Biology subjects

Uchida, K.

Publications and source records attributed to Uchida, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Genetic legacy in soil seedbanks after grassland conversion to plantation forests: evidence from Potentilla freyniana

Semi-natural grasslands are important ecosystems supporting biodiversity in Japan, but their area has declined rapidly due to land-use change and abandonment of traditional management practices such as mowing and burning. Although the conservation of genetic diversity is essential for the long-term persistence of grassland plants, little is known about the genetic diversity retained in soil seedbanks following conversion of grasslands to plantation forests. In this study, we compared the genetic diversity and population structure of above-ground and soil seedbank populations of the grassland perennial forb Potentilla freyniana across three sites in each of three land-use types: burned grasslands, deciduous plantation forests, and evergreen plantation forests (plantation ages approximately 21-62 years) on the Kaida Plateau, central Japan. Soil seedbank populations were obtained from soil samples through germination experiments, and genetic analyses were conducted using newly developed simple sequence repeat (SSR) markers. Genetic diversity was assessed using expected heterozygosity, allelic richness, and private allelic richness, population structure was evaluated using analysis of molecular variance (AMOVA), STRUCTURE analyses, and pairwise FST. Soil seedbank populations maintained levels of genetic diversity comparable to those of above-ground populations, and no significant differences were detected between the two population types. Furthermore, soil seedbank populations in evergreen plantation forests, where above-ground individuals of P. freyniana were absent, retained genetic diversity comparable to that observed in burned grasslands. AMOVA detected no significant genetic differentiation between above-ground and soil seedbank populations. These results suggest that high levels of genetic diversity can persist in soil seedbank populations for decades after forest establishment and highlight the potential importance of soil seedbanks as genetic resources for grassland restoration.

ecology↗

bioRxiv2026

Effects of Arachis hypogaea extract on TRPV4 activation and epidermal barrier function

Transient receptor potential vanilloid 4 (TRPV4) is a Ca2+-permeable non-selective cation channel and its activating stimuli include anandamide, bisandrographolide, citric acid, arachidonic acid metabolic products by epoxygenases, hypo-osmotic cell swelling, and warm temperature. TRPV4 is involved in Ca2+-dependent signal transduction in several tissues. Since the activation of TRPV4 facilitates adherens junction formation in the skin epithelium, compounds that activate TRPV4 are expected to maintain or improve the barrier function of epidermal cells. In this study, we found that the extract of Arachis hypogaea (A. hypogaea) activate human TRPV4 (hTRPV4). In the Ca2+-imaging experiment, the application of A. hypogaea extract exhibited an increase in intracellular Ca2+ concentration ([Ca2+]i) in HEK293T cells expressing hTRPV4. The [Ca2+]i increases by application of A. hypogaea extract were not observed in HEK293T cells expressing hTRPV1, mouse TRPV2, hTRPV3, hTRPM8, or hTRPA1. We then examined the physicochemical properties of the components responsible for TRPV4 activation. Ethanol extracts of A. hypogaea caused an increase in [Ca2+]i in hTRPV4-expressing HEK293JN cells, whereas water, chloroform, and hexane extracts showed no activity. Moreover, the application of A. hypogaea extract enhanced transepithelial electrical resistance in the keratinocyte monolayer. These results suggest that A. hypogaea extract may contribute to the maintenance and improvement of the epidermal barrier function.

pharmacology and toxicology↗

bioRxiv2026

Cardiac microtubules mediate transverse (t)-tubule growth and homeostasis

Transverse (t)-tubules enable rapid, synchronous Ca release required for efficient cardiac contraction by bringing L-type Ca channels into close apposition with ryanodine receptors. In heart failure with reduced ejection fraction (HFrEF), t-tubule disorganisation and loss occur alongside cardiac microtubule remodelling, contributing to impaired Ca handling and contractile dysfunction. Despite their canonical function in contraction, how t-tubules develop is unknown. Microtubules support delivery of L-type Ca channels to t-tubules via Amphiphysin-II/BIN1, yet whether microtubules directly regulate t-tubule formation and maintenance is unclear. Here, we investigated a role for microtubules in t-tubule development and homeostasis. Neonatal rat ventricular myocytes (NRVMs), which lack endogenous t-tubules, were used as a reductionist model in which BIN1 overexpression induces nascent membrane tubules. Microtubule depolymerisation with nocodazole before BIN1 overexpression impaired BIN1-driven tubule formation, reducing tubule density and length. Dynein inhibition with EHNA produced similar effects, indicating a requirement for microtubule-based motor activity during tubule elongation. Knockdown of the microtubule +TIP tracking protein CLIP-170 also reduced BIN1-driven tubule density, implicating BIN1-CLIP-170-dependent microtubule capture in tubule initiation. Microtubules were also required to maintain existing tubules. In NRVMs with established BIN1-driven tubules, microtubule depolymerisation, microtubule stabilisation or dynein inhibition each reduced tubule density and length. Consistent with this, acute microtubule depolymerisation or stabilisation disrupted native t-tubule networks in isolated adult sheep left atrial myocytes. Together, these findings identify cardiac microtubules as active regulators of t-tubule architecture. We propose that BIN1-dependent tubule formation requires CLIP-170-mediated microtubule plus-end capture and dynein-dependent elongation, while ongoing microtubule dynamics are necessary to preserve mature t-tubule structure.

physiology↗

bioRxiv2026

Identification of a transient receptor potential channel that is regulated by phospholipid asymmetry

AbstractPhospholipid asymmetry is a hallmark of mammalian cell membranes and reflects the selective distribution of distinct phospholipid species between the two leaflets of the lipid bilayer. Although this asymmetry is tightly maintained, the membrane proteins whose functions depend on it remain largely unknown. To perturb phospholipid asymmetry experimentally, we expressed a constitutively active phospholipid scramblase and thereby identified transient receptor potential melastatin 8 (TRPM8) as an ion channel regulated by this membrane property. Activation of TRPM8 by both l-menthol and innocuous cold was markedly suppressed following disruption of phospholipid asymmetry. Likewise, selective depletion of phosphatidylserine (PS), a phospholipid enriched in the cytoplasmic leaflet, using a cytosolically targeted PS decarboxylase attenuated TRPM8 activation, indicating that cytoplasmic PS is required for proper TRPM8 function. Mechanistically, our findings suggest that cytoplasmic PS supports efficient TRPM8 activation by maintaining the biochemical state of the channel. Together, these findings identify TRPM8 as a phospholipid asymmetry-dependent ion channel and establish an experimental strategy for systematically identifying membrane proteins regulated by phospholipid asymmetry. This work provides a foundation for future studies investigating the biological significance of this fundamental membrane property.

biochemistry↗

bioRxiv2026

Bidirectional Extracellular Vesicle-Mediated Maternal-Embryonic Exchange in the Lecithotrophic Teleost Guppy

The placenta is defined as an organ that mediates the exchanges of nutrients, hormones, and other substances between the mother and embryo in viviparous animals. Its structure is diverse due to interspecific differences in fetal tissues and variation in the forms of maternal-fetal interfaces. Matrotrophic poeciliid teleosts, in which the embryo develops within the maternal ovarian follicle, possess functional placentas formed from maternal follicular and embryonic tissues. However, the physiological mechanisms underlying substrate exchange between mother and embryo remain unclear. Furthermore, although similar embryonic-maternal interfaces are observed in lecithotrophic poeciliids, it is unknown whether nutrient exchange occurs in these species. Therefore, this study investigated whether substance exchange occurs between the mother and embryo in the lecithotrophic teleost guppy (Poecilia reticulata) and identified the underlying physiological mechanisms. Histological analysis revealed that guppies have embryonic-maternal interfaces consisting of the maternal follicle and the embryonic yolk sac and pericardial sac. Additionally, tracking of 2,000 kDa fluorescein isothiocyanate-dextran injected into pregnant guppies confirmed its transport from mother to embryo. Immunofluorescence staining and electron microscopy revealed that substance transport from mother to embryo occurs via extracellular vesicles. Moreover, immunofluorescence staining and pharmacological experiments revealed exosome transport from embryo to mother. This study demonstrates that lecithotrophic guppies possess a functional placenta that mediates maternal-embryonic substrate transfer via extracellular vesicles. These findings provide fundamental insight into the evolution of placental strategies within the Poeciliidae family. Significance StatementViviparity, in which embryos develop within the maternal body, has evolved independently across diverse animal lineages. In lecithotrophic viviparity, embryos are thought to rely primarily on yolk-derived nutrients, with maternal-fetal exchange limited to small molecules such as gases. Here, using macromolecular tracer experiments and ultrastructural analyses in guppies, we show that large macromolecules (2000 kDa) are exchanged bidirectionally between mother and fetus via extracellular vesicles, despite the absence of direct tissue attachment. These findings challenge the conventional view of lecithotrophic viviparity and reveal a previously unrecognized mechanism of maternal-fetal communication. Our results suggest that extracellular vesicle-mediated exchange may represent a widespread and evolutionarily conserved strategy for maternal-fetal interaction across viviparous animals.

developmental biology↗

bioRxiv2026

Genomic basis of rapid urban evolution revealed by the subgenome-resolved genome of octoploid Oxalis corniculata

Urbanization is a major driver of contemporary evolution, yet the genomic basis of urban adaptation remains poorly understood, particularly in non-model plants with complex polyploid genomes. Here, we investigate the genetic mechanisms underlying leaf color variation in the octoploid Oxalis corniculata, a phenotype associated with heat tolerance in urban environments. By integrating high-fidelity long-read sequencing and chromosome conformation capture, we generated the subgenome-resolved, chromosome-scale genome assemblies for both red- and green-leaved lines, resolving four distinct subgenomes. LTR insertion timing revealed a two-step hybridization history that established this octoploid genome within the last 1 million years. Leveraging a nationwide citizen science initiative, we collected and analyzed over 1,700 samples across a broad geographic range. We identified a major locus on one subgenome underlying this variation and implicate a coding-sequence repeat-length polymorphism in a MYB transcription factor as the candidate causal variant. This simple sequence repeat likely acts as a molecular "tuning knob" for rapid adaptation to urban heat islands. This study provides a new baseline for evolutionary ecological genomics in plants and highlights the power of integrating advanced genomics with public participation to forecast evolutionary responses in an increasingly urbanized world.

genomics↗

bioRxiv2026

Cerebral Oxygen Budgeting: Network-Level BOLD Dynamics During Acute Hypoxia

Hypoxia constrains cerebral oxygen availability and challenges brain function. Previous work showed that functional connectivity reorganizes early during acute hypoxia, preceding cognitive deterioration, but the functional changes accompanying more severe hypoxic stress remain incompletely understood. We examined dynamic amplitude of low-frequency fluctuations (dALFF) in blood-oxygenation-level dependent (BOLD) fMRI during normoxia, sustained mild hypoxia, and transient severe hypoxia in healthy adults performing a continuous Go/No-go task with concurrent physiological monitoring. We characterized dALFF at whole-brain and network levels using a causal sliding-window approach, principal component analysis, and Schaefer's 17-network parcellation. Severe hypoxia elicited a non-monotonic, phase-dependent dALFF response that was not observed during normoxia or sustained mild hypoxia. Relative preservation during early hypoxia was followed by late-hypoxia suppression, which we operationally defined as a decompensation phase, and by a pronounced rebound after reoxygenation. Within this global response, dALFF became increasingly differentiated across intrinsic brain networks: DefaultA showed marked suppression, whereas SomMotB exhibited relative preservation or enhancement during decompensation. These changes were neither spatially uniform nor tightly synchronized with systemic oxygenation, while broadly overlapping temporally with cognitive deterioration. Together, these findings indicate that dALFF captures a complementary aspect of the brain's response to acute hypoxic stress, characterized by reversible, phase- and network-dependent reorganization of ongoing low-frequency BOLD dynamics under constrained oxygen availability.

neuroscience↗

bioRxiv2026

Immune and psychogenic fever arise through UCP1-independent thermogenic mechanisms

Brown adipose tissue (BAT) thermogenesis is essential for cold defense, but its contribution to fever and emotionally induced hyperthermia (psychogenic fever) remains disputed. Here we address this issue using genetic, surgical, physiological, and molecular approaches in rats. We generated UCP1 knockout rats, in which classical BAT thermogenesis is abolished, and examined body temperature responses to systemic inflammation induced by lipopolysaccharide and to emotional stressors such as restraint and cage exchange. Despite profound impairment of cold-induced and {beta}3-adrenergic-induced thermogenesis, UCP1 deletion did not affect LPS- or stress-evoked elevations in core or interscapular temperature. Surgical removal of interscapular BAT in wild-type rats likewise failed to alter these hyperthermic responses. Consistent with these findings, LPS and emotional stress induced only small, strain-dependent changes in the expression of thermogenic genes in BAT and minimally affected BAT mass, in marked contrast to the robust BAT activation elicited by {beta}3-adrenergic stimulation. Notably, emotional stress induced UCP3 expression in neck muscles, suggesting a potential contribution of skeletal muscle metabolic processes to stress-induced hyperthermia. Together, these findings demonstrate that both immune-induced and psychogenic fever occur independently of BAT thermogenesis and point to non-BAT tissues - likely including skeletal muscle - as candidate peripheral effectors supporting fever and emotional hyperthermia.

physiology↗

bioRxiv2026

Active transport of tRNAs facilitates distributed protein synthesis

Large, polarized cells such as cardiomyocytes, skeletal myofibers, and neurons rely on localized protein synthesis to sustain size, remodel and adapt to stress. The subcellular architecture of these cells is also inherently unfavorable for long-range, diffusion-based transport, which may promote their heavy reliance on active transport mechanisms for the localization of RNA and proteins. Transfer RNAs (tRNAs) function as essential regulators of protein synthesis by linking transcription and translation. Since their discovery in the 1950s, tRNA subcellular distribution has been assumed to occur through passive diffusion. Here, we report that there are pools of tRNAs that depend on the microtubule network for distribution in cardiomyocytes, skeletal myofibers and neurons. Employing dual-color live and fixed-cell super-resolution imaging, we demonstrate that active transport of tRNAs involves hitchhiking on the exterior of endolysosomal vesicles (ELV). We establish that leucyl-tRNA synthetase (LeuRS), the tRNA-binding protein that charges leucine to its cognate tRNA and interacts with Rag GTP on the surface of ELVs, is essential for tRNA transport. Disruption of LeuRS-ELV interactions is sufficient to impair long-range, microtubule-dependent tRNA transport, without affecting mRNA or rRNA transport. We also show that preventing tRNA transport is sufficient to impair translation at sites distal from the nucleus as well as globally impair protein synthesis, ultimately reducing cell size. These findings redefine tRNAs as actively trafficked cargo and establish their directed transport as a fundamental layer of translation regulation required for myocyte homeostasis. Highlights1. Pools of tRNAs require an intact microtubule network for distribution in large, complex cells. 2. tRNAs undergo active transport by hitchhiking on endolysosomal vesicles trafficked by microtubule motors. 3. The tRNA synthetase LeuRS functions as a tRNA-specific adaptor linking tRNAs to endolysosomal vesicles, and disrupting this interaction selectively impairs tRNA, but not mRNA or rRNA transport. 4. Loss of tRNA transport reduces distal translation, global protein synthesis, and cell size.

cell biology↗

bioRxiv2025

Outbreak of leaf spot disease of Cavendish banana caused by Nigrospora spp. in Mindanao Island, Philippines

Banana leaf diseases are a significant threat to Cavendish banana production. In the Philippines, the main disease has been diagnosed as Black sigatoka disease caused by Pseudocercospora fijiensis based on symptoms. However, our study showed that the main pathogen in Mindanao island, the largest banana-producing region in the Philippines, belongs to the genus Nigrospora, contradicting previous assumptions. We clarified the phylogenetic positions of 160 Nigrospora isolates based on molecular phylogenetic analyses using ITS, {beta}-tubulin, and tef1 sequences, and compared their morphology with known species. Molecular phylogenetic and morphological analysis revealed that Nigrospora isolates comprised N. chinensis, N. lacticolonia, N. cf. singularis, N. sphaerica, N. vesicularifera, and a novel species, N. nigrocolonia. Pathogenicity tests on banana leaves confirmed that these species are pathogenic. Species other than N. sphaerica were for the first time reported as pathogens of banana leaf. The results of the fungicide sensitivity test using 14 fungicides, including pyrimethanil, spiroxamine, and tebuconazole, for the Sigatoka disease showed 100% inhibition of all isolates at 100 ppm of active ingredients. However, low-sensitivity isolates were observed for the remaining 11 fungicides. Our findings indicated the need for a comprehensive review of banana leaf disease prevention strategies.

molecular biology↗

bioRxiv2025

Mechanosensitive Stanniocalcin-1 Attenuates Pulmonary Arterial Hypertension by Suppressing Smooth Muscle Cell Proliferation

BackgroundIdiopathic pulmonary arterial hypertension (IPAH) is driven by progressive pulmonary vascular remodeling, particularly pulmonary arterial smooth muscle cell (PASMC) proliferation. Current combination vasodilator therapies have markedly improved outcomes; however, prognosis remains poor in subgroups such as patients with respiratory comorbidities, highlighting the need for novel therapies. Elevation of intravascular hydrostatic pressure is a hallmark of IPAH, yet its direct role in PASMC pathobiology remains largely unexplored. We developed a cyclic hydrostatic pressurization culture system to model hypertensive hemodynamic stress in vitro and identify pressure-responsive mediators. MethodsPASMCs from 4 patients with IPAH were exposed to high hydrostatic pressure (70/40 mmHg, 60 bpm). Transcriptomic profiling identified differentially expressed genes, validated by qPCR. Functional studies included piezo-type mechanosensitive ion channel component 1 (PIEZO1) modulation, recombinant human stanniocalcin-1 (rhSTC1) treatment, bromodeoxyuridine (BrdU) incorporation, and western blotting for cell-cycle regulators. In vivo, chronic hypoxia-induced pulmonary hypertension was assessed in wild-type and Stc1-/- mice by hemodynamic and histological analyses, with or without intratracheal rhSTC1 administration. ResultsRNA sequencing revealed STC1 as a robustly pressure-induced gene in IPAH PASMCs. PIEZO1 activation upregulated STC1, whereas knockdown blunted this response. Elevated STC1 expression was observed in PASMCs of IPAH lung tissues, and rhSTC1 reduced PASMC proliferation and increased p-p53, p21, and p27 expression. In the chronic hypoxia model, Stc1-/- mice exhibited higher right ventricular systolic pressure (RVSP) (43.7 {+/-} 1.3 vs. 30.6 {+/-} 0.9 mmHg) and greater pulmonary arterial medial thickness (39.1 {+/-} 2.5% vs. 26.5 {+/-} 1.3%) than wild-type mice. CD68-positive macrophages were increased in Stc1-/- mice under normoxia and further elevated with hypoxia. In wild-type and Stc1-/- PAH models, intratracheal administration of rhSTC1 markedly reduced medial thickening, CD68-positive macrophage accumulation, and RVSP in both wild-type and Stc1-/- mice. ConclusionsWe demonstrate that elevated hydrostatic pressure drives STC1 expression via PIEZO1, conferring potent anti-remodeling effects in IPAH. STC1 supplementation represents a potential therapeutic strategy that addresses an urgent medical need not fulfilled by conventional therapies. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIWe established a novel hydrostatic pressurization system to recapitulate idiopathic pulmonary arterial hypertension (IPAH) hemodynamic conditions in vitro. C_LIO_LIStanniocalcin-1 (STC1) is a hydrostatic pressure-responsive gene in pulmonary arterial smooth muscle cells (PASMCs) from IPAH patients, induced via the mechanosensitive receptor piezo type mechanosensitive ion channel component 1 (PIEZO1). C_LIO_LIExogenous STC1 suppresses PASMC proliferation and attenuates pulmonary vascular remodeling in chronic hypoxia-induced PAH models. C_LI What Are the Clinical Implications?O_LISTC1 supplementation represents a potential therapeutic strategy for IPAH, acting through a non-vasodilatory mechanism. C_LIO_LISTC1 supplementation may offer benefit in patients with limited response to current vasodilator therapies or with comorbid respiratory disease. C_LIO_LITargeting mechanotransduction pathways could expand treatment options for pulmonary hypertension. C_LI

molecular biology↗

bioRxiv2025

Brain functional connectivity initiates structured reorganization at a critical oxygen threshold during hypoxia

The human brain dynamically adapts to hypoxia, a reduction in oxygen essential for metabolism. The brains adaptive response to hypoxia, however, remains unclear. We investigated dynamic functional connectivity (FC) in healthy adults under acute hypoxia (FiO2 = 7.7%, 11.8%) using BOLD fMRI, physiological monitoring (PetO2, PetCO2, SpO2), and a Go/No-Go task. Principal component analysis identified a hypoxia-responsive FC component involving 400 cerebral parcels. This component emerged with a critical drop in PetO2 ([~]53 mmHg), preceding changes in SpO2, BOLD signals, and behavior. These FC changes were network-specific and centered on the default mode network (DMN), which selectively synchronized with other high-level cognitive networks. In contrast, visual networks remained stable and segregated from the DMN. These results suggest that the brain proactively reorganizes its functional architecture in anticipation of oxygen decline, rather than in response to it. FC-based markers may offer early indicators of vulnerability in neurological or neurodegenerative conditions.

neuroscience↗

bioRxiv2025

Methionine oxidation of TRPV2 regulates thermogenesis in brown adipocytes

Transient receptor potential vanilloid 2 (TRPV2) is a non-selective cation channel activated by mechanical stimuli and temperatures above 52{degrees}C. Although we have previously reported that TRPV2 regulates non-shivering thermogenesis through facilitating the expression of genes related to thermogenesis, how TRPV2 activity is regulated in brown adipocytes under physiological conditions remains unclear. Recently, it was reported that methionine oxidation was shown to reduce the temperature threshold of TRPV2 for activation to core body temperature or lower. In the present study, we investigated whether methionine oxidation activates TRPV2 and regulates thermogenesis in the differentiated brown adipocytes. As a result, treatment with Chloramine-T (ChT, a methionine oxidant) activated TRPV2 at temperatures of >30{degrees}C in mouseTRPV2-expressing HEK293T cells and in the differentiated brown adipocytes. Moreover, ChT treatment enhanced the expression of genes related to thermogenesis in the differentiated brown adipocytes. These results suggest that methionine oxidation might activate TRPV2 at around body temperature and increase thermogenesis-related gene expression.

physiology↗

bioRxiv2025

Database Chemistry for Genomics-Based Safety and Quality Evaluation of Biologics

Genomics-based safety and quality evaluation studies are advancing the bioindustry by enhancing various aspects, including viral safety, host cell protein (HCP) control, product heterogeneity control, cellular heterogeneity control, and process reproducibility. High-throughput instruments and genome-scale databases are essential in genomics, with the reference genome sequence being the most critical database. The completeness and accuracy of these genome sequences depend on DNA quality, sequencing instruments, read coverage, and assembly strategies. Significant efforts are being made to perfect genome assembly and continuously improve it. However, the quantitative impact of reference genome sequence accuracy on the safety of biologics is not yet fully understood. In this study, we compared and benchmarked six Chinese hamster genomes, including four newly sequenced genomes derived from Chinese hamster cell lines, from an industrial perspective. We also developed database assembly techniques to enhance the safety of biologics. We recommend using two or more independent reference genomes for viral safety studies. For HCP control, we suggest using protein sequences in which trypsin degradation peptides that overlap with high-risk proteins should be masked and unified. Additionally, we can predict microenvironments using single-cell transcriptome data. In bioengineering processes, any nucleotide samples have potential commercial benefits.

bioengineering↗

bioRxiv2025

Dual Translational Control in Cardiomyocytes by Heterogeneous mTORC1 and Hypertrophic ERK Activation

BackgroundCardiac hypertrophy allows post-mitotic cardiomyocytes to meet increased hemodynamic demands but can predispose the heart to adverse clinical outcomes. Despite its central role in cardiac adaptation, the translational control mechanisms that drive cardiac hypertrophy are poorly understood. In this study, we elucidate the relative contributions of the various translational control mechanisms operant during homeostasis and hypertrophic growth. MethodsA combination of immunofluorescence and single myocyte protein synthesis assays were used to dissect the single-cardiomyocyte mechanisms of translational control under basal and hypertrophic conditions in isolated adult rat cardiomyocytes. Translational control mechanism were examined in a mouse model of acute hypertrophic phenylephrine (PE) stimulation prior to overt cardiac growth. ResultsWe observed strikingly heterogeneous activity of mTORC1, the master regulator of translation, across cardiomyocytes both in situ and ex vivo. Heterogeneous mTORC1 activity drove heterogeneous protein synthesis, with translation primarily controlled via canonical mTORC1-dependent 4EBP1 phosphorylation at Thr36/Thr45/Thr69 under baseline conditions. Hypertrophic PE stimulation recruited more cardiomyocytes into a high mTORC1 activity state. PE induced a switch in 4EBP1 phosphorylation by increasing mTORC1-dependent phosphorylation at Thr36/Thr45, but not Thr69. Further, PE induced a novel mTORC1-independent, but MEK-ERK-dependent, pathway driving 4EBP1 phosphorylation at Ser64 in both isolated cardiomyocytes and in vivo. Ribosome biogenesis was also observed within hours upon hypertrophic stimulation, while the mTORC1-S6K-eEF2K-eEF2 pathway was not found to be a major driver of protein translation. ConclusionsProtein synthesis is heterogeneous across cardiomyocytes driven by heterogeneous mTORC1 activity. MEK-ERK signaling directly controls 4EBP1 phosphorylation to augment translation during cardiac hypertrophy and challenges the canonical model of translation initiation. Key pointsO_LImTORC1 activity is low and heterogeneous across cardiomyocytes at baseline. C_LIO_LIHeterogeneous mTORC1 activity drives variable 4EBP1 phosphorylation at Thr36/Thr45/Thr69, resulting in heterogeneous protein translation. C_LIO_LIPhenylephrine stimulation recruits more cardiomyocytes into a high mTORC1 activity state to augment protein synthesis through 4EBP1 phosphorylation at Thr36/Thr45, but not Thr69. C_LIO_LIPhenylephrine stimulation boosts protein translation through mTORC1-independent but ERK-dependent phosphorylation of 4EBP1 at Ser64. C_LIO_LImTORC1-S6K-eEF2K-eEF2 pathway is not a major driver of global protein translation. C_LIO_LIRibosome biogenesis is observed within hours after hypertrophic stimulation. C_LIO_LIThe data demonstrates a dual input model of mTORC1 and ERK dependent phosphorylation of 4EBP1 to regulate protein translation in cardiomyocytes. C_LI

physiology↗

bioRxiv2025

ROR2 drives right ventricular heart failure via disruption of proteostasis

BackgroundNo therapies exist to reverse right ventricular failure (RVF), and the molecular mechanisms that drive RVF remain under studied. We recently reported that the developmentally restricted noncanonical WNT receptor ROR2 is upregulated in human RVF in proportion to severity of disease. Here we test the mechanistic role of ROR2 in RVF pathogenesis. MethodsROR2 was overexpressed or knocked down in neonatal rat ventricular myocytes (NRVMs) and then characterized using confocal microscopy, RNAseq, proteomics, proteostatic functional assays, and pacing to assess contractile properties. The impact of cardiac ROR2 expression was evaluated in mice by AAV9-mediated overexpression and by AAV9-mediated delivery of shRNA to knockdown ROR2 in a pulmonary artery banded pressure overload model of RVF. ROR2-modified mice were evaluated by echocardiography, histology, and RV protein synthesis and proteasome capacity. ResultsIn NRVMs, we find that ROR2 profoundly dysregulates the coordination between protein translation and folding. This imbalance leads to excess protein clearance by the ubiquitin proteasome system (UPS) with dramatic impacts on sarcomere and cytoskeletal structure and function. Inhibiting the UPS or restoring chaperone expression is sufficient to partially rescue ROR2-induced structural and contractile deficits in cardiomyocytes. In mice, forced cardiac ROR2 expression is sufficient to disrupt proteostasis and drive RVF, while conversely ROR2 knockdown partially rescues proteostasis and RV structure and function in a pressure overload model of RVF. ConclusionsIn sum, ROR2 is a key driver of RVF pathogenesis through proteostatic disruption and, thus, provides a promising target to treat RVF.

molecular biology↗

bioRxiv2025

CYP722A1-driven 16-hydroxylation of carlactonoic acid regulates the floral transition in Arabidopsis

Strigolactones (SLs) are multifunctional plant hormones and rhizosphere signals with diverse structures, broadly categorized as canonical or noncanonical SLs. In Arabidopsis thaliana, SL biosynthesis mutants exhibit increased shoot branching and early flowering, underscoring their roles in developmental regulation. Shoot branching inhibition in Arabidopsis depends on the methylation of carlactonoic acid (CLA), a key intermediate classified as a noncanonical SL, catalyzed by CLA methyltransferase (CLAMT). Canonical SLs primarily function as rhizosphere signals, with their biosynthesis in dicots mediated by CYP722C enzymes. It is hypothesized that Arabidopsis does not produce canonical SL because of the lack of the CYP722C genes in its genome. Instead, Arabidopsis possesses CYP722A1, a member of the previously uncharacterized CYP722A subfamily, distinct from the CYP722C subfamily. This study demonstrates that Arabidopsis cyp722a1 mutants exhibit an earlier floral transition without excessive shoot branching. Biochemical analysis revealed that CYP722A1 catalyzes the hydroxylation of CLA to produce 16-hydroxy-CLA (16-HO-CLA), which is subsequently methylated by CLAMT to form 16-HO-MeCLA. 16-HO-CLA and 16-HO-MeCLA were detected in the wild-type; however, these compounds were absent in max1-4 mutant, which is deficient in CLA synthesis, and in cyp722a1 mutant. These findings indicate the presence of CYP722A1-dependent 16-hydroxylation activity of CLA in Arabidopsis. Moreover, they suggest that hydroxylated CLA specifically regulates floral transition, distinct from branching inhibition. By identifying CYP722A1 as a regulator of floral transition, which is the distinct role of the CYP722A subfamily, this work provides insights into the adaptation of SL structures for specialized biological functions in plant development.

plant biology↗

bioRxiv2024

Genetic diversity and differentiation of a mycoheterotrophic orchid (Cymbidium macrorhizon) under urbanization

Urbanization exerts substantial pressures on genetic diversity of plant species. However, depending on species-specific life history, the direction/magnitude of urbanization impacts can vary. To elucidate relationships between life history and urbanization effects, there are needs to accumulate the knowledge on genetic diversity/differentiation along urban-rural gradients for species with unique traits. We examined these facets based on genome-wide single nucleotide polymorphisms of a mycoheterotrophic and vegetative-dormant orchid, Cymbidium macrorhizon, for eleven populations in remnant forests along an urban gradient within the Tokyo metropolitan area, Japan. The reduced inbreeding coefficient and increased genetic differentiation were observed with increased proportion of surrounding urban land-use 50 years ago rather than in recent years. This pattern might reflect lowest heterozygosity under intensive urbanization due to population bottleneck and genetic drift due to habitat shrinkage and fragmentation. The significant impacts of past landscape on the indices might indicate time lags of genetic erosion, namely intra-specific extinction debt, due to longevity and dormancy. Therefore, 30% increase of urban land-use since 1970s has not yet affected genetic erosion, resulting in its progression in the future. We emphasize the importance not only to assess genetic diversity but also to connect the assessments with life history and spatiotemporal urbanization impacts.

ecology↗