Endocytic Profiling of Cancer Cell Models Reveals Critical Factors Influencing LNP-Mediated mRNA Delivery and Protein Expression.

Lipid nanoparticles have great potential for delivering nucleic-acid-based therapeutics, but low efficiency limits their broad clinical translation. Differences in transfection capacity between in vitro models used for nanoparticle pre-clinical testing are poorly understood. To address this, using a clinically relevant lipid nanoparticle (LNP) delivering mRNA, we highlight specific endosomal characteristics in in vitro tumor models that impact protein expression. A 30-cell line LNP-mRNA transfection screen identified three cell lines having low, medium, and high transfection that correlated with protein expression when they were analyzed in tumor models. Endocytic profiling of these cell lines identified major differences in endolysosomal morphology, localization, endocytic uptake, trafficking, recycling, and endolysosomal pH, identified using a novel pH probe. High-transfecting cells showed rapid LNP uptake and trafficking through an organized endocytic pathway to lysosomes or rapid exocytosis. Low-transfecting cells demonstrated slower endosomal LNP trafficking to lysosomes and defective endocytic organization and acidification. Our data establish that efficient LNP-mRNA transfection relies on an early and narrow endosomal escape window prior to lysosomal sequestration and/or exocytosis. Endocytic profiling should form an important pre-clinical evaluation step for nucleic acid delivery systems to inform model selection and guide delivery-system design for improved clinical translation.

Evaluating child helmet protection and testing standards: A study using PIPER child head models aged 1.5, 3, 6, and 18 years.

The anatomy of children's heads is unique and distinct from adults, with smaller and softer skulls and unfused fontanels and sutures. Despite this, most current helmet testing standards for children use the same peak linear acceleration threshold as for adults. It is unclear whether this is reasonable and otherwise what thresholds should be. To answer these questions, helmet-protected head responses for different ages are needed which is however lacking today. In this study, we apply continuously scalable PIPER child head models of 1.5, 3, and 6 years old (YO), and an upgraded 18YO to study child helmet protection under extensive linear and oblique impacts. The results of this study reveal an age-dependence trend in both global kinematics and tissue response, with younger children experiencing higher levels of acceleration and velocity, as well as increased skull stress and brain strain. These findings indicate the need for better protection for younger children, suggesting that youth helmets should have a lower linear kinematic threshold, with a preliminary value of 150g for 1.5-year-old helmets. However, the results also show a different trend in rotational kinematics, indicating that the threshold of rotational velocity for a 1.5YO is similar to that for adults. The results also support the current use of small-sized adult headforms for testing child helmets before new child headforms are available.

Evidence of predation pressure on sensitive species by raccoons based on parasitological studies.

To demonstrate predation and potential impacts of raccoons on various species, a total of 108 raccoons from aquatic-associated nature reserves and natural areas in three federal states of Germany, Hesse (n = 36), Saxony-Anhalt (n = 36) and Brandenburg (n = 36), were investigated from a dietary ecological perspective in the present study. Fecal analyses and stomach content examinations were conducted for this purpose. Additionally, as a supplementary method for analyzing the dietary spectrum of raccoons, the parasite fauna was considered, as metazoan parasites, in particular, can serve as indicators for the species and origin of food organisms. While stomach content analyses allow for a detailed recording of trophic relationships solely at the time of sampling, parasitological examinations enable inferences about more distant interaction processes. With their different developmental stages and heteroxenous life cycles involving specific, sometimes obligate, intermediate hosts, they utilize the food web to reach their definitive host. The results of this study clearly demonstrate that spawning areas of amphibians and reptiles were predominantly utilized as food resources by raccoons in the study areas. Thus, common toad (Bufo bufo), common newt (Lissotriton vulgaris), grass frog (Rana temporaria), and grass snake (Natrix natrix) were identified as food organisms for raccoons. The detection of the parasite species Euryhelmis squamula, Isthmiophora melis, and Physocephalus sexalatus with partially high infestation rates also suggests that both amphibians and reptiles belong to the established dietary components of raccoons from an ecological perspective, as amphibians and reptiles are obligate intermediate hosts in the respective parasitic life cycles of the detected parasites. The study clearly demonstrates that raccoons have a significant impact on occurrence-sensitive animal species in certain areas and, as an invasive species, can exert a negative influence on native species and ecosystems.

A comparative study of blood cell count in four automated hematology analyzers: An evaluation of the impact of preanalytical factors.

Differential white blood cell counts are frequently used in diagnosis, patient stratification, and treatment selection to optimize therapy responses. Referral laboratories are often used but challenged with use of different hematology platforms, variable blood shipping times and storage conditions, and the different sensitivities of specific cell types. To extend the scientific literature and knowledge on the temporal commutability of blood samples between hematology analyzers, we performed a comparative ex-vivo study using four of the most utilized commercial platforms, focusing on the assessment of eosinophils given its importance in asthma management. Whole blood from healthy volunteers with and without atopy (n = 6+6) and participants with eosinophilic asthma (n = 6) were stored under different conditions (at 4, 20, 30, and 37°C, with or without agitation) and analyzed at different time points (3, 6, 24, 48 and 72h post-sampling) in parallel on the Abbott CELL-DYN Sapphire, Beckman Coulter DxH900, Siemens ADVIA 2120i and Sysmex XN-1000V. In the same blood samples, eosinophil-derived neurotoxin (EDN), eosinophil activation and death markers were analyzed. All platforms gave comparable measurements of cell differentials on fresh blood within the same day of sampling. However, by 24 hours, significant temporal and temperature-dependent differences were observed, most markedly for eosinophils. None of the platforms performed perfectly across all temperatures tested during the 72 hours, showing that handling conditions should be optimized depending on the cell type of interest and the hematology analyzer. Neither disease status (healthy vs. asthma) nor agitation of the sample affected the cell quantification result or EDN release. The eosinophil activation markers measured by flow cytometry increased with time, were influenced by temperature, and were higher in those with asthma versus healthy participants. In conclusion, hematology analyzer, time window from sampling until analysis, and temperature conditions must be considered when analyzing blood cell differentials, particularly for eosinophils, via central labs to obtain counts comparable to the values obtained in freshly sampled blood.

The hidden threat: Exploring the parasite burden and feeding habits of invasive raccoon dogs (Nyctereutes procyonoides) in central Europe.

Originally from Asia, the raccoon dog Nyctereutes procyonoides is an invasive alien species in Europe, listed since 2019 on the List of invasive alien species of Union concern. The raccoon dog is considered to have negative impact on native biodiversity, as well as a crucial role in hosting and transmitting diverse parasites and pathogens of human and veterinary importance. In the present study, stomach content analyses and parasitological examinations were performed on 73 raccoon dogs from Germany. In addition, fecal samples were analyzed. The results of the study confirm the assumption that the examined raccoon dogs were infested with a various ecto- and endoparasite fauna. A total of 9 ecto- and 11 endoparasites were detected, with 6 of the endoparasites having human pathogenic potential. Trichodectes canis (P = 53.42%), Toxocara canis (P = 50.68%) and Uncinaria stenocephala (P = 68.49%) were the most abundant parasite species. The stomach contents consisted of approximately one-third vegetable and two-thirds animal components, composed of various species of amphibians, fish, insects, mammals and birds. Among them were specially protected or endangered species such as the grass frog Rana temporaria. The study shows that the raccoon dog exerts predation pressure on native species due to its omnivorous diet and, as a carrier of various parasites, poses a potential risk of infection to wild, domestic and farm animals and humans.

Raccoons contraband - The metazoan parasite fauna of free-ranging raccoons in central Europe.

The invasive raccoon (Procyon lotor) is an abundant carnivore and considered as an important potential vector of infectious diseases and parasites in Europe. Raccoons show a broad, opportunistic, omnivorous food spectrum. Food supply and habitat quality in urban areas are very attractive for the generalist raccoon. This inevitably leads to increased interaction with humans, domestic animals and livestock, making the raccoon a potentially suitable zoonosis vector. In its autochthonous range, especially in the Eastern and Midwestern United States, the raccoon has been studied very intensively since the beginning of the 20th century. Whereas, basic field biology and parasitology studies in Germany and Europe are lacking and have only been conducted sporadically, regionally and on small sample sizes. In the presented study 234 raccoons from central Germany were comprehensively examined for their metazoan parasite fauna. The present study shows for the first time an extremely diverse parasite fauna in raccoons outside their native range and proves their essential role as intermediate hosts and hosts for ecto- and endoparasites. A total of 23 different parasite species were identified, five of which are human pathogens, 14 of which are new for the parasite fauna of raccoons in Europe. The human pathogenic raccoon roundworm Baylisascaris procyonis is the most common parasite species in this study, with a prevalence of up to 95%. The digenetic trematode Plagiorchis muris, another human pathogenic parasite species, was detected for the first time in raccoons. The ongoing spread of invasive carnivores and the associated spread and transmission of their parasites and other pathogens increases the potential health risk of wild and farmed animals as well as humans. An increase in parasitic diseases in humans (e.g. raccoon roundworm) is to be expected, especially in urban areas, where raccoons are becoming more and more abundant.

SARS-CoV-2 and West Nile Virus Prevalence Studies in Raccoons and Raccoon Dogs from Germany.

Unlike farm animals, wild animals are not subject to continuous health surveillance. Individual projects designed to screen wildlife populations for specific pathogens are, therefore, also of great importance for human health. In this context, the possible formation of a reservoir for highly pathogenic zoonotic pathogens is a focus of research. Two of these pathogens that have received particular attention during the last years are the novel severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), due to its fast global spread and high impact to the human health, and, since its introduction into Germany, the flavivirus West Nile virus (WNV). Especially in combination with invasive vertebrate species (e.g., raccoons (Procyon lotor) and raccoon dogs (Nyctereutes procyonoides) in Germany), risk analysis must be done to enable health authorities to assess the potential for the establishment of new wild life reservoirs for pathogens. Therefore, samples were collected from raccoons and raccoon dogs and analyzed for the presence of SARS-CoV-2 and WNV infections in these populations. Molecular biological and serological data obtained imply that no SARS-CoV-2 nor WNV reservoir has been established in these two wild life species yet. Future investigations need to keep an eye on these invasive carnivore populations, especially since the close contact of these animals to humans, mainly in urban areas, would make animal-human transmission a challenge for human health.

Development of an ObLiGaRe Doxycycline Inducible Cas9 system for pre-clinical cancer drug discovery.

The CRISPR-Cas9 system has increased the speed and precision of genetic editing in cells and animals. However, model generation for drug development is still expensive and time-consuming, demanding more target flexibility and faster turnaround times with high reproducibility. The generation of a tightly controlled ObLiGaRe doxycycline inducible SpCas9 (ODInCas9) transgene and its use in targeted ObLiGaRe results in functional integration into both human and mouse cells culminating in the generation of the ODInCas9 mouse. Genomic editing can be performed in cells of various tissue origins without any detectable gene editing in the absence of doxycycline. Somatic in vivo editing can model non-small cell lung cancer (NSCLC) adenocarcinomas, enabling treatment studies to validate the efficacy of candidate drugs. The ODInCas9 mouse allows robust and tunable genome editing granting flexibility, speed and uniformity at less cost, leading to high throughput and practical preclinical in vivo therapeutic testing.

Structure of Lipid Nanoparticles Containing siRNA or mRNA by Dynamic Nuclear Polarization-Enhanced NMR Spectroscopy.

Here, we show how dynamic nuclear polarization (DNP) NMR spectroscopy experiments permit the atomic level structural characterization of loaded and empty lipid nanoparticles (LNPs). The LNPs used here were synthesized by the microfluidic mixing technique and are composed of ionizable cationic lipid (DLin-MC3-DMA), a phospholipid (distearoylphosphatidylcholine, DSPC), cholesterol, and poly(ethylene glycol) (PEG) (dimyristoyl phosphatidyl ethanolamine (DMPE)-PEG 2000), as well as encapsulated cargoes that are either phosphorothioated siRNA (50 or 100%) or mRNA. We show that LNPs form physically stable complexes with bioactive drug siRNA for a period of 94 days. Relayed DNP experiments are performed to study 1H-1H spin diffusion and to determine the spatial location of the various components of the LNP by studying the average enhancement factors as a function of polarization time. We observe a striking feature of LNPs in the presence and in the absence of encapsulating siRNA or mRNA by comparing our experimental results to numerical spin-diffusion modeling. We observe that LNPs form a layered structure, and we detect that DSPC and DMPE-PEG 2000 lipids form a surface rich layer in the presence (or absence) of the cargoes and that the cholesterol and ionizable cationic lipid are embedded in the core. Furthermore, relayed DNP 31P solid-state NMR experiments allow the location of the cargo encapsulated in the LNPs to be determined. On the basis of the results, we propose a new structural model for the LNPs that features a homogeneous core with a tendency for layering of DSPC and DMPE-PEG at the surface.