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Total RNA-sequencing Detects Transcriptomic Differences in Mouse Preosteoblast Response to Bulk Nanostructured Titanium.

ABSTRACT: Rational: Titanium is the most widely used alloy family in dental and orthopedic implants due to its natural ability to integrate into bone, but cytotoxic alloying elements are required in titanium for its mechanical properties to match the functionality of natural bone in high-load bearing applications. Recent advances in nanostructuring, such as Continuous Equal Channel Angular Pressing (C-ECAP), reduce the grain size and increase the strength of pure grades of titanium, thereby eliminating the need for cytotoxic elements and increasing cytocompatibilty as measured by traditional cell biology techniques. Transcriptomic profiling of cells grown on conventional coarse grain (CG) versus nanostructured ultrafine grain (UG) surfaces can simultaneously enhance our understanding of genomics and biomaterials, facilitating the development of a new generation of implantable materials. Objective: We initiated this pilot project to develop a workflow for transcriptional profiling of both protein coding and non-coding RNAs (ncRNAs) in cells grown on Ti discs that are characterized for grain size, 12.1 μm for CG Ti and 0.28 μm for UG Ti. Methods: Two Ti substrates were designed for cell growth in 24-well culture dishes, CG and UG. Mouse pre-osteoblasts were cultured on Ti disc for 72 hours. Cells were harvested and total-RNA extracted. Ribosomal RNA subtraction was performed prior to Illumina sequencing library construction. Single-end 50 base pairs reads were subjected to Illumina quality control and the resulting reads were mapped to the mouse genome and annotations with Genomic Short-read Nucleotide Alignment Program (GSNAP) and HTseq-count. Read count files were normalized and statistically analyzed with empirical analysis of digital gene expression data in R (EdgeR) and filtered for low expression levels using Zipf’s Power Law. Transcripts with adequate coverage and significant expression differences were subjected to GO Biological Process term enrichment and network analyses with the Cytoscape App ClueGO. Results: Gene expression differences with an FDR≤0.05 are limited to six genes, five of which are likely ncRNAs. Of the 432 transcripts with significant differences at p≤0.05 117 form an interconnected network of genes and 12 GO biological processes (P(adj) ≤ 0.05). Biological processes that connect to predominately upregulated genes include cell division, mRNA metabolic processes, chromatin modification and protein localization to the cytoskeleton, while downregulated processes include regulation of apoptosis, of p38MAK cascade, and response to molecules of bacterial origin. Conclusions: After 72 hours, the transcriptional profile of cells on UG Ti confirms more rapid cell division than on CG. These results demonstrate that informative transcriptional profiling of cells growing on substrates that differ only in their crystallographic structure can be accomplished by a workflow that includes standard cell biology techniques, transcriptomic profiling, data mining, and network analyses.

ORGANISM(S): Mus musculus

PROVIDER: GSE123282 | GEO | 2020/04/09

REPOSITORIES: GEO

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Project description:An abrupt transition from high forage to high fermentable diet may induce digestive disorders in the rumen. To date, the host mechanisms regulate the adaption to such dietary transition are largely unknown. To understand the molecular mechanisms involved in such phenomena, RNA-sequencing was performed to identify the changes in the transcriptomes of ruminal epithelia during a rapid high grain transition from 3% to 92% grain. In total, the expression of 11,044, 11,322 and 11,282 genes was detected in ruminal epithelia of beef heifers (n=15) fed 3%, 75% and 92% barley grain diet, respectively. When diet was changed from 75% to 92% grain, a significant difference was observed in the mean ruminal pH change among the individuals with five heifers decreased (DG; pH from 6.30 ± 0.09 to 5.87 ± 0.15, P < 0.01) and five other ones increased (UG; pH from 5.84 ± 0.42 to 6.35 ± 0.37, P < 0.05). In the ruminal epithelia of DG animals, the expression of genes involved in ketogenesis (HMGCL, P < 0.1) and lipid synthesis (SREBF2, P < 0.1; FABP4, P < 0.05) was increased, the abundance of genes involved in short chain fatty acid transporters (SMCT1, P < 0.1) and monocarboxylate transporters (MCT4, P < 0.1) was decreased. The function of “Concentration of lipid” was activated after 75% to 92% grain diet transition. Comparing to DG animals, the expression of ketogenesis (ACAT2, P < 0.05; HMGCS P < 0.1) and cholesterol synthesis related genes (HMGC and FDPs, P < 0.1) was deceased, while the expression of proton and cholesterol efflux related genes NHE3 and ABCA1 were increased (P < 0.05), and the function of “Storage of lipid” was inhibited in the ruminal epithelial of UG heifers. In addition, the function of differentially expressed (DE) genes in DG were involved in “T cell receptor signaling” and “complement and coagulation cascades” pathways, while the functions “metabolism of xenobiotics by cytochrome P450” and “p53 signaling pathway” were enriched for DE genes in UG animals. Furthermore, the single nucleotide polymorphisms (SNP) discovery by RNA-seq was performed, and SNP (g.46834311A > G) in FABP4 identified between two groups of animals (P < 0.1). The expression of genes involved in regulating lipid transport and fatty acid metabolism varied between DG and UG heifers during rapid 75% to 92% grain transition. The identified genes and SNP could be potential markers and may account for the varied ruminal pH responses between DG and UG heifers, which can be potentially applied in breeding and selecting beef cattle with more tolerance in dietary transition stress.

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Total RNA-sequencing Detects Transcriptomic Differences in Mouse Preosteoblast Response to Bulk Nanostructured Titanium.

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