Journal: International Journal of Molecular Sciences

Article Title: Deduction of Novel Genes Potentially Involved in Osteoblasts of Rheumatoid Arthritis Using Next-Generation Sequencing and Bioinformatic Approaches

doi: 10.3390/ijms18112396

Figure Lengend Snippet: Identification of differentially expressed microRNAs and potential microRNA–mRNA interactions in rheumatoid arthritis primary osteoblasts. ( A ) The next generation sequencing (NGS) identified 35 differentially expressed microRNAs (thresholds of >2.0-fold change and reads per million (RPM) >10) in rheumatoid arthritis (RA) osteoblasts, compared to normal osteoblasts. The heat map analysis with z-score values is shown here. ( B ) The 16 up-regulated and 19 down-regulated microRNAs predicted 435 and 391 putative targets, respectively, using the miRmap database with selection threshold of miRmap score ≥99.0. Additionally, 434 protein-coding genes with >2.0-fold change and >0.3 fragments per kilobase of transcript per million (FPKM) were identified from the NGS, where 199 genes were up-regulated and 235 genes were down-regulated in RA osteoblasts. The putative targets of up-regulated (down-regulated) microRNAs were matched to the down-regulated (up-regulated) protein-coding genes by the Venn diagram analysis. Finally, thirteen genes (eight down-regulated and five up-regulated) with potential miRNA–mRNA interactions were identified.

Article Snippet: Osteoblasts were grown in human osteoblast growth medium (Cell Applications, Inc.) and maintained in 37 °C incubator containing 5% CO 2 until confluence.

Techniques: Next-Generation Sequencing, Selection

Journal: International Journal of Molecular Sciences

Article Title: Deduction of Novel Genes Potentially Involved in Osteoblasts of Rheumatoid Arthritis Using Next-Generation Sequencing and Bioinformatic Approaches

doi: 10.3390/ijms18112396

Figure Lengend Snippet: Gene ontology terms involved in the 13 candidate genes of rheumatoid arthritis primary osteoblasts. Using the functional annotation analysis in the DAVID database, the 13 candidate genes were classified by terms of the Gene Ontology in: the molecular function domain ( A ); and the cellular component domain ( B ). The related genes are listed below each term. The selected criteria for functional annotation analysis was EASE = 1.0.

Article Snippet: Osteoblasts were grown in human osteoblast growth medium (Cell Applications, Inc.) and maintained in 37 °C incubator containing 5% CO 2 until confluence.

Techniques: Functional Assay

Journal: International Journal of Molecular Sciences

Article Title: Deduction of Novel Genes Potentially Involved in Osteoblasts of Rheumatoid Arthritis Using Next-Generation Sequencing and Bioinformatic Approaches

doi: 10.3390/ijms18112396

Figure Lengend Snippet: Analysis of 13 genes with potential microRNA–mRNA interactions in the Gene Expression Omnibus (GEO) database. The expression values of: five up-regulated genes ( A ); and eight down-regulated genes ( B ) identified from normal and rheumatoid arthritis (RA) osteoblasts were validated in a representative array (GSE77298) of normal and RA synovial tissues from the GEO database. Significant up-regulation of LRRC15 and down-regulation of AKAP12 and RGS5 were observed in the synovial tissues of patients with RA, compared to the normal subjects. * indicated p < 0.05, and n.s. indicated no statistical significance. (Probe ID reference: BDKRB2 , 205870_at; CREB5 , 229228_at; FGFRL1 , 223321_s_at; LRRC15 , 213909_at; SESN3 , 242899_at; ADAMTS12 , 221421_s_at; AKAP12 , 231067_s_at; COL5A3 , 218975_at; FAM26E , 230254_at; KCTD20 , 228299_at; KLHL3 , 221221_s_at; RGS5 , 209071_s_at; and SERPINB9 , 242814_at).

Article Snippet: Osteoblasts were grown in human osteoblast growth medium (Cell Applications, Inc.) and maintained in 37 °C incubator containing 5% CO 2 until confluence.

Techniques: Gene Expression, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Deduction of Novel Genes Potentially Involved in Osteoblasts of Rheumatoid Arthritis Using Next-Generation Sequencing and Bioinformatic Approaches

doi: 10.3390/ijms18112396

Figure Lengend Snippet: Network analysis by Ingenuity ® Pathway Analysis (IPA) indicated molecules associated with rheumatic disease and inflammation of joint. The network analysis was performed by IPA software to indicate networks involved in the 13 candidate genes from rheumatoid arthritis (RA) osteoblasts. Ten of the 13 candidate genes were grouped in one network associated with cardiovascular system development and function, cellular development, cellular growth and proliferation. Molecules including ADAMTS12 , BDKRB1 , BDKRB2 , BMP1 , FGF2 , FOXO1 , KCTD20 , NOTCH4 , PPARG , TGFB1 and miR-146a-5p were associated with rheumatic disease and inflammation of joint in the network, as indicated in purple frames. Molecules in green indicated down-regulated expressions, and molecules in red indicated up-regulated expressions in RA osteoblasts compared to normal osteoblasts. The green and red color scales disclosed the relative gene expression values of RA to normal osteoblasts.

Article Snippet: Osteoblasts were grown in human osteoblast growth medium (Cell Applications, Inc.) and maintained in 37 °C incubator containing 5% CO 2 until confluence.

Techniques: Software, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Deduction of Novel Genes Potentially Involved in Osteoblasts of Rheumatoid Arthritis Using Next-Generation Sequencing and Bioinformatic Approaches

doi: 10.3390/ijms18112396

Figure Lengend Snippet: Analysis of the interconnection between AKAP12 and the merged networks of related joint diseases and functions. The 434 differentially expressed genes identified in normal and rheumatoid arthritis (RA) osteoblasts were analyzed by the IPA to be categorized into 25 networks. Diseases and functions related to joint destruction in RA microenvironment, including inflammation of joint, chemotaxis, damage of connective tissue, migration of connective tissue cells, proliferation of osteoblasts, and neovascularization were selected to identify related genes. AKAP12 , one of the genes involved in chemotaxis, was connected to HGF and ARRB1 , molecules involved in damage of connective tissue and neovascularization. In addition, the overlay canonical pathway analysis indicated AKAP12 , MAP3K1 , VASP , PTK2B , TGFB2 , ITPR3 , and NFATC1 (marked in light blue) to be involved in the protein kinase A signaling, and participated in various indicated networks.

Article Snippet: Osteoblasts were grown in human osteoblast growth medium (Cell Applications, Inc.) and maintained in 37 °C incubator containing 5% CO 2 until confluence.

Techniques: Chemotaxis Assay, Migration

Journal: International Journal of Molecular Sciences

Article Title: Deduction of Novel Genes Potentially Involved in Osteoblasts of Rheumatoid Arthritis Using Next-Generation Sequencing and Bioinformatic Approaches

doi: 10.3390/ijms18112396

Figure Lengend Snippet: The biological process analysis of differentially expressed genes in rheumatoid arthritis osteoblasts. The 434 differentially expressed genes in rheumatoid arthritis osteoblasts were analyzed in the DAVID database for the identification of involved biological processes. The results indicated these genes were potentially involved in cell adhesion (38 genes), extracellular matrix organization (23 genes), positive regulation of cell migration (21 genes), skeletal system development (18 genes), angiogenesis (22 genes), type I interferon signaling pathway (12 genes), positive regulation of cell proliferation (31 genes), response to hypoxia (18 genes), heart development (18 genes), and positive regulation of PI3K signaling (11 genes). The selected criteria for functional annotation analysis were EASE = 0.1 and fold enrichment >1.3. The proportions of the pie chart were drawn according to the numbers of genes involved in each biological term, and the numbers within the pie chart indicated −log ( p -value) of each biological term.

Article Snippet: Osteoblasts were grown in human osteoblast growth medium (Cell Applications, Inc.) and maintained in 37 °C incubator containing 5% CO 2 until confluence.

Techniques: Migration, Functional Assay

Journal: International Journal of Molecular Sciences

Article Title: Deduction of Novel Genes Potentially Involved in Osteoblasts of Rheumatoid Arthritis Using Next-Generation Sequencing and Bioinformatic Approaches

doi: 10.3390/ijms18112396

Figure Lengend Snippet: The proposed novel molecular signatures and microRNA regulations in rheumatoid arthritis osteoblasts.

Article Snippet: Osteoblasts were grown in human osteoblast growth medium (Cell Applications, Inc.) and maintained in 37 °C incubator containing 5% CO 2 until confluence.

Techniques:

Journal: Cellular and Molecular Bioengineering

Article Title: Remote-Controlled Gene Delivery in Coaxial 3D-Bioprinted Constructs using Ultrasound-Responsive Bioinks

doi: 10.1007/s12195-024-00818-x

Figure Lengend Snippet: Effect of varying the number of ultrasound pulses on ultrasound-controlled osteoblast cellular transfection in coaxially-bioprinted constructs. A Coaxially-bioprinted osteoblast-laden (hFOB 1.19) 4% alginate bioink containing GFP-coupled microbubbles before ultrasound, 0 hr post-ultrasound, and 48 hr post-ultrasound with varying ultrasound exposure (10, 40, or 80 pulses). B Number of transfected cells in bioprinted constructs at 48 hr post-ultrasound for varying numbers of ultrasound pulses (*p < 0.05, ****p < 0.0001, one-way ANOVA, Tukey’s multiple comparisons test, error bars denote standard deviation). C Diameter of zone containing transfected cells at 48 hr post-ultrasound for varying numbers of ultrasound pulses (one-way ANOVA, Tukey’s multiple comparisons test, error bars denote standard deviation)

Article Snippet: They were then suspended in complete media containing Human Osteoblast Media (Cell Applications, Inc.), 10% FBS (HyClone Characterized Fetal Bovine Serum CA Origin, Cytiva), and 1% pen-strep (penicillin-streptomycin, 10,000 μg/mL, Gibco) then concentrated to 3.85 × 10 7 cell/mL in complete media.

Techniques: Transfection, Construct, Standard Deviation

Journal: International Journal of Nanomedicine

Article Title: Electrospun nanofiber blend with improved mechanical and biological performance

doi: 10.2147/IJN.S175619

Figure Lengend Snippet: ( A ) ALP activity showed an increase of calcification of the extracellular matrix after inclusion of GelMA. ( B ) Calcium deposition demonstrated a further influence of GelMA to enhance the functions of osteoblasts. ( C ) MTS assay showing that osteoblastic cells were further influenced by hydrophilic properties after inclusion of PEG and GelMA. Data plotted in mean and SD (N=5). Values of P <0.01 were considered significant. Data were normalized by the cells, and the y-axis was multiplied by 10 4 . For the ALP and calcium deposition, the data were compared to control (cells) and between each time. For cellular proliferation assays, the data were compared to pure PCL. N=5. ** P <0.01, *** P <0.001, and **** P <0.0001 mean statistical differences. SEM of hFOBs cultivated on scaffolds after 7 days. ( D ) (i) PCL and (ii) magnified view. ( E ) (i) PCL-PEG and (ii) magnified view. ( F ) (i) PCL-PEG-GelMA without UV crosslinking and (ii) magnified view. ( G ) (i) PCL-PEG-GelMA after UV crosslinking and (ii) magnified view. The cells are spreading on all produced scaffolds presenting filopodium and cytoplasmic extension. Abbreviations: ALP, alkaline phosphatase; GelMA, gelatin methacryloyl; hFOB, human osteoblasts; MTS, (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium); NS, no significance; PCL, polycaprolactone; PEG, poly(ethylene glycol); SEM, scanning electron microscopy.

Article Snippet: Totally 5,000 cells/cm 2 of human osteoblast cells line (hFOB 1.19, bone-forming cells; Lonza, CRL-11372, second passage) were cultured using C27015 media (Osteoblast Basal Medium, Promocell GmbH), 1% penicillin/streptomycin (P/S; Hyclone), and an Osteoblast Growth Medium Supplement Mix under standard cell culture conditions (37°C, 5% CO 2 , and 95% air).

Techniques: Activity Assay, MTS Assay, Control, Produced, Electron Microscopy