Search Results (610)

Background/Objectives: Triple-negative breast cancer (TNBC) is frequently characterized by notably elevated Ki-67 expression, a hallmark of uncontrolled rapid cell-cycle progression. However, the underlying mechanisms remain unclear, leading to limited therapeutic options. Methods: In this study, hub gene was identified through integrated bioinformatic analysis of public datasets (TCGA-BRCA and METABRIC). Subsequent functional validation was performed both in vitro and in vivo using siRNA-mediated knockdown and small-molecule inhibitors. Phenotypic effects—including cell viability, cell cycle distribution, DNA synthesis, and clonogenic survival—were comprehensively assessed using MTT assays, flow cytometry, EdU, and colony formation assays. Protein-level changes were confirmed by Western blotting and immunohistochemistry (IHC). To dissect the transcriptional regulation of the key hub gene TTK, we first predicted potential upstream transcription factors using the JASPAR database; binding specificity was then validated through in silico motif analysis, luciferase reporter assays, and chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR). Results: The mitotic kinase TTK is significantly overexpressed in TNBC compared with non-TNBC breast cancers. Notably, TTK overexpression exhibited a strong positive correlation with elevated Ki-67 indices and reduced overall survival in TNBC patients. Functional validation demonstrated that pharmacological or genetic inhibition of TTK effectively induced G2/M cell-cycle arrest and potently suppressed TNBC proliferation in both in vitro cell cultures and in vivo xenograft models. Mechanistically, TTK overexpression stems from enhanced transcriptional initiation driven by the transcription factor NFYA binding to the CCAAT box in the TTK promoter—an interaction newly identified here. Concurrently, TTK blockade disrupted spindle assembly checkpoint (SAC) signaling via BUB1B/MAD1L1 downregulation, triggering mitotic arrest and catastrophe. Conclusions: Collectively, these findings establish TTK as a key cell-cycle regulator driving TNBC proliferation. More importantly, targeting mitotic control through TTK inhibition represents an efficient strategy to impede the aberrantly fast cell cycle progression in TNBC. Full article

Fragment-based drug discovery (FBDD) has emerged as a primary approach for identifying low molecular weight leads that can be systematically optimized into high-affinity compounds. Because fragments bind inherently weakly, their detection relies on highly sensitive biophysical tools. Nuclear magnetic resonance (NMR) spectroscopy is uniquely qualified for fragment screening due to its capability in detecting weak interactions across a broad affinity range while providing site-specific binding information that supports structure-guided optimization. While FBDD is a mature field for protein targets, structured and disease-relevant RNAs have transitioned from ‘undruggable’ molecules to viable therapeutic targets for small-molecule intervention. Recent studies demonstrate that NMR-based screening can identify authentic RNA binders and guide their evolution into potent, selective ligands. This review summarizes the practical and methodological pipelines for RNA-targeted small molecule NMR screening, covering RNA construct design, sample preparation, and library pooling strategies. We evaluate both ligand- and RNA-observed NMR assays for primary hit screening and validation, integration of NMR restraints with structural modeling, and representative case studies. Finally, we discuss current bottlenecks in the field and highlight emerging strategies to accelerate the discovery of RNA-directed therapeutics. Full article

Post-infectious Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a chronic disease with unresolved pathophysiology and limited diagnostic options. Extracellular vesicles (EVs) carry disease-specific protein and miRNA signatures and may enable improved disease profiling. We aimed to identify novel protein and miRNA markers as potential biomarkers in plasma EVs from female ME/CFS patients, including post-COVID-19 ME/CFS and post-infectious ME/CFS of other origins, compared with healthy controls. EVs were isolated from plasma by size-exclusion chromatography and characterized for number, size, morphology, and surface marker expression. Flow cytometry showed that small EVs strongly expressed tetraspanins, with only minor differences between ME/CFS patients and healthy donors. Proteomic profiling of EVs from ME/CFS patients identified altered cargo proteins, including hemoglobin subunit alpha and insulin-like growth factor-binding protein acid labile subunit compared with healthy controls (n ≤ 10/cohort). Small RNA sequencing followed by qPCR revealed significant downregulation of hsa-let-7b-5p in EVs from post-COVID-19 ME/CFS patients (n = 12) versus healthy controls (n = 15). Reduced hsa-let-7b-5p expression correlated with impaired physical functioning and increased fatigue, pain, and immune activation. These findings indicate that EV cargo differences, particularly hemoglobin subunit alpha and insulin-like growth factor-binding protein acid labile subunit, as well as hsa-let-7b-5p, represent promising candidates for ME/CFS diagnosis and patient stratification. Full article

Human Pegivirus (HPgV) is an understudied flavivirus that is highly prevalent and often persists in the blood and tissues of humans. HPgV-infected brain tissue from individuals with Parkinson’s disease has shown significant transcriptomic and immune signaling differences compared to non-infected Parkinson’s brains. The HPgV genome is similar to Hepatitis C Virus (HCV), a well-characterized flavivirus with multiple approved small-molecule therapeutics. Here, we used HCV crystal structures to create homology models for two HPgV non-structural (NS) proteins, the serine protease (NS3) and the RNA-dependent RNA polymerase (NS5B), and performed molecular dynamic simulations. HCV and HPgV proteins had minimal structural differences, as seen by the Root Mean Square Deviation (RMSD) difference between NS3 (1.00 Å) and NS5B (1.26 Å). FDA-approved small molecules were then docked in silico to the NS3 and NS5B subunits of HCV and HPgV. HCV had weak to moderate correlated docking scores with HPgV NS3 (R² = 0.21, p < 0.001) and NS5B (R² = 0.58, p < 0.001). The predicted protein–ligand interactions showed potential binding between HCV antivirals and conserved residues of HPgV, including the catalytic triad for NS3 or the GDD motif for NS5B. Together, these results provide structural insights for key HPgV proteins and highlight possibilities for therapeutic repurposing of HCV antivirals. Full article

Background/Objectives: Alzheimer’s disease (AD) is characterized by progressive neurodegeneration driven by interconnected mechanisms, including oxidative stress, mitochondrial dysfunction, neuroinflammation, synaptic impairment, and abnormal protein aggregation. MicroRNAs (miRNAs) have emerged as post-transcriptional regulators of these complex pathways; however, efficient delivery remains a major limitation. Small extracellular vesicles (sEVs) have been proposed as biologically compatible carriers for miRNA delivery. Methods: In this study, milk-derived sEVs were isolated, characterized, and loaded with microRNA-137-5p (miR-137-5p). Their effects were evaluated in an amyloid-β (Aβ)-induced in vitro AD model using SH-SY5Y human neuroblastoma cells. Oxidative stress markers, including reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), lactate dehydrogenase (LDH), and glutathione peroxidase 1 (GPX1), were assessed. Inflammation- and neuroprotection-related gene expression analyses included intercellular adhesion molecule 1 (ICAM1), tumor necrosis factor alpha (TNF-α), and brain-derived neurotrophic factor (BDNF). Cytoskeletal injury was evaluated using neurofilament light chain (NfL). Mitochondrial stress markers included cytochrome c (Cyt-c), 8-hydroxy-2′-deoxyguanosine (8-OHdG), PTEN-induced kinase 1 (PINK1), dynamin-1-like protein (DNM1L), and mitochondrial transcription factor A (TFAM). Synaptic and extracellular matrix-associated proteins, including complexin-2 (CPLX2), SPARC-related modular calcium-binding protein 1 (SMOC1), and receptor tyrosine kinase-like orphan receptor 1 (ROR1), as well as AD-related biomarkers, including total tau, phosphorylated tau at threonine 181 (pTau-181), phosphorylated tau at threonine 217 (pTau-217), and amyloid-β 1–40 (Aβ1–40), were evaluated using molecular and biochemical approaches. Results: Aβ exposure was associated with increased oxidative stress, inflammatory activation, mitochondrial and cytoskeletal alterations, synaptic-related disturbances, and elevations in tau- and amyloid-associated proteins. Treatment with unloaded sEVs was associated with partial modulation of several parameters, whereas miR-137-5p-loaded sEVs were consistently associated with normalization of multiple pathological markers toward control levels. Conclusions: These findings indicate that miR-137-5p-enriched sEVs may represent a useful experimental platform for multi-target modulation of AD-related cellular alterations. Further mechanistic and in vivo studies are required to clarify translational relevance. Full article

G3BP2 is an important RNA-binding protein that belongs to the mammalian Ras-GAP SH3 domain-binding protein (G3BP) family. Its structure enables it to bind to RNA or proteins, regulate nuclear–cytoplasmic shuttling, and participate in various functions, including cell growth, differentiation, migration, and RNA and protein metabolism. Studies have found that G3BP2 is involved in the occurrence and development of various human diseases, such as high expression across multiple tumor diseases, including gastric cancer, breast cancer, non-small-cell lung cancer, esophageal squamous cell carcinoma, colorectal cancer, and pancreatic ductal adenocarcinoma, driving the occurrence of human tumors, participating in tumor progression, and playing an essential role in promoting the proliferation, invasion, and migration of tumor cells. Additionally, G3BP2 is closely associated with various non-tumor diseases, including viral infections, as well as cardiovascular and cerebrovascular diseases. This review elucidates the role of G3BP2 in the development and progression of various diseases, identifying biomarkers and therapeutic targets for clinical diagnosis and treatment based on G3BP2. Full article

Ophthalmic diseases, including inherited retinal dystrophies, age-related macular degeneration (AMD), and glaucomatous neuropathies, are often driven by the expression of pathogenic proteins or dysfunctional non-coding RNAs that are currently considered ‘undruggable’ with conventional small-molecule therapeutics. The emerging strategy of Ribonuclease-Targeting Chimeras (RIBOTACs) offers a revolutionary approach to address this therapeutic gap. RIBOTACs are heterobifunctional small molecules designed to bind a specific target RNA with one moiety and recruit a latent endogenous ribonuclease, such as RNase L, with the other, thereby catalyzing the RNA’s degradation. This targeted degradation can potentially halt the production of mutant proteins, eliminate toxic gain-of-function RNAs, or modulate key regulatory pathways involved in angiogenesis, inflammation, and apoptosis—core processes in many blinding diseases. This review explores the immense potential of applying RIBOTAC technology to ophthalmology, discussing prospective targets such as mutant alleles in retinitis pigmentosa, VEGF transcripts in neovascular AMD, and inflammatory mediators in uveitis. We will also address the unique challenges and opportunities for RIBOTAC development in the eye, including delivery strategies to overcome ocular barriers, the need for high specificity to avoid off-target RNA degradation, and the optimization of pharmacokinetic properties for intraocular administration. With continued innovation, RIBOTACs are poised to evolve into a robust therapeutic platform, expanding the druggable genome and enabling precise, durable treatments for a range of currently intractable ophthalmic conditions. Full article

Post-transcriptional regulation of gene expression is influenced by RNA-binding proteins (RBPs) and small non-coding RNAs that bind to conserved mRNA sequences to modulate mRNA processing. These regulatory molecules affect the structural conformation of mRNAs, creating formations like G-quadruplexes (G4s), which alter translation initiation and regulatory-factor site accessibility. Recent studies have highlighted Nuclear factor erythroid 2–related factor 2 (NRF2) as a key regulator of cellular redox homeostasis and cellular response to oxidative stress. An intriguing feature of NRF2 is the structural formation of its 5′ untranslated region (UTR), which may promote or inhibit translation initiation depending on the cellular context. In this study with minigenes, we provide in vitro evidence of RNA G4s in the NRF2 mRNA’s 5′ UTR under basal (no stress) conditions. Achieved via electrophoretic mobility shift assay and fluorescence spectra in the presence of Pyridostatin. Understanding how structural motifs within NRF2 5′ UTRs influence mRNA function provides insights into a common molecular mechanism underlying diseases where NRF2 is dysregulated, like cancers, cardiovascular disease, and neurodegeneration, and highlights potential therapeutic avenues through regulation of NRF2. Full article

Spermatogenesis is a tightly coordinated differentiation program that sustains male fertility while transmitting genetic and epigenetic information to the next generation. This review consolidates mechanistic evidence showing how RNA-centered regulation integrates with the epitranscriptome and three-dimensional (3D) genome architecture to orchestrate germ-cell fate transitions from spermatogonial stem cells through meiosis and spermiogenesis. Recent literature is critically surveyed and synthesized, with particular emphasis on human and primate data and on stage-resolved maps generated by single-cell and multi-omics technologies. Collectively, available studies support a layered regulatory model in which RNA-binding proteins and RNA modifications coordinate transcript processing, storage, translation, and decay; small and long noncoding RNAs shape post-transcriptional programs and transposon defense; and dynamic chromatin remodeling and 3D reconfiguration align transcriptional competence with recombination, sex-chromosome silencing, and genome packaging. Convergent nodes implicated in spermatogenic failure are highlighted, including defects in RNA metabolism, piRNA pathway integrity, epigenetic reprogramming, and nuclear architecture, and the potential of these frameworks to refine molecular phenotyping in male infertility is discussed. Finally, key gaps and priorities for causal testing in spatially informed, stage-specific experimental systems are outlined. Full article

Extracellular vesicles (EVs) are emerging as key factors in maintaining cellular homeostasis, critical mediators of intercellular communication, potential biomarkers, and therapeutic tools. While small EVs have been extensively characterized, the molecular signatures of large EVs (including those generated during regulated cell death pathways) remain poorly defined. Here, we investigated the characteristics of large EVs released during apoptosis and pyroptosis by human monocytic cell lines (THP-1 and U937). Apoptosis was induced by staurosporine and blocked using the pan-caspase inhibitor Q-VD-OPh, whereas pyroptosis was triggered by LPS/nigericin and inhibited with a selective NLRP3 inhibitor. We found that both forms of regulated cell death markedly enhanced the release of large EVs. Both apoptotic and pyroptotic large EVs showed increased Annexin V binding and decreased CD9 expression compared with those released by healthy cells. Large EVs derived from apoptotic and pyroptotic cells exhibited distinct proteomic profiles. Pyroptotic large EVs carried interacting protein networks of RNA-binding proteins and chromatin-associated proteins many of which are known damage-associated molecular patterns or alarmins. In contrast, we found that a subpopulation of apoptotic large EVs was characterized by the presence of dsDNA, and active caspase-3/7. Together, our data shed light on the specific protein cargo of large EVs released by cells during apoptosis and pyroptosis. This study identifies candidate markers of large EVs released by dying cells and may enhance our understanding of the role of EVs in regulated cell death. Full article

Although oxidants are known to be deleterious for cellular homeostasis by oxidizing macromolecules like DNA or proteins, they are also involved in signaling processes essential for cellular proliferation and survival. Here, we investigated the role of superoxide anion (O₂⁻) and hydrogen peroxide (H₂O₂) homeostasis for the proliferation and survival of classical Hodgkin’s lymphoma (cHL) cell lines. Inhibition of NADPH oxidases (NOX) using apocynin (Apo) and diphenylene iodonium (DPI), or treatment with the antioxidant butylated hydroxyanisole (BHA), significantly reduced proliferation and induced apoptosis in HL cell lines. These effects correlated with transcriptomic alterations involving redox regulation, immune signaling, and cell cycle control. Interestingly, treatment with DPI or antioxidants attenuated constitutive Signal Transducer and Activator of Transcription (STAT) activity, as seen by decreased phospho-STAT6 levels and reduced STAT6 DNA binding. This suggests a sensitivity of the Janus kinase (JAK)/STAT pathway in cHL cell lines to O₂⁻ and H₂O₂ depletion. Functional assays confirmed this by demonstrating partial restoration of proliferation or apoptosis in L428 cells that expressed constitutively active STAT6 or were transfected with small interfering RNAs (siRNAs) that targeted STAT regulators. These findings highlight that oxidants, particularly H₂O₂, act as both general oxidative stressors and essential modulators of oncogenic signaling pathways. Specifically, maintenance of oxidant homeostasis is critical for sustaining JAK/STAT-mediated growth and survival programs in cHL cells. Targeting redox homeostasis might offer a promising therapeutic strategy to impair JAK/STAT-driven proliferation and survival in cHL. Full article

The SARS-CoV-2 nucleocapsid protein (Np) plays multifunctional roles in the viral life cycle. By interacting with host cellular proteins, Np regulates viral RNA transcription, replication, and immune evasion. It controls genome packaging and counteracts host RNA interference mediated antiviral responses through its RNA binding activity. Previous studies revealed a physical interaction between Np and DDX3X, a human DEAD-box RNA helicase that facilitates the replication of several viruses. This interaction enhances Np affinity for double-stranded RNA and inhibits DDX3X helicase activity. Since Np-RNA binding activity promotes ribonucleoprotein complex formation, targeting this interaction is a promising antiviral strategy. We generated truncated protein variants to define interaction regions between Np and DDX3X. Using AlphaFold modelling, we identified RecA2 as the key DDX3X domain involved in Np binding. Finally, to disrupt Np-RNA complex formation, we screened a small molecule library of putative binders of Np N-terminal region and identified two candidate inhibitors for further development. Full article

Upon transcription, most mRNAs associate with the small ribosomal subunit, after which a fully translating ribosome assembles. Under starvation or stress, however, mRNA–ribosome associations are blocked and many mRNAs are instead sequestered with specific RNA-binding proteins into stress granules or other subcellular condensates, a process that has been extensively studied. In contrast, much less attention has been paid to the fate of ribosomes under these same conditions. Ribosomes can remain fully assembled but unbound to mRNA, entering an inactive, dormant state. Dormancy is often supported by specific protein factors which protect ribosomes from degradation and facilitate reactivation once growth conditions improve. In this review, we highlight that dormant ribosome states are well defined in prokaryotes, in part because they possess distinct and experimentally tractable features, such as stable vacant 100S dimers. In eukaryotes, by contrast, analogous disomes are largely absent, making their discovery more indirect and method-dependent. We therefore focus on how evidence for eukaryotic dormant ribosomes has been assembled through multiple independent findings and how their interpretation depends critically on the experimental approaches used to study them. Finally, we consider atypical ribosomal states, such as translationally inactive polysomes in neurons, which underscore the context-dependent nature of ribosome activity. Full article

Antibiotic-resistant infections remain a major challenge in cystic fibrosis (CF), where chronic Pseudomonas aeruginosa colonization drives lung infection. The overexpression of adhesion-related proteins and extracellular matrix components, including fibronectin (Fn), facilitates bacterial colonization. Recent evidence identifies the RNA-binding protein Human Antigen R (HuR) as a key regulator of this process, as it stabilizes Vav3 mRNA, promoting Fn deposition and the formation of bacterial docking platforms. Here, we report the synthesis, optimization, and functional evaluation of the HuR-targeted small-molecule (2S,3S)-BOPC1. Functional assays in CF human airway epithelial cells demonstrated that (2S,3S)-BOPC1 significantly reduced P. aeruginosa adhesion in a dose-dependent manner without detectable cytotoxic effects. These findings provide the first evidence that targeting HuR can disrupt the HuR–Vav3–Fn axis, reducing bacterial attachment. This host-directed approach represents a promising strategy to prevent chronic infections in CF without promoting antibiotic resistance. Full article

Tomato (Solanum lycopersicum L.), one of the most widely consumed horticultural crops worldwide, is also a notable source of food allergens, with a higher prevalence of allergic reactions in pollen-sensitized patients. Profilin, a small actin-binding protein, is a major allergen in tomato fruits encoded by two homologous genes, Lyc e1.01 and Lyc e1.02. Although various strategies have been proposed to reduce allergenicity in foods, no prior study has successfully achieved the complete elimination of profilin proteins in tomato using precise genome editing. In this study, we designed a single-guide RNA targeting a conserved region of both genes and employed the CRISPR/Cas9 system to generate loss-of-function mutations. We first evaluated single-guide RNA editing efficiency in tomato protoplasts and then performed Agrobacterium-mediated stable transformation, which yielded 13 transgenic T₀ lines. Genotyping and Western blot analyses confirmed successful editing at both target loci and significantly decreased profilin accumulation in mutant tomato fruits. Notably, two homozygous Cas9-free lines were identified in the T1 generation, and one of these (line 23-15) showed significantly decreased profilin protein levels in the fruit. These findings demonstrate that the CRISPR/Cas9-mediated disruption of allergenic Lyc e1 genes effectively removes profilin from tomato fruits. This strategy provides a promising framework for developing hypoallergenic tomato cultivars that may be extended to other related crop species. Full article