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1.
Int J Legal Med ; 136(6): 1913-1923, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-35710956

RESUMO

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.


Assuntos
Proteínas Musculares , Mudanças Depois da Morte , Animais , Patologia Legal/métodos , Humanos , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Proteólise , Ratos , Reprodutibilidade dos Testes
2.
Diagnostics (Basel) ; 11(7)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201836

RESUMO

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.

3.
PLoS One ; 15(12): e0243395, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33296399

RESUMO

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.


Assuntos
Medicina Legal/métodos , Patologia Legal/métodos , Músculo Esquelético/química , Proteólise , Animais , Sepultamento , Morte , Exumação , Humanos , Insetos/fisiologia , Microbiota , Modelos Animais , Mudanças Depois da Morte
4.
Diagnostics (Basel) ; 10(12)2020 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-33256203

RESUMO

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.

5.
6.
Int J Legal Med ; 134(5): 1775-1782, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32632798

RESUMO

In recent years, protein decomposition has become of increasing interest for the use in forensic estimation of the postmortem interval (PMI). Especially skeletal muscle tissue has proven to be a prime target tissue, among other reasons, due to its large abundance in the human body. In this regard, it is important to know whether there are any intra- and intermuscular differences in the behavior of protein degradation. Thus, samples from different locations within several skeletal muscles as well as from cardiac and smooth muscle tissue samples were collected from three autopsy cases with varying degree of decomposition. Samples were analyzed by SDS-PAGE and Western blotting and compared for protein degradation patterns. Intramuscular variations turned out to be minimal and without major influence for the use of the method. Observed intermuscular differences provide possibilities for future improvement of the precision and temporal application range. The results of this study show the strengths and current limitations of protein degradation-based PMI estimation and provide a deeper understanding of intraindividual postmortem protein degradation processes.


Assuntos
Actinina/análise , Músculo Esquelético/química , Músculo Liso/química , Miocárdio/química , Proteólise , Tubulina (Proteína)/análise , Vinculina/análise , Adulto , Idoso , Western Blotting , Eletroforese em Gel de Poliacrilamida , Patologia Legal , Humanos , Masculino , Pessoa de Meia-Idade , Mudanças Depois da Morte
7.
Int J Legal Med ; 134(4): 1361-1373, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32248308

RESUMO

Estimating the postmortem interval (PMI) is one of the major tasks and a continuous challenge in forensic pathology. It is often an exclusion process of available methods, which ultimately can lead to an unsatisfactory outcome due to poor reliability. This problem is most acute in the late PMI, when decomposition proceeds and some methods (such as rigor, livor, and algor mortis) are no longer applicable. Several methods, such as forensic entomology, skeletal muscle protein degradation, and the study of body decomposition by application of a morphological scoring, are expected to provide further information; however, all have certain limitations and weaknesses. Availability of a tool-box of methods allows a case-specific selection of the most appropriate one(s), or eventually provides improvements in the overall accuracy and precision of the PMI estimation by merging and combining methods. To investigate practical (field) application, eventual interferences, and/or synergetic effects, as well as the robustness of these methods towards specific influencing factors, a field study was conducted, using eight pig cadavers of different body weights and physical coverage, left to decompose under natural conditions for 16 days. Morphological changes during decomposition were assessed using the total body score (TBS), muscle samples were collected to analyze protein degradation, and insect colonization was evaluated. The results reveal strengths and current limitations of all tested methods, as well as promising synergistic effects, and thus, provide a baseline for targeted future research.


Assuntos
Restos Mortais/patologia , Patologia Legal/métodos , Modelos Animais , Mudanças Depois da Morte , Suínos , Animais , Projetos Piloto
8.
Int J Legal Med ; 133(3): 899-908, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30864069

RESUMO

The assessment of postmortem degradation of skeletal muscle proteins has emerged as a novel approach to estimate the time since death in the early to mid-postmortem phase (approximately 24 h postmortem (hpm) to 120 hpm). Current protein-based methods are limited to a small number of skeletal muscle proteins, shown to undergo proteolysis after death. In this study, we investigated the usability of a target-based and unbiased system-wide protein analysis to gain further insights into systemic postmortem protein alterations and to identify additional markers for postmortem interval (PMI) delimitation. We performed proteomic profiling to globally analyze postmortem alterations of the rat and mouse skeletal muscle proteome at defined time points (0, 24, 48, 72, and 96 hpm), harnessing a mass spectrometry-based quantitative proteomics approach. Hierarchical clustering analysis for a total of 579 (rat) and 896 (mouse) quantified proteins revealed differentially expressed proteins during the investigated postmortem period. We further focused on two selected proteins (eEF1A2 and GAPDH), which were shown to consistently degrade postmortem in both rat and mouse, suggesting conserved intra- and interspecies degradation behavior, and thus preserved association with the PMI and possible transferability to humans. In turn, we validated the usefulness of these new markers by classical Western blot experiments in a rat model and in human autopsy cases. Our results demonstrate the feasibility of mass spectrometry-based analysis to discover novel protein markers for PMI estimation and show that the proteins eEF1A2 and GAPDH appear to be valuable markers for PMI estimation in humans.


Assuntos
Biomarcadores/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Fator 1 de Elongação de Peptídeos/metabolismo , Mudanças Depois da Morte , Proteômica , Idoso , Animais , Cromatografia Líquida , Análise por Conglomerados , Feminino , Patologia Legal/métodos , Humanos , Masculino , Espectrometria de Massas , Camundongos Endogâmicos ICR , Modelos Animais , Ratos Sprague-Dawley
9.
Int J Legal Med ; 131(6): 1615-1621, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28721468

RESUMO

A most precise determination of the postmortem interval (PMI) is a crucial aspect in forensic casework. Although there are diverse approaches available to date, the high heterogeneity of cases together with the respective postmortal changes often limit the validity and sufficiency of many methods. Recently, a novel approach for time since death estimation by the analysis of postmortal changes of muscle proteins was proposed. It is however necessary to improve the reliability and accuracy, especially by analysis of possible influencing factors on protein degradation. This is ideally investigated on standardized animal models that, however, require legitimization by a comparison of human and animal tissue, and in this specific case of protein degradation profiles. Only if protein degradation events occur in comparable fashion within different species, respective findings can sufficiently be transferred from the animal model to application in humans. Therefor samples from two frequently used animal models (mouse and pig), as well as forensic cases with representative protein profiles of highly differing PMIs were analyzed. Despite physical and physiological differences between species, western blot analysis revealed similar patterns in most of the investigated proteins. Even most degradation events occurred in comparable fashion. In some other aspects, however, human and animal profiles depicted distinct differences. The results of this experimental series clearly indicate the huge importance of comparative studies, whenever animal models are considered. Although animal models could be shown to reflect the basic principles of protein degradation processes in humans, we also gained insight in the difficulties and limitations of the applicability of the developed methodology in different mammalian species regarding protein specificity and methodic functionality.


Assuntos
Modelos Animais , Proteínas Musculares/metabolismo , Mudanças Depois da Morte , Proteólise , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Patologia Legal , Humanos , Masculino , Camundongos , Suínos
10.
Am J Sports Med ; 45(3): 676-684, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27729321

RESUMO

BACKGROUND: Muscle injuries are among the most common sports-related lesions in athletes; however, optimal treatment remains obscure. Extracorporeal shock wave therapy (ESWT) may be a promising approach in this context, because it has gained increasing importance in tissue regeneration in various medical fields. HYPOTHESIS: ESWT stimulates and accelerates regenerative processes of acute muscle injuries. STUDY DESIGN: Controlled laboratory study. METHODS: Adult Sprague-Dawley rats were divided into 4 experimental groups (2 ESWT+ groups and 2 ESWT- groups) as well as an uninjured control group (n ≥ 6 in each group). An acute cardiotoxin-induced injury was set into the quadriceps femoris muscle of rats in the experimental groups. A single ESWT session was administered to injured muscles of the ESWT+ groups 1 day after injury, whereas ESWT- groups received no further treatment. At 4 and 7 days after injury, 1 each of the ESWT+ and ESWT- groups was euthanized. Regenerating lesions were excised and analyzed by histomorphometry and immunohistochemistry to assess fiber size, myonuclear content, and recruitment of satellite cells. RESULTS: The size and myonuclear content of regenerating fibers in ESWT+ muscle was significantly increased compared with ESWT- muscle fibers at both 4 and 7 days after injury. Similarly, at both time points, ESWT+ muscles exhibited significantly higher contents of pax7-positive satellite cells, mitotically active H3P+ cells, and, of cells expressing the myogenic regulatory factors, myoD and myogenin, indicating enhanced proliferation and differentiation rates of satellite cells after ESWT. Mitotic activity at 4 days after injury was doubled in ESWT+ compared with ESWT- muscles. CONCLUSION: ESWT stimulates regeneration of skeletal muscle tissue and accelerates repair processes. CLINICAL RELEVANCE: We provide evidence for accelerated regeneration of damaged skeletal muscle after ESWT. Although further studies are necessary, our findings support the view that ESWT is an effective method to improve muscle healing, with special relevance to sports injuries.


Assuntos
Litotripsia , Músculo Esquelético/lesões , Músculo Esquelético/fisiologia , Regeneração/fisiologia , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Modelos Animais de Doenças , Masculino , Mitose/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Ratos Sprague-Dawley , Células Satélites de Músculo Esquelético/fisiologia , Cicatrização/fisiologia
11.
Int J Legal Med ; 131(2): 479-483, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27770266

RESUMO

Awareness of postmortem degradation processes in a human body is fundamental to develop methods for forensic time since death estimation (TDE). Currently, applied approaches are all more or less limited to certain postmortem phases, or have restrictions on behalf of circumstances of death. Novel techniques, however, rarely exceed basic research phases due to various reasons. We report the first application of a novel method, based on decay of muscle proteins, in a recent case of murder-suicide, where other TDE methods failed to obtain data. We detected considerably different protein degradation profiles in both individuals involved and compared the data to our presently available database. We obtained statistical evidence for un-simultaneous death and therefore received valuable information to trace the progression of events based on protein degradation. Although we could not sensibly convert the data to respective times of death, this case highlights the potential for future application and elucidates the necessary further steps to develop a viable TDE method.


Assuntos
Músculo Esquelético/metabolismo , Mudanças Depois da Morte , Proteólise , Idoso , Calpaína/metabolismo , Desmina/metabolismo , Feminino , Homicídio , Humanos , Masculino , Suicídio , Tropomiosina/metabolismo , Troponina T/metabolismo
12.
Int J Legal Med ; 130(6): 1547-1555, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26951243

RESUMO

Forensic estimation of time since death relies on diverse approaches, including measurement and comparison of environmental and body core temperature and analysis of insect colonization on a dead body. However, most of the applied methods have practical limitations or provide insufficient results under certain circumstances. Thus, new methods that can easily be implemented into forensic routine work are required to deliver more and discrete information about the postmortem interval (PMI). Following a previous work on skeletal muscle degradation in the porcine model, we analyzed human postmortem skeletal muscle samples of 40 forensic cases by Western blotting and casein zymography. Our results demonstrate predictable protein degradation processes in human muscle that are distinctly associated with temperature and the PMI. We provide information on promising degradation markers for certain periods of time postmortem, which can be useful tools for time since death delimitation. In addition, we discuss external influencing factors such as age, body mass index, sex, and cause of death that need to be considered in future routine application of the method in humans.


Assuntos
Músculo Esquelético/metabolismo , Mudanças Depois da Morte , Proteólise , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Índice de Massa Corporal , Calpaína/metabolismo , Criança , Pré-Escolar , Desmina/metabolismo , Feminino , Patologia Legal/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Temperatura , Tropomiosina/metabolismo , Troponina T/metabolismo , Adulto Jovem
13.
Int J Legal Med ; 130(2): 421-31, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26041514

RESUMO

Estimating the time since death is a very important aspect in forensic sciences which is pursued by a variety of methods. The most precise method to determine the postmortem interval (PMI) is the temperature method which is based on the decrease of the body core temperature from 37 °C. However, this method is only useful in the early postmortem phase (~0-36 h). The aim of the present work is to develop an accurate method for PMI determination beyond this present limit. For this purpose, we used sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and casein zymography to analyze the time course of degradation of selected proteins and calpain activity in porcine biceps femoris muscle until 240 h postmortem (hpm). Our results demonstrate that titin, nebulin, desmin, cardiac troponin T, and SERCA1 degraded in a regular and predictable fashion in all samples investigated. Similarly, both the native calpain 1 and calpain 2 bands disintegrate into two bands subsequently. This degradation behavior identifies muscular proteins and enzymes as promising substrates for future molecular-based PMI determination technologies.


Assuntos
Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Mudanças Depois da Morte , Animais , Western Blotting , Calpaína/metabolismo , Conectina/metabolismo , Desmina/metabolismo , Eletroforese em Gel de Poliacrilamida , Patologia Legal , Modelos Animais , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Suínos , Troponina T/metabolismo
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