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  • 1
    Online Resource
    Online Resource
    Identification of Pri-miRNA Stem-Loop Interacting Proteins in Plants Using a Modified Version of the Csy4 CRISPR Endonuclease
    MDPI AG ; 2022
    In:  International Journal of Molecular Sciences Vol. 23, No. 16 ( 2022-08-11), p. 8961-
    Details
    In: International Journal of Molecular Sciences, MDPI AG, Vol. 23, No. 16 ( 2022-08-11), p. 8961-
    Abstract: Regulation at the RNA level by RNA-binding proteins (RBPs) and microRNAs (miRNAs) is key to coordinating eukaryotic gene expression. In plants, the importance of miRNAs is highlighted by severe developmental defects in mutants impaired in miRNA biogenesis. MiRNAs are processed from long primary-microRNAs (pri-miRNAs) with internal stem-loop structures by endonucleolytic cleavage. The highly structured stem-loops constitute the basis for the extensive regulation of miRNA biogenesis through interaction with RBPs. However, trans-acting regulators of the biogenesis of specific miRNAs are largely unknown in plants. Therefore, we exploit an RNA-centric approach based on modified versions of the conditional CRISPR nuclease Csy4* to pull down interactors of the Arabidopsis pri-miR398b stem-loop (pri-miR398b-SL) in vitro. We designed three epitope-tagged versions of the inactive Csy4* for the immobilization of the protein together with the pri-miR398b-SL bait on high affinity matrices. After incubation with nucleoplasmic extracts from Arabidopsis and extensive washing, pri-miR398b-SL, along with its specifically bound proteins, were released by re-activating the cleavage activity of the Csy4* upon the addition of imidazole. Co-purified proteins were identified via quantitative mass spectrometry and data sets were compared. In total, we identified more than 400 different proteins, of which 180 are co-purified in at least two out of three independent Csy4*-based RNA pulldowns. Among those, the glycine-rich RNA-binding protein AtRZ-1a was identified in all pulldowns. To analyze the role of AtRZ-1a in miRNA biogenesis, we determined the miR398 expression level in the atrz-1a mutant. Indeed, the absence of AtRZ-1a caused a decrease in the steady-state level of mature miR398 with a concomitant reduction in pri-miR398b levels. Overall, we show that our modified Csy4*-based RNA pulldown strategy is suitable to identify new trans-acting regulators of miRNA biogenesis and provides new insights into the post-transcriptional regulation of miRNA processing by plant RBPs.
    Type of Medium: Online Resource
    ISSN: 1422-0067
    URL: Article
    DOI: 10.3390/ijms23168961
    Language: English
    Publisher: MDPI AG
    Publication Date: 2022
    detail.hit.zdb_id: 2019364-6
    SSG: 12
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  • 2
    Online Resource
    Online Resource
    Abdominal Obesity-Related Disturbance of Insulin Sensitivity Is Associated with CD8+ EMRA Cells in the Elderly
    MDPI AG ; 2021
    In:  Cells Vol. 10, No. 5 ( 2021-04-23), p. 998-
    Details
    In: Cells, MDPI AG, Vol. 10, No. 5 ( 2021-04-23), p. 998-
    Abstract: Aging and overweight increase the risk of developing type 2 diabetes mellitus. In this cross-sectional study, we aimed to investigate the potential mediating role of T-EMRA cells and inflammatory markers in the development of a decreased insulin sensitivity. A total of 134 healthy older volunteers were recruited (age 59.2 (SD 5.6) years). T cell subpopulations were analyzed by flow cytometry. Furthermore, body composition, HOMA-IR, plasma tryptophan (Trp) metabolites, as well as cytokines and adipokines were determined. Using subgroup and covariance analyses, the influence of BMI on the parameters was evaluated. Moreover, correlation, multiple regression, and mediation analyses were performed. In the subgroup of participants with obesity, an increased proportion of CD8+EMRA cells and elevated concentrations of plasma kynurenine (KYN) were found compared to the lower-weight subgroups. Linear regression analysis revealed that an elevated HOMA-IR could be predicted by a higher proportion of CD8+EMRA cells and KYN levels. A mediation analysis showed a robust indirect effect of the Waist-to-hip ratio on HOMA-IR mediated by CD8+EMRA cells. Thus, the deleterious effects of abdominal obesity on glucose metabolism might be mediated by CD8+EMRA cells in the elderly. Longitudinal studies should validate this assumption and analyze the suitability of CD8+EMRA cells as early predictors of incipient prediabetes.
    Type of Medium: Online Resource
    ISSN: 2073-4409
    URL: Article
    DOI: 10.3390/cells10050998
    Language: English
    Publisher: MDPI AG
    Publication Date: 2021
    detail.hit.zdb_id: 2661518-6
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  • 3
    Online Resource
    Online Resource
    An eDiary App Approach for Collecting Physiological Sensor Data from Wearables together with Subjective Observations and Emotions
    MDPI AG ; 2022
    In:  Sensors Vol. 22, No. 16 ( 2022-08-16), p. 6120-
    Details
    In: Sensors, MDPI AG, Vol. 22, No. 16 ( 2022-08-16), p. 6120-
    Abstract: Field measurement campaigns with traffic participants using wearable sensors and questionnaires can be challenging to carry out because of inconsistent interfaces across manufacturers for accessing sensor data and campaign-specific questionnaire contents bear large potential for errors. We present an app able to consolidate data from multiple technical sensors and questionnaires. The functionality includes providing feedback for correct sensor platform mounting, accessing and storing all sensor and questionnaire data in a uniform data structure. To do this, the app implements a sensor data bus class that unifies data from technical sensors and questionnaires. The app can also be extended to accommodate other sensor platforms provided they have a suitable API. We also describe a database structure holding the data from multiple campaigns and test subjects in a privacy preserving fashion. Finally, we identify some potential issues and hints that practitioners may encounter when conducting a measurement campaign.
    Type of Medium: Online Resource
    ISSN: 1424-8220
    URL: Article
    DOI: 10.3390/s22166120
    Language: English
    Publisher: MDPI AG
    Publication Date: 2022
    detail.hit.zdb_id: 2052857-7
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  • 4
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    Online Resource
    Immunomodulating Effect of the Consumption of Watercress (Nasturtium officinale) on Exercise-Induced Inflammation in Humans
    MDPI AG ; 2021
    In:  Foods Vol. 10, No. 8 ( 2021-07-30), p. 1774-
    Details
    In: Foods, MDPI AG, Vol. 10, No. 8 ( 2021-07-30), p. 1774-
    Abstract: The vegetable watercress (Nasturtium officinale R.Br.) is, besides being a generally nutritious food, a rich source of glucosinolates. Gluconasturtiin, the predominant glucosinolate in watercress, has been shown to have several health beneficial properties through its bioactive breakdown product phenethyl isothiocyanate. Little is known about the immunoregulatory effects of watercress. Moreover, anti-inflammatory effects have mostly been shown in in vitro or in animal models. Hence, we conducted a proof-of-concept study to investigate the effects of watercress on the human immune system. In a cross-over intervention study, 19 healthy subjects (26.5 ± 4.3 years; 14 males, 5 females) were given a single dose (85 g) of fresh self-grown watercress or a control meal. Two hours later, a 30 min high-intensity workout was conducted to promote exercise-induced inflammation. Blood samples were drawn before, 5 min after, and 3 h after the exercise unit. Inflammatory blood markers (IL-1β, IL-6, IL-10, TNF-α, MCP-1, MMP-9) were analyzed in whole blood cultures after ex vivo immune cell stimulation via lipopolysaccharides. A mild pro-inflammatory reaction was observed after watercress consumption indicated by an increase in IL-1β, IL-6, and TNF-α, whereas the immune response was more pronounced for both pro-inflammatory and anti-inflammatory markers (IL-1β, IL-6, IL-10, TNF-α) after the exercise unit compared to the control meal. During the recovery phase, watercress consumption led to a stronger anti-inflammatory downregulation of the pro-inflammatory cytokines IL-6 and TNF-α. In conclusion, we propose that watercress causes a stronger pro-inflammatory response and anti-inflammatory counter-regulation during and after exercise. The clinical relevance of these changes should be verified in future studies.
    Type of Medium: Online Resource
    ISSN: 2304-8158
    URL: Article
    DOI: 10.3390/foods10081774
    Language: English
    Publisher: MDPI AG
    Publication Date: 2021
    detail.hit.zdb_id: 2704223-6
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  • 5
    Online Resource
    Online Resource
    A Mechanistic Pharmacodynamic Modeling Framework for the Assessment and Optimization of Proteolysis Targeting Chimeras (PROTACs)
    MDPI AG ; 2023
    In:  Pharmaceutics Vol. 15, No. 1 ( 2023-01-05), p. 195-
    Details
    In: Pharmaceutics, MDPI AG, Vol. 15, No. 1 ( 2023-01-05), p. 195-
    Abstract: The field of targeted protein degradation is growing exponentially. Yet, there is an unmet need for pharmacokinetic/pharmacodynamic models that provide mechanistic insights, while also being practically useful in a drug discovery setting. Therefore, we have developed a comprehensive modeling framework which can be applied to experimental data from routine projects to: (1) assess PROTACs based on accurate degradation metrics, (2) guide compound optimization of the most critical parameters, and (3) link degradation to downstream pharmacodynamic effects. The presented framework contains a number of first-time features: (1) a mechanistic model to fit the hook effect in the PROTAC concentration-degradation profile, (2) quantification of the role of target occupancy in the PROTAC mechanism of action and (3) deconvolution of the effects of target degradation and target inhibition by PROTACs on the overall pharmacodynamic response. To illustrate applicability and to build confidence, we have employed these three models to analyze exemplary data on various compounds from different projects and targets. The presented framework allows researchers to tailor their experimental work and to arrive at a better understanding of their results, ultimately leading to more successful PROTAC discovery. While the focus here lies on in vitro pharmacology experiments, key implications for in vivo studies are also discussed.
    Type of Medium: Online Resource
    ISSN: 1999-4923
    URL: Article
    DOI: 10.3390/pharmaceutics15010195
    Language: English
    Publisher: MDPI AG
    Publication Date: 2023
    detail.hit.zdb_id: 2527217-2
    SSG: 15,3
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  • 6
    Online Resource
    Online Resource
    Potential and Limits of Kidney Cells for Evaluation of Renal Excretion
    MDPI AG ; 2021
    In:  Pharmaceuticals Vol. 14, No. 9 ( 2021-09-07), p. 908-
    Details
    In: Pharmaceuticals, MDPI AG, Vol. 14, No. 9 ( 2021-09-07), p. 908-
    Abstract: A large number of therapeutic drugs, herbal components and their metabolites are excreted by the kidneys. Therefore, generally applied models for estimating renal excretion, including freshly isolated rat proximal tubule cells, cultured tubule cells and immortalized kidney cell lines MDCKII, NRK-52E, IHKE-1 and Caki-1, were investigated regarding their predictive potential for active renal transport. Cultured proximal tubule cells showed an epithelial cell-like morphology and formed tight monolayers. However, mRNA expression analyses and immunohistochemical studies revealed patterns of tight junction proteins that were notably different from freshly isolated cells and distinct from those in vivo. High levels of mannitol permeation were found in NRK-52E, IHKE-1 and Caki-1 cells, suggesting that they are not suitable for bidirectional transport studies. Cultured cells and freshly isolated cells also differed in proximal tubule markers and transport proteins, indicating that cultured primary cells were in a state of dedifferentiation. Cell lines MDCKII, NRK-52E, IHKE-1 and Caki-1 did not accurately reflect the characteristics of proximal tubules. The expression patterns of marker and transport proteins differed from freshly isolated primary cells. In summary, each of these models has profound disadvantages to consider when adopting them reliable models for the in vivo situation. Thus, they should not be used alone but only in combination.
    Type of Medium: Online Resource
    ISSN: 1424-8247
    URL: Article
    DOI: 10.3390/ph14090908
    Language: English
    Publisher: MDPI AG
    Publication Date: 2021
    detail.hit.zdb_id: 2193542-7
    SSG: 15,3
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  • 7
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    Online Resource
    The Microtubule-Targeting Agent Pretubulysin Impairs the Inflammatory Response in Endothelial Cells by a JNK-Dependent Deregulation of the Histone Acetyltransferase Brd4
    MDPI AG ; 2023
    In:  Cells Vol. 12, No. 16 ( 2023-08-21), p. 2112-
    Details
    In: Cells, MDPI AG, Vol. 12, No. 16 ( 2023-08-21), p. 2112-
    Abstract: The anti-inflammatory effects of depolymerizing microtubule-targeting agents on leukocytes are known for a long time, but the potential involvement of the vascular endothelium and the underlying mechanistic basis is still largely unclear. Using the recently synthesized depolymerizing microtubule-targeting agent pretubulysin, we investigated the anti-inflammatory potential of pretubulysin and other microtubule-targeting agents with respect to the TNF-induced leukocyte adhesion cascade in endothelial cells, to improve our understanding of the underlying biomolecular background. We found that treatment with pretubulysin reduces inflammation in vivo and in vitro via inhibition of the TNF-induced adhesion of leukocytes to the vascular endothelium by down-regulation of the pro-inflammatory cell adhesion molecules ICAM-1 and VCAM-1 in a JNK-dependent manner. The underlying mechanism includes JNK-induced deregulation and degradation of the histone acetyltransferase Bromodomain-containing protein 4. This study shows that depolymerizing microtubule-targeting agents, in addition to their established effects on leukocytes, also significantly decrease the inflammatory activation of vascular endothelial cells. These effects are not based on altered pro-inflammatory signaling cascades, but require deregulation of the capability of cells to enter constructive transcription for some genes, setting a baseline for further research on the prominent anti-inflammatory effects of depolymerizing microtubule-targeting agents.
    Type of Medium: Online Resource
    ISSN: 2073-4409
    URL: Article
    DOI: 10.3390/cells12162112
    Language: English
    Publisher: MDPI AG
    Publication Date: 2023
    detail.hit.zdb_id: 2661518-6
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  • 8
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    Online Resource
    Collection, Standardization and Attribution of Robust Disaster Event Information—A Demonstrator of a National Event-Based Loss and Damage Database in Austria
    MDPI AG ; 2022
    In:  Geosciences Vol. 12, No. 8 ( 2022-07-22), p. 283-
    Details
    In: Geosciences, MDPI AG, Vol. 12, No. 8 ( 2022-07-22), p. 283-
    Abstract: Loss and damage databases are essential tools within the disaster risk management cycle for making informed decisions. However, even in data-rich countries such as Austria, no consistent and curated multi-hazard database is available. Based on the requirements of the United Nations, the European Union, as well as on national demands to deal with disaster impacts, we conceived and set up a demonstrator for a consistent multi-hazard national event-based loss and damage database that addresses event identification, loss accounting and disaster forensics according to international standards. We built our database on already existing data from administration and federal agencies and formulated a process to combine those data in a synergetic way. Furthermore, we tested how earth observation and weather data could help to derive more robust disaster event information. Our demonstrator focuses on two Austrian federal provinces, three hazard types—floods, storms and mass movements—and the period between 2005 and 2018. By analyzing over 140.000 single event descriptions, we conclude that—despite some limitations in retrospective data harmonization—the implementation of a curated event-based national loss and damage database is feasible and adds significant value compared to the usage of single national datasets or existing international databases such as EM-DAT or the Risk Data Hub. With our demonstrator, we are able to support the national risk assessment, the national Sendai Monitoring and federal disaster risk management with the provision of best possible harmonized loss and damage information, tailored indicators and statistics as well as hazard impact maps on the municipality scale.
    Type of Medium: Online Resource
    ISSN: 2076-3263
    URL: Article
    DOI: 10.3390/geosciences12080283
    Language: English
    Publisher: MDPI AG
    Publication Date: 2022
    detail.hit.zdb_id: 2655946-8
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  • 9
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    Correlation of Growth and Surface Properties of Poly(\(p\)-xylylenes) to Reaction Conditions
    MDPI AG ; 2015
    In:  Coatings Vol. 5, No. 2 ( 2015-05-08), p. 142-171
    Details
    In: Coatings, MDPI AG, Vol. 5, No. 2 ( 2015-05-08), p. 142-171
    Type of Medium: Online Resource
    ISSN: 2079-6412
    URL: Article
    DOI: 10.3390/coatings5020142
    Language: English
    Publisher: MDPI AG
    Publication Date: 2015
    detail.hit.zdb_id: 2662314-6
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