Biology subjects

Nakajima, T.

Publications and source records attributed to Nakajima, T..

16 recordsLinked to original sources

bioRxiv2026

Crop-associated differences in soil chemical properties and root-associated bacterial communities between Welsh onion and sweet potato

Crop species may shape soil chemical properties and root-associated microbiota, but direct comparisons between contrasting crops remain limited. We compared soils and root-associated bacterial communities of Welsh onion (Allium fistulosum) and sweet potato (Ipomoea batatas) under the same field context. Sweet potato soil showed significantly lower electrical conductivity, inorganic nitrogen, and Mg saturation than control soil. Root-associated communities differed between crops, whereas alpha diversity did not. Proteobacteria-related taxa were more represented in Welsh onion roots, whereas Actinomycetia-related taxa were more represented in sweet potato roots, providing a basis for future studies on crop-specific soil microbial management.

microbiology↗

bioRxiv2026

Topology-dependent FRET efficiency in living cells via N-C swapping of fluorescent protein fusions

Forster resonance energy transfer (FRET) is a physicochemical phenomenon involving non-radiative energy transfer between donor and acceptor fluorophores. While FRET efficiency primarily depends on the proximity between fluorophores, additional factors substantially influence the efficiency in living cells. However, how non-distance factors modulate live-cell FRET efficiency remains poorly understood. Here, we report the significant role of N- and C-terminal topology in determining live-cell FRET efficiency, independent of fluorophore proximity, donor variants, and subcellular compartment. Using acceptor photobleaching and sensitized emission measurements in living cells, we found that FRET efficiencies of mCherry-EGFP or mCherry-EYFP (acceptor-donor) were significantly higher than those of EGFP-mCherry or EYFP-mCherry (donor-acceptor), respectively. FRET efficiencies were higher with EYFP than with EGFP as the donor. These efficiencies were comparable between the nucleus and cytoplasm. AlphaFold2-based structural modeling suggested similar proximity between donor and acceptor fluorophores despite structurally heterogeneous and loosely constrained geometries. In contrast, ensemble FRET simulations suggested that dynamic conformational sampling amplifies the apparent asymmetry between donor-acceptor and acceptor-donor configurations observed in live-cell FRET measurements. Collectively, these results demonstrate that topological arrangement, rather than static structural geometry alone, plays a significant role in FRET efficiency in living cells, providing molecular implications for the design of intramolecular FRET-based biosensors.

biophysics↗

bioRxiv2026

Lipidated ApoE is found in nanoscale proximity to Aβ aggregates in human Alzheimer brains.

Apolipoprotein E (APOE) associates with amyloid plaques (A{beta}) in Alzheimer disease (AD). The {varepsilon}4 allele of apolipoprotein E (APOE{varepsilon}4) is the strongest genetic risk factor for sporadic AD and exacerbates A{beta} plaque burden relative to APOE{varepsilon}3 and APOE{varepsilon}2. The majority of ApoE associates with multiple lipid classes to form lipoproteins both in the brain and the periphery. However, the lipidation status of A{beta} plaque-associated ApoE is not yet fully defined. Here, we use fluorescence lifetime imaging microscopy coupled with Forster resonance energy transfer (FLIM-FRET) to determine the lipidation status of ApoE in plaques, as well as the nanoscale spatial proximity of ApoE and A{beta} to anionic lipids and cholesterol within human AD brain tissue. We demonstrate that lipids are in close nanoscale proximity to ApoE and A{beta} within A{beta} plaques. Our results reveal that lipidated ApoE complexes enriched in anionic lipids and cholesterol are core constituents of AD plaques in-situ. We propose a pathological mechanism in which the surface presentation of anionic lipids on ApoE lipoproteins facilitates initial interaction with and subsequent aggregation of A{beta}.

neuroscience↗

bioRxiv2026

Excess Met1-ubiquitination leads to solid aggregate formation

The ubiquitin ligase HOIL-1 has a unique role in controlling quantity of Met1-linked/ linear ubiquitin chains in cells by coordinating action with the ubiquitin ligase HOIP. Both ligases are components of the Linear UBiquitin chain Assembly Complex (LUBAC), the only known ligase complex that is able to generate Met1-linked ubiquitin chains. Although importance of Met1-linked ubiquitin chains in inflammation and immunity is well established, physiological relevance of quantity of these chains remain unknown. Here, we demonstrate that cells expressing catalytically inactive HOIL-1 exhibited significantly higher numbers of -Synuclein, tau, and amyloid beta aggregates. This phenotype is associated with a disruption in late-stage autophagic flux, wherein p62-positive aggregates fail to colocalize with lysosomal markers, leading to impaired clearance. Additionally, a biophysical transition in aggregate properties was observed in vitro, with mutant cells forming more rigid solid-like inclusions, shifting from dynamic and liquid-like condensates. Elevated Met1-linked ubiquitin chains, either through HOIL-1 catalytic inactivation or knockdown of the Met1-linked chain-specific deubiquitinase OTULIN, phenocopied the defects in aggregate clearance. These findings reveal a critical role of HOIL-1 catalytic activity in modulating aggregate clearance through autophagy and maintaining the quantity of Met1-ubiquitin chains, highlighting HOIL-1 as a key factor in proteostasis in neurodegenerative diseases. HighlightsO_LIHOIL-1 catalytic activity prevents neurodegenerative protein aggregate accumulation C_LIO_LIInactive HOIL-1 impairs late-stage autophagic clearance of protein aggregates C_LIO_LILoss of HOIL-1 shifts aggregates to rigid, solid-like states C_LIO_LIExcess Met1-ubiquitin chains drive aggregate solidification C_LI

cell biology↗

bioRxiv2025

Lateral plate mesoderm directs human amnion and ventral skin organoid formation

Summary ParagraphEngineering organoids that faithfully replicate the intricate architecture and region-specific features of bodily organs and extraembryonic tissues remains a significant scientific challenge. Previously, we demonstrated that craniofacial skin organoids (cSkOs)--containing epidermis, dermis, and hair--could be generated by co-developing epidermal progenitors with cranial mesenchyme. Building on this approach, we precisely adjusted cellular composition and signaling environments to generate ventral skin organoids (vSkOs) with lateral plate mesoderm (LPM) progenitors, successfully recapitulating features of abdominal or groin skin. Modulating early BMP and FGF signaling redirected these vSkOs toward an extraembryonic fate, producing human amnion-like tissues, termed Amnioids. Like native human amnion, Amnioids rapidly expanded into large, avascular, hairless cysts, in sharp contrast to the primitive vasculature and abundant hair follicles of vSkOs. Single-cell RNA sequencing identified divergent molecular signatures and developmental trajectories, highlighting key roles for NOTCH, WNT, and YAP/Hippo signaling pathways. Functional studies further underscored mesenchymal-epithelial interactions and mechanical forces as critical regulators of epithelial expansion. Together, these models provide potent tools to investigate human development at the embryonic-extraembryonic interface, offering critical insights into congenital skin and amniotic disorders and opening new avenues for precision regenerative therapies.

developmental biology↗

bioRxiv2024

Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

Lipogenesis-driven metabolic flux is crucial for differentiation of pathogenic Th17 cells. Although our previous CRISPR-based screening identified PLA2G12A as a key player in this process, it has remained obscure how this secreted phospholipase A2 isoform controls Th17 differentiation. Here we show that global, T cell-specific, or fibroblast-specific deletion of PLA2G12A prevents Th17 differentiation and associated diseases including psoriasis and arthritis. PLA2G12A acts on Th17-derived extracellular vesicles (EVs) to produce lysophospholipids including the ROR{gamma}t activator 1-oleoyl-lysophosphatidylethanolamine. These lysophospholipids are further converted by autotaxin to lysophosphatidic acid (LPA), which assists Th17 differentiation mainly via LPA2 receptor. Moreover, PLA2G12A promotes the secretion and uptake of EVs by Th17 cells and alters their cargo contents. Defective Th17 differentiation by PLA2G12A deficiency is rescued by supplementation with PLA2G12A-modified EVs. Importantly, a PLA2G12A-blocking antibody prevents Th17 differentiation and ameliorates psoriasis and arthritis models. Thus, targeting the PLA2G12A-EV-lysophospholipid axis may be useful for treatment of Th17-related diseases.

immunology↗

bioRxiv2023

APOE2 gene therapy reduces amyloid deposition, and improves markers of neuroinflammation and neurodegeneration in a mouse model of Alzheimer disease

Epidemiological studies show that individuals who carry the relatively uncommon APOE {varepsilon}2 allele rarely develop Alzheimer disease, and if they do they have a later age of onset, milder clinical course, and less severe neuropathological findings than others with Alzheimer disease. The contrast is especially stark in comparison to the phenotype associated with the major genetic risk factor for Alzheimer disease, APOE {varepsilon}4, which has an age of onset several decades earlier, as well as a more aggressive clinical course and notably more severe neuropathological findings, especially in terms of the amount of amyloid deposition. Even one APOE {varepsilon}2 allele improves phenotype, but it is uncertain if that is due to the replacement of a more toxic allele by APOE {varepsilon}2, or if APOE {varepsilon}2 has a protective, neuro-modulatory effect. Here, we demonstrate that brain exposure to APOE2 via a gene therapy approach which bathes the entire cortical mantle in the gene product after transduction of the ependyma, rapidly ameliorates established A{beta} plaque deposition, neurodegenerative synaptic loss, and, remarkably, reduces microglial activation in an APP/PS1 mouse model despite continued expression of human APOE4. This result suggests a promising protective effect of exogenous APOE2, revealing a cell non-autonomous effect of the protein on microglial activation. We also show that plaque associated microglia in the brain of patients who inherit APOE2 similarly have less microglial reactivity to plaques. These data raise the potential that an APOE2 therapeutic could be effective in Alzheimer disease even in individuals born with the risk {varepsilon}4 allele. One Sentence SummaryIntroduction of ApoE2 using an AAV that transduces the ependymal cells of the ventricle causes a reduction in amyloid load and plaque associated synapse loss, and reduces neuroinflammation by modulating microglial responsiveness to plaques.

neuroscience↗

bioRxiv2023

Thrombospondin 2 is a key determinant of fibrogenesis in NAFLD

Hepatic overexpression of the thrombospondin 2 gene (THBS2) and elevated levels of circulating thrombospondin 2 (TSP2) have been observed in patients with chronic liver disease. The current study aimed to identify the specific cells expressing THBS2/TSP2 in non-alcoholic fatty liver disease (NAFLD) and investigate the underlying mechanism behind THBS2/TSP2 up-regulation. Comprehensive NAFLD liver gene datasets, including single-cell RNA sequencing (scRNA-seq), in-house NAFLD liver tissue, and LX-2 cells derived from human hepatic stellate cells (HSCs), were analyzed using a combination of computational biology, genetic, immunological, and pharmacological approaches. Analysis of the genetic dataset revealed the presence of 1433 variable genes in patients with advanced fibrosis NAFLD, with THBS2 ranked among the top 2 genes. Quantitative polymerase chain reaction examination of NAFLD livers showed a significant correlation between THBS2 expression and fibrosis stage (r=0.349, p<0.001). In support of this, scRNA-seq data and in situ hybridization demonstrated that the THBS2 gene was highly expressed in HSCs of NAFLD patients with advanced fibrosis. Pathway analysis of the gene dataset revealed THBS2 expression to be associated with the transforming growth factor beta (TGF{beta}) pathway and collagen gene activation. Moreover, the activation of LX-2 cells with TGF{beta} increased THBS2/TSP2 and collagen expression independently of the TGF{beta}-SMAD2/3 pathway. THBS2 gene knockdown significantly decreased collagen expression in LX-2 cells. In conclusion, THBS2/TSP2 is highly expressed in HSCs and plays a role in regulating fibrogenesis in NAFLD patients. THBS2/TSP2 may therefore represent a potential target for anti-fibrotic therapy in NAFLD. (241 words) One-sentence summariesThrombospondin 2 represent a potential target for anti-fibrotic therapy in NAFLD.

molecular biology↗

bioRxiv2021

Anticipating future SARS-CoV-2 variants of concern through ab initio quantum mechanical modeling

Evolved SARS-CoV-2 variants are currently challenging the efficacy of first-generation vaccines, largely through the emergence of spike protein mutants. Among these variants, Delta is presently the most concerning. We employ an ab initio quantum mechanical model based on Density Functional Theory to characterize the spike protein Receptor Binding Domain (RBD) interaction with host cells and gain mechanistic insight into SARS-CoV-2 evolution. The approach is illustrated via a detailed investigation of the role of the E484K RBD mutation, a signature mutation of the Beta and Gamma variants. The simulation is employed to: predict the depleting effect of the E484K mutation on binding the RBD with select antibodies; identify residue E484 as a weak link in the original interaction with the human receptor hACE2; and describe SARS-CoV-2 Wuhan strand binding to the bat Rhinolophus macrotis ACE2 as more optimized than the human counterpart. Finally, we predict the hACE2 binding efficacy of a hypothetical E484K mutation added to the Delta variant RBD, identifying a potential future variant of concern. Results can be generalized to other mutations, and provide useful information to complement existing experimental datasets of the interaction between randomly generated libraries of hACE2 and viral spike mutants. We argue that ab initio modeling is at the point of being aptly employed to inform and predict events pertinent to viral and general evolution.

biophysics↗

bioRxiv2021

A glycosylphosphatidylinositol-anchored α-amylase encoded by amyD contributes to a decrease in the molecular mass of cell wall α-1,3-glucan in Aspergillus nidulans

-1,3-Glucan is one of the main polysaccharides in the cell wall of Aspergillus nidulans. We previously revealed that it plays a role in hyphal aggregation in liquid culture, and that its molecular mass (MM) in an agsA-overexpressing (agsAOE) strain was larger than that in an agsB-overexpressing (agsBOE) strain. The mechanism that regulates the MM of -1,3-glucan is poorly understood. Although the gene amyD, which encodes glycosyl-phosphatidylinositol (GPI)-anchored -amylase (AmyD), is involved in the biosynthesis of -1,3-glucan in A. nidulans, how it regulates this biosynthesis remains unclear. Here we constructed strains with disrupted amyD ({Delta}amyD) or overexpressed amyD (amyDOE) in the genetic background of the ABPU1 (wild-type), agsAOE, or agsBOE strain, and characterized the chemical structure of -1,3-glucans in the cell wall of each strain, focusing on their MM. The MM of -1,3-glucan from the agsBOE amyDOE strain was smaller than that in the parental agsBOE strain. In addition, the MM of -1,3-glucan from the agsAOE {Delta}amyD strain was greater than that in the agsAOE strain. These results suggest that AmyD is involved in decreasing the MM of -1,3-glucan. We also found that the C-terminal GPI-anchoring region is important for these functions.

microbiology↗

bioRxiv2021

Retinoids rescue ceruloplasmin secretion and alleviate oxidative stress in Wilson's disease-specific hepatocytes

Wilsons disease (WD) is a copper metabolic disorder, which is caused by defective ATP7B function. Here, we have generated induced pluripotent stem cells (iPSCs) from WD patients carrying compound heterozygous mutations on ATP7B. ATP7B loss- and gain-of-functions were further manifested with ATP7B-deficient iPSCs and heterozygously-corrected R778L WD patient-derived iPSCs using CRISPR-Cas9-based gene editing. Transcriptome analysis identified abnormalities of retinoid signaling pathway and lipid metabolism in WD-specific hepatocytes. Although the expression level of ATP7B protein was variable among WD-specific hepatocytes, the expression and secretion of ceruloplasmin (Cp), which is a downstream copper carrier in plasma, were consistently decreased. Cp secretion-based drug screening identified all-trans retinoic acid (ATRA) as promising candidates for rescuing Cp secretion. ATRA also alleviated reactive oxygen species (ROS) production induced by lipid accumulation in WD-specific hepatocytes. Our patient-derived iPSC-based hepatic models provide potential therapeutics for liver steatosis in WD and other fatty liver diseases.

cell biology↗

bioRxiv2021

Discovery of SARS-CoV-2 Mpro Peptide Inhibitors from Modelling Substrate and Ligand Binding

The main protease (Mpro) of SARS-CoV-2 is central to its viral lifecycle and is a promising drug target, but little is known concerning structural aspects of how it binds to its 11 natural cleavage sites. We used biophysical and crystallographic data and an array of classical molecular mechanics and quantum mechanical techniques, including automated docking, molecular dynamics (MD) simulations, linear-scaling DFT, QM/MM, and interactive MD in virtual reality, to investigate the molecular features underlying recognition of the natural Mpro substrates. Analyses of the subsite interactions of modelled 11-residue cleavage site peptides, ligands from high-throughput crystallography, and designed covalently binding inhibitors were performed. Modelling studies reveal remarkable conservation of hydrogen bonding patterns of the natural Mpro substrates, particularly on the N-terminal side of the scissile bond. They highlight the critical role of interactions beyond the immediate active site in recognition and catalysis, in particular at the P2/S2 sites. The binding modes of the natural substrates, together with extensive interaction analyses of inhibitor and fragment binding to Mpro, reveal new opportunities for inhibition. Building on our initial Mpro-substrate models, computational mutagenesis scanning was employed to design peptides with improved affinity and which inhibit Mpro competitively. The combined results provide new insight useful for the development of Mpro inhibitors.

bioinformatics↗

bioRxiv2021

Mechanistic Insight from Full Quantum Mechanical Modeling: Laccase as a Detoxifier of Aflatoxins

We demonstrate a path towards full Quantum Mechanics (QM) characterization of enzymatic activity. As a case-study, we investigate the detoxification of aflatoxin, a carcinogenic food contaminant, by laccase, a versatile oxidase capable of--but not efficient for--degrading aflatoxin. We use a combination of quantitative experimentation and QM modeling to show that low enzymatic steric affinity for aflatoxin is the main bottleneck, rather that the oxidative activity of laccase. To identify the structural elements responsible for low reaction rates, we perform a density functional theory (DFT) based modeling of both the substrate and the enzyme in a full QM simulation of more than 7,000 atoms. Thanks to our approach we point to amino acid residues that determine the affinity of laccase for aflatoxin. We show that these residues are substrate-dependent, making a full QM approach necessary for enzyme optimization. Altogether, we establish a roadmap for rational enzyme engineering applicable beyond our case study.

biophysics↗

bioRxiv2020

Synthesis and preclinical evaluation of MTP38 as a novel PET ligand for phophodiesterase 7 in the brain

PurposePhosphodiesterase (PDE) 7 is a potential therapeutic target for neurological and inflammatory diseases, although in-vivo visualization of PDE7 has not been successful. In this study, we aimed to develop [11C]MTP38 as a novel positron emission tomography (PET) ligand for PDE7. Methods[11C]MTP38 was radiosynthesized by 11C-cyanation of a bromo precursor with [11C]HCN. PET scans of rat and rhesus monkey brains and in-vitro autoradiography of brain sections derived from these species were conducted with [11C]MTP38. In monkeys, dynamic PET data were analyzed with an arterial input function to calculate the total distribution volume (VT). The non-displaceable binding potential (BPND) in the striatum was also determined by a reference tissue model with cerebellar reference. Finally, striatal occupancy of PDE7 by an inhibitor was calculated in monkeys according to changes in BPND. Results[11C]MTP38 was synthesized with radiochemical purity [≥] 99.4% and molar activity of 38.6 {+/-} 12.6 GBq/mol. Autoradiography revealed high radioactivity in the striatum and its reduction by non-radiolabeled ligands, in contrast with unaltered autoradiographic signals in other regions. In-vivo PET after radioligand injection to rats and monkeys demonstrated that radioactivity was rapidly distributed to the brain and intensely accumulated in the striatum relative to the cerebellum. Correspondingly, estimated VT values in the monkey striatum and cerebellum were 3.59 and 2.69 mL/cm3, respectively. The cerebellar VT value was unchanged by pretreatment with unlabeled MTP38. Striatal BPND was reduced in a dose-dependent manner after pretreatment with MTP-X, a PDE7 inhibitor. Relationships between PDE7 occupancy by MTP-X and plasma MTP-X concentration could be described by Hills sigmoidal function. ConclusionWe have provided the first successful preclinical demonstration of in-vivo PDE7 imaging with a specific PET radioligand. [11C]MTP38 is a feasible radioligand for evaluating PDE7 in the brain and is currently being applied to a first-in-human PET study.

neuroscience↗

bioRxiv2020

Full quantum mechanical modeling of the enzyme-substrate system: how laccase detoxifies aflatoxin

This work focuses on: 1) the development of a methodology to perform a full Quantum Mechanics (QM) characterization of enzymatic activity; 2) the development of a rational approach to laccase engineering as a food bioremediator. Aflatoxins are among the most dangerous natural carcinogens, and regularly contaminate reserves of staple crops worldwide. Decontamination of aflatoxin-polluted food is of great interest for ensuring food safety, and bioremediation is regarded as the most promising solution. The fungal isoforms of laccase display the rare potential to detoxify aflatoxin by tackling its aromatic moieties.. Yet, because of a generally low efficiency, large-scale application of naturally occurring isoforms has so far been unfeasible. We perform a combination of quantitative experimentation and quantum mechanical modeling on aflatoxin and reveal that: (1) detoxification efficiency is limited by the low enzymatic affinity for the substrate; and (2) aflatoxin is not detoxified by oxidative activity of laccase alone, but requires additional stimulation from the environment. QM modeling also allowed identification of the residues in the laccase tertiary structure that determine affinity of the enzymatic pocket for aflatoxin. We conclude that, for our case-study, a full QM approach is mandatory as a first step towards rational optimization. We detail a feasible approach towards this endeavor and argue that our full QM characterization can serve as a roadmap for enzyme development in other applications pertaining laccase as well as other enzymes.

biophysics↗

bioRxiv2019

Both galactosaminogalactan and α-1,3-glucan contribute to aggregation of Aspergillusoryzae hyphae in liquid culture

Filamentous fungi generally form aggregated hyphal pellets in liquid culture. We previously reported that -1,3-glucan-deficient mutants of Aspergillus nidulans did not form hyphal pellets and their hyphae were fully dispersed, and we suggested that -1,3-glucan functions in hyphal aggregation. Yet, Aspergillus oryzae -1,3-glucan-deficient (AG{Delta}) mutants still form small pellets; therefore, we hypothesized that another factor responsible for forming hyphal pellets remains in these mutants. Here, we identified an extracellular matrix polysaccharide galactosaminogalactan (GAG) as such a factor. To produce a double mutant of A. oryzae (AG-GAG{Delta}), we disrupted the genes required for GAG biosynthesis in an AG{Delta} mutant. Hyphae of the double mutant were fully dispersed in liquid culture, suggesting that GAG is involved in hyphal aggregation in A. oryzae. Addition of partially purified GAG fraction to the hyphae of the AG-GAG{Delta} strain resulted in formation of mycelial pellets. Acetylation of the amino group in galactosamine of GAG weakened GAG aggregation, suggesting that hydrogen bond formation by this group is important for aggregation. Genome sequences suggest that -1,3-glucan, GAG, or both are present in many filamentous fungi and thus may function in hyphal aggregation in these fungi. We also demonstrated that production of a recombinant polyesterase, CutL1, was higher in the AG-GAG{Delta} strain than in the wild-type and AG{Delta} strains. Thus, controlling hyphal aggregation factors of filamentous fungi may increase productivity in the fermentation industry.\n\nImportanceProduction using filamentous fungi is an important part of the fermentation industry, but hyphal aggregation in these fungi in liquid culture limits productivity compared with that of yeast or bacterial cells. We found that galactosaminogalactan and -1,3-glucan both function in hyphal aggregation in Aspergillus oryzae, and that the hyphae of a double mutant deficient in both polysaccharides become fully dispersed in liquid culture. We also revealed the relative contribution of -1,3-glucan and galactosaminogalactan to hyphal aggregation. Recombinant protein production was higher in the double mutant than in the wild-type strain. Our research provides a potential technical innovation for the fermentation industry that uses filamentous fungi, as regulation of the growth characteristics of A. oryzae in liquid culture may increase productivity.

cell biology↗