Synonymous Codons and Hydrophobicity Optimization of Post-translational Signal Peptide PelB Increase Phage Display Efficiency of DARPins

DsbA leader peptide targets proteins for cotransla-tional translocation by signal recognition particle (SRP) pathway and has been the standard signal sequence for filamentous phage display of fast-folding Designed Ankyrin Repeat Proteins (DARPins). In contrast, translocation of DARPins via the post-translational pathway, for example, with the commonly used PelB leader, has been reported to be highly inefficient. In this study, two PelB signal sequence libraries were screened covering different regions of the leader peptide for identifying mutants with improved display of DARPins on phage. A PelB variant with the most favorable combination of synonymous mutations in the n-region and hydrophobic substitutions in the h-region increased the display efficiency of a DARPin library 44-and 12-fold compared to PelBWT and DsbA, respectively. Based on thioredoxin-1 (TrxA) export studies the triple valine mutant PelB DN5 V3 leader was capable of more efficient cotranslational translocation than PelBWT, but the overall display efficiency improvement over DsbA suggests that besides increased cotranslational translocation other factors contribute to the observed enhancement in DARPin display efficiency

Maternal IL-1β Production Prevents Lung Injury in a Mouse Model of Bronchopulmonary Dysplasia

Little is known about the influence of maternal inflammation on neonatal outcome. Production of IL-1β in the lungs of newborn infants is associated with bronchopulmonary dysplasia. Using bitransgenic (bi-TG) mice in which human (h) IL-1β is expressed with a doxycycline-inducible system controlled by the Clara cell secretory protein promoter, we have shown that hIL-1β expression causes a bronchopulmonary dysplasia–like illness in infant mice. To study the hypothesis that maternal hIL-1β production modifies the response of the newborn to hIL-1β, doxycycline was administered to bi-TG and control dams from Embryonic Day 0, inducing production of hIL-1β by the bi-TG dams before hIL-1β production started in their bi-TG fetuses, or from Embryonic Day 15, inducing simultaneous production of hIL-1β by both the bi-TG dams and their bi-TG fetuses. In addition to the lungs, hIL-1β was expressed at low levels in the uteri of bi-TG dams. Maternal inflammation preceding fetal inflammation increased the survival and growth of hIL-1β–expressing pups, enhanced alveolarization, and protected the airways against remodeling and goblet cell hyperplasia. Maternal hIL-1β production preceding fetal hIL-1β production caused silencing of several inflammatory genes, including CXC and CC chemokines, murine IL-1β, serum amyloid A3, and Toll-like receptors 2 and 4, and suppressed the expression of chitinase-like lectins Ym1 and Ym2 in the lungs of infant mice. Maternal inflammation protects the newborn against subsequent hIL-1β–induced lung inflammation and injury. In contrast, induction of hIL-1β production simultaneously in bi-TG dams and their fetuses offered no protection against inflammatory lung disease in the neonate

IL-1β Disrupts Postnatal Lung Morphogenesis in the Mouse

Pulmonary inflammation and increased production of the inflammatory cytokine IL-1β are associated with the development of bronchopulmonary dysplasia (BPD) in premature infants. To study the actions of IL-1β in the fetal and newborn lung in vivo, we developed a bitransgenic mouse in which IL-1β is expressed under conditional control in airway epithelial cells. Perinatal pulmonary expression of IL-1β caused respiratory insufficiency that was associated with increased postnatal mortality. While intrauterine growth of IL-1β–expressing mice was normal, their postnatal growth was impaired. IL-1β disrupted alveolar septation and caused abnormalities in α-smooth muscle actin and elastin deposition in the septa of distal airspaces. IL-1β disturbed capillary development and inhibited the production of vascular endothelial growth factor in the lungs of infant mice. IL-1β induced the expression of CXC chemokines KC (CXCL1) and macrophage inflammatory protein-2 (CXCL2) and of CC chemokines monocyte chemotactic protein (MCP)-1 (CCL2) and MCP-3 (CCL7), consistent with neutrophilic and monocytic infiltration of the lungs. IL-1β caused goblet cell metaplasia and bronchial smooth muscle hyperplasia. Perinatal expression of IL-1β in epithelial cells of the lung caused a lung disease that was clinically and histologically similar to BPD

Synonymous Codons and Hydrophobicity Optimization of Post-translational Signal Peptide PelB Increase Phage Display Efficiency of DARPins

DsbA leader peptide targets proteins for cotranslational translocation by signal recognition particle (SRP) pathway and has been the standard signal sequence for filamentous phage display of fast-folding Designed Ankyrin Repeat Proteins (DARPins). In contrast, translocation of DARPins via the post-translational pathway, for example, with the commonly used PelB leader, has been reported to be highly inefficient. In this study, two PelB signal sequence libraries were screened covering different regions of the leader peptide for identifying mutants with improved display of DARPins on phage. A PelB variant with the most favorable combination of synonymous mutations in the n-region and hydrophobic substitutions in the h-region increased the display efficiency of a DARPin library 44- and 12-fold compared to PelBWT and DsbA, respectively. Based on thioredoxin-1 (TrxA) export studies the triple valine mutant PelB DN5 V3 leader was capable of more efficient cotranslational translocation than PelBWT, but the overall display efficiency improvement over DsbA suggests that besides increased cotranslational translocation other factors contribute to the observed enhancement in DARPin display efficiency

Inflammatory cytokines in gastric fluid at birth and the development of bronchopulmonary dysplasia

Aim:  To assess whether the levels of inflammatory and anti-inflammatory proteins in gastric fluid of premature infants shortly after birth are associated with the development of bronchopulmonary dysplasia (BPD). Methods:  Gastric fluid retrieved within 1 h of birth of premature infants (gestational age <29 weeks) was analysed for interleukin (IL)-8, growth-related oncogene (Gro)-α, epithelial cell-derived neutrophil-activating peptide (ENA)-78, IL-1β and Clara cell secretory protein with ELISA. Results:  Of 51 enrolled infants, 86% had BPD. Of these, 54% had mild BPD, 30% had moderate BPD and 16% had severe BPD. Clinical chorioamnionitis was associated with high levels of IL-8, Gro-α, Epithelial cell-derived neutrophil-activating peptide-78 (ENA-78) and IL-1β in gastric fluid. Gastric fluid levels of IL-8, Gro-α, ENA-78 and IL-1β were higher in infants with moderate or severe BPD than in those with no or mild BPD. Ligation of the patent ductus arteriosus was associated with the development of moderate or severe BPD. These associations were no longer significant after adjustment for gestational age. Conclusion:  The levels of inflammatory mediators in gastric fluid samples retrieved soon after birth from intubated or nonintubated infants can be used to assess the infants' perinatal exposure to inflammatory mediators and its association with neonatal outcome