Biology subjects

Watanabe, S.

Publications and source records attributed to Watanabe, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

N-acetylcarnosine attenuates age-associated declines in multi-organ systems to improve survival

Histidine containing dipeptides (HCDs) such as N-acetylcarnosine are endogenous metabolites that are ergogenic and mitigate metabolic dysfunction. We previously demonstrated that short-term N-acetylcarnosine treatment is highly efficacious in protecting muscle atrophy induced by disuse. Here we demonstrate that a 6-months treatment of N-acetylcarnosine attenuates a broad spectrum of age-associated maladies and improved survival by [~]50% in female mice. A comprehensive survey of organ systems revealed that N-acetylcarnosine prevents decline in adiposity, diastolic function, vasodilation, muscle strength, and bone density. Together, N-acetylcarnosine substantially delays the onset of system-wide end-stage pathology to prolong lifespan. As an endogenously present metabolite, treatment with N-acetylcarnosine may be a safe and promising intervention to promote healthy aging in humans.

physiology↗

bioRxiv2025

Efficient gene disruption with CRISPR-Cas3 in human T cells

The CRISPR-Cas9 system has been widely adopted as a genome editing tool due to its high efficiency and versatility, contributing to the development of various therapeutic strategies. However, its clinical application remains limited by safety concerns, including off-target effects and large-scale chromosomal rearrangements such as translocations and inversions. Recently, the CRISPR-Cas3 system, a Class 1 CRISPR effector complex with unidirectional DNA degradation activity, has gained attention as a potential alternative, offering reduced off-target activity. In this study, we applied the CRISPR-Cas3 system to human T cells and successfully disrupted two clinically relevant genes, T cell receptor alpha constant (TRAC) and beta-2 microglobulin (B2M). These gene deletions were associated with a reduction in both graft-versus-host disease (GVHD) risk and host immune rejection. Importantly, no off-target mutations were detected in CRISPR-Cas3-edited cells, in contrast to the off-target effects observed with CRISPR-Cas9. Furthermore, CAR-T cells generated by deleting TRAC or B2M using CRISPR-Cas3 maintained their antigen-specific cytotoxicity against tumor cells, while exhibiting reduced alloreactivity. These results suggest that CRISPR-Cas3 provides a safer and promising platform for genome editing in T cell engineering, with potential applications in the development of next-generation allogeneic T cell therapies.

cancer biology↗

bioRxiv2025

Comparative Genomic Analysis of Cyanobacteria as Amphibian Food Sources: Insights into High-Temperature Tolerance Potential

Hot-springs represent one of the most accessible yet extreme environments on the Earths surface. We isolated and characterized two novel cyanobacterial strains of the genus Leptolyngbya, L. sp. Akita and L. sp. Seranma from geothermal hot-springs in Japan. These strains showed distinct morphological features; L. sp. Akita exhibited linear filamentous structures and blue-green color when cultured under white light, whereas L. sp. Seranma exhibited loosely coiled structures and a brown color under the same conditions. Whole-genome sequencing and comprehensive genomic analyses of their genomes identified the acquisition of gene clusters related to metabolic processes, which may be associated with strain-specific phenotypes. In addition, intestinal metagenomic analysis of tadpoles (Buergeria buergeri and B. japonica) sympatrically living with these strains in the hot-springs suggested that the tadpoles utilized these Leptolyngbya as temporal or regular foods in high-temperature environments. These findings provide insights into the ecological significance of these cyanobacteria in extreme environments and their potential applications in biotechnology and the ecological conservation of primary consumers, including amphibians.

microbiology↗

bioRxiv2025

Fetal reversion from diverse lineages sustains the intestinal stem cell pool and confers stress resilience

Plasticity is a central mechanism underlying the robust regenerative capacity of the intestinal epithelium. Two major plasticity have been described: spatial plasticity, in which differentiated cells revert to crypt base columnar cells (CBCs), and fetal reversion into revival stem cells (revSCs). However, the relationship among these two stem cell populations and differentiated cells remains to be clarified. Here, we demonstrated the bidirectional interconversion between CBCs and revSCs. Using lineage tracing, injury models and villus culture, we show that absorptive enterocytes can reprogram into revSCs and regenerate CBCs. These findings position fetal reversion as an entry point to spatial plasticity, establishing a regenerative hierarchy where CBCs, revSCs, and enterocytes collectively orchestrate intestinal repair. Furthermore, we identified revSCs as a highly stress-tolerant stem cell population, whose emergence is crucial for preserving the stem cell pool. Our results establish fetal reversion as a cellular escape mechanism safeguarding epithelial regeneration under inflammatory conditions.

cell biology↗

bioRxiv2025

Ultrafast endocytosis in mouse cortical inhibitory synapses

Neural circuitry depends on excitatory and inhibitory regulation of activity to yield functional outputs. However, examinations of synaptic vesicle recycling have focused heavily on excitatory synapses, leaving many questions unanswered for inhibitory synapse dynamics. Here we show that both excitatory and inhibitory cortical synapses contain depots of a protein essential for ultrafast endocytosis, Dynamin 1xA, at a region immediately next to the active zone where ultrafast endocytosis takes place. Using zap- and-freeze time-resolved electron microscopy in mouse acute cortical slices, we observe uncoated pits reminiscent of ultrafast endocytic intermediates appearing post-stimulus at putative inhibitory synapses. These findings suggest that excitatory and inhibitory synapses may perform similar modes of endocytosis.

neuroscience↗

bioRxiv2025

ARID1A regulates histone octamer transfer activity of human canonical BAF complex

Mutations that impact subunits of mammalian SWI/SNF (mSWI/SNF or BAF) chromatin remodeling complexes are found in over 20% of human cancers. Among these subunits, ARID1A is the most frequently mutated gene, occurring in over 8% of various cancers. The majority of ARID1A mutations are frameshift or nonsense mutations, causing loss of function. Previous studies have suggested that ARID1A may facilitate interactions between BAF complexes and various transcriptional coactivators, but a biochemical role for ARID1A in BAF remodeling activity has not been identified. Here, we describe the in vitro reconstitution of the cBAF, PBAF, and ncBAF complexes, and we compare their biochemical activities. In addition, we reconstitute a variety of cBAF subcomplexes, defining roles for several subunits in high affinity nucleosome binding and nucleosome sliding activity. Remarkably, we find that the ARID1A subunit of cBAF is largely dispensable for nucleosome binding, nucleosome sliding, and ATPase activity, but ARID1A is required for cBAF to transfer histone octamers between DNA templates. These data suggest a model in which the histone octamer transfer activity of BAF complexes is key for cancer prevention.

biochemistry↗

bioRxiv2025

A genus-wide interaction atlas across NS4B orthologues identifies a conserved role for UFMylation in orthoflavivirus replication

Orthoflavivirus infections represent an increasing public health burden, with several members of the genus emerging or re-emerging globally. Despite the availability of few vaccines, no antiviral drugs are currently licensed for the treatment of orthoflavivirus infections. Several pre-clinical studies identified the non-structural protein 4B (NS4B), one of the least characterized viral proteins within the orthoflavivirus genus, as the most promising target for the development of potent direct-acting antivirals. However, its functional roles in viral replication are still elusive. Here, we employ an integrated proteomic approach to systematically identify cellular targets of NS4B across eight prototypic orthoflaviviruses and characterize their influence on the human proteome. Using this approach, we mapped high-confidence NS4B-interacting human proteins across the genus, underlying potentially divergent mechanisms of host adaptation across orthoflaviviruses spanning diverse pathologies and vector preferences. Among these, we unveil a novel function for UBA5, the E1-activating enzyme of the UFMylation pathway, in orthoflavivirus replication. Mechanistically, we map associations of distinct viral proteins with multiple members of the UFMylation pathway, which are selectively recruited to sites of viral replication to promote mitochondrial respiration. Finally, we demonstrate that pharmacological inhibition of UFMylation exerts potent antiviral activity in vitro and in vivo. This integrative study provides a rational framework for a system-level understanding of orthoflavivirus NS4B effector functions and sheds light on a conserved and unconventional role for UFMylation in orthoflavivirus replication.

microbiology↗

bioRxiv2025

TBK1 eliminates aggregation-prone monomeric TDP-43 through the IFNβ-immunoproteasome pathway

Loss-of-function mutations in TANK-binding kinase 1 (TBK1) are genetically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), and induce cytoplasmic aggregation of TAR DNA-binding protein 43 (TDP-43), known as TDP-43 pathology. Although TBK1 deficiency is thought to contribute to TDP-43 pathology primarily through impaired autophagy, the full spectrum of its pathological impact remains unclear. Given the multifunctional nature of TBK1, alternative pathways beyond autophagy are possibly involved in TDP-43 pathology. Here, we found that TBK1 alleviates TDP-43 pathology in neuronal cells via induction of interferon-beta (IFN{beta}), and that the IFN{beta} receptor is downregulated in spinal motor neurons from ALS patients with TDP-43 pathology. We further demonstrated that IFN{beta} induces the immunoproteasome by upregulating its subunits, thereby promoting the degradation of aggregation-prone monomeric TDP-43. Furthermore, heterozygous deletion of Tbk1 in SOD1G93A ALS model mice resulted in reduced immunoproteasome induction and increased polyubiquitinated protein accumulation in the spinal cord. These findings suggest that impairment of the TBK1-IFN{beta}-immunoproteasome axis may contribute to the development of TDP-43 pathology in ALS and FTD.

neuroscience↗

bioRxiv2025

Proteomic profiling of tissue extracellular vesicles (EVs) identifies tissue-specific EV markers and predicts the accessibility of tissue EVs to the circulation

Extracellular vesicles (EVs) mediate cell-to-cell communication in endocrine, paracrine, and autocrine fashions, but their roles and transport in the body remain incompletely understood. It is expected that EVs circulate in the body through the bloodstream, where EVs derived from various tissues are present. However, how much each tissue contributes to circulating EVs is largely unknown. Therefore, identifying tissue-specific EV markers is of importance for elucidating in vivo dynamics of tissue EVs. A key issue that hampers studying EVs in vivo is the lack of methodologies for collecting them from tissues. Here, we developed methods to isolate EVs from four different tissues, skeletal muscle, heart, liver, and adipose tissue, and performed proteomic analysis on their EVs. Unbiased and quantitative proteomic analysis revealed protein signatures of EVs from the four tissues and identified marker proteins specific to EVs of each tissue. Furthermore, through comprehensive comparisons of the proteome from tissue and plasma EVs, we predicted the accessibility of EVs from the four tissues to the circulation. Our data suggests that, among the four tissues, adipose tissue-derived EVs more readily enter the circulation, while skeletal muscle-derived EVs exhibit poor accessibility to the blood. Collectively, our tissue EV proteome identified tissue-specific EV markers and suggests that the accessibility of tissue EVs to circulation highly depends on the original tissues.

physiology↗

bioRxiv2024

Ultrastructural membrane dynamics of mouse and human cortical synapses

Live human brain tissues provide unique opportunities for understanding synaptic transmission. Investigations have been limited to anatomy, electrophysiology, and protein localization--while crucial parameters such as synaptic vesicle dynamics were not visualized. Here we utilize zap-and-freeze time-resolved electron microscopy to overcome this hurdle. First, we validate the approach with acute mouse brain slices to demonstrate that slices can be stimulated to produce calcium signaling. Next, we show that synaptic vesicle endocytosis is induced in both mouse and human brain slices. Crucially, clathrin-free endocytic pits appear immediately next to the active zone, where ultrafast endocytosis normally occurs, and can be trapped at this location by a dynamin inhibitor. In both species a protein essential for ultrafast endocytosis, Dynamin 1xA, localizes to the region peripheral to the active zone, the putative endocytic zone, indicating a possible conserved mechanism between mouse and human. This approach has the potential to reveal dynamic, high-resolution information about synaptic membrane trafficking in intact human brain slices.

neuroscience↗

bioRxiv2024

Monitoring in vivo transcription with synthetic serum markers

Understanding transcription profiles of living tissues is critical for biology and medicine. However, measurement of the transcript levels is typically done in homogenized tissues post-mortem. Here, we present a new platform that enables non-invasive monitoring of specific mRNA levels in vivo, without tissue destruction. We achieved this by combining two cutting-edge tools - synthetic serum markers, called Released Markers of Activity (RMAs), and RNA-based sensors of transcription. We call this platform IN-vivo Tracking of ACtive Transcription, or INTACT. In INTACT, when the target mRNA is expressed, the RNA sensor detects it and triggers the production and release of RMA reporters into the blood. Once in blood, the RMAs can be easily measured through a simple blood draw. Our data shows that INTACT can measure transcription of transgenes, as well as endogenous transcripts, such as c-Fos or Arc, both in vivo in the brain and in tissue culture. INTACT enables simple measurement of transcript level histories in genetically-targetable cell populations of living animals.

bioengineering↗

bioRxiv2024

Structural basis for phosphatidylcholine synthesis by bacterial phospholipid N-methyltransferases

In phosphatidylcholine (PC)-containing bacteria, PC is synthesized by phospholipid N-methyltransferases (Pmts) and plays an important role in the interactions between symbiotic and pathogenic bacteria and their eukaryotic host cells. Pmts catalyze the SAM dependent three methylation reactions of the head group of phosphatidylethanolamine (PE) to form PC through monomethyl PE and dimethyl PE. However, the precise molecular mechanisms underlying PC biosynthesis by PmtA remain largely unclear, owing to the lack of structural information. Here, we determined the crystal structures of Agrobacterium tumefaciens Pmt (AtPmtA) in complex with SAH or 5'-methylthioadenosine. Crystal structures and NMR analysis revealed the binding mode of AtPmtA to SAH in solution. Structure-based mutational analyses showed that a conserved tyrosine residue in the substrate-binding groove is involved in methylation. Furthermore, we showed that differences in substrate specificity among Pmt homologs were determined by whether the amino acid residues comprising the substrate-binding groove were isoleucine or phenylalanine. These findings provide a structural basis for understanding the mechanisms underlying Pmts-mediated PC biosynthesis.

biochemistry↗

bioRxiv2024

Neutralizing antibody evasion of SARS-CoV-2 JN.1 derivatives KP.3, KP.3.1.1, LB.1, and XEC

The emergence of SARS-CoV-2 variants poses ongoing challenges to vaccine efficacy. We evaluated neutralizing antibody responses against JN.1 and its derivatives (KP.3, KP.3.1.1, LB.1, and XEC) in healthcare workers who received seven doses of BNT162b2, including XBB.1.5 monovalent vaccine. In COVID-19-naive individuals, KP.3.1.1 and LB.1 showed substantial immune escape, while previously infected individuals maintained neutralization activity against all variants. We also demonstrated that JN.1-based immunization induces robust cross-neutralizing activity against emerging variants. A single amino acid deletion at position 31 in the spike protein significantly impacted immune evasion. These findings support the potential effectiveness of JN.1-based vaccines while highlighting the need for continued surveillance and vaccine optimization.

immunology↗

bioRxiv2024

Cardiolipin deficiency disrupts CoQ redox state and induces steatohepatitis

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a progressive disorder marked by lipid accumulation, leading to metabolic dysfunction-associated steatohepatitis (MASH). A key feature of the transition to MASH involves oxidative stress resulting from defects in mitochondrial oxidative phosphorylation (OXPHOS). Here, we show that pathological alterations in the lipid composition of the inner mitochondrial membrane (IMM) directly instigate electron transfer inefficiency to promote oxidative stress. Specifically, mitochondrial cardiolipin (CL) was downregulated with MASLD/MASH in humans and in mice. Hepatocyte-specific CL synthase knockout (CLS-LKO) led to spontaneous and robust MASH with extensive steatotic and fibrotic phenotype. Loss of CL paradoxically increased mitochondrial respiratory capacity but also promoted electron leak primarily at sites IIIQO and IIF of the electron transport chain, reduced the formation of I+III2+IV respiratory supercomplex, and disrupted the propensity of coenzyme Q (CoQ) to become reduced. Thus, low mitochondrial CL disrupts electron transport chain to promote oxidative stress and contributes to pathogenesis of MASH.

biochemistry↗

bioRxiv2024

Muscle-specific Keap1 deletion enhances force production but does not prevent inactivity-induced muscle atrophy in mice

Immobilization-associated muscle atrophy and weakness appear to be driven in part by oxidative stress. Nuclear Factor Erythroid 2-Related Factor 2 (NRF2) is a critical redox rheostat that regulates oxidative stress responses, and its deletion is known to accelerate muscle atrophy and weakness during aging (sarcopenia) or denervation. Conversely, pharmacologic activation of NRF2 extends mouse lifespan and attenuates sarcopenia. Similarly, deletion of Kelch-like ECH-associated Protein 1 (Keap1), negative regulator of NRF2, enhances exercise capacity. The purpose of this study was to determine whether muscle-specific Keap1 deletion is sufficient to prevent muscle atrophy and weakness in mice following 7-days of hindlimb unloading (HU). To test this hypothesis, control (Ctrl) and tamoxifen inducible, muscle-specific Keap1 knockout (mKO) mice were subjected to either normal housing (Sham) or HU for 7 days. Activation of NRF2 in muscle was confirmed by increased mRNA of NRF2 targets thioredoxin 1 (Txn1) and NAD(P)H quinone dehydrogenase 1 (NQO1) in mKO mice. Keap1 deletion had an effect to increase force-generating capacity at baseline. However, muscle masses, cross sectional area, and ex vivo force were not different between mKO and Ctrl HU mice. In addition, muscle 4-hydroxynonenal-modified proteins and protein carbonyls were unaffected by Keap1 deletion. These data suggest NRF2 activation improves muscle force production during ambulatory conditions but is not sufficient prevent muscle atrophy or weakness following 7-days of HU. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/616570v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@433d1eorg.highwire.dtl.DTLVardef@61996dorg.highwire.dtl.DTLVardef@19fe734org.highwire.dtl.DTLVardef@104205f_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2024

Epigenetic control of tetrapyrrole biosynthesis by m4C DNA methylation in a cyanobacterium

Epigenetic DNA modifications are pivotal in eukaryotic gene expression, but their regulatory significance in bacteria is less understood. In Synechocystis 6803, the DNA methyltransferase M.Ssp6803II modifies the first cytosine in the GGCC motif, forming N4-methylcytosine (GGm4CC). Deleting the sll0729 gene ({Delta}sll0729) caused a bluish phenotype due to reduced chlorophyll levels, which was reversed by suppressor mutations. Re-sequencing of seven suppressor clones revealed a common GGCC to GGTC mutation in the slr1790 promoters discriminator sequence, encoding protoporphyrinogen IX oxidase, HemJ, crucial for tetrapyrrole biosynthesis. Transcriptomic and qPCR analyses indicated aberrant slr1790 expression in {Delta}sll0729 mutants. This aberration led to the accumulation of coproporphyrin III and protoporphyrin IX, indicative of impaired HemJ activity. To confirm the importance of DNA methylation in hemJ expression, native and mutated hemJ promoter variants were introduced into the wild type, followed by sll0729 deletion. The sll0729 deletion segregated in strains with the GGTC motif in the hemJ promoter, resulting in wild-type-like pigmentation, whereas freshly prepared {Delta}sll0729 mutants with the native hemJ promoter exhibited the bluish phenotype. These findings demonstrate that hemJ is tightly regulated in Synechocystis and that N4-methylcytosine is essential for proper hemJ expression.

microbiology↗

bioRxiv2024

Ultrastructure of immature synaptic inputs in the lateral superior olive of the rodent brainstem

Neurons of the lateral superior olive (LSO), which compute intensity differences between the two ears, receive two primary inputs, an ipsilaterally arising excitatory input and a contralaterally arising inhibitory input, that are precisely matched for stimulus frequency. Circuit refinement to establish this precise match takes place within the first few postnatal weeks through elimination of single-fiber inputs and concomitant strengthening of the remaining inputs. However, little is known about the ultrastructure of these young synapses and about how changes in physical features of these synapses could contribute to refinement. To characterize pre-hearing postnatal development of somatic synapses in the LSO, we performed transmission electron microscopy and examined synapses in the rodent LSO from birth to hearing onset at postnatal day 13. Synaptic vesicles and mitochondria in putative synaptic boutons were surprisingly scarce at birth. During the second week, bouton enlargement was accompanied by an increase in the number of vesicles and mitochondria. The size of mitochondria also increased, pointing to changes in functional and metabolic needs of synapses. Our results reveal extensive remodeling at individual presynaptic terminals that could strengthen single-fiber inputs and contribute to the development of robust synaptic transmission.

neuroscience↗

bioRxiv2024

Rapid neutralizing assay for circulating H5N1 influenza virus in dairy cows

A rapid and safe neutralization assay is required for emerging highly pathogenic avian influenza viruses, including the H5N1 subtype, which was recently found in cows. Herein, we report a novel neutralization assay using HiBiT-tagged virus-like particles (hiVLPs). Our hiVLP-based neutralization test demonstrated a higher quantitative value and shorter assay time than conventional methods. We used this assay to evaluate whether the neutralizing antibodies induced by the candidate vaccine virus (NIID-002) were cross-reactive with cow-derived H5N1. Our results suggest that the circulating H5N1 virus in cows shares antigenic characteristics with NIID-002, providing significant implications for the development and preparation of vaccines.

microbiology↗