[show abstract][hide abstract] ABSTRACT: The rice U-box/ARM E3 ubiquitin ligase SPL11 negatively regulates programmed cell death (PCD) and disease resistance, and controls flowering time through interacting with the novel RNA/DNA binding KH domain protein SPIN1. Overexpression of Spin1 causes late flowering in transgenic rice under short-day (SD) and long-day (LD) conditions. In this study, we characterized the function of the RNA-binding and SPIN1-interacting 1 (RBS1) protein in flowering time regulation. Rbs1was identified in a yeast-two-hybrid screen using the full-length Spin1 cDNA as a bait and encodes an RNA binding protein with three RNA recognition motifs. The protein binds RNA in vitro and interacts with SPIN1 in the nucleus. Rbs1 overexpression causes delayed flowering under SD and LD conditions in rice. Expression analyses of flowering marker genes show that Rbs1 overexpression represses the expression of Hd3a under SD and LD conditions. Rbs1 is upregulated in both Spin1 overexpression plants and in the spl11 mutant. Interestingly, Spin1 expression is increased but Spl11 expression is repressed in the Rbs1 overexpression plants. Western blot analysis revealed that the SPIN1 protein level is increased in the Rbs1 overexpression plants and that the RBS1 protein level is also up-regulated in the Spin1 overexpression plants. These results suggest that RBS1 is a new negative regulator of flowering time that itself is positively regulated by SPIN1 but negatively regulated by SPL11 in rice.
PLoS ONE 01/2014; 9(1):e87258. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: In the oral and craniofacial complex, tooth loss is the most commonly acquired disfiguring injury. Among the most formidable challenges of reconstructing tooth-supporting osseous defects in the oral cavity is the regeneration of functional multi-tissue complexes involving bone, ligament, and tooth cementum. Furthermore, periodontal multi-tissue engineering with spatiotemporal orientation of the periodontal ligament (PDL) remains the most challenging obstacle for restoration of physiological loading and homeostasis. We report on the ability of a hybrid computer-designed scaffold - developed utilizing computed tomography - to predictably facilitate the regeneration and integration of dental supporting tissues. Here we provide the protocol for rapid prototyping, manufacture, surgical implantation, and evaluation of dual-architecture scaffolds for controlling fiber orientation and facilitating morphogenesis of bone-ligament complexes. In contrast to conventional single-system methods of fibrous tissue formation, our protocol supports rigorous control of multi-compartmental scaffold architecture using computational scaffold design and manufacturing by 3D printing, as well as the evaluation of newly-regenerated tissue physiology for clinical implementation.
Tissue Engineering Part C Methods 11/2013; · 4.64 Impact Factor
[show abstract][hide abstract] ABSTRACT: Background: Stem cell therapy offers potential in the regeneration of craniofacial bone defects however, has it not been studied clinically. Tissue repair cells (TRCs) isolated from bone marrow represent a mixed stem and progenitor population enriched in CD90 and CD14 positive cells. In this Phase I/II, randomized, controlled, feasibility trial, we investigated TRC cell therapy to reconstruct localized craniofacial bone defects. Methods: 24 patients requiring localized reconstruction of jawbone defects participated in this longitudinal trial. For regenerative therapy, patients were randomized to receive either guided bone regeneration (GBR) or TRC transplantation. 6 or 12 weeks following treatment, clinical and radiographic assessments of bone repair were performed. Bone biopsies were harvested and underwent quantitative micro-computed tomographic (μCT) and bone histomorphometric analyses. Oral implants were installed, subsequently restored and functionally loaded with tooth restorations. Reconstructed sites were assessed for one year following therapy. Results: No study-related, serious adverse events were reported. Following therapy, clinical, radiographic, tomographic, and histological measures demonstrated that TRC therapy accelerated alveolar bone regeneration compared to GBR therapy. Additionally, TRC treatment significantly reduced the need for secondary bone grafting at the time of oral implant placement with a 5-fold decrease in implant bony dehiscence exposure (residual bone defects) as compared to GBR-treated sites (p < 0.01). Conclusions: Transplantation of TRCs for treatment of alveolar bone defects appears safe and accelerates bone regeneration, enabling jawbone reconstruction with oral implants. The results from this trial support expanded studies of TRC therapy in the treatment of craniofacial deformities (ClinicalTrials.gov number CT00755911).
[show abstract][hide abstract] ABSTRACT: Regeneration of bone-ligament complexes destroyed due to disease or injury is a clinical challenge due to complex topologies and tissue integration required for functional restoration. Attempts to reconstruct soft-hard tissue interfaces have met with limited clinical success. In this investigation, we manufactured biomimetic fiber-guiding scaffolds using solid free-form fabrication methods that custom fit complex anatomical defects to guide functionally-oriented ligamentous fibers in vivo. Compared to traditional, amorphous or random-porous polymeric scaffolds, the use of perpendicularly oriented micro-channels provides better guidance for cellular processes anchoring ligaments between two distinct mineralized structures. These structures withstood biomechanical loading to restore large osseous defects. Cell transplantation using hybrid scaffolding constructs with guidance channels resulted in predictable oriented fiber architecture, greater control of tissue infiltration, and better organization of ligament interface than random scaffold architectures. These findings demonstrate that fiber-guiding scaffolds drive neogenesis of triphasic bone-ligament integration for a variety of clinical scenarios.
[show abstract][hide abstract] ABSTRACT: Ubiquitin-regulated protein degradation is a critical regulatory mechanism that controls a wide range of biological processes in plants. Here, we report that OsDIS1 (for Oryza sativa drought-induced SINA protein 1), a C3HC4 RING finger E3 ligase, is involved in drought-stress signal transduction in rice (O. sativa). The expression of OsDIS1 was up-regulated by drought treatment. In vitro ubiquitination assays showed that OsDIS1 possessed E3 ubiquitin ligase activity and that the conserved region of the RING finger was required for the activity. Transient expression assays in Nicotiana benthamiana leaves and rice protoplasts indicated that OsDIS1 was localized predominantly in the nucleus. Overexpression of OsDIS1 reduced drought tolerance in transgenic rice plants, while RNA interference silencing of OsDIS1 enhanced drought tolerance. Microarray analysis revealed that a large number of drought-responsive genes were induced or suppressed in the OsDIS1 overexpression plants under normal and drought conditions. Yeast two-hybrid screening showed that OsDIS1 interacted with OsNek6 (for O. sativa NIMA-related kinase 6), a tubulin complex-related serine/threonine protein kinase. Coexpression assays in N. benthamiana leaves indicated that OsNek6 was degraded by OsDIS1 via the 26S proteasome-dependent pathway and that this degradation was abolished by the OsDIS1(H71Y) mutation, which is essential for its E3 ligase activity. Together, these results demonstrate that OsDIS1 plays a negative role in drought stress tolerance through transcriptional regulation of diverse stress-related genes and possibly through posttranslational regulation of OsNek6 in rice.
[show abstract][hide abstract] ABSTRACT: Inflammatory periodontal diseases are a leading cause of tooth loss and are linked to multiple systemic conditions, such as cardiovascular disease and stroke. Reconstruction of the support and function of affected tooth-supporting tissues represents an important therapeutic endpoint for periodontal regenerative medicine. An improved understanding of periodontal biology coupled with current advances in scaffolding matrices has introduced novel treatments that use cell and gene therapy to enhance periodontal tissue reconstruction and its biomechanical integration. Cell and gene delivery technologies have the potential to overcome limitations associated with existing periodontal therapies, and may provide a new direction in sustainable inflammation control and more predictable tissue regeneration of supporting alveolar bone, periodontal ligament, and cementum. This review provides clinicians with the current status of these early-stage and emerging cell- and gene-based therapeutics in periodontal regenerative medicine, and introduces their future application in clinical periodontal treatment. The paper concludes with prospects on the application of cell and gene tissue engineering technologies for reconstructive periodontology.
Journal of Periodontology 02/2011; 82(9):1223-37. · 2.40 Impact Factor
[show abstract][hide abstract] ABSTRACT: The aims of this article were to perform a detailed evaluation of the healing of extraction sockets covered with a resorbable collagen membrane 12 weeks following exodontia and to determine if this device had ossifying properties. Ten consecutive subjects in need of extraction of maxillary premolars were recruited. Each subject had a hopeless maxillary premolar extracted with minimal trauma. Sockets were then covered with a collagen barrier membrane alone. At 12 weeks, reentry surgery was performed, clinical measurements were repeated, and bone core biopsies were obtained prior to dental implant placement for histologic and microcomputed tomography (micro-CT) analysis. Study sites showed mean bone regeneration horizontally of 7.7 mm (buccopalatally) and 4.6 mm (mesiodistally). Vertical bone repair showed a mean gain of 10.9 mm. Subtraction radiography showed a mean apical shift of the crestal bone at the center of the socket of 2.1 mm (range, 0.7 to 4.3 mm). Micro-CT and histology revealed formation of well-mineralized tissue at 12 weeks, with a mean percentage of vital bone of 45.87% ± 12.35%. No signs of membrane ossification were observed. A detailed analysis of tissue neogenesis in extraction sites protected by this barrier membrane has demonstrated that adequate bone formation for implant placement occurs as early as 12 weeks following exodontia, with minimal changes in alveolar ridge dimensions. No evidence of membrane ossification was observed.
The International journal of periodontics & restorative dentistry 01/2011; 31(5):457-69. · 1.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: A major clinical challenge in the reconstruction of large oral and craniofacial defects is the neogenesis of osseous and ligamentous interfacial structures. Currently, oral regenerative medicine strategies are unpredictable for repair of tooth-supporting tissues destroyed as a consequence of trauma, chronic infection or surgical resection. Here, we demonstrate multi-scale computational design and fabrication of composite hybrid polymeric scaffolds for targeted cell transplantation of genetically modified human cells for the formation of human tooth dentin-ligament-bone complexes in vivo. The newly-formed tissues demonstrate the interfacial generation of parallel- and obliquely-oriented fibers that grow and traverse within the polycaprolactone (PCL)-poly(glycolic acid) (PGA) designed constructs forming tooth cementum-like tissue, ligament, and bone structures. This approach offers potential for the clinical implementation of customized periodontal scaffolds that may enable regeneration of multi-tissue interfaces required for oral, dental and craniofacial engineering applications.
[show abstract][hide abstract] ABSTRACT: For the determination of key factors or devices that promote periodontal regeneration, preclinical investigations using in vivo animal models are critical for evaluating the biological responses before human clinical trial testing. In this chapter, we provide an overview on the commonly used preclinical animals for the study of reconstructive procedures to promote bone and soft tissue repair of tooth-supporting periodontal defects. Steps are provided on the animal management for evaluation of outcome measures using descriptive histology, histomorphometry, three-dimensional imaging, and safety assessments. The use of these key measures of periodontal regeneration should aid investigators in the selection of appropriate surrogate endpoints to be utilized in the clinical arena, which are not practical or ethical in humans. These methods will prepare investigators and assist them in identifying endpoints that can then be adapted to human clinical trial planning.
Methods in molecular biology (Clifton, N.J.) 01/2010; 666:285-307.
[show abstract][hide abstract] ABSTRACT: Rice blast disease, caused by the fungus Magnaporthe oryzae, is a leading constraint to rice production and is a serious threat to food security worldwide. To elucidate the function
of effector proteins from M. oryzae in pathogenesis and interaction with the host, we have performed RL-SAGE and MPSS approaches to study the gene expression
profiles of M. oryzae during the interaction. The RL-SAGE and MPSS analyses identified 3,441 and 3,004 annotated M. oryzae genes from blast infected rice leaf tissues, respectively. Among them, 217 genes encoding putative secreted proteins, which
may play important roles as effectors, were identified. We developed a highly efficient transient protoplast system for gene
functional analysis in rice. We present here the detailed procedure of the rice protoplast transient expression system and
show the examples of using the system for functional analysis of putative effectors from M. oryzae. The combination of RL-SAGE/MPSS genomic profiling approaches with a protoplast functional assay system has provided an efficient
approach for large-scale isolation and analysis of effectors from M. oryzae.
[show abstract][hide abstract] ABSTRACT: Ubiquitin ligases play a central role in determining the specificity of the ubiquitination system by selecting a myriad of appropriate candidate proteins for modification. The U-box is a recently identified, ubiquitin ligase activity-related protein domain that shows greater presence in plants than in other organisms. In this study, we identified 77 putative U-box proteins from the rice genome using a battery of whole genome analysis algorithms. Most of the U-box protein genes are expressed, as supported by the identification of their corresponding expressed sequence tags (ESTs), full-length cDNAs, or massively parallel signature sequencing (MPSS) tags. Using the same algorithms, we identified 61 U-box proteins from the Arabidopsis genome. The rice and Arabidopsis U-box proteins were classified into nine major classes based on their domain compositions. Comparison between rice and Arabidopsis U-box proteins indicates that the majority of rice and Arabidopsis U-box proteins have the same domain organizations. The inferred phylogeny established the homology between rice and Arabidopsis U-box/ARM proteins. Cell death assay using the rice protoplast system suggests that one rice U-box gene, OsPUB51, might act as a negative regulator of cell death signaling. In addition, the selected U-box proteins were found to be functional E3 ubiquitin ligases. The identification and analysis of rice U-box proteins hereby at the genomic level will help functionally characterize this class of E3 ubiquitin ligase in the future.
[show abstract][hide abstract] ABSTRACT: The RANKL-OPG axis is a key regulator of osteoclastogenesis and bone turnover activity. Its contribution to bone resorption under altered mechanical states, however, has not been fully elucidated. Here we examined the role of OPG in regulating mechanically induced bone modeling in a rat model of orthodontic tooth movement.
The maxillary first molars of male Sprague-Dawley rats were moved mesially using a calibrated nickel-titanium spring attached to the maxillary incisor teeth. Two different doses (0.5 mg/kg, 5.0 mg/kg) of a recombinant fusion protein (OPG-Fc), were injected twice weekly mesial to the first molars. Tooth movement was measured using stone casts that were scanned and magnified. Changes in bone quantity were measured using micro-computed tomography and histomorphometric analysis was used to quantify osteoclasts and volumetric parameters. Finally, circulating levels of TRAP-5b (a bone resorption marker) was measured using enzyme-linked immunosorbent assay.
The 5.0 mg/kg OPG-Fc dose showed a potent reduction in mesial molar movement and osteoclast numbers compared to controls (p<0.01). The molar movement was inhibited by 45.7%, 70.6%, and 78.7% compared to controls at days 7, 14, and 21 respectively, with the high dose of OPG. The 0.5 mg dose also significantly (p<0.05) inhibited molar movement at days 7 (43.8%) and 14 (31.8%). While incisor retraction was also decreased by OPG-Fc, the ratio of incisor to molar tooth movement was markedly better in the high-dose OPG group (5.2:1, p<0.001) compared to the control group (2.3:1) and the low-dose OPG group (2.0:1).
Local delivery of OPG-Fc inhibits osteoclastogenesis and tooth movement at targeted dental sites.
[show abstract][hide abstract] ABSTRACT: Prevention of alveolar bone destruction is a clinical challenge in periodontal disease treatment. The receptor activator of nuclear factor-kappa B ligand (RANKL) inhibitor osteoprotegerin (OPG) inhibits osteoclastogenesis and suppresses bone resorption.
To study the effects of RANKL inhibition on alveolar bone loss, an experimental ligature-induced model of periodontitis was used. A total of 32 rats were administered human OPG-Fc fusion protein (10 mg/kg) or vehicle by subcutaneous delivery twice weekly for 6 weeks. Negative or positive controls received no treatment or disease through vehicle delivery, respectively. Biopsies were harvested after 3 and 6 weeks, and mandibulae were evaluated by microcomputed tomography (microCT) and histology. Serum levels of human OPG-Fc and tartrate-resistant acid phosphatase-5b (TRAP-5b) were measured throughout the study by enzyme-linked immunosorbent assay (ELISA). Statistical analyses included analysis of variance (ANOVA) and Tukey tests.
Human OPG-Fc was detected in the sera of OPG-Fc-treated animals by 3 days and throughout the study. Serum TRAP-5b was sharply decreased by OPG-Fc treatment soon after OPG-Fc delivery and remained low for the observation period. Significant preservation of alveolar bone volume was observed among OPG-Fc-treated animals compared to the controls at weeks 3 and 6 (P <0.05). Descriptive histology revealed that OPG-Fc significantly suppressed osteoclast surface area at the alveolar crest.
Systemic delivery of OPG-Fc inhibits alveolar bone resorption in experimental periodontitis, suggesting that RANKL inhibition may represent an important therapeutic strategy for the prevention of progressive alveolar bone loss.
Journal of Periodontology 07/2007; 78(7):1300-8. · 2.40 Impact Factor
[show abstract][hide abstract] ABSTRACT: Micro-computed tomography (micro-CT) offers significant potential for identifying mineralized structures. However, three-dimensional (3-D) micro-CT of alveolar bone has not been adapted readily for quantification. Moreover, conventional methods are not highly sensitive for analyzing bone loss or bone gain following periodontal disease or reconstructive therapy. The objective of this investigation was to develop a micro-CT methodology for quantifying tooth-supporting alveolar bone in 3-D following experimental preclinical situations of periodontitis or reconstructive therapy.
Experimental in vivo bone loss or regeneration situations were developed to validate the micro-CT imaging techniques. Twenty mature Sprague-Dawley rats were divided into two groups: bone loss (Porphyromonas gingivalis lipopolysaccharide-mediated bone resorption) and regenerative therapy. Micro-CT and software digitized specimens were reconstructed three-dimensionally for linear and volumetric parameter assessment of alveolar bone (linear bone height, bone volume, bone volume fraction, bone mineral content, and bone mineral density). Intra- and interexaminer reproducibility and reliability were compared for methodology validation.
The results demonstrated high examiner reproducibility for linear and volumetric parameters with high intraclass correlation coefficient (ICC) and coefficient of variation (CV). The ICC showed that the methodology was highly reliable and reproducible (ICC >0.99; 95% confidence interval, 0.937 to 1.000; CV <1.5%), suggesting that 3-D measurements may provide better alveolar bone analysis than conventional 2-D methods.
The developed methods allow for highly accurate and reproducible static measurements of tooth-supporting alveolar bone following preclinical situations of bone destruction or regeneration. Future investigations should focus on using in vivo micro-CT imaging for real-time assessments of alveolar bone changes.
Journal of Periodontology 02/2007; 78(2):273-81. · 2.40 Impact Factor