Regeneration of Craniofacial Bone Induced by Periosteal Pumping.

A variety of surgical techniques and tissue engineering strategies utilizing osteogenic potential of the periosteum have been developed for the repair of extended bone deficiencies. The aim of the present study was to assess the impact of an alternating protocol of periosteal distraction osteogenesis (PDO) on bone regeneration in an intraoral model. Eight adult, male Beagle dogs were used for the study. Two distraction devices were placed on each side of the mandible. After a 7-day latency period, distraction devices in all animals were manipulated at the rate of 0.5 mm for a total of 8 days. The pumping protocol in two test groups proceeded twice daily by alternating activation with relaxation. In the periosteal pumping/distraction (PPDO) group, the distraction screws were activated two times (at 12 and 24 h) and then turned back (at 36 h), and in the periosteal pumping (PP) group repeatedly activated and turned back (at 12 h). In the PDO group, only activation was performed once daily (positive control). Devices were left inactivated in the negative control (NC) group. The samples were harvested after 8 weeks of consolidation period and investigated by micro-CT and histological analysis. New mature, lamellar bone was formed over the pristine bone in all groups. PPDO and PDO groups showed more new bone area (NBA) compared to the PP (p < 0.001 and p < 0.001, respectively) and to the NC group (p = 0.032 and p = 0.031, respectively). Furthermore, greater NBA was found in the PP group than the NC group (p = 0.006). PDO demonstrated higher relative connective tissue area than the PPDO group (p = 0.005) and lower relative new bone volume than the NC group (p = 0.025). Pumping protocol of periosteal distraction may successfully induce the endogenous regeneration of the mandibular bone in dogs. Impact Statement Repair of extended bone defects impose a significant challenge to oral and maxillofacial surgeons. In this article, a principle of distraction osteogenesis was applied to stimulate bone regeneration in the mandible. A periosteum-based regeneration approach may represent a valuable step toward creating a significant volume of hard and soft tissues, without need for autogenous bone harvesting or application of biomaterials.

Dynamic subcellular partitioning of the nucleolar transcription factor TIF-IA under ribotoxic stress.

TIF-IA is a basal transcription factor of RNA polymerase I (Pol I) that is a major target of the JNK2 signaling pathway in response to ribotoxic stress. Using advanced fluorescence microscopy and kinetic modeling we elucidated the subcellular localization of TIF-IA and its exchange dynamics between the nucleolus, nucleoplasm and cytoplasm upon ribotoxic stress. In steady state, the majority of (GFP-tagged) TIF-IA was in the cytoplasm and the nucleus, a minor portion (7%) localizing to the nucleoli. We observed a rapid shuttling of GFP-TIF-IA between the different cellular compartments with a mean residence time of approximately 130 s in the nucleus and only approximately 30 s in the nucleoli. The import rate from the cytoplasm to the nucleus was approximately 3-fold larger than the export rate, suggesting an importin/exportin-mediated transport rather than a passive diffusion. Upon ribotoxic stress, GFP-TIF-IA was released from the nucleoli with a half-time of approximately 24 min. Oxidative stress and inhibition of protein synthesis led to a relocation of GFP-TIF-IA with slower kinetics while osmotic stress had no effect. The observed relocation was much slower than the nucleo-cytoplasmic and nucleus-nucleolus exchange rates of GFP-TIF-IA, indicating a time-limiting step upstream of the JNK2 pathway. In support of this, time-course experiments on the activity of JNK2 revealed the activation of the JNK kinase as the rate-limiting step.

TAF12 recruits Gadd45a and the nucleotide excision repair complex to the promoter of rRNA genes leading to active DNA demethylation.

Many studies have detailed the repressive effects of DNA methylation on gene expression. However, the mechanisms that promote active demethylation are just beginning to emerge. Here, we show that methylation of the rDNA promoter is a dynamic and reversible process. Demethylation of rDNA is initiated by recruitment of Gadd45a (growth arrest and DNA damage inducible protein 45 alpha) to the rDNA promoter by TAF12, a TBP-associated factor that is contained in Pol I- and Pol II-specific TBP-TAF complexes. Once targeted to rDNA, Gadd45a triggers demethylation of promoter-proximal DNA by recruiting the nucleotide excision repair (NER) machinery to remove methylated cytosines. Knockdown of Gadd45a, XPA, XPG, XPF, or TAF12 or treatment with drugs that inhibit NER causes hypermethylation of rDNA, establishes heterochromatic histone marks, and impairs transcription. The results reveal a mechanism that recruits the DNA repair machinery to the promoter of active genes, keeping them in a hypomethylated state.

Nuclear myosin I acts in concert with polymeric actin to drive RNA polymerase I transcription.

Actin is associated with all three nuclear RNA polymerases and acts in concert with nuclear myosin I (NM1) to drive transcription. Practically nothing is known regarding the state of actin and the functional interplay of actin and NM1 in transcription. Here we show that actin and NM1 act in concert to promote RNA polymerase I (Pol I) transcription. Drugs that prevent actin polymerization or inhibit myosin function inhibit Pol I transcription in vivo and in vitro. Mutants that stabilize the polymeric state actin are tightly associated with Pol I and activate transcription, whereas a polymerization-deficient mutant does not bind to Pol I and does not promote rDNA transcription. Consistent with nuclear actin and myosin synergizing in transcription activation, NM1 mutants that lack specific functions, such as binding to ATP, actin, or calmodulin, are incapable of associating with Pol I and rDNA. The results show that actin polymerization and the motor function of NM1 are required for association with the Pol I transcription machinery and transcription activation. These observations provide insights into the cooperative action of actin and myosin in the nucleus and reveal an actomyosin-based mechanism in transcription.

Polo-like kinase-2 is required for centriole duplication in mammalian cells.

Centriole duplication initiates at the G1-to-S transition in mammalian cells and is completed during the S and G2 phases. The localization of a number of protein kinases to the centrosome has revealed the importance of protein phosphorylation in controlling the centriole duplication cycle. Here we show that the human Polo-like kinase 2 (Plk2) is activated near the G1-to-S transition of the cell cycle. Endogenous and overexpressed HA-Plk2 localize with centrosomes, and this interaction is independent of Plk2 kinase activity. In contrast, the kinase activity of Plk2 is required for centriole duplication. Overexpression of a kinase-deficient mutant under S-phase arrest blocks centriole duplication. Downregulation of endogenous Plk2 with small hairpin RNAs interferes with the ability to reduplicate centrioles. Furthermore, centrioles failed to duplicate during the cell cycle of human fibroblasts and U2OS cells after overexpression of a Plk2 dominant-negative mutant. These results show that Plk2 is a physiological centrosomal protein and that its kinase activity is likely to be required for centriole duplication near the G1-to-S phase transition.

Assessment of video tracking usability for training simulators.

OBJECTIVE: A simulator was developed to mimic commercial CAS systems in implementing most tasks required to carry out a surgical operation. As tracking systems are generally expensive components, an alternative solution based on low-cost video-based tracking was used. Video tracking accuracy was assessed to determine whether or not this kind of approach was suitable for use in the training domain. Ultimately, video-based tracking should enable sufficiently accurate registration between a bony model and its virtual 3D representation. MATERIALS AND METHODS: Video tracking was assessed using two types of camera. For each one, common accuracy tests were realized as a series of 10 trials at ranges of 0.5-1.0 m from the camera lens. The pointer used as a digitizer was equipped with tracked video markers. Three sizes of marker were evaluated to estimate the impact of marker size on accuracy. RESULTS: For the better of the two cameras tested, results were encouraging. Results are presented as rounded whole-number values in millimeters. The noise test gave accuracies of 2 mm for the 80-mm marker, 3 mm for the 60-mm marker and 5 mm for the 40-mm marker. Relative accuracies, as evaluated on a grid of equally spaced dots, were 4 mm with the 80-mm marker, 7 mm with the 60-mm marker and 12 mm with the 40-mm marker. A pivoting test around the pointer tip gave 3 mm of accuracy for the 80-mm marker, 5 mm for the 60-mm marker and 11 mm for the 40-mm marker. An additional pivoting test was completed on increasing the distance of the marker from the pointer tip, giving accuracies of 5 mm for the 80-mm marker, 6 mm for the 60-mm marker and 13 mm for the 40-mm marker. The registration test gave accuracies of 8 mm for the 80-mm marker, 9 mm for the 60-mm marker and 11 mm for the 40-mm marker. CONCLUSIONS: The video-based approach offers sufficient accuracy to achieve registration in the domain of CAS training.

Analyzing intracellular binding and diffusion with continuous fluorescence photobleaching.

Transport and binding of molecules to specific sites are necessary for the assembly and function of ordered supramolecular structures in cells. For analyzing these processes in vivo, we have developed a confocal fluorescence fluctuation microscope that allows both imaging of the spatial distribution of fluorescent molecules with confocal laser scanning microscopy and probing their mobility at specific positions in the cell with fluorescence correlation spectroscopy and continuous fluorescence photobleaching (CP). Because fluorescence correlation spectroscopy is restricted to rapidly diffusing particles and CP to slower processes, these two methods complement each other. For the analysis of binding-related contributions to mobility we have derived analytical expressions for the temporal behavior of CP curves from which the bound fraction and/or the dissociation rate or residence time at binding sites, respectively, can be obtained. In experiments, we investigated HeLa cells expressing different fluorescent proteins: Although enhanced green fluorescent protein (EGFP) shows high mobility, fusions of histone H2B with the yellow fluorescent protein are incorporated into chromatin, and these nuclei exhibit the presence of a stably bound and a freely diffusing species. Nonpermanent binding was found for mTTF-I, a transcription termination factor for RNA polymerase I, fused with EGFP. The cells show fluorescent nucleoli, and binding is transient. CP yields residence times for mTTF-I-EGFP of approximately 13 s.

A kinetic framework for a mammalian RNA polymerase in vivo.

We have analyzed the kinetics of assembly and elongation of the mammalian RNA polymerase I complex on endogenous ribosomal genes in the nuclei of living cells with the use of in vivo microscopy. We show that components of the RNA polymerase I machinery are brought to ribosomal genes as distinct subunits and that assembly occurs via metastable intermediates. With the use of computational modeling of imaging data, we have determined the in vivo elongation time of the polymerase, and measurements of recruitment and incorporation frequencies show that incorporation of components into the assembling polymerase is inefficient. Our data provide a kinetic and mechanistic framework for the function of a mammalian RNA polymerase in living cells.

Multiple interactions between RNA polymerase I, TIF-IA and TAF(I) subunits regulate preinitiation complex assembly at the ribosomal gene promoter.

In mammals, growth-dependent regulation of rRNA synthesis is brought about by the transcription initiation factor TIF-IA. TIF-IA is associated with a fraction of the TBP-containing factor TIF-IB/SL1 and the initiation-competent form of RNA polymerase I (Pol I). We investigated the mechanisms that down-regulate cellular pre-rRNA synthesis and demonstrate that nutrient starvation, density arrest and protein synthesis inhibitors inactivate TIF-IA and impair the association of TIF-IA with Pol I. Moreover, we used a panel of TIF-IA deletion mutants to map the domains that mediate the interaction of TIF-IA with Pol I and TIF-IB/SL1. We found that amino acids 512-609 interact with two subunits of Pol I, RPA43 and PAF67, whereas a short, conserved motif (LARAK, amino acids 411-415) is required for the association of TIF-IA with TAF(I)95 and TAF(I)68. The results uncover an interphase for essential protein-protein interactions that facilitate Pol I preinitiation complex formation.