Assessing various Infrared (IR) microscopic imaging techniques for post-mortem interval evaluation of human skeletal remains.

Due to the influence of many environmental processes, a precise determination of the post-mortem interval (PMI) of skeletal remains is known to be very complicated. Although methods for the investigation of the PMI exist, there still remains much room for improvement. In this study the applicability of infrared (IR) microscopic imaging techniques such as reflection-, ATR- and Raman- microscopic imaging for the estimation of the PMI of human skeletal remains was tested. PMI specific features were identified and visualized by overlaying IR imaging data with morphological tissue structures obtained using light microscopy to differentiate between forensic and archaeological bone samples. ATR and reflection spectra revealed that a more prominent peak at 1042 cm-1 (an indicator for bone mineralization) was observable in archeological bone material when compared with forensic samples. Moreover, in the case of the archaeological bone material, a reduction in the levels of phospholipids, proteins, nucleic acid sugars, complex carbohydrates as well as amorphous or fully hydrated sugars was detectable at (reciprocal wavelengths/energies) between 3000 cm-1 to 2800 cm-1. Raman spectra illustrated a similar picture with less ν2PO43-at 450 cm-1 and ν4PO43- from 590 cm-1 to 584 cm-1, amide III at 1272 cm-1 and protein CH2 deformation at 1446 cm-1 in archeological bone material/samples/sources. A semi-quantitative determination of various distributions of biomolecules by chemi-maps of reflection- and ATR- methods revealed that there were less carbohydrates and complex carbohydrates as well as amorphous or fully hydrated sugars in archaeological samples compared with forensic bone samples. Raman- microscopic imaging data showed a reduction in B-type carbonate and protein α-helices after a PMI of 3 years. The calculated mineral content ratio and the organic to mineral ratio displayed that the mineral content ratio increases, while the organic to mineral ratio decreases with time. Cluster-analyses of data from Raman microscopic imaging reconstructed histo-anatomical features in comparison to the light microscopic image and finally, by application of principal component analyses (PCA), it was possible to see a clear distinction between forensic and archaeological bone samples. Hence, the spectral characterization of inorganic and organic compounds by the afore mentioned techniques, followed by analyses such as multivariate imaging analysis (MIAs) and principal component analyses (PCA), appear to be suitable for the post mortem interval (PMI) estimation of human skeletal remains.

Evidence for frequent and tissue-specific sequence heteroplasmy in human mitochondrial DNA.

Mitochondrial point heteroplasmy is a common event observed not only in patients with mitochondrial diseases but also in healthy individuals. We here report a comprehensive investigation of heteroplasmy occurrence in human including the whole mitochondrial control region from nine different tissue types of 100 individuals. Sanger sequencing was used as a standard method and results were supported by cloning, minisequencing, and massively parallel sequencing. Only 12% of all individuals showed no heteroplasmy, whereas 88% showed at least one heteroplasmic position within the investigated tissues. In 66% of individuals up to 8 positions were affected. The highest relative number of heteroplasmies was detected in muscle and liver (79%, 69%), followed by brain, hair, and heart (36.7%-30.2%). Lower percentages were observed in bone, blood, lung, and buccal cells (19.8%-16.2%). Accumulation of position-specific heteroplasmies was found in muscle (positions 64, 72, 73, 189, and 408), liver (position 72) and brain (partial deletion at position 71). Deeper analysis of these specific positions in muscle revealed a non-random appearance and position-specific dependency on age. MtDNA heteroplasmy frequency and its potential functional importance have been underestimated in the past and its occurrence is ubiquitous and dependent at least on age, tissue, and position-specific mutation rates.

Molecular genetic investigations on Austria's patron saint Leopold III.

The successful marriage policy of margrave Leopold III increased the importance of the House of Babenberg in late medieval Austria (12th century). Historical documentation is inconclusive in providing evidence whether or not his eldest son Adalbert derived from an earlier relationship or from the marriage with King Henry IV's daughter Agnes of Waiblingen, with whom Leopold is considered to have had 17 children. As a matter of fact Adalbert was ignored in the line of succession in favor of a younger brother, Leopold IV, which has led to long term historical discussions. Human remains attributed to these individuals were subjected to DNA analysis. Autosomal, Y-chromosomal and mitochondrial DNA analyses brought successful results, which suggested that Leopold III, Agnes and Adalbert were related in parent-son constellation, in contrast to historical considerations. A possible mix-up of Adalbert's remains with those of his younger brother Ernst could not be confirmed by DNA analysis.

Another 'soberade' on the market: does Outox keep its promise?

OBJECTIVE: Several products are being widely promoted for reduction of the concentration of alcohol in the human body. One of these preparations, the fructose soft drink Outox, claims to noticeably increase the alcohol elimination rate (beta 60). Theories to explain this 'fructose effect' are based on the assumption that NAD+, the coenzyme for alcohol dehydrogenase, is regenerated faster in the presence of fructose. METHOD: A randomized double-blind, placebo-controlled cross-over study was performed with 30 volunteers in two drinking sessions each. Under strictly identical conditions, the same amount of alcohol was consumed, followed by the consumption of either 250 ml Outox or 250 ml placebo. Periodical measurements of blood (BAC), breath (BrAC) and urine alcohol concentration (UAC) were performed. RESULTS: Analyses revealed a significant difference (P<0.0001) between the mean alcohol levels of the Outox and the placebo drinking sessions. The overall mean BAC difference was 0.077 g/l (BAC 0.748 g/l without vs 0.671 g/l with Outox), equivalent to 10.3%. The mean BrAC difference was 0.045 mg/l (BrAC 0.314 mg/l without vs 0.269 mg/l with Outox), equivalent to 14.3%. Differences were lower for women than for men. A significant difference between the alcohol elimination rates (beta 60) was not found. CONCLUSIONS: The results show that the soft drink Outox may decrease the alcohol concentration by about 10%. However, BAC and BrAC differences are rather a consequence of slower gastric absorption of alcohol, because Outox does not increase the alcohol elimination rate. Our study demonstrates that the claim of Outox or other fructose drinks to work as a 'soberade' cannot be proven from a scientific point of view. It should be the task of physicians to warn potential consumers, especially in connection with drinking and driving.

Medicolegal aspects of tetrazepam metabolism.

The benzodiazepine tetrazepam is primarily muscle relaxant with comparably lower central sedating effects and is therefore commonly prescribed for muscle spasms of different origins. To evaluate tetrazepam metabolism, a study was conducted with ten healthy volunteers. Blood and urine samples were regularly collected after the intake of 50 mg tetrazepam. Toxicological analyses revealed that tetrazepam is also metabolized to diazepam and further to nordazepam, which has not yet been reported. Tetrazepam and diazepam could be detected in urine samples at least 72 h after intake, the diazepam concentration being 33% (+/-14% SD), on average, of the tetrazepam concentration. On the basis of three case histories, the importance of the detection of these newly described metabolites is shown as necessary to prevent false accusations and potential negative legal consequences for examined persons.

An announced suicide with ecstasy.

Most cases of ecstasy overdose turn out to be accidental, whereas suicide attempts with designer drugs occur only sporadically. We report an announced suicide by means of a combination of 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethylamphetamine (MDEA). During autopsy, sampling for toxicological investigation (peripheral blood, urine, cerebrospinal fluid, bile and gastric contents) occurred. Serum concentrations as high as 13.33 mg/l for MDMA, 7.32 mg/l for MDEA and 0.43 mg/l for 3,4-methylenedioxyamphetamine were found. Ecstasy tablets, which were confiscated by the police a few days earlier, showed also a combination of MDMA and MDEA. This fact suggests that the ingested tablets probably came from the same source as the seized pills.

Generating population data for the EMPOP database - an overview of the mtDNA sequencing and data evaluation processes considering 273 Austrian control region sequences as example.

The European DNA profiling group (EDNAP) mtDNA population database (EMPOP) is an international collaborative project between DNA laboratories performing mtDNA analysis and the DNA laboratory of the Institute of Legal Medicine (GMI) in Innsbruck, Austria. The goal is to set up a directly accessible mtDNA population database, which can be used in routine forensic casework for frequency investigations. Here we describe a safe laboratory scheme involving electronical data handling and computer-aided data transfer, which help to minimize errors originating from potential sample mix-up, data misinterpretation and incorrect transcription. The procedure is demonstrated by example of an mtDNA control region population study on 273 unrelated individuals from Austria. Our population sample was compared with five other European populations via an analysis of molecular variance (AMOVA). The inclusion of regions outside HVS-I and HVS-II increased the amount of information on the haplogroup diagnostic sites in the control region. Most of the haplotypes in Austrians fell into haplogroups H, J, K, T, and U. The random match probability in Austrians was 1:125; the average number of nucleotide differences between individuals in the Austrian database was 9.32.

Unravelling the mystery of Nanga Parbat.

Since the disappearance of Günther Messner, brother of the world-famous mountaineer Reinhold Messner, on an expedition to Nanga Parbat in 1970, the circumstances of his death have given rise to controversy. Reinhold Messner claimed that he and Günther descended the Diamir face together when an avalanche killed his brother, while other expedition members argued that Günther was abandoned by Reinhold to descend the Rupal face. Now, 35 years after the event, Günther's remains have been found at the Diamir face and have been identified by forensic DNA fingerprinting. The location of the remains supports Messner's version, thus putting to rest one of the climbing communities' most publicised controversies.

Elimination rates of breath alcohol.

Legal driving limits are set coequally with 0.5 g/L blood alcohol concentration (BAC) or 0.25 mg/L breath alcohol concentration (BrAC) in Austria as well as in other European countries. As mostly some time elapses between BrAC measurement and driving offence, a back calculation of alcohol concentrations is often required. The calculation of hourly BrAC elimination rates can thereby help to avoid unnecessary variances. A study with 59 participants was performed under social conditions. BrAC was determined with the legally accredited Alcotest 7110 MK III A every 30 min, and concomitantly venous blood samples were drawn. Five hundred and four BrAC/BAC value pairs were evaluated. The overall mean peak BrAC was calculated with 0.456 mg/L (+/-0.119 mg/L standard deviation). The mean hourly BrAC elimination rate was overall determined with 0.082 mg/L per h (0.050-0.114, 95% range). Mean rate of females (0.087 mg/L h(-1)) and the according 95% limits were statistically significantly higher than of males (mean rate 0.078 mg/L h(-1), p<0.04). Our results confirm the possibility to implement hourly BrAC elimination rates, provided that adequate statistical ranges and basic forensic scientific rules that have been set up for alcohol back calculations are observed.

A modular real-time PCR concept for determining the quantity and quality of human nuclear and mitochondrial DNA.

We developed a modular real-time (rt) PCR system for absolute quantification of human nuclear (n) and mitochondrial (mt) DNA. For determination of the number of amplifiable template molecules with a minimum length required for downstream genotyping and assessment of the PCR-relevant degradation grade of the template DNA, primers yielding differently sized PCR products (nDNA: 79, 156, and 246 bp; mtDNA: 102, 143, 283, and 404 bp) and TaqMan hybridization probes were used for amplification and on-line product detection. DNase-degraded DNA served as model to demonstrate the effects of DNA fragmentation on rtPCR quantification and subsequent genotyping. Introduction of cloned internal amplification positive controls (IPCs)--generated by in vitro mutagenesis of primer-binding sites of the wild-type nDNA and mtDNA targets--enabled functionality-testing of the reaction mixture and detection of PCR inhibitors in DNA extracts, without a need for additional TaqMan probes. A hematin model was used to test the ability of the quantitative real-time (rtq) PCR system to predict the effects of inhibitors in downstream PCR-based genotyping.

Characterization of mtDNA SNP typing and mixture ratio assessment with simultaneous real-time PCR quantification of both allelic states.

We performed a study on the forensic utility of allele-discriminatory quantitative real-time PCR (rtPCR) using Minor Groove Binder TaqMan probes, targeting the highly variable mitochondrial single nucleotide polymorphism 16519T/C. The apparent single-cycle PCR efficiency was virtually 100% for both 16519 alleles. The allele designations made by rtPCR were concordant with the results obtained in a previous study by sequencing analysis. In heteroplasmic samples, minor allele proportions down to 9% were unambiguously detected and quantified. The variation in allele proportion estimates was essentially the same within and between different rtPCR runs, and the differences between total copy number estimates found for rerun samples were comparable to those found with non-allele-discriminatory quantitative rtPCR assays.

Ecstasy--deadly risk even outside rave parties.

Fatalities due to 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") are rare in Austria, although the use of designer drugs has become quite common. This is the first published case of a fatal MDMA intoxication in Austria. A 19-year-old girl died after the consumption of ecstasy tablets in the apartment of a friend. Blood analysis gave a concentration of MDMA as 3.8 mg/L and traces of its metabolite MDA. Cannabinoids were found as well. This case shows that the consumption of MDMA, without physical stress, can lead to death.

Y-STR typing of an Austrian population sample using a 17-loci multiplex PCR assay.

Y-chromosomal STR haplotypes were determined from a sample of 135 unrelated men and 70 sons from Tirol (Austria) using the AmpFlSTR Yfiler PCR amplification kit (Applied Biosystems) that coamplifies 17 Y-STRs. The panel of markers includes the 9-loci European minimal haplotype (minHt) and, in addition, the markers DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635 (Y GATA C4) and Y GATA H4. A total of 130 different haplotypes (125 were unique) were identified by the 17 Y-STR markers, an increase of 19 compared with the minHt. The gene diversity of DYS635, DYS456 and DYS458 exceeded 0.75 and only that of the duplicated marker DYS385 (0.86) was higher. Consistently high haplotype diversity values were found in all tested Y-SNP haplogroups. Because the simultaneous analysis of 17 Y-STR systems offers a high power of discrimination at minimum sample consumption, the Yfiler kit is a promising tool for forensic applications.

Systematic study on STR profiling on blood and saliva traces after visualization of fingerprint marks.

This paper describes a systematic study of the influence of optical, physical, and chemical methods used for fingerprint enhancement on subsequent DNA analysis of biological stains. Latent fingerprints as well as fingerprints in contact with blood and saliva on different surfaces were treated with dactyloscopic methods. As a general finding, subsequent STR profiling of the blood/saliva traces led to good results after all the enhancement methods included in this study. Concerning blood enhancement procedures, the airbrush technique showed deleterious effects on subsequent STR analysis in some cases. We therefore recommend the implementation of the layer technique, as it brings advantages for fingerprint enhancement as well. It could also be shown that, as can be necessary in practical casework, two enhancement methods can be performed on a single stain without having influence on STR profiling. In terms of methodological variety, this paper reflects a comprehensive study performed on STR profiling after fingerprint enhancement methods, including rare methods and variations of techniques, which can be a useful alternative in certain case scenarios.