Intravesicular Genomic DNA Enriched by Size Exclusion Chromatography Can Enhance Lung Cancer Oncogene Mutation Detection Sensitivity

Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RNA and DNA as potential templates for molecular diagnostics, e.g., in cancer genotyping. While it has been established that DNA templates appear on the outside of EVs, no consensus exists on which nucleic acid species inside small EVs (<200 nm, sEVs) are sufficiently abundant and accessible for developing genotyping protocols. We investigated this by extracting total intravesicular nucleic acid content from sEVs isolated from the conditioned cell medium of the human NCI-H1975 cell line containing the epidermal growth factor (EGFR) gene mutation T790M as a model system for non-small cell lung cancer. We observed that mainly short genomic DNA (<35–100 bp) present in the sEVs served as a template. Using qEV size exclusion chromatography (SEC), significantly lower yield and higher purity of isolated sEV fractions were obtained as compared to exoEasy membrane affinity purification and ultracentrifugation. Nevertheless, we detected the EGFR T790M mutation in the sEVs’ lumen with similar sensitivity using digital PCR. When applying SEC-based sEV separation prior to cell-free DNA extraction on spiked human plasma samples, we found significantly higher mutant allele frequencies as compared to standard cell-free DNA extraction, which in part was due to co-purification of circulating tumor DNA. We conclude that intravesicular genomic DNA can be exploited next to ctDNA to enhance EGFR T790M mutation detection sensitivity by adding a fast and easy-to-use sEV separation method, such as SEC, upstream of standard clinical cell-free DNA workflows.     

Immunogenicity of the Envelope Surface Unit of Human Endogenous Retrovirus K18 in Mice

The triggers for the development of multiple sclerosis (MS) have not been fully understood to date. One hypothesis proposes a viral etiology. Interestingly, viral proteins from human endogenous retroviruses (HERVs) may play a role in the pathogenesis of MS. Allelic variants of the HERV-K18 env gene represent a genetic risk factor for MS, and the envelope protein is considered to be an Epstein–Barr virus-trans-activated superantigen. To further specify a possible role for HERV-K18 in MS, the present study examined the immunogenicity of the purified surface unit (SU). HERV-K18(SU) induced envelope-specific plasma IgG in immunized mice and triggered proliferation of T cells isolated from these mice. It did not trigger phenotypic changes in a mouse model of experimental autoimmune encephalomyelitis. Further studies are needed to investigate the underlying mechanisms of HERV-K18 interaction with immune system regulators in more detail.     

Palladium‐Catalyzed C?N Cross‐Coupling of N′‐Monosubstituted Sulfondiimines with Aryl Bromides

A general method for the N‐arylation of sulfondiimines with aryl bromides using tris(dibenzylideneacetone)dipalladium(0) [Pd₂(dba)₃] and 2‐dicyclohexylphosphino‐2′,6′‐diisopropoxybiphenyl (RuPhos) as catalyst system was developed. A new benzothiazine was obtained, and a protocol for the cleavage of para‐methoxyphenyl (PMP) groups in PMP‐protected sulfondiimines has been found, which provides access to synthetically useful NH‐derivatives, that are difficult to prepare by other means.     

Integrating Archaeology and Landscape Analysis for the Cultural Heritage Management of a World War I Militarised Landscape: The German Field Defences in Antwerp

The approaching centenary of the start of World War I and the booming cultural tourism at the former Western Front in Belgium, combined with recent urbanisation and agricultural intensification processes, have promoted the demand for a more effective and sustainable heritage management. In addition, there is need for interdisciplinary research on how war and socio-natural landscapes reciprocally reproduce each other in time and space. The focus of this paper is a WWI defence system in the Province of Antwerp (Belgium), some 100 km to the east of the actual Western frontline. Research included the inventory and evaluation of the remaining above-ground relics of military features in a landscape archaeological perspective, based on WWI aerial photographs, historical maps and fieldwork. Landscape types and dynamics were identified from 1918 to 2011, based on a time series of aerial photos and maps, complemented with fieldwork. Second, an overall vision was formulated for sustainable heritage management of the militarised landscape. Both vision and practical recommendations are immediately useful for policy makers and stakeholders.     

Environmental toxicology and risk assessment of pharmaceuticals from hospital wastewater

In this paper, we evaluated the ecotoxicological potential of the 100 pharmaceuticals expected to occur in highest quantities in the wastewater of a general hospital and a psychiatric center in Switzerland. We related the toxicity data to predicted concentrations in different wastewater streams to assess the overall risk potential for different scenarios, including conventional biological pretreatment in the hospital and urine source separation. The concentrations in wastewater were estimated with pharmaceutical usage information provided by the hospitals and literature data on human excretion into feces and urine. Environmental concentrations in the effluents of the exposure scenarios were predicted by estimating dilution in sewers and with literature data on elimination during wastewater treatment. Effect assessment was performed using quantitative structure-activity relationships because experimental ecotoxicity data were only available for less than 20% of the 100 pharmaceuticals with expected highest loads. As many pharmaceuticals are acids or bases, a correction for the speciation was implemented in the toxicity prediction model.The lists of Top-100 pharmaceuticals were distinctly different between the two hospital types with only 37 pharmaceuticals overlapping in both datasets. 31 Pharmaceuticals in the general hospital and 42 pharmaceuticals in the psychiatric center had a risk quotient above 0.01 and thus contributed to the mixture risk quotient. However, together they constituted only 14% (hospital) and 30% (psychiatry) of the load of pharmaceuticals. Hence, medical consumption data alone are insufficient predictors of environmental risk. The risk quotients were dominated by amiodarone, ritonavir, clotrimazole, and diclofenac. Only diclofenac is well researched in ecotoxicology, while amiodarone, ritonavir, and clotrimazole have no or very limited experimental fate or toxicity data available. The presented computational analysis thus helps setting priorities for further testing.Separate treatment of hospital wastewater would reduce the pharmaceutical load of wastewater treatment plants, and the risk from the newly identified priority pharmaceuticals. However, because high-risk pharmaceuticals are excreted mainly with feces, urine source separation is not a viable option for reducing the risk potential from hospital wastewater, while a sorption step could be beneficial.     

Capturing and incorporating expert knowledge into machine learning models for quality prediction in manufacturing

Journal of Intelligent Manufacturing - Increasing digitalization enables the use of machine learning (ML) methods for analyzing and optimizing manufacturing processes. A main application of ML is...     

Chromogenic cross-link formation in green fluorescent protein

Recent advances in our understanding of the mechanism of chromophore formation in green fluorescent protein (GFP) are presented. GFP is the best-studied member of the family of GFP-like proteins, proteins that exhibit bright coloration spanning most of the visible spectrum. GFPs undergo a post-translational self-modification process that yields an intrinsic fluorophore constructed from an internal main-chain cross-link that is susceptible to air oxidation. A combination of protein X-ray crystallographic and kinetic experiments has led to the development of a mechanistic model that entails conformational pre-organization, electrophilic and base catalysis, and production of hydrogen peroxide upon protein oxidation. The process is concluded by a slow proton abstraction step from a tyrosine-derived carbon acid.