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.

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.

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.

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.

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.

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.

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.

Distortion in fingerprints: a statistical investigation using shape measurement tools.

Friction ridge impression appearance can be affected due to the type of surface touched and pressure exerted during deposition. Understanding the magnitude of alterations, regions affected, and systematic/detectable changes occurring would provide useful information. Geometric morphometric techniques were used to statistically characterize these changes. One hundred and fourteen prints were obtained from a single volunteer and impressed with heavy, normal, and light pressure on computer paper, soft gloss paper, 10-print card stock, and retabs. Six hundred prints from 10 volunteers were rolled with heavy, normal, and light pressure on soft gloss paper and 10-print card stock. Results indicate that while different substrates/pressure levels produced small systematic changes in fingerprints, the changes were small in magnitude: roughly the width of one ridge. There were no detectable changes in the degree of random variability of prints associated with either pressure or substrate. In conclusion, the prints transferred reliably regardless of pressure or substrate.

Shape measurement tools in footwear analysis: a statistical investigation of accidental characteristics over time.

Presence of accidental characteristics on footwear strengthens the linkage of a given piece of footwear to a footwear impression left at a crime-scene. Thus an understanding of rate of appearance and disappearance of these characteristics is of importance. Artificial cut-marks, 1-3mm in depth, were cut into outsoles of 11 pairs of athletic shoes. Loss of these cut-marks and acquisition of new accidental characteristics/wear patterns were monitored over a seven-week time-span. Feature-vector methods were used to acquire multivariate data on wear/acquisition rates. A repeatability study indicated the feature vector method could detect small differences among shoes relative to measurement uncertainty. The shoes displayed a strong retention of artificial cut-marks over the study interval. Net rate of wear was 0.1% of the textured area of the shoe per week, predominantly in the heel and ball area. Results indicate accidental characteristics can reasonably be expected to persist over time.

Effect of systematic dental shape modification in bitemarks.

Studies on human cadaver models have reported significant levels of distortion of bitemarks in skin, indicating that tooth characteristics are not reliably transferred and recorded in the bitten subject. Moreover, matches among the anterior biting dentition in open population studies have been found. This prompts the question as to what degree of difference in shape will distinguish one dentition from another as reflected in a bitemark. In order to understand how these variables appear on skin, 10 dental casts with systematic variations in tooth positions were produced. The height of the lateral incisors was systematically altered in 1mm increments up to 3mm and lateral incisor/canines were altered in facial/lingual displacement in 1mm increments up to 5mm. Each of the models was used to produce a series of 10 repeated bites, distributed over arms and legs of un-embalmed cadavers. Landmark-based geometric morphometrics were used for analysis of digital images of the bitemarks. Results indicate that alterations of height and displacement of particular teeth affected the position of impressions created by the adjacent teeth. Displacement of one lateral incisor/canine led to a relative shift in impressions of the central incisors and unaltered canines, while height alteration of the lateral incisors led to a shift in relative position of central incisors as recorded in the bitemark. The prominence of displacements was more pronounced in the bitemarks than in images of the dentition used to make the bites, thus the bitemarks tended to exaggerate the differences. It was found that a displacement of 5mm between teeth allowed for reliable distinction between bitemarks. No such threshold of distinction could be established for differences in height of teeth under these experimental conditions. The effect of distortion was more significant in the mandibular than maxillary arch, suggesting that the mandible exhibits higher variation than the maxilla, as impressed in skin. Numerous bitemarks also exhibited arch flattening, consistent with recent studies showing arch width as the principal variable in a bitemark.

Patterns of variation and match rates of the anterior biting dentition: characteristics of a database of 3D-scanned dentitions.

An understanding of the variability of the anterior human dentition is essential in bitemark analysis. A collection of 1099 3D laser scans of paired maxillary and mandibular arches were studied using geometric morphometric methods. Analyses were performed without scale (shape only) and with scale (shape and size). Specimens differing by no more than experimentally obtained measurement error were counted as matches, or as indistinguishable. A total of 487 maxillary (396 size preserved), 131 mandibular (83 size preserved), and one paired dentition (two size preserved) matches were found. Principal component analysis and partial least squares revealed interpretable patterns of variation and covariation in dental shape, principally dominated by variation in dental arch width. The sensitivity of match rate to assumed degree of measurement error was also determined showing rapid increases in match rate as measurement error increased. In conclusion, the concept of dental uniqueness with regard to bitemark analysis should be approached with caution.

Bitemarks: distortion and covariation of the maxillary and mandibular dentition as impressed in human skin.

In bitemark analysis the extent of distortion of both maxillary and mandibular arches and how one affects the other has not been studied. A single dentition was used to create 49 bites on unembalmed cadavers. Landmarks were placed on digital images of the bitemarks and scanned images of the biting dentition. A sample of 297 randomly acquired dental models was used for comparison purposes. Geometric morphometric techniques were utilized to statistically describe size and shape change, as well as the correlation between the two arches. Results indicate that the predominant distortion seen was in arch width, at 7-28 times as large as measurement error in the biting dentition and roughly 50% of the variation seen in the random population of dentitions. The correlation of arch width distortion between arches was very low (∼0.03). However, the principal patterns of all shape variation did co-vary in the bitemarks produced by the maxillary and mandibular dentition, an effect indicating independence of size and shape distortion. In conclusion, bitemark analysis should be approached with caution when the principal difference between suspects is arch width.

A study of multiple bitemarks inflicted in human skin by a single dentition using geometric morphometric analysis.

Criticisms of the forensic discipline of bitemark analysis state that the range of distortion in the shape of bitemark impressions in skin has not been scientifically established. No systematic statistical studies exist that explore this problem. As a preliminary investigation of this issue, a single dentition was mounted in a mechanical apparatus and used to create 89 bitemarks in human cadaver skin, both parallel and perpendicular to tension lines. Impressions of the same dentition were also created in wax. 2D scanned images of the biting dentition were obtained. Locations of incisal edges of all 6 anterior teeth as well as the midpoint of the canine were captured as landmarks in all specimens. This set of landmark data was then studied using established geometric morphometric methods. All specimen shapes were compared using Procrustes superimposition methods, and by a variation of Procrustes superimposition which preserves scale information. Match criteria were established by examining the range of variation produced by repeated measurements of the dentition for each class of specimen. The bitemarks were also compared to a population of 411 digitally scanned dentitions, again using the match criteria. Results showed that bitemarks in wax had lower measurement error than scanned images of the dentition, and both were substantially lower than measurement error as recorded in skin. None of the 89 bitemarks matched the measured shape of the biting dentition or bitemarks in wax, within the repeated measurements error level, despite the fact that all bitemarks were produced by this dentition. Comparison of the bitemarks to the collection of 411 dentitions showed that the closest match to the bitemarks was not always the same dentition that produced the bitemarks. Examination of Procrustes plots of matched shapes showed non-overlapping distributions of measurements of bitemarks in skin, wax, and the dentition. All had statistically significant differences in mean shape. Principal component analysis (PCA) and canonical variates analysis (CVA) both showed clear segregation of the three types of data. The patterns of variance revealed by PCA showed several distinct patterns produced by skin distortion; alteration of relative arch width, and varying displacement of non-aligned teeth in the dentition. These initial results indicate that when multiple suspects possess similar dentitions, bitemark analysis should be approached with caution.

Dental shape match rates in selected and orthodontically treated populations in New York State: a two-dimensional study.

Forensically identifying a suspect's dentition from a bitemark in an open population requires the supposition that every person's dental alignment is different. There have been few studies that have tested this claim. Four hundred and ten lower anterior dentitions from a selected population and 110 lower anterior dentitions from one that was orthodontically treated were measured using geometric morphometric analysis, allowing comparison of arch shape. Dental match rates of 1.46% and 42.7% of individuals were found in the respective populations, given an established measurement error. Orthodontic treatment had a strong effect on match rate suggesting that treated or naturally well-aligned dentitions may be indistinguishable. Sexual dimorphism was found to be only slightly significant. Principal shape variation in both populations was degree of arch curvature. Results of studying these populations show that dental matches can occur, and that statements of certainty concerning individualization in such populations should be approached with caution.

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.

Statistical evidence for the similarity of the human dentition.

Recent scrutiny of forensic science has focused on unreliability of expert witness testimony when based on statements of individuality. In bitemark analysis, assumptions regarding uniqueness of the dentition have been based on use of the product rule while ignoring correlation and nonuniformity of dental arrangement. To examine the effect of these factors, two separate sets of scanned dental models (n=172 and n=344) were measured and statistically tested to determine match rates. Results were compared to those of a prior study. Seven and 16 matches of the six anterior lower teeth were found in the respective data sets. Correlations and nonuniform distributions of tooth positions were observed. Simulation tests were performed to verify results. Results indicate that given experimental measurement parameters, statements of dental uniqueness with respect to bitemark analysis in an open population are unsupportable and that use of the product rule is inappropriate.

Photoactivated disinfection of Streptococcus intermedius through dentin disc at clinically relevant intervals: an in vitro study.

In this present study we have tested the impact of porfimer sodium (Photofrin, AXCAN PHARMA Inc., Quebec, Canada) photoactivated disinfection (PD) on cells of Streptococcus intermedius in suspension. In order to provide basic data to support future clinical studies of PD in dentistry the study used exposure to Quartz-tungsten-halogen (QTH) dental curing light for clinically relevant time periods to activate Photofrin and measured its effectiveness under a variety of conditions including activation through dentin hard tissue. S. intermedius was grown in planktonic suspension for 48h. Nine groups were formed: three control groups (1-3) and six experimental groups (4-9). Groups 4-6 tested the use of Photofrin treatment combined with QTH light at various intervals of irradiation (5, 15 and 60s). Groups 7-9 were similar to groups 4-6 with the exception that irradiation commenced through a dentin disc. Following treatment, bacteria were plated. Colony counts were measured following 72h incubation at 37 degrees C. Statistical analysis was carried out by one-way ANOVA at a 95% confidence level. A significant reduction in S. intermedius colony counts was observed for all experimental groups and one control group. The reduction in numbers of colonies in the experimental groups varied from 79.28 to 99.40% with an average of 94.61%. Reduction in viable bacterial cells indicated a strong relationship between power density and irradiation interval. When curing light energy density was lower due to the irradiation through the 1mm dentin disc, prolonged irradiation interval enhanced bacterial kill. In conclusion, where direct irradiation is not possible for PD treatment, irradiation through dentin may still be done successfully within a clinically relevant interval.

The response of skin to applied stress: investigation of bitemark distortion in a cadaver model.

Knowledge of distortional properties of skin is important in bitemark analysis. Thus, the response of skin to stress from bites was investigated. Four sets of models were created from the dentition of one individual. Anterior teeth were systematically removed to vary contact surface area. A biting apparatus was constructed with an integrated load cell. Forty-six bites were created perpendicular to Langer lines on six cadavers. Rate of force application and bite pressure were controlled. Metric/angular measurement and hollow volume overlays were employed. Distortion produced by each dentition was calculated and assessed. Results showed that as teeth impressed loose tissue, mesial/distal distance increased, angles of rotation flattened, and inter-canine distance lengthened. An opposite effect was seen in tight tissue. When the surface area of the dentition was reduced, a mixture of these effects was observed. Conclusions indicated that stiffness of the tissue was the most important variable in bitemark distortion.