Effects of spring temperature and volcanic eruptions on wader productivity

Key demographic parameters often show substantial annual variation that can have important consequences for rates of population growth. Since 2011 we have conducted annual estimates of the productivity of Icelandic Black‐tailed Godwits Limosa limosa islandica over a large part of their breeding range. During this period, a volcanic eruption resulted in extensive dust deposition across the region. We show that Godwit productivity varies with spring temperatures but in the year of the volcanic eruption, productivity was reduced to almost zero. This rare but extreme event is likely to have had only a short‐term influence, whereas ongoing warming of sub‐Arctic regions is potentially a more substantial driver of the continued growth of this population.     

Identification of an IgG CDR sequence contributing to co‐purification of the host cell protease cathepsin D

Recombinant therapeutic monoclonal antibodies (mAbs) must be purified from host cell proteins (HCPs), DNA, and other impurities present in Chinese hamster ovary (CHO) cell culture media. HCPs can potentially result in adverse clinical responses in patients and, in specific cases, have caused degradation of the final mAb product. As reported previously, residual traces of cathepsin D caused particle formation in the final product of mAb‐1. The current work was focused on identification of a primary sequence in mAb‐1 responsible for the binding and consequent co‐purification of trace levels of CHO cathepsin D. Surface plasmon resonance (SPR) was used to detect binding between immobilized CHO cathepsin D and a panel of mAbs. Out of 13 mAbs tested, only mAb‐1 and mAb‐6 bound to cathepsin D. An LYY motif in the HC CDR2 was common, yet unique, to only these two mAbs. Mutation of LYY to AAA eliminated binding of mAb‐1 to cathepsin D providing confirmation that this sequence motif was involved in the binding to CHO cathepsin D. Interestingly, the binding between mAb‐1 and cathepsin D was weaker than that of mAb‐6, which may be related to the fact that two aspartic acid residues near the LYY motif in mAb‐1 are replaced with neutral serine residues in mAb‐6. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:140–145, 2017     

Biophysical characterization of the catalytic domain of guanine nucleotide exchange factor BopE from Burkholderia pseudomallei

BopE is a type III secreted protein from Burkholderia pseudomallei, the aetiological agent of melioidosis. Like its Salmonella homologues SopE and SopE2, BopE is a guanine nucleotide exchange factor for Rho GTPases. It is thought that, in order to be secreted by the type III system, proteins must be unfolded or only partially folded. As part of a study of B. pseudomallei virulence proteins, we have expressed, purified and characterized the catalytic domain of BopE (amino acids 78–261). Analytical ultracentrifugation experiments in conjunction with analytical size exclusion chromatography show that BopE_78–261 is monomeric in aqueous solution. CD spectroscopy indicates that the protein is predominantly α-helical, with predicted secondary structure composition of 59% α-helix and 7% β-strand. NMR spectroscopy confirms that BopE_78–261 adopts a single, stable conformation. In differential scanning calorimetry experiments, thermal denaturation of BopE_78–261 (T_m 52 °C) is reversible. Also, the secondary structure composition of BopE_78–261 changes little over a range of pH values from 3.5 to 10.5. BopE may therefore fold spontaneously to a functional form upon secretion into the host cell cytoplasm, and retains a native or native-like fold in varied environments. These properties are likely to be advantageous for a secreted bacterial effector protein.     

Composition and Diversity of Microbial Communities Recovered from Surrogate Minerals Incubated in an Acidic Uranium-Contaminated Aquifer

Our understanding of subsurface microbiology is hindered by the inaccessibility of this environment, particularly when the hydrogeologic medium is contaminated with toxic substances. In this study, surrogate geological media contained in a porous receptacle were incubated in a well within the saturated zone of a pristine region of an aquifer to capture populations from the extant communities. After an 8-week incubation, the media were recovered, and the microbial community that developed on each medium was compared to the community recovered from groundwater and native sediments from the same region of the aquifer, using 16S DNA coding for rRNA (rDNA)-based terminal restriction fragment length polymorphism (T-RFLP). The groundwater and sediment communities were highly distinct from one another, and the communities that developed on the various media were more similar to groundwater communities than to sediment communities. 16S rDNA clone libraries of communities that developed on particles of a specular hematite medium incubated in the same well as the media used for T-RFLP analysis were compared with those obtained from an acidic, uranium-contaminated region of the same aquifer. The hematite-associated community formed in the pristine area was highly diverse at the species level, with 25 distinct phylotypes identified, the majority of which (73%) were affiliated with the β-Proteobacteria. Similarly, the hematite-associated community formed in the contaminated area was populated in large part by β-Proteobacteria (62%); however, only 13 distinct phylotypes were apparent. The three numerically dominant clones from the hematite-associated community from the contaminated site were affiliated with metal- and radionuclide-tolerant or acidophilic taxa, consistent with the environmental conditions. Only two populations were common to both sites.     

Longevity and turnover of roots in the shortgrass steppe: influence of diameter and depth

We used minirhizotrons to determine patterns of root longevity andturnover for the perennial bunchgrass Bouteloua gracilisinthe shortgrass steppe of eastern Colorado, USA. We hypothesized that rootlongevity would be partially controlled by root diameter, following previouslyobserved patterns in woody plants. In addition, we hypothesized that rootturnover would be greatest in surface soil horizons and decrease with depth dueto variation in soil moisture availability and temperature. Root longevity wascorrelated with root diameter. Median life span of roots > 0.4mm was approximately 320 days, while roots < 0.2mmhad a median life span of 180 days. There was approximately a 6%decreasein the likelihood of mortality with a 0.10-mm increase inroot diameter, controlling for the effect of depth in the soil profile. Rootlength production and mortality were highest in the upper20 cm of the soil profile and decreased with depth.However,because root length density also decreased with depth, there were nosignificantdifferences in turnover rate of root length among sampling intervals. Turnoverwas approximately 0.86 yr^–1 based on root length production,while turnover was 0.35 yr^–1 using root length mortality as ameasurement of flux. The imbalance between turnover estimates may be aconsequence of the time the minirhizotrons were in place prior to imaging or mayresult from our lack of over-winter measures of mortality. Our worksuggests that Bouteloua gracilis roots have complex lifehistory strategies, similar to woody species. Some portion of the root systemishighly ephemeral, while slightly larger roots persist much longer. Thesedifferences have implications for belowground carbon and nitrogen cycles in theshortgrass steppe.     

The dynamics of prey choice in fish: the importance of prey size and satiation

Over a number of decades the process of prey choice has been investigated using fishes as model predators. Using fishes for the model has allowed the proximate factors that determine how a mobile predator finds and chooses to eat the prey encountered within a variable 3‐D environment to be estimated. During prey choice a number of constraints exist, in particular most fish predators will eat their prey whole thus their jaws and gut create functional limitations once a prey has been attacked. By considering the relationship between the size of the prey and the predator's feeding apparatus and feeding motivation this study explores the link between mechanistic studies and theoretical, optimal foraging based predictions. How the prediction of prey choices made by the fish following prey encounter can be reconciled with what is likely to be found in the fish's stomach is discussed. This study uses a progression of empirical examples to illustrate how the limits of functional constraints and prey choice at different stages of motivation to feed can be taken into account to improve predictions of predator prey choice.     

Effect of soil moisture at different temperatures on Rhizoctonia root rot of wheat seedlings

The effect of soil moisture at different temperatures on root rot of wheat seedlings caused by Rhizoctonia solani AG-8 was studied in temperature controlled water tanks under glasshouse conditions. Four moisture levels (15, 30, 50 and 75% of soil water holding capacity at saturation which were equal to –10, –7, –5 and –3 kPa, respectively) were tested in tanks maintained 10, 15, 20 or 25 °C. The role of microbial activity in the effect of soil moisture and temperature on disease severity was also studied by including treatments of steam treated soil. Results showed that at soil moisture levels optimum for plant growth (50 and 75% WHC) disease was more severe at a lower temperature (10 °C), but under relatively dry conditions (15% WHC) disease levels were similar at all temperatures tested. In warm soils (20 and 25 °C) at high soil moisture levels (50 and 75% WHC), disease was more severe in steam treated soil than in non-steam treated soil, indicating that the suppression of disease in natural soil under these conditions was associated with high soil microbial activity.     

Structural features of LIM kinase that control effects on the actin cytoskeleton

LIM kinase phosphorylates and inactivates the actin binding/depolymerizing factor cofilin and induces actin cytoskeletal changes. Several unique structural features within LIM kinase were investigated for their roles in regulation of LIM kinase activity. Disruption of the second LIM domain or the PDZ domain or deletion of the entire amino terminus increased activity in vivo measured as increasing aggregation of the actin cytoskeleton. A kinase-deleted alternate splice product was identified and characterized. This alternate splice product and a kinase inactive mutant inhibited LIM kinase in vivo, indicating that the amino terminus suppresses activity of the kinase domain. Mutation of threonine 508 in the activation loop to valine abolished activity whereas replacement with 2 glutamic acid residues resulted in a fully active enzyme. Dephosphorylation of LIM kinase inhibited cofilin phosphorylation. Mutation of the basic insert in the activation loop inhibited activity in vivo, but not in vitro. These results indicate phosphorylation is an essential regulatory feature of LIM kinase and indicate that threonine 508 and the adjacent basic insert sequences of the activation loop are required for this process. A combination of structural features are thus involved in receiving upstream signals that regulate LIM kinase-induced actin cytoskeletal reorganization.