Establishment of a multiplex PCR-CE assay for the simultaneous and rapid analysis of age markers for Calliphora vicina pupae.

Insects, especially blow flies, are forensically relevant to determine the minimal postmortem interval (PMImin), based on the fact that they are usually the first colonisers of a body. By estimating the age of immature blow flies, interferences can be made about the time since death. Whilst morphological parameters are valuable for age estimation of blow fly larvae, gene expression profiling is more applicable for blow fly pupae. Here, the age-dependent changes in the gene expression levels during the development are analysed. 28 temperature-independent markers have already been described for the age estimation of pupae of the forensically important blow fly Calliphora vicina and are analysed by RT-qPCR. To allow simultaneous analysis of these age markers, a multiplex assay was developed in the present study. After reverse transcription, the markers are analysed simultaneously in an endpoint PCR and subsequently separated by capillary electrophoresis (CE). This method is highly attractive due to its quick and easy procedure and interpretation. The present age prediction tool was adapted and validated. The multiplex PCR assay reproduced the same expression profiles as the RT-qPCR assay based on the same markers. The statistical evaluation shows that the new assay has a lower precision but a better trueness for age determination compared to the RT-qPCR assay. Since the new assay is also qualified to estimate the age of C. vicina pupae and is practical, cost-effective and, even more importantly, time-saving, it is attractive for use in forensic casework.

It is all about the insects: a retrospective on 20 years of forensic entomology highlights the importance of insects in legal investigations.

This study highlights the importance of insect evidence by evaluating 949 insect-associated cases, including 139 entomological reports, from 2001 to 2019 at the Institute of Legal Medicine Frankfurt/Germany. With a high number of cases in the summer months and a low number in the colder season, 78.5% of the bodies were found indoors, regardless of year or month. In more than 80% of the cases, where PMI information was available (n = 704), the presumed PMI ranged from 1 to 21 days, a period during which entomological evidence can provide a day-specific estimate of PMImin. In cases where insects have been identified to species level (n = 279), most bodies were infested by one or two species with a maximum of 10 different species. Overall, a total of 55 insect species were found. Information on biology, activity and distribution of the most abundant taxa is given and applied for 5 case histories estimating different PMImins of up to over 6 months. Despite proved importance and scientific development of forensic entomology, insects are still rarely considered as a tool in forensic case work. The main reasons are a lack of awareness and (too) late involvement of a forensic entomologist. Our work shows that forensic entomology is an independent discipline that requires specialist expertise.

Age determination of the adult blow fly Lucilia sericata (Diptera: Calliphoridae) through quantitative pteridine fluorescence analysis.

Determination of a minimal postmortem interval via age estimation of necrophagous diptera has been restricted to the juvenile stages and the time until emergence of the adult fly, i.e. up until 2-6 weeks depending on species and temperature. Age estimation of adult flies could extend this period by adding the age of the fly to the time needed for complete development. In this context pteridines are promising metabolites, as they accumulate in the eyes of flies with increasing age. We studied adults of the blow fly Lucilia sericata at constant temperatures of 16 °C and 25 °C up to an age of 25 days and estimated their pteridine levels by fluorescence spectroscopy. Age was given in accumulated degree days (ADD) across temperatures. Additionally, a mock case was set up to test the applicability of the method. Pteridine increases logarithmically with increasing ADD, but after 70-80 ADD the increase slows down and the curve approaches a maximum. Sex had a significant impact (p < 4.09 × 10-6) on pteridine fluorescence level, while body-size and head-width did not. The mock case demonstrated that a slight overestimation of the real age (in ADD) only occurred in two out of 30 samples. Age determination of L. sericata on the basis of pteridine levels seems to be limited to an age of about 70 ADD, but depending on the ambient temperature this could cover an extra amount of time of about 5-7 days after completion of the metamorphosis.

Interdisciplinary teaching and training - A medicolegal specialty.

The Frankfurt model is described to exemplify the teaching and training concepts implemented at the Institute of Legal Medicine in Frankfurt am Main up to 2015. The Frankfurt model describes a comprehensive, networked teaching system aiming at an interdisciplinary training. Interdisciplinarity is a domain of forensic medicine as a broadly diversified subject related to various scientific disciplines. The importance of the medicolegal triad (research, teaching, services) rooted in the university setting, on which the success of this interdisciplinary teaching and training concept is based, is illustrated. Sufficient funding is required to maintain this medicolegal triad, and the consequences of potential reductions due to fiscal reasons are outlined.

Application of DNA barcoding for identifying forensically relevant Diptera from northern Thailand.

In recent decades, forensic entomology has become a useful tool in criminal investigations all over the world. Species-specific identification of flies plays an important role in this field and is obligatory for accurate calculation of the post-mortem interval. However, not all important colonizers of a corpse can be identified by common morphological keys. Due to similar morphology and the lack of keys for some taxa, especially for immature stages, DNA barcoding has become more popular during the last recent years. This development is particularly important for countries like Thailand, in which forensic entomology is a newly developing research area and which faces several challenges such as a high biodiversity of fly species. The most commonly used barcoding region in forensic entomology, the mitochondrial cytochrome oxidase subunit 1 (coI) gene, as well as a 1000-bp-long region of the 28S nuclear rRNA gene, was used to analyze and establish the molecular barcodes of 13 different species of flies of forensic relevance in northern Thailand.

,,Flora and fauna" in criminalistics - an analysis of the current use and relevance of non-human biological trace materials in criminal proceedings.

The analysis of biological, non-human trace specimens can contribute significantly to solving a criminal case. The present study searches the relevant German criminal, forensic, legal and biological literature, focusing on animal hairs, insects and plant fragments, and assesses the current opportunities of this special forensic branch and its acceptance and relevance for the evidence in court. It turns out that the analysis of these trace materials has an enormous range of potential applications which should not only be reflected in the forensic sciences, but also in the criminal trials. However, in the legal literature and legal annotations the topic of biological, non-human trace materials is addressed only sporadically. To derive the greatest practical benefit from the developments of forensic biology, the knowledge about the use of biological, non-human trace specimens should be promoted for the criminal proceedings. Investigators, judges, prosecutors and defense lawyers should be more thoroughly informed and become trained by forensic biologists.

Descriptive analyses of differentially expressed genes during larval development of Calliphora vicina (Diptera: Calliphoridae).

The analysis of insect evidence is often used in death investigations as the development of necrophagous insect species can be used to determine a minimum post-mortem interval (PMImin). Usually, the PMImin estimations are based on the age estimation of larvae developed on the corpse. So far, age estimation mostly relies on length or weight measurement of the larvae. These measurements are then compared to species-specific reference growth data obtained from laboratory studies. However, length and weight do not always represent the best developmental markers to provide accurate and precise age estimates for PMImin calculation, especially for post-feeding third-instar larvae which decrease in size. Therefore, more growth markers are required to improve age estimation not only for post-feeding larvae but also for all larval stages. As the analysis of temporally regulated genes proved suitable for age prediction in blow fly pupae, we examined the gene expression patterns of six genes (15_2, 2014192, EcdR, AR, hsp90 and actin) during larval development of Calliphora vicina at three constant temperatures and analysed the effect of a combination of morphological and molecular age markers on statistical models of development. EcdR, AR and 15_2 showed reliable tendencies to classify the post-feeding stage more precisely, and inclusion of gene expression data in models of development improved the statistical fit of the model. Nevertheless, for depicting the first larval stages and for quantifying the onset of the post-feeding stage more accurately, results of this preliminary study should be supported by searching for more informative genes.

Diapause-specific gene expression in Calliphora vicina (Diptera: Calliphoridae)--a useful diagnostic tool for forensic entomology.

Estimating the post mortem interval (PMImin) by age determination of blow fly larvae has been well-established for moderate temperatures. Low-temperature developmental data is only available sparsely and usually does not take overwintering strategies into account. The blow fly Calliphora vicina hibernates by diapausing in the third larval stage extending the duration of this developmental stage up to several weeks or even months. As the diagnosis of the diapause status is not possible by morphological characteristics, PMImin estimations might be biased during the cold season if only based on age determination of third instar larvae of C. vicina. Molecular markers were searched for which allows one to identify diapause in larvae. Expression analysis of 19 genes was performed in diapausing and non-diapausing larvae. Three genes encoding for heat shock proteins (hsp23, hsp24 and hsp70) were found to be up-regulated distinctly in diapausing larvae and at 1 day in non-diapausing larvae. If several larvae are subjected to an analysis, a high variance in the expression level of the gene encoding for the anterior fat body protein is a further marker for diapause. The present study proves the potential use of gene expression analysis as a suitable diagnosis tool for diapause in C. vicina.

The analysis of temporal gene expression to estimate the age of forensically important blow fly pupae: results from three blind studies.

The estimation of the minimum postmortem interval can be implemented by age estimation of corpse-associated primary colonizers such as the blow flies (Diptera: Calliphoridae). In cases where pupae represent the oldest stages found on a body, their age must be added to the duration of prepupal development to estimate the minimum postmortem interval. Although methods to age blow fly larvae have been well established using morphology, length or weight and age estimation of pupae has proved challenging. In a previous work, we quantified the changes in mRNA levels of four differentially expressed genes during the metamorphosis of Calliphora vicina pupae, hence representing molecular markers for pupal age (i.e., time elapsed since pupariation). Here, we demonstrate how these data can be used to estimate pupal age with inverse prediction. We present three blind studies conducted under various conditions and show that age of C. vicina pupae can be well estimated based on gene expression data. As these data are quantitative and can be processed automatically, gene expression has the potential to outperform morphological analysis in age estimation of forensically relevant blow fly pupae.

Differential gene expression during metamorphosis: a promising approach for age estimation of forensically important Calliphora vicina pupae (Diptera: Calliphoridae).

Necrophagous blow fly larvae can provide accurate estimates of the minimum postmortem interval in death investigations. During larval development, predictable morphological changes occur and measurements of weight, length, and width are compared to species-specific growth curves for reliable age estimates. However, aging blow fly pupae is more challenging because morphological and anatomical changes are not visible with the naked eye. Thus, delicate preparation of the pupae or rearing to the adult stage seems unavoidable. Conversely, metamorphosis evokes a remodelling of the larval shape to adult structures, and gene expression analysis potentially serves as a molecular tool to mirror the ageing process of a pupa. The present study focuses on the differential expression of two newly described, arbitrarily named genes (15_2, 2014192) and two previously identified genes (actin, arylphorin receptor) during Calliphora vicina (Diptera: Calliphoridae) metamorphosis. Quantification through real-time PCR revealed significant up- and downregulation of these transcripts found to be temperature dependent and age specific, hence, a new possibility to age forensically important blow fly pupae.

Identification of novel antiviral drug candidates using an optimized SARS-CoV-2 phenotypic screening platform.

Reliable, easy-to-handle phenotypic screening platforms are needed for the identification of anti-SARS-CoV-2 compounds. Here, we present caspase 3/7 activity as a readout for monitoring the replication of SARS-CoV-2 isolates from different variants, including a remdesivir-resistant strain, and of other coronaviruses in numerous cell culture models, independently of cytopathogenic effect formation. Compared to other models, the Caco-2 subline Caco-2-F03 displayed superior performance. It possesses a stable SARS-CoV-2 susceptibility phenotype and does not produce false-positive hits due to drug-induced phospholipidosis. A proof-of-concept screen of 1,796 kinase inhibitors identified known and novel antiviral drug candidates including inhibitors of phosphoglycerate dehydrogenase (PHGDH), CDC like kinase 1 (CLK-1), and colony stimulating factor 1 receptor (CSF1R). The activity of the PHGDH inhibitor NCT-503 was further increased in combination with the hexokinase II (HK2) inhibitor 2-deoxy-D-glucose, which is in clinical development for COVID-19. In conclusion, caspase 3/7 activity detection in SARS-CoV-2-infected Caco-2-F03 cells provides a simple phenotypic high-throughput screening platform for SARS-CoV-2 drug candidates that reduces false-positive hits.

The use of COI barcodes for molecular identification of forensically important fly species in Germany.

Deoxyribonucleic acid (DNA)-based insect identification has become a routine and accurate tool in forensic entomology. In the present study, we demonstrate the utility of the mitochondrial DNA cytochrome oxidase I gene "barcoding region" as a universal marker for molecular identification of forensically important Diptera. We analyzed 111 specimens belonging to 13 species originating from Frankfurt am Main, Germany (Calliphoridae: Calliphora vicina, Calliphora vomitoria, Lucilia ampullacea, Lucilia caesar, Lucilia illustris, Lucilia sericata, Lucilia silvarum, Phormia regina, Protophormia terraenovae; Piophilidae: Parapiophila vulgaris; Muscidae: Hydrotaea dentipes, Hydrotaea ignava, Hydrotaea similis). Intraspecific variation ranged from 0 to 1.17% and interspecific variation occurred between 1.17% and 15.21%. Although differences within species were generally less than among species, divergence percentages overlapped due to low interspecific nucleotide divergence of the recently separated sister species L. caesar and L. illustris. However, all species formed distinct monophyletic clades and thus the cytochrome oxidase 1 (COI) barcode has been shown suitable for clear differentiation and identification of forensically relevant Diptera in Germany.

Influence of storage on larval length and age determination of the forensically important blow fly Lucilia sericata (Diptera: Calliphoridae).

One of the main tasks in forensic entomology is the determination of the minimum post-mortem interval (PMImin) based on the age of the juvenile insects feeding and developing on the dead body. An important task is to store the evidence appropriately so that the evaluation and expert report can be used in court. However, existing recommendations can be contradictory or lacking scientific validation, e.g. by proposing various preservation liquids without knowing whether and to what extent the period of storage in such a liquid has an effect on the length of the preserved larvae. Storage time can be an issue since, due to technical and procedural circumstances, killed larvae may be stored for hours, days, weeks or even longer prior length measurement. A changed body length would have consequences for the entomological report, as the age of the larvae is usually derived from their length. This study investigates the effect of four differently concentrated ethanol solutions (70%, 80%, 90% and 96%) during a storage period of up to 196 days on the body length of stored larvae of the forensically important blow fly species L. sericata (Diptera: Calliphoridae). Larvae of different ages (24 h, 48 h and 72 h after hatching) were killed by immersion in hot, non-boiling water (≥80 °C) for at least 30 s. Their lengths were measured immediately. Subsequently samples were stored in ethanol of appropriate concentration at room temperature (approx. 22 °C). Further length measurements were made at 16 different storage intervals between 1 and 196 days. Many specimens showed a length decrease for most storage conditions and all larval ages. However, there was a tendency for 48 h- and 72 h-old larvae to increase in length after the first days of storage of up to 1.1 mm which may lead to an erroneous overestimation of the PMImin using this kind of specimens. All changes in length within each cohort over total time were in the range of +7% to -9.1%. Significant differences in length changes within the first days of storage were found mainly in larvae stored in 70%- and 80%-ethanol, but larvae stored in 90%- and 96%-ethanol showed first significant differences on day 56 at the earliest. Our results lead to the recommendation that the measurements of fly larvae samples should be taken immediately after killing and before storage to avoid any effects. Ethanol ≥90% should be used for storage.

Molecular identification of carrion-breeding scuttle flies (Diptera: Phoridae) using COI barcodes.

Entomological evidence is often used in forensic cases for post-mortem interval (PMI) calculation. The most dominant species present on a corpse are typically blowflies. However, several cases have been reported where access to a corpse has been restricted for blowflies (e.g., on a buried or wrapped cadavers) but species of the family Phoridae were abundant. It has also been reported that some phorid species that exploit human corpses may also feature in cases of myiasis acquired ante-mortem. In all these cases, they may provide decisive evidence. As for blowflies, the precise identification of a phorid species collected from a corpse is necessary when estimating the PMI. Since morphological determination is often hampered due to similar characteristics especially in the larval and pupal stage, we used DNA-based methods to identify six phorid species (Megaselia scalaris, Megaselia giraudii, Megaselia abdita, Megaselia rufipes, Conicera tibialis, and Puliciphora borinquenensis) on the molecular level. We focused on a 658-bp-long region of the cytochrome oxidase I gene (COI), the most common molecular marker in forensic entomology. The amplified fragment is also used in DNA barcode approaches and was found to be suitable for identification of a wide range of insect taxa. The present study demonstrates that this region is also sufficient to distinguish between several species of scuttle flies.

Revisited - Failure of tetrodotoxin to protect red-spotted newts, Notophthalmus viridescens, from endoparasites.

Red-spotted newts, Notophthalmus viridescens, contain tetrodotoxin (TTX) and its analogue 6-epiTTX in variable concentrations. In a follow-up study, newts were sampled from a pond in Pennsylvania, USA, in 2010, 2014, and 2018. Their toxin levels were assayed by liquid-chromatography-fluorescence detection (LC-FLD), and assessment of their infection with endoparasites such as nematodes and helminths was performed by histological examination of internal organs. In the 2010 and 2014 samples, average prevalence of parasite infection was 53 and 60%, respectively, but reached 100% in the 2018 sample, where metacercaria stages of the digenean trematode genus Australapatemon/Apatemon (family: Strigeidae) were predominant causing severe tissue damage in liver and kidney. Mean values of TTX and 6-epiTTX were not significantly different in parasitized or parasite-free newts over the study period, confirming previous findings that host toxicity and parasite load are not negatively correlated. Whereas the role of TTX in defence against predators is undisputed, its efficacy to prevent parasitic infections is less obvious. Toxin-resistance of various metazoan parasites may promote their widespread occurrence in poisonous newts.

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.

Intact-Cell MALDI-ToF Mass Spectrometry for the Authentication of Drug-Adapted Cancer Cell Lines.

The use of cell lines in research can be affected by cell line misidentification. Short tandem repeat (STR) analysis is an effective method, and the gold standard, for the identification of the genetic origin of a cell line, but methods that allow the discrimination between cell lines of the same genetic origin are lacking. Here, we use intact cell MALDI-ToF mass spectrometry analysis, routinely used for the identification of bacteria in clinical diagnostic procedures, for the authentication of a set of cell lines consisting of three parental neuroblastoma cell lines (IMR-5, IMR-32 and UKF-NB-3) and eleven drug-adapted sublines. Principal component analysis (PCA) of intact-cell MALDI-ToF mass spectrometry data revealed clear differences between most, but not all, of the investigated cell lines. Mass spectrometry whole-cell fingerprints enabled the separation of IMR-32 and its clonal subline IMR-5. Sublines that had been adapted to closely related drugs, for example, the cisplatin- and oxaliplatin-resistant UKF-NB-3 sublines and the vincristine- and vinblastine-adapted IMR-5 sublines, also displayed clearly distinctive patterns. In conclusion, intact whole-cell MALDI-ToF mass spectrometry has the potential to be further developed into an authentication method for mammalian cells of a common genetic origin.

Kit-dependent discrepancy in D16S539 and general considerations for database matches.

Throughout the last decade more companies have been offering multiplex PCR kits for forensic STR typing. As a consequence, it has been demonstrated, that an observed genotype may unexpectedly vary at a single locus when different STR kits have been used. Analysing STR profiles which have to be entered in a national database, unknown or undetected primer binding site mutations, insertions or deletions within the flanking region of STR loci may hinder matches and therefore have far-reaching consequences. The current study is a further example indicating that sequence variations in flanking regions are a common problem within STR typing which should not be underestimated. A deletion of 16 nucleotides close to the primer binding site downstream of the repeat sequence resulted in deviant genotypes at the D16S539 locus according to different STR kits used.

Dating Pupae of the Blow Fly Calliphora vicina Robineau-Desvoidy 1830 (Diptera: Calliphoridae) for Post Mortem Interval-Estimation: Validation of Molecular Age Markers.

Determining the age of juvenile blow flies is one of the key tasks of forensic entomology when providing evidence for the minimum post mortem interval. While the age determination of blow fly larvae is well established using morphological parameters, the current study focuses on molecular methods for estimating the age of blow flies during the metamorphosis in the pupal stage, which lasts about half the total juvenile development. It has already been demonstrated in several studies that the intraspecific variance in expression of so far used genes in blow flies is often too high to assign a certain expression level to a distinct age, leading to an inaccurate prediction. To overcome this problem, we previously identified new markers, which show a very sharp age dependent expression course during pupal development of the forensically-important blow fly Calliphora vicina Robineau-Desvoidy 1830 (Diptera: Calliphoridae) by analyzing massive parallel sequencing (MPS) generated transcriptome data. We initially designed and validated two quantitative polymerase chain reaction (qPCR) assays for each of 15 defined pupal ages representing a daily progress during the total pupal development if grown at 17 °C. We also investigated whether the performance of these assays is affected by the ambient temperature, when rearing pupae of C. vicina at three different constant temperatures-namely 17 °C, 20 °C and 25 °C. A temperature dependency of the performance could not be observed, except for one marker. Hence, for each of the defined development landmarks, we can present gene expression profiles of one to two markers defining the mentioned progress in development.