Biology subjects

Hashimoto, T.

Publications and source records attributed to Hashimoto, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Machine-learning-guided enzyme discovery and redox-system engineering for efficient production of the nylon monomer methyl 12-aminododecanoate in Escherichia coli

Methyl 12-aminododecanoate (ADAME) is a key precursor for Nylon 12 synthesis and an attractive target for sustainable microbial production. However, efficient biosynthesis of ADAME requires the coordinated oxidation and transamination of methyl dodecanoate (DAME), and identifying compatible enzymes for this multistep pathway remains challenging. In this study, we applied a machine learning-based bioinformatic screening approach to identify alternative enzymes for DAME-to-ADAME bioconversion. Among 18 selected distantly related AlkB homologs, AlkBGp01 from Alcanivorax sp. P2S70 showed activity toward DAME, producing 12-hydroxydodecanoic acid methyl ester (HDAME) and 12-oxododecanoic acid methyl ester (ODAME) despite low sequence identity to Pseudomonas putida AlkB. Further screening identified compatible redox partners, AlkG60 from the same species as AlkBGp01 and AlkTp04, whose combination with AlkBGp01 resulted in almost sixfold higher ODAME production than that achieved with P. putida AlkBFGJLT. ODAME production was further increased by more than threefold by conjugating these three newly identified proteins. The optimized single-plasmid system comprising conjugated AlkBGp01, AlkG60, and AlkTp04, together with AlaD and newly identified omega-transaminases EAV41574 produced 0.28 mM gDCW-1 ADAME, representing a 5.6-fold improvement over the previously reported P. putida AlkBFGJLT-based system with AlaD-CV2025. These results demonstrate that machine learning-guided enzyme discovery can identify functional distantly related homologs and compatible enzyme combinations for constructing efficient synthetic pathways. This study also highlights the importance of optimizing pathway architecture, including enzyme conjugation and plasmid configuration, for improving microbial production of bio-based monomer precursors.

bioengineering↗

bioRxiv2026

From introduction to eradication: reconstructing population size and removal history of an invasive species

O_LIUnderstanding the processes underlying successful eradication of invasive species is essential for achieving global island conservation goals. Despite the widespread availability of capture records from eradication programs, modeling frameworks that utilize these datasets to elucidate spatio-temporal population dynamics remain underdeveloped. C_LIO_LIIn this study, we reconstructed the spatio-temporal population dynamics of the small Indian mongoose on Amami-Oshima Island (712 km{superscript 2}), Japan, where the species was introduced in 1979 and officially declared eradicated in 2024 after more than 30 years of systematic removal. We integrated introduction records, capture data, and monitoring data using a hierarchical harvest-based model (HBM). To evaluate the models capacity to support management decisions and assess eradication success, we conducted retrospective analyses and compared estimated eradication probabilities with those obtained from a rapid eradication assessment (REA; Samaniego-Herrera et al., 2013). C_LIO_LIThe estimated population size (before reproduction) peaked at 5,449 individuals (95% CI: 4,703, 6,175) in 2000 and subsequently declined almost monotonically. The maximum invaded area was 547.78 km{superscript 2} (posterior median, 95% CI: 496.47, 566.04) in 2009, indicating that the removal program successfully prevented island-wide expansion. Retrospective analyses showed that population estimates remained within the 95% credible intervals of the full dataset estimates, demonstrating temporal consistency. Eradication probabilities estimated by the HBM were substantially higher than those from the REA, highlighting the sensitivity of estimates to fine-scale heterogeneity in detection processes. C_LIO_LISynthesis and applications: Hierarchical HBMs provide a powerful framework for reconstructing, predicting, and evaluating invasive species eradication dynamics. Being aware of the limitations for application to eradication evaluations, HBMs can support adaptive management in long-term eradication programs and improve our understanding of the mechanisms underlying successful eradication. C_LI

ecology↗

bioRxiv2025

Blockade of associative spine enlargement impairs awake cortical function

Awake cognition is usually attributed to arousal systems and ongoing electrical activity, yet these do not explain how cortical processing is coupled to prior context and memory formation. Associative synaptic plasticity provides a cellular mechanism for such coupling, but is viewed as memory-related rather than an online requirement for cognition. Here we developed SynC, an A/C-activated chemogenetic perturbation that selectively and reversibly blocks associative spine enlargement while sparing non-associative spine dynamics, excitability, synaptic transmission and NMDA receptor-mediated responses. In mice with broad frontoparietal SynC expression, acute A/C reversibly impaired open-field centre exploration, abolished laser-dot chasing, and delayed feeding initiation. During quiet-wake immobile epochs, cortical firing rates and {gamma}-band power were preserved, but pairwise spike correlations were reduced. Mice also intermittently showed sudden behavioural arrest with reduced {gamma} activity but without the robust {delta} elevation of slow-wave sleep. Critically, in vivo two-photon glutamate uncaging induced seconds-scale associative spine enlargement in [~]24% of neocortical spines, acutely blocked by SynC-A/C. These A/C-induced behavioural, circuit and spine-enlargement effects recovered within 1 h. Thus, wake-like firing and {gamma} activity are not sufficient to sustain awake cognitive function. Rapid associative spine enlargement is required to maintain functional cortical coupling and stable wakefulness.

neuroscience↗

bioRxiv2025

OGDHL regulates nucleotide metabolism, tumor growth, and neuroendocrine marker expression in prostate cancer

As cancer cells evade therapeutic pressure and adopt alternate lineage identities not commonly observed in the tissue of origin, they likely adopt alternate metabolic programs to support their evolving demands. Targeting these alternative metabolic programs in distinct molecular subtypes of aggressive prostate cancer may lead to new therapeutic approaches to combat treatment-resistance. We identify the poorly studied metabolic enzyme Oxoglutarate Dehydrogenase-Like (OGDHL), named for its structural similarity to the tricarboxylic acid (TCA) cycle enzyme Oxoglutarate Dehydrogenase (OGDH), as an unexpected regulator of tumor growth, treatment-induced lineage plasticity, and DNA Damage in prostate cancer. While OGDHL has been described as a tumor-suppressor in various cancers, we find that its loss impairs prostate cancer cell proliferation and tumor formation. Loss of OGDHL profoundly alters Androgen Receptor inhibition-induced plasticity, including suppressing the neuroendocrine markers DLL3 and HES6, induces accumulation of the DNA damage response marker {gamma}H2AX, and reduces nucleotide synthesis. Our data suggest that OGDHL has minimal impact on TCA cycle activity, and that mitochondrial localization is not required for its regulation of prostate cancer plasticity and nucleotide metabolism. Finally, we demonstrate that OGDHL expression is tightly correlated with neuroendocrine differentiation in clinical prostate cancer. These findings underscore the importance of investigating poorly characterized metabolic genes as potential regulators of distinct molecular subtypes of aggressive cancer.

cancer biology↗

bioRxiv2025

Immunological characterization of peritoneal exudate cells in liver cirrhosis patients

BACKGROUND AND AIMSLiver cirrhosis (LC) is the end stage of liver fibrosis caused by various chronic liver diseases. Patients with LC often develop ascites containing peritoneal exudate cells (PECs). However, those cells have not been fully immunologically characterized. In this study, we clarify immune cell profiles of PECs from patients with LC. APPROACH AND RESULTSPECs were collected from patients with LC or patients receiving continuous ambulatory peritoneal dialysis (CAPD) as a non-cirrhotic control and subjected to single-cell RNA sequencing (scRNA-seq), bulk RNA sequencing (RNA-seq) and flowcytometry analyses. Analysis of scRNA-seq revealed that dendritic cells (DCs) and macrophages were major populations in CAPD patient-derived PECs, while those cells were decreased and T cells were most abundant in LC patient-derived PECs. Notably, FCGR3A-expressing macrophages were dominant over DCs and GATA6-expressing macrophages in LC patient-derived PECs. Bulk RNA-seq analysis further clarified expression of a set of genes was up- or down-regulated along with LC severity. Especially, expression of T cell signature genes was featured by its increase at Child-Pugh class B, but decrease at Child-Pugh class C. Flowcytometry analysis showed increase of T cells, decrease of DCs and macrophages, and increased expression of CD16 and CD163 in CD1cintCD14high cells corresponding to FCGR3A-expressing macrophages in LC patient-derived PECs. CONCLUSIONSIn LC patient-derived PECs, myeloid and T cell populations and their gene expression profiles were fluctuated with severity. Our findings should contribute to further development of diagnosis or therapeutic maneuver for LC.

immunology↗

bioRxiv2024

IgG autoantibodies in bullous pemphigoid directly induce a pathogenic MyD88-dependent pro-inflammatory response in keratinocytes:

AbstractWhile autoantibodies in bullous pemphigoid (BP) are known to activate the innate immune response, their direct effect on keratinocytes, and the contribution of BP-IgG autoantibody-dependent keratinocyte responses to BP pathology is largely unknown. Herein, we performed multiplex immunoassays and bulk RNA-seq on primary keratinocytes treated with IgG from BP patients or controls. We identified a pro-inflammatory and proteolytic response with release of several cytokines (IL-6, IL-24, TGF-{beta}1), chemokines (CXCL16, CTACK, MIP-3{beta}, RANTES), C1s, DPP4, and MMP-9. We further validated this response using spatial transcriptomics and scRNA-seq of diseased and control skin. Blistering itself appeared to be major driver of this inflammatory response, with attached BP skin and spongiotic dermatitis revealing highly similar transcriptomes. Based on elevated levels of MyD88 and MyD88-dependent cytokines, we studied the impact of MyD88 deficiency in keratinocytes and demonstrated that MyD88 regulates BP-IgG-induced expression of IL-8, IL-24, and MMP-9. Induction of experimental BP in mice with Krt14-specific Myd88 knockout revealed significantly decreased disease severity with decreased serum levels of IL-1{beta}, IL-4, and IL-9 indicating the contributory role of keratinocyte-derived skin inflammation towards systemic response. Our work demonstrates the key contributions of keratinocyte and MyD88 dependent signaling in response to autoantibodies in BP. Key Messages-IgG antibodies from bullous pemphigoid (BP) patients induce significant upregulation of several inflammatory markers in keratinocytes including cytokines (IL-6, IL-24, TGF-{beta}1), chemokines (CXCL16, CTACK, MIP-3{beta}, RANTES), C1s, DPP4, and MMP9. Several of these markers, including IL-8, IL-24, and MMP9 are regulated by MyD88. -Spatial transcriptomics reveals that BP patient blistered skin demonstrated similar transcriptomic profiles to BP-IgG-treated keratinocytes. With attached skin demonstrating a comparable transcriptome to that seen in spongiotic dermatitis. -In a mouse BP model, keratinocyte-specific MyD88 deficiency results in decreased disease severity with a subsequent decrease in serum IL-1{beta}, IL-4, and IL-9 levels. Capsule summaryIgG from patients with bullous pemphigoid (BP) induces a pro-inflammatory response in keratinocytes, indicating their direct role in driving the inflammatory response in BP.

immunology↗

bioRxiv2024

The lysosomal membrane protein LAMP2B mediates microlipophagy to target obesity-related disorders

Lifestyle diseases, such as obesity, diabetes, and metabolic syndrome, are leading health problems, most of which are related to abnormal lipid metabolism. Lysosomes can degrade lipid droplets (LDs) via microautophagy. Here, we report the molecular mechanism and pathophysiological roles of microlipophagy, regulated by the lysosomal membrane protein LAMP2B. Our study revealed that LAMP2B interacts with phosphatidic acid, facilitating lysosomal-LD interactions and enhancing lipid hydrolysis via microlipophagy depending on endosomal sorting complexes required for transport. Correlative light and electron microscopy demonstrated direct LDs uptake into lysosomes at contact sites. Moreover, LAMP2B overexpression in mice prevents high-fat diet-induced obesity, insulin resistance, and adipose tissue inflammation; liver lipidomics analysis suggested enhanced triacylglycerol hydrolysis. Overall, the findings of this study elucidated the mechanism of microlipophagy, which could be promising for the treatment of obesity and related disorders.

cell biology↗

bioRxiv2024

Orthogonalization of spontaneous and stimulus-driven activity by hierarchical neocortical areal network in primates

Understanding how biological neural networks perform reliable information processing in the presence of intensive spontaneous activity (1-3) is an essential question in biological computation. Stimulus-evoked and spontaneous activities show orthogonal (dissimilar) patterns in the primary visual cortex (V1) of mice (4-6), which is likely to be beneficial for separating sensory signals from internally generated noise (5, 7-12); however, those in V1 of carnivores and primates show highly similar patterns (3, 13-17). Consequently, the mechanism of segregation of stimulus information and internally generated noise in carnivores and primates remain unclear. To address this question, we used primate-optimized functional imaging (18) to precisely compare spontaneous and stimulus-evoked activities in multiple areas of the marmoset visual cortical pathway (19). In marmoset V1, but not in mouse V1, spontaneous and stimulus-evoked activity shared similar activity patterns, suggesting a function of spontaneous activity specific to mammals with functional columns. However, in the higher-order visual areas of marmosets, spontaneous and stimulus-evoked activities exhibited dissimilar patterns. Analysis of neural activity geometry further revealed progressive orthogonalization of the two types of activities along the cortical hierarchy in marmosets, which reached a level comparable to that of mouse V1. Thus, orthogonalization of spontaneous and stimulus-evoked activity is a general principle of cortical computation, which, in primates, is implemented by the hierarchical areal network.

neuroscience↗

bioRxiv2024

Discovery of anti-SARS-CoV-2 S2 protein antibody CV804 with broad-spectrum reactivity with various beta coronaviruses and analysis of its pharmacological properties in vitro and in vivo

SARS-CoV-2 pandemic alerts us that spillovers of various animal coronaviruses to human in the future may bring us enormous damages. Thus, there is a significant need of antibody-based drugs to treat patients infected with previously unseen coronaviruses.CV804 against the S2 domain of the spike protein, which is less prone to mutations. CV804 shows not only broad cross-reactivities with representative 20 animal-origin coronaviruses but also with diseases-associated human beta coronaviruses including SARS-CoV, MERS-CoV, HCoV-OC43, HCoV-HKU1 and mutant strains of SARS-CoV-2. Other than that, the main characteristics of CV804 are that it has strong antibody-dependent cellular cytotoxicity (ADCC) activity to SARS-CoV2 spike protein-expressed cells in vitro and completely lacks virus-neutralization activity. Comprehensively in animal models, CV804 suppressed disease progression by SARS-CoV-2 infection. Structural studies using HDX-MS and point mutations of recombinant spike proteins revealed that CV804 binds to a unique epitope within the highly conserved S2 domain of the spike proteins of various coronaviruses. Based on the overall data, we suggest that the non-neutralizing CV804 antibody recognizes the conformational structure of the spike protein expressed on the surface of the infected cells and weakens the viral virulence by supporting host immune cells attack through ADCC activity in vivo. CV804 epitope identified in this study is not only useful for the design of pan-corona antibody therapeutics but also to design next-generation coronavirus vaccines and antiviral drugs.

microbiology↗

bioRxiv2023

Development of a fast feature extraction method for SARS-CoV-2 spike sequences using amino acid physicochemical properties

COVID-19 continues to spread today, leading to an accumulation of SARS-CoV-2 virus mutations in databases, and large amounts of genomic datasets are currently available. However, due to these large datasets, utilizing this amount of sequence data without random sampling is challenging. Major difficulties for downstream analyses include the increase in the dimension size along with the conversion of sequences into numerical values when using conventional amino acid representation methods, such as one-hot encoding and k-mer-based approaches that directly reflect sequences. Moreover, these sequences are deficient in physicochemical characteristics, such as structural information and hydrophilicity; hence, they fail to accurately represent the inherent function of the given sequences. In this study, we utilized the physicochemical properties of amino acids to develop a rapid and efficient approach for extracting feature parameters that are suitable for downstream processes of machine learning, such as clustering. A fixed-length feature vector representation of a spike sequence with reduced dimensionality was obtained by converting amino acid residues into physicochemical parameters. Next, t-distributed stochastic neighbor embedding (t- SNE), a method for dimensionality reduction and visualization of high-dimensional data, was performed, followed by density-based spatial clustering of applications with noise (DBSCAN). The results show that by using the physicochemical properties of amino acids rather than conventional methods that directly represent sequences into numerical values, SARS-CoV-2 spike sequences can be clustered with sufficient accuracy and a shorter runtime. Interestingly, the clusters obtained by using amino acid properties include subclusters that are distinct from those produced utilizing the method for the direct representation of amino acid sequences. A more detailed analysis indicated that the contributing parameters of this novel cluster identified exclusively when utilizing the physicochemical properties of amino acids significantly differ from one another. This suggests that representing amino acid sequences by physicochemical properties might enable the identification of clusters with enhanced sensitivity compared to conventional methods. Author summaryOne of the major causes of the global threat of SARS-CoV-2 is the rapid emergence of its variants. While analyzing these variants is crucial for understanding the mechanism of outbreaks, the expansion of database size is becoming a barrier for effective analysis. In this study, we provide an approach that allows researchers without vast computational resources to comprehensively analyze the variants of SARS-CoV-2 spike by representing the sequences using the physicochemical properties of amino acids. The result of clusters derived using this method demonstrates not only an accuracy comparable to the conventional approaches of directly converting sequences into numerical values but also indicates the potential for more detailed clustering outcomes. The results suggest that our approach is valuable for the rapid identification of characteristic residues in new variants of SARS-CoV-2 and other viruses that may arise in the future.

bioinformatics↗

bioRxiv2023

Enhanced Ca2+ Binding to EF-Hands through Phosphorylation of Conserved Serine Residues Activates MpRBOHB and Chitin-Triggered ROS Production

NADPH oxidases/RBOHs catalyze apoplastic ROS production and act as key signaling nodes, integrating multiple signal transduction pathways regulating plant development and stress responses. Although RBOHs have been suggested to be activated by Ca2+ binding and phosphorylation by various protein kinases, a mechanism linking Ca2+ binding and phosphorylation in the activity regulation remained elusive. Chitin-triggered ROS production required cytosolic Ca2+ elevation and Ca2+ binding to MpRBOHB in a liverwort Marchantia polymorpha. Heterologous expression analysis of truncated variants revealed that a segment of the N-terminal cytosolic region highly conserved among land plant RBOHs encompassing the two EF-hand motifs is essential for the activation of MpRBOHB. Within the conserved regulatory domain, we have identified two Ser residues whose phosphorylation is critical for the activation in planta. Isothermal titration calorimetry analyses revealed that phosphorylation of the two Ser residues increased the Ca2+ binding affinity of MpRBOHB, while Ca2+ binding is indispensable for the activation, even if the two Ser residues are phosphorylated. Our findings shed light on a mechanism through which phosphorylation potentiates the Ca2+-dependent activation of MpRBOHB, emphasizing the pivotal role of Ca2+ binding in mediating the Ca2+ and phosphorylation-driven activation of MpRBOHB, which is likely to represent a fundamental mechanism conserved among land plant RBOHs.

molecular biology↗

bioRxiv2023

Catalytic antibodies in arrhythmogenic cardiomyopathy patients cleave desmoglein 2 and N-cadherin and impair cardiomyocyte cohesion

AimsArrhythmogenic cardiomyopathy (AC) is a severe heart disease predisposing to ventricular arrhythmias and sudden cardiac death caused by mutations affecting intercalated disc (ICD) proteins and aggravated by physical exercise. Recently, autoantibodies targeting ICD proteins, including the desmosomal cadherin desmoglein 2 (DSG2), were reported in AC patients and were considered relevant for disease development and progression, particularly in patients without underlying pathogenic mutations. However, it is unclear at present whether these autoantibodies are pathogenic and by which mechanisms show specificity for DSG2 and thus can be used as a diagnostic tool. Methods and ResultsIgG fractions were purified from 15 AC patients and 4 healthy controls. Immunostainings dissociation assays, atomic force microscopy (AFM), western blot analysis and Triton-X-100 assays were performed utilizing human heart left ventricle tissue, HL-1 cells, and murine cardiac slices. Immunostainings revealed that autoantibodies against ICD proteins are prevalent in AC and most autoantibody fractions have catalytic properties and cleave the ICD adhesion molecules DSG2 and N-cadherin, thereby reducing cadherin interactions as revealed by AFM. Furthermore, most of the AC-IgG fractions causing loss of cardiomyocyte cohesion activated p38MAPK, which is known to contribute to a loss of desmosomal adhesion in different cell types, including cardiomyocytes. In addition, p38MAPK inhibition rescued the loss of cardiomyocyte cohesion induced by AC-IgGs. ConclusionOur study demonstrates that catalytic autoantibodies play a pathogenic role by cleaving ICD cadherins and thereby reducing cardiomyocyte cohesion by a mechanism involving p38MAPK activation. Finally, we conclude that DSG2 cleavage by autoantibodies could be used as a diagnostic tool for AC.

cell biology↗

bioRxiv2022

Microwell-based flow culture reduces hypoxia-induced phenotype and restores drug response in prostate cancer spheroids

3D cancer spheroids represent a highly promising model for study of cancer progression and therapeutic development. Wide-scale adoption of cancer spheroids, however, remains a challenge due to the lack of control over hypoxic gradients that may cloud the assessment of cell morphology and drug response. Here, we present a Microwell Flow Device (MFD) that generates in-well laminar flow around 3D tissues via repetitive tissue sedimentation. Using a prostate cancer cell line, we demonstrate the spheroids in the MFD exhibit improved cell growth, reduced necrotic core formation, enhanced structural integrity, and down-regulated expression of cell stress genes. The flow-cultured spheroids also exhibit an improved sensitivity to chemotherapy with greater transcriptional response. These results demonstrate how fluidic stimuli reveal the cellular phenotype previously masked by severe necrosis. Our platform advances 3D cellular models and enables study into hypoxia modulation, cancer metabolism, and drug screening within pathophysiological conditions.

bioengineering↗

bioRxiv2022

Androgen receptor inhibition induces metabolic reprogramming and increased reliance on oxidative mitochondrial metabolism in prostate cancer

Prostate cancer cells that survive clinical androgen receptor (AR) blockade mediate disease progression and lethality. Reprogrammed metabolic signaling is one mechanism by which tumor cells can survive treatment. However, how AR inhibition reprograms metabolism, and whether altered metabolism can be exploited to eradicate cells that survive AR blockade, remains unclear. Here, we comprehensively characterized the effect of AR blockade on prostate cancer metabolism using transcriptomics, metabolomics, and bioenergetics approaches. AR inhibition maintains oxidative mitochondrial metabolism and reduces glycolytic signaling, through hexokinase II downregulation and decreased MYC activity. Robust elongation of mitochondria via reduced DRP1 activity supports cell fitness after AR blockade. In addition, AR inhibition enhances sensitivity to complex I inhibitors in several models, suggesting that AR blockade increases reliance on oxidative mitochondrial metabolism. Our study provides an enhanced understanding of how AR inhibition alters metabolic signaling and highlights the potential of therapies that target metabolic vulnerabilities in AR-inhibited cells.

cancer biology↗

bioRxiv2021

Phylogenomics invokes the clade housing Cryptista, Archaeplastida, and Microheliella maris.

As-yet-undescribed branches in the tree of eukaryotes are potentially represented by some of "orphan" protists (unicellular micro-eukaryotes), of which phylogenetic affiliations have not been clarified in previous studies. By clarifying the phylogenetic positions of orphan protists, we may fill the previous gaps in the diversity of eukaryotes and further uncover the novel affiliation between two (or more) major lineages in eukaryotes. Microheliella maris was originally described as a member of the phylum Heliozoa, but a pioneering large-scale phylogenetic analysis failed to place this organism within the previously described species/lineages with confidence. In this study, we analyzed a 319-gene alignment and demonstrated that M. maris represents a basal lineage of one of the major eukaryotic lineages, Cryptista. We here propose a new clade name "Pancryptista" for Cryptista plus M. maris. The 319-gene analyses also indicated that M. maris is a key taxon to recover the monophyly of Archaeplastida and the sister relationship between Archaeplastida and Pancryptista, which is collectively called as "CAM clade" here. Significantly, Cryptophyceae tend to be attracted to Rhodophyta depending on the taxon sampling (ex., in the absence of M. maris and Rhodelphidia) and the particular phylogenetic "signal" most likely hindered the stable recovery of the monophyly of Archaeplastida in previous studies. We hypothesize that many cryptophycean genes (including those in the 319-gene alignment) recombined partially with the homologous genes transferred from the red algal endosymbiont during secondary endosymbiosis and bear a faint phylogenetic affinity to the rhodophytan genes.

evolutionary biology↗

bioRxiv2021

Compositional analysis of ALS-linked stress granule-like structures reveals factors and cellular pathways dysregulated by mutant FUS under stress

Formation of cytoplasmic RNA-protein structures called stress granules (SGs) is a highly conserved cellular response to stress. Abnormal metabolism of SGs may contribute to the pathogenesis of (neuro)degenerative diseases such as amyotrophic lateral sclerosis (ALS). Many SG proteins are affected by mutations causative of these conditions, including fused in sarcoma (FUS). Mutant FUS variants have high affinity to SGs and also spontaneously form de novo cytoplasmic RNA granules. Mutant FUS-containing assemblies (mFAs), often called "pathological SGs", are proposed to play a role in ALS-FUS pathogenesis. However, global structural differences between mFAs and physiological SGs remain largely unknown, therefore it is unclear whether and how mFAs may affect cellular stress responses. Here we used affinity purification to characterise the protein and RNA composition of normal SGs and mFAs purified from stressed cells. Comparison of the SG and mFA proteomes revealed that proteasome subunits and certain nucleocytoplasmic transport factors are depleted from mFAs, whereas translation elongation, mRNA surveillance and splicing factors as well as mitochondrial proteins are enriched in mFAs, as compared to SGs. Validation experiments for a hit from our analysis, a splicing factor hnRNPA3, confirmed its RNA-dependent sequestration into mFAs in cells and into pathological FUS inclusions in a FUS transgenic mouse model. Furthermore, silencing of the Drosophila hnRNPA3 ortholog dramatically enhanced FUS toxicity in transgenic flies. Comparative transcriptomic analysis of SGs and mFAs revealed that mFAs recruit a significantly less diverse spectrum of RNAs, including reduced recruitment of transcripts encoding proteins involved in protein translation, DNA damage response, and apoptotic signalling. However mFAs abnormally sequester certain mRNAs encoding proteins involved in stress signalling cascades. Overall, our study establishes molecular differences between physiological SGs and mFAs and identifies the spectrum of proteins, RNAs and respective cellular pathways affected by mFAs in stressed cells. In conclusion, we show that mFAs are compositionally distinct from SGs and that they cannot fully substitute for SG functions while gaining novel, potentially toxic functions in cellular stress response. Results of our study support a pathogenic role for stress-induced cytoplasmic FUS assemblies in ALS-FUS.

cell biology↗

bioRxiv2021

Evaluating the tea bag method as a potential tool for detecting the effects of added nutrients and their interactions with climate on litter decomposition

It is acknowledged that exogenous nutrient addition often stimulates early-stage litter decomposition in forests and late-stage decomposition is generally suppressed by nitrogen addition, whereas the interactive effects of nutrient addition and abiotic environmental factors, such as climate, on decomposition remain unclear. The tea bag method, which was developed to provide the decomposition rate constant k of early-stage decomposition and stabilization factor S of labile materials in the late stage, is a potentially useful tool for examining the impacts of nutrient addition on both early- and late-stage litter decomposition and their interactions with climate. At a long-term (38-year) continuous fertilization experimental site (an Abies sachalinensis Fr. Schmidt stand) in Hokkaido, Japan, we examined whether a standard tea bag method protocol was sufficiently sensitive to reveal any impacts of nutrient addition on early- and late-stage decomposition. In addition, we tested the interactive effects of nutrient addition and climate on litter decomposition. The short incubation period of the tea bag method (ca. 90 days) enabled us to obtain decomposition data from the same location at three different times in a year, i.e., early summer, midsummer, and winter, providing an opportunity to test interactive effects. We demonstrated that the decomposition rate of rooibos tea and the decomposition rate constant k of early-stage decomposition were clearly stimulated by fertilization in midsummer, but no impacts were detected in other seasons, probably because the relative importance of nutrient availability was elevated in midsummer, during which decomposition rates were less constrained by temperature and moisture. The green tea decomposition rate and stabilization factor S, an index related to late-stage decomposition, were unaffected by fertilization. This was probably because the tea bag method does not take into account lignin degradation, which is considered a key factor controlling late-stage litter decomposition. Overall, the present study (i) successfully determined the interactive effects of nutrient addition and climate factors on litter decomposition by making full use of the tea bag method, and (ii) the results suggest that the tea bag method can be a suitable tool for examining the direct effects of nutrient addition and their interactions with environmental factors on early-stage litter decomposition, but not those on late-stage decomposition.

ecology↗

bioRxiv2020

Evaluating the fitness of PA/I38T-substituted influenza A viruses with reduced baloxavir susceptibility in a competitive mixtures ferret model

Baloxavir is approved in several countries for the treatment of uncomplicated influenza in otherwise-healthy and high-risk patients. Treatment-emergent viruses with reduced susceptibility to baloxavir have been detected in clinical trials, but the likelihood of widespread occurrence depends on replication capacity and onward transmission. We evaluated the fitness of A/H3N2 and A/H1N1pdm09 viruses with the polymerase acidic I38T-variant conferring reduced susceptibility to baloxavir relative to wild-type (WT) viruses, using a competitive mixture ferret model, recombinant viruses and patient-derived virus isolates. The A/H3N2 I38T virus showed a reduction in within-host fitness but comparable between-host fitness to the WT virus, while the A/H1N1pdm09 I38T virus had broadly similar within-host fitness but substantially lower between-host fitness. Although I38T viruses replicate and transmit between ferrets, our data suggest that viruses with this amino acid substitution have lower fitness relative to WT and this relative fitness cost was greater in A/H1N1pdm09 viruses than in A/H3N2 viruses.

microbiology↗