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.

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.

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.

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.

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.

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.

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.

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.

A molecular, morphological, and physiological comparison of English and German populations of Calliphora vicina (Diptera: Calliphoridae).

The bluebottle blow fly Calliphora vicina is a common species distributed throughout Europe that can play an important role as forensic evidence in crime investigations. Developmental rates of C. vicina from distinct populations from Germany and England were compared under different temperature regimes to explore the use of growth data from different geographical regions for local case work. Wing morphometrics and molecular analysis between these populations were also studied as indicators for biological differences. One colony each of German and English C. vicina were cultured at the Institute of Legal Medicine in Frankfurt, Germany. Three different temperature regimes were applied, two constant (16°C & 25°C) and one variable (17-26°C, room temperature = RT). At seven time points (600, 850, 1200, 1450, 1800, 2050, and 2400 accumulated degree hours), larval lengths were measured; additionally, the durations of the post feeding stage and intrapuparial metamorphosis were recorded. For the morphometric and molecular study, 184 females and 133 males from each C. vicina population (Germany n = 3, England n = 4) were sampled. Right wings were measured based on 19 landmarks and analyzed using canonical variates analysis and discriminant function analysis. DNA was isolated from three legs per specimen (n = 61) using 5% chelex. A 784 bp long fragment of the mitochondrial cytochrome b gene was sequenced; sequences were aligned and phylogenetically analyzed. Similar larval growth rates of C. vicina were found from different geographic populations at different temperatures during the major part of development. Nevertheless, because minor differences were found a wider range of temperatures and sampling more time points should be analyzed to obtain more information relevant for forensic case work. Wing shape variation showed a difference between the German and English populations (P<0.0001). However, separation between the seven German and English populations at the smaller geographic scale remained ambiguous. Molecular phylogenetic analysis by maximum likelihood method could not unambiguously separate the different geographic populations at a national (Germany vs England) or local level.

Molecular Analysis of Forensically Important Blow Flies in Thailand.

Blow flies are the first insect group to colonize on a dead body and thus correct species identification is a crucial step in forensic investigations for estimating the minimum postmortem interval, as developmental times are species-specific. Due to the difficulty of traditional morphology-based identification such as the morphological similarity of closely related species and uncovered taxonomic keys for all developmental stages, DNA-based identification has been increasing in interest, especially in high biodiversity areas such as Thailand. In this study, the effectiveness of long mitochondrial cytochrome c oxidase subunit I and II (COI and COII) sequences (1247 and 635 bp, respectively) in identifying 16 species of forensically relevant blow flies in Thailand (Chrysomya bezziana, Chrysomya chani, Chrysomya megacephala, Chrysomya nigripes, Chrysomya pinguis, Chrysomya rufifacies, Chrysomya thanomthini, Chrysomya villeneuvi, Lucilia cuprina, Lucilia papuensis, Lucilia porphyrina, Lucilia sinensis, Hemipyrellia ligurriens, Hemipyrellia pulchra, Hypopygiopsis infumata, and Hypopygiopsis tumrasvini) was assessed using distance-based (Kimura two-parameter distances based on Best Match, Best Close Match, and All Species Barcodes criteria) and tree-based (grouping taxa by sequence similarity in the neighbor-joining tree) methods. Analyses of the obtained sequence data demonstrated that COI and COII genes were effective markers for accurate species identification of the Thai blow flies. This study has not only demonstrated the genetic diversity of Thai blow flies, but also provided a reliable DNA reference database for further use in forensic entomology within the country and other regions where these species exist.

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.

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.

Wing morphometrics as a tool in species identification of forensically important blow flies of Thailand.

BACKGROUND: Correct species identification of blow flies is a crucial step for understanding their biology, which can be used not only for designing fly control programs, but also to determine the minimum time since death. Identification techniques are usually based on morphological and molecular characters. However, the use of classical morphology requires experienced entomologists for correct identification; while molecular techniques rely on a sound laboratory expertise and remain ambiguous for certain taxa. Landmark-based geometric morphometric analysis of insect wings has been extensively applied in species identification. However, few wing morphometric analyses of blow fly species have been published. METHODS: We applied a landmark-based geometric morphometric analysis of wings for species identification of 12 medically and forensically important blow fly species of Thailand. Nineteen landmarks of each right wing of 372 specimens were digitised. Variation in wing size and wing shape was analysed and evaluated for allometric effects. The latter confirmed the influence of size on the shape differences between species and sexes. Wing shape variation among genera and species were analysed using canonical variates analysis followed by a cross-validation test. RESULTS: Wing size was not suitable for species discrimination, whereas wing shape can be a useful tool to separate taxa on both, genus and species level depending on the analysed taxa. It appeared to be highly reliable, especially for classifying Chrysomya species, but less robust for a species discrimination in the genera Lucilia and Hemipyrellia. Allometry did not affect species separation but had an impact on sexual shape dimorphism. CONCLUSIONS: A landmark-based geometric morphometric analysis of wings is a useful additional method for species discrimination. It is a simple, reliable and inexpensive method, but it can be time-consuming locating the landmarks for a large scale study and requires non-damaged wings for analysis.

Quantitative pteridine fluorescence analysis: A possible age-grading technique for the adult stages of the blow fly Calliphora vicina (Diptera: Calliphoridae).

Age estimation of adult flies could extend the possible window of time for calculating the minimal postmortem interval (PMImin) by means of entomological methods. Currently, this is done by estimating the time required by necrophagous Diptera to reach certain juvenile developmental landmarks, and the method only works until the end of metamorphosis and emergence of the adult fly. Particularly at indoor crime scenes, being able to estimate the age of trapped adult flies would be an important tool with which to extend the calculable PMI beyond the developmental period. Recently, several promising age-dependent morphological and physiological characteristics of adult insects have been investigated in medical and forensic entomology, but the results are still preliminary and restricted to a few species. We examined adults of the forensically relevant blow fly species Calliphora vicina and investigated the fluorescence levels of pteridine, a group of metabolites that accumulates in the eyes during aging. From Day 1 to Day 25 post-emergence, flies were kept at three different temperature regimes (20°C, 25°C, and fluctuating temperatures in the context of a field study) and 12:12 L:D. From Day 1 until Day 7, the fluorescence of pteridine was determined on a daily basis, and thereafter, every three days. The achieved fly age was multiplied with the relevant temperature and converted into accumulated degree-days (ADD). The fluorescence level of pteridine increased linear with increasing ADD (females: R2=0.777; males: R2=0.802). The difference between sexes was significant (p<0.001). Neither head weight nor temperature had an effect on pteridine fluorescence. Because the variation in pteridine fluorescence increased with increasing ADD, it seems favorable to combine several aging methods for more precise results. In context, we emphasize that different body parts of the same specimen can be used to analyze cuticular hydrocarbons (legs), pteridine fluorescence (head/eyes), and gonotrophic stage (female abdomen).

Acquired resistance to oxaliplatin is not directly associated with increased resistance to DNA damage in SK-N-ASrOXALI4000, a newly established oxaliplatin-resistant sub-line of the neuroblastoma cell line SK-N-AS.

The formation of acquired drug resistance is a major reason for the failure of anti-cancer therapies after initial response. Here, we introduce a novel model of acquired oxaliplatin resistance, a sub-line of the non-MYCN-amplified neuroblastoma cell line SK-N-AS that was adapted to growth in the presence of 4000 ng/mL oxaliplatin (SK-N-ASrOXALI4000). SK-N-ASrOXALI4000 cells displayed enhanced chromosomal aberrations compared to SK-N-AS, as indicated by 24-chromosome fluorescence in situ hybridisation. Moreover, SK-N-ASrOXALI4000 cells were resistant not only to oxaliplatin but also to the two other commonly used anti-cancer platinum agents cisplatin and carboplatin. SK-N-ASrOXALI4000 cells exhibited a stable resistance phenotype that was not affected by culturing the cells for 10 weeks in the absence of oxaliplatin. Interestingly, SK-N-ASrOXALI4000 cells showed no cross resistance to gemcitabine and increased sensitivity to doxorubicin and UVC radiation, alternative treatments that like platinum drugs target DNA integrity. Notably, UVC-induced DNA damage is thought to be predominantly repaired by nucleotide excision repair and nucleotide excision repair has been described as the main oxaliplatin-induced DNA damage repair system. SK-N-ASrOXALI4000 cells were also more sensitive to lysis by influenza A virus, a candidate for oncolytic therapy, than SK-N-AS cells. In conclusion, we introduce a novel oxaliplatin resistance model. The oxaliplatin resistance mechanisms in SK-N-ASrOXALI4000 cells appear to be complex and not to directly depend on enhanced DNA repair capacity. Models of oxaliplatin resistance are of particular relevance since research on platinum drugs has so far predominantly focused on cisplatin and carboplatin.

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.

Molecular identification and phylogenetic analysis of the forensically important family Piophilidae (Diptera) from different European locations.

Species identification plays an important role in forensic entomology and is mandatory for an accurate calculation of the minimum post-mortem interval. Many important Diptera and Coleoptera taxa of the cadaver community can already be identified by common barcoding approaches, i.e., by sequencing a 658bp region in the mitochondrial cytochrome c oxidase subunit I (coI) gene. Nevertheless, there is still a lack of reference barcodes for species, in particular, that can be found on cadavers at later decomposition stages. Flies of the family Piophilidae illustrate this gap of knowledge perfectly. Due to the fact that a reliable morphological identification key for the immature stages of this flies is still missing and the immature stages of many piophilids cannot be assigned to a certain species, there is need for additional tools to identify forensically relevant taxa. We collected adult piophilid specimens at 10 locations in five European countries: Spain (n=3 locations), Germany (n=3), Portugal (n=2), Poland (n=1) and Switzerland (n=1). Apart from the coI barcoding region, we additionally analyzed a 398bp long region of the nuclear elongation factor 1 alpha (ef1a) and subsequently established the molecular identifier for nine piophilid species. In addition, we present the molecular phylogeny of the examined taxa.

,,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.