Validation and optimization of the diatom L/D ratio as a diagnostic marker for drowning.

If a dead body is discovered in water, it nearly always raises the question about the cause of death, often associated with the persistent problem to differentiate between a drowning incident and post-mortem immersion. In numerous cases, a reliable confirmation of death by drowning is often only possible by a combination of diagnoses obtained from autopsy and additional investigations. As to the latter, the use of diatoms has been suggested (and debated) since decades. Based on the consideration that diatoms are present in almost every natural waterbody and are unavoidably incorporated when water is inhaled, their presence in the lung and other tissues can provide evidence of drowning. However, the traditional diatom test methods are still subject of controversial discussion and suspected of erroneous outcome, predominantly through contamination. A promising alternative to minimize the risk of erroneous outcome seems to be disclosed by the recently suggested MD-VF-Auto SEM technique. Especially the establishment of a new diagnostic marker (L/D ratio), which represents the factorial proportion between the diatom concentration in lung tissue and the drowning medium, allows for clearer distinction of drowning and post-mortal immersion and is largely robust to contamination. However, this highly elaborated technique requires specific devices which are frequently unavailable. We therefore developed a modified method of SEM-based diatom testing to enable the use on more routinely available equipment. Process steps such as digestion, filtration, and image acquisition were thoroughly broken down, optimized, and ultimately validated in five confirmed drowning cases. Taking certain limitations into consideration, L/D ratio analysis provided promising results, even in cases of advanced decomposition. We conclude that our modified protocol indeed opens a way for a broader use of the method in forensic drowning investigation.

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.

Dismembered porcine limbs as a proxy for postmortem muscle protein degradation.

The estimation of the postmortem interval (PMI) is of critical importance in forensic routine. The most frequently applied methods, however, are all restricted to specific time periods or must be excluded under certain circumstances. In the last years it has been shown that the analysis of muscle protein degradation has the potential to contribute to according delimitations in practice. In particular, upon biochemical analysis, the specific time points of degradation events provide reasonable markers for PMI delimitation. Nevertheless, considerable research is yet required to increase the understanding of protein decomposition and how it is affected by individual and environmental influencing factors. This is best investigated under standardized conditions, however, a considerate selection of proxies, regarding costs, effort, and expected outcome is required. Here, we use pigs to compare muscle protein decomposition in whole bodies and dismembered body parts (amputated hind limbs). Not only do experiments on body parts reduce the costs and allow easier handling in basic research, but also they aid to investigate the practical application of PMI estimation in dismembered body parts, or other extensive injuries, which are not unusual scenarios in crime investigation. Specifically, we investigated whether there are differences in the degradation rates of selected muscle proteins, sampled from dismembered legs and from hind limbs attached to whole pig bodies. Our results show distinct time-dependent degradation patterns of muscle proteins in a predictable manner regardless of sample origin. We are able to demonstrate that amputated hind limbs are suitable proxies for the analysis of muscle protein degradation, especially to investigate certain influencing factors and establish according standardized models.

Intra- and intermuscular variations of postmortem protein degradation for PMI estimation.

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.

Correction to: Intra- and intermuscular variations of postmortem protein degradation for PMI estimation.

The above article was published online with an error in the article title.

A field study to evaluate PMI estimation methods for advanced decomposition stages.

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.

Biallelic variants in the transcription factor PAX7 are a new genetic cause of myopathy.

PURPOSE: Skeletal muscle growth and regeneration rely on muscle stem cells, called satellite cells. Specific transcription factors, particularly PAX7, are key regulators of the function of these cells. Knockout of this factor in mice leads to poor postnatal survival; however, the consequences of a lack of PAX7 in humans have not been established. METHODS: Here, we study five individuals with myopathy of variable severity from four unrelated consanguineous couples. Exome sequencing identified pathogenic variants in the PAX7 gene. Clinical examination, laboratory tests, and muscle biopsies were performed to characterize the disease. RESULTS: The disease was characterized by hypotonia, ptosis, muscular atrophy, scoliosis, and mildly dysmorphic facial features. The disease spectrum ranged from mild to severe and appears to be progressive. Muscle biopsies showed the presence of atrophic fibers and fibroadipose tissue replacement, with the absence of myofiber necrosis. A lack of PAX7 expression was associated with satellite cell pool exhaustion; however, the presence of residual myoblasts together with regenerating myofibers suggest that a population of PAX7-independent myogenic cells partially contributes to muscle regeneration. CONCLUSION: These findings show that biallelic variants in the master transcription factor PAX7 cause a new type of myopathy that specifically affects satellite cell survival.

Postmortem proteomics to discover biomarkers for forensic PMI estimation.

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.

Are animal models predictive for human postmortem muscle protein degradation?

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.

First application of a protein-based approach for time since death estimation.

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.

Postmortem muscle protein degradation in humans as a tool for PMI delimitation.

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.

Postmortem degradation of skeletal muscle proteins: a novel approach to determine the time since death.

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.

A comparative study on the antibacterial photodynamic efficiency of a curcumin derivative and a formulation on a porcine skin model.

INTRODUCTION: The propagation of pathogens resistant to antibiotics around the globe has induced an urgent call for action: alternatives to conventional antibiotic therapy have to be developed to prevent a post-antibiotic catastrophe. This study focuses on the enhancement of Photodynamic Inactivation (PDI) of Gram(+) versus Gram(-) bacteria comparing a cationic derivative of curcumin (SACUR-3) to curcumin bound to polyvinylpyrrolidone (PVP-CUR) using an ex vivo porcine skin model to simulate an application on the human skin and foodstuff. EXPERIMENTAL: Porcine skin samples were inoculated with either Staphylococcus aureus or Escherichia coli and treated with either SACUR-3 or PVP-CUR at concentrations of 50 or 100 µM, respectively. Subsequent to blue light illumination (435 nm, 33.8 J cm(-2)) quantitative analyses were performed by counting the colony forming units. Furthermore, the localization of both photoactive compounds in the porcine skin was determined by fluorescence microscopy. PDI of S. aureus resulted in a reduction of 2.2 log10 steps if employing 50 µM of SACUR-3 and of 1.7 log10 steps with 50 µM of PVP-CUR. Phototoxicity towards E. coli was 3.3 log10 steps using 100 µM of SACUR-3 and 0.3 log10 steps for 100 µM of PVP-CUR. Both compounds do not exceed the stratum corneum of the skin. CONCLUSION: A direct comparison of both approaches yields that the cationic curcumin derivative SACUR-3 is effective against Gram(+) and Gram(-) pathogens, whereas the formulation of PVP-CUR has a photokilling effect on the Gram(+) model strain only, but leaves the approval of curcumin as a food additive E100 unaffected. Our results suggest the applicability of SACUR-3-based PDI in dermatology, hand hygiene and food production.

Neutrophil extracellular trap (NET) formation characterises stable and exacerbated COPD and correlates with airflow limitation.

BACKGROUND: COPD is a progressive disease of the airways that is characterized by neutrophilic inflammation, a condition known to promote the excessive formation of neutrophil extracellular traps (NETs). The presence of large amounts of NETs has recently been demonstrated for a variety of inflammatory lung diseases including cystic fibrosis, asthma and exacerbated COPD. OBJECTIVE: We test whether excessive NET generation is restricted to exacerbation of COPD or whether it also occurs during stable periods of the disease, and whether NET presence and amount correlates with the severity of airflow limitation. PATIENTS, MATERIALS AND METHODS: Sputum samples from four study groups were examined: COPD patients during acute exacerbation, patients with stable disease, and smoking and non-smoking controls without airflow limitation. Sputum induction followed the ECLIPSE protocol. Confocal laser microscopy (CLSM) and electron microscopy were used to analyse samples. Immunolabelling and fluorescent DNA staining were applied to trace NETs and related marker proteins. CLSM specimens served for quantitative evaluation. RESULTS: Sputum of COPD patients is clearly characterised by NETs and NET-forming neutrophils. The presence of large amounts of NET is associated with disease severity (p < 0.001): over 90 % in exacerbated COPD, 45 % in stable COPD, and 25 % in smoking controls, but less than 5% in non-smokers. Quantification of NET-covered areas in sputum preparations confirms these results. CONCLUSIONS: NET formation is not confined to exacerbation but also present in stable COPD and correlates with the severity of airflow limitation. We infer that NETs are a major contributor to chronic inflammatory and lung tissue damage in COPD.

Age-related changes in the composition of the cornified envelope in human skin.

The main function of the epidermis is to protect us against a multitude of hostile attacks from the environment. Its main cell type, the keratinocytes have a sophisticated system of different proteins and lipids available to form the cornified envelope, which is responsible for the barrier function of the skin. During ageing, dramatic changes are taking place. Some proteins of the SPRR-, S100- and LCE3-family are massively up-regulated, whereas others like loricrin, filaggrin and the LCE1&2 protein families are significantly down-regulated. The latter ones are known to be under control of calcium and/or 'calcium response elements'. We were able to show that the calcium peak specific for the stratum granulosum, which is the site where loricrin and the LCE1&2 families are synthesized, is reduced during ageing. The resulting cornified envelope in old skin has an extensively changed composition on the molecular level compared to young skin. This knowledge is of critical importance to understand chronic wound formation and ulcers in old age.

Distinct modes of vertebrate hypaxial muscle formation contribute to the teleost body wall musculature.

The formation of the body wall musculature in vertebrates is assumed to be initiated by direct ventral extension of the somites/myotomes. This contrasts to the formation of limb muscles and muscles involved in feeding or respiration/ventilation, which are founded by migratory muscle precursors (MMPs) distant to the somites. Here, we present evidence from morphology and expression of molecular markers proposing that the formation of the two muscle layers of the teleost body wall involves both of the above mechanisms: (1) MMPs from somites 5 and 6 found an independent muscle primordium-the so-called posterior hypaxial muscle (PHM)-which subsequently gives rise to the most anterior two segments of the medial obliquus inferioris (OI) muscle. (2) Direct epithelial extension of the hypaxial myotomes generates the OI segments from somite 7 caudalward and the entire lateral obliquus superioris (OS) muscle. The findings are discussed in relation to the evolution of hypaxial myogenic patterning including functional considerations. We hypothesise that the potential of the most anterior somites to generate migratory muscle precursors is a general vertebrate feature that has been differently utilised in the evolution in vertebrate groups.

Temperature-dependent modification of muscle precursor cell behaviour is an underlying reason for lasting effects on muscle cellularity and body growth of teleost fish.

Temperature is an important factor influencing teleost muscle growth, including a lasting ('imprinted') influence of embryonic thermal experience throughout all further life. However, little is known about the cellular processes behind this phenomenon. The study reported here used digital morphometry and immunolabelling for Pax7, myogenin and H3P to quantitatively examine the effects of thermal history on muscle precursor cell (MPC) behaviour and muscle growth in pearlfish (Rutilus meidingeri) until the adult stage. Fish were reared at three different temperatures (8.5, 13 and 16°C) until hatching and subsequently kept under the same (ambient) thermal conditions. Cellularity data were combined with a quantitative analysis of Pax7+ MPCs including those that were mitotically active (Pax7+/H3P+) or had entered differentiation (Pax7+/myogenin+). The results demonstrate that at hatching, body lengths, fast and slow muscle cross-sectional areas and fast fibre numbers are lower in fish reared at 8.5 and 13°C than at 16°C. During the larval period, this situation changes in the 13°C-fish, so that these fish are finally the largest. The observed effects can be related to divergent cellular mechanisms at the MPC level that are initiated in the embryo during the imprinting period. Embryos of 16°C-fish have reduced MPC proliferation but increased differentiation, and thus give rise to larger hatchlings. However, their limited MPC reserves finally lead to smaller adults. By contrast, embryos of 13°C-fish and, to a lesser extent, 8.5°-fish, show enhanced MPC proliferation but reduced differentiation, thus leading to smaller hatchlings but allowing for a larger MPC pool that can be used for enhanced post-hatching growth, finally resulting in larger adults.

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.

The life in a gradient: calcium, the lncRNA SPRR2C and mir542/mir196a meet in the epidermis to regulate the aging process.

The turnover of the epidermis beginning with the progenitor cells in the basal layer to the fully differentiated corneocytes is tightly regulated by calcium. Calcium more than anything else promotes the differentiation of keratinocytes which implies the need for a calcium gradient with low concentrations in the stratum basale and high concentrations in the stratum granulosum. One of the hallmarks of skin aging is a collapse of this gradient that has a direct impact on the epidermal fitness. The rise of calcium in the stratum basale reduces cell proliferation, whereas the drop of calcium in the stratum granulosum leads to a changed composition of the cornified envelope. We showed that keratinocytes respond to the calcium induced block of cell division by a large increase of the expression of several miRNAs (hsa-mir542-5p, hsa-mir125a, hsa-mir135a-5p, hsa-mir196a-5p, hsa-mir491-5p and hsa-mir552-5p). The pitfall of this rescue mechanism is a dramatic change in gene expression which causes a further impairment of the epidermal barrier. This effect is attenuated by a pseudogene (SPRR2C) that gives rise to a lncRNA. SPRR2C specifically resides in the stratum granulosum/corneum thus acting as a sponge for miRNAs.

Postembryonic fast muscle growth of teleost fish depends upon a nonuniformly distributed population of mitotically active Pax7+ precursor cells.

Muscle development in teleost embryos has been shown to depend on myogenic cell recruitment from the dermomyotome (DM). However, little is known as to the cellular mechanisms that account for myotome growth after the dissociation of the DM. Here we combine immunolabeling for cell-specific markers with quantitative analysis to determine the sources and patterns of activation of myogenic cells in pearlfish larvae. Results demonstrate that appearance of mitotically active myogenic precursors inside the myotome coincides with the dissociation of the DM. Such cells are preferentially aggregated within the posterior lateral fast muscle. We therefore propose a growth model in which a pool of proliferative DM-derived precursors transferred to the posterior lateral fast muscle functions as an important source of myogenic cell spread to carry forward stratified fast muscle hyperplasia. This indicates that postembryonic teleost muscle growth includes a cellular mechanism that has no direct equivalent in the amniotes.