Search Results (55)

Vibrio alginolyticus, a common Gram-negative opportunistic pathogen of marine animals and humans, is known for its rapid growth in organic-matter-rich environments. However, it remains unclear how it incorporates metabolic pathways in response to diverse carbon and nitrogen sources and rapidly alters gene expression. Increasing evidence suggests that post-transcriptional regulation by RNA-binding proteins and small RNAs (sRNAs) plays a crucial role in bacterial adaptation and metabolism. CsrA (carbon storage regulator A), a conserved post-transcriptional regulator in Gammaproteobacteria, is poorly characterized in Vibrio species. Using integrated transcriptomic and proteomic analyses, we found that CsrA alters the expression of 661 transcripts and 765 protein transcripts in V. alginolyticus, influencing key pathways including central carbon metabolism, amino acid metabolism and transport, quorum sensing, and bacterial secretion systems. Through directed CsrA-RNA EMSAs, we identified several direct mRNA targets of CsrA, including gltB, gcvP, aceE, and tdh, as well as secretion system components (tagH, tssL, yopD, and sctC). Notably, CsrA also directly regulates rraA, a key modulator of ribonuclease activity, suggesting a broader role in RNA metabolism. Our findings establish CsrA as a global regulator in V. alginolyticus, expanding the known targets of CsrA and providing new insights into its regulatory roles. Full article

Despite the essential role of phosphorus (P) in rice growth, P-use efficiency (PUE) remains low due to limited bioavailable P in soils and an over-reliance on chemical fertilizers, leading to resource waste and environmental risks, such as eutrophication. This study investigates whether and how alternating wetting and moderate drying (AWMD) irrigation promotes P absorption and transport in rice. This study was conducted over two years using a pot experiment. Conventional flooding (CF) irrigation was applied throughout the growing season, while AWMD irrigation was imposed from two weeks after transplanting to one week before harvest. AWMD improved shoot biomass by 8.7–9.4% and the photosynthetic rate by 12–15%, significantly enhanced PUE, and optimized root traits and enzyme activities related to P uptake. It also promoted leaf acid phosphatase and ribonuclease activities, facilitating P remobilization to grains. In conclusion, AWMD enhanced the ability of roots to absorb P and optimized the redistribution of P between vegetative organs and grains, synergistically increasing grain yield and PUE in rice. Full article

The human coronavirus 229E (HCoV-229E) is a member of the human coronavirus family that includes SARS-CoV-2, the causative agent of COVID-19. Developing antiviral strategies and compounds is crucial to treat and prevent HCoV-229E infections and the associated diseases. Ribozymes derived from ribonuclease P (RNase P) catalytic RNA represent a novel class of promising gene-targeting agents by cleaving their target mRNA and knocking down the expression of the target mRNA. However, it has not been reported whether RNase P ribozymes block the infection and replication of HCoV-229E. We report here the engineering of an anti-HCoV-229E RNase P ribozyme to target an overlapping region of viral genomic RNA and the mRNA encoding the nucleocapsid (N) protein, which is vital for viral replication and growth. The engineered ribozyme actively hydrolyzed the viral RNA target in vitro. HCoV-229E-infected cells expressing the engineered, catalytically active ribozyme exhibited a reduction of about 85% in viral RNA levels and N protein expression, and a reduction of about 750-fold in infectious particle production, compared to cells expressing no ribozymes or a control, catalytically inactive ribozyme. Our study provides the first direct evidence of the therapeutic potential of RNase P ribozymes against human coronaviruses such as HCoV-229E. Full article

Pancreatic (PC), colorectal (CRC), hepatocellular (HCC), and gallbladder (GC) cancers together account for nearly 20% of all cancer cases. However, specific biomarkers and therapeutic targets for these cancers are lacking. Diagnosing these cancers early and providing timely, appropriate treatment to improve patient outcomes is crucial. In this context, previous studies, including ours, have highlighted the potential of non-coding RNAs, particularly long non-coding RNAs (lncRNAs), in diagnosing and prognosis of various cancers. This review focuses on the mechanistic role of the recently identified lncRNA LINC00261 in PC, CRC, HCC, and GC. Our comprehensive literature analysis revealed that LINC00261 functions as a tumor suppressor, and its reduced expression is associated with larger tumor size, advanced tumor-node-metastasis (TNM) stages, lymphatic metastasis, and poorer overall survival rates. Additionally, we discovered that LINC00261 acts as a molecular sponge for miRNAs, such as miR-550a-3p, miR-23a-3p, miR-148a, miR-324-3p, and miR-105-5p, regulating critical cancer-related signaling pathways, including PI3K/Akt/mTOR, Protein kinase B, and Mammalian target of rapamycin (mTOR). Further bioinformatic analysis revealed that LINC00261 regulates key cellular processes, such as protein-DNA complex formation, ribonuclease complex activity, histone deacetylase complexes, and nuclear matrix interactions. Overall, we believe that LINC00261 holds significant promise as a future biomarker and, when combined with existing treatment strategies, may enhance cancer patient care and survival. Full article

Background: External guide sequences (EGSs) are small RNA molecules capable of hybridizing to a target mRNA and rendering the target RNA susceptible to degradation by ribonuclease P (RNase P), a tRNA processing enzyme. Methods: In this study, natural tRNA-originated and engineered variant EGSs were constructed to target the mRNA encoding human CC-chemokine receptor 5 (CCR5), an HIV co-receptor. Results: The EGS variant was about 100-fold more efficient in inducing RNase P-mediated cleavage of the CCR5 mRNA sequence in vitro than a natural tRNA-derived EGS. Furthermore, the expressed variant and natural tRNA-originated EGSs decreased CCR5 expression by 98% and 73–77% and reduced infection by the CCR5-tropic HIV_Ba-L strain in cells by more than 900- and 50-fold, respectively. By contrast, cells expressing these EGSs exhibited no change in the expression of CXCR4, another HIV co-receptor, and showed no reduction in infection by the CXCR4-tropic HIV_IIIB strain, which uses CXCR4 instead of CCR5 as the co-receptor. Thus, the EGSs specifically targeted CCR5 but not CXCR4. Conclusions: Our results demonstrate that EGSs are effective and specific in diminishing HIV infection and represent a novel class of gene-targeting agents for anti-HIV therapy. Full article

Reticuloendotheliosis virus (REV) is an oncogenic retrovirus that affects both commercial and free-ranging birds. To date, only two complete REV genome sequences have been identified in chickens from South America, with no records in other avian species. This study reports the first complete genome of REV detected in a mallard duck (Anas platyrhynchos domesticus) in South America. In 2021, a mallard duck in Brazil died from severe lymphoproliferative disease affecting multiple organs. Molecular detection and histopathological analysis confirmed REV as the causative agent. Using dideoxy sequencing and phylogenetic analysis, the virus was classified as subtype 3 (REV-3). The phylogenetic analysis identified three clades, each with a bootstrap value of 100, corresponding to the three REV subtypes. Furthermore, a comprehensive comparative genomic analysis revealed two distinct REV-3 subclusters—‘East’ (38 strains) and ‘West’ (24 strains)—with notable geographical associations. Additionally, 27 genomes in chimeric states with fowlpox virus (FWPV) were distributed across the phylogenetic tree, emphasizing the critical role of FWPV in the dissemination of REV. Selective pressure analysis revealed evidence of positive selection acting on several codons within the gag, pol, and env genes, particularly in domains such as matrix, p18, reverse transcriptase/ribonuclease H, and surface. These findings provide valuable insights into REV evolution and underscore the importance of genomic surveillance for detecting REV circulation in diverse hosts. Full article

During virus infection, the activation of the antiviral endoribonuclease, ribonuclease L (RNase L), by a unique ligand 2′-5′-oilgoadenylate (2-5A) causes the cleavage of single-stranded viral and cellular RNA targets, restricting protein synthesis, activating stress response pathways, and promoting cell death to establish broad antiviral effects. The immunostimulatory dsRNA cleavage products of RNase L activity (RL RNAs) recruit diverse dsRNA sensors to activate signaling pathways to amplify interferon (IFN) production and activate inflammasome, but the sensors that promote cell death are not known. In this study, we found that DEAH-box polypeptide 15 (DHX15) and retinoic acid-inducible gene I (Rig-I) are essential for apoptosis induced by RL RNAs and require mitochondrial antiviral signaling (MAVS), c-Jun amino terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK) for caspase-3-mediated intrinsic apoptosis. In RNase L-activated cells, DHX15 interacts with Rig-I and MAVS, and cells lacking MAVS expression were resistant to apoptosis. RL RNAs induced the transcription of genes for IFN and proinflammatory cytokines by interferon regulatory factor 3 (IRF-3) and nuclear factor kB (NF-kB), while cells lacking both DHX15 and Rig-I showed a reduced induction of cytokines. However, apoptotic cell death is independent of both IRF-3 and NF-kB, suggesting that cytokine and cell death induction by RL RNAs are uncoupled. The RNA binding of both DHX15 and Rig-I is required for apoptosis induction, and the expression of both single proteins in cells lacking both DHX15 and Rig-I is insufficient to promote cell death by RL RNAs. Cell death induced by RL RNAs suppressed Coxsackievirus B3 (CVB3) replication, and inhibiting caspase-3 activity or cells lacking IRF-3 showed that the induction of apoptosis directly resulted in the CVB3 antiviral effect, and the effects were independent of the role of IRF-3. Full article

Viral diseases of potatoes are among the main problems causing deterioration in the quality of tubers and loss of yield. The growth and development of potato plants largely depend on soil moisture. Prevention strategies require comprehensive protection against pathogens and abiotic stresses, including modeling the beneficial microbiome of agroecosystems combining microorganisms and immunostimulants. Chitosan and its derivatives have great potential for use in agricultural engineering due to their ability to induce plant immune responses. The effect of chitosan conjugate with caffeic acid (ChCA) in combination with Bacillus subtilis 47 on the transcriptional activity of PR protein genes and changes in the proteome of potato plants during potato virus Y (PVY) infection and drought was studied. The mechanisms of increasing the resistance of potato plants to PVY and lack of moisture are associated with the activation of transcription of genes encoding PR proteins: the main protective protein (PR-1), chitinase (PR-3), thaumatin-like protein (PR-5), protease inhibitor (PR-6), peroxidase (PR-9), and ribonuclease (PR-10), as well as qualitative and quantitative changes in the plant proteome. The revealed activation of the expression of marker genes of systemic acquired resistance and induced systemic resistance under the influence of combined treatment with B. subtilis and chitosan conjugate indicate that, in potato plants, the formation of resistance to viral infection in drought conditions proceeds synergistically. By two-dimensional electrophoresis of S. tuberosum leaf proteins followed by MALDI-TOF analysis, 10 proteins were identified, the content and composition of which differed depending on the experiment variant. In infected plants treated with ChCA, the synthesis of proteinaceous RNase P 1 and oxygen-evolving enhancer protein 2 was enhanced in conditions of normal humidity, and 20 kDa chaperonin and TMV resistance protein N-like was enhanced in conditions of lack of moisture. The virus coat proteins were detected, which intensively accumulated in the leaves of plants infected with potato Y-virus. ChCA treatment reduced the content of these proteins in the leaves, and in plants treated with ChCA in combination with Bacillus subtilis, viral proteins were not detected at all, both in conditions of normal humidity and lack of moisture, which suggests the promising use of chitosan derivatives in combination with B. subtilis bacteria in the regulation of plant resistance. Full article

Aim: Hyperglycemia damages mitochondria and downregulates transcription of mtDNA-encoded genes and the long noncoding RNA LncCytB, causing mitochondrial genomic instability. The genes encoded by mtDNA are transcribed as large polycistronic transcripts, and the 5′ ends of precursor tRNAs are processed by mitochondrial-targeted ribonuclease P (MRPPs). Our aim was to investigate the role of MRPP1 in the downregulation of LncCytB in diabetic retinopathy. Methods: Using human retinal endothelial cells incubated in 20 mM D-glucose for 96 h, the gene expression and mitochondrial localization (immunofluorescence) of MRPP1 and the interaction between MRPP1 and LncCytB (determined by RNA-FISH and RNA immunoprecipitation) were quantified. The results were confirmed in retinal microvessels from streptozotocin-induced diabetic mice and from human donors with documented diabetic retinopathy. Results: Compared to normal glucose, high glucose decreased mRNA and mitochondrial localization of MRPP1 and its interaction with LncCytB. While MRPP1 overexpression prevented glucose-induced decrease in MRPP1–LncCytB interaction, LncCytB expression and mitochondrial damage (reduction in protective nucleoids in mtDNA), MRPP1-siRNA further worsened them. Similar results were obtained from retinal microvessels from diabetic mice and from human donors with diabetic retinopathy. Conclusions: Downregulation of MRPP1 in diabetes suppresses LncCytB transcription, resulting in mitochondrial functional and genomic instability, ultimately leading to the development of diabetic retinopathy. Thus, preventing MRPP1 downregulation has the potential to inhibit retinopathy and prevent the fear of vision loss in diabetic patients. Full article

Ribonuclease P (RNase P) complexed with an external guide sequence (EGS) represents a promising nucleic acid-based gene targeting approach for gene expression knock-down and modulation. The RNase P-EGS strategy is unique as an EGS can be designed to basepair any mRNA sequence and recruit intracellular RNase P for hydrolysis of the target mRNA. In this study, we provide the first direct evidence that the RNase P-based approach effectively blocks the gene expression and replication of herpes simplex virus 2 (HSV-2), the causative agent of genital herpes. We constructed EGSs to target the mRNA encoding HSV-2 single-stranded DNA binding protein ICP8, which is essential for viral DNA genome replication and growth. In HSV-2 infected cells expressing a functional EGS, ICP8 levels were reduced by 85%, and viral growth decreased by 3000 folds. On the contrary, ICP8 expression and viral growth exhibited no substantial differences between cells expressing no EGS and those expressing a disabled EGS with mutations precluding RNase P recognition. The anti-ICP8 EGS is specific in targeting ICP8 because it only affects ICP8 expression but does not affect the expression of the other viral immediate-early and early genes examined. This study shows the effective and specific anti-HSV-2 activity of the RNase P-EGS approach and demonstrates the potential of EGS RNAs for anti-HSV-2 applications. Full article

The gaseous phytohormone ethylene plays a crucial role in plant growth, development, and stress responses. In the ethylene signal transduction cascade, the F-box proteins EIN3-BINDING F-BOX 1 (EBF1) and EBF2 are identified as key negative regulators governing ethylene sensitivity. The translation and processing of EBF1/2 mRNAs are tightly controlled, and their 3′ untranslated regions (UTRs) are critical in these regulations. However, despite their significance, the exact mechanisms modulating the processing of EBF1/2 mRNAs remain poorly understood. In this work, we identified the gene DCP1-ASSOCIATED NYN ENDORIBONUCLEASE 1 (DNE1), which encodes an endoribonuclease and is induced by ethylene treatment, as a positive regulator of ethylene response. The loss of function mutant dne1-2 showed mild ethylene insensitivity, highlighting the importance of DNE1 in ethylene signaling. We also found that DNE1 colocalizes with ETHYLENE INSENSITIVE 2 (EIN2), the core factor manipulating the translation of EBF1/2, and targets the P-body in response to ethylene. Further analysis revealed that DNE1 negatively regulates the abundance of EBF1/2 mRNAs by recognizing and cleaving their 3′UTRs, and it also represses their translation. Moreover, the dne1 mutant displays hypersensitivity to 1,4-dithiothreitol (DTT)-induced ER stress and oxidative stress, indicating the function of DNE1 in stress responses. This study sheds light on the essential role of DNE1 as a modulator of ethylene signaling through regulation of EBF1/2 mRNA processing. Our findings contribute to the understanding of the intricate regulatory process of ethylene signaling and provide insights into the significance of ribonuclease in stress responses. Full article

Messenger RNA (mRNA) therapies have emerged as potent and personalized alternatives to conventional DNA-based therapies. However, their therapeutic potential is frequently constrained by their molecular instability, susceptibility to degradation, and inefficient cellular delivery. This study presents the nanoparticle “ChargeSome” as a novel solution. ChargeSomes are designed to protect mRNAs from degradation by ribonucleases (RNases) and enable cell uptake, allowing mRNAs to reach the cytoplasm for protein expression via endosome escape. We evaluated the physicochemical properties of ChargeSomes using ¹H nuclear magnetic resonance, Fourier-transform infrared, and dynamic light scattering. ChargeSomes formulated with a 9:1 ratio of mPEG-b-PLL to mPEG-b-PLL-SA demonstrated superior cell uptake and mRNA delivery efficiency. These ChargeSomes demonstrated minimal cytotoxicity in various in vitro structures, suggesting their potential safety for therapeutic applications. Inherent pH sensitivity enables precise mRNA release in acidic environments and structurally protects the encapsulated mRNA from external threats. Their design led to endosome rupture and efficient mRNA release into the cytoplasm by the proton sponge effect in acidic endosome environments. In conclusion, ChargeSomes have the potential to serve as effective secure mRNA delivery systems. Their combination of stability, protection, and delivery efficiency makes them promising tools for the advancement of mRNA-based therapeutics and vaccines. Full article

This study aimed to evaluate the proteomic profile of seminal plasma from young Nellore bulls. We used 20 bulls aged between 19.8 and 22.7 months, divided into two groups according to the results of the Breeding Soundness Evaluation (BSE): approved (FIT n = 10) and not approved (UNFIT n = 10). The scrotal perimeter was measured and a semen collection was performed through electroejaculation. The percentage of sperm motility, mass motility, and sperm vigor were calculated using conventional microscopy, and the percentage of sperm abnormalities was calculated using phase-contrast microscopy of all ejaculates. Seminal plasma was separated from spermatozoa using centrifugation and processed for proteomic analysis by LC-MS/MS. Seminal plasma proteins were identified using MASCOT Daemon software v.2.4.0 and label-free quantification analysis was carried out by SCAFFOLD Q+ software v.4.0 using the Exponentially Modified Protein Abundance Index (emPAI) method. Functional classification of proteins was performed based on their genetic ontology terms using KOG. Functional cluster analysis was performed on DAVID. There were no differences in scrotal perimeter and physical semen characteristics between FIT and UNFIT groups of bulls. The percentage of sperm abnormalities was higher (p < 0.05) in the UNFIT group of bulls. A total of 297 proteins were identified for the two groups. There were a total of 11 differentially abundant proteins (p < 0.05), two of them more abundant in FIT bulls (Spermadhesin-1 and Ig gamma-1 chain C region) and nine in UNFIT bulls (Vasoactive intestinal peptide, Metalloproteinase inhibitor 2, Ig lambda-1 chain C regions, Protein FAM3C, Hemoglobin beta, Seminal ribonuclease, Spermadhesin 2, Seminal plasma protein BSP-30kDa, and Spermadhesin Z13). Spermadhesin-1 was the protein with the highest relative abundance (36.7%) in the seminal plasma among all bulls, corresponding to 47.7% for the FIT bulls and 25,7% for the UNFIT bulls. Posttranslational modification, protein turnover, and chaperones were the functional categories with the highest number of classified proteins. Protein functional annotation clusters were related to Phospholipid efflux, ATP binding, and chaperonin-containing T-complex. The differentially abundant proteins in the group of FIT bulls were related to sperm capacitation and protection against reactive species of oxygen. In contrast, differentially expressed proteins in the group of UNFIT bulls were related to motility inhibition, intramembrane cholesterol removal and oxidative stress. In conclusion, the proteomic profile of the seminal plasma of FIT bulls presents proteins with participation in several biological processes favorable to fertilization, while the proteins of the seminal plasma of UNFIT bulls indicate a series of alterations that can compromise the fertilizing capacity of the spermatozoa. In addition, the relative abundance of spermadhesin-1 found in the seminal plasma of young Nellore bulls could be studied as a reproductive parameter for selection. Full article

Ribotoxin-like proteins (RL-Ps) are specific ribonucleases found in mushrooms that are able to cleave a single phosphodiester bond located in the sarcin–ricin loop (SRL) of the large rRNA. The cleaved SRL interacts differently with some ribosomal proteins (P-stalk). This action blocks protein synthesis because the damaged ribosomes are unable to interact with elongation factors. Here, the amino acid sequences of eryngitin 3 and 4, RL-Ps isolated from Pleurotus eryngii fruiting bodies, were determined to (i) obtain structural information on this specific ribonuclease family from edible mushrooms and (ii) explore the structural determinants which justify their different biological and antipathogenic activities. Indeed, eryngitin 3 exhibited higher toxicity with respect to eryngitin 4 against tumoral cell lines and model fungi. Structurally, eryngitin 3 and 4 consist of 132 amino acids, most of them identical and exhibiting a single free cysteinyl residue. The amino acidic differences between the two toxins are (i) an additional phenylalanyl residue at the N-terminus of eryngitin 3, not retrieved in eryngitin 4, and (ii) an additional arginyl residue at the C-terminus of eryngitin 4, not retrieved in eryngitin 3. The 3D models of eryngitins show slight differences at the N- and C-terminal regions. In particular, the positive electrostatic surface at the C-terminal of eryngitin 4 is due to the additional arginyl residue not retrieved in eryngitin 3. This additional positive charge could interfere with the binding to the SRL (substrate) or with some ribosomal proteins (P-stalk structure) during substrate recognition. Full article

The aberrant activation of signaling pathways contributes to cancer cells with metabolic reprogramming. Thus, targeting signaling modulators is considered a potential therapeutic strategy for cancer. Subcellular fractionation, coimmunoprecipitation, biochemical analysis, and gene manipulation experiments revealed that decreasing the interaction of kirsten rat sarcoma viral oncogene homolog (KRAS) with p110α in lipid rafts with the use of naringenin (NGN), a citrus flavonoid, causes lipid raft-associated phosphatidylinositol 3-kinase (PI3K)−GTP-ras-related C3 botulinum toxin substrate 1 (Rac1)−protein kinase B (Akt)-regulated metabolic dysfunction of glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), leading to apoptosis in human nasopharyngeal carcinoma (NPC) cells. The use of lethal-7g (let-7g) mimic and let-7g inhibitor confirmed that elevated let-7g resulted in a decrease in KRAS expression, which attenuated the PI3K−Rac1−Akt−BCL-2/BCL-x_L-modulated mitochondrial energy metabolic functions. Increased let-7g depends on the suppression of the RNA-specificity of monocyte chemoattractant protein-induced protein-1 (MCPIP1) ribonuclease since NGN specifically blocks the degradation of pre-let-7g by NPC cell-derived immunoprecipitated MCPIP1. Converging lines of evidence indicate that the inhibition of MCPIP1 by NGN leads to let-7g upregulation, suppressing oncogenic KRAS-modulated PI3K–Rac1–Akt signaling and thereby impeding the metabolic activities of aerobic glycolysis and mitochondrial OXPHOS. Full article