Analyse von Regulatorproteinen des Yersinia Typ III Sekretionssystems

Bei Yersinia enterocolitica handelt es sich um einen humanpathogenen Keim, der ein Typ III Sekretionssystem (T3SS) zur Translokation von Effektorproteinen in Wirtszellen nutzt, um die Zellen zu paralysieren und der angeborenen Immunantwort zu entgehen. Die homologen Proteine YscM1 und YscM2, die bisher als funktionell äquivalent galten, wurden als Interaktionspartner der Phosphoenolpyruvatcarboxylase (PEPC) identifiziert. Die PEPC trägt durch die Bildung von Oxalacetat zur Auffüllung des Citratzyklus bei. Daher wurde die Interaktion der PEPC mit den YscM Proteinen in vivo und in vitro untersucht. Darüber hinaus wurden verschiedene weitere T3SS-Proteine aus Y. enterocolitica wie die Chaperone SycO, SycE und SycD und YscY/YscX charakterisiert und auf eine mögliche Verknüpfung zu den YscM Regulatoren überprüft. Das Ziel dieser Arbeit war die Charakterisierung verschiedener Regulatorproteine des Typ III Sekretionssystems, die ein regulatorisches Netzwerk bilden könnten

The Yersinia enterocolitica type three secretion chaperone SycO is integrated into the Yop regulatory network and binds to the Yop secretion protein YscM1

<p>Abstract</p> <p>Background</p> <p>Pathogenic yersiniae (<it>Y. pestis</it>, <it>Y. pseudotuberculosis</it>, <it>Y. enterocolitica</it>) share a virulence plasmid encoding a type three secretion system (T3SS). This T3SS comprises more than 40 constituents. Among these are the transport substrates called Yops (<it>Yersinia </it>outer proteins), the specific Yop chaperones (Sycs), and the Ysc (Yop secretion) proteins which form the transport machinery. The effectors YopO and YopP are encoded on an operon together with SycO, the chaperone of YopO. The characterization of SycO is the focus of this study.</p> <p>Results</p> <p>We have established the large-scale production of recombinant SycO in its outright form. We confirm that <it>Y. enterocolitica </it>SycO forms homodimers which is typical for Syc chaperones. SycO overproduction in <it>Y. enterocolitica </it>decreases secretion of Yops into the culture supernatant suggesting a regulatory role of SycO in type III secretion. We demonstrate that <it>in vitro </it>SycO interacts with YscM1, a negative regulator of Yop expression in <it>Y. enterocolitica</it>. However, the SycO overproduction phenotype was not mediated by YscM1, YscM2, YopO or YopP as revealed by analysis of isogenic deletion mutants.</p> <p>Conclusion</p> <p>We present evidence that SycO is integrated into the regulatory network of the <it>Yersinia </it>T3SS. Our picture of the <it>Yersinia </it>T3SS interactome is supplemented by identification of the SycO/YscM1 interaction. Further, our results suggest that at least one additional interaction partner of SycO has to be identified.</p

Tumor Cells Develop Defined Cellular Phenotypes After 3D-Bioprinting in Different Bioinks

Malignant melanoma is often used as a model tumor for the establishment of novel therapies. It is known that two-dimensional (2D) culture methods are not sufficient to elucidate the various processes during cancer development and progression. Therefore, it is of major interest to establish defined biofabricated three-dimensional (3D) models, which help to decipher complex cellular interactions. To get an impression of their printability and subsequent behavior, we printed fluorescently labeled melanoma cell lines with Matrigel and two different types of commercially available bioinks, without or with modification (RGD (Arginine-Glycine-Aspartate)-sequence/laminin-mixture) for increased cell-matrix communication. In general, we demonstrated the printability of melanoma cells in all tested biomaterials and survival of the printed cells throughout 14 days of cultivation. Melanoma cell lines revealed specific differential behavior in the respective inks. Whereas in Matrigel, the cells were able to spread, proliferate and form dense networks throughout the construct, the cells showed no proliferation at all in alginate-based bioink. In gelatin methacrylate-based bioink, the cells proliferated in clusters. Surprisingly, the modifications of the bioinks with RGD or the laminin blend did not affect the analyzed cellular behavior. Our results underline the importance of precisely adapting extracellular matrices to individual requirements of specific 3D bioprinting applications

Quantitative and Discrete Evolutionary Changes in the Egg-Laying Behavior of Single Drosophila Females

How a nervous system assembles and coordinates a suite of elementary behavioral steps into a complex behavior is not well understood. While often presented as a stereotyped sequence of events, even extensively studied behaviors such as fly courtship are rarely a strict repetition of the same steps in a predetermined sequence in time. We are focusing on oviposition, the act of laying an egg, in flies of the genus Drosophila to describe the elementary behavioral steps or microbehaviors that a single female fly undertakes prior to and during egg laying. We have analyzed the hierarchy and relationships in time of these microbehaviors in three closely related Drosophila species with divergent egg-laying preferences and uncovered cryptic differences in their behavioral patterns. Using high-speed imaging, we quantified in depth the oviposition behavior of single females of Drosophila suzukii, Drosophila biarmipes and Drosophila melanogaster in a novel behavioral assay. By computing transitions between microbehaviors, we identified a common ethogram structure underlying oviposition of all three species. Quantifying parameters such as relative time spent on a microbehavior and its average duration, however, revealed clear differences between species. In addition, we examined the temporal dynamics and probability of transitions to different microbehaviors relative to a central event of oviposition, ovipositor contact. Although the quantitative analysis highlights behavioral variability across flies, it reveals some interesting trends for each species in the mode of substrate sampling, as well as possible evolutionary differences. Larger datasets derived from automated video annotation will overcome this paucity of data in the future, and use the same framework to reappraise these observed differences. Our study reveals a common architecture to the oviposition ethogram of three Drosophila species, indicating its ancestral state. It also indicates that Drosophila suzukii’s behavior departs quantitatively and qualitatively from that of the outgroup species, in line with its known divergent ethology. Together, our results illustrate how a global shift in ethology breaks down in the quantitative reorganization of the elementary steps underlying a complex behavior

Data quality and information loss in standardised interpolated path analysis : quality measures and guidelines

Standardised interpolated path analysis (SIPA) is a method to investigate negotiation processes making different negotiation histories comparable. Due to its interpolation approach, researchers employing SIPA must take data quality and potential information loss into account to maximise the methods explanatory power. This paper presents quality measures and applies them to two negotiation datasets for deriving meaningful boundaries. Using these quality measures enables researchers to compare SIPA across segmentations, variables, and datasets also providing outlier analysis

Influence of the autotaxin-lysophosphatidic acid axis on cellular function and cytokine expression in different breast cancer cell lines

Previous studies provide high evidence that autotaxin (ATX)-lysophosphatidic acid (LPA) signaling through LPA receptors (LPAR) plays an important role in breast cancer initiation, progression, and invasion. However, its specific role in different breast cancer cell lines remains to be fully elucidated to offer improvements in targeted therapies. Within this study, we analyzed in vitro the effect of LPA 18:1 and the LPAR1, LPAR3 (and LPAR2) inhibitor Ki16425 on cellular functions of different human breast cancer cell lines (MDA-MB-231, MDA-MB-468, MCF-7, BT-474, SKBR-3) and the human breast epithelial cell line MCF-10A, as well as Interleukin 8 (IL-8), Interleukin 6 (IL-6) and tumor necrosis factor (TNF)-alpha cytokine secretion after LPA-incubation. ATX-LPA signaling showed a dose-dependent stimulatory effect especially on cellular functions of triple-negative and luminal A breast cancer cell lines. Ki16425 inhibited the LPA-induced stimulation of triple-negative breast cancer and luminal A cell lines in variable intensity depending on the functional assay, indicating the interplay of different LPAR in those assays. IL-8, IL-6 and TNF-alpha secretion was induced by LPA in MDA-MB-468 cells. This study provides further evidence about the role of the ATX-LPA axis in different breast cancer cell lines and might contribute to identify subtypes suitable for a future targeted therapy of the ATX-LPA axis

Pflanzliche Proteine als Fleischersatz: eine Betrachtung für die Schweiz

Soll die Eigenversorgung an pflanzlichem Protein für die menschliche Ernährung ausgebaut werden, bedarf es einer möglichst gesamthaften Betrachtung. In dieser Studie wird die Situation in der Schweiz systemisch analysiert. Es wird aufgezeigt, welche proteinreichen Pflanzen sich besonders für einen nachhaltigen und ökologischen Anbau eignen, welches ernährungsphysiologische Potenzial sie mitbringen und welche Prozessschritte notwendig sind, um sie zu Proteinkonzentraten und -isolaten aufzuarbeiten, die sich wiederum zur Herstellung von Fleischersatzprodukten eignen

Total Psoas Muscle Area as a Marker for Sarcopenia Is Related to Outcome in Children With Neuroblastoma

Background: Sarcopenia describes a generalized loss of skeletal muscle mass, strength, or function. Determined by measuring the total psoas muscle area (tPMA) on cross-sectional imaging, sarcopenia is an independent marker for poor post-surgical outcomes in adults and children. Children with cancer are at high risk for sarcopenia due to immobility, chemotherapy, and cachexia. We hypothesize that sarcopenic children with neuroblastoma are at higher risk for poor post-operative outcomes. Patients and Methods: Retrospective analysis of children with neuroblastoma ages 1–15 years who were treated at our hospital from 2008 to 2016 with follow-up through March 2021. Psoas muscle area (PMA) was measured from cross-sectional images, using computed tomography (CT) and magnetic resonance imaging (MRI) scans at lumbar disc levels L3-4 and L4-5. tPMA is the sum of the left and right PMA. Z-scores were calculated using age- and gender-specific reference values. Sarcopenia was defined as a tPMA z-score below −2. A correlation of tPMA z-scores and sarcopenia with clinical variables and outcome was performed. Results: One hundred and sixty-four children with workup for neuroblastoma were identified, and 101 children fulfilled inclusion criteria for further analysis, with a mean age of 3.92 years (SD 2.71 years). Mean tPMA z-score at L4-5 was −2.37 (SD 1.02). Correlation of tPMA z-score at L4-5 with weight-for-age z-score was moderate (r = 0.54; 95% CI, 0.38, 0.66). No association between sarcopenia and short-term outcome was observed. Sarcopenia had a sensitivity of 0.82 (95% CI, 0.62–0.93) and a specificity of 0.48 (95% CI 0.36–0.61) in predicting 5-year survival. In a multiple regression analysis, pre-operative sarcopenia, pre-operative chemotherapy in the NB2004 high-risk group, unfavorable tumor histology, and age at diagnosis were associated with 5-year survival after surgery, with hazard ratios of 4.18 (95% CI 1.01–17.26), 2.46 (95% CI 1.02–5.92), 2.39 (95% CI 1.03–5.54), and 1.01 (95% CI 1.00–1.03), respectively. Conclusion: In this study, the majority of children had low tPMA z-scores and sarcopenia was a risk factor for decreased 5-year survival in children with neuroblastoma. Therefore, we suggest measuring the tPMA from pre-surgical cross-sectional imaging as a biomarker for additional risk stratification in children with neuroblastoma