Accelerated aging of forensically relevant biological materials on swabs.

The preservation of DNA in biological samples is important for forensic testing, as samples can be tested years or even decades after collection. Generally, the DNA within biological evidence is stable over shorter time frames but can degrade over extended periods. In this work, we evaluated accelerated aging as a method to reduce the duration of studies examining the stability of DNA in forensic evidence-type samples. Evaluation of the DNA extracted from cells stored at 37 and 50°C for 194 or 79 days, respectively, showed similar quality metrics to cells stored at 25°C for 548 days.

Identification of skeletal remains of Communist Armed Forces victims during and after World War II: combined Y-chromosome (STR) and MiniSTR approach.

AIM: To report on the use of STR, Y-STRs, and miniSTRs typing methods in the identification of victims of revolutionary violence and crimes against humanity committed by the Communist Armed Forces during and after World War II in which bodies were exhumed from mass and individual graves in Slovenia. METHODS: Bone fragments and teeth were removed from human remains found in several small and closely located hidden mass graves in the Skofja Loka area (Lovrenska Grapa and Zolsce) and 2 individual graves in the Ljubljana area (Podlipoglav), Slovenia. DNA was isolated using the Qiagen DNA extraction procedure optimized for bone and teeth. Some DNA extracts required additional purification, such as N-buthanol treatment. The QuantifilerTM Human DNA Quantification Kit was used for DNA quantification. Initially, PowerPlex 16 kit was used to simultaneously analyze 15 short tandem repeat (STR) loci. The PowerPlex S5 miniSTR kit and AmpF/STR MiniFiler PCR Amplification Kit was used for additional analysis if preliminary analysis yielded weak partial or no profiles at all. In 2 cases, when the PowerPlex 16 profiles indicated possible relatedness of the remains with reference samples, but there were insufficient probabilities to call the match to possible male paternal relatives, we resorted to an additional analysis of Y-STR markers. PowerPlex Y System was used to simultaneously amplify 12 Y-STR loci. Fragment analysis was performed on an ABI PRISM 310 genetic analyzer. Matching probabilities were estimated using the DNA-View software. RESULTS: Following the Y-STR analysis, 1 of the "weak matches" previously obtained based on autosomal loci, was confirmed while the other 1 was not. Combined standard STR and miniSTR approach applied to bone samples from 2 individual graves resulted in positive identifications. Finally, using the same approach on 11 bone samples from hidden mass grave Zolosce, we were able to obtain 6 useful DNA profiles. CONCLUSION: The results of this study, in combination with previously obtained results, demonstrate that Y-chromosome testing and mini-STR methodology can contribute to the identification of human remains of victims of revolutionary violence from World War II.

Spatial impacts of a multi-individual grave on microbial and microfaunal communities and soil biogeochemistry.

Decomposing vertebrates, including humans, result in pronounced changes in surrounding soil biogeochemistry, particularly nitrogen (N) and carbon (C) availability, and alter soil micro- and macrofauna. However, the impacts of subsurface human decomposition, where oxygen becomes limited and microbial biomass is generally lower, are far less understood. The goals of this study were to evaluate the impact of human decomposition in a multi-individual, shallow (~70 cm depth) grave on soil biogeochemistry and soil microbial and nematode communities. Three individuals were interred and allowed to decay for four years. Soils were collected from two depths (0‒5 and 30‒35 cm) along linear transects radiating from the grave as well as from within and below (85‒90 cm depth) the grave during excavation to assess how decomposition affects soil properties. Along radiating surface transects, several extracellular enzymes rates and nematode richness increased with increasing distance from the grave, and likely reflect physical site disruption due to grave excavation and infill. There was no evidence of carcass-sourced C and N lateral migration from the grave, at least at 30‒35 cm depth. Within the grave, soils exhibited significant N-enrichment (e.g., ammonium, dissolved organic N), elevated electrical conductivity, and elevated respiration rates with depth. Soil biogeochemistry within the grave, particularly in the middle (30‒35 cm) and base (70‒75 cm depth), was significantly altered by human decomposition. Mean microbial gene abundances changed with depth in the grave, demonstrating increased microbial presence in response to ongoing decomposition. Human-associated Bacteroides were only detected at the base of the grave where anoxic conditions prevailed. Nematode community abundance and richness were reduced at 70‒75 cm and not detectable below 85‒90 cm. Further, we identified certain Plectus spp. as potential indicators of enrichment due to decomposition. Here we demonstrate that human decomposition influences soil biogeochemistry, microbes, and microfauna up to four years after burial.

Highly effective DNA extraction method for nuclear short tandem repeat testing of skeletal remains from mass graves.

AIM: To quantitatively compare a silica extraction method with a commonly used phenol/chloroform extraction method for DNA analysis of specimens exhumed from mass graves. METHODS: DNA was extracted from twenty randomly chosen femur samples, using the International Commission on Missing Persons (ICMP) silica method, based on Qiagen Blood Maxi Kit, and compared with the DNA extracted by the standard phenol/chloroform-based method. The efficacy of extraction methods was compared by real time polymerase chain reaction (PCR) to measure DNA quantity and the presence of inhibitors and by amplification with the PowerPlex 16 (PP16) multiplex nuclear short tandem repeat (STR) kit. RESULTS: DNA quantification results showed that the silica-based method extracted on average 1.94 ng of DNA per gram of bone (range 0.25-9.58 ng/g), compared with only 0.68 ng/g by the organic method extracted (range 0.0016-4.4880 ng/g). Inhibition tests showed that there were on average significantly lower levels of PCR inhibitors in DNA isolated by the organic method. When amplified with PP16, all samples extracted by silica-based method produced 16 full loci profiles, while only 75% of the DNA extracts obtained by organic technique amplified 16 loci profiles. CONCLUSIONS: The silica-based extraction method showed better results in nuclear STR typing from degraded bone samples than a commonly used phenol/chloroform method.

DNA identification of skeletal remains from the World War II mass graves uncovered in Slovenia.

AIM: To present the joint effort of three institutions in the identification of human remains from the World War II found in two mass graves in the area of Skofja Loka, Slovenia. METHODS: The remains of 27 individuals were found in two small and closely located mass graves. The DNA was isolated from bone and teeth samples using either standard phenol/chloroform alcohol extraction or optimized Qiagen DNA extraction procedure. Some recovered samples required the employment of additional DNA purification methods, such as N-buthanol treatment. Quantifiler Human DNA Quantification Kit was used for DNA quantification. PowerPlex 16 kit was used to simultaneously amplify 15 short tandem repeat (STR) loci. Matching probabilities were estimated using the DNA View program. RESULTS: Out of all processed samples, 15 remains were fully profiled at all 15 STR loci. The other 12 profiles were partial. The least successful profile included 13 loci. Also, 69 referent samples (buccal swabs) from potential living relatives were collected and profiled. Comparison of victims' profile against referent samples database resulted in 4 strong matches. In addition, 5 other profiles were matched to certain referent samples with lower probability. CONCLUSION: Our results show that more than 6 decades after the end of the World War II, DNA analysis may significantly contribute to the identification of the remains from that period. Additional analysis of Y-STRs and mitochondrial DNA (mtDNA) markers will be performed in the second phase of the identification project.

Evaluating differential nuclear DNA yield rates and osteocyte numbers among human bone tissue types: A synchrotron radiation micro-CT approach.

Molecular human identification has conventionally focused on DNA sampling from dense, weight-bearing cortical bone tissue, typically from femora or tibiae. A comparison of skeletal elements from three contemporary individuals demonstrated that elements with high quantities of cancellous bone yielded nuclear DNA at the highest rates, suggesting that preferentially sampling cortical bone may be suboptimal (Mundorff & Davoren, 2014). Despite these findings, the reason for the differential DNA yields between cortical and cancellous bone tissues remains unknown. The primary goal of this work is to ascertain whether differences in bone microstructure can be used to explain differential nuclear DNA yield among bone tissue types observed by Mundorff and Davoren (2014), with a focus on osteocytes and the three-dimensional (3D) quantification of their associated lacunae. Osteocytes and other bone cells are recognized to house DNA in bone tissue, thus examining the density of their lacunae may explain why nuclear DNA yield rates differ among bone tissue types. Lacunae were visualized and quantified using synchrotron radiation-based micro-Computed Tomographic imaging (SR micro-CT). Volumes of interest (VOIs) from cortical and cancellous bone tissues (n=129) were comparatively analyzed from the three skeletons sampled for Mundorff and Davoren's (2014) study. Analyses tested the primary hypothesis that the abundance and density of osteocytes (inferred from their lacunar spaces) vary between cortical and cancellous bone tissue types. Results demonstrated that osteocyte lacunar abundance and density vary between cortical and cancellous bone tissue types, with cortical bone VOIs containing a higher lacunar abundance and density. We found that the osteocyte lacunar density values are independent of nuclear DNA yield, suggesting an alternative explanation for the higher nuclear DNA yields from bones with greater quantities of cancellous bone tissue. The use of SR micro-CT allowed for a scale of analysis that revealed a high range of variation in lacunar abundance in both tissue types. Moreover, high-resolution SR micro-CT imaging revealed potential soft tissue remnants within marrow spaces not visible macroscopically. It is hypothesized that soft tissue remnants observed among the trabeculae of skeletal elements with high quantities of cancellous bone tissue are responsible for the high nuclear DNA yields. These findings have significant implications for bone-sample selection for nuclear DNA analysis in a forensic context when skeletal remains are recovered from the ground surface.

Population data at two short tandem repeat loci D2S1338 and D19S433 in the sample of multinational Bosnia and Herzegovina residents.

POPULATION: We have analyzed the distribution of allele frequencies at two short tandem repeats loci (D2S1338 and D19S433) in a multinational sample of Bosnia and Herzegovina (B&H) residents. A total of 110 unrelated male and female individuals (Caucasians) from different regions of B&H were sampled for the analysis. We ensured that the sample reflected approximate proportional participation of the three main ethnic groups in the population of B&H (Bosniacs-Muslim [45%], Serbs [34%], Croats [21%]).

The effect of common imaging and hot water maceration on DNA recovery from skeletal remains.

Identifying human remains often begins with cleaning and imaging the material. Hot water maceration is used to remove adherent soft tissue from bone and radiographs are taken to better visualize osseous details. Heat and radiation are known to have harmful effects on DNA, but their ability to degrade DNA when used for cleaning and imaging has not been well studied. To better understand their individual and combined effects on the recoverability of DNA from bone, skeletal samples were subjected to (1) hot water maceration (62 °C for 45 min); (2) CT scanning (0.6mm slices, 120 kV, 10.4s); (3) X-ray (50 kVp, 150 mA, 0.03 s, 40 in); and (4) all 3 treatments combined. Forty-eight DNA samples were extracted, quantified and amplified with the AmpFLSTR(®) Identifiler(®) system. Nearly all of the processed samples had reduced RFU values relative to the unprocessed samples, indicating some amount of genetic loss. This loss did not always translate into loss of profile completeness, since only a few samples had a reduction in the number of loci detected after processing. DNA yields were not significantly reduced by any one of the processing methods, however the results indicate that the damaging effects are additive. It is possible that processing may reduce a bone's DNA reservoir and as more procedures are preformed, the pool of available genetic information might be diminished. Many intrinsic and extrinsic factors can affect the recoverability of DNA from bone. Collecting a DNA sample prior to processing avoids the negative effects from hot water maceration and radiological imaging.

Examination of DNA yield rates for different skeletal elements at increasing post mortem intervals.

Identification of contemporary human remains by DNA STR testing is mainly limited by the ability to isolate sufficient amounts of DNA from the skeletal samples. A key part of this work relies on selection of the skeletal element with the best chance of obtaining a DNA STR profile. DNA was extracted from 55 bone samples, from 3 recently skeletonized individuals, representing most element types in the human body. Comparison of DNA yields from samples within an individual showed that the small cancellous bones on average have much higher amounts of DNA per unit mass than dense cortical bones. Complete 16 locus STR profiles were obtained for all 3 individuals from 36 of the element types, 10 had full profiles for 2 of the 3 individuals, 3 had full profiles for 1 of the 3 and 5 did not have any full profiles. The sample types with the least STR loci were from the arms. Ten skeletal elements were tested from 12 additional skeletons ranging from 3-21 years post mortem interval (PMI). At increasing PMI the small cancellous bones continued to yield more DNA and STR loci than the cortical bones. These findings suggest that the current recommendation for selection of long cortical bone samples for DNA testing of skeletal remains should be re-evaluated.

Development and optimization of a PCR assay for detection of Dobrava and Puumala hantaviruses in Bosnia and Herzegovina.

Hantavirus-specific serology tests are the main diagnostic technique for detection of hantavirus infection in Bosnia and Herzegovina. In order to enhance hantavirus infections monitoring a sensitive PCR based assay was developed to detect Dobrava (DOBV) and Puumala (PUUV) hantaviruses. Nested primer sets were designed within three different regions of the viral RNA (S and M segment of DOBV and M segment of PUUV) based on highly similar regions from a number of different European hantavirus strains. Assay conditions were optimized using cell cultures infected with DOBV Slovenia, PUUV Sotkamo and PUUV CG 18-20. This sensitive and specific assay has proven to be useful for detection of both Puumala and Dobrava hantaviruses.

Application of novel "mini-amplicon" STR multiplexes to high volume casework on degraded skeletal remains.

The International Commission on Missing Persons (ICMP) conducts high throughput STR profiling on degraded skeletal remains, primarily recovered from mass graves relating to conflicts from 1992 to 1999 in the former Yugoslavia. To date, over 11,000 individuals have been identified through comparison of bone profiles to a large database of profiles from family members of the missing. To increase success rates in STR recovery, three short amplicon STR multiplexes (a 7-plex, a 6-plex, and a 5-plex) have been devised and implemented. These target loci from large commercial multiplexes, with an average decrease in amplicon size of 144 bp. The ICMP "miniplexes" have proven to provide substantially greater recovery of DNA data from a certain subset of difficult samples. However, the circumstances under which miniplexes provide additional data are restricted, and their advantages do not outweigh those of large commercial multiplexes for a majority of cases. The miniplexes, however, also have a very powerful use in DNA testing to support large scale reassociation of commingled, partial skeletons recovered from secondary mass graves.

Characterization of the bovine ampkgamma1 gene.

AMP-activated protein kinase (AMPK) represents the mammalian form of the core component of a kinase cascade that is conserved between fungi, plants, and animals. AMPK plays a major role in protecting mammalian cells from metabolic stress by switching off biosynthetic pathways that require ATP and switching on ATP-regenerating pathways. In this report, we describe the isolation and characterization of the gene for the noncatalytic bovine gamma1 subunit of AMPK. The bovine ampkgamma1 (PRKAG1) gene spans in excess of 14 kb and is located at BTA 5q21-q22. It consists of 12 exons ranging in size from 38 b to 166 b, interspersed with 11 introns that range between 97 b and 6753 b in length. The coding region of the bovine gene shares 93% and 90% nucleotide sequence similarity with its human and rat counterparts, and the bovine AMPKgamma1 protein is 98% and 95% identical to its human and rat homologs, respectively, in amino acid sequence. SNP discovery using a cattle DNA panel revealed a number of polymorphisms that may be useful for the evaluation of ampkgamma1 as a candidate gene for energy metabolism-related production traits.