An Investigation of the Dimensional Stability of Dental Alginates

Dimensional stability was defined by Nicholls (1977) as “the ability (of a material) to maintain accuracy over time”, and the result of loss of accuracy, “distortion”, as “the relative movement of a single point, or group of points, away from some originally specified reference position such that permanent deformation is apparent”. Maintaining dimensional stability of dental impression materials is vital if the impression cannot be cast (in stone) soon after removal from the mouth. Dental irreversible hydrocolloid (alginate) is a major dental impression material used worldwide in many clinical procedures. However, alginate is dimensionally unstable and changes its dimensions (suffers “distortion”) after removal from the mouth. With storage times of more than ten minutes, alginate begins to distort, and after one to three hours (depending on the product and storage conditions) cannot be used for many clinical purposes, especially fixed prosthodontics such as crowns and bridges (Hampson 1955, Skinner & Hoblit 1956, Wilson & Smith 1963, Rudd et al. 1969, Miller 1975, Inohara 1977, Schoen et al. 1978, Coleman et al. 1979, Linke et al. 1985, Habu et al. 1986, Peutfeldt & Asmussen 1989, Mathilde & Peters 1992, Khan & Aziz Sahu 1995, Eriksson et al. 1998, Schleier et al. 2001, and Donovan & Chee 2004). This loss of accuracy, due to dimensional instability, manifests as a time-dependent distortion of the poured stone cast, and thus any prosthesis fabricated will not fit in the mouth. With the introduction of the more stable elastomers in the 1950s (Stackhouse 1970, Glenner 1997, Brown 2003) that could be stored for days if necessary, without loss of accuracy, the alginates fell out of favour for fixed prosthodontics. Recently, there has been a resurgence of interest in alginate for use in dental procedures where dimensional stability is critical (Peutzfeldt and Asmussen 1989, Eriksson et al. 1998). This in part is due to the favourable properties of alginate not found in the elastomers. Of greatest significance is that alginate hydrocolloid is hydrophilic, whereas elastomers are hydrophobic (Phillips & Ito 1958, Glenner 2004). Thus, alginate materials are able to reproduce wet oral areas with greater precision and to produce a superior "fit" of, say, a gold casting produced by the Lost Wax technique (Skinner and Phillips 1982). A number of reports have been published which investigate newer alginate materials that are claimed to be more dimensionally stable than older formulations. Puetzfeldt and Asmussen (1989) found that a newer alginate , if stored at 100% relative humidity, retained accuracy over 24 hours that was equivalent to that of the elastomers. More recently, the manufacturer of another alginate has claimed equivalent dimensional stability to the elastomers for up to 100 hours, and, whilst this claim has not been reported on in the literature, the present thesis will show that, under favourable conditions of storage, the material maintained clinically useful accuracy for up to 100 hours. Another approach to improving the accuracy of alginate impressions has been to combine reversible hydrocolloid with alginate (the “Bilaminar” technique). Frederick and Caputo (1997) confirmed that the new agar reversible hydrocolloids are just as accurate (at the time of removal from the mouth) as the new elastomers. Mathilde et al. (1992) and Eriksson et al. (1998) have shown that several of the “bilaminar” impression techniques for fixed prosthodontics, where alginate is used as a tray material supporting a reversible hydrocolloid (agar) wash, are as accurate and dimensionally stable as elastomers for up to three hours. However, these studies are difficult to interpret due to lack of uniformity in the testing methods, and the fact that there is no regulatory standard available to measure dimensional stability for dental alginates. The International Standard (IS) for alginate impression materials (ISO 1563:1990E) contains no specification for dimensional stability, and thus places no requirement for manufacturers to state dimensional stability properties on their labels. In contrast, ISO 4823:1992(E) specifies the IS for elastomeric dental impression materials, and it does specify a requirement for dimensional stability (less than 1.5% distortion after 24 hours). Further, the IS sets a method for determination of dimensional stability. Briefly, this method (the Optical Method) uses a travelling optical microscope to measure the accuracy of the distance between score lines on an impression of a test grid, at various time periods. The American Dental Association Specification No. 19 for dental elastomeric impression materials is identical to the IS. There is currently no specific Australian Standard (AS) for the dimensional stability of any dental impression material. Overview of Experimental Methods A. The Optical Method The aim of Part A of this investigation was to: 1. Adapt the Optical Method of the IS for elastomers to be reproducible for dental alginates. This was achieved by using a perforated test tray (to simulate clinical conditions), and measuring the grid pattern on a dental stone button after casting the test impression, rather than direct measurement of the impression, as for the IS. 2. To measure and rank the dimensional stability of a number of locally available dental alginates. Measurements of the test stone buttons proved reproducible, and the results were different for each sample, allowing them to be ranked according to dimensional stability after 50 and 100 hours of storage. The results show that the traditional optical method for measuring dimensional stability, as specified in the IS for dental elastomers, can be adapted to measure the dimensional stability of dental alginates However, the Optical Method of measuring dimensional stability of dental alginates is cumbersome and time-consuming. It was hypothesised that dimensional stability of dental alginates could be measured more conveniently by finding a thermal property that is directly proportional to dimensional stability. This method could be useful for the rapid determination of relative performance, and allow comparison with a determined benchmark. B. The Thermal Method Recently, modern methods of Thermal Analysis, Thermal Gravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC) have been used to rapidly age various polymers, including food alginates (Chinachoti 1996), in order to measure thermal stability. This thesis shows that thermal stability is an indicator of dimensional stability. The aim of Part B of this investigation was therefore to adapt thermal analysis techniques to dental alginates, and develop a method to measure their thermal stability. These results were then compared with those for dimensional stability measured by the Optical Method to determine the relationship between thermal and dimensional stability for dental alginates. The results show that current thermal analysis methods of TGA and DSC can be adapted to measure relative dental alginate dimensional stability, and are both rapid and convenient. This study also provides evidence that commercial products differ as regards the property of dimensional stability, and can be ranked accordingly. C. Practical Application of the Methods The aim of part C of this thesis was to validate the methods (both optical and thermal) developed in this study by using them to investigate the effect of varying the water/powder ratio on the dimensional stability of dental alginates. It was shown that dimensional stability is affected by changes to the recommended water/powder ratio, that both the methods detected and measured the changes, and that the results were proportional, in that any percentage change detected by the optical method, was mirrored by the thermal method, confirming that the more convenient thermal methods can be used to measure dimensional stability

Molecular characterization of Sin3 PAH-domain interactor specificity and identification of PAH partners

Sin3 is the central component of a multisubunit co-repressor complex. A number of DNA-binding proteins are targeted by the Sin3 complex to chromatin through association with its paired amphipathic helix (PAH) domains. Here, we performed a yeast two-hybrid screening using a peptide aptamer library and identified peptides that interact with either PAH1 or PAH2. Analysis of PAH2 interacting peptides uncovered motifs similar to previously characterized PAH2 interacting proteins, Mad, Ume6 and kruppel-like members, while analysis of PAH1 interacting peptides revealed an LXXLL motif. In addition, a tandem affinity purification (TAP)-tagging approach of Sin3b resulted in the isolation of known and novel interactors amongst which neural retina leucine (NRL) zipper. Strikingly, one of the identified PAH2 interacting peptide showed strong resemblance to the NRL region amino acids 125–150. Direct association between PAH2 and NRL was shown and NRL(125–150) mediated transcriptional repression in reporter assays. Finally, we reveal that PAH1 and PAH2 amino acids 7, 14 and 39 shown previously to be important for Mad–PAH2 interaction, also play an important role in the specificity of interaction between PAH1, PAH2 and identified aptamers. Our results provide novel insights into the molecular determinant of the specificity of PAH1 and PAH2 for their interacting partners

DNA methylation immediately adjacent to active histone marking does not silence transcription

Active promoters generally contain histone H3/H4 hyperacetylation and tri-methylation at H3 lysine 4, whereas repressed promoters are associated with DNA methylation. Here we show that the repressed erythroid-specific carbonic anhydrase II (CAII) promoter has active histone modifications localized around the transcription start, while high levels of CpG methylation are present directly upstream from these active marks. Despite the presence of active histone modifications, the repressed promoter requires hormone-induced activation for efficient preinitiation complex assembly. Transient and positional changes in histone H3/H4 acetylation and local changes in nucleosome density are evident during activation, but the bipartite epigenetic code is stably maintained. Our results suggest that active histone modifications may prevent spreading of CpG methylation towards the promoter and show that repressive DNA methylation immediately adjacent to a promoter does not necessarily repress transcription

Role of p53 Serine 46 in p53 Target Gene Regulation

The tumor suppressor p53 plays a crucial role in cellular growth control inducing a plethora of different response pathways. The molecular mechanisms that discriminate between the distinct p53-responses have remained largely elusive. Here, we have analyzed the p53-regulated pathways induced by Actinomycin D and Etoposide treatment resulting in more growth arrested versus apoptotic cells respectively. We found that the genome-wide p53 DNA-binding patterns are almost identical upon both treatments notwithstanding transcriptional differences that we observed in global transcriptome analysis. To assess the role of post-translational modifications in target gene choice and activation we investigated the genome-wide level of phosphorylation of Serine 46 of p53 bound to DNA (p53-pS46) and of Serine 15 (p53-pS15). Interestingly, the extent of S46 phosphorylation of p53 bound to DNA is considerably higher in cells directed towards apoptosis while the degree of phosphorylation at S15 remains highly similar. Moreover, our data suggest that following different chemotherapeutical treatments, the amount of chromatin-associated p53 phosphorylated at S46 but not at pS15 is higher on certain apoptosis related target genes. Our data provide evidence that cell fate decisions are not made primarily on the level of general p53 DNA-binding and that post-translationally modified p53 can have distinct DNA-binding characteristics

Controlled Assembly of Macromolecular β-Sheet Fibrils

Construction of functional molecular devices by directed assembly processes is one of the main challenges in the field of nanotechnology. Many approaches to this challenge use biological assembly as a source of inspiration for the build up of new materials with controlled organization at the nanoscale. In particular, the self-assembly properties of β-sheet peptides have been used in the design of supramolecular materials, such as tapes, nanotubes, and fibrils

A SILAC-Based Screen for Methyl-CpG Binding Proteins Identifies RBP-J as a DNA Methylation and Sequence-Specific Binding Protein

Contains fulltext : 91489.pdf (publisher's version ) (Open Access)10 p

Genome-wide modeling of transcription kinetics reveals patterns of RNA production delays

Genes with similar transcriptional activation kinetics can display very different temporal mRNA profiles because of differences in transcription time, degradation rate, and RNA-processing kinetics. Recent studies have shown that a splicing-associated RNA production delay can be significant. To investigate this issue more generally, it is useful to develop methods applicable to genome-wide datasets. We introduce a joint model of transcriptional activation and mRNA accumulation that can be used for inference of transcription rate, RNA production delay, and degradation rate given data from high-throughput sequencing time course experiments. We combine a mechanistic differential equation model with a nonparametric statistical modeling approach allowing us to capture a broad range of activation kinetics, and we use Bayesian parameter estimation to quantify the uncertainty in estimates of the kinetic parameters. We apply the model to data from estrogen receptor alpha activation in the MCF-7 breast cancer cell line. We use RNA polymerase II ChIP-Seq time course data to characterize transcriptional activation and mRNA-Seq time course data to quantify mature transcripts. We find that 11% of genes with a good signal in the data display a delay of more than 20 min between completing transcription and mature mRNA production. The genes displaying these long delays are significantly more likely to be short. We also find a statistical association between high delay and late intron retention in pre-mRNA data, indicating significant splicing-associated production delays in many genes.Peer reviewe

ChIP-Chip Designs to Interrogate the Genome of Xenopus Embryos for Transcription Factor Binding and Epigenetic Regulation

Chromatin immunoprecipitation combined with genome tile path microarrays or deep sequencing can be used to study genome-wide epigenetic profiles and the transcription factor binding repertoire. Although well studied in a variety of cell lines, these genome-wide profiles have so far been little explored in vertebrate embryos. genome. In particular, a whole-genome microarray design was used to identify active promoters by close proximity to histone H3 lysine 4 trimethylation. A second microarray design features these experimentally derived promoter regions in addition to currently annotated 5′ ends of genes. These regions truly represent promoters as shown by binding of TBP, a key transcription initiation factor. embryos

An RNA-Seq Strategy to Detect the Complete Coding and Non-Coding Transcriptome Including Full-Length Imprinted Macro ncRNAs

Imprinted macro non-protein-coding (nc) RNAs are cis-repressor transcripts that silence multiple genes in at least three imprinted gene clusters in the mouse genome. Similar macro or long ncRNAs are abundant in the mammalian genome. Here we present the full coding and non-coding transcriptome of two mouse tissues: differentiated ES cells and fetal head using an optimized RNA-Seq strategy. The data produced is highly reproducible in different sequencing locations and is able to detect the full length of imprinted macro ncRNAs such as Airn and Kcnq1ot1, whose length ranges between 80–118 kb. Transcripts show a more uniform read coverage when RNA is fragmented with RNA hydrolysis compared with cDNA fragmentation by shearing. Irrespective of the fragmentation method, all coding and non-coding transcripts longer than 8 kb show a gradual loss of sequencing tags towards the 3′ end. Comparisons to published RNA-Seq datasets show that the strategy presented here is more efficient in detecting known functional imprinted macro ncRNAs and also indicate that standardization of RNA preparation protocols would increase the comparability of the transcriptome between different RNA-Seq datasets

Dynamic CpG methylation delineates subregions within super-enhancers selectively decommissioned at the exit from naive pluripotency.

Clusters of enhancers, referred as to super-enhancers (SEs), control the expression of cell identity genes. The organisation of these clusters, and how they are remodelled upon developmental transitions remain poorly understood. Here, we report the existence of two types of enhancer units within SEs typified by distinctive CpG methylation dynamics in embryonic stem cells (ESCs). We find that these units are either prone for decommissioning or remain constitutively active in epiblast stem cells (EpiSCs), as further established in the peri-implantation epiblast in vivo. Mechanistically, we show a pivotal role for ESRRB in regulating the activity of ESC-specific enhancer units and propose that the developmentally regulated silencing of ESRRB triggers the selective inactivation of these units within SEs. Our study provides insights into the molecular events that follow the loss of ESRRB binding, and offers a mechanism by which the naive pluripotency transcriptional programme can be partially reset upon embryo implantation