47 research outputs found
Development of Cysteine Protease Assays for the Validation of Cysteine Protease Inhibitors with Special Emphasis on SARS-CoV Main Protease
Cysteinproteasen stellen bedeutsame Targets in der Arzneistoffentwicklung dar. Um die Wirksamkeit potenzieller Inhibitoren an diesen Zielstrukturen zu testen und somit eine geeignete Leitstruktur für die Arzneistoffentwicklung zu finden, gibt es zahlreiche Möglichkeiten biologische Wertbestimmungen (sog. Assays) zu entwickeln. In der vorliegenden Arbeit wurde die Methodik eines kontinuierlichen fluorimetrischen Assays angewandt. Für einen validen Assay ist es wesentlich, die chemischen und physikalischen Eigenschaften der potenziellen Inhibitoren zu betrachten, um falsch positive Ergebnisse zu vermeiden. Solche falsch positiven Ergebnisse treten zum einen durch den sogenannten inneren Filtereffekt oder auch durch Aggregation der Testsubstanz auf. Um diese Effekte zu detektieren und somit richtig positive von falsch positiven Ergebnissen abgrenzen zu können, wurde ein Arbeitsablauf exemplarisch anhand der Cysteinprotease Cathepsin B und der Serinprotease Chymotrypsin erarbeitet. Dabei wurden einfache Methoden zur Detektion und Korrektur des inneren Filtereffektes und der Detektion und Größenbestimmung von Aggregaten erarbeitet. Ebenso wurde die Detektion unspezifisch kovalenter Inhibitoren eingebunden sowie Messtechniken zur Bestimmung der Art der Inhibition (reversibel, irreversibel) und der Kinetik von Inhibitoren (kompetitiv, nicht-kompetitiv, unkompetitiv).
Nach Erarbeitung des Assay-Arbeitsablaufs an den beiden genannten Proteasen, konnte dieser erfolgreich auf das betrachtete Target, die SARS-CoV Hauptprotease, übertragen werden und damit eine Hitvalidierung bereits publizierter Inhibitoren durchgeführt werden.
Mit ausgewählten falsch positiven sowie unselektiv kovalent bindenden Testsubstanzen aus der Hitvalidierung der SARS-CoV MPro wurden an möglichst diversen Cysteinproteasen wie Cathepsin B, Clostripain, Calpain-1 und Caspase-1 Selektivitätstestungen durchgeführt.Cysteine proteases are important targets in drug development. There are many different ways to develop enzymatic assays to test the inhibitory potency of potential inhibitors in the search for new lead structures. Here the methodology of a continuous fluorimetric assay was applied.
For the development of a valid assay it is essential to consider the physiochemical properties of the potential inhibitors to avoid false positive results. Those false positive results emerge from the so-called inner filter effect or from aggregation of the compounds. To detect these effects and thus to distinguish true positive from false positive results an assay workflow was developed with cathepsin B as a prototype cysteine protease and chymotrypsin as a prototype of a serin protease. This workflow consists of simple methods for detection and correction of the inner filter effect, for detection and determining the size distribution of aggregates and for detection of unspecific covalent inhibitors. Furthermore, methods to determine whether or not inhibition is reversible were applied in addition to the determination of the exact mechanism of inhibition (competitive, noncompetitive, uncompetitive).
After the development of the assay workflow with these two proteases, it could successfully be transferred to the target, the SARS-CoV main protease, to perform a hit validation with previously published inhibitors.
Selected compounds yielding false positives results as well as unspecific covalent inhibitors from the hit validation of SARS-CoV MPro were tested at diverse cysteine proteases such as cathepsin B, clostripain, calpain-1 and caspase-1 for selectivity
Land Cover Changes (1963–2010) and Their Environmental Factors in the Upper Danube Floodplain
To analyze the changes in the Upper Danube Floodplain, we used aerial photos to quantify the change of landscape pattern from 1963 to 2010. We focused on typical floodplain habitats, i.e., riparian forest and floodplain grassland. We used landscape metrics and transformation matrix to explore changes in land cover structure and composition. The active floodplain experienced increasing fragmentation from 1963 to 2010. Despite an increase of aggregation, riparian forest suffered a 2.3% area loss from 1995 to 2010. Arable land in the active floodplain declined by 28.5%, while its patch size significantly increased. Elevation, distance to river and soil quality were the most relevant environmental factors for the land cover change in the floodplain. Higher soil quality or longer distance to river led to an increase of conversion from grassland into arable land; grassland patches with poorer soil quality were likely to change into riparian forest; riparian forest closer to the river and with a lower height above mean water level tended to remain stable. This comprehensive understanding of historical land cover change and environmental factors is needed for the enhancement of landscape functions and sustainable development in the floodplain
Biological Flora of Central Europe–Lupinus polyphyllus Lindl
The invasive herb Lupinus polyphyllus has been focus of a number of fact sheets worldwide but a comprehensive summary of the species’ taxonomy and morphology, distribution, habitat requirements, and biology has been lacking. This paper gives a thorough account of the species’ systematic position and taxonomy, highlighting the difficulties to delimit taxa, which is related to interbreeding among members of this genus. However, L. polyphyllus var. polyphyllus is apparently the taxon that has naturalized and is regionally invasive in temperate-humid climates worldwide. We also present an updated distribution map of L. polyphyllus in the native and invaded ranges, which highlights seven regions in the world where the species has been established. We show that the climatic niche of L. polyphyllus in the invaded range shifts towards higher summer precipitation and lower isothermality, probably because the invaded range includes subcontinental regions of eastern Europe and western Siberia. The habitats of L. polyphyllus range from rather dry to wet, have moderately acidic to strongly acidic soils, and the species’ indicator values across Europe suggest that it occurs along a gradient from very nutrient poor sites to intermediate to rich sites from northern to southern Europe. The species shows high resistance to both drought and frost. In Central Europe, the species has a stronghold in alpic mountain hay meadows, abandoned meadows and pastures, low and medium altitude hay meadows, anthropogenic herb stands and temperate thickets and scrubs. In northern Europe, the species occurs in anthropogenic herb stands along roads and railroads as well as in abandoned pastures and fields. We also found some doubtful information about L. polyphyllus in the literature. This refers to its description as “rhizomatous perennial” although it lacks rhizomes; an apparently very high longevity of its seeds, which may only be true under artificial conditions in an ex situ seed repository; and a very deep rooting depth, which may not represent the average rooting depth but rather an extreme value. Knowledge about the interrelationships between the species’ future population dynamics and spread and ongoing climate warming is lacking. Finally, our review points out that there is currently no evidence-based strategy for a cost-efficient management of L. polyphyllus although it is among the most problematic non-native plant species in Europe due to its environmental and socio-economic impacts
Postvaccination anti-S IgG levels predict anti-SARS-CoV-2 neutralising activity over 24 weeks in patients with RA
OBJECTIVES
To correlate immune responses following a two-dose regimen of mRNA anti-SARS-CoV-2 vaccines in patients with rheumatoid arthritis (RA) to the development of a potent neutralising antiviral activity.
METHODS
The RECOVER study was a prospective, monocentric study including patients with RA and healthy controls (HCs). Assessments were performed before, and 3, 6, 12 and 24 weeks, after the first vaccine dose, respectively, and included IgG, IgA and IgM responses (against receptor binding domain, S1, S2, N), IFN-γ ELISpots as well as neutralisation assays.
RESULTS
In patients with RA, IgG responses developed slower with lower peak titres compared with HC. Potent neutralising activity assessed by a SARS-CoV-2 pseudovirus neutralisation assay after 12 weeks was observed in all 21 HCs, and in 60.3% of 73 patients with RA. A significant correlation between peak anti-S IgG levels 2 weeks after the second vaccine dose and potent neutralising activity against SARS-CoV-2 was observed at weeks 12 and 24. The analysis of IgG, IgA and IgM isotype responses to different viral proteins demonstrated a delay in IgG but not in IgA and IgM responses. T cell responses were comparable in HC and patients with RA but declined earlier in patients with RA.
CONCLUSION
In patients with RA, vaccine-induced IgG antibody levels were diminished, while IgA and IgM responses persisted, indicating a delayed isotype switch. Anti-S IgG levels 2 weeks after the second vaccine dose correlate with the development of a potent neutralising activity after 12 and 24 weeks and may allow to identify patients who might benefit from additional vaccine doses or prophylactic regimen
Influence of flooding duration and aeration on saplings of ten hardwood floodplain forest species.
Alluvial floodplain forests have been reduced drastically in many parts of Europe, due to deforestation, the transformation to settlement and expansion of agricultural areas. Although they have been heavily modified for centuries, generalized frameworks for their management are scarce and the complex interactions between the physical environment and biological processes are often not fully understood. As the zonation of woody species in floodplains is mainly determined by hydrological conditions, flooding tolerance can be regarded as a key factor for the successful establishment of woody species. Furthermore, the oxygen level of the flooding water might affect the responses to flooding. We examined the influence of flooding duration in combination with oxygen supply by aeration on the foliar injury and growth of six-week-old saplings of ten woody species, under controlled common garden conditions. Six of them are considered to be flooding tolerant whereas four are intolerant. In addition, seven are native whereas three are non-native species. During the experiment, the saplings were exposed to partial flooding of different durations (k = 3; three, six and nine weeks) and oxygen levels (k = 2; aerated and not aerated). For comparison, we included an unflooded control. We recorded foliar injury, plant height, number of leaves and stem diameter. We also included a long-term recovery period. Whereas foliar injury decreased for most species with increasing flooding duration, the typical floodplain forest species, classified as flooding tolerant developed better. The differences in species response to flooding could be most likely explained by their ability to react to the resulting stress in morphological, physiological and metabolic terms irrespective whether they are native or not. In addition, the inclusion of a recovery period seems to be important for the assessment of flooding tolerance
Mouse Hepatitis Virus Liver Pathology Is Dependent on ADP-Ribose-1″-Phosphatase, a Viral Function Conserved in the Alpha-Like Supergroup▿
Viral infection of the liver can lead to severe tissue damage when high levels of viral replication and spread in the organ are coupled with strong induction of inflammatory responses. Here we report an unexpected correlation between the expression of a functional X domain encoded by the hepatotropic mouse hepatitis virus strain A59 (MHV-A59), the high-level production of inflammatory cytokines, and the induction of acute viral hepatitis in mice. X-domain (also called macro domain) proteins possess poly-ADP-ribose binding and/or ADP-ribose-1′′-phosphatase (ADRP) activity. They are conserved in coronaviruses and in members of the “alpha-like supergroup” of phylogenetically related positive-strand RNA viruses that includes viruses of medical importance, such as rubella virus and hepatitis E virus. By using reverse genetics, we constructed a recombinant murine coronavirus MHV-A59 mutant encoding a single-amino-acid substitution of a strictly conserved residue that is essential for coronaviral ADRP activity. We found that the mutant virus replicated to slightly reduced titers in livers but, strikingly, did not induce liver disease. In vitro, the mutant virus induced only low levels of the inflammatory cytokines tumor necrosis factor alpha and interleukin-6 (IL-6). In vivo, we found that IL-6 production, in particular, was reduced in the spleens and livers of mutant virus-infected mice. Collectively, our data demonstrate that the MHV X domain exacerbates MHV-induced liver pathology, most likely through the induction of excessive inflammatory cytokine expression
Invasive legume affects species and functional composition of mountain meadow plant communities
Plant invasions are among the key drivers of global biodiversity and ecosystem change. They often cause reductions in native species richness and overall biodiversity. Nitrogen-fixing plants are problematic as they affect soil nutrient availability and outcompete species of nutrient-poor sites. Here we assessed the impacts of the legumeLupinus polyphylluson species and functional diversity of mountain meadow communities in the UNESCO Biosphere Reserve Rhon. We compared species diversity (richness, evenness and effective species number), functional diversity (functional richness, evenness, divergence and dispersion) and similarity of plots in three characteristic vegetation types (Nardusgrassland, mesic and wet mountain hay meadows) between different lupine cover classes. We calculated community weighted means (CWMs) of single plant traits and plotted them against lupine cover classes. The invasion ofL. polyphyllushomogenizes vegetation composition since the similarity among plots of the different vegetation types increased with increasing lupine cover. It significantly affected species diversity in terms of richness and effective species number and the functional divergence of the vegetation. The trait set of species occurring together with lupine was shifted towards more competitive trait values. We demonstrate strongly negative impacts ofL. polyphylluson different mountain meadow vegetation types sinceL. polyphyllus, fosters the growth of competitive species and leads to overall more productive plant communities