Search Results (910)

Most land plants engage in a mutualistic interaction with arbuscular mycorrhizal fungi (AMF), for which Rhizophagus irregularis is a model species. Like plant pathogenic fungi, AMF genomes encode hundreds of putative effector proteins. However, for only a few, the molecular mechanisms by which they alter the host’s physiology are known. Here, we combined several reverse genetic approaches to unravel the role of the RIRG190 effector protein in arbuscular mycorrhiza (AM) symbiosis. Using multiple heterologous tools, evidence is provided that the RIRG190 effector is secreted and localizes to the plant nucleus. Moreover, by means of yeast two-hybrid (Y2H) and ratiometric bimolecular fluorescence complementation (rBIFC) assays, the data demonstrate that RIRG190 interacts with the protein Something About Silencing (SAS10), known to be involved in rRNA biogenesis in the nucleolus of cortical cells. Our findings suggest that rRNA biogenesis is a key process modulated by AMF, potentially to enhance plant metabolic activity, facilitating cell cycle progression, and to support the establishment of the symbiosis. Full article

N⁶-methyladenosine (m⁶A), the most prevalent internal mRNA modification in eukaryotes, is also present in plants and is known to influence plant–virus interactions. However, its specific role in regulating Potato virus Y (PVY; Potyvirus yituberosi) infection, a major pathogen of potatoes, remains unclear. This study identified 16 potential m⁶A regulator genes in Nicotiana benthamiana through homology screening of Arabidopsis thaliana AlkB family members. Based on expression profiles in leaves at various developmental stages and following PVY infection, NbALKBH1L was selected for further analysis. Enzyme assays confirmed its m⁶A demethylase activity. Experiments with NbALKBH1L mutants, using RT-qPCR and m⁶A-IP-qPCR, demonstrated that it regulates PVY infection via the m⁶A pathway. Further investigation revealed that NbALKBH1L interacts with the PVY-encoded cylindrical inclusion (CI) protein. An interaction network constructed through immunoprecipitation–mass spectrometry (IP-MS) and RNA sequencing (RNA-seq) suggested that NbALKBH1L may serve as a central node in plant antiviral immunity, potentially linking metabolic processes with the regulation of viral infection. In summary, this study advances our understanding of plant m⁶A modifications in antiviral defense and provides valuable insights for future antiviral breeding strategies. Full article

Efficient reverse genetics systems are essential for understanding SARS-CoV-2 pathogenesis, host–virus interactions, and potential therapeutic interventions. Here, we developed a cost-effective PCR-based reverse genetics platform that splits the SARS-CoV-2 genome into only six bacterial plasmids, enabling cloning, manipulation, and the rescue of recombinant SARS-CoV-2 (rSARS-CoV-2) with high fidelity and high viral titers after a single passage. Using this system, we generated and characterized spike protein mutants Y453F and N501Y, as well as a U76G mutation in the 5′-UTR. Y453F showed reduced replication kinetics, lower cell binding, and diminished fitness, while N501Y exhibited comparable replication and fitness, highlighting the distinct effects of these spike protein mutations. The U76G mutation is located within a novel NSP9 binding site in the 5′-UTR and leads to impaired RNA synthesis and reduced viral replication efficiency, suggesting an important role in transcription and replication. Our findings highlight the robustness and adaptability of this reverse genetics system, providing a versatile, cost-effective tool for studying SARS-CoV-2 mutations and their effects on replication and fitness, with potential applications in vaccine and therapeutic development. Full article

Cytosine base editors (CBEs) enable precise C-to-T (G-to-A) conversions in genomic DNA, offering significant potential for specific gene editing. This study compared the prototypical Base Editor 3 (BE3) and a modified variant, BE3-Y130F, which utilizes an hA3A deaminase with the Y130F mutation, focusing on their editing efficiency and editing window characteristics using bovine zygotes. Following in vitro fertilization (IVF), sgRNA and Cas9 mRNA were injected as a targeting efficiency control, which resulted in 100% editing with no wild-type sequence. Then, either BE3 or BE3-Y130F mRNA, synthesized via in vitro transcription, and an sgRNA targeting exon 4 of the MYO7A gene was injected into zygotes. Genomic DNA was extracted from both blastocysts and developmentally arrested embryos, and Sanger sequencing was performed to evaluate C-to-T conversion efficiency and editing window. Both BE3 and BE3-Y130F achieved 100% C-to-T conversion efficiency at the primary target cytosine. BE3 displayed a defined editing window, primarily affecting cytosines at positions 7 and 8, indicating a predictable profile. In contrast, BE3-Y130F maintained high efficiency but had a less clearly defined editing window, resulting in incomplete editing and a remaining cytosine on the target sequence. Full article

Background: Circulating microRNAs (miRNAs) have emerged as potential biomarkers of platelet reactivity and thrombotic risk. Among them, miR-223-3p regulates P2Y12 receptor expression and may influence response to antiplatelet therapy. This study aimed to evaluate the prognostic value of selected circulating miRNAs in post-stroke patients receiving antiplatelet treatment. Methods: Sixty ischemic stroke survivors were prospectively enrolled and followed for 18 months for recurrent vascular events (stroke, transient ischemic attack, or myocardial infarction). Plasma levels of miR-126-3p, miR-223-3p, miR-24-3p, and miR-199a-5p were quantified using reverse transcription real-time PCR. Clinical data, antiplatelet regimen, statin use, and Essen Stroke Risk Scores (ESRS) were recorded. Logistic regression was applied to identify independent predictors of thrombotic events. Results: Expression of all examined miRNAs differed significantly across treatment groups. The dual antiplatelet therapy (DAPT) group showed the highest levels of miR-126-3p and miR-199a-5p (p < 0.01). Within the statin-naïve DAPT subgroup, lower miR-199a-5p levels (p < 0.001) were observed among patients who experienced ischemic events (n = 7/60; 12%; stroke = 4, TIA = 2, ACS = 1) during 18 months of follow-up. In multivariate analysis, reduced miR-223-3p remained the only independent predictor of recurrent thrombotic events (OR 1.18, 95% CI 1.01–1.37, p = 0.036), independent of ESRS and platelet reactivity. Elevated miR-126-3p and miR-199a-5p were associated with favorable treatment response, particularly among statin users. Conclusions: This study identifies low circulating miR-223-3p as an independent biomarker of thrombotic risk in post-stroke patients, potentially reflecting enhanced platelet activation via P2Y12 signaling. In contrast, higher miR-126-3p and miR-199a-5p levels may indicate more effective antiplatelet response. These findings support the potential utility of miRNA profiling for individualized antiplatelet therapy and long-term risk stratification after ischemic stroke. Full article

The potyvirus helper component proteinase (HcPro) is a multifunctional protein, with one of its most documented functions being host antiviral RNA silencing suppression. This study shows that the HcPro of potato virus Y (PVY), an important member of the potyvirus group, prevents the replication of a related competing secondary virus. This phenomenon, referred to as superinfection exclusion (SIE), is common in bacterial, human, and plant virus infections. We also report that HcPro’s induction of SIE is strain-specific and that this specificity is provided by the first four amino acid residues of the protein. Consistent with the mechanism of SIE, the study found that HcPro does not exclude a resident virus. Additionally, HcPro’s induction of SIE was observed to function independently of its ability to suppress antiviral RNA silencing. HcPro’s induction of SIE is relevant given the prevalence of multiple PVY strains that routinely co-infect the same cell and that may lead to recombination and emergence of new and more virulent strains. Furthermore, cross-protection or systemic acquired resistance (SAR) that is employed in plant virus disease management occurs when SIE moves from the cellular level and spreads systemically, emphasizing the importance of studying SIE. Full article

This study explores the role of human visceral white adipocytes (hv-WAD) in latent tuberculosis infection (LTBI). While granulomas and macrophages are traditionally viewed as central to TB latency, emerging evidence highlights adipocytes as significant non-canonical host cells that may facilitate bacterial persistence by providing a protective niche. Unlike the immune-driven environment within granulomas, adipocytes can shield Mycobacterium tuberculosis (Mtb) from immune surveillance, promoting survival. In vitro experiments showed that Mtb invades approximately 39% of hv-WAD within 48–72 h post-infection (hpi). Both avirulent H37Ra and virulent H37Rv Mtb strains, when infecting adipocytes, expressed RNA for key virulence factors (19 kDa, 30 kDa, Ag85b, 5KST, CFP10, and ESAT6) and dormancy-associated genes (Icl1, LipY, WhiB3, SodA, and Tgs1) at 72 hpi. Infection stimulated the production of inflammatory cytokines, notably leading to a fivefold increase in TNF-α with H37Rv (p < 0.01). Additionally, we detected Mtb RNA transcripts (IS6110, 5KST, 30 kDa, CFP10, Ag85) in 68% of biopsies from TB asymptomatic patients. The transcripts suggest a metabolically heterogeneous state of mycobacteria. These findings position visceral fat as a potential reservoir for Mtb in latent TB infection and underscore the development of novel diagnostic strategies targeting adipose tissue. Full article

Centronuclear and myotubular myopathies (CNMs) are rare, inherited muscle disorders characterized by muscle atrophy, weakness, and altered muscle fiber structure, primarily caused by mutations in MTM1, DNM2, or BIN1. The molecular mechanisms driving CNM are only partially understood, and no curative therapies are available. To elucidate molecular pathways involved in CNMs, we present an integrative multi-omics analysis across several CNM mouse models untreated or treated with pre-clinical strategies, combining transcriptomic, proteomic, and metabolomic datasets with curated interaction, metabolic, tissue, and phenotype knowledge using network-based approaches. Weighted Gene Co-expression Network Analysis (WGCNA) identified gene modules commonly altered in three CNM genetic forms. Modules correlated with improved muscle function were enriched for processes such as muscle contraction, RNA metabolism, and oxidative phosphorylation, whereas modules linked to disease severity were enriched for immune response, innervation, vascularization, and fatty acid oxidation. We further integrated transcriptomic, proteomic, and metabolomic data from the Mtm1^−/y mouse model with public knowledge bases into a multilayer network, and explored it using a random walk with restart approach. These analyses highlighted metabolites closely connected to CNM phenotypes, some of which may represent candidates for nutritional or pharmacological modulation. Our findings illustrate how integrative multi-omics and network analyses reveal both pathogenic and protective pathways in CNM and provide a foundation for identifying novel therapeutic opportunities. Full article

Background: Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, remains a leading cause of viral encephalitis. Current management is largely supportive, with no specific antivirals. This study evaluated the antiviral efficacy and mechanism of action of CC-90009 against JEV in vitro and in vivo. Methods: Five targeted protein degraders (TPDs) were screened for anti-JEV activity in the human neuroblastoma cell line SH-SY5Y. Time-of-addition, binding, and endocytosis assays were used to delineate the phase of action of CC-90009, a cereblon (CRBN) E3 ligase modulator (CELMoD) and molecular glue degrader. Small interfering RNA knockdown and co-immunoprecipitation (Co-IP) confirmed targets essential for its antiviral effects. The broad-spectrum activity of CC-90009 against other mosquito-borne viruses was also evaluated. In vivo efficacy was tested in a murine JEV model. Results: Of the five TPDs tested, only CC-90009 significantly inhibited JEV infection in SH-SY5Y cells, acting during both viral entry and post-entry phases without reducing adsorbed or internalised virions. CC-90009 reduced JEV RNA and non-structural protein accumulation. Knockdown of G1-to-S phase transition 1 (GSPT1), a key target of CC-90009, suppressed JEV infection and translation; Co-IP confirmed GSPT1 interaction with JEV non-structural protein 5 (NS5). CC-90009 disrupted JEV translation and replication by inducing proteasomal degradation of the GSPT1/NS5 complex, further demonstrating its broad-spectrum antiviral activity through the effective inhibition of West Nile virus and chikungunya virus. In vivo, it protected mice from JEV-induced mortality, reducing viral load, antigen levels, and brain pathology. Conclusions: CC-90009 exerts potent anti-JEV activity both in vitro and in vivo by inducing proteasomal degradation of the GSPT1/NS5 complex, thereby disrupting viral translation and replication. This targeted protein degradation strategy represents a novel host-directed antiviral approach with promising therapeutic potential against mosquito-borne viral encephalitis. Full article

Soybean cyst nematode (SCN, Heterodera glycines) is one of the major pathogens of soybean worldwide. We utilized the CHIP-Seq (chromatin immunoprecipitation sequencing) and RNA-Seq (RNA sequencing) data from the transgenic GmMYB29 strain (Glycine Max roots). We then performed enrichment analysis using KEGG and GO to identify potential candidate genes within the promoter-binding region. A targeted regulatory relationship between the GmMYB29 and GmPP2C-37like genes was further identified using the dual-luciferase Assay (Luciferase, LUC) and yeast one-hybrid Assay (Y1H). Hairy roots with target gene overexpression and gene-edited hairy roots were generated, and their resistance to soybean cyst nematode (SCN) was evaluated. Meanwhile, the presence of reciprocal genes with GmPP2C-37like was determined by the yeast two-hybrid library screening method. The targeting relationship between GmMYB29 and GmPP2C-37like genes was further validated through the Y1H assay and LUC assay. Based on phenotypic assessments of SCN, transgenic soybean roots overexpressing GmPP2C-37like exhibited significantly enhanced resistance to SCN 3 compared to wild-type. Further analysis revealed that GmPP2C-37like collaborates with other regulatory factors to modulate soybean resistance against SCN. Yeast two-hybrid library (Y2H) screening identified 18 interacting proteins. These findings not only illuminate the functional role of GmPP2C-37like but also provide a foundation for dissecting its molecular network. Moreover, the results offer promising candidate genes for enhancing SCN resistance and optimizing soybean resilience through targeted genetic strategies. Full article

Infertility affects about 17.5% of couples, with male factors accounting for approximately 50% of cases. Cytoskeletal remodeling is increasingly recognized as a critical component of male reproductive function, particularly in the regulation of testosterone synthesis by Leydig cells. However, the underlying molecular mechanisms remain poorly defined. Rho-associated coiled-coil-containing kinase (ROCK), a key cytoskeletal regulator, influences actin dynamics, impacting intracellular trafficking. In this study, we investigated the roles of ROCK1 and ROCK2 in Leydig cells using the TM3 cell model. Pharmacological inhibition of ROCK activity with Y-27632 impaired actin cytoskeleton organization, reduced the phosphorylation of LIMK, COFILIN, and MLC2, and disrupted the colocalization of F-actin with StAR and cholesterol, thereby decreasing testosterone production. Furthermore, RNA-seq revealed that hCG promotes transcription of steroidogenesis-related genes, while ROCK inhibition reverses this effect. Silencing of ROCK1 via siRNA mimicked the effects of ROCK-i, suppressing steroidogenic gene expression and testosterone synthesis. In contrast, ROCK2 knockdown enhanced testosterone secretion, promoted F-actin remodeling, and increased traffic of cholesterol targeting mitochondria. These opposing effects triggered distinct responses in the SCAP–SREBP2 axis, indicating a feedback mechanism regulating cholesterol homeostasis. Collectively, our findings uncover the isoform-specific roles of ROCK1 and ROCK2 in coordinating cytoskeletal dynamics and steroidogenic activity, providing new insights into the regulation of male reproductive endocrinology and identifying potential therapeutic targets for androgen deficiency and male infertility. Full article

Background/Objectives: Parkinson’s disease (PD) is the second most common neurodegenerative disease (NDD), marked by the progressive loss of dopaminergic neurons in the substantia nigra that causes motor dysfunction. Growing evidence indicates that neuroinflammation plays a crucial role in the onset and progression of PD, though the exact mechanisms are still unclear. In this study, we examined the anti-inflammatory and neuroprotective effects of 4-[3-oxo-3-(2-trifluoromethyl-phenyl)-propyl]-morpholinium chloride (OTPM), a fluoxetine derivative and selective serotonin reuptake inhibitor, in both lipopolysaccharide (LPS)-stimulated BV-2 microglial cells and an MPTP-induced mouse model of PD. Methods: C57BL/6 mice were orally administered OTPM (10 mg/kg b.w.) for 7 days and intraperitoneally injected with MPTP (20 mg/kg b.w.) for one day, with four injections at 2 h intervals. Bradykinesia was assessed using the Y-maze and Pole tests. Protein and mRNA levels were examined in vitro and in vivo using Western blotting and RT-PCR. Immunofluorescence was used to assess microglial and astrocyte activation. Results: In vitro, OTPM significantly decreased nitric oxide (NO) production (p < 0.001) and suppressed the protein and mRNA expression of iNOS (p < 0.001), COX-2 (p < 0.001), and pro-inflammatory cytokines, including IL-β (p < 0.001), IL-6 (p < 0.001), and TNF-α (p < 0.01), in LPS-activated BV-2 microglia. Further mechanistic studies showed that OTPM inhibited NF-κB phosphorylation and blocked its nuclear translocation, thereby reducing inflammatory signaling. In vivo, treatment with OTPM (10 mg/kg for 7 days) significantly reduced the MPTP-induced activation of microglia (MAC-1) and astroglia (GFAP) in the brain and improved behavioral deficits associated with PD, as assessed in the Y-maze and pole tests. Conclusions: Overall, these results reveal that OTPM has strong anti-neuroinflammatory and neuroprotective properties, suggesting its potential as a new therapeutic candidate for PD and other disorders associated with neuroinflammation. Full article

Treatment-induced neuroendocrine prostate cancer (t-NEPC) is a highly aggressive and therapy-resistant subtype of prostate cancer characterized by lineage plasticity and poor response to standard chemotherapy and androgen deprivation therapy. Although transcriptional mechanisms driving t-NEPC have been extensively studied, the contribution of post-transcriptional regulation remains less defined. Here, we report fibroblast growth factor 12 (FGF12) as a critical post-transcriptional regulator of t-NEPC progression. Transcriptomic analyses of patient biopsies, patient-derived xenografts, and prostate cancer cell models consistently demonstrated elevated FGF12 expression in t-NEPC, which was further validated by immunohistochemistry in archival specimens. Functional assays revealed that FGF12 expression conferred survival of cancer cells to chemotherapeutic agents, including etoposide and camptothecin. Integrative RNA sequencing and affinity purification–mass spectrometry showed that FGF12 mediates these functions mainly through interaction with the RNA-binding protein YB1, leading to stabilization of oncogenic long noncoding RNAs, including NEAT1 and MALAT1, whereas RNA silencing of YB1 abrogated the ability of FGF12 to upregulate these transcripts. Collectively, these findings uncover a previously unrecognized FGF12-YB1-lncRNA signaling axis that drives t-NEPC progression. Targeting this pathway may provide new therapeutic opportunities for patients with this aggressive disease. Full article

Platelets are increasingly recognized as multifunctional cells with roles extending beyond hemostasis to immune regulation, inflammation, and neurodegeneration. Here, we performed RNA-Seq profiling of platelets from patients with idiopathic REM sleep behavior disorder (IRBD), dementia with Lewy bodies (DLB), Parkinson disease (PD), Alzheimer disease (AD), and healthy controls (CTRLs) to explore disease-specific transcriptomic signatures. Across all groups, the RNA class distribution was similar, dominated by mRNAs (78–80%) and long non-coding RNAs (lncRNAs; 15–16%). DLB platelets displayed a reduced proportion of lncRNAs, suggesting an impaired RNA regulation, whereas IRBD concentrated the highest number of disease-specific lncRNAs, half of which were Y-linked, consistent with the male predominance observed in alpha-synucleinopathies. Differential expression analysis (DEA) revealed extensive transcriptomic remodeling in IRBD and DLB, particularly affecting RNA processing, cytoskeletal organization, and platelet activation pathways, while PD and AD showed minimal changes. These findings suggest a progressive impairment of platelet activation and signaling across the DLB continuum, potentially mirroring neuronal dysfunction. The limited transcriptional deregulation in PD may reflect its pronounced biological heterogeneity, consistent with recent multidimensional disease models. Overall, our study highlights platelets as accessible indicators of early and disease-stage-specific molecular alterations in α-synucleinopathies. Full article

Brassica juncea is an important leafy vegetable, and flowering time is a key determinant of its yield and quality. In this study, one significantly up-regulated gene, BjuAGL9-2, was identified from RNA-Seq data. qRT-PCR analysis confirmed that BjuAGL9-2 expression was significantly elevated in reproductive organs and reproductive stages. Further five BjuAGL9-2 over-expression (OE) lines were subsequently generated, which showed an early-flowering and pale-yellow leaf phenotype compared to the wild type. qRT-PCR assays found that the mRNA of core floral integrator genes was changed in Arabidopsis OE lines. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays indicated that BjuAGL9-2 interacted with BjuTUA5, BjuZFP7, BjuGSTU5, and BjuMAPK16 in vivo. Sub-cellular localization assays showed that BjuAGL9-2 localizes in the nucleus, whereas its interacting partners localize in the cytoplasm. qRT-PCR assays further revealed that BjuTUA5 and BjuGSTU5 were up-regulated in flower buds, while BjuZFP7 and BjuMAPK16 were down-regulated. During vegetative stages, all four genes were up-regulated in B. juncea. As for BjuAGL9-2 interaction protein-encoding homolog genes, except AtGSTU5, the other three genes were up-regulated in Arabidopsis OE lines. Additionally, qRT-PCR analysis of chlorophyll biosynthesis-related genes showed that 19 of 27 genes were up-regulated, while 8 genes were down-regulated, in Arabidopsis OE lines. Collectively, these findings suggest that BjuAGL9-2 promotes flowering and contributes to the pale-yellow phenotype by regulating its interacting protein-coding genes, floral integrators, and chlorophyll biosynthesis genes. Full article