Effect of temporary freezing on postmortem protein degradation patterns.

BACKGROUND: A precise determination of time since death plays a major role in forensic routine. Currently available techniques for estimating the postmortem interval (PMI) are restricted to specific time periods or cannot be applied for individual case-specific reasons. During recent years, it has been repeatedly demonstrated that Western blot analysis of postmortem muscle protein degradation can substantially contribute to overcome these limitations in cases with different background. Enabling to delimit time points at which certain marker proteins undergo distinct degradation events, the method has become a reasonable new tool for PMI delimitation under various forensic scenarios. However, additional research is yet required to improve our understanding of protein decomposition and how it is affected by intrinsic and extrinsic factors. Since there are temperature limits for proteolysis, and investigators are confronted with frozen corpses, investigation of the effects of freezing and thawing on postmortem protein decomposition in the muscle tissue is an important objective to firmly establish the new method. It is also important because freezing is often the only practical means to intermittently preserve tissue samples from both true cases and animal model research. METHODS: Sets of dismembered pig hind limbs, either freshly detached non-frozen, or thawed after 4 months of freeze-storage (n = 6 each), were left to decompose under controlled conditions at 30 °C for 7 days and 10 days, respectively. Samples of the M. biceps femoris were regularly collected at predefined time points. All samples were processed via SDS-PAGE and Western blotting to identify the degradation patterns of previously characterized muscle proteins. RESULTS: Western blots show that the proteins degrade predictably over time in precise patterns that are largely unaffected by the freeze-and-thaw process. Investigated proteins showed complete degradation of the native protein band, partly giving rise to degradation products present in distinct time phases of the decomposition process. CONCLUSION: This study provides substantial new information from a porcine model to assess the degree of bias that freezing and thawing induces on postmortem degradation of skeletal muscle proteins. Results support that a freeze-thaw cycle with prolonged storage in frozen state has no significant impact on the decomposition behavior. This will help to equip the protein degradation-based method for PMI determination with a robust applicability in the normal forensic setting.

Morphological changes and protein degradation during the decomposition process of pig cadavers placed outdoors or in tents-a pilot study.

The delimitation of the postmortem interval (PMI) is of utmost importance in forensic science. It is especially difficult to determine the PMI in advanced decomposition stages and/or when dead bodies are found under uncommon circumstances, such as tents, or other (semi-) enclosed environments. In such cases, especially when insect access is restricted, morphological assessment of body decomposition is one of the remaining approaches for delimitation of the PMI. However, as this method allows only vague statements/indications about the PMI, it is required to develop new and more reliable methods. One of the most important candidates is the biochemical analysis of protein degradation. In this regard, it has been demonstrated that specific skeletal muscle protein degradation patterns characterize certain time points postmortem and thus can be used as markers for PMI estimation. In order to test this method in different micro-environments, a pilot study using ten pig carcasses was conducted in summer in Northern Germany. The cadavers were openly placed outside (freely accessible for insects), as well as enclosed in tents nearby, and left to decompose to investigate decomposition processes over a time course of 10 days. Muscle samples of the M. biceps femoris were collected on a regular basis and processed via SDS-PAGE and degradation patterns of selected proteins identified by Western blotting. In addition, morphological changes of the cadavers during decomposition were assessed using the total body score (TBS). Results showed that postmortem protein degradation patterns are largely consistent between treatment groups (open field versus tents) despite major morphological differences in the decomposition rate. This field study provides evidence that muscle protein degradation is mostly unaffected by different levels of exposure, making it a sufficient candidate for PMI delimitation under various circumstances.

Dismembered porcine limbs as a proxy for postmortem muscle protein degradation.

The estimation of the postmortem interval (PMI) is of critical importance in forensic routine. The most frequently applied methods, however, are all restricted to specific time periods or must be excluded under certain circumstances. In the last years it has been shown that the analysis of muscle protein degradation has the potential to contribute to according delimitations in practice. In particular, upon biochemical analysis, the specific time points of degradation events provide reasonable markers for PMI delimitation. Nevertheless, considerable research is yet required to increase the understanding of protein decomposition and how it is affected by individual and environmental influencing factors. This is best investigated under standardized conditions, however, a considerate selection of proxies, regarding costs, effort, and expected outcome is required. Here, we use pigs to compare muscle protein decomposition in whole bodies and dismembered body parts (amputated hind limbs). Not only do experiments on body parts reduce the costs and allow easier handling in basic research, but also they aid to investigate the practical application of PMI estimation in dismembered body parts, or other extensive injuries, which are not unusual scenarios in crime investigation. Specifically, we investigated whether there are differences in the degradation rates of selected muscle proteins, sampled from dismembered legs and from hind limbs attached to whole pig bodies. Our results show distinct time-dependent degradation patterns of muscle proteins in a predictable manner regardless of sample origin. We are able to demonstrate that amputated hind limbs are suitable proxies for the analysis of muscle protein degradation, especially to investigate certain influencing factors and establish according standardized models.

Does altered protein metabolism interfere with postmortem degradation analysis for PMI estimation?

An accurate estimation of the postmortem interval (PMI) is a central aspect in forensic routine. Recently, a novel approach based on the analysis of postmortem muscle protein degradation has been proposed. However, a number of questions remain to be answered until sensible application of this method to a broad variety of forensic cases is possible. To evaluate whether altered in vivo protein metabolism interferes with postmortem degradation patterns, we conducted a comparative study. We developed a standardized animal degradation model in rats, and collected additional muscle samples from animals recovering from muscle injury and from rats with developed disuse muscle atrophy after induced spinal cord injury. All samples were analyzed by SDS-PAGE and Western blot, labeling well-characterized muscle proteins. Tropomyosin was found to be stable throughout the investigated PMI and no alterations were detected in regenerating and atrophic muscles. In contrast, significant predictable postmortem changes occurred in desmin and vinculin protein band patterns. While no significant deviations from native patterns were detected in at-death samples of disuse muscle atrophy, interestingly, samples of rats recovering from muscle injury revealed additional desmin and vinculin degradation bands that did not occur in this form in any of the examined postmortem samples regardless of PMI. It remains to be investigated whether in vivo-altered metabolism influences postmortem degradation kinetics or if such muscle samples undergo postmortem degradation in a regular fashion.

Four-year Survival of Endodontically Treated Premolars Restored with Fiber Posts.

The objective of this prospective clinical trial was to investigate the influence of the residual coronal structure of endodontically treated teeth and the type of cement used for luting fiber posts on four-year clinical survival. Two groups (n = 60) were defined, depending on the amount of residual coronal dentin after abutment build-up and final preparation: (1) more than 50% of coronal residual structure; and (2) equal to or less than 50% of coronal residual structure. Within each group, teeth were randomly divided into 2 subgroups (n = 30) according to the material used for luting fiber posts: (A) resin core build-up material, Gradia Core; or (B) self-adhesive universal cement GCem Automix. The rate of success was assessed based on clinical and intra-oral radiographic examinations at the follow-up after 6, 12, 24, 36, and 48 months. The highest 48-month success and survival rates were recorded in group 1A (90% and 100%, respectively), whereas teeth in group 2B exhibited the lowest performance (63.3% success rate, 86.6% survival rate). Cox regression analysis revealed that neither the amount of coronal residual structure nor the luting material significantly influenced the failure risk (p > .05) (ClinicalTrials.gov, NCT01532947).

Psychometric validation of the Spanish version of the Dundee Ready Education Environment Measure applied to dental students.

AIM: To carry out a psychometric evaluation of the Spanish-language version of the Dundee Ready Education Environment Measure (DREEM) applied to dental students. METHODS: A total of 1,391 students from nine Spanish public schools of dentistry responded to the DREEM questionnaire. To analyse the reliability of the DREEM questionnaire, the internal consistency was assessed and a 'test-retest' carried out. Validity was evaluated through analysis of item response rate, floor and ceiling effects, corrected item-total and item-subscale correlations and factor structure. A confirmatory factor analysis was performed to analyse the structure of the original DREEM scale. RESULTS: Cronbach's alpha coefficient for the 'Educational Climate' (EC) global scale was 0.92. In the subscales, the 'observed' Cronbach's alpha coefficients ranged between 0.57 and 0.79 and were higher than the 'expected' ones; except for the Social subscale. In the DREEM questionnaire, all of the corrected correlation coefficients between the items and the EC global scale, and the items and their corresponding subscales, were >0.2; except for items 50 and 17. All goodness-of-fit indices of confirmatory factor analysis showed acceptable values (close to one or zero, depending on the case), and there was consistency in the results. CONCLUSIONS: The Spanish-language version of the DREEM questionnaire is a reliable and valid instrument for analysing the EC for dental students and its factor structure is supported by the data. Although our findings indicate that the DREEM may be as culturally independent as was originally stated, more research should be directed at verifying the factor structure in various languages and cultural environments.

Analysis of the 'Educational Climate' in Spanish Public Schools of Dentistry using the Dundee Ready Education Environment Measure: a multicenter study.

AIM: To analyse the 'Educational Climate' (EC) of dental students in Spain. METHODS: The study group consisted of 1391 students from nine Spanish Public Schools of Dentistry, who responded to the questionnaire based on 'Dundee Ready Education Environment Measure' (DREEM). This questionnaire has 50 items that are grouped into five domains: Learning, Teachers, Academic, Atmosphere and Social. RESULTS: The global score on the EC was 123.1 (interpretation: 'EC more positive than negative'). The scores obtained in the different domains were: 28.0 in Learning (interpretation: 'a generally positive perception of learning'), 26.8 in Teachers (interpretation: 'teachers are going in the right direction'), 20.8 in Academic (interpretation: 'feeling more on the positive side'), 29.7 in Atmosphere (interpretation: 'a generally positive atmosphere') and 17.7 in Social (interpretation: 'social perception acceptable'). In seven items (14%), an average of <2 was detected, showing that there are some educational problem areas. Regarding the EC in the different Schools of Dentistry, an average of >100 was achieved in all of them, although there were two centres that showed significantly higher values of EC. CONCLUSIONS: Spanish dental students felt that their EC was more positive than negative and considered that the different domains were positive and acceptable. However, they pointed out the existence of several educational problem areas associated with the development of a traditional curriculum. Accordingly, and in parallel with the implementation of an innovative curriculum in all Spanish Dental Schools in the coming years, immediate educational goals must address the problem areas identified, thereby further promoting a more positive perception of EC.

Fatal aconitine intoxication or thyroid storm? A case report.

The case of a female in the latter half of her teens found dead in her father's apartment is reported. A glass containing liquid and plant remnants was found at the death scene. There were no indications of any intervention or the application of force by a third party. Autopsy showed unremarkable findings. Toxicological investigations revealed lethal doses of aconitine, a highly poisonous alkaloid and the major active compound of Aconitum napellus, in all specimens. Plant remnants were identified as A. napellus leaves by a botanist and toxicological examination of the liquid in the glass on site showed extremely high concentrations of aconitine. Additionally, laboratory results revealed that the young female was suffering from thyrotoxicosis factitia, an uncommon form of hyperthyroidism caused by misuse or overdosing of thyroid hormones in order to loose weight. A rare but serious and often fatal complication of hyperthyroidism is thyroid storm. Eventually the condition of thyroid storm due to thyrotoxicosis factitia could have contributed to the woman's death from aconitine poisoning.

ElectroBond application may improve wetting characteristics of etched dentine.

OBJECTIVE: To assess the efficacy of an electrically assisted application technique on dentine wettability (water contact angle measurements) and to determine the promoted surface roughness of dentine after applying 37% phosphoric acid etching, a self-etching primer or a self-etching adhesive system. METHODS: Wettability was assessed on 30 caries-free extracted human third molars. Specimens were sectioned parallel to the occlusal surface to expose moderately deep dentine and ground flat (water-wet 180-grit SiC) to provide uniform flat surfaces. 37% H3PO4, Clearfil SE Bond (CSEB) primer or Prompt-L-Pop (PLP) adhesive system were applied on dentine surfaces. In half of the specimens, the electric impulse-assisted application technique/ElectroBond-assisted application was followed. Contact angle measurements were performed to assess wettability using the Axisymmetric Drop Shape Analysis technique. Additional surfaces were conditioned for atomic force microscopy (AFM) analysis. Two-way ANOVA, Student's t and Student-Newman-Keuls tests were performed (P<0.05). RESULTS: PLP-treated dentine showed the highest intertubular roughness and the lowest dentine wettability. ElectroBond application reduced water contact angles when dentine was treated with H3PO4 or Clearfil SE Bond primer, but not when dentine was treated with PLP. CONCLUSIONS: The use of electric current improved wettability of dentine surface following application of phosphoric acid and a mild self-etch primer, but not self-etch adhesive.

Limited decalcification/diffusion of self-adhesive cements into dentin.

Resin cement diffusion into dentin may differ as a function of the pre-treatment regimen. Since self-adhesive cements do not require substrate pre-treatment for luting, penetration of and interaction with the underlying dentin are questioned. We hypothesized that differences in the resin cement diffusion into dentin may exist among current commercial adhesive cements. Composite cylinders were luted on mid-coronal dentinal surfaces by an etch-and-rinse cement (Calibra), a self-etching system (Panavia F 2.0), and 4 self-adhesive cements (Multilink Sprint, Rely X Unicem, G-Cem, Bis-Cem). Dentin/cement interfacial characteristics were analyzed by a staining technique (Masson's trichrome) and by scanning electron microscopy. Conventional acid etching resulted in partially infiltrated adhesive interfaces differing from those achieved with the application of self-etching primer. No hybrid layer and/or resin tag formation was detectable at the interfaces bonded with self-adhesive cements. Limited decalcification/infiltration was observed for self-adhesive cements into the underlying dentin. Self-adhesive cements were not able to demineralize/dissolve the smear layer completely.

Surface roughness analysis of fiber post conditioning processes.

The chemo-mechanical surface treatment of fiber posts increases their bonding properties. The combined use of atomic force and confocal microscopy allows for the assessment and quantification of the changes on surface roughness that justify this behavior. Quartz fiber posts were conditioned with different chemicals, as well as by sandblasting, and by an industrial silicate/silane coating. We analyzed post surfaces by atomic force microscopy, recording average roughness (R(a)) measurements of fibers and resin matrix. A confocal image profiler allowed for the quantitative assessment of the average superficial roughness (R(a)). Hydrofluoric acid, potassium permanganate, sodium ethoxide, and sandblasting increased post surface roughness. Modifications of the epoxy resin matrix occurred after the surface pre-treatments. Hydrofluoric acid affected the superficial texture of quartz fibers. Surface-conditioning procedures that selectively react with the epoxy-resin matrix of the fiber post enhance roughness and improve the surface area available for adhesion by creating micro-retentive spaces without affecting the post's inner structure.

Sealing properties of two contemporary single-cone obturation systems.

AIM: To compare the sealing of root canals filled with two single-cone obturation systems and a warm vertical compaction technique. METHODOLOGY: Forty-two single-rooted teeth were decoronated to obtain 17-mm-long root segments. The root canals were cleaned and shaped to size 40, 0.06 taper and filled with: (i) warm vertical compaction with AH Plus (control); (ii) ActiV GP and (iii) GuttaFlow with single master cones. Leakage was evaluated by fluid filtration at 10 psi before root resection, and after 3, 6, 9 and 12 mm apical resections. Repeated measures anovas on ranks and Dunn's multiple comparison tests were performed to examine differences in fluid flow rates amongst different resection lengths for each filling technique. The surface and interior aspects of glass-ionomer filler-coated ActiV GP gutta-percha cones was evaluated with SEM. RESULTS: No statistical difference amongst the filling techniques was seen at 0 and 3 mm root resections. ActiV GP and GuttaFlow exhibited more leakage than AH Plus at 6, 9 and 12 mm resections. AH Plus recorded the best overall results. A nonhomogeneous coating of glass-ionomer fillers on the surface of ActiV GP cones was detected. CONCLUSIONS: The two single-cone techniques examined are as effective in sealing the apex as AH Plus when the latter was used with warm vertical compaction. It is further hypothesized that the inferior coronal seal of these single-cone techniques may be improved with the placement of accessory cones to reduce sealer thickness or an immediate coronal adhesive restoration.

The adhesion between fibre posts and composite resin cores: the evaluation of microtensile bond strength following various surface chemical treatments to posts.

AIM: To evaluate the influence of various surface treatments to fibre posts on the microtensile bond strength with different composite resins. METHODOLOGY: A total of 110 fibre posts were randomly divided into five groups, according to the surface pre-treatment performed. Group 1: immersion in 24% H(2)O(2) for 10 min and silanization for 60 s; group 2: immersion in 10% H(2)O(2) for 20 min and silanization for 60 s; group 3: immersion in 4% hydrofluoric acid gel for 60 s and silanization for 60 s; group 4: silanization of the post surface for 60 s and application of the bonding agent G-Bond; group 5: silanization of the post surface for 60 s (control group). After treatment, two posts were randomly selected from each group to evaluate the morphological aspect of the post surface with scanning electron microscopy. The remaining posts in each group were divided into five subgroups of five posts each, which differed in the type of composite resin used for the core build-up. Post-core strength were calculated and the differences among experimental groups were analysed with two-way anova and the Tukey test (alpha = 0.05). RESULTS: The post-core strengths achieved in groups 1 and 2 were significantly higher (P < 0.05), than those of groups 3, 4 and 5. The post-core strength in the control group was significantly lower (P < 0.05) than all other groups. CONCLUSIONS: Hydrogen peroxide and hydrofluoric acid both modified the surface morphology of fibre posts and with silane, significantly enhanced the interfacial strength between them and core materials.

Fatal pulmonary cement embolism following percutaneous vertebroplasty (PVP).

A case of fatal pulmonary embolism with acrylic cement occuring during a percutaneous vertebroplasty on account of a fracture of L1 is described. Autopsy confirmed the presence of large amounts of surgical cement in the paravertebral venous system and in the pulmonary arteries. This rare complication occurred because perivertebral venous cement migration was not recognized during vertebroplasty. To our knowledge, this is the first case of fatal pulmonary embolism caused by surgical cement following a percutaneous vertebroplasty.

A one step procedure for luting glass fibre posts: an SEM evaluation.

AIM: To evaluate the ability of two luting procedures for bonding translucent fibre posts to form resin tags, adhesive lateral branches, and resin dentine interdiffusion zones (RDIZ). METHODOLOGY: Forty root filled teeth, extracted for periodontal reasons, were selected for the study. The teeth were randomly divided into four groups of 10 each. Group 1: One Step, light-cured (LC) 20 s + dual link resin cement (LC 20 s); group 2: One Step, not light-cured (NLC) + dual link resin cement (LC 30 s); group 3: One Step (NLC) + dual link resin cement (LC 60 s); group 4: One Step (NLC) + dual link resin cement (LC 90 sec). Forty translucent fibre posts (DT posts, RTD) were inserted. Then, root specimens were processed for scanning electron microscope (SEM) observations to assess the continuity of the RDIZ, the presence or absence of gaps and the density and morphology of resin tags using a four-step scale method. RESULTS: With all luting procedures the formation of a RDIZ occurred. However, the microscopic examination of adhesive interfaces revealed that the percentage of RDIZ was significantly higher in group 1 than in the other three groups (P < 0.05). Group 4 had a higher percentage of RDIZ than group 3, which in turn exhibited a greater percentage than group 2, but these differences were not statistically significant (P > 0.05). Resin tag formation was evident in all the groups. The characteristic reverse cone shape of resin tags was always noted in the coronal and middle third of all groups, and in the apical third of group 1. In the apical third a shorter length and a less uniform appearance of resin tags were noted in groups 2, 3 and 4. CONCLUSIONS: In this laboratory study, when bonding a translucent fibre post into a root canal, the luting procedure including light curing of the adhesive solution before the placement of the cement was more satisfactory, from a SEM standpoint, than the procedure involving the simultaneous curing of adhesive and cement (one step procedure).

Crossbow homicides.

A total of eight cases of homicide by crossbow are reported, including six intentional, assault-like killings and one hired killer. The bolts showed a high penetration capacity despite the rather low kinetic energy (<100 J): a field-tip traversed one upper arm and the thorax (36 cm) and two broadheads caused perforating injuries of the thorax (25-26 cm). This was due to the high sectional density and the split-like penetration mechanism. Wound morphology was especially important if the perpetrator had extracted the bolt, which occurred in half of the cases. The shape of the entrance wound depended on the type of arrowhead: broadheads produced star-shaped to triangular wounds, field-tips caused circular, oval or slit-like injuries. Foreign material from the arrowhead was found inside two injuries. In assaults, the crossbow was used to hunt the victim down from a short distance which does not require practice but still has the advantage of a distance weapon. However, immediate incapacitation occurred rarely so that additional violence was frequently applied. The noiseless character of the weapon explains why many victims were taken by surprise and why the corpses initially remained unnoticed. Crossbows can therefore be considered ideal weapons for man hunting and some were bought for the very purpose of the killing.

In vivo and in vitro permeability of one-step self-etch adhesives.

Adhesive dentistry should effectively restore the peripheral seal of dentin after enamel removal. We hypothesize that non-rinsing, simplified, one-step self-etch adhesives are effective for minimizing dentin permeability after tooth preparation procedures. Crown preparations in vital human teeth were sealed with Adper Prompt, Xeno III, iBond, or One-Up Bond F. Epoxy resin replicas were produced from polyvinyl siloxane impressions for SEM examination. Dentin surfaces from extracted human teeth were bonded with these adhesives and connected to a fluid-transport model for permeability measurements and TEM examination. Dentinal fluid droplets were observed from adhesive surfaces in resin replicas of in vivo specimens. In vitro fluid conductance of dentin bonded with one-step self-etch adhesives was either similar to or greater than that of smear-layer-covered dentin. TEM revealed water trees within the adhesives that facilitate water movement across the polymerized, highly permeable adhesives. Both in vitro and in vivo results did not support the proposed hypothesis.

Air rifle injury with an entrance through the nose: a case report and review of the literature.

A case of attempted homicide is reported where a 31-year-old woman was shot in the left nostril with a pellet from an air rifle. The projectile channel reconstruction showed penetration of the nasal septum, the maxillary and sphenoid cavities and the dura mater, with the pellet finally lodging in the anterior cranial fossa between the sinus cavernosus and the internal carotid artery. The patient was finally discharged from hospital in a good physical condition without any neurological symptoms. Although the muzzle velocity of the air rifle was within the legal limits, the present case demonstrates the potential lethality of air weapons considering the site of entrance of the pellet.

Cytokine pattern in patients with infections after liver transplantation.

Severe infections are the most frequent cause of death after liver transplantation. Determination of new parameters may increase the knowledge of pathophysiological mechanisms of infection. For this purpose, 81 patients with 85 liver transplants were monitored for various new parameters on a daily basis. Patients with severe infections (n = 10) were compared with patients with mild or asymptomatic cholangitis (n = 11) and with patients with an uneventful postoperative course (n = 37). One-year patient survival was 88.9%; in five patients, death was related to serious infections. Mean neopterin, soluble tumor necrosis factor-RII (sTNF-RII), and hyaluronic acid levels were significantly elevated in patients with serious infections compared with the other two groups (P < or = 0.01). A further increase in sTNF-RII and neopterin levels was observed in patients with lethal infections (P < or = 0.01 versus surviving patients with serious infection). An increase in neopterin levels was observed prior to severe infection, and in six of ten patients, this increase occurred as early as during the reperfusion period. Soluble TNF-RII and hyaluronic acid levels also increased significantly prior to severe infection. Interleukin (IL)-6, soluble intercellular adhesion molecule-1 (sICAM-1), and sIL-2R increased in patients with serious infection and cholangitis to a similar extent. As part of an overwhelming immune response, a significant increase in IL-6, sIL-2R, and also IL-1 beta levels occurred during the late phase of lethal infection (P < or = 0.01 versus surviving patients with serious infection). Routine monitoring of these parameters may improve current diagnostic tools and possibly lead to earlier detection of patients at risk after liver transplantation.