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Background Bacteria inhabit the in- and outside of the human body, such as skin, gut or the oral cavity where they play an innoxious, beneficial, e.g. as probiotic bacteria, or even pathogenic role, e.g. during a sepsis. Recent studies have shown that bacteria can secrete membrane vesicles (MVs) like eukaryotic cells with extracellular vesicles (EVs). Several studies indicate that bacterial membrane vesicles (bMVs) play a crucial role in microbiome-host interactions. However, the composition of such bMVs and their functionality under different culture conditions are still largely unknown. Methods To get more insights into bMVs, we investigated the composition and functionality of E. coli (DSM 105380) bMVs from the culture media Lysogeny broth (LB) and RPMI 1640 throughout the different phases of growth (lag-, log- and stationary-phase). bMVs from 3 time points from 2 media (LB_8h, LB_54h, LB_168h, RPMI_8h, RPMI_54h and RPMI_168h) were isolated by ultracentrifugation and analyzed using nanoparticle tracking analysis (NTA), EV flow-cytometry, cryogenic electron microscopy (Cryo-EM), conventional transmission electron microscopy (TEM) and mass spectrometry-based proteomics (LC-MS/MS). Furthermore, we examined pro-inflammatory cytokines IL-1 and IL-8 in the human monocyte cell line THP-1 by treatment with bMVs. Results Particle numbers increased with inoculation periods. The bMV morphologies in Cryo-EM/TEM were similar at each time point and condition. EV flow-cytometry showed a similarity of the common bMV markers OmpA+ GroEL- and OmpA- GroEL+ in each group. We found 140 proteins to be consistently expressed in the 6 groups with LC-MS/MS proteomics while we could also observe unique proteins throughout these treatments. Treatment of THP-1 cells with bMVs of all 6 groups lead to significantly high IL-1 and IL-8 expressions. Conclusions Our study showed that the choice of medium and the duration of culturing significantly influences E.coli bMV protein composition. Moreover, our flow-cytometry results indicate that different bMV subpopulations may be shed. Irrespective of the medium used, we observed an accumulation of E. coli bMVs over time, possibly due to increase of bacterial cells. Our cell culture experiments/functional assays imply that bMVs isolated from the 6 groups by ultracentrifugation retain their function and lead to comparable cytokine induction.

The availability of genetically modified mice has facilitated the study of mammalian T cells. No model has yet been developed to study these cells in chicken, an important livestock species with a high availability of γδ T cells. To investigate the role of γδ and αβ T cell populations in birds, we generated chickens lacking these T cell populations. This was achieved by genomic deletion of the constant region of the T cell receptor γ or αβ chain, leading to a complete loss of either γδ or αβ T cells. Our results show that a deletion of αβ T cells but not γδ T cells resulted in a severe phenotype in knockout chickens. The αβ T cell knockout chickens exhibited granulomas associated with inflammation of the spleen and the proventriculus. Immunophenotyping of αβ T cell knockout chickens revealed a significant increase in monocytes and the absence of CD4+T cells and FoxP3+regulatory T cells compared to wild type chickens. In addition, we observed a significant decrease in immunoglobulins, B lymphocytes, and changes in the bursa morphology. Our data reveal the consequences of T cell knockouts in chickens and provide new insights into their function in vertebrates.Significance statementThe lack of genetically modified chickens has severely limited research in avian immunology compared to other animal models. Here, we report the generation of two T cell knockout chicken lines that will contribute significantly to the understanding of T cell biology as a very important research model as well as an important livestock species. The generated animals reveal the function of different T cell populations in chickens and will help to better understand the role of these cells during the interaction with various pathogens in birds.

Background & Aims: Necrotizing enterocolitis (NEC) is a life-threatening disease, and the most common gastrointestinal emergency in premature infants. Accurate early diagnosis is challenging. Modified Bell’s staging is routinely used to guide diagnosis, but early diagnostic signs are non-specific, potentially leading to unobserved disease progression, which is problematic given the often rapid deterioration observed. We investigated faecal cytokine levels, coupled with gut microbiota profiles, as a non-invasive method to discover specific NEC-associated signatures that can be applied as potential diagnostic markers. Methods: Premature babies born below 32 weeks of gestation were admitted to the 2-site neonatal intensive care unit (NICU) of Imperial College hospitals (St. Mary’s or Queen Charlotte’s & Chelsea) between January 2011 and December 2012. During the NICU stay, expert neonatologist grouped individuals by modified Bell’s staging (healthy, NEC1, NEC2/3) and faecal samples from diapers were collected consecutively. Microbiota profiles were assessed by 16S rRNA gene amplicon sequencing and cytokine concentrations were measured by V-Plex multiplex assays. Results: Early evaluation of microbiota profiles revealed only minor differences. However, at later time points, significant changes in microbiota structure were observed for Bacillota (adj. p=0.0396), with Enterococcus being the least abundant in Bell stage 2/3 NEC. Evaluation of faecal cytokine levels revealed significantly higher concentrations of IL-1α (p=0.045), IL-5 (p=0.0074), and IL-10 (p=0.032) in Bell stage 1 NEC compared to healthy individuals. Conclusions: Differences in certain faecal cytokine profiles in patients with NEC indicate their potential use as diagnostic biomarkers to facilitate earlier diagnosis. Additionally, associations between microbial and cytokine profiles contribute to improving knowledge about NEC pathogenesis.

ObjectivesNecrotizing enterocolitis (NEC) is a life-threatening disease, and the most common gastrointestinal emergency in premature infants. Accurate early diagnosis is challenging. Modified Bell’s staging is routinely used to guide diagnosis, but early diagnostic signs are non-specific, potentially leading to unobserved disease progression, which is problematic given the often rapid deterioration observed in NEC infants. New techniques, using biomarkers as diagnostic tool to improve diagnosis of NEC, are emerging. Here we investigated faecal cytokine levels, coupled with gut microbiota profiles, as a non-invasive method to discover specific NEC-associated signatures that can be applied as potential diagnostic markers.Study designPremature babies born below 32 weeks of gestation were admitted to the 2-site neonatal intensive care unit (NICU) of Imperial College hospitals (St. Mary’s or Queen Charlotte’s & Chelsea) between January 2011 and December 2012. All but two babies received a first course of antibiotics from birth onwards. Faecal samples from diapers were collected consecutively during the NICU stay.ResultsEvaluation of microbiota profiles between the study groups revealed only minor differences. However, at later time points, significant changes in microbiota structure were observed for Firmicutes, withEnterococcusbeing the least abundant in Bell stage 2/3 NEC. Faecal cytokine levels were similar to those found in previous studies evaluating systemic cytokine concentrations in NEC settings, but measurement in faeces represents a non-invasive method to evaluate the early onset of the disease. For IL-1α, IL-5 and IL-10, a significantly rising gradient of levels were observed from healthy to NEC1 to NEC2/3.ConclusionsDifferences in certain faecal cytokine profiles in patients with NEC indicate their potential use as diagnostic biomarkers to facilitate earlier diagnosis. Additionally, associations between microbial and cytokine profiles, contribute to improving knowledge about NEC pathogenesis.

MSystems 6(3), e01300-20 (2021). doi:10.1128/mSystems.01300-20, Published by American Society for Microbiology, Washington, DC

Simple Summary Feed for laboratory mice is available in different physical forms. However, there is insufficient standardization in nutrient composition and physical forms. Results pertaining to energy and nutrient digestibility show that differentially processed feed (pelleted vs. extruded) and even batches from the same provider (pelleted vs. pelleted) differ in starch gelatinization. Here we show that feed processing impacts the mice’s gastrointestinal microbiome. Reproducibility and comparability between experiments with differently processed feeds in laboratory mice should not be taken for granted. Therefore, details on dietary ingredients and feed processing should be specified in studies that include animal experiments. Abstract The composition of the microbiome is subject to the host’s diet. In commercial laboratory mouse diets, different physical forms of the same diets are available, containing—according to their labels—identical ingredients and nutrient compositions. However, variations in nutrient composition and starch gelatinization due to production processes and their impact on digestibility have been described. In this study, a total of 48 C57BL/J6 mice were assigned to two equal groups and were fed diets (produced with different processes—extruded vs. pelleted) for eight weeks in two biological replicates. At the end of the experiment, samples were collected from five different gastrointestinal regions, including the stomach, small intestine, cecum, large intestine, and an extracorporeal region (feces), and the microbiome was analyzed with 16S rRNA gene amplicon sequencing. The replicates in both experiments differed significantly in their relative abundances of Muribaculaceae species. Furthermore, the gastrointestinal content of pellet-fed mice contained larger numbers of Lactobacillus species. These results indicate that starch gelatinization and ingredient composition significantly influence microbial makeup. In conclusion, different feed processing methods may affect fundamental digestive and metabolic processes, impacting animal experiments and biasing microbiome data.

Im naturwissenschaftlichen Unterricht sind Schulervorstellungen zu zentralen Konzepten ein wichtiger Ausgangspunkt fur die Gestaltung von Lernprozessen. Schulervorstellungen konnen als Hypothesen formuliert und in Experimenten uberpruft werden. Im Rahmen einer Interventionsstudie wurden Hypothesen sowie Experimentplanungen hinsichtlich des von Hadfield [1] beschriebenen Kupfer-Problems erhoben und analysiert. Im vorliegenden Beitrag wird dargestellt, welche Hypothesen bezuglich des schwarzen Belags auf einem erhitzten Kupferblech von Schulerinnen und Schulern aufgestellt werden, welche dieser Hypothesen in Experimentplanungen von ihnen uberpruft werden und von welcher Qualitat die Experimentplanungen sind. Die Ergebnisse deuten darauf hin, dass Schulerinnen und Schuler bestimmte Hypothesen bevorzugt uberprufen und dass die Qualitat der Experimentplanungen von hoherer Qualitat ist, wenn Hypothesen auf einem niedrigen Verstandnisniveau uberpruft werden.

Kurzfassung Die Digitale Fabrik wird immer wichtiger. Dies zeigt auch das kontinuierlich wachsende Interesse von Groß- und Kleinunternehmen, insbesondere in der Automobilindustrie. Dabei ist die Digitale Fabrik zunehmend als Wettbewerbsfaktor zu betrachten, welcher die notwendige Reaktion auf sich rasch verändernde Produktionsrahmenbedingungen erst ermöglicht. Der vorliegende Beitrag zeigt die Notwendigkeit der differenzierenden Betrachtung der Digitalen Fabrik, die weder als reines Software-Werkzeug noch als reine Methodik verstanden werden darf.