Rab11 mediates selective recycling and endocytic trafficking in Trypanosoma brucei.

Trypanosoma brucei possesses a streamlined secretory system that guarantees efficient delivery to the cell surface of the critical glycosyl-phosphatidylinositol (GPI)-anchored virulence factors, variant surface glycoprotein (VSG) and transferrin receptor (TfR). Both are thought to be constitutively endocytosed and returned to the flagellar pocket via TbRab11+ recycling endosomes. We use conditional knockdown with established reporters to investigate the role of TbRab11 in specific endomembrane trafficking pathways in bloodstream trypanosomes. TbRab11 is essential. Ablation has a modest negative effect on general endocytosis, but does not affect turnover, steady state levels or surface localization of TfR. Nor are biosynthetic delivery to the cell surface and recycling of VSG affected. TbRab11 depletion also causes increased shedding of VSG into the media by formation of nanotubes and extracellular vesicles. In contrast to GPI-anchored cargo, TbRab11 depletion reduces recycling of the transmembrane invariant surface protein, ISG65, leading to increased lysosomal turnover. Thus, TbRab11 plays a critical role in recycling of transmembrane, but not GPI-anchored surface proteins. We proposed a two-step model for VSG turnover involving release of VSG-containing vesicles followed by GPI hydrolysis. Collectively, our results indicate a critical role of TbRab11 in the homeostatic maintenance of the secretory/endocytic system of bloodstream T. brucei.

Controlling transferrin receptor trafficking with GPI-valence in bloodstream stage African trypanosomes.

Bloodstream-form African trypanosomes encode two structurally related glycosylphosphatidylinositol (GPI)-anchored proteins that are critical virulence factors, variant surface glycoprotein (VSG) for antigenic variation and transferrin receptor (TfR) for iron acquisition. Both are transcribed from the active telomeric expression site. VSG is a GPI2 homodimer; TfR is a GPI1 heterodimer of GPI-anchored ESAG6 and ESAG7. GPI-valence correlates with secretory progression and fate in bloodstream trypanosomes: VSG (GPI2) is a surface protein; truncated VSG (GPI0) is degraded in the lysosome; and native TfR (GPI1) localizes in the flagellar pocket. Tf:Fe starvation results in up-regulation and redistribution of TfR to the plasma membrane suggesting a saturable mechanism for flagellar pocket retention. However, because such surface TfR is non-functional for ligand binding we proposed that it represents GPI2 ESAG6 homodimers that are unable to bind transferrin-thereby mimicking native VSG. We now exploit a novel RNAi system for simultaneous lethal silencing of all native TfR subunits and exclusive in-situ expression of RNAi-resistant TfR variants with valences of GPI0-2. Our results conform to the valence model: GPI0 ESAG7 homodimers traffick to the lysosome and GPI2 ESAG6 homodimers to the cell surface. However, when expressed alone ESAG6 is up-regulated ~7-fold, leaving the issue of saturable retention in the flagellar pocket in question. Therefore, we created an RNAi-resistant GPI2 TfR heterodimer by fusing the C-terminal domain of ESAG6 to ESAG7. Co-expression with ESAG6 generates a functional heterodimeric GPI2 TfR that restores Tf uptake and cell viability, and localizes to the cell surface, without overexpression. These results resolve the longstanding issue of TfR trafficking under over-expression and confirm GPI valence as a critical determinant of intracellular sorting in trypanosomes.

Similarity and match rates of the human dentition in three dimensions: relevance to bitemark analysis.

Uniqueness of the human dentition is a fundamental premise in bitemark analysis. Despite the importance of this key aspect of bitemark methodology, systematic studies of large populations have been limited. Furthermore, there have been no investigations of the significance of the third dimension with regard to dental uniqueness. One hundred digitally scanned mandibular models were analyzed in both 2D and three dimension (3D) using Landmark software. Additionally, 500 3D maxillary and mandibular sets were investigated for determining dental match rate. Statistical analysis was performed with geometric morphometric methods. Results show that measurements in 3D preserve more information about the dentition, reducing but not eliminating random matches in a sample population of 100 mandibular dentitions. Examination of pairs of maxillary and mandibular dentitions showed a substantial number of random matches (197 maxillary, 51 mandibular, one of both maxillary and mandibular). Conclusions indicate that a zero match rate cannot be claimed for the population studied.

Turnover of Variant Surface Glycoprotein in Trypanosoma brucei Is a Bimodal Process.

African trypanosomes utilize glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG) to evade the host immune system. VSG turnover is thought to be mediated via cleavage of the GPI anchor by endogenous GPI-specific phospholipase C (GPI-PLC). However, GPI-PLC is topologically sequestered from VSG substrates in intact cells. Recently, A. J. Szempruch, S. E. Sykes, R. Kieft, L. Dennison, et al. (Cell 164:246-257, 2016, https://doi.org/10.1016/j.cell.2015.11.051) demonstrated the release of nanotubes that septate to form free VSG+ extracellular vesicles (EVs). Here, we evaluated the relative contributions of GPI hydrolysis and EV formation to VSG turnover in wild-type (WT) and GPI-PLC null cells. The turnover rate of VSG was consistent with prior measurements (half-life [t1/2] of ∼26 h) but dropped significantly in the absence of GPI-PLC (t1/2 of ∼36 h). Ectopic complementation restored normal turnover rates, confirming the role of GPI-PLC in turnover. However, physical characterization of shed VSG in WT cells indicated that at least 50% is released directly from cell membranes with intact GPI anchors. Shedding of EVs plays an insignificant role in total VSG turnover in both WT and null cells. In additional studies, GPI-PLC was found to have no role in biosynthetic and endocytic trafficking to the lysosome but did influence the rate of receptor-mediated endocytosis. These results indicate that VSG turnover is a bimodal process involving both direct shedding and GPI hydrolysis. IMPORTANCE African trypanosomes, the protozoan agent of human African trypanosomaisis, avoid the host immune system by switching expression of the variant surface glycoprotein (VSG). VSG is a long-lived protein that has long been thought to be turned over by hydrolysis of its glycolipid membrane anchor. Recent work demonstrating the shedding of VSG-containing extracellular vesicles has led us to reinvestigate the mode of VSG turnover. We found that VSG is shed in part by glycolipid hydrolysis but also in approximately equal part by direct shedding of protein with intact lipid anchors. Shedding of exocytic vesicles made a very minor contribution to overall VSG turnover. These results indicate that VSG turnover is a bimodal process and significantly alter our understanding of the "life cycle" of this critical virulence factor.

An in vitro comparison of ultraviolet versus white light in the detection of adhesive remnants during orthodontic debonding.

OBJECTIVES: To assess the effectiveness and efficiency of ultraviolet (UV) illumination compared to conventional white light in the detection of fluorescent-tagged adhesive remnants during orthodontic debonding. MATERIALS AND METHODS: Orthodontic brackets were bonded to extracted human premolars using one of two bonding resins having fluorescent properties (Pad Lock, Reliance Orthodontics, Itasca, Ill; Opal Bond MV, Opal Orthodontics, South Jordan, Utah; n = 40 each). The brackets were then debonded and, in each adhesive group, half the teeth had the remaining adhesive resin removed under illumination using the operatory light and the other half using a UV (395 nm) light emitting diode (LED) flashlight (n = 20/group). Time for teeth cleanup was recorded. Follow-up images were obtained under a dissecting microscope using UV illumination, and the surface area of adhesive remnants was calculated. Effectiveness of adhesive removal was also assessed using scanning electron microscopy imaging. Analysis of variance and Kruskal-Wallis tests were used to analyze time and adhesive remnants, respectively. RESULTS: Assessment using the dissecting microscope found groups using UV light during adhesive removal had statistically significantly lower amounts of adhesive remnants than groups using white light (P ≤ .01). Time for adhesive removal was significantly lower with Opal Bond MV adhesive using UV light when compared with the white light (P ≤ .01). Assessment by scanning electron microscopy showed that thin remnants of adhesive (<2 µm) remained undetected by UV illumination. CONCLUSIONS: UV light is more effective and tends to be more efficient than white light in the detection of fluorescent adhesive during orthodontic debonding. Although there are limitations, the use of UV LED lighting is a practical tool that aids in adhesive detection.

Publisher Correction: Metabolic reprogramming of stromal fibroblasts by melanoma exosome microRNA favours a pre-metastatic microenvironment.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Ultraviolet illumination as an adjunctive aid in dental inspection.

Tooth-colored resin fillings have become increasingly popular as restorative materials. Their presence in the dentition presents a challenge to the clinician and the forensic odontologist, as detection of the fillings can be difficult both visually and radiographically. As they necessarily form part of the unique dentition of an individual, recognition of the resins is important for forensic identification. Alternative light sources have been used with success in various fields of forensic science. In recent years small LED flashlights emitting at specific wavelengths in the ultraviolet light (UV) range have been developed. Their low cost, small size, and ready availability makes their use practical in both forensic dental inspection and clinical settings. UV inspection is of interest because enamel, dentin and dental materials all have differing fluorescent properties when illuminated by UV light. It was one goal of this research to quantitatively assess the fluorescence properties of modern restorative resins in order to predict their behavior during inspection using UV illumination. The second goal was to demonstrate practical use of UV in dental inspection with examples of how different materials fluoresce. Quantitative measurements were obtained for optical emission wavelength and intensity for 15 modern resins using a spectrophotometer. Results indicated that resin brands fluoresce at different wavelengths and with varying intensities. Practical use and comparison of the flashlights revealed that the most useful excitation wavelengths for resin detection were in the UVA range (365 and 380 nm). Porcelain restorations and composite resin fillings exhibited different responses to these two wavelengths and thus use of both is recommended for forensic dental inspection.

Analytical survey of restorative resins by SEM/EDS and XRF: databases for forensic purposes.

Frequently in forensic cases, unknown substances must be identified. Automated databases can ease the burden of comparison as materials may be compared against many known standards in a relatively short period of time. It has been shown that dental resins can be named according to brand or brand group even in conditions as harsh as cremation. Databases are already in use for many materials, but no such database exists for dental resins. Thus, two databases were generated. One utilized a laboratory-based method, scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM/EDS), in conjunction with the Spectral Library Identification and Classification Explorer (SLICE) software. The other was based on portable X-ray fluorescence (XRF). The ability to perform database comparison with portable instrumentation can thus be brought directly to the field. Both the SLICE and XRF databases were evaluated by testing unknown resins. EDS is a well-established technique and the SLICE program was demonstrated to be a good tool for unknown resin identification. Portable XRF is a relatively new instrument in this regard and its databases have been constructed mostly for metal alloy comparison and environmental soil testing. However, by creation of a custom spectral library, it was possible to distinguish resin brand and bone and tooth from other substances.

Identification of incinerated root canal filling materials after exposure to high heat incineration.

With the increase in global terrorism there is a higher probability of having to identify victims of incineration events secondary to incendiary explosive devices. The victims of incineration events challenge forensic odontologists when coronal restorations are no longer present to compile postmortem data. With 40 million root canals being completed annually in the United States, a very large pool of antemortem data is available to the forensic odontologist to make positive identifications. When complete and thorough dental records exist, individuals that have undergone surgical and nonsurgical root canal therapy may have materials present in the canal that may aid in identification. This study provides elemental fingerprints of root canal obturation materials to be utilized as a forensic identification aid. This study used scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDS) to assess the elemental composition of materials before and after high temperature incineration. Sixteen endodontic materials were analyzed pre-incineration and placed in extracted teeth. The filled teeth were subjected to incineration at 900 degrees C for 30 min to simulate incineration events or cremation. Incinerated materials were radiographed and re-analyzed to determine if they retained their original elemental composition. Endodontic sealers, gutta percha, root-end filling materials, silver points, and separated files were distinguishable in the canal and traceable after incineration. The authors present a fingerprint of the endodontic obturation materials that are capable of withstanding high heat incineration to be used as an aid for postmortem identification. This work represents the initial stage of database generation for root canal filling materials for use as an aid in forensic identification.

Metabolic reprogramming of stromal fibroblasts by melanoma exosome microRNA favours a pre-metastatic microenvironment.

Local acidification of stroma is proposed to favour pre-metastatic niche formation but the mechanism of initiation is unclear. We investigated whether Human Melanoma-derived exosomes (HMEX) could reprogram human adult dermal fibroblasts (HADF) and cause extracellular acidification. HMEX were isolated from supernatants of six melanoma cell lines (3 BRAF V600E mutant cell lines and 3 BRAF wild-type cell lines) using ultracentrifugation or Size Exclusion Chromatography (SEC). Rapid uptake of exosomes by HADF was demonstrated following 18 hours co-incubation. Exposure of HDAF to HMEX leads to an increase in aerobic glycolysis and decrease in oxidative phosphorylation (OXPHOS) in HADF, consequently increasing extracellular acidification. Using a novel immuno-biochip, exosomal miR-155 and miR-210 were detected in HMEX. These miRNAs were present in HMEX from all six melanoma cell lines and were instrumental in promoting glycolysis and inhibiting OXPHOS in tumour cells. Inhibition of miR-155 and miR-210 activity by transfection of miRNA inhibitors into HMEX reversed the exosome-induced metabolic reprogramming of HADF. The data indicate that melanoma-derived exosomes modulate stromal cell metabolism and may contribute to the creation of a pre-metastatic niche that promotes the development of metastasis.

Identification through X-ray fluorescence analysis of dental restorative resin materials: a comprehensive study of noncremated, cremated, and processed-cremated individuals.

Tooth-colored restorative materials are increasingly being placed in the practice of modern dentistry, replacing traditional materials such as amalgam. Many restorative resins have distinct elemental compositions that allow identification of brand. Not only are resins classifiable by elemental content, but they also survive extreme conditions such as cremation. This is of significance to the forensic odontologist because resin uniqueness adds another level of certainty in victim identification, especially when traditional means are exhausted. In this three-part study, unique combinations of resins were placed in six human cadavers (total 70 restorations). Simulated ante-mortem dental records were created. In a blind experiment, a portable X-ray fluorescence (XRF) unit was used to locate and identify the resin brands placed in the dentition. The technique was successful in location and brand identification of 53 of the restorations, which was sufficient to enable positive victim identification among the study group. This part of the experiment demonstrated the utility of portable XRF in detection and analysis of restorative materials for victim identification in field or morgue settings. Identification of individuals after cremation is a more difficult task, as the dentition is altered by shrinkage and fragmentation, and may not be comparable with a dental chart. Identification of processed cremains is a much greater challenge, as comminution obliterates all structural relationships. Under both circumstances, it is the nonbiological artifacts that aid in identification. Restorative resin fillings can survive these conditions, and can still be named by brand utilizing elemental analysis. In a continuation of the study, the cadavers were cremated in a cremation retort under standard mortuary conditions. XRF was again used to analyze retrieved resins and to identify the individuals based on restorative materials known to exist from dental records. The cremains were then processed and the analysis was repeated to determine whether restorative resins could be found under this extreme condition. Under both circumstances, sufficient surviving resin material was found to distinguish positively each individual in the study group. This study showed the utility of XRF as an analytical tool for forensic odontology and also the significance of the role of restorative resins in victim identification, even after cremation.

Detection and classification of composite resins in incinerated teeth for forensic purposes.

The great demand for esthetic restorations has resulted in placement of large numbers of composite resin fillings. The popularity of these materials is reflected in the quantity and variety of resin brands currently on the market. The ability to distinguish resin brands can aid in positive identification of burn victims, assuming that appropriate dental records exist. Scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDS) was used to characterize the structure and composition of 10 modern resins. The structure of each resin was unique to manufacturer, and elemental analysis allowed separation into distinct groups. These 10 resins were also placed in extracted teeth and incinerated at 900 degrees C for 30 min, simulating near cremation conditions. The resins were identifiable by SEM/EDS after incineration, and the elemental composition remained almost unchanged. The data produced are immediately useful for resin identification in forensics, and comparative analysis can be readily performed using standard equipment. This work represents the initial stage of database generation.

Practical Considerations in Trace Element Analysis of Bone by Portable X-ray Fluorescence.

Forensic anthropologists are more often turning to nondestructive methods to assist with skeletal analyses, specifically for trace elemental analyses. Portable XRF (pXRF) instruments are versatile and are able to be used in diverse settings or for specimens of a shape and size that cannot be accommodated by laboratory-based instruments. Use of XRF requires knowledge of analysis parameters such as X-ray penetration and exit depth. Analysis depth was determined by examining pure elements through known thicknesses of equine bone slices. Correlation between the element's X-ray emission energy and the depth of reading was observed. Bone surfaces from a small unknown historic cemetery were analyzed before and after sanding of the periosteal surface to observe possible changes in XRF readings based on potential diagenesis. Results validate the pXRF device as a powerful and convenient instrument for nondestructive analysis, while highlighting limitations and considerations for the analysis of osseous materials.

Forensic bitemark identification: weak foundations, exaggerated claims.

Several forensic sciences, especially of the pattern-matching kind, are increasingly seen to lack the scientific foundation needed to justify continuing admission as trial evidence. Indeed, several have been abolished in the recent past. A likely next candidate for elimination is bitemark identification. A number of DNA exonerations have occurred in recent years for individuals convicted based on erroneous bitemark identifications. Intense scientific and legal scrutiny has resulted. An important National Academies review found little scientific support for the field. The Texas Forensic Science Commission recently recommended a moratorium on the admission of bitemark expert testimony. The California Supreme Court has a case before it that could start a national dismantling of forensic odontology. This article describes the (legal) basis for the rise of bitemark identification and the (scientific) basis for its impending fall. The article explains the general logic of forensic identification, the claims of bitemark identification, and reviews relevant empirical research on bitemark identification-highlighting both the lack of research and the lack of support provided by what research does exist. The rise and possible fall of bitemark identification evidence has broader implications-highlighting the weak scientific culture of forensic science and the law's difficulty in evaluating and responding to unreliable and unscientific evidence.

A new model for nanoscale enamel dissolution.

The dissolution kinetics of human tooth enamel surfaces was investigated using nanomolar-sensitive constant composition (CC) and in situ atomic force microscopy (AFM) under simulated caries formation conditions (relative undersaturation with respect to hydroxyapatite = 0.902, pH = 4.5). Scanning electron microscopic (SEM) examination of the resulting etched enamel surfaces showed that deminerzalization, initiated at core/wall interfaces of rods, developed anisotropically along the c-axes. After an initial rapid removal of surface polishing artifacts, the dissolution rate decreased as the reaction proceeded in accordance with our recently proposed crystal dissolution model, resulting in hollow enamel cores and nanosized remaining crystallites, resistant to further dissolution. Generally, dissolution of minerals is regarded as a spontaneous reaction in which all the solid phase can be dissolved in undersaturated solutions. However, the dissolution of some biominerals may be suppressed when the crystallites approach nanometer size. This study shows that CC demineralization of enamel in acidic medium follows this new model that can be used to mimic carious lesion formation. In dissolution studies, nanosized enamel crystallites exhibit a remarkable degree of self-preservation in the fluctuating physiological milieu.

Evaluation of varied repair protocols applied to aged composite resin.

PURPOSE: This paper aims to examine varied surface treatments combined with adhesive bonding to ascertain the highest bond strength of the composite resin repair. MATERIALS AND METHODS: A total of 100 specimens was made using an increment technique. The prepared specimens were aged for 20 days in 37 degrees +/- 20 degrees C water and then kept for 24 h at room temperature. The samples were separated into 5 groups of 20, and each group then received a different surface treatment (control group, phosphoric acid, diamond bur, air abrasion, silane primer combined with a diamond bur treatment). The effect of each surface treatment on the bond strength was determined by a shear bond test. RESULTS: Specimens that received surface treatment with a diamond bur exhibited higher shear bond strengths than any of the other repaired groups (p < 0.05). Those treated with air abrasion had the second highest shear bond strengths, but without a statistically significant difference when compared to the control group (p > 0.05). Specimens treated with phosphoric acid showed virtually no difference compared to the specimens of the control group (p > 0.05). CONCLUSION: Statistical analysis revealed that surface treatment with a diamond bur results in higher shear bond strength than other tested methods.

Complex layered dental restorations: Are they recognizable and do they survive extreme conditions?

Recent research has shown that restorative dental materials can be recognized by microscopy and elemental analysis (scanning electron microscopy/energy dispersive X-ray spectroscopy and X-ray fluorescence; SEM/EDS and XRF) and that this is possible even in extreme conditions, such as cremation. These analytical methods and databases of dental materials properties have proven useful in DVI (disaster victim identification) of a commercial plane crash in 2009, and in a number of other victim identification cases. Dental materials appear on the market with ever expanding frequency. With their advent, newer methods of restoration have been proposed and adopted in the dental office. Methods might include placing multiple layers of dental materials, where they have different properties including adhesion, viscosity, or working time. These different dental materials include filled adhesives, flowable resins, glass ionomer cements, composite resins, liners and sealants. With possible combinations of different materials in these restorations, the forensic odontologist is now confronted with a new difficulty; how to recognize each individual material. The question might be posed if it is even possible to perform this task. Furthermore, an odontologist might be called upon to identify a victim under difficult circumstances, such as when presented with fragmented or incinerated remains. In these circumstances the ability to identify specific dental materials could assist in the identification of the deceased. Key to use of this information is whether these new materials and methods are detailed in the dental chart. Visual or radiographic inspection may not reveal the presence of a restoration, let alone the possible complex nature of that restoration. This study demonstrates another scientific method in forensic dental identification.

Engineering a bioactive matrix by modifications of calcium sulfate.

The goal of this study was to define the conditions for the fabrication of a bioactive matrix that induces and supports cell proliferation and tissue regeneration. The proposed hypothesis was that a composite graft could be engineered by the absorption of platelet-rich plasma (PRP) onto calcium sulfate (CS). Evaluation of the biological activity of the engineered grafts was based on osteoblast proliferation studies and scanning electron microscopy (SEM) analyses. Graft samples were created in a standard size and shape so that the surface available for attachment and cell proliferation was always identical. Proliferation data were expressed as counts per minute per group and differences among groups were statistically analyzed by analysis of variance followed by the Scheffé test (alpha = 0.1). SEM analysis showed that the combination of CS and PRP presents a preserved crystalline structure well integrated by organic matrix. This combination showed the highest cell proliferation levels (p < 0.001). Further evaluations demonstrated that PRP is activated when combined with CS. When tested as a possible carrier for biologically active molecules such as platelet-derived growth factor (PDGF), CS showed increased cell proliferation (p < 0.001). SEM revealed adherent osteoblasts with broad flattened edges on CS-PRP. This study proposes CS as an efficient carrier for PRP or PDGF and supports the use of these combinations as bioactive matrices in clinical or laboratory applications.

Noncarious cervical lesions and abfractions: a re-evaluation.

BACKGROUND: The concept of abfraction is controversial. The authors present the fundamental basis of abfraction and review studies that describe the condition. TYPES OF STUDIES REVIEWED: The authors used data on masticatory forces, enamel and dentin properties, as well as stress studies related to abfraction, for background information. They also analyzed the genesis of the abfraction theory, experimental evidence, case presentations, clinical investigations and restorative studies. RESULTS: The theory of abfraction is based primarily on engineering analyses that demonstrate theoretical stress concentration at the cervical areas of teeth. While some recent stress studies support earlier findings, others have provided significant deviating information. Few controlled studies exist that demonstrate the relationship between occlusal loading and abfraction lesions. The role of occlusal loading in noncarious cervical lesions (as shown by clinical data) appears to be part of a multifactorial event that may not necessarily follow the proposed classic abfraction mechanism, and other mechanisms or factors may explain cervical restoration failure just as well. CLINICAL IMPLICATIONS: There is little direct evidence supporting the theory of abfraction as the primary factor in the formation of noncarious cervical lesions. Controlled clinical trials are necessary to elucidate more fully the etiology of those lesions.

Toothbrushing and gingival recession.

AIM: To review the hypothesis that toothbrushing leads to gingival recession. Gingival recession develops due to anatomical and pathological factors. The prevalence of recession is dependent on the age and characteristic of the population because it usually presents in individuals with periodontal disease or those who practise zealous or improper oral hygiene methods. Gingival trauma and gingival abrasion from toothbrushing is thought to progress directly to gingival recession. Case studies documenting recession from toothbrush trauma are speculative. Short-term studies suggest that gingival trauma and gingival abrasion may result from toothbrushing, but the direct relationship between traumatic home care and gingival recession is inconclusive. Long-term studies remain elusive or do not support the development of recession following toothbrushing. Nevertheless, tooth abrasion may be an integral part in the aetiology of recession. Toothbrush abrasion also may cause wear at the cemento-enamel junction resulting in the destruction of the supporting periodontium leading to recession.