Search Results (206)

Osteoarthritis (OA) is a chronic joint disorder characterized by progressive degeneration of articular cartilage, pain, synovial inflammation, and bone remodeling. Post-transcriptional RNA modifications, known as epitranscriptome, are a group of biochemical alterations in the primary RNA transcript that might influence RNA structure, stability, and function. Different kinds of RNA modifications have been recognized, such as methylation, acetylation, pseudouridylation, and phosphorylation. N6-methyladenosine (m6A), 5-methylcytosine (m5C), N7-methylguanosine (m7G), 2′-O-ribose methylation (2′-O-Me), and pseudouridylation (Ψ) are the most prevalent RNA modifications. Recent studies have shown that disruption in these modifications can interfere with gene expression and protein function. Here, we will review all types of RNA modifications and how they contribute to the onset and progression of OA. To the best of our knowledge, this is the first review comprehensively addressing all epitranscriptomic modifications in OA. Full article

Rolling circle amplification (RCA) at ambient temperature is prone to false positive signals during nucleic acid detection, which makes it challenging to establish an efficient RCA detection method. The false positive signals are primarily caused by binding of non-target nucleic acids to the circular single-stranded template, leading to non-specific amplification. Here, we present an RCA method for miRNA detection at 37 °C using two circular ssDNAs, each of which is formed by ligating the intramolecularly formed nick (without any splint) in a secondary structure. The specific target recognition is realized by utilizing low concentrations (0.1 nM) of circular ssDNA1 (C1). A phosphorothioate modification is present at G*AATTC on C1 to generate a nick for primer extension during the primer self-generated rolling circle amplification (PG-RCA). The fragmented amplification products are used as primers for the following RCA that serves as signal amplification using circular ssDNA2 (C2). Notably, the absence of splints and the low concentration of C1 significantly inhibits non-target binding, thus minimizing false positive signals. A high concentration (10 nM) of C2 is used to carry out linear rolling circle amplification (LRCA), which is highly specific. This strategy demonstrates a good linear response to 0.01–100 pM of miRNA with a detection limit of 7.76 fM (miR-155). Moreover, it can distinguish single-nucleotide mismatch in the target miRNA, enabling the rapid one-pot detection of miRNA at 37 °C. Accordingly, this method performs with high specificity and sensitivity. This approach is suitable for clinical serum sample analysis and offers a strategy for developing specific biosensors and diagnostic tools. Full article

Background/Objective: Oligodeoxynucleotides (ODNs) containing base-labile modifications such as N4-acetyldeoxycytidine (4acC), N6-acetyladenosine (6acA), N2-acetylguanosine (2acG), and N4-methyoxycarbonyldeoxycytidine (4mcC) are highly challenging to synthesize because standard ODN synthesis methods require deprotection and cleavage under strongly basic and nucleophilic conditions, and there is a lack of ideal alternative methods to solve the problem. The objective of this work is to explore the capability of the recently developed 1,3-dithian-2-yl-methoxycarbonyl (Dmoc) method for the incorporation of multiple 4acC modifications into a single ODN molecule and the feasibility of using the method for the incorporation of the 6acA, 2acG and 4mcC modifications into ODNs. Methods: The sensitive ODNs were synthesized on an automated solid phase synthesizer using the Dmoc group as the linker and the methyl Dmoc (meDmoc) group for the protection of the exo-amino groups of nucleobases. Deprotection and cleavage were achieved under non-nucleophilic and weakly basic conditions. Results: The 4acC, 6acA, 2acG, and 4mcC were all found to be stable under the mild ODN deprotection and cleavage conditions. Up to four 4acC modifications were able to be incorporated into a single 19-mer ODN molecule. ODNs containing the 6acA, 2acG, and 4mcC modifications were also successfully synthesized. The ODNs were characterized using RP HPLC, capillary electrophoresis, gel electrophoresis and MALDI MS. Conclusions: Among the modified nucleotides, 4acC has been found in nature and proven beneficial to DNA duplex stability. A method for the synthesis of ODNs containing multiple 4acC modifications is expected to find applications in biological studies involving 4acC. Although 6acA, 2acG, and 4mcC have not been found in nature, a synthetic route to ODNs containing them is expected to facilitate projects aimed at studying their biophysical properties as well as their potential for antisense, RNAi, CRISPR, and mRNA therapeutic applications. Full article

Background: The global burden of obesity and type 2 diabetes mellitus is a significant contributor to mortality and disability in the modern world. In this regard, the modification of adipocyte metabolism has been identified as a promising approach to develop new genetic and cellular engineering therapeutics. In this study, we activate the expression of creatine kinase B (CKB), a key enzyme of a non-canonical futile cycle and the regulator of energy storage, to promote catabolic processes in mature adipocytes. Methods: The protein-coding sequence of CKB was amplified by PCR from Mus musculus brain mRNA. Lentiviral transduction was used to transfer the CKB sequence into mature adipocytes. Adipocyte metabolism was analyzed by radioisotope monitoring of labeled [3H]-2-deoxyglucose and [14C]-glucose. Confocal microscopy was applied to estimate lipid droplets morphology (BODIPY493/503 dye), mitochondrial membrane potential (JC-1 dye), and thermogenesis (ERthermAC dye). Results: After lentiviral delivery of the CKB-coding sequence, CKB mRNA level increased 75-fold and protein expression fivefold. CKB overexpression does not cause significant changes in lipid droplet morphology. Despite this, enhanced glucose uptake and reduced lipid synthesis under adrenergic stimulation are detected during CKB overexpression. CKB causes an increase in mitochondrial potential with no effect on thermogenesis in adipocytes. Conclusions: In this study, we have shown that CKB overexpression in mature adipocytes allows us to obtain adipocytes with high glucose uptake, potency of ATP synthesis, and suppressed lipogenesis. These genetically modified cells may potentially exhibit a favorable metabolic effect in the context of excessive nutrient utilization. Full article

Severe dental pulp inflammation can lead to tissue lysis and destruction, underscoring the necessity for effective treatment of pulpitis. N-acetyltransferase 10 (NAT10)-mediated N4-acetylcytidine (ac⁴C) modification has recently emerged as a key regulator in inflammatory processes. However, whether NAT10 affects the inflammatory response in human dental pulp stem cells (hDPSCs) remains unelucidated. In this study, elevated NAT10 expression was observed in pulpitis tissues and LPS-stimulated hDPSCs. Knockdown of NAT10 led to reduced inflammatory gene expression and lower reactive oxygen species (ROS) production in LPS-stimulated hDPSCs, while the chemotactic migration of macrophages was also suppressed. Similar results were observed when hDPSCs were treated with Remodelin, an inhibitor of NAT10. Differentially expressed genes identified through RNA sequencing were significantly enriched in inflammatory signaling pathways after NAT10 depletion. Among the differential genes, pentraxins 3 (PTX3) was identified as the potential target gene due to the presence of the ac⁴C modification site and its known ability to regulate dental pulp inflammation. The mRNA and protein levels of PTX3 were reduced in NAT10-deficient cells, along with a decrease in its mRNA stability. Exogenous PTX3 supplementation partially reversed the inflammatory inhibition induced by NAT10 knockdown. Further evidence in vivo revealed that Remodelin treatment attenuated the severity of dental pulp inflammation in rats with pulpitis. In summary, these data indicated that NAT10 deficiency inhibited the stability of PTX3 mRNA and further inhibited hDPSC inflammation, while Remodelin might be a potential therapeutic agent for pulp capping. Full article

RNA methylation plays a critical role in regulating all aspects of RNA function, which are implicated in the pathogenesis of various human diseases. Recent studies emphasize the role of 5-methylcytosine (m5C), an RNA modification, in key biological functions. Metabolic dysfunction-associated steatotic liver disease (MASLD) has emerged as the leading chronic liver condition globally. However, the relationship between m5C methylation and MASLD remains unclear. This study aimed to investigate m5C modification in a mouse model of MASLD. In this research, using RNA transcriptome sequencing (RNA-Seq) and methylated RNA bisulfite sequencing (RNA-BS-Seq) in leptin receptor-deficient mice, we found that genes associated with hypermethylation were primarily linked to lipid metabolism. We identified 156 overlapping and differentially expressed genes (DEGs) that changed at both the mRNA expression level and the m5C modification level. Among them, 72 genes showed elevated expression and m5C modification. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses indicated that these genes were significantly associated with lipid metabolism-related signaling pathways. Our results demonstrate that m5C methylation modifications may play an important role in the development of MASLD. Full article

Background/Objectives: Lipid nanoparticles (LNPs) are leading mRNA delivery vehicles, with ionizable lipids (ILs) as their key component. However, the relationship between the IL structure and LNP endogenous organ-targeting is not well understood. In this study, we developed a novel library of biodegradable ILs featuring beta-propionate linkers, which, when incorporated into a four-component LNP formulation, show excellent extrahepatic selectivity and high protein expression. Methods: We explored the impact of structural modifications in the hydrophobic chains and polar-head groups in the ILs while keeping the linkers unchanged. In vivo results were evaluated to examine how structural changes influence the biodistribution to spleen or lungs. LNP formulations were assessed for their protein expression levels and organ-specific targeting. Additionally, protein corona formation by the best-performing LNPs was examined to provide further mechanistic insights. Results: Organ targeting was significantly influenced by structural changes in the ILs, allowing for precise control of the biodistribution between the spleen and lungs. Branched hydrophobic chains demonstrated a higher propensity for spleen targeting, while modifications in the polar-head group could drastically shift biodistribution from the lung to the spleen. This led to the identification of LNPs’ zeta potential as a key determinant of their extrahepatic targeting properties. Notably, ionizable lipid A3T2C7, also known as CP-LC-1495, displayed strong lung selectivity (97%) and high protein expression in lung tissue (1.21 × 10⁸ p/s). Similarly, several promising candidates for spleen-targeting LNPs displayed protein expression levels exceeding 1 × 10⁷ p/s (selectivity >80%). Conclusions: This study elucidates the structure–function relationships of ILs in passive organ-specific mRNA delivery, highlighting how the fine-tuning of hydrophobic chains, polar-head groups, and surface charge (zeta potential) allows for the precise control of LNP endogenous biodistribution, a mechanism influenced by protein corona formation. These findings enable the rational design of targeted LNP systems, enhancing their therapeutic potential for specific organs, such as the spleen and lungs. Full article

Obesity-related cardiac dysfunction is a significant global health challenge. High-fat diets (HFDs) are well-established models of obesity. HFD has been reported to induce cardiac dysfunction and alter cardiac miRNA expression, DNA methylation and histone modifications. Nevertheless, it remains unclear whether cardiac miRNAs altered due to HFD target epigenetic regulator enzymes and function as epigenetic regulator miRNAs (epi-miRNAs), thereby contributing to HFD-induced epigenetic changes and cardiac dysfunction. To address this gap in our knowledge, this study aimed to identify putative cardiac epi-miRNAs and their potential epigenetic targets through an in silico analysis of a previously published miRNA dataset from Sprague Dawley rats subjected to HFD. Using two independent databases, miRDB and miRWalk, predicted miRNA-mRNA interactions were analyzed. A total of 71 miRNAs were identified in our present study as putative epi-miRNAs. A total of 34 epi-miRNAs were upregulated (e.g., miR-92b-3p, let-7c-5p, miR-132-3p), and 37 were downregulated (e.g., miR-21-3p, miR-29c-3p, miR-199a-3p) in response to HFD. Epi-miRNAs targeted 81 individual epigenetic regulators (e.g., Dnmt3a, Ezh2, Hdac4, Kdm3a) with 202 possible miRNA–target interactions. Most of the targeted epigenetic regulators were involved in histone modification. An epi-miRNA–target analysis indicated increased DNA methylation and histone acetylation and decreased histone methylation in the hearts of HFD-fed rats. These findings suggest the importance of epi-miRNA-induced epigenetic changes in HFD-related cardiac dysfunction. Full article

Objective: Non-small cell lung cancer (NSCLC) remains one of the most significant contributors to cancer-related mortality. This investigation explores the influence and underlying mechanisms of the USP1 inhibitor SJB2-043 on A549 cells, with the aim of advancing the development of anti-NSCLC therapeutics. Methods: Publicly available databases were utilized to assess USP1 expression and its association with the progression of NSCLC. Gene expression variations were ascertained through RNA sequencing, followed by the Kyoto Encyclopedia of Genes and Genomes and Gene Ontology pathway enrichment evaluations. Various doses of SJB2-043 were administered to A549 cells to evaluate its impact on cell multiplication, motility, apoptosis, and the cell cycle using CCK-8 assays, colony formation, wound healing, flow cytometry, and Western blotting (WB). Results: USP1 was found to be overexpressed in NSCLC specimens and linked to adverse prognosis. Treatment with SJB2-043 markedly inhibited A549 cell proliferation and migration, diminished clonogenic potential, and triggered apoptosis in a dose-dependent manner. Modifications in the cell cycle were observed, showing an elevated percentage of cells in the G2 phase while exhibiting a parallel decline in the G₁ phase. WB examination demonstrated diminished protein levels of N-cadherin, CyclinB₁, CDK1, C-myc, Bcl-2, p-ERK/ERK, p-p38/p38, p-JNK/JNK, p-AKT/AKT, and p-mTOR/mTOR, alongside an upregulation of E-cadherin, ZO-1, occludin, p53, Bax, p-β-catenin/β-catenin, and GSK3β. Conclusions: SJB2-043 exerts a suppressive effect on A549 cell proliferation, migration, and epithelial–mesenchymal transition while enhancing apoptosis. These cellular effects appear to be mediated through the inhibition of the MAPK, Wnt/β-catenin, and PI3K/AKT/mTOR signaling cascades, in addition to modulation of the cell cycle. Full article

Chinese tongue sole (Cynoglossus semilaevis) is an important mariculture fish in China, and female individuals present a growth advantage. However, genetic females (ZW) can sex reverse to phenotypic males, designated pseudomales. The pseudomale shows abnormal spermatogenesis and produces only Z sperm. Histone is pivotal in spermatogenesis, and post-translational modification could regulate its function. A comparison of testis phosphorylated and ubiquitinated proteins revealed 8 and 12 differentially phosphorylated and ubiquitinated histones in the testes of male and pseudomale Chinese tongue soles, respectively, but there was no difference in the translation level of these proteins. We selected four histone genes, h1.1-like, h1.2-like, h3, and h3.3-like, for further analysis. The expression levels of the h1.1-like, h3, and h3.3-like genes reached their highest levels at 2 years post-hatching (yph), and the expression level of h1.2-like reached its highest level at 1.5 years post-hatching (1.5 yph), indicating that its role began during the late stage of gonadal development. Promoter activity verification revealed that the promoters of the h1.1-like, h1.2-like, h3, and h3.3-like genes were located approximately upstream 2000 bp and six histone-related transcription factor sites were predicted. YY1A, YY1B, C-JUN, and JUNB may have negative regulatory effects on h1.1-like, h1.2-like, h3, and h3.3-like; AR and ETS-2 may have positive regulatory effects on h3 and h3.3-like. The ISH results revealed that h1.1-like, h1.2-like, h3, and h3.3-like mRNAs were located mainly in the sperm cells in the testes and the oocytes at various stages in the ovaries. After siRNA knockdown, the expression of dmrt1 in testis cell lines and the expression of tesk1 and neurl3 in males was downregulated, suggesting that the h1.1-like, h1.2-like, h3, and h3.3-like genes may have a negative regulatory role in spermatogenesis. The regulatory role in female fish remains to be explored. Mass spectrometry analysis revealed that histones have an important role in chromosome remodeling. These results provide a genetic basis for the molecular mechanism of gonadal development and spermatogenesis in Chinese tongue sole. Full article

Gapmer-type antisense oligonucleotides (ASOs) are an emerging class of therapeutic agents that directly inhibit pathogenic mRNA. In this study, three new 4′-C-substituted thymidine analogs were generated using a synthetic strategy recently established by our group, namely, 4′-C-(N-ethyl) aminoethyl (4′-EAE-T), 4′-C-(N-butyl) aminoethyl (4′-BAE-T), and 4′-C-(N-octyl) aminoethyl (4′-OAE-T). Their properties were evaluated and compared with those of previously reported analogs, including 4′-C-aminoethyl (4′-AE-T) and 4′-C-(N-methyl) aminoethyl (4′-MAE-T). The novel nucleoside analogs were subsequently incorporated into gapmer-type ASOs featuring phosphorothioate (PS) linkages and locked nucleic acids (LNAs) in the wing regions. The incorporation of 4′-EAE-T and 4′-BAE-T analogs resulted in RNA binding affinities similar to that of the previously reported 4′-MAE-T analog, whereas a marked decrease in RNA affinity was noted for 4′-OAE-T, however, this reduction was mitigated when combined with other chemical modifications. Furthermore, the structural modifications conferred enhanced nuclease resistance under bovine serum conditions, with 4′-EAE-T resulting in the highest stability, followed by 4′-BAE-T and 4′-OAE-T. Additionally, oligonucleotides modified with the developed analogs preserved their RNase H cleavage susceptibility, albeit inducing minor alterations in the cleavage pattern. Finally, the oligonucleotides were applied in a gene silencing experiment targeting the KRAS gene, conducted without the use of transfection agents, displaying gene silencing activities comparable to that of the control, with the exception of the 4′-OAE-modified nucleotide, which exhibited low activity. Full article

Pharmaceutical advancements and an improved understanding of pathophysiology have enabled innovative therapies for chronic conditions like dyslipidemia. This condition is marked by abnormalities in lipid homeostasis. Nucleic acid therapeutics, including antisense oligonucleotides and small interfering RNAs, are novel management strategies that silence genes by targeting mRNA. Antisense oligonucleotides modify mRNA to inhibit protein production, whereas small interfering RNAs induce mRNA degradation via the RNA-induced silencing complex (RISC), thus offering promising treatments for dyslipidemia and atherosclerotic cardiovascular disease. Chemical modifications improve their stability and mRNA targeting. RNA-based therapies targeting PCSK9, Lp(a), ApoC-III, and ANGPTL3 hold transformative potential for treating dyslipidemia effectively. This article discusses the latest data from completed and ongoing trials on RNA therapies for dyslipidemia, including inclisiran, pelacarsen, olpasiran, zerlasiran, lepodisiran, volanesorsen, olezarsen, plozasiran, zodasiran, and solbinsiran. Each therapy targets specific molecules while also significantly impacting other lipid parameters. The promising results of these trials indicate potential improvements in lipid therapy and cardiovascular risk reduction, with ongoing studies expected to further refine the role of the novel RNA-based agents in effective lipid management. Full article

Melatonin (MLT), produced by the pineal gland and other tissues, is known for its anti-inflammatory effects, particularly in regulating inflammatory markers and cytokines in intestinal cells. Our study aimed to investigate how MLT influences the expression of inflammatory genes through histone modification in canine ileum epithelial cells (cIECs). In our experiment, cIECs were cultured and divided into a control group (CON) and an MLT-treatment group. MLT did not significantly affect cell growth or death in cIECs compared to the CON. However, MLT treatment led to an upregulation of CD40, ZAP70, and IL7R and a downregulation of LCK, RPL37, TNFRSF13B, CD4, CD40LG, BLNK, and CIITA at the mRNA expression level. Moreover, MLT significantly altered the NF-kappa B signaling pathway by upregulating genes, such as CD40, ZAP70, TICAM1, VCAMI, GADD45B, IRAK1, TRADD, RELA, RIPK1, and RELB, and downregulating PRKCB, LY96, CD40LG, ILIB, BLNK, and TNFRSF11A. Using ChIP-qPCR, we discovered that MLT treatment enhanced histone acetylation marks H3K9ac, H3K18ac, H3K27ac, and methylation marks H3K4me1 and H3K4me3 at the ZAP70 and CD40 gene loci (p < 0.05). Additionally, the enrichment of RNA polymerase II and phosphorylated Ser5 pol-II at these loci was increased in MLT-treated cells (p < 0.05), indicating heightened transcriptional activity. In conclusion, our findings suggest that MLT mitigates inflammation in cIECs by modulating the transcription of ZAP70 and CD40 through histone modifications, offering potential therapeutic insights for inflammatory bowel diseases. Full article

Background/Objectives: MIDN (midnolin) is newly discovered method for critically regulating a ubiquitin-independent proteasomal degradation pathway. This study aims to examine the expression, prognostic value, genomic changes, interacting proteins, methylation status, and correlations with the tumor immune microenvironment of MIDN in various cancers. Methods: The GTEx, Depmap, GEPIA2, and Kaplan–Meier Plotter databases are applied to evaluate the MIDN level in tumor and normal tissues and the MIDN prognostic value in cancers. The genetic alterations of MIDN in cancers are investigated using the cBioPortal database. The STRING, GeneMANIA, DAVID, and Human Protein Atlas are harnessed to identify and analyze MIDN-interacted proteins. The Sangerbox 3.0 platform (a pan-cancer analysis module) is used to measure the correlations between the MIDN level and the tumor immune microenvironment, stemness, immune cell infiltration, tumor mutational burden, immune checkpoint genes, and RNA modification genes. Immunofluorescence, qRT-PCR, and Western blotting assays were used to evaluate the biological roles of MIDN in breast and gastric cancer cells. Results: MIDN expression was dysregulated in many cancers and associated with prognosis in several cancers, such as esophageal cancer. MIDN was mutated in 1.7% of cancers, and deep deletion was the dominant mutation type. NR4A1, PSMC1, and EGR1 were selected as MIDN-interacted proteins, and these four molecules were co-expressed in pancreatic cancer, liver cancer, urothelial cancer, melanoma, and breast cancer. MIDN expression was significantly correlated with the infiltration of CD8+ T cell, CD4+ T cell, B cell, macrophage, neutrophil, and DC both in prostate adenocarcinoma and liver hepatocellular carcinoma. The MIDN level was correlated with several immune checkpoint genes, such as VEGFA, and RNA modification genes such as YTHDF1, YTHDF2, YTHDF3, and YTHDC1 in cancers. Furthermore, in breast cancer cells, the downregulation of MIDN suppressed the colony formation abilities and lessened cell-cycle-associated and stemness-associated genes; in gastric cancer, the knockdown of MIDN diminished the mRNA levels of Nanog and LDHA. Strikingly, silence of MIDN upregulated FTO protein expression in both breast and gastric cancer cells. Conclusions: Our findings demonstrate the expression, prognostic value, mutation status, interacting proteins, methylation status, and correlations with the tumor immune microenvironment of MIDN. MIDN will be developed as a potential therapeutic target and a prognosis biomarker. Full article

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants and children. mRNA vaccines based on the lipopolyplex (LPP) platform have been previously reported, but they remain unapplied in RSV vaccine development. In this study, we developed a novel LPP-delivered mRNA vaccine that expresses the respiratory syncytial virus prefusion protein (RSV pre-F) to evaluate its immunogenicity and protective effect in a mouse model. We synthesized mRNAs with gene modification for RSV pre-F and prepared mRNA vaccines using the LPP delivery platform, referred to as RSV pre-F LPP-mRNA. RSV pre-F protein expression in mRNA vaccines was characterized in vitro. Then, we evaluated the effects of the immune response and protection of this mRNA vaccine in mice up to 24 weeks post-vaccination. Following booster immunization, robust and long-lasting RSV pre-F-specific IgG antibodies were detected in the serum of mice, which exhibited Th1/Th2 balanced IgG response and cross-neutralizing antibodies against different subtypes (RSV A2, B18537, and clinical isolate hRSV/C-Tan/BJ 202301), with a clear dose–response relationship observed. RSV pre-F-specific IgG antibodies were maintained in the mice for an extended period, lasting up to 18 weeks post-immunization. Concurrently, multifunctional RSV F-specific CD8⁺ T cells (IFN–γ, IL-2, and TNF-α) were detected in the mice. After RSV A2 challenge, the RSV pre-F LPP-mRNA vaccine led to a significant reduction in viral replication, while reduced pathological damage was observed in lung tissue. The LPP-delivered mRNA vaccine expressing RSV pre-F induces a robust and long-lasting immune response and protection, indicating good prospects for further development and application. Full article