Biology subjects

Nakamura, T.

Publications and source records attributed to Nakamura, T..

At least 73 records · Page 4Linked to original sources

bioRxiv2023

Endothelial-fibroblast interactions during Scarb1 accelerate heart failure

Endothelial cells (ECs) maintain cardiac homeostasis and EC dysfunction causes heart failure progression. Moreover, pathological changes occur via interactions between multiple cells, including ECs. Here, we conducted single-cell RNA-seq analysis of non-cardiomyocytes in mouse hearts during heart failure progression to elucidate the pathological changes in ECs and fibroblasts (FBs) mediated by cell-cell interactions. We show that capillary and arterial ECs exhibit mesenchymal gene expression changes with heart failure progression, indicating that endothelial-to-mesenchymal transition (EndMT) is a major pathological alteration in ECs. We also found that the interaction between ECs and FBs was enriched during heart failure, particularly when involving Scavenger Receptor Class B Member 1 (Scarb1) in ECs. FBs induce mesenchymal gene alterations in ECs in the EC-FB co-culture system, which is inhibited by blocking SCARB1. RNA-seq analysis showed that administration of a SCARB1 inhibitor blocked mesenchymal gene expression, and inflammatory changes, suggesting that the EC-FB interaction via Scarb1 is important for EndMT induction in ECs. Systemic administration of a SCARB1 inhibitor attenuated heart failure progression and cardiac fibrosis. EC-specific Scarb1 knockout mouse showed improved cardiac function, suggesting a crucial role of Scarb1 in heart failure progression. Our results suggest that Scarb1 is a promising candidate for novel heart failure treatments that target ECs.

molecular biology↗

bioRxiv2023

S-Nitrosylation of CRTC1 in Alzheimer's disease impairs CREB-dependent gene expression induced by neuronal activity

CREB-regulated transcription coactivator 1 (CRTC1) plays an important role in synaptic plasticity, learning and long-term memory formation through regulation of neuronal activity-dependent gene expression, and CRTC1 dysregulation is implicated in Alzheimers disease (AD). Here, we show that increased S-nitrosylation of CRTC1 (forming SNO-CRTC1), as seen in cell-based, animal-based, and human induced pluripotent stem cell (hiPSC)-derived cerebrocortical neuron-based AD models, disrupts its binding with CREB and diminishes the activity-dependent gene expression mediated by the CRTC1/CREB pathway. We identified Cys216 of CRTC1 as the primary target of S-nitrosylation by nitric oxide (NO)-related species. Using CRISPR/Cas9 techniques, we mutated Cys216 to Ala in hiPSC-derived cerebrocortical neurons bearing one allele of the APPSwe mutation (AD-hiPSC neurons). Introduction of this non-nitrosylatable CRTC1 construct rescued defects in AD-hiPSC neurons, including decreased neurite length and increased neuronal cell death. Additionally, expression of non-nitrosylatable CRTC1 in vivo in the hippocampus rescued synaptic plasticity in the form of long-term potentiation (LTP) in 5XFAD mice. Taken together, these results demonstrate that formation of SNO-CRTC1 contributes to the pathogenesis of AD by attenuating the neuronal activity-dependent CREB transcriptional pathway, and suggests a novel therapeutic target for AD.

neuroscience↗

bioRxiv2023

Copy number of the eukaryotic rRNA gene can be counted comprehensively

Gene sequence has been widely used in molecular ecology. For instance, the ribosomal RNA (rRNA) gene has been widely used as a biological marker to understand microbial communities. The variety of the detected rRNA gene sequences reflects the diversity of the microorganisms existing in the analyzed sample. Their biomass also can be estimated by applying quantitative sequencing with the information of rRNA gene copy numbers in genomes, however, the information on rRNA gene copy number is still limited. Especially, the copy number in microbial eukaryotes is much less understood than that of prokaryotes, possibly because of the large and complex structure of eukaryotic genomes. In this communication, we report an alternative approach that is more appropriate from the existing method of quantitative sequencing and demonstrate that the copy number of eukaryotic rRNA can be measured efficiently and comprehensively. By applying this approach widely, information on the eukaryotic rRNA copy number can be determined, and their community structures can be depicted and compared more efficiently.

microbiology↗

bioRxiv2023

Determining the structure of protein-bound ceramides, essential lipids for skin barrier function

Protein-bound ceramides, specialized ceramides covalently bound to corneocyte surface proteins, are essential for skin permeability barrier function. However, their exact structure and target amino acid residues are unknown. Here, we found that epoxy-enone (EE) ceramides, precursors of protein-bound ceramides, as well as their synthetic analog, formed stable conjugates only with Cys among nucleophilic amino acids. NMR spectroscopy revealed that the {beta}-carbon of the enone was attached by the thiol group of Cys via a Michael addition reaction. We confirmed the presence of Cys-bound EE ceramides in mouse epidermis by mass spectrometry analysis of protease-digested epidermis samples. EE-ceramides were reversibly released from protein-bound ceramides via sulfoxide elimination. We found that protein-bound ceramides with reversible release properties accounted for approximately 60% of total protein-bound ceramides, indicating that Cys-bound EE ceramides are the predominant protein-bound ceramides. Our findings provide clues to the molecular mechanism of skin barrier formation by protein-bound ceramides. HighlightsO_LIEpoxy-enone (EE) ceramides form stable conjugates with Cys. C_LIO_LICys binds to the {beta}-carbon of the enone via a Michael addition reaction. C_LIO_LICys-bound EE ceramides exist in the mouse epidermis. C_LIO_LICys-bound EE ceramides are the predominant protein-bound ceramides. C_LI In BriefProtein-bound ceramides are specialized ceramides essential for skin permeability barrier function. In the present study, we determined the exact structure of protein-bound ceramides as Cys-bound epoxy-enone ceramides where the {beta}-carbon of the epoxy-enone ceramides is attached by Cys residues of corneocyte surface proteins via a Michael addition reaction.

cell biology↗

bioRxiv2023

Distinct ossification trade-offs illuminate the shoulder girdle reconfiguration at the water-to-land transition.

The mechanisms of the pectoral girdle transformation at the origin of terrestrial locomotion in vertebrates remains an outstanding problem in evolutionary biology1. The loss of dermal bones and the enlargement of endochondral bones resulted in the disarticulation of the pectoral girdle from the skull and the formation of the neck during the fish-to-tetrapod transition2-5. Despite the functional implications of this skeletal shift in the emergence of terrestrial vertebrates, the underlying genetic-developmental alterations have remained enigmatic. Here, we discovered that in zebrafish pectoral girdle mesodermal cells expressing gli3, a transcription factor in the Hedgehog signaling pathway, contribute to both dermal and endochondral bones. We show that Gli3 regulates expression of activin A receptor type 1-like, a BMP type 1 receptor lost in tetrapod lineages, and thereby determines endochondral and dermal ossification. Intriguingly, Gli and Hedgehog compound knockout fish exhibited an unexpected combination of actinopterygian fish and stem-tetrapod pectoral girdle characteristics. These ontogenetic and anatomical data suggest that a trade-off between the two distinct ossification pathways is a deeply embedded developmental program in bony fishes, with potential for tuning of this trade-off to generate novel pectoral girdle forms akin to stem-tetrapods at the dawn of vertebrate terrestrialization.

evolutionary biology↗

bioRxiv2023

Multiple embryonic sources converge to form the pectoral girdle skeleton in zebrafish

The morphological transformation of the pectoral/shoulder girdle is fundamental to the water-to-land transition in vertebrate evolution. Although previous studies have resolved the embryonic origins of the tetrapod shoulder girdle, those of the fish pectoral girdle remain uncharacterized, creating a gap in the understanding of girdle transformation mechanisms from fish to modern tetrapods. Here, we identified the embryonic origins of the pectoral girdle of zebrafish (Danio rerio), including the cleithrum as an ancestral pectoral girdle element lost in extant tetrapods. Our combinatorial approach of photoconversion and genetic cell lineage tracing mapped that cleithrum development combines three adjoining embryonic populations: cranial neural crest cells and lateral plate mesoderm-derivatives (trunk lateral plate mesoderm and cardiopharyngeal mesoderm-associated cells). The topographical position of the cleithrum at the head/trunk interface is a shared characteristic among cleithrum-bearing fish, thus its multiple embryonic origins are likely a conserved feature. Moreover, a comparison of the pectoral girdle progenitors between aquatic fish and extant amniotes suggests that cleithrum loss is associated with the disappearance of its unique developmental environment by the insertion of the neck lateral plate mesoderm into the head/trunk interface. Overall, our study establishes an embryological framework for pectoral/shoulder girdle formation and their evolutionary trajectories from their origin in water to diversification on land.

developmental biology↗

bioRxiv2023

Similarity and dissimilarity in alteration of gene expression profile associated with inhalational anesthesia between sevoflurane and desflurane

Although sevoflurane is the most commonly used inhalational anesthetic agent, the popularity of desflurane is increasing to a similar level. The main beneficial property of desflurane is the relatively fast emergence of the patient from the anesthetic state after halting its supply compared with anesthesia using other anesthetic agents. However, there has been no comprehensive comparison of the effects of these two anesthetic agents on alterations in liver gene expression profiles in animals, including humans, to assess the levels of hepatotoxicity that is induced at least in some extent by inhalational anesthesia. Thus, we compared alterations in the global gene expression profiles in the livers of rats subjected to inhalational anesthesia by sevoflurane or desflurane by a next-generation sequencing method. Our data revealed that both anesthetic agents significantly activated a similar set of genes including those related to drug metabolism and circadian rhythm. Furthermore, many genes downregulated by sevoflurane were also downregulated by desflurane. However, many of the genes related to the cholesterol biosynthetic process were specifically repressed by sevoflurane, but not by desflurane.

molecular biology↗

bioRxiv2023

UBL3 interaction with α-synuclein is downregulated by silencing MGST3

Ubiquitin-like 3 (UBL3) is a membrane-anchored protein that has been discovered to function as a protein post-translational modifier, helping to sort proteins into small extracellular vesicles (sEVs). Aggregations of alpha-synuclein (-syn) are associated with the pathology of neurodegenerative diseases such as Parkinsons disease (PD). The aggregation and toxicity of -syn can be influenced by its interactions with specific proteins. Recently, the interactions between UBL3 and -syn was uncovered. It is believed to play a role in eliminating excess -syn from neurons, or in the spread of -syn pathology in the brain associated with neurodegenerative diseases. However, the regulator that can mediate the interaction between UBL3 and -syn remains unclear. In this study, we employed the split gaussian luciferase complementation assay and RNA interference (RNAi) technology to discover that QSOX2, HTATIP2, UBE3C, MGST3, NSF, HECTD1, SAE1 and ATG3 are involved in downregulating the interaction between UBL3 and -syn. Among these proteins, silencing MGST3 had the most significant impact on the UBL3--syn interaction (with a fold change log2 of less than - 1). MGST3 is a part of the antioxidant system, and silencing MGST3 is believed to contribute to oxidative stress. We used hydrogen peroxide (H2O2) to induce oxidative stress and observed its effect on the UBL3--syn interaction. Our finding showed that an 800M concentration of H2O2 could also downregulate this interaction. However, the effect of oxidative stress caused by 800M H2O2 on the UBL3--syn interaction did not be enhanced by silencing MGST3. In conclusion, the interaction between UBL3 and -syn is downregulated by silencing MGST3.

molecular biology↗

bioRxiv2023

UBL3 Interacts with Alpha-synuclein in Cells and the Interaction is Downregulated by the EGFR Pathway Inhibitor Osimertinib

Ubiquitin-like 3 (UBL3) acts as a post-translational modification (PTM) factor and regulates protein sorting into small extracellular vesicles (sEVs). sEVs have been reported as vectors for the pathology propagation of neurodegenerative diseases, such as -synucleinopathies. Alpha-synuclein (-syn) has been widely studied for its involvement in -synucleinopathies. However, it is still unknown whether UBL3 interacts with -syn, and is influenced by drugs or compounds. In this study, we investigated the interaction between UBL3 and -syn, and any ensuing possible functional and pathological implications. We found that UBL3 can interact with -syn by the Gaussia princeps based split luciferase complementation assay in cells and immunoprecipitation, while cysteine residues at its C-terminal, which are considered important as PTM factors for UBL3, were not essential for the interaction. The interaction was upregulated by 1-methyl-4-phenylpyridinium exposure. In drug screen results, the interaction was significantly downregulated by the treatment of osimertinib. These results suggest that UBL3 interacts with -syn in cells and be significantly downregulated by epidermal growth factor receptor (EGFR) pathway inhibitor osimertinib. Therefore, the UBL3 pathway may be a new therapeutic target for -synucleinopathies in the future.

molecular biology↗

bioRxiv2023

In Vivo mRNA Hacking with Staple Oligomers Prevents Myocardial Hypertrophy

Summary paragraphThe elucidation of gene-silencing mechanisms by RNA interference (RNAi) and antisense oligomers has drawn increasing attention to nucleic acid medicine. However, several challenges remain to be overcome, such as in vivo stability1, target selectivity 2,3, drug delivery4,5, and induced innate immunity6. Here, we report a new, versatile, and highly-selective method to hack RNA by controlling RNA structure using short oligonucleotides (RNA hacking: RNAh) in living cells. The oligonucleotide, named Staple oligomer, hybridizes specifically to a target mRNA and artificially induces an RNA higher-order structure, RNA G-quadruplex (RGq)7, on the mRNA. As a result, the RGq allows effective suppression of the target protein translation. This technology does not require cooperation with bioprocesses including enzymatic reactions as in RNAi or antisense technologies, permitting the introduction of artificial nucleic acids into Staple oligomers to increase their in vivo stability without compromising their effectiveness. The method was validated by translational regulation of the mRNAs of TPM3, MYD88, and TRPC6, in a cell-free system and in living mammalian cells. In vivo application of the technology to TRPC6 mRNA allowed us to prevent cardiac hypertrophy in transverse aortic constriction (TAC)-treated mice with no detectable off-target effects. This technology provides new insights into gene therapy after RNAi and antisense technologies.

molecular biology↗

bioRxiv2023

Viruses encode tRNA and anti-retron to evade bacterial immunity

Retrons are bacterial genetic retroelements that encode reverse transcriptase capable of producing multicopy single-stranded DNA (msDNA) and function as antiphage defense systems. Phages employ several strategies to counter the host defense systems, but no mechanisms for evading retrons are known. Here, we show that tRNATyr and Rad (retron anti-defense) of T5 phage family inhibit the defense activity of retron 78 and a broad range of retrons, respectively. The effector protein of retron 78, ptuAB, specifically degraded tRNATyr leading abortive infection, but phage countervailed this defense by supplying tRNATyr. Rad inhibited retron function by degrading noncoding RNA, the precursor of msDNA. In summary, we demonstrated that viruses encode at least two independent strategies for overcoming bacterial defense systems: anti-defense, such as Rad, and defense canceler, like tRNA.

microbiology↗

bioRxiv2023

Unsupervised decomposition and description of nonhuman primate behavior

Nonhuman primates (NHPs) exhibit complex and diverse behavior that typifies advanced cognitive function and social communication, but quantitative and systematical measure of this natural nonverbal processing has been a technical challenge. Specifically, a method is required to automatically segment time series of behavior into elemental motion motifs, much like finding meaningful words in character strings. Here, we propose a solution called SyntacticMotionParser (SMP), a general-purpose unsupervised behavior parsing algorithm using a non-parametric Bayesian model. Using three-dimensional posture-tracking data from NHPs, SMP automatically outputs an optimized sequence of latent motion motifs classified into the most likely number of states. When applied to behavioral datasets from common marmosets and rhesus monkeys, SMP outperformed conventional posture-clustering models and detected a set of behavioral ethograms from publicly available data. SMP also quantified and visualized the behavioral effects of chemogenetic neural manipulations. SMP thus has the potential to dramatically improve our understanding of natural NHP behavior in a variety of contexts.

neuroscience↗

bioRxiv2023

Emergence of rhythmic chunking in complex stepping of mice

Motor chunking is important for motor execution, allowing atomization and efficiency of movement sequences. However, it remains unclear why and how chunks contribute to motor execution. To analyze the structure of naturally occurring chunks, we trained mice to run in a complex series of steps and identified the formation of chunks. We found that intervals (cycle) and the positional relationship between the left and right limbs (phase) of steps inside the chunks, unlike those outside the chunks, were consistent across occurrences. Further, licking by the mice was also more periodic and linked to the specific phases of limb movements within the chunk. Based on these findings, we propose the rhythm chunking hypothesis, whereby within chunks, the repetitive movements of many body parts are linked by the rhythm parameters: cycle and phase. The computational complexity of movement may thereby be reduced by adjusting movements as the combination of rhythms.

animal behavior and cognition↗

bioRxiv2023

Selective bacteriophages reduce the emergence of resistant bacteria in the bacteriophage-antibiotic combination therapy

Escherichia coli O157:H7 is a globally important foodborne pathogen that affects food safety. Antibiotic administration against O157:H7 may contribute to the exacerbation of hemolytic uremic syndrome (HUS) and antibiotic-resistant strains increase; therefore, bacteriophage therapy (phage therapy) is considered a useful alternative. In the treatment of resistant bacterial infections, combination therapy with bacteriophages and antibiotics, taking advantage of the benefits of both agents, has been suggested to be effective in inhibiting the emergence of antimicrobial-resistant strains; however, its effectiveness against O157:H7 is not well understood. In this study, we isolated SP015, a phage that infects O157:H7, and compared the combined effect of the bacteriophage and fosfomycin (FOM) with that of the PP01 phage. Genomic analysis revealed that FOM exerts its antibacterial activity through glycerol-3-phosphate transporter (GlpT) and hexose phosphate transporter (UhpT) proteins, and the receptors of PP01 and SP015 phages are the outer membrane protein C (OmpC) and ferrichrome outer membrane transporter protein (FhuA), respectively. Experiments with knockout strains have suggested that FOM also uses OmpC, the receptor for PP01, as a transporter. This may explain why the combination treatment with PP01 resulted in a faster emergence of resistance than the combination treatment with SP015. We propose that phage-antibiotic combination therapy requires careful selection of the phage to be used.

microbiology↗

bioRxiv2022

Novel monoclonal antibodies showing broad neutralizing activity for SARS-CoV-2 variants including Omicrons BA.5 and BA.2.75

We identified novel neutralizing monoclonal antibodies against SARS-CoV-2 variants (including Omicron) from individuals received two doses of mRNA vaccination after they had been infected with wildtype. We named them MO1, MO2 and MO3. MO1 shows high neutralizing activity against authentic variants: D614G, Delta, BA.1, BA.1.1, BA.2, and BA.2.75 and BA.5. Our findings confirm that the wildtype-derived vaccination can induce neutralizing antibodies that recognize the epitopes conserved among the SARS-CoV-2 variants (including BA.5 and BA.2.75). The monoclonal antibodies obtained herein could serve as novel prophylaxis and therapeutics against not only current SARS-CoV-2 viruses but also future variants that may arise.

microbiology↗

bioRxiv2022

Identification of lactic acid bacteria with reduced inflammatory and enhanced protective properties against influenza virus

Lactic acid bacteria (LAB) exert beneficial effects on health by regulating innate immunity in the intestinal tract. Many LAB that demonstrate anti-viral properties depend on the hosts inflammatory responses; however, a few LAB strains have low inflammatory and anti-viral properties. In this study, the LAB strain D279 (NITE_BP-03645, Latilactobacillus sakei) was isolated from among 741 LAB strains that were analyzed for their ability to induce interleukin 12 (IL-12), and was subsequently characterized. D279 induced the highest levels of IL-12 among the screened LAB. Further, D279 significantly activated anti-viral genes and preferentially induced IFN{lambda} in vitro, which plays a critical role in the epithelial tissue, conferring strong anti-influenza potency without inflammation, whereas IFN levels were relatively lower. Administration of pasteurized D279 to mice resulted in strong anti-influenza potency, including higher natural killer (NK) cell activity, and a reduced number of viruses in the lung compared to the control. Importantly, no D279-fed mice were sacrificed during the viral infection tests. Taken together, these results suggest that D279 administration confers beneficial effects with reduced inflammation, regulates innate immunity, and may be relevant for commercial use in the future.

microbiology↗

bioRxiv2022

Wnt signalling, cell fate determination and anteroposterior polarity of the skate gill arch skeleton

The gill skeleton of cartilaginous fishes (sharks, skates, rays and holocephalans) exhibits a striking anterior-posterior polarity, with a series of fine appendages called branchial rays projecting from the posterior margin of the gill arch cartilages. We previously demonstrated in the skate (Leucoraja erinacea) that branchial rays derive from a posterior domain of pharyngeal arch mesenchyme that is responsive to Shh signalling from a distal gill arch epithelial ridge (GAER) signalling centre. However, how branchial ray progenitors are specified exclusively within posterior gill arch mesenchyme is not known. Here we show that genes encoding several Wnt ligands are expressed in the ectoderm immediately adjacent to the skate GAER, and that these Wnt signals are transduced largely in the anterior arch environment. Using pharmacological manipulation, we show that inhibition of Wnt signalling results in an anterior expansion of Shh signal transduction in developing skate gill arches, and in the formation of ectopic anterior branchial ray cartilages. Our findings demonstrate that ectodermal Wnt signalling contributes to gill arch skeletal polarity in skate by restricting Shh signal transduction and chondrogenesis to the posterior arch environment and highlights the importance of signalling interactions at embryonic tissue boundaries for cell fate determination in vertebrate pharyngeal arches.

developmental biology↗

bioRxiv2022

Resolution of the curse of dimensionality in single-cell RNA sequencing data analysis

Single-cell RNA sequencing (scRNA-seq) can determine gene expression in numerous individual cells simultaneously, promoting progress in the biomedical sciences. However, scRNA-seq data are high-dimensional with substantial technical noise, including dropouts. During analysis of scRNA-seq data, such noise engenders a statistical problem known as the curse of dimensionality (COD). Based on high-dimensional statistics, we herein formulate a noise reduction method, RECODE (resolution of the curse of dimensionality), for high-dimensional data with random sampling noise. We show that RECODE consistently eliminates COD in relevant scRNA-seq data with unique molecular identifiers. RECODE does not involve dimension reduction and recovers expression values for all genes, including lowly expressed genes, realizing precise delineation of cell-fate transitions and identification of rare cells with all gene information. Compared to other representative imputation methods, RECODE employs different principles and exhibits superior overall performance in cell-clustering and single-cell level analysis. The RECODE algorithm is parameter-free, data-driven, deterministic, and high-speed, and notably, its applicability can be predicted based on the variance normalization performance. We propose RECODE as a general strategy for preprocessing noisy high-dimensional data.

bioinformatics↗