Rapidly mutating Y-STR analyses of compromised forensic samples.

Rapidly mutating Y-chromosomal short tandem repeats (RM Y-STRs) were identified to improve differentiation of unrelated males and also to enable separating closely and distantly related males in human identity testing in forensic and other applications. RM-Yplex assay was developed as a single multiplex that is capable of simultaneously amplifying all currently known RM Y-STRs, and reproducibility and sensitivity testing were performed on reference samples. Additional analyses are necessary to test its suitability for analysing compromised forensic samples. For this purpose, we applied the RM-Yplex assay to approximately 70-year-old skeletons that were used as a model for poorly preserved, challenging forensic samples. We analysed 57 male skeletal remains (bones and teeth) from 55 skeletons excavated from the Second World War (WWII) mass graves in Slovenia. The RM-Yplex typing was successful in all 57 samples; there were 56% full profiles obtained, and in partial profiles, up to 7 locus drop-outs were observed and they appeared correlated with low DNA quantities and degradation of DNA obtained from WWII bone and tooth samples. The longest loci, DYS403S1b, DYS547, DYS627 and DYS526b, were the most often dropped-out RM Y-STRs. In spite of high frequency of drop-out events, the RM-Yplex typing was successful in all WWII samples, showing the possibility of successful amplification of at least half of the RM Y-STRs even from the most compromised samples analysed.

Prediction of autosomal STR typing success in ancient and Second World War bone samples.

Human-specific quantitative PCR (qPCR) has been developed for forensic use in the last 10 years and is the preferred DNA quantification technique since it is very accurate, sensitive, objective, time-effective and automatable. The amount of information that can be gleaned from a single quantification reaction using commercially available quantification kits has increased from the quantity of nuclear DNA to the amount of male DNA, presence of inhibitors and, most recently, to the degree of DNA degradation. In skeletal remains samples from disaster victims, missing persons and war conflict victims, the DNA is usually degraded. Therefore the new commercial qPCR kits able to assess the degree of degradation are potentially able to predict the success of downstream short tandem repeat (STR) typing. The goal of this study was to verify the quantification step using the PowerQuant kit with regard to its suitability as a screening method for autosomal STR typing success on ancient and Second World War (WWII) skeletal remains. We analysed 60 skeletons excavated from five archaeological sites and four WWII mass graves from Slovenia. The bones were cleaned, surface contamination was removed and the bones ground to a powder. Genomic DNA was obtained from 0.5g of bone powder after total demineralization. The DNA was purified using a Biorobot EZ1 device. Following PowerQuant quantification, DNA samples were subjected to autosomal STR amplification using the NGM kit. Up to 2.51ng DNA/g of powder were extracted. No inhibition was detected in any of bones analysed. 82% of the WWII bones gave full profiles while 73% of the ancient bones gave profiles not suitable for interpretation. Four bone extracts yielded no detectable amplification or zero quantification results and no profiles were obtained from any of them. Full or useful partial profiles were produced only from bone extracts where short autosomal (Auto) and long degradation (Deg) PowerQuant targets were detected. It is concluded that STR typing of old bones after quantification with the PowerQuant should be performed only when both Auto and Deg targets are detected simultaneously with no respect to [Auto]/[Deg] ratio. Prediction of STR typing success could be made according to successful amplification of Deg fragment. The PowerQuant kit is capable of identifying bone DNA samples that will not yield useful STR profiles using the NGM kit, and it can be used as a predictor of autosomal STR typing success of bone extracts obtained from ancient and WWII skeletal remains.

Bringing colour back after 70 years: Predicting eye and hair colour from skeletal remains of World War II victims using the HIrisPlex system.

Retrieving information about externally visible characteristics from DNA can provide investigative leads to find unknown perpetrators, and can also help in disaster victim and other missing person identification cases. Aiming for the application to both types of forensic casework, we previously developed and forensically validated the HIrisPlex test system enabling parallel DNA prediction of eye and hair colour. Although a recent proof-of-principle study demonstrated the general suitability of the HIrisPlex system for successfully analysing DNA from bones and teeth of various storage times and conditions, practical case applications to human remains are scarce. In this study, we applied the HIrisPlex system to 49 DNA samples obtained from bones or teeth of World War II victims excavated at six sites, mostly mass graves, in Slovenia. PCR-based DNA quantification ranged from 4pg/µl to 313pg/µl and on an average was 41pg/µl across all samples. All 49 samples generated complete HIrisPlex profiles with the exception of one MC1R DNA marker (N29insA) missing in 83.7% of the samples. In 44 of the 49 samples (89.8%) complete 15-loci autosomal STR (plus amelogenin) profiles were obtained. Of 5 pairs of skeletal remains for which STR profiling suggested an origin in the same individuals, respectively, 4 showed the same HIrisPlex profiles and predicted eye and hair colours, respectively, while discrepancies in one pair (sample 26 and 43) are likely to be explained by DNA quantity and quality issues observed in sample 43. Sample 43 had the lowest DNA concentration of only 4pg/µl, producing least reliable STR results and could be misleading in concluding that samples 43 and 26 originate from the same individual. The HIrisPlex-predicted eye and hair colours from two skeletal samples, suggested to derive from two brothers via STR profiling together with a living sister, were confirmed by the living sister's report. Overall, we demonstrate that after more than 70 years, HIrisPlex-based eye and hair colour prediction from skeletal remains is feasible with high success rate. Our results further encourage the use of the HIrisPlex system in missing person/disaster victim identification to aid the identification process in cases where ante-mortem samples or putative relatives are not directly available, and DNA predicted eye and hair colour information provides leads for locating them, allowing STRbased individual identification.

Searching for the mother missed since the Second World War.

The aim of the study was to perform the genetic identification of a human cranium from a Second World War gravesite in Slovenia and find out if it belonged to the mother of a woman used as a family reference. Both genetic and anthropological examinations were carried out. The genetic examination was performed on 2 molars and petrous bone. Prior to DNA isolation 0.5 g of tooth and bone powder was decalcified. The DNA was purified in a Biorobot EZ1 (Qiagen) device. The nuclear DNA of the samples was quantified and short tandem repeat (STR) typing performed using two different autosomal and Y-STR kits. Up to 22.4 ng DNA/g of powder was obtained from samples analyzed. We managed to obtain nuclear DNA for successful STR typing from the left second molar and from the petrous bone. Full autosomal genetic profile including amelogenin locus revealed the male origin of the cranium that was further confirmed by the analyses of Y-STRs. The same conclusions were adopted after the anthropological analysis which identified the cranium as that of a very young Caucasoid male. The male origin of the cranium rejected the possibility of motherhood for the compared daughter. For traceability in the event of contamination, we created an elimination database including genetic profiles of the nuclear and Y-STRs of all persons that had been in contact with the analyzed cranium and no match was found.

Highly efficient automated extraction of DNA from old and contemporary skeletal remains.

We optimised the automated extraction of DNA from old and contemporary skeletal remains using the AutoMate Express system and the PrepFiler BTA kit. 24 Contemporary and 25 old skeletal remains from WWII were analysed. For each skeleton, extraction using only 0.05 g of powder was performed according to the manufacturer's recommendations (no demineralisation - ND method). Since only 32% of full profiles were obtained from aged and 58% from contemporary casework skeletons, the extraction protocol was modified to acquire higher quality DNA and genomic DNA was obtained after full demineralisation (FD method). The nuclear DNA of the samples was quantified using the Investigator Quantiplex kit and STR typing was performed using the NGM kit to evaluate the performance of tested extraction methods. In the aged DNA samples, 64% of full profiles were obtained using the FD method. For the contemporary skeletal remains the performance of the ND method was closer to the FD method compared to the old skeletons, giving 58% of full profiles with the ND method and 71% of full profiles using the FD method. The extraction of DNA from only 0.05 g of bone or tooth powder using the AutoMate Express has proven highly successful in the recovery of DNA from old and contemporary skeletons, especially with the modified FD method. We believe that the results obtained will contribute to the possibilities of using automated devices for extracting DNA from skeletal remains, which would shorten the procedures for obtaining high-quality DNA from skeletons in forensic laboratories.

Diffuse Axonal Injury-A Distinct Clinicopathological Entity in Closed Head Injuries.

The knowledge about the diffuse axonal injury (DAI) as a clinicopathological entity has matured in the last 30 years. It has been defined clinically (immediate and prolonged unconsciousness leading to death or severe disability) and pathologically (the triad of DAI specific changes). In terms of its biomechanics, DAI is occurring as a result of acceleration forces of longer duration and has been fully reproduced experimentally.In the process of diagnosing DAI, the performance of a complete forensic neuropathological examination is essential and the immunohistochemistry method using antibodies against ß-amyloid precursor protein (ß-APP) has been proved to be highly sensitive and specific, selectively targeting the damaged axons.In this review, we are pointing to the significant characteristics of DAI as a distinct clinicopathological entity that can cause severe impairment of the brain function, and in the forensic medicine setting, it can be found as the concrete cause of death. We are discussing not only its pathological feature, its mechanism of occurrence, and the events on a cellular level but also the dilemmas about DAI that still exist in science: (1) regarding the strict criteria for its diagnosis and (2) regarding its biomechanical significance, which can be of a big medicolegal importance.

Frequency and number of resuscitation related rib and sternum fractures are higher than generally considered.

AIM OF THE STUDY: This study aimed to establish the incidence, number and location of CPR-related skeletal chest injuries (SCI) and to investigate the influence of age, gender, changes in resuscitation guidelines and technique of resuscitation. METHODS: We analysed SCI in 2148 patients who had undergone resuscitation for non-traumatic cardiac arrest, as shown by autopsies performed at the Institute of Forensic Medicine in Ljubljana in the period 2004-2013. RESULTS: External cardiac massage caused SCI in 86% of males and in 91% of females; sternum fractures occurred in 59% of males and 79% of females, rib fractures in 77% of males and 85% of females and sternocostal separations in 33% of males and 12% of females. The average number of all SCI per person was thus almost the same in males and females: 10.95 vs. 10.96. The percentage of patients injured and the number of SCI increased with age. Changes in resuscitation guidelines were also identified as a factor contributing to the incidence and number of SCI. No adverse effect of the use of LUCAS was found. CONCLUSION: It is generally considered that at least 1/3 of resuscitated patients sustain rib fractures and at least 1/5 sustains sternum fractures. However, our study showed that these injuries are much more frequent and that increased compression rate and depth cause more SCI. Since in the period 2011-2013 accompanying severe injuries occurred in only 1.85% of cases, the resuscitation technique has not yet jeopardised patient's safety, but further close monitoring is needed.

Secular trends in body height in Balkan populations from 1945 to 1995.

The aim of this study was to look for any secular trend in the stature of Balkan populations from the time of World War II (1939-1945) to the Balkans War (1991-1995). The research was based on the examination of exhumed skeletons of 202 men killed in World War II in the area of the Republic of Slovenia, and 243 men killed in the Bosnian War in Bosnia and Herzegovina. The length measurements of the right and left humerus, femur, tibia and fibula were taken. Since the results revealed no significant differences and the left-sided bones were more complete and recurrent in the sample, the bones of the left side were used in the analysis. Since the increase in height depends mostly on the increase in length of the long bones, with an average absolute change of about 0.28 cm for humerus, 0.55 cm for femur, 0.49 cm for tibia and 0.20 cm for fibula per decade in our case, these results suggest a significant increase of the height of the Balkans population. The difference of the sum of the average femur and tibia length for the study period was 4.13 cm. Recalculated average length increase of the sum length of femur and tibia per decade was 0.88 cm for the left side. Our study revealed that there was a trend towards increased long bone lengths, at least in the male population analyzed.

Toward male individualization with rapidly mutating y-chromosomal short tandem repeats.

Relevant for various areas of human genetics, Y-chromosomal short tandem repeats (Y-STRs) are commonly used for testing close paternal relationships among individuals and populations, and for male lineage identification. However, even the widely used 17-loci Yfiler set cannot resolve individuals and populations completely. Here, 52 centers generated quality-controlled data of 13 rapidly mutating (RM) Y-STRs in 14,644 related and unrelated males from 111 worldwide populations. Strikingly, >99% of the 12,272 unrelated males were completely individualized. Haplotype diversity was extremely high (global: 0.9999985, regional: 0.99836-0.9999988). Haplotype sharing between populations was almost absent except for six (0.05%) of the 12,156 haplotypes. Haplotype sharing within populations was generally rare (0.8% nonunique haplotypes), significantly lower in urban (0.9%) than rural (2.1%) and highest in endogamous groups (14.3%). Analysis of molecular variance revealed 99.98% of variation within populations, 0.018% among populations within groups, and 0.002% among groups. Of the 2,372 newly and 156 previously typed male relative pairs, 29% were differentiated including 27% of the 2,378 father-son pairs. Relative to Yfiler, haplotype diversity was increased in 86% of the populations tested and overall male relative differentiation was raised by 23.5%. Our study demonstrates the value of RM Y-STRs in identifying and separating unrelated and related males and provides a reference database.

Slovenian population data for five new European Standard Set short tandem repeat loci and SE33 locus.

AIM: To establish the allele distribution and statistical parameters of forensic interest for the D10S1248, D22S1045, D2S441, D1S1656, D12S391, and SE33 loci in Slovenian population and to compare allele frequencies with those from other populations. METHODS: We analyzed blood and buccal swab samples from 333 unrelated, healthy Slovenian individuals. All samples were genotyped using the AmpFlSTR NGM Kit to obtain the allele frequency data for the loci D10S1248, D22S1045, D2S441, D1S1656, and D12S391. Samples from 113 individuals were also analyzed using the PowerPlex ESX 17 system to obtain the allele frequency data for the SE33 locus. Allele frequencies and statistical parameters of forensic interest were determined and frequency profiles compared between Slovenian and other European Caucasian populations using the Arlequin software, version 3.5.1.3. RESULTS: The investigated short tandem repeat (STR) loci in Slovenian population had a great discriminating potential with a combined discrimination power of 0.99999998. The highest discrimination power and polymorphism information content were observed for the SE33 locus, followed by loci D1S1656, D12S391, D10S1248, D2S441, and D22S1045. When Slovenian allele frequency distribution was compared with other European populations, deviations were found only for Spanish and Italian population for D2S441 and D12S391. CONCLUSION: Slovenian population does not differ significantly from other European populations in terms of allele frequency distributions for the six analyzed STR loci. Based on forensic efficiency values, SE33 may be considered the most informative locus, which makes it especially useful in forensic investigations.

Viability of human articular chondrocytes harvested postmortem: changes with time and temperature of in vitro culture conditions.

Different studies of long-term chondrocytes viability have shown a gradual reduction as a function of time and ambient temperature. The aim of our in vitro study was to establish chondrocyte postmortem viability curves for 4°C, 11°C, 23°C, 35°C during 63 days after the donors' death. Osteochondral cylinders were procured from the knees of 16 male donors (20-47 years), stored in preservation media that was not changed, and analyzed in 3-day intervals using a confocal laser scanning microscope. A significant influence of time on viability was found from Day 9 (p = 0.0029) and onwards (p < 0.0001). The lowest overall chondrocyte viability was at 35°C, followed by 4°C (p < 0.0001). The conditions used in this in vitro analysis suggest that similar viabilities may occur while in situ in the decedent. Further studies of chondrocyte viability from individuals with known postmortem intervals may show premise to help evaluate time since death in the late postmortem interval.

Identical human papillomavirus (HPV) genomic variants persist in recurrent respiratory papillomatosis for up to 22 years.

Seventy initial and 125 follow-up tissue specimens of laryngeal papillomas, obtained from 70 patients who had had recurrent respiratory papillomatosis for from 1-22 years, were investigated for the presence of human papillomavirus (HPV) DNA and HPV E5a, LCR and/or full-length genomic variants. HPV-6 was found in 130/195, HPV-11 in 63/195, and HPV-6/HPV-11 in 2/195 samples. Within 67/70 (95.7%) patients, all follow-up HPV isolates genetically matched completely initial HPV isolate over the highly variable parts of the genome or over the entire genome. Frequent recurrence of laryngeal papillomas is a consequence of long-term persistence of the identical initial HPV genomic variant.

The optimal combination of cartilage source and apparatus for long-term in vitro chondrocyte viability analysis.

Most studies of long-term chondrocytes survival were for tissue banks. They showed a gradual reduction in the viable chondrocytes percentage as a function of time and ambient temperature, but the samples were harvested under optimal conditions. The aim of our study was to determine the most reliable combination of cartilage source and assay for the in vitro postmortem chondrocyte viability analysis in the conditions that imitate a dead body. Osteochondral cylinders were procured from femoral condyles and talar trochleas of three male donors and stored in the cell culture media at 4 ± 2°C and 23 ± 2°C. The samples were analyzed by a cell viability analyzer and a confocal laser scanning microscope (CLSM) initially 24-36 h after death and then in 4-week intervals. The results reconfirmed the significant influence of time (p = 0.0002), but not of the temperature (p = 0.237). The largest reproducibility was presented for the knee joint and the CLSM.

Highly efficient nuclear DNA typing of the World War II skeletal remains using three new autosomal short tandem repeat amplification kits with the extended European Standard Set of loci.

AIM: To perform an efficiency study of three new amplification kits with the extended European Standard Set (ESS) of loci for autosomal short tandem repeat (STR) typing of skeletal remains excavated from the World War II mass graves in Slovenia. METHODS: In the beginning of the 2011, we analyzed 102 bones and teeth using the PowerPlex ESX 17 System (Promega), AmpFiSTR NGM PCR Amplification Kit (Applied Biosystems), and Investigator ESSplex Kit (Qiagen). We cleaned the bones and teeth, removed surface contamination, and ground them into a powder using liquid nitrogen. Prior to DNA isolation with Biorobot EZ1 (Qiagen), 0.5 g bone or tooth powder was decalcified. Nuclear DNA of the samples was quantified using real-time polymerase chain reaction. All three kits used the same extract with the amplification conditions recommended by the manufacturers. RESULTS: We extracted up to 131 ng DNA/g of powder from the bones and teeth. All three amplification kits showed very similar efficiency, since DNA typing was successful with all amplification kits in 101 out of 102 bones and teeth, which represents a 99% success rate. CONCLUSION: The commercially available ESX 17, ESSplex, and NGM kits are highly reliable for STR typing of World War II skeletal remains with the DNA extraction method optimized in our laboratory.

An MRI study of the differences in the rate of thrombolysis between red blood cell-rich and platelet-rich components of venous thrombi ex vivo.

PURPOSE: To test whether T(1)-weighted MRI can detect the differences in the rate of thrombolysis induced by recombinant tissue plasminogen activator (rt-PA) between platelet-rich regions and red blood cell (RBC)-rich regions of venous thrombi ex vivo. MATERIALS AND METHODS: Each of 21 venous thrombi ex vivo (8 pulmonary emboli and 13 in situ thrombi) was dissected along the longitudinal axis. Half of it was analyzed for the presence of platelet, fibrin, and RBC components by immunohistochemistry and the other half was imaged serially by high-resolution T(1)-weighted three-dimensional MRI to assess the progression of thrombolysis. The MR images were analyzed for proportions of the remaining platelet-rich and RBC-rich regions. RESULTS: Laminated platelet-rich regions, corresponding to Zahn lines, were confirmed immunohistochemically and by MRI in 18/21 venous thrombi. In T(1)-weighted MR images (TE/TR = 10/105 ms) the mean signal intensity of platelet-rich regions was on average 2.3 higher than that of RBC-rich regions. The rate of thrombolysis in platelet-rich regions was on average 30% lower than in RBC-rich regions. After 120 min of thrombolysis the proportion of lysed platelet-rich regions was 0.27 ± 0.04 versus 0.40 ± 0.08 in RBC regions, which resulted in 1.4% decrease of lysed thrombus volume per 1% increase of platelet-rich content. CONCLUSION: Venous thrombi are most often composed of interspersed platelet-rich and RBC-rich regions. T(1) -weighted MRI is capable of noninvasive discrimination between those two components of venous thrombi ex vivo which have a different susceptibility to thrombolysis by rt-PA.

The association between brain-derived neurotrophic factor polymorphism (BDNF Val66Met) and suicide.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) mediates neural plasticity, mood, different behaviours, and stress response. A functional BDNF polymorphism (BDNF Val66Met) was reported to influence the effects of stressful life events or childhood adversity on depression and suicidal behaviour in various psychopathologies. The study evaluated the association between BDNF Val66Met variants and suicide, committed with violent or non-violent methods, in victims with or without stressful childhood experience. METHODS: BDNF Val66Met polymorphism was genotyped on 560DNA samples from 359 suicide victims and 201 control subjects collected on autopsy from unrelated Caucasian subjects and subdivided according to gender, method of suicide, and influence of childhood adversity. RESULTS: A similar frequency of BDNF Val66Met variants was found between all included suicide victims and the control groups, and also between the male groups. The frequency of the combined Met/Met and Met/Val genotypes and the homozygous Val/Val genotype was significantly different between the female suicide victims and female controls, between the female suicide victims who used violent suicide methods and female controls, and between all included suicide victims with or without stressful life events. The combined Met/Met and Met/Val genotypes contributed to this significance. LIMITATION: A small group of suicide victims with available data on childhood adversity was studied. CONCLUSIONS: The combined Met/Met and Met/Val genotypes of the BDNF Val66Met variant could be the risk factor for violent suicide in female subjects and for suicide in victims exposed to childhood trauma. These results confirm a major role of BDNF in increased vulnerability to suicide.

Increased BDNF promoter methylation in the Wernicke area of suicide subjects.

CONTEXT: Brain-derived neurotrophic factor (BDNF) plays a pivotal role in the pathophysiology of suicidal behavior and BDNF levels are decreased in the brain and plasma of suicide subjects. So far, the mechanisms leading to downregulation of BDNF expression are poorly understood. OBJECTIVES: To test the hypothesis that alterations of DNA methylation could be involved in the dysregulation of BDNF gene expression in the brain of suicide subjects. DESIGN: Three independent quantitative methylation techniques were performed on postmortem samples of brain tissue. BDNF messenger RNA levels were determined by quantitative real-time polymerase chain reaction. SETTING: Academic medical center. PATIENTS OR OTHER PARTICIPANTS: Forty-four suicide completers and 33 nonsuicide control subjects of white ethnicity. MAIN OUTCOME MEASURES: The DNA methylation degree at BDNF promoter IV and the genome-wide DNA methylation levels in the brain's Wernicke area. RESULTS: Postmortem brain samples from suicide subjects showed a statistically significant increase of DNA methylation at specific CpG sites in BDNF promoter/exon IV compared with nonsuicide control subjects (P < .001). Most of the CpG sites lying in the -300/+500 region, on both strands, had low or no methylation, with the exception of a few sites located near the transcriptional start site that had differential methylation, while genome-wide methylation levels were comparable among the subjects. The mean methylation degree at the 4 CpG sites analyzed by pyrosequencing was always less than 12.9% in the 33 nonsuicide control subjects, while in 13 of 44 suicide victims (30%), the mean methylation degree ranged between 13.1% and 34.2%. Higher methylation degree corresponded to lower BDNF messenger RNA levels. CONCLUSIONS: BDNF promoter/exon IV is frequently hypermethylated in the Wernicke area of the postmortem brain of suicide subjects irrespective of genome-wide methylation levels, indicating that a gene-specific increase in DNA methylation could cause or contribute to the downregulation of BDNF expression in suicide subjects. The reported data reveal a novel link between epigenetic alteration in the brain and suicidal behavior.

Molecular genetic identification of skeletal remains from the Second World War Konfin I mass grave in Slovenia.

This paper describes molecular genetic identification of one third of the skeletal remains of 88 victims of postwar (June 1945) killings found in the Konfin I mass grave in Slovenia. Living relatives were traced for 36 victims. We analyzed 84 right femurs and compared their genetic profiles to the genetic material of living relatives. We cleaned the bones, removed surface contamination, and ground the bones into powder. Prior to DNA isolation using Biorobot EZ1 (Qiagen), the powder was decalcified. The nuclear DNA of the samples was quantified using the real-time polymerase chain reaction method. We extracted 0.8 to 100 ng DNA/g of bone powder from 82 bones. Autosomal genetic profiles and Y-chromosome haplotypes were obtained from 98% of the bones, and mitochondrial DNA (mtDNA) haplotypes from 95% of the bones for the HVI region and from 98% of the bones for the HVII region. Genetic profiles of the nuclear and mtDNA were determined for reference persons. For traceability in the event of contamination, we created an elimination database including genetic profiles of the nuclear and mtDNA of all persons that had been in contact with the skeletal remains. When comparing genetic profiles, we matched 28 of the 84 bones analyzed with living relatives (brothers, sisters, sons, daughters, nephews, or cousins). The statistical analyses showed a high confidence of correct identification for all 28 victims in the Konfin I mass grave (posterior probability ranged from 99.9% to more than 99.999999%).

Characteristics of the number of odontoblasts in human dental pulp post-mortem.

Estimation of the time since death is important in forensic medicine, and so far not much is known in employing dental pulp for such purposes. The tooth organ is the hardest organ in the human body, with a loose connective tissue of dental pulp situated within a rigid encasement of mineralized surrounding tissues. Human material was obtained from 31 corpses of people who died in car and train accidents and had healthy oral statuses. Samples were divided into two groups at different environmental temperatures. During the autopsy, the jaws were resected to keep teeth in situ, and every day one tooth was extracted. After decalcification, serial thin sections stained with hematoxylin and eosin were cut. Odontoblasts in the dental pulp were counted and data analysed. Statistical analysis showed that the number of odontoblasts drops during the time after death, and no odontoblasts remain in the pulp after 5 days.

Promoter and functional polymorphisms of HTR2C and suicide victims.

In Europe, the countries with the highest suicide rates form a so-called J-curve, which starts in Finland and extends down to Slovenia-a country with one of the world's highest suicide rates. So far, the strongest association between suicide and genes has been shown for the serotonergic system. A functional polymorphism 68G>C (Cys23Ser) and a promoter polymorphism-995G>A of serotonin receptor 2C (HTR2C) have already been investigated, but no associations with suicide were determined. In the present study 334 suicide victims and 211 controls of Slovenian origin were genotyped for the above-mentioned polymorphisms using standard methods. In the case of the polymorphism-995G>A no association with suicide was found. However, a significant association was observed between female suicide victims and polymorphism 68G>C. The significance remained when we combined alleles of female and male populations. An excess of GG genotype and allele G was observed. However, no statistically important differences were present when only males were analyzed. Haplotype analysis on female population showed marginal association of haplotype G-C with suicide. The present study speaks for the plausible implication of the HTR2C in suicide susceptibility.