Effect of repeated heating and cooling cycles on the degree of conversion and microhardness of four resin composites.

OBJECTIVE: This study evaluated the effect of repeatedly heating and cooling four resin-based composites (RBCs) for up to six cycles. MATERIALS AND METHODS: Four commercial RBCs were heated to 68°C and cooled to room temperature for up to six cycles before photocuring at 30°C. Specimens spent a total of 0, 30, 60, 90, 120, 150 min, or 7 days at 68°C. The degree of conversion (DC) was measured at the bottom of the specimens immediately after photocuring. The Vickers microhardness was measured at the top and bottom of the RBC surfaces 24 h after photocuring. The data were analyzed using one-way analysis of variance, Dunnett's or Bonferroni post-hoc tests, and Spearman correlation analysis (α = 0.05). RESULTS: For two brands of RBC, the DC decreased at various time points; however, these decreases were small, and there was no correlation (negative or positive) between the number of heating cycles and the DC for any of the RBCs. Repeated heated and cooling resulted in small changes in the hardness (compared to the control) in both directions (Dunnett; p < 0.05). Two of the RBCs showed a significant, positive correlation between the number of heating cycles and their hardness at the bottom surface. CONCLUSION: Repeated heating, cooling, and then reheating the RBCs for up to 1 week had little overall effect on their DC and microhardness values. The 2 mm thick specimens of all four RBCs achieved a bottom: top hardness ratio exceeding 0.8 after a 20 s exposure to light from a commercial LED curing light CLINICAL SIGNIFICANCE: Six repeated dry heating and cooling cycles of up to 1 week in duration had little effect on the DC and the microhardness of four commercial resin-based composites.

Induction of Rhodobacter capsulatus Gene Transfer Agent Gene Expression Is a Bistable Stochastic Process Repressed by an Extracellular Calcium-Binding RTX Protein Homologue.

Bacteriophage-like gene transfer agents (GTAs) have been discovered in both of the prokaryotic branches of the three-domain phylogenetic tree of life. The production of a GTA (RcGTA) by the phototrophic alphaproteobacterium Rhodobacter capsulatus is regulated by quorum sensing and a phosphorelay homologous to systems in other species that control essential functions such as the initiation of chromosome replication and cell division. In wild-type strains, RcGTA is produced in <3% of cells in laboratory cultures. Mutants of R. capsulatus that exhibit greatly elevated production of RcGTA were created decades ago by chemical mutagenesis, but the nature and molecular consequences of the mutation were unknown. We show that the number of cells in a population that go on to express RcGTA genes is controlled by a stochastic process, in contrast to a genetic process. We used transposon mutagenesis along with a fluorescent protein reporter system and genome sequence data to identify a gene, rcc00280, that encodes an RTX family calcium-binding protein homologue. The Rc280 protein acts as an extracellular repressor of RcGTA gene expression by decreasing the percentage of cells that induce the production of RcGTA.IMPORTANCE GTAs catalyze horizontal gene transfer (HGT), which is important for genomic evolution because the majority of genes found in bacterial genomes have undergone HGT at some point in their evolution. Therefore, it is important to determine how the production of GTAs is regulated to understand the factors that modulate the frequency of gene transfer and thereby specify the tempo of evolution. This work describes a new type of genetic regulation in which an extracellular calcium-binding protein homologue represses the induction of the Rhodobacter capsulatus GTA, RcGTA.

Functional and Evolutionary Characterization of a Gene Transfer Agent's Multilocus "Genome".

Gene transfer agents (GTAs) are phage-like particles that can package and transfer a random piece of the producing cell's genome, but are unable to transfer all the genes required for their own production. As such, GTAs represent an evolutionary conundrum: are they selfish genetic elements propagating through an unknown mechanism, defective viruses, or viral structures "repurposed" by cells for gene exchange, as their name implies? In Rhodobacter capsulatus, production of the R. capsulatus GTA (RcGTA) particles is associated with a cluster of genes resembling a small prophage. Utilizing transcriptomic, genetic and biochemical approaches, we report that the RcGTA "genome" consists of at least 24 genes distributed across five distinct loci. We demonstrate that, of these additional loci, two are involved in cell recognition and binding and one in the production and maturation of RcGTA particles. The five RcGTA "genome" loci are widespread within Rhodobacterales, but not all loci have the same evolutionary histories. Specifically, two of the loci have been subject to frequent, probably virus-mediated, gene transfer events. We argue that it is unlikely that RcGTA is a selfish genetic element. Instead, our findings are compatible with the scenario that RcGTA is a virus-derived element maintained by the producing organism due to a selective advantage of within-population gene exchange. The modularity of the RcGTA "genome" is presumably a result of selection on the host organism to retain GTA functionality.

Genome-wide identification and characterization of small RNAs in Rhodobacter capsulatus and identification of small RNAs affected by loss of the response regulator CtrA.

Small non-coding RNAs (sRNAs) are involved in the control of numerous cellular processes through various regulatory mechanisms, and in the past decade many studies have identified sRNAs in a multitude of bacterial species using RNA sequencing (RNA-seq). Here, we present the first genome-wide analysis of sRNA sequencing data in Rhodobacter capsulatus, a purple nonsulfur photosynthetic alphaproteobacterium. Using a recently developed bioinformatics approach, sRNA-Detect, we detected 422 putative sRNAs from R. capsulatus RNA-seq data. Based on their sequence similarity to sRNAs in a sRNA collection, consisting of published putative sRNAs from 23 additional bacterial species, and RNA databases, the sequences of 124 putative sRNAs were conserved in at least one other bacterial species; and, 19 putative sRNAs were assigned a predicted function. We bioinformatically characterized all putative sRNAs and applied machine learning approaches to calculate the probability of a nucleotide sequence to be a bona fide sRNA. The resulting quantitative model was able to correctly classify 95.2% of sequences in a validation set. We found that putative cis-targets for antisense and partially overlapping sRNAs were enriched with protein-coding genes involved in primary metabolic processes, photosynthesis, compound binding, and with genes forming part of macromolecular complexes. We performed differential expression analysis to compare the wild type strain to a mutant lacking the response regulator CtrA, an important regulator of gene expression in R. capsulatus, and identified 18 putative sRNAs with differing levels in the two strains. Finally, we validated the existence and expression patterns of four novel sRNAs by Northern blot analysis.

Mimicry, deception and competition: The life of competing endogenous RNAs.

Since their discovery, small regulatory RNAs (sRNAs) were thought to be regulated exclusively at the transcriptional level. However, accumulating data from recent reports indicate that posttranscriptional signals can also modulate the function and stability of sRNAs. One of these posttranscriptional signals are competing endogenous RNAs (ceRNAs). Commonly called RNA sponges, ceRNAs can effectively sequester sRNAs and prevent them from binding their cognate target messenger RNAs (mRNAs). Subsequently, they prevent sRNA-dependent regulation of translation and stability of mRNA targets. While some ceRNAs seem to be expressed constitutively, others are intricately regulated according to environmental conditions. The outcome of ceRNA binding to a sRNA reaches beyond simple sequestration. Various effects observed on sRNA functions extend from reducing transcriptional noise to promote RNA turnover. Here, we present a historical perspective of the discovery of ceRNAs in eukaryotic organisms and mainly focus on the synthesis and function of select, well-described, ceRNAs in bacterial cells. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Small Molecule-RNA Interactions Translation > Translation Regulation RNA Turnover and Surveillance > Regulation of RNA Stability.

CD24 induces changes to the surface receptors of B cell microvesicles with variable effects on their RNA and protein cargo.

The CD24 cell surface receptor promotes apoptosis in developing B cells, and we recently found that it induces B cells to release plasma membrane-derived, CD24-bearing microvesicles (MVs). Here we have performed a systematic characterization of B cell MVs released from WEHI-231 B lymphoma cells in response to CD24 stimulation. We found that B cells constitutively release MVs of approximately 120 nm, and that CD24 induces an increase in phosphatidylserine-positive MV release. RNA cargo is predominantly comprised of 5S rRNA, regardless of stimulation; however, CD24 causes a decrease in the incorporation of protein coding transcripts. The MV proteome is enriched with mitochondrial and metabolism-related proteins after CD24 stimulation; however, these changes were variable and could not be fully validated by Western blotting. CD24-bearing MVs carry Siglec-2, CD63, IgM, and, unexpectedly, Ter119, but not Siglec-G or MHC-II despite their presence on the cell surface. CD24 stimulation also induces changes in CD63 and IgM expression on MVs that is not mirrored by the changes in cell surface expression. Overall, the composition of these MVs suggests that they may be involved in releasing mitochondrial components in response to pro-apoptotic stress with changes to the surface receptors potentially altering the cell type(s) that interact with the MVs.