Biology subjects

Sasaki, M.

Publications and source records attributed to Sasaki, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Interleukin-34 Promotes Activation of Disease-associated Microglia-like Cells and Attenuates Disease Progression in West Nile Virus Infection

West Nile virus (WNV) is a mosquito-borne orthoflavivirus that causes severe encephalitis, for which no approved antiviral therapies or human vaccines are currently available. Disease-associated microglia (DAM) represent a microglial state associated with neuroprotective functions in neurodegenerative diseases. We previously showed that DAM-like cells are localized in the vicinity of WNV-infected cells in the mouse brain and surmised that these cells may respond to WNV-infected cells. However, the functional significance of this spatial association has since remained unclear. Given the previous reports linking interleukin-34 (IL-34) to the activation of DAM-like cells, we investigated whether IL-34 promotes DAM-like responses and whether these responses are associated with protection against WNV infection. Transcriptomic analysis of IL-34-treated HMC3 human microglial cells revealed upregulation of markers characteristic of DAM, including TREM2, APOE, FABP5, and FTH1. IL-34-treated HMC3 cells suppressed WNV replication in co-culture with WNV-infected SH-SY5Y human neuroblastoma cells apparently in a cell-cell contact-dependent manner, independent of secreted factors. In WNV-infected mice, IL-34 administration improved survival, reduced viral titers, and decreased neuronal apoptosis. IL-34 also increased the abundance of CD11c- and SPP1-positive DAM-like cells, predominantly in the vicinity of WNV-infected cells, in the mouse brain. These findings indicate that IL-34 promotes the activation of DAM-like cells and enhances protective responses in WNV encephalitis. IMPORTANCEWest Nile virus (WNV) is a major cause of viral encephalitis worldwide, with no approved antiviral treatment or human vaccine currently available. Microglia, the resident immune cells of the brain, play key roles in responding to viral infection. However, the mechanism through which specific microglial activation states contribute to protection in viral encephalitis remains poorly understood. In this study, we show that interleukin-34 promotes microglial responses resembling disease-associated microglia (DAM) and is associated with enhanced protection against WNV infection in cell culture and mouse models. The study findings suggest that activation of DAM-like cells may contribute to protective host responses against neurotropic viral infection and provide new insights into the role of specific microglial states in viral encephalitis.

microbiology↗

bioRxiv2026

Rabies virus antagonizes interferon signaling by targeting phosphorylated STAT1 tetramers

AO_SCPLOWBSTRACTC_SCPLOWRabies virus phosphoprotein (P protein) antagonizes interferon signalling by selectively targeting activated STAT1 protein, but the structural basis for the selective recognition of activated STAT1 has remained unclear. Here we report cryo-EM structures of tyrosine-phosphorylated STAT1 (pY-STAT1) in complex with the C-terminal domain of P protein (P-CTD). P-CTD engages the pY-STAT1 tetramer through three discrete interfaces, contacting two DNA-binding domains within a pY-mediated STAT1 dimer and the N-terminal domain of a third STAT1 protomer. This binding mode explains how P-CTD selectively recognizes activated STAT1, occludes surfaces required for importin 5 and DNA binding, and stabilizes the compact tetrameric state, thereby restricting the conformational transition required for cooperative DNA binding. Cell-based assays further indicate that distinct P-CTD surfaces differentially contribute to antagonism of type I and type II interferon signalling. Further, by acting as an anchoring molecule, P-CTD also enabled structural elucidation of the complete STAT1 structures in the tetramer. Together, these data reveal critical mechanisms in viral immune evasion and pathogenesis, and fundamental innate immune signaling.

molecular biology↗

bioRxiv2026

Biomechanical regulation of Ca2+ dynamics during muscle stem cell activation

Muscle satellite cells (MuSCs) are muscle-resident stem cells that are responsible for myofiber regeneration. Although the importance of calcium ions (Ca2+) in muscle physiology has been well established, the mechanism by which Ca2+ mobilization governs MuSC function remains poorly understood. In this study, we aimed to systematically characterize Ca2+ dynamics in MuSCs and to define the mechanisms regulating these signals during muscle regeneration. By employing modified protocols for mouse MuSC isolation and Ca2+ measurement, we observed spontaneous Ca2+ fluctuations in MuSCs isolated from regenerating muscle after cardiotoxin-induced myofiber injury. Our detailed analysis using chemical Ca2+ indicators and a genetically encoded Ca2+ indicator revealed that the frequency and amplitude of Ca2+ fluctuations increased significantly during the activated and proliferative stages of MuSCs in muscle regeneration. This effect was more pronounced in MuSCs isolated from dystrophic and aged mice. Mechanistically, these Ca2+ fluctuations were at least partially mediated by mechanosensitive ion channels, including PIEZO1 and TRPM7, which promote MuSC migration. Collectively, our findings demonstrate that Ca2+ fluctuations through mechanosensitive ion channels act as a key regulator of MuSC activation during muscle regeneration and may provide new insights into the role of Ca2+ influx in muscle biology and the pathogenesis of muscle diseases.

cell biology↗

bioRxiv2026

A surrogate marker of protection confirms the efficacy of an AddaS03-adjuvanted West Nile virus subunit vaccine

West Nile virus (WNV) is the causative agent of fatal West Nile encephalitis. To date, no human vaccine against WNV has been approved. Adjuvants are important for developing effective and affordable vaccines that enhance the immunogenicity and decrease the required antigen doses. In this study, we assessed the efficacy of AddaS03, a synthetic adjuvant analogous to AS03, in a WNV subunit vaccine composed of soluble recombinant envelope protein (sEnv). Using a passive immunization mouse model, we defined the neutralizing antibody titer threshold required for protection against lethal WNV infections and applied this threshold as a surrogate marker to evaluate adjuvant efficacy. AddaS03-adjuvanted formulations elicited markedly higher neutralizing antibody titers compared to Alhydrogel adjuvant 2% (Alhydrogel), even at suboptimal antigen doses, and consistently exceeded the defined protective threshold titer. Moreover, in a sequential challenge mouse model, AddaS03-adjuvanted vaccines completely protected mice from symptomatic WNV infections, whereas Alhydrogel-adjuvanted vaccines failed to confer full protection. Collectively, these findings demonstrate that AddaS03 is a promising adjuvant for WNV subunit vaccine development and highlights the utility of a passive immunization model for defining protective antibody thresholds as a surrogate marker for vaccine evaluation.

microbiology↗

bioRxiv2026

Integrated in vivo and transcriptomic analyses of lethal Oropouche virus infection reveal suppression of pathogenic host responses by antiviral therapy

Oropouche virus (OROV) is an emerging arbovirus responsible for large outbreaks of febrile illness in Central and South America, with increasing reports of severe neurological disease and fatal outcomes. Despite its growing public health impact, no approved antiviral therapies or vaccines are currently available. Here, we show that favipiravir, a broad-spectrum nucleoside analogue, robustly suppresses OROV replication and disease in vivo. In a lethal Syrian hamster model, favipiravir treatment provided complete protection against OROV infection, preventing viral dissemination to peripheral organs and the central nervous system, and remained highly effective when administration was initiated after infection. In contrast, insufficient antiviral control resulted in viral neuroinvasion and fatality. To define host responses associated with OROV pathogenesis and their modulation by antiviral therapy, we performed transcriptomic profiling of liver and brain tissues. OROV infection induced interferon-driven inflammatory programs accompanied by marked disruption of metabolic and tissue homeostatic pathways, whereas these transcriptional signatures were largely abrogated by favipiravir treatment. Together, our findings identify favipiravir as a potent antiviral candidate against OROV and provide the first in vivo, tissue-resolved transcriptomic framework of OROV infection, linking effective viral suppression with the prevention of neuroinvasion and pathogenic host responses. These results highlight antiviral intervention as a viable strategy to mitigate OROV-associated disease and mortality.

microbiology↗

bioRxiv2026

Structural basis of lenacapavir-induced HIV-1 capsid disruption during virion maturation

Long-acting lenacapavir (LEN) has emerged as a highly effective, potentially game-changing therapy for HIV treatment and prevention1-5. Although its mechanism of action in the early phase of HIV-1 replication, when the capsid directs key post-entry steps such as reverse transcription, nuclear import, and integration, has been well characterized1,2,6-8, its effects during the late phase of replication, when the capsid assembles and matures within budding virions, remain poorly understood. Here, we determine the cryo-electron microscopy structure of the mature HIV-1 capsid lattice assembled within virus-like particles in the presence of LEN. Our structural analyses revealed that LEN alters interhexamer interactions, perturbs capsid lattice curvature, and thereby prevents the formation of a functional cone-shaped capsid. Biochemical analyses further demonstrated that LEN-containing cores lose reverse transcriptase due to a compromised capsid integrity, whereas integrase and viral RNA remain associated. Functionally, viruses produced in the presence of LEN exhibited markedly reduced infectivity, low reverse transcription activity, and poor integration. Together, these findings provide mechanistic insight into the late-phase action of LEN and provide key directions for the design of future inhibitors.

microbiology↗

bioRxiv2026

Prostaglandin E2 Reverses Myofibroblast Differentiation in Eosinophilic Esophagitis

Background & AimsUnchecked inflammation in Eosinophilic esophagitis (EoE) leads to esophageal fibrosis and eventual stricture. Differentiated fibroblasts, termed myofibroblasts, are the main effector cells in fibrosis, responsible for secreting extracellular matrix proteins leading to tissue stiffness. Regulating myofibroblasts has not been explored as a therapeutic possibility in the fibrostenotic esophagus. Herein, we aim to investigate the efficacy of Prostaglandin E2 (PGE2) in dedifferentiation of the EoE myofibroblast. MethodsWe evaluated the efficacy and mechanism of myofibroblast dedifferentiation using fetal esophageal fibroblasts (FEF3), patient-derived fibroblasts, and a murine model of EoE. ResultsFibrosis markers (SMA, FN1, and COL1A1) and contractility of myofibroblasts were significantly decreased by PGE2 via the cAMP pathway. PGE2 treatment decreased nuclear accumulation of phospho-Smad2/3-YAP complex and induced phospho-YAP proteasomal degradation. Transcriptome analyses of FEF3 treated with TGF{beta} or PGE2 revealed that the Integrin1 pathway, and specifically thrombospondin 1 (THBS-1), was significantly upregulated by TGF{beta} and downregulated by PGE2, as supported by pseudo-bulk single-cell RNA-seq of EoE biopsies. THBS-1 was shown to be regulated by PGE2 via the cAMP/YAP pathway, and its knockdown induced myofibroblasts dedifferentiation. In a murine model of EoE, Butaprost, agonist of the E-prostanoid G protein-coupled receptor 2, treatment significantly reduced the expression of THBS-1, SMA, and FN1 along with a decrease in YAP nuclear translocation. Additionally, collagen fiber organization in the lamina propria was markedly reduced. ConclusionPGE2 promotes dedifferentiation of myofibroblasts in EoE via the cAMP/YAP/ THBS-1 pathway. Our data suggest that PGE2 is a promising treatment strategy for EoE with stenosis. What You Need to KnowO_ST_ABSBackground and ContextC_ST_ABSIn Eosinophilic esophagitis, unchecked inflammation and tissue stiffness drives fibroblast differentiation and fibrostenosis of the esophagus, yet targeting myofibroblasts as regulators of extracellular matrix deposition in fibrostenotic disease remains clinically unexplored. New FindingsProstaglandin E2 promotes dedifferentiation of myofibroblasts in eosinophilic esophagitis via the cAMP/YAP pathway, with Thrombospondin-1 identified as a critical YAP regulated target driving fibrostenosis. LimitationsThis study focused on fibroblast-specific mechanisms. The effects of PGE2 on esophageal epithelial differentiation, barrier function, and immune cell recruitment in EoE remain to be determined. Clinical Research RelevanceThis study demonstrates proof-of-concept that pharmacological reversal of established fibrosis is achievable in EoE. PGE2 and its EP2-selective agonists represent translatable therapeutic targets for fibrostenotic EoE--a patient population that remains treatment-refractory to current immunosuppressive approaches. Basic Research RelevanceThe cAMP/YAP/THBS-1 signaling in fibroblasts emerges as a critical therapeutic target for esophageal fibrosis. Importantly, this work demonstrates that terminally differentiated myofibroblasts retain remarkable plasticity and can dedifferentiate--challenging the paradigm that fibrosis is irreversible.

cell biology↗

bioRxiv2025

Structural and mechanistic basis for membrane recognition and activation of the vacuolar lipase Atg15

Summary/Abstract Atg15 is a vacuolar phospholipase B essential for the degradation of intravacuolar vesicles such as autophagic bodies. Despite its central role in cellular membrane turnover, the molecular basis of how Atg15 is activated and selectively acts on internal membranes has remained elusive. Here, by combining all-atom and coarse-grained molecular dynamics (MD) simulations with in vitro and in vivo analyses, we elucidate the structural and mechanistic principles underlying Atg15 activation and substrate recognition. Our simulations revealed that disulfide bonds are critical for maintaining the structural integrity of the catalytic core, while the C-terminal region locks the catalytic center in a closed state that prevents activation. Membrane binding induces a transition to an open state, enabling catalysis. Through MD-guided mutational analysis, we identified three regions crucial for catalytic locking, membrane binding, and substrate recognition, and experimentally confirmed that mutations in these regions inhibit activity. Furthermore, Atg15 preferentially associates with positively curved membranes, providing a potential basis for its selective action on internal vesicular membranes. These findings suggest that Atg15s activity is controlled through multiple regulatory layers to ensure safe and selective membrane degradation.

cell biology↗

bioRxiv2025

Caveolin-1 controls lineage fidelity of adult neural stem cells via lysosomal degradation of PDGFRα

Caveolae are cell-surface signaling hubs involved in stem cell proliferation and differentiation. However, the molecular details of how they influence stem cell quiescence remain poorly understood. In this study, we show that most caveolae components, particularly caveolin-1 (Cav-1), are specifically upregulated in quiescent neural stem cells (NSCs). Furthermore, we demonstrate that Cav-1 restricts platelet-derived growth factor receptor (PDGFR) signaling, a key regulator for NSC proliferation and oligodendrocyte differentiation in the adult brain, through lysosomal degradation. Genetic ablation of Cav-1 leads to aberrant NSC activation, reduced neurogenesis, and enhanced ectopic oligodendrocyte differentiation on the ventricular surface in the ventral side of the ventricular-subventricular zone (V-SVZ), a key NSC niche responsible for neuroblast differentiation. Mechanistically, we show that Cav-1 deficiency suppresses the endo-lysosomal degradation of PDGFR, leading to PDGFR accumulation and excessive signaling for oligodendrocyte differentiation. Taken together, our results uncover a crucial homeostatic mechanism wherein caveolae function as a safeguard to maintain adult NSC quiescence and lineage fidelity by controlling lysosomal turnover of PDGFR in a niche-specific manner.

neuroscience↗

bioRxiv2025

Application of the CPER reverse genetics system for genetic engineering of rabies virus

Reverse genetics (RG) systems are essential tools for basic virological studies and applied studies using engineered recombinant viruses in various research fields. While the circular polymerase extension reaction (CPER) has been widely applied to prepare a full-length infectious complementary DNA (cDNA) of positive-sense RNA viruses, its use for negative-sense RNA viruses (mononegaviruses) remains limited. Here, we report the first CPER-based RG system for rabies virus (RABV), a member of mononegaviruses. Infectious RABV was successfully rescued from cells transfected with helper plasmids and the CPER product, the assembled overlapping DNA fragments encoding the full-length viral genome cDNA and regulatory elements. Using this system, we generated wild-type, point-mutant, reporter-expressing, and chimeric RABVs, all of which retained their expected biological properties. Deep sequencing revealed that CPER-derived viruses occasionally harbor low-frequency mutations undetectable by Sanger sequencing, highlighting PCR-related artifacts as a limitation. In addition, CPER products with a pUC19 backbone could be directly applied for E. coli transformation and cloning of RABV full genome cDNA plasmids, offering a flexible, ligase-free cloning strategy for conventional RG. Our work establishes CPER as a versatile platform for engineering recombinant RABVs, facilitating rapid generation and genetic manipulation of RABV with potential applications for research on other mononegaviruses.

microbiology↗

bioRxiv2025

Lysine lactylation regulates ATF4-mediated stress responses under glucose starvation in canine hemangiosarcoma

Hemangiosarcoma (HSA) is a malignant endothelial tumor that occurs frequently in dogs but is rare in other species including humans. Due to its aggressive behavior and limited therapeutic options, patient prognosis is generally poor. Tumor cells produce excess lactate via anerobic glycolysis, and it regulate gene expressions through histone lactylation in response to cellular metabolic conditions. However, how histone lactylation affects biological behavior under glucose-limited conditions in HSA remains unknown. Here, we established canine HSA cell lines and patient-derived xenograft models and investigated the role of histone lactylation during glucose deprivation. HSA cells exhibited higher global histone lactylation levels than normal endothelial cells. Although glucose restriction reduced global histone lactylation levels, Cleavage Under Targets and Tagmentation (CUT&Tag) analysis revealed enrichment of lactylation peaks at transcription-start sites (TSSs) of ATF4-regulated stress-response, asparagine biosynthesis and immune-related genes. TSSs of stress-response genes were co-occupied with RNA polymerase II phosphorylated at serine 5 and showed increased gene expressions, suggesting that lactylation at TSSs activated transcription under glucose-deprived conditions. [U-13C]glutamine tracing indicated that HSA cells synthesized asparagine from glutamine when glucose was scarce. Asparagine supplementation modestly activated cell proliferation. In HSA patient tissues, H3K18la levels were heterogeneous, and M2-like macrophages preferentially infiltrated tumor regions showing low histone lactylation levels. Consistently, glucose-starved HSA cells attracted macrophages and induced M2-like polarization in vitro. These findings demonstrate that lysine lactylation, possibly histone lactylation, persists even under glucose-deprived conditions and regulate transcription that supports tumor cell survival and fosters a pro-tumor microenvironment. One Sentence SummaryLysine lactylation is enriched at TSSs of stress-response genes under glucose starvation and associated with their transcription in canine hemangiosarcoma.

cancer biology↗

bioRxiv2025

FOXM1 Modulation Alleviates Epithelial Remodeling and Inflammation in Eosinophilic Esophagitis

BackgroundEosinophilic esophagitis (EoE) is a chronic allergic disease characterized by esophageal epithelial remodeling, barrier dysfunction, and inflammation. Despite histologic remission, molecular and structural changes in the epithelium persist, contributing to ongoing symptoms and relapse. The transcription factor FOXM1 has been shown to be a key regulator of epithelial proliferation and inflammation in allergic asthma. ObjectiveTo investigate the role of FOXM1 in epithelial disruption in EoE and to evaluate the therapeutic potential of FOXM1 inhibition. DesignFOXM1 expression was analyzed in human esophageal biopsies, patient-derived organoids, and murine EoE models. IL-13 stimulation was used to model EoE in vitro. The effects of FOXM1 inhibition via the small molecule RCM-1 and siRNA-mediated knockdown were assessed by histology, gene expression profiling, organoid formation rates, and barrier integrity assays. RNA sequencing and chromatin immunoprecipitation were performed to elucidate molecular mechanisms. ResultsFOXM1 was significantly upregulated in patients with active EoE and localized to the basal epithelium. IL-13 increased FOXM1 expression, which impaired epithelial differentiation and enhanced basal cell hyperplasia. FOXM1 inhibition restored differentiation markers, reduced basal hyperplasia, and improved barrier function. In murine models, RCM-1 ameliorated epithelial changes and decreased eosinophil infiltration. Mechanistically, FOXM1 directly regulated cell cycle gene, CCNB1, which was upregulated in EoE and downregulated upon FOXM1 inhibition. FOXM1 expression was driven by an IL-13-PI3K/AKT axis. ConclusionFOXM1 plays a pivotal role in epithelial disruption in EoE by driving proliferation and impairing differentiation. Targeting FOXM1 restores epithelial homeostasis, mitigates inflammation, and offers a novel therapeutic approach for EoE. Key MessagesWhat is already known on this topic: Eosinophilic esophagitis is marked by epithelial remodeling and barrier dysfunction driven by Th2 inflammation. Despite remission, molecular and histologic changes in the esophageal epithelium persist, contributing to symptoms and relapse. The mechanisms underlying this epithelial dysregulation remain poorly understood. What this study adds: This study identifies FOXM1 as a key transcriptional regulator of epithelial disruption in EoE, demonstrating that FOXM1 inhibition restores epithelial differentiation, reduces basal cell hyperplasia, improves barrier integrity, and mitigates inflammation. How this study might affect research, practice, or policy: Targeting FOXM1 offers a novel therapeutic strategy to restore epithelial homeostasis and reduce inflammation in EoE. This dual approach, addressing both epithelial and immune dysregulation, may guide future therapeutic development and improve patient outcomes.

molecular biology↗

bioRxiv2025

Simple and quantitative detection of double-stranded RNA as a signature of pan-virus infections using a luciferase-based biosensor

Viral infections produce double-stranded RNA (dsRNA) during replication, which trigger host innate immune responses. Immunoassays using anti-dsRNA antibodies have been widely employed to detect viral dsRNA. In this study, we used a luciferase-based dsRNA biosensor which consists of protein kinase R (PKR)-derived dsRNA binding domains fused to split luciferase subunits. Here, we demonstrate the use of the dsRNA biosensor to measure viral dsRNA in RNA specimens extracted from cells infected with Japanese encephalitis virus (JEV). Moreover, the biosensor reacts to a broad-spectrum of dsRNAs from infection with representatives of various viral families including positive- and negative-sense single-stranded RNA (ssRNA) viruses, dsRNA viruses, and DNA viruses. We validated the specific interaction between the dsRNA biosensor and viral RNA including subgenomic flavivirus RNA (sfRNA) through RNA immunoprecipitation. Additionally, we observed luminescence signals directly from lysates of JEV-infected cells after cell lysis and phase separation with Triton X-114. Finally, we used the biosensor to assess the activity of antiviral compounds. In summary, our results demonstrate that the luciferase-based dsRNA biosensor offers a simple, homogeneous, and high-throughput platform for quantifying viral replication, presenting a promising alternative to antibody-based dsRNA detection methods.

microbiology↗

bioRxiv2025

Rad27/FEN1 prevents accumulation of unprocessed Okazaki fragments and ribosomal DNA copy number changes

DNA copy number changes are the most frequent genomic alterations in cancer cells. The ribosomal DNA (rDNA) region is particularly vulnerable to such changes due to its repetitive nature. Here, we demonstrate that Rad27/FEN-1, a structure-specific nuclease in budding yeast, plays a crucial role in maintaining rDNA stability. The production of extrachromosomal rDNA circles and severe chromosomal rDNA instability are observed in the rad27{Delta} mutant, independently of Fob1-mediated DNA replication fork arrest and DNA double-strand break (DSB) formation in the rDNA. The rad27{Delta} mutant accumulates unprocessed Okazaki fragments in the rDNA region, without inducing DSB formation. Similar rDNA instability is observed in DNA ligaseCdc9-deficient cells. Furthermore, we show that Exonuclease 1 and PCNA can compensate for the loss of Rad27 function in the rDNA stabilization. These findings highlight the importance of proper Okazaki fragment processing in preventing non-DSB-induced rDNA copy number changes.

molecular biology↗

bioRxiv2024

Membrane potential modulates ERK activity and cell proliferation

Plasma membrane potential has been linked to cell proliferation for over 40 years. In this study, we experimentally demonstrated that membrane depolarization promotes mitosis and that this process depends on the voltage-dependent activation of extracellular signal-regulated kinase (ERK). Notably, ERK activity showed a clear dependence on the membrane potential, independent of growth factor stimulation. This voltage dependence was observed even near the resting membrane potential, indicating that small shifts in the resting potential can influence proliferative activity. Voltage-dependent ERK activity is derived from the altered dynamics of phosphatidylserine and is not mediated by calcium influx from the extracellular space. These findings suggest that fundamental biological processes such as cell proliferation are regulated by the physicochemical properties of membrane lipids. This study highlights the broader physiological roles of membrane potentials beyond action potentials, which are well-established in neural systems.

cell biology↗

bioRxiv2024

The peroxiredoxin Tsa1 extends the lifespan of budding yeast by maintaining the stability of the ribosomal RNA gene cluster.

Tsa1 is a major budding yeast peroxiredoxin that also has nonperoxidase functions. Here we demonstrate that Tsa1 is required for stabilizing the ribosomal RNA gene (rDNA) cluster. Tsa1 deficiency led to lower replication initiation and an elevated recombination frequency in the rDNA. However, the absence of Tsa1 did not affect Fob1-dependent replication fork arrest at the replication fork barrier (RFB) site, indicating that forks at this site are arrested stably. Tsa1 deficiency did not affect the frequency of DNA double-strand breaks (DSBs) but enhanced transcription from the regulatory promoter E-pro toward the RFB. Because transcription from E-pro inhibits cohesin association and DSB end resection, elevated transcription may cause rDNA instability. We showed that the shortened lifespan and rDNA instability in the tsa1{Delta} mutant were largely suppressed by the fob1 mutation. Therefore, Tsa1-mediated rDNA stabilization in response to replication fork arrest is important for prolonging lifespan. The tsa1{Delta} mutant accumulated single-strand breaks in the rDNA in a Fob1-independent manner. Taken together, these findings suggest that Tsa1 plays a crucial role in maintaining rDNA stability in both Fob1-dependent and Fob1-independent manners and contributes to lifespan extension.

molecular biology↗

bioRxiv2024

The histone chaperone CAF-1 maintains the stability of the ribosomal RNA gene cluster by suppressing end resection of replication-associated DNA double-strand breaks.

DNA replication-coupled chromatin assembly is crucial to maintain genome integrity. Here, we demonstrate that absence of the budding yeast histone chaperone CAF-1 induces chromosomal ribosomal RNA gene (rDNA) copy number changes as well as the production of extrachromosomal rDNA circles in a manner dependent on Fob1 that causes replication fork arrest in the rDNA, the homologous recombination protein Rad52, and its interaction with Proliferating Cell Nuclear Antigen. CAF-1 deficiency enhances transcription from the regulatory promoter E-pro, contributing to rDNA instability, but it also stabilizes the rDNA independently through its regulation of E-pro. Absence of CAF-1 induces end resection of DNA double-strand breaks (DSBs) formed at arrested replication forks, which are repaired in a manner dependent on the Mre11-Rad50-Xrs2 complex. CAF-1 deficiency causes partial defects in lagging strand synthesis coupled to nucleosome spacing in the rDNA. Our findings suggest that CAF-1 suppresses Rad52-mediated rDNA instability during repair of replication-coupled DSBs.

molecular biology↗

bioRxiv2024

Wolbachia infection in Aedes aegypti does not affect its vectorial capacity for Dirofilaria immitis

Mosquito-borne diseases such as dengue and filariasis are a growing public health concern in endemic countries. Biological approaches, such as the trans-infection of Wolbachia pipientis in mosquitoes, are an alternative vector control strategy, especially for arthropod-borne viruses such as dengue. In the present study, the effect of Wolbachia (wMel strain) on the vectorial capacity of Aedes aegypti for Dirofilaria immitis was studied. Our results showed that Wolbachia does not affect the phenotype of mosquito survival or the prevalence, number, and molting rate of third-stage larvae in both susceptible and resistant strains of Ae. aegypti. RNA-seq analysis of Malpighian tubules at 2 days post-infection with D. immitis showed the differentially expressed genes (DEGs) with and without wMel infection. No characteristic immune-related gene expression patterns were observed among the DEGs. No significant change in the amount of Wolbachia was observed in the Ae. aegypti after D. immitis infection. Our results suggest that infection of D. immitis in Ae. aegypti populations will not interfere with Wolbachia-based vector control strategies in dengue-endemic areas where cases of D. immitis are present. This study demonstrated the veterinary medical validity of a dengue control program using Wolbachia.

microbiology↗