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Institute
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.
Novel treatment options are needed for the successful therapy of patients with high-risk neuroblastoma. Here, we investigated the cyclin-dependent kinase (CDK) inhibitor SNS-032 in a panel of 109 neuroblastoma cell lines consisting of 19 parental cell lines and 90 sublines with acquired resistance to 14 different anticancer drugs. Seventy-three percent of the investigated neuroblastoma cell lines and all four investigated primary tumor samples displayed concentrations that reduce cell viability by 50% in the range of the therapeutic plasma levels reported for SNS-032 (<754 nM). Sixty-two percent of the cell lines and two of the primary samples displayed concentrations that reduce cell viability by 90% in this concentration range. SNS-032 also impaired the growth of the multidrug-resistant cisplatin-adapted UKF-NB-3 subline UKF-NB-3rCDDP1000 in mice. ABCB1 expression (but not ABCG2 expression) conferred resistance to SNS-032. The antineuroblastoma effects of SNS-032 did not depend on functional p53. The antineuroblastoma mechanism of SNS-032 included CDK7 and CDK9 inhibition-mediated suppression of RNA synthesis and subsequent depletion of antiapoptotic proteins with a fast turnover rate including X-linked inhibitor of apoptosis (XIAP), myeloid cell leukemia sequence 1 (Mcl-1), baculoviral IAP repeat containing 2 (BIRC2; cIAP-1), and survivin. In conclusion, CDK7 and CDK9 represent promising drug targets and SNS-032 represents a potential treatment option for neuroblastoma including therapy-refractory cases.
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.
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.
Intact-cell maldi-tof mass spectrometry for the authentication of drug-adapted cancer cell lines
(2019)
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.
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.
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 read-out for monitoring the replication of SARS-CoV-2 isolates from different variants, including a remdesivir-resistant strain, and of other coronaviruses in a broad range of cell culture models, independently of cytopathogenic effect formation. Compared to other cell culture models, the Caco-2 subline Caco-2-F03 displayed superior performance, as it possesses a stable SARS-CoV-2 susceptible phenotype and does not produce false-positive hits due to drug-induced phospholipidosis. A proof-of-concept screen of 1796 kinase inhibitors identified known and novel antiviral drug candidates including inhibitors of PHGDH, CLK-1, and CSF1R. The activity of the PHGDH inhibitor NCT-503 was further increased in combination with the 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-2F03 cells provides a simple phenotypic high-throughput screening platform for SARS-CoV-2 drug candidates that reduces false positive hits.
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.
Aurora kinase inhibitors displayed activity in pre-clinical neuroblastoma models. Here, we studied the effects of the pan-aurora kinase inhibitor tozasertib (VX680, MK-0457) and the aurora kinase inhibitor alisertib (MLN8237) that shows some specificity for aurora kinase A over aurora kinase B in a panel of neuroblastoma cell lines with acquired drug resistance. Both compounds displayed anti-neuroblastoma activity in the nanomolar range. The anti-neuroblastoma mechanism included inhibition of aurora kinase signalling as indicated by decreased phosphorylation of the aurora kinase substrate histone H3, cell cycle inhibition in G2/M phase, and induction of apoptosis. The activity of alisertib but not of tozasertib was affected by ABCB1 expression. Aurora kinase inhibitors induced a p53 response and their activity was enhanced in combination with the MDM2 inhibitor and p53 activator nutlin-3 in p53 wild-type cells. In conclusion, aurora kinases are potential drug targets in therapy-refractory neuroblastoma, in particular for the vast majority of p53 wild-type cases.
Background: Various kinase inhibitors are known to be ATP-binding cassette (ABC) transporter substrates and resistance acquisition to kinase inhibitors has been associated to increased ABC transporter expression. Here, we investigated the role of the ABC transporters ABCB1, ABCC1, and ABCG2 during melanoma cell resistance acquisition to the V600-mutant BRAF inhibitors PLX4032 (vemurafenib) and PLX4720. PLX4032 had previously been shown to interfere with ABCB1 and ABCG2. PLX4720 had been demonstrated to interact with ABCB1 but to a lower extent than PLX4032.
Findings: PLX4032 and PLX4720 affected ABCC1- and ABCG2-mediated drug transport in a similar fashion. In a panel of 16 V600E BRAF-mutated melanoma cell lines consisting of four parental cell lines and their sub-lines with acquired resistance to PLX4032, PLX4720, vincristine (cytotoxic ABCB1 and ABCC1 substrate), or mitoxantrone (cytotoxic ABCG2 substrate), we detected enhanced ABC transporter expression in 4/4 cytotoxic ABC transporter substrate-resistant, 3/4 PLX4720-resistant, and 1/4 PLX4032-resistant melanoma cell lines.
Conclusion: PLX4032 has the potential to induce ABC transporter expression but this potential is lower than that of PLX4720 or cytotoxic ABC transporter substrates. Since ABC transporters confer multi-drug resistance, this is of relevance for the design of next-line therapies.