Identification of a novel amelogenin gene splicing transcript in <i>Ctenosaura similis</i>.

<p>Chromatograms showed the partial sequence of two <i>Ctenosaura similis</i> amelogenin transcripts identified by sequencing PCR-amplified products. The cDNA clones corresponding to the transcripts were designed <i>as C. similis</i>-T2L (UP) and <i>C. similis</i>-T2S (low), respectively. In relation to <i>C. similis</i>-T2S, <i>C. similis</i>-T2L contains additional nucleotides (UP).</p

Effect of exon X sequence on the amelogenin protein structures.

<p>(A) The hydrophilicity-plot analysis using the Kyte and Doolittle algorithm. The hydrophilicity-plots of T2S and T2L and mouse (XM_136160) were generated and compared. In relation to T2S and mouse amelogenin, the region underlined with black line (middle panel) is exon X sequence, which is highly hydrophobic. (B) Exon X affects the secondary structure of T2L predicted by Psipred. Two potential helixes regions for T2S (aa 7∼16; aa 41∼43) and T2L (aa 6∼17; aa 51∼57) in relation to one potential helix region for mouse amelogenin (aa 4∼12) were revealed by Psipred prediction. A strand region (aa 32–35) was also revealed in relation to T2S and mouse amelogenin. (C) Exon X sequence effects on the tertiary structures of T2L. T2S and T2L were used as query sequence for homology detection and structure prediction by HMM-HMM comparison using HHpred. A bar graph summarizes the positions and color-coded significances of the database matches with the probability. A tabular hit lists with probabilities, E-values, scores, and match regions in query and templates.</p

Novel isoforms of amelogenin gene in

<p><b><i>Ctenosaura similis</i></b><b>.</b> (A) Alignment analysis of the full-length novel iguana amelogenin cDNA sequence <i>C. similis</i>-T2S, C. <i>similis</i>-T2L, and <i>I. iguana</i>. The full-length of <i>C. similis</i>-T2S transcript is 825 bp, while <i>C. similis</i>-T2L transcript is 873 bp. In relation to <i>C. similis</i>-T2S and <i>I. iguana</i>, the <i>C. similis</i>-T2L contains additional 48 bp located between nucleotide 217 and 218. The 5′-untranslated region contains 69 bp upstream of translation start codon ATG. The 3′-untranslated region contains 183 bp. (B) Sequence analysis of deduced amino acid of <i>I. iguana</i>, <i>C. similis</i>-T2S, and <i>C. similis</i>-T2L. The <i>C. similis</i>-T2S encodes 190 amino acid residues, while <i>C. similis</i>-T2L encodes 206 amino acid residues. Additional 16 amino acid residues were revealed in the deduced <i>C. similis</i>-T2L amino acid sequence located between amino acid residue 49 and 50 in relation to <i>C. similis</i>-T2S. Similar to <i>I. iguana</i>, the T2S and T2L also have a deletion of 3 amino acid residues in exon 3. The nucleotides/amino acid sequence variations were indicated and highlighted as red color.</p

Novel iguana amelogenin gene cDNA structure.

<p>Analysis of novel iguana amelogenin sequence revealed the full-length amelogenin cDNA containing 7 exons including exon 1, 2, 3, 5, X, 6, and 7 (exon numbers is relative to published mammalian amelogenin exon numbers). Different from amelogenin genes of all species so far, an unique exon named exon X between exon 5 and exon 6 was detected; Similar to other species, no corresponding sequence elements were detected resembling exon 4, suggesting that amelogenin exon 4 is skipped or deleted in <i>Ctenosaura similis</i>.</p

Alignment of genomic sequences spanning exon X of <i>C. similis</i>-T2L amelogenin gene with the selected species.

<p>The black spiny-tailed iguana genomic sequence spanning the exon X was amplified by PCR and analyzed by alignment with the Carolina anole in the same region of the black spiny-tailed iguana exon X sequence. Like the majority of exons, a conserved sequence feature, the presence of AG at the 5′ splice site and GT at the 3′ splice site were observed spanning the exon X, while the same sequence region of the Carolina anole share a high sequence identity except the lack of G in AG at the 5′ splice site, which fail to generate a exon X observed in the black spiny-tailed iguana. The 5′ and 3′ splice sites in the black spiny-tailed iguana genomic sequence spanning the exon X are highlighted as red. Alignment gaps are indicated by a dash (−).</p

Primers used for determination of the exon and intron boundaries.

<p>S refers to sense; A stands for antisense.</p

Expression of exon X protein in tooth organ. (

<p>A) The microphotograph illustrates the components of the developing iguana tooth organ, including the enamel layer (en), dental layer (de) and dental pulp (pl). (B) Expression of amelogenin protein isoform containing exon X in the <i>Ctenosaura similis</i> tooth organs by immunohistochemistry staining using antibody against iguana amelogenin exon X peptide. Immunoreaction with antibody against exon X was detected in the enamel layer (en) and dental pulp.</p

Lower Extent but Similar Rhythm of Osteogenic Behavior in hBMSCs Cultured on Nanofibrous Scaffolds <i>versus</i> Induced with Osteogenic Supplement

Nanotopographic cues from biomaterials exert powerful effects on the osteogenic differentiation of mesenchymal stem cells because of their niche-mimicking features. However, the biological mechanisms underlying cell lineage determination by surface nanotopography have not been clearly elucidated. Here, we explored the osteogenic behavior of human bone marrow mesenchymal stem cells (hBMSCs) on poly-l-lactide nanofibers with different orientations and monitored the dynamic changes in global gene expression triggered by topographical cues. RT-PCR analysis of osteogenic marker genes and ALP activity assays demonstrated that hBMSCs cultured on random nanofibers showed enhanced osteogenic-specific fate compared with those on aligned nanofibers. Microarray analysis demonstrated a similar temporal change in gene expression patterns between hBMSCs cultured on random nanofibers and those induced with an osteogenic supplement (OS). However, the extent of osteogenic differentiation on the fibrous scaffold was much lower than that driven by chemical OS. In-depth pathway analysis revealed that focal adhesion kinase, TGF-β, Wnt, and MAPK pathways were involved in the activation of osteogenic differentiation in hBMSCs on random nanofibers. These findings suggested that a lower extent but similar rhythm of dynamic cellular behavior was induced on random nanofibers when compared with the OS condition and that mechanotransduction could trigger nonspecific and multilevel responses in hBMSCs. This study provides insight into the regulation of osteogenesis directed by substratum surfaces

DataSheet1_Chirality-biased protein expression profile during early stages of bone regeneration.docx

Introduction: Chirality is a crucial mechanical cue within the extracellular matrix during tissue repair and regeneration. Despite its key roles in cell behavior and regeneration efficacy, our understanding of chirality-biased protein profile in vivo remains unclear.Methods: In this study, we characterized the proteomic profile of proteins extracted from bone defect areas implanted with left-handed and right-handed scaffold matrices during the early healing stage. We identified differentially-expressed proteins between the two groups and detected heterogenic characteristic signatures on day 3 and day 7 time points.Results: Proteomic analysis showed that left-handed chirality could upregulate cell adhesion-related and GTPase-related proteins on day 3 and day 7. Besides, interaction analysis and in vitro verification results indicated that the left-handed chiral scaffold material activated Rho GTPase and Akt1, ultimately leading to M2 polarization of macrophages.Discussion: In summary, our study thus improved understanding of the regenerative processes facilitated by chiral materials by characterizing the protein atlas in the context of bone defect repair and exploring the underlying molecular mechanisms of chirality-mediated polarization differences in macrophages.</p

Synthesis and Fluorescent Property of Biodegradable Polyphosphazene Targeting Long-Term <i>in Vivo</i> Tracking

The importance of developing photoluminescent biodegradable scaffolding materials for tissue engineering is obvious, but it meets challenges with conventional biodegradable polymers such as aliphatic polyesters. In this study, photoluminescent biodegradable polyphosphazenes (PTA) were suggested as alternatives to target for long-term in vivo tracking applications. The PTA polymers were synthesized via nucleophilic cosubstitution of linear poly­(dichloro­phosphazene) with a fluorescent compound (TPCA) and alanine ethyl ester. The TPCA, with high fluorescent intensity and high quantum yield (∼0.5), was synthesized from citric acid and 2-amino­ethanethiol. The resulted PTA polymers demonstrated adjustable degradation rates and fluorescent intensities in relating to their chemical compositions. In comparison with TPCA, the photostability of PTA polymers has been significantly improved, which made the long-term <i>in vivo</i> tracking feasible. PTA polymers were proven biocompatible and noncytotoxic for biomedical applications via both <i>in vitro</i> cell culture and <i>in vivo</i> implantation evaluations. During the 24-week subcutaneous implantation in mouse, the location and the degradation of PTA polymer were clearly visualized with the aid of fluorescent excitation and emission. In summary, PTA polymers were envisioned as good choices for tissue regeneration as scaffolding materials with <i>in situ</i> bioimaging potentials