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Viruses Apr 2024The cucumber mosaic virus (CMV) 2b protein is a suppressor of plant defenses and a pathogenicity determinant. Amongst the 2b protein's host targets is the RNA silencing...
The cucumber mosaic virus (CMV) 2b protein is a suppressor of plant defenses and a pathogenicity determinant. Amongst the 2b protein's host targets is the RNA silencing factor Argonaute 1 (AGO1), which it binds to and inhibits. In , if 2b-induced inhibition of AGO1 is too efficient, it induces reinforcement of antiviral silencing by AGO2 and triggers increased resistance against aphids, CMV's insect vectors. These effects would be deleterious to CMV replication and transmission, respectively, but are moderated by the CMV 1a protein, which sequesters sufficient 2b protein molecules into P-bodies to prevent excessive inhibition of AGO1. Mutant 2b protein variants were generated, and red and green fluorescent protein fusions were used to investigate subcellular colocalization with AGO1 and the 1a protein. The effects of mutations on complex formation with the 1a protein and AGO1 were investigated using bimolecular fluorescence complementation and co-immunoprecipitation assays. Although we found that residues 56-60 influenced the 2b protein's interactions with the 1a protein and AGO1, it appears unlikely that any single residue or sequence domain is solely responsible. In silico predictions of intrinsic disorder within the 2b protein secondary structure were supported by circular dichroism (CD) but not by nuclear magnetic resonance (NMR) spectroscopy. Intrinsic disorder provides a plausible model to explain the 2b protein's ability to interact with AGO1, the 1a protein, and other factors. However, the reasons for the conflicting conclusions provided by CD and NMR must first be resolved.
Topics: Argonaute Proteins; Cucumovirus; Arabidopsis; Arabidopsis Proteins; Protein Binding; Viral Proteins; Host-Pathogen Interactions; Viral Replicase Complex Proteins; Plant Diseases; RNA-Dependent RNA Polymerase; Methyltransferases
PubMed: 38793558
DOI: 10.3390/v16050676 -
Microbiology Spectrum May 2024Sweet orange () is one of the most important fruit crops worldwide. Virus infections in this crop can interfere with cellular processes, causing dramatic economic...
Sweet orange () is one of the most important fruit crops worldwide. Virus infections in this crop can interfere with cellular processes, causing dramatic economic losses. By performing RT-qPCR analyses, we demonstrated that citrus psorosis virus (CPsV)-infected orange plants exhibited higher levels of unprocessed microRNA (miRNA) precursors than healthy plants. This result correlated with the reported reduction of mature miRNAs species. The protein 24K, the CPsV suppressor of RNA silencing (VSR), interacts with miRNA precursors . Thus, this protein becomes a candidate responsible for the increased accumulation of unprocessed miRNAs. We analyzed 24K RNA-binding and protein-protein interaction domains and described patterns of its subcellular localization. We also showed that 24K colocalizes within nuclear D-bodies with the miRNA biogenesis proteins DICER-LIKE 1 (DCL1), HYPONASTIC LEAVES 1 (HYL1), and SERRATE (SE). According to the results of bimolecular fluorescence complementation and co-immunoprecipitation assays, the 24K protein interacts with HYL1 and SE. Thus, 24K may inhibit miRNA processing in CPsV-infected citrus plants by direct interaction with the miRNA processing complex. This work contributes to the understanding of how a virus can alter the regulatory mechanisms of the host, particularly miRNA biogenesis and function.IMPORTANCESweet oranges can suffer from disease symptoms induced by virus infections, thus resulting in drastic economic losses. In sweet orange plants, CPsV alters the accumulation of some precursors from the regulatory molecules called miRNAs. This alteration leads to a decreased level of mature miRNA species. This misregulation may be due to a direct association of one of the viral proteins (24K) with miRNA precursors. On the other hand, 24K may act with components of the cell miRNA processing machinery through a series of predicted RNA-binding and protein-protein interaction domains.
PubMed: 38785434
DOI: 10.1128/spectrum.03513-23 -
Journal of Orthopaedic Surgery and... May 2024Elderly patients suffering from osteoporotic fractures are more susceptible to delayed union or nonunion, and their bodies then are in a state of low-grade chronic...
BACKGROUND
Elderly patients suffering from osteoporotic fractures are more susceptible to delayed union or nonunion, and their bodies then are in a state of low-grade chronic inflammation with decreased antioxidant capacity. Tanshinone IIA is widely used in treating cardiovascular and cerebrovascular diseases in China and has anti-inflammatory and antioxidant effects. We aimed to observe the antioxidant effects of Tanshinone IIA on mesenchymal stem cells (MSCs), which play important roles in bone repair, and the effects of local application of Tanshinone IIA using an injectable biodegradable hydrogel on osteoporotic fracture healing.
METHODS
MSCs were pretreated with or without different concentrations of Tanshinone IIA followed by HO treatment. Ovariectomized (OVX) C57BL/6 mice received a mid-shaft transverse osteotomy fracture on the left tibia, and Tanshinone IIA was applied to the fracture site using an injectable hydrogel.
RESULTS
Tanshinone IIA pretreatment promoted the expression of nuclear factor erythroid 2-related factor 2 and antioxidant enzymes, and inhibited HO-induced reactive oxygen species accumulation in MSCs. Furthermore, Tanshinone IIA reversed HO-induced apoptosis and decrease in osteogenic differentiation in MSCs. After 4 weeks of treatment with Tanshinone IIA in OVX mice, the bone mineral density of the callus was significantly increased and the biomechanical properties of the healed tibias were improved. Cell apoptosis was decreased and Nrf2 expression was increased in the early stage of callus formation.
CONCLUSIONS
Taken together, these results indicate that Tanshinone IIA can activate antioxidant enzymes to protect MSCs from HO-induced cell apoptosis and osteogenic differentiation inhibition. Local application of Tanshinone IIA accelerates fracture healing in ovariectomized mice.
Topics: Animals; Abietanes; Female; Mesenchymal Stem Cells; Apoptosis; Mice, Inbred C57BL; Fracture Healing; Ovariectomy; Mice; Antioxidants; Hydrogen Peroxide; Osteogenesis; Osteoporotic Fractures
PubMed: 38783358
DOI: 10.1186/s13018-024-04793-x -
BioRxiv : the Preprint Server For... May 2024PCIF1 (hosphorylated TD-nteracting actor ) is the mRNA (2'-O-methyladenosine-N(6)-)-methyltransferase that catalyzes the formation of cap-adjacent...
PCIF1 (hosphorylated TD-nteracting actor ) is the mRNA (2'-O-methyladenosine-N(6)-)-methyltransferase that catalyzes the formation of cap-adjacent N,2'-O-dimethyladenosine (m6Am) by methylating adenosines at the first transcribed position of capped mRNAs. While previous studies assumed that PCIF1 was nuclear, cell fractionation and immunofluorescence both show that a population of PCIF1 is localized to the cytoplasm. Further, PCIF1 redistributes to stress granules upon oxidative stress. Immunoprecipitation studies with stressed cells show that PCIF1 also physically interacts with G3BP and other stress granule components. In addition, PCIF1 behaves as a stress granule component as it disassociates from stress granules upon recovery from stress. Overexpressing full-length PCIF1 also inhibits stress granule formation, while knocking out PCIF1 slows stress granule disassembly. Next, our enhanced crosslinking and immunoprecipitation (eCLIP) data show that PCIF1 binds mRNAs in their coding sequences rather than cap-proximal regions. Further PCIF1's association with mRNAs increased upon NaAsO stress. In contrast to eCLIP data, ChIP-Seq experiments show that PCIF1 is predominantly associated with transcription start sites rather than gene bodies, indicating that PCIF1's association with mature mRNA is not co-transcriptional. Collectively, our data suggest that PCIF1 has cytoplasmic RNA surveillance role(s) independent of transcription-associated cap-adjacent mRNA modification, particularly during the stress response.
PubMed: 38766247
DOI: 10.1101/2024.05.08.593175 -
BioRxiv : the Preprint Server For... May 2024Precision nuclear run-on (PRO) sequencing (PRO-seq) is a powerful technique for mapping polymerase active sites with nucleotide resolution and measuring newly...
Precision nuclear run-on (PRO) sequencing (PRO-seq) is a powerful technique for mapping polymerase active sites with nucleotide resolution and measuring newly synthesized transcripts at both promoters and enhancer elements. The current PRO-seq protocol is time-intensive, technically challenging, and requires a large amount of starting material. To overcome these limitations, we developed rapid PRO-seq (rPRO-seq) which utilizes pre-adenylated single-stranded DNAs (AppDNA), a dimer blocking oligonucleotide (DBO), on-bead 5' RNA end repair, and column-based purification. These modifications enabled efficient transcriptome mapping within a single day (∼12 hours) increasing ligation efficiency, abolished adapter dimers, and reduced sample loss and RNA degradation. We demonstrate the reproducibility of rPRO-seq in measuring polymerases at promoters, gene bodies, and enhancers as compared to original PRO-seq protocols. Additionally, rPRO-seq is scalable, allowing for transcriptome mapping with as little as 25,000 cells. We apply rPRO-seq to study the role of Integrator in mouse hematopoietic stem and progenitor cell (mHSPC) homeostasis, identifying as an essential component of transcriptional regulation and RNA processing in mHSPC homeostasis. Overall, rPRO-seq represents a significant advance in the field of nascent transcript analyses and will be a valuable tool for generating patient-specific genome-wide transcription profiles with minimal sample requirements.
PubMed: 38766081
DOI: 10.1101/2024.05.08.593182 -
BioRxiv : the Preprint Server For... May 2024Biomolecular condensates play pivotal roles in many cellular processes, yet predicting condensate growth dynamics within the complex intracellular environment is...
Biomolecular condensates play pivotal roles in many cellular processes, yet predicting condensate growth dynamics within the complex intracellular environment is challenging. While chromatin mechanics are known to influence condensate coarsening in the nucleus, the effect of condensate properties remains unclear. Our study demonstrates that the interplay between condensate properties and chromatin mechanics dictates condensate growth dynamics. Through chemical dimerization, we induced condensates of various properties in the cell nuclei, revealing distinct growth mechanisms: diffusion-driven or ripening-dominated. To explain experimental observations, we developed a quantitative theory that uncovers the role of chromatin in modulating condensate growth via size-dependent pressure. We find that surface tension is a critical factor in determining whether condensates undergo elastic or Ostwald ripening. Our model predicts that different condensates are affected differently by chromatin heterogeneity, validated by experimentally perturbing chromatin organization. Taken together, our work elucidates how condensate surface tension and chromatin heterogeneity govern nuclear condensate ripening.
PubMed: 38766065
DOI: 10.1101/2024.05.07.593010 -
BioRxiv : the Preprint Server For... May 2024Chromatin is organized into compartments enriched with functionally-related proteins driving non-linear biochemical activities. Some compartments, transcription foci,...
Chromatin is organized into compartments enriched with functionally-related proteins driving non-linear biochemical activities. Some compartments, transcription foci, behave as liquid condensates. While the principles governing the enrichment of proteins within condensates are being elucidated, mechanisms that coordinate condensate dynamics with other nuclear processes like DNA replication have not been identified. We show that at the G1/S cell cycle transition, large transcription condensates form at histone locus bodies (HLBs) in a cyclin-dependent kinase 1 and 2 (CDK1/2)-dependent manner. As cells progress through S phase, ataxia-telangiectasia and Rad3-related (ATR) accumulates within HLBs and dissolves the associated transcription condensates. Integration of CDK1/2 and ATR signaling creates a phosphorylation code within the intrinsically-disordered region of mediator subunit 1 (MED1) coordinating condensate dynamics with DNA replication. Disruption of this code results in imbalanced histone biosynthesis, and consequently, global DNA damage. We propose the spatiotemporal dynamics of transcription condensates are actively controlled via phosphorylation and essential for viability of proliferating cells.
PubMed: 38765978
DOI: 10.1101/2024.05.10.593572 -
Chemosphere May 2024The decommissioning and normal functioning of nuclear facilities can result in the production and release of airborne particles in the environment. Aquatic biota are...
The decommissioning and normal functioning of nuclear facilities can result in the production and release of airborne particles in the environment. Aquatic biota are expected to be exposed to these particles considering that nuclear facilities are often located near water bodies. Aerosols, such as cement dust, can interact with radionuclides as well as with heavy metals, and therefore elicit not only radiological impacts but also chemical toxicity. In the present study, we aimed to determine the effects of hydrogenated cement particles (HCPs) as a first step before evaluating any radiotoxicity of tritiated cement particles in the marine mussels, Mytilus galloprovincialis. Responses at different levels of biological organisation were assessed, including clearance rate (CR), tissue specific accumulation, DNA damage and transcriptional expression of key stress related genes. Acute (5 h) and medium-term, chronic (11 d) exposures to 1000 μg L HCPs showed that bioaccumulation, assessed using Cu as a proxy and determined by inductively coupled plasma mass spectrometry, was time and tissue dependent. The highest levels of Cu were found in the digestive gland (DG) after 11 d. HCP exposure caused changes in the expression of oxidative and other stress-related genes, including mt20 in DG and gst and sod in the gill after 5 h exposure, while an overexpression of hsp70 in the gill was observed after 11 d. Genotoxic effects in haemocytes were observed after 11 d of HCP exposure. Multivariate analysis indicated that oxidative stress is the most probable factor contributing to overall physiological dysfunction. Our results provide a baseline to perform further studies employing tritiated cement particles. Specifically, future work should focus on the DG since only this tissue showed significant bioaccumulation when compared to the negative control.
PubMed: 38759810
DOI: 10.1016/j.chemosphere.2024.142243 -
Burns & Trauma 2024Severe burn injury causes a hypermetabolic response, resulting in muscle protein catabolism and multiple organ damage syndrome. However, this response has not yet been...
BACKGROUND
Severe burn injury causes a hypermetabolic response, resulting in muscle protein catabolism and multiple organ damage syndrome. However, this response has not yet been continuously characterized by metabolomics in patients. This study aims to quantify temporal changes in the metabolic processes of patients with severe burns.
METHODS
We employed H-nuclear magnetic resonance (NMR) spectroscopy to scrutinize metabolic alterations during the initial 35 days following burn injury in a cohort of 17 adult patients with severe burns, with 10 healthy individuals included as controls. Plasma specimens were collected from patients on postburn days 1, 3, 7, 14, 21, 28 and 35. After performing multivariate statistical analysis, repeated-measures analysis of variance and time-series analysis, we quantified changes in metabolite concentrations.
RESULTS
Among the 36 metabolites quantified across 119 samples from burn patients, branched-chain amino acids, glutamate, glycine, glucose, pyruvate, lactate, trimethylamine -oxide and others exhibited obvious temporal variations in concentration. Notably, these metabolites could be categorized into three clusters based on their temporal characteristics. The initial response to injury was characterized by changes in lactate and amino acids, while later changes were driven by an increase in fatty acid catabolism and microbial metabolism, leading to the accumulation of ketone bodies and microbial metabolites.
CONCLUSIONS
Metabolomics techniques utilizing NMR have the potential to monitor the intricate processes of metabolism in patients with severe burns. This study confirmed that the third day after burn injury serves as the boundary between the ebb phase and the flow phase. Furthermore, identification of three distinct temporal patterns of metabolites revealed the intrinsic temporal relationships between these metabolites, providing clinical data for optimizing therapeutic strategies.
PubMed: 38756185
DOI: 10.1093/burnst/tkae007 -
Heliyon May 2024This research introduces an innovative framework for addressing the escalating issue of greenhouse gas emissions through the integration of game theory with differential...
This research introduces an innovative framework for addressing the escalating issue of greenhouse gas emissions through the integration of game theory with differential equations, proposing a novel model to simulate the regulatory dynamics between emission sources and legislative actions. By blending advanced mathematical modeling with environmental science, this paper underscores the critical necessity for pioneering, proactive strategies in environmental management and policy formulation. Central to our approach is the simulation of interactions within a game-theoretic context, aiming to delineate optimal strategies for emission sources and regulatory bodies, factoring in legislative constraints and environmental ramifications. The methodology employs a system of ordinary differential equations, capturing the dynamic, non-stationary nature of atmospheric processes and offering a realistic portrayal of the challenges in mitigating greenhouse gas emissions. Furthermore, the study introduces a fee-based regulatory mechanism designed to encourage emission reductions, highlighting the economic implications of such strategies. Significantly contributing to environmental management, this research presents a detailed model capable of predicting the trajectory of greenhouse gas emissions over a decade, considering the potential impact of technological innovations in emission control. The conclusion emphasizes the promising role of artificial intelligence in refining environmental governance, acknowledging the complexities and limitations inherent in predictive modeling. Aimed at policymakers and environmental scientists, this paper serves as a strategic tool for informed decision-making, advocating for a multidisciplinary approach to develop sustainable, effective solutions to combat one of the most critical environmental challenges facing the globe today.
PubMed: 38726135
DOI: 10.1016/j.heliyon.2024.e30549