Distinct binding sites of Ala48-hirudin1-47 and Ala48-hirudin48-65 on alpha-thrombin

The interaction of alpha-thrombin with Ala48-hirudin, Ala48-hirudin1-47, and Ala48-hirudin48-65 was analyzed. Mutations at Pro48 were found to cause only slight changes in the kon (human: 3.1 +/- 0.3 x 10(8) M-1 s-1; bovine: 1.03 +/- 0.3 x 10(8) M-1 s-1) and koff (human: 0.4 +/- 0.2 x 10(-3) s-1; bovine: 2.9 +/- 0.4 x 10(-3) s-1) rate constants for the formation of the thrombin-hirudin complex. The amino-terminal fragment Ala48-hirudin1-47 containing the three disulfide bridges and the carboxyl-terminal fragment Ala48-hirudin48-65 were derived from the Ala48 mutant by proteolysis with endoproteinase Lys-C. These fragments inhibit bovine alpha-thrombin clotting activity with IC50 values of 0.6 and 4.9 microM, respectively (2.4 nM for r-hirudin). By mapping the interaction of Ala48-hirudin-derived fragments with bovine alpha-thrombin by limited proteolysis with trypsin and pancreatic elastase distinct binding sites for each fragment were determined. The carboxyl-terminal fragment was found to bind to the proposed anion-binding exosite in the region B62-74, whereas the amino-terminal fragment binds to a region around the elastase cleavage site at residues 150-151 of the alpha-thrombin B-chain.     

Multiple Stressors in Agricultural Streams: A Mesocosm Study of Interactions among Raised Water Temperature,Sediment Addition and Nutrient Enrichment

Changes to land use affect streams through nutrient enrichment, increased inputs of sediment and, where riparian vegetation has been removed, raised water temperature. We manipulated all three stressors in experimental streamside channels for 30 days and determined the individual and pair-wise combined effects on benthic invertebrate and algal communities and on leaf decay, a measure of ecosystem functioning. We added nutrients (phosphorus+nitrogen; high, intermediate, natural) and/or sediment (grain size 0.2 mm; high, intermediate, natural) to 18 channels supplied with water from a nearby stream. Temperature was increased by 1.4°C in half the channels, simulating the loss of upstream and adjacent riparian shade. Sediment affected 93% of all biological response variables (either as an individual effect or via an interaction with another stressor) generally in a negative manner, while nutrient enrichment affected 59% (mostly positive) and raised temperature 59% (mostly positive). More of the algal components of the community responded to stressors acting individually than did invertebrate components, whereas pair-wise stressor interactions were more common in the invertebrate community. Stressors interacted often and in a complex manner, with interactions between sediment and temperature most common. Thus, the negative impact of high sediment on taxon richness of both algae and invertebrates was stronger at raised temperature, further reducing biodiversity. In addition, the decay rate of leaf material (strength loss) accelerated with nutrient enrichment at ambient but not at raised temperature. A key implication of our findings for resource managers is that the removal of riparian shading from streams already subjected to high sediment inputs, or land-use changes that increase erosion or nutrient runoff in a landscape without riparian buffers, may have unexpected effects on stream health. We highlight the likely importance of intact or restored buffer strips, both in reducing sediment input and in maintaining cooler water temperatures.     

Scale‐down simulators for mammalian cell culture as tools to access the impact of inhomogeneities occurring in large‐scale bioreactors

During the scale‐up of a bioprocess, not all characteristics of the process can be kept constant throughout the different scales. This typically results in increased mixing times with increasing reactor volumes. The poor mixing leads in turn to the formation of concentration gradients throughout the reactor and exposes cells to varying external conditions based on their location in the bioreactor. This can affect process performance and complicate process scale‐up. Scale‐down simulators, which aim at replicating the large‐scale environment, expose the cells to changing environmental conditions. This has the potential to reveal adaptation mechanisms, which cells are using to adjust to rapidly fluctuating environmental conditions and can identify possible root causes for difficulties maintaining similar process performance at different scales. This understanding is of utmost importance in process validation. Additionally, these simulators also have the potential to be used for selecting cells, which are most robust when encountering changing extracellular conditions. The aim of this review is to summarize recent work in this interesting and promising area with the focus on mammalian bioprocesses, since microbial processes have been extensively reviewed.     

Development,characterization, and application of a 2‐Compartment system to investigate the impact of pH inhomogeneities in large‐scale CHO‐based processes

Large‐scale bioreactors for the production of monoclonal antibodies reach volumes of up to 25 000 L. With increasing bioreactor size, mixing is however affected negatively, resulting in the formation of gradients throughout the reactor. These gradients can adversely affect process performance at large scale. Since mammalian cells are sensitive to changes in pH, this study investigated the effects of pH gradients on process performance. A 2‐Compartment System was established for this purpose to expose only a fraction of the cell population to pH excursions and thereby mimicking a large‐scale bioreactor. Cells were exposed to repeated pH amplitudes of 0.4 units (pH 7.3), which resulted in decreased viable cell counts, as well as the inhibition of the lactate metabolic shift. These effects were furthermore accompanied by increased absolute lactate levels. Continuous assessment of molecular attributes of the expressed target protein revealed that subunit assembly or N‐glycosylation patterns were only slightly influenced by the pH excursions. The exposure of more cells to the same pH amplitudes further impaired process performance, indicating this is an important factor, which influences the impact of pH inhomogeneity. This knowledge can aid in the design of pH control strategies to minimize the effects of pH inhomogeneity in large‐scale bioreactors.     

Provenance- and life-history stage-specific responses of the dwarf shrub Calluna vulgaris to elevated vapour pressure deficit

Climate change may alter microscale-effective ecosystem properties such as atmospheric water vapour pressure, but consequences for plant growth are insufficiently understood. Within a northwest German heathland an open-top chamber experiment was established to analyse the effects of elevated vapour pressure deficit (eVPD) on growth responses of Calluna vulgaris considering both plant origin (Atlantic (AP), sub-Atlantic (SAP), sub-Continental (SCP)) and life-history stage (1-year vs. 10-year old plants). We hypothesised that the plants’ sensitivity to eVPD decreases (i) from AP to SCP and (ii) with progressing life-history stage. Elevated VPD caused a provenance-specific decrease of shoot increment whilst aboveground biomass productivity remained unaffected. AP and SAP responded with increasing belowground biomass δ¹³C signatures to eVPD, whereas δ¹³C values decreased for SCP. Moreover, eVPD increased and decreased belowground biomass δ¹³C signatures of 1- and 10-year old plants, respectively. These responses to eVPD were related to differences in morphological-chemical traits and the plants’ trait plasticity in response to eVPD. SCP showed the highest aboveground tissue mass density and significantly increased tissue C:N ratios under eVPD. One-year old plants had a tenfold higher shoot:root ratio than 10-year old plants, making young plants more sensitive to eVPD. Our findings demonstrate that the atmospheric water status affects the morphology and physiology of Calluna independent of the soil water status. The results have implications for the conservation of heathlands under climate change: (i) SCP may constitute an appropriate ecotype for assisted migration-approaches, and (ii) management needs to weigh different options for heathland rejuvenation.

     

Comparing diversity levels in environmental samples: DNA sequence capture and metabarcoding approaches using 18S and COI genes

Environmental DNA studies targeting multiple taxa using metabarcoding provide remarkable insights into levels of species diversity in any habitat. The main drawbacks are the presence of primer bias and difficulty in identifying rare species. We tested a DNA sequence‐capture method in parallel with the metabarcoding approach to reveal possible advantages of one method over the other. Both approaches were performed using the same eDNA samples and the same 18S and COI regions, followed by high throughput sequencing. Metabarcoded eDNA libraries were PCR amplified with one primer pair from 18S and COI genes. DNA sequence‐capture libraries were enriched with 3,639 baits targeting the same gene regions. We tested amplicon sequence variants (ASVs) and operational taxonomic units (OTUs) in silico approaches for both markers and methods, using for this purpose the metabarcoding data set. ASVs methods uncovered more species for the COI gene, whereas the opposite occurred for the 18S gene, suggesting that clustering reads into OTUs could bias diversity richness especially using 18S with relaxed thresholds. Additionally, metabarcoding and DNA sequence‐capture recovered 80%–90% of the control sample species. DNA sequence‐capture was 8x more expensive, nonetheless it identified 1.5x more species for COI and 13x more genera for 18S than metabarcoding. Both approaches offer reliable results, sharing ca. 40% species and 72% families and retrieve more taxa when nuclear and mitochondrial markers are combined. eDNA metabarcoding is quite well established and low‐cost, whereas DNA‐sequence capture for biodiversity assessment is still in its infancy, is more time‐consuming but provides more taxonomic assignments.     

Single and combined effects of Drosophila suzukii and Drosophila melanogaster on sour rot development in viticulture

Sour rot is a disease complex that causes serious damage in viticulture. The common vinegar fly Drosophila melanogaster (Diptera: Drosophilidae) is associated with sour rot in overripe or otherwise damaged grapes. Drosophila suzukii (Diptera: Drosophilidae) is an invasive species, which is suspected to induce sour rot in previously undamaged grapes due to the flies' ability to infest healthy, undamaged soft fruits with its serrated ovipositor. As a consequence, infection of healthy grapes by D. suzukii may facilitate the colonization by D. melanogaster. We investigated the single and combined effects of D. suzukii and D. melanogaster on sour rot development by measuring volatile acidity under near-natural conditions in the vineyard, along with laboratory experiments under controlled climate. In 2017, the combined field and laboratory experiments suggested that the presence of D. suzukii and D. melanogaster increased the volatile acidity levels at a similar rate. In 2018, the field experiments showed an only marginal increase in sour rot development in treatments with both Drosophila species. Under more favourable laboratory conditions, the presence of D. suzukii, but not D. melanogaster triggered sour rot emergence. A facilitating effect of D. suzukii infestation for D. melanogaster was not detectable. These findings suggest that D. suzukii does in fact have the potential to trigger sour rot, but will probably rarely do so under field conditions in the vineyard, at least in the studied region. Instead, our study showed that D. melanogaster can have a similar impact on sour rot development as D. suzukii, emphasizing the need of comparative studies.