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.

Decomposition and insect succession of pig cadavers in tents versus outdoors - A preliminary study.

The pattern of insect succession and the species composition on cadavers can be influenced by several parameters, one of which is concealment. It has been previously shown for cadavers inside containers (e.g. suitcases or vehicles) or various indoor situations, that concealment can lead to a delayed arrival, altered species composition and decreased number of taxa present at the cadaver. As no data is yet available for the specific environment of tents on these processes, we placed five pig cadavers inside closed two-person tents during the summer of 2021 in a mixed forest in Germany. Five control cadavers were freely accessible for insects. To minimize disturbances, tents were only opened every fifth day over a period of 25 days for estimating the temperature profiles, insect diversity and quantification of the cadavers' decomposition using the total body score (TBS). The temperature inside the tents was only slightly elevated compared to the ambient temperature during the study. Although the tents prevented direct access of adult flies and beetles, the cadavers were colonized because the flies oviposited on the zippers and fly screens of the inner tents. However, the resulting infestation of the cadavers by fly larvae was reduced and delayed, compared to the exposed cadavers. The dominant fly species on both the tent and exposed cadavers was the blow fly Lucilia caesar. Open cadavers showed the expected decomposition patterns with large larval masses. 25 days after placement, only bones and hair remained from the exposed pigs (TBS = 32), while most of the tissue of the cadavers inside the tents was still present (TBS = 22.5) and post feeding larvae were not able to leave the tents. Regarding the attraction of beetles to both treatments, open cadavers were predominately colonized by the silphid Oiceoptoma thoracicum, while the silphid Necrodes littoralis was the most dominant species in the pitfall traps surrounding the tents. Considering the prolonged pre-appearance interval of fly larvae colonizing the cadavers inside tents, the entomological evidence of forensic cases dealing with concealed bodies should be handled with caution, since the post mortem interval might be significantly underestimated.

A standard protocol for the analysis of postmortem muscle protein degradation: process optimization and considerations for the application in forensic PMI estimation.

The analysis of postmortem protein degradation has become of large interest for the estimation of the postmortem interval (PMI). Although several techniques have been published in recent years, protein degradation-based techniques still largely did not exceed basic research stages. Reasons include impractical and complex sampling procedures, as well as highly variable protocols in the literature, making it difficult to compare results. Following a three-step procedure, this study aimed to establish an easily replicable standardized procedure for sampling and processing, and further investigated the reliability and limitations for routine application. Initially, sampling and processing were optimized using a rat animal model. In a second step, the possible influences of sample handling and storage on postmortem protein degradation dynamics were assessed on a specifically developed human extracorporeal degradation model. Finally, the practical application was simulated by the collection of tissue in three European forensic institutes and an international transfer to our forensic laboratory, where the samples were processed and analyzed according to the established protocol.

Influencing Factors on Postmortem Protein Degradation for PMI Estimation: A Systematic Review.

The present review provides an overview of the current research status on the effects of influencing factors on postmortem protein degradation used to estimate the PMI (postmortem interval). Focus was set on characteristics of internal and external influencing factors and the respective susceptibility and/or robustness of protein degradation. A systematic literature search up to December 2020 was conducted on the effect of influencing factors investigated in the context of postmortem protein degradation in the tissues of animals and humans using the scientific databases PubMed and Google Scholar, as well as the reference lists of eligible articles. We identified ten studies investigating a total of seven different influencing factors in degrading tissues/organs (n = 7) of humans and animals using six different methodological approaches. Although comparison of study outcomes was impeded by the high variety of investigated factors, and by high risk of bias appraisals, it was evident that the majority of the influencing factors concerned affected protein degradation, thus being able to modulate the precision of protein degradation-based PMI estimation. The results clearly highlight the need for a thorough screening for corresponding factors to enable the introduction of appropriate correction factors and exclusion criteria. This seems especially relevant for the protein degradation-based study of human PMI to increase the reliability and precision of the method and to facilitate a broader applicability in routine forensic casework.

Postmortem Protein Degradation as a Tool to Estimate the PMI: A Systematic Review.

Objectives: We provide a systematic review of the literature to evaluate the current research status of protein degradation-based postmortem interval (PMI) estimation. Special attention is paid to the applicability of the proposed approaches/methods in forensic routine practice. Method: A systematic review of the literature on protein degradation in tissues and organs of animals and humans was conducted. Therefore, we searched the scientific databases Pubmed and Ovid for publications until December 2019. Additional searches were performed in Google Scholar and the reference lists of eligible articles. Results: A total of 36 studies were included. This enabled us to consider the degradation pattern of over 130 proteins from 11 different tissues, studied with different methods including well-established and modern approaches. Although comparison between studies is complicated by the heterogeneity of study designs, tissue types, methods, proteins and outcome measurement, there is clear evidence for a high explanatory power of protein degradation analysis in forensic PMI analysis. Conclusions: Although only few approaches have yet exceeded a basic research level, the current research status provides strong evidence in favor of the applicability of a protein degradation-based PMI estimation method in routine forensic practice. Further targeted research effort towards specific aims (also addressing influencing factors and exclusion criteria), especially in human tissue will be required to obtain a robust, reliable laboratory protocol, and collect sufficient data to develop accurate multifactorial mathematical decomposition models.

The applicability of forensic time since death estimation methods for buried bodies in advanced decomposition stages.

Estimation of the postmortem interval in advanced postmortem stages is a challenging task. Although there are several approaches available for addressing postmortem changes of a (human) body or its environment (ecologically and/or biochemically), most are restricted to specific timeframes and/or individual and environmental conditions. It is well known, for instance, that buried bodies decompose in a remarkably different manner than on the ground surface. However, data on how established methods for PMI estimation perform under these conditions are scarce. It is important to understand whether and how postmortem changes are affected under burial conditions, if corrective factors could be conceived, or if methods have to be excluded for respective cases. We present the first multi-methodological assessment of human postmortem decomposition carried out on buried body donors in Europe, at the Amsterdam Research Initiative for Sub-surface Taphonomy and Anthropology (ARISTA) in the Netherlands. We used a multidisciplinary approach to investigate postmortem changes of morphology, skeletal muscle protein decomposition, presence of insects and other necrophilous animals as well as microbial communities (i.e., microbiomes) from August to November 2018 associated with two complete body exhumations and eight partial exhumations. Our results clearly display the current possibilities and limitations of methods for PMI estimation in buried remains and provide a baseline for future research and application.