFC and VS, respectively. These intimate dynamics suggest that cOFC and VS compute the expected-values with unique time constants, as distinct, partially overlapping processes.

neuroscience↗

bioRxiv2020

Demonstration of resistance to satyrization behavior in Aedes aegypti (Linnaeus) from La Reunion island

Aedes aegypti and Aedes albopictus are competent vectors of arboviruses such as dengue and chikungunya viruses which co-exist in some areas including La Reunion island. A kind of reproductive interference called satyrization has been described in sympatric species where a dominant species tends to control the spread of the other species. Here, we investigated satyrization in laboratory experiments to provide insights on the potential impact on Ae. aegypti of a control campaign including a sterile insect technique component against Ae. albopictus. Different mating crosses were used to test sympatric, conspecific-interspecific and allopatric effects of irradiated and non-irradiated male Ae. albopictus on female Ae. aegypti, including in a situation of skewed male ratio. Our results suggest that there was only a low level of satyrization between sympatric populations of Ae. aegypti and Ae. albopictus colonised from La Reunion island. A male Ae. albopictus to female Ae. aegypti ratio of 3:1 did not increase the level of satyrization. Female Ae. aegypti previously mated to male Ae. albopictus were not prevented from being inseminated by conspecific male Ae. aegypti. A satyrization effect was not seen between allopatric Ae. albopictus and Ae. aegypti strains from La Reunion Island either. The tested Ae. aegypti strain from La Reunion island has therefore developed full resistance to satyrization and so releasing sterile male Ae. albopictus may not suppress Ae. aegypti populations if an overflooding of irradiated male Ae. albopictus leads to similar results. The management strategy of two competent species in a sympatric area is discussed.

ecology↗

bioRxiv2018

Six species of non-tuberculosis mycobacteria carry non-identical 16S rRNA gene copies

Non-tuberculosis mycobacteria (NTM) can carry two or more 16S rRNA gene copies that are, in some instances, non-identical. In this study, we used a combined cloning and sequencing approach to analyze the 16S rRNA gene sequences of six NTM species, Mycobacterium cosmeticum, M. pallens, M. hodleri, M. crocinum, M. flavescens, and M. xenopi. The approach facilitated the identification of two distinct gene copies in each species. The two M. cosmeticum genes had a single nucleotide difference, whereas two nucleotide polymorphisms were identified in M. hodleri, M. flavescens, and M. xenopi. M. pallens had a difference in four nucleotides and M. crocinum in 23. Hence, we showed that the six NTM species possess at least two non-identical 16S rRNA gene copies.\n\nImportanceThe presence of multiple 16S rRNA gene copies with nucleotide polymorphisms represents a challenge for species identification using 16S rRNA as the target sequence. Our analysis was focused on six NTM species, M. cosmeticum, M. pallens, M. hodleri, M. crocinum, M. flavescens, and M. xenopi. As a result, we generated the full-length sequences of two non-identical 16S rRNA copies for each NTM species. The data will be helpful for the sequence analysis of specimens or other samples.

bioinformatics↗

bioRxiv2017

Dynamic clustering of dynamin-amphiphysin rings regulates membrane constriction and fission coupled with GTP hydrolysis

Dynamin is a mechanochemical GTPase essential for membrane fission during clathrin mediated endocytosis. Dynamin forms washer ring-shaped/helical complexes at the neck of clathrin-coated pits and their structural changes coupled with GTP hydrolysis drive membrane fission. Dynamin and its binding protein amphiphysin cooperatively regulates membrane remodeling during fission, but its precise mechanism remains elusive. In this study, we analyze structural changes of dynamin-amphiphysin complexes during membrane fission using electron microscopy (EM) and high-speed atomic force microscopy (HS-AFM). Interestingly, HS-AFM analyses show that the dynamin-amphiphysin rings are rearranged to form clusters upon GTP hydrolysis and membrane constriction occurs at protein-uncoated regions flanking the clusters. We also show a novel function of amphiphysin in size control of the clusters to enhance biogenesis of endocytic vesicles. Our new approaches using combination of EM and HS-AFM clearly demonstrates dynamics of dynamin-amphiphysin complexes during membrane fission suggesting a novel \"clusterase\" model of dynamin-mediated membrane fission.

biophysics↗