Lydgate's Danse Macabre and the Trauma of the Hundred Years War.

This essay argues that the foundational traumatic lacuna behind John Lydgate's Danse Macabre is the social agon between those who wage the Hundred Years War and those who fight in it. Drawing from the insights of trauma theory to discuss the poem's form, the essay uncovers Lydgate's persistent concern with the damage caused by the war and the concomitant political unrest it causes. It argues further that Lydgate theorized this agon using the emergent genre of tragedy, which is beginning to be practiced anew in late-medieval England. Tragic discourse is riven by concerns about the efficacy of human action and the radical contingency of fortune, creating a crisis of agency that can be used as a form of political critique. Ultimately, Lydgate blends the genre of tragedy with the mirror for princes and estates satire genres to argue that, while everyone must eventually dance with death, during war some estates lead the dance.

Lydgate's Danse Macabre and the Trauma of the Hundred Years War.

This essay argues that the foundational traumatic lacuna behind John Lydgate's Danse Macabre is the social agon between those who wage the Hundred Years War and those who fight in it. Drawing from the insights of trauma theory to discuss the poem's form, the essay uncovers Lydgate's persistent concern with the damage caused by the war and the concomitant political unrest it causes. It argues further that Lydgate theorized this agon using the emergent genre of tragedy, which is beginning to be practiced anew in late-medieval England. Tragic discourse is riven by concerns about the efficacy of human action and the radical contingency of fortune, creating a crisis of agency that can be used as a form of political critique. Ultimately, Lydgate blends the genre of tragedy with the mirror for princes and estates satire genres to argue that, while everyone must eventually dance with death, during war some estates lead the dance.

Unexpected results from the application of signature-tagged mutagenesis to identify Yersinia pestis genes required for adherence and invasion.

The signature-tagged mutagenesis (STM) method was applied in a protocol designed to identify genes required for Yersinia pestis invasion into epithelial cells. A library of 3060 mutants of Y. pestis CO99-3015 was made and assayed using an in vitro invasion assay with gentamicin protection. Initial results from the screen identified a set of 23 genes that might be required for invasion; however, screening of individual mutants for decreased invasion, even in a competition assay with the parent strain, failed to reveal obvious invasion defects. Altered colony character or size might have imposed a growth disadvantage for two of the mutants, which could possibly account for apparently decreased invasion. The sensitivity of the mutants to gentamicin was assayed to determine if the presence of the kanamycin-resistance cassette in the STM transposon changed the gentamicin resistance of the individual mutants. It was discovered that the mutants exhibited a variable range of resistance to killing by gentamicin, suggesting that the presence of the kanamycin-resistance cassette or the particular insertion mutation did in many cases affect the bactericidal potency of gentamicin. However, all mutants remained highly sensitive to growth inhibition in a disk assay on plates. These results may warrant precautions for use of kanamycin-resistance markers in studies with fully virulent Y. pestis, since gentamicin has been recommended for treatment of plague. Further, to use STM in the context of invasion assays, a selection other than gentamicin should be applied.

Identification and characterization of the hemophore-dependent heme acquisition system of Yersinia pestis.

Yersinia pestis possesses a heme-protein acquisition system (Hmu) that allows it to utilize heme and heme-protein complexes as the sole sources of iron. Analysis of the Y. pestis CO92 genomic sequence revealed a second heme-protein acquisition gene cluster that shares homology with the hemophore-dependent heme acquisition system (Has system) of Serratia marcescens. This locus consisted of the hasR(yp) receptor gene, the hasA(yp) hemophore gene, and genes encoding components of the HasA(yp) dedicated ABC transporter factor (hasDE(yp)), as well as a tonB homologue (hasB(yp)). By using a reconstituted secretion system in Escherichia coli, we showed that HasA(yp) is a secreted heme-binding protein and that expression of HasA(yp) is iron regulated in E. coli. The use of a transcriptional reporter fusion showed that the hasRADEB promoter is Fur regulated and has increased activity at 37 degrees C. Hemoglobin utilization via the Has(yp) system was studied with both E. coli and Y. pestis, for which has and has hmu mutant strains were used. No contribution of the Has system to heme utilization was observed in either E. coli or Y. pestis under the conditions we tested. Previously it was shown that a deletion of the Hmu system had no effect on the virulence of Y. pestis in a mouse model of bubonic plague. An Hmu(-) Has(-) double mutant also retained full virulence in this model of infection. This report constitutes the first attempt to investigate the contribution of the hemophore-dependent heme acquisition system in bacterial pathogenicity.

Characterization of the Yersinia pestis Yfu ABC inorganic iron transport system.

In Yersinia pestis, the causative agent of plague, two inorganic iron transport systems have been partially characterized. The yersiniabactin (Ybt) system is a siderophore-dependent transport system required for full virulence. Yfe is an ABC transport system that accumulates both iron and manganese. We have identified and cloned a Y. pestis yfuABC operon. The YfuABC system is a member of the cluster of bacterial ABC iron transporters that include Sfu of Serratia, Hit of Haemophilus, and Yfu of Yersinia enterocolitica. The Y. pestis KIM6+ system is most homologous to that in Y. enterocolitica, showing identities of 84% for YfuA (periplasmic binding protein), 87% for YfuB (inner membrane permease), and 75% for YfuC (ATP hydrolase). We constructed a yfuABC promoter-lacZ fusion to examine regulation of transcription. This promoter contains a potential Fur binding sequence and is iron and Fur regulated. Significant expression from the yfuABC promoter occurred during iron-deficient growth conditions. In vitro transcription and translation of a recombinant plasmid encoding yfuABC indicates that YfuABC proteins are expressed. Escherichia coli 1017 (an enterobactin-deficient mutant) carrying this plasmid was able to grow in an iron-restrictive complex medium. We constructed a deletion encompassing the yfuABC promoter and most of yfuA. This mutation was introduced into strains with mutations in Ybt, Yfe, or both systems to examine the role of Yfu in iron acquisition in Y. pestis. Growth of the yfu mutants in a deferrated, defined medium (PMH2) at 26 and 37 degrees C failed to identify a growth or iron transport defect due to the yfu mutation. Fifty percent lethal dose studies in mice did not demonstrate a role for the Yfu system in mammalian virulence.

The tc genes of Photorhabdus: a growing family.

The toxin complex (tc) genes of Photorhabdus encode insecticidal, high molecular weight Tc toxins. These toxins have been suggested as useful alternatives to those derived from Bacillus thuringiensis for expression in insect-resistant transgenic plants. Although Photorhabdus luminescens is symbiotic with nematodes that kill insects, tc genes have recently been described from other insect-associated bacteria such as Serratia entomophila, an insect pathogen, and Yersinia pestis, the causative agent of bubonic plague, which has a flea vector. Here, recent advances in our understanding of the tc gene family are reviewed in view of their potential development as insect-control agents.

Yersinia pestis YbtU and YbtT are involved in synthesis of the siderophore yersiniabactin but have different effects on regulation.

One prerequisite for the virulence of Yersinia pestis, causative agent of bubonic plague, is the yersiniabactin (Ybt) siderophore-dependent iron transport system that is encoded within a high-pathogenicity island (HPI) within the pgm locus of the Y. pestis chromosome. Several gene products within the HPI have demonstrated functions in the synthesis or transport of Ybt. Here we examine the roles of ybtU and ybtT. In-frame mutations in ybtT or ybtU yielded strains defective in siderophore production. Mutant strains were unable to grow on iron-deficient media at 37 degrees C but could be cross-fed by culture supernatants from a Ybt-producing strain of Y. pestis. The ybtU mutant failed to express four indicator Ybt proteins (HMWP1, HMWP2, YbtE, and Psn), a pattern similar to those for other ybt biosynthetic mutants. In contrast, strains carrying mutations in ybtT or ybtS (a previously identified gene required for Ybt biosynthesis) produced all four proteins at wild-type levels under iron-deprived conditions. To assess the effects of ybtT, -U, and -S mutations on transcription of ybt genes, reporter plasmids with ybtP or psn promoters controlling lacZ expression were introduced into these mutants. Normal iron-regulated beta-galactosidase activity was observed in the ybtT and ybtS mutants, whereas a significant loss of expression occurred in the DeltaybtU strain. These results show that ybtT and ybtU genes are involved in the biosynthesis of the Ybt siderophore and that a ybtU mutation but not ybtT or ybtS mutations affects transcription from the ybtP and psn promoters.

Invasion of epithelial cells by Yersinia pestis: evidence for a Y. pestis-specific invasin.

The causative agent of plague, Yersinia pestis, is regarded as being noninvasive for epithelial cells and lacks the major adhesins and invasins of its enteropathogenic relatives Yersinia enterocolitica and Yersinia pseudotuberculosis. However, there are studies indicating that Y. pestis invades and causes systemic infection from ingestive and aerogenic routes of infection. Accordingly, we developed a gentamicin protection assay and reexamined invasiveness of Y. pestis for HeLa cells. By optimizing this assay, we discovered that Y. pestis is highly invasive. Several factors, including the presence of fetal bovine serum, the configuration of the tissue culture plate, the temperature at which the bacteria are grown, and the presence of the plasminogen activator protease Pla-encoding plasmid pPCP1, were found to influence invasiveness strongly. Suboptimal combinations of these factors may have contributed to negative findings by previous studies attempting to demonstrate invasion by Y. pestis. Invasion of HeLa cells was strongly inhibited by cytochalasin D and modestly inhibited by colchicine, indicating strong and modest respective requirements for microfilaments and microtubules. We found no significant effect of the iron status of yersiniae or of the pigmentation locus on invasion and likewise no significant effect of the Yops regulon. However, an unidentified thermally induced property (possibly the Y. pestis-specific capsular protein Caf1) did inhibit invasiveness significantly, and the plasmid pPCP1, unique to Y. pestis, was essential for highly efficient invasion. pPCP1 encodes an invasion-promoting factor and not just an adhesin, because Y. pestis lacking this plasmid still adhered to HeLa cells. These studies have enlarged our picture of Y. pestis biology and revealed the importance of properties that are unique to Y. pestis.

Disinfection and communication practices: a survey of U.S. dental laboratories.

BACKGROUND: The need to disinfect impressions is crucial to prevent the transmission of infectious diseases. The authors report the results of a survey of U.S. dental laboratory directors. The survey was designed to determine how well dental laboratory personnel are communicating with dentists regarding the disinfection of impressions, and, in turn, what laboratory technicians are doing to protect themselves against microbial cross-contamination. METHODS: Four hundred dental laboratory directors were selected in a blinded and random manner. To create a geographically representative sample, an equal number of laboratory directors from the East, Midwest and West were interviewed. A survey consisting of 16 open-ended questions was conducted by trained interviewers via 10- to 15-minute telephone interviews. All dental laboratory directors stated that they were thoroughly familiar with their laboratory's disinfection protocol. RESULTS: The survey documented that the majority of impressions were made of polyvinyl (57 percent) or polyether (27 percent) materials. Only 44 percent of the respondents stated that they knew if the impressions they received had been disinfected. Twenty-three percent of the laboratory directors did not know the method of disinfection used, and 47 percent did not know the length of time involved. Forty-five percent of the respondents reported that they receive inadequate instruction in regard to disinfection techniques. No one class of impression materials was found to be more problematic than others by the laboratory directors. CONCLUSIONS: The results indicate a significant and problematic lack of communication between these team members. The responses also suggested that laboratory-perceived problems with impressions were not linked to any particular type of material, but more to the disinfection technique used. PRACTICE IMPLICATIONS: Lack of communication between dentists, staff members and dental laboratory personnel, along with poor training of laboratory personnel in disinfection techniques, may have a direct effect on the prosthetic results achieved in dental practices.

Two-year clinical evaluation of a high-density posterior restorative material.

The purpose of this study was to clinically evaluate SureFil high-density posterior composite for Class 2 restorations of permanent first and second molars. All teeth were restored in occlusion with the natural dentition. After cavity preparation, the enamel was etched for 15 seconds with 34% phosphoric acid. No liner/base material was used for the dentin. Prime & Bond 2.1 (two applications) was applied for adhesion to the exposed dentin and etched enamel. The first coat of adhesive was cured for 20 seconds with a curing light and the next coat was only spread thin with a gentle jet of air. The cavity was restored in 3-mm to 5-mm increments. Clinical evaluation (US Public Health Service method) and wear analysis (indirect cast comparison method) were conducted immediately after placement (baseline) and at 3 months, 6 months, 9 months, 1 year, and 2 years. A total of 25 restorations were inserted. At baseline and after 3, 6, and 9 months (n = 24), all restorations were graded Alfa in all categories. At the 1-year recall, 3 restorations (n = 22) were graded Bravo for surface staining. At the 2-year recall, an additional 3 restorations (n = 22) were graded Bravo for surface staining. No restoration was graded Charlie in any category at any time. Wear analysis revealed an average of 13.8 microns of wear at the 2-year recall (n = 22). No evidence of recurrent caries was observed. In conclusion, after 2 years, this high-density composite material demonstrated clinical acceptability and excellent wear characteristics in all categories, making it suitable for posterior restorations.

Molecular characterization of the hemin uptake locus (hmu) from Yersinia pestis and analysis of hmu mutants for hemin and hemoprotein utilization.

Sequence analysis of the hemin uptake locus (hmu) of Yersinia pestis revealed five genes, hmuRSTUV, required for use of hemin and hemoproteins as iron sources. The translated gene products have homologies with proteins of the hemin transport genes of several gram-negative bacteria. Promoters were identified upstream of hmuP'R (p1) and upstream of hmuS (p2); p1, which contains a Fur box, is regulated by iron and Fur, while p2 exhibits weak, but constitutive, activity. HmuR, which has homology with TonB-dependent outer membrane (OM) receptors, is localized to the OM of Y. pestis and is required for utilizing hemin and all hemoproteins under iron-depleted conditions. The proposed ABC transporter, HmuTUV, is necessary for use of hemin, hemin-albumin, and myoglobin, but not hemoglobin, hemoglobin-haptoglobin, or heme-hemopexin, as iron sources. In the absence of HmuTUV, HmuS, a cytoplasmic protein, is involved in use of hemoglobin and heme-hemopexin. In mice, the 50% lethal doses of Y. pestis DeltahmuP'RSTUV mutants injected subcutaneously or retro-orbitally did not differ from that of the Hmu(+) parent strain. Thus, the hmu system is not essential for infection in mice via these routes. Growth studies showed that a DeltahmuP'RSTUV mutant could grow in iron-depleted medium containing high concentrations of hemoglobin, suggesting that an Hmu-independent, lower-affinity hemoglobin uptake system may exist.

YbtP and YbtQ: two ABC transporters required for iron uptake in Yersinia pestis.

Yersinia pestis, the causative agent of plague, makes a siderophore termed yersiniabactin (Ybt), which it uses to obtain iron during growth at 37 degrees C. The genes required for the synthesis and utilization of Ybt are located within a large, unstable region of the Y. pestis chromosome called the pgm locus. Within the pgm locus, just upstream of a gene (ybtA) that regulates expression of the Ybt receptor and biosynthetic genes, is an operon consisting of 4 genes - ybtP, ybtQ, ybtX and ybtS. Transcription of the ybtPQXS operon is repressed by Fur and activated by YbtA. The product of ybtX is predicted to be an exceedingly hydrophobic cytoplasmic membrane protein that does not appear to contribute any vital function to Ybt biosynthesis or utilization in vitro. ybtP and ybtQ encode putative members of the traffic ATPase/ABC transporter family. YbtP and YbtQ are structurally unique among the subfamily of ABC transporters associated with iron transport, in that they both contain an amino-terminal membrane-spanning domain and a carboxy-terminal ATPase. Cells with mutations in ybtP or ybtQ still produced Ybt but were impaired in their ability to grow at 37 degrees C under iron-deficient conditions, indicating that YbtP and YbtQ are needed for iron uptake. In addition, a ybtP mutant showed reduced iron accumulation and was avirulent in mice by a subcutaneous route of infection that mimics flea transmission of bubonic plague.

The Yfe system of Yersinia pestis transports iron and manganese and is required for full virulence of plague.

Iron acquisition in Yersinia pestis is fundamental to the success of plague pathogenesis. We have previously identified an approximately 5.6 kb region (yfe) of Y. pestis genomic DNA, capable of restoring iron-deficient growth but not siderophore production to an Escherichia coli mutant (SAB11) incapable of synthesizing the siderophore, enterobactin. The yfe locus of Y. pestis, found in both pigmented (Pgm+) and nonpigmented (Pgm-) strains, comprises five genes arranged in two distinct operons (yfeA-D and yfeE ). The larger of these, yfeABCD, encodes an ABC transport system, whose expression is iron and Fur regulated and is repressed in cells grown in the presence of manganese. Cells from a Pgm-, Yfe- (DeltayfeAB ) mutant strain of Y. pestis exhibited reduced transport of both 55Fe and 54Mn. Furthermore, cells containing an intact yfe locus showed reduced 55Fe uptake when competing amounts of MnCl2 or ZnCl2 were present, whereas 54Mn uptake was inhibited by FeCl3 but not by ZnCl2. Similarly, yfe mutants of Y. pestis exhibited growth defects on media supplemented with the iron chelators 2,2'-dipyridyl or conalbumin. These growth defects were not relieved by supplementation with MnCl2. A ybt-, DeltayfeAB mutant of Y. pestis was completely avirulent in mice infected intravenously (LD50 > 1.7 x 107 cfu) compared with its parental ybt-, yfe+ strain, which had an LD50 of < 12. In addition, compared with its ybt+, yfe+ parent, a ybt+, DeltayfeAB mutant of Y. pestis had an approximately 100-fold increase in the LD50 from a subcutaneous route of infection. These data suggest that the Yfe and Ybt systems may function effectively to accumulate iron during different stages of the infectious process of bubonic plague.

Iron acquisition in plague: modular logic in enzymatic biogenesis of yersiniabactin by Yersinia pestis.

BACKGROUND: Virulence in the pathogenic bacterium Yersinia pestis, causative agent of bubonic plague, has been correlated with the biosynthesis and transport of an iron-chelating siderophore, yersiniabactin, which is induced under iron-starvation conditions. Initial DNA sequencing suggested that this system is highly conserved among the pathogenic Yersinia. Yersiniabactin contains a phenolic group and three five-membered thiazole heterocycles that serve as iron ligands. RESULTS: The entire Y. pestis yersiniabactin region has been sequenced. Sequence analysis of yersiniabactin biosynthetic regions (irp2-ybtE and ybtS) reveals a strategy for siderophore production using a mixed polyketide synthase/nonribosomal peptide synthetase complex formed between HMWP1 and HMWP2 (encoded by irp1 and irp2). The complex contains 16 domains, five of them variants of phosphopantetheine-modified peptidyl carrier protein or acyl carrier protein domains. HMWP1 and HMWP2 also contain methyltransferase and heterocyclization domains. Mutating ybtS revealed that this gene encodes a protein essential for yersiniabactin synthesis. CONCLUSIONS: The HMWP1 and HMWP2 domain organization suggests that the yersiniabactin siderophore is assembled in a modular fashion, in which a series of covalent intermediates are passed from the amino terminus of HMWP2 to the carboxyl terminus of HMWP1. Biosynthetic labeling studies indicate that the three yersiniabactin methyl moieties are donated by S-adenosylmethionine and that the linker between the thiazoline and thiazolidine rings is derived from malonyl-CoA. The salicylate moiety is probably synthesized using the aromatic amino-acid biosynthetic pathway, the final step of which converts chorismate to salicylate. YbtS might be necessary for converting chorismate to salicylate.

DNA sequencing and analysis of the low-Ca2+-response plasmid pCD1 of Yersinia pestis KIM5.

The low-Ca2+-response (LCR) plasmid pCD1 of the plague agent Yersinia pestis KIM5 was sequenced and analyzed for its genetic structure. pCD1 (70,509 bp) has an IncFIIA-like replicon and a SopABC-like partition region. We have assigned 60 apparently intact open reading frames (ORFs) that are not contained within transposable elements. Of these, 47 are proven or possible members of the LCR, a major virulence property of human-pathogenic Yersinia spp., that had been identified previously in one or more of Y. pestis or the enteropathogenic yersiniae Yersinia enterocolitica and Yersinia pseudotuberculosis. Of these 47 LCR-related ORFs, 35 constitute a continuous LCR cluster. The other LCR-related ORFs are interspersed among three intact insertion sequence (IS) elements (IS100 and two new IS elements, IS1616 and IS1617) and numerous defective or partial transposable elements. Regional variations in percent GC content and among ORFs encoding effector proteins of the LCR are additional evidence of a complex history for this plasmid. Our analysis suggested the possible addition of a new Syc- and Yop-encoding operon to the LCR-related pCD1 genes and gave no support for the existence of YopL. YadA likely is not expressed, as was the case for Y. pestis EV76, and the gene for the lipoprotein YlpA found in Y. enterocolitica likely is a pseudogene in Y. pestis. The yopM gene is longer than previously thought (by a sequence encoding two leucine-rich repeats), the ORF upstream of ypkA-yopJ is discussed as a potential Syc gene, and a previously undescribed ORF downstream of yopE was identified as being potentially significant. Eight other ORFs not associated with IS elements were identified and deserve future investigation into their functions.

The nonribosomal peptide synthetase HMWP2 forms a thiazoline ring during biogenesis of yersiniabactin, an iron-chelating virulence factor of Yersinia pestis.

Pathogenic Yersinia species have been shown to synthesize a siderophore molecule, yersiniabactin, as a virulence factor during iron starvation. Here we provide the first biochemical evidence for the role of the Yersinia pestis high molecular weight protein 2 (HMWP2), a nonribosomal peptide synthetase homologue, and YbtE in the initiation of yersiniabactin biosynthesis. YbtE catalyzes the adenylation of salicylate and the transfer of this activated salicyl group to the N-terminal aryl carrier protein domain (ArCP; residues 1-100) of HMWP2. A fragment of HMWP2, residues 1-1491, can adenylate cysteine and with the resulting cysteinyl-AMP autoaminoacylate the peptidyl carrier protein domain (PCP1; residues 1383-1491) either in cis or in trans. Catalytic release of hydroxyphenylthiazoline carboxylic acid (HPT-COOH) and/or N-(hydroxyphenylthiazolinylcarbonyl)cysteine (HPT-cys) is observed upon incubation of YbtE, HMWP2 1-1491, L-cysteine, salicylate, and ATP. These products presumably arise from nucleophilic attack by water or cysteine of a stoichiometric hydroxyphenylthiazolinylcarbonyl-S-PCP1-HMWP2 intermediate. Detection of the heterocyclization capacity of HMWP2 1-1491 implies salicyl-transferring and thiazoline-forming activity for the HMWP2 condensation domain (residues 101-544) and is the first demonstration of such heterocyclization ability in a nonribosomal peptide synthetase enzyme.

An ABC transporter system of Yersinia pestis allows utilization of chelated iron by Escherichia coli SAB11.

The acquisition of iron is an essential component in the pathogenesis of Yersinia pestis, the agent of bubonic and pneumonic plague. A cosmid library derived from the genomic DNA of Y. pestis KIM6+ was used for transduction of an Escherichia coli mutant (SAB11) defective in the biosynthesis of the siderophore enterobactin. Recombinant plasmids which had a common 13-kb BamHI fragment were isolated from SAB11 transductants in which growth but not enterobactin synthesis was restored on media containing the iron chelator EDDA [ethylenediamine-di(o-hydroxyphenyl acetic acid)]. Subcloning and transposon mutagenesis revealed a 5.6-kb region, designated yfe, essential for SAB11 growth stimulation. In vitro transcription-translation analysis identified polypeptides of 18, 29.5, 32, and 33 kDa encoded by the yfe locus. Sequence analysis shows this locus to be comprised of five genes in two separate operons which have potential Fur-binding sequences in both promoters. A putative polycistronic operon, yfeABCD, is Fur regulated and responds to iron and manganese. A functional Fur protein is required for the observed manganese repression of this operon. This operon encodes polypeptides which have strong similarity to the ATP-binding cassette (ABC) family of transporters and include a periplasmic binding protein (YfeA), an ATP-binding protein (YfeB), and two integral membrane proteins (YfeC and -D), which likely function in the acquisition of inorganic iron and possibly other ions. The approximately 21-kDa protein encoded by the separately transcribed yfeE gene may be located in the cell envelope, since a yfeE::TnphoA fusion is PhoA+. Mutations in this gene abrogate growth of SAB11 on iron-chelated media.

Dyract compomer: comparison of total etch vs. no etch technique.

Different dental materials and methods can influence the integrity of the marginal seal of restorations. To evaluate the microleakage of Dyract AP Light Cured Compomer, a polyacid modified resin (Caulk), using etched and unetched techniques, standardized trapezoidal Class V restorations were placed on facial or lingual surfaces of 20 human molars with the gingival margin in the cementum. Each restoration was scored at the cervical by two independent, double blinded operators, with reference to the DEJ, for dye penetration on a ranking system of: 0 = no evidence of dye penetration; 1 = dye penetration up to one-half the distance to the axial wall; 2 = dye penetration beyond one-half the distance to the axial wall but short of the axial wall; 3 = dye penetration to the axial wall or beyond. Statistical analysis (Fisher Exact Test) indicated that the etched compomer demonstrated significantly less microleakage when compared to the unetched compomer (p < 0.05).