Search Results (437)

Potyvirus genomes are expressed as a single large open reading frame, which is translated into a polyprotein that is post-translationally cleaved by three virus-encoded proteases into 10 functional proteins. Several of these potyviral proteins, including nuclear inclusion protein b (NIb), are multifunctional. Here, using the classic GFP silencing in Nicotiana benthamiana gfp-transgenic plants, we show that potato virus Y (PVY) NIb, in addition to its canonical role as the viral RNA-dependent RNA polymerase (RdRP), functions as a suppressor of RNA silencing. Mutational analyses reveal a previously unreported NIb nuclear localization signal (NLS) consisting of a triple-lysine motif. NIb suppression of RNA silencing activity was lost when the NLS was mutated, suggesting that nuclear localization is required for NIb suppression of RNA silencing activity. Analysis of sequenced GFP siRNAs revealed three reproducible hotspot regions at ≈175 nt, ≈320–330 nt, and a broader 3′-proximal region spanning ≈560–700 nt that contains multiple local maxima. These data show differences in the positional distribution of siRNAs between samples expressing NIb and those expressing NIb^Del3×2, the NIb null mutant that does not suppress RNA silencing. However, the positional distribution of GFP-derived small RNAs across the transgene differed modestly between NIb and NIb^Del3×2, while both treatments showed the same three reproducible hotspot regions. Furthermore, NIb was found to interact with four key RNA silencing pathway proteins—AGO4, HSP70, HSP90, and SGS3. Except for HSP90, each of these proteins showed degradation products that were absent in NIb mutants that did not suppress RNA silencing. These findings support a role for NIb in countering host defense during virus infection. Full article

Leukocyte-specific protein 1 (LSP1) is known as an endothelial gatekeeper because it controls endothelial permeability and transendothelial cell migration, including that of leukocytes and potentially metastatic cancer cells. In endothelial cells, LSP1 is predominantly in the nucleus under resting conditions but translocates to extranuclear compartments upon stimulation with TNF-α. The discrepancy between its predicted molecular weight (~37 kDa) and its observed migration on SDS-PAGE (≥52 kDa), along with its dynamic subcellular distribution, suggests a possible post-translational modification by SUMOylation. To investigate this, we examined endogenous LSP1 in murine primary endothelial cells and overexpressed recombinant LSP1 in murine endothelial (SVEC4-10EE2) and HEK293T cells. Our results demonstrate that LSP1 is SUMOylated by SUMO1, with Ubc9 serving as the conjugating enzyme and SENP1 as the deSUMOylating protease. Site-directed mutagenesis of lysines K270 and K318 abolished SUMOylation, resulting in a marked reduction in LSP1 steady-state levels. This reduction was attributed to enhanced ubiquitination and accelerated proteasomal degradation of LSP1 in the SUMOylation-deficient state. Furthermore, deSUMOylation impaired the TNF-α-induced translocation of LSP1 from the nucleus to extranuclear compartments, particularly the cytoskeleton. In summary, our findings establish that LSP1 is a SUMO1-modified protein. SUMOylation stabilizes LSP1 by preventing proteasomal degradation and is essential for its proper subcellular trafficking in endothelial cells in response to inflammatory stimuli. Full article

MicroRNAs (miRNAs) are small non-coding RNA molecules approximately 20–22 nucleotides in length that regulate gene expression at the post-transcriptional level. By binding to target messenger RNAs (mRNAs), miRNAs inhibit translation or induce degradation, thus influencing a wide array of biological processes including development, inflammation, apoptosis, and tissue remodeling. Owing to their remarkable stability and tissue specificity, miRNAs have emerged as promising biomarkers in both clinical and forensic settings. In recent years, increasing evidence has demonstrated their utility in cardiovascular diseases, where they may serve as diagnostic, prognostic, and therapeutic tools. This systematic review aims to comprehensively summarize the role of miRNAs in cardiovascular pathology, focusing on their diagnostic potential in myocardial infarction, sudden cardiac death (SCD), and cardiomyopathies, and their applicability in post-mortem investigations. Following PRISMA guidelines, we screened PubMed, Scopus, and Web of Science databases for studies up to December 2024. The results highlight several miRNAs—including miR-1, miR-133a, miR-208b, miR-499a, and miR-486-5p—as robust markers for ischemic injury and sudden death, even in degraded or formalin-fixed autopsy samples. The high stability of miRNAs under extreme post-mortem conditions reinforces their potential as molecular tools in forensic pathology. Nevertheless, methodological heterogeneity and limited standardization currently hinder their routine application. Future studies should aim to harmonize analytical protocols and validate diagnostic thresholds across larger, well-characterized cohorts to fully exploit miRNAs as reliable molecular biomarkers in both clinical cardiology and forensic medicine. Full article

Chronic and complex wounds, including diabetic foot ulcers, venous leg ulcers, burns and post-surgical defects, remain difficult to manage due to persistent inflammation, impaired angiogenesis, microbial colonization and insufficient extracellular matrix (ECM) remodeling. Conventional dressings provide protection, but they do not supply the necessary biochemical and structural signals for effective tissue repair. This review examines recent advances in hydroxyapatite–collagen (HAp–Col) composite dressings, which combine the architecture of collagen with the mechanical reinforcement and ionic bioactivity of hydroxyapatite. Analysis of the literature indicates that in situ and biomimetic mineralization, freeze-drying, electrospinning, hydrogel and film processing, and emerging 3D printing approaches enable precise control of pore structure, mineral dispersion, and degradation behavior. Antimicrobial functionalization remains critical: metallic ions and locally delivered antibiotics offer robust early antibacterial activity, while plant-derived essential oils (EOs) provide broad-spectrum antimicrobial, antioxidant and anti-inflammatory effects with reduced risk of resistance. Preclinical studies consistently report enhanced epithelialization, improved collagen deposition and reduced bacterial burden in HAp–Col systems; however, translation is limited by formulation variability, sterilization sensitivity and the lack of standardized clinical trials. Overall, HAp–Col composites represent a versatile framework for next-generation wound dressings that can address both regenerative and antimicrobial requirements. Full article

Background/Objectives: Osteoarthritis (OA) is a multifactorial degenerative joint disease characterized by synovial inflammation, oxidative stress, and progressive cartilage degeneration. This study investigated whether intra-articular N-acetylcysteine (NAC) attenuates synovial inflammation and oxidative stress and whether these effects translate into structural cartilage protection. Methods: OA was induced in rats by anterior cruciate ligament transection (ACLT). NAC (5 mg/50 µL) was administered intra-articularly once weekly for three weeks post-ACLT. Inflammatory cytokines (IL-1β, IL-6, TNF-α), oxidative stress markers (iNOS, TAS, TOS, OSI), and cartilage degradation markers (MMP-13, COMP, CTX-II) were quantified in synovial fluid and cartilage homogenates using ELISA. Cartilage integrity was evaluated histologically using the modified Mankin scoring system. Results: Compared with controls, NAC significantly reduced synovial IL-1β, IL-6, TNF-α, MMP-13, and iNOS levels and improved the synovial redox profile by increasing TAS and reducing TOS and OSI (all p < 0.05). In contrast, NAC did not significantly alter cartilage homogenate levels of inflammatory cytokines, oxidative stress indices, or degradation markers (COMP, CTX-II, MMP-13). Histological analysis demonstrated persistent cartilage fissuring, hypocellularity, and proteoglycan loss in both groups (p > 0.05). Conclusions: Intra-articular NAC exerts potent anti-inflammatory and antioxidative effects within the synovial compartment but fails to prevent cartilage degeneration in the ACLT model. These findings indicate a compartment-specific therapeutic profile, suggesting that NAC may function as a symptom-modifying agent in synovitis-dominant OA rather than a structure-modifying therapy. Future studies should focus on optimized delivery systems or combination strategies targeting cartilage and subchondral bone to achieve disease modification. Full article

Protein post-translational modifications (PTMs), as an important biological process of plants responding to environmental stimuli, can regulate the chemical decoration and properties of translated proteins by altering amino acid side chains or protein terminal structures, thereby affecting the synthesis, assembly, localization, function, and degradation of proteins. Notably, PTMs regulate protein function without changing protein expression levels. Two dozen types of PTMs have been identified. This review summarizes the molecular mechanisms of major types of PTMs, including phosphorylation, ubiquitination, SUMOylation, glycosylation, methylation, and acetylation, with a focus on their regulatory roles in plant responses to abiotic stresses. Under heat stress, phosphorylation activates transcription factors such as HSFA1 (heat shock transcription factor 1), while SUMOylation regulates the activity of HSFA1/HSFA2 in the heat stress signaling pathway. Upon cold stress, phosphorylation, ubiquitination, and S-acylation collectively regulate the expression of cold tolerance-related genes. The drought stress response relies on SnRK2s (Sucrose 321 non-Fermenting 1-related protein kinase 2s) -mediated phosphorylation, regulation of ARF7 (auxin response factor 7) by SUMOylation, and ubiquitination. In salt stress, the coupling of phosphorylation of SOS (salt overly sensitive) pathway-related proteins, ubiquitination, and phospholipid metabolism maintains ion homeostasis. Additionally, PTMs play a key role in ABA-mediated abiotic stress responses by regulating core components of signal transduction, such as PYR (pyrabactin resistance)/PYL (PYR1-LIKE)/RCAR (regulatory components of ABA receptor) receptors, PP2Cs (protein phosphatases type 2C), and SnRK2s. On the basis of the synthesis of the regulatory mechanisms of PTMs, we discuss how PTMs can be manipulated to breed abiotic stress resilient crops and the issues to be addressed to achieve the goal, such as crosstalk between PTMs, technical challenges in investigating PTMs and identifying PTM substrates. Full article

Background: In high-dimensional, small-sample omics studies such as metabolomics, feature selection not only determines the discriminative performance of classification models but also directly affects the reproducibility and translational value of candidate biomarkers. However, most existing methods primarily optimize classification accuracy and treat stability as a post hoc diagnostic, leading to considerable fluctuations in selected feature sets under different data splits or mild perturbations. Methods: To address this issue, this study proposes FRL-TSFS, a feature selection framework synergistically driven by filter-based Robust Rank Aggregation and L1-sparse modeling. Five complementary filter methods—variance thresholding, chi-square test, mutual information, ANOVA F test, and ReliefF—are first applied in parallel to score features, and Robust Rank Aggregation (RRA) is then used to obtain a consensus feature ranking that is less sensitive to the bias of any single scoring criterion. An L1-regularized logistic regression model is subsequently constructed on the candidate feature subset defined by the RRA ranking to achieve task-coupled sparse selection, thereby linking feature selection stability, feature compression, and classification performance. Results: FRL-TSFS was evaluated on six representative metabolomics and gene expression datasets under a mildly perturbed scenario induced by 10-fold cross-validation, and its performance was compared with multiple baselines using the Extended Kuncheva Index (EKI), Accuracy, and F1-score. The results show that RRA substantially improves ranking stability compared with conventional aggregation strategies without degrading classification performance, while the full FRL-TSFS framework consistently attains higher EKI values than the other feature selection schemes, markedly reduces the number of selected features to several tens of metabolites or genes, and maintains competitive classification performance. Conclusions: These findings indicate that FRL-TSFS can generate compact, reproducible, and interpretable biomarker panels, providing a practical analysis framework for stability-oriented feature selection and biomarker discovery in untargeted metabolomics. Full article

RACK1 (Receptor for Activated C Kinase 1) is a highly conserved scaffold protein that functions as a central integrator within diverse cellular signaling pathways. Initially identified as a receptor for activated Protein Kinase C, it is now recognized as a dynamic platform coordinating processes such as cell proliferation, migration, apoptosis, and immune responses. A defining feature of RACK1 is its ability to direct cellular fate by determining whether proteins are synthesized or degraded. However, a unified model explaining this functional pleiotropy has been lacking. In this review, we synthesize current knowledge to propose an integrative model centered on a functional dimorphism driven by RACK1’s localization and post-translational modifications. We posit that RACK1 operates in two primary, mutually exclusive states: a ribosome-associated monomer that supports the translation of specific mRNAs and quality control, and a free monomer or dimer that governs signaling cascades and gene expression. Phosphorylation at key sites, such as Thr50 and Ser146, acts as a molecular switch, spatiotemporally redistributing RACK1 between these pools. This mechanism allows the cell to rapidly reprogram its proteomic landscape in response to stimuli, pivoting between protein synthesis and stress adaptation. Our model resolves the apparent dichotomy of RACK1’s roles by framing it as a cellular “resource manager,” whose regulated switching between functional states ensures an optimal response to the extracellular environment, with significant implications for understanding cancer and neurodegenerative diseases. 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

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by uncontrolled proliferation of myeloid cells. MicroRNAs (miRNAs), small noncoding RNAs, regulate post-transcriptional gene expression by degrading target mRNAs or repressing translation. Dysregulated miRNA expression has been implicated in various malignancies, including CML, where they can function as oncogenes or tumor suppressors. This study aimed to investigate the relationship between miR-122-5p and cell division cycle 25A (CDC25A) in CML and to elucidate the regulatory mechanisms of miR-122-5p. This study integrates bioinformatics analysis with in vitro RT-qPCR validation in K562 chronic myeloid leukemia cells to explore the potential regulatory relationship between miR-122-5p and CDC25A. mRNA expression profiles were retrieved from the GSE100026 dataset in the Gene Expression Omnibus (GEO), and differentially expressed genes were identified using GEO2R. Quantitative real-time PCR (RT-qPCR) was performed to measure miR-122-5p, CDC25A, and cyclin-dependent kinase 4 (CDK4) expression levels. Bioinformatics analyses (miRNeT, miRDIP, TargetScan, BioGPS, GeneMANIA, STRING) were applied to predict molecular interactions and functional pathways. Public RNA-seq datasets and in silico tools were used to prioritize candidates; RT-qPCR in a single CML cell line (K562) provided in vitro expression validation. In K562 cells, miR-122-5p expression was significantly reduced, while CDC25A and CDK4 were markedly upregulated. Bioinformatics tools confirmed CDC25A as a potential miR-122-5p target. Functional enrichment indicated CDC25A involvement in cell cycle regulation and apoptosis. These findings suggest that miR-122-5p functions as a tumor suppressor in CML by targeting CDC25A. Modulating the miR-122-5p/CDC25A axis may provide potential molecular targets for inhibiting CML progression through regulation of cell cycle pathways. Findings are exploratory and based on bioinformatics with limited in vitro expression confirmation; functional studies are required to establish causality. Full article

Underwater debris poses a significant threat to marine ecosystems, fisheries, and the tourism industry, necessitating the development of automated vision-based detection systems. Although recent studies have sought to enhance detection performance through underwater image enhancement, improvements in visual quality do not necessarily translate into higher detection accuracy and may, in some cases, degrade performance. To address this discrepancy between perceptual quality and detection reliability, we propose DiCAF, a dual-input co-attention fusion network built upon the latest You Only Look Once v11 detector. The proposed architecture processes both original and enhanced images in parallel and fuses their complementary features through a co-attention module, thereby improving detection stability and consistency. To mitigate high-frequency noise amplified during the enhancement process, a lightweight Gaussian filter is applied as a post-processing step, enhancing robustness against speckle noise commonly introduced by suspended particles in underwater environments. Furthermore, DiCAF incorporates a non-maximum suppression (NMS)-based ensemble that integrates detection outputs from three branches—original, enhanced, and fused—enabling complementary detection of objects missed by individual models and maximizing overall detection performance. Experimental results demonstrate that the proposed single-model DiCAF with Gaussian post-processing achieves an AP@0.5 of 0.87 and an AP@0.5:0.95 of 0.71 on a marine trash dataset. With the NMS-based ensemble, performance improves to 0.91 and 0.75, respectively. Under artificially injected speckle noise conditions, the proposed method maintains superior robustness, achieving an AP@0.5 of 0.62 and consistently outperforming conventional enhancement-based models. Full article

Background/Objectives: Approximately 20% of familial ALS (fALS) cases are linked to mutations in Cu/Zn superoxide dismutase (SOD1). Through a gain function, SOD1 misfolding exerts a toxic effect on motor neurons, leading to their degradation and ALS symptomology in both fALS cases and sporadic ALS (sALS) cases with no known genetic cause. To further our understanding of SOD1-ALS etiology, identifying motor neuron-specific SOD1 post-translational modifications (PTMs) and studying their structural influence is necessary. To this end, we have conducted a study on the influence of the deamidation of Asn53, a PTM proximal to key stabilizing motifs in SOD1, which has scarcely been addressed in the literature to date. Methods: Deamidation to N53 was identified by tandem mass spectrometry of SOD1 immunoprecipitated from motor neuron (MN) cultures derived from wild-type (WT) human induced pluripotent stem cells (iPSCs). WT SOD1 and N53D SOD1, a mutant mimicking the deamidation, were expressed in Escherichia coli and purified for in vitro analyses. Differences between species were measured by experiments probing metal cofactors, relative monomer populations, and aggregation propensity. Furthermore, molecular dynamics experiments were conducted to model and determine the influence of the PTM on SOD1 structure. Results: In contrast to WT, N53D SOD1 showed non-native incorporation of metal cofactors, coordinating more Zn²⁺ cofactors than total Zn-binding sites, and more readily adopted monomeric forms, unfolded, and aggregated with heating, possibly while releasing coordinated metals. Conclusions: Deamidation to N53 in SOD1 encourages the adoption of non-native conformers, and its detection in WT MN cultures suggests relevance to sALS pathophysiology. Full article

Huntingtin (HTT) is a large, ubiquitously expressed scaffold protein that participates in multiple cellular processes, including vesicular transport, transcriptional regulation, and energy metabolism. The mutant form of HTT (mHTT), characterized by an abnormal polyglutamine (polyQ) expansion in its N-terminal region, is the causative agent of Huntington’s disease (HD), a progressive neurodegenerative disorder. Current therapeutic efforts for HD have primarily focused on lowering HTT levels through gene silencing or promoting mHTT degradation. However, accumulating evidence suggests that post-translational modifications (PTMs) of HTT—such as phosphorylation, ubiquitination, acetylation, and SUMOylation—play pivotal roles in modulating HTT’s conformation, aggregation propensity, subcellular localization, and degradation pathways. These modifications regulate the balance between HTT’s physiological functions and pathological toxicity. Importantly, dysregulation of PTMs has been linked to mHTT accumulation and selective neuronal vulnerability, highlighting their relevance as potential therapeutic targets. A deeper understanding of how individual PTMs and their crosstalk regulate HTT homeostasis may not only provide mechanistic insights into HD pathogenesis but also uncover novel, more specific strategies for intervention. In this review, we summarize recent understanding on HTT PTMs, discuss their implications for disease modification, and outline critical knowledge gaps that remain to be addressed. Full article

The human Ccr4–Not complex is a central regulator of post-transcriptional gene regulation, impacting on translation and mRNA degradation. In mRNA degradation, Ccr4–Not participates in the shortening of the mRNA poly(A)-tail via two catalytic subunits. The Caf1 nuclease is encoded by the highly similar paralogues CNOT7 or CNOT8. In addition to its poly(A)-specific ribonuclease activity, this subunit also provides a structural role by binding Ccr4, the second catalytic nuclease subunit encoded by the paralogues CNOT6 or CNOT6L. To facilitate investigations into the roles of the Caf1 subunit, and to complement genetic tools, we set out to identify inhibitors of the enzymatic activity of Caf1/CNOT7. To this end, we screened a library of 10,880 chemically diverse, drug-like compounds using a fluorescence-based biochemical assay. This effort led to the discovery of 15 inhibitors of Caf1/CNOT7 with biochemical IC₅₀ values below 25 μM. Molecular docking was performed to explore potential binding modes of these compounds. The compounds reported here may be useful to differentiate between catalytic and non-catalytic roles of Caf1/CNOT7. In addition, they may be valuable starting points for the development of more potent inhibitors of the Caf1/CNOT7 poly(A)-selective ribonuclease. Full article

Background: Photostability assessment is a critical component in the development of drug products, particularly for antibody–drug conjugates (ADCs) containing light-sensitive small molecules such as camptothecin (CPT) and its derivatives. ADCs conjugated with CPT derivative payloads often require extensive formulation and drug product development to ensure product stability due to their unique light-induced degradation pathways. In this study, we assessed the photostability of two ADC molecules with a CPT derivative payload (deruxtecan, DXd). Methods: Following light exposure, the stability of ADCs was assessed by examining critical quality attributes, such as aggregation and photodegradation products of the antibody, payload, and formulation excipients, using advanced liquid chromatography and mass spectrometry techniques. Results: Our results revealed key degradation pathways, including the formation of high-molecular-weight (HMW) species, payload degradation, and post-translational modifications (PTMs) on amino acid residues in the antibodies. Additionally, the DXd payload amplified the photosensitivity of the formulation solution, leading to histidine degradation in the formulation buffer and subsequent pH changes. To enhance the stability of ADCs for manufacturing and therapeutic use, we developed a robust formulation by systematic buffer screening and a targeted evaluation of selected antioxidant excipients. Further investigations into light conditions revealed that DXd ADCs are particularly sensitive to short-wavelength light. When evaluating the container closure system, it was demonstrated that using amber vials is a viable option for protecting against light-induced degradation. Conclusions: This report outlines a comprehensive strategy to address photo instability in DXd ADC drug product development, focusing on formulation optimization, controlled manufacturing light settings, and the option of using protective containers to ensure product stability. Full article