Functional analysis of cell-free-produced human endothelin B receptor reveals transmembrane segment 1 as an essential area for ET-1 binding and homodimer formation

The functional and structural characterization of G-protein-coupled receptors (GPCRs) still suffers from tremendous difficulties during sample preparation. Cell-free expression has recently emerged as a promising alternative approach for the synthesis of polytopic integral membrane proteins and, in particular, for the production of G-protein-coupled receptors. We have now analyzed the quality and functional folding of cell-free produced human endothelin type B receptor samples as an example of the rhodopsin-type family of G-protein-coupled receptors in correlation with different cell-free expression modes. Human endothelin B receptor was cell-free produced as a precipitate and subsequently solubilized in detergent, or was directly synthesized in micelles of various supplied mild detergents. Purified cell-free-produced human endothelin B receptor samples were evaluated by single-particle analysis and by ligand-binding assays. The soluble human endothelin B receptor produced is predominantly present as dimeric complexes without detectable aggregation, and the quality of the sample is very similar to that of the related rhodopsin isolated from natural sources. The binding of human endothelin B receptor to its natural peptide ligand endothelin-1 is demonstrated by coelution, pull-down assays, and surface plasmon resonance assays. Systematic functional analysis of truncated human endothelin B receptor derivatives confined two key receptor functions to the membrane-localized part of human endothelin B receptor. A 39 amino acid fragment spanning residues 93-131 and including the proposed transmembrane segment 1 was identified as a central area involved in endothelin-1 binding as well as in human endothelin B receptor homo-oligomer formation. Our approach represents an efficient expression technique for G-protein-coupled receptors such as human endothelin B receptor, and might provide a valuable tool for fast structural and functional characterizations.     

Enhanced CHO Clone Screening: Application of Targeted Locus Amplification and Next‐Generation Sequencing Technologies for Cell Line Development

Early analytical clone screening is important during Chinese hamster ovary (CHO) cell line development of biotherapeutic proteins to select a clonally derived cell line with most favorable stability and product quality. Sensitive sequence confirmation methods using mass spectrometry have limitations in throughput and turnaround time. Next‐generation sequencing (NGS) technologies emerged as alternatives for CHO clone analytics. We report an efficient NGS workflow applying the targeted locus amplification (TLA) strategy for genomic screening of antibody expressing CHO clones. In contrast to previously reported RNA sequencing approaches, TLA allows for targeted sequencing of genomic integrated transgenic DNA without prior locus information, robust detection of single‐nucleotide variants (SNVs) and transgenic rearrangements. During clone selection, TLA/NGS revealed CHO clones with high‐level SNVs within the antibody gene and we report in another case the utility of TLA/NGS to identify rearrangements at transgenic DNA level. We also determined detection limits for SNVs calling and the potential to identify clone contaminations by TLA/NGS. TLA/NGS also allows to identify genetically identical clones. In summary, we demonstrate that TLA/NGS is a robust screening method useful for routine clone analytics during cell line development with the potential to process up to 24 CHO clones in less than 7 workdays.     

Bears benefit plants via a cascade with both antagonistic and mutualistic interactions

Predators can influence primary producers by generating cascades of effects in ecological webs. These effects are often non‐intuitive, going undetected because they involve many links and different types of species interactions. Particularly, little is understood about how antagonistic (negative) and mutualistic (positive) interactions combine to create cascades. Here, we show that black bears can benefit plants by consuming ants. The ants are mutualists of herbivores and protect herbivores from other arthropod predators. We found that plants near bear‐damaged ant nests had greater reproduction than those near undamaged nests, due to weaker ant protection for herbivores, which allowed herbivore suppression by arthropod predators. Our results highlight the need to integrate mutualisms into trophic cascade theory, which is based primarily on antagonistic relationships. Predators are often conservation targets, and our results suggest that bears and other predators should be managed with the understanding that they can influence primary producers through many paths.     

TRAIL+ monocytes and monocyte‐related cells cause lung damage and thereby increase susceptibility to influenza–Streptococcus pneumoniae coinfection

Streptococcus pneumoniae coinfection is a major cause of influenza-associated mortality; however, the mechanisms underlying pathogenesis or protection remain unclear. Using a clinically relevant mouse model, we identify immune-mediated damage early during coinfection as a new mechanism causing susceptibility. Coinfected CCR2^−/− mice lacking monocytes and monocyte-derived cells control bacterial invasion better, show reduced epithelial damage and are overall more resistant than wild-type controls. In influenza-infected wild-type lungs, monocytes and monocyte-derived cells are the major cell populations expressing the apoptosis-inducing ligand TRAIL. Accordingly, anti-TRAIL treatment reduces bacterial load and protects against coinfection if administered during viral infection, but not following bacterial exposure. Post-influenza bacterial outgrowth induces a strong proinflammatory cytokine response and massive inflammatory cell infiltrate. Depletion of neutrophils or blockade of TNF-α facilitate bacterial outgrowth, leading to increased mortality, demonstrating that these factors aid bacterial control. We conclude that inflammatory monocytes recruited early, during the viral phase of coinfection, induce TRAIL-mediated lung damage, which facilitates bacterial invasion, while TNF-α and neutrophil responses help control subsequent bacterial outgrowth. We thus identify novel determinants of protection versus pathology in influenza–Streptococcus pneumoniae coinfection.     

Enantioselective degradation of dufulin pesticide in water: Uptake,thermodynamics, and kinetics studies

Kudzu (Pueraria thunbergiana) plant extract impregnated sediments were used for abiotic and biotic uptakes and biodegradation. The optimized conditions were 25 μg L^?1 concentration, 7 days for abiotic uptake and 56 days for biotic uptake and biodegradation, dose 2 g L^?1, 7 pH, and 35°C temperature. The amount removed of dufulin was 32.6% in abiotic conditions while these were 90% in the case of biotic uptake and biodegradation. Enantioselective biodegradation indicated that S‐(+)‐enantiomer degraded faster (90%) than R‐(?)‐enantiomer (87%). The data for abiotic and biotic uptakes and biodegradation followed well Langmuir, thermodynamics, and kinetics models. All these processes followed pseudo first‐order kinetics. It was observed that biodegradation was three times responsible for dufulin removal than simple sorption uptake (abiotic and biotic). The abiotic and biotic uptakes and biodegradation were quite fast and endothermic nature. The developed method may be used to remove the racemic and enantiomeric dufulin in water.     

Entomopathogenic fungi naturally infecting the eucalypt bronze bug,Thaumastocoris peregrinus (Heteroptera: Thaumastocoridae), in Uruguay

Thaumastocoris peregrinus Carpintero & Dellapé (Heteroptera: Thaumastocoridae) is a sap‐sucking insect that feeds on leaves of Eucalyptus. In Uruguay, it was detected in 2008 causing significant economic losses in Eucalyptus plantations. At present, there is no efficient control for this pest; thus, the use of biological control agents seems to be an environmentally friendly alternative to reduce the damage caused by this insect. The aims of this study were to isolate and identify the species of entomopathogenic fungi that naturally infect T. peregrinus in Uruguay and to characterize and select the most virulent isolates towards this pest. Individuals of T. peregrinus were collected in eight Eucalyptus plantations infested by the pest. The entomopathogenic fungi were isolated and identified by observation of their micromorphological characteristics, and their identity was confirmed by molecular methods. The pathogenicity and virulence against T. peregrinus of the isolated entomopathogenic fungi were evaluated. Isolates causing the highest insect mortality were selected to evaluate the effect of temperature and water activity on conidial viability. Entomopathogenic fungi were found in very low number with the prevalence of less than 3%. Isolates corresponded to Beauveria, Fusarium, Isaria, Lecanicillium, Paecilomyces, Pochonia, Purpureocillium and Simplicillium genera. Two species, B. pseudobassiana and L. muscarium, were first recorded in Uruguay. Among all the tested species, an isolate of B. bassiana (FI 2403) showed the highest virulence followed by an isolate of B. pseudobassiana. The isolate of B. bassiana presented the highest percentage of spore germination at the three temperatures and the highest viability at low water activities. Isolate FI 2403 was selected as a promissory candidate for the development of a commercial formulation against T. peregrinus.