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BACKGROUND: With the unprecedented rapid growth rate (up to 2.75 cm/day), velvet antler is an invaluable model for the identification of potent growth factors and signaling networks for extremely fast growing tissues, mainly cartilage. Antler growth center (AGC) locates in its tip and consists of five tissue layers: reserve mesenchyme (RM), precartilage (PC), transition zone (TZ), cartilage (CA) and mineralized cartilage (MC). The aim of this study was to investigate the transcription dynamics in the AGC using RNA-seq technology. RESULTS: Five tissue layers in the AGC were collected from three 3-year-old male sika deer using our previously reported sampling method (morphologically distinguishable). After sequencing (15 samples; triplicates/tissue layer), we assembled a reference transcriptome de novo and used RNA-seq to measure gene expression profiles across these five layers. Nine differentially expressed genes (DEGs) were selected from our data and subsequently verified using qRT-PCR. The results showed a high consistency with the RNA-seq results (R2 = 0.80). Nine modules were constructed based on co-expression network analysis, and these modules contained 370 hub genes. These genes were found to be mainly involved in mesenchymal progenitor cell proliferation, chondrogenesis, osteogenesis and angiogenesis. Combination of our own results with the previously published reports, we found that Wnt signaling likely plays a key role not only in stimulating the antler stem cells or their immediate progeny, but also in promoting chondrogenesis and osteogenesis during antler development. CONCLUSION: We have successfully assembled a reference transcriptome, generated gene expression profiling across the five tissue layers in the AGC, and identified nine co-expressed modules that contain 370 hub genes and genes predorminantly expressed in and highly relevant to each tissue layer. We believe our findings have laid the foundation for the identification of novel genes for rapid proliferation and chondrogenic differentiation of antler cells.
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Diferenciación Celular/genética , Ciervos/genética , Perfilación de la Expresión Génica , Transcriptoma/genética , Animales , Cuernos de Venado/crecimiento & desarrollo , Cartílago/crecimiento & desarrollo , Condrogénesis/genética , Ciervos/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica/genética , Masculino , Especificidad de Órganos/genética , Osteogénesis/genéticaRESUMEN
In recent years, conductive polymer composites have been widely studied for their electrical conductivity and electromagnetic shielding effects due to their advantages of light weight, simple preparation methods, and structural design versatility. In this study, oxidized multi-walled carbon nanotubes/waterborne polyurethane composites (OCNT/WPU) were prepared by grafting oxidized carbon nanotubes onto polyurethane molecular chains through in situ polymerization, using environmentally friendly waterborne polyurethane as the polymer matrix. Then, the OCNT/WPU structure was broken by high shear force, and the loading of CNTs was increased by adsorption, and a new composite structure was designed (denoted by OCWPU). The structure and morphology of OCNT/WPU and OCWPU were characterized by FT-IR and SEM. The structure and morphology of OCWPU with different multi-walled carbon nanotube loadings (CNTs/OCWPU) were characterized by SEM, Raman. Finally, the electrical conductivity and the electromagnetic shielding properties of the composites were investigated. It was found that after application of high shear force, the structure of OCWPU was disrupted and the surface activity of the material increased. With the increase in CNTs content, CNTs formed a rosette structure in the polyurethane matrix and covered the surface, and its electromagnetic shielding effect in X-bond (8.2-12.4 Ghz) would be able to reach 23 dB at 5% CNTs/OCWPU and 66.5 dB at 50% CNTs/OCWPU to meet the commercial needs. With 50% CNTs/OCWPU, an electrical conductivity of 5.1 S/cm could be achieved. This work provides a novel idea for the structural design of conductive polymer composites, which can achieve greater performance with the same carbon nanotube content.
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S100A4 is a multiple-function protein highly expressed in tumor or stem cells. We found S100A4 was a novel protein partner for heat shock protein 47 (HSP47) in deer antlerogenic periosteum cells (AP cells), indicating that S100A4 could bind with HSP47. S100A4 had both calcium-dependent and calcium-independent patterns (labeled as SCd and SCi, respectively) to execute different biological activities. Homology models of HSP47, SCd and SCi were constructed. HSP47:collagen model, HSP47:collagen I-V, HSP47:SCd and HSP47:SCi complexes were built using ZDOCK software. Together with free SCd and SCi, 200 ns molecular dynamic (MD) simulations were performed to analyze binding free energies and SCi/SCd conformational changes. The energetic results showed that SCi had the strongest affinity to HSP47, and followed by collagens. SCd had little interaction with HSP47. Decomposition energy results showed that collagen model interacted with HSP47 mainly though neutral amino acids. When SCi bound with HSP47, the majority of mediated amino acids were charged. These results indicated that SCi could compete with collagen on the binding site of HSP47. Root mean square fluctuation (RMSF) values and cross-correlation matrices of principal component analysis (PCA) were calculated to evaluate the SCi/SCd structural variation during MD simulation. Both HSP47 and Ca2+ could stabilize the conformation of SCi/SCd. The loops interacting with Ca2+s and linking the two EF-hand motifs were impacted particularly. The relative moving directions of α-helices in EF-hands were distinct by the binding effect of HSP47 and Ca2+. We found that SCi may regulate the differentiation of AP cells by disturbing the interaction between HSP47 and collagen. Communicated by Ramaswamy H. Sarma.
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Cuernos de Venado , Calcio/química , Proteínas del Choque Térmico HSP47/química , Proteína de Unión al Calcio S100A4/química , Células Madre , Animales , Cuernos de Venado/citología , CiervosRESUMEN
BACKGROUND: Deer antlers are the only known mammalian organ with vascularized cartilage that can completely regenerate. Antlers are of real significance as a model of mammalian stem cell-based regeneration with particular relevance to the fields of chondrogenesis, angiogenesis, and regenerative medicine. Recent research found that thymosin beta 10 (TMSB10) is highly expressed in the growth centers of growing antlers. The present study reports here the expression, functions, and molecular interactions of deer TMSB10. METHODS: The TMSB10 expression level in both tissue and cells in the antler growth center was measured. The effects of both exogenous (synthetic protein) and endogenous deer TMSB10 (lentivirus-based overexpression) on antlerogenic periosteal cells (APCs; nonactivated antler stem cells with no basal expression of TMSB10) and human umbilical vein endothelial cells (HUVECs; endothelial cells with no basal expression of TMSB10) were evaluated to determine whether TMSB10 functions on chondrogenesis and angiogenesis. Differences in deer and human TMSB10 in angiogenesis and molecular structure were determined using animal models and molecular dynamics simulation, respectively. The molecular mechanisms underlying deer TMSB10 in promoting angiogenesis were also explored. RESULTS: Deer TMSB10 was identified as a novel proangiogenic factor both in vitro and in vivo. Immunohistochemistry revealed that TMSB10 was widely expressed in the antler growth center in situ, with the highest expression in the reserve mesenchyme, precartilage, and transitional zones. Western blot analysis using deer cell lines further supports this result. Both exogenous and endogenous deer TMSB10 significantly decreased proliferation of APCs (P < 0.05), while increasing the proliferation of HUVECs (P < 0.05). Moreover, deer TMSB10 enhanced chondrogenesis in micromass cultures and nerve growth as assessed using a dorsal root ganglion model (P < 0.05). Deer TMSB10 was proangiogenic using models of chicken chorioallantoic membrane, tube formation, and aortic arch assay. At the molecular level, endogenous deer TMSB10 elevated the expression of vascular endothelial growth factor (VEGF), VEGF-B, VEGF-C, and VEGF-D, and VEGFR2 and VEGFR3 in HUVECs (P < 0.05). CONCLUSIONS: Deer TMSB10, in contrast to its human counterpart, was identified as a novel stimulating factor for angiogenesis, cartilage formation, and nerve growth, which is understandable given that deer antlers (as the arguably fastest mammalian growing tissue) may require this extra boost during a period of rapid growth and regeneration.
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Morfogénesis/fisiología , Regeneración/fisiología , Timosina/uso terapéutico , Animales , Cuernos de Venado , Ciervos , Timosina/farmacologíaRESUMEN
Developing an innovative "Test Paper" based on virgin nanoporous silicon (NPSi) which shows intense visible emission and excellent fluorescence stability. The visual fluorescence quenching "Test Paper" was highly selective and sensitive recognizing Cu2+ at µmol/L level. Within the concentration range of 5 × 10-7 ~50 × 10-7mol/L, the linear regression equation of IPL = 1226.3-13.6[CCu2+] (R = 0.99) was established for Cu2+ quantitative detection. And finally, Cu2+ fluorescence quenching mechanism of NPSi prober was proposed by studying the surface chemistry change of NPSi and metal ions immersed-NPSi using XPS characterization. The results indicate that SiHx species obviously contribute to the PL emission of NPSi, and the introduce of oxidization state and the nonradiative recombination center are responsible for the PL quenching. These results demonstrate how virgin NPSi wafer can serve as Cu2+ sensor. This work is of great significant to promote the development of simple instruments that could realize rapid, visible and real-time detection of various toxic metal ions.
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In order to understand the binding mechanism between Barrier-to-autointegration factor (BAF) and DNA, two DNA:BAF complexes with wild type (WT) BAF and its Gly25Glu point mutate type (MT) were generated by molecular docking on the basis of the crystal structures of BAF (PDB code: 2ODG, chain A) and DNA (PDB code: 2BZF, chain B and C). Then, molecular dynamics (MD) simulations were performed on the two docked structures, as well as BAF (WT) and BAF (MT). The results show that monomer BAF is more flexible than BAF in DNA:BAF complex, suggesting that DNA is effective to stabilize conformation of BAF, which is in good agreement with the experimental results. Besides, the mutated Glu25 in DNA:BAF (MT) can change the BAF conformation to some extent. With deeper investigation on the DNA:BAF structures, the hydrogen bonds are found to make great contribution to the interaction between DNA and BAF. The hydrogen bonds in DNA:BAF (MT) are fewer than those in DNA:BAF (WT), indicating that the Gly25Glu mutation in BAF has an important effect on the hydrogen bonds in the DNA:BAF complex. Besides, the binding free energy in DNA:BAF (MT) is also higher than that in DNA:BAF (WT). It results from the influence of Glu25 side chain on the orientation of Lys6 and Lys33 in the interface between DNA and BAF. The binding free energy of Lys72, another key residue, decreases a lot in DNA:BAF (MT) anomalously. The decreasing energy causes the destruction of hydrophobic pocket in the binding site between DNA and BAF (MT). Our results are helpful for further experimental investigations.
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Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Mutación Puntual , Sitios de Unión , ADN/química , Proteínas de Unión al ADN/química , Transferencia de Energía , Glutamina , Glicina , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas Nucleares/química , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Electricidad Estática , Relación Estructura-Actividad , Propiedades de SuperficieRESUMEN
The interaction between barrier-to-autointegration factor dimer (BAF2) and LEM domain of emerin (EmLEM) was studied by molecular simulation methods. Nonspecific fragment of double-strand DNA molecule was docked with each chain of BAF2 by ZDOCK program. The model of DNA2:BAF2:EmLEM was thus constructed. The mutant Gly25Glu of BAF2 was manually constructed to explore the detailed effect of the mutation on the binding of BAF2 and EmLEM. It has been experimentally suggested that point mutation Gly25Glu can disturb the binding between BAF2 and EmLEM. Then, molecular dynamics (MD) simulations were performed on DNA2:BAF2(WT):EmLEM and DNA2:BAF2(MT):EmLEM complexes. 30ns trajectories revealed that the trajectory fluctuations of MT complex are more violent than that of the WT complex. Further, the binding free energy analysis showed that the electronegative residues Asp57, Glu61 and Asp65 from chain A, glu36 from chain B of BAF2 mainly contribute to interact with EmLEM. Besides, a stable π-π stack between trp62 and phe39 from BAF2(WT) chain B is destroyed by Glu25 in BAF2(MT). As a result, trp62 forms an interaction with glu25, and phe39 converts to strengthen affinity to EmLEM. On the other hand, Trp62 from chain A also forms a strong interaction with MT Glu25. Thus, with the docking of DNA, BAF2(MT) has higher affinity with EmLEM than BAF2(WT).
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Based on the chain A structures of hemoglobin (PDB code: 1HDS, 1IBE, 1FAW, 3AT5), the three dimensional (3D) structure of natural velvet antler polypeptide (nVAP) was constructed by homology modeling and molecular dynamics (MD) method. The structural rationality was further checked by Profile-3D and Procheck, both of which confirmed that the 3D structure of nVAP was reasonable. The modeled structure indicates that the stable conformation of nVAP is composed of two α-helixes. The extracellular domains of transforming growth factor-ß1 receptor I (TßRI-ED) and II (TßRII-ED) were docked with nVAP, respectively. The results show that both of TßR-EDs have high affinity with nVAP which locates near the active center of TßRII-ED integrating with transforming growth factor-ß1 (TGF-ß1). Otherwise, nVAP can also insert near the "pre-helix extension" of TßRI-ED, which is the key domain to interact on TGF-ß1 and TßRII-ED. With the perturbation of nVAP, TßRI-ED can not be recruited by TGF-ß1:TßRII-ED complex rigorously. The intracellular domain of TßRI (TßRI-ID) is not phosphorylated and activated by TßRII. This study shows that nVAP prefers tethering TßRI-ED which is more crucial in TGF-ß1:TßRII-ED:TßRI-ED complex. Thus nVAP can disturb the TGF-ß1 binding pattern by interacting on TßRs (TßRI and TßRII), further intercepting TGF-ß1 pathway downstream.