Versatile Design of Intracellular Protein-Responsive Translational Regulation System for Synthetic mRNA

Synthetic mRNA (mRNA) enables transgene expression without the necessity of nuclear import and the risk of insertional mutagenesis, which makes it an attractive tool for medical applications such as vaccination and protein replacement therapy. For further improvement of mRNA therapeutics, cell-selective translation is desirable, because transgene expression in nontarget cells sometimes causes adverse effects. In this study, we developed an intracellular protein-responsive translational regulation system based on Caliciviral VPg-based translational activator (CaVT) combined with inteins and target protein-binding nanobodies. This system enabled both translational activation and repression in a target protein-dependent manner. Importantly, the target protein can be altered by simply exchanging the nanobodies. The versatile design for target protein-responsive translational regulation holds promise for producing mRNA therapeutics with high safety

MOESM1 of Combined CatWalk Index: an improved method to measure mouse motor function using the automated gait analysis system

Additional file 1. Example spreadsheet with CCI coefficients. An annotated example spreadsheet containing CatWalk and BMS data from impact force optimization experiments. This spreadsheet shows how to calculate CCI coefficients and scores

Table2_Unbiased comparison and modularization identify time-related transcriptomic reprogramming in exercised rat cartilage: Integrated data mining and experimental validation.XLSX

Exercise is indispensable for maintaining cartilage integrity in healthy joints and remains a recommendation for knee osteoarthritis. Although the effects of exercise on cartilage have been implied, the detailed mechanisms, such as the effect of exercise time which is important for exercise prescription, remain elusive. In this study, bioinformatic analyses, including unbiased comparisons and modularization, were performed on the transcriptomic data of rat cartilage to identify the time-related genes and signaling pathways. We found that exercise had a notable effect on cartilage transcriptome. Exercise prominently suppressed the genes related to cell division, hypertrophy, catabolism, inflammation, and immune response. The downregulated genes were more prominent and stable over time than the upregulated genes. Although exercise time did not prominently contribute to the effects of exercise, it was a factor related to a batch of cellular functions and signaling pathways, such as extracellular matrix (ECM) homeostasis and cellular response to growth factors and stress. Two clusters of genes, including early and late response genes, were identified according to the expression pattern over time. ECM organization, BMP signaling, and PI3K-Akt signaling were early responsive in the exercise duration. Moreover, time-related signaling pathways, such as inositol phosphate metabolism, nicotinate/nicotinamide metabolism, cell cycle, and Fc epsilon RI signaling pathway, were identified by unbiased mapping and polarization of the highly time-correlated genes. Immunohistochemistry staining showed that Egfr was a late response gene that increased on day 15 of exercise. This study elucidated time-related transcriptomic reprogramming induced by exercise in cartilage, advancing the understanding of cartilage homeostasis.</p

Image1_Unbiased comparison and modularization identify time-related transcriptomic reprogramming in exercised rat cartilage: Integrated data mining and experimental validation.TIF

Exercise is indispensable for maintaining cartilage integrity in healthy joints and remains a recommendation for knee osteoarthritis. Although the effects of exercise on cartilage have been implied, the detailed mechanisms, such as the effect of exercise time which is important for exercise prescription, remain elusive. In this study, bioinformatic analyses, including unbiased comparisons and modularization, were performed on the transcriptomic data of rat cartilage to identify the time-related genes and signaling pathways. We found that exercise had a notable effect on cartilage transcriptome. Exercise prominently suppressed the genes related to cell division, hypertrophy, catabolism, inflammation, and immune response. The downregulated genes were more prominent and stable over time than the upregulated genes. Although exercise time did not prominently contribute to the effects of exercise, it was a factor related to a batch of cellular functions and signaling pathways, such as extracellular matrix (ECM) homeostasis and cellular response to growth factors and stress. Two clusters of genes, including early and late response genes, were identified according to the expression pattern over time. ECM organization, BMP signaling, and PI3K-Akt signaling were early responsive in the exercise duration. Moreover, time-related signaling pathways, such as inositol phosphate metabolism, nicotinate/nicotinamide metabolism, cell cycle, and Fc epsilon RI signaling pathway, were identified by unbiased mapping and polarization of the highly time-correlated genes. Immunohistochemistry staining showed that Egfr was a late response gene that increased on day 15 of exercise. This study elucidated time-related transcriptomic reprogramming induced by exercise in cartilage, advancing the understanding of cartilage homeostasis.</p

Table4_Unbiased comparison and modularization identify time-related transcriptomic reprogramming in exercised rat cartilage: Integrated data mining and experimental validation.XLSX

Exercise is indispensable for maintaining cartilage integrity in healthy joints and remains a recommendation for knee osteoarthritis. Although the effects of exercise on cartilage have been implied, the detailed mechanisms, such as the effect of exercise time which is important for exercise prescription, remain elusive. In this study, bioinformatic analyses, including unbiased comparisons and modularization, were performed on the transcriptomic data of rat cartilage to identify the time-related genes and signaling pathways. We found that exercise had a notable effect on cartilage transcriptome. Exercise prominently suppressed the genes related to cell division, hypertrophy, catabolism, inflammation, and immune response. The downregulated genes were more prominent and stable over time than the upregulated genes. Although exercise time did not prominently contribute to the effects of exercise, it was a factor related to a batch of cellular functions and signaling pathways, such as extracellular matrix (ECM) homeostasis and cellular response to growth factors and stress. Two clusters of genes, including early and late response genes, were identified according to the expression pattern over time. ECM organization, BMP signaling, and PI3K-Akt signaling were early responsive in the exercise duration. Moreover, time-related signaling pathways, such as inositol phosphate metabolism, nicotinate/nicotinamide metabolism, cell cycle, and Fc epsilon RI signaling pathway, were identified by unbiased mapping and polarization of the highly time-correlated genes. Immunohistochemistry staining showed that Egfr was a late response gene that increased on day 15 of exercise. This study elucidated time-related transcriptomic reprogramming induced by exercise in cartilage, advancing the understanding of cartilage homeostasis.</p

Table1_Unbiased comparison and modularization identify time-related transcriptomic reprogramming in exercised rat cartilage: Integrated data mining and experimental validation.XLSX

Exercise is indispensable for maintaining cartilage integrity in healthy joints and remains a recommendation for knee osteoarthritis. Although the effects of exercise on cartilage have been implied, the detailed mechanisms, such as the effect of exercise time which is important for exercise prescription, remain elusive. In this study, bioinformatic analyses, including unbiased comparisons and modularization, were performed on the transcriptomic data of rat cartilage to identify the time-related genes and signaling pathways. We found that exercise had a notable effect on cartilage transcriptome. Exercise prominently suppressed the genes related to cell division, hypertrophy, catabolism, inflammation, and immune response. The downregulated genes were more prominent and stable over time than the upregulated genes. Although exercise time did not prominently contribute to the effects of exercise, it was a factor related to a batch of cellular functions and signaling pathways, such as extracellular matrix (ECM) homeostasis and cellular response to growth factors and stress. Two clusters of genes, including early and late response genes, were identified according to the expression pattern over time. ECM organization, BMP signaling, and PI3K-Akt signaling were early responsive in the exercise duration. Moreover, time-related signaling pathways, such as inositol phosphate metabolism, nicotinate/nicotinamide metabolism, cell cycle, and Fc epsilon RI signaling pathway, were identified by unbiased mapping and polarization of the highly time-correlated genes. Immunohistochemistry staining showed that Egfr was a late response gene that increased on day 15 of exercise. This study elucidated time-related transcriptomic reprogramming induced by exercise in cartilage, advancing the understanding of cartilage homeostasis.</p

Table6_Unbiased comparison and modularization identify time-related transcriptomic reprogramming in exercised rat cartilage: Integrated data mining and experimental validation.XLSX

Exercise is indispensable for maintaining cartilage integrity in healthy joints and remains a recommendation for knee osteoarthritis. Although the effects of exercise on cartilage have been implied, the detailed mechanisms, such as the effect of exercise time which is important for exercise prescription, remain elusive. In this study, bioinformatic analyses, including unbiased comparisons and modularization, were performed on the transcriptomic data of rat cartilage to identify the time-related genes and signaling pathways. We found that exercise had a notable effect on cartilage transcriptome. Exercise prominently suppressed the genes related to cell division, hypertrophy, catabolism, inflammation, and immune response. The downregulated genes were more prominent and stable over time than the upregulated genes. Although exercise time did not prominently contribute to the effects of exercise, it was a factor related to a batch of cellular functions and signaling pathways, such as extracellular matrix (ECM) homeostasis and cellular response to growth factors and stress. Two clusters of genes, including early and late response genes, were identified according to the expression pattern over time. ECM organization, BMP signaling, and PI3K-Akt signaling were early responsive in the exercise duration. Moreover, time-related signaling pathways, such as inositol phosphate metabolism, nicotinate/nicotinamide metabolism, cell cycle, and Fc epsilon RI signaling pathway, were identified by unbiased mapping and polarization of the highly time-correlated genes. Immunohistochemistry staining showed that Egfr was a late response gene that increased on day 15 of exercise. This study elucidated time-related transcriptomic reprogramming induced by exercise in cartilage, advancing the understanding of cartilage homeostasis.</p

Table7_Unbiased comparison and modularization identify time-related transcriptomic reprogramming in exercised rat cartilage: Integrated data mining and experimental validation.XLSX

Exercise is indispensable for maintaining cartilage integrity in healthy joints and remains a recommendation for knee osteoarthritis. Although the effects of exercise on cartilage have been implied, the detailed mechanisms, such as the effect of exercise time which is important for exercise prescription, remain elusive. In this study, bioinformatic analyses, including unbiased comparisons and modularization, were performed on the transcriptomic data of rat cartilage to identify the time-related genes and signaling pathways. We found that exercise had a notable effect on cartilage transcriptome. Exercise prominently suppressed the genes related to cell division, hypertrophy, catabolism, inflammation, and immune response. The downregulated genes were more prominent and stable over time than the upregulated genes. Although exercise time did not prominently contribute to the effects of exercise, it was a factor related to a batch of cellular functions and signaling pathways, such as extracellular matrix (ECM) homeostasis and cellular response to growth factors and stress. Two clusters of genes, including early and late response genes, were identified according to the expression pattern over time. ECM organization, BMP signaling, and PI3K-Akt signaling were early responsive in the exercise duration. Moreover, time-related signaling pathways, such as inositol phosphate metabolism, nicotinate/nicotinamide metabolism, cell cycle, and Fc epsilon RI signaling pathway, were identified by unbiased mapping and polarization of the highly time-correlated genes. Immunohistochemistry staining showed that Egfr was a late response gene that increased on day 15 of exercise. This study elucidated time-related transcriptomic reprogramming induced by exercise in cartilage, advancing the understanding of cartilage homeostasis.</p

Table5_Unbiased comparison and modularization identify time-related transcriptomic reprogramming in exercised rat cartilage: Integrated data mining and experimental validation.XLSX

Exercise is indispensable for maintaining cartilage integrity in healthy joints and remains a recommendation for knee osteoarthritis. Although the effects of exercise on cartilage have been implied, the detailed mechanisms, such as the effect of exercise time which is important for exercise prescription, remain elusive. In this study, bioinformatic analyses, including unbiased comparisons and modularization, were performed on the transcriptomic data of rat cartilage to identify the time-related genes and signaling pathways. We found that exercise had a notable effect on cartilage transcriptome. Exercise prominently suppressed the genes related to cell division, hypertrophy, catabolism, inflammation, and immune response. The downregulated genes were more prominent and stable over time than the upregulated genes. Although exercise time did not prominently contribute to the effects of exercise, it was a factor related to a batch of cellular functions and signaling pathways, such as extracellular matrix (ECM) homeostasis and cellular response to growth factors and stress. Two clusters of genes, including early and late response genes, were identified according to the expression pattern over time. ECM organization, BMP signaling, and PI3K-Akt signaling were early responsive in the exercise duration. Moreover, time-related signaling pathways, such as inositol phosphate metabolism, nicotinate/nicotinamide metabolism, cell cycle, and Fc epsilon RI signaling pathway, were identified by unbiased mapping and polarization of the highly time-correlated genes. Immunohistochemistry staining showed that Egfr was a late response gene that increased on day 15 of exercise. This study elucidated time-related transcriptomic reprogramming induced by exercise in cartilage, advancing the understanding of cartilage homeostasis.</p

Lactosylated Poly(ethylene glycol)-siRNA Conjugate through Acid-Labile β-Thiopropionate Linkage to Construct pH-Sensitive Polyion Complex Micelles Achieving Enhanced Gene Silencing in Hepatoma Cells

The remarkably enhanced gene silencing in hepatoma cells was achieved by assembling lactosylated-PEG−siRNA conjugates bearing acid-labile β-thiopropionate linkages into polyion complex (PIC) micelles through the mixing with poly(l-lysine). The PIC micelles with clustered lactose moieties on the periphery were successfully transported into hepatoma cells in a receptor-mediated manner, releasing hundreds of active siRNA molecules into the cellular interior responding to the pH decrease in the endosomal compartment. Eventually, almost 100 times enhancement in gene silencing activity compared to that of the free conjugate was achieved for the micelle system, facilitating the practical utility of siRNA therapeutics