Search Results (64)

Background/Objectives: Osteotoxicity is a severe complication of Methotrexate (MTX) chemotherapy, characterized by oxidative stress and disrupted bone remodeling. The primary objective of this study was to investigate the cytoprotective mechanisms of the antioxidant Alpha-Lipoic Acid (ALA) against MTX-induced osteotoxicity, specifically focusing on its modulation of oxidative stress, apoptosis, and Mitogen-Activated Protein Kinase (MAPK) signaling pathways. Methods: Murine osteocyte-like MLO-Y4 cells were cultured and exposed to a fixed dose of MTX (10⁻⁵ M), either alone or concurrently with ALA (50 μmol/L) for 48 h. Biochemical profiling was performed using specific enzyme-linked immunosorbent assays (ELISA) and colorimetric kits to evaluate pro- and anti-apoptotic proteins (Caspase-3, Bax, Bcl-2, Wee1, GRP78, GADD153, AIF), active MAPK components (p-JNK, p-ERK), and standard oxidative stress parameters (TAS, TOS, SOD, GPx). Results: MTX treatment induced significant cellular stress, evidenced by elevated Caspase-3, Bax, p-JNK, and p-ERK levels, alongside a critical reduction in Bcl-2 expression. MTX also markedly increased TOS while depleting TAS, SOD, and GPx levels. Conversely, co-treatment with ALA significantly mitigated these cytotoxic responses. ALA restored the Bax/Bcl-2 balance, effectively downregulated both p-JNK and p-ERK activation, and substantially reinforced the cellular antioxidant defense system by enhancing TAS, SOD, and GPx activities, although recovery to baseline control levels was partial. Conclusions: ALA exerts robust in vitro cytoprotective effects against MTX-induced osteotoxicity in MLO-Y4 cells by counteracting oxidative stress and inhibiting aberrant apoptotic and MAPK signaling. These findings establish a mechanistic baseline, underscoring the need for subsequent in vivo dose–response studies to validate ALA’s therapeutic potential in chemotherapy management. Full article

Long non-coding RNAs (lncRNAs) and RNA–protein complexes (RNPs) are increasingly recognized as central to the regulatory complexity of modern eukaryotes. This review proposes that the remarkable diversity of eukaryotic systems arises from the long-term integration of ancient RNA/RNP mechanisms, layered with innovations introduced by successive symbioses. We outline four interconnected levels of symbiosis contributing to this process: (1) molecular symbiosis, involving dynamic assemblies of RNAs, proteins, and membraneless organelles (MLOs); (2) genome symbiosis, driven by the expansion of non-coding and repetitive DNA; (3) intracellular symbiosis, initiated by mitochondria acquisition; and (4) intercellular symbiosis, rooted in the cellular cooperation that enables multicellularity. We highlight lncRNAs and intrinsically disordered proteins (IDPs) as versatile mediators that interweave interactions across scales, predominantly within phase-separated condensates. Building upon these multi-level processes, we propose the framework of integrated symbiotic pleiotropy—a concept where molecular components acquire layered functional roles as a direct consequence of successive symbiotic acquisitions. This paradigm unites information layering, functional moonlighting, molecular tinkering, and exaptation into a coherent trajectory for eukaryotic evolution. Full article

Purpose: Osteotoxicity is a well-recognized adverse effect of Methotrexate (MTX) therapy, primarily driven by oxidative stress and impaired bone remodeling. This study aimed to investigate the protective effects of Tempol, a membrane-permeable nitroxide antioxidant, against MTX-induced osteotoxicity, and to assess how these effects are influenced by ML210, a glutathione peroxidase 4 (GPX4) inhibitor. Methods: Murine osteocyte-like MLO-Y4 cells were treated with MTX alone, Tempol alone, or a combination of MTX with Tempol and ML210. Apoptotic markers (caspase-3, Bax, Bcl-2), MAPK signaling proteins (p-JNK, p-ERK), and oxidative stress parameters (TAS, TOS, SOD, GPx) were measured via ELISA to evaluate the redox and apoptotic responses. Results: MTX significantly induced apoptosis, as evidenced by increased caspase-3 activity and Bax expression, along with decreased Bcl-2 levels. MTX also activated the MAPK pathway by upregulating p-JNK and p-ERK. Furthermore, MTX decreased TAS, SOD, and GPx levels, while increasing TOS. Tempol treatment successfully reversed these effects, restoring apoptotic balance, inhibiting MAPK activation, and enhancing antioxidant capacity. However, co-treatment with ML210 markedly attenuated Tempol’s protective effects, resulting in sustained oxidative stress, elevated apoptotic markers, and persistent MAPK pathway activation. This suggests that Tempol’s cytoprotective actions are dependent on functional GPX4 activity. Conclusion: Tempol exhibits strong potential as an adjunctive antioxidant therapy to counteract MTX-induced osteotoxicity. Nevertheless, its efficacy is significantly influenced by the status of the endogenous antioxidant enzyme GPX4. These findings underscore the need for further investigation into Tempol’s mechanism of action in redox-dependent pathways and its suitability in clinical settings, especially where GPX4 function may be compromised. Full article

MLO (Mildew Resistance Locus O) genes encode seven-transmembrane proteins that function as critical regulators of powdery mildew resistance and abiotic stress responses. Despite their established importance, the MLO gene family in Gossypium hirsutum L. has not been systematically investigated under salt stress conditions. Here, we performed genome-wide identification of 46 GhMLO members using Hidden Markov Model and BLAST searches based on the latest cotton genome assembly. Phylogenetic analysis classified these genes into four distinct subfamilies. Transmembrane topology and conserved domain analyses revealed that all GhMLO proteins contain typical MLO domains and transmembrane structures, maintaining high structural similarity with dicotyledonous model plants. Synteny analysis demonstrated that the expansion of the GhMLO family was primarily driven by segmental and tandem duplications. Integration of transcriptomic data from the COTTONOMICS database revealed tissue-specific expression patterns, with higher transcript abundance in receptacles, stems, and roots, but lower levels in stamens and petals. Salt, drought, and cold stress treatments induced upregulation of GhMLO family members, with most genes showing increased expression over time. RT-qPCR analysis validated that five candidate GhMLO genes were significantly upregulated under salt stress. In summary, this study provides a comprehensive genome-wide characterization of the GhMLO gene family, elucidating their phylogenetic relationships and expression dynamics, which establishes a theoretical basis for identifying key regulatory genes involved in abiotic stress responses and offers novel genetic resources for improving stress tolerance in cotton molecular breeding. Full article

Barley grown in temperate regions is often challenged by powdery mildew disease. An effective solution is mildew resistance locus o (mlo)-based resistance, which is monogenic, durable, and broad-spectrum. While the pleiotropic effects of mlo mutations on above-ground tissues are well documented, their impact on the root-associated microbiome remains underexplored. We utilized CRISPR/Cas9 to generate novel mlo mutant lines and evaluated their resistance to causal fungus Blumeria hordei. We further examined if mlo knockout has any impact on the overall root microbiome diversity and composition under field-like conditions and applied DESeq2 to compare the abundance of microbial taxa between mutants and wild type. We created five novel resistant mlo lines, including the first mutants with amino acid alterations in the protein’s extracellular region. Mutant lines showed significantly reduced B. hordei colony formation (0.5–5%). While microbial alpha and beta diversity were not significantly altered, a few microbial taxa displayed time-dependent shifts in abundance. Overall, our study demonstrates the effectiveness of CRISPR/Cas9 in generating mlo-based resistance. Moreover, the study revealed functionally important residues in the protein’s extracellular region. Finally, we present the first evidence of limited mlo-associated effects on root microbiome diversity and relative abundance of microbial taxa. Full article

Bone remodeling is maintained through the coordinated actions of osteoblasts, osteoclasts, and osteocytes, among which osteocytes serve as major regulators of osteoclast-mediated bone resorption through the receptor activator of the nuclear factor-κB ligand (RANKL)–osteoprotegerin (OPG) signaling axis. While molecular signals regulating osteocytic RANKL-OPG expression are fairly understood, how post-transcriptional mechanisms impact osteocyte function remains poorly defined. HuR (human antigen R) encoded by Elavl1 (embryonic lethal abnormal vision-like 1), a ubiquitously expressed RNA-binding protein, is known for stabilizing AU-rich element-containing transcripts involved in inflammatory and stress responses; however, its role in osteocyte-derived bone resorption is unknown. In this study, we examined the effect of HuR loss on osteocyte–osteoclastogenesis. Short hairpin RNA (shRNA)-mediated HuR knockdown in MLO-Y4 osteocyte-like cells resulted in a significant increase in OPG mRNA and its protein expression, whereas RANKL levels remained unchanged, leading to a significantly reduced RANKL/OPG ratio. Both co-culture and conditioned-medium assays demonstrated that HuR-deficient osteocytes produced a markedly diminished osteoclastogenic environment. Actinomycin D chase experiments showed no alteration in OPG mRNA decay kinetics, and RNA immunoprecipitation (RIP)-PCR failed to detect HuR–OPG interactions, indicating that HuR regulates OPG expression through indirect mechanisms rather than mRNA binding. These findings identify HuR as an indirect regulator of osteocyte-derived OPG expression that impacts osteoclast differentiation and reveal a previously unrecognized mechanism by which HuR contributes to bone remodeling. Full article

Powdery mildew poses a significant threat to watermelon production. The development of disease-resistant varieties through gene editing represents a major focus in current breeding research. In this study, we identified an MLO family gene in watermelon, denoted by ClMLO5b, which is phylogenetically closely related to cucumber CsaMLO8 and melon CmMLO5. Homology modeling revealed high conservation of the three-dimensional protein structures among these orthologs. Expression analysis demonstrated that ClMLO5b is significantly up-regulated upon powdery mildew infection, and the protein localizes to the plasma membrane. To validate its function, we first employed an Agrobacterium rhizogenes-mediated hairy root transformation system to rapidly verify the editing efficiency of two CRISPR/Cas9 targets designed for ClMLO5b. Subsequently, stable transgenic watermelon plants were generated via Agrobacterium tumefaciens-mediated transformation, and a mutant line with homozygous substitutions at target site 2 was obtained. Disease resistance assays showed that, compared to wild-type plants, the Clmlo5b exhibited strongly inhibited mycelial growth, significantly reduced disease severity, and a substantial decrease in spore production after inoculation with powdery mildew. Our findings confirm that ClMLO5b is a key susceptibility gene in watermelon and provide both a promising genetic target and valuable breeding material for developing powdery mildew-resistant watermelon varieties. Full article

Liquid–liquid phase separation (LLPS) is a fundamental biophysical process in which proteins and nucleic acids dynamically demix from the cellular milieu to form membraneless organelles (MLO) with liquid-like properties. Environmental cues, such as light, temperature fluctuations, and pathogen interactions, induce LLPS of fungal proteins with intrinsically disordered regions (IDRs) or multimerization domains, thereby regulating fungal hyphal growth, sexual reproduction, pathogenesis, and adaptation. Recently, LLPS has emerged as a powerful tool for biomolecular research, innovative biotechnological application, biosynthesis and metabolic engineering. This review focuses on the current advances in environmental cue-triggered fungal condensates assembled by LLPS, with a focus on their roles in regulating the fungal physical biology and cellular processes including transcription, RNA modification, translation, posttranslational modification process (PTM), transport, and stress response. It further discusses the strategies of engineering synthetic biomolecular condensates in microbial cell factories to enhance production and metabolic efficiency. Full article

Liquid–liquid phase separation (LLPS) in cell biology has revolutionized our understanding of how cells organize biochemical reactions and structures through dynamic, membraneless organelles (MLOs). In neurons, LLPS-driven processes are particularly important for regulating synaptic plasticity, RNA metabolism, and responses to environmental stressors. Over the past decade, LLPS has gained increasing attention in neurobiology as a framework to interpret altered synaptic functions in various neurodevelopmental disorders (NDDs). These diseases comprise a diverse spectrum of clinical and pathological symptoms (e.g., global developmental delay, impaired cognitive and mental functions, as well as social withdrawal). Recent studies have highlighted how mutations in proteins containing intrinsically disordered regions (IDRs)—key drivers of LLPS—can alter condensate properties, resulting in persistent or defective MLO formation. These aberrant assemblies may disrupt RNA transport, splicing, and translation in developing neurons, thereby contributing to disorder pathology. IDRs are known to be enriched in membraneless components, such as stress granules, nuclear paraspeckles, and P-bodies, where they play crucial role in the formation, maintenance, and function of protein–RNA networks. This review explores the role of stress-induced MLOs in the nervous system, the molecular principles governing their formation, and how their dysfunction bridges the gap between environmental stress responses and neurodevelopmental impairment. Full article

Promyelocytic leukemia nuclear bodies (PML-NBs) are dynamic membrane-less organelles (MLOs) located in the nucleus that serve as regulatory hubs for multiple cellular processes. This review examines current understanding of PML-NB structure, assembly mechanisms, and their diverse functional roles. We discuss how PML-NBs interact with chromatin to influence gene expression, regulate transcription factors, and participate in protein quality control. The review highlights their critical functions in tumor suppression, particularly in acute promyelocytic leukemia, and their role in intrinsic antiviral defense against various pathogens. Despite significant advances in the field, key questions remain regarding the mechanistic triggers of PML-NB formation and their common roles across different pathologies. Further elucidation of these aspects may provide valuable insights for developing therapeutic approaches targeting the PML-NB axis in disease treatment. Full article

Powdery mildew (PM), caused by Sphaerotheca phaseoli, severely threatens mungbean (Vigna radiata) productivity and quality, yet the molecular basis of resistance remains poorly defined. This study employed transcriptome profiling to compare defense responses in a resistant genotype, SUPER5, and a susceptible variety, CN84-1, following pathogen infection. A total of 1755 differentially expressed genes (DEGs) were identified, with SUPER5 exhibiting strong upregulation of genes encoding pathogenesis-related (PR) proteins, disease resistance proteins, and key transcription factors. Notably, genes involved in phenylpropanoid and flavonoid biosynthesis, pathways associated with antimicrobial compound and lignin production, were markedly induced in SUPER5. In contrast, CN84-1 showed limited activation of defense genes and downregulation of essential regulators such as MYB14. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses highlighted the involvement of plant–pathogen interaction pathways, MAPK signaling, and reactive oxygen species (ROS) detoxification in the resistant response. Quantitative real-time PCR validated 11 candidate genes, including PAL3, PR2, GSO1, MLO12, and P21, which function in pathogen recognition, signaling, the biosynthesis of antimicrobial metabolites, the production of defense proteins, defense regulation, and the reinforcement of the cell wall. Co-expression network analysis revealed three major gene modules linked to flavonoid metabolism, chitinase activity, and responses to both abiotic and biotic stresses. These findings offer valuable molecular insights for breeding PM-resistant mungbean varieties. Full article

The Mildew Resistance Locus (Mlo) gene family is reported in various species as regulators of powdery mildew (PM) resistance. However, the Mlo genes in cucurbit crops remain limited. In this study, a genome-wide investigation of Mlo genes was conducted in eight Cucurbitaceae species and in rice, maize, arabidopsis, and barley, and a total of 202 Mlo genes were identified. The phylogenetic analysis showed that 202 Mlo genes can be classified into six clades, and the Mlo genes from clades I and III are likely pivotal in regulating PM resistance in dicotyledonous and monocotyledonous plants, respectively. The Ka/Ks ratios for these homologous Mlo gene pairs ranged from 0 to 0.6, revealing that they underwent substantial purifying selection during evolution. Among 12 crops, there were the most Mlo genes (22 Cm-mlo) in melon. An expression analysis revealed that six Cm-mlo genes showed expression responses to PM infection in which Cm-mlo38 and Cm-mlo44 were phylogenetically close to Mlo genes that regulated PM resistance. Using the VIGS system for silencing, Cm-mlo38 and Cm-mlo44 enhanced resistance to PM in susceptible material. A protein interaction analysis indicated that Cm-mlo38 might regulate PM resistance through interactions with PR5 and CML proteins. These results provide a comprehensive understanding of the Mlo family in Cucurbitaceae and pave the way for future functional analysis and genetic improvement for improving PM resistance in Cucurbitaceae. Full article

The venom of Macrovipera lebetinus obtusa (MLO) has remarkable properties that are hard to overlook. This venom’s described 38 protein components work synergistically, forming complexes that greatly enhance their combined effectiveness. Previous studies have shown that both crude venom and one of its components, obtustatin, can reduce sarcoma tumors by 50% and 30%, respectively. Obtustatin, a member of the short disintegrin family, inhibits the angiogenic activity of α1β1 integrin, the adhesive receptor of collagen IV. However, the mechanisms of the greater efficacy of the crude venom compared to its isolated components remain unclear. To investigate this, we propose an experimental work to explore the activity of certain low-molecular-weight components of MLO venom. Our in vitro tests on fibrosarcoma (HT-1080) cells using six venom fractions revealed cytotoxic fractions, which, through mass spectrometry, were identified as containing protein classes such as dimeric and short disintegrins, acidic phospholipase A2, and serine proteinases. Notably, these fractions exhibited minimal toxicity to human dermal microvascular endothelial (HDEC) cells, suggesting their potential as a promising candidate for oncotherapy in the future. Full article

European plum production is affected by mostly harm Monilinia spp., causing full pathogen brown-rot infections. The plums are the susceptible to the Monilinia fructigena pathogen, which is the most common in Europe. This study aims to analyze the gene expression profiles and signaling pathways of the European plum, cv. Victoria, inoculated with the M. fructigena pathogen at 24, 48, and 72 h post inoculation. By transcriptome sequencing, the number of differentially expressed genes (DEGs) increased over time, with the highest number at 72 hpi, showing the tendency to involve more genes in the response to prolonged exposure to the pathogen. Pathogenesis-related (PR) family and mildew resistance locus O (MLO-like) proteins were expressed the most during plum response to the pathogen. The plum initiates complex defense responses by significantly activating 23 pathways according to Kyoto Encyclopedia of Genes and Genomes (KEGG). In this study, expressed genes over the infection were in response to stress, defense, cell death, and disease resistance. The findings of this study could be used as the basis for further research of markers linked to resistance or susceptibility to disease in plum hybrids at an early age, which will improve the plum breeding process. Full article

Background/Objective: Osteocytes are the most abundant cell type in the skeleton, with key endocrine functions, particularly in regulating osteoblast and osteoclast activity to maintain bone quality. Angiotensin II (Ang II), a critical component of the renin–angiotensin–aldosterone system, is well-known for its role in vasoconstriction during hypertension. Beyond its cardiovascular functions, Ang II participates in various biological processes, including bone metabolism. While its influence on osteoblast proliferation, differentiation, and osteoclastogenesis has been documented, its effects on osteocytes remain unexplored. This study hypothesized that Ang II enhances the osteoclastogenic activity of osteocytes. Methods: Mouse calvariae were cultured ex vivo in an Ang II-containing medium, analyzed via immunohistochemistry, and evaluated for osteoclastogenic gene expression through real-time PCR. Western blotting was employed to assess protein levels and signaling pathway activation in the MLO-Y4 osteocytic cell line in vitro. Results: Ang II significantly increased the expression of receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). These effects were abrogated by azilsartan, a blocker targeting Ang II type 1 receptors (AT1R). p38 and ERK1/2 in the MAPK pathway were also activated by Ang II. Conclusions: Ang II enhances osteocyte-mediated osteoclastogenesis via AT1R activation, highlighting its potential as a therapeutic target for bone diseases. Full article