Biology subjects

Sasaki, R.

Publications and source records attributed to Sasaki, R..

16 recordsLinked to original sources

bioRxiv2026

Proximity Labeling-Assisted Flow Cytometry Improves Fluorescence Contrast in Extracellular Vesicle-Enriched Fractions

Extracellular vesicles (EVs) are potential sources of circulating biomarkers, but disease-associated EVs are often scarce and their nanoscale dimensions limit analysis by conventional flow cytometry. To improve the detection of disease-associated EV signals using widely available instruments, we developed a workflow combining preparation of serum EV-enriched fractions by size-exclusion chromatography, CHL1-initiated enzyme-mediated activation of radical sources (EMARS) proximity labeling, and conventional flow cytometry. EMARS deposits fluorescein on molecules proximal to antibody-bound CHL1, thereby increasing target-initiated fluorescence. In a representative comparison, EMARS increased the signal-to-background ratio from 1.21 with conventional antibody labeling to 6.28. Because unlabeled EVs and debris interfered with analysis, we used an AI-assisted procedure to explore FITC-H cutoffs for retained P1 events. ChatGPT compared 65 candidate cutoffs across statistical and event-retention metrics and recommended 1000; the authors reviewed the complete results before adopting this value. At this data-selected cutoff, mean fluorescence intensity was higher in interstitial lung disease (ILD) than in non-ILD samples among 28 evaluable samples (P = 0.002; area under the receiver operating characteristic curve [AUC] = 0.846); neither value was adjusted for cutoff selection. Post hoc nested leave-oneout cross-validation (LOOCV), in which cutoff selection was repeated within each outer fold, yielded an AUC of 0.716 with 29 of 34 samples evaluable. The association with ILD requires validation in independent cohorts, but these exploratory findings suggest that proximity labeling may enhance measurable fluorescence contrast in serum EV-enriched fractions and support further evaluation of disease-associated EV signals using conventional flow cytometers.

biochemistry↗

bioRxiv2026

From photoprotection to growth: shifts in high-light acclimation strategies associated with molecular evolutionary rates in duckweeds

Molecular evolutionary rates vary widely among lineages, yet the biological processes generating this variation remain poorly understood. In plants, which lack an early-segregated germline, growth- and environment-dependent physiology could itself influence mutation accumulation and, ultimately, substitution rates. Here we examine this possibility in duckweeds (Araceae, Lemnoideae), whose closely related lineages differ severalfold in sequence divergence. Nonsynonymous and synonymous divergence scaled proportionally across all six lineages examined, indicating that this heterogeneity does not reflect differences in selective constraint and instead points to variation in mutation rate. Lineages without prominent anthocyanin accumulation occupied lower latitudes and, under high light, sustained higher relative growth rates and higher photosynthetic efficiency. Comparing the early-diverging, anthocyanin-accumulating Spirodela polyrhiza with the late-diverging, anthocyanin-free Wolffia australiana revealed contrasting acclimation strategies: S. polyrhiza preferentially induced photoprotective pathways, including anthocyanin biosynthesis and non-photochemical quenching, whereas W. australiana instead expanded mitochondrial oxidative phosphorylation together with the cytosolic translational machinery, and broadly remodeled primary metabolism. Duckweed lineages therefore differ in how they balance photoprotection against photochemical energy use and downstream metabolic capacity, a divergence that may be associated with their lineage-specific molecular evolutionary rates.

evolutionary biology↗

bioRxiv2026

Tubulin Monoglutamylation is Sufficient to Rescue the Ciliary Motility Defects in a Chlamydomonas Polyglutamylation Deficient Mutant

The axonemes of eukaryotic cilia and flagella display high tubulin glutamylation heterogeneity, yet the functional significance of this variation remains elusive. We previously showed that long-chain polyglutamylation is crucial for ciliary motility in Chlamydomonas. However, the respective contributions of long-chain polyglutamylation versus short-chain species to motility remain unclear, as existing mutants did not allow for a clear functional dissection of these two modification states. Here, we generated mutants deficient in deglutamylases, cytosolic carboxypeptidases (CCPs) 1, 2, and 5. Importantly, CCP5 is known to remove the branch-point glutamate residue, the final step in deglutamylation. While axonemal polyglutamylation levels remained largely unaffected in these mutants, abundance of short-chain glutamylation was significantly increased in both the axonemal and cytoplasmic microtubules of ccp5-1, consistent with CCP5s role as a branch-point deglutamylase. Although each single mutant exhibited slightly reduced swimming velocity, the loss of CCP5 in the tpg1 background lacking long polyglutamate side chains resulted in a significant restoration of motility. These findings indicate that the abundance of short-chain species, regulated by CCP5, plays a distinct role in modulating ciliary motility, particularly in the absence of long polyglutamate side chains. This suggests that even minimal glutamylation can functionally support dynein-driven microtubule sliding.

cell biology↗

bioRxiv2025

NADPH phosphatase activity of Mesh1 controls sleep in Drosophila

Recent findings have shown that metazoans accumulate the bacterial second messenger guanosine tetraphosphate (ppGpp) and possess Mesh1, a gene encoding a ppGpp hydrolase domain. Mesh1 deficiency affects sleep behavior and eclosion under starvation in Drosopila, and ferroptosis in human cells. However, human Mesh1 also exhibits NADPH/NADP+ phosphatase activity in vitro, making it unclear whether these phenotypes result from loss of ppGpp hydrolysis, NADPH/NADP+ dephosphorylation, or both. To address this, we performed biochemical and genetic analysis of Drosophila Mesh1. We first found that Drosophila Mesh1 dephosphorylates NADPH, but not NADP+, in vitro. We subsequently sought to generate Drosophila Mesh1 point mutants specifically impaired in NADPH phosphatase activity. Based on structural data, we mutated W138 and R142, residues that are predicted to interact with NADPH but not ppGpp. W138 was replaced with phenylalanine results in complete loss of NADPH phosphatase activity with retained ppGpp hydrolase activity. Flies carrying the W138F mutation, introduced via genome editing, exhibited shortened total sleep and increased sleep fragmentation in behavioral assays, without changes in intracellular ppGpp levels. These results indicate that the NADPH phosphatase activity of Mesh1, and specifically the W138 residue, is essential for normal sleep regulation in Drosophila.

molecular biology↗

bioRxiv2025

Tubulin Acetylation Deficiency Promotes Axonemal Turnover and Increases Cytoplasmic Microtubules

Tubulin post-translational modifications regulate microtubule dynamics; among these, -tubulin acetylation has been linked to microtubule stability. We generated a Chlamydomonas mutant lacking the acetyltransferase TAT1, which completely abolished -tubulin K40 acetylation. Surprisingly, the lengths of normally acetylated structures, axonemes and rootlets, were largely unaffected. TAT1 localized to the flagellar tip, suggesting that it is the primary site of acetylation. Loss of acetylation caused an increase in axonemal tubulin turnover, as revealed by dikaryon-fusion assays. Unexpectedly, the tat1 mutant displayed an increased number of dynamic cytoplasmic microtubules and could regenerate long flagella after amputation, even when protein synthesis was inhibited. Despite these cytoskeletal changes, steady-state flagellar length, cell growth, and cell division remained essentially normal. These findings suggest that acetylation modulates microtubule behavior by regulating axonemal tubulin turnover and cytoplasmic microtubule dynamics, while cellular morphology is buffered against variations in microtubule content.

cell biology↗

bioRxiv2025

Pseudovirus-Mediated Proximity Labeling Identifies Candidate Host Cell Membrane Proteins Involved in Viral Attachment

Viral infections represent a significant threat to humanity, as exemplified by the COVID-19 pandemic. To mitigate the associated damage, it is essential to understand the biological characteristics of causative viruses. In this study, we report a novel method for identifying host cell molecules (including virus receptor) required for viral attachment, referred to here as "Host Cell Attachment Factors (HCAFs)". Our approach utilizes proximity labeling (PL) technology: pseudoviruses engineered to express a PL enzyme are applied to host cells, where they bind and initiate proximity labeling. Since HCAFs are expected to be localized near viral attachment sites, candidate HCAFs are specifically tagged by the PL process. Analysis of influenza HCAFs enabled the identification of multiple candidate molecules. Subsequent virus attachment experiments using CHO cells suggested that NRP1 may serve as an HCAF for influenza viruses. Given its simplicity and rapid turnaround, this method is adaptable to a wide range of enveloped viruses, including pandemic viruses that require emergency analysis.

microbiology↗

bioRxiv2025

Mild traumatic brain injury alters function in the dorsolateral prefrontal cortex: a TMS-EEG study.

Numerous studies highlight alterations in motor cortical neurophysiology after mild traumatic brain injury (mTBI). In contrast, understanding of effects in cognitive brain areas, which potentially underpin more impactful effects of injury, remains limited. The current study addressed this limitation by using electroencephalography to index responses to transcranial magnetic stimulation (TMS-EEG) applied over the dorsolateral prefrontal cortex (DLPFC). In 18 mTBI patients and 22 healthy controls, TMS-EEG was used to index cortical inhibition and long-term depression (LTD)-like plasticity within DLPFC. These were assessed using paired-pulse TMS (short-[SICI] and long-[LICI] interval intracortical inhibition) and continuous theta burst stimulation (cTBS), respectively. Functional effects of injury were assessed via the N-back and Trail Making Test. Responses were quantified in both temporal (i.e., TMS-evoked potentials; TEPs) and spectral (i.e., TMS-related oscillations; TROs) domains. For TEPs, measures of inhibition and plasticity both tended to be reduced by mTBI. In contrast, TRO data suggested a contradictory increased response of patients to both paired-pulse stimulation and cTBS that was specific to high-frequency bands. In addition, while TMT performance was reduced in the mTBI group, this was unrelated to neurophysiological effects of injury. These findings highlight novel effects of mTBI on DLPFC neurophysiology and emphasise the importance of considering both evoked and induced activity when examining neurophysiological effects of mTBI.

neuroscience↗

bioRxiv2024

Dimerization GAS2 mediates microtubule and F-actin crosslinking

GAS2 was originally identified as a growth arrest-specific protein, and recent studies have revealed its involvement in multiple cellular processes. Its dual interaction with actin filaments and microtubules highlights its essential role in cytoskeletal organization, such as cell division, apoptosis, and possibly tumorigenesis. However, the structural bases by which GAS2 regulates cytoskeletal dynamics remain unclear. In this study, we present cryo-EM structures of the GAS2- CH3 domain in complex with F-actin at 2.8 [A] resolution, representing the first type 3 CH domain structure bound to F-actin, confirming its actin-binding activity. We also provide the first near- atomic resolution cryo-EM structure of the GAS2-GAR domain bound to microtubules and identified conserved microtubule-binding residues. Our biochemical experiments show that GAS2 promotes microtubule nucleation and polymerization and its C-terminal region is essential for dimerization, bundling of both F-actin and microtubules, and microtubule nucleation. Based on these results, we propose how GAS2 controls cytoskeletal organization.

biochemistry↗

bioRxiv2024

Thermodynamics Underpinning the Microbial Community-Level Nitrogen Networks

Nitrogen species often serve as crucial electron donors or acceptors in microbial catabolism, enabling the synthesis of adenosine triphosphate (ATP). Although theoretically any nitrogen redox reactions could be an energy source, it remains unclear why specific reactions are predominantly utilized. This study evaluates energetically superior reactions from 988 theoretically plausible combinations involving 11 nitrogen species, oxygen gas, hydrogen ion, and water. Our analysis of the similarity between this model-based energetically superior network and the actual microbial community-level nitrogen network, reconstructed as a combination of enzymatic reactions, showed increased link overlap rates with thermodynamic weighting on reaction rates. In particular, existing microbial reactions involving solely nitrogen species and additionally oxygen, such as anaerobic ammonia oxidation (ANAMMOX) and complete and partial nitrification, were frequently identified as energetically superior among the examined reactions. The alignment of these reactions with thermodynamically favorable outcomes underscores the critical role of thermodynamics not only in individual metabolic processes but also in shaping the broader network interactions within ecosystems, consequently affecting biodiversity and ecological functions. Significance StatementThis study advances our understanding of how thermodynamics governs energy metabolism at the community level within microbial ecosystems by systematically analyzing 988 potential redox reactions involving inorganic nitrogen species, oxygen gas, hydrogen ion, and water. We uncover that existing microbial reactions, such as anaerobic ammonia oxidation (ANAMMOX) and nitrification, stand out as energetically superior over other examined reactions. The robust alignment between model-predicted energetically favorable reactions and actual microbial nitrogen reactions underscores the predictive power of thermodynamic principles, even in ecological networks. Our findings extend the traditional applications of thermodynamics in biology, highlighting how thermodynamic constraints shape ecological networks and influence biodiversity and ecosystem functions in natural ecosystems.

ecology↗

bioRxiv2024

Investigating the effects of repetitive paired-pulse transcranial magnetic stimulation on visuomotor training using TMS-EEG.

ObjectivesI-wave periodicity repetitive paired-pulse transcranial magnetic stimulation (iTMS) can modify acquisition of a novel motor skill, but the associated neurophysiological effects remain unclear. The current study therefore used combined TMS-electroencephalography (TMS-EEG) to investigate the neurophysiological effects of iTMS on subsequent visuomotor training (VT). MethodsSixteen young adults (26.1 {+/-} 5.1 years) participated in three sessions including real iTMS and VT (iTMS + VT), control iTMS and VT (iTMSsham + VT), or iTMS alone. Motor-evoked potentials (MEPs) and TMS-evoked potentials (TEPs) were measured before and after iTMS, and again after VT, to assess neuroplastic changes. ResultsIrrespective of the intervention, MEP amplitude was not changed after iTMS or VT (P = 0.211). Motor skill was improved compared with baseline (P < 0.001), but no differences were found between stimulus conditions. In contrast, the P30 peak was altered by VT when preceded by sham iTMS (P < 0.05), but this effect was not apparent when VT was preceded by iTMS or following iTMS alone (all P > 0.15). ConclusionIn contrast to expectations, iTMS was unable to modulate MEP amplitude or influence motor learning. Despite this, changes in P30 amplitude suggested that motor learning was associated with altered cortical reactivity. Furthermore, this effect was abolished by priming with iTMS, suggesting an influence of priming that failed to impact learning. Authorship statementsConceptualization: JGS; Data curation: RS, BJH, and WL; Formal analysis: RS; Funding acquisition: RS; Investigation: RS, BJH, and WL; Methodology: RS, GMO, BJH and JGS; Project administration: GMO and JGS; Supervision: GMO and JGS; Roles/Writing - original draft: RS and GMO; Writing - review & editing: BJH, WL, and JGS.

neuroscience↗

bioRxiv2023

Tau Filaments from Amyotrophic Lateral Sclerosis/Parkinsonism-Dementia Complex (ALS/PDC) adopt the CTE Fold

The amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) of the island of Guam and the Kii peninsula of Japan is a fatal neurodegenerative disease of unknown cause that is characterised by the presence of abundant filamentous tau inclusions in brains and spinal cords. Here we used electron cryo-microscopy (cryo-EM) to determine the structures of tau filaments from the cerebral cortex of three cases of ALS/PDC from Guam and eight cases from Kii, as well as from the spinal cord of two of the Guam cases. Tau filaments had the chronic traumatic encephalopathy (CTE) fold, with variable amounts of Type I and Type II filaments. Paired helical tau filaments were also found in two Kii cases. We also identified a novel Type III CTE tau filament, where protofilaments pack against each other in an anti-parallel fashion. ALS/PDC is the third known tauopathy with CTE-type filaments and abundant tau inclusions in cortical layers II/III, the others being CTE and subacute sclerosing panencephalitis. Because these tauopathies are believed to have environmental causes, our findings support the hypothesis that ALS/PDC is caused by exogenous factors. SIGNIFICANCEA neurodegenerative disease of unknown cause on the island of Guam and the Kii peninsula of Japan has been widely studied, because patients can suffer from the combined symptoms of motor neuron disease, parkinsonism and dementia. Abnormal filamentous inclusions made of tau protein characterise this amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) and their formation closely correlates with neurodegeneration. Here we have used electron cryo-microscopy (cryo-EM) to show that tau filaments from ALS/PDC are identical to those from chronic traumatic encephalopathy (CTE), a disease caused by repetitive head impacts or blast waves. CTE tau filaments are also found in subacute sclerosing panencephalitis, which is a rare consequence of measles infection. ALS/PDC may therefore also be caused by environmental factors.

neuroscience↗

bioRxiv2023

Intronic TNR-retained ISOPROPYLMALATE ISOMERASE LARGE SUBUNIT1 transcripts impair leaf development in Arabidopsis

Intronic trinucleotide repeat (TNR) is widely distributed in plant genomes. In Arabidopsis accession Bur-0, abnormally expanded TTC repeat in intron-3 of the ISOPROPYLMALATE ISOMERASE LARGE SUBUNIT1 (IIL1) gene causes growth defects called the irregularly impaired leaves (iil) phenotype, triggered by DNA methylation-mediated IIL1 gene silencing at elevated temperature. However, little is known about how the reduced expression of IIL1 causes the iil phenotype. We demonstrated that the iil phenotype was resulted from the relative increase of intron-3-retained IIL1 transcripts through the experiments where the iil phenotype was reproduced by introducing the IIL1 gene harboring 100 copies of TTC repeat into Col-0. The iil phenotype appeared when the total amount of the IIL1 transcripts was decreased by co-suppression and the percentage of intron-3-retained IIL1 transcripts was increased. The IIL1 gene encodes an isopropylmalate isomerase large subunit, forming heterodimers with small subunits (AtLeuD1, AtLeuD2, or AtLeuD3). In the myb28 myb29 mutant lacking AtLeuD1 and AtLeuD2, the iil phenotype was almost completely suppressed regardless of higher percentage of intron-3-retained IIL1 transcripts. The results indicated that the iil phenotype was associated with interaction with AtLeuDs, suggesting that intronic TNR-containing transcripts were translated into abnormal proteins and perturbed the metabolic pathway supporting the leaf development.

plant biology↗

bioRxiv2022

Modulation of I-wave generating pathways with repetitive paired-pulse transcranial magnetic stimulation: A TMS-EEG study

ObjectivesRepetitive paired-pulse transcranial magnetic stimulation (iTMS) at indirect (I) wave intervals increases motor-evoked potentials (MEPs) produced by TMS to primary motor cortex (M1). However, the effects of iTMS at early and late intervals on the plasticity of specific I-wave circuits remains unclear. The current study therefore aimed to assess how the timing of iTMS influences intracortical excitability within early and late I-wave circuits. To investigate the cortical effects of iTMS more directly, changes due to the intervention were also assessed using combined TMS-electroencephalography (EEG). Material and MethodsEighteen young adults (24.6 {+/-} 4.2 years) participated in four sessions in which iTMS targeting early (1.5 ms interval; iTMS1.5) or late (4.0 ms interval; iTMS4.0) I-waves was applied over M1. Neuroplasticity was assessed using both posterior-to-anterior (PA) and anterior-to-posterior (AP) stimulus directions to record MEPs and TEPs before and after iTMS. SICF at inter-stimulus intervals of 1.5 and 4.0 ms was also used to index I-wave activity. ResultsMEP amplitude was increased after iTMS (P < 0.01) and this was greater for PA responses (P < 0.01), but not different between iTMS intervals (P = 0.9). Irrespective of iTMS interval and coil current, SICF was facilitated after the intervention (P < 0.01). While the N45 produced by AP stimulation was reduced by iTMS1.5 (P = 0.04), no other changes in TEP amplitude were observed. ConclusionThe timing of iTMS failed to influence which I-wave circuits were potentiated by the intervention. In contrast, reductions in the N45 suggest that the neuroplastic effects of iTMS may include disinhibition of intracortical inhibitory processes.

neuroscience↗

bioRxiv2022

Utilising TMS-EEG to assess the response to cerebellar-brain inhibition

BackgroundCerebellar-brain inhibition (CBI) is a transcranial magnetic stimulation (TMS) paradigm indexing excitability of cerebellar projections to motor cortex (M1). Stimulation involved with CBI is often considered to be uncomfortable, and alternative ways to index connectivity between cerebellum and the cortex would be valuable. Utilising electroencephalography in conjunction with TMS (combined TMS-EEG) to record the response to CBI has the potential to achieve this, but has not been attempted previously. ObjectiveTo investigate the utility of TMS-EEG for characterising cerebellar-cortical interactions recruited by CBI. MethodsA total of 33 volunteers (25.7 {+/-} 4.9 years, 20 females) participated across three experiments. These investigated EEG responses to CBI induced with a figure-of-eight (F8; experiment 1) or double cone (DC; experiment 2) conditioning coil over cerebellum, in addition to multisensory sham stimulation (experiment 3). ResultsBoth F8 and DC coils suppressed early TMS-evoked EEG potentials (TEPs) produced by TMS to M1 (P < 0.05). Furthermore, the TEP produced by CBI stimulation was related to the motor inhibitory response to CBI recorded in a hand muscle (P < 0.05), but only when using the DC coil. Multisensory sham stimulation failed to modify the M1 TEP. ConclusionsCerebellar conditioning produced changes in the M1 TEP that were not apparent following sham stimulation, and that were related to the motor inhibitory effects of CBI. Our findings therefore suggest it is possible to index the response to CBI using TMS-EEG. In addition, while both F8 and DC coils appear to recruit cerebellar projections, the nature of these may be different.

neuroscience↗

bioRxiv2022

Cerebellar transcranial direct current stimulation disrupts neuroplasticity of intracortical motor circuits.

While previous research using transcranial magnetic stimulation (TMS) suggest that cerebellum (CB) influences the neuroplastic response of primary motor cortex (M1), the role of different indirect (I) wave inputs in M1 mediating this interaction remains unclear. The aim of this study was therefore to assess how CB influences neuroplasticity of early and late I-wave circuits. 22 young adults (22 {+/-} 2.7 years) participated in 3 sessions in which I-wave periodicity repetitive transcranial magnetic stimulation (iTMS) was applied over M1 during concurrent application of cathodal transcranial direct current stimulation over CB (tDCSCB). In each session, iTMS either targeted early I-waves (1.5 ms interval; iTMS1.5), late I-waves (4.5 ms interval; iTMS4.5), or had no effect (variable interval; iTMSSham). Changes due to the intervention were examined with motor evoked potential (MEP) amplitude using TMS protocols measuring corticospinal excitability (MEP1mV) and the strength of CB-M1 connections (CBI). In addition, we indexed I-wave activity using short-interval intracortical facilitation (SICF) and low-intensity single-pulse TMS applied with posterior-anterior (MEPPA) and anterior-posterior (MEPAP) current directions. Following both active iTMS sessions, there was no change in MEP1mV, CBI or SICF (all P > 0.05), suggesting that tDCSCB broadly disrupted the excitatory response that is normally seen following iTMS. However, although MEPAP also failed to facilitate after the intervention (P > 0.05), MEPPA potentiated following both active iTMS sessions (both P < 0.05). This differential response between current directions suggests that the disruptive effects of CB modulation on M1 plasticity may be selectively mediated by AP-sensitive circuits (also likely recruited with MEP1mV, CBI, and SICF).

neuroscience↗

bioRxiv2020

Modulation of motor cortex plasticity by repetitive paired-pulse TMS at late I-wave intervals is influenced by intracortical excitability

The late indirect (I) waves recruited by transcranial magnetic stimulation (TMS) over primary motor cortex (M1) can be modulated using I-wave periodicity repetitive TMS (iTMS). The purpose of this study was to determine if the response to iTMS is influenced by different interstimulus intervals (ISIs) targeting late I-waves, and whether these responses were associated with individual variations in intracortical excitability. 17 young (27.2 {+/-} 6.4 years, 12 females) healthy adults received iTMS at late I-wave intervals (4.0, 4.5 & 5.0 ms) in three separate sessions. Changes due to each intervention were examined with motor evoked potential (MEP) amplitudes and short-interval intracortical facilitation (SICF) using both posterior-anterior (PA) and anterior-posterior (AP) TMS current directions. Changes in MEP amplitude and SICF were influenced by iTMS ISI, with the greatest facilitation for ISIs at 4 and 5 ms with PA TMS, and 4 ms with AP TMS. Maximum SICF at baseline (irrespective of ISI) was associated with increased iTMS response, but only for PA stimulation. These results suggest that modifying iTMS parameters targeting late I-waves can influence M1 plasticity. They also suggest that maximum SICF may be a means by which responders to iTMS targeting the late I-waves could be identified.

neuroscience↗