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mRNA vaccines are entering a period of rapid development. However, their synthesis is still plagued by challenges related to mRNA impurities and fragments (incomplete mRNA). Most impurities of mRNA products transcribed in vitro are mRNA fragments. Only full-length mRNA transcripts containing both a 5′-cap and a 3′-poly(A) structure are viable for in vivo expression. Therefore, RNA fragments are the primary product-related impurities that significantly hinder mRNA efficacy and must be effectively controlled; these species are believed to originate from either mRNA hydrolysis or premature transcriptional termination. In the manufacturing of commercial mRNA vaccines, T7 RNA polymerase-catalyzed in vitro transcription (IVT) synthesis is a well-established method for synthesizing long RNA transcripts. This study identified a pivotal domain on the T7 RNA polymerase that is associated with erroneous mRNA release. By leveraging the advantageous properties of a T7 RNA polymerase mutant and precisely optimized IVT process parameters, we successfully achieved an mRNA integrity exceeding 91%, thereby further unlocking the immense potential of mRNA therapeutics. [ABSTRACT FROM AUTHOR]

• This study found that adipose-derived stem cell exosomes can promote M2 polarization of macrophages through the transmission of LncRNA DLEU2. • The aim of this study was to explore the molecular mechanism of adipose-derived stem cell exosomes in sepsis-induced lung injury. • Through high-throughput sequencing, this study found that LncRNA DLEU2 is enriched in exosomes of sepsis lung tissue. • This study confirmed that ADSC-Exos can promote M2 polarization of macrophages and alleviate inflammation and apoptosis of lung injury cells. This study investigates the molecular mechanisms by which extracellular vesicles (EVs) derived from adipose-derived mesenchymal stem cells (ADSCs) promote M2 polarization of macrophages and thus reduce lung injury caused by sepsis. High-throughput sequencing was used to identify differentially expressed genes related to long non-coding RNA (lncRNA) in ADSC-derived EVs (ADSC-EVs) in sepsis lung tissue. Weighted gene co-expression network analysis (WGCNA) was employed to predict the downstream target genes of the lncRNA DLEU2. The RNAInter database predicted miRNAs that interact with DLEU2 and LXN. Functional and pathway enrichment analyses were performed using GO and KEGG analysis. A mouse model of sepsis was established, and treatment with a placebo or ADSC-EVs was administered, followed by RT-qPCR analysis. ADSC-EVs were isolated and identified. In vitro cell experiments were conducted using the mouse lung epithelial cell line MLE-12, mouse macrophage cell line RAW264.7, and mouse lung epithelial cell line (LEPC). ADSC-EVs were co-cultured with RAW264.7 and MLE-12/LEPC cells to study the regulatory mechanism of the lncRNA DLEU2. Cell viability, proliferation, and apoptosis of lung injury cells were assessed using CCK-8, EdU, and flow cytometry. ELISA was used to measure the levels of inflammatory cytokines in the sepsis mouse model, flow cytometry was performed to determine the number of M1 and M2 macrophages, lung tissue pathology was evaluated by H&E staining, and immunohistochemistry was conducted to examine the expression of proliferation- and apoptosis-related proteins. High-throughput sequencing and bioinformatics analysis revealed enrichment of the lncRNA DLEU2 in ADSC-EVs in sepsis lung tissue. Animal and in vitro cell experiments showed increased expression of the lncRNA DLEU2 in sepsis lung tissue after treatment with ADSC-EVs. Furthermore, ADSC-EVs were found to transfer the lncRNA DLEU2 to macrophages, promoting M2 polarization, reducing inflammation response in lung injury cells, and enhancing their viability, proliferation, and apoptosis inhibition. Further functional experiments indicated that lncRNA DLEU2 promotes M2 polarization of macrophages by regulating miR-106a-5p/LXN, thereby enhancing the viability and proliferation of lung injury cells and inhibiting apoptosis. Overexpression of miR-106a-5p could reverse the biological effects of ADSC-EVs-DLEU2 on MLE-12 and LEPC in vitro cell models. Lastly, in vivo animal experiments confirmed that ADSC-EVs-DLEU2 promotes high expression of LXN by inhibiting the expression of miR-106a-5p, further facilitating M2 macrophage polarization and reducing lung edema, thus alleviating sepsis-induced lung injury. lncRNA DLEU2 in ADSC-EVs may promote M2 polarization of macrophages and enhance the viability and proliferation of lung injury cells while inhibiting inflammation and apoptosis reactions, thus ameliorating sepsis-induced lung injury in a mechanism involving the regulation of the miR-106a-5p/LXN axis. [ABSTRACT FROM AUTHOR]

Delivering functional gene into targeted skin cells or tissues to modulate the genes expression, has the potential to treat various hereditary cutaneous disorders. Nevertheless, the lack of safe and effective gene delivery vehicles greatly limits the clinical translation of gene therapy for inherited skin diseases. Herein, we developed a facile elution fractionation strategy to isolate eight HPAEs with M w ranging from 7.6 to 131.8 kg/mol and Đ < 2.0 from the one crude HPAE 23.7k , and investigated the expression efficiency for TGM1 and COL7A1 plasmids. Gene transfection results revealed that the intermediate MW HPAEs, HPAE 20.6k , exhibited the highest gene transfection efficiency (46.4%) and the strongest mean fluorescence intensity (143,032 RLU), compared to other isolated components and the crude product. Importantly, best-performing isolated HPAE effectively delivered COL7A1 (15,974 bp) and TGM1 (7181 bp) plasmids, promoting the efficient expression of type VII collagen (C7) and transglutaminase-1 proteins in cutaneous cells. Our study establishes a straightforward step-by-step elution fractionation strategy for the development of HPAEs gene delivery vectors, expediting their clinical translation in inherited skin diseases. HPAEs crude polymer is fractionated to eight HPAEs with M w ranging from 7.6 to 131.8 kg/mol and Đ < 2.0. The best-performing isolated HPAE effectively delivered COL7A1 (15,974 bp) and TGM1 (7181 bp) plasmids, promoting the efficient expression of type VII collagen (C7) and transglutaminase-1 proteins in cutaneous cells. [Display omitted] • We establish a straightforward step-by-step elution fractionation strategy to isolate HPAEs with different molecular weight. • Isolated intermediate molecular weight HPAEs exhibit high transfection efficiency and biocompatibility in diverse cell types. • The top-performing HPAEs efficiently deliver TGM and COL7A1 DNA to potentially rescue hereditary cutaneous disorders. [ABSTRACT FROM AUTHOR]

Osteonecrosis of the femoral head (ONFH) is a elaborate hip disease characterized by collapse of femoral head and osteoarthritis. RNA N6-methyladenosine (m6A) plays a crucial role in a lot of biological processes within eukaryotic cells. However, the role of m6A in the regulation of ONFH remains unclear. In this study, we identified the m6A regulators in ONFH and performed subtype classification. We identified 7 significantly differentially expressed m6A regulators through the analysis of differences between ONFH and normal samples in the Gene Expression Omnibus (GEO) database. A random forest algorithm was employed to monitor these regulators to assess the risk of developing ONFH. We constructed a nomogram based on these 7 regulators. The decision curve analysis suggested that patients can benefit from the nomogram model. We classified the ONFH samples into two m6A models according to these 7 regulators through consensus clustering algorithm. After that, we evaluated those two m6A patterns using principal component analysis. We assessed the scores of those two m6A patterns and their relationship with immune infiltration. We observed a higher m6A score of type A than that of type B. Finally, we performed a cross-validation of crucial m6A regulatory factors in ONFH using external datasets and femoral head bone samples. In conclusion, we believed that the m6A pattern could provide a novel diagnostic strategy and offer new insights for molecularly targeted therapy of ONFH. [ABSTRACT FROM AUTHOR]

[Display omitted] • The KAT5 Tudor-knot motif is an essential domain for cell survival. • The Tudor-knot domain is important for KAT5-dependent transcriptional regulation. • The Tudor-knot domain regulates KAT5 HAT activity on nucleosomes. • The Tudor-knot domain may regulate accessibility of histone tails in nucleosomes. The lysine acetyltransferase KAT5 is a pivotal enzyme responsible for catalyzing histone H4 acetylation in cells. In addition to its indispensable HAT domain, KAT5 also encompasses a conserved Tudor-knot domain at its N-terminus. However, the function of this domain remains elusive, with conflicting findings regarding its role as a histone reader. In our study, we have employed a CRISPR tiling array approach and unveiled the Tudor-knot motif as an essential domain for cell survival. The Tudor-knot domain does not bind to histone tails and is not required for KAT5's chromatin occupancy. However, its absence leads to a global reduction in histone acetylation, accompanied with genome-wide alterations in gene expression that consequently result in diminished cell viability. Mechanistically, we find that the Tudor-knot domain regulates KAT5's HAT activity on nucleosomes by fine-tuning substrate accessibility. In summary, our study uncovers the Tudor-knot motif as an essential domain for cell survival and reveals its critical role in modulating KAT5's catalytic efficiency on nucleosome and KAT5-dependent transcriptional programs critical for cell viability. [ABSTRACT FROM AUTHOR]