Circular RNAs (circRNAs) are ubiquitous endogenous RNA found in various organisms that can regulate gene expression in eukaryotes. However, little is known about potential roles for circRNAs in ...muscle development. We analyzed circRNA sequencing data of bovine skeletal muscle tissue and found differential expression of circTitin (circTTN) in fetal and adult bovine muscle tissue. We then further studied the role of circTTN in bovine muscle development. Overexpression and inhibition of circTTN together elicited its promoting roles in proliferation and differentiation of bovine primary myoblasts. Mechanistically, circTTN showed interaction with miR-432 by luciferase screening and RNA immunoprecipitation (RIP) assays. Additionally, miR-432 is a regulator of insulin-like growth factor 2 (IGF2), as indicated by luciferase activity, quantitative real-time PCR, and western blotting assays. Increased miR-432 expression inhibited the expression of IGF2, but this effect was remitted by circTTN. Conclusively, our results showed that circTTN promoted proliferation and differentiation of bovine primary myoblasts via competitively combining with miR-432 to activate the IGF2/phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway.
Adipose development is regulated by a series of complex processes, and non-coding RNAs (ncRNAs), including circular RNAs (circRNAs), play important roles in regulating proliferation and ...differentiation of adipocytes. In this study, we profiled circRNA expression in cattle fat tissue during calf and adult developmental stages and detected 14,274 circRNA candidates. Some circRNAs are differentially expressed between two developmental stages. We characterized circFUT10, named for its host gene FUT10, a highly expressed and abundant circRNA. Luciferase screening, an RNA-binding protein immunoprecipitation (RIP) assay, quantitative real-time PCR, and western blotting assays indicated that circFUT10 directly binds let-7c/let-e, and PPARGC1B (peroxisome proliferator-activated receptor γ coactivator 1-β) is identified as a target of let-7c. Flow cytometry, EdU (5-ethynyl-2′-deoxyuridine) incorporation, a CCK-8 (cell counting kit-8) assay, oil red O staining, and western blotting assays demonstrated that circFUT10 promotes adipocyte proliferation and inhibits cell differentiation by sponging let-7c. The results demonstrate that circFUT10 binding of let-7c promotes cell proliferation and inhibits cell differentiation by targeting PPARGC1B in cattle adipocytes.
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In order to verify the potential function of circRNA in adipose tissue, high-throughput sequencing of Qinchuan subcutaneous adipose tissue was performed, and circFUT10 was differentially expressed in calf and adult cattle adipose tissue. Experimental verification showed that circFUT10 promoted the proliferation of adipose cells and inhibited their differentiation by combining with let-7 family members.
Many novel non-coding RNAs, such as microRNAs (miRNAs) and circular RNAs (circRNAs), are involved in various physiological and pathological processes. The PI3K/AKT signaling pathway is important for ...its role in regulating skeletal muscle development. In this study, molecular and biochemical assays were used to confirm the role of miRNA-145 (miR-145) in myoblast proliferation and apoptosis. Based on sequencing data and bioinformatics analysis, we identified a new circRILPL1, which acts as a sponge for miR-145. The interactions between circRILPL1 and miR-145 were examined by bioinformatics, a luciferase assay, and RNA immunoprecipitation. Mechanistically, knockdown or exogenous expression of circRILPL1 in the primary myoblasts was performed to prove the functional significance of circRILPL1. We investigated the inhibitory effect of miR-145 on myoblast proliferation by targeting IGF1R to regulate the PI3K/AKT signaling pathway. A novel circRILPL1 was identified that could sponge miR-145 and is related to AKT activation. In addition, circRILPL1 was positively correlated with muscle proliferation and differentiation in vitro and could inhibit cell apoptosis. The newly identified circRILPL1 functions as a miR-145 sponge to regulate the IGF1R gene and rescue the inhibitory effect of miR-145 on the PI3K/AKT signaling pathway, thereby promoting myoblast growth.
Most current methods to assess T-cell-dependent antibody responses (TDAR) are semi-quantitative and based on measures of antibody titer generated against a standard antigen like keyhole limpet ...hemocyanin (KLH). The precision, sensitivity, and convenience of TDAR assays might be improved by applying rapid, sensitive, specific cytometric bead assays (CBA). In the study here, KLH antigen was covalently coupled onto the surface of cytometric beads using immune microsphere technology, and IgM antibody capture spheres were prepared for use in pretreatment processing of samples. The working parameters associated with this novel TDAR-CBA system were optimized in orthogonal experiments. The optimal concentration of the KLH coating solution in this system was 160 μg/ml, that of the anti-KLH IgG capture spheres 6.0 × 10
5
/ml, and the optimal dilution of fluorescein isothiocyanate (FITC)-conjugated Affini-Pure Goat Anti-Mouse IgG (H + L) was 60 μg/ml. Repeated tests indicated that this approach yielded good linearity (r
2
= 0.9937) method, with a within-run precision of 3.1-4.9%, and a between-run precision of 4.4-4.9%. This new approach had a limit of detection of 113.43 ng/ml (linear range = 390.63-50 000), and an interference rate of just 0.04-3.51%. Based on these findings, it seems that a new mouse TDAR assay based on CBA can be developed that would appear to be more sensitive, accurate, and precise than the current TDAR assay approaches based on traditional ELISA.
Long noncoding RNAs (lncRNAs) are involved in the regulation of skeletal muscle development. In the present study, differentially expressed lncRNAs were identified from RNA-seq data derived from ...myoblasts and myotubes. We conducted studies to elucidate the function and molecular mechanism of action of Linc-smad7 during skeletal muscle development. Our findings show that Linc-smad7 is upregulated during the early phase of myoblasts differentiation. In in vitro studies, we showed that overexpression of Linc-smad7 promoted the arrest of myoblasts in G1 phase, inhibited DNA replication, and induced myoblast differentiation. Our in vivo studies suggest that Linc-smad7 stimulates skeletal muscle regeneration in cardiotoxin-induced muscle injury. Mechanistically, Linc-smad7 overexpression increased smad7 and IGF2 protein levels. On the contrary, overexpression of miR-125b reduced smad7 and IGF2 protein levels. Results of RNA immunoprecipitation analysis and biotin-labeled miR-125b capture suggest that Linc-smad7 could act as a competing endogenous RNA (ceRNA) for miRNA-125b. Taken together, our findings suggest that the novel noncoding regulator Linc-smad7 regulates skeletal muscle development.
MicroRNAs (miRNAs), belonging to a class of evolutionarily conserved small noncoding RNA of ∼22 nucleotides, are widely involved in skeletal muscle growth and development by regulating gene ...expression at the post-transcriptional level. While the expression feature and underlying function of miR-216a in mammal skeletal muscle development, especially in cattle, remains to be further elucidated. The aim of this study was to investigate the function and mechanism of miR-216a during bovine primary muscle cells proliferation and differentiation. Herein, we found that the expression level of miR-216a both presented a downward trend during the proliferation and differentiation phases, which suggested that it might have a potential role in the development of bovine skeletal muscle. Functionally, during the cells proliferation phase, overexpression of miR-216a inhibited the expression of proliferation-related genes, reduced the cell proliferation status, and resulted in cells G1 phase arrest. In cells differentiation stages, overexpression of miR-216a suppressed myogenic maker genes mRNA, protein, and myotube formation. Mechanistically, we found that
and
were the directly targets of miR-216a in regulating bovine primary muscle cells proliferation and differentiation, respectively. Altogether, these findings suggested that miR-216a functions as a suppressive miRNA in development of bovine primary muscle cells
targeting
and
.
Immune checkpoint blockade (ICB) therapy potently revives T cell's response to cancer. However, patients suffered with tumors that had inadequate infiltrated immune cells only receive limited ...therapeutic benefits from ICB therapy. Synthetic biology promotes the alternative strategy of harnessing tumor-targeting bacteria to synthesize therapeutics to modulate immunity in situ. Herein, we engineered attenuated Salmonella typhimurium VNP20009 with gene circuits to synthetize granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 7 (IL-7) within tumors, which recruited dendritic cells (DCs) and enhanced T cell priming to elicit anti-tumor response. The bacteria-produced GM-CSF stimulated the maturation of bone marrow-derived dendritic cells (BMDCs), while IL-7 promoted the proliferation of spleen isolated T cells and inhibited cytotoxicity T cell apoptosis in vitro. Virtually, engineered VNP20009 prefer to colonize in tumors, and inhibited tumor growth by enhancing DCs and T cell infiltration. Moreover, the tumor-toxic GZMB+ CD8+ T cell and IFN-γ+ CD8+ T cell populations conspicuously increased with the treatment of engineered bacteria. The combination of GM–CSF–IL-7-VNP20009 with PD-1 antibody synergistically stunted the tumor progress and metastasis.
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•An inducible protein-secreting engineered Salmonella was constructed.•Bacteria-produced GM-CSF and IL-7 had their biological activity in vitro.•Engineered Salmonella enriched within tumors.•Engineered Salmonella strongly inhibited the progression of melanoma.
The role of long non-coding RNA (lncRNA) in the regulation of bovine skeletal muscle development remains poorly understood. The present study investigated the function and regulatory mechanism of a ...novel lncRNA, insulin-like growth factor 2 antisense transcript (IGF2 AS), in bovine myoblast proliferation and differentiation. Gain or loss of IGF2 AS was performed using an expression plasmid or small interfering RNA (siRNA), respectively. Bovine myoblasts were used to investigate the biological function and mechanisms of IGF2 AS in vitro. Results were conjointly analyzed by celluar and molecular biology experiments as well as bioinformatics. Functionally, IGF2 AS could promote proliferation and differentiation of bovine myoblasts. The preliminary mechanism suggests, on the one hand, that IGF2 AS could complement the IGF2 gene intron region and affect the stability and expression of IGF2 mRNA. On the other hand, RNA pull-down and immunoprecipitation assays demonstrated that IGF2 AS could directly bind to the interleukin enhancer binding factor 3 (ILF3) protein and maybe partly though it to regulate myogenesis. In conclusion, the novel identified lncRNA IGF2 AS promoted proliferation and differentiation of bovine myoblasts through various pathways.
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Song et al. found a novel antisense non-coding RNA, IGF2 AS, in the bovine IGF2 gene location. IGF2 AS could form a double-stranded complex with IGF2 gene transcription precursors and enhance IGF2 expression. IGF2 AS could also bind to the protein ILF3 to regulate the proliferation and differentiation of bovine myoblasts.
Although T-cell-dependent antibody response (TDAR) assays with keyhole limpet hemocyanin (KLH) as specific antigen have many advantages, most experiments produce qualitative results based on antibody ...titers. It was hypothesized that if experimental conditions (like antigen coating concentration, serum dilution, and detecting here, horseradish peroxidase-goat anti-mouse IgG antibody dilution) could be optimized, the resulting measured value (here, optical density) could be used to directly analyze and evaluate the experimental results. This means specifically that the assay OD values could be used for approximate quantitative statistical analysis; it does not require a further conversion of the data into qualitative forms or require obtaining further titer data from additional experiments. As such, the use of this "improved" assay would: greatly reduce the complexity of experimental operations; improve overall sensitivity and practicality of traditional TDAR assays; and, allow for direct assessing of any immunosuppression caused by a test drug in a host. Here, KLH-immunized serum was obtained from BALB/c mice, and the means to detect serum anti-KLH antibodies by an indirect ELISA were optimized. The results indicated that in this system, the optimal KLH coating concentration was 80 μg/ml, the optimal dilution range of the serum (at immunization dose of 5 mg KLH/kg) was 1:200-1:800, and the optimal dilution of HRP-goat anti-mouse IgG antibody was 1:16,000. Methodology verification was performed and a regression model of y = 144.16x + 0.9815 (R
2
= 0.9571, indicating very good linearity) was obtained. Intragroup precision was 7.51-9.40%; the intergroup coefficient of variation was 9.83-14.22%. The lower limit of detection was 0.1385. The present results showed this indirect ELISA exhibited very good linearity, accuracy, and precision.
Blockchain, as the underlying technology of crypto-currencies, has attracted significant attention. It has been adopted in numerous applications, such as smart grid and Internet-of-Things. However, ...there is a significant scalability barrier for blockchain, which limits its ability to support services with frequent transactions. On the other side, edge computing is introduced to extend the cloud resources and services to be distributed at the edge of the network, but currently faces challenges in its decentralized management and security. The integration of blockchain and edge computing into one system can enable reliable access and control of the network, storage, and computation distributed at the edges, hence providing a large scale of network servers, data storage, and validity computation near the end in a secure manner. Despite the prospect of integrated blockchain and edge computing systems, its scalability enhancement, self organization, functions integration, resource management, and new security issues remain to be addressed before widespread deployment. In this survey, we investigate some of the work that has been done to enable the integrated blockchain and edge computing system and discuss the research challenges. We identify several vital aspects of the integration of blockchain and edge computing: motivations, frameworks, enabling functionalities, and challenges. Finally, some broader perspectives are explored.
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