Biology subjects

Harada, M.

Publications and source records attributed to Harada, M..

15 recordsLinked to original sources

bioRxiv2026

A phosphorylation-dependent partner-switching-like module controls heterocyst polysaccharide layer formation in Anabaena sp. strain PCC 7120

Heterocysts are specialized cells formed by filamentous cyanobacteria to enable nitrogen fixation under aerobic conditions. These cells are surrounded by a multilayered envelope, including a heterocyst-specific polysaccharide (Hep) layer that restricts oxygen diffusion and protects nitrogenase activity. Although several genes required for Hep biosynthesis have been identified, the regulatory mechanisms controlling Hep layer formation remain poorly understood. Here, we show that Hep layer formation in Anabaena sp. PCC 7120 is regulated by a phosphorylation-dependent partner-switching-like system. The phosphatase HenR and the STAS domain-containing protein All4160 are both required for Hep formation and nitrogen fixation. A nonphosphorylatable All4160 variant restores Hep formation and nitrogen fixation in a henR disruptant, indicating that phosphorylation negatively regulates All4160 function. Using a bacterial two-hybrid assay, we identified two candidate kinases, Alr3423 and All2284, that interact with All4160, and both proteins phosphorylate All4160 in vitro. Genetic analysis revealed that deletion of alr3423, but not all2284, suppresses the nitrogen fixation defect of the henR disruptant, suggesting that Alr3423 is a major kinase regulating All4160 in vivo. These findings uncover a phosphorylation switch that controls heterocyst polysaccharide layer formation and expand the role of partner-switching systems beyond {sigma} factor-dependent transcription to the direct regulation of a biosynthetic enzyme involved in exopolysaccharide (EPS) synthesis. ImportanceExopolysaccharides (EPS) are involved in diverse aspects of bacterial physiology, including environmental acclimation, motility, host association, and cellular differentiation, yet their regulatory mechanisms remain poorly understood. Cyanobacteria play central roles in global carbon and nitrogen cycles through photosynthesis and nitrogen fixation, the latter requiring a micro-oxic environment provided by specialized cells called heterocysts. This study reveals a previously unrecognized mechanism for controlling polysaccharide biosynthesis, in which a partner-switching-like system directly regulates the activity of a biosynthetic enzyme. By expanding the role of partner-switching systems beyond {sigma} factor-dependent transcription, these findings provide a framework for understanding how bacteria control EPS production in response to environmental and developmental cues.

microbiology↗

bioRxiv2026

Identification of a microRNA with a mutation in the loop structure in the silkworm Bombyx mori

MicroRNAs (miRNAs) play essential roles in the posttranscriptional regulation of gene expression in organisms. In the process of synthesizing mature miRNAs from miRNA precursors, the miRNA precursors are cleaved via Dicer at their loop structure, after which the miRNA precursors become mature and regulate transcription. However, the consequences of altering the loop sequence are not fully understood. The silkworm Bombyx mori is a lepidopteran insect with many genetic strains. We identified a mutant of the miRNA miR-3260 whose the part of the loop structure was lacking in a silkworm strain with translucent larval skin. Here, we aimed to analyze the role of wild-type miR-3260 and the influence of the mutation of the loop structure in B. mori. First, we identified the genomic region responsible for the translucent larval skin phenotype and determined that the mutated miR-3260 nucleotide sequences. Then, we predicted the binding partners of wild-type miR-3260 using the RNA hybrid tool and found two juvenile hormone (JH)-related genes as targets of wild-type miR-3260. Next, we assessed the relationships between miR-3260 and JH and found that miR-3260 was highly expressed in the Corpora allata and its expression responded to JH treatment. Meanwhile, miR-3260 mimic and inhibitor did not induce the typical phenotypes associated with JH in B. mori. Then, we compared the dicing products from wild-type and mutant miR-3260 precursors and observed that neither form underwent Dicer-mediated cleavage when the loop structure was altered. These results suggest that loop mutations in the miR-3260 precursor may not influence dicing activity, consistent with the lack of observable phenotypic effects.

molecular biology↗

bioRxiv2025

A scalable filtration-based method for isolating exomeres and other nanoscale extracellular particles

Extracellular particles, including extracellular vesicles (EVs) and non-vesicular extracellular particles (NVEPs), enable intercellular communication by transferring regulatory miRNAs and other biomolecules. While EVs have been studied for drug delivery, NVEPs remain relatively unexplored. Exomeres, a recently discovered class of NVEPs enriched in RNAi proteins, preferentially carry miRNAs and deliver them to cells more effectively than EVs, underscoring their potential as vehicles for therapeutic RNAs. One current limitation to studying and applying exomeres for therapeutic RNA delivery is the shortage of scalable, cost-effective, and rapid isolation methods. Here, we investigated whether tangential flow filtration (TFF), a common bioseparation approach that separates species by size, would effectively isolate exomeres from conditioned media with comparable purity and identity to those isolated by differential ultracentrifugation. TFF successfully isolated exomeres that were enriched in RNAi components including AGO2, HSP90AB1, and a unique set of miRNAs not abundant in EVs. Remarkably, exomere miRNAs were resistant to nuclease degradation even after treatment with protease and surfactant, suggesting that exomeres are highly stable, non-vesicular complexes with potentially extended circulating half-lives. Together, our results establish TFF as an efficient method for isolating exomeres, and further demonstrate that TFF could be applied in a bioprocess for exomere-based RNA therapeutics production. This study is also the first to demonstrate that exomere miRNAs are highly resistant to nuclease degradation, suggesting that exomeres could complement and potentially outperform current clinical standards for RNA delivery.

bioengineering↗

bioRxiv2025

Generation of Cellular Biofactories for Scalable Production of Surface-Engineered Extracellular Vesicles via CRISPR Genome Editing

Extracellular vesicles (EVs) are versatile biological nanoparticles with applications in therapeutics, diagnostics, and biotechnology. Current production methods using transient transfection or chemical conjugation suffer from high variability, limited scalability, and heterogeneous EV populations. Here, we developed CRISPR-Cas9 engineered HEK293T cell lines with stable integration of mCherry-C1C2 fusion proteins at the AAVS1 locus for continuous production of surface-modified EVs. The engineered cell lines demonstrated significantly higher surface display efficiency compared to transient transfection, with reduced batch-to-batch variability. EVs maintained native characteristics including size distribution (120-130 nm) and marker expression while showing efficient cellular uptake. The platform maintained consistent production of uniformly modified EVs with stable transgene expression over at least 25 passages (~3 months), eliminating the need for repeated transfections and reducing batch-to-batch variability inherent to transient expression systems.

bioengineering↗

bioRxiv2025

VesicleVoyager: In vivo selection of surface displayed proteins that direct extracellular vesicles to tissue-specific targets

The development of technologies for screening proteins that bind to specific tissues in vivo and facilitate delivery of large cargos remains challenging, with most approaches limited to cell culture systems that often yield clinically irrelevant hits. To overcome this limitation, we developed a novel molecular screening platform using an extracellular vesicle (EV) display library. EVs are natural molecular carriers capable of delivering diverse cargos, which can be engineered to enhance specificity and targeting through surface modifications. We constructed an EV-display library presenting monobody repertoires on EV surfaces, with genetic cargo inside the EVs corresponding to the displayed proteins. These libraries were screened for tissue specific delivery through serial passage in mice via sequential intravenous administration in and recovery of tissue-selected EVs and amplification of their encapsulated monobody genes at each passage. Our results demonstrated successful selection of tissue-specific targeting proteins, as revealed by fluorescence and bioluminescence imaging followed by DNA sequencing. To understand the stochastic relationship between displayed proteins and packaged genes, we developed a Markov chain model that quantified selection dynamics and predicted enrichment patterns despite the imperfect correlation between phenotype and genotype. This EV-based monobody screening approach, combined with mathematical modeling, is a significant advancement in targeted drug delivery by leveraging the natural capabilities of EVs with the selection of targeting proteins in a physiologically relevant environment.

bioengineering↗

bioRxiv2024

RecombiCraft Library construction: A novel method for DNA Library cloning and expansion using non-enzymatic single-step DNA recombination and liquid culture

In this study, we introduce RecombiCraft, an innovative, rapid, and cost-efficient method for constructing DNA libraries in E. coli. This method uses seamless ligation cloning extract (SLiCE) coupled with liquid culture amplification to effectively minimize sequence biases. The technique capitalizes on the natural homologous recombination capabilities of E. coli cell lysates, eliminating the need for multiple purified enzymes and reducing costs. We first synthesized the library backbone and inserts via PCR, employing high-fidelity polymerase to minimize sequence bias. The SLiCE technique was then used to assemble the DNA fragments introduced into E. coli through electroporation. To ensure the integrity of the library, we optimized culture times based on next-generation sequencing analysis which confirmed the minimal sequence bias. The RecombiCraft method demonstrates that this approach is economical and maintains the librarys uniformity. It is a promising tool for genetic research and biotechnological applications with a significantly shorter library generation period.

molecular biology↗

bioRxiv2024

Scalable Isolation of Surface-Engineered Extracellular Vesicles and Separation of Free Proteins via Tangential Flow Filtration and Size Exclusion Chromatography (TFF-SEC)

BackgroundExtracellular vesicles (EVs) represent small lipid bilayer structures pivotal in mediating intercellular communication via biomolecular transfer. Their inherent characteristics, including packaging, non-immunogenicity, and biofluid stability, position EVs as promising drug delivery vectors. However, developing clinical quality EVs requires multifaceted technological advancement. MethodsIn this study, a method is introduced for engineering extracellular vesicles (eEVs) from cultured cells and their subsequent isolation using lab-scale tangential flow filtration (TFF). This is the first study to evaluate DNA loading efficacy into EVs isolated by TFF, marking a significant milestone in the field of targeted drug delivery. Initially, cells are transfected with EV-display constructs to facilitate the secretion of eEVs bearing the desired coding molecules. Following brief centrifugation, the cell culture media undergoes filtration using hollow fiber filters. TFF, by applying a constant flow, effectively segregates molecules based on designated molecular weight cut-off (MWCO), enriching particles between 50 nm and 650 nm. ResultsCompared to conventional methods like ultracentrifugation, TFF demonstrates higher efficiency in removing undesired molecules/aggregates while exerting less stress on EVs. Characterization of eEVs through various assays confirms TFFs superiority in isolating pure EV populations. Additionally, the necessity of size-exclusion chromatography (SEC) after tangential flow filtration (TFF) becomes evident for effectively removing unbound protein contaminants. ConclusionIn conclusion, TFF-SEC emerges as a scalable and superior approach for eEV isolation, promising significant advancements in clinical applications.

bioengineering↗

bioRxiv2023

Missing view completion for multi-timepoints omics data via representation disentanglement and temporal knowledge transfer

Longitudinal multi-view omics data offer unique insights into the temporal dynamics of individual-level physiology, which provides opportunities to advance personalized healthcare. However, the common occurrence of incomplete views makes extrapolation tasks difficult, and there is a lack of tailored methods for this critical issue. Here, we introduce LEOPARD, an innovative approach specifically designed to complete missing views in multi-timepoint omics data. By disentangling longitudinal omics data into content and temporal representations, LEOPARD transfers the temporal knowledge to the omics-specific content, thereby completing missing views. The effectiveness of LEOPARD is validated on three benchmark datasets constructed with data from the MGH COVID study and the KORA cohort, spanning periods from 3 days to 14 years. Compared to conventional imputation methods, such as missForest, PMM, GLMM, and cGAN, LEOPARD yields the most robust results across the benchmark datasets. LEOPARD-imputed data also achieve the highest agreement with observed data in our analyses for age-associated metabolites detection, estimated glomerular filtration rate-associated proteins identification, and chronic kidney disease prediction. Our work takes the first step toward a generalized treatment of missing views in longitudinal omics data, enabling comprehensive exploration of temporal dynamics and providing valuable insights into personalized healthcare.

bioinformatics↗

bioRxiv2023

AtDREB2G is a novel regulator of riboflavin biosynthesis under low-temperature stress and abscisic acid treatment in Arabidopsis thaliana

Riboflavin (RF) serves as a precursor to FMN and FAD, crucial cofactors in various metabolic processes. Strict regulation of cellular flavin homeostasis is imperative, yet information regarding the factors governing this regulation remains largely elusive. In this study, we first examined the impact of external flavin treatment on the Arabidopsis transcriptome to identify novel regulators of cellular flavin levels. Our analysis revealed alterations in the expression of 49 putative transcription factors. Subsequent reverse genetic screening highlighted a member of the Dehydration-Responsive Element Binding (DREB) family, AtDREB2G, as a potential regulator of cellular flavin levels. Knockout mutants of AtDREB2G (dreb2g) exhibited reduced flavin levels and decreased expression of RF biosynthetic genes compared to wild-type plants. Conversely, conditional overexpression of AtDREB2G led to an increase in the expression of RF biosynthetic genes and elevated flavin levels. In wild-type plants, exposure to low temperatures and abscisic acid treatment stimulated enhanced flavin levels and upregulated the expression of RF biosynthetic genes, concomitant with the induction of AtDREB2G. Notably, these responses were significantly attenuated in dreb2g mutants. Our findings establish AtDREB2G as a novel positive regulator of flavin biosynthesis in Arabidopsis, particularly under conditions of low temperature and abscisic acid treatment.

plant biology↗

bioRxiv2023

Stimulation of VTA dopamine inputs to LH upregulates orexin neuronal activity in a DRD2-dependent manner

Dopamine and orexins (hypocretins) play important roles in regulating reward-seeking behaviors. It is known that hypothalamic orexinergic neurons project to dopamine neurons in the ventral tegmental area (VTA), where they can stimulate dopaminergic neuronal activity. Although there are reciprocal connections between dopaminergic and orexinergic systems, whether and how dopamine regulates the activity of orexin neurons is currently not known. Here we implemented an opto-Pavlovian task in which mice learn to associate a sensory cue with optogenetic dopamine neuron stimulation to investigate the relationship between dopamine release and orexin neuron activity in the LH. We found that dopamine release can be evoked in LH upon optogenetic stimulation of VTA dopamine neurons, and is also naturally evoked by cue presentation after opto-Pavlovian learning. Furthermore, orexin neuron activity could also be upregulated by local stimulation of dopaminergic terminals in the LH in a way that is partially dependent on dopamine D2 receptors (DRD2). Our results reveal previously unknown orexinergic coding of reward expectation and unveil an orexin-regulatory axis mediated by local dopamine inputs in the LH. HighlightsO_LIOptical VTA DA neuron stimulation is sufficient to elicit a Pavlovian-like dopamine transient in the NAc C_LIO_LIDopamine in the LH encodes both negative and positive reward prediction errors C_LIO_LIDopamine in the LH positively modulates orexin neuronal activity locally in a D2R dependent way C_LI

neuroscience↗

bioRxiv2022

Morphogenesis of bullet-shaped rabies virus particles requires a functional interplay between the viral matrix protein and ESCRT-I component TSG101

Viral protein assembly and virion budding are tightly regulated to enable the proper formation of progeny virions. At this late stage in the virus life cycle, some enveloped viruses take advantage of the host ESCRT (endosomal sorting complex required for transport) machinery, which contributes to the physiological functions of membrane modulation and abscission. Bullet-shaped viral particles are unique morphological characteristics of rhabdoviruses; however, the involvement of host factors in rhabdovirus infection, and specifically the molecular mechanisms underlying virion formation are not fully understood. In the present study, we used a siRNA screening approach and found that the ESCRT-I component TSG101 contributes to the propagation of rabies virus (RABV). We demonstrated that the matrix protein (M) of RABV interacts with TSG101 via the late-domain containing the PY and YL motifs, which are conserved in various viral proteins. Loss of the YL motif in the RABV M or the downregulation of host TSG101 expression resulted in the intracellular aggregation of viral proteins and abnormal virus particle formation, indicating a defect in the RABV assembly and budding processes. These results indicate that the interaction of the RABV M and TSG101 is pivotal for not only the efficient budding of progeny RABV from infected cells but also for the bullet-shaped virion morphology. ImportanceEnveloped-viruses bud from cells with the host lipid bilayer. Generally, the membrane modulation and abscission are mediated by host ESCRT (endosomal sorting complex required for transport) complexes. Some enveloped-viruses utilize their late (L)-domain to interact with ESCRTs, which promotes viral budding. Rhabdoviruses form characteristic bullet-shaped enveloped-virions, but the underlying molecular mechanisms involved remain elusive. Herein, we showed that TSG101, one of ESCRT components, supports rabies virus (RABV) budding and proliferation. TSG101 interacted with RABV matrix protein via L-domain, and the absence of this interaction resulted in intracellular virion accumulation and distortion of the morphology of progeny virions. Our study reveals that virion formation of RABV is highly regulated by TSG101 and the virus matrix protein.

microbiology↗

bioRxiv2022

Evaluation of EV Storage Buffer for Efficient Preservation of Engineered Extracellular Vesicles

Extracellular vesicles (EVs), detectable in all bodily fluids, mediate intercellular communication by transporting molecules between cells. The capacity of EVs to transport molecules between distant organs has drawn interest for clinical applications in diagnostics and therapeutics. Although EVs hold potential for nucleic acid-based and other molecular therapeutics, the lack of standardized technologies, including isolation, characterization, and storage, leaves many challenges for clinical applications, potentially resulting in misinterpretation of crucial findings. Previously, several groups demonstrated the problems of commonly used storage methods that distort EV integrity. This work aims to evaluate the process to optimize the storage conditions of EVs and then characterize them according to the experimental conditions and the models used previously. Our study reports a highly efficient EV storage condition, focusing on EVs capacity to protect their molecular cargo from biological, chemical, and mechanical damage. Compared with commonly used EV storage conditions, our EV storage buffer leads to less size and particle number variation at both 4{degrees}C and -80 {degrees}C, enhancing the ability to protect EVs while maintaining targeting functionality.

bioengineering↗

bioRxiv2019

Cortico-striatal synaptic plasticity underlying compulsive reward seeking

Addicted individuals compulsively seek drugs. Cortico-striatal projections have been implicated in persevering to seek rewards even when punished. The temporo-spatial determinants of the activity underlying the compulsive reward seeking however remains elusive. Here we trained mice in a seek-take chain, rewarded by optogenetic dopamine neuron self-stimulation (oDASS). Mice that persevered when seeking was punished, exhibited an increased AMPA/NMDA ratio selectively at orbitofrontal cortex (OFC) to dorsal striatum (DS) synapses. In addition, an activity peak of spiny projection neurons (SPNs) in the DS at the moment of signalled reward availability was detected. Chemogenetic inhibition of OFC neurons curbed the activity peak and reduced punished reward seeking, as did optogenetic hyperpolarization of SPNs time locked to the cue predicting reward availability, establishing a causal link. Taken together, we conclude that the strengthening of OFC-DS synapses drives SPNs activity when a reward predictive cue is delivered, thus encouraging reward seeking in subsequent trials.

neuroscience↗

bioRxiv2019

Highly reliable, targeted photothermal cancer therapy combined with thermal dosimetry using indocyanine green lactosome

Indocyanine green (ICG) is a near-infrared light-absorbing substance. Thus, when a tumor in which ICG has accumulated is irradiated with a near-infrared (NIR) laser, only the tumor can be heated by a photothermal reaction. We developed ICG lactosome, a novel drug delivery system (DDS) composed of polymeric micelles and ICG that shows selective accumulation in tumor based on an enhanced permeability and retention (EPR) effect. We showed that ICG lactosome accumulated in a tumor by using an intradermal tumor mouse model of a murine colon cancer cell line (Colon26) transfected with Nano lantern luminescent protein (NLC26). Two days after the administration of ICG lactosome, the tumor was irradiated with an 808-nm diode-laser while monitoring tumor temperature. The results showed that the treated tumors were cured when the peak of tumor temperature during NIR irradiation reached 43{degrees}C or higher. To verify these results, photothermal therapy (PTT) using ICG lactosome was carried out using a newly developed system that can control the temperature at the NIR irradiation site at a constant level. All of the tumors that had been kept at 43{degrees}C during irradiation were cured, while 2 of 5 tumors that had been kept at 42{degrees}C were not cured, and none of tumors that had been kept at a temperature below 41{degrees}C were cured. ICG lactosome-assisted PTT combined with thermal dosimetry is a highly reliable method for cancer treatment and may afford further clinical opportunities for PTT.

bioengineering↗

bioRxiv2018

A survey of public attitudes towards third-party reproduction in Japan in 2014

Objective: The objective of this study was to examine public attitudes towards third-party reproduction and the disclosure of conception through third-party reproduction.\n\nMethods: We conducted the web-based survey for the public attitude towards third-party reproduction in February 2014. Twenty-five hundred people were recruited with equal segregation of age (20s, 30s, 40s, and 50s) and gender. We analyzed the association between gender, age, infertility, and ethical view using a questionnaire regarding donor sperm, donor oocyte, donor embryo, gestational surrogacy, and disclosure to offspring.\n\nResults: Of the respondents, 36.2% approved and 26.6% disapproved with gamete or embryo donation. The frequency of those who approved was the lowest in females in the 50-59 years age group, and was significantly higher in males or females with infertility. Secondly, 40.9% approved and 21.8% disapproved with gestational surrogacy. The frequency of those who approved gestational surrogacy was higher in males or females with infertility. Thirdly, 46.3% of respondents agreed and 20.4% disagreed with \"offspring have the right to know their origin\". Those who disagreed were primarily in the 50-59 age group of both genders, and disagreement was significantly higher in the infertility group compared with non-infertility group.\n\nConclusion: In this study, public attitudes were affected by gender, age, and experience of infertility. These study findings are important in understanding the attitude towards third-party reproduction and disclosure to the offspring. Respondents having indecisive attitudes were >30%, which might indicate an increased requirement for information and education to enhance the discussion on the ethical consensus on third-party reproduction in Japan.

scientific communication and education↗