Biology subjects

Suzuki, T.

Publications and source records attributed to Suzuki, T..

At least 73 records · Page 4Linked to original sources

bioRxiv2025

Changes over a 10-year Period in the Distribution Ranges and Genetic Hybridization of Three Pelophylax Pond Frogs in Central Japan

In this study, we focused on interspecial interactions in Central Japan where three closely related pond frog species, Pelophylax nigromaculatus, Pelophylax porosus porosus and Pelophylax porosus brevipodus (two species and two subspecies) are distributed and are in contact and/or overlap. The presence or absence of hybridization between these species was evaluated by comparing the sequences of the mitochondrial DNA Cyt-b (586-bp) and the nuclear gene Tyrosinase (747-bp) regions of each specimen collected. By comparing interspecific hybridization in the same area in both 2010 (119 specimens) and 2020 (152 specimens), we clarified population distributions and corresponding genetic dynamics over a 10-year period; there was no major change in the degree of interspecific hybridization between P. nigromaculatus and P. p. brevipodus in the Ina Basin, but it was revealed that the proportion of "pure"-bred Pelophylax p. brevipodus remained low. On the other hand, in the Matsumoto Basin, interspecific hybridization between P. nigromaculatus and P. p. porosus was evaluated to have progressed slightly. The proportion of "pure"-bred Pelophylax p. porosus declined to less than half, while the proportion of hybrid individuals showed a tendency to increase. Moreover, the dispersal of P. nigromaculatus was confirmed in areas along the Sai River, where only the pure-bred P. p. porosus was previously known to inhabit. Since the three frogs targeted in this study are all "Red List" species that are endangered, continuous monitoring of population structures and genetic dynamics is required.

ecology↗

bioRxiv2025

Macromolecular Crowding-Affected Mobility of Bcnt/Cfdp1, a Disordered Component of the Srcap Complex, from the Nucleus to Cytosol in Subcellular Fractionation

Metazoan Bucentaur/Craniofacial Development Protein 1 (Bcnt/Cfdp1) is crucial for embryonic development, and its yeast homolog protein, SWC5, functions as part of the SWR1 complex in histone exchange reactions. Bcnt/Cfdp1, an intrinsically disordered protein (IDP) subject to various post-translational modifications, contains a putative nuclear localization sequence. Nevertheless, its subcellular localization remains ambiguous. We investigated its intracellular trafficking using diverse approaches. In C2C12 myoblasts, fluorescent protein-tagged Bcnt/Cfdp1 was predominantly localized within the nucleus, preferentially in regions of low DAPI density in myoblasts, and the localization persisted in differentiated multinucleated myotubes. Conversely, detergent-based biochemical fractionation of C2C12 and HEK293T cells indicated a substantial presence of Bcnt/Cfdp1 in cytoplasmic fractions. Quantitative proteomic analysis of fractionated proteins revealed that six IDPs, including a PEST-containing nuclear protein, demonstrated similar distribution patterns to Bcnt/Cfdp1; despite being annotated as nuclear proteins, they were detected in cytoplasmic fractions. Direct treatment of both cell lines with digitonin enabled the collection of released proteins in three steps to accurately isolate cytoplasmic proteins. Bcnt/Cfdp1 exhibited distinct extraction patterns compared to cytoplasmic soluble protein extracts in Ficoll/sucrose solution, while no differences were noted when cells underwent digitonin treatment in an isotonic solution. Quantitative proteomic analysis of these extracts highlighted the mobility or integrity of each protein within the cytoplasm. Consequently, although Bcnt/Cfdp1 is essential within the nucleus, our findings suggest its additional presence in the cytoplasm. We hypothesize that the elastic properties of Bcnt/Cfdp1 facilitate its translocation from the nucleus during subcellular fractionation, which is affected by macromolecular crowding.

biochemistry↗

bioRxiv2025

Structural dynamics of mixed-subunit CaMKIIα/β heterododecamers filmed by high-speed AFM

CaMKII predominantly assembles into a 12-meric ring assembly, primarily consisting of CaMKII and CaMKII{beta} variants in the brain. Previous biochemical studies have reported varying ratios of these CaMKII variants across different brain regions and developmental stages. However, direct evidence for the formation of CaMKII/{beta} heterooligomers within a 12-meric ring assembly has been lacking at the single-molecule level. Here, we employed high-speed atomic force microscopy to visualize the conformational dynamics of forebrain-mimicked CaMKII/{beta} at a 3:1 ratio. Our findings revealed that the CaMKII and CaMKII{beta} subunits are intermixed within the 12-meric ring assembly, with more than 83% probability that CaMKII{beta} subunits adjacent to one another. Furthermore, in the activated state, CaMKII/{beta} heterooligomers form a stable kinase domain complex via interactions between adjacent CaMKII{beta} subunits, resulting in a long-lasting structure with an exposed target binding site. Collectively, our observations provide insights into the structural role of CaMKII{beta} subunits within the CaMKII/{beta} heterododecamer.

neuroscience↗

bioRxiv2025

Non-DNA-damaging DNA-PK activation improving hearing and prolonging life due to NAD+ and SIRT upregulation

Emerging evidence strongly supports a close relationship between age-related hearing loss and frailty, highlighting the importance of early detection and intervention. Recently, we invented a mitochondria-homing drug named mitochonic acid 5 (MA-5), that increases the adenosine triphosphate (ATP) levels, rescue mitochondrial function, and protect tissue damages. Currently, the phase I clinical trial has been finished in Japan (jRCT2031210495) and the phase 2 clinical trial has already been approved by PMDA. Here we show that MA-5 improved various types of hearing loss in mouse models. Structural chemical bioanalysis revealed that MA-5 is a mixture of equal amount of S- and R- enantiomer and both S- and R- enantiomer increase ATP by binding mitochondrial protein, mitofilin. However, S-enantiomer significantly increased the NAD+ levels by binding to the NAD+-producing key enzyme nicotinamide phosphoribosyltransferase (NAMPT). Moreover, the S-enantiomer increased the sirtuin 1 protein by suppressing polyubiquitination induced by tripartite motif containing 28 (TRIM28) phosphorylation which was triggered by DNA-dependent protein kinase (DNA-PK) activation in the absence of DNA damage. Transcriptomic signatures showed that the signature of MA-5 shows an inverse correlation with aging and mortality and is oriented in the same direction as the OSKM-related iPSCs, suggesting the modification of aging pathways. Oral administration of MA-5 to mitochondrial disease model mouse showed increased survival. Our findings suggest that, in addition to enhancing ATP levels, the coordinated regulation of NAD+ metabolism, SIRT protein expression, and DNA-PK activity-constituting a novel therapeutic triad may contribute to the amelioration of hearing impairment and mitochondrial dysfunction, thereby improving life prognosis.

cell biology↗

bioRxiv2025

Survey of hippocampal responses to sound in naive mice reveals widespread activation by broadband noise onsets

Recent studies suggest some hippocampal (HC) neurons respond to passively presented sounds in naive subjects, but the specificity and prevalence of these responses remain unclear. We used Neuropixels probes to record unit activity in HC and auditory cortex (ACtx) of awake, untrained mice during presentation of diverse sound stimuli. A subset of HC neurons exhibited reliable, short-latency responses to passive sounds, including tones and broadband noise. HC units showed evidence of tuning for tone frequency but not spectrotemporal features in continuous dynamic moving ripples. Across sound types, HC responses overwhelmingly occurred at stimulus onset; they quickly adapted to continuous sounds and did not respond at sound offset. Among all sounds tested, broadband noise was by far most effective at driving HC activity, with response prevalence scaling with increasing spectral bandwidth and density. Responses to noise were also far more common than visual flash stimuli. Sound-evoked face movements, quantified by total facial motion energy (FME), correlated with population-level HC activity, but many individual units responded regardless of movement, indicating both auditory and motor-related inputs. These results show that abrupt, acoustic events are sufficient to activate HC neurons in the absence of learning or behavioral engagement. This suggests a possible role for HC in detecting salient environmental changes and supports the idea that auditory inputs contribute directly to HC function. Given emerging links between hearing loss and dementia, these findings highlight a potential pathway by which auditory deafferentation could impact cognitive health.

neuroscience↗

bioRxiv2025

Immune phenotype-guided identification of disease-associated pathobionts in Crohn's disease

Aberrant immune activation within the gut mucosa and gut dysbiosis have been implicated in the pathogenesis of Crohns disease (CD). However, the specific immune responses triggered by dysbiotic microbiota, as well as the bacteria responsible for this activation, remain incompletely understood. Here, using the human microbiota-associated (HMA) mouse system, we demonstrated that colonization with dysbiotic gut microbiota from CD patients specifically induces the accumulation of mononuclear phagocytes, which may drive an interleukin-1 (IL-1)-driven inflammatory signature. Moreover, we identified pathobiont strains with a potent IL-1{beta}-inducing capacity, termed IL-1{beta}-inducing pathobionts (IBIP). Isolated IBIP strains exhibit genetic and functional similarities to adherent-invasive Escherichia coli but harbor unique virulence-associated genes. Colonization with the IBIP E. coli strain exacerbated experimental colitis in an IL-1 signal-dependent manner. Notably, the colonization of IBIP E. coli can be detected by measuring the levels of specific immunoglobulin A (IgA) in their stool samples. Moreover, the level of IBIP-reactive IgA in stool may serve as a predictive biomarker for treatment response to anti-TNF therapies in treatment-naive pediatric CD patients. Altogether, IBIP colonization could help identify CD patients with inflammatory dysbiosis who are likely to be refractory to anti-TNF therapies.

immunology↗

bioRxiv2025

A transfer RNA methyltransferase with an unusual domain composition catalyzes 2'-O-methylation at position 6 in tRNA.

Thermococcus kodakarensis tRNATrp contains 2'-O-methylcytidine at position 6 (Cm6). However, the tRNA methyltransferase responsible for the modification has not been identified. Using comparative genomics we predicted TK1257 as a candidate for the modification. Biochemical and mass spectrometry studies of purified recombinant TK1257 gene product show it to possess a tRNA methyltransferase activity for Cm6 formation. This protein has a highly unusual composition of domains, containing N-terminal ferredoxin-like, SPOUT catalytic and THUMP domains. Previous to this study, all known THUMP-related tRNA methyltransferases were shown to contain a Rossmann fold catalytic domain and the nucleosides they produced were N2-methylguanosine and/or N2, N2-dimethylguanosine. Therefore, our findings extend the knowledge of architecture of tRNA methyltransferases. We named the TK1257 gene product TrmTS and showed it can synthesize Am6 and Um6 as well as Cm6. A trmTS gene deletion strain showed slight growth retardation at high temperatures. Site-directed mutagenesis studies based on structural model revealed catalytically and structurally important amino acid residues in TrmTS and identified a TrmTS-specific linker is structurally essential. We showed that TrmTS recognizes the 3'-CCA terminal region and a stretch loop connected with at least two stems in RNA. Finally, we constructed a model of the binding between TrmTS and tRNA.

biochemistry↗

bioRxiv2025

Intermediate Evolutionary State of Motile Sperm and Pollen Tubes in the Extant Gymnosperm Cycas revoluta

The transformation of male reproductive cells is a significant event in the evolution of land plant fertilization systems from zooidogamy to siphonogamy. Basal plants such as bryophytes and pteridophytes have motile sperm, whereas most seed plants have non-motile sperm, delivered by a pollen tube. Despite being seed plants, gymnosperm cycads and ginkgo uniquely form highly multi-flagellated and large motile sperm within pollen tubes. However, the evolutionary state of these male reproductive cells remains unknown. We clarified the gene expression profiles of Cycas revoluta pollen tubes and motile sperm that swam toward female reproductive cells. Male cycad cells had fewer genes associated with transcription, translation, and related processes, which is consistent across land plants. Similar to other male gametes, a sperm-specific histone variant may contribute to transcriptional regulation via chromatin condensation. Plasma membrane-localized proteins probably involving male-female interactions revealed that cycad sperm express both factors found in motile plant sperm and seed plant sperm cells, suggesting a transitional state from motile to non-motile male gametes. In contrast, cycad pollen tubes lacked plasma membrane-localized receptor genes previously reported in angiosperms and cell wall-associated factors involved in pollen tube tip growth. These results support the hypothesis that the function of the cycad pollen tube is confined to the haustoria as previously reported. These results indicate a molecular intermediate state of the cycad fertilization mechanism with motile sperm and pollen tubes, providing important insights into the evolution of land plant fertilization. Significant StatementCycads and ginkgo, which possess primitive motile sperm and derived pollen tubes, are important for understanding the evolution of land plant fertilization from zooidogamy to siphonogamy. We explored gene expression in living, capacitated motile sperm and pollen tubes of Cycas revoluta in a tissue-specific transcriptome analysis. Similar to other land plants, the transcriptional repression mechanism has been suggested in these male cells. Plasma membrane-localized genes revealed the characteristics of motile and non-motile sperm, while molecular evidence of pollen tube guidance and tip growth, as seen in angiosperms, was lacking in cycads. A molecular intermediate state of the cycad fertilization mechanism was clarified, providing important insights into the evolutionary process of land plant fertilization.

plant biology↗

bioRxiv2025

Distribution of myogenic stem cell activator, hepatocyte growth factor, in skeletal muscle extracellular matrix and effect of short-term disuse and reloading

Hepatocyte growth factor (HGF) is a key myogenic stem cells (satellite cells) activator, that resides in the extracellular matrix (ECM). However, HGF distribution in the ECM varies depending on the muscle fiber type. Furthermore, aging impedes the binding of HGF to its receptors due to nitration by peroxynitrite (ONOO-). Though oxidative stress increases rapidly during muscle disuse atrophy, satellite cells are rapidly activated upon reloading. In this study, we investigated the distribution of HGF in the ECM in various muscle fiber types, and examined nitration of HGF in disuse and reloading models. Immunofluorescence staining was performed on the soleus (Sol), plantaris (Pla), and gastrocnemius (Gas) muscles of 10-week-old mice. Three mice were used to assess HGF distribution, while 12 mice, divided into control, disuse, and reloading groups were used for qualitative evaluation of nitrated HGF (nitroHGF). In Sol muscle, type IIa and IIx muscle fibers exhibited higher HGF distribution in the ECM (70.7{+/-}1.9% and 63.9{+/-}2.6%, respectively) than type I fibers (28.0{+/-}1.7%; p<0.001). In Pla and Gas muscle, type IIa (65.4{+/-}2.3% and 70.3{+/-}2.2%, respectively) and type IIx fibers (55.3{+/-}1.8% and 61.4{+/-}3.2%, respectively) had significantly higher HGF distribution in the ECM than type IIb fibers (13.1{+/-}1.8% and 5.9{+/-}1.6%; p<0.001, respectively). The amount of nitroHGF increased in the disuse group compared to that in the control group but decreased in the reloading group compared to that in the disuse group. This preferential HGF distribution around type IIa and IIx muscle fibers indicates a distinct mechanism for satellite cell activation, differing from the satellite cell-rich environment associated with type I fibers and the lower HGF association with type IIb fibers. Disuse-induced HGF nitration may inhibit satellite cell activation. Reloading likely triggers mechanisms that counteract nitration, enabling satellite cell reactivation in young muscle.

biochemistry↗

bioRxiv2025

Systematic Discovery and Design of Synthetic Immune Receptors in Plants

Plants deploy a diverse array of pattern recognition receptors (PRRs) that perceive microbe-associated molecular patterns (MAMPs) to activate immune responses. Leucine-rich repeat receptor-like kinase subgroup XII (LRR-RLK-XII) represents one of the largest PRR families due to lineage-specific diversification. Bioinformatics, synthetic biology, and biochemical approaches were integrated to characterize PRRs from 285 plant species. We identified a receptor, named "SCORE", that perceives cold-shock protein (CSP) peptides. SCORE orthologs from multiple angiosperm lineages exhibit remarkable CSP recognition polymorphisms, indicating recurrent selection for pathogen recognition through substitutions at key amino acid residues. Through functional phylogenomics and protein structure predictions, we generated synthetic SCORE variants capable of detecting multiple phytopathogen CSP peptides, thus revealing the diverse PRR recognition landscape in plants. Our strategy hold promises for engineering plant immune receptors, particularly for perennial crops.

plant biology↗

bioRxiv2025

Longitudinal analysis of antibody titers after primary and booster mRNA COVID-19 vaccination can identify individuals at risk for breakthrough infection

A key issue in the post-COVID-19 era is the ongoing administration of COVID-19 vaccines. Repeated vaccination is essential for preparing against currently circulating and newly emerging SARS-CoV-2 variants while enabling people to continue with daily life. Optimizing vaccination strategies is crucial to efficiently manage medical resources and establish an effective vaccination framework. Therefore, it is important to quantitatively understand vaccine-induced immunity dynamics and to be able to identify poor responders with lower sustained antibody titers as potential priorities for revaccination. We investigated longitudinal antibody titer data in a cohort of 2,526 people in Fukushima, Japan, from April 2021 to November 2022 for whom basic demographic and health information was available. Using mathematical modeling and machine learning, we stratified the time-course patterns of antibody titers after 2 primary doses and 1 booster dose of mRNA COVID-19 vaccines. We identified 3 notable populations, which we refer to as the durable, the vulnerable, and the rapid-decliner populations, approximately half of which remained in the same population after the booster dose. Notably, the rapid-decliner population experienced earlier infections than the others. Furthermore, when comparing IgG(S) titers, IgA(S) titers, and T-spot counts between participants who experienced breakthrough infections after booster vaccination and those who did not, we found that IgA(S) titers were significantly lower in breakthrough infected participants during the early stage after booster vaccination. Our computational approach is adaptable to various types of vaccinations. This flexibility can inform policy decisions on vaccine distribution to enhance immunity both in future pandemics and in the post-COVID-19 era.

immunology↗

bioRxiv2025

Arabidopsis TITAN LIKE is required for U12-type intron splicing, especially for AT-AC subtypes

Many eukaryotes possess two types of spliceosomes: the U2-dependent and U12-dependent spliceosomes. The U2-dependent spliceosome processes more than 99% of all introns, whereas the U12-dependent spliceosome acts on only about 0.3% of introns, one-third of which start with AT and end with AC, with the remainder having GT-AG termini. How the U12-dependent spliceosome splices two types of introns with different terminal sequences remains poorly understood. Human CENATAC is a subunit of the U12-dependent spliceosome that is particularly required for the splicing of the AT-AC subtype. The Arabidopsis genome contains a single homolog, TITAN LIKE (TTL), but its function in splicing remains unknown. Here, we generated TTL mutants and isolated two viable alleles, of which we analyzed one, designated ttl-142, to investigate TTL function in splicing. ttl-142 carries a 42-nucleotide deletion that removes 14 amino acid residues from the predicted protein, and homozygous mutants exhibit morphological abnormalities. Most U12-dependent introns were less efficiently spliced in ttl-142 than in the wild-type, with the splicing of AT-AC introns particularly suppressed. Splicing suppression in ttl-142 was more extensive than that in a DROL1 mutant, which carries a mutation in a gene specifically required for AT-AC intron splicing. Conversely, fewer genes showed altered expression levels in ttl-142 than in drol1, and most differentially expressed genes differed between the two mutants. These results suggest that the phenotypes of ttl-142 and drol1 mutants may reflect the impairment of distinct spliceosomal functions.

plant biology↗

bioRxiv2025

Cell-penetrating peptide-mediated mouse oocyte activation

IntroductionThe cell cycle of ovulated oocytes from various animal species, including mice, is arrested in the second meiotic metaphase until fertilization. The meiotic cell cycle must be initiated to initiate embryonic development. Besides natural fertilization, several other methods have been developed to activate unfertilized oocytes without using sperm. These methods are not only utilized for animal production but have also proven effective in studying the molecular mechanisms that regulate the meiotic cell cycle, oocyte activation, and embryonic development. This study aimed to develop a method to activate mouse oocytes using a cell-penetrating peptide based on the knowledge that the C-terminal domain of the meiotic protein Emi2 can resume the arrested meiotic cell cycle. MethodsThis study used female B6D2F1 mice to investigate the effects of a CPP-fused Emi2 peptide on oocyte activation. Second meiotic metaphase oocytes were collected, cultured, and treated with the peptide or strontium chloride. Pronuclear formation, second polar body extrusion, and blastocyst development were assessed, and statistical significance was determined using Fishers exact test. ResultsThe cell-penetrating peptide activated zona-intact oocytes in a manner dependent on specific amino acid residues and peptide concentrations, which are critical components for cell membrane penetration. It has also been confirmed that oocytes activated using this method can develop to the blastocyst stage. DiscussionThe introduction of peptides or functional amino acid sequences using CPP or related methods could be an alternative for easily performing functional analyses of the activity of target proteins in oocytes.

developmental biology↗

bioRxiv2025

Salivary proteins in spider mites: dual roles in feeding and silk fiber coating

Spider mites are plant cell-sucking herbivores that spin nanoscale adhesive silk fibers released from their mouthpart appendages. Previous whole-genome sequencing predicted 17 silk genes in the two-spotted spider mite, Tetranychus urticae, while silk proteomics detected two candidates, Fibroin-1 and Fibroin-2. However, saliva proteomics also detected Fibroin-1, Fibroin-2, and sFibroin-1 (with high sequence similarity to Fibroin-1). We investigated whether these three proteins function in silk, saliva, or both. We found Fibroin-1, sFibroin-1, and Fibroin-2 genes expressed in the salivary glands but found no direct internal connection between salivary and silk glands. Our silk proteomics detected 8 silk proteins previously predicted and 66 salivary proteins, including Fibroin-1, sFibroin-1, and Fibroin-2. We detected a bead-on-a-string pattern formed by fluid (hypothesized to be saliva) on the silk fiber and a fluid patch that remains at the piercing site on the leaf surface after mite feeding. RNAi-mediated silencing of Fibroin-1 and sFibroin-1 genes reduced mite feeding duration, survival, and fecundity, and silencing of Fibroin-2 gene showed a tendency to reduce the thickness of individual silk fibers. These results suggest that Fibroin-1, sFibroin-1, and Fibroin-2 are secreted from the salivary glands and then adhere to silk fibers. Fibroin-1 and sFibroin-1 may assist mites to adhere the tip of the mouthparts to the leaf surface when sucking with their stylets, while Fibroin-2 may coat silk fibers. This study reveals the dual role of saliva in plant cell-sucking herbivorous mites, namely, stabilizing the mouthparts during feeding and coating silk fibers to provide them with adhesive properties. HighlightsThree salivary proteins of spider mites are abundantly detected in their silk fibers. Though the salivary glands and silk glands are not connected, saliva appears to adhere to silk fibers after secretion. Knockdown of two of the three salivary protein genes suppressed the feeding behavior of spider mites, resulting in reduced survival and fecundity. Knockdown of the remaining salivary protein gene showed a tendency to reduce the thickness of the silk filament.

molecular biology↗

bioRxiv2025

Arabidopsis root-type ferredoxin:NADP(H) oxidoreductases are crucial for root growth and ferredoxin-dependent processes

Root-type ferredoxin:NADP(H) oxidoreductase (RFNR) is believed to reduce ferredoxin using NADPH in nonphotosynthetic tissues, facilitating ferredoxin-dependent biological processes. However, the physiological functions of RFNR remain unclear due to the difficulty in obtaining mutants lacking redundant RFNR isoproteins. The present study successfully generated Arabidopsis homozygous rnfr1;2 double mutants by traditional crossing and selection. However, they displayed severely stunted roots, challenging subsequent growth and abundant seed recovery. Notably, grafted plants combining mutant scions with wild-type rootstocks exhibited normal growth and produced abundant mutant seeds. Growth analysis employing reciprocal grafts with the wild-type and mutant plants showed that primary root growth was inhibited only when the rootstock was derived from the mutants. Meanwhile, the absence of RFNR1 and 2 in the scion had no apparent impact on shoot and root growth. Root transcriptome analysis revealed that RFNR1 and 2 deficiency upregulated genes encoding ferredoxin-dependent enzymes and root-type ferredoxin, leading to genome-wide reprogramming associated with cell walls, lipids, photosynthesis, secondary metabolism, and biotic/abiotic stress responses. Thus, Arabidopsis RFNR1 and 2 are crucial for root growth and various ferredoxin-dependent biological processes.

plant biology↗

bioRxiv2025

A trehalase-derived MAMP triggers LecRK-V-mediated immune responses in Arabidopsis

Plant-parasitic nematodes (PPNs) cause major agricultural losses worldwide, yet the molecular basis of plant immunity against these pathogens remains poorly understood. To investigate how plants recognize PPNs, we aimed to identify microbe-associated molecular patterns (MAMPs) from nematodes and the corresponding plant immune components. Due to the limited availability of material from obligate PPNs, we used Caenorhabditis elegans, a free-living nematode, as a MAMP source. C. elegans extracts activated MAMP-triggered immune responses in Arabidopsis Col-0. Through chromatography-based purification, we identified a secreted trehalase and pinpointed a conserved peptide region essential for its MAMP activity. A corresponding peptide from root-knot nematode trehalase enabled the identification of lectin receptor kinases LecRK-V.5 and LecRK-V.6 as key components in immune induction. Notably, this peptide region is conserved across insect and fungal pathogens, with LecRK-Vs required for immune responses to these peptides, highlighting the role of LecRK-V-mediated mechanism for broad-spectrum pathogen detection via trehalase-derived peptides. TeaserTrehalase-derived peptides trigger LecRK-V-mediated immune responses in plants, enabling broad detection of nematodes, insects, and fungi.

plant biology↗

bioRxiv2025

Risk Prediction of RNA Off-Targets of CRISPR Base Editors in Tissue-Specific Transcriptomes Using Language Models

Base-editing technologies, particularly cytosine base editors (CBEs), allow precise gene modification without introducing double-strand breaks; however, unintended RNA off-target effects remain a critical concern and are under-studied. To address this gap, we developed PiCTURE, a standardized computational pipeline for detecting and quantifying transcriptome-wide CBE-induced RNA off-target events. PiCTURE identifies both canonical ACW (W = A or T/U) motif-dependent and non-canonical RNA off-targets, revealing a broader WCW motif that underlies many unanticipated edits. Additionally, we developed two machine learning models based on the DNABERT-2 language model, termed STL and SNL, which outperformed motif-only approaches in terms of accuracy, precision, recall, and F1 score. To demonstrate the practical application of our predictive model for CBE-induced RNA off-target risk, we integrated PiCTURE outputs with the PROTECTiO pipeline and estimated RNA off-target risk for each transcript showing tissue-specific expression. The analysis revealed differences among tissues: while the brain and ovaries exhibited relatively low off-target burden, the colon and lungs displayed relatively high risks. Our study provides a comprehensive frame-work for RNA off-target profiling, emphasizing the importance of advanced machine learning-based classifiers in CBE safety evaluations and offering valuable insights for the development of safer genome-editing therapies.

genomics↗

bioRxiv2025

Cleavage cascade of the sigma regulator FecR orchestrates TonB-dependent signal transduction

TonB-dependent signal transduction is a versatile mechanism observed in gram-negative bacteria, integrating energy-dependent substrate transport with signal relay. In Escherichia coli, the TonB-ExbBD motor complex energizes the TonB-dependent transporter FecA, facilitating ferric citrate import. FecA also functions as a sensor, transmitting signals to the cytoplasmic membrane protein FecR. We previously demonstrated that FecR undergoes a three-step cleavage process, culminating in the activation of the cytoplasmic sigma factor FecI, which drives fec gene transcription. Here, we describe the complete mechanism of FecR cleavage-mediated ferric citrate signaling involving FecA and TonB. The cleavage cascade begins with FecR autoproteolysis prior to membrane integration. The soluble C-terminal domain (CTD) fragment of FecR is co-translocated with the N-terminal domain (NTD) fragment through a Tat system-mediated process. In the periplasm, the interaction between the CTD and NTD fragments prevents further cleavage. This inhibition is lifted by TonB-mediated motor function, which releases the CTD, allowing the cleavage cascade to proceed. This process is essential for ferric citrate signal-induced activation of fec gene expression. Our findings reveal that the regulation of FecR cleavage, relying on the TonB-FecA axis, plays a central role in bacterial response to ferric citrate signals. Significance StatementScarcity of iron, an essential element for life, has driven bacteria to evolve intricate acquisition systems, yet the molecular basis of their signal transduction mechanisms remains elusive. Unlike conventional pathways, iron transport systems employ outer membrane receptors that mediate signal transduction across both the outer and cytoplasmic membranes, powered by TonB. Using the Escherichia coli Fec system, we uncovered the mechanism by which FecRs cleavage cascade orchestrates ferric citrate signaling through TonB-mediated energy transfer. These findings shed light on similar mechanisms in gram-negative bacteria and have significant implications for understanding bacterial adaptation and pathogenesis.

immunology↗