Nitrogen Fertilization and Native C4 Grass Species Alter Abundance, Activity, and Diversity of Soil Diazotrophic Communities

Native C4 grasses have become the preferred species for native perennial pastures and bioenergy production due to their high productivity under low soil nitrogen (N) status. One reason for their low N requirement is that C4 grasses may benefit from soil diazotrophs and promote biological N fixation. Our objective was to evaluate the impact of N fertilization rates (0, 67, and 202 kg N ha−1 ) and grass species (switchgrass [Panicum virgatum] and big bluestem [Andropogon gerardii]) on the abundance, activity, diversity, and community composition of soil diazotrophs over three agricultural seasons (grass green-up, initial harvest, and second harvest) in a field experiment in East Tennessee, United States. Nitrogen fertilization rate had a stronger influence on diazotroph population size and activity (determined by nifH gene and transcript abundances) and community composition (determined by nifH gene amplicon sequencing) than agricultural season or grass species. Excessive fertilization (202 kg N ha−1 ) resulted in fewer nifH transcripts compared to moderate fertilization (67 kg N ha−1 ) and decreased both richness and evenness of diazotrophic community, reflecting an inhibitory effect of high N application rates on soil diazotrophic community. Overall, cluster I and cluster III diazotrophs were dominant in this native C4 grass system. Diazotroph population size and activity were directly related to soil water content (SWC) based on structural equation modeling. Soil pH, SWC, and C and N availability were related to the variability of diazotrophic community composition. Our results revealed relationships between soil diazotrophic community and associated soil properties, adding to our understanding of the response of soil diazotrophs to N fertilization and grass species in native C4 grass systems

High-Throughput, Multispecies, Parallelized Plasma Stability Assay for the Determination and Characterization of Antibody–Drug Conjugate Aggregation and Drug Release

The stability of antibody–drug conjugates (ADCs) in circulation is critical for maximum efficacy and minimal toxicity. An ADC reaching the intended target intact can deliver the highest possible drug load to the tumor and reduce off-target toxicity from free drug in the blood. As such, assessment of ADC stability is a vital piece of data during development. However, traditional ADC stability assays can be manually intensive, low-throughput, and require large quantities of ADC material. Here, we introduce an automated, high-throughput plasma stability assay for screening drug release and aggregation over 144 h for up to 40 ADCs across five matrices simultaneously. The amount of ADC material during early drug development is often limited, so this assay was implemented in 384-well format to minimize material requirements to <100 μg of each ADC and 100 μL of plasma per species type. Drug release and aggregation output were modeled using nonlinear regression equations to calculate formation rates for each data type. A set of 15 ADCs with different antibodies and identical valine–citrulline–<i>p</i>-aminobenzylcarbamate–monomethylauristatin E linker-drug payloads was tested and formation rates were compared across ADCs and between species, revealing several noteworthy trends. In particular, a wide range in aggregation was found when altering only the antibody, suggesting a key role for plasma stability screening early in the development process to find and remove antibody candidates with the potential to create unstable ADCs. The assay presented here can be leveraged to provide stability data on new chemistry and antibody screening initiatives, select the best candidate for in vivo studies, and provide results that highlight stability issues inherent to particular ADC designs throughout all stages of ADC development

Interleukin-10 modulates the synthesis of inflammatory mediators in the sensory circumventricular organs: implications for the regulation of fever and sickness behaviors

<p>Abstract</p> <p>Background</p> <p>Whereas the role played by interleukin (IL)-10 in modulating fever and sickness behavior has been linked to it targeting the production of pro-inflammatory cytokines in the circulation, liver and spleen, it is not known whether it could directly target the local production of pro-inflammatory cytokines within the sensory circumventricular organs (CVOs) situated within the brain, but outside the blood–brain barrier. Using inactivation of IL-10, we, therefore, investigated whether IL-10 could modulate the synthesis of pro-inflammatory cytokines within the sensory CVOs, in particular the <it>organum vasculosum laminae terminalis</it> (OVLT) and <it>area postrema</it> (AP).</p> <p>Findings</p> <p>Primary OVLT and AP microcultures were established from topographically excised rat pup brain tissue. The microcultures were pretreated with either IL-10 antibodies (AB) (10 μl/350 μl medium) or phosphate-buffered saline (PBS) (10 μl/350 μl medium) before being incubated with lipopolysaccharide (LPS) (100 μg/ml) or PBS in complete medium for 6 h. Supernatants were removed from the microcultures after 6 h of incubation with LPS and used for the determination of IL-6 and tumor necrosis factor (TNF)-α. Pre-treating the OVLT and AP microcultures with IL-10 antibodies significantly enhanced the LPS-induced increase in TNF-α and IL-6 in the supernatant obtained from the microcultures.</p> <p>Conclusions</p> <p>Our results show for the first time that the LPS-induced release of pro-inflammatory cytokines in cells cultured from the AP and OVLT can be modulated in the presence of IL-10 antibodies. Thus, we have identified that the sensory CVOs may have a key role to play in both the initiation and modulation of neuroinflammation.</p