mRNA Research Articles

Isosteric 3D Bicyclo[1.1.1]Pentane (BCP) Core-Based Lipids for mRNA Delivery and CRISPR/Cas Gene Editing.

Lipid nanoparticles (LNPs) are an essential component of messenger RNA (mRNA) vaccines and genome editing therapeutics. Ionizable amino lipids, which play the most crucial role in enabling mRNA to overcome delivery barriers, have, to date, been restricted to two-dimensional (2D) architectures. Inspired by improved physicochemical properties resulting from the incorporation of three-dimensionality (3D) into small-molecule drugs, we report the creation of 3D ionizable lipid designs through the introduction of bicyclo[1.1.1]pentane (BCP) core motifs. BCP-based lipids enabled efficient in vivo mRNA delivery to the liver and spleen with significantly greater performance over 2D benzene- and cyclohexane-based analogues. Notably, lead BCP-NC2-C12 LNPs mediated ∼90% reduction in the PCSK9 serum protein level via CRISPR/Cas9 gene knockout, outperforming 2D controls and clinically used DLin-MC3-DMA LNPs at the same dose. Here, we introduce BCP-based designs with superior in vivo activity, thereby expanding the chemical scope of ionizable amino lipids from 2D to 3D and offering a promising avenue to improve mRNA and gene editing efficiency for the continued development of genetic medicines.

The Influence of Extra-Ribosomal Functions of Eukaryotic Ribosomal Proteins on Viral Infection

The eukaryotic ribosome is a large ribonucleoprotein complex consisting of four types of ribosomal RNA (rRNA) and approximately 80 ribosomal proteins (RPs), forming the 40S and 60S subunits. In all living cells, its primary function is to produce proteins by converting messenger RNA (mRNA) into polypeptides. In addition to their canonical role in protein synthesis, RPs are crucial in controlling vital cellular processes such as cell cycle progression, cellular proliferation, differentiation, DNA damage repair, genome structure maintenance, and the cellular stress response. Viruses, as obligate intracellular parasites, depend completely on the machinery of the host cell for their replication and survival. During viral infection, RPs have been demonstrated to perform a variety of extra-ribosomal activities, which are especially important in viral disease processes. These functions cover a wide range of activities, ranging from controlling inflammatory responses and antiviral immunity to promoting viral replication and increasing viral pathogenicity. Deciphering the regulatory mechanisms used by RPs in response to viral infections has greatly expanded our understanding of their functions outside of the ribosome. Furthermore, these findings highlight the promising role of RPs as targets for the advancement of antiviral therapies and the development of novel antiviral approaches. This review comprehensively examines the many functions of RPs outside of the ribosome during viral infections and provides a foundation for future research on the host–virus interaction.

3D printed β-TCP scaffolds loaded with SVVYGLR peptide for promoting revascularization and osteoinduction

It is crucial for the successful transplantation of large segmental bone defects to achieve rapid vascularization within bone scaffolds. However, there are certain limitations including uncontrolled angiogenesis and inadequate vascular function. Therefore, there is an urgent need to develop bone scaffolds with functional vascular networks. In our study, porousβ-tricalcium phosphate (β-TCP) scaffolds with varying pore sizes were prepared by 3D printing technology, loaded with osteopontin derived peptide Ser-Val-Val-Tyr-Gly-Leu-Arg (SVVYGLR) to induce osteoinduction and angiogenesis.In vitro, the proliferation and migration behaviors of human umbilical vein endothelial cell on scaffolds were assessed by Cell Counting Kit-8, confocal laser scanning microscopy and scanning electron microscopy. And the osteogenic ability of bone marrow mesenchymal stem cells was assessed using alkaline phosphatase staining and Alizarin Red S staining. The messenger ribonucleic acid (mRNA) expression levels of cell adhesion molecule (CD31), vascular endothelial growth factor and hypoxia inducible factor-1αin each group were detected by quantitative real-time fluorescence polymerase chain reaction (PCR) analysis.In vivo, cube scaffolds were subcutaneously implanted on the right hips of Sprague-Dawley (SD) rats for 6 weeks. Hematoxylin and Eosin staining, Masson's trichrome staining, and immunohistochemical analysis of osteocalcin and CD31 were performed on slices for every sample with three sections to explore the effect of SVVYGLR-loaded scaffolds on angiogenesis and osteogenic induction for bone reconstruction. The results indicate that 3D printedβ-TCP scaffolds loaded with the SVVYGLR peptide offer superior revascularization and osteoinduction to the scaffolds without the SVVYGLRin situ. Moreover, scaffolds with a pore size of 400 µm demonstrate higher effectiveness compared to those with a 150 µm pore size. The distinct hollow channel scaffolds and the specific SVVYGLR peptide substantially improve cell adhesion, spreading, and proliferation, as well as promote angiogenesis and bone formation. Furthermore, scaffolds with a pore size of 400 µm may exhibit greater efficacy compared to those with a pore size of 150 µm. The results of this study provide an idea for the development of practical applications for tissue-engineered bone scaffolds.

Evaluation of Breast Cancer Gene Type 1 (BRCA1) Protein Levels in Cancer Tissue Using Surface-Enhanced Raman Spectroscopy.

Raman spectroscopy is a chemical process that utilizes the interaction between light and matter to get significant insights into the structure or characteristics of matter. Raman spectroscopy techniques, such as quantitative evaluation, early diagnostic capabilities, and elucidation of the spectral properties of tissues, are excellent candidates for use in research. In cancer, changes in genes and proteins expressed by related genes are associated with a poor prognosis and aggressive tumor characteristics. Due to modifications and regulatory steps in protein translation, the results of the messenger RNA (mRNA) expression of genes may not correctly reflect the results of protein expression. For this reason, the mRNA and protein expressions of genes are studied in parallel in molecular studies on cancer. In our study, the breast cancer gene type 1 (BRCA1) gene, which is frequently studied in breast cancer and is relatively more difficult to measure by traditional methods due to its high molecular weight, was selected, and protein quantification was performed in tissue samples by Raman spectroscopy. With Raman spectroscopy, it is possible to obtain rapid and precise quantitative results even with a small amount of sample, so it is quite advantageous compared to traditional methods. In our study, we performed surface-enhanced Raman spectroscopy (SERS) to analyze the quantitative protein amount. SERS is a highly sensitive method for detecting compounds at low concentrations. For this purpose, magnetic nanoparticles modified with protein antibodies were used, and the target protein was withdrawn from the complex environment and transferred to an appropriate buffer environment. The calibration curve for BRCA1, which plots Raman intensity against concentration, was derived by calculating the average response reading from duplicate assays conducted under identical conditions. The BRCA1 protein levels of cells were determined from the regression curve of the BRCA1 protein. The relation between the concentration of BRCA1 protein and SERS spectrum intensity was determined to be logarithmic in the range of 300 µg·mL-1 to 292 ng·mL-1 (R2 = 0.9928, limit of detection = 10.41 µg·mL-1, and limit of quantitation = 31.24 µg·mL-1).

Early Detection of Hearing Impairment Signals Post-mRNA COVID-19 Vaccination: A Disproportionality Analysis Study on French Pharmacovigilance Database.

Improving adverse events following immunisation (AEFI) detection is vital for vaccine safety surveillance, as an early safety signal can help minimize risks. In February 2022, the World Health Organization reported a preliminary signal on sudden sensorineural hearing loss (SSNHL) following coronavirus disease 2019 (COVID-19) vaccination, 54 million persons in France received at least one dose, covering 78.8% of the population within a year. The primary objective of this study was to identify a method of disproportionality analysis capable to detect a safety signal for hearing impairment (HI) as early as possible during the initial phases of the COVID-19 vaccination campaign. Secondly, we described all cases of SSNHL reported during vaccine booster campaigns in France. Data from January 2011 to February 2022 were extracted from the French pharmacovigilance database. Cases were all spontaneous reports ofAEFI for elasomeran and tozinameran, while non-cases wereAEFIreported for other vaccines. Disproportionality analysis for HI was performed monthly during 2021, to estimate a reporting odds ratio (ROR). Four different methods were used for ROR estimation. Furthermore, we reviewed cases of SSNHL following messenger RNA COVID-19 vaccinations reported during booster campaigns, from 2 February 2022 to 1 March 2023, based on a comprehensive medical evaluation. Using a standard methodology, we identified a signal on 31 July 2021 (ROR 1.50, 95% confidence interval [CI] [1.06-2.18]). Multivariate analysis adjusted for sex, age, ototoxic drugs and excluding reference reports of common AEFI for vaccines allowed us to detect the HI signal as early as 31 March 2021 (ROR 2.67, 95% CI [1.36-5.57]). The SSNHL reporting rate was estimated to be 0.83/1,000,000 doses for tozinameran and 4.3/1,000,000 for elasomeran during the booster campaigns. Using a well-structured disproportionality analysis could have enhanced early detection of safety signals and contribute to risk minimizing measures. According to descriptive data, HI following mRNA COVID-19 vaccines remains rare.

Identification and Analysis of Autophagy-Related Genes as Diagnostic Markers and Potential Therapeutic Targets for Tuberculosis Through Bioinformatics.

According to the World Health Organization, Mycobacterium tuberculosis infections affect approximately 25% of the world's population. There is mounting evidence linking autophagy and immunological dysregulation to tuberculosis (TB). As a result, this research set out to discover TB-related autophagy-related biomarkers and prospective treatment targets. We used five autophagy databases to get genes linked to autophagy and Gene Expression Omnibus databases to get genes connected to TB. Then, functional modules associated with autophagy were obtained by analyzing them using weighted gene co-expression network analysis. Both Gene Ontology and Kyoto Encyclopedia of Genes and Genomes were used to examine the autophagy-related genes (ATGs) of important modules. Limma was used to identify differentially expressed ATGs (DE-ATGs), and the external datasets were used to further confirm their identification. We used DE-ATGs and a protein-protein interaction network to search the hub genes. CIBERSORT was used to estimate the kinds and amounts of immune cells. After that, we built a drug-gene interaction network and a network that included messenger RNA, small RNA, and DNA. At last, the differential expression of hub ATGs was confirmed by RT-qPCR, immunohistochemistry, and western blotting. The diagnostic usefulness of hub ATGs was evaluated using receiver operating characteristic curve analysis. Including 508 ATGs, four of the nine modules strongly linked with TB were deemed essential. Interleukin 1B (IL1B), CAPS1, and signal transducer and activator of transcription 1 (STAT1) were identified by intersection out of 22 DE-ATGs discovered by differential expression analysis. Research into immune cell infiltration found that patients with TB had an increased proportion of plasma cells, CD8 T cells, and M0 macrophages. A competitive endogenous RNA network utilized 10 long non-coding RNAs and 2 miRNAs. Then, the IL1B-targeted drug Cankinumad was assessed using this network. During bioinformatics analysis, three hub genes were validated in mouse and macrophage infection models. We found that IL1B, CASP1, and STAT1 are important biomarkers for TB. As a result, these crucial hub genes may hold promise as TB treatment targets.

Advances in the study of LNPs for mRNA delivery and clinical applications.

Messenger ribonucleic acid (mRNA) was discovered in 1961 as an intermediary for transferring genetic information from DNA to ribosomes for protein synthesis. The COVID-19 pandemic brought worldwide attention to mRNA vaccines. The emergency use authorization of two COVID-19 mRNA vaccines, BNT162b2 and mRNA-1273, were major achievements in the history of vaccine development. Lipid nanoparticles (LNPs), one of the most superior non-viral delivery vectors available, have made many exciting advances in clinical translation as part of the COVID-19 vaccine and therefore has the potential to accelerate the clinical translation of many gene drugs. In addition, due to these small size, biocompatibility and excellent biodegradability, LNPs can efficiently deliver nucleic acids into cells, which is particularly important for current mRNA therapeutic regimens. LNPs are composed cationic or pH-dependent ionizable lipid bilayer, polyethylene glycol (PEG), phospholipids, and cholesterol, represents an advanced system for the delivery of mRNA vaccines. Furthermore, optimization of these four components constituting the LNPs have demonstrated enhanced vaccine efficacy and diminished adverse effects. The incorporation of biodegradable lipids enhance the biocompatibility of LNPs, thereby improving its potential as an efficacious therapeutic approach for a wide range of challenging and intricate diseases, encompassing infectious diseases, liver disorders, cancer, cardiovascular diseases, cerebrovascular conditions, among others. Consequently, this review aims to furnish the scientific community with the most up-to-date information regarding mRNA vaccines and LNP delivery systems.

Qufeng Tongluo Decoction May Alleviate Podocyte Injury Induced by High Glucose and Hydrogen Peroxide by Regulating Autophagy

Background: Diabetic kidney disease is a significant complication of diabetes. Previous studies have confirmed that Qufeng Tongluo (QFTL) decoction can alleviate podocyte injury in a diabetic rat model, but its mechanism remains unclear. The present in vitro study investigated QFTL’s mechanism in protecting podocytes. Methods: The mouse podocyte clone 5 (MPC-5) cell line stimulated by high glucose and hydrogen peroxide was used as a model for podocyte injury. The cells were treated with QFTL, QFTL + SC79 (activator of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt), and C2 ceramide (inhibitor of the PI3K/Akt). Immunofluorescence, western blot and polymerase chain reaction (PCR) were employed to detect the expression of microtubule-associated protein 1 light chain 3 (LC3), sequestosome 1 (SQSTM1/p62), PI3K, Akt, and phosphatase and tensin homolog (PTEN) proteins and their messenger RNA (mRNA) levels. Results: High glucose and hydrogen peroxide stimulation may disrupt podocytes’ cytoskeletal structure, while QFTL may mitigate these structural changes. After the SC79 intervention, QFTL’s alleviation of the cytoskeletal structure damage disappeared. High glucose and hydrogen peroxide stimulation decreased the expression of LC3 in MPC-5 cells, whereas QFTL upregulated its expression, but this effect was reversed by the SC79 intervention. Interestingly, P62 decreased after high glucose + hydrogen peroxide stimulation, and QFTL failed to upregulate P62. The expression levels of PI3K, Akt, and PTEN in the model group decreased, which were improved by QFTL. Conclusion: These results suggest that QFTL can maintain the stability of autophagic flux in injured podocytes, which may be related to its ability to improve P62 expression but not to regulating the PI3K/Akt signaling pathway.

RNA-based Therapeutics: Past, Present and Future Prospects, Challenges in Cancer Treatment.

It is becoming more and harder in today's climate to disregard the impact of cancer on social health. Even though a significant amount of money is spent annually on cancer research, it still ranks as the second leading cause of death worldwide. Additionally, only about half of the patients suffering from complex forms of cancer survive a year after receiving traditional cancer therapies. A method for silencing genes is called RNA interference (RNAi). Such a method is very effective in focusing on genes linked to cancer. Most gene products implicated in cancer have recently been used as RNA interference (RNAi) therapeutic targets. According to the findings from this research, RNAi application is necessary for today's cancer treatment to target functioning carcinogenic molecules and tumor resistance to chemotherapy and radiation. Proapoptotic and antiproliferative activity has been reported from previous research studies on cell culture systems, animal models, and clinical trials through the knockdown of gene products from RNAi technology. Numerous novel RNAi-based medications are now in the clinical trial stages thanks to the discovery of the RNAi mechanism and advancements in the area. In the future, genomic-based personalized medicines can be developed through this RNAi therapy. Hopefully, cancer sufferers will find this sort of therapy to be one of the most effective ones. Various kinds of RNA-based treatments, such as aptamers, small interfering RNAs, microRNAs, antisense oligonucleotides, and messenger RNA, are covered in broad terms in this study. We also present an overview of the RNA-based therapies that have received regulatory approval in the past or are now undergoing clinical studies.

Identifying predictors of treatment response and molecular changes induced by neoadjuvant chemotherapy and endocrine therapy in hormone receptor-positive/HER2-negative breast cancer: the NEOENDO translational study

Predictors of response to neoadjuvant chemotherapy (NACT) and endocrine therapy (NET) in hormone receptor-positive (HoR+)/human epidermal growth factor receptor 2 (HER2)-negative breast cancer (BC) are required. Also, pathological and molecular changes induced by both strategies and their impact on patients' outcomes have not been reported so far. In a cohort of 186 patients with early-stage HoR+/HER2-negative BC treated with NACT or NET, we assessed the association of baseline main clinicopathological features and PAM50 gene expression (GE), intrinsic subtypes (IS) and risk-of-relapse (ROR-P) score with pathological outcomes according to treatment strategy. Molecular NACT/NET-induced changes were described and compared, along with their associations with event-free survival (EFS). Comparison of the two cohorts after propensity score matching (PSM) was used as sensitivity analysis. Molecular changes were confirmed in cell lines. NACT was associated with higher rates of residual cancer burden (RCB)-0/I than NET in the overall population (38.2% versus 13.5%, P < 0.001) and after PSM (P= 0.036). PAM50 non-luminal IS were the only independent and positive predictor of RCB-0/I (P= 0.024) in the NACT cohort, while MMP11 messenger RNA levels were the only independent and negative predictor (P= 0.014) in the NET cohort. Both treatments shifted the tumor types toward less aggressive forms (i.e. PAM50 luminal A/normal-like), lowered the risk of recurrence in terms of ROR-P, up-regulated selected immune genes and PAM50 basal-like-related genes/signature and significantly downregulated proliferation-/luminal-/HER2-related genes/signatures, though NACT more than NET. Molecular findings were confirmed after PSM. A net reduction in proliferation-related genes and ROR-P was confirmed in cell lines with chemotherapy and endocrine therapy. Different baseline molecular features associated with diverse kind of responses (ROR-P downstaging, Ki67 reduction or pathological responses) with NACT and NET. Decreasing ROR-P and transitioning the tumor subtype to resemble normal tissue (i.e. PAM50 normal-like) suggested improved EFS. NACT was more effective in the molecular and dimensional tumor 'downstaging' than NET but baseline molecular features associated with differential responses according to treatment strategy. Examining baseline and post-treatment GE might help tailor more personalized and effective care.

METTL3-VISTA axis-based combination immunotherapy for APC truncation colorectal cancer

ObjectiveAlthough immune checkpoint blockade (ICB) therapy represents a bright spot in antitumor immunotherapy, its clinical benefits in colorectal cancer (CRC) are limited. Therefore, a new target for mediating CRC immunosuppression...

Sequentially amplified integration of catalytic DNA circuits for high-performance intracellular imaging of miRNA and interpretation of mRNA-miRNA signalling pathway

The cascaded catalytic circuits are viable tools for improving the signal gain of biosensors, yet their sensing performance is still limited by the signal leakage from complex biological environment and unsatisfying reaction efficiency from inter-reactants steric hindrance. Herein, we proposed a catalytically localized DNA (CLD) circuit for the accurate and high-efficiency imaging of microRNA (miRNA) in living cells by virtue of the sequentially and successively amplified integration of catalytic DNA circuits. The compact CLD circuit was constructed by integrating two elemental catalytic circuits, cell-responsive EDR module and analyte-sensing CHA module, where CHA module was initially caged in EDR module for eliminating the unwanted off-site and off-target signal leakage. Only by cell-specific messenger RNA (mRNA)-activated EDR operation then the elemental CHA circuit could be successively connected to facilitate the highly efficient intramolecular reaction with low steric hindrance, thus leading to accelerated reaction efficiency for miRNA analyte. The multiple molecular recognition and the spatial self-confinement of the smart CLD circuit enable the accurate and high-efficiency imaging of intracellular miRNA. The interaction network of mRNA and miRNA was then investigated in situ through our CLD circuit, which provides a powerful tool for discovering the underlying signal pathways between these different RNAs in living cells.

METTL14 Regulates the m6A Modification of TRAF6 to Suppress Mitochondrial Dysfunction and Ferroptosis in Dopaminergic Neurons via the cGAS-STING Pathway.

The degeneration of dopaminergic (DA) neurons has emerged as a crucial pathological characteristic in Parkinson's disease (PD). To enrich the related knowledge, we aimed to explore the impact of the METTL14-TRAF6-cGASSTING axis in mitochondrial dysfunction and ferroptosis underlying DA neuron degeneration. 1-methyl-4-phenylpyridinium ion (MPP+) was used to treat DA neuron MN9D to develop the PD cell models. Afterward, a cell counting kit, flow cytometer, DCFH-DA fluorescent probe, and Dipyrromethene Boron Difluoride staining were utilized to measure the cell viability, iron concentration, ROS level, and lipid peroxidation, respectively. Meanwhile, the mitochondrial ultrastructure, the activity of mitochondrial respiratory chain complexes, and levels of malondialdehyde and glutathione were monitored. In addition, reverse transcription-quantitative polymerase chain reaction and western blot assays were adopted to measure the expression of related genes. cGAS ubiquitylation and TRAF6 messenger RNA (mRNA) N6-methyladenosine (m6A) levels, the linkages among METTL14, TRAF6, and the cGAS-STING pathway were also evaluated. METTL14 expression was low, and TRAF6 expression was high after MPP+ treatment. In MPP+-treated MN9D cells, METTL14 overexpression reduced ferroptosis, ROS generation, mitochondrial injury, and oxidative stress (OS) and enhanced mitochondrial membrane potentials. TRAF6 overexpression had promoting impacts on mitochondrial dysfunction and ferroptosis in MPP+-treated MN9D cells, which was reversed by further overexpression of METTL14. Mechanistically, METTL14 facilitated the m6A methylation of TRAF6 mRNA to down-regulate TRAF6 expression, thus inactivating the cGAS-STING pathway. METTL14 down-regulated TRAF6 expression through TRAF6 m6A methylation to inactivate the cGAS-STING pathway, thereby relieving mitochondrial dysfunction and ferroptosis in DA neurons.

Innovations in mRNA-based cancer immunotherapy: Challenges and future directions

Messenger RNA (mRNA) vaccines have quickly become a potent tool in cancer immunotherapy, with the potential to personalise cancer treatment and get around many of the drawbacks of conventional therapies. With an emphasis on their processes, technological developments, and clinical applications, this study examines the scientific breakthroughs and difficulties surrounding mRNA cancer vaccines. We begin by investigating the ways in which mRNA vaccines work to stimulate the immune system, encode antigens specific to tumours, and elicit a potent anti-tumor response. We also look at the variety of mRNA vaccines which are currently being deployed and emphasize their successes in clinical studies, especially when combined with immune checkpoint inhibitors. The effectiveness and safety of these vaccines have been significantly increased by technological developments, such as enhancements in mRNA stability, design, and delivery systems (such as lipid nanoparticles and polyplexes). Emerging technologies like circular RNA and self-amplifying present promising opportunities for enduring and more potent therapies. But there are still limitations with mRNA vaccines, such as stability, immunogenicity, degradation, and effective in vivo delivery. Concerns about possible adverse effects and safety in long-term applications are also covered in this review. Despite these obstacles, novel approaches are being developed to boost antigen expression, optimize delivery systems, and improve mRNA stability, establishing mRNA vaccines as a crucial component of tailored cancer immunotherapy. These developments provide the foundation for upcoming advances in mRNA vaccine technology and represent a major breakthrough in the fight against cancer.

Identification of mRNA biomarkers in extremely early hypertensive intracerebral hemorrhage (HICH)

IntroductionHypertensive intracerebral hemorrhage (HICH) stands out as a critical complication of primary hypertension. Consequently, investigating messenger RNA (mRNA) biomarkers becomes imperative, offering potential targets. This study is conducted for elucidating the expression profile of blood mRNA biomarkers in HICH.MethodsTwenty-five HICH patients were constituted the HICH group.Twenty-two healthy volunteers recruited and comprised the control group. Peripheral blood cells were extracted to identify candidate mRNA. The identified differential expressions of genes between the two groups were validated, and the potential associations between these differentially expressed genes and adverse events were analyzed. GO and KEGG enrichment of DEGs, Weighted Gene Co-expression Network and Protein Interaction Network were established. target mRNA was screened.ResultsThe study identified 3163 differentially expressed genes in HICH. 8 candidate mRNA (SPI1, HK3, HCK, SYK, CD14, FCER1G, CYBB, FGR) were pinpointed. Associations with pathways affecting HICH development included HIF-1 signaling, NF-kappa B signaling, and C-type lectin receptor signaling. In the HICH group, higher expressions of HK3, HCK, SYK, CD14, FCER1G, CYBB, and FGR, and lower SPI1 expression compared to the control group. HICH patients experienced high rates of complications: pulmonary infection (84%), epilepsy (16%), enlarged hematoma (20%), gastrointestinal bleeding (48%), malnutrition (84%), and lower limb deep vein thrombosis (DVT) (12%). Factors contributing to pulmonary infection included age and elevated expression of HCK, SYK, CD14, and FGR. SPI1 was associated with epilepsy, while its lower expression correlated with hematoma enlargement. Gastrointestinal bleeding was linked to increased cerebral hemorrhage. Malnutrition was associated with higher age, and expressions of HK3, HCK, SYK, CD14, FCER1G, CYBB, and FGR. Patients with lower limb DVT had elevated expressions of the identified genes.ConclusionIn hypertensive intracerebral hemorrhage, there are elevated expressions of HK3, HCK, SYK, CD14, FCER1G, CYBB, and FGR, along with reduced expression of SPI1. Furthermore, age, along with elevated expressions of HCK, SYK, CD14, and FGR, serves as influencing factors contributing to pulmonary infection in patients.

METTL14 Promotes Lipopolysaccharide-Induced Myocardial Damage via m6A-Dependent Stabilization of TRPM7 mRNA.

Sepsis-induced myocardial injury (SIMI) is a vital pathological component of severe sepsis and septic shock. As a prevalent internal mRNA modification in eukaryotic cells, N6-methyladenosine (m6A) modification is implicated in sepsis and immune disorders. Methyltransferase-like 14 (METTL14), a core subunit of the methyltransferase complex that catalyzes messenger RNA m6A modification, is involved in the regulation of human cardiomyocyte cell line (AC16) injury. This study aimed to explore the role and mechanism of METTL14 in lipopolysaccharide (LPS) -induced myocardial injury.Cell viability and apoptosis were analyzed via 3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT), flow cytometry, and TdT-mediated dUTP nick-end labeling (TUNEL) assay. The Tumor necrosis factor alpha (TNF-α) and Interleukin-1beta (IL-1β) levels were analyzed via Enzyme linked immunosorbent assay (ELISA). Caspase-3 activity, reactive oxygen species activity, malondialdehyde level, and glutathione level were assessed using special assay kits. The levels of transient receptor potential melastatin 7 (TRPM7) and METTL14 mRNA were determined via Real-time quantitative polymerase chain reaction (RT-qPCR). Meanwhile, the protein levels of TRPM7, METTL14, phospho-p65 (p-p65), total p65 (p65), p-IκBα, and total IκBα (IκBα) were examined via western blot assay.LPS treatment repressed AC16 cell viability and induced cell apoptosis, inflammatory response, oxidative stress, and ferroptosis in vitro. METTL14 and TRPM7 were upregulated in LPS-treated AC16 cells. At the molecular level, METTL14 could increase the stability of TRPM7 mRNA via m6A methylation. Moreover, METTL14 deficiency could abolish LPS-triggered AC16 cell injury and ferroptosis via TRPM7 regulation.METTL14 knockdown reversed LPS-caused myocardial cell damage mainly by regulating the stability of TRPM7 mRNA, providing a novel therapeutic target for septic cardiomyopathy treatment.

Comprehensive Analysis of circRNA and mRNA Revealing Potential Mechanism Underlying Neuroinflammation in BV2 Cells

Background: The significance of circular RNAs (circRNAs) in diabetic complications has been established. However, their role in basal and diabetic states, as well as cognitive dysfunction, requires further investigation. Methods: BV-2 microglial cells were exposed to high glucose (50mM) and insulin (2μM) for 48 hours. The levels of interleukin-1beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factoralpha (TNF-α) were assessed through quantitative polymerase chain reaction (qPCR), western blot, and ELISA. CircRNA and messenger RNA (mRNA) sequencing were performed, and the data were analyzed. Differentially expressed circRNAs and mRNAs were identified using qPCR. The circRNA-miRNA interaction was predicted using Miranda and TargetScan software, and their levels were quantified by qPCR. Results: The results demonstrated a significant increase in mRNA and protein levels of IL-1β, IL-6, and TNF-α in BV2 cells treated with glucose and insulin. Five circRNAs (four upregulated and one downregulated) were identified in both glucose and insulin groups compared to the control. Further qPCR analysis revealed marked increases in the levels of chr17:40159331- 40159711+ and chr2:72800499-72801858- (mmu_circ_0010164) in both treatment groups. Competitive endogenous RNA networks showed significant upregulation of mRNA levels of mitochondrial transcription termination factor 1b (Mterf1b) and G protein subunit gamma 4 (Gng4), accompanied by a decrease in mmu-miR-6918-3p and mmu-miR-7043-3p levels in the glucose and insulin groups compared to the control. Knockdown of mmu_circ_0010164 significantly inhibited the inflammatory response induced by glucose and insulin in BV-2 microglial cells. Conclusion: These findings indicate that both glucose and insulin can elicit inflammatory responses in BV2 cells through the modulation of mmu_circ_0010164 levels. The underlying mechanism may involve potential downstream targets of mmu_circ_0010164, specifically mmu-miR-7043-3p/Gng4 and mmu-miR-6918-3p/Mterf1b. This provides novel insights into the treatment of glucose-induced neuroinflammation.

Adenosine relaxes vagina smooth muscle through the cyclic guanosine monophosphate- and cyclic guanosine monophosphate-dependent pathways.

In males, adenosine (ADO) is known to relax penile smooth muscles, although its role in the vagina is not yet fully elucidated. This study investigated the effect of ADO on vagina smooth muscle activity, using a validated female Sprague-Dawley rat model. Contractility studies, using noradrenaline-precontracted vaginal strips, tested the effects of ADORA1/3 antagonists and ADORA2A/2B antagonists and agonists. Increasing doses of ADO were tested after in vivo or in vitro treatment with Nω-nitro-L-arginine-methyl-ester hydrochloride (L-NAME) or with guanylate or adenylate cyclase inhibitors. Immunopositivity for ADORA2A and ADORA2B was assessed, and messenger RNA (mRNA) analysis was performed. Cyclic ADO monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were quantified both in rat vagina smooth muscle cells (rvSMCs) and in vaginal tissues with increasing doses of ADO. Demonstrating ADO's role in the relaxing/contractile mechanism in distal vagina smooth muscle. All ADO receptors mRNAs were expressed in vaginal tissue, with a prevalent content of ADORA2B. A high expression of genes regulating ADO catabolism (ADK) and de novo synthesis (NT5E) was found. In vaginal strips, ADO induced relaxation with IC50 = 144.7μM and a flat pseudo-Hill coefficient value = -0.42, indicating an activity on heterogeneous receptors. Blocking ADORA1/3 shifted ADO response to the left and with a steeper slope. ADORA2A/2B agonists showed a higher potency than ADO in inducing relaxation. Immunolocalization confirmed the presence of ADORA2A/2B in vaginal musculature, in the blood vessels endothelium, and in the epithelium. ADO stimulation of vagina tissues induced a significant increase in cAMP and cGMP contents. Experiments on rvSMCs confirmed that ADO time- and dose-dependently stimulated cAMP production in these cells. However, ADORA2A/2B antagonists, although reducing the ADO-induced relaxation, did not completely block it. A similar inhibition was obtained by blocking adenylate cyclase. Overall, these findings suggest that ADO relaxation involves other pathways, eg, nitric oxide (NO)/cGMP. Accordingly, blocking NO formation through L-NAME substantially blunted ADO responsiveness, as it does the block of cGMP formation through 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one. Simultaneous incubation with cGMP and cAMP blockers completely inhibited ADO responsiveness. The study highlights ADO's role in regulating vaginal smooth muscle activity, suggesting its potential effect on the vagina. This is the first study on ADO in the vagina, although the results are preliminary and limited to the rat model. These results show that ADO acts as a vaginal relaxing modulator through selective activation of receptors involving not only cAMP but also cGMP.

An mRNA-encoded dominant-negative inhibitor of transcription factor RUNX1 suppresses vitreoretinal disease in experimental models.

Messenger RNA (mRNA)-based therapies are a promising approach to medical treatment. Except for infectious diseases, no other disease has mRNA-based therapies available. The eye is an ideal model for mRNA therapeutic development because it requires limited dosing. Proliferative vitreoretinopathy (PVR) is a blinding condition caused by retinal detachment that now lacks available medical treatment, with surgery as the only treatment option. We previously implicated runt-related transcription factor-1 (RUNX1) as a driver of epithelial-to-mesenchymal transition (EMT) in PVR and as a critical mediator of aberrant ocular angiogenesis when up-regulated. On the basis of these findings, an mRNA was designed to express a dominant-negative inhibitor of RUNX1 (RUNX1-Trap). We show that RUNX1-Trap delivered in polymer-lipidoid complexes or lipid nanoparticles sequestered RUNX1 in the cytosol and strongly reduced proliferation in primary cell cultures established from fibrotic membranes derived from patients with PVR. We assessed the preclinical efficacy of intraocular delivery of mRNA-encoded RUNX1-Trap in a rabbit model of PVR and in a laser-induced mouse model of aberrant angiogenesis often used to study wet age-related macular degeneration. mRNA-encoded RUNX1-Trap suppressed ocular pathology, measured as pathological scores in the rabbit PVR model and leakage and lesion size in the laser-induced choroidal neovascularization mouse model. mRNA-encoded RUNX1-Trap also strongly reduced proliferation in a human ex vivo explant model of PVR. These data demonstrate the therapeutic potential of mRNA-encoded therapeutic molecules with dominant-negative properties, highlighting the potential of mRNA-based therapies beyond standard gene supplementation approaches.

Transcriptome-wide mapping of internal mRNA N7-methylguanosine in sporulated and unsporulated oocysts of Eimeria tenella reveals stage-specific signatures

BackgroundGrowing evidence indicates that N7-methylguanosine (m7G) modification plays critical roles in epigenetic regulation. However, no data regarding m7G modification are currently available in Eimeria tenella, a highly virulent species causing coccidiosis in chickens.MethodsIn the present study, we explore the distribution of internal messenger RNA (mRNA) m7G modification in sporulated and unsporulated oocysts of E. tenella as well as its potential biological functions during oocyst development using methylated RNA immunoprecipitation sequencing (MeRIP-seq) and mRNA sequencing (mRNA-seq), and the mRNA-seq and MeRIP-seq data were verified by the quantitative reverse transcription polymerase chain reaction (RT–qPCR) and MeRIP–qPCR, respectively.ResultsOur data showed that m7G peaks were detected throughout the whole mRNA body, and the coding DNA sequence (CDS) region displayed the most methylation modification. Compared with unsporulated oocysts, 7799 hypermethylated peaks and 1945 hypomethylated peaks were identified in sporulated oocysts. Further combined analysis of differentially methylated genes (DMGs) and differentially expressed genes (DEGs) showed that there was a generally positive correlation between m7G modification levels and gene transcript abundance. Unsurprisingly, the mRNA-seq and MeRIP-seq data showed good consistency with the results of the RT–qPCR and MeRIP–qPCR, respectively. Gene Ontology (GO) and pathway enrichment analysis of DEGs with altered m7G-methylated peaks were involved in diverse biological functions and pathways, including DNA replication, RNA transport, spliceosome, autophagy-yeast, and cAMP signaling pathway.ConclusionsAltogether, our findings revealed the potential significance of internal m7G modification in E. tenella oocysts, providing some directions and clues for later in-depth research.Graphical abstract