Journal: eLife

Article Title: The cis -regulatory effects of modern human-specific variants

doi: 10.7554/eLife.63713

Figure Lengend Snippet: Relative percentage of bases in each chromHMM ( ; ) category throughout the entire genome ( a ), in fixed or nearly fixed modern human-derived variants ( b ), in active sequences ( c ), and in differentially active sequences ( d ), per cell type. See Discussion for cell-type specificity and enhancer enrichment. ( e ) Histogram of the number of tissues and number of sequences with transcription start site- (TSS) or enhancer-related chromHMM marks for all 14,042 sequences. Tissues and cell types investigated include embryonic stem cells (ESCs), osteoblasts, neural progenitor cells (NPCs), mesenchymal stem cells, monocytes, skin fibroblasts, brain hippocampus, skeletal muscle, heart left ventricle, sigmoid colon, ovary, fetal lung, and liver. Inset shows data for ESC, osteoblast, and NPC only.

Article Snippet: Human fetal osteoblasts were purchased from Cell Applications Inc (406 K-05f, tested negative for mycoplasma) and were maintained in Osteoblast Growth Medium (Cell Applications Inc).

Techniques: Derivative Assay

Journal: eLife

Article Title: The cis -regulatory effects of modern human-specific variants

doi: 10.7554/eLife.63713

Figure Lengend Snippet: ( a ) Overlap between cell types of active sequences. Super Exact test p-value is shown for the overlap of the three groups. ( b-d ) Enrichment levels of active and repressive histone modification marks within active sequences. Enrichment is computed compared to inactive sequences. The enrichment of H3K27me3 in embryonic stem cells (ESCs) possibly reflects the presence of this mark in bivalent genes, which become active in later stages of development . For confidence intervals, see . ( e ) Enrichment of differentially active sequences in various chromatin-based genomic annotations. Missing circles reflect no differentially active sequences in that category. Stars mark significant enrichments (false discovery rate [FDR] <0.05). ( f ) Violin plots of DNA methylation levels for active (green) vs. inactive (red) sequences in osteoblasts. Methylation levels per sequence were computed as the mean methylation across all modern and archaic human bone methylation samples. The circle marks mean methylation across all sequences in each group. t -test p-value is shown.

Article Snippet: Human fetal osteoblasts were purchased from Cell Applications Inc (406 K-05f, tested negative for mycoplasma) and were maintained in Osteoblast Growth Medium (Cell Applications Inc).

Techniques: Modification, DNA Methylation Assay, Methylation, Sequencing

Journal: eLife

Article Title: The cis -regulatory effects of modern human-specific variants

doi: 10.7554/eLife.63713

Figure Lengend Snippet: ( a–c ) Violin plots of DNA methylation levels in modern and archaic human bone methylation samples, for differentially active ( a ), promoter differentially active ( b ), and CpG-poor promoter differentially active ( c ) sequences in osteoblasts. Promoter sequences are sequences between 5 kb upstream and 1 kb downstream of a transcription start site (TSS). CpG-poor promoter sequences were defined as the bottom 50% promoter sequences. ( d ) Violin plots of absolute predicted TF binding score difference between modern and archaic sequences. Points show mean.

Article Snippet: Human fetal osteoblasts were purchased from Cell Applications Inc (406 K-05f, tested negative for mycoplasma) and were maintained in Osteoblast Growth Medium (Cell Applications Inc).

Techniques: DNA Methylation Assay, Methylation, Binding Assay

Journal: eLife

Article Title: The cis -regulatory effects of modern human-specific variants

doi: 10.7554/eLife.63713

Figure Lengend Snippet: (a–c) Expression fold-change vs. predicted TF binding fold-change for each sequence. Positive scores represent increased binding in the modern sequence. Parentheses show number of points in each quadrant with a score difference >0. ( d ) Pearson’s correlation between differential expression and predicted differential binding affinity. Only significant TFs (false discovery rate [FDR] ≤0.05, ) are shown for osteoblasts (yellow) and neural progenitor cells (NPCs) (red). ( e ) Expression fold-change vs. predicted TF binding fold-change for ZNF281 in NPCs. Pearson’s r and p-value are shown. ( f ) Enriched Gene Ontology terms for embryonic stem cells (ESCs) (blue), osteoblasts (yellow), and NPCs (red). ( g ) Expression fold-change of differentially active sequences compared to the cis -regulatory expression fold-change between human and chimpanzee of genes associated with these sequences. cis -regulatory expression changes were taken from hybrid human-chimpanzee induced pluripotent stem cells (iPSCs) . ( h ) RT-qPCR validation of NPCs at passage 1 (pink) and passage 10 (red). Expression levels are normalized to HPRT expression.

Article Snippet: Human fetal osteoblasts were purchased from Cell Applications Inc (406 K-05f, tested negative for mycoplasma) and were maintained in Osteoblast Growth Medium (Cell Applications Inc).

Techniques: Expressing, Binding Assay, Sequencing, Quantitative Proteomics, Quantitative RT-PCR, Biomarker Discovery

Journal: PLOS ONE

Article Title: Evaluation of decellularization process for developing osteogenic bovine cancellous bone scaffolds in-vitro

doi: 10.1371/journal.pone.0283922

Figure Lengend Snippet: Bar charts indicate comparison between DCC scaffold vs DCC scaffold + cells and DMB scaffolds vs DMB scaffold + cells by direct method (a). Bar charts indicate comparison between DMB conditioned media (CM) and DCC conditioned media (CM) vs complete culture media (Control) by indirect method (b). *p<0.05 is indicated. n = 4; CM: conditioned media. (c). HOB cells seeded on DMB and DCC scaffolds showing optimum cell attachment by DAPI staining. (i & iii) DMB scaffolds and (ii & iv) DCC scaffolds; images were captured by fluorescence and confocal microscopy, respectively. Scanning electron micrograph of HOB cells seeded onto DMB (d) and DCC (e) scaffolds. The osteoblast shows a healthy morphology residing on the ECM with evidence of filopodia spread (red arrows).

Article Snippet: Human osteoblast cells (HOB) used for biocompatibility evaluation were obtained from Addexbio (Cat no: P0004010; USA).

Techniques: Comparison, Control, Cell Attachment Assay, Staining, Fluorescence, Confocal Microscopy

Journal: PLOS ONE

Article Title: Evaluation of decellularization process for developing osteogenic bovine cancellous bone scaffolds in-vitro

doi: 10.1371/journal.pone.0283922

Figure Lengend Snippet: Photomicrographs of HOB cells stained with Alizarin Red S. HOB cells showed increased mineralized nodules in osteogenic medium (OS + ) compared to cells without supplements (OS - ). More significant mineralization was seen by day 14 (a). Quantification of alizarin Red S stained mineralized nodules in human osteoblast cells seeded on DMB and DCC scaffolds grown in the presence or absence of osteogenic induction media at days 7 and 14 in comparison to cells alone is shown; p<0.001 is indicated). The absorbance of the extracted Alizarin dye was measured at 405 nm. Data presented as mean ± SEM (n = 6) (b). Quantification of Alizarin Red S staining in osteoblast cell-seeded DMB and DCC scaffolds on day 14 in the presence and absence of osteogenic supplements. Data presented as mean ± SEM (n = 4) (c); p<0.001 is indicated.

Article Snippet: Human osteoblast cells (HOB) used for biocompatibility evaluation were obtained from Addexbio (Cat no: P0004010; USA).

Techniques: Staining, Comparison

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