Abstract
Introduction
Endodontic infections are very prevalent and have a polymicrobial etiology characterized by complex interrelationships between endodontic microorganisms and the host defenses. Proteomic analysis of endodontic infections can provide global insights into the invasion, pathogenicity mechanisms, and multifactorial interactions existing between root canal bacteria and the host in the initiation and progression of apical periodontitis. The purpose of this study was to apply proteomic techniques such as liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the identification of proteins of bacterial origin present in endodontic infections.
Methods
Endodontic specimens were aseptically obtained from seven patients with root canal infections. Protein mixtures were subjected to tryptic in-solution digestion and analysed by reverse-phase nano-LC–MS/MS followed by a database search.
Results
Proteins, mainly of cell wall or membrane origin, from endodontic bacteria especially Enterococcus faecalis, Enterococcus faecium, Porphyromonas gingivalis, Fusobacterium nucleatum, and Treponema denticola were identified from all the samples tested. Identified proteins included adhesins, autolysins, proteases, virulence factors, and antibiotic-resistance proteins.
Conclusions
LC–MS/MS offers a sensitive analytical platform to study the disease processes in the root canal environment. The array of proteins expressed in endodontic infections reflects the complex microbial presence and highlights the bacterial species involved in the inflammatory process.
Keywords: bacteria, endodontics, liquid chromatography/mass spectroscopy, proteomics, virulence
Endodontic infections are unique and have a complex polymicrobial etiology often predominated by obligate anaerobes. The exact bacteria that mediate the symptoms or persistence of disease are not known. The initiation and progression of apical periodontitis may involve complex inter-relationships between the root canal microbes and host defense (15). The former may involve virulence factors, toxins, antibiotic resistance proteins, adhesins bacteriocins, and quorum-sensing molecules. The latter might involve host cells, inflammatory mediators, metabolites, neuropeptides or other effector molecules, and immunoglobulins. Both culture-based and molecule-based studies have shown that the most prevalent bacterial species present in endodontic infections belong to genera such as Fusobacterium, Porphyromonas, Prevotella, Bacteroides, Peptostreptococcus and Enterococcus, especially Enterococcus faecalis, which is frequently associated with persistent cases (4, 6, 23, 25). The degree of microbial complexity and diversity often present in endodontic infections renders the investigation of individual microorganisms, specific virulence factors, or specific host effector molecules ineffective, or at best insufficient, to fully comprehend the disease process. Conversely, emerging proteomic technologies such as mass-spectrometry-based peptide sequence analysis can provide global insights into the invasion, virulence, pathogenicity mechanisms, and multifactorial interactions existing between the endodontic microorganisms and the host.
Among the proteomic techniques commonly used for analysis of protein expression in biological fluids, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is a popular technique for the separation of proteins. However, there are many limitations associated with this technique that have been well outlined (9). More recent efforts have focused on using liquid chromatography/mass spectrometry (LC/MS) with either a top-down (7) or bottom-up (27) approach. The bottom-up or shotgun approach involves the proteolytic digestion of all the proteins in the sample and relies on database analysis of the individual peptides to identify the source proteins in the sample. This method reduces sample handling time and eliminates the need for processing of individual proteins. It allows the direct analysis of extremely complex biological samples and rapidly generates protein profile and sequence information. Tandem mass spectrometry (MS/MS) has become increasingly important and indispensable for identifying complex protein mixtures in high-throughput proteomics experiments (1).
Within the field of oral biology, the use of proteomic methods has largely been confined to the use of 2D-PAGE. Proteomic approaches have been applied to analyse the physiological adaptations or mechanisms of survival, invasion or pathogenicity mainly in Streptococcus mutans (8), Streptococcus oralis (26), Fusobacterium nucleatum (2, 30), Porphyromonas gingivalis (13, 29) and E. faecalis (17). Developments in the proteomic analysis of oral pathogens were summarized by Macarthur and Jacques (14). No attempt has been made to identify the proteins that are expressed in vivo in the root canal in endodontic infections. In this context, we report for the first time the application of proteomic techniques such as liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) in a shotgun approach for the identification of proteins expressed in the root canal in endodontic infections.
All patient-related procedures used in this study conformed to protocols approved by the Institutional Review Board at the University of Maryland. Patients presenting for endodontic treatment for a tooth with either a primary or a persistent (previously endodontically treated) endodontic infection were included. The teeth involved had a negative pulp test result (for primary cases) and a periradicular lesion at least 3 mm in diameter.
Samples were collected from teeth with endodontic infections, primary (n = 4) or persistent (n = 2) as per the protocol described previously (4). One case was initially described as a primary infection but upon access it was determined to have undergone incomplete endodontic treatment years earlier and consequently was not assigned a primary/persistent category.
Following isolation with a rubber dam, the field was disinfected with 30% H2O2, followed by 5% tincture of iodine and 5.25% sodium hypochlorite. The halides were inactivated with 5% sodium thiosulfate. Following access preparation, microbial specimens were obtained from root canals using one sterile K-file or H-file, which was first used to disrupt canal wall biofilms, and three sterile paper points. The metallic portion of the file and the paper points were placed in sterile, DNA-free and RNA-free vials containing 1.5 ml sterile 10 mm Tris–HCl (pH 8.0) and 0.5 g sterile glass beads (0.71–1.18-mm diameter). The vials were frozen at −70°C until use. The vials containing paper point specimens were vortexed for 2 min to disperse microbial cellular material into suspension followed by centrifugation at 3144 g for 10 min. The supernatant was withdrawn and used for proteomic analysis.
The protein mixtures were initially subjected to a buffer exchange with 50 mm ammonium bicarbonate (pH 8.2) and reduced with 10 mm dithiothreitol for 1 h at 56°C. Cysteine residues in the protein samples were alkylated with 40 mm iodoacetamide for 45 min at room temperature with shaking in the dark. Following alkylation, the samples were neutralized by adding 200 mm dithiothreitol and incubated for 1 h. The samples were buffer exchanged with 50 mm ammonium bicarbonate and digested with sequencing-grade modified porcine trypsin (Promega, Madison, WI, USA) overnight at 37°C with a 1 : 50 (weight : weight) trypsin : protein ratio. The peptides were vacuum-dried and reconstituted with 0.1% formic acid before LC–MS/MS analysis.
Mass spectrometric analysis of the extracted peptides was carried out using a nanoscale reverse-phase high-performance liquid chromatography capillary column (10.5 cm × 75 µm inner diameter fused silica reverse-phase C18 column; Pico frit column (New Objective, Woburn, MA, USA). The sample peptides (10 µl injection volume) were separated by a linear gradient of 5%/90% acetonitrile/water mixture, containing 0.1% formic acid in 90 min at a flow rate of 300 nl/min. The gradient was provided by a Surveyor LC pump (Thermo-Finnegan, San Jose, CA, USA). The nanospray voltage was set to 2 kV. Mass spectrometric analysis was carried out on an LCQ Deca XP linear ion trap (Thermo-Finnegan, San Jose, CA, USA) which was operated on positive-ion mode. Peptide ions were detected in a survey scan (five most intense peaks) from 400 to 1800 atomic mass units (amu) (three microscans) followed by five data-dependent MS/MS scans (five microscans each, isolation width 2.5 amu, 35% normalized collision energy). The acquired spectra were further used for a database search.
After the acquisition of MS/MS spectra, they were searched against bacterial protein databases downloaded from the National Center for Biotechnology Information (NCBI), using the software algorithm sequest (28). The sequest criteria used for protein identifications were as follows: strict trypsin enzyme digestion, peptide mass tolerance of <2.5 Da, monoisotopic mass of the precursor and product ions <0.15 Da, possible modifications of methionine oxidation and carboxyamidomethylation. For the positive identification of any individual protein, at least two peptides were required. The threshold of cross-correlation (Xcorr) scores set for peptides were 1.5, 2.0, and 2.5 for +1, +2, and +3 charged fully digested peptides, respectively, and a threshold of 0.08 was required for ΔCn values for individual peptides (16). The same set of MS/MS spectra were searched against E. faecalis V583 protein sequences using the Sorcerer™ IDA server (SageN, Inc., San Jose, CA, USA). The validation of protein identification was performed with Trans-Peptide Pipeline (TPP; open source software: http://www.sourceforge.net) software as specified by the Peptide Prophet algorithm with a Peptide Prophet score higher than 0.9.
Preoperative specimen samples taken from seven patients with primary or persistent infections were used for proteomic analysis of endodontic infections. Diversity and prevalence of bacterial phylotypes in these samples were analysed by both broad-based polymerase chain reaction (PCR) cloning and sequencing and by genus-specific PCR and will be reported separately. Application of LC–MS/MS resulted in the identification of a number of bacterial proteins; these are listed in Table 1 and Table 2.
Table 1.
Identification of proteins of non-enterococcal origin present in endodontic infections
| UniProt acc. no. |
Protein | Bacteria | No. of peptides |
Biological function |
|---|---|---|---|---|
| Q39PI2 | Lipocalin-like protein (1)1 | Burkholderia sp. | 8 | Transport, Starvation response |
| A3I6E1 | Microbial collagenase metalloprotease (2) | Bacillus sp. | 4 | Invasion, virulence |
| Q5LBV7 | Hypothetical protein BF2715 (1) | Bacteroides fragilis | 2 | Unknown |
| Q5LB48 | Putative ABC transport system, membrane protein (1) | B. fragilis | 7 | Multidrug transport system |
| Q5LEF4 | Putative outer membrane protein (5) | Bacteroides vulgatus | 4 | Nutrient binding, Transport |
| A6L076 | Penicillin-binding protein (2) | B. vulgatus | 5 | Antibiotic resistance |
| A5CNU5 | Putative beta lactamase/penicillin-binding protein (1) | Clavibacter michiganensis | 4 | Antibiotic resistance |
| Q8FLL7 | Putative virulence-associated protein (1) | Corynebacterium sp | 2 | Virulence |
| B2QEG9 | Fibronectin-binding A domain protein (2) | Exiguobacterium sp | 5 | Adhesin |
| Q8RGG3 | Outer membrane porin F (2) | Fusobacterium nucleatum | 3 | Cell envelope biogenesis |
| Q8RIE8 | Oligopeptide-binding protein oppA (1) | F. nucleatum | 4 | Transport |
| Q8RI38 | Spermidine/putrescine ABC transporter permease (3) | F. nucleatum | 4 | Transport |
| Q8RHF5 | ABC transporter substrate-binding protein (1) | F. nucleatum | 3 | Transport |
| A5TVG7 | N-acylneuraminate cytidylyltransferase (1) | F. nucleatum | 4 | Lipopolysaccharide biosynthetic process |
| Q040U6 | Uncharacterized membrane-bound protein (4) | Lactobacillus gasseri | 3 | Unknown |
| Q9CIL7 | Penicillin-binding protein 2B (3) | Lactococcus lactis | 4 | Antibiotic resistance |
| Q2G7B1 | Conserved hypothetical membrane protein (1) | Novosphingobium aromaticivorans | 12 | Unknown |
| B1DLX6 | ABC transporter related (1) | Paenibacillus sp. | 3 | Transport |
| B1D7U4 | Coagulation factor 5/8 type domain protein (2) | Paenibacillus sp. | 8 | Cell adhesion |
| Q7MWV9 | YngK protein (1) | Porphyromonas gingivalis | 2 | Unknown |
| B2RKT2 | Preprotein translocase SecA subunit (1) | P. gingivalis | 3 | Protein export |
| Q7MUL8 | TraG family protein (1) | P. gingivalis | 3 | Conjugation |
| Q6ABI3 | Putative penicillin-binding protein (2) | Propionibacterium acnes | 4 | Antibiotic resistance |
| Q0S9R0 | Possible universal stress protein (2) | Rhodococcus sp. | 4 | Stress response |
| P95800 | groEL protein (1) | Stenotrophomonas maltophilia | 4 | Chaperonins |
| Q3D4N5 | PTS system, IIBC components (1) | Streptococcus agalactiae | 3 | Sugar transport |
| Q3CY15 | Fibrinogen-binding protein (3) | S. agalactiae | 7 | Adhesion |
| A8AXJ4 | Lipoprotein, putative (1) | Streptococcus gordonii | 5 | Adhesion |
| Q2ZXI8 | Binding-protein-dependent transport systems (1) | Streptococcus suis | 3 | Transport |
| Q73QT1 | Bacterial immunoglobulin-like domain protein (1) | Treponema denticola | 6 | Putative adhesin |
| A3CPZ1 | Thioredoxin reductase, putative (1) | Streptococcus sanguinis | 3 | Removal of superoxide radicals, chaperones |
Numbers in parenthesis indicate the number of patient samples where the protein was identified.
Table 2.
Identification of proteins of enterococcal origin present in endodontic infections
| UniProt acc. no. |
Protein | No. of peptides |
Probability score |
Biological function |
|---|---|---|---|---|
| Q82ZX8 | ABC transporter, ATP-binding protein (3)1 | 23 | 1 | Transport |
| Q837A1 | ABC transporter, ATP-binding/permease protein (2) | 28 | 1 | Transport |
| Q82YN1 | Aggregation substance PrgB (1) | 11 | 1 | Virulence factor |
| Q833M7 | ATP-dependent Clp protease, ATP-binding subunit ClpX (2) | 7 | 1 | Chaperone |
| P37710 | Autolysin (2) | 5 | 0.99 | Autolysin |
| Q47779 | BacB protein (3) | 6 | 0.99 | Bacteriocin, Immunity protein |
| Q3Y1R6 | Cell wall hydrolase/autolysin (1) | 7 | 1 | Peptidoglycan catabolic process |
| Q833P7 | Cell wall surface anchor family protein (3) | 8 | 1 | Adhesion |
| Q839K7 | Conjugal transfer protein, putative (2) | 9 | 1 | Conjugation |
| Q8KH16 | Conserved hypothetical protein (3) | 37 | 1 | Unknown |
| Q835K7 | Drug resistance transporter, EmrB/QacA family protein (1) | 6 | 1 | Tetracycline transport |
| Q835S9 | Endolysin (1) | 3 | 1 | Peptidoglycan catabolic process |
| Q836Z9 | Extracellular protein, putative (1) | 4 | 1 | Peptidoglycan turnover |
| A1YGU1 | Extracellular serine proteinase (1) | 7 | 0.99 | Proteolysis, Virulence |
| Q820V6 | FtsK/SpoIIIE family protein (3) | 7 | 1 | Cell division |
| Q49SF0 | Gls24 (1) | 6 | 1 | Stress response |
| Q832Q0 | Glycosyl transferase, group 2 family protein (1) | 7 | 1 | Cell envelope biogenesis |
| Q836W3 | Hemolysin A (1) | 11 | 1 | Virulence factor |
| Q838X9 | Lipoprotein, putative (2) | 8 | 1 | Unknown |
| Q835M5 | Magnesium-translocating P-type ATPase (2) | 8 | 1 | Magnesium ion transport |
| Q832Q4 | Membrane protein, putative (3) | 13 | 1 | Unknown |
| Q9RPP2 | Membrane-associated zinc metalloprotease, putative (1) | 8 | 1 | Pheromone production |
| Q834G2 | Metallo-beta-lactamase, AtsA/ElaC family (1) | 4 | 1 | Antibiotic resistance |
| Q830Y6 | N-acetylmuramoyl-l-alanine amidase, family 4 (3) | 13 | 1 | Peptidoglycan catabolic process |
| Q79A51 | Nickase (1) | 7 | 1 | Unidirectional conjugation |
| Q838B1 | Oligoendopeptidase F, putative (1) | 5 | 1 | Proteolysis |
| Q5G3N8 | PcfD (1) | 5 | 1 | Conjugation |
| Q9K3C9 | Penicillin-binding protein 4 (2) | 9 | 0.99 | Antibiotic resistance |
| Q82ZH7 | Peptidase T (1) | 3 | 1 | Proteolysis |
| Q839D6 | Peptidase, M20/M25/M40 family (2) | 4 | 0.99 | Proteolysis |
| Q830Q0 | Peptidase, M42 family (3) | 13 | 1 | Proteolysis |
| Q834B4 | Phosphate ABC transporter, ATP-binding protein (2) | 31 | 1 | Phosphate transport |
| Q82ZM8 | Polysaccharide lyase, family 8 (1) | 4 | 1 | Cell adhesion |
| Q82ZY7 | Potassium uptake protein (1) | 4 | 1 | Potassium ion transport |
| Q82YM8 | PrgE (1) | 29 | 1 | Pheromone response |
| Q837M0 | PTS system, IIC component (2) | 8 | 1 | Carbohydrate transport |
| Q8KI44 | Putative uncharacterized protein (1) | 34 | 1 | Unknown |
| Q82ZQ8 | Rhodanese family protein (2) | 13 | 1 | Cyanide detoxification |
| Q6WS02 | Rlx-like protein (1) | 8 | 0.99 | Conjugation |
| Q82ZJ9 | Sortase family protein (2) | 8 | 1 | Surface protein anchoring |
| Q832J2 | Sugar ABC transporter, sugar-binding protein (1) | 3 | 1 | Transport |
| Q833W2 | Sulfatase domain protein (1) | 5 | 1 | Cell envelope biogenesis |
| Q831L1 | Teichoic acid biosynthesis protein, putative (1) | 5 | 1 | Teichoic acid biosynthetic process |
| Q2UXR5 | Tetracycline resistance protein (1) | 10 | 0.99 | Tetracycline resistance |
| Q47810 | Tetracycline resistance protein tetM (3) | 71 | 1 | Tetracycline resistance |
| Q5GBH8 | TetT (1) | 4 | 0.99 | Tetracycline resistance |
| Q82YJ4 | Toxin ABC transporter, ATP-binding/permease protein (2) | 8 | 1 | Bacteriocin/lantibiotic exporters |
| Q832B2 | TraG family protein (1) | 5 | 1 | Unidirectional conjugation |
| O07108 | UDP-N-acetylmuramoylalanine-d-glutamate ligase (1) | 7 | 1 | Peptidoglycan biosynthetic process |
| Q3XXB2 | Uncharacterized protein conserved in bacteria (1) | 7 | 1 | Unknown |
| Q93A46 | VanE (1) | 3 | 1 | Peptidoglycan biosynthesis |
| Q47745 | VanSB, Sensor protein (2) | 7 | 1 | Response to vancomycin |
| Q30BF3 | VanSG2, Sensor protein (3) | 17 | 1 | Signal transduction |
| Q30BE9 | VanTG2 (1) | 6 | 0.99 | Response to vancomycin |
| Q30BF2 | VanWG2 (1) | 4 | 1 | Unknown |
| Q836M0 | Von Willebrand factor type A domain protein (1) | 3 | 1 | Adhesion |
| P0A4M1 | Zeta-toxin (1) | 5 | 1 | Programmed cell death of plasmid free cells |
Protein-Prophet scores (pp) above 0.9 were accepted; at this cut-off, the rate of false-positive protein identifications is >10%.
Numbers in parenthesis indicate the number of patient samples where the protein was identified.
Many of the identified proteins are outer membrane proteins probably involved directly in the pathogenic processes, for example, adhesins, autolysins, proteases, penicillin-binding proteins, and those with a predicted function of virulence, invasion, nutrient binding, and cell envelope biogenesis. A few of the proteins identified were hypothetical with no putative function. The majority of the proteins detected were from phylotypes that had been identified in endodontic infections such as E. faecalis, Enterococcus faecium, Treponema denticola, Bacteroides fragilis, Bacteroides vulgatus, F. nucleatum, Parvimonas micra, and P. gingivalis.
Among the proteins of non-enterococcal origin, proteins such as putative outer membrane protein probably involved in nutrient binding, spermidine/putrescine ABC transporter, and penicillin-binding protein 2B were found in more than one patient. Proteins with a putative function in antibiotic resistance, invasion/virulence, and nutrient binding were found in five of the seven patients. Chaperonins and those proteins involved in the stress response were identified in three specimens.
Among the proteins that were identified with E. faecalis as the source, proteins such as ABC transporter-ATP-binding protein, cell wall surface anchor family protein, N-acetylmuramoyl-l-alanine amidase, penicillin-binding protein, BacB protein, tetM, and VanSG2 sensor protein were very prevalent. Proteins involved in antibiotic resistance, adhesion, membrane transport, virulence, autolysins, bacteriocins, and conjugation were highly predominant and were present in three of the seven patients, highlighting a potentially active role of E. faecalis in the infectious processes of endodontic diseases. Other enterococcal species such as E. faecium produced proteins involved in the stress response and autolysins along with many uncharacterized proteins.
Even though no purposeful attempt was made to lyse the bacterial cells present in the sample, a few proteins of subcellular origin were also identified. These proteins might have resulted from cell lysis caused by vortexing with glass beads, which was performed to dislodge microbial cells from the paper points, or from autolysed cells.
In most cases, bacterial phylotypes from which the proteins were identified had been detected in the respective patient specimens by clonal analysis (data not shown). In four patients, three with primary disease and one with persistent disease, in whom no enterococci were detected by either broad-range PCR cloning and sequencing or by genus-specific PCR, the proteomic analysis could not detect any proteins of enterococcal origin. In the rest of the specimens where Enterococcus spp. were highly prevalent, many proteins of enterococcal origin were detected. Proteins were also identified from other bacteria that are not recognized as strict endodontic pathogens such as Burkholderia sp., Bacillus sp., and Lactobacillus sp. They were either detected in these patient specimens by clonal analysis or were listed in literature as present in periodontal infections, as part of the biofilm communities of dental water lines, or could even be food contaminants (11, 22).
P. gingivalis was found to express numerous potential virulence factors such as fimbriae, hemagglutinins, lipopolysaccharides, and various proteases (12). In the samples selected for this study, P. gingivalis was not prevalent; it was detected in only one case. Increased expression of chaperonins such as GroEL by F. nucleatum under conditions that mimic those in periodontal pockets has been reported (30). The identification of GroEL protein, as well as of other proteins with putative functions as chaperonins, is relevant because they have been reported to be immunodominant bacterial antigens that may play a role in pathogenesis by stimulating the host immune response (24). Proteins involved in cell wall biosynthesis in S. mutans (8), ampicillin resistance in F. nucleatum (2), and virulence in P. gingivalis (29) were also reported by proteomic analysis.
Enterococcal spp., especially E. faecalis, are reported to be highly prevalent (12–90%) in endodontic infections and are purported to play an important role in treatment failures (6, 10, 19). However, these organisms were also shown to be prevalent in endodontically treated teeth that have no periapical lesions (10, 31), thereby raising doubts about their role in the pathogenesis of the lesions in every case. A large number of virulence factors have been recognized among enterococci including gelatinase (21), cytolysin (3), enterococcal surface proteins (20), and E. faecalis collagen adhesin (18). Expression of cytolysin was found to be prevalent by proteomic analysis in enterococcal isolates from endodontic infections (17). The fact that the proteins from the tet and Van operons were predominant in all the patient cases where enterococci were detected by genomic analysis is important from a treatment perspective because antibiotics are known to be not very effective in treating chronic or localized acute endodontic infections or in preventing flare-ups. E. faecalis was highly prevalent in the patient sample in which VanE protein was identified by proteomic analysis. In addition, E. casseliflavus, known for its low resistance to vancomycin, was also highly prevalent in the same patient samples where vancomycin-resistance proteins were predominant.
Many studies have outlined the predominant taxa associated with endodontic infections with respect to the clinical manifestations (4, 5). However, drawing conclusions on the identity of the most pathogenic or causative organisms from the complex phylogenetic diversity by genomic analysis alone is not feasible. It is evident from proteomic analysis that a majority of the virulence and antibiotic-resistance proteins present in the samples analysed here were secreted by enterococci, stressing their role as potentially serious pathogens among the polymicrobial flora present in endodontic infections.
Proteomic techniques such as LC–MS/ MS offer a very good analytical platform with high sensitivity to study the disease processes in the root canal environment characterized by a complex microbial presence and very low sample concentrations. In addition, proteomic data clearly complement the genomic information gathered on individual specimens in terms of prevalent phylotypes. With all the challenges inherent in proteomic analyses, genomics and proteomics together could provide a better understanding of the etiology and pathogenesis of endodontic diseases. Such global information about both genes and protein expression provides a rational basis for identification of molecular targets that could be used for better diagnoses, prevention, and treatment of endodontic infections.
In conclusion, this study details the first direct identification of bacterial proteins expressed in endodontic infections. It is obvious that the proteins identified in this study represent only a fraction of the major protein components because of the small sample size and the fact that the genomes of many oral bacteria are not yet fully annotated. However, these results provide a new avenue for directly characterizing the virulence-associated proteins commonly present in endodontic infections and thereby for characterizing the pathogenic species among the diverse endodontic taxa that should ultimately lead to better treatment strategies.
Acknowledgments
This study was supported by grant DE014476 from NIDCR.
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