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Profiling of Human Neural Crest Chemoattractant Activity as a Replacement of Fetal Bovine Serum for In Vitro Chemotaxis Assays
Get PDFFetal bovine serum (FBS) is the only known stimulus for the migration of human neural crest cells (NCCs). Non-animal chemoattractants are desirable for the optimization of chemotaxis assays to be incorporated in a test battery for reproductive and developmental toxicity. We con-firmed here in an optimized transwell assay that FBS triggers directed migration along a con-centration gradient. The responsible factor was found to be a protein in the 30–100 kDa size range. In a targeted
approach, we tested a large panel of serum constituents known to be chem-otactic for NCCs in animal models (e.g., VEGF, PDGF, FGF, SDF-1/CXCL12, ephrins, endothelin, Wnt, BMPs). None of the
corresponding human proteins showed any effect in our chemotaxis assays based on human NCCs.
We then examined, whether human cells would produce any fac-tor able to trigger NCC migration in a broad screening approach. We found that HepG2 hepa-toma cells produced chemotaxis-triggering activity (CTA). Using chromatographic methods and by employing the NCC chemotaxis test as bioassay, the responsible protein was enriched by up to 5000-fold. We also explored human serum and platelets as a direct source, independent of any cell culture manipulations. A CTA was enriched
from platelet lysates several thousand-fold. Its temperature and protease sensitivity suggested also a protein component. The capacity of this factor to trigger chemotaxis was confirmed by single-cell
video-tracking analysis of migrating NCCs. The human CTA characterized here may be employed in the future for the setup of assays testing for the disturbance of directed NCC migration by toxicants
Verbraucherstudie zur Nutzung multifunktionaler Küchenmaschinen mit Kochfunktion
Get PDFÜber eine Beobachtungsstudie von 29 Haushalten wird das reale Nutzungsverhalten multifunktionaler Küchenmaschinen mit Kochfunktion analysiert, um relevante Bewertungsparameter für Vebrauchs-tauglichkeitsprüfungen abzuleiten: Von den durchschnittlichen 3,2 Nutzungen pro Woche entfallen je 50 % auf reine Verarbeitungsprozesse sowie Prozesse unter Nutzung der Kochfunktion. Bei Kochanwendungen sind das Erhitzen und Fortkochen sowie Dampfgaranwendungen am relevantesten, bei Verarbeitungsprozessen sind es Zerkleinerungsprozesse und die Zubereitung von Teigen für Backanwendungen. Die Nutzung einer Kochunterstützung über Schritt-für–Schritt-Kochanleitungen zählt nur bedingt zur gelebten Nutzungspraxis
A Ligand Selection Strategy Indentifies Chemical Probes Targeting the Proteases of SARS-CoV-2
No full textActivity-based probes are valuable tools for chem� ical biology. However, finding probes that specifically target the active site of an enzyme remains a challenging task. Herein, we present a ligand selection strategy that allows to rapidly tailor electrophilic probes to a target of choice and showcase its application for the two cysteine proteases of SARS-CoV-2 as proof of concept. The resulting probes were specific for the active site labeling of 3CLpro and PLpro with sufficient selectivity in a live cell model as well as in the background of a native human proteome. Exploiting the probes as tools for competitive profiling of a natural product library identified
salvianolic acid derivatives as promising 3CLpro inhibitors. We
anticipate that our ligand selection strategy will be useful to
rapidly develop customized probes and discover inhibitors for
a wide range of target proteins also beyond corona virus proteases
Synuclein Family Members Prevent Membrane Damage by Counteracting α-Synuclein Aggregation
Get PDFThe 140 amino acid protein α-synuclein (αS) is an intrinsically disordered protein (IDP) with various roles and locations in healthy neurons that plays a key role in Parkinson’s disease (PD). Contact with biomembranes can lead to α-helical conformations, but can also act as s seeding event for aggregation and a predominant β-sheet conformation. In PD patients, αS is found to aggregate in various fibrillary structures, and the shift in aggregation and localization is associated with disease progression. Besides full-length αS, several related polypeptides are present in neurons. The role of
many αS-related proteins in the aggregation of αS itself is not fully understood Two of these potential aggregation modifiers are the αS splicing variant αS ∆exon3 (∆3) and the paralog β-synuclein (βS).
Here, polarized ATR-FTIR spectroscopy was used to study the membrane interaction of these proteins individually and in various combinations. The method allowed a continuous monitoring of both the
lipid structure of biomimetic membranes and the aggregation state of αS and related proteins. The use of polarized light also revealed the orientation of secondary structure elements. While αS led to a
destruction of the lipid membrane upon membrane-catalyzed aggregation, βS and ∆3 aggregated significantly less, and they did not harm the membrane. Moreover, the latter proteins reduced the membrane damage triggered by αS. There were no major differences in the membrane interaction for the different synuclein variants. In combination, these observations suggest that the formation of particular protein aggregates is the major driving force for αS-driven membrane damage. The misbalance of αS, βS, and ∆3 might therefore play a crucial role in neurodegenerative disease
Integration of temporal single cell cellular stress response with logic-ODE modeling reveals activation of ATF4-CHOP axis as a critical predictor of drug-induced liver injury
Get PDFDrug-induced liver injury (DILI) is the most prevalent adversity encountered in drug development and clinical settings leading to urgent needs to understand the underlying mechanisms. In this study, we have systematically investigated the dynamics of the activation of cellular stress response pathways and cell death outcomes upon exposure of a panel of liver toxicants using live cell imaging of fluorescent reporter cell lines. We established a comprehensive temporal dynamic response profile of a large set of BAC-GFP HepG2 cell lines representing the following components of stress signaling: i) unfolded protein response (UPR) [ATF4, XBP1, BIP and CHOP]; ii) oxidative stress [NRF2, SRXN1, HMOX1]; iii) DNA damage [P53, P21, BTG2, MDM2]; and iv) NF-κB pathway [A20, ICAM1]. We quantified the single cell GFP expression as a surrogate for endogenous protein expression using live cell imaging over > 60 h upon exposure to 14 DILI compounds at multiple concentrations. Using logic-based ordinary differential equation (Logic-ODE), we modelled the dynamic profiles of the different stress responses and extracted specific descriptors potentially predicting the progressive outcomes. We identified the activation of ATF4-CHOP axis of the UPR as the key pathway showing the highest correlation with cell death upon DILI compound perturbation. Knocking down main components of the UPR provided partial protection from compound-induced cytotoxicity, indicating a complex interplay among UPR components as well as other stress pathways. Our results suggest that a systematic analysis of the temporal dynamics of ATF4-CHOP axis activation can support the identification of DILI risk for new candidate drugs.
Keywords: Drug-induced liver injury (DILI); GFP-reporter; Logic-ODE; Stress response pathway
Biobasierte Barrierebeschichtungen
No full textDas EU-Verbundprojekt BIOnTop (Grant Agreement Number GA 837761) wurde in der Plastverarbeiterausgabe 04|2020 erstmals vorgestellt. Das Ziel des EU-Projektes unter Beteiligung von 21 Expertenteams ist das Erforschen und Entwickeln von innovativen Verpackungskonzepten und Textilien mit maßgeschneiderten Eigenschaften sowie neuen End-of-Life Optionen auf Basis von biobasierten Polymeren
Biogenic Amine Detection Systems for Intelligent Packaging Concepts: Meat and Meat Products
Get PDFDuring meat and meat product spoilage metabolites are formed which can be detected by indicator systems to evaluate products’ deterioration, suitable to provide the consumer information about the freshness of a meat or meat product. Due to strong correlation between microbial count and biogenic amine concentration, biogenic amines were identified as suitable target metabolites for meat and meat product freshness determination in intelligent packaging concepts. The objective of this review is to provide an overview of different indication systems to detect biogenic amine formation during spoilage of meat and meat products. Different classes of bioreceptors, chromophores and fluorophores are outlined and discussed regarding their feasibility as indicator substances with a potential use for intelligent packaging concepts. Overall, this review points out that there is great potential for some of the discussed indication systems and their combination to be used as spoilage indicators for meat and meat products, though further research is necessary to overcome commercial application limitations, including precision, reliability, and low-cost producibility of the indicators
Cold Brew Coffee - Pilot Studies on Definition, Extraction, Consumer Preference, Chemical Characterization and Microbiological Hazards
Get PDFCold brew coffee is a new trend in the coffee industry. This paper presents pilot studies on several aspects of this beverage